Complete chloroplast genome sequence of the medicinal plant Arctium lappa.

Arctium lappa, commonly called burdock, has a long medicinal and edible history. It has recently gained increasing attention because of its economic value. In this study, we obtained the complete chloroplast genome of A. lappa by Illumina Hiseq. The complete chloroplast genome of A. lappa is a typical circular structure with 152 708 bp in length. The GC content in the whole chloroplast genome of A. lappa is 37.7%. A total of 37 tRNA genes, 8 rRNA genes, and 87 protein-coding genes were successfully annotated. And the chloroplast genome contains 113 unique genes, 19 of which are duplicated in the inverted repeat. The distribution of 39 simple sequence repeats was analysed, and most of them are in the large single-copy (LSC) sequence. An inversion comprising 16 genes was found in the LSC region, which is 26 283 bp long. We performed multiple sequence alignments using 72 common protein-coding genes of 29 species and constructed a Maximum Parsimony (MP) tree. The MP phylogenetic result shows that A. lappa grouped together with Carthamus tinctorius, Centaurea diffusa, and Saussurea involucrata. The chloroplast genome of A. lappa is a valuable resource for further studies in Asteraceae.

Gene Losses and Variations in Chloroplast Genome of Parasitic Plant Macrosolen and Phylogenetic Relationships within Santalales.

Macrosolen plants are parasitic shrubs, several of which are important medicinal plants, that are used as folk medicine in some provinces of China. However, reports on Macrosolen are limited. In this study, the complete chloroplast genome sequences of Macrosolen cochinchinensis, Macrosolen tricolor and Macrosolen bibracteolatus are reported. The chloroplast genomes were sequenced by Illumina HiSeq X. The length of the chloroplast genomes ranged from 129,570 bp (M. cochinchinensis) to 126,621 bp (M. tricolor), with a total of 113 genes, including 35 tRNA, eight rRNA, 68 protein-coding genes, and two pseudogenes (ycf1 and rpl2). The simple sequence repeats are mainly comprised of A/T mononucleotide repeats. Comparative genome analyses of the three species detected the most divergent regions in the non-coding spacers. Phylogenetic analyses using maximum parsimony and maximum likelihood strongly supported the idea that Loranthaceae and Viscaceae are monophyletic clades. The data obtained in this study are beneficial for further investigations of Macrosolen in respect to evolution and molecular identification.

Comparison and Phylogenetic Analysis of Chloroplast Genomes of Three Medicinal and Edible Amomum Species.

Amomum villosum is an important medicinal and edible plant with several pharmacologically active volatile oils. However, identifying A. villosum from A. villosum var. xanthioides and A. longiligulare which exhibit similar morphological characteristics to A. villosum, is difficult. The main goal of this study, therefore, is to mine genetic resources and improve molecular methods that could be used to distinguish these species. A total of eight complete chloroplasts (cp) genomes of these Amomum species which were collected from the main producing areas in China were determined to be 163,608-164,069 bp in size. All genomes displayed a typical quadripartite structure with a pair of inverted repeat (IR) regions (29,820-29,959 bp) that separated a large single copy (LSC) region (88,680-88,857 bp) from a small single copy (SSC) region (15,288-15,369 bp). Each genome encodes 113 different genes with 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. More than 150 SSRs were identified in the entire cp genomes of these three species. The Sanger sequencing results based on 32 Amomum samples indicated that five highly divergent regions screened from cp genomes could not be used to distinguish Amomum species. Phylogenetic analysis showed that the cp genomes could not only accurately identify Amomum species, but also provide a solid foundation for the establishment of phylogenetic relationships of Amomum species. The availability of cp genome resources and the comparative analysis is beneficial for species authentication and phylogenetic analysis in Amomum.

Functional elucidation of miR-494 in the tumorigenesis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma has very high incidence and high mortality worldwide. MiRNA is related to the tumorigenesis and metastasis of a variety of tumors. In the present study, we verify that the expression of miR-494 in NPC tissues and NPC-derived cells was down-regulated, respectively. The proliferation, colony formation, migration, and invasion of NPC-derived cells were suppressed, while the cell apoptosis was promoted, when miR-494 was over-expressed in these cells. GALNT7 and CDK16 were confirmed to be the direct targets of miR-494. These results suggested that miR-494 play an inhibitory role in the tumorigenesis of NPC.

