Low dose cadmium exposure regulates miR-381-ANO1 interaction in airway epithelial cells.

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death worldwide. Cigarette smoke which has approximately 2-3 µg of Cadmium (Cd) per cigarette contributes to the environmental exposure and development and severity of COPD. With the lack of a cadmium elimination mechanism in humans, the contribution of cadmium induced stress to lung epithelial cells remains unclear. Studies on cadmium responsive miRNAs suggest regulation of target genes with an emphasis on the critical role of miRNA-mRNA interaction for cellular homeostasis. Mir-381, the target miRNA in this study is negatively regulated by cadmium in airway epithelial cells. miR-381 is reported to also regulate ANO1 (Anoctamin 1) expression negatively and in this study low dose cadmium exposure to airway epithelial cells was observed to upregulate ANO1 mRNA expression via mir-381 inhibition. ANO1 which is a Ca2+-activated chloride channel has multiple effects on cellular functions such as proliferation, mucus hypersecretion and fibroblast differentiation in inflamed airways in chronic respiratory diseases. In vitro studies with cadmium at a high concentration range of 100-500 µM is reported to activate chloride channel, ANO1. The secretory epithelial cells are regulated by chloride channels like CFTR, ANO1 and SLC26A9. We examined "ever" smokers with COPD (n = 13) lung tissue sections compared to "never" smoker without COPD (n = 9). We found that "ever" smokers with COPD had higher ANO1 expression. Using mir-381 mimic to inhibit ANO1, we demonstrate here that ANO1 expression is significantly (p < 0.001) downregulated in COPD derived airway epithelial cells exposed to cadmium. Exposure to environmental cadmium contributes significantly to ANO1 expression.

Use of a pulmosphere model to evaluate drug antifibrotic responses in interstitial lung diseases.

BACKGROUND: Interstitial lung diseases (ILD) encompass a heterogenous group of diffuse parenchymal lung disorders characterized by variable degrees of inflammation and fibrosis. Pretherapeutic clinical testing models for such diseases can serve as a platform to test and develop effective therapeutic strategies. In this study, we developed patient derived 3D organoid model to recapitulate the disease process of ILDs. We characterized the inherent property of invasiveness in this model and tested for antifibrotic responses with an aim to develop a potential platform for personalized medicine in ILDs. METHODS: In this prospective study, 23 patients with ILD were recruited and underwent lung biopsy. 3D organoid-based models (pulmospheres) were developed from the lung biopsy tissues. Pulmonary functioning testing and other relevant clinical parameters were collected at the time of enrollment and follow up visits. The patient derived pulmospheres were compared to normal control pulmospheres obtained from 9 explant lung donor samples. These pulmospheres were characterized by their invasive capabilities and responsiveness to the antifibrotic drugs, pirfenidone and nintedanib. RESULTS: Invasiveness of the pulmospheres was measured by the zone of invasiveness percentage (ZOI%). The ILD pulmospheres (n = 23) had a higher ZOI% as compared to control pulmospheres (n = 9) (516.2 ± 115.6 versus 54.63 ± 19.6 respectively. ILD pulmospheres were responsive to pirfenidone in 12 of the 23 patients (52%) and responsive to nintedanib in all 23 patients (100%). Pirfenidone was noted to be selectively responsive in patients with connective tissue disease related ILD (CTD-ILD) at low doses. There was no correlation between the basal pulmosphere invasiveness, response to antifibrotics, and FVC change (Δ FVC). CONCLUSIONS: The 3D pulmosphere model demonstrates invasiveness which is unique to each individual subject and is greater in ILD pulmospheres as compared to controls. This property can be utilized to test responses to drugs such as antifibrotics. The 3D pulmosphere model could serve as a platform for the development of personalized approaches to therapeutics and drug development in ILDs and potentially other chronic lung diseases.

Cutaneous Exposure to Arsenicals Is Associated with Development of Constrictive Bronchiolitis in Mice.

