A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

Detecting approximate clones in business process model repositories

Empirical evidence shows that repositories of business process models used in industrial practice contain significant amounts of duplication. This duplication arises for example when the repository covers multiple variants of the same processes or due to copy-pasting. Previous work has addressed the problem of efficiently retrieving exact clones that can be refactored into shared subprocess models. This article studies the broader problem of approximate clone detection in process models. The article proposes techniques for detecting clusters of approximate clones based on two well-known clustering algorithms: DBSCAN and Hi- erarchical Agglomerative Clustering (HAC). The article also defines a measure of standardizability of an approximate clone cluster, meaning the potential benefit of replacing the approximate clones with a single standardized subprocess. Experiments show that both techniques, in conjunction with the proposed standardizability measure, accurately retrieve clusters of approximate clones that originate from copy-pasting followed by independent modifications to the copied fragments. Additional experiments show that both techniques produce clusters that match those produced by human subjects and that are perceived to be standardizable.

Temporal and spatial distribution and habitat associations of an urban population of New Zealand long-tailed bats (Chalinolobus tuberculatus)

Two ultrasound survey methods were used to determine the presence and activity patterns of New Zealand long-tailed bats (Chalinolobus tuberculatus) in the city of Hamilton. First, 13 monthly surveys conducted at 18 green spaces found C. tuberculatus in only one urban forest reserve, Hammond Bush, where they were found consistently throughout the year. Bat activity was strongly related to temperature. Second, twice-yearly citywide surveys conducted over 2 years determined the distribution and habitat associations of C. tuberculatus. Bats were found only in the southern part of the city and were strongly associated with the Waikato River. Bat activity was negatively correlated with housing and street light density and positively correlated with topographical complexity. In Hamilton, topographical complexity indicates the presence of gullies. Gullies probably provide foraging and roosting opportunities and connect the river to distant forest patches. These results suggest that urban habitats can be useful for bats if gullies can link these to distant habitat fragments.

Establishing prostate cancer patient derived xenografts: Lessons learned from older studies

Background Understanding the progression of prostate cancer to androgen-independence/castrate resistance and development of preclinical testing models are important for developing new prostate cancer therapies. This report describes studies performed 30 years ago, which demonstrate utility and shortfalls of xenografting to preclinical modeling. Methods We subcutaneously implanted male nude mice with small prostate cancer fragments from transurethral resection of the prostate (TURP) from 29 patients. Successful xenografts were passaged into new host mice. They were characterized using histology, immunohistochemistry for marker expression, flow cytometry for ploidy status, and in some cases by electron microscopy and response to testosterone. Two xenografts were karyotyped by G-banding. Results Tissues from 3/29 donors (10%) gave rise to xenografts that were successfully serially passaged in vivo. Two, (UCRU-PR-1, which subsequently was replaced by a mouse fibrosarcoma, and UCRU-PR-2, which combined epithelial and neuroendocrine features) have been described. UCRU-PR-4 line was a poorly differentiated prostatic adenocarcinoma derived from a patient who had undergone estrogen therapy and bilateral castration after his cancer relapsed. Histologically, this comprised diffusely infiltrating small acinar cell carcinoma with more solid aggregates of poorly differentiated adenocarcinoma. The xenografted line showed histology consistent with a poorly differentiated adenocarcinoma and stained positively for prostatic acid phosphatase (PAcP), epithelial membrane antigen (EMA) and the cytokeratin cocktail, CAM5.2, with weak staining for prostate specific antigen (PSA). The line failed to grow in female nude mice. Castration of three male nude mice after xenograft establishment resulted in cessation of growth in one, growth regression in another and transient growth in another, suggesting that some cells had retained androgen sensitivity. The karyotype (from passage 1) was 43–46, XY, dic(1;12)(p11;p11), der(3)t(3:?5)(q13;q13), -5, inv(7)(p15q35) x2, +add(7)(p13), add(8)(p22), add(11)(p14), add(13)(p11), add(20)(p12), -22, +r4[cp8]. Conclusions Xenografts provide a clinically relevant model of prostate cancer, although establishing serially transplantable prostate cancer patient derived xenografts is challenging and requires rigorous characterization and high quality starting material. Xenografting from advanced prostate cancer is more likely to succeed, as xenografting from well differentiated, localized disease has not been achieved in our experience. Strong translational correlations can be demonstrated between the clinical disease state and the xenograft model

Is there a bone-nail specific entry point? automated fit quantification of tibial nail designs during the insertion for six different nail entry points

Intramedullary nailing is the standard fixation method for displaced diaphyseal fractures of tibia. Selection of the correct nail insertion point is important for axial alignment of bone fragments and to avoid iatrogenic fractures. However, the standard entry point (SEP) may not always optimise the bone-nail fit due to geometric variations of bones. This study aimed to investigate the optimal entry for a given bone-nail pair using the fit quantification software tool previously developed by the authors. The misfit was quantified for 20 bones with two nail designs (ETN and ETN-Proximal Bend) related to the SEP and 5 entry points which were 5 mm and 10 mm away from the SEP. The SEP was the optimal entry point for 50% of the bones used. For the remaining bones, the optimal entry point was located 5 mm away from the SEP, which improved the overall fit by 40% on average. However, entry points 10 mm away from the SEP doubled the misfit. The optimised bone-nail fit can be achieved through the SEP and within the range of a 5 mm radius, except posteriorly. The study results suggest that the optimal entry point should be selected by considering the fit during insertion and not only at the final position.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.

