Hybrid Stepper Research Articles

NeuroArray, A Custom CGH Microarray to Decipher Copy Number Variants in Alzheimer's Disease.

Background: Copy Number Variants (CNVs) represent a prevailing type of structural variation (deletions or duplications) in the human genome. In the last few years, several studies have demonstrated that CNVs represent significant mutations in Alzheimer’s Disease (AD) hereditability. Currently, innovative high-throughput platforms and bioinformatics algorithms are spreading to screening CNVs involved in different neurological diseases. In particular, the use of custom arrays, based on libraries of probes that can detect significant genomic regions, have greatly improved the resolution of targeted regions and the identification of chromosomal aberrations.Objective: In this work, we report the use of NeuroArray, a custom CGH microarray useful to screening and further investigate the role of the recurring genomic aberrations in patients with confirmed or suspected AD.Methods:The custom oligonucleotide aCGH design includes 641 genes and 9118 exons, linked to AD. The genomic DNA was isolated from blood samples of AD affected patients. The entire protocol of custom NeuroArray included digestion, labelling and hybridization steps as a standard aCGH assay.Results: The NeuroArray analysis revealed the presence of amplifications in several genes associated with AD. In the coding regions of these genes, 14,586 probes were designed with a 348 bp median probe spacing. The majority of targeted AD genes map on chromosomes 1 and 10. A significant aspect of the NeuroArray design is that 95% of the total exon targets is covered by at least one probe, a resolution higher than CGH array platforms commercially available.Conclusion: By identifying with a high sensitivity the chromosomal abnormalities in a large panel of AD-related genes and other neurological diseases, the NeuroArray platform is a valid tool for clinical diagnosis.

The flash-small-pool PCR: how to transform blotting and numerous hybridization steps into a simple denatured PCR.

Numerous human diseases are associated with abnormal expansion of unstable trinucleotide repeats (TNRs). TNR instability mechanisms are complex, and remain only partially understood. Small-pool-PCR (SP-PCR) is the reference method to assess TNR instability. SP-PCR amplifies a low number of DNA molecules and is followed by Southern blot. However, SP-PCR remains expensive and time consuming. Here, we describe an optimized SP-PCR that can be done in a day, which reduces cost and experimental biases: the flash-small-pool PCR (FSP-PCR). This method consists of a fluorescent PCR on a few DNA molecules, followed by an alkaline gel electrophoresis revealed with a near infra-red detector system. With reduced experimental steps, cost, and time consumption, microsatellite analysis will become more accessible due to FSP-PCR.

Anharmonic acoustic effects during DNA hybridization on an electrochemical quartz crystal resonator

The paper describes a sensor for single-stranded DNA (ssDNA) biomarker based on anharmonic acoustic signals arising during hybridization with complementary thiolated ssDNA functionalised on the gold electrode of an electrochemical quartz crystal resonator. The steps of sensor preparation and hybridization are carried out in an electrochemical microfluidic flowcell. While the electrochemical impedance spectroscopy does not allow a definitive interpretation, the changes in resonance frequency and third Fourier harmonic current of the resonator on actuation at the fundamental mode indicate formation of a flexibly bound layer. The functionalization and hybridization steps monitored by the anharmonic detection technique (ADT) are described with a simple model based on Duffing nonlinear equation.

Spiky gold shells on magnetic particles for DNA biosensors

Combined separation and detection of biomolecules has the potential to speed up and improve the sensitivity of disease detection, environmental testing, and biomolecular analysis. In this work, we synthesized magnetic particles coated with spiky nanostructured gold shells and used them to magnetically separate out and detect oligonucleotides using SERS. The distance dependence of the SERS signal was then harnessed to detect DNA hybridization using a Raman label bound to a hairpin probe. The distance of the Raman label from the surface increased upon complementary DNA hybridization, leading to a decrease in signal intensity. This work demonstrates the use of the particles for combined separation and detection of oligonucleotides without the use of an extrinsic tag or secondary hybridization step.

Two-Color In Situ Hybridization: A Technique for Simultaneous Detection of Transcripts from Different Loci.

Over the years, in situ hybridization has been used for visualizing spatial gene expression patterns. Direct comparison of the expression patterns of different genes is useful for studying various biological activities in many situations. If we can distinguish signals derived from different probes in the same hybridization reaction, the localization of multiple gene transcripts can be detected simultaneously. In this chapter, we describe a two-color in situ hybridization procedure, which enables us to compare the expression patterns of two genes in a single tissue section. First, we explain how to prepare RNA probes and tissue samples. Then, we explain how to perform an in situ experiment, including pre-hybridization, hybridization, post-hybridization, and detection steps. A stepwise detection procedure is used to obtain the two color signals.

