Cyclic tetrapyrrolic photosensitizers from Cladophora patentiramea (Cladophoraceae, Chlorophyta) and Turbinaria conoides (Sargassaceae, Phaeophyta) for photodynamic therapy

In screening for novel photosensitizers for photodynamic therapy, 14 seaweed samples from Port Dickson in Malaysia were collected. Methanolic extracts of these samples were prepared and evaluated for phototoxicity using a short-term cell viability assay, where promyelocytic leukemia cells, HL60 were incubated with the extracts prior to irradiation with a broad spectrum light at 9.6?J?cm^?2 (equivalent to 10.5?mW?cm^?2 for 10?min). Four of the methanolic extracts demonstrated moderate to strong phototoxicity and bioassay-guided isolation of photosensitizers was carried out on two selected seaweeds to yield a total of eight cyclic tetrapyrrolic compounds which are derivatives of chlorophyll-a and -b. Seven of these compounds showed >50% phototoxicity at 5?μg?mL^?1 while exhibiting minimal cytotoxicity in the dark, which is an important characteristic of an ideal photosensitizer.     

Identification of functional elements of the GDP-fucose transporter SLC35C1 using a novel Chinese hamster ovary mutant

The GDP-fucose transporter SLC35C1 critically regulates the fucosylation of glycans. Elucidation of its structure-function relationships remains a challenge due to the lack of an appropriate mutant cell line. Here we report a novel Chinese hamster ovary (CHO) mutant, CHO-gmt5, generated by the zinc-finger nuclease technology, in which the Slc35c1 gene was knocked out from a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter (CST) gene (Slc35a1). Consequently, CHO-gmt5 harbors double genetic defects in Slc35a1 and Slc35c1 and produces N-glycans deficient in both sialic acid and fucose. The structure-function relationships of SLC35C1 were studied using CHO-gmt5 cells. In contrast to the CST and UDP-galactose transporter, the C-terminal tail of SLC35C1 is not required for its Golgi localization but is essential for generating glycans that are recognized by a fucose-binding lectin, Aleuria aurantia lectin (AAL), suggesting an important role in the transport activity of SLC35C1. Furthermore, we found that this impact can be independently contributed by a cluster of three lysine residues and a Glu-Met (EM) sequence within the C terminus. We also showed that the conserved glycine residues at positions 180 and 277 of SLC35C1 have significant impacts on AAL binding to CHO-gmt5 cells, suggesting that these conserved glycine residues are required for the transport activity of Slc35 proteins. The absence of sialic acid and fucose on Fc N-glycan has been independently shown to enhance the antibody-dependent cellular cytotoxicity (ADCC) effect. By combining these features into one cell line, we postulate that CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC effect.     

MicroRNA-10b pleiotropically regulates invasion, angiogenicity and apoptosis of tumor cells resembling mesenchymal subtype of glioblastoma multiforme

Glioblastoma multiforme (GBM) is a heterogeneous disease despite its seemingly uniform pathology. Deconvolution of The Cancer Genome Atlas''s GBM gene expression data has unveiled the existence of distinct gene expression signature underlying discrete GBM subtypes. Recent conflicting findings proposed that microRNA (miRNA)-10b exclusively regulates glioma growth or invasion but not both. We showed that silencing of miRNA-10b by baculoviral decoy vectors in a glioma cell line resembling the mesenchymal subtype of GBM reduces its growth, invasion and angiogenesis while promoting apoptosis in vitro. In an orthotopic human glioma mouse model, inhibition of miRNA-10b diminishes the invasiveness, angiogenicity and growth of the mesenchymal subtype-like glioma cells in the brain and significantly prolonged survival of glioma-bearing mice. We demonstrated that the pleiotropic nature of miRNA-10b was due to its suppression of multiple tumor suppressors, including TP53, FOXO3, CYLD, PAX6, PTCH1, HOXD10 and NOTCH1. In particular, siRNA-mediated knockdown experiments identified TP53, PAX6, NOTCH1 and HOXD10 as invasion regulatory genes in our mesenchymal subtype-like glioma cells. By interrogating the REMBRANDT, we noted that dysregulation of many direct targets of miRNA-10b was associated with significantly poorer patient survival. Thus, our study uncovers a novel role for miRNA-10b in regulating angiogenesis and suggests that miRNA-10b may be a pleiotropic regulator of gliomagenesis.     

Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons

Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. While genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs, the current application of PET sequencing with short insert size DNA can be insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We employed a recently developed procedure to generate PET sequencing data using large DNA inserts of 10–20 kb and compared their characteristics with short insert (1 kb) libraries for their ability to identify SVs. Our results suggest that although short insert libraries bear an advantage in identifying small deletions, they do not provide significantly better breakpoint resolution. In contrast, large inserts are superior to short inserts in providing higher physical genome coverage for the same sequencing cost and achieve greater sensitivity, in practice, for the identification of several classes of SVs, such as copy number neutral and complex events. Furthermore, our results confirm that large insert libraries allow for the identification of SVs within repetitive sequences, which cannot be spanned by short inserts. This provides a key advantage in studying rearrangements in cancer, and we show how it can be used in a fusion-point-guided-concatenation algorithm to study focally amplified regions in cancer.     

The interactive effect of alcohol and folic acid on genome stability in human WIL2-NS cells measured using the cytokinesis-block micronucleus cytome assay

Chromosomal mutations are commonly found in cancer cells, and can be caused by several factors including dietary insufficiency and exposure to environmental and life-style genotoxins. Folate (vitamin B9), one of the essential micronutrients, is required for DNA repair and synthesis and to maintain genome stability. Since excessive alcohol (ethanol) consumption may alter folate status and low folate might alter susceptibility to alcohol toxicity, a study was performed to investigate the individual and interactive impacts of folic acid (FA) and ethanol on genome stability in vitro. The experiments were performed using WIL2-NS cells cross-tested at three FA (20, 200 and 2000 nM) and four ethanol concentrations (0, 0.09, 0.36 and 1.34%, v/v) over a two-week culture time. Chromosomal damage and cytotoxicity were measured using the cytokinesis-block micronucleus cytome assay. The present study showed dose-related genotoxic effects of both decreasing folic acid concentration and increased ethanol on day 15 resulting in significant induction of micronuclei, nuclear buds and nucleoplasmic bridges which are biomarkers of chromosome breakage or loss, gene amplification and chromosomal rearrangement, respectively. Increased ethanol and FA deficiency interacted to further significantly increase micronuclei and nucleoplasmic bridges. However there was no evidence showing alcohol's ability to cleave FA. The findings from this study suggest a protective effect of FA against alcohol-induced DNA damage and that FA deficiency in the physiological range has a stronger impact on genome stability than exposure to cytotoxic doses of ethanol achievable in binge drinking.     

BRCA1 and BRCA2 germline mutations in Malaysian women with early-onset breast cancer without a family history

Background

In Asia, breast cancer is characterised by an early age of onset: In Malaysia, approximately 50% of cases occur in women under the age of 50 years. A proportion of these cases may be attributable, at least in part, to genetic components, but to date, the contribution of genetic components to breast cancer in many of Malaysia''s ethnic groups has not been well-characterised.

Methodology

Given that hereditary breast carcinoma is primarily due to germline mutations in one of two breast cancer susceptibility genes, BRCA1 and BRCA2, we have characterised the spectrum of BRCA mutations in a cohort of 37 individuals with early-onset disease (≤40 years) and no reported family history. Mutational analysis of BRCA1 and BRCA2 was conducted by full sequencing of all exons and intron-exon junctions.

Conclusions

Here, we report a total of 14 BRCA1 and 17 BRCA2 sequence alterations, of which eight are novel (3 BRCA1 and 5 BRCA2). One deleterious BRCA1 mutation and 2 deleterious BRCA2 mutations, all of which are novel mutations, were identified in 3 of 37 individuals. This represents a prevalence of 2.7% and 5.4% respectively, which is consistent with other studies in other Asian ethnic groups (4–9%).     

Real-time determination of kinetics of adsorption of lead(II) onto palm shell-based activated carbon using ion selective electrode

In this study, the kinetics of adsorption of Pb(II) from aqueous solution onto palm shell-based activated carbon (PSAC) were investigated by employing ion selective electrode (ISE) for real-time Pb(II) and pH monitoring. Usage of ISE was very appropriate for real-time adsorption kinetics data collection as it facilitated recording of adsorption data at very specific and short time intervals as well as provided consistent kinetics data. Parameters studied were initial Pb(II) concentration and agitation speed. It was found that increases in initial Pb(II) concentration and agitation speed resulted in higher initial rate of adsorption. Pseudo first-order, pseudo second-order, Elovich, intraparticle diffusion and liquid film diffusion models were used to fit the adsorption kinetics data. It was suggested that chemisorption was the rate-controlling step for adsorption of Pb(II) onto PSAC since the adsorption kinetics data fitted both the pseudo second-order and Elovich models well.     

