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Introduction
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Publications
Publications (82)
Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed i...
Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of “pieces” of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the...
A previous study identified kartogenin (KGN) as a potent modulator of bone marrow mesenchymal stem/stromal cell (BMSC) chondrogenesis. This initial report did not contrast KGN directly against transforming growth factor-beta 1 (TGF-β1), the most common growth factor used in chondrogenic induction medium. Herein, we directly compared the in vitro ch...
Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughp...
A mathematical model was developed for mesenchymal stromal cell (MSC) growth in a packed bed bioreactor that improves oxygen availability by allowing oxygen diffusion through a gas-permeable wall. The governing equations for oxygen, glucose and lactate, the inhibitory waste product, were developed assuming Michaelis-Menten kinetics, together with a...
Oxygen concertation (mM) at the centre and wall interface with varying wall thickness at day 7.
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Predicted shear stress as a function of medium flow rate.
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Seeding efficiency of using the rocker-roller method compared to straightforward injection of the cells, allowing them to attach under static conditions, (n = 4, mean + SD).
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Mesh refinement study, difference in final average cell density (cell/cm2) at different mesh sizes (cm).
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Predicted glucose concentration (mM) using the base model.
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Predicted lactate concentration (mM) using the base model.
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Predicted glucose and oxygen concentrations for different flow rates in scaled up bioreactor model.
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Predicted oxygen concentration (mM) in reactor vessel and PDMS wall of the base model.
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Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection worldwide and the leading cause of preventable blindness. Reports have emerged of treatment failure, suggesting a need to develop new antibiotics to battle Chlamydia infection. One possible candidate for a new treatment is the protease inhibitor JO146, which is an...
While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein,...
Multimedia video is rapidly becoming mainstream, and many studies indicate that it is a more effective communication medium than text. In this project we AIM to test if videos can be used, in place of text-based grant proposals, to improve communication and increase the reliability of grant ranking. We will test if video improves reviewer comprehen...
Polydimethylsiloxane (PDMS) is the most commonly used material in the manufacture of customized cell culture devices. While there is concern that uncured PDMS oligomers may leach into culture medium and/or hydrophobic molecules may be absorbed into PDMS structures, there is no consensus on how or if PDMS influences cell behaviour. We observed that...
Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture...
Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-di...
Polydimethylsiloxane (PDMS) is the most commonly used material in the manufacture of customized cell culture devices. While there is concern that uncured PDMS oligomers may leach into culture medium and/or hydrophobic molecules may be absorbed into PDMS structures, there is no consensus on how or if PDMS influences cell behaviour. We observed that...
Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The pac...
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Isolating, purifying, and identifying proteins in complex biological matrices are often difficult, time consuming, and unreliable. Herein we describe a rapid screening technique for proteins in biological matrices that combines selective protein isolation with direct surface enhanced Raman spectroscopy (SERS) detection. Magnetic core g...
Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and...
Microwell platforms are frequently described for the efficient and uniform manufacture of 3-dimensional (3D) multicellular microtissues. Multiple partial or complete medium exchanges can displace microtissues from discrete microwells, and this can result in either the loss of microtissues from culture, or microtissue amalgamation when displaced mic...
Chlamydia (C.) trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Genetic approaches to investigate C. trachomatis have been only recently developed due to the organism’s intracellular developmental cycle. HtrA is a critical stress response serine protease and chapero...
We and others have published on the rapid manufacture of micropellet tissues, typically formed from 100-500 cells each. The micropellet geometry enhances cellular biological properties, and in many cases the micropellets can subsequently be utilized as building blocks to assemble complex macrotissues. Generally, micropellets are formed from cells a...
Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like a...
Highlights
•Ultra senstive nanosensors for the detection of bio-moleculs.
•Ultra trace and rapid selective analysis of EPO and caffiene.
•Label free SERS detection of bio-active compounds.
•Low quantification limits of 3.5×10−13 M and 1×10−10 M of EPO and caffiene.
•The EPO sensor can screen natural levels of the protein in biological fluids.
We...
