Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis.

OBJECTIVE: Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. METHODS: Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. RESULTS: The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/µL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. CONCLUSION: This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.

Inside out: high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars.

KEY MESSAGE: Selection of pre-embryogenic callus from a core structure from mature seed-derived callus is the key for high-efficiency plant regeneration and transformation of switchgrass different cultivars. Switchgrass (Panicum virgatum L.) has been identified as a dedicated biofuel crop. For its trait improvement through biotechnological approaches, we have developed a highly efficient plant regeneration and genetic transformation protocol for both lowland and upland cultivars. We identified and separated a pre-embryogenic "core" structure from the seed-derived callus, which often leads to development of highly regenerative type II calluses. From the type II callus, plant regeneration rate of lowland cultivars Alamo and Performer reaches 95%, and upland cultivars Blackwell and Dacotah, 50 and 76%, respectively. The type II callus was also amenable for Agrobacterium-mediated transformation. Transformation efficiency of 72.8% was achieved for lowland cultivar Alamo, and 8.0% for upland cultivar Dacotah. PCR, Southern blot and GUS staining assays were performed to verify the transgenic events. High regenerative callus lines could be established in 3 months, and transgenic plants could be obtained in 2 months after Agrobacterium infection. To our knowledge, this is the first report on successful plant regeneration and recovery of transgenic plants from upland switchgrass cultivars by Agrobacterium-mediated transformation. The method presented here could be helpful in breaking through the bottleneck of regeneration and transformation of lowland and upland switchgrass cultivars and probably other recalcitrant grass crops.

Sirtuin 1 Activator SRT1720 Protects Against Lung Injury via Reduction of Type II Alveolar Epithelial Cells Apoptosis in Emphysema.

In chronic obstructive pulmonary disease (COPD), two major pathological changes that occur are the loss of alveolar structure and airspace enlargement. Type II alveolar epithelial cells (AECII) play a vital role in maintaining alveolar homeostasis and lung tissue repair. Sirtuin 1 (SIRT1), a NAD(+)-dependent histone deacetylase, regulates many pathophysiological processes including inflammation, apoptosis, cellular senescence and stress resistance. The main aim of this study was to investigate whether SRT1720, a pharmacological SIRT1 activator, could protect against AECII apoptosis in rats with emphysema caused by cigarette smoke exposure and intratracheal lipopolysaccharide instillation in vivo. During the induction of emphysema in rats, administration of SRT1720 improved lung function including airway resistance and pulmonary dynamic compliance. SRT1720 treatment up-regulated the levels of surfactant protein (SP)A, SPC, SIRT1 and forkhead box O 3, increased SIRT1 activity, down-regulated the level of p53 and inhibited AECII apoptosis. Lung injury caused by emphysema was alleviated after SRT1720 treatment. SRT1720 could protect against AECII apoptosis in rats with emphysema and thus could be used in COPD treatment.

ED50 value of remifentanil in inhibiting coughing during extubation in children with snoring.

Objective: This study aimed to determine the effective dose 50% (ED50) value of remifentanil in inhibiting coughing during extubation in children with snoring. Methods: The subjects were children who scored a grade I in the American Society of Anesthesiology (ASA) metric and who were undergoing tonsillectomy (with or without adenoidectomy) under general anesthesia. Using Dixon's up-and-down sequential method, the initial infusion rate of remifentanil was 0.06 µg/kg/min, and the difference between the infusion rates of the two adjacent groups was 0.01 µg/kg/min. If a child had no cough response during extubation, the infusion rate for the next child was reduced by 0.01 µg/kg/min. If that child had cough response, the infusion rate for the next child was increased by 0.01 µg/kg/min, and the test was terminated when seven pairs of children with positive-negative alternating results were obtained. The ED50 value and its 95% confidence interval (CI) were calculated by probit regression. The times for extubation, awakening, agitation, and respiratory complications after extubation were compared between the two groups. Results: 1) The ED50 value of a continuous infusion of remifentanil required to inhibit the cough response of children during extubation was 0.042 µg/kg/min, and the 95% confidence interval was 0.025-0.062 µg/kg/min. 2) The total dosage and infusion rate of remifentanil in the cough suppression group were higher than those in the cough group (p < 0.05), but the differences in the times for extubating and awakening between the two groups were not statistically significant (p > 0.05). 3) There was no correlation between the infusion rate of remifentanil and the time for extubating and awakening in the cough suppression group; the r values were 0.13 and 0.12, respectively, and p > 0.05. 4) The differences in postoperative respiratory complications between the two groups were not statistically significant (p > 0.05). Conclusion: The ED50 value of a continuous infusion of remifentanil required to inhibit the cough response of children during extubation after tonsillectomy (with or without adenoidectomy) was 0.042 µg/kg/min, and a low-dose infusion of remifentanil does not affect the times for awakening and extubating in children.

