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Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes severe viral hemorrhagic fever and thrombocytopenia syndrome with a fatality rate of up to 30%. No licensed vaccines or therapeutics are currently available for humans. Here, we develop seven monoclonal antibodies (mAbs) against SFTSV surface glycoprotein Gn. Mechanistic studies show that three neutralizing mAbs (S2A5, S1G3, and S1H7) block multiple steps during SFTSV infection, including viral attachment and membrane fusion, whereas another neutralizing mAb (B1G11) primarily inhibits the viral attachment step. Epitope binning and X-ray crystallographic analyses reveal four distinct antigenic sites on Gn, three of which have not previously been reported, corresponding to domain I, domain II, and spanning domain I and domain II. One of the most potent neutralizing mAbs, S2A5, binds to a conserved epitope on Gn domain I and broadly neutralizes infection of six SFTSV strains corresponding to genotypes A to F. A single dose treatment of S2A5 affords both pre- and post-exposure protection of mice against lethal SFTSV challenge without apparent weight loss. Our results support the importance of glycoprotein Gn for eliciting a robust humoral response and pave a path for developing prophylactic and therapeutic antibodies against SFTSV infection.
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Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos , Phlebovirus , Febre Grave com Síndrome de Trombocitopenia , Animais , Phlebovirus/imunologia , Camundongos , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Febre Grave com Síndrome de Trombocitopenia/imunologia , Febre Grave com Síndrome de Trombocitopenia/virologia , Febre Grave com Síndrome de Trombocitopenia/prevenção & controle , Humanos , Epitopos/imunologia , Feminino , Camundongos Endogâmicos BALB C , Proteínas do Envelope Viral/imunologia , Cristalografia por Raios X , Chlorocebus aethiops , Glicoproteínas/imunologia , Células VeroRESUMO
INTRODUCTION: malnutrition is a very frequent problem in oncology patients and can have serious repercussions. Adequate nutritional management is cost-effective in terms of health and survival in this population, but it requires multidisciplinary coordination, specific training, and continuous follow-up. OBJECTIVE: to validate the applicability and efficacy of a multidisciplinary nutritional support protocol in oncology patients. METHODS: a multidisciplinary nutritional protocol was developed for oncology patients, with guidelines for screening and assessment of malnutrition, treatment, re-evaluation, and management of side effects, as well as guidance on supplementation and eating patterns. The protocol would be implemented in various clinical centers, collecting data through a structured questionnaire, registering variables before and after implementation. RESULTS: the protocol and its impact were implemented and evaluated in 39 centers. An improvement in nutritional care was observed, evidenced by an earlier initiation of nutritional assessment and an increase in the number of patients receiving adequate care following the protocol implementation. Problems related to inadequate malnutrition coding in the centers, limited resources, and the need for greater interdepartmental collaboration were identified. CONCLUSIONS: the conduct of this study provides insights into how the implementation of a multidisciplinary nutritional support protocol can improve the nutritional care received by patients and informs about the main obstacles to adequate implementation.
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ETHNOPHARMACOLOGICAL RELEVANCE: Minimal persistent inflammation (MPI) is a major contributor to the recurrence of allergic rhinitis (AR). The traditional Chinese herbal medicine known as Bimin Kang Mixture (BMK) have been used in clinics for decades to treat AR, which can relieve AR symptoms, reduce inflammatory response and improve immune function. However, its mechanism in controlling MPI is still unclear. AIM OF THE STUDY: This study aims to assess the therapeutic effect of BMK on MPI, and elaborate the mechanism involved in BMK intervention in BCL11B regulation of type 2 innate lymphoid cell (ILC2) plasticity in the treatment of MPI. MATERIAL AND METHODS: The effect of BMK (9.1 ml/kg) and Loratadine (15.15 mg/kg) on MPI was evaluated based on symptoms, pathological staining, and ELISA assays. RT-qPCR and flow cytometry were also employed to assess the expression of BCL11B, IL-12/IL-12Rß2, and IL-18/IL-18Rα signaling pathways associated with ILC2 plasticity in the airway tissues of MPI mice following BMK intervention. RESULTS: BMK restored the airway epithelial barrier, and markedly reduced inflammatory cells (eosinophils, neutrophils) infiltration (P < 0.01) and goblet cells hyperplasia (P < 0.05). BCL11B expression positively correlated with the ILC2 proportion in the lungs and nasal mucosa of AR and MPI mice (P < 0.01). BMK downregulated BCL11B expression (P < 0.05) and reduced the proportion of ILC2, ILC3 and ILC3-like ILC2 subsets (P < 0.05). Moreover, BMK promoted the conversion of ILC2 into an ILC1-like phenotype through IL-12/IL-12Rß2 and IL-18/IL-18Rα signaling pathways in MPI mice. CONCLUSION: By downregulating BCL11B expression, BMK regulates ILC2 plasticity and decreases the proportion of ILC2, ILC3, and ILC3-like ILC2 subsets, promoting the conversion of ILC2 to ILC1, thus restoring balance of ILC subsets in airway tissues and control MPI.
