Yeast Two-Hybrid Assay for Investigating Antiviral Innate Immunity.

Yeast two-hybrid (YTH) technology is a powerful tool for studying protein interactions and has been widely used in various fields of molecular biology, including the study of antiviral innate immunity. This chapter presents detailed information and experimental procedures for identifying virus-host protein interactions involved in immune regulation using yeast two-hybrid technology.

Predictive risk factors for one-year mortality in idiopathic inflammatory myopathy patients with interstitial lung disease: A retrospective, single-center cohort study.

Objectives: This study aimed to analyze the risk factors for mortality of idiopathic inflammatory myopathy (IIM) patients admitted with interstitial lung disease (ILD) to guide rapid and accurate judgment of clinical prognosis. Patients and methods: This retrospective, single-center cohort study was conducted with 135 participants (37 males, 98 females; mean age: 54.8±11.1 years; range, 24 to 85 years) between June 1, 2016, and June 30, 2021. The participants were categorized into the survival group (n=111) and nonsurvivors (n=24) according to whether they survived during the one-year follow-up. The independent risk factors for mortality in one year after discharge were analyzed. Receiver operating characteristic curve analysis was used to determine the accuracy of oxygenation index at baseline combined with pulmonary infection (PI) at follow-up to indicate death in IIM-ILD patients. Results: Compared to the survival group, nonsurvivors were older (p=0.006) and had a higher proportion of anti-MDA5 (melanoma differentiation-associated protein 5) positivity (p<0.001). The ILD duration was shorter (p=0.006), the oxygenation index was lower (p<0.001), and the intensive care unit occupancy rate (p<0.001) and ventilator utilization rate (p<0.001) were elevated in nonsurvivors compared to the survival group. Oxygenation index at baseline (odds ratio [OR]=1.021, 95% confidence interval [CI]: 1.001-1.023, p=0.040) and PI (clinical judgment) at follow-up (OR=16.471, 95% CI: 1.565-173.365, p=0.020) were found as independent risk factors for death in the year after discharge in IIM inpatients with ILD. An oxygenation index ≤279 mmHg at baseline combined with PI at follow-up exhibited a promising predictive value for all-cause death in IIM-ILD patients within one year. Conclusion: Oxygenation index at baseline and PI during follow-up were independent risk factors for death of IIM-ILD patients within one year after discharge. Patients with an oxygenation index ≤279 mmHg at baseline had an increased risk of death once they developed PI during the one-year follow-up.

The SUMOylation of Human Cytomegalovirus Capsid Assembly Protein Precursor (UL80.5) Affects Its Interaction with Major Capsid Protein (UL86) and Viral Replication.

Human Cytomegalovirus Capsid Assembly Protein Precursor (pAP, UL80.5) plays a key role in capsid assembly by forming an internal protein scaffold with Major Capsid Protein (MCP, UL86) and other capsid subunits. In this study, we revealed UL80.5 as a novel SUMOylated viral protein. We confirmed that UL80.5 interacted with the SUMO E2 ligase UBC9 (58-93aa) and could be covalently modified by SUMO1/SUMO2/SUMO3 proteins. 371Lysine located within a ψKxE consensus motif on UL80.5 carboxy-terminal was the major SUMOylation site. Interestingly, the SUMOylation of UL80.5 restrained its interaction with UL86 but had no effects on translocating UL86 into the nucleus. Furthermore, we showed that the removal of the 371lysine SUMOylation site of UL80.5 inhibited viral replication. In conclusion, our data demonstrates that SUMOylation plays an important role in regulating UL80.5 functions and viral replication.

Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection.

Influenza virus has the ability to circumvent host innate immune system through regulating certain host factors for its effective propagation. However, the detailed mechanism is still not fully understood. Here, we report that a host sphingolipid metabolism-related factor, sphingosine kinase 2 (SPHK2), upregulated during influenza A virus (IAV) infection, promotes IAV infection in an enzymatic independent manner. The enhancement of the virus replication is not abolished in the catalytic-incompetent SPHK2 (G212E) overexpressing cells. Intriguingly, the sphingosine-1-phosphate (S1P) related factor HDAC1 also plays a crucial role in SPHK2-mediated IAV infection. We found that SPHK2 cannot facilitate IAV infection in HDAC1 deficient cells. More importantly, SPHK2 overexpression diminishes the IFN-ß promoter activity upon IAV infection, resulting in the suppression of type I IFN signaling. Furthermore, ChIP-qPCR assay revealed that SPHK2 interacts with IFN-ß promoter through the binding of demethylase TET3, but not with the other promoters regulated by TET3, such as TGF-ß1 and IL6 promoters. The specific regulation of SPHK2 on IFN-ß promoter through TET3 can in turn recruit HDAC1 to the IFN-ß promoter, enhancing the deacetylation of IFN-ß promoter, therefore leading to the inhibition of IFN-ß transcription. These findings reveal an enzymatic independent mechanism on host SPHK2, which associates with TET3 and HDAC1 to negatively regulate type I IFN expression and thus facilitates IAV propagation.