[XPS study on the influence of calcination conditions to cerium ion valence].

For the system of Ce(NO3)2.6H2O and urea solution during homogeneous precipitation method, X-ray diffraction (XRD), infrared spectrum (IR) and especially X-ray photoelectron spectroscopy (XPS) were used to study and characterize the product structure, variety of cerium ion valence, compound surface character and kernel electronic configurations. The results of XRD and IR showed that calcination temperature had a great effect on the cerium ion valence. The products are orthorhombic Ce2 O(CO3)2.H2O with valence III by using homogeneous precipitation method directly. When heated from the temperature 200 degrees C to 250 degrees C, the product of CeO(CO3)2.H2O with valence VI was finally changed into stable CeO2 with valence IV. XPS was used to study the surface character and kernel electronic configurations of the three different compounds through fine scanning of O(1s), Ce(3d) and Ce(4d) apices, and the results approved that the compounds with different valences are caused by the different valence electronic configurations of the products.

Apelin attenuates depressive-like behavior and neuroinflammation in rats co-treated with chronic stress and lipopolysaccharide.

Several experimental studies have proved that activation of neuroinflammation pathways may contribute to the development of depression, a neuropsychiatric disorder disease. Our previous studies have shown the antidepressant properties of apelin, but the mechanism was unkown. This study was performed to verify whether the antidepressant effect of apelin was related to its anti-inflammation effect in the central nervous system. To achieve our aim, we selected the co-treatment of chronic stress and LPS to induced an inflammatory process in rats. The effect of this co-treatment was evaluated through the expression of inflammatory markers and glial cell activation. LPS injection co-treated with unpredictable chronic mild stress resulted in the activation of microglial cell and astrocyte, expression of inflammatory markers and depressive behaviors. Treatment with apelin significantly attenuates the deleterious effects in these rats. Our results showed that apelin improved depressive phenotype and decreased the activation of glial cells in stress co-treatment group. The down-regulations of p-NF-κB and p-IKKß suggested that the effects are possibly mediated by inhibition of the NF-κB-mediated inflammatory response. These findings speculated that intracerebroventricular injection of apelin could be a therapeutic approach for the treatment of depression, and the antidepressant function of apelin may closely associated with its alleviation in neuroinflammation.

Resveratrol downregulates TNF-α-induced monocyte chemoattractant protein-1 in primary rat pulmonary artery endothelial cells by P38 mitogen-activated protein kinase signaling.

Background: To evaluate the effects of resveratrol to monocyte chemoattractant protein-1 (MCP-1) and the role of p38 mitogen-activated protein kinase (MAPK) in this process in vitro. Materials and methods: Animal acute pulmonary thromboembolism (PTE) model: rat model was established by infusion of an autologous blood clot into the pulmonary artery through a polyethylene catheter. One hundred and thirty-two rats were randomly and equally divided into ten groups: rats-control (untreated), rats-1% DMSO, rats-TNF-α, rats-TNF-α + resveratrol, rats-TNF-α +C1142, rats-TNF-α+SB203580, rats-TNF-α+resveratrol + SB203580, rats-resveratrol only, rats-C1142 only, and rats-SB203580 only. Rat pulmonary artery endothelial cells (RPAs) tests: RPAs were isolated from above animal and designated as: RPAs-control, RPAs-1% DMSO control, RPAs-TNF-α, RPAs-TNF-α + resveratrol, RPAs-TNF-α + C1142, RPAs-TNF-α + SB203580, RPAs-TNF-α + resveratrol + SB203580, RPAs-resveratrol only, RPAs-C1142 only, and RPAs-SB203580 only. Each group was further divided into 1, 4, and 8 hrs time point for evaluation (n=6 rats per time point) except RPAs-TNF-α + SB203580, RPAs-TNF-α + resveratrol + SB203580, RPAs-C1142 and RPAs-SB203580 only, which were evaluated at 8 hrs time point. At each time point, mRNA and protein expressions of RPAs of MCP-1 were measured. The phosphorylation of p38 MAPK (p-pMAPK) of RPAs was also detected. Results: We found that the RPAs-TNF-α elicited significant increases in MCP-1 expression and phosphorylation of p38 mitogen-activated protein kinase (p-p38 MAPK). Furthermore, the MCP-1 expressions of RPAs-Resveratrol, RPAs-C1142, and RPAs-SB203580 were significantly down-regulated, which was associated with robustly suppressed TNF-α-induced p-p38MAPK expression. Conclusion: Our findings suggested that MCP-1 was involved in the formation of TNF-α-induced inflammatory response, and resveratrol could down-regulate the expression of MCP-1 via TNF-α- inhibition, which might contribute to the decline of acute PTE-induced PH in vivo.