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Medicamentos de Ervas Chinesas , Linfócitos , Rinite Alérgica , Animais , Rinite Alérgica/tratamento farmacológico , Rinite Alérgica/imunologia , Camundongos , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos Endogâmicos BALB C , Imunidade Inata/efeitos dos fármacos , Inflamação/tratamento farmacológico , Feminino , Masculino , Transdução de Sinais/efeitos dos fármacos , Plasticidade Celular/efeitos dos fármacos , Proteínas Repressoras , Proteínas Supressoras de TumorRESUMO
Heartland virus (HRTV), an emerging tick-borne pathogenic bunyavirus, has been a concern since 2012, with an increasing incidence, expanding geographical distribution, and high pathogenicity in the United States. Infection from HRTV results in fever, thrombocytopenia, and leucopenia in humans, and in some cases, symptoms can progress to severe outcomes, including haemorrhagic disease, multi-organ failure, and even death. Currently, no vaccines or antiviral drugs are available for treatment of the HRTV disease. Moreover, little is known about HRTV-host interactions, viral replication mechanisms, pathogenesis and virulence, further hampering the development of vaccines and antiviral interventions. Here, we aimed to provide a brief review of HRTV epidemiology, molecular biology, pathogenesis and virulence on the basis of published article data to better understand this virus and provide clues for further study.
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Bunyaviridae , Replicação Viral , Humanos , Virulência , Animais , Infecções por Bunyaviridae/virologia , Thogotovirus/patogenicidade , Thogotovirus/genética , Thogotovirus/fisiologia , Estados Unidos/epidemiologia , Interações Hospedeiro-PatógenoRESUMO
Minimally invasive surgery can involve the use of robotics to improve patient outcomes. Some robotic systems require special instruments with a designated number of uses. In China, during the reprocessing of the robotic instruments, health care personnel determined that the existing tracking processes were inadequate. They conducted a quality improvement project with the goal of establishing a barcode-based standardized process for tracking robotic instruments. They implemented technology that generated a unique identifier each time a robotic instrument was reprocessed after use. Nurses scanned the identifier when surgeons used the instrument. The findings included the increased accuracy of use documentation and decreases in untraceable sterilization and use records, charging concerns, and average daily and monthly inventory times. An increase in adverse event reports associated with robotic instruments also was noted. The use of barcode technology for robotic instrument tracking continues at the facility and may be expanded for additional specialty instruments.
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Processamento Eletrônico de Dados , Processamento Eletrônico de Dados/métodos , Humanos , Robótica/instrumentação , Robótica/normas , Melhoria de Qualidade , Procedimentos Cirúrgicos Robóticos/métodos , Procedimentos Cirúrgicos Robóticos/instrumentação , ChinaRESUMO
Osteosarcoma, a common malignant tumor in children, has emerged as a major threat to the life and health of pediatric patients. Presently, there are certain limitations in the diagnosis and treatment methods for this disease, resulting in inferior therapeutic outcomes. Therefore, it is of great importance to study its pathogenesis and explore innovative approaches to diagnosis and treatment. In this study, a non-negative matrix decomposition method was employed to conduct a comprehensive investigation and analysis of aggregated autophagy-related genes within 331,394 single-cell samples of osteosarcoma. Through this study, we have elucidated the intricate communication patterns among various cells within the tumor microenvironment. Based on the classification of aggregated autophagy-related genes, we are not only able to more accurately predict patients' prognosis but also offer robust guidance for treatment strategies. The findings of this study hold promise for breakthroughs in the diagnosis and treatment of osteosarcoma, intervention of aggrephagy is expected to improve the survival rate and quality of life of osteosarcoma patients.
