Three-way junction structure-mediated reverse transcription-free exponential amplification reaction for pathogen RNA detection.

Since RNA is an important biomarker of many infectious pathogens, RNA detection of pathogenic organisms is crucial for disease diagnosis and environmental and food safety. By simulating the base mismatch during DNA replication, this study presents a novel three-way junction structure-mediated reverse transcription-free exponential amplification reaction (3WJ-RTF-EXPAR) for the rapid and sensitive detection of pathogen RNA. The target RNA served as a switch to initiate the reaction by forming a three-way junction (3WJ) structure with the ex-trigger strand and the ex-primer strand. The generated trigger strand could be significantly amplified through EXPAR to open the stem-loop structure of the molecular beacon to emit fluorescence signal. The proofreading activity of Vent DNA polymerase, in combination with the unique structure of 2+1 bases at the 3'-end of the ex-primer strand, could enhance the role of target RNA as a reaction switch to reduce non-specific amplification and ensure excellent specificity to differentiate target pathogen from those causing similar symptoms. Furthermore, detection of target RNA showed a detection limit of 1.0×104 copies/mL, while the time consumption was only 20 min, outperforming qRT-LAMP and qRT-PCR, the most commonly used RNA detection methods in clinical practice. All those indicates the great application prospects of this method in clinical diagnostic.

Denaturation bubble-mediated two-stage isothermal nucleic acid amplification in a single closed tube.

Here, we established a two-stage isothermal amplification method comprising strand exchange amplification and hyperbranched rolling circle amplification, which only employed one DNA polymerase and was performed in a single closed tube at a constant temperature, providing a promising signal amplification strategy for accurate and rapid pathogen detection in the clinic.

Combination of Cetylpyridinium Chloride and Chlorhexidine Acetate: A Promising Candidate for Rapid Killing of Gram-Positive/Gram-Negative Bacteria and Fungi.

Combined use of the present antimicrobial drugs has been proved to be an alternative approach for antimicrobial agents' development since the co-existed of the drugs working in different mechanism have been demonstrated potentially enhance their antimicrobial activity. In this work, antibacterial and antifungal activity of the cetylpyridinium chloride (CPC)/chlorhexidine acetate (CHA) combination was evaluated for the first time, while a universal concentration for the rapid killing of gram-positive/gram-negative bacteria and fungi was also proposed. The minimum inhibitory concentrations (MIC) of CPC and CHA used alone or in combination were first measured, showing that the combined treatment decreased the MIC against tested gram-positive/gram-negative bacteria and fungi to 1/8-1/2. Growth curve assays demonstrated CPC and CHA had dynamic combined effects against the tested microorganisms at the concentration equal to MIC. Besides, combined use of these two drugs could also enhance their biocidal activity, which was illustrated by fluorescence microscopy and SEM images, as well as soluble protein measurement. More importantly, in vitro acute eye and skin irritation tests showed short-term contact with CPC/CHA combination would not cause any damage to mammalian mucosa and skin. In a word, CPC/CHA combination exhibited broad-spectrum antibacterial and antifungal activity against tested gram-positive/gram-negative bacteria and fungi while without any acute irritation to mammalian mucosa and skin, providing a new perspective on the selection of personal disinfectants.

Hyperoside inhibits proinflammatory cytokines in human lung epithelial cells infected with Mycoplasma pneumoniae.

Mycoplasma pneumoniae pneumonia (MPP) is the most common respiratory infection in young children and its incidence has increased worldwide. In this study, high expression of chemokine ligand 5 (CCL5) was observed in the serum of MPP patients, and its expression was positively correlated to DNA of M. pneumoniae (MP-DNA). In vitro, M. pneumoniae (MP) infection to A549 cells induced the expression of CCL5, chemokines receptor 4 (CCR4), nuclear factor-κB (NF-κB) nuclear protein, and phosphorylation of NF-κB-p65 (p-NF-κB-p65), whereas NF-κB cytoplasmic protein was decreased. On the contrary, treatment of hyperoside counteracted the induction of MP infection and promoted the proliferation of MP-infected A549 cells. Similarly, MP-induced IL-8 and TNF-α production was also markedly reduced by hyperoside. And CCR4 inhibitor AZD2098 had a better effect than hyperoside. In addition, CCL5 recombinant protein inhibited the effect of hyperoside to promote IL-8 and TNF-α production and CCR4 expression. These results indicated that CCL5 may be involved in the progression of MPP, and hyperoside was beneficial for MPP probably through CCL5-CCR4 interactions, which may provide a potential effective therapy for MPP.

