Using three-dimensional rapid prototyping in the design and development of orthopaedic screws in standardised pull-out tests.

The majority of orthopaedic screws are designed, tested and manufactured by existing orthopaedics companies and are predominantly developed with healthy bone in mind. The timescales and costs involved in the development of a new screw design, for example, for osteoporotic bone, are high. In this study, standard wood screws were used to analyse the concept of using three-dimensional printing, or rapid prototyping, as a viable stage of development in the design of a new bone screw. Six wood screws were reverse engineered and printed in polymeric material using stereolithography. Three of the designs were also printed in Ti6Al4V using direct metal laser sintering; however, these were not of sufficient quality to test further. Both the original metal screws (metal) and polymeric rapid prototyping screws were then tested using standard pull-out tests from low-density polyurethane blocks (Sawbones). Results showed the highest pull-out strengths for screws with the longest thread length and the smallest inner diameter. Of the six screw designs tested, five showed no more than a 17% variance between the metal and rapid prototyping results. A similar pattern of results was shown between the screw designs for both the metal and rapid prototyping screws in five of the six cases. While not producing fully comparable pull-out results to orthopaedic screws, the results from this study do provide evidence of the potential usefulness and cost-effectiveness of rapid prototyping in the early stages of design and testing of orthopaedic screws.

Hi-fidelity discrimination of isomiRs using G-quadruplex gatekeepers.

Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DNA probe-miRNA target hybridization is the primary, or initial, mode of detection. These errors then become exacerbated through subsequent amplification steps. Here, we present the design of DNA probes modified with poly-guanine (PG) tracts that were induced to form G-quadruplexes (G4) for hi-fidelity discrimination of miRNA core target sequence from single-base mismatched isomiRs. We demonstrate that, when compared to unmodified probes, this G4 'gate-keeping' function within the G4-modified probes enables more stringent hybridization of complementary core miRNA target transcripts while limiting non-specific hybridizations. This increased discriminatory power of the G4-modified probes over unmodified probes is maintained even after further reverse transcriptase extension of probe-target hybrids. Enzymatic extension also enhanced the clarity and sensitivity of readouts and allows different isomiRs to be distinguished from one another via the relative positions of the mismatches.

Single nucleotide polymorphism discrimination with and without an ethidium bromide intercalator.

Single nucleotide polymorphism (SNP) genotyping is an important aspect in understanding genetic variations. Here, we discriminate SNPs using toe-hold mediated displacement reactions. The biological target is an 80 nucleotide long double-stranded-DNA from the mtDNA HV1 region, associated with maternal ancestry. This target has been specially designed with a pendant toehold and a cationic fluorophore, ATTO 647N, as a reporter, produced in a polymerase chain reaction. Rates of reaction for the toehold-polymerase chain reaction products (TPPs) with their corresponding complementary displacing sequences, labelled with a Black Hole Quencher 1, followed the order TPP-Cytosine > TPP-Thymine > TPP-Adenine ≥ TPP-Guanine. Non-complementary rates were the slowest with mismatches involving cytosine. These reactions, operating in a static/or contact mode, gave averaged readouts between SNPs within 15 min (with 80-90% quenching), compared to 25-30 min in previous studies involving fluorescence resonance energy transfer. Addition of an intercalating agent, ethidium bromide, retarded the rate of reaction in which cytosine was involved, presumably through stabilization of the base pairing, which resulted in markedly improved discrimination of cytosine containing SNPs.

A DNA Circuit for IsomiR Detection.

A synthetic DNA oligonucleotide has been programmed to function as a biological circuit to detect 5'-IsomiRs. The circuit consists of two integrated DNA switches. The first is "activated" when a DNA probe is enzymatically modified by a reverse transcriptase that incorporates nucleotides complementary to the 5'-region of a microRNA (miRNA). Addition of the correct number and sequence of nucleotides enables the probe to assemble into an asymmetric DNA hairpin. The reconfigured hairpin probe then primes an internal polymerisation reaction, resulting in the synthesis of a symmetrical DNA hairpin. This activates the second switch, which then initiates the amplification of reverse-transcribed miRNA through a continuous cycle of DNA nicking and polymerisation. The DNA circuit enables sensitive and rapid detection of femtomoles of a miRNA transcript under isothermal conditions.

Accurate detection of methylated cytosine in complex methylation landscapes.

