Metabolic improvements of novel microbial fermentation on black tea by Eurotium cristatum.

Due to its traditional fermentation, there are obvious limits on the quality improvements in black tea. However, microbial fermentation can provide an abundance of metabolites and improve the flavor of tea. The "golden flower" fungi are widely used in the microbial fermentation of tea and has unique uses in healthcare. To further explore the improvements in black tea quality achieved via microbial fermentation, we used widely targeted metabolomics and metagenomics analyses to investigate the changes in and effects of metabolites and other microorganisms during the interaction between the "golden flower" fungi and black tea. Five key flavor metabolites were detected, the levels of catechin, epigallocatechin gallate, (-)-epicatechin gallate were decreased by different degrees after the inoculation of the "golden flower" fungus, whereas the levels of caffeine and (+)-gallocatechin increased. Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes, Aspergillaceae, Trichocomaceae, and Lecanoromycetes play a positive role in the black tea fermentation process after inoculation with the "golden flower" fungi. D-Ribose can prevent hypoxia-induced apoptosis in cardiac cells, and it shows a strong correlation with Botryosphaeriaceae and Botryosphaeriales. The interaction between microorganisms and metabolites is manifested in tryptophan metabolism, starch and sucrose metabolism, and amino sugar and nucleotide sugar metabolism. In conclusion, the changes in metabolites observed during the fermentation of black tea by "golden flower" fungi are beneficial to human health. This conclusion extends the knowledge of the interaction between the "golden flower" fungi and black tea, and it provides important information for improving the quality of black tea.

Qing Fei Hua Xian Decoction ameliorates bleomycin-induced pulmonary fibrosis by suppressing oxidative stress through balancing ACE-AngII-AT1R/ACE2-Ang-(1-7)-Mas axis.

Objectives: We aimed to investigate the preventative effect of Qing Fei Hua Xian Decoction (QFHXD) against pulmonary fibrosis (PF) and its potential mechanisms. Materials and Methods: Bleomycin (BLM)-induced rats were respectively treated with 413.3, 826.6, and 1239.9 mg/kg of QFHXD and prednisone for 28 days. The lung tissues of rats were collected on day 28 for histological and western blotting analysis. Results: QFHXD significantly reduced alveolus inflammation, collagen accumulation, and fibrosis deposition in BLM-induced PF rats (P<0.05). Collagen I and III, vimentin, and α-smooth muscle actin(α-SMA) expression levels were likewise decreased in PF rats treated with QFHXD (P<0.05). Additionally, QFHXD increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) while decreasing NADPH oxidase 4 (NOX4) expression (P<0.05). Furthermore, QFHXD suppressed the PF progression by down-regulating Angiotensin-Converting Enzyme (ACE) -Angiotensin II (AngII) -Angiotensin II Type 1 Receptor (AT1R) axis (P<0.01) and up-regulating Angiotensin-Converting Enzyme 2 (ACE2) -Angiotensin-(1-7) (Ang-(1-7)) -Mas axis (P<0.05). Conclusion: QFHXD suppressed inflammatory infiltration and PF brought on by BLM in lung tissues through reducing oxidative stress by maintaining the equilibrium of ACE-AngII-AT1R and ACE2-Ang-(1-7) -Mas axes. This study may provide a novel clinical therapy option for PF.

Nontargeted metabolomics to characterize the effects of isotretinoin on skin metabolism in rabbit with acne.

Background: Acne vulgaris is a chronic inflammatory disease of the pilosebaceous unit. This study aimed to explore the pathogenesis of acne and the therapeutic mechanism of isotretinoin from the metabolic perspective in coal tar-induced acne in rabbits. Methods: Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-qTOF-MS) based metabolomics was used to identify skin metabolites in groups C (blank control), M (model group) and T (isotretinoin group). Multivariate statistical analysis was used to process the metabolomics data. Results: 98 differential metabolites in group C and group M were identified. The highest proportion of differential metabolites were organic acids and derivatives, lipid metabolites, organic heterocyclic compounds, and nucleoside metabolites. The most significant metabolic pathways included protein digestion and absorption, central carbon metabolism in cancer, ABC transporters, aminoacyl-tRNA biosynthesis, biosynthesis of amino acids, and sphingolipid signaling pathway. Isotretinoin treatment normalized eight of these metabolites. Conclusions: Our study will help to further elucidate the pathogenesis of acne, the mechanism of isotretinoin at the metabolite level, and identify new therapeutic targets for treating acne.