Exonuclease III-Assisted Upconversion Resonance Energy Transfer in a Wash-Free Suspension DNA Assay.

Sensitivity is the key in optical detection of low-abundant analytes, such as circulating RNA or DNA. The enzyme Exonuclease III (Exo III) is a useful tool in this regard; its ability to recycle target DNA molecules results in markedly improved detection sensitivity. Lower limits of detection may be further achieved if the detection background of autofluorescence can be removed. Here we report an ultrasensitive and specific method to quantify trace amounts of DNA analytes in a wash-free suspension assay. In the presence of target DNA, the Exo III recycles the target DNA by selectively digesting the dye-tagged sequence-matched probe DNA strand only, so that the amount of free dye removed from the probe DNA is proportional to the number of target DNAs. Remaining intact probe DNAs are then bound onto upconversion nanoparticles (energy donor), which allows for upconversion luminescence resonance energy transfer (LRET) that can be used to quantify the difference between the free dye and tagged dye (energy acceptor). This scheme simply avoids both autofluorescence under infrared excitation and many tedious washing steps, as the free dye molecules are physically located away from the nanoparticle surface, and as such they remain "dark" in suspension. Compared to alternative approaches requiring enzyme-assisted amplification on the nanoparticle surface, introduction of probe DNAs onto nanoparticles only after DNA hybridization and signal amplification steps effectively avoids steric hindrance. Via this approach, we have achieved a detection limit of 15 pM in LRET assays of human immunodeficiency viral DNA.

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has greatly advanced our understanding of biological processes, including developmental regulation. In situ protocols are today routinely followed in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acid sequence. This step is generally carried out at high temperatures and in a denaturing solution, called hybridization buffer, commonly containing 50% (v/v) formamide – a hazardous chemical. When applied to the soft-bodied hydrozoan medusa Clytia hemisphaerica, we found that this traditional hybridization approach was not fully satisfactory, causing extensive deterioration of morphology and tissue texture which compromised our observation and interpretation of results. We thus tested alternative solutions for in situ detection of gene expression and, inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8M urea solution in the hybridization buffer. Our new protocol not only yielded better morphologies and tissue consistency, but also notably improved the resolution of the signal, allowing more precise localization of gene expression and reducing aspecific staining associated with problematic areas. Given the improved results and reduced manipulation risks, we tested the urea protocol on other metazoans, two brachiopod species (Novocrania anomala and Terebratalia transversa) and the priapulid worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea during in situ hybridization offers a safer alternative, potentially of widespread use in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

Single-step, homogeneous and sensitive detection for microRNAs with dual-recognition steps based on luminescence resonance energy transfer (LRET) using upconversion nanoparticles

A single-step, homogeneous and sensitive LRET assay is presented for the detection of miRNAs. The amplification-free assay provides a unique combination of high specificity with dual-recognition approach of different hybridization and ligation steps and preventing background auto-fluorescence in biological samples using upconversion nanoparticles (UCNPs) as signal-producing nanoprobes. The assay probe is composed of signal-producing unit (a pair of homogeneous upconversion luminescence resonance energy transfer (UC-LRET)-based oligonucleotides) and recognition unit (two adaptor oligonucleotides). In the presence of target miRNAs, the probe and target miRNAs leads to the formation of stable double-strands and semi-stable adaptor-miRNAs complexes with an adaptor nick. Ligation of the nick using ligase cause the formation of stable double-strands, resulting in UCNPs-to-dye UC-LRET for detection of the miRNAs with near-infrared radiation (980nm). Sensitive detection of miRNA-21 at concentrations of 200pM to 1.4nM and detection limits of 0.095nM with good precision of 3.9% (RSD) for seven repeated measurements of 500pM miRNAs demonstrate the feasibility of both high throughput and point-of-care clinical diagnostics. The homogeneous UC-LRET assay without any washing can be extended to the application in other important types of nucleic acid analysis.

An algorithmic approach to diagnose haematolymphoid neoplasms in effusion by combining morphology, immunohistochemistry and molecular cytogenetics.

