Early detection of cancer biomarkers can reduce cancer mortality rate. miRNAs are small non-coding RNAs whose expression changes upon the onset of various types of cancer. Biosensors that specifically detect such biomarkers can be engineered and integrated into point-of-care devices (POC) using label-free detection, high sensibility and compactness. In this paper, a new engineered Molecular Beacon (MB) construct used to detect miRNAs is presented. Such a construct is immobilized onto biosensor surfaces in a covalent and spatially oriented way using the photonic technology Light Assisted Molecular Immobilization (LAMI). The construct consists of a Cy3 labelled MB covalently attached to a light-switchable peptide. One MB construct contains a poly-A sequence in its loop region while the other contains a sequence complementary to the cancer biomarker miRNA-21. The constructs have been characterized by UV-Vis spectroscopy, mass spectrometry and HPLC. LAMI led to the successful immobilization of the engineered constructs onto thiol functionalized optically flat quartz slides and Silicon on Insulator (SOI) sensor surfaces. The immobilized Cy3 labelled MB construct has been imaged using confocal fluorescence microscopy (CFM). The bioavailability of the immobilized engineered MB biosensors was confirmed through specific hybridization with the Cy5 labelled complementary sequence and imaged by CFM and FRET. Hybridization kinetics have been monitored using steady state fluorescence spectroscopy. The label-free detection of miRNA-21 was also achieved by using integrated photonic sensing structures. The engineered light sensitive constructs can be immobilized onto thiol reactive surfaces and are currently being integrated in a POC device for the detection of cancer biomarkers.

An experimental study of the influence of the conformational change suffered by molecular beacon (MB) probes—upon the biorecognition of nucleic acid target oligonucleotides over evanescent wave photonic sensors—is reported. To this end, high sensitivity photonic sensors based on silicon photonic bandgap (PBG) structures were used, where the MB probes were immobilized via their 5′ termination. Those MBs incorporate a biotin moiety close to their 3′ termination in order to selectively bind a streptavidin molecule to them. The different photonic sensing responses obtained toward the target oligonucleotide detection, when the streptavidin molecule was bound to the MB probes or not, demonstrate the conformational change suffered by the MB upon hybridization, which promotes the displacement of the streptavidin molecule away from the surface of the photonic sensing structure.

A label-free sensor, based on the combination of silicon photonic bandgap (PBG) structures with immobilized molecular beacon (MB) probes, is experimentally developed. Complementary target oligonucleotides are specifically recognized through hybridization with the MB probes on the surface of the sensing structure. This combination of PBG sensing structures and MB probes demonstrates an extremely high sensitivity without the need for complex PCR-based amplification or labelling methods.

This session will review the role of microRNAs in the molecular pathogenesis of osteoarthritis (OA). It will present data on the regulation of microRNAs by relevant factors and their impact on intracellular signalling. Key targets of microRNAs in OA will also be explored. MicroRNAs have also been reported as circulating biomarkers of disease, and the utility of this approach and how it informs on the functional role of microRNAs will be discussed. Finally, the potential for microRNAs to be used therapeutically and the hurdles which need to be overcome to do this will be addressed. The focus of the Clark lab has been on the role and function of microRNAs in cartilage, particularly miR-455, the miR-29 family, miR-140 and miR-3085. MicroRNA-455 is genomically located within an intron of COL27A1. Collagen XXVII is expressed in cartilage, suggesting function of the miR in this tissue. We initially described a role for miR-455 in TGFbeta signalling, but have more recently uncovered function in Wnt signalling and in the regulation of Sirt1. The miR-29 family have been well-researched and are known e.g. to regulate collagen gene expression, giving them a role in fibroses. We identified miR-29b as one of only two miRs which was regulated at an early time point after surgery in the murine DMM ('destabilisation of the medial meniscus') model of OA. Potential targets of miR-29 were regulated in the opposite direction to the miR, suggesting function. The miR-29 family were regulated in many models of chondrocyte differentiation and in human end-stage OA. In chondrocytes we have shown that miR-29 is negatively regulated by Sox9 and negatively regulates a number of key intracellular signalling pathways in OA. We have identified novel direct targets in the Wnt pathway and we have also shown that miR-29 directly targets a number of ADAMTS protease genes. We used RNA-Seq to explore the full range of miRs expressed by human articular chondrocytes from OA patients. This showed that the so-called passenger strand of miR-140, miR-140-3p was more highly expressed than the guide strand, miR-140-5p in newly isolated osteoarthritic chondrocytes. We have now shown that miR-140-3p directly targets a number of enzymes in the heparan sulphate proteoglycan synthesis pathway. These studies also identified miR-3085 in human chondrocytes. This miR had only previously been annotated in rodents where it was presumed intergenic. However, in man it is located in the final intron of the CRTAC1 gene, which codes for cartilage acidic protein 1. We have shown it to directly target ITGA5, the integrin alpha5 gene, but it also strongly induces interleukin-1 signalling in chondrocytes.