Preconcentration of liposoluble constituents in Salvia Miltiorrhiza using acid-assisted liquid phase microextraction based on a switchable deep eutectic solvent.

A switchable deep eutectic solvent-based liquid-phase microextraction was proposed and applied to the preconcentration and determination of liposoluble quality-markers of diterpenoid quinones (dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA) in traditional Chinese medicine coupled with high performance liquid chromatography-ultraviolet detection. In the procedure, the hydrophilic deep eutectic solvent of diethanolamine-hexanoic acid (molar ratio 1:1) was prepared and added into the sample phase as an extractant, and a homogeneous solution was formed under slight vortex stirring. After the addition of HCl solution, the deep eutectic solvent miscible with the sample phase was converted to hydrophobic form, and a cloudy solution was generated. Then, the upper hydrophobic layer enriching the target analytes was collected through centrifugation for high performance liquid chromatography analysis. Several critical parameters affecting the extraction performance including the composition and consumption of switchable deep eutectic solvent, the type and amount of acid, salt amount and extraction time were investigated and optimized. Moreover, the structures of the deep eutectic solvent and the recovered hydrophobic layer were both characterized using Fourier transform infrared spectroscopy, further demonstrating the switching mechanism of the extractant during the extraction process. Under the optimal conditions, enrichment factors of diterpenoid quinones ranged from 59 to 274. Good linearities (r≥0.9963), low detection limits (0.5-0.7 ng/mL), satisfactory precisions (relative standard deviations 0.5%-8.6%) and accuracies (recoveries 94.6%-104.6%) were also obtained. Comparing the proposed switchable deep eutectic solvent-based liquid-phase microextraction with other published methods, the characteristics of the procedure were summarized. The developed method was successfully applied for the preconcentration of four liposoluble diterpenoid quinones from a traditional Chinese herbal medicine of Salvia Miltiorrhiza.

Protein tyrosine phosphatase, receptor type B is a potential biomarker and facilitates cervical cancer metastasis via epithelial-mesenchymal transition.

Cervical cancer (CC) is one of the most common malignant tumors. This study analyzed the impact of protein tyrosine phosphatase, receptor type B (PTPRB) on malignant behavior of CC and explored its possible molecular mechanism. RT-PCR, western blot and Immunohistochemistry were applied to examine the expression of PTPRB in CC specimens and cells. Aberrant PTPRB expression in CC and survival outcomes were constructed using The Cancer Genome Atlas (TCGA) database and tissue microarray cervical squamous cell carcinoma cohort. Cultured human CC cells were assayed for viability, apoptosis, migration, and invasion in vitro and in vivo. Kyoto Encyclopedia of Genes and Genomes (KEGG) assays and gene set enrichment analysis (GSEA) assays were used to delve into PTPRB-related pathways using TCGA datasets. The levels of proteins associated with the epithelial-mesenchymal transition (EMT) pathway and modulated by PTPRB were examined through Western blot. We found that the levels of PTPRB in CC tissues and cells were distinctly up-regulated. PTPRB was also an unfavorable prognostic factor for CC patients. Functionally, PTPRB knockdown exhibits tumor-suppressive function via reducing cell proliferation and metastasis and inducing cell apoptosis. KEGG assays and GSEA assays suggested PTPRB overexpression was associated with several tumor-related pathways. The results of Western blot assays suggested that N-cadherin was decreased in the PTPRB-knockdown CC cells, while E-cadherin was increased. Overall, PTPRB is highly expressed in CC and can effectively enhance the proliferation, metastasis and EMT process of tumor cells. PTPRB is expected to be a therapeutic target for CC.

