Programmable Ligation-Transcription Circuit-Driven Cascade Amplification Machinery for Multiple Long Noncoding RNAs Detection in Lung Tissues.

The measurement of long noncoding RNAs (lncRNAs) is essential to diagnosis and treatment of various diseases such as cancers. Herein, we develop a simple method to simultaneously detect multiple lncRNAs using programmable ligation-transcription circuit-driven cascade amplification and single-molecule counting. The presence of targets lncRNA HOTAIR and lncRNA MALAT1 activates the ligation-transcription circuits to produce two corresponding functional RNAs. The functional RNAs then cyclically initiate the digestion of signal probes by duplex-specific nuclease to liberate Cy5 and Cy3 molecules. After magnetic separation, the liberated Cy5 and Cy3 molecules are measured by single-molecule counting. In this assay, a single lncRNA can activate ligation-transcription circuit to generate abundant functional RNAs, endowing this assay with high sensitivity. Integration of single-molecule counting ensures the high sensitivity. This method shows extremely high sensitivity with a limit of detection (LOD) of 0.043 aM for HOX gene antisense intergenic RNA (lncRNA HOTAIR) and 0.126 aM for mammalian metastasis-related lung adenocarcinoma transcript 1 (lncRNA MALAT1). Importantly, this method enables simultaneous measurement of multiple endogenous lncRNAs at the single-cell level, and it may discriminate the expressions of various lncRNA in lung tumor tissues of nonsmall cell lung cancer (NSCLC) patients and their corresponding healthy adjacent tissues, offering a promising platform for clinical diagnosis and biomedical research.

Necrostatin-1 attenuates Caspase-1-dependent pyroptosis induced by the RIPK1/ZBP1 pathway in ventilator-induced lung injury.

BACKGROUND: Ventilator-induced lung injury (VILI) is a complex pathophysiological process leading to acute respiratory distress syndrome (ARDS) and poor outcomes in affected patients. As a form of programmed cell death, pyroptosis is proposed to play an important role in the development of ARDS. Here we investigated whether treating mice with the specific RIPK1 inhibitor Necrostatin-1 (Nec-1) before mechanical ventilation could inhibit pyroptosis and alleviate lung injury in a mouse model. METHODOLOGYS: Anesthetized C57BL/6J mice received a transtracheal injection of Nec-1 (5 mg/kg) or vehicle (DMSO) 30 min before the experiment which was ventilated for up to 4 h. Lung damage was assessed macroscopically and histologically with oedema measured as the wet/dry ratio of lung tissues. The release of inflammatory mediators into bronchoalveolar lavage fluid (BALF) was assessed by ELISA measurements of TNF-α,interleukin-1ß (IL-1ß), and IL-6. The expression of RIPK1, ZBP1, caspase-1, and activated (cleaved) caspase-1 were analyzed using western blot and immunohistochemistry, and the levels of gasdermin-D (GSDMD) and IL-1ß were analyzed by immunofluorescence staining. RESULTS: High tidal ventilation produced time-dependent inflammation and lung injury in mice which could be significantly reduced by pretreatment with Nec-1. Notably, Nec-1 reduced the expression of key pyroptosis mediator proteins in lung tissues exposed to mechanical ventilation, including caspase-1, cleaved caspase-1, and GSDMD together with inhibiting the release of inflammatory cytokines. CONCLUSION: Nec-1 pretreatment alleviates pulmonary inflammatory responses and protects the lung from mechanical ventilation damage. The beneficial effects were mediated at least in part by inhibiting caspase-1-dependent pyroptosis through the RIPK1/ZBP1 pathway.

Influences of dexmedetomidine on stress responses and postoperative cognitive and coagulation functions in patients undergoing radical gastrectomy under general anesthesia.

