DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana

DOUBLE-STRANDED RNA BIN DIN G (DRB) proteins have been functionally characterized in viruses, prokaryotes and eukaryotes and are involved in all aspects of RNA biology. Arabidopsis thaliana (Arabidopsis) encodes five closely related DRB proteins, DRB1 to DRB5. DRB1 and DRB4 are required by DICER-LIKE (DCL) proteins DCL1 and DCL4 to accurately and efficiently process structurally distinct double-stranded RNA (dsRNA) precursor substrates in the microRNA (miRNA) and trans-acting small-interfering RNA (tasiRNA) biogenesis pathways respectively. We recently reported that DRB2 is also involved in the biogenesis of specific miRNA subsets. Furthermore, the severity of the developmental phenotype displayed by the drb235 triple mutant plant, compared with those expressed by either drb2, drb3 and drb5 single mutants, or double mutant combinations thereof, indicates that DRB3 and DRB5 function in the same non-canonical miRNA pathway as DRB2. Through the use of our artificial miRNA (amiRNA) plant expression vector, pBlueGreen 2,3 we demonstrate here that unlike DRB2, DRB3 and DRB5 are not involved in the dsRNA processing stages of the miRNA biogenesis pathway, but are required to mediate RNA silencing of target genes of DRB2-associated miRNA s. © 2012 Landes Bioscience.

miR-451 regulates zebrafish erythroid maturation in vivo via its target gata2

We demonstrate that in zebrafish, the microRNA miR-451 plays a crucial role in promoting erythroid maturation, in part via its target transcript gata2. Zebrafish miR-144 and miR-451 are processed from a single precursor transcript selectively expressed in erythrocytes. In contrast to other hematopoietic mutants, the ze-brafish mutant meunier (mnr) showed intact erythroid specification but diminished miR-144/451 expression. Although erythropoiesis initiated normally in mnr, erythrocyte maturation was morphologically retarded. Morpholino knockdown of miR-451 increased erythrocyte immaturity in wild-type embryos, and miR-451 RNA duplexes partially rescued erythroid maturation in mnr, demonstrating a requirement and role for miR-451 in erythro-cyte maturation. mnr provided a selectively miR-144/451-deficient background, facilitating studies to discern miRNA function and validate candidate targets. Among computer-predicted miR-451 targets potentially mediating these biologic effects, the pro-stem cell transcription factor gata2 was an attractive candidate. In vivo reporter assays validated the predicted miR-451/gata2-3'UTR interaction, gata2 down-regulation was delayed in miR-451-knockdown and mnr embryos, and gata2 knockdown partially restored erythroid maturation in mnr, collectively confirming gata2down-regulation as pivotal for miR-451-driven erythroid maturation. These studies define a new genetic pathway promoting erythroid maturation (mnr/miR-451/gata2) and provide a rare example of partial rescue of a mutant phenotype solely by miRNA overexpression. © 2009 by The American Society of Hematology.

Rice ragged stunt oryzavirus genome segment S4 could encode an RNA dependent RNA polymerase and a second protein of unknown function

The complete nucleotide sequence of genome segment S4 of rice ragged stunt oryzavirus (RRSV, Thai-isolate) was determined. The 3823 bp sequence contains two large open reading frames (ORFs). ORF1, spanning nucleotides 12 to 3776, is capable of encoding a protein of M(r) 141,380 (P4a). The P4a amino acid sequence predicted from the nucleotide sequence contains sequence motifs conserved in RNA-dependent RNA polymerases (RDRPs). When compared for evolutionary relationships with RDRPs of other reoviruses using the amino acid sequences around the conserved GDD motif, P4a was shown to be more related to Nilaparvata lugens reovirus and reovirus serotype 3 than to rice dwarf phytoreovirus, bovine rotavirus or bluetongue virus. The ORF2, spanning nucleotides 491 to 1468, is out of frame with ORF1 and is capable of encoding a protein of 36, 920 (P4b). Coupled in vitro transcription-translation from cloned ORF2 in wheat germ extract confirmed the existence of ORF2 but in vivo production and possible function of P4b is yet to be determined.