Organoarsenicals, such as lewisite and related chloroarsine, diphenylchloroarsine (DPCA), are chemical warfare agents developed during World War I. Stockpiles in Eastern Europe remain a threat to humans. The well-documented effects of cutaneous exposure to these organoarsenicals include skin blisters, painful burns, and life-threatening conditions such as acute respiratory distress syndrome. In survivors, long-term effects such as the development of respiratory ailments are reported for the organoarsenical sulfur mustard; however, no long-term pulmonary effects are documented for lewisite and DPCA. No animal models exist to explore the relationship between skin exposure to vesicants and constrictive bronchiolitis. We developed and characterized a mouse model to study the long-term effects of cutaneous exposure on the lungs after exposure to a sublethal dose of organoarsenicals. We exposed mice to lewisite, DPCA, or a less toxic surrogate organoarsenic chemical, phenyl arsine oxide, on the skin. The surviving mice were followed for 20 weeks after skin exposure to arsenicals. Lung microcomputed tomography, lung function, and histology demonstrated increased airway resistance, increased thickness of the smooth muscle layer, increased collagen deposition in the subepithelium, and peribronchial lymphocyte infiltration in mice exposed to arsenical on skin.

Fibrinogen mediates cadmium-induced macrophage activation and serves as a predictor of cadmium exposure in chronic obstructive pulmonary disease.

The etiologies of chronic obstructive pulmonary disease (COPD) remain unclear. Cadmium (Cd) causes both pulmonary fibrosis and emphysema; however, the predictors for Cd exposure and the mechanisms by which Cd causes COPD remain unknown. We demonstrated that Cd burden was increased in lung tissue from subjects with COPD and this was associated with cigarette smoking. Fibrinogen levels increased markedly in lung tissue of patients with smoked COPD compared with never-smokers and control subjects. Fibrinogen concentration also correlated positively with lung Cd load, but inversely with the predicted % of FEV1 and FEV1/FVC. Cd enhanced the secretion of fibrinogen in a cdc2-dependent manner, whereas fibrinogen further mediated Cd-induced peptidylarginine deiminase 2 (PAD2)-dependent macrophage activation. Using lung fibroblasts from CdCl2-treated Toll-like receptor 4 (TLR4) wild-type and mutant mice, we demonstrated that fibrinogen enhanced Cd-induced TLR4-dependent collagen synthesis and cytokine/chemokine production. We further showed that fibrinogen complexed with connective tissue growth factor (CTGF), which in turn promoted the synthesis of plasminogen activator inhibitor-2 (PAI-2) and fibrinogen and inhibited fibrinolysis in Cd-treated mice. The amounts of fibrinogen were increased in the bronchoalveolar lavage fluid (BALF) of Cd-exposed mice. Positive correlations were observed between fibrinogen with hydroxyproline. Our data suggest that fibrinogen is involved in Cd-induced macrophage activation and increases in fibrinogen in patients with COPD may be used as a marker of Cd exposure and predict disease progression.

NETosis in the pathogenesis of acute lung injury following cutaneous chemical burns.

Despite the high morbidity and mortality among patients with extensive cutaneous burns in the intensive care unit due to the development of acute respiratory distress syndrome, effective therapeutics remain to be determined. This is primarily because the mechanisms leading to acute lung injury (ALI) in these patients remain unknown. We test the hypothesis that cutaneous chemical burns promote lung injury due to systemic activation of neutrophils, in particular, toxicity mediated by the deployment of neutrophil extracellular traps (NETs). We also demonstrate the potential benefit of a peptidyl arginine deiminase 4 (PAD4) inhibitor to prevent NETosis and to preserve microvascular endothelial barrier function, thus reducing the severity of ALI in mice. Our data demonstrated that phenylarsine oxide (PAO) treatment of neutrophils caused increased intracellular Ca2+-associated PAD4 activity. A dermal chemical burn by lewisite or PAO resulted in PAD4 activation, NETosis, and ALI. NETs disrupted the barrier function of endothelial cells in human lung microvascular endothelial cell spheroids. Citrullinated histone 3 alone caused ALI in mice. Pharmacologic or genetic abrogation of PAD4 inhibited lung injury following cutaneous chemical burns. Cutaneous burns by lewisite and PAO caused ALI by PAD4-mediated NETosis. PAD4 inhibitors may have potential as countermeasures to suppress detrimental lung injury after chemical burns.