TREM1: A positive regulator for inflammatory response via NF-κB pathway in A549 cells infected with Mycoplasma pneumoniae.

Herein, we found that serum content of the triggering receptor expressed on myeloid cells-1 (TREM1) was increased, and positively correlated with Mycoplasma pneumoniae (MP)-DNA in children with MP infection. In this study, A549 cells, known as human lung epithelial cells, were co-cultured with 107 CCU/ml of MP to established in vitro model of MP infection. We studied the roles of TREM1 in inflammatory response of A549 cell by regulating the secretions of cytokine interleukin (IL)-8 and tumor necrosis factor (TNF)-α in cell culture supernatants. Moreover, transcriptional activity of nuclear factor kappa B (NF-кB) was assessed by measuring protein levels of NF-кB in the cytoplasm and nuclear. Our data suggested that sanguinarine chloride significantly decreased TREM1 expression, and alleviated inflammatory response of MP-infected A549 cells via preventing NF-кB nuclear translocation. To study the roles of TREM1 in inflammatory regulation in MP-infected A549 cells and the underlying mechanisms, we established TREM1 overexpression transfected A549 cells. PDTC was used for inhibiting NF-кB nuclear translocation. We found that TREM1 overexpression induced server inflammatory response of A549 cells. Besides, TREM1 overexpression attenuated anti-inflammatory effects of sanguinarine chloride in MP-infected cells. More importantly, pro-inflammatory effects of TREM1 overexpression was significantly reversed with additional PDTC treatment in MP-infected cells treated with sanguinarine chloride, suggesting that TREM1 was a pro-inflammatory factor via regulating NF-кB nuclear translocation in MP-infected A549 cells.

Hemodynamic Solution Computation and Pathological Analysis in the Circle of Willis.

Hemodynamic indexes will change significantly compared to the normal range of many vascular diseases, therefore it is necessary to establish hemodynamic computation model. Blood circulation is periodically forced huge fluid flow network, the heart is generator of the entire fluid network, based on this hemodynamic characteristics, the circle of Willis's structure is simplified from the perspective of network and hemodynamics. According to hemodynamic equations and circuit graph theory, models blood flow network of the periodically forced hemodynamic equation, obtains the approximate solution of the harmonic waves form based on averaging computation. We apply this model in the network of the circle of Willis, which may help explain the development processes of cerebral circulation disease. The simulation results show that computing results consistent with the clinical observation of blood flow changes in cerebral infarction.

Therapeutic effects of liver soothing pingchuan formula decoction on experimental asthma in BALB/c mice via regulation of nerve growth factor-tyrosine kinase A pathway.

The present study was designed to investigate the effects of liver soothing pingchuan formula decoction (LSPF) on experimental asthma in BALB/c mice and explore its potential molecular mechanisms. An animal model of asthma was established in BALB/c mice through sensitization and activation with intraperitoneal injection of 10% ovalbumin (OVA)/Al(OH)3 solution in addition to inhalation of a 5% OVA solution. LSPF (300 and 600 mg/kg/day) was initially administered orally prior to activation. Following this, bronchoalveolar lavage fluid (BALF) and lung tissues were collected for histopathalogical examination. Levels of inflammatory cells and cytokines were determined in the BALF, and levels of nerve growth factor (NGF) and tyrosine kinase A (TrkA) in the lung tissues were determined. The results of the present study indicated that increased inflammatory reactions were observed following OVA sensitization (P<0.05), and the expression levels of NGF (P<0.05) and TrkA (P<0.05) were significantly increased, compared with normal mice. Notably, compared with the asthma model group, immunohistochemical results revealed that LSPF treatment suppressed OVA induced inflammatory reactions (P<0.05) and NGF (P<0.05) and TrkA expression levels (P<0.05). In addition, the NGF (P<0.05) and TrkA (P<0.05) were revealed to be downregulated with LSPF treatment from the results of the ELISA and western blotting assay. Overall, the results of the present study demonstrated that LSPF exhibits therapeutic effects on experimental asthma in mice, via downregulation of the NGF-TrkA pathway.

