Ross Barnard

Comparison of Competitively Primed and Conventional Allele-Specific Nucleic Acid Amplification

Publisher: Elsevier BV

Date: 05-2001

DOI: 10.1006/ABIO.2001.5077

Development of an Oligonucleotide-Based SNP Detection Method on Lateral Flow Strips Using Hexapet Tags

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 09-2005

DOI: 10.1097/01.POC.0000178265.83599.83

A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus.

Publisher: Springer Science and Business Media LLC

Date: 09-07-2008

DOI: 10.1186/1743-422X-5-77

Abstract: The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and lification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes. Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region lified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y. Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.

The Tortoise and the Hare: Evolving Regulatory Landscapes for Biosimilars

Publisher: Elsevier BV

Date: 2016

DOI: 10.1016/J.TIBTECH.2015.10.009

Abstract: Challenges in demonstrating interchangeability and safety, as well as the ongoing evolution of regulations governing biosimilars, have meant that the development of the biosimilars industry has not been, and will not be, a carbon copy of the generics industry. Complexity in the development process reduces the cost advantages for biosimilars that generics offer over originators. There has been a marked difference in the number of biosimilars approved by the European Medicines Agency (EMA) and US FDA due to a lack of consensus and the different rates of progress in establishing both law and stable evidence-based regulatory guidelines for biosimilars. In this review, we provide a précis of the history and status of the regulatory regimes in the USA and Europe. Included is an assessment of market and nonmarket factors that may continue to influence the development of the biosimilars industry.

Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos: A role for GH in early embryogenesis?

Publisher: Proceedings of the National Academy of Sciences

Date: 13-05-1997

DOI: 10.1073/PNAS.94.10.5125

Abstract: The results of this study challenge the widely held view that growth hormone (GH) acts only during the postnatal period. RNA phenotyping shows transcripts for the GH receptor and GH-binding protein in mouse preimplantation embryos of all stages from fertilized eggs (day 1) to blastocysts (day 4). An antibody specific to the cytoplasmic region of the GH receptor revealed receptor protein expression, first in two-cell embryos, the stage of activation of the embryonic genome (day 2), and in all subsequent stages. In cleavage-stage embryos this immunoreactivity was localized mainly to the nucleus, but clear evidence of membrane labeling was apparent in blastocysts. GH receptor immunoreactivity was also observed in cumulus cells associated with unfertilized oocytes but not in the unfertilized oocytes. The blastocyst receptor was demonstrated to be functional, exhibiting the classic bell-shaped dose–response curves for GH stimulation of both 3- O -methyl glucose transport and protein synthesis. Maximal stimulation of 40–50% was seen for both responses at less than 1 ng/ml recombinant GH, suggesting a role for maternal GH. However mRNA transcripts for GH were also detected from the morula stage (day 3) by using reverse transcription–PCR, and GH immunoreactivity was seen in blastocysts. These observations raise the possibility of a paracrine/autocrine GH loop regulating embryonic development in its earliest stages.

Antiinflammatory and Anticancer Properties of Grewia asiatica Crude Extracts and Fractions: A Bioassay-Guided Approach

Publisher: Hindawi Limited

Date: 28-03-2022

DOI: 10.1155/2022/2277417

Abstract: The study was an extension of our earlier work on antiinflammatory and anticancer properties of G. asiatica fruit. We aimed to develop a bioassay guided multistep purification technique for producing bioactive fractions of G. asiatica crude extracts. Dried fruit powder was sequentially fractionated with 100% dichloromethane, 100% methanol (MeOH), and 50% MeOH. Active extracts were subjected to liquid-liquid partitioning followed by subfractionation using RP-HPLC. Antioxidant, antiinflammatory, and anticancer activities of the fruit extracts, and their potent fractions were evaluated in vitro, while identification of compounds from the bioactive fractions was performed by ESI-MS/MS analysis. The amount of the identified compounds present was confirmed using external standards adopting a simple, accurate, and rapid analytical HPLC method. The results showed that 100% and 50% MeOH extracts possessed bioactivity one of which (the 50% MeOH extract) displayed potent activity in all in vitro bioassays. MeOH extract (50%) derived fraction C and hydroalcoholic fraction 5 (GAHAF5) were observed to possess higher antioxidant, antiinflammatory, and in vitro anticancer activity. IC50 of GAHAF5 against MCF-7, HEp-2, and NCI-H522 cancer cells was recorded as 26.2, 51.4, and 63 μg/mL, respectively. ESI-MS/MS and HPLC analysis identified catechin, chlorogenic acid, caffeic acid, and morin as potential bioactive compounds in the GAHAF5 fraction with concentrations of 1230, 491, 957, and 130 μg/g, respectively. The findings indicated that G. asiatica bioactive fractions possessed antiinflammatory activity in vitro and were cytotoxic against breast cancer, lung cancer, and laryngeal cancer cell lines.