Impaired surface expression and conductance of the KCNQ4 channel lead to sensorineural hearing loss.

KCNQ4, a voltage-gated potassium channel, plays an important role in maintaining cochlear ion homoeostasis and regulating hair cell membrane potential, both essential for normal auditory function. Mutations in the KCNQ4 gene lead to DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss across all frequencies. Despite recent advances in the identification of pathogenic KCNQ4 mutations, the molecular aetiology of DFNA2 remains unknown. We report here that decreased cell surface expression and impaired conductance of the KCNQ4 channel are two mechanisms underlying hearing loss in DFNA2. In HEK293T cells, a dramatic decrease in cell surface expression was detected by immunofluorescent microscopy and confirmed by Western blot for the pathogenic KCNQ4 mutants L274H, W276S, L281S, G285C, G285S, G296S and G321S, while their overall cellular levels remained normal. In addition, none of these mutations affected tetrameric assembly of KCNQ4 channels. Consistent with these results, all mutants showed strong dominant-negative effects on the wild-type (WT) channel function. Most importantly, overexpression of HSP90ß, a key component of the molecular chaperone network that controls the KCNQ4 biogenesis, significantly increased cell surface expression of the KCNQ4 mutants L281S, G296S and G321S. KCNQ4 surface expression was restored or considerably improved in HEK293T cells mimicking the heterozygous condition of these mutations in DFNA2 patients. Finally, our electrophysiological studies demonstrated that these mutations directly compromise the conductance of the KCNQ4 channel, since no significant change in KCNQ4 current was observed after KCNQ4 surface expression was restored or improved.

The relationships of CD8+ T cell subsets in RA patients with disease activity and clinical parameters.

BACKGROUND: CD8+ T cells are plentiful in rheumatoid arthritis (RA) and have a important role in it's pathogenesis. Many subsets have been identified in CD8+ T cells, however, the relationship between CD8+ T subpopulations and disease activity of RA is poorly defined. Here we detected different CD8+ T cell subsets in peripheral blood and examined their relationships with clinical features and serological parameters in RA. METHODS: CD8+ T cell phenotypes and percentages in peripheral blood were determined by flow cytometry in 39 patients with RA. The clinical characteristics and serological parameters of RA patients were collected and DAS28-ESR was measured as indicator of disease activity. Linear regression was performed to assess the correlation of CD8+ T cell subsets with RA clinical variables. RESULTS: Naive CD8+ T cells were significantly and negatively correlated with RA disease activity indicator DAS28-ESR(r2 = 0.1027, p = 0.0468), erythrocyte sedimentation rate (ESR)(r2 = 0.1891, p = 0.0057), clinical disease activity index(CDAI)(r2 = 0.1474, p = 0.0158), simplified disease activity index(SDAI)(r2 = 0.1465, p = 0.0255), and duration(r2 = 0.1247, p = 0.0274). And the percent of naive CD8+ T cells were obviously decreased in RA with high disease activity when compared with RA in low disease activity(p < 0.01). In addition, Our results indicated significant positive correlations between CD8+ CD28- T cells and DAS28-ESR(r2 = 0.1881, p = 0.0058), ESR(r2 = 0.2279, p = 0.0021), c reaction protein (CRP)(r2 = 0.2203, p = 0.0051), CDAI (r2 = 0.1778, p = 0.0075), SDAI (r2 = 0.2618, p = 0.0020), rheumatoid factor(RF)(r2 = 0.1823, p = 0.0067), age(r2 = 0.1968, p = 0.0047), as well as similar positive correlations between CD8+ CD27- T cells and DAS28-ESR(r2 = 0.1661, p = 0.01), ESR(r2 = 0.1586, p = 0.012), CRP(r2 = 0.1778, p = 0.013), CDAI (r2 = 0.1622, p = 0.0110), SDAI(r2 = 0.2316, p = 0.0040), RF(r2 = 0.2097, p = 0.0034), age(r2 = 0.1932, p = 0.0051). Furthermore, interesting results showed observable positive correlations between activated CD8+ T cells and total cholesterol(TC)(r2 = 0.2757, p = 0.0007), triglyceride(TG)(r2 = 0.2886, p = 0.0005), low density lipoprotein(LDL-C)(r2 = 0.09643, p = 0.0264) and Krebs yon denlungen-6(KL-6)(r2 = 0.4171, p = 0.0002). And TCRγδ + CD8+ T cells were also found positively related with total cholesterol(TC)(r2 = 0.5015, p < 0.0001), triglyceride(TG)(r2 = 0.2031, p = 0.0045), and KL-6(r2 = 0.2122, p = 0.0136). CONCLUSIONS: Our results suggest that naive CD8+ T cells, CD8+ CD28- T cells, and CD8+ CD27- T cells are obviously correlated with inflammation and disease activity of RA. While activated CD8+ T cells and TCRγδ + CD8+ T cells may involve in lipidmetabolism and lung fibrosis of RA. These CD8+ T cell subsets may be new biomarkers and targets for RA disease evaluation, therapeutic target-selecting, curative effects and prognoses assessment.