BACKGROUND: Radical gastrectomy (RG) is commonly used in the treatment of patients with gastric cancer (GC), but this procedure may lead to stress responses, postoperative cognitive dysfunction, and blood coagulation abnormalities in patients. AIM: To investigate the influences of dexmedetomidine (DEX) on stress responses and postoperative cognitive and coagulation functions in patients undergoing RG under general anesthesia (GA). METHODS: One hundred and two patients undergoing RG for GC under GA from February 2020 to February 2022 were retrospectively reviewed. Of these, 50 patients had received conventional anesthesia intervention [control group (CG)] and 52 patients had received DEX in addition to routine anesthesia intervention [observation group (OG)]. Inflammatory factor (IFs; tumor necrosis factor-α, TNF-α; interleukin-6, IL-6), stress responses (cortisol, Cor; adrenocorticotropic hormone, ACTH), cognitive function (CF; Mini-Mental State Examination, MMSE), neurological function (neuron-specific enolase, NSE; S100 calcium-binding protein B, S100B), and coagulation function (prothrombin time, PT; thromboxane B2, TXB2; fibrinogen, FIB) were compared between the two groups before surgery (T0), as well as at 6 h (T1) and 24 h (T2) after surgery. RESULTS: Compared with T0, TNF-α, IL-6, Cor, ACTH, NSE, S100B, PT, TXB2, and FIB showed a significant increase in both groups at T1 and T2, but with even lower levels in OG vs CG. Both groups showed a significant reduction in the MMSE score at T1 and T2 compared with T0, but the MMSE score was notably higher in OG compared with CG. CONCLUSION: In addition to a potent inhibitory effect on postoperative IFs and stress responses in GC patients undergoing RG under GA, DEX may also alleviate the coagulation dysfunction and improve the postoperative CF of these patients.

Mismatched fluorescent probes with an enhanced strand displacement reaction rate for intracellular long noncoding RNA imaging.

We design mismatched fluorescent probes to directly monitor the long noncoding RNA (lncRNA) in living cells. The introduction of mismatched bases in the fluorescent probe greatly enhances the strand displacement reaction rate toward the target lncRNA. These mismatched probes can monitor the intracellular lncRNA expression level in various cell lines and discriminate cancer cells from normal cells, holding great potential in fundamental biomedical research and clinical disease diagnosis.

Clinical impact of type 2 diabetes mellitus on outcomes after one-lung ventilation during thoracic surgery: a retrospective cohort study.

BACKGROUND: The preoperative presence of diabetes mellitus (DM) has been recently demonstrated to be a risk factor for adverse events after thoracic surgery. However, the specific effects of presence of DM preoperatively on thoracic surgery is not known. This study aimed to investigate the association between preoperative DM and clinical outcomes and the short-term survival rates after thoracic surgery. METHODS: In this retrospective, observational, and matched-pair analysis study, patients receiving thoracic surgery from a tertiary university hospital in 2 consecutive years were grouped as either type 2 DM (T2DM) or controlled within the first 24 hours after surgery. Multivariate Cox regression was conducted to investigate the impact of T2DM within the first 24 hours of admission on in-intensive care unit (ICU) and hospital survival. RESULTS: Among the included thoracic patients, 41 (8.4%) had T2DM and 450 (91.6%) did not have T2DM. In the single-factor analyses, T2DM patients were shown to have a higher preoperative white blood cells (WBCs) count, increased release of immunoglobulin A, complement C3 and C4, impaired kidney function with high level of urea, and low expression of alanine aminotransferase (ALT) and monoamine oxidase (MAO). In multivariate analyses, the preoperative urea level was associated with a low-grade risk of dying for the ICU survival time. In contrast, preoperative complement C3 level favored a positive contribution in-ICU survival. Besides the complement C3 level, immunoglobulin A level remained a positive contribution in regression models of hospital survival. CONCLUSIONS: Pre-admission T2DM was not associated with an increased in-ICU and hospital mortality among patients with thoracic surgery. Furthermore, they were accompanied by impaired kidney function, activated inflammation and liver function.

Mitophagy-Mediated mtDNA Release Aggravates Stretching-Induced Inflammation and Lung Epithelial Cell Injury via the TLR9/MyD88/NF-κB Pathway.

