Expression of M3 acetylcholine receptor in asthmatic mice and bronchial airway remodeling prediction.

This study aimed to investigate the role of M3 acetylcholine receptor (M3-AChR) expression in airway remodeling. Additionally, we aimed to evaluate the effects of ipratropium bromide solution inhaled in an early phase of asthma on airway remodeling in ovalbumin (OVA)-sensitized and challenged mice. Thirty BALB/c mice were divided into three groups, namely, control group (saline sensitized/challenged mice), asthma group (OVA sensitized/challenged mice), and treatment group (OVA sensitized/challenged mice treated by ipratropium bromide). Pathological changes were detected by histological staining in the bronchopulmonary tissue of mice. WAt/Pbm (the airway wall area /basement membrane perimeter) ratio of the asthma group (25.37 ± 4.25) increased significantly (P < 0.05) when compared with that of the control (12.89 ± 1.71) and treatment group (15.82 ± 2.91). WAm/Pbm (smooth muscle wall area / basement membrane perimeter) ratio of the asthma group (7.58 ± 2.16) increased significantly (P < 0.05) when compared with that of the control (2.55 ± 0.72) and treatment group (3.36 ± 1.69). M3-AChR concentration increased in the treatment group (29.24 ± 3.59) and was significantly different (P < 0.05) from that of the control group (25.50 ± 1.83). During asthma treatment, SAMA can alleviate airway remodeling in murine model by lessening the thickness of bronchial walls and inhibiting the proliferation of smooth muscle cells. There were no obvious changes in M3-AChR density in the murine model of asthma characterized by airway remodeling. However, ipratropium bromide may up-regulate the expression of M3-AChR in bronchial walls of asthmatic murine model.

Complete mitochondrial genome sequence and gene organization of Chinese indigenous chickens with phylogenetic considerations.

In this study, we sequenced the complete mitochondrial DNA of Chinese indigenous Jinhu Black-bone and Rugao chickens. The two chicken mitochondrial genomes were deposited in GenBank under accession Nos. KP742951 and KR347464, respectively. The complete mitochondrial genomes of Jinhu Black-bone and Rugao chickens were sequenced and found to span 16,785 and 16,786 bp, respectively, and consisted of 22 tRNA genes, two rRNA genes (12S rRNA and 16S rRNA), 13 protein-coding genes, and one control region (D-loop). The majority of genes were positioned on the H-strand, and the ND6 and eight tRNA genes were found to be encoded on the L-strand. The mitogenomes showed a similar gene order to that of the published Gallus gallus genome, as neither included a control region. The overall base composition of the genome of the two chickens was A = 30.22/30.28%, G = 13.57/13.49%, T = 23.74/23.76%, and C = 32.48/32.48%. Nucleotide skewness of the coding strands of the two chicken genomes (AT-skew = 0.12, GC-skew = -0.41) was biased towards T and G. Phylogenetic analysis revealed 29 subspecies, and the molecular genetic relationship among the 29 subspecies was identical to that of traditional taxonomy.

Mutations in WT1 in boys with sporadic isolated steroid-resistant nephrotic syndrome.

Mutations in the Wilms' tumor gene, WT1, can lead to syndromic steroid-resistant nephrotic syndrome and isolated steroid-resistant nephrotic syndrome. WT1 mutations have been identified in the majority of children with Denys-Drash or Frasier syndrome. WT1 mutations have not previously been identified in boys with sporadic isolated steroid-resistant nephrotic syndrome, but, recently, four boys with isolated nephrotic syndrome were identified to have WT1 mutations. However, whether boys with sporadic isolated steroid-resistant nephrotic syndrome should be routinely subjected to mutation analysis of WT1 has not been established. We examined 35 boys with sporadic isolated steroid-resistant nephrotic syndrome for mutations in WT1. Mutation analysis of all 10 exons of WT1 was performed by polymerase chain reaction and direct sequencing. Karyotype analysis or Y chromosome identification was performed for all patients. A Y chromosome or a 46, XY karyotype was demonstrated for all 35 patients. No causative WT1 mutation was identified in any of the patients. The WT1 mutation, IVS4+14T>C, which is not predicted to affect splicing, was identified in one patient who achieved complete remission after 8 weeks of oral prednisone treatment, indicating that IVS4+14T>C is not a causative mutation. Five WT1 polymorphisms were also identified in some patients and controls. Our results suggest that mutation analysis of WT1 should not be routinely performed for genetically defined boys with sporadic isolated steroid-resistant nephrotic syndrome.

