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Sepsis is a major cause of acute lung injury (ALI) characterized by inflammatory responses. Ubiquitination plays a critical role in the pathogenesis of ALI. This study aimed to investigate the role of USP50, a deubiquitinating enzyme, in sepsis-induced ALI and its underlying molecular mechanisms. THP-1 cells were differentiated into macrophages and exposed to lipopolysaccharide (LPS) to establish an in vitro injury model. Pyroptosis was assessed using immunoblotting, flow cytometry, and enzyme-linked immunosorbent assay. The regulation of USP50 on NLRP3 deubiquitination was analyzed through immunoprecipitation, immunoblotting, and protein stability assays. The in vivo function of USP50 was evaluated using a cecal ligation and puncture (CLP)-induced septic mouse model. Results demonstrated that USP50 expression was significantly upregulated in the blood of patients with sepsis-induced ARDS and in the lungs of CLP-treated mice. USP50 knockdown suppressed pyroptosis in LPS-stimulated macrophages and septic mice. Furthermore, USP50 inhibition enhanced NLRP3 degradation by facilitating K48-linked ubiquitination. Overexpression of NLRP3 reversed the anti-pyroptotic effects induced by USP50 depletion in macrophages. In conclusion, USP50 suppression attenuates macrophage pyroptosis through inhibition of NLRP3 deubiquitination, thereby reducing lung injury in sepsis-induced models. These findings identify USP50 as a potential therapeutic target for sepsis-associated acute lung injury.

BST2/Tetherin is a restriction factor with broad antiviral activity against enveloped viruses, including coronaviruses. Specifically, BST2 traps nascent particles to membrane compartments, preventing their release and spread. In turn, viruses have evolved multiple mechanisms to counteract BST2. Here, we examined the interactions between BST2 and SARS-CoV-2. Our study shows that BST2 reduces SARS-CoV-2 virion release. However, the virus uses the Spike (S) protein to downregulate BST2. This requires a physical interaction between S and BST2, which routes BST2 for lysosomal degradation in a Clathtin- and ubiquitination-dependent manner. By surveying different SARS-CoV-2 variants of concern (Alpha-Omicron), we found that Omicron is more efficient at counteracting BST2, and that mutations in S account for its enhanced anti-BST2 activity. Mapping analyses revealed that several surfaces in the extracellular region of BST2 are required for an interaction with the Spike, and that the Omicron variant has changed its patterns of association with BST2 to improve its counteraction. Therefore, our study suggests that, besides enhancing receptor binding and evasion of neutralizing antibodies, mutations accumulated in the Spike afford more efficient counteraction of BST2, which highlights that BST2 antagonism is important for SARS-CoV-2 infectivity and spread.

In this study, we sought to identify the host dependency factors that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses for the generation of replication organelles: cellular membranous structures that SARS-CoV-2 builds in order to support the replication and transcription of its genome. We uncovered that RAB5 is an important dependency factor for SARS-CoV-2 replication and the generation of replication organelles, and that the viral protein NSP6 participates in this process. Hence, NSP6 represents a promising target to halt SARS-CoV-2 replication.

Background Autophagy plays an important role as a cellular defense mechanism against intracellular pathogens, like viruses. Specifically, autophagy orchestrates the recruitment of specialized cargo, including viral components needed for replication, for lysosomal degradation. In addition to this primary role, the cleavage of viral structures facilitates their association with pattern recognition receptors and MHC-I/II complexes, which assists in the modulation of innate and adaptive immune responses against these pathogens. Importantly, whereas autophagy restricts the replicative capacity of human immunodeficiency virus type 1 (HIV-1), this virus has evolved the gene nef to circumvent this process through the inhibition of early and late stages of the autophagy cascade. Despite recent advances, many details of the mutual antagonism between HIV-1 and autophagy still remain unknown. Here, we uncover the genetic determinants that drive the autophagy-mediated restriction of HIV-1 as well as the counteraction imposed by Nef. Additionally, we also examine the implications of autophagy antagonism in HIV-1 infectivity. Results We found that sustained activation of autophagy potently inhibits HIV-1 replication through the degradation of HIV-1 Gag, and that this effect is more prominent for nef -deficient viruses. Gag re-localizes to autophagosomes where it interacts with the autophagosome markers LC3 and SQSTM1. Importantly, autophagy-mediated recognition and recruitment of Gag requires the myristoylation and ubiquitination of this virus protein, two post-translational modifications that are essential for Gag’s central role in virion assembly and budding. We also identified residues T 48 and A 49 in HIV-1 NL4-3 Nef as responsible for impairing the early stages of autophagy. Finally, a survey of pandemic HIV-1 transmitted/founder viruses revealed that these isolates are highly resistant to autophagy restriction. Conclusions This study provides evidence that autophagy antagonism is important for virus replication and suggests that the ability of Nef to counteract autophagy may have played an important role in mucosal transmission. Hence, disabling Nef in combination with the pharmacological manipulation of autophagy represents a promising strategy to prevent HIV spread.