Genetically diverse RNA viruses like dengue viruses (DENVs) segregate into multiple, genetically distinct, lineages that temporally arise and disappear on a regular basis. Lineage turnover may occur through multiple processes such as, stochastic or due to variations in fitness. To determine the variation of fitness, we measured the distribution of...
Introduction: Natural product provenance is important in the food, beverage and pharmaceutical industries, for consumer confidence and with health implications. Raman spectroscopy has powerful molecular fingerprint abilities. Surface Enhanced Raman Spectroscopy’s (SERS) sharp peaks allow distinction between minimally different molecules, so it shou...
Multimedia communication capabilities are rapidly expanding, and visual information is easily shared electronically, yet funding bodies still rely on paper grant proposal submissions. Incorporating modern technologies will streamline the granting process by increasing the fidelity of grant communication, improving the efficiency of review, and redu...
Type 2 diabetes is the epidemic of our generation, and diabetic foot ulcers (DFUs) are a major complication. Although DFU formation itself can indicate disease progression, the failure to effectively treat ulcers contributes further to a decay in patient quality of life and increased mortality. Herein we discuss the development of next-generation D...
The format of grant applications should be updated to incorporate multimedia video. This would help researchers to convey complex topics to grant-review panels.
If time-poor research panels cannot quickly grasp the scientific ideas presented in a paper application, other factors, such as author affiliations and track records, may disproportionatel...
Recent literature suggests that mesenchymal stem/stromal cells (MSC) could be used as Trojan Horses to deliver “death-signals” to cancer cells. Herein, we describe the development of a novel multichannel cell migration device, and use it to investigate the relative migration rates of bone marrow-derived MSC and breast cancer cells (MCF-7) towards e...
Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality...
We have developed an explanation for ultra trace detection found when using Au/Ag SERS nanoparticles linked to biochemical affinity tags, e.g. antibodies. The nanoparticle structure is not as usually assumed and the aggregated nanoparticles constitute hot spots that are indispensable for these very low levels of analyte detection, even more so when...
Here we report an ultrasensitive method for detecting bio-active compounds in biological samples by means of functionalised nanoparticles interrogated by surface enhanced Raman spectroscopy (SERS). This method is applicable to the recovery and detection of many diagnostically important peptidyl analytes such as insulin, human growth hormone, growth...
Complementary sequences at the 5' and 3' ends of the dengue virus RNA genome are essential for viral replication, and are believed to cyclise the genome through long-range base pairing in cis. Although consistent with evidence in the literature, this view neglects possible biologically active multimeric forms that are equally consistent with the da...
Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×10(5)-5×10(5) chondrocytes are aggregated, resulting in "macro" pellets ha...
Primers used for gene expression analysis.
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Surface modification testing. To assess the stability and functionality of the surface modification after incubation in ethanol and PBS, a testing platform was set up as follows: flat 24 well plate PDMS disks were produced and half of them were multilayered as explained in the Materials and Methods section. The disks were incubated under conditions...
Mass production of PhD training compromises graduate quality. As PhD quality becomes more stratified, industry will increasingly turn to quality-branded institutions and programs when distinguishing among job candidates.
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Hepatitis C virus (HCV) core (C) protein is thought to bind to viral RNA before it undergoes oligomerization leading to RNA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal C protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site (IRES). This nucleotide seq...
Results of protease assays using the REPLI Library all wells which has detectable substrates for CtHtrA are shown on the graph. The relative rate of fluorescent accumulation per min per ug of CtHtrA is shown on the Y axis. The well descriptions are indicated on the x axis. The wells contain 4–8 peptides with similar functional residues grouped, wit...
Primers and amplification conditions used for site directed mutagenesis PCR.
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A. Weblog of the PICS results. B. A full listing of all PICs data (listed below).
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Characterisation of Bcas1 cleavage by CtHtrA. A. LC/MS plot of Bcas1 alone. B. B LC/MS plot of Bcas1 cleaved with CtHtrA shows addition of ions compared to Bcas1 alone. C. MS/MS of product ion 552.37 m/z, 26.3 min (circle in B) matches internal fragment of Bcas1.