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Neoplasias Ósseas , Osteossarcoma , Humanos , Criança , Macroautofagia , Qualidade de Vida , Microambiente Tumoral/genética , Apoptose , Osteossarcoma/diagnóstico , Osteossarcoma/genética , Comunicação Celular , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/genéticaRESUMO
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high case mortality rates, which is caused by Dabie bandavirus (DBV), a novel pathogen also termed as SFTS virus (SFTSV). Currently, no specific therapeutic drugs or vaccines are available for SFTS. Myxovirus resistance protein A (MxA) has been shown to inhibit multiple viral pathogens; however, the role of MxA in DBV infection is unknown. Here, we demonstrated that DBV stimulates MxA expression which, in turn, restricts DBV infection. Mechanistic target analysis revealed that MxA specifically interacts with the viral nucleocapsid protein (NP) in a manner independent of RNA. Minigenome reporter assay showed that in agreement with its targeting of NP, MxA inhibits DBV ribonucleoprotein (RNP) activity. In detail, MxA interacts with the NP N-terminal and disrupts the interaction of NP with the viral RNA-dependent RNA polymerase (RdRp) but not NP multimerization, the critical activities of NP for RNP formation and function. Furthermore, MxA N-terminal domain was identified as the functional domain inhibiting DBV infection, and, consistently, then was shown to interact with NP and obstruct the NP-RdRp interaction. Additionally, threonine 103 within the N-terminal domain is important for MxA inhibition to DBV, and its mutation (T103A) attenuates MxA binding to NP and obstruction of the NP-RdRp interaction. This study uncovers MxA inhibition of DBV with a series of functional and mechanistical analyses, providing insights into the virus-host interactions and probably helping inform the development of antiviral agents in the future.IMPORTANCEDBV/SFTSV is an emerging high-pathogenic virus. Since its first identification in China in 2009, cases of DBV infection have been reported in many other countries, posing a significant threat to public health. Uncovering the mechanisms of DBV-host interactions is necessary to understand the viral pathogenesis and host response and may advance the development of antiviral therapeutics. Here, we found that host factor MxA whose expression is induced by DBV restricts the virus infection. Mechanistically, MxA specifically interacts with the viral NP and blocks the NP-RdRp interaction, inhibiting the viral RNP activity. Further studies identified the key domain and amino acid residue required for MxA inhibition to DBV. Consistently, they were then shown to be important for MxA targeting of NP and obstruction of the NP-RdRp association. These findings unravel the restrictive role of MxA in DBV infection and the underlying mechanism, expanding our knowledge of the virus-host interactions.
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Phlebovirus , Febre Grave com Síndrome de Trombocitopenia , Humanos , Proteínas do Nucleocapsídeo , Ribonucleoproteínas/metabolismo , RNA Polimerase Dependente de RNA , Febre Grave com Síndrome de Trombocitopenia/metabolismo , Febre Grave com Síndrome de Trombocitopenia/virologia , Phlebovirus/fisiologia , Interações Hospedeiro-PatógenoRESUMO
Crimean-Congo hemorrhagic fever virus (CCHFV) is a biosafety level-4 pathogen requiring urgent research and development efforts. The glycoproteins of CCHFV, Gn and Gc, are considered to play multiple roles in the viral life cycle by interactions with host cells; however, these interactions remain largely unclear to date. Here, we analyzed the cellular interactomes of CCHFV glycoproteins and identified 45 host proteins as high-confidence Gn/Gc interactors. These host molecules are involved in multiple cellular biological processes potentially associated with the physiological actions of the viral glycoproteins. Then, we elucidated the role of a representative cellular protein, HAX1. HAX1 interacts with Gn by its C-terminus, while its N-terminal region leads to mitochondrial localization. By the strong interaction, HAX1 sequestrates Gn to mitochondria, thus depriving Gn of its normal Golgi localization that is required for functional glycoprotein-mediated progeny virion packaging. Consistently, the inhibitory activity of HAX1 against viral packaging and hence propagation was further elucidated in the contexts of pseudotyped and authentic CCHFV infections in cellular and animal models. Together, the findings provide a systematic CCHFV Gn/Gc-cell protein-protein interaction map, but also unravel a HAX1/mitochondrion-associated host antiviral mechanism, which may facilitate further studies on CCHFV biology and therapeutic approaches.