Biological clocks and incremental growth line formation in dentine.

Dentine- and enamel-forming cells secrete matrix in consistent rhythmic phases, resulting in the formation of successive microscopic growth lines inside tooth crowns and roots. Experimental studies of various mammals have proven that these lines are laid down in subdaily, daily (circadian), and multidaily rhythms, but it is less clear how these rhythms are initiated and maintained. In 2001, researchers reported that lesioning the so-called master biological clock, the suprachiasmatic nucleus (SCN), halted daily line formation in rat dentine, whereas subdaily lines persisted. More recently, a key clock gene (Bmal1) expressed in the SCN in a circadian manner was also found to be active in dentine- and enamel- secretory cells. To probe these potential neurological and local mechanisms for the production of rhythmic lines in teeth, we reexamined the role of the SCN in growth line formation in Wistar rats and investigated the presence of daily lines in Bmal1 knockout mice (Bmal1-/- ). In contrast to the results of the 2001 study, we found that both daily and subdaily growth lines persisted in rat dentine after complete or partial SCN lesion in the majority of individuals. In mice, after transfer into constant darkness, daily rhythms continued to manifest as incremental lines in the dentine of each Bmal1 genotype (wild-type, Bmal+/- , and Bmal1-/- ). These results affirm that the manifestation of biological rhythms in teeth is a robust phenomenon, imply a more autonomous role of local biological clocks in tooth growth than previously suggested, and underscore the need further to elucidate tissue-specific circadian biology and its role in incremental line formation. Investigations of this nature will strengthen an invaluable system for determining growth rates and calendar ages from mammalian hard tissues, as well as documenting the early lives of fossil hominins and other primates.

Saponin from Tupistra chinensis Bak Inhibits NF-κB Signaling in Sarcoma S-180 Cell Mouse Xenografts.

This study examined the effect of saponins from Tupistra chinensis Bak (STCB) on the growth of sarcoma S-180 cells in vitro and in mouse xenografts as well as the underlying mechanisms. Cell proliferation was assessed by MTT assay. Cell cycle distribution was determined by flow cytometry. Sarcoma S-180 tumor-bearing mice were treated with different doses of STCB with 10 µg/mL 5-fluorouracil (5-Fu) as a positive control. The activity of nuclear factor (NF)-κB was detected by gel mobility shift assay. The mRNA level of NF-κB was determined by real-time quantitative RT-PCR. The results showed that in vitro STCB inhibited the growth of S-180 cells in a concentration-dependent manner, which was accompanied by cell cycle arrest at S-phase. In vivo STCB significantly inhibited the growth of S-180 tumor mouse xenografts in a dose-dependent manner with apparent induction of cell apoptosis. Moreover, STCB inhibited the activity of NF-κB p65 and reduced the expression of NF-κB p65 mRNA in mouse xenografts. It was concluded that STCB inhibits the proliferation and cell cycle progression of S-180 cells by suppressing NF-κB signaling in mouse xenografts. Our findings suggest STCB is a promising agent for the treatment of sarcoma.

Montelukast and budesonide combination for children with chronic cough-variant asthma.

This study investigated the effectiveness and safety of montelukast combined budesonide (MCB) treatment for children with chronic cough-variant asthma (CCVA).In total, 82 cases of children with CCVA, aged 4 to 11 years were included in this study. All cases received either MCB or budesonide alone between May 2015 and April 2017. The primary outcome was lung function, measured by the peak expiratory flow rates (PEFRs) and forced expiratory volume in 1 second (FEV1). The secondary outcome was measured by the clinical assessment score. Furthermore, adverse events (AEs) were also recorded in this study. All outcomes were measured after 8-week treatment.After 8-week treatment, MCB showed greater effectiveness than did budesonide alone in improving the lung function, measured by PEFR V1 (P = .02), and FEV1 (P < .01). Similarly, the clinical assessment score also demonstrated significant difference between the 2 groups (P < .05). In addition, no serious AEs occurred in both groups.The results of this study demonstrate that the effectiveness of MCB is superior to budesonide alone in the treatment of children with CCVA.