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Characterisation of protease specificity in the presence of the activator peptide using PICS. A. Web Logo summary of all peptide cleavage sites identified during PICS protocol with CtHtrA and Act1. B. The table below lists all peptide cleavage sites identified during PICS protocol with CtHtrA and Act1.
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DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer...
Northern blot analysis of RNA from DENV 2 isolate (61452) and from C6-36 cells infected with this virus. (A) Blots probed with a DIG-labelled probe complementary to the DENV2 5′1-206 nt (5′-probe). Gen., genome length RNA transcribed from DENV 2 infectious clone pWSK601; SG, 290 nt sub-genomic DENV 2 RNA transcribed from plasmid pGEM-D2-DI; UC, cel...
Sub-genomic RNA detected in sera from dengue patients.
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Sequences of oligonucleotide primers employed in this study.
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While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been...
Infectious cDNA clones of RNA viruses are important research tools, but flavivirus cDNA clones have proven difficult to assemble and propagate in bacteria. This has been attributed to genetic instability and/or host cell toxicity, however the mechanism leading to these difficulties has not been fully elucidated. Here we identify and characterize an...
Serum ferritin and hepatic iron concentrations are frequently elevated in patients who are chronically infected with the hepatitis C virus (HCV), and hepatic iron concentration has been used to predict response to interferon therapy, but these correlations are not well understood. The HCV genome contains an RNA structure resembling an iron responsi...
Several studies have generated computer-based predictions of secondary structure of the 3' untranslated region (UTR) of Dengue virus (DEN); however, experimental verification of the formation of these structures in vitro is lacking. This study assessed the congruence of Mfold predictions of secondary structure of the core region of the DEN type 4 3...
To investigate the role of the hepatitis C virus internal ribosome entry site (HCV IRES) domain IV in translation initiation
and regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and classical
swine fever virus IRES elements. This permitted an examination of the role of domain IV in the...
A self-modulating mechanism by the hepatitis C virus (HCV) core protein has been suggested to influence the level of HCV replication, but current data on this subject are contradictory. We examined the effect of wild-type and mutated core protein on HCV IRES- and cap-dependent translation. The wild-type core protein was shown to inhibit both IRES-...
The effect of cyanocobalamin (CNCbl, vitamin B12) on hepatitis C virus internal ribosome entry site (HCV IRES)-dependent initiation of translation was studied by ribosomal toeprinting and sucrose gradient centrifugation analysis. These results suggested that CNCbl did not inhibit HCV IRES-dependent translation by a competitive binding mechanism. CN...
Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis
of negative-strand strong-stop DNA [(−)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors
affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevir...
Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly af...
An assay for the bovine viral diarrhoea virus (BVDV) replicase was developed using extracts from BVDV-infected cells. The replicase activity was maximal approximately 8 h post-infection as measured by the generation of a genomic length radiolabelled RNA. Using a semi-denaturing gel system, three virus-specific in vitro radiolabelled nascent RNA spe...
The rates of hydrolysis of benzoannelated vs nonbenzoannelated sulfuranes, viz. 5 vs 3 or 5 vs 4, were compared. Benzoannelation was found to provide very modest kinetic stabilization. Crystal structures of sulfuranes 3 and 4 were obtained and compared with each other, with a dibenzoannelated sulfurane, 17, and with a non-sulfurane analogue of 4. B...
Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrat...
Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium iso...
The radical pair that results from photolysis of adenosylcob(III)alamin (AdoCb1(III)) undergoes primary geminate recombination with a first-order rate constant of 1 x 10(9) s(-1). In contrast, methylcob(III)alamin (CH(3)Cb1(III)) and aristeromicylcob(III)alamin (AriCb1(III), the carbocyclic analogue of AdoCb1(III) in which the ribofuranose ring oxy...
The rate of singlet-to-triplet intersystem crossing in 1,4-didehydrobenzene (the biradical produced as a reactive intermediate in the thermal cycloaromatization of enediynes), cannot be increased by the application of an external magnetic field. The rate of product formation and the distribution of stable products of 2,3-di--propyl-1,4-didehydroben...
Thesis (Ph. D.)--Dept. of Chemistry, University of Utah, 1995. Includes bibliographical references.
