The use of artificial MicroRNA technology to control gene expression in Arabidopsis thaliana

In plants, double-stranded RNA (dsRNA) is an effective trigger of RNA silencing, and several classes of endogenous small RNA (sRNA), processed from dsRNA substrates by DICER-like (DCL) endonucleases, are essential in controlling gene expression. One such sRNA class, the microRNAs (miRNAs) control the expression of closely related genes to regulate all aspects of plant development, including the determination of leaf shape, leaf polarity, flowering time, and floral identity. A single miRNA sRNA silencing signal is processed from a long precursor transcript of nonprotein-coding RNA, termed the primary miRNA (pri-miRNA). A region of the pri-miRNA is partially self-complementary allowing the transcript to fold back onto itself to form a stem-loop structure of imperfectly dsRNA. Artificial miRNA (amiRNA) technology uses endogenous pri-miRNAs, in which the miRNA and miRNA*(passenger strand of the miRNA duplex) sequences have been replaced with corresponding amiRNA/ amiRNA*sequences that direct highly efficient RNA silencing of the targeted gene. Here, we describe the rules for amiRNA design, as well as outline the PCR and bacterial cloning procedures involved in the construction of an amiRNA plant expression vector to control target gene expression in Arabidopsis thaliana. © 2014 Springer Science+Business Media New York.

A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis

It is known that 22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger phased small-interfering RNA (phasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA)-mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA-mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA-DEPENDENT RNA POLYMERASE6-mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA-directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families.

Vectors and methods for hairpin RNA and artificial microRNA-mediated gene silencing in plants

In plant cells, DICER-LIKE4 processes perfectly double-stranded RNA (dsRNA) into short interfering (si) RNAs, and DICER-LIKE1 generates micro (mi) RNAs from primary miRNA transcripts (pri-miRNA) that form fold-back structures of imperfectly dsRNA. Both si and miRNAs direct the endogenous endonuclease, ARGONAUTE1 to cleave complementary target single-stranded RNAs and either small RNA (sRNA)-directed pathway can be harnessed to silence genes in plants. A routine way of inducing and directing RNA silencing by siRNAs is to express self-complementary single-stranded hairpin RNA (hpRNA), in which the duplexed region has the same sequence as part of the target gene's mRNA. Artificial miRNA (amiRNA)-mediated silencing uses an endogenous pri-miRNA, in which the original miRNA/miRNA* sequence has been replaced with a sequence complementary to the new target gene. In this chapter, we describe the plasmid vector systems routinely used by our research group for the generation of either hpRNA-derived siRNAs or amiRNAs.

Efficient silencing of endogenous microRNAs using artificial microRNAs in Arabidopsis thaliana

We report here that the expression of endogenous microRNAs (miRNAs) can be efficiently silenced in Arabidopsis thaliana (Arabidopsis) using artificial miRNA (amiRNA) technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted. Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis. This suggests that small RNA (sRNA)-guided RNA cleavage in plants occurs not only in the cytoplasm, but also in the nucleus. Many plant miRNA gene families have been identified via sequencing and bioinformatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of effective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants. © The Author 2010. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.

Portfolio selection using artificial intelligence

The application of artificial intelligence in finance is relatively new area of research. This project employed artificial neural networks (ANNs) that use both fundamental and technical inputs to predict future prices of widely held Australian stocks and use these predicted prices for stock portfolio selection over a long investment horizon. The research involved the creation and testing of a large number of possible network configurations and draws conclusions about ANN architectures and their overall suitability for the purpose of stock portfolio selection.