Lentivirus-mediated disintegrin and metalloproteinase 17 RNA interference reversed the acquired resistance to gefitinib in lung adenocarcinoma cells in vitro.

OBJECTIVE: The aim of the study is to evaluate the effects of silencing a disintegrin and metalloproteinase 17 (ADAM17) gene expression by lentivirus-mediated RNA interference (RNAi) in the gefitinib-resistant lung adenocarcinoma cells, and then to explore whether the recombinant lentivirus mediated ADAM17 RNAi reversed the acquired resistance of lung adenocarcinoma to gefitinib in vitro. METHODS: The gefitinib-resistant RPC-9 cells were established and the mutations of EGFR were detected by gene sequencing. The ADAM17 shRNA expression vectors were constructed and packaged to recombinant lentivirus. The cell proliferation viability was detected by MTT, and cellular apotosis was analyzed by flow cytometry assay. The expression levels of ADAM17, EGFR and the phosphorylated EGFR were respectively detected by reverse transcription polymerase chain reaction and western blot. TGF-α production in the supernatant was detected by enzyme-linked immunosorbent assay. RESULTS: The gefitinib-resistant RPC-9 cells in which mutated EGFR (exon 20) carried 790T > T/M mutation were established. When the concentrations of gefitinib were less than 10µmol/L, there were no significant changes in the apoptosis and cellular proliferation of RPC-9 with the dose-escalation of gefitinib. The cell proliferation viability of RPC-9 was significantly decreased by lentivirus mediated ADAM17 RNAi (P < 0.05). Gefitinib did not inhibit ADAM17 expression in both the gefitinib-sensitive PC-9 and gefitinib-resistant RPC-9 cells (P > 0.05). Gefitinib had no significant effects on TGF alpha production in the supernatants (P > 0.05). Gefitinib did not inhibit EGFR expression in gefitinib-sensitive PC-9 and gefitinib-resistant RPC-9 cells (P > 0.05). The phosphorylation of EGFR in gefitinib-sensitive PC-9 cells was significantly inhibited by gefitinib (P < 0.05), but that in gefitinib-resistant RPC-9 could not be inhibited by gefitinib (P > 0.05). Lentivirus mediated ADAM17 RNAi significantly inhibited the mRNA and protein expression of ADAM17 in gefitinib-resistant RPC-9 cells (P < 0.05), as well as TGF alpha production in the supernatants (P < 0.05). Also, the phosphorylation of EGFR was significantly reduced in gefitinib-resistant RPC-9 cells by lentivirus mediated ADAM17 RNAi (P < 0.05); however, the mRNA and protein expression of EGFR could not be inhibited. CONCLUSION: Lentivirus mediated ADAM17 RNAi may reverse the acquired resistance of lung adenocarcinoma to gefitinib via inhibiting the upstream of EGFR signal pathway, which may provide a new therapeutic target to solve the acquired resistance to EGFR tyrosine kinase inhibitors in lung adenocarcinoma. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:196-205, 2018.

[The synergism between Mtx1 from Bacillus sphaericus and Cyt1 Aa from Bacillus thuringiensis to Culex quinquefasciatus].

