Levels of Cry1Ab protein in several herbivores and their predators through food chain from transgenic Bt rice (KMD1) were studied in the laboratory. The results showed that the levels of Cry1Ab in stripped stem borer (SSB) Chilo suppressalis continuously feeding on KMD1 or on Xushui 11 control rice after feeding on KMD1 for 36 h declined with time at the booting stage as well as at the maturing stage. Levels of Cry1Ab in Mycalesis gotama continuously feeding on KMD1 or on Xushui 11 control rice after feeding on KMD1 for two days declined with time, but more dramatically than those of SSB. High levels of Cry1Ab were detected in faeces of both C. suppressalis and M. gotama, but no Cry1Ab detected in their controls. 3.5 ng/g Cry1Ab was detected in the haemolymph of C. suppressalis feeding on KMD1. Different levels of Cry1Ab were all detected in brown planthopper (BPH) Nilaparvata lugens, rice aphid (RA) Sitobion avenae reared on KMD1, and wolf spider (WS) Pirata subpiraticus fed with preys that had ingested KMD1. The concentration of Cry1Ab protein in P. subpiraticus fed with M. gotama larvae reared on KMD1 was about 60 times higher than that in P. subpiraticus fed with C. suppressalis reared on KMD1 for 36 h, and no Cry1Ab protein was detected in their controls. These results indicated Cry1Ab protein could be transferred along food chain.
Abstract Airway epithelial transcriptome analysis of asthma patients with different severity was used to disentangle the immune infiltration mechanisms affecting asthma exacerbation, which may be advantageous to asthma treatment. Here we introduce various bioinformatics methods and develop two models: an OVA/CFA-induced neutrophil asthma mouse model and an LPS-induced human bronchial epithelial cell damage model. Our objective is to investigate the molecular mechanisms, potential targets, and therapeutic strategies associated with asthma severity. Multiple bioinformatics methods identify meaningful differences in the degree of neutrophil infiltration in asthma patients with different severity. Then, PTPRC, TLR2, MMP9, FCGR3B, TYROBP, CXCR1, S100A12, FPR1, CCR1 and CXCR2 are identified as the hub genes. Furthermore, the mRNA expression of 10 hub genes is determined in vivo and in vitro models. Reperixin is identified as a pivotal drug targeting CXCR1, CXCR2 and MMP9. We further test the potential efficiency of Reperixin in 16HBE cells, and conclude that Reperixin can attenuate LPS-induced cellular damage and inhibit the expression of them. In this study, we successfully identify and validate several neutrophilic signatures and targets associated with asthma severity. Notably, Reperixin displays the ability to target CXCR1, CXCR2, and MMP9, suggesting its potential therapeutic value for managing deteriorating asthma.
Acute respiratory distress syndrome (ARDS) is a disease that seriously threatens human life and health. The aim of the study was to investigate the effects of ulinastatin combined with mechanical ventilation on oxygen metabolism, inflammation and stress response, as well as the antioxidant capacity of ARDS. Eighty patients with ARDS treated in Yiwu Central Hospital from January, 2015 to December, 2016 were enrolled in the present study and divided into the observation (n=40) and control (n=40) groups, using a random number table. The control group was treated with mechanical ventilation, while the observation group, based on treatment of the control group, was treated with ulinastatin for 14 consecutive days as one course of treatment. The changes in the relevant indexes of oxygen metabolism, lung function, time of ventilator treatment, total hospital stay, and St. George's Respiratory Questionnaire (SGRQ) score of the two groups after intervention were compared, and the changes in inflammatory cytokine levels, dopamine receptor-related hormone levels, superoxide dismutase (SOD), malondialdehyde (MDA) and total antioxidant capacity of the two groups before intervention and at 1 and 4 weeks after intervention were compared. After intervention, the arterial blood lactate in the observation group was significantly lower than that in the control group (P<0.05), the oxygen uptake rate was significantly higher than that in the control group (P<0.05) and the arterial oxygen content was significantly higher than that in the control group (P<0.05). In the lung function indexes, the FEV1 and FEV1/FVC levels in the observation group were smaller than those in the control group (P<0.05), the duration of ventilator treatment was significantly shorter than that in the control group (P<0.05), and the hospital stay was significantly less than that in the control group (P<0.05). Prior to intervention, SGRQ scores in the two groups were not statistically significant (P>0.05). At 1 and 4 weeks after intervention, the SGRQ scores of the observation group were significantly increased to those of the control group (P<0.05). The tumor levels of necrosis factor-α (TNF-α), interleukin-6 (IL-6) and CRP were significantly lower than those of the control group (P<0.05). The levels of adrenaline and norepinephrine were significantly lower than those of the control group (P<0.05). The levels of MDA, SOD and the total antioxidant capacity were significantly increased to those of control group (P<0.05). The application of ulinastatin combined with mechanical ventilation in ARDS patients is of great significance in improving the oxygen delivery‑consumption balance of body, increasing the lung function, reducing the inflammatory and stress response, and improving the antioxidant capacity.