Monitoring DNA methylation can be a useful biomarker for disease diagnosis and prognosis. However, monitoring the methylation status of a specific cytosine biomarker is often confounded by heterogeneous peripheral DNA methylation. To address this issue, molecular inversion probes were designed with inosine strategically positioned to complement suspected DNA methylation sites. This enabled the methylation status of a specific cytosine to be accurately measured with a high level of specificity, irrespective of adjacent epigenetic modifications.

Sensitive quantification of somatic mutations using molecular inversion probes.

Somatic mutations in DNA can serve as cancer specific biomarkers and are increasingly being used to direct treatment. However, they can be difficult to detect in tissue biopsies because there is often only a minimal amount of sample and the mutations are often masked by the presence of wild type alleles from nontumor material in the sample. To facilitate the sensitive and specific analysis of DNA mutations in tissues, a multiplex assay capable of detecting nucleotide changes in less than 150 cells was developed. The assay extends the application of molecular inversion probes to enable sensitive discrimination and quantification of nucleotide mutations that are present in less than 0.1% of a cell population. The assay was characterized by detecting selected mutations in the KRAS gene, which has been implicated in up to 25% of all cancers. These mutations were detected in a single multiplex assay by incorporating the rapid flow cytometric readout of multiplexable DNA biosensors.

Rapid DNA detection by beacon-assisted detection amplification.

This protocol describes a new and rapid isothermal reaction process designed to amplify and detect a specific DNA sequence in purified DNA extracted from cultured cells. The protocol uses a DNA nanomachine that comprises two molecular switches that function in concert to isothermally amplify and detect a DNA target. First, a molecular beacon detection switch is 'activated' only if a DNA target sequence is present. A DNA primer and DNA polymerase are used to lock the beacon in an activated conformation. Second, an amplification and signal-transduction switch is initiated following successful activation. A nicking endonuclease and the DNA polymerase are used to replicate the DNA target. Both switches operate simultaneously at 40 °C in a single reaction to rapidly generate multiple copies of the DNA target in a cyclic polymerization reaction. This protocol enables femtomole amounts of a DNA target to be reproducibly amplified and detected in <40 min. We demonstrate the successful use of this protocol in assays containing synthetic DNA components and purified DNA extracted from biological samples.

Epiallele quantification using molecular inversion probes.

The location and level of DNA methylation within a genome is emerging as an important biomarker for cancer diagnosis. Despite its potential, it is difficult to comprehensively analyze the epialleles that are often found in a biological sample. Therefore, an assay utilizing molecular inversion probes was designed and used to expose and quantify epialleles in heterogeneously methylated bisulphite treated genomic DNA. Different CpG dinucleotides were able to be rapidly quantified with high resolution, sensitivity and specificity over a large dynamic range using rapid flow cytometric readout of multiplexable microbead DNA biosensors.

Considerations of solid-phase DNA amplification.

Solid-phase (SP) polymerase chain reaction (PCR) is an increasingly popular tool used to produce immobilized DNA for a variety of applications, including high-throughput DNA sequencing and SNP analysis. Despite its usefulness, the mechanism of DNA amplification using immobilized primers has not been thoroughly explored. Herein, we describe a SP-PCR process that was designed to explore and better understand some limitations of SP-DNA amplification. The rate of SP-DNA amplification was measured, and the ability to exponentially amplify DNA on a surface was demonstrated. Approximately 50 amol of DNA was amplified to detectable levels using SP-PCR. The mechanism and some limitations of the reaction were investigated by measuring the density of the primer on the surface prior to amplification and the amount of immobilized amplicon produced after SP-PCR. This enabled some of the practical limitations of the reaction to be addressed within a logical theoretical framework.

Quantitative considerations for suspension array assays.

A novel format of a multiplex microbead assay that is capable of accurately and reproducibly measuring small changes in 10(8) copies of a nucleic acid is reported herein. The dynamic range of the assay was 32-fold greater than conventional multiplex microbead assays. The assay was modeled mathematically which enabled the accuracy of DNA quantification to be formally analysed using a logical theoretical framework. The assay was also evaluated on RNA extracts taken from stimulated macrophages, to demonstrate that changes in gene expression can be accurately monitored in a biological system. We believe the reproducibility and accuracy of the assay presented herein will be useful for high-throughput, multiplex sensitive detection of nucleic acids over a large dynamic range in a research or diagnostic setting.

Expression of survivin in lung eosinophils is associated with pathology in a mouse model of allergic asthma.