Influence of eye rotation on peripheral eye length measurement obtained with a partial coherence interferometry instrument

Purpose The eye rotation approach for measuring peripheral eye length leads to concern about whether the rotation influences results, such as through pressure exerted by eyelids or extra-ocular muscles. This study investigated whether this approach is valid. Methods Peripheral eye lengths were measured with a Lenstar LS 900 biometer for eye rotation and no-eye rotation conditions (head rotation for horizontal meridian and instrument rotation for vertical meridian). Measurements were made for 23 healthy young adults along the horizontal visual field (±30°) and, for a subset of eight participants along the vertical visual field (±25°). To investigate the influence of the duration of eye rotation, for six participants measurements were made at 0, 60, 120, 180 and 210 s after eye rotation to ±30° along horizontal and vertical visual fields. Results Peripheral eye lengths were not significantly different for the conditions along the vertical meridian (F1,7 = 0.16, p = 0.71). The peripheral eye lengths for the conditions were significantly different along the horizontal meridian (F1,22 = 4.85, p = 0.04), although not at individual positions (p ≥ 0.10) and were not important. There were no apparent differences between the emmetropic and myopic groups. There was no significant change in eye length at any position after maintaining position for 210 s. Conclusion Eye rotation and no-eye rotation conditions were similar for measuring peripheral eye lengths along horizontal and vertical visual field meridians at ±30° and ±25°, respectively. Either condition can be used to estimate retinal shape from peripheral eye lengths.

Putative full-length clones of the genomic DNA segments of subterranean clover stunt virus and identification of the segment coding for the viral coat protein

Subterranean clover stunt disease is an economically important aphid-borne virus disease affecting certain pasture and grain legumes in Australia. The virus associated with the disease, subterranean clover stunt virus (SCSV), was previously found to be representative of a new type of single-stranded DNA virus. Analysis of the virion DNA and restriction mapping of double-stranded cDNA synthesized from virion DNA suggested that SCSV has a segmented genome composed of 3 or 4 different species of circular ssDNA each of about 850-880 nucleotides. To further investigate the complexity of the SCSV genome, we have isolated the replicative form DNA from infected pea and from it prepared putative full-length clones representing the SCSV genome segments. Analysis of these clones by restriction mapping indicated that clones representing at least 4 distinct genomic segments were obtained. This method is thus suitable for generating an extensive genomic library of novel ssDNA viruses containing multiple genome segments such as SCSV and banana bunchy top virus. The N-terminal amino acid sequence and amino acid composition of the coat protein of SCSV were determined. Comparison of the amino acid sequence with partial DNA sequence data, and the distinctly different restriction maps obtained for the full-length clones suggested that only one of these clones contained the coat protein gene. The results confirmed that SCSV has a functionally divided genome composed of several distinct ssDNA circles each of about 1 kb.

A pre-clinical functional assessment of an acellular scaffold intended for the treatment of hard-to-heal wounds

Dermal wound healing is a biochemical and cellular process critical to life. While the majority of the population will only ever experience successful wound healing outcomes, some 1-3 % of those aged over 65 years will experience wound healing delay or perpetuation. These hard-to-heal wounds are comprised of degraded and dysfunctional extracellular matrix, yet the integrity of this structure is critical in the processes of normal wound healing. As such, extracellular matrix replacements have been devised that can replace dysfunctional extracellular matrix in hard-to-heal wounds with the aim of restoring normal wound healing processes. Here we evaluated a novel synthetic matrix protein for its ability to act as an acellular scaffold that can replace dysfunctional extracellular matrix. In this regard the synthetic protein demonstrated an ability to rapidly adsorb to the dermal surface, permit cell attachment and facilitate the cellular functions essential to wound healing. When applied to deep partial thickness wounds in a porcine animal model the matrix protein also demonstrated the ability to reduce wound duration. These data provide evidence that the synthetic matrix protein has the ability to function as an acellular scaffold for wound healing purposes.