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Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia , Animais , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/metabolismo , Febre Hemorrágica da Crimeia/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismoRESUMO
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses an unprecedented threat to human health since late 2019. Notably, the progression of the disease is associated with impaired antiviral interferon (IFN) responses. Although multiple viral proteins were identified as potential IFN antagonists, the underlying molecular mechanisms remain to be fully elucidated. In this study, we firstly demonstrate that SARS-CoV-2 NSP13 protein robustly antagonizes IFN response induced by the constitutively active form of transcription factor IRF3 (IRF3/5D). This induction of IFN response by IRF3/5D is independent of the upstream kinase, TBK1, a previously reported NSP13 target, thus indicating that NSP13 can act at the level of IRF3 to antagonize IFN production. Consistently, NSP13 exhibits a specific, TBK1-independent interaction with IRF3, which, moreover, is much stronger than that of NSP13 with TBK1. Furthermore, the NSP13-IRF3 interaction was shown to occur between the NSP13 1B domain and IRF3 IRF association domain (IAD). In agreement with the strong targeting of IRF3 by NSP13, we then found that NSP13 blocks IRF3-directed signal transduction and antiviral gene expression, counteracting IRF3-driven anti-SARS-CoV-2 activity. These data suggest that IRF3 is likely to be a major target of NSP13 in antagonizing antiviral IFN responses and provide new insights into the SARS-CoV-2-host interactions that lead to viral immune evasion.
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COVID-19 , Fator Regulador 3 de Interferon , Proteínas não Estruturais Virais , Humanos , COVID-19/imunologia , Evasão da Resposta Imune , Fator Regulador 3 de Interferon/genética , Interferons , SARS-CoV-2 , Proteínas não Estruturais Virais/genéticaRESUMO
There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.
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COVID-19 , SARS-CoV-2 , Animais , Humanos , Chlorocebus aethiops , SARS-CoV-2/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Células Vero , Replicação Viral , RNA ViralRESUMO
Coronary angiography (CAG) is the "gold standard" for diagnosing coronary artery disease (CAD). However, due to the limitation of current imaging methods, the CAG image has low resolution and poor contrast with a lot of artifacts and noise, which makes it difficult for blood vessels segmentation. In this paper, we propose a DBCU-Net for automatic segmentation of CAG images, which is an extension of U-Net, DenseNet with bi-directional ConvLSTM(BConvLSTM). The main contribution of our network is that instead of convolution in the feature extraction of U-Net, we incorporate dense connectivity and the bi-directional ConvLSTM to highlight salient features. We conduct our experiment on our private dataset, and achieve average Accuracy, Precision, Recall and F1-score for coronary artery segmentation of 0.985, 0.913, 0.847 and 0.879 respectively.
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Doença da Artéria Coronariana , Aprendizado Profundo , Humanos , Angiografia Coronária/métodos , Valor Preditivo dos Testes , Doença da Artéria Coronariana/diagnóstico por imagem , Artefatos , Processamento de Imagem Assistida por Computador/métodosRESUMO
Glaucoma is a chronic disease that requires lifelong treatment, whereas, discomfort caused by frequent medication may affect the quality of life. Moreover, the therapeutic efficacy of traditional local administration was unsatisfactory due to the rapid ocular clearance mechanism and the ocular barrier. Herein, a triple crosslinked micelle-hydrogel lacrimal implant with low polymer content was fabricated for localized and prolonged therapy of glaucoma. Latanoprost and timolol were simultaneously entrapped in the PEG-PLA micelles with high encapsulation efficiency and further loaded into the triple crosslinked hydrogel, facilitating a double sustained release of drugs. Subsequently, the implant was constructed by a unique molecular orientation fixation technology, which enables the implant to be fixed in the lacrimal duct. The triple crosslinked micelle-hydrogel lacrimal implant manifested a distinguished physicochemical characterization to sustain the release of latanoprost and timolol. In vitro release experiment demonstrated the duration of two drugs was extended for up to 28 days. The in vivo test of elevated intraocular pressure (IOP) in a rabbit model revealed that the IOP-lowering effects were sustained longer than 28 days as expected. The relative pharmacological availability (PA) of lacrimal implants was 5.7 times greater than that of the eye drops. The results of the studies on ocular irritation and histological examination demonstrated the good safety of the lacrimal implant. In conclusion, the triple crosslinked micelle-hydrogel lacrimal implant could effectively lower the IOP with splendid compatibility, demonstrating the promising prospect in the long-term noninvasive treatment of glaucoma.