Humans vary markedly in their propensity to develop asthma, despite often being exposed to similar environmental stimuli. Similarly, mouse strains vary in susceptibility to airways pathology in experimental asthma. Sensitization and aerosol challenge with ovalbumin (OVA) induces eosinophil accumulation, mucus production and airways obstruction in BALB/c and C57BL/6 mice. In contrast, CBA/Ca mice show relatively little pathology. Allergen-induced production of IL-4, IL-5, IL-10 and IFN-gamma was detected in all three strains, with BALB/c mice generating the highest levels of IL-4, IL-5 and IL-10. Microarray analysis was used to identify genes differentially regulated in lung tissue after OVA challenge. Differentially regulated genes in the lungs of the asthma-susceptible C57BL/6 and BALB/c strains numbered 242 and 145, respectively, whereas only 42 genes were differentially expressed in the resistant CBA/Ca strain. In C57BL/6 mice, transcripts were enriched for adhesion molecules and this was associated with high levels of eosinophil recruitment. Differentially regulated genes in the lungs of only the asthma-susceptible strains numbered 64 and several of these have not previously been associated with asthma. Many of the genes differentially regulated in the susceptible strains were enzymes involved in inflammation. Using network analysis, mRNA for the anti-apoptotic protein survivin was found to be up-regulated in the lungs following allergen challenge. Survivin mRNA and protein were also expressed at high levels in eosinophils recovered by bronchoalveolar lavage from BALB/c and C57BL/6 mice. We propose that rapid apoptosis of lung eosinophils due to low expression of survivin contributes to the limitation of pathology in CBA/Ca mice.

Differential rates of apoptosis and recruitment limit eosinophil accumulation in the lungs of asthma-resistant CBA/Ca mice.

The accumulation of eosinophils is a common feature of allergic airway inflammation and correlates with disease severity. In an ovalbumin (OVA)-induced murine model of allergic lung disease, CBA/Ca mice develop much lower levels of lung eosinophilia, lung oedema, mucus hypersecretion and airways obstruction than BALB/c and C57BL/6 strains. In this study these strains have been examined to identify mechanisms that control the recruitment and survival of eosinophils in the allergic lung. Following immunization with OVA, CBA/Ca mice developed a robust systemic allergic response, with high levels of total and OVA-specific IgE and increases in peripheral blood eosinophils. Lung eotaxin-1 levels and expression of CD18 on eosinophils recovered by bronchoalveolar lavage (BAL) were least pronounced in CBA/Ca mice, whereas mRNA for L-selectin was highest in eosinophils from C57BL/6 mice. Apoptosis of BAL eosinophils ex vivo was most pronounced in the CBA/Ca strain. BALB/c mice expressed the highest levels of the eosinophil growth and survival factor interleukin (IL)-5 in the lungs and BAL eosinophils from these animals expressed more of the anti-apoptotic proteins Bcl-xL and Bcl-2 than cells from the other strains. A combination of lower levels of recruitment and rapid apoptosis may therefore limit the accumulation of eosinophils and pathology in the lungs of CBA/Ca mice. In addition, although the level of pathology that developed in C57BL/6 and BALB/c mice was similar, some of the underlying mechanisms are likely to differ.

Heterologous microarray experiments used to identify the early gene response to heat stress in a coral reef fish.

Coral reef fishes are expected to experience rising sea surface temperatures due to climate change. How well tropical reef fishes will respond to these increased temperatures and which genes are important in the response to elevated temperatures is not known. Microarray technology provides a powerful tool for gene discovery studies, but the development of microarrays for individual species can be expensive and time-consuming. In this study, we tested the suitability of a Danio rerio oligonucleotide microarray for application in a species with few genomic resources, the coral reef fish Pomacentrus moluccensis. Results from a comparative genomic hybridization experiment and direct sequence comparisons indicate that for most genes there is considerable sequence similarity between the two species, suggesting that the D. rerio array is useful for genomic studies of P. moluccensis. We employed this heterologous microarray approach to characterize the early transcriptional response to heat stress in P. moluccensis. A total of 111 gene loci, many of which are involved in protein processing, transcription, and cell growth, showed significant changes in transcript abundance following exposure to elevated temperatures. Changes in transcript abundance were validated for a selection of candidate genes using quantitative real-time polymerase chain reaction. This study demonstrates that heterologous microarrays can be successfully employed to study species for which specific microarrays have not yet been developed, and so have the potential to greatly enhance the utility of microarray technology to the field of environmental and functional genomics.