Hypermethylation of Smad7 in CD4+ T cells is associated with the disease activity of rheumatoid arthritis.

Background: Smad7 is protective in a mouse model of rheumatoid arthritis. Here we investigated whether Smad7-expressing CD4+ T cells and the methylation of Smad7 gene in CD4+ T cells contribute to the disease activity of RA in patients. Methods: Peripheral CD4+ T cells were collected from 35 healthy controls and 57 RA patients. Smad7 expression by CD4+ T cells were determined and correlated with the clinical parameters of RA including RA score and serum levels of IL-6, CRP, ESR, DAS28-CRP, DAS28-ESR, Swollen joints and Tender joints. Bisulfite sequencing (BSP-seq) was used to determine the DNA methylation in Smad7 promoter (-1000 to +2000) region in CD4+ T cells. In addition, a DNA methylation inhibitor, 5-Azacytidine (5-AzaC), was added to CD4+ T cells to examine the possible role of Smad7 methylation in CD4+ T cell differentiation and functional activity. Results: Compared to the heath controls, Smad7 expression was significantly decreased in CD4+ T cells from RA patients and inversely correlated with the RA activity score and serum levels of IL-6 and CRP. Importantly, loss of Smad7 in CD4+ T cell was associated with the alteration of Th17/Treg balance by increasing Th17 over the Treg population. BSP-seq detected that DNA hypermethylation occurred in the Smad7 promoter region of CD4+ T cells obtained from RA patients. Mechanistically, we found that the DNA hypermethylation in the Smad7 promoter of CD4+ T cells was associated with decreased Smad7 expression in RA patients. This was associated with overreactive DNA methyltransferase (DMNT1) and downregulation of the methyl-CpG binding domain proteins (MBD4). Inhibition of DNA methylation by treating CD4+ T cells from RA patients with 5-AzaC significantly increased Smad7 mRNA expression along with the increased MBD4 but reduced DNMT1 expression, which was associated with the rebalance in the Th17/Treg response. Conclusion: DNA hypermethylation at the Smad7 promoter regions may cause a loss of Smad7 in CD4+ T cells of RA patients, which may contribute to the RA activity by disrupting the Th17/Treg balance.

Identification of Medicinal Bidens Plants for Quality Control Based on Organelle Genomes.

