Comparison of fluorescence biosensors and whole-cell patch clamp recording in detecting ACh, NE, and 5-HT.

The communication between neurons and, in some cases, between neurons and non-neuronal cells, through neurotransmission plays a crucial role in various physiological and pathological processes. Despite its importance, the neuromodulatory transmission in most tissues and organs remains poorly understood due to the limitations of current tools for direct measurement of neuromodulatory transmitters. In order to study the functional roles of neuromodulatory transmitters in animal behaviors and brain disorders, new fluorescent sensors based on bacterial periplasmic binding proteins (PBPs) and G-protein coupled receptors have been developed, but their results have not been compared to or multiplexed with traditional methods such as electrophysiological recordings. In this study, a multiplexed method was developed to measure acetylcholine (ACh), norepinephrine (NE), and serotonin (5-HT) in cultured rat hippocampal slices using simultaneous whole-cell patch clamp recordings and genetically encoded fluorescence sensor imaging. The strengths and weaknesses of each technique were compared, and the results showed that both techniques did not interfere with each other. In general, genetically encoded sensors GRABNE and GRAB5HT1.0 showed better stability compared to electrophysiological recordings in detecting NE and 5-HT, while electrophysiological recordings had faster temporal kinetics in reporting ACh. Moreover, genetically encoded sensors mainly report the presynaptic neurotransmitter release while electrophysiological recordings provide more information of the activation of downstream receptors. In sum, this study demonstrates the use of combined techniques to measure neurotransmitter dynamics and highlights the potential for future multianalyte monitoring.

SWEmean of Quadriceps, a Potential Index of Complication Evaluation to Patients with Chronic Obstructive Pulmonary Disease.

Purpose: To develop a potential quadriceps' index of complication evaluation for patients with chronic obstructive pulmonary disease (COPD) which is simple, convenient, and quantifiable. Patients and Methods: We conducted a prospective study of 59 patients with COPD and 56 healthy controls recruited by the Chengdu First People's Hospital. Grayscale ultrasound (US) of the rectus femoris was performed to measure thickness (RFthick) and cross-sectional area (RFcsa). Shear wave elastography was used to determine the mean elasticity index (SWEmean) of the rectus femoris (SWERFmean), vastus lateralis (SWEVLmean) and vastus medialis (SWEVMmean). Clinical features included dyspnea index score (modified British Medical Research Council (MMRC) score), COPD Assessment Test (CAT), the Five-Repetition Sit-to-Stand Test (5STS) and the Six-Minute Walk Test (6MWT). We compared the differences between US parameters and SWEmean in healthy controls and COPD patients. We also described the correlation between US parameters, SWEmean and clinical features of patients with COPD. Results: The intra-observer repeatability for the performance of using SWE to measure quadriceps stiffness was excellent (intraclass correlation coefficient (ICC)>0.75, p < 0.001). There was a statistically significant difference in the SWEmean of the quadriceps (p < 0.001), but no significant difference in terms of RFthic and RFcsa (p > 0.05) between healthy controls and COPD patients. The SWERFmean was positively correlated with the 6MWT (r = 0.959, p < 0.001), and negatively related to the mMRC (r=-0.825, p < 0.001), CAT (r=-0.993, p < 0.001) and 5STS (r=-0.936, p < 0.001). However, the RFthic, RFcsa, SWEVLmean and SWEVMmean were not correlated with clinical features (p > 0.05). Conclusion: As a supplement to US, SWE reflects changes of stiffness in the quadriceps of COPD patients, and can expanding the dimension of US for assessing the quadriceps. Furthermore, SWEmean was associated with clinical features, and represents a potential index with which to reflect the clinical features of COPD patients.

Circ_0001058 represses the progression of lung adenocarcinoma through governing of the miR-486-5p/TEK signaling axis.

