Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding β-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense ∼22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

A satellite RNA of barley yellow dwarf virus contains a novel hammerhead structure in the self-cleavage domain

An RNA molecule with properties of a satellite RNA was found in an isolate of barley yellow dwarf virus (BYDV), RPV serotype. It is 322 nucleotides long, single-stranded, and does not hybridize to the viral genome. Dimers of the RNA, which presumably represent replicative intermediates, were able to self-cleave into monomers. In vitro transcripts from cDNA clones were capable of self-cleavage in both the plus (encapsidated) and minus orientations. The sequence flanking the minus strand cleavage site contained a consensus " hammerhead" structure, similar to those found in other self-cleaving satellite RNAs. Although related to the hammerhead structure, sequences flanking the plus strand termini showed differences from the consensus and may be folded into a different structure containing a pseudoknot. © 1991.

Innovations in dietary interventions : Use of a photographic food diary method and telephone linked computerized care in chronic disease

Access to dietetic care is important in chronic disease management and innovative technologies assists in this purpose. Photographic dietary records (PhDR) using mobile phones or cameras are valid and convenient for patients. Innovations in providing dietary interventions via telephone and computer can also inform dietetic practice. Three studies are presented. A mobile phone method was validated by comparing energy intake (EI) to a weighed food record and a measure of energy expenditure (EE) obtained using the doubly labelled water technique in 10 adults with T2 diabetes. The level of agreement between mean (±sd) energy intake mobile phone (8.2±1.7 MJ) and weighed record (8.5±1.6 MJ) was high (p=0.392), however EI/EE for both methods gave similar levels of under-reporting (0.69 and 0.72). All subjects preferred using the mobile phone vs. weighed record. Nineteen individuals with Parkinsons disease kept 3-day PhDRs on three occasions using point-and-shoot digital cameras over a 12 week period. The camera was rated as easy to use by 89%, keeping a PhDR was considered acceptable by 94% and none would rather use a “pen and paper” method. Eighty-three percent felt confident to use the camera again to record intake. An interactive, automated telephone system designed to coach people with T2 diabetes to adopt and maintain diabetes self-care behaviours, including nutrition, showed trends for improvements in total fat, saturated fat and vegetable intake of the intervention group compared to control participants over 6 months. Innovative technologies are acceptable to patients with chronic conditions and can be incorporated into dietetic care.

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

Exploration of novel routes to azo-linked oligoporphyrins

This research explored new ways of chemically combining porphyrins, which are vital biomolecules, to produce new pigments known as azoporphyrins, for applications in advanced technologies. Although the final targets have not yet been realized, numerous novel compounds and known compounds lacking experimental data in the literature were fully characterised by a range of techniques. The data will facilitate subsequent studies of other novel routes to azoporphyrins and related molecules.

Integrin αvβ3 upregulates integrin-linked kinase expression in human ovarian cancer cells via enhancement of ILK gene transcription

We previously showed that integrin alphavbeta3 overexpression and engagement by its ligand vitronectin increased adhesion, motility, and proliferation of human ovarian cancer cells. In search of differentially regulated genes involved in these tumor biological events, we previously identified the integrin-linked kinase (ILK) to be under control of alphavbeta3. In the present investigation we demonstrated significantly upregulated ILK protein as a function of alphavbeta3 in two ovarian cancer cell lines, OV-MZ-6 and OVCAR-3, and proved co-localization at the surface of alphavbeta3-overexpressing cells adherent to vitronectin. Increase of ILK protein was reflected by enhanced ILK promoter activity, an effect, which we further characterized with regard to transcriptional response elements involved. Abrogation of NF-kappaB/c-rel or p53 binding augmented ILK promoter activity and preserved induction by alphavbeta3. The AP1-mutant exhibited decreased promoter activity but was also still inducible by alphavbeta3. Disruption of the two DNA consensus motifs for Ets proteins led to divergent observations: mutation of the Ets motif at promoter position -462 bp did not significantly alter promoter activity but still allowed response to alphavbeta3. In contrast, disruption of the second Ets motif at position -85 bp did not only lead to slightly diminished promoter activity but also, in that case, abrogated ILK promoter induction by alphavbeta3. Subsequent co-transfection studies with ets-1 in the presence of the second Ets motif led to additional induction of ILK promoter activity. Taken together, these data suggest that ets-1 binding to the second Ets DNA motif strongly contributes to alphavbeta3-mediated ILK upregulation. By increasing ILK as an important integrin-proximal kinase, alphavbeta3 may promote its intracellular signaling and tumor biological processes arising thereof in favor of ovarian cancer metastasis.