Interdisciplinary science research and education

Publisher: Informa UK Limited

Date: 06-2013

DOI: 10.1080/07294360.2012.686482

Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation

Publisher: Springer Science and Business Media LLC

Date: 19-08-2016

DOI: 10.1038/LEU.2015.231

Abstract: Current immunosuppressive/anti-inflammatory agents target the responding effector arm of the immune response and their nonspecific action increases the risk of infection and malignancy. These effects impact on their use in allogeneic haematopoietic cell transplantation and other forms of transplantation. Interventions that target activated dendritic cells (DCs) have the potential to suppress the induction of undesired immune responses (for ex le, graft versus host disease (GVHD) or transplant rejection) and to leave protective T-cell immune responses intact (for ex le, cytomegalovirus (CMV) immunity). We developed a human IgG1 monoclonal antibody (mAb), 3C12, specific for CD83, which is expressed on activated but not resting DC. The 3C12 mAb and an affinity improved version, 3C12C, depleted CD83(+) cells by CD16(+) NK cell-mediated antibody-dependent cellular cytotoxicity, and inhibited allogeneic T-cell proliferation in vitro. A single dose of 3C12C prevented human peripheral blood mononuclear cell-induced acute GVHD in SCID mouse recipients. The mAb 3C12C depleted CMRF-44(+)CD83(bright) activated DC but spared CD83(dim/-) DC in vivo. It reduced human T-cell activation in vivo and maintained the proportion of CD4(+) FoxP3(+) CD25(+) Treg cells and also viral-specific CD8(+) T cells. The anti-CD83 mAb, 3C12C, merits further evaluation as a new immunosuppressive agent in transplantation.

Development of a Multiplexed Microsphere PCR for Culture-Free Detection and Gram-Typing of Bacteria in Human Blood Samples

Publisher: American Chemical Society (ACS)

Date: 19-01-2018

DOI: 10.1021/ACSINFECDIS.7B00277

Abstract: Bloodstream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected bloodstream infection remain centered around blood culture (highly variable timing, in the order of hours to days to become positive), and empiric use of broad-spectrum antibiotics is therefore employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g., combinations containing vancomycin or teicoplanin for Gram-positives piperacillin-tazobactam or a carbapenem for Gram-negatives) however, current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Gram-positive from Gram-negative bacterial DNA in a 3.5 h time period. The combination of a simple assay design ( licon-dependent release of Gram-type specific Cy3-labeled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labeled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Publisher: Public Library of Science (PLoS)

Date: 24-10-2022

DOI: 10.1371/JOURNAL.PCBI.1010633

Abstract: Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

A novel insect-specific flavivirus replicates only in Aedes-derived cells and persists at high prevalence in wild Aedes vigilax populations in Sydney, Australia

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.VIROL.2015.07.021

Abstract: To date, insect-specific flaviviruses (ISFs) have only been isolated from mosquitoes and increasing evidence suggests that ISFs may affect the transmission of pathogenic flaviviruses. To investigate the ersity and prevalence of ISFs in Australian mosquitoes, s les from various regions were screened for flaviviruses by ELISA and RT-PCR. Thirty-eight pools of Aedes vigilax from Sydney in 2007 yielded isolates of a novel flavivirus, named Parramatta River virus (PaRV). Sequencing of the viral RNA genome revealed it was closely related to Hanko virus with 62.3% nucleotide identity over the open reading frame. PaRV failed to grow in vertebrate cells, with only Aedes-derived mosquito cell lines permissive to replication, suggesting a narrow host range. 2014 collections revealed that PaRV had persisted in A. vigilax populations in Sydney, with 88% of pools positive. Further investigations into its mode of transmission and potential to influence vector competence of A. vigilax for pathogenic viruses are warranted.

Gene expression phenotypes for lipid metabolism and intramuscular fat in skeletal muscle of cattle

Publisher: Oxford University Press (OUP)

Date: 03-2013

DOI: 10.2527/JAS.2012-5409

Abstract: Gene expression phenotypes were evaluated for intramuscular fat (IMF) in bovine skeletal muscle as an alternative to traditional estimates of IMF%. Gene expression data from a time course of LM development in high- and low-marbling Bos taurus cattle crosses were compared to identify genes involved in intramuscular adipocyte lipid metabolism with developmentally similar gene expression profiles. Three sets of genes were identified: triacylglyceride (TAG) synthesis and storage, fatty acid (FA) synthesis, and PPARγ-related genes. In an independent analysis in the LM of 48 Bos indicus cattle, TAG and FA gene sets were enriched in the top 100 genes of which expression was most correlated with IMF% (P = 1.2 × 10(-24) and 3.5 × 10(-9), respectively). In general, genes encoding enzymes involved in the synthesis of FA and TAG in the intramuscular adipocytes were present in the top 100 genes. In B. indicus, effects of a steroid hormone growth promotant (HGP), 2 experimental sites [New South Wales (NSW) and Western Australia (WA)], and 3 tenderness genotypes on the expression levels of genes in the TAG gene set and the correlation of gene expression with IMF% were investigated. Although correlation between expression of 12 in idual TAG genes and IMF% was observed in HGP-treated animals in both experimental sites (mean r = 0.43), correlation was not observed for untreated animals at the NSW site (mean r = -0.07, P < 3 × 10(-6)). However, TAG genes showed an average 1.6-fold (P 0.05) on the correlation of TAG genes with IMF%. In general, the interactions among genotype, treatment and location, and TAG gene set gene expression were consistent with the interactions among the same factors and IMF% detected using 255 animals, of which the 48 in this study were a subset. Thus, the TAG gene set constitutes a gene expression phenotype able to predict effects of different genotypes and treatments on IMF% using much smaller groups than current approaches, even in animals with very low IMF%.

