Genetic association analysis of miRNA SNPs implicates MIR145 in breast cancer susceptibility

Background MicroRNAs (miRNAs) are important small non-coding RNA molecules that regulate gene expression in cellular processes related to the pathogenesis of cancer. Genetic variation in miRNA genes could impact their synthesis and cellular effects and single nucleotide polymorphisms (SNPs) are one example of genetic variants studied in relation to breast cancer. Studies aimed at identifying miRNA SNPs (miR-SNPs) associated with breast malignancies could lead towards further understanding of the disease and to develop clinical applications for early diagnosis and treatment. Methods We genotyped a panel of 24 miR-SNPs using multiplex PCR and chip-based matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis in two Caucasian breast cancer case control populations (Primary population: 173 cases and 187 controls and secondary population: 679 cases and 301 controls). Association to breast cancer susceptibility was determined using chi-square (X 2 ) and odds ratio (OR) analysis. Results Statistical analysis showed six miR-SNPs to be non-polymorphic and twelve of our selected miR-SNPs to have no association with breast cancer risk. However, we were able to show association between rs353291 (located in MIR145) and the risk of developing breast cancer in two independent case control cohorts (p = 0.041 and p = 0.023). Conclusions Our study is the first to report an association between a miR-SNP in MIR145 and breast cancer risk in individuals of Caucasian background. This finding requires further validation through genotyping of larger cohorts or in individuals of different ethnicities to determine the potential significance of this finding as well as studies aimed to determine functional significance. Keywords: Association analysis; Breast cancer; microRNA; miR-SNPs; MIR145

miR-514a regulates the tumour suppressor NF1 and modulates BRAFi sensitivity in melanoma

To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E-targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.

Muscular activity and fatigue in lower-limb and trunk muscles during different sit-to-stand tests

Sit-to-stand (STS) tests measure the ability to get up from a chair, reproducing an important component of daily living activity. As this functional task is essential for human independence, STS performance has been studied in the past decades using several methods, including electromyography. The aim of this study was to measure muscular activity and fatigue during different repetitions and speeds of STS tasks using surface electromyography in lower-limb and trunk muscles. This cross-sectional study recruited 30 healthy young adults. Average muscle activation, percentage of maximum voluntary contraction, muscle involvement in motion and fatigue were measured using surface electrodes placed on the medial gastrocnemius (MG), biceps femoris (BF), vastus medialis of the quadriceps (QM), the abdominal rectus (AR), erector spinae (ES), rectus femoris (RF), soleus (SO) and the tibialis anterior (TA). Five-repetition STS, 10-repetition STS and 30-second STS variants were performed. MG, BF, QM, ES and RF muscles showed differences in muscle activation, while QM, AR and ES muscles showed significant differences in MVC percentage. Also, significant differences in fatigue were found in QM muscle between different STS tests. There was no statistically significant fatigue in the BF, MG and SO muscles of the leg although there appeared to be a trend of increasing fatigue. These results could be useful in describing the functional movements of the STS test used in rehabilitation programs, notwithstanding that they were measured in healthy young subjects.

