Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells.

Molecular engineering of biocatalysts has revolutionized complex synthetic chemistry and sustainable catalysis. Here, we show that it is also possible to use engineered biocatalysts to reprogram signal transduction in human cells. More specifically, we manipulate cellular hypoxia (low O2) signaling by engineering the gas-delivery tunnel of prolyl hydroxylase 2 (PHD2), an iron-dependent enzymatic O2 sensor. Using computational modeling and rational protein design techniques, we resolve PHD2's gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. Systematic modification of these residues open the constriction topology of PHD2's gas tunnel with the most effectively designed mutant displaying 11-fold enhanced hydroxylation efficiency. Furthermore, transfection of plasmids that express these engineered PHD2 mutants in HEK-293T cells reveal significant reduction in the levels of hypoxia inducible factor (HIF-1α) even under hypoxic conditions. Our studies reveal that activated PHD2 mutants can reprogram downstream HIF pathways in cells to simulate physiological O2-like conditions despite extreme hypoxia and underscores the potential of engineered biocatalysts in controlling cellular function.

Predictive value of the systemic immune-inflammation index for cancer-specific survival of osteosarcoma in children.

Background: Osteosarcoma (OS) is the primary malignant bone tumor that most commonly affects children and adolescents. Recent years effective chemotherapy have improved the 5-year survival in osteosarcoma patients to up to 60%-70%. Still, there is a lack of novel therapeutic strategies to enhance further survival. Our study aimed to evaluate the clinical significance of pretreatment inflammatory-based parameters, including PLT, NLR, and SII, as prognostic indicators of survival in pediatric osteosarcoma patients. Methods: A total of 86 pediatric osteosarcoma patients between 2012 and 2021 in the Department of Orthopedics or tumor Surgery of Children's Hospital affiliated to Chongqing Medical University were retrospectively analyzed. The clinicopathological variables and systematic inflammatory biomarkers, including NLR, PLR and SII, was performed by the A Receiver operating characteristic (ROC) curve and Cox proportional risk regression model. According to the results of multivariate analysis, a prognostic nomogram was generated, and the concordance index (C-index) was calculated to predict the performance of the established nomogram. The survival curve was plotted by the Kaplan-Meier method. Results: Univariate analysis showed that TNM stage, tumor size, NLR value, PLR value, SII value, neutrophil count and platelet count were related to CSS (p < 0.05). According to multivariate analysis, only TNM stage (p = 0.006) and SII values (p = 0.015) were associated with poor prognosis.To further predict survival in pediatric osteosarcoma patients, multivariate Cox regression analysis was used to predict cancer-specific survival at 1, 3 and 5 years. And constructed a nomogram model to predict children's CSS. The C-index of the nomogram is 0.776 (95%CI, 0.776-0.910), indicating that the model has good accuracy. Conclusion: Preoperative SII and TNM staging are independent prognostic markers for pediatric osteosarcoma patients. SII may be used in conjunction with TNM staging for individualized treatment of pediatric osteosarcoma patients in future clinical work.

Correction to: Efficacy and safety of tetramethylpyrazine phosphate on pulmonary hypertension: study protocol for a randomized controlled study.

After publication of our article [1] the authors have notified us of two typos in the Trial status.

Transplantation of Mesenchymal Stem Cells Attenuates Pulmonary Hypertension by Normalizing the Endothelial-to-Mesenchymal Transition.

For decades, stem cell therapies for pulmonary hypertension (PH) have progressed from laboratory hypothesis to clinical practice. Promising preclinical investigations have laid both a theoretical and practical foundation for clinical application of mesenchymal stem cells (MSCs) for PH therapy. However, the underlying mechanisms are still poorly understood. We sought to study the effects and mechanisms of MSCs on the treatment of PH. For in vivo experiments, the transplanted GFP+ MSCs were traced at different time points in the lung tissue of a chronic hypoxia-induced PH (CHPH) rat model. The effects of MSCs on PH pathogenesis were evaluated in both CHPH and sugen hypoxia-induced PH models. For in vitro experiments, primary pulmonary microvascular endothelial cells were cultured and treated with the MSC conditioned medium. The specific markers of endothelial-to-mesenchymal transition (EndMT) and cell migration properties were measured. MSCs decreased pulmonary arterial pressure and ameliorated the collagen deposition, and reduced the thickening and muscularization in both CHPH and sugen hypoxia-induced PH rat models. Then, MSCs significantly attenuated the hypoxia-induced EndMT in both the lungs of PH models and primary cultured rat pulmonary microvascular endothelial cells, as reflected by increased mesenchymal cell markers (fibronectin 1 and vimentin) and decreased endothelial cell markers (vascular endothelial cadherin and platelet endothelial cell adhesion molecule-1). Moreover, MSCs also markedly inhibited the protein expression and degradation of hypoxia-inducible factor-2α, which is known to trigger EndMT progression. Our data suggest that MSCs successfully prevent PH by ameliorating pulmonary vascular remodeling, inflammation, and EndMT. Transplantation of MSCs could potentially be a powerful therapeutic approach against PH.