Application of the PrimRglo Assay Chemistry to Multiplexed Bead Assays

Publisher: Wiley

Date: 07-2014

DOI: 10.1002/0471142956.CY1313S69

Abstract: In this unit, we describe a multiplex microsphere quantitative PCR. The system is based on the use of two additional oligonucleotides within a single tube PCR reaction. The first oligonucleotide is modified with a single base pair mismatch and is otherwise equivalent to a universal sequence added to the forward PCR primer. Further, this first extra oligonucleotide is coupled to Luminex microspheres. The second additional oligonucleotide is designed to be complementary to the universal sequence, and is modified with the fluorescent dye Cy3. As the PCR reaction proceeds, the second oligonucleotide is able to bind to the microspheres. Thus, quantitative monitoring of PCR progress takes place. The microsphere-mediated Cy3-detection is measured using flow cytometry directly after the PCR reaction. This allows a flow cytometer analysis from up to 150 different spheres and, therefore, multiple genes in one reaction. The multiplex microsphere qPCR is demonstrated using three target genes from Influenza A and Neisseria meningitidis. The multiplex microsphere system will enable a higher degree of multiplexing than is possible with currently available qPCR systems.

Signal Transduction by the Growth Hormone Receptor

Publisher: SAGE Publications

Date: 07-1994

DOI: 10.3181/00379727-206-43745

Abstract: It has been proposed that dimerization of identical receptor subunits by growth hormone (GH) is the mechanism of signal transduction across the cell membrane. We present here data with analogs of porcine GH (pGH), with GH receptors (GHR) mutated in the dimerization domain and with monoclonal antibodies to the GHR which indicate that dimerization is necessary but not sufficient for transduction. We also report nuclear uptake of GH both in vivo and in vitro, along with nuclear localization of the receptor and GH-binding protein (GHBP). This suggests that GH acts directly at the nucleus, and one possible target for this action is a rapid increase in transcription of C/EBP delta seen in 3T3-F442A cells in response to GH. This tyrosine kinase-dependent event may be an archetype for induction of other immediate early gene transcription factors which then interact to determine the programming of the subsequent transcriptional response to GH.

The first disulphide loop of the rabbit growth hormone receptor is required for binding to the hormone.

Publisher: Bioscientifica

Date: 12-1992

DOI: 10.1677/JME.0.0090213

Abstract: Residues within the first disulphide loop of the GH receptor are highly conserved, and the two cysteines forming this motif are conserved across many cytokine receptors. We have used site-directed mutagenesis and the polymerase chain reaction with splicing by overlap extension to show that these residues are essential for binding of bovine (b)GH and human (h)GH to the rabbit GH receptor. When all residues within this loop were replaced with an equivalent polyalanine sequence, hormone binding was abolished. Conversion of Arg 39 within the loop to aspartate (R39D) decreased affinity for bGH by up to 20-fold. Conversion of Glu 42 to lysine (E42K) also resulted in a fivefold loss of affinity for bGH. However, charge reversals at Lys 37, Glu 44 and the conversion of Leu 43 to an arginine (as in the human receptor) were without a major effect on bGH binding. The lack of effect of the L43R mutation on bGH affinity, despite a significant (threefold) decrease in hGH affinity, argues against a role for Arg 43 as a residue conferring primate GH-binding specificity on the human receptor. Examination of the affinities of poly Ala, R39D and E42K mutants for a variety of hormone-binding-site directed and other monoclonal antibodies (MAbs) to the GH receptor revealed that these mutations were without a major effect on tertiary structure. It is of interest that the epitopes for the hormone-binding inhibitory MAbs 263 and 7 are located within this first loop, since the poly Ala mutation abolished the binding of both MAbs, and the R39D mutation abolished binding of MAb 7. In conclusion, residues within the first disulphide loop of the GH receptor are critical for hormone binding, and are epitopes for the binding of inhibitory MAbs. These findings may be applicable to other cytokine receptors.

Chronic exposure to anabolic steroids induces the muscle expression of oxytocin and a more than fifty fold increase in circulating oxytocin in cattle