There are limited studies of cytology diagnosis of haematopoietic and lymphoid tumours in serosal effusion except for occasional case reports. We would like to demonstrate an algorithmic approach for accurate diagnosis, especially in patients without previous history. We reviewed 36 cases of lymphoma diagnosed in serosal effusion following an algorithmic approach. Suspected tumour cells were classified into small, intermediate and large sizes and two characteristic forms of plasmacytoid and Reed Sternberg-like on smears (step 1), followed by utilising panels of immunohistochemical markers and Epstein-Barr encoding region in situ hybridisation on cell blocks (step 2). A panel of CD3, CD20 and Ki-67 formed the basic workup, followed by pertinent batteries of immunostaining. Molecular tests were applied in 22 selected cases by fluorescence in situ hybridisation (step 3). There were 15 diffuse large B-cell lymphomas; 12 plasma cell myelomas; two mantle cell lymphomas; one anaplastic large cell lymphoma ALK +; one small lymphocytic lymphoma; one plasmablastic lymphoma; one peripheral T-cell lymphoma, not otherwise specified, one extranodal NK/T-cell lymphoma, nasal type and two T-cell lymphoblastic lymphomas. 14 cases with previous history had complete concordance in immunophenotype between cytology and histology. Another 14 cases were primarily diagnosed in patients with initial symptom of effusion based on immunophenotyping and cytogenetic test in selected cases. Eight cases were diagnosed based on morphology alone. An algorithmic approach based on morphology and immunohistochemistry is the key to making an accurate diagnosis of haematopoietic and lymphoid tumours in effusion. A molecular test is also important for confirmation and prognostic prediction. We reviewed 36 haematolymphoid neoplasms diagnosed in effusion including 14 cases primarily diagnosed in patients without previous history following an algorithmic approach by combining morphology, immunohistochemistry and molecular cytogenetics.

Abstract 4362: Coordinated regulation of several microRNAs in lung cancer patients

Abstract Background Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. NSCLCs are mainly classified adenocarcinoma and squamous cell carcinoma. It seems more and more obvious that specific molecular analyses are necessary to elucidate the complexity of clinically relevant phenotypes which determine the lung carcinogenesis. This includes the role of microRNAs (miRNAs) in the disease, involved in the complexity of gene expression regulation. Different modulator mechanisms are involved in the biogenesis and function of miRNAs, one of them is DNA methylation. Alterations in these mechanisms may contribute to the tumor genesis and progression. The purpose of this study was to assess miRNA methylation patterns in patients with NSCLC to study the potential of coordinate regulation of miRNAs as a relevant mechanism involved in this pathogenesis. Materials and Methods DNA methylation of gene clusters was analyzed by Illumina 70 subjects. The samples were divided into two cohorts. A first cohort constituted from 47 patients who had undergone surgical resection for clinical early stage NSCLC. A second group from 23 subjects was used as health cohort. DNA was extracted using the QIAamp DNA Mini Kit. For each assay, 500 ng of DNA was treated with sodium bisulfate using EZ DNA Methylation™ Kit and cleaned with ZR-96 DNA Clean-up Kit™, before standard Illumina amplification, hybridization, and imaging steps. Methylation data were processed using the RnBeads R package. Results We identified two clusters clearly corregulated by methylation in lung cancer, involving 49 miRNAs differentially hypomethylated in tumor samples respect to control tissue. Some of these miRNAs have been implicated in several pathways in cancer. Conclusions Our results strongly imply hypomethylation of two miRNA clusters, which represent key targets in unravelling of the mechanism of lung tumorigenesis. Citation Format: Irene Ferrer, Ana Salinas, Angela Marrugal, Jon Zugazagoitia, Amancio Carnero, Luis Paz-Ares, Sonia Molina-Pinelo. Coordinated regulation of several microRNAs in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4362. doi:10.1158/1538-7445.AM2017-4362

Detection of BRCA1 gross rearrangements by droplet digital PCR.