Abstract The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9‐ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9‐mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV‐infected human hepatoma cell line HepG2.2.15 with HBV‐specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene‐editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off‐target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA/Cas9 RNP‐based gene‐editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.
T cells are crucial for controlling viral infections; however, the mechanisms that dampen their responses during viral infections remain incompletely understood. Here, we studied the role and mechanisms of mitochondrial topoisomerase 1 (Top1mt) inhibition in mitochondrial dysfunction and T cell dysregulation using CD4 T cells from patients infected with HCV or HIV and compared it with CD4 T cells from healthy individuals following treatment with Top1 inhibitor - camptothecin (CPT). We found that Top1mt protein levels and enzymatic activity are significantly decreased, along with Top1 cleavage complex (Top1cc) formation, in mitochondria of CD4 T cells from HCV- and HIV-infected patients. Notably, treatment of healthy CD4 T cells with CPT caused similar changes, including inhibition of Top1mt, accumulation of Top1cc in mitochondria, increase in PARP1 cleavage, and decrease in mtDNA copy numbers. These molecular changes resulted in mitochondrial dysfunction, T cell dysregulation, and programmed cell death through multiple signaling pathways, recapitulating the phenotype we detected in CD4 T cells from HCV- and HIV-infected patients. Moreover, treatment of CD4 T cells from HCV or HIV patients with CPT further increased cellular and mitochondrial reactive oxygen species (ROS) production and cell apoptosis, demonstrating a critical role for Top1 in preventing mtDNA damage and cell death. These results provide new insights into the molecular mechanisms underlying immune dysregulation during viral infection and indicate that Top1 inhibition during chronic HCV or HIV infection can induce mtDNA damage and T cell dysfunction. Thus, reconstituting Top1mt protein may restore the mtDNA topology and T cell functions in humans with chronic viral infection.
Severe asthma (SA), a heterogeneous inflammatory disease characterized by immune cell infiltration, is particularly difficult to treat and manage. The airway epithelium is an important tissue in regulating innate and adaptive immunity, and targeting airway epithelial cell may contribute to improving the efficacy of asthma therapy.Bioinformatics methods were utilized to identify the hub genes and signaling pathways involved in SA. Experiments were performed to determine whether these hub genes and signaling pathways were affected by the differences in immune cell infiltration.The weighted gene coexpression network analysis identified 14 coexpression modules, among which the blue and salmon modules exhibited the strongest associations with SA. The blue module was mainly enriched in actomyosin structure organization and was associated with regulating stem cell pluripotency signaling pathways. The salmon module was mainly involved in cornification, skin development, and glycosphingolipid biosynthesis-lacto and neolacto series. The protein-protein interaction network and module analysis identified 11 hub genes in the key modules. The CIBERSORTx algorithm revealed statistically significant differences in CD8+ T cells (P = 0.013), T follicular helper cells (P = 0.002), resting mast cells (P = 0.004), and neutrophils (P = 0.002) between patients with SA and mild-moderate asthma patients. Pearson's correlation analysis identified 11 genes that were significantly associated with a variety of immune cells. We further predicted the utility of some potential drugs and validated our results in external datasets.Our results may help provide a better understanding of the relationship between the airway epithelial transcriptome and clinical data of SA. And this study will help to guide the development of SA-targeted molecular therapy.
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