Diporphyrins linked by two-atom bridges synthestic, structural and theoretical studies

The syntheses, properties and electronic structures of a series of porphyrin dimers connected by two-atom bridges were compared. The study found that an azo linker results in the most efficient electronic communication between the two porphyrin rings, and is the superior connector for dimers, trimers and oligomers in the design of nonlinear optical materials. This has implications for the design of molecular probes and sensors, photodynamic therapy, microfabrication, and three-dimensional optical data storage. The research led to the synthesis of a number of new porphyrin monomers and dimers, which were characterised using structural, spectroscopic and spectrometric techniques.

A Satellite Selection Algorithm for Achieving High Reliability of Ambiguity Resolution with GPS and Beidou Constellations

Reliability of carrier phase ambiguity resolution (AR) of an integer least-squares (ILS) problem depends on ambiguity success rate (ASR), which in practice can be well approximated by the success probability of integer bootstrapping solutions. With the current GPS constellation, sufficiently high ASR of geometry-based model can only be achievable at certain percentage of time. As a result, high reliability of AR cannot be assured by the single constellation. In the event of dual constellations system (DCS), for example, GPS and Beidou, which provide more satellites in view, users can expect significant performance benefits such as AR reliability and high precision positioning solutions. Simply using all the satellites in view for AR and positioning is a straightforward solution, but does not necessarily lead to high reliability as it is hoped. The paper presents an alternative approach that selects a subset of the visible satellites to achieve a higher reliability performance of the AR solutions in a multi-GNSS environment, instead of using all the satellites. Traditionally, satellite selection algorithms are mostly based on the position dilution of precision (PDOP) in order to meet accuracy requirements. In this contribution, some reliability criteria are introduced for GNSS satellite selection, and a novel satellite selection algorithm for reliable ambiguity resolution (SARA) is developed. The SARA algorithm allows receivers to select a subset of satellites for achieving high ASR such as above 0.99. Numerical results from a simulated dual constellation cases show that with the SARA procedure, the percentages of ASR values in excess of 0.99 and the percentages of ratio-test values passing the threshold 3 are both higher than those directly using all satellites in view, particularly in the case of dual-constellation, the percentages of ASRs (>0.99) and ratio-test values (>3) could be as high as 98.0 and 98.5 % respectively, compared to 18.1 and 25.0 % without satellite selection process. It is also worth noting that the implementation of SARA is simple and the computation time is low, which can be applied in most real-time data processing applications.

The reduced expression of the HADH2 protein causes X-linked mental retardation, choreoathetosis, and abnormal behavior

Recently, we defined a new syndromic form of X-linked mental retardation in a 4-generation family with a unique clinical phenotype characterized by mild mental retardation, choreoathetosis, and abnormal behavior (MRXS10). Linkage analysis in this family revealed a candidate region of 13.4 Mb between markers DXS1201 and DXS991 on Xp11; therefore, mutation analysis was performed by direct sequencing in most of the 135 annotated genes located in the region. The gene (HADH2) encoding L-3-hydroxyacyl-CoA dehydrogenase II displayed a sequence alteration (c.574 C-->A; p.R192R) in all patients and carrier females that was absent in unaffected male family members and could not be found in 2,500 control X chromosomes, including in those of 500 healthy males. The silent C-->A substitution is located in exon 5 and was shown by western blot to reduce the amount of HADH2 protein by 60%-70% in the patient. Quantitative in vivo and in vitro expression studies revealed a ratio of splicing transcript amounts different from those normally seen in controls. Apparently, the reduced expression of the wild-type fragment, which results in the decreased protein expression, rather than the increased amount of aberrant splicing fragments of the HADH2 gene, is pathogenic. Our data therefore strongly suggest that reduced expression of the HADH2 protein causes MRXS10, a phenotype different from that caused by 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, which is a neurodegenerative disorder caused by missense mutations in this multifunctional protein.