Purpose: Using deep sequencing to identify novel miRNAs in human osteoarthritis cartilage, a sequence annotated as miR-3085-3p in mice and rats, was identified for the first time in man. In man, miR-3085 is located within in intron of the CRTAC1 gene which is expressed in chondrocytes. The purpose of this study was to characterize this microRNA and to define its function. Methods: Northern blot and ectopic overexpression of a hairpin structure of miR-3085 followed by qRT-PCR were used to prove that miR-3085 is a miRNA. Expression of miR-3085 and CRTAC1 was investigated by qRT-PCR. Expression was analysed in SW1353 chondrosarcoma cells and primary human articular chondrocytes stimulated with TGFβ1, IL-1β or Wnt3A; or after manipulation of SOX9, p50, p65. Functional interaction between the miRNA and Smad and NFκB pathways was explored using luciferase reporters and confirmed by Western blot. Results: Expression of the mature miR-3085-3p was strongly increased when a hairpin structure consisting of mature miR-3085 strands together with the flanking regions was overexpressed. The expression of miR-3085-3p and CRTAC1 was regulated similarly. TGFβ1 and IL-1β increased expression of miR-3085-3p whilst Wnt3a decreased its expression in SW1353, and human primary articular chondrocytes in both monolayer and micromass culture. Ectopic expression of either P65 or SOX9 increased the expression of miR-3085-3p. Functionally, miR-3085-3p acts as a positive regulator of Smad and NFκB signalling. MicroRNA-3805-3p increased the phosphorylation of p65 and the expression of MMP13 upon IL-1β treatment. MicroRNA-3085-3p supressed the expression of COL2A1 and ACAN (aggrecan). Conclusions: These data experimentally prove miR-3085 as a miRNA. It is regulated by a number of factors known to have a role in cartilage homeostasis and it has functional impact on relevant signalling pathways. Future work will continue to investigate the factors controlling its expression and function in chondrocyte homeostasis and osteoarthritis.

Small non-coding RNAs known as microRNAs (miRs) have recently been recognised as important regulators of gene expression in human cells. Whilst originally mooted to fine-tune gene expression, it is now clear that they can have profound effects on physiology and disease. A role for microRNAs in chondrogenesis and osteoarthritis has become clear over the last decade. Deletion of the machinery for microRNA biogenesis specifically in the cartilage of mice has shown that, as a class, microRNAs influence the development of the skeleton and homeostasis of articular cartilage. Identifying roles for individual microRNAs has been more difficult, with the main in vivo data coming from miR-140. This session will review the role of microRNAs in the molecular pathogenesis of osteoarthritis (OA). It will present data on the regulation of microRNAs by relevant factors and their impact on intracellular signalling. Key targets of microRNAs in OA will also be explored. It will touch upon the use of microRNAs as circulating biomarkers of disease and the potential for microRNA-based therapy. The focus of the Clark lab has been on the role and function of microRNAs in cartilage, particularly miR-455, the miR-29 family, miR-140 and miR-3085. MicroRNA-455 is genomically located within an intron of COL27A1. Collagen XXVII is expressed in cartilage, suggesting function of the miR in this tissue. We initially described a role for miR-455 in TGFbeta signalling, but have more recently uncovered function in Wnt signalling and in the regulation of Sirt1. The miR-29 family have been well-researched and are known e.g. to regulate collagen gene expression, giving them a role in fibroses. We identified miR-29b as one of only two miRs which was regulated at an early time point after surgery in the murine DMM (“destabilisation of the medial meniscus”) model of OA. Potential targets of miR-29 were regulated in the opposite direction to the miR, suggesting function. The miR-29 family were regulated in many models of chondrocyte differentiation and in human end-stage OA. In chondrocytes we have shown that miR-29 is negatively regulated by Sox9 and negatively regulates a number of key intracellular signalling pathways in OA. We have identified novel direct targets in the Wnt pathway and we have also shown that miR-29 directly targets a number of ADAMTS protease genes. We used RNA-Seq to explore the full range of miRs expressed by human articular chondrocytes from OA patients. This showed that the so-called passenger strand of miR-140, miR-140-3p was more highly expressed than the guide strand, miR-140-5p in newly isolated osteoarthritic chondrocytes. We have now shown that miR-140-3p directly targets a number of enzymes in the heparan sulphate proteoglycan synthesis pathway. These studies also identified miR-3085 in human chondrocytes. This miR had only previously been annotated in rodents where it was presumed intergenic. However, in man it is located in the final intron of the CRTAC1 gene, which codes for cartilage acidic protein 1. We have shown it to directly target ITGA5, the integrin alpha5 gene, but it also strongly induces interleukin-1 signalling in chondrocytes. Disclosure of Interest None declared

