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.

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.

A suite of novel promoters and terminators for plant biotechnology II. The pPLEX series for use in monocots

A suite of plant expression vectors (pPLEX), constructed from the gene regulation signals from subterranean clover stunt virus (SCSV) genome, has previously been used in dicot transformation for a variety of applications in plant biotechnology. To assess their use for the transformation of monocots, a number of modifications were made to the basic vector series and assessed in rice. In their unmodified forms, the SCSV promoters directed low levels of gene expression, however, insertion of an intron between the promoter and the transgene open reading frame (analogous to the rice actin and maize ubiquitin promoter systems) increased transgene expression 50-fold. The expression patterns from the intron-modified SCSV (segments 4 and 7) promoters were very similar to those directed by the actin or ubiquitin promoters. All promoter systems investigated directed expression that appeared to be constitutive within leaf tissue, and localised to the epidermal and vascular tissues of the root. The pPLEX vectors described here are an important counterpart to the dicot pPLEX series and have the potential to be useful in monocot research and biotechnology.

Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

We report the first successful Agrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph (encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4-fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 carrying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher β-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity comparable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

High risk feet in subacute rehabilitation facilities : how many are there?

Background Australian subacute rehabilitation facilities face significant challenges from the ageing population with increased burden of chronic disease. High risk foot complications are a negative consequence of many chronic diseases. With the rapid expansion of subacute services, it seems imperative to investigate the prevalence of foot complications in this population. The primary aim of this study was to quantify the high risk foot complication prevalence in a subacute rehabilitation population. Methods Eligible participants were all adults admitted overnight, over two 4 week periods, into a large Australian subacute rehabilitation facility. Consenting participants underwent a short non-invasive foot examination by a podiatrist. The standard Queensland Health High Risk Foot Form collected data on age, sex, co-morbidities and foot complications. Descriptive statistics, logistic regression and odds ratios were used to determine the prevalence of foot complications and associations with explanatory variables. Results Overall, 85 of 97 eligible participants consented; mean age 80(9) and 71% were female. At least one foot complication was present in 56.5% participants; including 21.2% defined as high risk and 11.8% current foot ulcer. A previous diagnosis of neuropathy increased the risk of presenting with a high risk foot by 13-fold (OR 13.504, p = 0.001). Conclusion This study highlights the significance of foot complications in the subacute population. It appears that one in every two patients present with a foot complication and one in eight with a foot ulcer. It is suggested all patients admitted to subacute rehabilitation services should be screened for foot complications.

Tissue viability imaging of skin microcirculation following exposure to whole body cryotherapy (-110°C) and cold water immersion (8°C)

Cryotherapy is currently used in various clinical, rehabilitative, and sporting settings. However, very little is known regarding the impact of cooling on the microcirculatory response. Objectives: The present study sought to examine the influence of two commonly employed modalities of cryotherapy, whole body cryotherapy (WBC; -110°C) and cold water immersion(CWI; 8±1°C), on skin microcirculation in the mid- thigh region. Methods: The skin area examined was a 3 × 3 cm located between the most anterior aspect of the inguinal fold and the patella. Following 10 minutes of rest, 5 healthy, active males were exposed to either WBC for 3 minutes or CWI for 5 minutes in a randomised order. Volunteers lay supine for five minutes after treatment, in order to monitor the variation of red blood cell (RBC) concentration in the region of interest for a duration of 40 minutes. Microcirculation response was assessed using a non-invasive, portable instrument known as a Tissue Viability imaging system. After a minimum of seven days, the protocol was repeated. Subjective assessment of the volunteer’s thermal comfort and thermal sensation was also recorded. Results: RBC was altered following exposure to both WBC and CWI but appeared to stabilise approximately 35 minutes after treatments. Both WBC and CWI affected thermal sensation (p < 0.05); however no betweengroup differences in thermal comfort or sensation were recorded (p > 0.05). Conclusions: As both WBC and CWI altered RBC, further study is necessary to examine the mechanism for this alteration during whole body cooling.

Why Australian and Indian researchers should collaborate to advance wound management innovation

In 2011, 366 million people suffered from diabetes worldwide, resulting in 4.6 million deaths at a cost of US$465 billion in direct healthcare expenditures1. India has the world’s second largest diabetic population at 61.8 million (8.3% of total population)1, while in Australia 8.1% of the population have been diagnosed with diabetes1. Diabetic foot ulcers (DFUs) affect up to 25% of diabetic patients, precipitating 85% of all diabetic amputations2,3. DFUs have significant social and economic impacts associated with increased hospitalisation rates, cost of care, and the reduced capacity of patients and carers to work. In isolated regions of Australia and India the incidence of DFU and associated infection is substantially increased, resulting in hospitalisation rates up to 4- fold that of major cities...