Publisher: American Physiological Society

Date: 05-2011

DOI: 10.1152/PHYSIOLGENOMICS.00226.2010

Abstract: Molecular mechanisms in skeletal muscle associated with anabolic steroid treatment of cattle are unclear and we aimed to characterize transcriptional changes. Cattle were chronically exposed (68 ± 20 days) to a steroid hormone implant containing 200 mg trenbolone acetate and 20 mg estradiol (Revalor-H). Biopsy s les from 48 cattle (half treated) from longissimus dorsi (LD) muscle under local anesthesia were collected. Gene expression levels were profiled by microarray, covering 16,944 unique bovine genes: 121 genes were differentially expressed (DE) due to the implant (99.99% posterior probability of not being false positives). Among DE genes, a decrease in expression of a number of fat metabolism-associated genes, likely reflecting the lipid storage activity of intramuscular adipocytes, was observed. The expression of IGF1 and genes related to the extracellular matrix, slow twitch fibers, and cell cycle (including SOX8, a satellite cell marker) was increased in the treated muscle. Unexpectedly, a very large 21- (microarray) to 97 (real time quantitative PCR)-fold higher expression of the mRNA encoding the neuropeptide hormone oxytocin was observed in treated muscle. We also observed an ∼50-fold higher level of circulating oxytocin in the plasma of treated animals at the time of biopsy. Using a coexpression network strategy OXTR was identified as more likely than IGF1R to be a major mediator of the muscle response to Revalor-H. A re-investigation of in vivo cattle LD muscle s les during early to mid-fetal development identified a -fold increased expression of OXT, coincident with myofiber differentiation and fusion. We propose that oxytocin may be involved in mediating the anabolic effects of Revalor-H treatment.

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Publisher: Cold Spring Harbor Laboratory

Date: 31-12-2019

DOI: 10.1101/2019.12.30.891457

Abstract: Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (“indel”) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time. Massive sequencing projects expose the extent of natural, genetic ersity. Here, we describe a method with capacity to perform ancestor sequence reconstruction from data sets in excess of 10,000 sequences, poised to recover ancestral ersity, including the evolutionary events that determine present-time biological function and structure. We introduce a novel strategy for suggesting “indel variants” that are distinct from, but can be explored alongside, substitution variants for creating ancestral libraries. We demonstrate how indels can be used as building blocks to form “hybrid ancestors” based on this strategy, we synthesise ancestor variants, with varying enzymatic activities, for wide-ranging applications in the biotechnology sector.

Growth hormone binding protein in normal and aneuploid pregnancy: A paradoxical decrease in trisomy 18

Publisher: Elsevier BV

Date: 07-2001

DOI: 10.1016/S0306-5456(00)00179-0

Commensal viruses of mosquitoes: Host restriction, transmission, and interaction with arboviral pathogens

Publisher: SAGE Publications

Date: 2016

DOI: 10.4137/EBO.S40740

Abstract: Recent advances in virus detection strategies and deep sequencing technologies have enabled the identification of a multitude of new viruses that persistently infect mosquitoes but do not infect vertebrates. These are usually referred to as insect-specific viruses (ISVs). These novel viruses have generated considerable interest in their modes of transmission, persistence in mosquito populations, the mechanisms that restrict their host range to mosquitoes, and their interactions with pathogens transmissible by the same mosquito. In this article, we discuss studies in our laboratory and others that demonstrate that many ISVs are efficiently transmitted directly from the female mosquito to their progeny via infected eggs, and, moreover, that persistent infection of mosquito cell cultures or whole mosquitoes with ISVs can restrict subsequent infection, replication, and transmission of some mosquito-borne viral pathogens. This suggests that some ISVs may act as natural regulators of arboviral transmission. We also discuss viral and host factors that may be responsible for their host restriction.

Bcl-2 genes and growth factors in the pathology of ischaemic acute renal failure

Publisher: Wiley

Date: 06-1999

DOI: 10.1046/J.1440-1711.1999.00826.X

Abstract: For the past decade, an attempt has been made by many research groups to define the roles of the growing number of Bcl-2 gene family proteins in the apoptotic process. The Bcl-2 family consists of pro-apoptotic (or cell death) and anti-apoptotic (or cell survival) genes and it is the balance in expression between these gene lineages that may determine the death or survival of a cell. The majority of studies have analysed the role/s of the Bcl-2 genes in cancer development. Equally important is their role in normal tissue development, homeostasis and non-cancer disease states. Bcl-2 is crucial for normal development in the kidney, with a deficiency in Bcl-2 producing such malformation that renal failure and death result. As a corollary, its role in renal disease states in the adult has been sought. Ischaemia is one of the most common causes of both acute and chronic renal failure. The section of the kidney that is most susceptible to ischaemic damage is the outer zone of the outer medulla. Within this zone the proximal tubules are most sensitive and often die by necrosis or desquamate. In the distal nephron, apoptosis is the more common form of cell death. Recent results from our laboratory have indicated that ischaemia-induced acute renal failure is associated with up-regulation of two anti-apoptotic Bcl-2 proteins (Bcl-2 and Bcl-XL) in the damaged distal tubule and occasional up-regulation of Bax in the proximal tubule. The distal tubule is a known reservoir for several growth factors important to renal growth and repair, such as insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). One of the likely possibilities for the anti-cell death action of the Bcl-2 genes is that the protected distal cells may be able to produce growth factors that have a further reparative or protective role via an autocrine mechanism in the distal segment and a paracrine mechanism in the proximal cells. Both EGF and IGF-1 are also up-regulated in the surviving distal tubules and are detected in the surviving proximal tubules, where these growth factors are not usually synthesized. As a result, we have been using in vitro methods to test: (i) the relative sensitivities of renal distal and proximal epithelial cell populations to injury caused by mechanisms known to act in ischaemia-reperfusion (ii) whether a Bcl-2 anti-apoptotic mechanism acts in these cells and (iii) whether an autocrine and/or paracrine growth factor mechanism is initiated. The following review discusses the background to these studies as well as some of our preliminary results.