Objective: To use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function.Design: A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray and computational analysis, validated using 3'-UTR-luciferase reporter plasmids. Protein levels were assessed by western blot and functional analysis by cell adhesion.Results: We identified 990 known miRNAs and 1621 potential novel miRNAs in human osteoarthritic chondrocytes, 60 of the latter were expressed in all samples assayed. MicroRNA-140-3p was the most highly expressed microRNA in osteoarthritic cartilage. Sixteen novel candidate miRNAs were analysed further, of which six remained after northern blot analysis. Three novel miRNAs were regulated across models of chondrogenesis, chondrocyte differentiation or cartilage injury. One sequence (novel #11), annotated in rodents as microRNA-3085-3p, was preferentially expressed in cartilage, dependent on chondrocyte differentiation and, in man, is located in an intron of the cartilage-expressed gene CRTAC-1. This microRNA was shown to target the ITGA5 gene directly (which encodes integrin alpha5) and inhibited adhesion to fibronectin (dependent on alpha5beta1 integrin).Conclusion: Deep sequencing has uncovered many potential microRNA candidates expressed in human cartilage. At least three of these show potential functional interest in cartilage homeostasis and osteoarthritis (OA). Particularly, novel #11 (microRNA-3085-3p) which has been identified for the first time in man. (C) 2015 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International.

Purpose: Osteoarthritis is a painful and debilitating disease characterised by loss of cartilage, aberrant gene expression and bone remodelling. MicroRNAs are very small, 20-24 nucleotide non-coding RNA molecules that post-transcriptionally regulate gene expression. There is evidence to suggest that they can affect chondrogenesis and both the initiation and progression of osteoarthritis, however the exact mechanisms are still mostly undetermined. In this study we discovered novel microRNAs, using next generation deep sequencing, with a role in chondrogenesis and osteoarthritis, and subsequently elucidate their function. Methods: Novel microRNAs expressed in human osteoarthritic articular cartilage were identified using deep sequencing with Illumina's GAIIX system and validated in primary chondrocyte culture in vitro using real time PCR. Expression of the novel microRNAs were also measured in chondrogenesis assays and across diverse human tissues. Northern blotting was utilised to validate size of novel microRNAs, and bioinformatics analysis was employed in initial target searches to establish function. An Illumina total genome microarray will be used alongside the bioinformatics data to confirm microRNA targets and thereby elucidate function. Results: Deep sequencing of the small RNA pool from chondrocytes extracted from primary human osteoarthritic cartilage identified ∼1600 completely novel miRNAs. Sixteen of these have been validated in cultured primary human chondrocytes and across a human tissue panel using qRT-PCR. Three novel microRNAs were proven by northern blotting to be of correct size. A bioinformatics target search using RStudios combined with freely available online software has shown a range of targets for these three such as IL6ST, CD44, and MMP16 which are known to be involved in osteoarthritis and chondrocyte development. Conclusions: The entire microRNA population of cartilage is still not fully determined, however the sequencing used in this project provided substantially less bias than many deep sequencing experiments. We found a number of novel microRNAs to be regulated during chondrogenesis and to have differential expression between osteoarthritic and non-diseased articular cartilage tissue. Potential targets such as IL-6ST and CD44 further suggest a role in osteoarthritis or chondrogenesis. The significant number of novel microRNAs discovered and their subsequent validation provides new insights into how microRNAs may regulate both cartilage homeostasis and contribute to osteoarthritis.