Citrullinated vimentin mediates development and progression of lung fibrosis.

The mechanisms by which environmental exposures contribute to the pathogenesis of lung fibrosis are unclear. Here, we demonstrate an increase in cadmium (Cd) and carbon black (CB), common components of cigarette smoke (CS) and environmental particulate matter (PM), in lung tissue from subjects with idiopathic pulmonary fibrosis (IPF). Cd concentrations were directly proportional to citrullinated vimentin (Cit-Vim) amounts in lung tissue of subjects with IPF. Cit-Vim amounts were higher in subjects with IPF, especially smokers, which correlated with lung function and were associated with disease manifestations. Cd/CB induced the secretion of Cit-Vim in an Akt1- and peptidylarginine deiminase 2 (PAD2)-dependent manner. Cit-Vim mediated fibroblast invasion in a 3D ex vivo model of human pulmospheres that resulted in higher expression of CD26, collagen, and α-SMA. Cit-Vim activated NF-κB in a TLR4-dependent fashion and induced the production of active TGF-ß1, CTGF, and IL-8 along with higher surface expression of TLR4 in lung fibroblasts. To corroborate ex vivo findings, mice treated with Cit-Vim, but not Vim, independently developed a similar pattern of fibrotic tissue remodeling, which was TLR4 dependent. Moreover, wild-type mice, but not PAD2-/- and TLR4 mutant (MUT) mice, exposed to Cd/CB generated high amounts of Cit-Vim, in both plasma and bronchoalveolar lavage fluid, and developed lung fibrosis in a stereotypic manner. Together, these studies support a role for Cit-Vim as a damage-associated molecular pattern molecule (DAMP) that is generated by lung macrophages in response to environmental Cd/CB exposure. Furthermore, PAD2 might represent a promising target to attenuate Cd/CB-induced fibrosis.

Simultaneous characterisation of multiple Mahonia fortunei bioactive compounds in rat plasma by UPLC-MS/MS for application in pharmacokinetic studies and anti-inflammatory activity in vitro.

The stems of Mahonia fortunei (MF) are commonly used in Chinese Traditional Medicine and contain multiple bioactive compounds, including 3,4,5-trimethoxyphenol-1-O-ß-d-glucopyranoside (1), 5-hydroxypicolinic acid methyl ester (2), acortatarin A (3), syringic acid (4), 9-epi-acortatarin A (5), vomifoliol (6), corydaldine (7), noroxyhydrastinine (8), columbamine (9), jatrorrhizine (10), palmatine (11), berberine (12) and schisandrin (13). The pharmacokinetics of these 13 compounds in the rat plasma were assessed using a novel sensitive, rapid, and specific UPLC-ESI-MS/MS method after oral administration of an aqueous extract of MF stems. Carbamazepine was employed as the internal standard (IS) and all samples were precipitated with acetonitrile. Chromatographic separation was performed on a C18 column using a gradient elution at 0.3 mL/min, with the mobile phase consisting of acetonitrile and 0.06 % formic acid and 5 mM ammonium acetate aqueous solution. The calibration curves showed satisfactory linearity in the examination area (r2 ≥ 0.99). The accuracy, precision, extraction recovery, matrix effect, and stability were within acceptable ranges. The method successfully assessed the pharmacokinetics of these 13 compounds. In vitro, compound 12 exhibited potent inhibitory activity against production of nitric oxide (NO) in the RAW264.7 cell line when stimulated by lipopolysaccharide (LPS), while compounds 7, 12, and 13 were the most potent inhibitors of NO production in the BV2 cell line when stimulated by LPS. The IC50 values of compounds 7, 12 and 13 were 42.81, 20.55 and 22.74 µM. We conclude that these compounds have promise for clinical application, although their synergistic action may be more effective than that by any single compound alone.

[Characteristic fingerprint and multi-components quantitative determination for Fuke Qianjin Capsules by HPLC].

