Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants

RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 59 rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.

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.

Constructs and methods for hairpin RNA-mediated gene silencing in plants

Double-stranded RNA (dsRNA) induces an endogenous sequence-specific RNA degradation mechanism in most eukaryotic cells. The mechanism can be harnessed to silence genes in plants by expressing self-complementary single-stranded (hairpin) RNA in which the duplexed region has the same sequence as part of the target gene's mRNA. We describe a number of plasmid vectors for generating hairpin RNAs, including those designed for high-throughput cloning, and provide protocols for their use.

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.

Plant and animal microRNAs : similarities and differences

Plant and animal microRNAs (miRNAs) are evolutionarily ancient small RNAs, ∼19-24 nucleotides in length, that are generated by cleavage from larger highly structured precursor molecules. In both plants and animals, miRNAs posttranscriptionally regulate gene expression through interactions with their target mRNAs, and these targets are often genes involved with regulating key developmental events. Despite these similarities, plant and animal miRNAs exert their control in fundamentally different ways. Generally, animal miRNAs repress gene expression by mediating translational attenuation through (multiple) miRNA-binding sites located within the 3′ untranslated region of the target gene. In contrast, almost all plant miRNAs regulate their targets by directing mRNA cleavage at single sites in the coding regions. These and other differences suggest that the two systems may have originated independently, possibly as a prerequisite to the development of complex body plans. © Springer-Verlag 2005.

Virus resistance and gene silencing : killing the messenger

On occasion, virus-derived transgenes in plants can be poorly expressed and yet provide excellent virus resistance, and transgene constructs designed to supplement the expression of endogenous genes can have the effect of co-suppressing themselves and the endogenous genes. These two phenomena appear to result from the same post-transcriptional silencing mechanism, which operates by targeted-RNA degradation. Recent research into RNA-mediated virus resistance and co-suppression has provided insights into the interactions between plant viruses and their hosts, and spawned several models to explain the phenomenon.

Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens

In binary vectors, the antibiotic resistance gene used for selection of transformed plant cells is also usually expressed in the transforming Agrobacterium cells. This expression gives the bacterium antibiotic resistance, an unnecessary advantage on selective medium containing the antibiotic. Insertion of a castor bean catalase-1 (CAT-1) gene intron or a Parasponia andersonii haemoglobin gene intron into the coding region of the selectable marker gene, hph, completely abolished the expression of the gene in Agrobacterium, rendering it susceptible to hygromycin B. Use of these modified binary vectors minimized the overgrowth of Agrobacterium during plant transformation. Both of the introns were correctly spliced in plant cells and significantly enhanced hph gene expression in transformed rice tissue. The presence of these introns in the hph coding sequence not only maintained the selection efficiency of the hph gene, but with the CAT-1 intron also substantially increased the frequency of rice transformation. Transgenic lines with an intron-hph gene generally contained fewer gene copies and produced substantially more mRNA of the predicted size. Our results also indicate that transgenic plants with many copies of the transgene were more likely to show gene silencing than plants with 1-3 copies.

Development of an evidence-based framework to guide injury prevention interventions for off-road motorcyclists

There is little research on off-road motorcycle and all-terrain vehicle riders though injury levels are high. This thesis identified formal responsibility for monitoring injuries, targeting young male and recreational riders, promotion of family members as models, and controlled and accessible riding locations as ways to increase safety. These recommendations were based on analysis of Queensland hospitalisation records, rider personal reports and survey responses.

Watermemories : a swimming club for people with dementia

The purpose of this article is to describe the conceptual model and implementation strategies of an evidence-based, aquatic exercise program specifically targeting individuals with dementia—The Watermemories Swimming Club (WSC). Physical exercise not only improves the functional capacity of people with dementia but also has significant effects on other aspects of quality of life such as sleep, appetite, behavioral and psychological symptoms, depression, and falls. Additionally, exercise can improve a person’s overall sense of well-being and positively enhance their sociability. The WSC was designed to increase physical exercise while being easy to implement, safe, and pleasurable. Many challenges were faced along the way, and we discuss how these were overcome. Implications for nurses are also provided.