SERINC5 is a restriction factor that becomes incorporated into nascent retroviral particles, impairing their ability to infect target cells. In turn, retroviruses have evolved countermeasures against SERINC5. For instance, the primate lentiviruses (HIV and SIV) use Nef, Moloney Murine Leukemia Virus (MLV) uses GlycoGag, and Equine Infectious Anemia Virus (EIAV) uses S2 to remove SERINC5 from the plasma membrane, preventing its incorporation into progeny virions. Recent studies have shown that SERINC5 also restricts other viruses, such as Hepatitis B Virus (HBV) and Classical Swine Fever Virus (CSFV), although through a different mechanism, suggesting that SERINC5 can interfere with multiple stages of the virus life cycle. To investigate whether SERINC5 can also impact other steps of the replication cycle of HIV, the effects of SERINC5 on viral transcripts, proteins, and virus progeny size were studied. Here, we report that SERINC5 causes significant defects in HIV gene expression, which impacts virion production. While the underlying mechanism is still unknown, we found that the restriction occurs at the transcriptional level and similarly affects plasmid and non-integrated proviral DNA (ectopic or non-self-DNA). However, SERINC5 causes no defects in the expression of viral RNA, host genes, or proviral DNA that is integrated in the cellular genome. Hence, our findings reveal that SERINC5’s actions in host defense extend beyond blocking virus entry.

Objectives To investigate the clinical efficacy and mechanism of Jianpi Huatan Tongfu granule in treating acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Methods Sixty patients with AECOPD were enrolled in either of two groups: integrative treatment (Western medicine combined with Jianpi Huatan Tongfu granule) (n = 30) and Western medical treatment (n = 30). Thirty healthy individuals were included in the control group. Results Compared with healthy participants, patients with AECOPD had elevated clinical symptom and dyspnea severity scores. Patients with AECOPD had worsened lung function, compared with healthy participants. The therapeutic efficacy for integrative treatment was superior to Western medical treatment. Inflammatory proteins and cytokines were significantly elevated in patients with AECOPD, including C-reactive protein, interleukin-6, interleukin-8, and tumor necrosis factor-α; these were alleviated by both treatments, with more obvious effects for integrative treatment. Integrative treatment significantly changed the intestinal flora in patients with AECOPD, reaching levels comparable with those of healthy participants. Firmicutes abundance was significantly higher in healthy participants, whereas Bacteroidetes abundance was significantly higher in patients with AECOPD. After treatment, Verrucomicrobia abundance was significantly reduced in patients with AECOPD. Conclusion Jianpi Huatan Tongfu granule could alleviate inflammatory responses and improve clinical therapeutic efficacy in patients with AECOPD.

Breast cancer-associated gene 2 (BCA2) is an E3 ubiquitin and SUMO ligase with antiviral properties against HIV. Specifically, BCA2 (i) enhances the restriction imposed by BST2/Tetherin, impeding viral release; (ii) promotes the ubiquitination and degradation of the HIV protein Gag, limiting virion production; (iii) down-regulates NF-κB, which is necessary for HIV RNA synthesis; and (iv) activates the innate transcription factor IRF1. Due to its antiviral properties, ectopic expression of BCA2 in infected cells represents a promising therapeutic approach against HIV infection. However, BCA2 up-regulation is often observed in breast tumors. To date, the studies about BCA2 and cancer development are controversial, stating both pro- and anti-oncogenic roles. Here, we investigated the impact of BCA2 on cellular metabolic activity, cell proliferation, cell migration, and cell cycle progression. In addition, we also examined the ability of BCA2 to regulate NF-κB and IRF1 in transformed and non-tumor breast epithelial environments. Despite the fact that BCA2 promotes the transition from G1 to S phase of the cell cycle, it did not increase cell proliferation, migration nor metabolic activity. As expected, BCA2 maintains its enzymatic function at inhibiting NF-κB in different breast cancer cell lines. However, the effect of BCA2 on IRF1 differs depending on the cellular context. Specifically, BCA2 activates IRF1 in ER + breast cell lines while it inhibits this transcription factor in ER – breast cancer cells. We hypothesize that the distinct actions of BCA2 over IRF1 may explain, at least in part, the different proposed roles for BCA2 in these cancers.