Large genomic rearrangements (LGRs) constitute a significant share of pathogenic BRCA1 mutations. Multiplex ligation-dependent probe amplification (MLPA) is a leading method for LGR detection; however, it is entirely based on the use of commercial kits, includes relatively time-consuming hybridization step, and is not convenient for large-scale screening of recurrent LGRs. We developed and validated the droplet digital PCR (ddPCR) assay, which covers the entire coding region of BRCA1 gene and is capable to precisely quantitate the copy number for each exon. 141 breast cancer (BC) patients, who demonstrated evident clinical features of hereditary BC but turned out to be negative for founder BRCA1/2 mutations, were subjected to the LGR analysis. Four patients with LGR were identified, with three cases of exon 8 deletion and one women carrying the deletion of exons 5-7. Excellent concordance with MLPA test was observed. Exon 8 copy number was tested in additional 720 BC and 184 ovarian cancer (OC) high-risk patients, and another four cases with the deletion were revealed; MLPA re-analysis demonstrated that exon 8 loss was a part of a larger genetic alteration in two cases, while the remaining two patients had isolated defect of exon 8. Long-range PCR and next generation sequencing of DNA samples carrying exon 8 deletion revealed two types of recurrent LGRs. Droplet digital PCR is a reliable tool for the detection of large genomic rearrangements.

Are we fishing or catching? Evaluating the efficiency of bait capture of CODIS fragments

This study sought to document the efficiency of DNA bait capture (i.e., “fishing”) methods by two measures: (1) its ability to retain targeted DNA molecules, and (2) its ability to remove non-target DNA molecules from a pool containing both. DNA bait capture uses synthetic biotinylated DNA primers to bind target DNA, which are then immobilized onto streptavidin coated magnetic beads and drawn to a magnet. Bound DNA should, therefore, be isolated from non-target DNA and impurities (e.g., PCR inhibitors) and can be later eluted from the beads for downstream applications. Efficiencies were estimated by comparing the number of “copies in” to “copies out” with quantitative polymerase chain reaction (qPCR). Retention of target DNA molecules, ranging from 109 to 288 base pairs (bps) in length, averaged just 9.06–3.53% (i.e., loss of 90.94–96.47%) using the fishing protocol as originally described. Some improvement was achieved by employing a modified protocol (i.e., with a shortened hybridization time, use of twice the amount of M-270 streptavidin-coated beads, and modified bead washing), resulting in average retention of 31.41–12.08% of the same set of targeted molecules. Noted was the lack of efficacy in removing non-target DNA molecules as opposed to targeted molecules. It was also observed that most of the molecules (61.35–69.49%) are “lost” during the essential hybridization step of the fishing protocol, suggesting its suitability for high copy number samples only. While the bait capture method may be useful in the study of polymerase chain reaction (PCR) inhibited DNA samples as previously suggested, it is necessary to carefully weigh this possible advantage against the degree of expected DNA loss and the non-selectivity of the method for targeted over non-targeted DNA.

Rapid and effective isolation of candidate sequences for development of microsatellite markers in 30 fish species by using kit-based target capture and multiplexed parallel sequencing

Recent advances in next-generation sequencing (NGS) technology have accelerated the development of microsatellite markers for wildlife conservation genetics. Although the discovery of microsatellite-containing sequences based on NGS is more efficient with sequencing of a microsatellite-enriched library than with whole-genome shotgun sequencing, the process of constructing a microsatellite-enriched library is somewhat complicated. Therefore, many researchers prefer to use external services for the microsatellite-enrichment, which requires more time. To facilitate the rapid and effective development of novel microsatellite markers, we attempted to simplify the process of constructing a microsatellite-enriched library for multiplexed parallel sequencing. To capture microsatellite-containing sequences, we applied an easy-to-use commercially available kit for the hybridization and wash steps. After preparing shotgun libraries of 30 fish species for NGS, we captured microsatellite-containing DNA fragments directly from the shotgun libraries by using the commercially available kit. Next, three runs of multiplexed parallel sequencing were conducted on the 454 GS Junior platform. The resulting sequences for each species included high proportions of microsatellite-containing sequences (from 46 to 79%). Thus, sufficient numbers of primer sets, ranging from 1029 to 6606, were effectively designed for each species. Microsatellite capture and sequencing were completed in about a week, so the time required was substantially reduced. To validate the effectiveness of our strategy, we screened 44 potential primer sets designed for ayu (Plecoglossus altivelis). The results of polymorphisms revealed that allelic variability at 23 markers will be useful for studying population structure. These results prove the effectiveness of our improved approach for microsatellite marker development.

SERS-active metal-dielectric nanostructures integrated in microfluidic devices for label-free quantitative detection of miRNA.

