Identification, Cloning and Characterization of Selected Full-Length Fragrance-Related Transcripts from Orchid (Vanda Mimi Palmer)

Floral fragrance has important economical value in ornamental plants, crops and industries related to essential oils. However, the understanding of the molecular mechanisms underlying the biosynthesis of floral fragrance in monocotyledonous plants; in particular orchids, is still in its infancy. This study aimed to isolate and characterize fragrance-related genes from Vanda Mimi Palmer in order to enhance understanding of the molecular biology of fragrance in vandaceous orchid. Vanda Mimi Palmer is a tropical scented orchid with high economical value. In the effort to identify potential fragrance-related genes in Vanda Mimi Palmer, a floral cDNA library and a subtracted cDNA library were constructed. A total of 100 clones were selected from the cDNA library and their nucleotide sequences were determined, of which 83 clones showed homology to known amino acid sequences, comprising 6 contigs and 62 singletons, which were further assigned into 9 categories based on their functional roles. Two ESTs were identified as potential fragrance-related transcripts and they were 1-deoxy-Dxylulose 5-phosphate reductoisomerase (DXR) and lipoxygenase. From the Suppression Subtractive Hybridization (SSH) library, 107 clones were up-regulated in the full bloom flowers of Vanda Mimi Palmer where 33 clones (3 singletons and 30 contigs) showed similarities to known sequences in the public database and were classified based on their putative functional roles as secondary metabolism (97%) and hypothetical proteins (3%), and 32 of the clones were transcripts encoding fragrance-related transcripts. The fragrance-related transcripts code for sesquiterpene synthase, tyrosine decarboxylase and putative alcohol acyltransferase. However, only three ESTs were selected for fulllength gene isolation and characterization and they are putative alcohol acyltransferase (VMPAAT), sesquiterpene synthase (VMPSTS) and DXR (VMPDXR). Southern analyses showed that each of the isolated transcripts belongs to a large gene family, containing more than one copy in the Vanda Mimi Palmer genome. Real time RT-PCR indicated that VMPAAT and VMPSTS transcripts were expressed preferentially in floral tissues whereas VMPDXR was expressed differentially in different types of tissues (root, leaf, petal, sepal and column). All three clones showed higher transcript expressions in blooming and full bloom flowers compared to flower bud. VMPAAT and VMPDXR transcripts expressions showed no fluctuations whereas VMPSTS showed otherwise. In conclusion, the findings in this study have contributed to the GeneBank database resources for orchids and have opened some insights on molecular biology of fragrance in vandaceous orchids

Isolation, cloning and characterisation of new fragrance-related floral transcripts of Vanda Mimi Palmer

A subtracted cDNA library of open flower was constructed using the suppression subtractive hybridization (SSH) technique, to identify fragrance-related transcripts of Vanda Mimi Palmer. In total, 107 transcripts up-regulated during blooming were identified and sequenced. Only 33 clones (4 singletons and 29 contigs) showed similarities to known sequences in the public database. Of these, thirty-two clones were transcripts encoding fragrance-related enzymes including sesquiterpene synthase, (±)-germacrene D synthase, tyrosine decarboxylase and putative acyltransferase. Two fragrance-related transcripts, VMPAAT encoding a putative alcohol acyltransferase and VMPSTS encoding a sesquiterpene synthase, were subjected to full-length cDNA isolation and characterization. The full length cDNA of VMPAAT has a 1343bp open reading frame (ORF) of 448 amino acid residues whereas VMPSTS is predicted to encode a polypeptide of 561 amino acid residues with 1682bp ORF. VMPAAT and VMPSTS show high homologies with plant alcohol acyltransferase and terpene synthase, respectively. Real time RT-PCR indicated that both transcripts were expressed preferentially in floral tissues, with high levels in blooming and full bloom flowers. VMPAAT and VMPSTS transcripts were expressed in a rhythmic pattern. The results presented in this study will be potentially useful in providing additional insights into the fragrance-related pathways of Orchidaceae members, which until today is still limited

Molecular characterization of a new 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) transcript from Vanda Mimi Palmer.