Fusion transcript loci share many genomic features with non-fusion loci

Background Fusion transcripts are found in many tissues and have the potential to create novel functional products. Here, we investigate the genomic sequences around fusion junctions to better understand the transcriptional mechanisms mediating fusion transcription/splicing. We analyzed data from prostate (cancer) cells as previous studies have shown extensively that these cells readily undergo fusion transcription. Results We used the FusionMap program to identify high-confidence fusion transcripts from RNAseq data. The RNAseq datasets were from our (N = 8) and other (N = 14) clinical prostate tumors with adjacent non-cancer cells, and from the LNCaP prostate cancer cell line that were mock-, androgen- (DHT), and anti-androgen- (bicalutamide, enzalutamide) treated. In total, 185 fusion transcripts were identified from all RNAseq datasets. The majority (76 %) of these fusion transcripts were ‘read-through chimeras’ derived from adjacent genes in the genome. Characterization of sequences at fusion loci were carried out using a combination of the FusionMap program, custom Perl scripts, and the RNAfold program. Our computational analysis indicated that most fusion junctions (76 %) use the consensus GT-AG intron donor-acceptor splice site, and most fusion transcripts (85 %) maintained the open reading frame. We assessed whether parental genes of fusion transcripts have the potential to form complementary base pairing between parental genes which might bring them into physical proximity. Our computational analysis of sequences flanking fusion junctions at parental loci indicate that these loci have a similar propensity as non-fusion loci to hybridize. The abundance of repetitive sequences at fusion and non-fusion loci was also investigated given that SINE repeats are involved in aberrant gene transcription. We found few instances of repetitive sequences at both fusion and non-fusion junctions. Finally, RT-qPCR was performed on RNA from both clinical prostate tumors and adjacent non-cancer cells (N = 7), and LNCaP cells treated as above to validate the expression of seven fusion transcripts and their respective parental genes. We reveal that fusion transcript expression is similar to the expression of parental genes. Conclusions Fusion transcripts maintain the open reading frame, and likely use the same transcriptional machinery as non-fusion transcripts as they share many genomic features at splice/fusion junctions.

Development of innovative robust stability enhancement algorithms for distribution systems containing distributed generators

This project was a step forward in improving the voltage profile of traditional low voltage distribution networks with high photovoltaic generation or high peak demand. As a practical and economical solution, the developed methods use a Dynamic Voltage Restorer or DVR, which is a series voltage compensator, for continuous and communication-less power quality enhancement. The placement of DVR in the network is optimised in order to minimise its power rating and cost. In addition, new approaches were developed for grid synchronisation and control of DVR which are integrated with the voltage quality improvement algorithm for stable operation.

Distributed Robotic Vision as a service

The vision sense of standalone robots is limited by line of sight and onboard camera capabilities, but processing video from remote cameras puts a high computational burden on robots. This paper describes the Distributed Robotic Vision Service, DRVS, which implements an on-demand distributed visual object detection service. Robots specify visual information requirements in terms of regions of interest and object detection algorithms. DRVS dynamically distributes the object detection computation to remote vision systems with processing capabilities, and the robots receive high-level object detection information. DRVS relieves robots of managing sensor discovery and reduces data transmission compared to image sharing models of distributed vision. Navigating a sensorless robot from remote vision systems is demonstrated in simulation as a proof of concept.

Structural and functional characterization of dendritic arbors and GABAergic synaptic inputs on interneurons and principal cells in the rat basolateral amygdala

The basolateral amygdala (BLA) is a complex brain region associated with processing emotional states, such as fear, anxiety, and stress. Some aspects of these emotional states are driven by the network activity of synaptic connections, derived from both local circuitry and projections to the BLA from other regions. Although the synaptic physiology and general morphological characteristics are known for many individual cell types within the BLA, the combination of morphological, electrophysiological, and distribution of neurochemical GABAergic synapses in a three-dimensional neuronal arbor has not been reported for single neurons from this region. The aim of this study was to assess differences in morphological characteristics of BLA principal cells and interneurons, quantify the distribution of GABAergic neurochemical synapses within the entire neuronal arbor of each cell type, and determine whether GABAergic synaptic density correlates with electrophysiological recordings of inhibitory postsynaptic currents. We show that BLA principal neurons form complex dendritic arborizations, with proximal dendrites having fewer spines but higher densities of neurochemical GABAergic synapses compared with distal dendrites. Furthermore, we found that BLA interneurons exhibited reduced dendritic arbor lengths and spine densities but had significantly higher densities of putative GABAergic synapses compared with principal cells, which was correlated with an increased frequency of spontaneous inhibitory postsynaptic currents. The quantification of GABAergic connectivity, in combination with morphological and electrophysiological measurements of the BLA cell types, is the first step toward a greater understanding of how fear and stress lead to changes in morphology, local connectivity, and/or synaptic reorganization of the BLA.