Amyotrophic lateral sclerosis (ALS) is a progressive and seriously disabling adult-onset neurological disease. Ninety percent of ALS patients are sporadic cases (sALS) with no clear genetic linkage. Accumulating evidence indicates that various microRNAs (miRNAs), expressed in a spatially and temporally controlled manner in the brain, play a key role in neuronal development. In addition, microRNA dysregulation contributes to some mental disorders and neurodegeneration diseases. In our research, the expression of one selected miRNA, miR-338-3p, which previously we have found over-expressed in blood leukocytes, was studied in several different tissues from sALS patients. For the first time, we detected a specific microRNA disease-related upregulation, miR-338-3p, in blood leukocytes as well in cerebrospinal fluid, serum, and spinal cord from sALS patients. Besides, staining of in situ hybridization showed that the signals of miR-338-3p were localized in the grey matter of spinal cord tissues from sALS autopsied patients. We propose that miRNA profiles found in tissue samples from sALS patients can be relevant to understand sALS pathogenesis and lead to set up effective biomarkers for sALS early diagnosis.

OBJECTIVE:To use an in vitro model of chondrogenesis to identify microRNAs (miRNAs) with a functional role in cartilage homeostasis. METHODS:The expression of miRNAs was measured in the ATDC5 cell model of chondrogenesis using microarray and was verified using quantitative reverse transcription-polymerase chain reaction. MicroRNA expression was localized by in situ hybridization. Predicted miRNA target genes were validated using 3'-untranslated region-Luc reporter plasmids containing either wild-type sequences or mutants of the miRNA target sequence. Signaling through the Smad pathway was measured using a (CAGA)(12) -Luc reporter. RESULTS:The expression of several miRNAs was regulated during chondrogenesis. These included 39 miRNAs that are coexpressed with miRNA-140 (miR-140), which is known to be involved in cartilage homeostasis and osteoarthritis (OA). Of these miRNAs, miR-455 resides within an intron of COL27A1 that encodes a cartilage collagen. When human OA cartilage was compared with cartilage obtained from patients with femoral neck fractures, the expression of both miR-140-5p and miR-455-3p was increased in OA cartilage. In situ hybridization showed miR-455-3p expression in the developing limbs of chicks and mice and in human OA cartilage. The expression of miR-455-3p was regulated by transforming growth factor β (TGFβ) ligands, and miRNA regulated TGFβ signaling. ACVR2B, SMAD2, and CHRDL1 were direct targets of miR-455-3p and may mediate its functional impact on TGFβ signaling. CONCLUSION:MicroRNA-455 is expressed during chondrogenesis and in adult articular cartilage, where it can regulate TGFβ signaling, suppressing the Smad2/3 pathway. Diminished signaling through this pathway during the aging process and in OA chondrocytes is known to contribute to cartilage destruction. We propose that the increased expression of miR-455 in OA exacerbates this process and contributes to disease pathology.

Within the lateral organisation of plasma membranes of polarized cell types there exist heterogenous microdomains of distinct lipid composition, the small size of which (10–200nm) makes them difficult to discern with traditional microscopic techniques, but which can be distinguished on the basis of lipid packing. These microdomains or rafts can be concentrated in larger more visible liquid-ordered regions, particularly by cross-linking of their constituents as in the immunological synapse or in features of the polarized cell such as pseudopodia or flagella. One technique, Laurdan fluorescence microscopy, has proven very useful for distinguishing such regions but has hitherto relied on 2-photon confocal microscopy. This has to some extent limited its utility to living systems and its widespread adoption in studying membrane dynamics on the surface of living cells. Here we describe and validate the adaptation of a standard widefield fluorescence microscope for live imaging of Laurdan stained cell membranes.

microRNAs are short RNA molecules that are often expressed in specific tissues and regulate a variety of developmental processes. We used locked nucleic acid probes in in situ hybridisation reactions to study the distribution of microRNA-449 (miR449) during mouse embryonic development in order to obtain clues about its function/s. Between E9.75 and E11.5, miR449 was found to be expressed specifically in the developing roof plate of the fourth ventricle within the domain of roof plate marker, Lmx1a. From E12.5 onwards, this expression became restricted to the epithelial cell layer of the fourth ventricle choroid plexus. MiR449 also became detectable specifically in the choroid plexuses of the lateral and 3rd ventricles at E13.5 and E15.5, respectively. Northern blot analysis of adult brain also showed a selective and enriched expression in the choroid plexus tissue. Potential target genes regulated by miR449 were selected for experimental validation in luciferase-reporter assays and the transcription factor E2f5, which regulates CSF production, was verified as a miR449 target gene. Taken together, these findings suggest that miR449 has a specific role in the development and functioning of choroid plexuses.