Predictors of initiating and maintaining active commuting to work using transport and public health perspectives in Australia

Objective To identify predictors for initiating and maintaining active commuting (AC) to work following the 2003 Australia's Walk to Work Day (WTWD) campaign. Methods Pre- and post-campaign telephone surveys of a cohort of working age (18–65years) adults (n = 1100, 55% response rate). Two dependent campaign outcomes were assessed: initiating or maintaining AC (i.e., walk/cycle and public transport) on a single day (WTWD), and increasing or maintaining health-enhancing active commuting (HEAC) level (≥ 30min/day) in a usual week following WTWD campaign. Results A significant population-level increase in HEAC (3.9%) was observed (McNemar's χ2 = 6.53, p = 0.01) with 136 (19.0%) achieving HEAC at post campaign. High confidence in incorporating walking into commute, being active pre-campaign and younger age (< 46years) were positively associated with both outcomes. The utility of AC for avoiding parking hassles (AOR = 2.1, 95% CI: 1.2–3.6), for less expense (AOR = 1.8, 95% CI: 1.1–3.1), for increasing one's health (AOR = 2.5, 95% CI: 1.1–5.6) and for clean air (AOR = 2.2, 95% CI: 1.0–4.4) predicted HEAC outcome whereas avoiding the stress of driving (AOR = 2.6, 95% CI: 1.4–5.0) and the hassle of parking predicted the single-day AC. Conclusions Transportation interventions targeting parking and costs could be further enhanced by emphasizing health benefits of AC. AC was less likely to occur among inactive employees.

Concealed texting while driving : what are young people’s beliefs about this risky behaviour?

Making a conscious effort to hide the fact that one is texting while driving (i.e., concealed texting) is a deliberate and risky behaviour involving attention diverted from the road. As the most frequent users of text messaging services and mobile phones while driving, young people appear at heightened crash risk from engaging in this behaviour. First, several small focus group discussions (N = 12) were carried out to elicit the underlying salient beliefs regarding this behaviour, in accordance with the Theory of Planned Behaviour (TPB). Findings from these discussions, in conjunction with available prior evidence regarding general mobile phone use while driving, then informed questionnaire items that assessed young peoples’ beliefs regarding this behaviour, as well as intention to engage in this behaviour in the next week. In the questionnaire phase of the study, participants (N = 171) were aged 17–25 years, owned a mobile phone, and held a current driver’s licence. Results showed that there were significant differences between low and high intenders (to engage in concealed texting while driving) on the behavioural, normative, and control beliefs investigated. Specifically, high intenders were more likely to believe that concealed texting while driving would result in sharing information with others, using time effectively, and were less likely to think that free-flowing traffic would prevent their engagement in this behaviour. By targeting these beliefs, these findings may potentially inform the development of advertising and other public intervention strategies, aimed at ensuring young drivers reconsider their engagement in this risky behaviour.

Transcutaneous immunization with a novel lipid-based adjuvant protects against Chlamydia genital and respiratory infections

Mucosal adjuvants are important to overcome the state of immune tolerance normally associated with mucosal delivery and to enhance adaptive immunity to often-weakly immunogenic subunit vaccine antigens. Unfortunately, adverse side effects of many experimental adjuvants limit the number of adjuvants approved for vaccination. Lipid C is a novel, non-toxic, lipid oral vaccine-delivery formulation, developed originally for oral delivery of the live Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine. In the present study, murine models of chlamydial respiratory and genital tract infections were used to determine whether transcutaneous immunization (TCI) with Lipid C-incorporated protein antigens could elicit protective immunity at the genital and respiratory mucosae. BALB/c mice were immunized transcutaneously with Lipid C containing the chlamydial major outer membrane protein (MOMP), with and without addition of cholera toxin and CpG-ODN 1826 (CT/CpG). Both vaccine combinations induced mixed cell-mediated and mucosal antibody immune responses. Immunization with Lipid C-incorporated MOMP (Lipid C/MOMP), either alone or with CT/CpG resulted in partial protection following live challenge with Chlamydia muridarum as evidenced by a significant reduction in recoverable Chlamydia from both the genital secretions and lung tissue. Protection induced by immunization with Lipid C/MOMP alone was not further enhanced by the addition of CT/CpG. These results highlight the potential of Lipid C as a novel mucosal adjuvant capable of targeting multiple mucosal surfaces following TCI. Protection at both the respiratory and genital mucosae was achieved without the requirement for potentially toxic adjuvants, suggesting that Lipid C may provide a safe effective mucosal adjuvant for human vaccination.