Effects of Dangguibuxue decoction on rat glomerular mesangial cells cultured under high glucose conditions.

BACKGROUND: Dysfunction of glomerular mesangial cells (GMCs) plays an important role in pathogenesis of diabetic nephropathy. Here, we investigated the effects of Dangguibuxue decoction (DBD), an herbal traditional Chinese medicinal (TCM) formula composed of Astragali Radix and Angelicae Sinensis Radix, on GMC proliferation and fibrogenesis under high-glucose (HG) conditions. METHODS: Sixty male Sprague Dawley rats were divided into 5 groups and administered intragastric 0.9% saline, low concentration DBD (DBD-L, 1.75 g/kg/d), middle concentration DBD (DBD-M, 3.5 g/kg/d), high concentration DBD (DBD-H, 7.0 g/kg/d) and gliclazide (GL, 2 mg/kg/d), respectively, for 1 week, and then their sera were obtained. Rat mesangial cells (HBZY-1 cells) were treated with these sera under HG condition (30 mmol/L). RESULTS: The proliferation of GMCs under HG conditions was significantly greater than that under normal glucose condition. Low concentration DBD (DBD-L) inhibited proliferation of GMCs after 72-h incubation (P < 0.01), while high concentration DBD (DBD-H) inhibited GMCs proliferation at 24, 48 and 72 time points (P < 0.01). There was no significant difference between the inhibitory effect of DBD-H and GL sera on GMC proliferation (P > 0.05). Furthermore, all concentrations of DBD (DBD-L, DBD-M and DBD-H) significantly decreased the protein expression of α-SMA(α-smooth muscle actin) (P < 0.01), an indicator of interstitial fibrosis of GMCs. Finally, DBD-L, DBD-M, DBD-H sera obviously inhibited the increase of HYP (hydroxyproline)secretion under HG condition (P < 0.01). CONCLUSION: Our results demonstrate an inhibitory effect of DBD extract on proliferation and fibrogenesis of GMCs under HG conditions. The potential role of DBD in the treatment of diabetic neuropathy merits further investigation.

Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs.

To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO) and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE65524). The associated study on the regulation of Hoxa1 and Hoxc13 genes by YAP was published in Molecular Cellular Biology in 2015 [Liu et al., 2015].

YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues.

Yes-associated protein (YAP) is a Hippo signaling transcriptional coactivator that plays pivotal roles in stem cell proliferation, organ size control, and tumor development. The downstream targets of YAP have been shown to be highly context dependent. In this study, we used the embryonic mouse tooth germ as a tool to search for the downstream targets of YAP in ectoderm-derived tissues. Yap deficiency in the dental epithelium resulted in a small tooth germ with reduced epithelial cell proliferation. We compared the gene expression profiles of embryonic day 14.5 (E14.5) Yap conditional knockout and YAP transgenic mouse tooth germs using transcriptome sequencing (RNA-Seq) and further confirmed the differentially expressed genes using real-time PCR and in situ hybridization. We found that YAP regulates the expression of Hoxa1 and Hoxc13 in oral and dental epithelial tissues as well as in the epidermis of skin during embryonic and adult stages. Sphere formation assay suggested that Hoxa1 and Hoxc13 are functionally involved in YAP-regulated epithelial progenitor cell proliferation, and chromatin immunoprecipitation (ChIP) assay implies that YAP may regulate Hoxa1 and Hoxc13 expression through TEAD transcription factors. These results provide mechanistic insights into abnormal YAP activities in mice and humans.

[Spatiotemporal expression pattern of E-cadherin and P-cadherin during mouse tooth development].