Characterization of the banana streak virus capsid protein and mapping of the immunodominant continuous B-cell epitopes to the surface-exposed N terminus

Publisher: Microbiology Society

Date: 16-12-2016

DOI: 10.1099/JGV.0.000643

Abstract: This study identified the structural proteins of two badnavirus species, Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV), and mapped the distribution of continuous B-cell epitopes. Two different capsid protein (CP) isoforms of about 44 and 40 kDa (CP1 and CP2) and the virion-associated protein (VAP) were consistently associated with purified virions. For both viral species, the N terminus of CP2 was successfully sequenced by Edman degradation but that of CP1 was chemically blocked. De novo peptide sequencing of tryptic digests suggested that CP1 and CP2 derive from the same region of the P3 polyprotein but differ in the length of either the N or the C terminus. A three-dimensional model of the BSMYV-CP was constructed, which showed that the CP is a multi-domain structure, containing homologues of the retroviral capsid and nucleocapsid proteins, as well as a third, intrinsically disordered protein region at the N terminus, henceforth called the NID domain. Using the Pepscan approach, the immunodominant continuous epitopes were mapped to the NID domain for five different species of banana streak virus. Anti-peptide antibodies raised against these epitopes in BSMYV were successfully used for detection of native virions and denatured CPs in serological assays. Immunoelectron microscopy analysis of the virion surface using the anti-peptide antibodies confirmed that the NID domain is exposed on the surface of virions, and that the difference in mass of the two CP isoforms is due to variation in length of the NID domain.

A New Clade of Insect-Specific Flaviviruses from Australian Anopheles Mosquitoes Displays Species-Specific Host Restriction

Publisher: American Society for Microbiology

Date: 30-08-2017

DOI: 10.1128/MSPHERE.00262-17

Abstract: Flaviviruses like dengue, Zika, or West Nile virus infect millions of people each year and are transmitted to humans via infected-mosquito bites. A subset of flaviviruses can only replicate in the mosquito host, and recent studies have shown that some can interfere with pathogenic flaviviruses in mosquitoes and limit the replication and transmission of the latter. The insect-specific flaviviruses (ISFs) reported here form a new Anopheles mosquito-associated clade separate from the Aedes - and Culex -associated ISF clades. The identification of distinct clades for each mosquito genus provides new insights into the evolution and ecology of flaviviruses. One of these viruses was shown to replicate in the midgut of the mosquito host and exhibit the most specialized host restriction reported to date for ISFs. Understanding this unprecedented host restriction in ISFs could help identify the mechanisms involved in the evolution of flaviviruses and their emergence as mosquito-borne pathogens.

Evolution of the magic bullet: Single chain antibody fragments for the targeted delivery of immunomodulatory proteins

Publisher: SAGE Publications

Date: 19-12-2017

DOI: 10.1177/1535370217748575

Abstract: Immunocytokines are fusion proteins that combine the specific antigen binding capacities of an antibody or derivative thereof and the potent bioactivity of a cytokine partner. These novel biopharmaceuticals have been directed to various targets of oncological as well as non-oncological origin and a handful of promising constructs are currently advancing in the clinical trial pipeline. Several factors such as the choice of a disease specific antigen, the antibody format and the modulatory nature of the payload are crucial, not only for therapeutic efficacy and safety but also for the commercial success of such a product. In this review, we provide an overview of the basic principles and obstacles in immunocytokine design with a specific focus on single chain antibody fragment-based constructs that employ interleukins as the immunoactive component. Selective activation of the immune system in a variety of malignancies represents an attractive approach when existing strategies have failed to provide adequate treatment options. Immunocytokines as a novel class of bifunctional protein therapeutics have emerged recently and generated promising results in preclinical and clinical studies. In order to harness their full potential, multiple different aspects have to be taken into consideration. Several key points of these fusion constructs are discussed here and should provide an outline for the development of novel products based on an overview of selected formats.

Syzygium cumini(L.),Skeels fruit extracts: In vitro and in vivo anti-inflammatory properties

Publisher: Elsevier BV

Date: 05-2021

DOI: 10.1016/J.JEP.2021.113805

In silico molecular dynamics of human glycophorin A (GPA) extracellular structure

Publisher: AME Publishing Company

Date: 06-2021

DOI: 10.21037/AOB-20-51

Molecular Phylogeny of Edge Hill Virus Supports its Position in the Yellow Fever Virus Group and Identifies a New Genetic Variant

Publisher: SAGE Publications

Date: 2010

DOI: 10.4137/EBO.S4966

Abstract: Edge Hill virus (EHV) is a mosquito-borne flavivirus isolated throughout Australia during mosquito surveillance programs. While not posing an immediate threat to the human population, EHV is a taxonomically interesting flavivirus since it remains the only member of the yellow fever virus (YFV) sub-group to be detected within Australia. Here we present both an antigenic and genetic investigation of collected isolates, and confirm taxonomic classification of the virus within the YFV-group. Isolates were not clustered based on geographical origin or time of isolation, suggesting that minimal genetic evolution of EHV has occurred over geographic distance or time within the EHV cluster. However, two isolates showed significant differences in antigenic reactivity patterns, and had a much larger ergence from the EHV prototype (19% nucleotide and 6% amino acid ergence), indicating a distinct subtype or variant within the EHV subgroup.