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Glaucoma , Aparelho Lacrimal , Hipertensão Ocular , Prostaglandinas F Sintéticas , Animais , Coelhos , Micelas , Timolol , Latanoprosta/uso terapêutico , Hidrogéis/química , Anti-Hipertensivos , Qualidade de Vida , Pressão Intraocular , Glaucoma/tratamento farmacológico , Prostaglandinas F Sintéticas/química , Prostaglandinas F Sintéticas/uso terapêutico , Resultado do Tratamento , Hipertensão Ocular/tratamento farmacológicoRESUMO
Autophagy is emerging as a critical player in host defense against diverse infections, in addition to its conserved function to maintain cellular homeostasis. Strikingly, some pathogens have evolved strategies to evade, subvert or exploit different steps of the autophagy pathway for their lifecycles. Here, we present a new viral mechanism of manipulating autophagy for its own benefit with severe fever with thrombocytopenia syndrome bunyavirus (SFTSV, an emerging high-pathogenic virus) as a model. SFTSV infection triggers autophagy, leading to complete autophagic flux. Mechanistically, we show that the nonstructural protein of SFTSV (NSs) interacts with mTOR, the pivotal regulator of autophagy, by targeting its kinase domain and captures mTOR into viral inclusion bodies (IBs) induced by NSs itself. Furthermore, NSsimpairs mTOR-mediated phosphorylation of unc-51-like kinase 1 (ULK1) at Ser757, disrupting the inhibitory effect of mTOR on ULK1 activity and thus contributing to autophagy induction. Pharmacologic treatment and Beclin-1 knockout experimental results establish that, in turn, autophagy enhances SFTSV infection and propagation. Moreover, the minigenome reporter system reveals that SFTSV ribonucleoprotein (the transcription and replication machinery) activity can be bolstered by autophagy. Additionally, we found that the NSs proteins of SFTSV-related bunyaviruses have a conserved function of targeting mTOR. Taken together, we unravel a viral strategy of inducing pro-viral autophagy by interacting with mTOR, sequestering mTOR into IBs and hence provoking the downstream ULK1 pathway, which presents a new paradigm for viral manipulation of autophagy and may help inform future development of specific antiviral therapies against SFTSV and related pathogens.
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Corpos de Inclusão , Phlebovirus , Humanos , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Corpos de Inclusão/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Phlebovirus/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
Crimean-Congo hemorrhagic fever virus (CCHFV) is a biosafety level-4 (BSL-4) pathogen that causes Crimean-Congo hemorrhagic fever (CCHF) characterized by hemorrhagic manifestation, multiple organ failure and high mortality rate, posing great threat to public health. Despite the recently increasing research efforts on CCHFV, host cell responses associated with CCHFV infection remain to be further characterized. Here, to better understand the cellular response to CCHFV infection, we performed a transcriptomic analysis in human kidney HEK293 âcells by high-throughput RNA sequencing (RNA-seq) technology. In total, 496 differentially expressed genes (DEGs), including 361 up-regulated and 135 down-regulated genes, were identified in CCHFV-infected cells. These regulated genes were mainly involved in host processes including defense response to virus, response to stress, regulation of viral process, immune response, metabolism, stimulus, apoptosis and protein catabolic process. Therein, a significant up-regulation of type III interferon (IFN) signaling pathway as well as endoplasmic reticulum (ER) stress response was especially remarkable. Subsequently, representative DEGs from these processes were well validated by RT-qPCR, confirming the RNA-seq results and the typical regulation of IFN responses and ER stress by CCHFV. Furthermore, we demonstrate that not only type I but also type III IFNs (even at low dosages) have substantial anti-CCHFV activities. Collectively, the data may provide new and comprehensive insights into the virus-host interactions and particularly highlights the potential role of type III IFNs in restricting CCHFV, which may help inform further mechanistic delineation of the viral infection and development of anti-CCHFV strategies.