BACKGROUND: In animal models of ventilation-induced lung injury, mitophagy triggers mitochondria damage and the release of mitochondrial (mt) DNA, which activates inflammation. However, the mechanism of this process is unclear. METHODS: A model of cyclic stretching (CS)-induced lung epithelial cell injury was established. The genetic intervention of phosphatase and tensin homolog-induced kinase 1 (PINK1) expression via lentivirus transfection was used to identify the relationship between PINK1-mediated mitophagy and mtDNA release in stretching-induced inflammatory response and injury. Pharmacological inhabitation of Toll-like receptor 9 (TLR9) and myeloid differentiation factor 88 (MyD88) expression was performed via their related inhibitors, while pre-treatment of exogenous mtDNA was used to verify the role of mtDNA in stretching-induced inflammatory response and injury. RESULTS: Using a cell culture model of CS, we found that knocking down PINK1 in lung epithelial cells reduced mitophagy activation and mtDNA release, leading to milder inflammatory response and injury; conversely, up-regulating PINK1 exacerbated stretching-induced inflammation and injury, and similar effects were observed by upregulating TLR9 to induce expression of MyD88 and nuclear factor-κB (NF-κB)/p65. Down-regulating MyD88 protected lung epithelial cells from stretching injury and decreased NF-κB/p65 expression. CONCLUSION: These findings suggest that PINK1-dependent mitophagy and associated TLR9 activation is indeed a major factor in stretch-induced cell injury via a mechanism in which released mtDNA activates TLR9 and thereby the MyD88/NF-κB pathway. Inhibiting this process may be a therapeutic approach to prevent inflammation and cell injury in patients on mechanical ventilation.

[Effect of activation of nuclear factor-KappaB on modulation of secretion of intercellular adhesion molecule-1 by A549 cell].

OBJECTIVE: To investigate the effects of interleukin-1beta (IL-1beta) on intercellular adhesion molecule-1 (ICAM-1) expression on A549 cell and underlying signal transduction pathways. METHODS: A549 cells were pre-incubated with SC-514 [nuclear factor-KappaB (NF-KappaB) inhibitor (IKappaB) kinase-2 (IKK-2) inhibitor] and/or pre-treated with 1 microg/L IL-1beta. The phosphorylated IKappaBalpha (pIKappaBalpha) and degradation of IKappaBalpha were determined by Western blotting with specific antibody at 5, 10, 30 and 60 minutes. Laser scanning confocal microscope (LSCM) was used to examine the nuclear translocation of p65 at 30 minutes after stimulation. The DNA binding activity of p65 in nuclear extracts was detected at 1 hour following IL-1beta treatment. Chromatin immunoprecipitation (ChIP) assays combined with polymerase chain reaction (PCR) were used to evaluate interaction between p65 and ICAM-1 promoter site DNA at 1 hour after stimulation. The expression of ICAM-1 mRNA was assessed by reverse transcription (RT)-PCR at 4 hours, and the ICAM-1 expression on A549 cell surface was measured by enzyme linked immunosorbent assay (ELISA) at 24 hours after IL-1beta was added. RESULTS: IL-1beta induced rapid pIKappaBalpha augmentation and its subsequent degradation. LSCM graphs showed that IL-1beta stimulated the translocation of p65 from the cytosol to the nucleus. IL-1beta significantly increased the DNA binding ability of p65 (P<0.01) in cell nuclear extracts. ChIP-PCR suggested that both acetylated histone 4 and p65 were recruited to ICAM-1 promoter. IL-1beta significantly augmented ICAM-1 mRNA level at 4 hours and expression of ICAM-1 on A549 cell surface at 24 hours (both P<0.01). The IKK-2 inhibitor, SC-514, inhibited IL-1beta induced IKappaBalpha protein activity, blocked p65 nuclear translocation, caused a significant reduction in IL-1beta induced DNA binding activity for p65 and ICAM-1 mRNA expression, and suppressed ICAM-1 expression on A549 cell surface (all P<0.01). CONCLUSION: These results suggest that the activation of NF-KappaB mediates IL-1beta induced ICAM-1 expression in A549 cells.

Necroptosis in pulmonary macrophages mediates lipopolysaccharide-induced lung inflammatory injury by activating ZBP-1.