MicroRNAs (miRNAs) are recently discovered short regulatory RNA molecules representing a new layer in posttranscriptional gene expression regulation. Although more than 450 human miRNAs have been identified, only a very few of them have been characterized in detail. The precise understanding of miRNA-mediated processes requires the reliable spatial and temporal analyses of miRNA accumulation at tissue/cell level. However, the detection of miRNAs by in situ hybridization (ISH) is technically challenging because of the small size of target sequences. It was shown recently that locked nucleic acid nucleotide-containing probes can anneal to short nucleic acids with high specificity. This enabled several research groups to analyze the expression patterns of miRNAs in both plant and animal tissues. This review focuses on the results of recent publications on the detection of miRNAs by ISH.

MicroRNAs (miRNAs) are small regulatory molecules suppressing mRNA activity in metazoans. Here we describe two new miRNAs cloned from brain tissue of mouse embryos. These miRNAs are expressed mainly during embryogenesis and specifically in the central nervous system. We also established the expression patterns of three recently identified miRNAs that were found in our short RNA library. All of them were expressed in the brain and spinal chord but while miR-410 and miR-431 were central nervous system specific, miR-500 was also expressed in limb buds. In addition, the expression of miR-500 in limb buds showed very strong asymmetry in favour of the left hand side.

The microRNAs (miRNAs) are recently discovered short, noncoding RNAs, that regulate gene expression in metazoans. We have cloned short RNAs from chicken embryos and identified five new chicken miRNA genes. Genome analysis identified 17 new chicken miRNA genes based on sequence homology to previously characterized mouse miRNAs. Developmental Northern blots of chick embryos showed increased accumulation of most miRNAs analyzed from 1.5 days to 5 days except, the stem cell-specific mir-302, which was expressed at high levels at early stages and then declined. In situ analysis of mature miRNAs revealed the restricted expression of mir-124 in the central nervous system and of mir-206 in developing somites, in particular the developing myotome. In addition, we investigated how miR-206 expression is controlled during somite development using bead implants. These experiments demonstrate that fibroblast growth factor (FGF) -mediated signaling negatively regulates the initiation of mir-206 gene expression. This may be mediated through the effects of FGF on somite differentiation. These data provide the first demonstration that developmental signaling pathways affect miRNA expression. Thus far, miRNAs have not been studied extensively in chicken embryos, and our results show that this system can complement other model organisms to investigate the regulation of many other miRNAs.

MicroRNAs (miRNA) are short RNA molecules regulating the expression of specific mRNAs. We investigated the expression pattern and potential targets of mouse miR-140 and found that miR-140 is specifically expressed in cartilage tissues of mouse embryos during both long and flat bone development. MiR-140 expression was detected in the limbs of E11.5 embryos in the primorida of future bones both in the fore and hindlimb and across autopod, zeugopod and stylopod. All digits of E14.5 fore- and hindlimbs showed accumulation of miR-140, except the first digit of the hindlimb. MiR-140 expression was also detected in the cartilagenous base of E17.5 skulls and in the sternum, the proximal rib heads and the developing vertebral column of E15.5 embryos. A potential target of miR-140, histone deacetylase 4, was validated experimentally and the possible role of miR-140 in long bone development is discussed.

Background Many new types of expanding or fragmenting handgun ammunition have been developed. Knowledge of these unusual bullets may aid in the management of patients and their wounds. Methods Eleven different expanding or fragmenting.45 caliber bullets and a nondeforming, full metal jacketed bullet for comparison were fired multiple times from the same handgun into both a water reservoir and ordnance gelatin. Performance was observed and recorded. Muzzle velocities were measured using a chronograph. Bullets were disassembled and cross-sectioned to facilitate inspection. Results The distinguishing surface and internal features of each bullet are described. When fired into water and ordnance gelatin, the bullets reliably expanded to 1.49 to 1.89 times their prefired diameters. Rates of kinetic energy loss of bullets of equal mass fired into ordnance gelatin were plotted. Full metal jacketed bullets penetrated twice as deeply as deforming bullets. Jackets of some of the expanding bullets separated when fired into water. Conclusion Expanding/fragmenting bullets produce larger, shallower wounds than do full metal jacketed bullets. Recognition of the wound and roentgenographic appearances of these unusual bullets will help the trauma surgeon to properly treat gunshot victims. Because of the occurrence of jacket separation in water, ordnance gelatin should be used for optimal evaluation of bullet performance.