Effects of increasing neuromuscular electrical stimulation current intensity on cortical sensorimotor network activation: A time domain fNIRS study

Neuroimaging studies have shown neuromuscular electrical stimulation (NMES)-evoked movements activate regions of the cortical sensorimotor network, including the primary sensorimotor cortex (SMC), premotor cortex (PMC), supplementary motor area (SMA), and secondary somatosensory area (S2), as well as regions of the prefrontal cortex (PFC) known to be involved in pain processing. The aim of this study, on nine healthy subjects, was to compare the cortical network activation profile and pain ratings during NMES of the right forearm wrist extensor muscles at increasing current intensities up to and slightly over the individual maximal tolerated intensity (MTI), and with reference to voluntary (VOL) wrist extension movements. By exploiting the capability of the multi-channel time domain functional near-infrared spectroscopy technique to relate depth information to the photon time-of-flight, the cortical and superficial oxygenated (O₂Hb) and deoxygenated (HHb) hemoglobin concentrations were estimated. The O₂Hb and HHb maps obtained using the General Linear Model (NIRS-SPM) analysis method, showed that the VOL and NMES-evoked movements significantly increased activation (i.e., increase in O₂Hb and corresponding decrease in HHb) in the cortical layer of the contralateral sensorimotor network (SMC, PMC/SMA, and S2). However, the level and area of contralateral sensorimotor network (including PFC) activation was significantly greater for NMES than VOL. Furthermore, there was greater bilateral sensorimotor network activation with the high NMES current intensities which corresponded with increased pain ratings. In conclusion, our findings suggest that greater bilateral sensorimotor network activation profile with high NMES current intensities could be in part attributable to increased attentional/pain processing and to increased bilateral sensorimotor integration in these cortical regions.

Direct measurement of inner prosthesis loading during activities of daily living: Proposal for classification of functional outcome

Background The purpose of this presentation is to outline the relevance of the categorization of the load regime data to assess the functional output and usage of the prosthesis of lower limb amputees. The objectives are • To highlight the need for categorisation of activities of daily living • To present a categorization of load regime applied on residuum, • To present some descriptors of the four types of activity that could be detected, • To provide an example the results for a case. Methods The load applied on the osseointegrated fixation of one transfemoral amputee was recorded using a portable kinetic system for 5 hours. The load applied on the residuum was divided in four types of activities corresponding to inactivity, stationary loading, localized locomotion and directional locomotion as detailed in previously publications. Results The periods of directional locomotion, localized locomotion, and stationary loading occurred 44%, 34%, and 22% of recording time and each accounted for 51%, 38%, and 12% of the duration of the periods of activity, respectively. The absolute maximum force during directional locomotion, localized locomotion, and stationary loading was 19%, 15%, and 8% of the body weight on the anteroposterior axis, 20%, 19%, and 12% on the mediolateral axis, and 121%, 106%, and 99% on the long axis. A total of 2,783 gait cycles were recorded. Discussion Approximately 10% more gait cycles and 50% more of the total impulse than conventional analyses were identified. The proposed categorization and apparatus have the potential to complement conventional instruments, particularly for difficult cases.

The mechanisms of human renal epithelial cell modulation of autologous dendritic cell phenotype and function