OBJECTIVE: To investigate the expression patterns of E-cadherin and P-cadherin in murine-tooth germs at early developmental stages. METHODS: Mandible samples of CD1 mice from embryonic day 12.5 to postnatal day 3.5 were collected. The expressions of E-cadherin and P-cadherin in murine mandibular first molar germs were detected by immunofluorescence and observed under confocal fluorescence microscope. HE staining was performed for tissue morphology. RESULTS: Both E-cadherin and P-cadherin were widely expressed in the epithelial tissues through early developmental stages. The E-cadherin expression was increased in polarizing pre-ameloblasts, whereas the P-cadherin expression declined. The expression of the P-cadherin could be detected in epithelial tissues before bud stage, and expressed in mature ameloblasts at secretory stage. CONCLUSION: The E-cadherin and P-cadherin expressed in different spatiotemporal expression patterns, indicating their individual functions during tooth development. P-cadherin might function in the secretion and mineralization of enamel.

[Start-up Characteristics of Four-zone Integrated Reactor for Nitrogen Removal in Winter and Analysis of Nitrobacteria Community].

The treatment of decentralized sewage has gained more and more attention in China in recent years. A four-zone integrated reactor was designed by combining biofilm system and activated sludge system and start-up by gradient shorten the HRT. The removal of COD, NH4+ -N and TN were studied at 8-15°C. Fluorescence in situ hybridization(FISH) was used to detect nitrobacteria population (AOB,NOB) so as to study the relationship between the reactor effect and functional micro-bacteria. The results showed that, when the HRT was 9.2 h, the removal efficiencies of COD, ammonia and TN were 92. 11%, 99. 21% and 61. 63%, respectively. Compared to the initial stage, the numbers of AOB and NOB in the late phase were increased by 5. 82 and 6. 14 times, respectively. In addition, the proportion of nitrobacteria was increased from 6. 12% to 16. 38% , which became the dominant bacteria in biofilms. Moreover, the nitrification efficiency was increased from 78. 49% to 97. 52% , while the number of NOB was 5. 61-fold increased and the value of AOB/NOB was optimized to 1. 47. The effluent quality is guaranteed by the enrichment of AOB and NOB and suitable value of AOB/NOB.

Lifespan extension by dietary intervention in a mouse model of Cockayne syndrome uncouples early postnatal development from segmental progeria.

Cockayne syndrome (CS) is a rare autosomal recessive segmental progeria characterized by growth failure, lipodystrophy, neurological abnormalities, and photosensitivity, but without skin cancer predisposition. Cockayne syndrome life expectancy ranges from 5 to 16 years for the two most severe forms (types II and I, respectively). Mouse models of CS have thus far been of limited value due to either very mild phenotypes, or premature death during postnatal development prior to weaning. The cause of death in severe CS models is unknown, but has been attributed to extremely rapid aging. Here, we found that providing mutant pups with soft food from as late as postnatal day 14 allowed survival past weaning with high penetrance independent of dietary macronutrient balance in a novel CS model (Csa(-/-) | Xpa(-/-)). Survival past weaning revealed a number of CS-like symptoms including small size, progressive loss of adiposity, and neurological symptoms, with a maximum lifespan of 19 weeks. Our results caution against interpretation of death before weaning as premature aging, and at the same time provide a valuable new tool for understanding mechanisms of progressive CS-related progeroid symptoms including lipodystrophy and neurodysfunction.

[The 50% effective dose (ED50a) of seasonal spilt influenza vaccine in mice].

OBJECTIVE: To evaluate the seasonal influenza spilt vaccine's immunogenicity and the 50% effective dose (ED50a) of hemagglutin (HA) that can make 50% of the mice hemagglutination inhibition antibody (HI) titers to 40. METHODS: The 2008-2009 seasonal influenza spilt vaccine's two components, with HA from H1N1 and H3N2 influenza virus respectively, were used as a model. Mice were immunized once or twice with different doses, and the HI antibody titers were tested to determine the immunization procedure and to evaluate the immugenicity of seasonal influenza spilt vaccine in mice; Consequently, HI antibody response kinetics of the two components were observed to determine the time point when the HI antibody titer reached the peak point; Finally, mice were immunized with different doses of HA to evaluate the ED50a that can make 50% of mice HI titers reach 40. RESULTS: Immunization procedures study showed that one-dose of seasonal influenza vaccine induced the HI antibody titers ranged from 10 to 120, while two-dose of influenza vaccine improved the HI antibody titer 10-100 times as compared with one dose; antibody kinetics study suggested that the time point of HI antibody produced to peak is 28-35 days post one dose immunization; and the ED50a detection results indicated that one dose of 1.5 microg HA could make 50% of the mice HI antibody titer reach 40. CONCLUSION: Seasonal influenza spilt vaccine is very immunogenic in mouse; the time point of HI antibody produced to peak is 28-35 days post one dose immunization; and the ED50a of HA is 1.5 microg, which can make 50% of the mice HI titer reach 40. The experimental results provided foundation for the establishment of influenza vaccine evaluation system based on seasonal influenza vaccine.