Comparative analysis of phylogenetic relationships, morphologies, and pathogenicities among Curvularia lunata isolates from maize in China.

To understand the effects of disease-resistant maize varieties and new cropping systems on the population of Curvularia lunata, 52 isolates of C. lunata were collected in China from 2011 to 2013. The isolates were analyzed in terms of phylogenetic relationships, morphology, and pathogenicity. Phylogenetic analysis showed that the 52 isolates clustered into 2 distinct clusters with further subdivisions, suggesting the emergence of new genetic divergence within C. lunata. Results of morphology and pathogenicity analyses demonstrated that there were significant differences among these isolates: 27 isolates were classified as fast growing, 5 as slow growing, and 20 as moderate growing. Three isolates had white-colored colonies, 13 had yellowish green-colored colonies, and the remaining isolates had dark green-colored colonies. Furthermore, conidiation rates were assessed: 30 isolates were characterized as having low conidiation rates, 15 as having medium conidiation rates, and the remaining 7 isolates as having high conidiation rates. Eleven of the isolates appeared to be strongly pathogenic against maize, 15 isolates proved to be weakly pathogenic against maize, and the remaining isolates were regarded to be moderately pathogenic. Interestingly, correlation analysis demonstrated a negative correlation between the growth rate and the pathogenicity of the isolates, while a positive correlation was observed between the conidiation rate and the pathogenicity. No correlation was observed between the colony color and the pathogenicity of the isolates.

Effect of Xin Mai Jia on atherosclerosis in rats.

We investigated the therapeutic effect of Xin Mai Jia (XMJ) on atherosclerosis (AS) in rats. Rat models of AS were established by peritoneally injecting vitamin D, feeding a high-fat diet, and inducing balloon injuries in rats. The stomachs of the rats were irrigated continuously for 10 weeks with XMJ. Blood lipid- and hemorheology-related indices of blood samples were detected. Pathological changes in the right common carotid arterial tissues were also determined. The protein expression levels of endothelial nitric oxide synthase, angio-tensin-1, and endothelin-1 were determined by western blotting. XMJ reduced cholesterol, trigylecride, and low-density lipoprotein levels as well as blood viscosity, sedimentation, and hematocrit. Furthermore, XMJ alleviated vascular endothelial injury and reduced/eliminated atherosclerotic plaques. In contrast, XMJ significantly increased the endothelium-dependent relaxing response of the AS rat models. The western blotting results showed that XMJ upregulated endothelial nitric oxide synthase but downregulated angiotensin-1 and endothelin-1. XMJ prevented the development of AS by regulating blood lipid levels, hemorheology, and vascular function.

A simplified genomic DNA extraction protocol for pre-germination genotyping in rice.

Genotyping is a critical step for molecular marker-assisted selection in rice. Rice genomic DNA samples for genotyping are typically isolated from living tissues such as seedlings. This requires the germination of all candidate seeds and extraction of DNA from the seedlings. Currently, an ideal individual is selected from a very large number of plants, which is time- and labor-consuming, requiring several transplantations of materials and sampling processes. In this study, we developed a simplified genomic DNA extraction protocol in rice by using amylase to treat half-seeds. The yields of genomic DNA from a half-seed of Indica and Japonica rice were greater than 203.8 ± 32.5 and 143.2 ± 25.5 ng, respectively, and the 260/280 nm absorbance ratio was 1.75-2.10. The DNA was confirmed to be sufficient for polymerase chain reaction amplification and can be used in a marker-assisted selection program.

Effectiveness of bacterial infection-related cytokine profile (BIRCP) determination for monitoring pathogen infections in children with hemopathy in the bone marrow inhibition phase.

Serum cytokine profiles were analyzed before and after infection in children with hemopathy in the bone marrow inhibition phase to explore the utility of cytokine variations for detecting infections. Serum Th1/Th2 cytokine levels, including tumor necrosis factor, interleukin (IL)-2, IL-4, IL-6, IL-10, and interferon, were quantitatively determined by cytometric bead array technology in 480 cases (230 children) of children with hemopathy in the bone marrow inhibition phase with signs of infection, such as fever, and without, to establish baseline and affected levels for comparison with healthy control children. We used the cytokine profile of infected, blood culture-positive children to establish a bacterial infection-related cytokine profile (BIRCP) for predicting infections by pathogens in blood culture-negative children. Overall, 82.9% of children with Gram-negative bacterial infections were accompanied by marked increases of IL-6 and IL-10 levels [>10 times (means ± SD)], whereas only a mild increase of IL-6 levels occurred in Gram-positive bacteria-infected children [>2 times (means ± SD)] and only a mild increase of IFN-γ levels occurred in fungal culture-positive children [>2 times (means ± SD)]. Gram-positive bacterial and fungal infections did not cause a marked increase in IL-6 or IL- 10 levels. The effective rate (86.05%, N = 43) of infectious cases predicted by BIRCP was significantly higher than that obtained using traditional methods for selecting antibiotics based on clinical indications (65.45%, N = 55, P < 0.05). In summary, BIRCP can be used to predict the infections by pathogens in children with hemopathy and to select appropriate antibiotics.