Antimicrobial Silver in Medicinal and Consumer Applications: A Patent Review of the Past Decade (2007–2017)

Publisher: MDPI AG

Date: 26-10-2018

DOI: 10.3390/ANTIBIOTICS7040093

Abstract: The use of silver to control infections was common in ancient civilizations. In recent years, this material has resurfaced as a therapeutic option due to the increasing prevalence of bacterial resistance to antimicrobials. This renewed interest has prompted researchers to investigate how the antimicrobial properties of silver might be enhanced, thus broadening the possibilities for antimicrobial applications. This review presents a compilation of patented products utilizing any forms of silver for its bactericidal actions in the decade 2007–2017. It analyses the trends in patent applications related to different forms of silver and their use for antimicrobial purposes. Based on the retrospective view of registered patents, statements of prognosis are also presented with a view to heightening awareness of potential industrial and health care applications.

Human Blood CD1c+ Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4+ T Cells

Publisher: Frontiers Media SA

Date: 17-08-2017

DOI: 10.3389/FIMMU.2017.00971

Effects of magnesium deficiency – More than skin deep

Publisher: SAGE Publications

Date: 13-06-2014

DOI: 10.1177/1535370214537745

Abstract: Dead Sea and magnesium salt therapy are two of the oldest forms of treatment for skin disease and several other disorders, supported by a body of largely anecdotal evidence. In this paper we review possible pathways for penetration of magnesium ions through the epidermis to reach the circulation, in turn replenishing cellular magnesium levels. We also discuss mechanisms for intercellular movement of magnesium ions and possible mechanisms for the interaction between magnesium ions and inflammatory mediators. Upon addition of magnesium ions in vitro, the expression of inflammatory mediators such as tumour necrosis factor α (TNFα) and nuclear factor κβ (NFκβ) is down regulated. Dysregulation of these and other inflammatory mediators has been linked to several inflammatory disorders, including asthma, arthritis, atherosclerosis and neuroinflammation.

Multiplex–microsphere–quantitative polymerase chain reaction: Nucleic acid amplification and detection on microspheres

Publisher: Elsevier BV

Date: 2013

DOI: 10.1016/J.AB.2012.09.017

Abstract: We report the development of a new system to monitor the lification of nucleic acids on microspheres. This was realized by the design of (i) a "universal" oligonucleotide "tagged" polymerase chain reaction (PCR) forward primer, (ii) a sensor sequence complementary to the universal sequence on the forward PCR primer modified with a fluorescent dye, and (iii) a universal oligonucleotide coupled to Luminex microspheres. The PCR takes place with the microspheres present in the reaction tube. With the consumption of the universal oligonucleotide tagged forward primer, the fluorescently labeled sequences can bind to the universal oligonucleotide on the microspheres. We tested the microsphere quantitative PCR system with up to three different target genes (Neisseria meningitides porA and ctrA and influenza A M gene segment) as templates in a single PCR tube. The analytical sensitivity of this quantitative PCR system was tested and compared with the TaqMan system. The multiplex-microsphere-quantitative PCR system does not require design of unique internal probes for each target and has potential for a high degree of multiplexing, greater than the limited multiplexing achievable with current real-time protocols.

The interaction between Glycophorin A (GPA) and Band 3 in the formation of the Wright b (Wr^b) antigen

Publisher: Wiley

Date: 18-12-2020

DOI: 10.1111/VOX.13055

Detection of Influenza A Virus Neuraminidase and PB2 Gene Segments by One Step Reverse Transcription Polymerase Chain Reaction

Publisher: Humana Press

Date: 2010

DOI: 10.1007/978-1-60761-629-0_5

Abstract: We describe a single step reverse transcription polymerase chain reaction protocol that can be used to lify part of the neuraminidase gene segment (segment 6) from all nine subtypes of influenza A virus. The method has also been applied to lify gene segment 1 of influenza A, which encodes the basic polymerase protein 2 (PB2). The method combines the use of mixed base primers with a "touchdown" thermal cycling program and is applicable to a wide range of nucleic acid targets in which there is genetic variability in the regions complementary to the PCR primers.

Genomic characterisation of Wongabel virus reveals novel genes within the Rhabdoviridae.

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1016/J.VIROL.2008.03.004

Abstract: Viruses belonging to the family Rhabdoviridae infect a variety of different hosts, including insects, vertebrates and plants. Currently, there are approximately 200 ICTV-recognised rhabdoviruses isolated around the world. However, the majority remain poorly characterised and only a fraction have been definitively assigned to genera. The genomic and transcriptional complexity displayed by several of the characterised rhabdoviruses indicates large ersity and complexity within this family. To enable an improved taxonomic understanding of this family, it is necessary to gain further information about the poorly characterised members of this family. Here we present the complete genome sequence and predicted transcription strategy of Wongabel virus (WONV), a previously uncharacterised rhabdovirus isolated from biting midges (Culicoides austropalpalis) collected in northern Queensland, Australia. The 13,196 nucleotide genome of WONV encodes five typical rhabdovirus genes N, P, M, G and L. In addition, the WONV genome contains three genes located between the P and M genes (U1, U2, U3) and two open reading frames overlapping with the N and G genes (U4, U5). These five additional genes and their putative protein products appear to be novel, and their functions are unknown. Predictive analysis of the U5 gene product revealed characteristics typical of viroporins, and indicated structural similarities with the alpha-1 protein (putative viroporin) of viruses in the genus Ephemerovirus. Phylogenetic analyses of the N and G proteins of WONV indicated closest similarity with the avian-associated Flanders virus however, the genomes of these two viruses are significantly erged. WONV displays a novel and unique genome structure that has not previously been described for any animal rhabdovirus.