First cervical vertebra (atlas) fracture mechanism studies using finite element method.

Injury mechanisms and stress distribution patterns are important in the clinical evaluation of spinal injuries. Recognition and interpretation of the failure patterns help to determine spinal instability and consequently the choice of treatment. Although, the biomechanics responses of the atlas have received much attention, it has not been investigated using theoretical modeling. Mathematical techniques such as finite element model will provide further understanding to the injury mechanisms of the atlas, which is important for the prevention, diagnosis, and treatment of spinal injuries. In the present study, a detailed three-dimensional finite element model of the human atlas (C1) was constructed, with the geometrical data obtained using a three-dimensional digitizer. Anterior arch, superior/inferior articular processes, transverse processes, posterior arch and posterior tubercule were modeled using eight-noded brick elements. Using the material properties from literature, the 7808-finite element model was exercised under three simulated axial compressive mode of pressure loading and boundary conditions to investigate the sites of failure reported in vivo and in vitro. This report demonstrates high concentration of localized stress at the anterior and posterior archs of the atlas, which agrees well with those reported in the literature. Furthermore, under simulated hyperextension, our results agreed well with the experimental findings, which show that the groove of the posterior arch is subjected to enormous bending moment. The close agreement of the failure location provided confidence to perform further analysis and in vitro experiments. These results may be potentially used to supplement experimental research in understanding the clinical biomechanics of the atlas.     

Src kinases relax adherens junctions between the neighbors of apoptotic cells to permit apical extrusion

Epithelia can eliminate apoptotic cells by apical extrusion. This is a complex morphogenetic event where expulsion of the apoptotic cell is accompanied by rearrangement of its immediate neighbors to form a rosette. A key mechanism for extrusion is constriction of an actomyosin network that neighbor cells form at their interface with the apoptotic cell. Here we report a complementary process of cytoskeletal relaxation that occurs when cortical contractility is down-regulated at the junctions between those neighbor cells themselves. This reflects a mechanosensitive Src family kinase (SFK) signaling pathway that is activated in neighbor cells when the apoptotic cell relaxes shortly after injury. Inhibiting SFK signaling blocks both the expulsion of apoptotic cells and the rosette formation among their neighbor cells. This reveals the complex pattern of spatially distinct contraction and relaxation that must be established in the neighboring epithelium for apoptotic cells to be extruded.     

Quantitative secretome and glycome of primary human adipocytes during insulin resistance

Adipose tissue is both an energy storage depot and an endocrine organ. The impaired regulation of the secreted proteins of adipose tissue, known as adipocytokines, observed during obesity contributes to the onset of whole-body insulin resistance and the pathobiology of type 2 diabetes mellitus (T2DM). In addition, the global elevation of the intracellular glycosylation of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) via either genetic or pharmacological methods is sufficient to induce insulin resistance in both cultured cells and animal models. The elevation of global O-GlcNAc levels is associated with the altered expression of many adipocytokines. We have previously characterized the rodent adipocyte secretome during insulin sensitive and insulin resistant conditions. Here, we characterize and quantify the secretome and glycome of primary human adipocytes during insulin responsive and insulin resistant conditions generated by the classical method of hyperglycemia and hyperinsulinemia or by the pharmacological manipulation of O-GlcNAc levels. Using a proteomic approach, we identify 190 secreted proteins and report a total of 20 up-regulated and 6 down-regulated proteins that are detected in both insulin resistant conditions. Moreover, we apply glycomic techniques to examine (1) the sites of N-glycosylation on secreted proteins, (2) the structures of complex N- and O-glycans, and (3) the relative abundance of complex N- and O-glycans structures in insulin responsive and insulin resistant conditions. We identify 91 N-glycosylation sites derived from 51 secreted proteins, as well as 155 and 29 released N- and O-glycans respectively. We go on to quantify many of the N- and O-glycan structures between insulin responsive and insulin resistance conditions demonstrating no significant changes in complex glycosylation in the time frame for the induction of insulin resistance. Thus, our data support that the O-GlcNAc modification is involved in the regulation of adipocytokine secretion upon the induction of insulin resistance in human adipocytes.