A 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) transcript was successfully isolated from the floral cDNA library of Vanda Mimi Palmer (VMPDXR). The full-length cDNA of clone VMPDXR was predicted to encode a polypeptide of 473 amino acid residues with 15 bp of 5′ UTR and 230 bp of 3′ UTR including a poly-A tail. VMPDXR was predicted to have a molecular mass of 51.4 kD and a pI value of 6.04. It has two conserved domains, an N-terminal NADPH binding site (GSTGSIG) and an N-terminal proline-rich region(PPPPAWPGR). It also contains two highly homologous regions, a 78–207 amino acids stretch at the N-terminal and a 221–304 amino acids stretch at the C-terminal domain. The putative plastid transit peptide is not found in VMPDXR and it is clustered into the plant DXRs in the phylogenetic tree. VMPDXR was differentially expressed in roots, leaves, sepals, petals and column. The VMPDXR transcript levels were preferentially high in blooming and fully bloomed flowers compared to the bud. The expression of VMPDXR at different times did not appear in a rhythmic manner and no drastic fluctuation was observed at night except at 2 pm during the day

A Multidisciplinary Primary Care Team Consultation In a Socio-economically Deprived Community: An Exploratory Randomised Controlled Trial

Background: Psychosocial problems in socioeconomically deprived communities are not always amenable to traditional medical approaches. Mothers living in these areas are a particularly vulnerable group. The objective of this study was to evaluate the effectiveness of a lengthened multi-disciplinary team consultation in primary care in reducing anxiety and depression in mothers.Methods: This was a prospective randomised controlled trial of a multidisciplinary team consultation against normal care. 94 mothers were recruited from three general practices from an area of extreme socio-economic deprivation. Mothers randomised into the intervention group attended a multidisciplinary consultation with up to four case-specific health care professionals. Consultations addressed medical, psychological and social problems and lasted up to one hour. Conventional primary care continued to be available to the intervention families. Control group families received normal primary care services. The outcomes measured were anxiety and depression as using the Hospital Anxiety and Depression Scale (HADS), health status using SF36v2, and quality of life using the abbreviated Schedule for the Evaluation of Individual Quality of Life (SEIQoL-DW) at baseline, 6 months and 12 months.Results: Ordered logistic regression was used to analyse the data. There was no significant difference found between intervention and control groups after 6 months and 12 months in all of the measured outcomes.Conclusions: The new lengthened multi-disciplinary team consultation did not have any impact on the mental health, general health, and quality of life of mothers after 6 and 12 months. Other methods of primary health care delivery in socio-economically deprived communities need to be evaluated

Green Building Rating Systems and Construction Waste in High Density Urban Environment: The Case Study of Hong Kong

Hong Kong, as many other countries, is currently facing a waste management crisis with the shortage of reclamation sites and landfill space. Examination of construction waste management (CWM) criteria within Hong Kong’s BEAM Plus is limited and lacking. In this paper, CWM criteria were assessed and a case study was conducted on a recent project, pursuing BEAM Plus, to assess the effectiveness of CWM criteria to facilitate waste reduction. The findings revealed that the overall impact of BEAM Plus on construction waste reduction is negligible. The case study showed that the reasons material/waste-related credits are not commonly attempted is due to the lack of minimum thresholds and a low weighting relative to credits in other categories. In the case study, waste generated per constructed floor area was 0.21 tons/m2. This paper also makes recommendations to improve CWM criteria in BEAM Plus in order to promote a more sustainable building industry

An Uncertainty Aided Framework for Learning based Liver T1ρT_1\rho Mapping and Analysis

Objective: Quantitative $T_1\rho$ imaging has potential for assessment of biochemical alterations of liver pathologies. Deep learning methods have been employed to accelerate quantitative $T_1\rho$ imaging. To employ artificial intelligence-based quantitative imaging methods in complicated clinical environment, it is valuable to estimate the uncertainty of the predicated $T_1\rho$ values to provide the confidence level of the quantification results. The uncertainty should also be utilized to aid the post-hoc quantitative analysis and model learning tasks. Approach: To address this need, we propose a parametric map refinement approach for learning-based $T_1\rho$ mapping and train the model in a probabilistic way to model the uncertainty. We also propose to utilize the uncertainty map to spatially weight the training of an improved $T_1\rho$ mapping network to further improve the mapping performance and to remove pixels with unreliable $T_1\rho$ values in the region of interest. The framework was tested on a dataset of 51 patients with different liver fibrosis stages. Main results: Our results indicate that the learning-based map refinement method leads to a relative mapping error of less than 3% and provides uncertainty estimation simultaneously. The estimated uncertainty reflects the actual error level, and it can be used to further reduce relative $T_1\rho$ mapping error to 2.60% as well as removing unreliable pixels in the region of interest effectively. Significance: Our studies demonstrate the proposed approach has potential to provide a learning-based quantitative MRI system for trustworthy $T_1\rho$ mapping of the liver