To establish an HPLC characteristic fingerprint method of Fuke Qianjin Capsules,and determine the contents of its main components. The analysis was carried out on a Kromasil 100-5-C18 analytical column(4. 6 mm ×250 mm,5 µm) with gradient elution by acetonitrile(A)-0. 1% phosphoric acid aqueous solution(B),a flow rate at 1. 0 m L·min-1 and the detection wavelength of 254 nm.The column temperature was 30 ℃,and the injection volume was 10 µL. The determination method of genistin,jatrorrhizine,andrographolide and 14-deoxy-11,12-didehydroandrographolide index components were studied methodologically. The common mode of the characteristic fingerprint of Fuke Qianjin Capsules was set up with 8 common peaks,which were identified as genistin,jatrorrhizine,palmatine,berberine,andrographolide,14-deoxy-11,12-didehydroandrographolide,Z-ligustilide,and Z-3-butylidenephthalide,respectively,in comparison with the references. The similarities of 20 batches of Fuke Qianjin Capsules samples were above 0. 95. All of the above-mentioned 4 analytes could be well separated under the optimized chromatographic conditions. RSD of precision and repeatability experiment were both less than 1. 5%,and the sample solution was stable during 72 h. All of the compounds had a good linearity and linear range. The contents of genistin,jatrorrhizine,andrographolide,and 14-deoxy-11,12-didehydroandrographolide in 20 batches of Fuke Qianjin Capsules samples were 28. 66-56. 04,94. 77-197. 92,1 705. 33-4 148. 93 and 462. 16-1 225. 96 µg in each capsule,respectively. The developed HPLC characteristic fingerprint and quantitative analysis methods were reliable,accurate and sensitive,and could be used effectively evaluate the quality of Fuke Qianjin Capsules samples.

Vimentin intermediate filament assembly regulates fibroblast invasion in fibrogenic lung injury.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease, with a median survival of 3-5 years following diagnosis. Lung remodeling by invasive fibroblasts is a hallmark of IPF. In this study, we demonstrate that inhibition of vimentin intermediate filaments (VimIFs) decreases the invasiveness of IPF fibroblasts and confers protection against fibrosis in a murine model of experimental lung injury. Increased expression and organization of VimIFs contribute to the invasive property of IPF fibroblasts in connection with deficient cellular autophagy. Blocking VimIF assembly by pharmacologic and genetic means also increases autophagic clearance of collagen type I. Furthermore, inhibition of expression of collagen type I by siRNA decreased invasiveness of fibroblasts. In a bleomycin injury model, enhancing autophagy in fibroblasts by an inhibitor of VimIF assembly, withaferin A (WFA), protected from fibrotic lung injury. Additionally, in 3D lung organoids, or pulmospheres, from patients with IPF, WFA reduced the invasiveness of lung fibroblasts in the majority of subjects tested. These studies provide insights into the functional role of vimentin, which regulates autophagy and restricts the invasiveness of lung fibroblasts.

Molecular Identification of ten species of stored-product psocids through microarray method based on ITS2 rDNA.

Stored-product psocids (Psocoptera: Liposcelididae) are cosmopolitan storage pests that can damage stored products and cause serious economic loss. However, because of the body size (~1 mm) of eggs, nymphs, and adults, morphological identification of most stored-product psocids is difficult and hampers effective identification. In this study, 10 economically important stored-product Liposcelis spp. psocids (Liposcelis brunnea, L. entomophila, L. decolor, L. pearmani, L. rufa, L.mendax, L. bostrychophila, L. corrodens, L. paeta, and L. tricolor) were collected from 25 geographic locations in 3 countries (China, Czech Republic, and the United States). Ten species-specific probes for identifying these 10 psocid species were designed based on ITS2 sequences. The microarray method and reaction system were optimized. Specificity of each of the ten probes was tested, and all probes were found suitable for use in identification of the respective10 Liposcelis spp. psocids at 66 °C. This method was also used to identify an unknown psocid species collected in Taian, China. This work has contributed to the development of a molecular identification method for stored-product psocids, and can provide technical support not only to facilitate identification of intercepted samples in relation to plant quarantine, but also for use in insect pest monitoring.