Chapter 2 Complexity and mastery in shaping interdisciplinarity

Publisher: Emerald Group Publishing Limited

Date: 08-11-2010

DOI: 10.1108/S1479-3628(2010)0000005005

PrimRglo: A multiplexable quantitative real-time polymerase chain reaction system for nucleic acid detection

Publisher: Elsevier BV

Date: 03-2012

DOI: 10.1016/J.AB.2011.12.038

Abstract: We report the development of a new real-time polymerase chain reaction (PCR) detection system that uses oligonucleotide "tagged" PCR primers, a fluorophore-labeled "universal" detection oligonucleotides, and a complementary quenching oligonucleotide. The fluorescence signal decreases as PCR product accumulates due to the increase in detection/quencher hybrid formation as the tagged primer is consumed. We use plasmids containing the influenza A matrix gene and the porA and ctrA genes of Neisseria meningitidis as targets for developing the system. Cycle threshold (Ct) values were generated, and the sensitivity of the new system (dubbed "PrimRglo") compared favorably with the commonly used SYBR green and Taqman detection systems and, unlike the latter system, does not require the design of a new dual-labeled detection oligonucleotide for each new target sequence.

A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies

Publisher: Elsevier BV

Date: 03-2010

DOI: 10.1016/J.JIM.2010.02.001

Abstract: Recombinant monoclonal antibodies currently dominate the protein biologics marketplace. The path from target antigen discovery and screening, to a recombinant therapeutic antibody can be time-consuming and laborious. We describe a set of expression vectors, termed mAbXpress, that enable rapid and sequence-independent insertion of antibody variable regions into human constant region backbones. This method takes advantage of the In Fusion cloning system from Clontech, which allows ligation-free, high-efficiency insertion of the variable region cassette without the addition of extraneous amino acids. These modular vectors simplify the antibody reformatting process during the preliminary evaluation of therapeutic or diagnostic candidates. The resulting constructs can be used directly for transient or lifiable, stable expression in mammalian cells. The effectiveness of this method was demonstrated by the creation of a functional, fully human anti-human CD83 monoclonal antibody.

Discovery and characterisation of a new insect-specific bunyavirus from Culex mosquitoes captured in northern Australia

Publisher: Elsevier BV

Date: 02-2016

DOI: 10.1016/J.VIROL.2015.11.003

Abstract: Insect-specific viruses belonging to significant arboviral families have recently been discovered. These viruses appear to be maintained within the insect population without the requirement for replication in a vertebrate host. Mosquitoes collected from Badu Island in the Torres Strait in 2003 were analysed for insect-specific viruses. A novel bunyavirus was isolated in high prevalence from Culex spp. The new virus, provisionally called Badu virus (BADUV), replicated in mosquito cells of both Culex and Aedes origin, but failed to replicate in vertebrate cells. Genomic sequencing revealed that the virus was distinct from sequenced bunyavirus isolates reported to date, but phylogenetically clustered most closely with recently discovered mosquito-borne, insect-specific bunyaviruses in the newly proposed Goukovirus genus. The detection of a functional furin cleavage motif upstream of the two glycoproteins in the M segment-encoded polyprotein suggests that BADUV may employ a unique strategy to process the virion glycoproteins.

Hydroxyl substituted benzoic acid/cinnamic acid derivatives: Tyrosinase inhibitory kinetics, anti-melanogenic activity and molecular docking studies

Publisher: Elsevier BV

Date: 2020

DOI: 10.1016/J.BMCL.2019.126722

Abstract: The inhibition of tyrosinase is an established strategy for treating hyperpigmentation. Our previous findings demonstrated that cinnamic acid and benzoic acid scaffolds can be effective tyrosinase inhibitors with low toxicity. The hydroxyl substituted benzoic and cinnamic acid moieties of these precursors were incorporated into new chemotypes that displayed in vitro inhibitory effect against mushroom tyrosinase. The most active compound, (2-(3-methoxyphenoxy)-2-oxoethyl (E)-3-(4-hydroxyphenyl) acrylate) 6c, inhibited tyrosinase with an IC

Towards definition of the global biotechnology value chain using cases from Australian biotechnology SMEs

Publisher: Inderscience Publishers

Date: 2004

DOI: 10.1504/IJGSB.2004.005619

Tyrosinase inhibitors as potential antibacterial agents

Publisher: Elsevier BV

Date: 02-2020

DOI: 10.1016/J.EJMECH.2019.111892

Precision Medicines Have Faster Approvals Based On Fewer And Smaller Trials Than Other Medicines