Status of the world's remaining closed forests : an assessment using satellite data and policy options

Historically, it appears that some of the WRCF have survived because i) they lack sufficient quantity of commercially valuable species; ii) they are located in remote or inaccessible areas; or iii) they have been protected as national parks and sanctuaries. Forests will be protected when people who are deciding the fate of forests conclude than the conservation of forests is more beneficial, e.g. generates higher incomes or has cultural or social values, than their clearance. If this is not the case, forests will continue to be cleared and converted. In the future, the WRCF may be protected only by focused attention. The future policy options may include strategies for strong protection measures, the raising of public awareness about the value of forests, and concerted actions for reducing pressure on forest lands by providing alternatives to forest exploitation to meet the growing demands of forest products. Many areas with low population densities offer an opportunity for conservation if appropriate steps are taken now by the national governments and international community. This opportunity must be founded upon the increased public and government awareness that forests have vast importance to the welfare of humans and ecosystems' services such as biodiversity, watershed protection, and carbon balance. Also paramount to this opportunity is the increased scientific understanding of forest dynamics and technical capability to install global observation and assessment systems. High-resolution satellite data such as Landsat 7 and other technologically advanced satellite programs will provide unprecedented monitoring options for governing authorities. Technological innovation can contribute to the way forests are protected. The use of satellite imagery for regular monitoring and Internet for information dissemination provide effective tools for raising worldwide awareness about the significance of forests and intrinsic value of nature.

Unintended consequences : satellite policy and Indigenous television

This article examines two instances of media policy involving satellite transmission and Indigenous television: the introduction of the Viewer Access Satellite Television (VAST) platform in 2010 and the introduction of AUSSAT in the mid-1980s. The government’s failure to provide community and Indigenous broadcasters with an access regime at the time of AUSSAT resulted in Australia’s first and only Indigenous commercial television licensee, Imparja. Over a quarter of a century later, Imparja now forms part of the joint-venture company that runs VAST, a key component of Australia’s digital switchover planning. During the passage of the legislative amendments required to establish VAST, the issue of access resurfaced – this time in relation to Australia’s national and community Indigenous television channels. The article recounts the events leading up to the 2010 Bill, and examines the intended and unintended consequences of satellite policy in relation to Indigenous media, including equalisation and transparency of government funding programs.

A new plasmid display technology for the in vitro selection of functional phenotype–genotype linked proteins

Background: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype–genotype link between the displayed protein and the encoding gene. Results: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50–DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein–plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype–genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Coincident sequence-specific RNA degradation of linked transgenes in the plant genome

The expression of transgenes in plant genomes can be inhibited by either transcriptional gene silencing or posttranscriptional gene silencing (PTGS). Overexpression of the chalcone synthase-A (CHS-A) transgene triggers PTGS of CHS-A and thus results in loss of flower pigmentation in petunia. We previously demonstrated that epigenetic inactivation of CHS-A transgene transcription leads to a reversion of the PTGS phenotype. Although neomycin phosphotransferase II (nptII), a marker gene co-introduced into the genome with the CHS-A transgene, is not normally silenced in petunia, even when CHS-A is silenced, here we found that nptII was silenced in a petunia line in which CHS-A PTGS was induced, but not in the revertant plants that had no PTGS of CHS-A. Transcriptional activity, accumulation of short interfering RNAs, and restoration of mRNA level after infection with viruses that had suppressor proteins of gene silencing indicated that the mechanism for nptII silencing was posttranscriptional. Read-through transcripts of the CHS-A gene toward the nptII gene were detected. Deep-sequencing analysis revealed a striking difference between the predominant size class of small RNAs produced from the read-through transcripts (22 nt) and that from the CHS-A RNAs (21 nt). These results implicate the involvement of read-through transcription and distinct phases of RNA degradation in the coincident PTGS of linked transgenes and provide new insights into the destabilization of transgene expression.

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

Background Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. Results A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

The organisation and expression of the genes encoding the mitochondrial glycine decarboxylase complex and serine hydroxymethyltransferase in pea (Pisum sativum)

Restriction fragment length polymorphisms have been used to determine the chromosomal location of the genes encoding the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) of pea leaf mitochondria. The genes encoding the H subunit of GDC and the genes encoding SHMT both show linkage to the classical group I marker i. In addition, the genes for the P protein of GDC show linkage to the classic group I marker a. The genes for the L and T proteins of GDC are linked to one another and are probably situated on the satellite of chromosome 7. The mRNAs encoding the five polypeptides that make up GDC and SHMT are strongly induced when dark-grown etiolated pea seedlings are placed in the light. Similarly, when mature plants are placed in the dark for 48 h, the levels of both GDC protein and SHMT mRNAs decline dramatically and then are induced strongly when these plants are returned to the light. During both treatments a similar pattern of mRNA induction is observed, with the mRNA encoding the P protein of GDC being the most rapidly induced and the mRNA for the H protein the slowest. Whereas during the greening of etiolated seedlings the polypeptides of GDC and SHMT show patterns of accumulation similar to those of the corresponding mRNAs, very little change in the level of the polypeptides is seen when mature plants are placed in the dark and then re-exposed to the light.