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Fenômenos Biológicos , Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia , Humanos , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Febre Hemorrágica da Crimeia/metabolismo , Interferon lambda , Células HEK293 , Antivirais/metabolismoRESUMO
Oncolytic viruses (OVs), including oncolytic herpes simplex viruses (oHSVs), are promising therapeutics against cancer. Here, we report two ICP6-mutated HSVs (type I) generated by CRISPR/Cas9, rHSV1/∆RR (with ICP6 ribonucleotide reductase [RR] domain deleted) and rHSV1/∆ICP6 (with a complete deletion of ICP6), exhibiting potent antitumor efficacy against lung adenocarcinoma. Both the mutants showed strong cytotoxicity in vitro, comparable with the control viruses expressing intact ICP6, but in relatively lower titers. Moreover, these mutant viruses exhibited preferential killing ability against lung tumor cells rather than normal lung fibroblast cells. Further, unlike the control HSV-1 causing severe illness or death in the mouse model, the ICP6-mutated viruses did not induce significant pathogenicity but instead effectively reduced tumor burden in vivo and led to 100% survival of the animals, indicating notable antitumor activity and attenuated virulence. In addition, rHSV1/∆RR seemed to have even better antitumor efficacy than rHSV1/∆ICP6, albeit no statistical significance in inhibition of tumor volume. Histopathologically, rHSV1/∆RR induced massive neutrophil infiltration to the tumor microenvironment and consistently, triggered more antitumor immune and neutrophil chemotactic cytokines or higher expression levels of them (indicated by quantitative polymerase chain reaction and transcriptome analyses). These results demonstrate the anti-adenocarcinoma potential of the CRISPR/Cas9-engineered ICP6 mutant HSV1, especially the rHSV1/∆RR, which likely induces stronger innate antitumor immune response. Together, these findings may provide new valuable clues for further development of OV-based therapeutics against lung adenocarcinoma or other types of tumors.
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Adenocarcinoma de Pulmão , Herpesvirus Humano 1 , Neoplasias Pulmonares , Ribonucleotídeo Redutases , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/terapia , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Herpesvirus Humano 1/genética , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Camundongos , Ribonucleotídeo Redutases/genética , Microambiente TumoralRESUMO
Background: The notch signal pathway is important in the development of both tumor-associated macrophages (TAMs) and stomach cancer, but how Notch signaling affects TAMs in stomach cancer is barely understood. Methods: The expressions of Notch1, Notch2, Notch3, Notch4, hes family bHLH transcription factor 1 (Hes1), and delta-like canonical Notch ligand 3 (DLL3) were detected by Western blot and the expressions of interleukin (IL)-10, IL-12, and IL1-ß were detected using enzyme-linked immunosorbent assay after the co-culture of macrophages and stomach-cancer cells. The proliferation and migration of cancer cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scratch assay, respectively, and the cell cycle was detected using Annexin V/propidium iodide assay. The protein interactions with DLL3 were detected using co-immunoprecipitation and mass spectrometry. Results: The co-culture of macrophages and stomach-cancer cells MKN45 and BGC823 could enhance cell proliferation accompanied by the activation of Notch1/Notch2 signaling and upregulation of DLL3. Notch signaling gamma-secretase inhibitor (DAPT) blocked this process. The overexpression of DLL3 in stomach-cancer cells could promote the proliferation of cancer cells, enhance the activation of Notch1/Notch2 signaling, induce the expression of IL-33, lead to the degradation of galectin-3-binding protein (LG3BP) and heat shock cognate 71 kDa protein (HSPA8), and result in elevated IL-1ß, IL-12, and IL-10 secretion by macrophages. Higher expression of DLL3 or IL-33 could lead to a lower survival rate based on University of California, Santa Cruz Xena Functional Genomics Explorer and The Cancer Genome Atlas data set. Conclusions: This is evidence that DLL3 regulates macrophages in stomach cancer, suggesting that DLL3 may be a novel and potential target for stomach-cancer therapy.