Proximal tubule epithelial cells (PTEC) of the kidney line the proximal tubule downstream of the glomerulus and play a major role in the re-absorption of small molecular weight proteins that may pass through the glomerular filtration process. In the perturbed disease state PTEC also contribute to the inflammatory disease process via both positive and negative mechanisms via the production of inflammatory cytokines which chemo-attract leukocytes and the subsequent down-modulation of these cells to prevent uncontrolled inflammatory responses. It is well established that dendritic cells are responsible for the initiation and direction of adaptive immune responses. Both resident and infiltrating dendritic cells are localised within the tubulointerstitium of the renal cortex, in close apposition to PTEC, in inflammatory disease states. We previously demonstrated that inflammatory PTEC are able to modulate autologous human dendritic cell phenotype and functional responses. Here we extend these findings to characterise the mechanisms of this PTEC immune-modulation using primary human PTEC and autologous monocyte-derived dendritic cells (MoDC) as the model system. We demonstrate that PTEC express three inhibitory molecules: (i) cell surface PD-L1 that induces MoDC expression of PD-L1; (ii) intracellular IDO that maintains the expression of MoDC CD14, drives the expression of CD80, PD-L1 and IL-10 by MoDC and inhibits T cell stimulatory capacity; and (iii) soluble HLA-G (sHLA-G) that inhibits HLA-DR and induces IL-10 expression by MoDC. Collectively the results demonstrate that primary human PTEC are able to modulate autologous DC phenotype and function via multiple complex pathways. Further dissection of these pathways is essential to target therapeutic strategies in the treatment of inflammatory kidney disorders.

Functional evaluation of genetic variants associated with endometriosis near GREB1

STUDY QUESTION: Do DNA variants in the growth regulation by estrogen in breast cancer 1 (GREB1) region regulate endometrial GREB1 expression and increase the risk of developing endometriosis in women? SUMMARY ANSWER: We identified new single nucleotide polymorphisms (SNPs) with strong association with endometriosis at the GREB1 locus although we did not detect altered GREB1 expression in endometriosis patients with defined genotypes. WHAT IS ALREADY KNOWN: Genome-wide association studies have identified the GREB1 region on chromosome 2p25.1 for increasing endometriosis risk. The differential expression of GREB1 has also been reported by others in association with endometriosis disease phenotype. STUDY DESIGN, SIZE, DURATION: Fine mapping studies comprehensively evaluated SNPs within the GREB1 region in a large-scale data set (>2500 cases and >4000 controls). Publicly available bioinformatics tools were employed to functionally annotate SNPs showing the strongest association signal with endometriosis risk. Endometrial GREB1 mRNA and protein expression was studied with respect to phases of the menstrual cycle (n = 2-45 per cycle stage) and expression quantitative trait loci (eQTL) analysis for significant SNPs were undertaken for GREB1 [mRNA (n = 94) and protein (n = 44) in endometrium]. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants in this study are females who provided blood and/or endometrial tissue samples in a hospital setting. The key SNPs were genotyped using Sequenom MassARRAY. The functional roles and regulatory annotations for identified SNPs are predicted by various publicly available bioinformatics tools. Endometrial GREB1 expression work employed qRT-PCR, western blotting and immunohistochemistry studies. MAIN RESULTS AND THE ROLE OF CHANCE: Fine mapping results identified a number of SNPs showing stronger association (0.004 < P < 0.032) with endometriosis risk than the original GWAS SNP (rs13394619) (P = 0.034). Some of these SNPs were predicted to have functional roles, for example, interaction with transcription factor motifs. The haplotype (a combination of alleles) formed by the risk alleles from two common SNPs showed significant association (P = 0.026) with endometriosis and epistasis analysis showed no evidence for interaction between the two SNPs, suggesting an additive effect of SNPs on endometriosis risk. In normal human endometrium, GREB1 protein expression was altered depending on the cycle stage (significantly different in late proliferative versus late secretory, P < 0.05) and cell type (glandular epithelium, not stromal cells). However, GREB1 expression in endometriosis cases versus controls and eQTL analyses did not reveal any significant changes. LIMITATIONS, REASONS FOR CAUTION: In silico prediction tools are generally based on cell lines different to our tissue and disease of interest. Functional annotations drawn from these analyses should be considered with this limitation in mind. We identified cell-specific and hormone-specific changes in GREB1 protein expression. The lack of a significant difference observed following our GREB1 expression studies may be the result of moderate power on mixed cell populations in the endometrial tissue samples. WIDER IMPLICATIONS OF THE FINDINGS: This study further implicates the GREB1 region on chromosome 2p25.1 and the GREB1 gene with involvement in endometriosis risk. More detailed functional studies are required to determine the role of the novel GREB1 transcripts in endometriosis pathophysiology. STUDY FUNDING/COMPETING INTERESTS: Funding for this work was provided by NHMRC Project Grants APP1012245, APP1026033, APP1049472 and APP1046880. There are no competing interests.