The most common type of lung cancer is lung adenocarcinoma. Emerging views believe that circular RNA (circRNA) participates in its pathogenesis. The objective of this study is to find out the potential functions and mechanisms of circ_0001058 in lung adenocarcinoma pathogenesis. To detect circ_0001058, miR-486-5p and TEK tyrosine kinase (TEK) receptor tyrosine kinase expressions, real-time quantitative PCR (RT-qPCR) and western blotting were performed. Cell functions, including proliferation, apoptosis and invasion, were then evaluated using cell counting kit-8, caspase-3 activity and transwell assays, respectively. To establish the role of circ_0001058 in tumorigenesis, nude mice were utilized as in-vivo models. The predicted binding relationships of miR-486-5p to circ_0001058 or TEK were further verified by performing a dual-luciferase assay and ribonucleoprotein immunoprecipitation (RIP) analysis. Decreased circ_0001058 expression was observed in lung adenocarcinoma cells and tissue specimens. Circ_0001058 was predominantly situated in the cytoplasm and was greatly resistant to RNase R digestion. Circ_0001058 overexpression restrained A549 and PC9 cells' abilities to proliferate, survive and invade, and it also repressed tumorigenesis in the animal models. Circ_0001058 directly targeted miR-486-5p and depleted its expression. Restoring miR-486-5p could invert the inhibitory effects of circ_0001058 in the cancer cell phenotypes. Furthermore, miR-486-5p targeted TEK, so the inhibitory effects of TEK overexpression on the malignant behaviors of A549 and PC9 cells could also be abolished by miR-486-5p restoration. Circ_0001058 overexpression blocked the malignant development of lung adenocarcinoma via modulation of the miR-486-5p/TEK pathway. These results contribute new insights on the pathogenesis of lung adenocarcinoma.

Effect of Autoimmune Cell Therapy on Immune Cell Content in Patients with COPD: A Randomized Controlled Trial.

OBJECTIVE: To explore the effect of autoimmune cell therapy on immune cells in patients with chronic obstructive pulmonary disease (COPD) and to provide a reference for clinical treatment of COPD. METHODS: Sixty patients with stable COPD were randomly divided into control group and treatment group (n = 30). The control group was given conventional treatment, and the treatment group was given one autoimmune cell therapy on the basis of conventional treatment. The serum levels of CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the peripheral blood were detected by flow cytometry. Possible adverse reactions were detected at any time during treatment. RESULTS: There were no significant differences in the contents of CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the serum of the control group (P > 0.05). Compared with before treatment, the contents of CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the serum of the treatment group were significantly increased (P < 0.05). The ratio of CD4 + /CD8+ T cells in both control and treatment groups did not change significantly during treatment (P > 0.05). There were no significant differences in serum CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the treatment group at 30 days and 90 days after treatment (P > 0.05), but they were significantly higher than those in the control group (P < 0.05). CONCLUSION: Autoimmune cell therapy can significantly increase the level of immune cells in the body and can be maintained for a long period of time, which has certain clinical benefits for recurrent respiratory tract infections and acute exacerbation in patients with COPD.

Circular RNA MAT2B promotes migration, invasion and epithelial-mesenchymal transition of non-small cell lung cancer cells by sponging miR-431.

Circular RNAs (circRNAs) have recently been described as key regulators in the progression of non-small cell lung cancer (NSCLC), and this study aimed to investigate the functional role of circMAT2B in NSCLC. CircMAT2B expression in NSCLC tissues and cell lines was investigated using RT-qPCR analysis. A series of functional experiments, including MTT assay, colony formation assay, wound healing assay and transwell assay, were carried out to investigate the effects of circMAT2B knockdown/overexpression on the malignant traits of NSCLC cells. Western blot analysis was performed to detect the expression of EMT-related proteins. Dual-luciferase reporter assay and RIP assay were further carried out to analyze the interaction between circMAT2B and miR-431 in NSCLC. We observed that circMAT2B was overexpressed in NSCLC tissues and cell lines, and high expression of circMAT2B was closely associated with large tumor size, advanced TNM stage and poor prognosis of NSCLC patients. Further functional experiments showed that circMAT2B knockdown markedly inhibited the proliferation, migration, invasion and EMT of NSCLC cells, whereas circMAT2B overexpression led to the opposing results. Mechanistically, circMAT2B could directly interact with miR-431, and subsequently decrease miR-431 expression in NSCLC. The effects of circMAT2B overexpression in NSCLC cells were abrogated by miR-431 restoration. Our findings revealed the novel oncogenic roles of circMAT2B in NSCLC by sponging miR-431.

SNHG17 upregulates WLS expression to accelerate lung adenocarcinoma progression by sponging miR-485-5p.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been uncovered to be essential regulators in the biological processes of human cancers, including lung adenocarcinoma (LUAD). Recently, small nucleolar RNA host gene 17 (SNHG17) has been identified as one novel oncogenic lncRNA in gastric cancer. However, it remains unclear whether SNHG7 exert functions in LUAD progression. METHODS: The expression levels of SNHG17, miR-485-5p and Wnt ligand secretion mediator (WLS) in LUAD cells was evaluated by RT-qPCR. The effect of SNHG7 silencing on LUAD cell proliferation was assessed by colony formation and EdU assays. The apoptosis of LUAD cells was measured by flow cytometry analysis. Transwell assays were applied to detect cell migration and invasion. The relationship between SNHG17 and miR-485-5p was validated by RIP, RNA pull down and luciferase reporter assays. RESULTS: SNHG17 and WLS were up-regulated in LUAD cell lines. Down-regulation of SNHG17 curbed LUAD cell proliferation, migration and invasion but facilitated apoptosis. SNHG17 acted as miR-485-5p sponge to upregulate WLS expression. CONCLUSION: SNHG17 triggers the progression of LUAD via sponging miR-485-5p to upregulate WLS expression.