Fusion of worlds : consumers’ self-narrative in a hybrid physical/digital world

As the Internet becomes deeply embedded into consumers’ daily life, the digital virtual world brings significant influence to consumers’ self and narrative. Prior studies look at consumer self from either from a certain online space or comparing consumers’ physical and digital virtual selves but not the integration of the physical/digital world. This paper aims to explore the meanings of the digital virtual space on consumers’ narrative as a whole (their interests, dreams, or subjectivity). We utilise a postmodern concept of the cyborg to understand the cultural complexity, subjective meanings of, and the extent to which the digital virtual space plays a role in consumers’ self-narrative. We conducted in-depth interviews and gathered three consumer narratives. Our findings indicate that consumers’ narrative contains important fragments from both physical and digital virtual worlds and their physical and digital virtual selves form a feedback loop that strengthen their overall narrative.

Sub-genomic RNA of defective interfering (D.I.) dengue viral particles is replicated in the same manner as full length genomes

The predicted secondary structure of sub-genomic RNA in dengue virus defective interfering (D.I.) particles from patients, or generated in vitro, resembled that of the 3′ and 5′ regions of wild type dengue virus (DENV) genomes. While these structures in the sub-genomic RNA were found to be essential for its replication, their nucleotide sequences were not, so long as any new sequences maintained wild type RNA secondary structure. These observations suggested that these sub-genomic fragments of RNA from dengue viruses were replicated in the same manner as the full length genomes of their wild type, “helper”, viruses and that they probably represent the smallest fragments of DENV RNA that can be replicated during a natural infection. While D.I. particles containing sub-genomic RNA are completely parasitic, the relationship between wild type and D.I. DENV may be symbiotic, with the D.I. particles enhancing the transmission of infectious DENV.

Silylation of mechanically ground kaolinite

Silylated kaolinites were synthesized at 80°C without the use of inert gas protection. The method presented started with mechanical grinding of kaolinite, followed by grafting with 3-aminopropyltriethoxysilane (APTES). The mechanical grinding treatment destroyed the ordered sheets of kaolinite, formed fine fragments and generated broken bonds (undercoordinated metal ions). These broken bonds served as new sites for the condensation with APTES. Fourier transform infrared spectroscopy (FTIR) confirmed the existence of –CH2 from APTES. 29Si cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy (29Si CP/MAS NMR) showed that the principal bonding mechanism between APTES and kaolinite fitted a tridentate silylation model (T3) with a chemical shift at 66.7 ppm. The silane loadings of the silylated samples were estimated from the mass loss obtained by TG-DTG curves. The results showed that the 6-hour ground kaolinite could be grafted with the most APTES (7.0%) using cyclohexane as solvent. The loaded amount of APTES in the silylated samples obtained in different solvents decreased in the order as: nonpolar solvent > polar solvent with low dielectric constant (toluene) > polar solvent with high dielectric constant (ethanol).

EphB4 localises to the nucleus of prostate cancer cells

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

Increased intra-cortical porosity reduces bone stiffness and strength in pediatric patients with osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable disease occurring in one out of every 20,000 births. Although it is known that Type I collagen mutation in OI leads to increased bone fragility, the mechanism of this increased susceptibility to fracture is not clear. The aim of this study was to assess the microstructure of cortical bone fragments from patients with osteogenesis imperfecta (OI) using polarized light microscopy, and to correlate microstructural observations with the results of previously performed mechanical compression tests on bone from the same source. Specimens of cortical bone were harvested from the lower limbs of three (3) OI patients at the time of surgery, and were divided into two groups. Group 1 had been subjected to previous micro-mechanical compression testing, while Group 2 had not been subjected to any prior testing. Polarized light microscopy revealed disorganized bone collagen architecture as has been previously observed, as well as a large increase in the areal porosity of the bone compared to typical values for healthy cortical bone, with large (several hundred micron sized), asymmetrical pores. Importantly, the areal porosity of the OI bone samples in Group 1 appears to correlate strongly with their previously measured apparent Young's modulus and compressive strength. Taken together with prior nanoindentation studies on OI bone tissue, the results of this study suggest that increased intra-cortical porosity is responsible for the reduction in macroscopic mechanical properties of OI cortical bone, and therefore that in vivo imaging modalities with resolutions of ~ 100 μm or less could potentially be used to non-invasively assess bone strength in OI patients. Although the number of subjects in this study is small, these results highlight the importance of further studies in OI bone by groups with access to human OI tissue in order to clarify the relationship between increased porosity and reduced macroscopic mechanical integrity.

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.

Development of a method to assess the quality of ankle joint reduction in 3D

This project developed a quantitative method for determining the quality of the surgical alignment of the bone fragments after an ankle fracture. The research examined the feasibility of utilising MRI-based bone models versus the gold standard CT-based bone models in order to reduce the amount of ionising radiation the patient is exposed to. In doing so, the thesis reports that there is potential for MRI to be used instead of CT depending on the scanning parameters used to obtain the medical images, the distance of the implant relative to the joint surface, and the implant material.

Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing tile PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive width MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.