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Bunyaviruses are members of the Bunyavirales order, which is the largest group of RNA viruses, comprising 12 families, including a large group of emerging and re-emerging viruses. These viruses can infect a wide variety of species worldwide, such as arthropods, protozoans, plants, animals, and humans, and pose substantial threats to the public. In view of the fact that a better understanding of the life cycle of a highly pathogenic virus is often a precondition for developing vaccines and antivirals, it is urgent to develop powerful tools to unravel the molecular basis of the pathogenesis. However, biosafety level -3 or even -4 containment laboratory is considered as a necessary condition for working with a number of bunyaviruses, which has hampered various studies. Reverse genetics systems, including minigenome (MG), infectious virus-like particle (iVLP), and infectious full-length clone (IFLC) systems, are capable of recapitulating some or all steps of the viral replication cycle; among these, the MG and iVLP systems have been very convenient and effective tools, allowing researchers to manipulate the genome segments of pathogenic viruses at lower biocontainment to investigate the viral genome transcription, replication, virus entry, and budding. The IFLC system is generally developed based on the MG or iVLP systems, which have facilitated the generation of recombinant infectious viruses. The MG, iVLP, and IFLC systems have been successfully developed for some important bunyaviruses and have been widely employed as powerful tools to investigate the viral replication cycle, virus-host interactions, virus pathogenesis, and virus evolutionary process. The majority of bunyaviruses is generally enveloped negative-strand RNA viruses with two to six genome segments, of which the viruses with bipartite and tripartite genome segments have mostly been characterized. This review aimed to summarize current knowledge on reverse genetic studies of representative bunyaviruses causing severe diseases in humans and animals, which will contribute to the better understanding of the bunyavirus replication cycle and provide some hints for developing designed antivirals.
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COVID-19/imunologia , Interferons/metabolismo , SARS-CoV-2/imunologia , Fator de Transcrição STAT1/metabolismo , Proteínas não Estruturais Virais/metabolismo , COVID-19/virologia , Humanos , Ligação Proteica/imunologia , Mapeamento de Interação de Proteínas , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Transdução de Sinais/imunologiaRESUMO
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the tick-borne SFTS bunyavirus (SFTSV) resulting in a high fatality rate up to 30%. SFTSV is a negative-strand RNA virus containing three single-stranded RNA genome segments designated as L, M, and S, which respectively, encode the RNA-dependent RNA polymerase (RdRp), glycoproteins Gn and Gc, and nucleoprotein (N) and non-structural proteins (NSs). NSs can form inclusion bodies (IBs) in infected and transfected cells. A previous study has provided a clue that SFTSV NSs may be involved in virus-like or viral RNA synthesis; however, the details remain unclear. Our work described here reveals that SFTSV NSs can downregulate virus-like RNA synthesis in a dose-dependent manner within a cDNA-derived viral RNA synthesis system, i.e., minigenome (-) and minigenome (+) systems based on transfection, superinfection, and luciferase reporter activity determination; meanwhile, NSs also show a weak inhibitory effect on virus replication. By using co-immunoprecipitation (Co-IP) and RT-PCR combined with site-directed mutagenesis, we found that NSs suppress virus-like RNA or virus replication through interacting with N but not with RdRp, and the negative regulatory effect correlates closely with the IB structure it formed but is not associated with its role of antagonizing host innate immune responses. When the cytoplasmic structure of IB formed by SFTSV NSs was deprived, the inhibitory effect of NSs on virus-like RNA synthesis would weaken and even disappear. Similarly, we also evaluated other bandavirus NSs that cannot form IB in neither infected nor transfected cells, and the results showed that the NSs of Heartland bandavirus (HRTV) did not show a significant inhibitory effect on virus-like RNA synthesis within a minigenome system. Our findings provide experimental evidence that SFTSV NSs participate in regulating virus-like or viral RNA synthesis and the negative effect may be due to the NSs-N interaction.