LINC00680 Promotes the Progression of Non-Small Cell Lung Cancer and Functions as a Sponge of miR-410-3p to Enhance HMGB1 Expression.

PURPOSE: LINC00680 was reported to be involved in various cancers through multiple mechanisms. Therefore, we intended to investigate its role in the progression of non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Firstly, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test LINC00680 in NSCLC tissue and cell lines. Subsequently, A549 and H1299 cells were transfected with LINC00680 overexpressing plasmids and their proliferation and colony formation and apoptosis was tested by Transwell assay and flow cytometry. In addition, xenograft tumor experiments in nude mice also affirmed. Meanwhile, we predicted that miR-410-3p, LINC00680 and high-mobility group protein box 1(HMGB1) relationship by Starbase, dual-luciferase reporter and RIP assay. Finally, the carcinogenic effects of LINC00680 were reversed by ethyl pyruvate (EP), a specific inhibitor of HMGB1. RESULTS: LINC00680 was upregulated in NSCLC and was closely related to the malignancy and poor prognosis of NSCLC patients. LINC00680 promoted proliferation and colony formation and inhibited apoptosis of A549 and H1299 cells. In addition, overexpressing LINC00680 accelerated the growth of NSCLC cells in xenograft tumor experiments in nude mice also affirmed. Meanwhile, high-mobility group protein box 1(HMGB1) was astoundingly amplified in NSCLC and was negatively regulated by miR-410-3p. Further, HMGB1 acted as a downstream target of miR-410-3p, upregulating miR-410-3p to attenuate HMGB1, while LINC00680 strengthened the expression of HMGB1 in A549 and H1299 cells. DISCUSSION: Thus, these results indicated that LINC00680 was cancerogenic in NSCLC by upregulating HMGB1 via sponging miR-410-3p.

[The Clinical Study of Comorbidities and Systemic Inflammation in COPD].

OBJECTIVE: To assess the association between chronic obstructive pulmonary disease (COPD) comorbidities and clinical characteristics, and to explore the inflammation mechanism. METHODS: 220 stable COPD patients were included. Clinical characteristics and comorbidities were recorded, and blood samples were collected. The relationship among the number and type of comorbidities, Charlson comorbidity index (CCI), clinical characteristics and the levels of plasma inflammatory markers [interleukin (IL)-6, high sensitivity C-reaction protein (hs-CRP), tumor necrosis factor-α (TNF-α), IL-8] were studied. RESULTS: The top five comorbidities were hypertension, metabolic syndrome and diabetes osteoporosis, bronchiectasis and peripheral vascular diseases. The level of plasma IL-6 was greater in higher CCI score (≥4) group compared with lower CCI score (<4) group ( P=0.011). Levels of IL-6 and IL-8 and the number of hospitalization in prior year were positively correlated with CCI and age adjusted CCI (r<0.03, P<0.05). There was a correlation between the COPD comorbidities and systemic inflammatory response (r<0.3, P<0.05). CONCLUSION: Patients with a higher CCI score had more severe symptoms, functional impairment and higher level of inflammatory factors and high frequency of hospital admission due to acute exacerbation. The mechanism by which COPD may play a role in systemic inflammatory response deserves further study.

Par3 is essential for the establishment of planar cell polarity of inner ear hair cells.

In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3-deficient cochleae. Thus, a Par3-GEF-Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling.

Stimulation of the Hippocampal POMC/MC4R Circuit Alleviates Synaptic Plasticity Impairment in an Alzheimer's Disease Model.

