Chemical and Biomolecular Engineering - Research Publications

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    A comparative study of the coagulation behaviour of marine microalgae
    Eldridge, RJ ; Hill, DRA ; Gladman, BR (SPRINGER, 2012-12)
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    Electrokinetic flow in connected channels: a comparison of two circuit models
    Biscombe, CJC ; Davidson, MR ; Harvie, DJE (SPRINGER HEIDELBERG, 2012-09)
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    Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin
    Lauridsen, LH ; Shamaileh, HA ; Edwards, SL ; Taran, E ; Veedu, RN ; Antopolsky, M (PUBLIC LIBRARY SCIENCE, 2012-07-30)
    BACKGROUND: Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period. PRINCIPAL FINDINGS: Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM. CONCLUSION: We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.
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    Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells
    Faille, D ; El-Assaad, F ; Mitchell, AJ ; Alessi, M-C ; Chimini, G ; Fusai, T ; Grau, GE ; Combes, V (WILEY, 2012-08)
    Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca(2+) chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.
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    An "Escape Clock'' for Estimating the Turnover of SIV DNA in Resting CD4+T Cells
    Reece, J ; Petravic, J ; Balamurali, M ; Loh, L ; Gooneratne, S ; De Rose, R ; Kent, SJ ; Davenport, MP ; Silvestri, G (PUBLIC LIBRARY SCIENCE, 2012-04)
    Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an "escape clock" approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT) SIV DNA present in early infection by CTL escape mutant (EM) strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication.
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    Comparison of Influenza and SIV Specific CD8 T Cell Responses in Macaques
    Jegaskanda, S ; Reece, JC ; De Rose, R ; Stambas, J ; Sullivan, L ; Brooks, AG ; Kent, SJ ; Sexton, A ; Ambrose, Z (PUBLIC LIBRARY SCIENCE, 2012-03-05)
    Macaques are a potentially useful non-human primate model to compare memory T-cell immunity to acute virus pathogens such as influenza virus and effector T-cell responses to chronic viral pathogens such as SIV. However, immunological reagents to study influenza CD8(+) T-cell responses in the macaque model are limited. We recently developed an influenza-SIV vaccination model of pigtail macaques (Macaca nemestrina) and used this to study both influenza-specific and SIV-specific CD8(+) T-cells in 39 pigtail macaques expressing the common Mane-A*10(+) (Mane-A01*084) MHC-I allele. To perform comparative studies between influenza and SIV responses a common influenza nucleoprotein-specific CD8(+) T-cell response was mapped to a minimal epitope (termed RA9), MHC-restricted to Mane-A*10 and an MHC tetramer developed to study this response. Influenza-specific memory CD8(+) T-cell response maintained a highly functional profile in terms of multitude of effector molecule expression (CD107a, IFN-γ, TNF-α, MIP-1β and IL-2) and showed high avidity even in the setting of SIV infection. In contrast, within weeks following active SIV infection, SIV-specific CD8(+) effector T-cells expressed fewer cytokines/degranulation markers and had a lower avidity compared to influenza specific CD8(+) T-cells. Further, the influenza specific memory CD8 T-cell response retained stable expression of the exhaustion marker programmed death-marker-1 (PD-1) and co-stimulatory molecule CD28 following infection with SIV. This contrasted with the effector SIV-specific CD8(+) T-cells following SIV infection which expressed significantly higher amounts of PD-1 and lower amounts of CD28. Our results suggest that strategies to maintain a more functional CD8(+) T-cell response, profile may assist in controlling HIV disease.
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    Increased Gut Permeability and Microbiota Change Associate with Mesenteric Fat Inflammation and Metabolic Dysfunction in Diet-Induced Obese Mice
    Lam, YY ; Ha, CWY ; Campbell, CR ; Mitchell, AJ ; Dinudom, A ; Oscarsson, J ; Cook, DI ; Hunt, NH ; Caterson, ID ; Holmes, AJ ; Storlien, LH ; Zhang, RR (PUBLIC LIBRARY SCIENCE, 2012-03-23)
    We investigated the relationship between gut health, visceral fat dysfunction and metabolic disorders in diet-induced obesity. C57BL/6J mice were fed control or high saturated fat diet (HFD). Circulating glucose, insulin and inflammatory markers were measured. Proximal colon barrier function was assessed by measuring transepithelial resistance and mRNA expression of tight-junction proteins. Gut microbiota profile was determined by 16S rDNA pyrosequencing. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA levels were measured in proximal colon, adipose tissue and liver using RT-qPCR. Adipose macrophage infiltration (F4/80⁺) was assessed using immunohistochemical staining. HFD mice had a higher insulin/glucose ratio (P = 0.020) and serum levels of serum amyloid A3 (131%; P = 0.008) but reduced circulating adiponectin (64%; P = 0.011). In proximal colon of HFD mice compared to mice fed the control diet, transepithelial resistance and mRNA expression of zona occludens 1 were reduced by 38% (P<0.001) and 40% (P = 0.025) respectively and TNF-α mRNA level was 6.6-fold higher (P = 0.037). HFD reduced Lactobacillus (75%; P<0.001) but increased Oscillibacter (279%; P = 0.004) in fecal microbiota. Correlations were found between abundances of Lactobacillus (r = 0.52; P = 0.013) and Oscillibacter (r = -0.55; P = 0.007) with transepithelial resistance of the proximal colon. HFD increased macrophage infiltration (58%; P = 0.020), TNF-α (2.5-fold, P<0.001) and IL-6 mRNA levels (2.5-fold; P = 0.008) in mesenteric fat. Increased macrophage infiltration in epididymal fat was also observed with HFD feeding (71%; P = 0.006) but neither TNF-α nor IL-6 was altered. Perirenal and subcutaneous adipose tissue showed no signs of inflammation in HFD mice. The current results implicate gut dysfunction, and attendant inflammation of contiguous adipose, as salient features of the metabolic dysregulation of diet-induced obesity.