PITX2 and non-canonical Wnt pathway interaction in metastatic prostate cancer

The non-canonical Wnt pathway, a regulator of cellular motility and morphology, is increasingly implicated in cancer metastasis. In a quantitative PCR array analysis of 84 Wnt pathway associated genes, both non-canonical and canonical pathways were activated in primary and metastatic tumors relative to normal prostate. Expression of the Wnt target gene PITX2 in a prostate cancer (PCa) bone metastasis was strikingly elevated over normal prostate (over 2,000-fold) and primary prostate cancer (over 200-fold). The elevation of PITX2 protein was also evident on tissue microarrays, with strong PITX2 immunostaining in PCa skeletal and, to a lesser degree, soft tissue metastases. PITX2 is associated with cell migration during normal tissue morphogenesis. In our studies, overexpression of individual PITX2A/B/C isoforms stimulated PC-3 PCa cell motility, with the PITX2A isoform imparting a specific motility advantage in the presence of non-canonical Wnt5a stimulation. Furthermore, PITX2 specific shRNA inhibited PC-3 cell migration toward bone cell derived chemoattractant. These experimental results support a pivotal role of PITX2A and non-canonical Wnt signaling in enhancement of PCa cell motility, suggest PITX2 involvement in homing of PCa to the skeleton, and are consistent with a role for PITX2 in PCa metastasis to soft and bone tissues. Our findings, which significantly expand previous evidence that PITX2 is associated with risk of PCa biochemical recurrence, indicate that variation in PITX2 expression accompanies and may promote prostate tumor progression and metastasis.

The effect of nitroxides on swarming motility and biofilms, multicellular behaviors in Pseudomonas aeruginosa

The ability of NO to induce biofilm dispersion has been well established. Here we investigated the effect of nitroxides (sterically hindered nitric oxide analogues) on biofilm formation and swarming motility in Pseudomonas aeruginosa. A transposon mutant unable to produce nitric oxide endogenously (nirS) was deficient in swarming motility relative to wild type and the complemented strain. Moreover, expression of the nirS gene was up-regulated by 9.65-fold in wild type swarming cells when compared to planktonic cells. Wild type swarming levels were substantially restored upon exogenous addition of nitroxide containing compounds, consistent with the hypothesis that NO is necessary for swarming motility. Here, we showed that nitroxides not only mimicked the dispersal activity of NO, but also prevented biofilms from forming in flow cell chambers. In addition, a nirS transposon mutant was deficient in biofilm formation relative to wild type and the complemented strain, thus implicating NO in the formation of biofilms. Intriguingly despite its stand alone action in inhibiting biofilm formation and promoting dispersal, a nitroxide partially restored the ability of a nirS mutant to form biofilms.

Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin

Background Ghrelin is a 28 amino acid peptide hormone that is expressed in the stomach and a range of peripheral tissues, where it frequently acts as an autocrine/paracrine growth factor. Ghrelin is modified by a unique acylation required for it to activate its cognate receptor, the growth hormone secretagogue receptor (GHSR), which mediates many of the actions of ghrelin. Recently, the enzyme responsible for adding the fatty acid residue (octanoyl/acyl group) to the third amino acid of ghrelin, GOAT (ghrelin O-acyltransferase), was identified. Methods We used cell culture, quantitative real-time reverse transcription (RT)-PCR and immunohistochemistry to demonstrate the expression of GOAT in prostate cancer cell lines and tissues from patients. Real-time RT-PCR was used to demonstrate the expression of prohormone convertase (PC)1/3, PC2 and furin in prostate cancer cell lines. Prostate-derived cell lines were treated with ghrelin and desacyl ghrelin and the effect on GOAT expression was measured using quantitative RT-PCR. Results We have demonstrated that GOAT mRNA and protein are expressed in the normal prostate and human prostate cancer tissue samples. The RWPE-1 and RWPE-2 normal prostate-derived cell lines and the LNCaP, DU145, and PC3 prostate cancer cell lines express GOAT and at least one other enzyme that is necessary to produce mature, acylated ghrelin from proghrelin (PC1/3, PC2 or furin). Finally, ghrelin, but not desacyl ghrelin (unacylated ghrelin), can directly regulate the expression of GOAT in the RWPE-1 normal prostate derived cell line and the PC3 prostate cancer cell line. Ghrelin treatment (100nM) for 6 hours significantly decreased GOAT mRNA expression two-fold (P < 0.05) in the PC3 prostate cancer cell line, however, ghrelin did not regulate GOAT expression in the DU145 and LNCaP prostate cancer cell lines. Conclusions This study demonstrates that GOAT is expressed in prostate cancer specimens and cell lines. Ghrelin regulates GOAT expression, however, this is likely to be cell-type specific. The expression of GOAT in prostate cancer supports the hypothesis that the ghrelin axis has autocrine/paracrine roles. We propose that the RWPE-1 prostate cell line and the PC3 prostate cancer cell line may be useful for investigating GOAT regulation and function.