Autoimmunity to Vimentin Is Associated with Outcomes of Patients with Idiopathic Pulmonary Fibrosis.

Autoimmunity has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF); however, the repertoire of autoantigens involved in this disease and the clinical relevance of these autoimmune responses are still being explored. Our initial discovery assays demonstrated that circulating and intrapulmonary vimentin levels are increased in IPF patients. Subsequent studies showed native vimentin induced HLA-DR-dependent in vitro proliferation of CD4 T cells from IPF patients and enhanced the production of IL-4, IL-17, and TGF-ß1 by these lymphocytes in contrast to normal control specimens. Vimentin supplementation of IPF PBMC cultures also resulted in HLA-DR-dependent production of IgG with anti-vimentin specificities. Circulating anti-vimentin IgG autoantibody levels were much greater in IPF subjects from the University of Alabama at Birmingham (n = 102) and the University of Pittsburgh (U. Pitt., n = 70) than in normal controls. Anti-vimentin autoantibody levels in IPF patients were HLA biased and inversely correlated with physiological measurements of lung function (i.e., forced expiratory volumes and diffusing capacities). Despite considerable intergroup differences in transplant-free survival between these two independent IPF cohorts, serious adverse outcomes were most frequent among the patients within each population that had the highest anti-vimentin autoantibody levels (University of Alabama at Birmingham: hazard ratio 2.5, 95% confidence interval 1.2-5.3, p = 0.012; University of Pittsburgh: hazard ratio 2.7, 95% confidence interval 1.3-5.5, p = 0.006). These data show that anti-vimentin autoreactivity is prevalent in IPF patients and is strongly associated with disease manifestations. These findings have implications with regard to the pathogenesis of this enigmatic disease and raise the possibility that therapies specifically directed at these autoimmune processes could have therapeutic efficacy.

Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin.

Exposure to cadmium (Cd) has been associated with development of chronic obstructive lung disease (COPD). The mechanisms and signaling pathways whereby Cd causes pathological peribronchiolar fibrosis, airway remodeling, and subsequent airflow obstruction remain unclear. We aimed to evaluate whether low-dose Cd exposure induces vimentin phosphorylation and Yes-associated protein 1 (YAP1) activation leading to peribronchiolar fibrosis and subsequent airway remodeling. Our data demonstrate that Cd induces myofibroblast differentiation and extracellular matrix (ECM) deposition around small (<2 mm in diameter) airways. Upon Cd exposure, α-smooth muscle actin (α-SMA) expression and the production of ECM proteins, including fibronectin and collagen-1, are markedly induced in primary human lung fibroblasts. Cd induces Smad2/3 activation and the translocation of both Smad2/3 and Yes-associated protein 1 (YAP1) into the nucleus. In parallel, Cd induces AKT and cdc2 phosphorylation and downstream vimentin phosphorylation at Ser39 and Ser55, respectively. AKT and cdc2 inhibitors block Cd-induced vimentin fragmentation and secretion in association with inhibition of α-SMA expression, ECM deposition, and collagen secretion. Furthermore, vimentin silencing abrogates Cd-induced α-SMA expression and decreases ECM production. Vimentin-deficient mice are protected from Cd-induced peribronchiolar fibrosis and remodeling. These findings identify two specific sites on vimentin that are phosphorylated by Cd and highlight the functional significance of vimentin phosphorylation in YAP1/Smad3 signaling that mediates Cd-induced peribronchiolar fibrosis and airway remodeling.

3D pulmospheres serve as a personalized and predictive multicellular model for assessment of antifibrotic drugs.