Site-specific SUMOylation of viral polymerase processivity factor: a way of localizingtoND10 subnuclear domains for restricted and self-controlled reproduction of herpesvirus.

Lytic replication of human cytomegalovirus (HCMV), a member of ß-herpesvirus, is a highly complicated and organized process that requires its DNA polymerase processivity factor, UL44, the first-reported HCMV replication protein subjected to SUMO post-translational modification (PTM). SUMOylation plays a pleiotropic role in protein functions of host cells and infecting viruses. Particularly, formation of herpesviral replication compartments (RCs) upon infection is induced in proximity to ND10 subnuclear domains, the host cell's intrinsic antiviral immune devices and hot SUMOylation spots, relying just on SUMOylation of their protein components to become mature and functional in restriction of the viral replication. In this study, to unveil the exact role of SUMO PTM on UL44 involved in HCMV replication, we screened and identified PIAS3, an annotated E3 SUMO ligase, as a novel UL44-interacting protein engaged in cellular SUMOylation pathway. Co-existence of PIAS3 could enhance the UBC9-based SUMO modification of UL44 specifically at its conserved 410lysine residue lying within the single canonical ψKxE SUMO Conjugation Motif (SCM). Intriguingly, we found this SCM-specific SUMOylation contributes to UL44 co-localization and interaction with subnuclear ND10 domains during infection, which in turn exerts an inhibitory effect on HCMV replication and growth. Together, these results highlight the importance of SUMOylation in regulating viral protein subnuclear localization, representing a novel way of utilizing ND10-based restriction to achieve the self-controlled slower replication and reproduction of herpesviruses.

Relationship between serum 25-hydroxyvitamin D3 levels and severity of chronic periodontitis in type 2 diabetic patients: A cross-sectional study.

BACKGROUND AND OBJECTIVES: Serum 25-hydroxyvitamin D3 (25(OH)D3 ), a newly emerged immune regulator, is considered to be involved in type 2 diabetic periodontitis (T2DCP). However, the risk factors and genes with altered expression that influence the progression and severity of T2DCP remain unknown. Accordingly, the aim of the present study was to elucidate the relationship between 25(OH)D3 deficiency and severity of T2DCP as well as the potential mechanisms. MATERIAL AND METHODS: A total of 182 subjects were divided into two groups: chronic periodontitis without diabetes (P group, n = 88) and type 2 diabetes mellitus with periodontitis (DM+P group, n = 94). Patients in both groups were further classified according to age as young (Y) and elderly (E) for a total of four groups: P/Y, P/E, DM+P/Y, and DM+P/E. Periodontal status was evaluated based on the probing depth (PD) and clinical attachment loss (CAL). The serum levels of human 25(OH)D3 , interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α were measured by enzyme-linked immunosorbent assays. Immunohistochemistry was used to measure the expression of protein tyrosine phosphatase non-receptor type 2 (PTPN2), vitamin D receptor (VDR), and JAK/STAT proteins in the gingival tissue. RESULTS: Serum 25(OH)D3 levels were lower in the DM+P group than those in the P group (P < 0.001). When the patients were subgrouped according to age, 25(OH)D3 deficiency was more commonly found in DM+P/E than in DM+P/Y (67% vs 51%), with a significant difference detected in the 25(OH)D3 quartile of 15-20 ng/mL (P = 0.007). The 25(OH)D3 level showed a significant negative correlation with fasting blood glucose (FBG) (r = -0.623), serum IL-1ß (r = -0.392), serum TNF-α (r = -0.218), PD (r = -0.269), and CAL (r = -0.305) in the DM+P group (all P < 0.05), but not with hemoglobin A1c (P = 0.123). Additionally, reduced VDR and PTPN2 expression levels were observed in DM+P patients, whereas JAK1 and p-STAT5 protein levels were increased in this group. CONCLUSIONS: Vitamin D3 deficiency is strongly associated with T2DCP, and age mediates this relationship. Abnormal FBG and IL-1ß levels should be considered as important potential risk factors for the progression and severity of T2DCP. Moreover, 25(OH)D3 deficiency may be related to the immune function of T2DCP by weakening PTPN2 signaling.

Metformin ameliorates experimental diabetic periodontitis independently of mammalian target of rapamycin (mTOR) inhibition by reducing NIMA-related kinase 7 (Nek7) expression.

