Functional Bioinformatics Analyses of the Matrisome and Integrin Adhesome.

The extracellular matrix (ECM) is the noncellular compartment of living organisms and is formed of a complex network of cross-linked proteins, which is collectively known as the matrisome. Apart from providing the structure for an organism, cells interact and thereby communicate with the ECM. Cells interact with their surrounding ECM using cell-surface receptors, such as integrins. Upon integrin engagement with the ECM, cytoskeletal proteins are recruited to integrins and form a molecular protein complex known as the integrin adhesome. Global descriptions of the matrisome and integrin adhesome have been proposed using in silico bioinformatics approaches, as well as through biochemical enrichment of matrisome and adhesome fractions coupled with mass spectrometry-based proteomic analyses, providing inventories of their compositions in different contexts. Here, methods are described for the computational downstream analyses of matrisome and adhesome mass spectrometry datasets that are accessible to wet lab biologists, which include comparing datasets to in silico descriptions, generating interaction networks and performing functional ontological analyses.

Applications and Caveats on the Utilization of DNA-Specific Probes in Cell-Based Assays.

To perform cell-based assays using fluorescence as the readout there is a fundamental need to identify individual cellular objects. In the majority of cases this requires the addition of a DNA dye or so-called nuclear counterstain and these have become integral to assay design. End-point assays can use live or fixed cells and thus it is beneficial if such reagents are cell membrane-permeant.Further, membrane-permeant DNA dyes can open new opportunities in dynamic real time assays with caveats according to the impact of their interaction with the chromatin in live cells. As cell-based assays offer information on the in vitro toxicity of treatments, cell viability has become a basic readout and cell membrane-impermeant fluorescent DNA-specific dyes can provide this information.In the case of both nuclear counterstaining and viability reporting, it is beneficial if the DNA dyes employed are suitably spectrally separated to permit multi-color experimental design. Methods will be described for these two important assay readouts.

Influence of different intravenous lipid emulsions on growth, development and laboratory and clinical outcomes in hospitalised paediatric patients: A systematic review.

BACKGROUND & AIMS: Fats in the form of lipid emulsions (LEs) are an integral part of intravenous nutrition. The fatty acid composition of different LEs varies. The exact composition of a LE may influence cell and tissue function and clinical outcome. Currently, it is not clear which LE might be best for paediatric patients. We conducted a systematic review of the effects of different intravenous LEs in hospitalised paediatric patients. METHODS: Randomised controlled trials published in a peer reviewed journal, written in the English language, and comparing two or more different intravenous LEs in hospitalised paediatric patients were included. Data on outcomes of relevance (growth, development, laboratory and clinical outcomes) were extracted, collated and interpreted. RESULTS: Thirty-one articles involving 1522 infants or children were included. Most outcomes were not affected by the nature of the LE used. LEs containing fish oil, a source of omega-3 fatty acids, improved outcome of retinopathy of prematurity, decreased liver cholestasis and increased blood omega-3 fatty acid levels. LEs containing olive oil increased blood oleic acid level and had a cholesterol lowering effect. CONCLUSION: Blood fatty acids are influenced by the nature of the intravenous LE used in hospitalised paediatric patients. Most studies suggest limited differences in relevant laboratory or clinical outcomes or in growth in paediatric patients receiving different LEs, although several studies do find benefits from including fish oil or olive oil. There is a need for larger trials to fully evaluate the effects of the available LE types in hospitalised paediatric patients.

Red/far-red fluorescing DNA-specific anthraquinones for nucl:cyto segmentation and viability reporting in cell-based assays.

The advent and wide use of image-based, high-content screening assay formats demands reliable solutions for cellular compartment segmentation to track critical events-for example, those reported by GFP fusions within cell cycle control pathways, signaling pathways, protein translocations, and those associated with drug-induced toxicity such as mitochondrial membrane depolarization, plasma membrane permeabilization, and reactive oxygen species. To meet this need, a series of nuclear/cytoplasmic discriminating probes has been developed: the supravital dyes DRAQ5™ and CyTRAK Orange™ and most recently the viability dye DRAQ7™. These are all spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. As red/far-red emitting dyes, they provide convenient fluorescent emission signatures which are spectrally separated from the majority of commonly used reporter proteins (e.g., eGFP, YFP, mRFP), and a wide range of fluorescent tags such as Alexafluor 488, fluorescein, and Cy2 and fluorescent functional probes used to report cell health status or demark organellar structures. In addition, they are not excited by UV wavelengths thus avoiding complications of the frequently seen pharmacophore UV-autofluorescence in drug discovery. Conversely, their preferential red excitation reduces interference by biological sample autofluorescence. High water solubility and high-affinity DNA-binding properties provide a convenient means of stoichiometrically labeling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Powerfully, they permit the simultaneous and differential labeling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions, and most recently compound in vitro toxicology testing. In one case, DRAQ7™, the core structure has been chemically derivatized to render it intact-cell-membrane impermeant. This far-red viability dye can be more widely combined with other fluorescent reporters to reveal temporally separated events and shows negligible cytotoxicity as determined by sensitive bioassays.

Cyclin D1 expression in acral melanoma: a case control study in Sarawak.

Acral melanoma has been reported to have distinctive clinical presentation and ethnic distribution compared to other histological types of malignant melanoma. Acral melanoma also exhibits distinctive focused gene amplifications, including cyclin D1 overexpression. We reviewed archived histological material of malignant melanoma in the Sarawak General Hospital from year 2004 to 2010. 43 tumours, comprising 28 acral melanoma and 15 non-acral melanoma, had sufficient material to be included in the study. The majority (36%) of acral melanoma tumours occurred in the heel. The tumours were analyzed for cyclin D1 expression by immunohistochemistry. 68% of acral melanoma were cyclin D1 positive compared to a positivity of 33% in non-acral tumours. This difference was statistically significant (p < 0.05). This finding may improve the histological diagnosis of acral melanoma and detection of positive resection margins.

Cyclin D1 expression in acral melanoma: a case control study in Sarawak

Acral melanoma has been reported to have distinctive clinical presentation and ethnic distribution compared to other histological types of malignant melanoma. Acral melanoma also exhibits distinctive focused gene amplifi cations, including cyclin D1 overexpression. We reviewed archived histological material of malignant melanoma in the Sarawak General Hospital from year 2004 to 2010. 43 tumours, comprising 28 acral melanoma and 15 non-acral melanoma, had suffi cient material to be included in the study. The majority (36%) of acral melanoma tumours occurred in the heel. The tumours were analyzed for cyclin D1 expression by immunohistochemistry. 68% of acral melanoma were cyclin D1 positive compared to a positivity of 33% in non-acral tumours. This difference was statistically signifi cant (p <0.05). This fi nding may improve the histological diagnosis of acral melanoma and detection of positive resection margins.

Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells.

Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events--for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5 and CyTRAK Orange. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.

Disposable hydrophobic surface on MALDI targets for enhancing MS and MS/MS data of peptides.

Automated analyses in MALDI MS are complicated by the uneven distribution of analyte over the sample spot, resulting in areas of analyte localization, or "sweet spots". Hence, the ability to concentrate and localize samples is advantageous, especially for automated studies involving low concentrations of analyte. A method for rapidly creating a removable and affordable hydrophobic surface that is free from memory effect is presented. The potential for such compounds to serve as a practical coating for MALDI targets is examined. An example compound with a complete methodology is shown to increase sample homogeneity, peak intensity, and resolution when used for peptide mixtures with CHCA and DHB.