Hippocampal synaptic plasticity is modulated by neuropeptides, the disruption of which might contribute to cognitive deficits observed in Alzheimer's disease (AD). Although pro-opiomelanocortin (POMC)-derived neuropeptides and melanocortin 4 receptor (MC4R) are implicated in hippocampus-dependent synaptic plasticity, how the POMC/MC4R system functions in the hippocampus and its role in synaptic dysfunction in AD are largely unknown. Here, we mapped a functional POMC circuit in the mouse hippocampus, wherein POMC neurons in the cornu ammonis 3 (CA3) activate MC4R in the CA1. Suppression of hippocampal MC4R activity in the APP/PS1 transgenic mouse model of AD exacerbates long-term potentiation impairment, which is alleviated by the replenishment of hippocampal POMC/MC4R activity or activation of hippocampal MC4R-coupled Gs signaling. Importantly, MC4R activation rescues amyloid-ß-induced synaptic dysfunction via a Gs/cyclic AMP (cAMP)/PKA/cAMP-response element binding protein (CREB)-dependent mechanism. Hence, disruption of this hippocampal POMC/MC4R circuit might contribute to synaptic dysfunction observed in AD, revealing a potential therapeutic target for the disease.

Determining the optimal 5-FU therapeutic dosage in the treatment of colorectal cancer patients.

Fluorouracil (5-FU) has been wildly used as a primary medication in the treatment of solid tumors including colorectal cancer. The treatment efficacy and toxicity of 5-FU varies greatly among individuals, suggesting a need for individualized regimen for cancer patients. The present study analyzed the blood concentration of 5-FU and its therapeutic efficacy and toxicity, evaluated the relationship of AUC (area under the plasma concentration-time curve), and the protein expression of DPD (dihydropyrimidine dehydrogenase) and TS (thymidylate synthetase), and therapeutic efficacy and toxicity. It was found that the AUC of 5-FU was 34.16±14.83mgmg·h/L in this cohort of study. The immunohistochemical analysis revealed 38.96% and 81.82% positive staining for DPD and TS in colorectal cancer tissues, respectively. We demonstrated that the expression of TS is positively correlated with the expression of DPD. There was a positive correlation between AUC and therapeutic efficacy, and gastrointestinal tract and neural toxicity. The expression of neither DPD nor TS had significant correlations with therapeutic efficacy and toxicity. Based on the blood 5-FU concentration and its relationship with treatment efficacy and toxicity, we determined an optimal therapeutic dosage of 5-FU to be equivalent to an AUC=28.03-38.94mgmg·h/L. Our study will be helpful in providing an individualized medical regimen for the treatment of colorectal cancer patients.

Effect of Tanreqing Injection on treatment of acute exacerbation of chronic obstructive pulmonary disease with Chinese medicine syndrome of retention of phlegm and heat in Fei.

OBJECTIVE: To explore the effect of Tanreqing Injection (TRQI) on the treatment of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) with Chinese medicine syndrome of retention of phlegm and heat in Fei (RPHF). METHODS: In a prospective randomized controlled clinical trial, 90 patients with AECOPD of RPHF syndrome were randomly assigned to 3 groups, TRQI and controls A and B, each with 30 cases. The TRQI group was administered with the intravenous injections of 20 mL TRQI once a day and conventional Western medicine treatment. Control group A was administered with the intravenous injection of 15 mg ambroxol hydrochloride twice a day and conventional Western medicine treatment, and control group B was administered with conventional Western medicine treatment only. The treatments were administered for 10 days. Chinese medical symptoms and signs were scored, and plasma concentrations of interleukin (IL)-8 and neutrophil elastase (NE) were recorded. RESULTS: (1) The Chinese medical symptoms (cough, sputum amount, expectoration, dyspnea and fever) and signs (tongue and pulse) improved significantly in the TRQI group (P<0.05 or P<0.01), and improvements in cough, sputum amount and expectoration were better in the TRQI group than control group B (P<0.05); there was no significant difference between the TRQI group and control group A (P>0.05). The sign of tongue was also improved significantly in the TRQI group (P<0.05). (2) The overall effects in the TRQI group and control group A were significantly better than in control group B (P<0.05), with no significant differences between the TRQI group and control group A (P>0.05). There was no significant difference in the total effective rate among the three groups (P>0.05). (3) After treatment, the plasma concentrations of IL-8 and NE decreased in the TRQI group and control group A (P<0.05), and the concentration of IL-8 in control group B decreased (P<0.05). The difference in IL-8 was greater in the TRQI group than in control group A and B before and after treatment, and the change in NE was greater in control group A than in the TRQI group and control group B, but there was no statistical significance among the three groups with regards to the change in IL-8 or NE (P>0.05). CONCLUSION: TRQI could improved the Chinese medical signs and symptoms in the patients with AECOPD, possibly because of the decreasing plasma levels of IL-8 and NE which could improve response to airway inflammation and mucus hypersecretion.