Z-DNA combined protein-1 (ZBP-1), an important necroptosis regulator, activates necrosis-associated inflammation and immune response. Increased ZBP-1 expression in necroptosis-associated inflammation correlates with activation of receptor interacting protein kinase (RIPK1)/RIPK3 and nuclear factor (NF)-κB. Here we explored the role of ZBP-1-mediated necroptosis in lipopolysaccharide (LPS)-induced lung injury. Bone marrow-derived macrophages (BMDMs) transfected with a small interfering RNA against ZBP-1 or scrambled control RNA were administered to mice that had been depleted of alveolar macrophages (AMs). Then the animals were treated with E. coli LPS (2.0 mg/kg) or phosphate-buffered saline by intratracheal instillation for 48 h. LPS-induced lung inflammatory injury was verified, and the mRNA and protein expression of ZBP-1, RIPK1/RIPK3 and NF-κB in AMs were then assessed by Western blot and real time-quantitative polymerase chain reaction. In mechanistic studies in vitro, BMDM cultures were treated with different concentrations of LPS for 24 h, and the expression of ZBP-1, RIPK1/RIPK3 and NF-κB were assessed. LPS activated ZBP-1-mediated necroptosis, primarily in AMs. This activation and associated lung inflammatory injury were much weaker after AMs depletion or silencing of ZBP-1 in BMDMs, which correlated with down-regulation of RIPK1/RIPK3. These in vivo findings were confirmed in experiments with cultures of BMDMs. In conclusion, LPS induces lung inflammation and injury by activating ZBP-1-mediated necroptosis and release of pro-inflammatory cytokines by macrophages.

Non-intubated anesthesia in patients undergoing video-assisted thoracoscopic surgery: A systematic review and meta-analysis.

INTRODUCTION: Non-intubated anesthesia (NIA) has been proposed for video-assisted thoracoscopic surgery (VATS), although how the benefit-to-risk of NIA compares to that of intubated general anesthesia (IGA) for certain types of patients remains unclear. Therefore, the aim of the present meta-analysis was to understand whether NIA or IGA may be more beneficial for patients undergoing VATS. METHODS: A systematic search of Cochrane Library, Pubmed and Embase databases from 1968 to April 2019 was performed using predefined criteria. Studies comparing the effects of NIA or IGA for adult VATS patients were considered. The primary outcome measure was hospital stay. Pooled data were meta-analyzed using a random-effects model to determine the standard mean difference (SMD) with 95% confidence intervals (CI). RESULTS AND DISCUSSION: Twenty-eight studies with 2929 patients were included. The median age of participants was 56.8 years (range 21.9-76.4) and 1802 (61.5%) were male. Compared to IGA, NIA was associated with shorter hospital stay (SMD -0.57 days, 95%CI -0.78 to -0.36), lower estimated cost for hospitalization (SMD -2.83 US, 95% CI -4.33 to -1.34), shorter chest tube duration (SMD -0.32 days, 95% CI -0.47 to -0.17), and shorter postoperative fasting time (SMD, -2.76 days; 95% CI -2.98 to -2.54). NIA patients showed higher levels of total lymphocytes and natural killer cells and higher T helper/T suppressor cell ratio, but lower levels of interleukin (IL)-6, IL-8 and C-reactive protein (CRP). Moreover, NIA patients showed lower levels of fibrinogen, cortisol, procalcitonin and epinephrine. CONCLUSIONS: NIA enhances the recovery from VATS through attenuation of stress and inflammatory responses and stimulation of cellular immune function.

Adjuvant sorafenib in hepatocellular carcinoma: A cautionary comment of STORM trial.

Recurrence rate of hepatocellular carcinoma (HCC) is very high even after curative surgery, and no postoperative therapies have been definitively shown to prevent HCC recurrence. Sorafenib is proved to be effective for advanced HCC by two large randomized controlled trials in 2008 and 2009. Therefore it stands to reason to expect that adjuvant sorafenib may improve post-surgery outcomes of patients with HCC. However, many questions still exist about the value of sorafenib for patients with HCC after surgery or transarterial chemoembolization. In this editorial, we complehensively reviewed the safety and efficacy of adjuvant sorafenib for patients with hepatocellar carcinoma after surgery or transarterial chemoembolization. We emphasized the positive and negative role of sorafenib.