Semi-binary based video features for activity representation

Efficient and effective feature detection and representation is an important consideration when processing videos, and a large number of applications such as motion analysis, 3D scene understanding, tracking etc. depend on this. Amongst several feature description methods, local features are becoming increasingly popular for representing videos because of their simplicity and efficiency. While they achieve state-of-the-art performance with low computational complexity, their performance is still too limited for real world applications. Furthermore, rapid increases in the uptake of mobile devices has increased the demand for algorithms that can run with reduced memory and computational requirements. In this paper we propose a semi binary based feature detectordescriptor based on the BRISK detector, which can detect and represent videos with significantly reduced computational requirements, while achieving comparable performance to the state of the art spatio-temporal feature descriptors. First, the BRISK feature detector is applied on a frame by frame basis to detect interest points, then the detected key points are compared against consecutive frames for significant motion. Key points with significant motion are encoded with the BRISK descriptor in the spatial domain and Motion Boundary Histogram in the temporal domain. This descriptor is not only lightweight but also has lower memory requirements because of the binary nature of the BRISK descriptor, allowing the possibility of applications using hand held devices.We evaluate the combination of detectordescriptor performance in the context of action classification with a standard, popular bag-of-features with SVM framework. Experiments are carried out on two popular datasets with varying complexity and we demonstrate comparable performance with other descriptors with reduced computational complexity.

Traffic state estimation from partial Bluetooth and volume observations : case study in the Brisbane metropolitan area

The application of the Bluetooth (BT) technology to transportation has been enabling researchers to make accurate travel time observations, in freeway and arterial roads. The Bluetooth traffic data are generally incomplete, for they only relate to those vehicles that are equipped with Bluetooth devices, and that are detected by the Bluetooth sensors of the road network. The fraction of detected vehicles versus the total number of transiting vehicles is often referred to as Bluetooth Penetration Rate (BTPR). The aim of this study is to precisely define the spatio-temporal relationship between the quantities that become available through the partial, noisy BT observations; and the hidden variables that describe the actual dynamics of vehicular traffic. To do so, we propose to incorporate a multi- class traffic model into a Sequential Montecarlo Estimation algorithm. Our framework has been applied for the empirical travel time investigations into the Brisbane Metropolitan region.

Innovation & workplace culture in the Australian interactive entertainment industry : the Halfbrick story

A recent success story of the Australian videogames industry is Brisbane based Halfbrick Studios, developer of the hit game for mobile devices, Fruit Ninja. Halfbrick not only survived the global financial crisis and an associated downturn in the Australian industry, but grew strongly, moving rapidly from developing licensed properties for platforms such as Game Boy Advance, Nintendo DS, and Playstation Portable (PSP) to becoming an independent developer and publisher of in-house titles, generating revenue both through App downloads and merchandise sales. Amongst the reasons for Halfbrick’s success is their ability to adaptively transform by addressing different technical platforms, user dynamics, business models and market conditions. Our ongoing case-study research from 2010 into Halfbrick’s innovation processes, culminating with some 10 semi-structured interviews with senior managers and developers, has identified a strong focus on workplace organisational culture, with staff reflecting that the company is a flat, team-based organisation devolving as much control as possible to the development teams directly, and encouraging a work-life balance in which creativity can thrive. The success of this strategy is evidenced through Halfbrick’s low staff turnover; amongst our interviewees most of the developers had been with the company for a number of years, with all speaking positively of the workplace culture and sense of creative autonomy they enjoyed. Interviews with the CEO, Shainiel Deo, and team leaders highlighted the autonomy afforded to each team and the organisation and management of the projects on which they work. Deo and team leaders emphasised the collaboration and communication skills they require in the developers that they employ, and that these characteristics were considered just as significant in hiring decisions as technical skills. Halfbrick’s developers celebrate their workplace culture and insist it has contributed to their capacity for innovation and to their commercial success with titles such as Fruit Ninja. This model of organisational management is reflected in both Stark’s (2009) idea of heterarchy, and Neff’s (2012) concept of venture labour, and provides a different perspective on the industry than the traditional political economy critique of precarious labour exploited by gaming conglomerates. Nevertheless, throughout many of the interviews and in our informal discussions with Halfbrick developers there is also a sense that this rewarding culture is quite tenuous and precarious in the context of a rapidly changing and uncertain global videogames industry. Whether such a workplace culture represents the future of the games industry, or is merely a ‘Prague Spring’ before companies such as Halfbrick are swallowed by traditional players’ remains to be seen. However, as the process of rapid and uncertain transformation plays out across the videogames industry, it is important to pay attention to emerging modes of organisation and workplace culture, even whilst they remain at the margins of the industry. In this paper we investigate Halfbrick’s workplace culture and ask how sustainable is this kind of rewarding and creative workplace?