[Immune protective effectiveness of seasonal influenza spilt vaccine against homologous and heterogonous subtypes of influenza virus in mice].

To investigate the seasonal influenza split vaccine's immune protective effectiveness against the homologous and heterogonous subtypes of influenza A virus challenge and the relationship between the protective effectiveness and hemagglutination inhibition (HI) antibody titer in mice. Two components of H1N1 and H3N2 in Chinese 2008-2009 seasonal influenza spilt vaccine, were derived from vaccine strain A/Brisbane/59/2007 (H1N1)-like virus and A/Brisbane/10/2007 (H3N2)-like virus respectively, and were used to immune BALB/c mice. Firstly, different doses of the vaccines were used to immunize mice and the HA immunization dosage that can induce the HI antibody titer of 40 in mice was identified; Secondly, H1N1 vaccine immunized mice were challenged with different doses of influenza virus mouse adaptation strains of A/Brisbane/59/2007 (H1N1)-like virus (MA) (referred to as A1 virus, well matched-strain in the homologous subtype) and A/Purto Rico/8/34 (H1N1) (referred to as PR8 virus, poor matched-strain in the homologous subtype) respectively, and H3N2 vaccine immunized mice were challenged with H1N1 influenza virus of A1 strain (Heterogonous subtype), body weight changes and survival rates were observed to explore the immune protective effectiveness of influenza split vaccine against the homologous and heterogonous subtypes of influenza A virus in mice. Results indicated that HI antibody titers were elevated as the HA protein immunization dosages increased from 0.15 microg, 0.5 microg, 1.5 microg, 5 microg to 15 microg in mice, and 1.5 microg HA of the seasonal influenza split vaccine could induced HI antibody titer of 40 in mice; 3LD50, 10LD50, 30LD50, 100LD50, 300LD50,1000LD50 and 3000LD50 of influenza virus strain A1 were used to challenge the H1N1 immunization mice, 1.5 microg HA of H1N1 vaccine could 100% protect mice against challenge with 1000LD50 of matched and homologous subtype of influenza virus strains A1, mice immunized with 15 microg HA of H1N1 vaccine even could 100% protect mice against challenge with 3000LD50 of influenza virus strains A1; but mice immunized with both the 1.5 microg and 15 microg HA of H1N1 vaccine were all sacrificed when challenged with 3LD50 of the mismatched and homologous subtype of influenza virus strain PR8, and mice immunized with the high dosage of 15 microg HA of H3N2 vaccine also were all sacrificed when challenged with 3LD50 of the heterogonous subtype of influenza virus strain A1. These results suggest that 1.5 microg HA of seasonal influenza split vaccine could induced HI antibody titer of 40 after one dose in mice, this dosage of HA can effectively protect mice against matched homologous subtype of influenza virus strain, but hardly to protect mice against mismatched homologous or heterogonous subtype of influenza virus strain. These results provide materials for the establishment of influenza vaccine evaluation system based on seasonal influenza vaccine.

[Efficient soluble expression and purification of influenza A nucleoprotein in Escherichia coli].