Biobanking of blood and bone marrow : emerging challenges for custodians of public resources

The Australian Bone Marrow Donor Registry (ABMDR) is a publicly funded company that is part of an international network that facilitates unrelated bone marrow transplantation. This role means that the ABMDR has access to a large biospecimen repository, therefore making it a highly valuable research resource. Recognising the potential value of these biospecimens for research purposes, the ABMDR is in the process of determining whether, and how, to share its biospecimens with other biobanks. While this would undoubtedly be of value to the scientific community, and ultimately to the wider community, it would also inevitably transform the role of an institution whose primary role is therapeutic, and would compromise the degree of control that a custodian has over donated material. This article describe the challenges confronting the ABMDR, and organisations like it, in balancing their duties to donors, patients, researchers and the general public. These problems have led inevitably to the use of "property" rights language in the discussion of these issues but notions of gift, ownership, trusteeship and transfer might also be considered.

A preliminary study of the effects of Tai Chi and Qigong medical exercise on indicators of metabolic syndrome, glycaemic control, health-related quality of life, and psychological health in adults with elevated blood glucose

Objectives To evaluate the feasibility, acceptability and effects of a Tai Chi and Qigong exercise programme in adults with elevated blood glucose. Design, Setting, and Participants A single group pre–post feasibility trial with 11 participants (3 male and 8 female; aged 42–65 years) with elevated blood glucose. Intervention Participants attended Tai Chi and Qigong exercise training for 1 to 1.5 h, 3 times per week for 12 weeks, and were encouraged to practise the exercises at home. Main Outcome Measures Indicators of metabolic syndrome (body mass index (BMI), waist circumference, blood pressure, fasting blood glucose, triglycerides, HDL-cholesterol); glucose control (HbA1c, fasting insulin and insulin resistance (HOMA)); health-related quality of life; stress and depressive symptoms. Results There was good adherence and high acceptability. There were significant improvements in four of the seven indicators of metabolic syndrome including BMI (mean difference −1.05, p<0.001), waist circumference (−2.80 cm, p<0.05), and systolic (−11.64 mm Hg, p<0.01) and diastolic blood pressure (−9.73 mm Hg, p<0.001), as well as in HbA1c (−0.32%, p<0.01), insulin resistance (−0.53, p<0.05), stress (−2.27, p<0.05), depressive symptoms (−3.60, p<0.05), and the SF-36 mental health summary score (5.13, p<0.05) and subscales for general health (19.00, p<0.01), mental health (10.55, p<0.01) and vitality (23.18, p<0.05). Conclusions The programme was feasible and acceptable and participants showed improvements in metabolic and psychological variables. A larger controlled trial is now needed to confirm these promising preliminary results.

Mainstream monsters : the otherness of humans in Twilight, the Vampire Diaries and True Blood

The representation of vampires in horror movies and television programmes has changed considerably over the last two decades. No longer is the vampire portrayed simply as a monster or representation of death. Now, the vampire on our screen such as True Blood’s Bill Compton, or Twilight’s Edward Cullen, passes as human, chooses to make morally sound decisions, becomes an upstanding assimilated citizen, works in the community, and aspires to be a husband to mortal women. The success of recent series such as The Twilight Saga (2009, 2010, 2011, 2012), The Vampire Diaries (2009 - ) and True Blood (2008 - ) has popularised the idea of vampires who cling to remnants of their humanity (or memories of what it means to be human) and attempt to live as human, which builds upon similar – albeit embryonic – themes which emerged from the vampire sub-genre in the 1990s. Within these narratives, representations of the other have shifted from the traditional idea of the monster, to alternative and surprising loci. As this chapter argues, humans themselves, and the concept of the human body, now represent, in many instances, both abject and other. The chapter begins by considering the nature of the abject and otherness in relation to representations of classical vampires and how they have traditionally embodied the other. This provides a backdrop against which to examine the characteristics of the contemporary mainstreaming vampire ‘monster’. An examination of the broad thematic and representational shifts from other to mainstream vampire demonstrates how mainstream monsters are increasingly assimilating into mortal lifestyles with trappings that many viewers may find appealing. The same shifts in theme and representation also reveal that humans are frequently cast as mundane and unappealing in contemporary vampire narratives.