Effect of treatment with geraniol on ovalbumin-induced allergic asthma in mice.

BACKGROUND: Asthma, a complex highly prevalent airway disease, is a major public health problem for which current treatment options are inadequate. OBJECTIVE: To evaluate the antiasthma activity of geraniol and investigate its underlying molecular mechanisms. METHODS: In a standard experimental asthma model, Balb/c mice were sensitized with ovalbumin, treated with geraniol (100 or 200 mg/kg) or a vehicle control, during ovalbumin challenge. RESULTS: Treatment of ovalbumin-sensitized/challenged mice with geraniol significantly decreased airway hyperresponsiveness to inhaled methacholine. Geraniol treatment reduced eotaxin levels in bronchoalveolar lavage fluid and attenuated infiltration of eosinophils induced by ovalbumin. Geraniol treatment reduced TH2 cytokines (including interleukins 4, 5, and 13), increased TH1 cytokine interferon γ in bronchoalveolar lavage fluid, and reduced ovalbumin-specific IgE in serum. In addition, treatment of ovalbumin-sensitized/challenged mice with geraniol enhanced T-bet (TH1 response) messenger RNA expression and reduced GATA-3 (TH2 response) messenger RNA expression in lungs. Furthermore, treatment of ovalbumin -sensitized/challenged mice with geraniol further enhanced Nrf2 protein expression and activated Nrf2-directed antioxidant pathways, such as glutamate-cysteine ligase, superoxide dismutase, and glutathione S-transferase, and enhanced formation of reduced glutathione and reduced formation of malondialdehyde in lungs. CONCLUSION: Geraniol attenuated important features of allergic asthma in mice, possibly through the modulation of TH1/TH2 balance and activation the of Nrf2/antioxidant response element pathway.

Pingchuan formula improves asthma via restoration of the Th17/Treg balance in a mouse model.

BACKGROUND: Pingchuan Formula (PCF) is a traditional Chinese recipe. PCF improves chronic airway inflammation by correcting the imbalance of T-helper cell ratio. The purpose of this study was to investigate the effect of PCF on pathological changes in the lungs of asthmatic mice in terms of Treg/Th17 balance. METHODS: A bronchial asthma BALB/c mouse model was established using the ovalbumin excitation method. Distilled water (for MDL group) and drugs (for DEX or PCF group) were administered by gavage immediately after the first excitation. Mice were sacrificed after 7 and 28 d treatment. Lung tissues and bronchoalveolar lavage fluid were collected and lung pathological changes were observed after hematoxylin and eosin staining. Differential cell counts, concentrations of interleukins-6, -17, -23 and TGF-ß in bronchoalveolar lavage fluid were determined by enzyme-linked immunosorbent assay. Expression of transcriptional factors Foxp3 and RORγt was determined by immunohistochemistry and immunoblot. RESULTS: An asthma model was successfully established. After 7 or 28 d treatment, lung pathological changes were improved and concentration of interleukins-6, -17, -23 and TGF-ß in bronchoalveolar lavage fluid significantly decreased in the PCF group. RORγt expression in lung tissue was decreased in the PCF group, while Foxp3 expression increased (all P values<0.05 compared with the MDL group). There was no significant difference between the PCF and DEX group except that mice in the PCF group lost less bodyweight. CONCLUSIONS: Treatment with PCF downregulates RORγt, elevates Foxp3 expression, reduces interleukins-6, -17, -23 and TGF-ß in bronchoalveolar lavage fluid, thus restoring Th17/Treg balance, improving airway inflammation and reducing asthma symptoms.