Bidens plants are annuals or perennials of Asteraceae and usually used as medicinal materials in China. They are difficult to identify by using traditional identification methods because they have similar morphologies and chemical components. Universal DNA barcodes also cannot identify Bidens species effectively. This situation seriously hinders the development of medicinal Bidens plants. Therefore, developing an accurate and effective method for identifying medicinal Bidens plants is urgently needed. The present study aims to use phylogenomic approaches based on organelle genomes to address the confusing relationships of medicinal Bidens plants. Illumina sequencing was used to sequence 12 chloroplast and eight mitochondrial genomes of five species and one variety of Bidens. The complete organelle genomes were assembled, annotated and analysed. Phylogenetic trees were constructed on the basis of the organelle genomes and highly variable regions. The organelle genomes of these Bidens species had a conserved gene content and codon usage. The 12 chloroplast genomes of the Bidens species were 150,489 bp to 151,635 bp in length. The lengths of the eight mitochondrial genomes varied from each other. Bioinformatics analysis revealed the presence of 50-71 simple sequence repeats and 46-181 long repeats in the organelle genomes. By combining the results of mVISTA and nucleotide diversity analyses, seven candidate highly variable regions in the chloroplast genomes were screened for species identification and relationship studies. Comparison with the complete mitochondrial genomes and common protein-coding genes shared by each organelle genome revealed that the complete chloroplast genomes had the highest discriminatory power for Bidens species and thus could be used as a super barcode to authenticate Bidens species accurately. In addition, the screened highly variable region trnS-GGA-rps4 could be also used as a potential specific barcode to identify Bidens species.

Attenuated expression of HRH4 in colorectal carcinomas: a potential influence on tumor growth and progression.

BACKGROUND: Earlier studies have reported the production of histamine in colorectal cancers (CRCs). The effect of histamine is largely determined locally by the histamine receptor expression pattern. Recent evidence suggests that the expression level of histamine receptor H4 (HRH4) is abnormal in colorectal cancer tissues. However, the role of HRH4 in CRC progression and its clinical relevance is not well understood. The aim of this study is to evaluate the clinical and molecular phenotypes of colorectal tumors with abnormal HRH4 expression. METHODS: Immunoblotting, real-time PCR, immunofluorescence and immunohistochemistry assays were adopted to examine HRH4 expression in case-matched CRC samples (n = 107) and adjacent normal tissues (ANTs). To assess the functions of HRH4 in CRC cells, we established stable HRH4-transfected colorectal cells and examined cell proliferation, colony formation, cell cycle and apoptosis in these cells. RESULTS: The protein levels of HRH4 were reduced in most of the human CRC samples regardless of grade or Dukes classification. mRNA levels of HRH4 were also reduced in both early-stage and advanced CRC samples. In vitro studies showed that HRH4 over-expression caused growth arrest and induced expression of cell cycle proteins in CRC cells upon exposure to histamine through a cAMP -dependent pathway. Furthermore, HRH4 stimulation promoted the 5-Fu-induced cell apoptosis in HRH4-positive colorectal cells. CONCLUSION: The results from the current study supported previous findings of HRH4 abnormalities in CRCs. Expression levels of HRH4 could influence the histamine-mediated growth regulation in CRC cells. These findings suggested a potential role of abnormal HRH4 expression in the progression of CRCs and provided some new clues for the application of HRH4-specific agonist or antagonist in the molecular therapy of CRCs.

Alterations of the preoperative coagulation profile in patients with acute appendicitis.

BACKGROUND: Acute appendicitis (AA) is usually associated with a systemic inflammatory response that often leads to activation of coagulation. However, limited data about coagulation changes in AA are available. METHODS: Results of preoperative coagulation testing in 702 patients with confirmed AA and 697 patients undergoing minor elective surgery (control) during the same period were analyzed retrospectively. Coagulation activity of factors VII, IX (FVII:C, FIX:C) and the concentration of plasma endotoxin from 40 patients with AA and 15 control subjects were measured. RESULTS: Compared with control subjects, prothrombin time (PT), fibrinogen (Fib) and endotoxin increased (all p<0.01), FVII:C decreased (p<0.05), and thrombin time shortened (p<0.01) significantly in patients with AA, which showed trends with increasing severity of disease. Areas under the receiver operating characteristic curve of Fib for discriminating complicated appendicitis or acute perforated appendicitis from enrolled patients were larger than those for leukocyte parameters. The concentration of endotoxin correlated negatively with FVII:C (r=-0.860, p<0.001), positively with PT (0.713, <0.001), and FVII:C negatively with PT (-0.729, <0.001) in individuals that were evaluated. The change in activated partial thromboplastin time and difference in FIX:C among patients with various pathological types of appendicitis were not significant. CONCLUSIONS: Endotoxin-induced activation of the extrinsic coagulation pathway was present in patients with AA. Fib may be useful as a potential indicator for excluding complicated appendicitis.