Efficacy and safety of tetramethylpyrazine phosphate on pulmonary hypertension: study protocol for a randomized controlled study.

BACKGROUND: Tetramethylpyrazine (TMP), an active ingredient in the traditional Chinese herbal medicine Rhizoma Chuanxiong, has been used clinically for the prevention and treatment of cardiovascular disease. The benefits of TMP are largely attributed to its anti-oxidative and vasodilative properties. However, the efficacy of TMP in the treatment of pulmonary hypertension (PH) is unknown. We hypothesized that TMP may have a therapeutic effect in patients with PH. METHODS/DESIGN: A randomized, single-blinded, clinical study with a TMP treatment group and a control group will be conducted to evaluate the efficacy and safety of TMP intervention in patients with PH. The recruitment target is 120 subjects meeting the following criteria: (i) at rest and at sea level, mean pulmonary artery pressure above 20 mmHg and pulmonary capillary wedge pressure below 15 mmHg; (ii) type 1 or 4 PH in the stable phase; (iii) age 15-70 years; (iv) 6-min walk distance between 100 and 450 m; (v) World Health Organization (WHO) functional classification of pulmonary hypertension of II, III, or IV. Subjects will be assigned randomly into two groups at a ratio of 1:2 (control:TMP). Both groups will receive routine treatment, and the treatment group will also receive oral TMP (100 mg) three times a day for 16 weeks. All patients will be followed up for 4, 8, 12, and 16 weeks; symptoms and patient compliance will be recorded. DISCUSSION: We aimed to determine the efficacy and safety of TMP for the treatment of PH. TRIAL REGISTRATION: Chinese Clinical Trial Register, ChiCTR1800018664. Registered on 2 October 2018.

Effects of ozone stimulation of bronchial epithelial cells on proliferation and collagen synthesis of co-cultured lung fibroblasts.

Ozone (O3) as a major air pollutant is widely recognized for causing pathological changes of the airway system. However, it is not clear whether O3 exposure of bronchial epithelial cells (BECs) influences the proliferation and collagen synthesis of submucosal fibroblasts and contributes to the pathogenesis of airway remodeling in diseases, including asthma. In the present study, a co-culture method was applied to culture human lung fibroblasts (HLFs) with human bronchial epithelial cells (HBECs) that were pre-stimulated with O3. Following co-culture for up to 24 h, the proliferation of HLFs was measured using MTT colorimetry. Furthermore, the collagen synthesis capacity of HLFs was determined by the level of hydroxyproline. In addition, the protein expression levels of cytokines, including transforming growth factor (TGF)-ß1, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2) were assessed. Results indicated that the proliferation of HLFs co-cultured with HBECs was significantly inhibited when compared with HLFs cultured alone (P<0.05). By contrast, co-culture with O3-stimulated HBECs significantly promoted the proliferation of HLFs compared with the HLFs cultured alone or those cultured with HBECs but no O3 stimulation, respectively (P<0.05 and P<0.01). Furthermore, similar effects were observed regarding the collagen synthesis capacity of HLFs co-cultured with HBECs for 24. In the supernatant, TGF-ß1 concentration was continuously increased over 24 h, whereas the concentration of PGE2 increased and plateaued between 12 to 24 h and TNF-α concentration was not significantly altered during the assessed time period. To conclude, the present results suggest that O3 pre-exposure of HBECs may promote the transformation of HLFs from the typical inhibitory state into a promoting state with respect to proliferation and collagen synthesis, which may likely occur through a mechanism that influences the balance between pro- and anti-inflammatory factors, including TGF-ß1 and PGE2. The present findings may improve the understanding of the mechanism involved in O3-induced airway remodeling from a novel perspective of maintenance/loss of steady-state function of the airway epithelium.