Molecular cloning, sequence analysis, prokaryotic expression, and function prediction of foot-specific peroxidase in Hydra magnipapillata Chinese strain.

The cDNA sequence of foot-specific peroxidase PPOD1 from the Chinese strain of Hydra magnipapillata was cloned by reverse transcription-polymerase chain reaction. The cDNA sequence contained a coding region with an 873-bp open reading frame, a 31-bp 5'-untranslated region, and a 36-bp 3'-untranslated region. The structure prediction results showed that PPOD1 contains 10.34% of α-helix, 38.62% of extended strand, 12.41% of ß-turn, and 38.62% of random coil. The structural core was α-helix at the N terminus. The GenBank protein blast server showed that PPOD1 contains 2 fascin-like domains. In addition, high-level PPOD1 activity was only present in the ectodermal epithelial cells located on the edge of the adhesive face of the basal disc, and that these cells extended lamellipodia and filopodia when the basal disc was tightly attached to a glass slide. The fascin-like domains of Hydra PPOD1 might contribute to the bundling of the actin filament of these cells, and hence, the formation of filopodia. In conclusion, these cells might play an important role in strengthening the adsorbability of the basal disc to substrates.

Treatment of seawater immersion-complicated open-knee joint fracture.

The current study aimed to select suitable remedies for seawater immersion-complicated open-knee joint fracture by exploring the effects of different treatment methods. Forty adult rabbits weighing 2.20 ± 0.25 kg were divided equally into internal fracture fixation group (A), seawater-immersed group with primary internal fixation (B), seawater-immersed group with secondary internal fixation (C), and seawater-immersed group with external fixation (D), using the random-digit table method. Open-femoral internal condylar fracture models were established. Group A was left untreated for 2 h, whereas the other three groups were subjected to seawater immersion for 2 h. Afterwards, groups A and B underwent debridement and steel plate and screw internal fixation. Group C underwent debridement and external fixation, which was followed by secondary steel plate and screw internal fixation after the wound healed. Group D underwent transarticular arthrodesis. Wound infection, joint functional rehabilitation, and radiological and histopathological changes in fracture healing in each group were assessed. The results showed that delayed internal fixation effectively reduces the infection rate of seawater immersion-complicated open fracture and benefits joint function rehabilitation.

Adrenomedullin and adrenotensin regulate collagen synthesis and proliferation in pulmonary arterial smooth muscle cells.

To understand the pathophysiological mechanisms of pulmonary arterial smooth muscle cell (PASMC) proliferation and extracellular-matrix accumulation in the development of pulmonary hypertension and remodeling, this study determined the effects of different doses of adrenomedullin (ADM) and adrenotensin (ADT) on PASMC proliferation and collagen synthesis. The objective was to investigate whether extracellular signal-regulated kinase (ERK1/2) signaling was involved in ADM- and ADT-stimulated proliferation of PASMCs in 4-week-old male Wistar rats (body weight: 100-150 g, n=10). The proliferation of PASMCs was examined by 5-bromo-2-deoxyuridine incorporation. A cell growth curve was generated by the Cell Counting Kit-8 method. Expression of collagen I, collagen III, and phosphorylated ERK1/2 (p-ERK1/2) was evaluated by immunofluorescence. The effects of different concentrations of ADM and ADT on collagen I, collagen III, and p-ERK1/2 protein expression were determined by immunoblotting. We also investigated the effect of PD98059 inhibition on the expression of p-ERK1/2 protein by immunoblotting. ADM dose-dependently decreased cell proliferation, whereas ADT dose-dependently increased it; and ADM and ADT inhibited each other with respect to their effects on the proliferation of PASMCs. Consistent with these results, the expression of collagen I, collagen III, and p-ERK1/2 in rat PASMCs decreased after exposure to ADM but was upregulated after exposure to ADT. PD98059 significantly inhibited the downregulation by ADM and the upregulation by ADT of p-ERK1/2 expression. We conclude that ADM inhibited, and ADT stimulated, ERK1/2 signaling in rat PASMCs to regulate cell proliferation and collagen expression.