Cells of adult brain germinal zone have properties akin to hair cells and can be used to replace inner ear sensory cells after damage.

Auditory hair cell defect is a major cause of hearing impairment, often leading to spiral ganglia neuron (SGN) degeneration. The cell loss that follows is irreversible in mammals, because inner ear hair cells (HCs) have a limited capacity to regenerate. Here, we report that in the adult brain of both rodents and humans, the ependymal layer of the lateral ventricle contains cells with proliferative potential, which share morphological and functional characteristics with HCs. In addition, putative neural stem cells (NSCs) from the subventricular zone of the lateral ventricle can differentiate into functional SGNs. Also important, the NSCs can incorporate into the sensory epithelia, demonstrating their therapeutic potential. We assert that NSCs and edendymal cells can undergo an epigenetic functional switch to assume functional characteristics of HCs and SGNs. This study suggests that the functional plasticity of renewable cells and conditions that promote functional reprogramming can be used for cell therapy in the auditory setting.

Comparative and phylogenetic analyses of the chloroplast genomes of species of Paeoniaceae.

Plants belonging to family Paeoniaceae are not only economically important ornamental plants but also medicinal plants used as an important source of traditional Chinese medicine. Owing to the complex network evolution and polyploidy evolution of this family, its systematics and taxonomy are controversial and require a detailed investigation. In this study, three complete chloroplast genomes of sect. Paeonia, one of the sections of Paeonia, were sequenced and then analysed together with 16 other published chloroplast genomes of Paeoniaceae species. The total lengths of the chloroplast genomes of these species were 152,153-154,405 bp. A total of 82-87 protein-coding genes, 31-40 tRNA genes and 8 rRNA genes were annotated. Bioinformatics analysis revealed 61-74 simple sequence repeats (SSRs) in the chloroplast genomes, most of which have A/T base preference. Codon usage analysis showed that A/U-ending codons were more positive than C/G-ending codons, and a slight bias in codon usage was observed in these species. A comparative analysis of these 19 species of Paeoniaceae was then conducted. Fourteen highly variable regions were selected for species relationship study. Phylogenetic analysis revealed that the species of sect. Paeonia gathered in one branch and then divided into different small branches. P. lactiflora, P. anomala, P. anomala subsp. veitchii and P. mairei clustered together. P. intermedia was related to P. obovata and P. obovata subsp. willmottiae. P. emodi was the sister to all other species in the sect. Paeonia.

Development and regeneration of hair cells share common functional features.

The structural phenotype of neural connections in the auditory brainstem is sculpted by spontaneous and stimulus-induced neural activities during development. However, functional and molecular mechanisms of spontaneous action potentials (SAPs) in the developing cochlea are unknown. Additionally, it is unclear how regenerating hair cells establish their neural ranking in the constellation of neurons in the brainstem. We have demonstrated that a transient Ca(2+) current produced by the Ca(v)3.1 channel is expressed early in development to initiate spontaneous Ca(2+) spikes. Ca(v)1.3 currents, typical of mature hair cells, appeared later in development. Moreover, there is a surprising disappearance of the Ca(v)3.1 current that coincides with the attenuation of the transient Ca(2+) current as the electrical properties of hair cells transition to the mature phenotype. Remarkably, this process is recapitulated during hair-cell regeneration, suggesting that the transient expression of Ca(v)3.1 and the ensuing SAPs are signatures of hair cell development and regeneration.

The complete chloroplast genome sequence of Rhus potaninii.