Comparison and evaluation of two different methods to establish the cigarette smoke exposure mouse model of COPD.

Animal model of cigarette smoke (CS) -induced chronic obstructive pulmonary disease (COPD) is the primary testing methodology for drug therapies and studies on pathogenic mechanisms of disease. However, researchers have rarely run simultaneous or side-by-side tests of whole-body and nose-only CS exposure in building their mouse models of COPD. We compared and evaluated these two different methods of CS exposure, plus airway Lipopolysaccharides (LPS) inhalation, in building our COPD mouse model. Compared with the control group, CS exposed mice showed significant increased inspiratory resistance, functional residual capacity, right ventricular hypertrophy index, and total cell count in BALF. Moreover, histological staining exhibited goblet cell hyperplasia, lung inflammation, thickening of smooth muscle layer on bronchia, and lung angiogenesis in both methods of CS exposure. Our data indicated that a viable mouse model of COPD can be established by combining the results from whole-body CS exposure, nose-only CS exposure, and airway LPS inhalation testing. However, in our study, we also found that, given the same amount of particulate intake, changes in right ventricular pressure and intimal thickening of pulmonary small artery are a little more serious in nose-only CS exposure method than changes in the whole-body CS exposure method.

The nonstructural protein 11 of porcine reproductive and respiratory syndrome virus inhibits NF-κB signaling by means of its deubiquitinating activity.

Since its emergence in the late 1980s, porcine reproductive and respiratory syndrome (PRRS) has been devastating the swine industry worldwide. The causative agent is an Arterivirus, referred to as PRRS virus (PRRSV). The pathogenic mechanisms of PRRS are poorly understood, but are believed to correlate with the ability of PRRSV to inhibit immune responses of the host. However, precisely how the virus is capable of doing so remains obscure. In this study, we showed that PRRSV infection led to reduced ubiquitination of cellular proteins. Screening all of the 12 nonstructural proteins (Nsps) encoded by PRRSV revealed that, apart from the Nsp2 which contains the deubiqintinating (DUB) ovarian tumor (OTU) domain, Nsp11, which encodes a unique and conserved endoribonuclease (NendoU) throughout the Nidovirus order, also possesses DUB activity. In vivo assay demonstrated that Nsp11 specifically removed lysine 48 (K48)-linked polyubiquitin chains and the conserved sites C112, H144, D173, K180, and Y219 were critical for its DUB activity. Remarkably, DUB activity was responsible for the capacity of Nsp11 to inhibit nuclear factor κB (NF-κB) activation. Mutations abrogating the DUB activity of Nsp11 toward K48-linked polyubiquitin chains of IκBα nullified the suppressive effect on NF-κB. Our data add Nsp11 to the list of DUBs encoded by PRRSV and uncover a novel mechanism by which PRRSV cripples host innate immune responses.

Mycobacterium tuberculosis Rv2185c contributes to nuclear factor-κB activation.

Tuberculosis caused by Mycobacterium tuberculosis has a detrimental impact on public health worldwide, especially in developing countries. The nuclear factor-κB (NF-κB) signaling pathway is reportedly involved in the innate immune response against M. tuberculosis infections. We screened the secreted proteins, membrane proteins, and lipoproteins of the M. tuberculosis H37Rv strain using luciferase activity assays. The Rv2185c protein exhibited the potential to activate NF-κB in HeLa and A549 cells. Overexpression of Rv2185c-induced IκBα degradation and nuclear translocation of NF-κB; it also induced NF-κB-dependent inflammatory factors, including interleukin (IL)-6, IL-8, IL-1ß and tumor necrosis factor (TNF)-α. The intact binding site for the NF-κB element is required for the activation of Rv2185c-induced IL-6 and IL-8 gene expression. NF-κB activation and NF-κB-regulated genes encoding TNF-α and TNF-related apoptosis-inducing ligand have also been shown to be involved in Rv2185c-induced apoptosis.

Ubiquitin-specific proteases 25 negatively regulates virus-induced type I interferon signaling.

Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-ß, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), as well as the phosphorylation of IRF3 and NF-κB subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-ß. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.