Publisher: Health Affairs (Project Hope)

Date: 05-2018

DOI: 10.1377/HLTHAFF.2017.1580

Abstract: Precision medicines can benefit patients by increasing the probability of a successful treatment response in selected patient populations. The potential for more immediate signals of efficacy during clinical trials suggests such medicines will reach the market more rapidly than traditional drugs will. Using data from the Food and Drug Administration (FDA), we examined the regulatory review and pivotal trial characteristics of precision medicines. We found that in the period January 2013-June 2017, precision medicines were developed and reviewed almost two years faster than nonprecision medicines. In addition, approximately 48 percent of the precision medicines in our study qualified for the FDA's breakthrough therapy designation. Precision medicines were also approved based on fewer pivotal trials with fewer patients, and the trials were less likely to be randomized, blinded, or controlled with either an active or placebo comparator.

Permeation of topically applied Magnesium ions through human skin is facilitated by hair follicles

Publisher: John Libbey Eurotext

Date: 06-2016

DOI: 10.1684/MRH.2016.0402

Abstract: Magnesium is an important micronutrient essential for various biological processes and its deficiency has been linked to several inflammatory disorders in humans. Topical magnesium delivery is one of the oldest forms of therapy for skin diseases, for ex le Dead Sea therapy and Epsom salt baths. Some anecdotal evidence and a few published reports have attributed amelioration of inflammatory skin conditions to the topical application of magnesium. On the other hand, transport of magnesium ions across the protective barrier of skin, the stratum corneum, is contentious. Our primary aim in this study was to estimate the extent of magnesium ion permeation through human skin and the role of hair follicles in facilitating the permeation. Upon topical application of magnesium solution, we found that magnesium penetrates through human stratum corneum and it depends on concentration and time of exposure. We also found that hair follicles make a significant contribution to magnesium penetration.

University biotechnology education in Australia in an international context

Publisher: Elsevier BV

Date: 10-2008

DOI: 10.1016/J.JBIOTEC.2008.07.1665

Placental growth hormone is not suppressed by oral glucose loading in normal human pregnancy

Publisher: Georg Thieme Verlag KG

Date: 05-2002

DOI: 10.1055/S-2002-32138

Abstract: Placental growth hormone (PGH) progressively replaces pituitary growth hormone in the maternal circulation from mid-gestation onwards in human pregnancy. Our previous investigations have shown that placental growth hormone concentrations correlate well with foetal growth. Despite the apparent correlation between PGH and birthweight, the physiology of its secretion during pregnancy has not been well defined. We investigated the response of maternal serum PGH to oral glucose loading in pregnant women (n = 24) who demonstrated normal glucose tolerance at a mean gestation of 29 weeks. Mean (SEM) fasting PGH concentrations were high (36.9 [6.4] ng/ml). No suppression of PGH was noted at one, two or three hours after a 75 g oral glucose load. Similarly, no changes were noted in growth hormone binding protein or in calculated free PGH over the course of the glucose tolerance test. As expected, insulin concentrations rose sixfold and insulin like growth factor binding protein 1 concentrations fell by 20 % with glucose loading. Correlation analysis showed maternal weight, BMI, fasting serum glucose serum insulin to be significantly correlated with the babies' birthweight. Our results support the proposition that PGH concentrations in maternal serum are not suppressed by oral glucose loading in non-diabetic mothers.

RNA and DNA Diagnostics on Microspheres: Current and Emerging Methods

Publisher: Springer International Publishing

Date: 2015

DOI: 10.1007/978-3-319-17305-4_10

A New, Multiplex, Quantitative Real-Time Polymerase Chain Reaction System for Nucleic Acid Detection and Quantification

Publisher: Humana Press

Date: 2013

DOI: 10.1007/978-1-62703-535-4_4

Abstract: Quantitative real-time polymerase chain reaction (qPCR) has emerged as a powerful investigative and diagnostic tool with potential to generate accurate and reproducible results. qPCR can be designed to fulfil the four key aspects required for the detection of nucleic acids: simplicity, speed, sensitivity, and specificity. This chapter reports the development of a novel real-time multiplex quantitative PCR technology, dubbed PrimRglo™, with a potential for high-degree multiplexing. It combines the capacity to simultaneously detect many viruses, bacteria, or nucleic acids, in a single reaction tube, with the ability to quantitate viral or bacterial load. The system utilizes oligonucleotide-tagged PCR primers, along with complementary fluorophore-labelled and quencher-labelled oligonucleotides. The analytic sensitivity of PrimRglo technology was compared with the widely used Taqman(®) and SYBR green detection systems.

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: Correlations with fetal growth

Publisher: The Endocrine Society

Date: 03-2000

DOI: 10.1210/JC.85.3.1143

Linkage Projects - Grant ID: LP0211022

Start Date: 2002

End Date: 12-2004

Amount: $194,344.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0348038

Start Date: 2003

End Date: 08-2006

Amount: $240,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354702

Start Date: 2004

End Date: 12-2004

Amount: $10,000.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC160100027

Start Date: 03-2017

End Date: 03-2023

Amount: $4,340,802.00

Funder: Australian Research Council