Mosquitocidal toxin 1 (Mtx1) was synthesized during vegetative phase of Bacillus sphaericus and it had been proved to have higher activity to Aedes spp. larvae and Binary toxin (Bin) resistance Culex larvae. The truncated 97 kDa Mtx1 with a deletion of the signal peptide and the Cyt1 Aa crystal protein, a 27.3 kDa delta-endotoxin from Bacillus thuringiensis subsp. israelensis (Bti), were purified from Escherichia coli and B. thuringiensis recombinant strains respectively. Both purified toxins had high toxicity against Culex quinquefasciatus larvae. Bioassay result revealed the purified Mtx1 toxin had high toxicity against the target mosquito larvae, with LCso of 45.2 ng/mL. However, the mixture of Mtx1 and Cytl Aa exhibited higher toxicity against the mosquito larvae, with a lowest LC50 value of 20.19 ng/mL at the ratio of 1:3. (Mtx1:Cyt1 Aa). The calculated synergistic factor of different mixtures suggested a strong synergistic effect between Cyt1 Aa toxin and Mtx1. Furthermore, the presence of Cyt1Aa in the mixture could induce early larval mortality, enhancing the activity of Mtx1 to the target mosquito larvae. The synergistic effect of Cyt1 Aa on mortality of Mtx1 to mosquito larvae might be caused by the damage of the larval midgut-hemocoel barrier induced by the activated CytlAa toxin, which enhanced the specific pathogenesis of Mtx1 on mosquito larvae. It is suggested that the co-application of Mtx1 and Cyt1 Aa in future will be integrated for mosquito management.

[Expression of mosquitocidal Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis in Asticcacaulis excentricus].

Asticcacaulis excentricus, who lives in upper-layer waters providing food resource to the mosquito larvae and has been proven to be a successful host to produce the mosquitocidal binary toxins or Cry11Aa toxin from Bacilli (Liu et al., 1996, Nat Biotech 14: 343; Armengol, et al. , 2005, Curr Microbiol 51: 430), was developed to express cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti). Two A. excentricus transformants were constructed with the attempt of producing CytlAa alone and alongside with Cry11Aa, repectively. Detection of expressed Cry11Aa and CytlAa proteins by immunoblot in the recombinant A. excentricus clones showed that either cry11Aa or cyt1Aa was expressed well solely but not simultaneously although both restriction analyses of plasmid DNA and DNA sequencing showed that the transformed plasmid was identical to scheme. To investigate the reason why the recombinant A. excentricus harboring both genes and their ribosome binding site (RBS) sequences expressed only Cry11Aa, the total RNA of A. excentricus cells was extracted and revealed three-band pattern in which all RNA molecule weights are not greater than 16S RNA of Escherichia coli by formamide agarose gel electrophoresis, indicating that different RNA systems within these two Gram-negative strains required distinguishingly organised constructs to express multiple foreign genes. It is hypothesized that an extra promoter upstream of RBS sequence is required to express cyt1Aa in the cry11Aa-cyt1Aa tandom plasmid.

ADAM17 mediates MMP9 expression in lung epithelial cells.

The purposes were to study the role of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α/nuclear factor-κB (NF-κB) signaling in matrix metalloproteinase 9 (MMP9) expression in A549 cells and to investigate the effects of lentivirus-mediated RNAi targeting of the disintegrin and metalloproteinase 17 (ADAM17) gene on LPS-induced MMP9 expression. MMP9 expression induced by LPS in A549 cells was significantly increased in a dose- and time-dependent manner (p<0.05). Pyrrolidine dithiocarbamate (PDTC) and a TNFR1 blocking peptide (TNFR1BP) significantly inhibited LPS-induced MMP9 expression in A549 cells (p<0.05). TNFR1BP significantly inhibited LPS-induced TNF-α production (p<0.05). Both PDTC and TNFR1BP significantly inhibited the phosphorylation of IκBα and expression of phosphorylation p65 protein in response to LPS (p<0.05), and the level of IκBα in the cytoplasm was significantly increased (p<0.05). Lentivirus mediated RNA interference (RNAi) significantly inhibited ADAM17 expression in A549 cells. Lentivirus-mediated RNAi targeting of ADAM17 significantly inhibited TNF-α production in the supernatants (p<0.05), whereas the level of TNF-α in the cells was increased (p<0.05). Lentiviral ADAM17 RNAi inhibited MMP9 expression, IκBα phosphorylation and the expression of phosphorylation p65 protein in response to LPS (p<0.05). PDTC significantly inhibited the expression of MMP9 and the phosphorylation of IκBα, as well as the expression of phosphorylation p65 protein in response to TNF-α (p<0.05). Lentiviral RNAi targeting of ADAM17 down-regulates LPS-induced MMP9 expression in lung epithelial cells via inhibition of TNF-α/NF-κB signaling.

Single nucleotide deletion of cqm1 gene results in the development of resistance to Bacillus sphaericus in Culex quinquefasciatus.

