Characterization of zebrafish polymerase iii promoters for the expression of short hairpin RNA interference molecules

RNA interference (RNAi) is a powerful, sequence specific, and long-lasting method of gene knockdown, and can be elicited by the expression of short-hairpin RNA (shRNA) molecules driven via polymerase III type 3 promoters from a DNA vector or transgene. To further develop RNAi as a tool in zebrafish, we have characterized the zebrafish U6 and H1 snRNA promoters and compared the efficiency of each of the promoters to express an shRNA and silence a reporter gene, relative to previously characterized U6 promoters from pufferfish, chicken, and mouse. Our results show that the zebrafish polymerase III promoters were capable of effective gene silencing in the zebrafish ZF4 cell line, but were ineffective in mammalian Vero cells. In contrast, mouse and chicken promoters were active in Vero but not ZF4 cells, highlighting the importance of homologous promoters to achieve effective silencing.

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

Characterisation of RNA in milk and prediction of microRNA functions

 Milk is considered on of the world’s most ‘complete’ food. To characterise milk composition, Amit investigated RNA present of milk form 8 different species ranging from platypus to human. By applying latest RNA sequencing and bioinformatic techniques, his work led to uncover hundreds of novel milk RNAs.

Characteristics of Mentally Disordered Youth Referred to a Forensic Satellite Clinic for Violence Risk Assessment: A Case Control Study

There is a lack of appropriate services to manage youth with comorbid mental health problems and violence risks. To address this gap, we implemented a forensic satellite clinic in a youth mental health service. This paper characterises offending histories among 45 young patients referred to the clinic, and compares them with matched clinical controls (n = 45). Levels of prior risk taking and aggression were prominent among referred patients. Forensic cases and controls did not differ on demographic and clinical variables, with the exception of psychiatric inpatient admissions, which were higher among referred patients. Group differences were observed for prior offending variables (e.g., physical aggression), which were significantly higher among referred patients than controls. Findings suggest that referrals were made to the clinic based on challenging and aggressive behaviour rather than specific clinical characteristics. The role of specialist assessment, treatment and management of violence risks in youth mental health services are discussed.

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

During assembly of HIV-1 particles in infected cells, the viral Pr55(Gag) protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55(Gag) achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55(Gag) binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55(Gag) to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.

Transcriptomics-based analysis using RNA-Seq of the coconut (Cocos nucifera) leaf in response to yellow decline phytoplasma infection

Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host–phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.

Developing RNA aptamer technology for targeting cancer stem cells

 This thesis successfully developed a novel strategy using chemical antibodies for targeting cancer stem cells, suggesting that this novel strategy of effective targeting the roots of cancer could potentially revolutionize the future cancer treatment.

Mutational interference mapping experiment (MIME) for studying RNA structure and function

RNA regulates many biological processes; however, identifying functional RNA sequences and structures is complex and time-consuming. We introduce a method, mutational interference mapping experiment (MIME), to identify, at single-nucleotide resolution, the primary sequence and secondary structures of an RNA molecule that are crucial for its function. MIME is based on random mutagenesis of the RNA target followed by functional selection and next-generation sequencing. Our analytical approach allows the recovery of quantitative binding parameters and permits the identification of base-pairing partners directly from the sequencing data. We used this method to map the binding site of the human immunodeficiency virus-1 (HIV-1) Pr55(Gag) protein on the viral genomic RNA in vitro, and showed that, by analyzing permitted base-pairing patterns, we could model RNA structure motifs that are crucial for protein binding.

Satellite imagery as a single source of predictor variables for habitat suitability modelling: how Landsat can inform the conservation of a critically endangered lemur

1. Statistical modelling of habitat suitability is an important tool for planning conservation interventions, particularly for areas where species distribution data are expensive or hard to collect. Sometimes however the predictor variables typically used in habitat suitability modelling are themselves difficult to obtain or not meaningful at the geographical extent of the study, as is the case for the Alaotran gentle lemur Hapalemur alaotrensis, a critically endangered lemur confined to the marshes of Lake Alaotra in Madagascar.2. We developed a habitat suitability model where all predictor variables, including vegetation indices and image texture measures at different scales (as surrogates for habitat structure), were derived from Landsat7 satellite imagery. Using relatively few presence records, the maximum entropy (Maxent) approach and AUC were used to assess the performance of candidate predictor variables, for studying the effect of scale, model selection and mapping suitable habitat.3. This study demonstrated the utility of satellite imagery as a single source of predictor variables for a Maxent habitat suitability model at the landscape level, within a restricted geographical extent and with a fine grain, in a case where predictor variables typically used at the macro-scale level (e.g. climatic and topographic) were not applicable.4. In the case of H. alaotrensis, the methodology generated a habitat suitability map to inform conservation management in Lake Alaotra and a replicable protocol to allow rapid updates to habitat suitability maps in the future. The exploration of candidate predictor variables allowed the identification of scales that appear ecologically relevant for the species.5. Synthesis and applications. This study presents a cost-effective combination of maximum entropy habitat suitability modelling and satellite imagery, where all predictor variables are derived solely from Landsat7 images. With a habitat modelling method like Maxent that shows good performance with few presence samples and Landsat images now freely available, the methodology can play an important role in rapid assessments of the status of species at the landscape level in data-poor regions, when typical macro-scale environmental predictors are of little use or difficult to obtain.

Tunnelling current recognition through core-satellite gold nanoparticles for ultrasensitive detection of copper ions

We report a new method for ultrasensitive detection of Cu(2+), which is based on changes in the tunnelling recognition current across self-assembled core-satellite gold nanoparticles (GNPs) networks functionalised with amino acids (l-cysteine). The addition of copper ions induces the formation of GNP/l-cysteine/Cu(2+)/l-cysteine/GNP molecular junctions and generates a significant decrease in the resistance through the networks. The networks are ultrasensitive to over ten orders range of copper ion concentrations.

Combining Satellite And Gauge Precipitation Data With Co-Kriging Method For Jakarta Region

Jakarta is vulnerable to flooding mainly caused by prolonged and heavy rainfall and thus a robust hydrological modeling is called for. A good quality of spatial precipitation data is therefore desired so that a good hydrological model could be achieved. Two types of rainfall sources are available: satellite and gauge station observations. At-site rainfall is considered to be a reliable and accurate source of rainfall. However, the limited number of stations makes the spatial interpolation not very much appealing. On the other hand, the gridded rainfall nowadays has high spatial resolution and improved accuracy, but still, relatively less accurate than its counterpart. To achieve a better precipitation data set, the study proposes cokriging method, a blending algorithm, to yield the blended satellite-gauge gridded rainfall at approximately 10-km resolution. The Global Satellite Mapping of Precipitation (GSMaP, 0.1⁰×0.1⁰) and daily rainfall observations from gauge stations are used. The blended product is compared with satellite data by cross-validation method. The newly-yield blended product is then utilized to re-calibrate the hydrological model. Several scenarios are simulated by the hydrological models calibrated by gauge observations alone and blended product. The performance of two calibrated hydrological models is then assessed and compared based on simulated and observed runoff.