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    Fabrication of Polypyrrole/Graphene Oxide Composite Nanosheets and Their Applications for Cr(VI) Removal in Aqueous Solution
    Li, S ; Lu, X ; Xue, Y ; Lei, J ; Zheng, T ; Wang, C ; Docoslis, A (PUBLIC LIBRARY SCIENCE, 2012-08-22)
    In this paper, we report on the simple, reliable synthesis of polypyrrole (PPy)/graphene oxide (GO) composite nanosheets by using sacrificial-template polymerization method. Herein, MnO(2) nanoslices were chosen as a sacrificial-template to deposit PPy, which served as the oxidant as well. During the polymerization of pyrrole on surface of GO nanosheets, MnO(2) component was consumed incessantly. As a result, the PPy growing on the surface of GO nanosheets has the morphology just like the MnO(2) nanoslices. This method can provide the fabrication of PPy nanostructures more easily than conventional route due to its independence of removing template, which usually is a complex and tedious experimental process. The as-prepared PPy/GO composite nanosheets exhibited an enhanced properties for Cr(VI) ions removal in aqueous solution based on the synergy effect. The adsorption capacity of the PPy/GO composite nanosheets is about two times as large as that of conventional PPy nanoparticles. We believe that our findings can open a new and effective avenue to improve the adsorption performance in removing heavy metal ions from waste water.
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    The reservoir-wave paradigm introduces error into arterial wave analysis: a computer modelling and in-vivo study
    Mynard, JP ; Penny, DJ ; Davidson, MR ; Smolich, JJ (LIPPINCOTT WILLIAMS & WILKINS, 2012-04)
    OBJECTIVES: Arterial wave reflection has traditionally been quantified from pressure and flow measurements using wave separation and wave intensity (WI) analysis. In the recently proposed reservoir-wave paradigm, these analyses are performed after dividing pressure into 'reservoir' and 'excess' components, yielding a modified wave intensity (WI(RW)). This new approach has led to controversial conclusions about the nature and significance of arterial wave reflection. Our aim was to assess whether WI or WI(RW) more accurately represent wave phenomena. METHODS: We studied two computer models (a simple network and a full model of the systemic arterial tree) in which all systolic forward waves and reflection properties were known a priori. Results of these models were compared with haemodynamic measurements in the ascending aorta of five adult sheep at baseline and after incremental arterial constriction. RESULTS: The key findings of model studies were that the reservoir-wave approach markedly underestimated or eliminated reflected compression waves, overestimated or artefactually introduced forward and backward expansion waves, and displayed nonphysical interactions between distal reflection sites and early systolic waves. These errors arose because, contrary to a key assumption of the reservoir-wave approach, reservoir pressure was not spatially uniform during systole. In-vivo results were qualitatively similar to model results, with baseline WI and WI(RW) suggesting that the arterial network was dominated by positive and negative wave reflection, respectively, while under all conditions, reflected WI(RW) compression waves were substantially smaller than corresponding WI waves. CONCLUSION: We conclude that the reservoir-wave paradigm introduces error into arterial wave analyses.
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    Molecular properties of lysozyme-microbubbles: towards the protein and nucleic acid delivery
    Melino, S ; Zhou, M ; Tortora, M ; Paci, M ; Cavalieri, F ; Ashokkumar, M (SPRINGER WIEN, 2012-08)
    Microbubbles (MBs) have specific acoustic properties that make them useful as contrast agents in ultrasound imaging. The use of the MBs in clinical practice led to the development of more sensitive imaging techniques both in cardiology and radiology. Protein-MBs are typically obtained by dispersing a gas phase in the protein solution and the protein deposited/cross-linked on the gas-liquid interface stabilizes the gas core. Innovative applications of protein-MBs prompt the investigation on the properties of MBs obtained using different proteins that are able to confer them specific properties and functionality. Recently, we have synthesized stable air-filled lysozyme-MBs (LysMBs) using high-intensity ultrasound-induced emulsification of a partly reduced lysozyme in aqueous solutions. The stability of LysMBs suspension allows for post-synthetic modification of MBs surface. In the present work, the protein folded state and the biodegradability property of LysMBs were investigated by limited proteolysis. Moreover, LysMBs were coated and functionalized with a number of biomacromolecules (proteins, polysaccharides, nucleic acids). Remarkably, LysMBs show a high DNA-binding ability and protective effects of the nucleic acids from nucleases and, further, the ability to transform the bacteria cells. These results highlight on the possibility of using LysMBs for delivery of proteins and nucleic acids in prophylactic and therapeutic applications.