In this work, SERS-based microfluidic PDMS chips integrating silver-coated porous silicon membranes were used for the detection and quantitation of microRNAs (miRNAs), which consist of short regulatory non-coding RNA sequences typically over- or under-expressed in connection with several diseases such as oncogenesis. In detail, metal-dielectric nanostructures which provide noticeable Raman enhancements were functionalized according to a biological protocol, adapted and optimized from an enzyme-linked immunosorbent assay (ELISA), for the detection of miR-222. Two sets of experiments based on different approaches were designed and performed, yielding a critical comparison. In the first one, the labelled target miRNA is revealed through hybridization to a complementary thiolated DNA probe, immobilized on the silver nanoparticles. In the second one, the probe is halved into shorter strands (half1 and half2) that interact with the complementary miRNA in two steps of hybridization. Such an approach, taking advantage of the Raman labelling of half2, provides a label-free analysis of the target. After suitable optimisation of the procedures, two calibration curves allowing quantitative measurements were obtained and compared on the basis of the SERS maps acquired on the samples loaded with several miRNA concentrations. The selectivity of the two-step assay was confirmed by the detection of target miR-222 mixed with different synthetic oligos, simulating the hybridization interference coming from similar sequences in real biological samples. Finally, that protocol was applied to the analysis of miR-222 in cellular extracts using an optofluidic multichamber biosensor, confirming the potentialities of SERS-based microfluidics for early-cancer diagnosis.

Detection of Transcriptionally Active Mycotoxin Gene Clusters: DNA Microarray.

Various bioanalytical tools including DNA microarrays are frequently used to map global transcriptional changes in mycotoxin producer filamentous fungi. This effective hybridization-based transcriptomics technology helps researchers to identify genes of secondary metabolite gene clusters and record concomitant gene expression changes in these clusters initiated by versatile environmental conditions and/or gene deletions. Such transcriptional data are of great value when future mycotoxin control technologies are considered and elaborated. Giving the readers insights into RNA extraction and DNA microarray hybridization steps routinely used in our laboratories and also into the normalization and evaluation of primary gene expression data, we would like to contribute to the interlaboratory standardization of DNA microarray based transcriptomics studies being carried out in many laboratories worldwide in this important field of fungal biology.

Dual signal amplification for highly sensitive hybridization microassays on chemically activated surfaces

A highly sensitive hybridization assay is developed for detection of specific sequences of genomic DNA without thermal or isothermal chain reaction cycling. The assays in microarray format were performed on polymeric surfaces derived from the audio-video disc industry. In order to achieve this challenge, a photo-chemical activation method for the covalent immobilization of DNA oligomers is developed. In addition, dual signal amplification approach, by integrating tyramide with silver-assisted enhancement for the detection of DNA at low pg/μL level, is presented.The methodology was two orders of magnitude more sensitive in comparison with the non-tyramide amplified hybridization assay. To demonstrate the feasibility of this approach, complementary oligomers and PCR products as a model targets were employed, achieving a quantification limit of 0.30pM and 0.2pg/μL, respectively. The reproducibility was good (RSD <18%) and the hybridization assay took 65min.As a proof of concept, the detection of genomic DNA of Salmonella spp., as food-borne pathogen model, was addressed. The principle of assay is based on massive biotinylation and fragmentation of genomic DNA prior the hybridization step. The biotinylated fragments, ranging from 400 to 750bp hybridize with the covalently immobilized specific probes (38 mer long), generating an analytical signal after tyramide amplification proportional to the concentration of DNA. The signal is quantified with a disc drive as detector. The results showed that the methodology is a promising alternative for DNA sensing without thermal or isothermal cycling based amplification.