We reported the complete chloroplast genome sequences of Rhus potaninii which was characterized by de novo assembly with Illumina sequencing data. The size of R. potaninii complete chloroplast genome is 159,620 bp in length and includes a large single copy region of 87,722 bp, a small single copy region of 18,948 bp, and a pair of inverted repeats of 26,475 bp. Its GC content is 37.9%. A total of 133 genes were predicted, including 86 protein-coding genes, 8 rRNA genes, 37 tRNA genes, and 2 pseudogenes. Maximum-likelihood (ML) phylogenetic tree indicates that R. potaninii is sister to R. chinensis.

Molecular structure and phylogenetic analyses of the complete chloroplast genomes of three original species of Pyrrosiae Folium.

Pyrrosia petiolosa, Pyrrosia lingua and Pyrrosia sheareri are recorded as original plants of Pyrrosiae Folium (PF) and commonly used as Chinese herbal medicines. Due to the similar morphological features of PF and its adulterants, common DNA barcodes cannot accurately distinguish PF species. Knowledge of the chloroplast (cp) genome is widely used in species identification, molecular marker and phylogenetic analyses. Herein, we determined the complete cp genomes of three original species of PF via high-throughput sequencing technologies. The three cp genomes exhibited a typical quadripartite structure with sizes ranging from 158 165 to 163 026 bp. The cp genomes of P. petiolosa and P. lingua encoded 130 genes, whilst that of P. sheareri encoded 131 genes. The complete cp genomes were compared, and five highly divergent regions of petA-psbJ, matK-rps16, ndhC-trnM, psbM-petN and psaC-ndhE were screened as potential DNA barcodes for identification of Pyrrosia genus species. The phylogenetic tree we obtained indicated that P. petiolosa and P. lingua are clustered in a single clade and, thus, share a close relationship. This study provides invaluable information for further studies on the species identification, taxonomy and phylogeny of Pyrrosia genus species.

Comparative and Phylogenetic Analysis of the Complete Chloroplast Genomes of Three Paeonia Section Moutan Species (Paeoniaceae).

Analysis of the relationships among wild species of section Moutan in the plant genus Paeonia has traditionally been problematic. Interspecies relationships cannot be effectively determined using phenotypic traits alone or through analysis of nuclear or chloroplast DNA fragments. Elucidation of complete chloroplast genome sequences will aid the identification and phylogeny of these species. In this study, the complete chloroplast genomes of three sect. Moutan plants were sequenced and analyzed. Comparative and phylogenetic analyses of the complete chloroplast genomes of all eight species of sect. Moutan were then conducted. The three complete chloroplast genomes gained in this study showed four-part annular structures, and the genome length, structure, GC content, codon usage, and gene distribution were highly similar. There was greater variation in the noncoding regions of the sequences than in the conserved protein-coding regions. Sequence variations in the small single copy (SSC) regions and large single copy (LSC) regions were considerably greater than those in the inverted repeat (IR) regions. Phylogenetic analysis revealed that the species of sect. Moutan clustered in one branch and then subdivided into smaller branches. As for the three complete chloroplast genome sequences obtained in this study, Paeonia jishanensis clustered with another P. jishanensis sequence from the GenBank database, Paeonia qiui clustered with Paeonia rockii, and Paeonia delavayi var. lutea clustered with Paeonia ludlowii. It was also found that the complete chloroplast genomes, LSC regions, and SSC regions all showed great abilities in identification and phylogenetic analysis of the species of sect. Moutan, while IRs regions and highly variable regions were not suitable for the species of sect. Moutan.

Genome-wide identification of abscisic acid (ABA) receptor pyrabactin resistance 1-like protein (PYL) family members and expression analysis of PYL genes in response to different concentrations of ABA stress in Glycyrrhiza uralensis.