[Effects of Chinese herbal medicine Pingchuan Formula on airway inflammation, interferon-γ and interleukin-4 in mice with asthma].

OBJECTIVE: To observe the changes of interferon-γ (IFN-γ) and interleukin-4 (IL-4), factors related to T helper 1/T helper 2 (Th1/Th2), in airway inflammation in an asthmatic model of mice and the effects of Pingchuan Formula (PCF), a compound traditional Chinese herbal medicine. METHODS: Eighty BALB/c mice were randomly divided into normal group, model group, dexamethasone group and PCF group, with 20 mice in each group. For inducing asthma, the BALB/c mice were sensitized by intraperitoneal injection of mixed ovalbumin, and then activated by inhaling ovalbumin. After 2 weeks of sensitizing, asthmatic mice were intragastrically administered with the drugs once a day for four weeks. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected respectively after 7-day activation or 2 weeks after activation. The general histological changes were observed by hematoxylin and eosin staining and collagen staining, meanwhile the levels of IFN-γ and IL-4 in BALF were detected by enzyme-linked immunosorbent assay. RESULTS: After 7 d of activation, compared with the normal group, significant decrease in IFN-γ and increase in IL-4 were detected in the model group; compared with the model group, IL-4 level of the two treated groups was decreased and the IFN-γ/IL-4 ratio was increased significantly; no significant difference was found between the two treated groups. Two weeks after activation, compared with the normal group, IFN-γ was decreased, IL-4 was increased, and the IFN-γ/IL-4 ratio was decreased in the model group; compared with the model group, IL-4 level in the dexamethasone group was significantly decreased and IFN-γ/IL-4 ratio was significantly elevated, while the level of IL-4 in the PCF group was significantly decreased, the level of IFN-γ was increased simultaneously, and the IFN-γ/IL-4 ratio was significantly elevated. There was no significant difference between the two treated groups in IFN-γ and IL-4 levels, nor in the IFN-γ/IL-4 ratio. The area of collagen fiber in airway wall epithelium in the model group was smaller than that in the normal group, and those in the treated groups were lower than that in the model group; there was no significant difference between the two treated groups. CONCLUSION: PCF has regulating function in correcting the imbalance of Th1/Th2 so as to improve airway inflammation. The therapeutic effects of PCF are basically similar to dexamethasone.

[Effects of Chinese herbal medicine Bushen Gubiao Recipe on toll-like receptor 4 and CD4(+)CD25(+)foxp3(+) regulatory T cells in mice with recurrent respiratory tract infections].

OBJECTIVE: To evaluate the effects of Bushen Gubiao Recipe (BGR), a compound traditional Chinese herbal medicine, on toll-like receptor 4 (TLR4) signaling pathway and CD4(+)CD25(+)foxp3(+)regulatory T cells (CD4(+)CD25(+)foxp3(+)Tregs) in mice with recurrent respiratory tract infections (RRTIs). METHODS: A mouse model of kidney-yang deficiency which simulated physical characteristics of RRTIs was established by intraperitoneal injection of hydrocortisone for 14 d. The model mice were divided into 4 groups, model group, high-dose BGR group, low-dose BGR group, and nucleic acid and casein oral solution group. They were administered respectively with distilled water, high-dose BGR (50 g/kg body weight), low-dose BGR (25 g/kg body weight) and nucleic acid and casein oral solution. Besides, a normal control group was set up and gastrogavage with distilled water. The effect of intervention was evaluated 4 weeks later by estimating the changes in behaviors of mice. Expressions of TLR4 and nuclear factor-kappa B (NF-κB) p65 in lung tissue were detected by immunohistochemical SABC method, the expression of TLR4 mRNA in lung tissue was detected by fluorescence quantitative-polymerase chain reaction (FQ-PCR), and the level of blood CD4(+)CD25(+)foxp3(+) Tregs was determined by flow cytometry. RESULTS: A kidney-yang deficiency mouse model with RRTIs was successfully established by intraperitoneal injection of hydrocortisone. BGR could improve the abnormal behavioral condition of the mice and enhance the expressions of TLR4 and NF-κB p65 in the lung tissue. Expression of TLR4 mRNA in high-dose BGR group was higher than that in model group (P<0.05), while the difference was not statistically significant between high-dose BGR group and low-dose BGR group (P>0.05). Levels of CD4(+)CD25(+)foxp3(+)Tregs in high-dose BGR group and nucleic acid and casein oral solution group were lower than that in model group (P<0.05), while the difference was not statistically significant between high-dose BGR group and low-dose BGR group (P>0.05). CONCLUSION: BGR can improve the behavior of the kidney-yang deficiency mice, and improve the innate immune function by up-regulating TLR/NF-κB signaling pathway. BGR can adjust the immune imbalance of T-helper cell (Th) 1/Th2 through reducing the activity of CD4(+)CD25(+)foxp3(+)Tregs and enhancing the immune response of Th1 type.