How accurate is the identification of serious traffic injuries by Police? The concordance between Police and hospital reported traffic injuries

Police reported crash data are the primary source of crash information in most jurisdictions. However, the definition of serious injury within police-reported data is not consistent across jurisdictions and may not be accurate. With the Australian National Road Safety Strategy targeting the reduction of serious injuries, there is a greater need to assess the accuracy of the methods used to identify these injuries. A possible source of more accurate information relating to injury severity is hospital data. While other studies have compared police and hospital data to highlight the under-reporting in police-reported data, little attention has been given to the accuracy of the methods used by police to identify serious injuries. The current study aimed to assess how accurate the identification of serious injuries is in police-reported crash data, by comparing the profiles of transport-related injuries in the Queensland Road Crash Database with an aligned sample of data from the Queensland Hospital Admitted Patients Data Collection. Results showed that, while a similar number of traffic injuries were recorded in both data sets, the profile of these injuries was different based on gender, age, location, and road user. The results suggest that the ‘hospitalisation’ severity category used by police may not reflect true hospitalisations in all cases. Further, it highlights the wide variety of severity levels within hospitalised cases that are not captured by the current police-reported definitions. While a data linkage study is required to confirm these results, they highlight that a reliance on police-reported serious traffic injury data alone could result in inaccurate estimates of the impact and cost of crashes and lead to a misallocation of valuable resources.

Differential Regulation of Clusterin Isoforms by the Androgen Receptor

Clusterin (CLU) was initially reported as an androgen-repressed gene which is now shown to be an androgen-regulated ATP-independent cytoprotective molecular chaperone. CLU binds to a wide variety of client proteins to potently inhibit stress-induced protein aggregation and chaperone or stabilise conformations of proteins at times of cell stress. CLU is an enigmatic protein, being ascribed both pro- and anti-apoptotic roles. Recent evidence has shown that both secreted (sCLU) and nuclear (nCLU) isoforms can be produced, and that protein function is dependent on the sub-cellular localisation. We and others have shown that sCLU is cytoprotective, while nCLU is pro-apoptotic. It now seems likely that the apparently dichotomous functions of CLU result from the expression of different but related CLU isoforms and splice variants, and that cell survival depends in part on the relative expression of pro- versus anti-apoptotic CLU proteins. In cancer cells, increased sCLU expression is associated with increased resistance to apoptotic triggers and treatment resistance. CLU is a stress-induced protein upregulated after apoptotic triggers like androgen ablation and chemotherapy. Treatment strategies targeting stress-associated increases in sCLU expression enhance treatment-induced apoptosis and delay the emergence of androgen independence. Differential regulation of CLU isoforms and splice variants by androgens may be a pathway whereby cancer cells develop treatment resistance and evade apoptosis.

Spatiotemporal patterns of Aedes aegyptipopulations in Cairns, Australia : assessing drivers of dengue transmission

OBJECTIVES To identify the meteorological drivers of dengue vector density and determine high- and low-risk transmission zones for dengue prevention and control in Cairns, Australia. METHODS Weekly adult female Ae. aegypti data were obtained from 79 double sticky ovitraps (SOs) located in Cairns for the period September 2007-May 2012. Maximum temperature, total rainfall and average relative humidity data were obtained from the Australian Bureau of Meteorology for the study period. Time series-distributed lag nonlinear models were used to assess the relationship between meteorological variables and vector density. Spatial autocorrelation was assessed via semivariography, and ordinary kriging was undertaken to predict vector density in Cairns. RESULTS Ae. aegypti density was associated with temperature and rainfall. However, these relationships differed between short (0-6 weeks) and long (0-30 weeks) lag periods. Semivariograms showed that vector distributions were spatially autocorrelated in September 2007-May 2008 and January 2009-May 2009, and vector density maps identified high transmission zones in the most populated parts of Cairns city, as well as Machans Beach. CONCLUSION Spatiotemporal patterns of Ae. aegypti in Cairns are complex, showing spatial autocorrelation and associations with temperature and rainfall. Sticky ovitraps should be placed no more than 1.2 km apart to ensure entomological coverage and efficient use of resources. Vector density maps provide evidence for the targeting of prevention and control activities. Further research is needed to explore the possibility of developing an early warning system of dengue based on meteorological and environmental factors.