Idiopathic pulmonary fibrosis (IPF) is a fatal progressive fibrotic lung disease characterized by the presence of invasive myofibroblasts in the lung. Currently, there are only two FDA-approved drugs (pirfenidone and nintedanib) for the treatment of IPF. There are no defined criteria to guide specific drug therapy. New methodologies are needed not only to predict personalized drug therapy, but also to screen novel molecules that are on the horizon for treatment of IPF. We have developed a model system that exploits the invasive phenotype of IPF lung tissue. This ex vivo 3D model uses lung tissue from patients to develop pulmospheres. Pulmospheres are 3D spheroids composed of cells derived exclusively from primary lung biopsies and inclusive of lung cell types reflective of those in situ, in the patient. We tested the pulmospheres of 20 subjects with IPF and 9 control subjects to evaluate the responsiveness of individual patients to antifibrotic drugs. Clinical parameters and outcomes were also followed in the same patients. Our results suggest that pulmospheres simulate the microenvironment in the lung and serve as a personalized and predictive model for assessing responsiveness to antifibrotic drugs in patients with IPF.

WISP-1 overexpression upregulates cell proliferation in human salivary gland carcinomas via regulating MMP-2 expression.

BACKGROUND: WISP-1 is a member of the CCN family of growth factors and has been reported to play an important role in tumorigenesis by triggering downstream events via integrin signaling. However, little is known about the role of WISP-1 in proliferation of salivary gland carcinoma (SGC) cells. METHODS: In this study, we investigated the WISP-1 expression in SGC tissues via immunohistochemical staining, Western blotting assay, and real-time quantitative polymerase chain reaction method, and then evaluated the regulatory role of WISP-1 in the growth of SGC A-253 cells. In addition, the role of MMP-2 in the WISP-1-mediated growth regulation was also investigated. RESULTS: It was demonstrated that the WISP-1 expression was upregulated at both mRNA and protein levels in 15 of 21 SGC tumor tissues, compared to the non-tumor tissues (five of 21), associated with the lymph node dissection and bone invasion. The in vitro CCK-8 assay and colony-forming assay demonstrated that the exogenous WISP-1 treatment or the WISP-1 overexpression promoted the growth of A-253 cells. In addition, we confirmed that the WISP-1 overexpression upregulated the MMP-2 expression in A-253 cells with the gain-of-function and loss-of-function strategies, and that the MMP-2 knockdown attenuated the WISP-1-mediated growth promotion of A-253 cells. CONCLUSION: We found that WISP-1 was overexpressed in the human SGCs, and the WISP-1 overexpression promoted the salivary gland cell proliferation via upregulating MMP-2 expression. Our study recognized the oncogenic role of WISP-1 in human SGCs, which could serve as a potential target for anticancer therapy.

Attenuated heme oxygenase-1 responses predispose the elderly to pulmonary nontuberculous mycobacterial infections.

Pulmonary infections with nontuberculous mycobacteria (P-NTM), such as by Mycobacterium avium complex (M. avium), are increasingly found in the elderly, but the underlying mechanisms are unclear. Recent studies suggest that adaptive immunity is necessary, but not sufficient, for host defense against mycobacteria. Heme oxygenase-1 (HO-1) has been recognized as a critical modulator of granuloma formation and programmed cell death in mycobacterial infections. Old mice (18-21 mo) infected with M. avium had attenuated HO-1 response with diffuse inflammation, high burden of mycobacteria, poor granuloma formation, and decreased survival (45%), while young mice (4-6 mo) showed tight, well-defined granuloma, increased HO-1 expression, and increased survival (95%). To further test the role of HO-1 in increased susceptibility to P-NTM infections in the elderly, we used old and young HO-1+/+ and HO-1-/- mice. The transcriptional modulation of the JAK/STAT signaling pathway in HO-1-/- mice due to M. avium infection demonstrated similarities to infected wild-type old mice with upregulation of SOCS3 and inhibition of Bcl2. Higher expression of SOCS3 with downregulation of Bcl2 resulted in higher macrophage death via cellular necrosis. Finally, peripheral blood monocytes (PBMCs) from elderly patients with P-NTM also demonstrated attenuated HO-1 responses after M. avium stimulation and increased cell death due to cellular necrosis (9.69% ± 2.02) compared with apoptosis (4.75% ± 0.98). The augmented risk for P-NTM in the elderly is due, in part, to attenuated HO-1 responses, subsequent upregulation of SOCS3, and inhibition of Bcl2, leading to programmed cell death of macrophages, and sustained infection.