[Study on extracting process for Tiao ZhiAn mixture].

OBJECTIVE: To study the technology of optimal extraction for Tiao ZhiAn mixture. METHODS: Optimization of the influence of the mixed volatile oil extracting of Ligusticum chuanxiong Hort., Angelica sinensis (Oliv.) Diels and Curcuma longa L. was chosen by single-index test. Orthogonal test was employed for selecting the optimum of extraction technology by colligation score as the indexes of the contents of stilbene glucoside of Polygunum multiflorum Thunb, total solid matters in water extraction. RESULTS: The best technology for extracting Ligusticum chuanxiong Hort., Angelica sinensis (oliv.) Diels and Curcuma longa L. was to add water nine times within eight hours. The way of adding water six times to the medicine sediment and other herbs, extracting 3 times in one hour each time was considered the optimum extraction technology. CONCLUSION: The considerable extraction rate of active components in the drugs is achieved by applying the selected technology, and the simple method is fit for manufacturing Tiao ZhiAn mixture.

[Expression and significance of PTEN/PI3K signal transduction-related proteins in non-small cell lung cancer].

BACKGROUND & OBJECTIVE: PTEN/PI3K signal transduction pathway regulates cell proliferation and survival, and is closely associated with the development and progress of various tumors. However, the relationship between the function of this pathway with lung carcinoma has not been completely elucidated. This study was to investigate the expression and clinical significance of PTEN/PI3K signal transduction-related proteins, such as insulin-like growth factor-1 receptor (IGF-1R), PTEN and phosphatidylinositol 3-kinase (PI3K), in non-small cell lung carcinoma (NSCLC). METHODS: The expression of IGF-1R, PTEN, and PI3K in 59 specimens of primary NSCLC, 19 specimens of metastatic lymph nodes, 16 specimens of para-cancerous hyperplastic lung tissues, and 7 specimens of normal lung tissues were detected by SP immunohistochemistry. The difference of positive rates of IGF-1R,PTEN and PI3K was evaluated by Chi-square test and Fisher's exact test. RESULTS: The positive rates of IGF-1R, PTEN, and PI3K in NSCLC were 72.88%, 27.12%, and 84.75%, respectively. The positive rates of IGF-1R and PI3K were not associated with the clinicopathologic features of NSCLC (P>0.05). The positive rate of PTEN was highly associated with lymph node metastasis of NSCLC (P=0.009), but was not associated with pathologic type and differentiation grade of NSCLC (P>0.05). The positive rate of PTEN was significantly lower and that of PI3K was significantly higher in NSCLC and metastatic lymph node tissues than in para-cancerous hyperplastic and normal lung tissues (P<0.05). The expression of IGF-1R in NSCLC was positively correlated to that of PI3K (r=0.432, P=0.001); while the expression of PTEN was negatively correlated to that of PI3K (r=0.505, P<0.001). CONCLUSION: Overexpression of IGF-1R and PI3K, and low expression of PTEN are closely correlated to the development, invasion and metastasis of NSCLC.