To efficiently express nucleoprotein (NP) of influenza A virus A/Jingke/30/95 (H3N2) in E. coli for further immunogenicity study, three forms of NP gene, NP(His) (NP fused with 6 x His tag), NPwt (wild type NP, non-fused NP with native codon) and NP(O) (codon optimized, non-fused NP) were cloned by the technologies of restriction enzyme digestion, PCR, codon optimization and gene synthesis. Three recombinant plasmids were subsequently constructed based on the prokaryotic vector pET-30a, respectively. The comparative studies with these plasmids were carried out on the gene expression efficiency, induction temperature and time, purification process and immune reactivity. It was confirmed by restriction enzyme digestion and sequencing analysis that the three NP genes were inserted into the expression plasmid pET-30a correctly. SDS-PAGE showed that all three forms of NP gene could be efficiently ex pressed in E. coli, among which NP(O) was expressed with the highest expression level. The lower temperature fermentation (T=25 degrees C) and longer time induction (t=10 h) were necessary for high-level expression of protein in soluble form. The purity of tag-free NP was up to 90% through the two-step purification process with anion-exchange and gel filtration chromatography. It was indicated by Western blot that purified NP reacted well with the serum from mice immunized with PR8 virus. These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli and the expressed NP protein with specific immune reactivity could be purified from the supernatant of bacterial lysate.

Molecular genetics of supernumerary tooth formation.

Despite advances in the knowledge of tooth morphogenesis and differentiation, relatively little is known about the aetiology and molecular mechanisms underlying supernumerary tooth formation. A small number of supernumerary teeth may be a common developmental dental anomaly, while multiple supernumerary teeth usually have a genetic component and they are sometimes thought to represent a partial third dentition in humans. Mice, which are commonly used for studying tooth development, only exhibit one dentition, with very few mouse models exhibiting supernumerary teeth similar to those in humans. Inactivation of Apc or forced activation of Wnt/ß(catenin signalling results in multiple supernumerary tooth formation in both humans and in mice, but the key genes in these pathways are not very clear. Analysis of other model systems with continuous tooth replacement or secondary tooth formation, such as fish, snake, lizard, and ferret, is providing insights into the molecular and cellular mechanisms underlying succesional tooth development, and will assist in the studies on supernumerary tooth formation in humans. This information, together with the advances in stem cell biology and tissue engineering, will pave ways for the tooth regeneration and tooth bioengineering.

Genetic interactions between Pax9 and Msx1 regulate lip development and several stages of tooth morphogenesis.

Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene-gene interactions. Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein-protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller "incisor field" forms in Pax9(+/-);Msx1(+/-) mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia.

Up-regulation of substance P in the lungs during acute myocardial ischemia and infarction in rats.

OBJECTIVE: Previous studies indicate that disturbance of respiratory functions during acute myocardial ischemia and infarction was not always parallel with decline of cardiac functions, indicating that some non-hemodynamic mechanism might be underlying the changes in the lungs. METHODS: The current study was designed to investigate the changes in the expression of substance P and its mRNA in the lungs and dorsal root ganglia using assays of immunohistochemistry, enzyme immunoassay, in situ hybridization and real-time quantitative reverse transcription-polymerase chain reaction, with a rodent model of acute myocardial infarction induced by permanent coronary artery occlusion, without and with epidural anesthesia in the upper thoracic segments of the spinal cord. RESULTS: Marked up-regulation of substance P in the lungs and the dorsal root ganglia of upper thoracic segments (T1-T5) was detected at 15, 30, 60, 180 and 360 min of coronary artery occlusion. The peptide was observed in the alveolar epithelium, mainly in type II pneumocytes, epithelium of bronchiole and the vascular walls. The preprotachykinin mRNA was mainly observed in the dendrites of nerve cells in the alveolar wall, the bronchiole and the pulmonary vessels and the basal lamina of the bronchiole. Pre-treatment of the animals with epidural local anesthetic could completely abolish the up-regulation of SP in the lungs and the dorsal root ganglia. CONCLUSIONS: The findings may suggest the involvement of substance P in the pathology of cross-talk between the heart and lungs in acute myocardial ischemia and infarction. Neural mechanism may be involved in the up-regulation of substance P in the lungs.