As abscisic acid (ABA) receptor, the pyrabactin resistance 1-like (PYR/PYL) protein (named PYL for simplicity) plays an important part to unveil the signal transduction of ABA and its regulatory mechanisms. Glycyrrhiza uralensis, a drought-tolerant medicinal plant, is a good model for the mechanism analysis of ABA response and active compound biosynthesis. However, knowledge about PYL family in G. uralensis remains largely unknown. Here, 10 PYLs were identified in G. uralensis genome. Characterization analysis indicated that PYLs in G. uralensis (GuPYLs) are relatively conserved. Phylogenetic analysis showed that GuPYL1-3 belongs to subfamily I, GuPYL4-6 and GuPYL10 belong to subfamily II and GuPYL7-9 belongs to subfamily III. In addition, transcriptome data presented various expression levels of GuPYLs under different exogenous ABA stresses. The expression pattern of GuPYLs was verified by Quantitative real-time polymerase chain reaction (qRT-PCR). The study proved that GuPYL4, GuPYL5, GuPYL8 and GuPYL9 genes are significantly up-regulated by ABA stress and the response process is dynamic. This study paves the way for elucidating the regulation mechanism of ABA signal to secondary metabolites and improving the cultivation and quality of G. uralensis using agricultural strategies.

Synthesis of biotin-labeled RNA for gene expression measurements using oligonucleotide arrays.

Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest. Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with T7 RNA polymerase, subsequent in vitro transcription in the presence of biotin-labeled ribonucleotides, and fractionation of the RNA to approximately 500 bp fragments, suitable for oligonucleotide array experiments. Because the membranous labyrinth is composed of functionally interdependent cellular structures, which themselves contain numerous, highly differentiated cell types, comprehensive analysis of gene expression in the cochlea is best complemented by immunohistochemical studies or, if no suitable antibodies are available, by in situ hybridization studies. Either one of these techniques will identify the specific cell types that express the genes of interests.

Identification of transcription factor-DNA interactions using chromatin immunoprecipitation assays.

Expression of almost every gene is regulated at the transcription level. Therefore, transcriptional factor Transcription factors, consequently, have marked effects on the fate of a cell by establishing the gene expression patterns that determine biological processes. In the auditory and vestibular systems, transcription factors have been found to be responsible for development, cell growth, and apoptosis. It is vital to identify the transcription factor target genes and the mechanisms by which transcription factors control and guide gene expression and regulation pathways. Compared with earlier methods devised to study transcription factor-DNA interactions, the advantage of the chromatin immunoprecipitation (ChIP) assay is that the interaction of a transcription factor with its target genes is captured in the native context of chromatin in living cells. Therefore, ChIP base assays are powerful tools to identify the direct interaction of transcription factors and their target genes in vivo. More importantly, ChIP assays have been used in combination with molecular biology techniques, such as PCR and real time PCR, gene cloning, and DNA microarrays, to determine the interaction of transcription factor-DNA from a few potential individual targets to genome-wide surveys.

Age-associated developmental changes in the activated partial thromboplastin time (APTT) and causes of prolonged APTT values in healthy Chinese children.

BACKGROUND: The concept of developmental hemostasis has been universally accepted. Physiological reference ranges for coagulation tests are available for infants and children of different ages. However, on Oriental children they are rare. METHODS: Results of preoperative activated partial thromboplastin time (APTT) in neonates, infants, children aged 1-18 years and adults with minor elective surgery in a university affiliated hospital were reviewed retrospectively. Plasma activity of factors VIII, IX, XI, XII (FVIII:C, FIX:C, FXI:C, FXII:C) and lupus anticoagulants (LAC) in 47 children with prolonged APTT and 34 adult controls were measured to investigate the causes of prolongation. RESULTS: Compared with adults, APTT values were prolonged significantly and were age-dependent in children, especially in neonates and infants aged 1-6 months. Mean values for FXII:C and FIX:C in children with prolonged APTT values were significantly lower than those in adults (p<0.001). Prolonged APTT values correlated negatively with FXII:C and FIX:C, and weakly with the LAC Screen ratio (LAC-SR) (r(0.01)=-0.808, -0.705 and 0.372, p=0.000, 0.000 and 0.001, respectively). There was weak negative correlation between FXII:C and LAC-SR (r(0.01)=-0.277, p=0.012). No significant correlation was seen between prolonged APTT values and FVIII:C or FXI:C. CONCLUSIONS: APTT values change dynamically with age during childhood and display a distinct pattern of evolution in children. Lower values of FXII:C and FIX:C, and presence of LAC contribute to the prolongation of APTT values in Chinese children.