DNA barcoding, species-specific PCR and real-time PCR techniques for the identification of six Tribolium pests of stored products.

Flour beetles of the genus Tribolium Macleay (Coleoptera: Tenebrionidae) are important stored product pests in China and worldwide. They are often found or are intercepted in grain depots, flour mills, and entry-exit ports, etc. Traditionally, Tribolium species are identified according to the morphological characteristics of the adult. However, it is almost impossible to rapidly identify adult fragments and non-adult stages based on external morphological characteristics. Molecular techniques for the rapid and accurate identification of Tribolium species are required, particularly for pest monitoring and the quarantine of stored products pests. Here, we establish DNA barcoding, species-specific PCR, and real-time PCR techniques for the identification of six stored-product pest Tribolium species including T. castaneum, T. confusum, T. destructor, T. madens, T. freemani and T. brevicornis. We detected the mitochondrial DNA cytochrome oxidase subunit I (COI) barcodes for Tribolium from 18 geographic populations and 101 individuals, built a Tribolium DNA barcode library, and designed species-specific primers and TaqMan probes for the above six Tribolium species. The three techniques were applied to identify Tribolium collected from stored samples and samples captured from quarantine ports. The results demonstrated that three techniques were all able to identify the six species of Tribolium both rapidly and accurately.

Th17 expression and IL-17 levels in laryngeal squamous cell carcinoma patients.

CONCLUSION: The Th17 cell frequency in peripheral blood and levels of IL-17 showed significant differences between patients with laryngeal squamous cell carcinoma and those with vocal cords polyps. Serum levels of IL-17 were correlated with laryngocarcinoma staging. OBJECTIVES: To investigate associations among the frequency of Th17 cells, levels of IL-17, and laryngeal squamous cell carcinoma. METHOD: Eighty in-patients with laryngeal squamous cell carcinoma and 114 in-patients with polypus of the vocal cord were enrolled. Th17 cell frequencies in peripheral blood and serum levels of IL-17 were measured by flow cytometry and enzyme-linked immunosorbent assay, respectively. The tissue expression levels of IL-17 mRNA transcripts and protein were measured using quantitative RT-PCR or immunohistochemical detection, respectively. RESULTS: Th17 cell frequencies in peripheral blood and serum concentrations of IL-17 were significantly higher in patients with laryngocarcinoma compared with those in patients with polyps (p < 0.01 for both Th17 cells and IL-17 levels). Serum concentrations of IL-17 were significantly higher in patients with advanced laryngocarcinoma than in patients with early laryngocarcinoma (p < 0.01). The mRNA and protein levels of IL-17 were significantly higher in laryngocarcinoma tissues than in adjacent normal tissues (p < 0.01 for mRNA levels, p < 0.05 for protein levels).

The establishment of species-specific primers for the molecular identification of ten stored-product psocids based on ITS2 rDNA.

Psocids are important stored product pests found worldwide that can be spread through grain trade. Most stored-product psocids, including eggs, nymphs, and adults, are very small (~1 mm) and difficult to identify morphologically. Here, we collected 10 economically important stored-product Liposcelis spp. psocids (L. bostrychophila, L. entomophila, L. decolor, L. paeta, L. brunnea, L. corrodens, L. mendax, L. rufa, L. pearmani, and L. tricolor) from 35 geographical locations in 5 countries (China, Czech Republic, Denmark, Germany, and the United States). The ITS2 rDNA gene was extracted and sequenced. The interspecific genetic distance of the stored-product psocids was significantly higher than the intraspecific genetic distance according to the barcoding gap analysis. Ten pairs of species-specific primers based on the ITS2 rDNA were developed for psocid identification. The sensitivity estimation indicated that the species-specific primers could correctly amplify the target ITS2 gene and successfully identify psocids at 1.0 ng/mL. Additionally, these species-specific primers could quantify specificity and identify 10 stored-product psocids; this approach could also be used to accurately identify other stored-product psocids. This work provides a practical approach for the precise examination of 10 stored-product psocid species and also contributes to the development of an identification method using ITS2 rDNA.