Stored blood products augment inflammation in a two-event model of Transfusion Related Acute Lung Injury (TRALI)

Aim/Background TRALI is hypothesised to develop via a two-event mechanism involving both the patieint's underlying morbidity and blood product factors. The storage of cellular products has been implicated in cases of non-antibody mediated TRALI, however the pathophysiological mechanisms are undefined. We investigated blood product storage-related modulation of inflmmatory cells and medicators involved in TRALI. Methods In an in vitro mode, fresh human whole blood was mixed with culture media (control) or LPS as a 1st event and "transfused" with 10% (v/v) pooled supernatant (SN) from Day 1 (d1, n=75) or Day 42 (D42, n=113) packed red blood cells (PRBCs) as a 2nd event. Following 6hrs, culture SN was used to assess the overall inflammatory response (cytometric bead array) and a duplicate assay containing protein transport inhibitor was used to assess neutrophil- and monocyte-specific inflmamatory responses using multi-colour flow cytometry. Panels: IL-6, IL-8, IL-10, IL-12, IL-1, TNF, MCP-1, IP-10, MIP-1. One-way ANOVA 95% CI. Results In the absence of LPS, exposure to D1 or D42 PRBC-SN reduced monocyte expression of IL-6, IL-8 and Il-10. D42 PRBC-SN also reduced monocyte IP-10, and the overall IL-8 production was increased. In the presence of LPS, D1-PRBC SN only modified overall IP-10 levels which were reduced. However, cf LPS alone, the combination of LPS and D42 PRBC-SN resulted in increased neutrophil and monocyte productionof IL-1 and IL-8 as well as reduced monocyte TNF production. Additionally, LPS and D42 PRBC-SN resulted in overall inflmmatory changes: elevated IL-8, blood product storage duration as a factor contributing to the development of inflmamatory responses in non-antibody mediated TRALI.

Health and guardianship law : the decision of X v the Sydney Children's Hospitals Network [2013] NSWCA 320 : mature minors and the refusal of blood products

This recent decision of the New South Wales Court of Appeal considers the scope of the parens patriae jurisdiction in cases where the jurisdiction is invoked for the protection of a Gillick competent minor. As outlined below, in certain circumstances the law recognises that mature minors are able to make their own decisions concerning medical treatment. However, there have been a number of Commonwealth decisions which have addressed the issue of whether mature minors are able to refuse medical procedures in circumstances where refusal will result in the minor dying. Ultimately, this case confirms that the minor does not necessarily have a right to make autonomous decisions; the minor’s right to exercise his or her autonomous decision only exists when such decision accords with what is deemed to be in his or her best interests.

Joseph Maiden and the national and transnational circulation of Wattle Acacia spp

During the nineteenth century and in the early years of the twentieth century wattle was circulated by botanists, botanical institutions, interested individuals, commercial seedsmen and government authorities. Wattle bark was used in the production of leather and was the subject of debate regarding its commercial development and conservation in Australia. It was also trialled in many other locations including America, New Zealand, Hawaii and Russia. In the process, South Africa became a major producer of wattle bark for a global market. At the same time wattle was also promoted as a symbol of Australian nationalism. This paper considers this movement of wattles, wattle material and wattle information by examining the career of one active agent in these botanical transfers: Joseph Maiden. In doing so it demonstrates that these seemingly different uses of the wattle overlap transnational and national spheres.

Recent advances in the design of artificial corneas

Purpose of review: Artificial corneas are being developed to meet a shortage of donor corneas as well as to address cases where allografting is contraindicated. A range of artificial corneas has been developed. Here we review several newer designs and especially those inspired by naturally occurring biomaterials found with the human body and elsewhere. Recent findings: Recent trends in the development of artificial corneas indicate a move towards the use of materials derived from native sources including decellularized corneal tissue and tissue substitutes synthesized by corneal cells in vitro when grown either on their own, or in conjunction with novel protein-based scaffolds. Biologically inspired materials are also being considered for implantation on their own with the view to promoting endogenous corneal tissue. Summary: More recent attempts at making artificial corneas have taken a more nature-based or nature-inspired approach. Several will in the near future be likely to be available clinically.

