Background Although traditional diagnostic techniques of infection are mature and price favorable at present, most of them are time-consuming and with a low positivity. Metagenomic next⁃generation sequencing (mNGS) was studied widely because of identification and typing of all pathogens not rely on culture and retrieving all DNA without bias. Based on this background, we aim to detect the difference between mNGS and traditional culture method, and to explore the relationship between mNGS results and the severity, prognosis of infectious patients. Methods 109 adult patients were enrolled in our study in Shanghai Tenth People’s Hospital from October 2018 to December 2019. The diagnostic results, negative predictive values, positive predictive values, false positive rate, false negative rate, pathogen and sample types were analyzed by using both traditional culture and mNGS methods. Then, the samples and clinical information of 93 patients in the infected group (ID) were collected. According to whether mNGS detected pathogens, the patients in ID group were divided into the positive group of 67 cases and the negative group of 26 cases. Peripheral blood leukocytes, C-reactive protein (CRP), procalcitonin (PCT) and neutrophil counts were measured, and the concentrations of IL-2, IL-4, IL-6, TNF-α, IL-17A, IL-10 and INF-γ in the serum were determined by ELISA. The correlation between the positive detection of pathogens by mNGS and the severity of illness, hospitalization days, and mortality were analyzed. Results 109 samples were assigned into infected group (ID, 92/109, 84.4%), non-infected group (NID, 16/109, 14.7%), and unknown group (1/109, 0.9%). Blood was the most abundant type of samples with 37 cases, followed by bronchoalveolar lavage fluid in 36 cases, tissue, sputum, pleural effusion, cerebrospinal fluid (CSF), pus, bone marrow and nasal swab. In the ID group, the majority of patients were diagnosed with lower respiratory system infections (73/109, 67%), followed by bloodstream infections, pleural effusion and central nervous system infections. The sensitivity of mNGS was significantly higher than that of culture method (67.4% vs 23.6%; P < 0.001), especially in sample types of bronchoalveolar lavage fluid (P = 0.002), blood (P < 0.001) and sputum (P = 0.037), while the specificity of mNGS was not significantly different from culture method (68.8% vs 81.3%; P = 0.41). The number of hospitals stays and 28-day-motality in the positive mNGS group were significantly higher than those in the negative group, and the difference was statistically significant (P < 0.05). Age was significant in multivariate logistic analyses of positive results of mNGS. Conclusions The study found that mNGS had a higher sensitivity than the traditional method, especially in blood, bronchoalveolar lavage fluid and sputum samples. And positive mNGS group had a higher hospital stay, 28-day-mortality, which means the positive of pathogen nucleic acid sequences detection may be a potential high-risk factor for poor prognosis of adult patients and has significant clinical value. MNGS should be used more in early pathogen diagnosis in the future.

Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-β and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis in vitro induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment.

The transcription factor Bach1 impairs angiogenesis after ischemic injury by suppressing Wnt/b-catenin signaling; however, the specific domains responsible for the anti-angiogenic effects of Bach1 remain unclear. This study determined the role of the BTB domain of Bach1 in ischemic angiogenesis. Bach1 is highly expressed in circulating endothelial cells from acute myocardial infarction patients and is the early induction gene after ischemia. Mice were treated with adenoviruses coding for GFP (AdGFP), Bach1 (AdBach1), or a Bach1 mutant lacking the BTB domain (AdBach1-DBTB) after surgically induced hind-limb ischemia. Measures of blood-flow recovery, capillary density, and the expression of vascular endothelial growth factor (VEGF) and heme oxygenase-1 (HO-1) were significantly lower and ROS levels were higher in the AdBach1 group, but not in AdBach1-DBTB animals. Furthermore, transfection with AdBach1, but not AdBach1-DBTB, in human endothelial cells was associated with significant declines in 1) capillary density and hemoglobin content in the Matrigel-plug assay, 2) proliferation, migration, tube formation, and VEGF and HO-1 expression in endothelial cells. Bach1 binds directly with TCF4, and this interaction is mediated by residues 81À89 of the Bach1 BTB domain and the N-terminal domain of TCF4. Bach1, but not Bach1-DBTB, also co-precipitated with histone deacetylase 1 (HDAC1), while the full-length HDAC1 proteins, but not HDAC1 mutants lacking the protein-interaction domain, co-precipitated with Bach1. Collectively, these results demonstrate that the anti-angiogenic activity of Bach1 is crucially dependent on molecular interactions that are mediated by the protein's BTB domain, and this domain could be a drug target for angiogenic therapy.