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of approximately 2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 x 10(-8) to P = 4 x 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

New Functional Biomaterials for Medicine and Healthcare

The book begins with an overview of the use of biomaterials in contemporary healthcare and the process of developing novel biomaterials; the key issues and challenges associated with the design of complex implantable systems are also highlighted. The book then reviews the main materials used in functional biomaterials, particularly their properties and applications. Individual chapters focus on both natural and synthetic polymers, metallic biomaterials, and bio-inert and bioactive ceramics.

Wetting, solubility and chemical characteristics of plasma-polymerized 1-isopropyl-4-methyl-1,4-cyclohexadiene thin films

Investigations on the wetting, solubility and chemical composition of plasma polymer thin films provide an insight into the feasibility of implementing these polymeric materials in organic electronics, particularly where wet solution processing is involved. In this study, thin films were prepared from 1-isopropyl-4-methyl-1,4-cyclohexadiene (γ-Terpinene) using radio frequency (RF) plasma polymerization. FTIR showed the polymers to be structurally dissimilar to the original monomer and highly cross-linked, where the loss of original functional groups and the degree of cross-linking increased with deposition power. The polymer surfaces were hydrocarbon-rich, with oxygen present in the form of O–H and C=O functional groups. The oxygen content decreased with deposition power, with films becoming more hydrophobic and, thus, less wettable. The advancing and receding contact angles were investigated, and the water advancing contact angle was found to increase from 63.14° to 73.53° for thin films prepared with an RF power of 10 W to 75 W. The wetting envelopes for the surfaces were constructed to enable the prediction of the surfaces’ wettability for other solvents. The effect of roughness on the wetting behaviour of the films was insignificant. The polymers were determined to resist solubilization in solvents commonly used in the deposition of organic semiconducting layers, including chloroform and chlorobenzene, with higher stability observed in films fabricated at higher RF power.

Feasibility of a stepped wedge cluster RCT and concurrent observational sub-study to evaluate the effects of modified ward night lighting on inpatient fall rates and sleep quality: A protocol for a pilot trial

Background: Falls among hospitalised patients impose a considerable burden on health systems globally and prevention is a priority. Some patient-level interventions have been effective in reducing falls, but others have not. An alternative and promising approach to reducing inpatient falls is through the modification of the hospital physical environment and the night lighting of hospital wards is a leading candidate for investigation. In this pilot trial, we will determine the feasibility of conducting a main trial to evaluate the effects of modified night lighting on inpatient ward level fall rates. We will test also the feasibility of collecting novel forms of patient level data through a concurrent observational sub-study. Methods/design: A stepped wedge, cluster randomised controlled trial will be conducted in six inpatient wards over 14 months in a metropolitan teaching hospital in Brisbane (Australia). The intervention will consist of supplementary night lighting installed across all patient rooms within study wards. The planned placement of luminaires, configurations and spectral characteristics are based on prior published research and pre-trial testing and modification. We will collect data on rates of falls on study wards (falls per 1000 patient days), the proportion of patients who fall once or more, and average length of stay. We will recruit two patients per ward per month to a concurrent observational sub-study aimed at understanding potential impacts on a range of patient sleep and mobility behaviour. The effect on the environment will be monitored with sensors to detect variation in light levels and night-time room activity. We will also collect data on possible patient-level confounders including demographics, pre-admission sleep quality, reported vision, hearing impairment and functional status. Discussion: This pragmatic pilot trial will assess the feasibility of conducting a main trial to investigate the effects of modified night lighting on inpatient fall rates using several new methods previously untested in the context of environmental modifications and patient safety. Pilot data collected through both parts of the trial will be utilised to inform sample size calculations, trial design and final data collection methods for a subsequent main trial.