PAP-LMPCR for improved, allele-specific footprinting and automated chromatin fine structure analysis

The analysis of chromatin fine structure and transcription factor occupancy of differentially expressed genes by in vivo footprinting and ligation-mediated-PCR (LMPCR) is a powerful tool to understand the impact of chromatin on gene expression. However, as with all PCR-based techniques, the accuracy of the experiments has often been reduced by sequence similarities and the presence of GC-rich or repeat sequences, and some sequences are completely refractory to analysis. Here we describe a novel method, pyrophosphorolysis activated polymerization LMPCR or PAP-LMPCR, which is capable of generating accurate and reproducible footprints specific for individual alleles and can read through sequences previously not accessible for analysis. In addition, we have adapted this technique for automation, thus enabling the simultaneous and rapid analysis of chromatin structure at many different genes

Integrated analyses of chromatin accessibility and gene expression data for elucidating the transcriptional regulatory mechanisms during early hematopoietic development in mouse

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The Effects of Fractions from Shiitake Mushroom on Composition and Cariogenicity of Dental Plaque Microcosms in an In Vitro Caries Model

The aim of the current study was to investigate the anticariogenic potential of the (sub)fractions obtained from the edible mushroom shiitake (Lentinula edodes) in in vitro caries model. We used a modified constant depth film fermentor (CDFF) with pooled saliva as the inoculum and bovine dentin as a substratum. The test compounds were low molecular weight fraction (MLMW) of the shiitake extract and subfractions 4 and 5 (SF4 and SF5) of this fraction. Chlorhexidine (CHX) and water served as a positive and a negative control, respectively. Dentin mineral loss was quantified (TMR), microbial shifts within the microcosms were determined (qPCR), and the acidogenicity of the microcosms was assessed (CIA). From the compounds tested, the SF4 of shiitake showed strong inhibiting effect on dentin demineralization and induced microbial shifts that could be associated with oral health. The acid producing potential was increased, suggesting uncoupling of the glycolysis of the microbiota by the exposure to SF4. In conclusion, the results suggest that SF4 of shiitake has an anticariogenic potential

LMO2 is required for TAL1 DNA binding activity and initiation of definitive haematopoiesis at the haemangioblast stage

LMO2 is a bridging factor within a DNA binding complex and is required for definitive haematopoiesis to occur. The developmental stage of the block in haematopoietic specification is not known. We show that Lmo2-/- mouse embryonic stem cells differentiated to Flk-1+ haemangioblasts, but less efficiently to haemogenic endothelium, which only produced primitive haematopoietic progenitors. Genome-wide approaches indicated that LMO2 is required at the haemangioblast stage to position the TAL1/LMO2/LDB1 complex to regulatory elements that are important for the establishment of the haematopoietic developmental program. In the absence of LMO2, the target site recognition of TAL1 is impaired. The lack of LMO2 resulted in altered gene expression levels already at the haemangioblast stage, with transcription factor genes accounting for ∼15% of affected genes. Comparison of Lmo2-/- with Tal1-/- Flk-1+ cells further showed that TAL1 was required to initiate or sustain Lmo2 expression

Ocean Colour Remote Sensing of Flood Plumes in the Great Barrier Reef

The objective of the research reported in this thesis was to develop a technique to monitor the dynamics of sediments and nutrients entering the coastal ocean with river plumes associated with high intensity low frequency events (e.g. floods), using ocean colour remote sensing. To achieve this objective, an inverse bio-optical model was developed, based on analytical and empirical relationships between concentrations of optically significant substances and remote sensing of water-leaving radiance. The model determines concentrations of water-colouring substances such as chlorophyll, suspended sediments, and coloured dissolved organic matter, as well as the values of optical parameters using water-leaving radiances derived from the Sea-viewing Wide Field-of-view Sensor (SeaWiFS). To solve atmospheric correction in coastal waters, the aerosol type over clear waters is transferred to adjacent turbid water pixels. The vicinity of the Herbert River, central Great Barrier Reef zone, Australia, was used as a case study for the application of the algorithm developed. The satellite ocean colour technique was successfully validated using sea-truth measurements of water-colouring constituents acquired in the area during various seasons throughout 2002-2004. A high correlation between chlorophyll and dissolved organic matter was found in the coastal waters of the region, and when the bio-optical model was constrained to make chlorophyll a function of dissolved organic matter, the relationship between in situ and satellite-derived data was substantially improved. With reliable retrieval of the major water-colouring constituents, the technique was subsequently applied to study fluxes of particulate and dissolved organic and inorganic matter following a flood event in the Herbert River during the austral summer of 1999. Extensive field observations covering a seasonal flood in the Herbert River in February 2004 revealed high sediment and nutrient exports from the river to the adjacent coastal waters during the flood event. Due to rapid settling, the bulk of the sediment-rich influx was deposited close inshore, while the majority of nutrients exported from the river were consumed by phytoplankton in a relatively small area of the coastal ocean. With the help of ocean colour remote sensing, it was demonstrated that river-borne sediments and nutrients discharged by a typical flood in the Herbert River are mostly precipitated or consumed within the first 20 km from the coast and therefore are unlikely to reach and possibly affect the midshelf coral reefs of this section of the Great Barrier Reef lagoon

Use of natural antioxidants from lyophilized water extracts of Borago officinalis in dry fermented sausages enriched in ω-3 PUFA.

An evaluation of the capacity of a lyophilized water extract of borage leaves to delay the lipid oxidation process in dry fermented sausages enriched with ω-3 PUFAs has been performed. Lyophilized extract (340ppm) showed an antioxidant capacity equivalent to 200ppm of a butylhydroxyanisol (BHA) and butylhydroxytoluene (BHT) mixture. Two batches of dry fermented sausages enriched in ω-3 PUFA were developed. One of them was supplemented with a synthetic antioxidants mixture (200ppm of BHA+BHT) and the other one with natural antioxidants (340ppm of lyophilized water extract of borage leaves). Furthermore, a traditional formulation of this type of dry fermented sausage (Control), was also manufactured. The natural extract gave rise to lower amount of volatile compounds (including hexanal), than the mixture of synthetic antioxidants (2202 and 2713ng dodecane/g dry matter, respectively). TBARS and Cholesterol Oxidation Products (COPs) did not show significant differences between products with different antioxidants. The sensorial analysis showed that lyophilized water extracts of borage leaves did not affect the sensorial properties of the products. From the economical and safety standpoints, the use of a by-product (borage leaves) and water as extracting solvent are valuable alternatives for obtaining natural antioxidants to be added to dry fermented sausages enriched in ω-3 PUF