As an innate immune system, autophagy becomes prominent for its ability to eliminate pathogens and modulate other immune responses. Therefore, taking advantage of autophagy offers a promising strategy to develop new antiviral treatments. However, viruses have evolved different approaches to antagonizing it, and some even leverage autophagy machinery for their replication, pathogenesis, and evasion of other immune responses. Therefore, a better understanding of how viruses and autophagy interact would help identify new drug targets, and the enhanced ability to manipulate autophagy would help improve antiviral therapies. This thesis focuses on the interplay between autophagy and two human pathogens, HIV and SARS-CoV-2.As for autophagy and HIV, I primarily focus on the autophagy effect on HIV viral proteins. In this thesis, I further identified that besides HIV-1 gag, HIV-1 Vpr is a novel autophagy target, while Vif downregulation by mTOR-mediated inhibition is autophagy-independent but due to increased proteasomal function. Moreover, I found that, unlike lab-adapted HIV-1, Vpr proteins from 11 HIV-1 M transmitted/founder viruses (TFVs) are resistant to autophagy-mediated elimination, and this resistance is important for HIV-1 transmission. Finally, I identify residues in lab-adapted Vpr as responsible for its autophagy susceptibility, which facilitate interaction with autophagy factors.As for autophagy and SARS-CoV-2, I primarily focus on the use of autophagosomes as the membrane source for the generation of Replication Organelles (ROs) – membrane structures where coronaviruses assemble their replication-transcription complex. However, I found that autophagy is dispensable for SARS-CoV-2 ROs biogenesis, while RAB5 is a host dependency factor in this process. Furthermore, I propose a model in which ER-resident viral protein NSP6 recruits RAB5+ endosomes to build SARS-CoV-2 ROs.In summary, through my work on the interplay between autophagy with HIV and SARS-CoV-2, my study demonstrates that: (i) HIV Vpr is an autophagy target and that Vpr susceptibility to autophagy plays an important role in HIV-1 transmission. (ii) Instead of using autophagosomes, SARS-CoV-2 NSP6 takes advantage of RAB5+ membranes to build ROs for viral genome replication. Altogether, these results build a strong connection between host-pathogen interaction and provide a basic foundation for antiviral treatment.

Autophagy plays an important role as a cellular defense mechanism against intracellular pathogens, like viruses. Specifically, autophagy orchestrates the recruitment of specialized cargo, including viral components needed for replication, for lysosomal degradation. In addition to this primary role, the cleavage of viral structures facilitates their association with pattern recognition receptors and MHC-I/II complexes, which assists in the modulation of innate and adaptive immune responses against these pathogens. Importantly, whereas autophagy restricts the replicative capacity of human immunodeficiency virus type 1 (HIV-1), this virus has evolved the gene nef to circumvent this process through the inhibition of early and late stages of the autophagy cascade. Despite recent advances, many details of the mutual antagonism between HIV-1 and autophagy still remain unknown. Here, we uncover the genetic determinants that drive the autophagy-mediated restriction of HIV-1 as well as the counteraction imposed by Nef. Additionally, we also examine the implications of autophagy antagonism in HIV-1 infectivity. We found that sustained activation of autophagy potently inhibits HIV-1 replication through the degradation of HIV-1 Gag, and that this effect is more prominent for nef-deficient viruses. Gag re-localizes to autophagosomes where it interacts with the autophagosome markers LC3 and SQSTM1. Importantly, autophagy-mediated recognition and recruitment of Gag requires the myristoylation and ubiquitination of this virus protein, two post-translational modifications that are essential for Gag's central role in virion assembly and budding. We also identified residues T and A in HIV-1 NL4-3 Nef as responsible for impairing the early stages of autophagy. Finally, a survey of pandemic HIV-1 transmitted/founder viruses revealed that these isolates are highly resistant to autophagy restriction. This study provides evidence that autophagy antagonism is important for virus replication and suggests that the ability of Nef to counteract autophagy may have played an important role in mucosal transmission. Hence, disabling Nef in combination with the pharmacological manipulation of autophagy represents a promising strategy to prevent HIV spread.

Objective. To investigate the effects of Baduanjin exercise for type 2 diabetes mellitus. Methods. Literature retrieval was performed in several databases, including PubMed, EMBASE, Cochrane Library, CNKI, Wanfang Data Information Site, CBM, and VIP from inception to April 2017. Randomized controlled trials on evaluating the effects of Baduanjin exercise were identified. The primary outcomes were glycosylated hemoglobin, fasting blood-glucose, and postprandial plasma glucose. Review Manager 5.2 (RevMan 5.2) and Stata V.13.0 software were conducted for data analysis. Results. The results of the meta-analysis indicated that the effects of type 2 diabetes mellitus were favoring Baduanjin plus conventional therapy, when compared with the routine treatment. Baduanjin plus conventional therapy lowered the level of glycosylated hemoglobin, fasting blood-glucose, postprandial plasma glucose, TC, TG, and LDL-C and improved HDL-C. Adverse events were not mentioned in all included studies. No publication bias was detected by Begg’s and Egger’s test and no single study affected the overall result by influence analysis. Conclusions. Evidence from meta-analysis suggested that Baduanjin exercise plus conventional therapy has a positive effect on type 2 diabetes mellitus. However, more rigorously designed and large sample RCTs are required to confirm the efficacy and safety in further studies.