The entomopathogen Bacillus sphaericus is one of the most effective biolarvicides used to control the Culex species of mosquito. The appearance of resistance in mosquitoes to this bacterium, however, remains a threat to its continuous use in integrated mosquito control programs. Previous work showed that the resistance to B. sphaericus in Culex colonies was associated with the absence of the 60-kDa binary toxin receptor (Cpm1/Cqm1), an alpha-glucosidase present in the larval midgut microvilli. In this work, we studied the molecular basis of the resistance developed by Culex quinquefasciatus to B. sphaericus C3-41. The cqm1 genes were cloned from susceptible (CqSL) and resistant (CqRL/C3-41) colonies, respectively. The sequence of the cDNA and genomic DNA derived from CqRL/C3-41 colony differed from that of CqSL one by a one-nucleotide deletion which resulted in a premature stop codon, leading to production of a truncated protein. Recombinant Cqm1S from the CqSL colony expressed in Escherichia coli specifically bound to the Bin toxin and had α-glucosidase activity, whereas the Cqm1R from the CqRL/C3-41 colony, with a deletion of three quarters of the receptor's C-terminal lost its α-glucosidase activity and could not bind to the binary toxin. Immunoblotting experiments showed that Cqm1 was undetectable in CqRL/C3-41 larvae, although the gene was correctly transcribed. Thus, the cqm1R represents a new allele in C. quinquefasciatus that confers resistance to B. sphaericus.

Expression and clinical significance of ezrin in non--small-cell lung cancer.

BACKGROUND: Ezrin is known to regulate cellular survival, adhesion, migration, and invasion and has been identified as 1 of the key components of tumor progression and metastasis. The purpose of this study was to evaluate the clinicopathologic and prognostic significance of ezrin expression in non-small-cell lung cancer (NSCLC). MATERIALS AND METHODS: We investigated the expression pattern of ezrin immunohistochemically in 89 paraffin samples of NSCLC between January 1998 and December 2006 and conducted survival analyses. In addition, 73 frozen specimens (including tumorous and precancerous tissues) of NSCLC and 28 frozen specimens of benign pneumonic diseases collected between January 2009 and December 2009 were analyzed by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In 89 paraffin samples, ezrin was expressed in 40 cases, either in the cytoplasm or on the membrane. Ezrin-positive expression was significantly associated with increased tumor stage and lymph node (LN) metastasis. The positive rate of cytoplasm expression was significantly associated with LN metastasis. Importantly, ezrin-positive expression independently predicted inferior overall survival (OS) and disease-free survival (DFS). In 73 frozen specimens of NSCLC and 28 frozen specimens of benign pneumonic diseases, the ezrin mRNA, protein, and phospho-ezrin protein expressions in tumorous tissues were higher than they were in precancerous tissues and benign pneumonic tissues. CONCLUSION: These results suggested that high-level ezrin expression contributed to NSCLC progression and that phosphorylation and subcellular translocation of ezrin might be the important mechanisms. Ezrin might be a potential prognostic marker of progression in NSCLC.

Molecular characterization of the Tax-containing HTLV-1 enhancer complex reveals a prominent role for CREB phosphorylation in Tax transactivation.

Transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1) is mediated by the viral oncoprotein Tax, which utilizes cellular transcriptional machinery to perform this function. The viral promoter carries three cyclic AMP-response elements (CREs), which are recognized by the cellular transcription factor cAMP-response element-binding protein (CREB). Tax binds to GC-rich sequences that immediately flank the CREs. The coactivator CREB-binding protein (CBP)/p300 binds to this promoter-bound ternary complex, which promotes the initiation of HTLV-1 transcription. Protein kinase A phosphorylation of CREB at serine 133 facilitates transcription from cellular CREs by recruiting CBP/p300 via its KIX domain. However, it remains controversial whether CREB phosphorylation plays a role in Tax transactivation. In this study, we biochemically characterized the quaternary complex formed by Tax, CREB, KIX, and the viral CRE by examining the individual molecular interactions that contribute to Tax stabilization in the complex. Our data show KIX, Ser(133)-phosphorylated CREB, and vCRE DNA are all required for stable Tax incorporation into the complex in vitro. Consonant with a fundamental role for CREB phosphorylation in Tax recruitment to the complex, we found that CREB is highly phosphorylated in a panel of HTLV-1-infected human T-cell lines. Significantly, we show that Tax is directly responsible for promoting elevated levels of CREB phosphorylation. Together, these data support a model in which Tax promotes CREB phosphorylation in vivo to ensure availability for Tax transactivation. Because pCREB has been implicated in leukemogenesis, enhancement of CREB phosphorylation by the virus may play a role in the etiology of adult T-cell leukemia.