Association between xenobiotic gene polymorphisms and non-Hodgkin's lymphoma risk

The last four decades have seen a significant increase in the incidence of non-Hodgkin's lymphoma (NHL) as a possible result of increasing environmental carcinogen exposure, particularly pesticides and solvents. Based on the increasing evidence for an association between carcinogen exposure-related cancer risk and xenobiotic gene polymorphisms, we have undertaken a case-control study of xenobiotic gene polymorphisms in individuals with a diagnosis of NHL. Polymorphisms of six xenobiotic genes (CYP1A1, GSTT1, GSTM1, PON1, NAT1, NAT2) were characterized in 169 individuals with NHL and 205 normal controls using polymerase chain reaction-based methods. Polymorphic frequencies were compared using Fisher's exact tests, and odds ratios for NHL risk were calculated. Among the NHL group, the incidence of GSTT1 null and PON1 BB genotypes were significantly increased compared with controls, 34% vs 14%, and 24% vs 11% respectively. Adjusted odds ratios calculated from multivariate analyses demonstrated that GSTT1 null conferred a fourfold increase in NHL risk (OR = 4.27; 95% CI, 2.40-7.61, P < 0.001) and PON1 BB a 2.9-fold increase (OR = 2.92; 95% CI, 1.49-5.72, P = 0.002). Furthermore, GSTT1 null combined with PON1 BB or GSTM1 null conferred an additional risk of NHL. This is the first time that a PON1 gene polymorphism has been shown to be associated with cancer risk. We conclude that the two polymorphisms, GSTT1 null and PON1 BB, are common genetic traits that pose low individual risk but may be important determinants of overall population NHL risk, particularly among groups exposed to NHL-related carcinogens.

A threshold analysis of dengue transmission in terms of weather variables and imported dengue cases in Australia

Dengue virus (DENV) transmission in Australia is driven by weather factors and imported dengue fever (DF) cases. However, uncertainty remains regarding the threshold effects of high-order interactions among weather factors and imported DF cases and the impact of these factors on autochthonous DF. A time-series regression tree model was used to assess the threshold effects of natural temporal variations of weekly weather factors and weekly imported DF cases in relation to incidence of weekly autochthonous DF from 1 January 2000 to 31 December 2009 in Townsville and Cairns, Australia. In Cairns, mean weekly autochthonous DF incidence increased 16.3-fold when the 3-week lagged moving average maximum temperature was <32 °C, the 4-week lagged moving average minimum temperature was ≥24 °C and the sum of imported DF cases in the previous 2 weeks was >0. When the 3-week lagged moving average maximum temperature was ≥32 °C and the other two conditions mentioned above remained the same, mean weekly autochthonous DF incidence only increased 4.6-fold. In Townsville, the mean weekly incidence of autochthonous DF increased 10-fold when 3-week lagged moving average rainfall was ≥27 mm, but it only increased 1.8-fold when rainfall was <27 mm during January to June. Thus, we found different responses of autochthonous DF incidence to weather factors and imported DF cases in Townsville and Cairns. Imported DF cases may also trigger and enhance local outbreaks under favorable climate conditions.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

Optimisation of lateral load-resisting systems in composite high-rise buildings

This research is carried out by using finite element modelling of building prototypes with three different layouts (rectangular, octagonal and L-shaped) for three different heights (98.0 m, 147.0 m and 199.5 m) for the optimization of lateral load-resisting systems in composite high-rise buildings. Variations of lateral bracings (different number and varied placement along model height of belt-truss and outrigger floors) with RCC (reinforced cement concrete) core wall are used in composite high-rise building models. Prototypes of composite buildings are analysed for dynamic wind and seismic loads. The effects on serviceability (deflection and frequency) of models are studied and conclusions are deduced.

Development and validation of a pregnancy symptoms inventory

Background Physical symptoms are common in pregnancy and are predominantly associated with normal physiological changes. These symptoms have a social and economic cost, leading to absenteeism from work and additional medical interventions. There is currently no simple method for identifying common pregnancy related problems in the antenatal period. A validated tool, for use by pregnancy care providers would be useful. The aim of this study was to develop and validate a Pregnancy Symptoms Inventory for use by health professionals. Methods A list of symptoms was generated via expert consultation with health professionals. Focus groups were conducted with pregnant women. The inventory was tested for face validity and piloted for readability and comprehension. For test-re-test reliability, the tool was administered to the same women 2 to 3 days apart. Finally, midwives trialled the inventory for 1 month and rated its usefulness on a 10cm visual analogue scale (VAS). Results A 41-item Likert inventory assessing how often symptoms occurred and what effect they had, was developed. Individual item test re-test reliability was between .51 to 1, the majority (34 items) scoring ≥0.70. The top four “often” reported symptoms were urinary frequency (52.2%), tiredness (45.5%), poor sleep (27.5%) and back pain (19.5%). Among the women surveyed, 16.2% claimed to sometimes or often be incontinent. Referrals to the incontinence nurse increased > 8 fold during the study period. Conclusions The PSI provides a comprehensive inventory of pregnancy related symptoms, with a mechanism for assessing their effect on function. It was robustly developed, with good test re-test reliability, face validity, comprehension and readability. This provides a validated tool for assessing the impact of interventions in pregnancy.