BACKGROUND: Metformin, a classical treatment for diabetes mellitus, has shown sound anti-inflammatory effects. Emerging studies have focused on the mechanism underlying inflammation-related diabetic complications, such as diabetic periodontitis. Herein, we investigated the anti-inflammatory effects of metformin on the NIMA-related kinase 7 (Nek7)/nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) pathway both in vivo and in vitro in experimental diabetic periodontitis. METHODS: All procedures were conducted in Porphyromonas gingivalis (Pg)-infected streptozotocin (STZ)-induced diabetic mice and in lipopolysaccharide (LPS)-treated RAW 264.7 cells under high-glucose conditions. A range of techniques were performed in this study: microcomputed tomography, western blotting, and immunofluorescence were used to analyze periodontal tissues. Enzyme-linked immunosorbent assay (ELISA) was for serum interleukin-1ß (IL-1ß) detection. Specific pharmacological inhibition was used to stimulate cells. Flow cytometry was implemented to analyze cell cycle. RESULTS: We found that metformin treatment can robustly ameliorate periodontal infection and tissue destruction and reduce blood glucose and serum IL-1ß levels in mice with diabetic periodontitis. Moreover, gingival tissue exhibited less macrophage infiltration and decreased expression of Nek7, NLRP3, caspase-1, and mammalian target of rapamycin (mTOR), which were simultaneously observed in RAW 264.7 cell models stimulated with metformin. Metformin also affected the cell cycle in a dose-dependent way. Furthermore, after stimulation with the mTOR inhibitor rapamycin, additional metformin treatment could still downregulate Nek7/NLRP3. CONCLUSIONS: Our research indicated that metformin significantly attenuated experimental diabetic periodontitis both in vivo and in vitro. Metformin suppressed the inflammatory state by inhibiting Nek7 expression to decrease NLRP3 inflammasome activity. Interestingly, mTOR inhibition was not involved in metformin-induced Nek7 downregulation. The observed Nek7 reduction could be related to metformin-mediated cell cycle arrest.

Tight control of genomic phosphorothioate modification by the ATP-modulated autoregulation and reusability of DndB.

DNA phosphorothioate (PT) modification was recently identified to occur naturally in diverse bacteria and to be governed by DndABCDE proteins. The nuclease resistance as well as the redox and nucleophilic properties of PT sulfur make PT modification a versatile player in restriction-modification (R-M) defense, epigenetic regulation, environmental fitness and the maintenance of cellular redox homeostasis. In this study, we discovered that tight control of PT levels is mediated by the ATPase activity of DndB. The ATP-binding activity of DndB stimulates the dissociation of the DndB-DNA complex, allowing transcriptional initiation, whereas its ATP hydrolysis activity promotes the conversion of DndB-ATP to free DndB that is capable of rebinding to promoter DNA for transcriptional inhibition. Since sulfur incorporation is an ATP-consuming process, these activities provide an economical way to fine-tune PT modification in an ATP-sensing manner. To our knowledge, this ATP-mediated regulation is a rare example among DNA epigenetic modification systems; the features of autoregulation and the repeated usage of DndB allow the dedicated regulation of PT levels in response to cellular ATP concentrations, providing insight into PT function and its role in physiology.

25-Hydroxyvitamin D3 Alleviates Experimental Periodontitis via Promoting Expression of Cathelicidin in Mice with Type 2 Diabetic Mellitus.

Type 2 diabetic mellitus is manifested by metabolic impairments with high prevalence worldwide, of which periodontitis represents a typical oral complication (also called diabetic periodontitis). Oral epithelia bear the brunt of periodontal damage from microscopic intruders; thus the defense function of epithelial cells is of vital significance. We have previously proved that 25-hydroxyvitamin D3 (25-OHD3) altered the expression of cathelicidin antimicrobial peptide in oral epithelial cells in vitro. Herein, we discovered that 25-OHD3 intraperitoneal injection attenuated periodontal inflammation by promoting cathelicidin production in gingival epithelia and reducing fasting glucose of diabetic mice. Dotblotting of serum showed cathelicidin secretion was consistent with 25-OHD3 treatment. Immunochemistry exhibited enhanced expression of cathelicidin and vitamin D receptors along with reduced expression of TLR4 in diabetic mice. Stereomicroscope showed less alveolar bone loss when injected with 25-OHD3.These results showed 25-OHD3 can promote cathelicidin and ameliorate the severity of diabetic periodontitis. Our study complemented the mechanism of cathelicidin and extended knowledge of 25-OHD3's role in diabetic periodontitis.

STAT1 activation represses IL-22 gene expression and psoriasis pathogenesis.

IL-22 plays an important role in tissue repair and inflammatory responses, and is implicated in the pathogenesis of psoriasis, ulcerative colitis, as well as liver and pancreas damage. The molecular mechanisms of its regulation have been actively studied. Here, we show that the differential regulation of IL-22 expression in CD4+ T cells by IL-6 and IL-27 was detected rapidly after stimulation. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that both STAT1 and STAT3 directly bind to the STAT responsive elements (SRE) of the IL-22 promoter, and the balance between activated STAT3 and STAT1 determines IL-22 promoter activities. We further show that the heterozygous mutation of the STAT1 gene results in elevated levels of IL-22 production and induces much severer skin inflammation in an imiquimod (IMQ)-induced murine psoriasis model. Together, our results reveal a novel regulatory mechanism of IL-22 expression by STAT1 through directly antagonizing STAT3, and the importance of the balance between STAT3 and STAT1 in IL-22 regulation and psoriasis pathogenesis.