The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

BACKGROUND: The murine ghrelin gene (Ghrl), originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0). We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. -----FINDINGS: 5' RACE revealed two transcription start sites (TSSs) in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR), we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. -----CONCLUSION: We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1) raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

Making visible the coding process : Using qualitative data software in a post-structural study

Qualitative research methods require transparency to ensure the ‘trustworthiness’ of the data analysis. The intricate processes of organizing, coding and analyzing the data are often rendered invisible in the presentation of the research findings, which requires a ‘leap of faith’ for the reader. Computer assisted data analysis software can be used to make the research process more transparent, without sacrificing rich, interpretive analysis by the researcher. This article describes in detail how one software package was used in a poststructural study to link and code multiple forms of data to four research questions for fine-grained analysis. This description will be useful for researchers seeking to use qualitative data analysis software as an analytic tool.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Mutation frequency of non-ESBL phenotype SENTRY (Asia-Pacific) isolates of Klebsiella pneumoniae conversion to an ESBL positive phenotype

Extended spectrum β-lactamases or ESBLs, which are derived from non-ESBL precursors by point mutation of β-lactamase genes (bla), are spreading rapidly all over the world and have caused considerable problems in the treatment of infections caused by bacteria which harbour them. The mechanism of this resistance is not fully understood and a better understanding of these mechanisms might significantly impact on choosing proper diagnostic and treatment strategies. Previous work on SHV β-lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains which contain plasmid-borne blaSHV are able to mutate to phenotypically ESBL-positive strains and there was also evidence of an increase in blaSHV copy number. Therefore, it was hypothesised that although specific point mutation is essential for acquisition of ESBL activity, it is not yet enough, and blaSHV copy number amplification is also essential for an ESBL-positive phenotype, with homologous recombination being the likely mechanism of blaSHV copy number expansion. In this study, we investigated the mutation rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive status by using the MSS-maximum likelihood method. Our data showed that blaSHV mutation rate of a non-ESBL expressing isolate is lower than the mutation rate of the other single base changes on the chromosome, even with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive (≥16 µg/mL for cefotaxime) is very high. This is because only gene copy number increase is needed which is probably mediated by homologous recombination that typically takes place at a much higher frequencies than point mutations. Using a subinhibitory concentration of novobiocin, as a homologous recombination inhibitor, revealed that this is the case.

Effect of female sex hormones on Chlamydia trachomatis growth and gene expression

Transmissible diseases are re-emerging as a global problem, with Sexually Transmitted Diseases (STDs) becoming endemic. Chlamydia trachomatis is the leading cause of bacterially-acquired STD worldwide, with the Australian cost of infection estimated at $90 - $160 million annually. Studies using animal models of genital tract Chlamydia infection suggested that the hormonal status of the genital tract epithelium at the time of exposure may influence the outcome of infection. Oral contraceptive use also increased the risk of contracting chlamydial infections compared to women not using contraception. Generally it was suggested that the outcome of chlamydial infection is determined in part by the hormonal status of the epithelium at the time of exposure. Using the human endolmetrial cell line ECC-1 this study investigated the effects of C. trachomatis serovar D infection, in conjunction with the female sex hormones, 17β-estradiol and progesterone, on chlamydial gene expression. While previous studies have examined the host response, this is the first study to examine C.trachomatis gene expression under different hormonal conditions. We have highlighted a basic model of C. trachomatis gene regulation in the presence of steroid hormones by identifying 60 genes that were regulated by addition of estradiol and/or progesterone. In addition, the third chapter of this thesis discussed and compared the significance of the current findings in the context of data from other research groups to improve our understanding of the molecular basis of chlamydial persistence under hormonal different conditions. In addition, this study analysed the effects of these female sex hormones and C. trachomatis Serovar D infection, on host susceptibility and bacterial growth. Our results clearly demonstrated that addition of steroid hormones not only had a great impact on the level of infectivity of epithelial cells with C.trachomatis serovar D, but also the morphology of chlamydial inclusions was affected by hormone supplementation.

A re-examination of the Ghrelin and Ghrelin receptor genes

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

User-centered video quality assessment for scalable video coding of H.264/AVC standard

Scalable video coding of H.264/AVC standard enables adaptive and flexible delivery for multiple devices and various network conditions. Only a few works have addressed the influence of different scalability parameters (frame rate, spatial resolution, and SNR) on the user perceived quality within a limited scope. In this paper, we have conducted an experiment of subjective quality assessment for video sequences encoded with H.264/SVC to gain a better understanding of the correlation between video content and UPQ at all scalable layers and the impact of rate-distortion method and different scalabilities on bitrate and UPQ. Findings from this experiment will contribute to a user-centered design of adaptive delivery of scalable video stream.