The impact of risk-reducing hysterectomy and bilateral salpingo-oophorectomy on survival in patients with a history of breast cancer – a population-based data linkage study

Prophylactic surgery including hysterectomy and bilateral salpingo-oophorectomy (BSO) is recommended in BRCA positive women, while in women from the general population, hysterectomy plus BSO may increase the risk of overall mortality. The effect of hysterectomy plus BSO on women previously diagnosed with breast cancer is unknown. We used data from a population-base data linkage study of all women diagnosed with primary breast cancer in Queensland, Australia between 1997 and 2008 (n=21,067). We fitted flexible parametric breast cancer specific and overall survival models with 95% confidence intervals (also known as Royston-Parmar models) to assess the impact of risk-reducing surgery (removal of uterus, one or both ovaries). We also stratified analyses by age 20-49 and 50-79 years, respectively. Overall, 1,426 women (7%) underwent risk-reducing surgery (13% of premenopausal women and 3% of postmenopausal women). No women who had risk-reducing surgery, compared to 171 who did not have risk-reducing surgery developed a gynaecological cancer. Overall, 3,165 (15%) women died, including 2,195 (10%) from breast cancer. Hysterectomy plus BSO was associated with significantly reduced risk of death overall (adjusted HR = 0.69, 95% CI 0.53-0.89; P =0.005). Risk reduction was greater among premenopausal women, whose risk of death halved (HR, 0.45; 95% CI, 0.25-0.79; P < 0.006). This was largely driven by reduction in breast cancer-specific mortality (HR, 0.43; 95% CI, 0.24-0.79; P < 0.006). This population-based study found that risk-reducing surgery halved the mortality risk for premenopausal breast cancer patients. Replication of our results in independent cohorts, and subsequently randomised trials are needed to confirm these findings.

The efficacy of Aloe vera, tea tree oil and saliva as first aid treatment for partial thickness burn injuries

Many alternative therapies are used as first aid treatment for burns, despite limited evidence supporting their use. In this study, Aloe vera, saliva and a tea tree oil impregnated dressing (Burnaid) were applied as first aid to a porcine deep dermal contact burn, compared to a control of nothing. After burn creation, the treatments were applied for 20 min and the wounds observed at weekly dressing changes for 6 weeks. Results showed that the alternative treatments did significantly decrease subdermal temperature within the skin during the treatment period. However, they did not decrease the microflora or improve re-epithelialisation, scar strength, scar depth or cosmetic appearance of the scar and cannot be recommended for the first aid treatment of partial thickness burns.

The optimal temperature of first aid treatment for partial thickness burn injuries

Using our porcine model of deep dermal partial thickness burn injury, various cooling techniques (15 degrees C running water, 2 degrees C running water, ice) of first aid were applied for 20 minutes compared with a control (ambient temperature). The subdermal temperatures were monitored during the treatment and wounds observed and photographed weekly for 6 weeks, observing reepithelialization, wound surface area and cosmetic appearance. Tissue histology and scar tensile strength were examined 6 weeks after burn. The 2 degrees C and ice treatments decreased the subdermal temperature the fastest and lowest, however, generally the 15 and 2 degrees C treated wounds had better outcomes in terms of reepithelialization, scar histology, and scar appearance. These findings provide evidence to support the current first aid guidelines of cold tap water (approximately 15 degrees C) for 20 minutes as being beneficial in helping to heal the burn wound. Colder water at 2 degrees C is also beneficial. Ice should not be used.