1529P - DLK1-DIO3 imprinted cluster in lung cancer

According to classical Mendelian laws, the majority of genes in a human cell are inherited in two functionally equivalent parental copies. However, there is a small subset of genes with one turned-off copy in a parent-of-origin-dependent manne. This phenomenon, known as genomic imprinting, is an epigenetic process that involves monoallelic expression. Some genes are imprinted across the complete genome sequence, but the vast majority of imprinted genes are clustered. In this sense, the DLK1-DIO3 imprinted cluster is located on chromosome 14q32.2. This region includes protein-coding genes (DLK1, RTL1 and DIO3), long non-coding RNAs (MEG3, MEG8, MEG9 and LINC00524), two large clusters of miRNAs, two families of small nucleolar RNAs (SNORD113 and SNORD114) and several pseudogenes. Aberrations involving some components of the DLK1-DIO3 cluster have been linked to pathological processes. The purpose of this study was to assess the role of the DLK1-DIO3 cluster in non-small cell lung cancer. DNA methylation of gene clusters was analyzed by Illumina in tumour and matched control (healthy) tissue from 47 patients with lung cancer. DNA was extracted using the QIAamp DNA Mini Kit. For each assay, 500 ng of DNA was treated with sodium bisulfate using EZ DNA Methylation™ Kit and cleaned with ZR-96 DNA Clean-up Kit™, before standard Illumina amplification, hybridization, and imaging steps. Methylation data were processed using the RnBeads R package. Patients with lung cancer showed three deregulated protein-coding genes: one was hypermethylated (DIO3) and two were hypomethylated (DLK1 and RTL1). Statistically significant differences (adjusted p-value < 0.05) were also detected in two different families of SNORDs (SNORD113 and SNORD114), with the exception of SNORD114-30 that showed similar but non-significant pattern of DNA hypomethylation (p = 0.07. Similarly, two miRNA clusters and four lncRNAs (MEG3, MEG8, MEG9 and LINC00524) were found to be significantly less methylated in tumors as compared to non-tumoral tissue. Our results strongly imply that hypomethylation of this cluster may be a key target in unravelling of the mechanism of lung cancer.

DNA Electrochemical Hybridization Detection in Droplets Using Gold Ultramicroelectrodes in a Two-Electrode Configuration

A 23-base DNA probe monolayer was self-assembled on a 25-μm gold microelectrode via thiol adsorption. Long-range electron transfer and the use of the redox methylene blue as DNA intercalator were chosen for the monitoring of the hybridization step. The electrochemical properties of the sensor were screened with the [Fe(III)(CN)6]3-/[Fe(II)(CN)6]4- redox couple in cyclic voltammetry in a two-electrode configuration well adapted in the case of microliter biological samples. A study of the stability of the self-assembled monolayer is included in this work. The femtomolar limit of detection for DNA target quantification was deduced from the current density measured and blank measurements depicting the desorption rate of the thiolated DNA probes.

Experimental and finite element studies of bolted, bonded and hybrid step lap joints of thick carbon fibre/epoxy panels used in aircraft structures

Abstract A prior study conducted by the authors had investigated the behaviour of thin composite double lap joint repairs. This investigation was an extension to the previous study and focused on analysing the behaviour of thick step lap joint repairs which are more complicated in nature. Finite element analysis (FEA) was performed to verify the static and fatigue strength of bolted, bonded and hybrid joint configurations. Thick carbon fibre/epoxy laminates and aerospace grade film adhesive and fasteners were selected. Several configurations were considered in this case. These include varying the number of fasteners in the joint region as well as inclusion of bondline defects. Adhesive non-linear material properties, fastener contacts and frictional forces were all included in the three dimensional (3D) finite element (FE) models. The progressive failure process was simulated and the Multicontinuum Theory (MCT) was used to determine the stress states in all specimens considered. The strain energy release rate (SERR) as a function of crack length was also utilised to gain further understanding into the fatigue behaviour. Overall the FE models are able to accurately predict the bonded, bolted and hybrid joint strengths. SERR results suggest it is vital to place fasteners closer to the ends of the overlap to suppress the peak peeling stresses and to delay the effects of early crack initiation. This overall improves the longevity of a conventional bonded joint design.

Spectral imaging to visualize higher-order genomic organization

ABSTRACTA concern in the field of genomics is the proper interpretation of large, high-throughput sequencing datasets. The use of DNA FISH followed by high-content microscopy is a valuable tool for validation and contextualization of frequently occurring gene pairing events at the single-cell level identified by deep sequencing. However, these techniques possess certain limitations. Firstly, they do not permit the study of colocalization of many gene loci simultaneously. Secondly, the direct assessment of the relative position of many clustered gene loci within their respective chromosome territories is impossible. Thus, methods are required to advance the study of higher-order nuclear and cellular organization. Here, we describe a multiplexed DNA FISH technique combined with indirect immunofluorescence to study the relative position of 6 distinct genomic or cellular structures. This can be achieved in a single hybridization step using spectral imaging during image acquisition and linear unmixing. Here, we detail the use of this method to quantify gene pairing between highly expressed spliceosomal genes and compare these data to randomly positioned in silico simulated gene clusters. This is a potentially universally applicable approach for the validation of 3C-based technologies, deep imaging of spatial organization within the nucleus and global cellular organization.