Controlling whole blood activation and resultant clot properties by carboxyl and alkyl functional groups on material surfaces : a possible therapeutic approach for enhancing bone healing

Most research virtually ignores the important role of a blood clot in supporting bone healing. In this study, we investigated the effects of surface functional groups carboxyl and alkyl on whole blood coagulation, complement activation and blood clot formation. We synthesised and tested a series of materials with different ratios of carboxyl (–COOH) and alkyl (–CH3, –CH2CH3 and –(CH2)3CH3) groups. We found that surfaces with –COOH/–(CH2)3CH3 induced a faster coagulation activation than those with –COOH/– CH3 and –CH2CH3, regardless of the –COOH ratios. An increase in –COOH ratios on –COOH/–CH3 and –CH2CH3 surfaces decreased the rate of coagulation activation. The pattern of complement activation was entirely similar to that of surface-induced coagulation. All material coated surfaces resulted in clots with thicker fibrin in a denser network at the clot/material interface and a significantly slower initial fibrinolysis when compared to uncoated glass surfaces. The amounts of platelet-derived growth factor-AB (PDGF-AB) and transforming growth factor-b (TGF-b1) released from an intact clot were higher than a lysed clot. The release of PDGF-AB was found to be correlated with the fibrin density. This study demonstrated that surface chemistry can significantly influence the activation of blood coagulation and complement system, resultant clot structure, susceptibility to fibrinolysis as well as release of growth factors, which are important factors determining the bone healing process.

An Artificial Neutral Network (ANN) model for predicting biodiesel kinetic viscosity as a function of temperature and chemical compositions

An Artificial Neural Network (ANN) is a computational modeling tool which has found extensive acceptance in many disciplines for modeling complex real world problems. An ANN can model problems through learning by example, rather than by fully understanding the detailed characteristics and physics of the system. In the present study, the accuracy and predictive power of an ANN was evaluated in predicting kinetic viscosity of biodiesels over a wide range of temperatures typically encountered in diesel engine operation. In this model, temperature and chemical composition of biodiesel were used as input variables. In order to obtain the necessary data for model development, the chemical composition and temperature dependent fuel properties of ten different types of biodiesels were measured experimentally using laboratory standard testing equipments following internationally recognized testing procedures. The Neural Networks Toolbox of MatLab R2012a software was used to train, validate and simulate the ANN model on a personal computer. The network architecture was optimised following a trial and error method to obtain the best prediction of the kinematic viscosity. The predictive performance of the model was determined by calculating the absolute fraction of variance (R2), root mean squared (RMS) and maximum average error percentage (MAEP) between predicted and experimental results. This study found that ANN is highly accurate in predicting the viscosity of biodiesel and demonstrates the ability of the ANN model to find a meaningful relationship between biodiesel chemical composition and fuel properties at different temperature levels. Therefore the model developed in this study can be a useful tool in accurately predict biodiesel fuel properties instead of undertaking costly and time consuming experimental tests.

Integrin-functionalized artificial membranes as test platforms for monitoring small integrin ligand binding by surface plasmon-enhanced fluorescence spectroscopy

The design and synthesis of molecularly or supramolecularly defined interfacial architectures have seen in recent years a remarkable growth of interest and scientific research activities for various reasons. On the one hand, it is generally believed that the construction of an interactive interface between the living world of cells, tissue, or whole organisms and the (inorganic or organic) materials world of technical devices such as implants or medical parts requires proper construction and structural (and functional) control of this organism–machine interface. It is still the very beginning of generating a better understanding of what is needed to make an organism tolerate implants, to guarantee bidirectional communication between microelectronic devices and living tissue, or to simply construct interactive biocompatibility of surfaces in general. This exhaustive book lucidly describes the design, synthesis, assembly and characterization, and bio-(medical) applications of interfacial layers on solid substrates with molecularly or supramolecularly controlled architectures. Experts in the field share their contributions that have been developed in recent years.