The optimal duration and delay of first aid treatment for deep partial thickness burn injuries

Using our porcine model of deep dermal partial thickness burn injury, various durations (10min, 20min, 30min or 1h) and delays (immediate, 10min, 1h, 3h) of 15 degrees C running water first aid were applied to burns and compared to untreated controls. The subdermal temperatures were monitored during the treatment and wounds observed weekly for 6 weeks, for re-epithelialisation, wound surface area and cosmetic appearance. At 6 weeks after the burn, tissue biopsies were taken of the scar for histological analysis. Results showed that immediate application of cold running water for 20min duration is associated with an improvement in re-epithelialisation over the first 2 weeks post-burn and decreased scar tissue at 6 weeks. First aid application of cold water for as little as 10min duration or up to 1h delay still provides benefit.

A porcine deep dermal partial thickness burn model with hypertrophic scarring

We developed a reproducible model of deep dermal partial thickness burn injury in juvenile Large White pigs. The contact burn is created using water at 92 degrees C for 15s in a bottle with the bottom replaced with plastic wrap. The depth of injury was determined by a histopathologist who examined tissue sections 2 and 6 days after injury in a blinded manner. Upon creation, the circular wound area developed white eschar and a hyperaemic zone around the wound border. Animals were kept for 6 weeks or 99 days to examine the wound healing process. The wounds took between 3 and 5 weeks for complete re-epithelialisation. Most wounds developed contracted, purple, hypertrophic scars. On measurement, the thickness of the burned skin was approximately 1.8 times that of the control skin at week 6 and approximately 2.2 times thicker than control skin at 99 days after injury. We have developed various methods to assess healing wounds, including digital photographic analysis, depth of organising granulation tissue, immunohistochemistry, electron microscopy and tensiometry. Immunohistochemistry and electron microscopy showed that our porcine hypertrophic scar appears similar to human hypertrophic scarring. The development of this model allows us to test and compare different treatments on burn wounds.

The α5 subunit regulates the expression and function of α4*-containing neuronal nicotinic acetylcholine receptors in the ventral-tegmental area

Human genetic association studies have shown gene variants in the α5 subunit of the neuronal nicotinic receptor (nAChR) influence both ethanol and nicotine dependence. The α5 subunit is an accessory subunit that facilitates α4* nAChRs assembly in vitro. However, it is unknown whether this occurs in the brain, as there are few research tools to adequately address this question. As the α4*-containing nAChRs are highly expressed in the ventral tegmental area (VTA) we assessed the molecular, functional and pharmacological roles of α5 in α4*-containing nAChRs in the VTA. We utilized transgenic mice α5+/+(α4YFP) and α5-/-(α4YFP) that allow the direct visualization and measurement of α4-YFP expression and the effect of the presence (α5+/+) and absence of α5 (-/-) subunit, as the antibodies for detecting the α4* subunits of the nAChR are not specific. We performed voltage clamp electrophysiological experiments to study baseline nicotinic currents in VTA dopaminergic neurons. We show that in the presence of the α5 subunit, the overall expression of α4 subunit is increased significantly by 60% in the VTA. Furthermore, the α5 subunit strengthens baseline nAChR currents, suggesting the increased expression of α4* nAChRs to be likely on the cell surface. While the presence of the α5 subunit blunts the desensitization of nAChRs following nicotine exposure, it does not alter the amount of ethanol potentiation of VTA dopaminergic neurons. Our data demonstrates a major regulatory role for the α5 subunit in both the maintenance of α4*-containing nAChRs expression and in modulating nicotinic currents in VTA dopaminergic neurons. Additionally, the α5α4* nAChR in VTA dopaminergic neurons regulates the effect of nicotine but not ethanol on currents. Together, the data suggest that the α5 subunit is critical for controlling the expression and functional role of a population of α4*-containing nAChRs in the VTA.