Wilms' tumor suppressor gene mutations in girls with sporadic isolated steroid-resistant nephrotic syndrome.

Mutations in the Wilms' tumor suppressor gene (WT1) can lead to syndromic forms of steroid-resistant nephrotic syndrome (SRNS) such as Denys-Drash or Frasier syndrome and can cause isolated SRNS. A mutation within WT1 is a frequent cause of sporadic isolated SRNS in girls. In a worldwide cohort of girls, the rate of occurrence was 10.8%. Previous reports have indicated that in Chinese girls, the detection rate of WT1 mutations is 16.7% for early onset isolated nephrotic syndrome. The detection rate of WT1 mutations in Chinese girls with sporadic isolated SRNS is unknown. We examined WT1 mutations in 14 Chinese girls with sporadic isolated SRNS using polymerase chain reaction and direct sequencing and studied a control group of 38 boys with sporadic isolated SRNS. We identified a WT1 mutation in 1 of 14 (7.1% detection rate) Chinese girls with sporadic isolated SRNS. No mutations occurred in WT1 in the remaining 13 girls or the control group. Our investigation supports the necessity of genetic examination for mutations in WT1 in girls with sporadic isolated SRNS.

Adrenomedullin and adrenotensin regulate collagen synthesis and proliferation in pulmonary arterial smooth muscle cells

To understand the pathophysiological mechanisms of pulmonary arterial smooth muscle cell (PASMC) proliferation and extracellular-matrix accumulation in the development of pulmonary hypertension and remodeling, this study determined the effects of different doses of adrenomedullin (ADM) and adrenotensin (ADT) on PASMC proliferation and collagen synthesis. The objective was to investigate whether extracellular signal-regulated kinase (ERK1/2) signaling was involved in ADM- and ADT-stimulated proliferation of PASMCs in 4-week-old male Wistar rats (body weight: 100-150 g, n=10). The proliferation of PASMCs was examined by 5-bromo-2-deoxyuridine incorporation. A cell growth curve was generated by the Cell Counting Kit-8 method. Expression of collagen I, collagen III, and phosphorylated ERK1/2 (p-ERK1/2) was evaluated by immunofluorescence. The effects of different concentrations of ADM and ADT on collagen I, collagen III, and p-ERK1/2 protein expression were determined by immunoblotting. We also investigated the effect of PD98059 inhibition on the expression of p-ERK1/2 protein by immunoblotting. ADM dose-dependently decreased cell proliferation, whereas ADT dose-dependently increased it; and ADM and ADT inhibited each other with respect to their effects on the proliferation of PASMCs. Consistent with these results, the expression of collagen I, collagen III, and p-ERK1/2 in rat PASMCs decreased after exposure to ADM but was upregulated after exposure to ADT. PD98059 significantly inhibited the downregulation by ADM and the upregulation by ADT of p-ERK1/2 expression. We conclude that ADM inhibited, and ADT stimulated, ERK1/2 signaling in rat PASMCs to regulate cell proliferation and collagen expression.

Proteomic and bioinformatic analysis of outer membrane proteins of the protobacterium Bartonella henselae (Bartonellaceae).

Bartonella henselae, an infectious agent causing cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen. The outer membrane proteins of B. henselae are key molecules that play a primary role in host-cell interactions. We isolated B. henselae outer membrane proteins, using the ionic detergent N-lauroyl sarcosine sodium salt and sodium carbonate, purification by two-dimensional (2-D) gel electrophoresis, and protein identification using mass spectrometry. Treatment with buffers containing ASB-14 and ZWITTERGENT 3-10 increased solubilization of B. henselae proteins, particularly proteins with basic pI. Three hundred and sixty-eight spots were detected from the sarcosine-insoluble outer membrane fraction; 94 distinct protein species were identified from 176 spots. In the outer membrane fraction from carbonate incubation, 471 spots were calculated and 259 spots were identified, which included 139 protein entries. There were six outer membrane proteins in the sarcosine-insoluble outer membrane fraction compared with nine outer membrane proteins from samples subjected to carbonate incubation. We used bioinformatic analysis to identify 44 outer membrane proteins by prediction of their domains and tertiary structures and documented the potential virulence factors. We established the 2-D reference maps of the outer membrane subproteome of B. henselae using the two different extraction methods, which were partly complementary to each other. Sodium carbonate extraction isolated low-abundance and basic proteins better than the lauroyl sarcosine sodium salt extraction, which enriched high-abundance porins.