Small RNA sequencing of potato leafroll virus-infected plants reveals an additional subgenomic RNA encoding a sequence-specific RNA-binding protein

Potato leafroll virus (PLRV) is a positive-strand RNA virus that generates subgenomic RNAs (sgRNA) for expression of 3' proximal genes. Small RNA (sRNA) sequencing and mapping of the PLRV-derived sRNAs revealed coverage of the entire viral genome with the exception of four distinctive gaps. Remarkably, these gaps mapped to areas of PLRV genome with extensive secondary structures, such as the internal ribosome entry site and 5' transcriptional start site of sgRNA1 and sgRNA2. The last gap mapped to ~500. nt from the 3' terminus of PLRV genome and suggested the possible presence of an additional sgRNA for PLRV. Quantitative real-time PCR and northern blot analysis confirmed the expression of sgRNA3 and subsequent analyses placed its 5' transcriptional start site at position 5347 of PLRV genome. A regulatory role is proposed for the PLRV sgRNA3 as it encodes for an RNA-binding protein with specificity to the 5' of PLRV genomic RNA. © 2013.

Role of short RNAs in gene silencing

Recent research has revealed the existence of an elegant defence mechanism in plants and lower eukaryotes. The mechanism, known in plants as post-transcriptional gene silencing, works through sequence-specific degradation of RNA. It appears to be directed by double-stranded RNA, associated with the production of short 21-25 nt RNAs, and spread through the plant by a diffusible signal. The short RNAs are implicated as the guides for both a nuclease complex that degrades the mRNA and a methyltransferase complex that methylates the DNA of silenced genes. It has also been suggested that these short RNAs might be the mobile silencing signal, a suggestion that has been challenged recently.

Expression patterns of vascular-specific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants

The expression patterns of GUS fusion constructs driven by the Agrobacterium rhizogenes RolC and the maize Sh (Shrunken: sucrose synthase-1) promoters were examined in transgenic potatoes (cv. Atlantic). RolC drove high-level gene expression in phloem tissue, bundle sheath cells and vascular parenchyma, but not in xylem or non-vascular tissues. Sh expression was exclusively confined to phloem tissue. Potato leafroll luteovirus (PLRV) replicates only in phloem tissues, and we show that when RolC is used to drive expression of the PLRV coat protein gene, virus-resistant lines can be obtained. In contrast, no significant resistance was observed when the Sh promoter was used.

Reconsidering the agency of a privately appointed receiver and manager in three specific circumstances

Where a secured lender elects to appoint a receiver and manager, the appointment document standardly provides for the receiver and manager to act as the agent of the debtor. This article considers the significance of this agency in the context of three specific issues that have the potential to arise in the receivership of a corporate borrower across all Australian jurisdictions.

Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity

Introduction The clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes. Methods We investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague-Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining. Results Sex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p = 0.028). Throughout the follow-up, the cross-sectional area of callus relative to bone was reduced in females (p ≤ 0.01), and the bridging of callus was delayed (p 2weeks = 0.041). μCT revealed a reduced callus size (p = 0.003), mineralization (p = 0.003) and polar moment of inertia (p = 0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae (p femur = 0.017, p tibia = 0.010). Functional characteristics of male and female MSCs were similar. Conclusion Biomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.

Second harmonic generation and multiphoton microscopic detection of collagen without the need for species specific antibodies

High-resolution, high-contrast, three-dimensional images of live cell and tissue architecture can be obtained using second harmonic generation (SHG), which comprises non-absorptive frequency changes in an excitation laser line. SHG does not require any exogenous antibody or fluorophore labeling, and can generate images from unstained sections of several key endogenous biomolecules, in a wide variety of species and from different types of processed tissue. Here, we examined normal control human skin sections and human burn scar tissues using SHG on a multi-photon microscope (MPM). Examination and comparison of normal human skin and burn scar tissue demonstrated a clear arrangement of fibers in the dermis, similar to dermal collagen fiber signals. Fluorescence-staining confirmed the MPM-SHG collagen colocalization with antibody staining for dermal collagen type-I but not fibronectin or elastin. Furthermore, we were able to detect collagen MPM-SHG signal in human frozen sections as well as in unstained paraffin embedded tissue sections that were then compared with hematoxylin and eosin staining in the identical sections. This same approach was also successful in localizing collagen in porcine and ovine skin samples, and may be particularly important when species-specific antibodies may not be available. Collectively, our results demonstrate that MPM SHG-detection is a useful tool for high resolution examination of collagen architecture in both normal and wounded human, porcine and ovine dermal tissue.

Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients

Recent studies suggest that genetic and environmental factors do not account for all the schizophrenia risk and epigenetics also plays a role in disease susceptibility. DNA methylation is a heritable epigenetic modification that can regulate gene expression. Genome-Wide DNA methylation analysis was performed on post-mortem human brain tissue from 24 patients with schizophrenia and 24 unaffected controls. DNA methylation was assessed at over 485 000 CpG sites using the Illumina Infinium Human Methylation450 Bead Chip. After adjusting for age and post-mortem interval (PMI), 4 641 probes corresponding to 2 929 unique genes were found to be differentially methylated. Of those genes, 1 291 were located in a CpG island and 817 were in a promoter region. These include NOS1, AKT1, DTNBP1, DNMT1, PPP3CC and SOX10 which have previously been associated with schizophrenia. More than 100 of these genes overlap with a previous DNA methylation study of peripheral blood from schizophrenia patients in which 27 000 CpG sites were analysed. Unsupervised clustering analysis of the top 3 000 most variable probes revealed two distinct groups with significantly more people with schizophrenia in cluster one compared to controls (p = 1.74x10-4). The first cluster was composed of 88% of patients with schizophrenia and only 12% controls while the second cluster was composed of 27% of patients with schizophrenia and 73% controls. These results strongly suggest that differential DNA methylation is important in schizophrenia etiology and add support for the use of DNA methylation profiles as a future prognostic indicator of schizophrenia.

Context-specific stressors, work-related social support and work-family conflict : a mediation study

Understanding the antecedents of work-family conflict is important as it allows organisations to effectively engage in work design for professional employees. This study examines the impact of sources of social support as antecedents of work-family conflict. The hypotheses were tests using Partial Least Squares modelling on a sample of 366 professional employees. The path model showed that context-specific stressors impacted positively on job demand, which led to higher levels of work-family conflict. Contrary to our expectation, non-work related social support did not have any statistical relationship with job demand and work-family conflict. In addition, individuals experiencing high job demands were found to obtain more social support from both work and non-work-related sources. Individuals with more work-related social support were less likely to have less work-family conflict. Surprisingly, non-work social support sources had no statistically significant relationship with work-family conflict.

Lesions in the bed nucleus of the stria terminalis disrupt corticosterone and freezing responses elicited by a contextual but not by a specific cue-conditioned fear stimulus

The bed nucleus of the stria terminalis (BNST) is believed to be a critical relay between the central nucleus of the amygdala (CE) and the paraventricular nucleus of the hypothalamus in the control of hypothalamic–pituitary– adrenal (HPA) responses elicited by conditioned fear stimuli. If correct, lesions of CE or BNST should block expression of HPA responses elicited by either a specific conditioned fear cue or a conditioned context. To test this, rats were subjected to cued (tone) or contextual classical fear conditioning. Two days later, electrolytic or sham lesions were placed in CE or BNST. After 5 days, the rats were tested for both behavioral (freezing) and neuroendocrine (corticosterone) responses to tone or contextual cues. CE lesions attenuated conditioned freezing and corticosterone responses to both tone and con- text. In contrast, BNST lesions attenuated these responses to contextual but not tone stimuli. These results suggest CE is indeed an essential output of the amygdala for the expres- sion of conditioned fear responses, including HPA re- sponses, regardless of the nature of the conditioned stimu- lus. However, because lesions of BNST only affected behav- ioral and endocrine responses to contextual stimuli, the results do not support the notion that BNST is critical for HPA responses elicited by conditioned fear stimuli in general. Instead, the BNST may be essential specifically for contex- tual conditioned fear responses, including both behavioral and HPA responses, by virtue of its connections with the hippocampus, a structure essential to contextual condition- ing. The results are also not consistent with the hypothesis that BNST is only involved in unconditioned aspects of fear and anxiety.

Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone

The development of effective therapeutic strategies against prostate cancer bone metastases has been impeded by the lack of adequate animal models that are able to recapitulate the biology of the disease in humans. Bioengineered approaches allow researchers to create sophisticated experimentally and physiologically relevant in vivo models to study interactions between cancer cells and their microenvironment under reproducible conditions. The aim of this study was to engineer a morphologically and functionally intact humanized organ bone which can serve as a homing site for human prostate cancer cells. Transplantation of biodegradable tubular composite scaffolds seeded with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone construct including a large number of human mesenchymal cells which were shown to be metabolically active and capable of producing extracellular matrix components. Micro-CT analysis demonstrated that the newly formed ossicle recapitulated the morphological features of a physiological organ bone with a trabecular network surrounded by a cortex-like outer structure. This microenvironment was supportive of the lodgement and maintenance of murine haematopoietic cell clusters, thus mimicking a functional organ bone. Bioluminescence imaging demonstrated that luciferase-transduced human PC3 cells reproducibly homed to the humanized tissue engineered bone constructs, proliferated, and developed macro-metastases. This model allows the analysis of interactions between human prostate cancer cells and a functional humanized bone organ within an immuno-incompetent murine host. The system can serve as a reproducible platform to study effects of therapeutics against prostate cancer bone metastases within a humanized microenvironment.

Mining specific features for acquiring user information needs

Term-based approaches can extract many features in text documents, but most include noise. Many popular text-mining strategies have been adapted to reduce noisy information from extracted features; however, text-mining techniques suffer from low frequency. The key issue is how to discover relevance features in text documents to fulfil user information needs. To address this issue, we propose a new method to extract specific features from user relevance feedback. The proposed approach includes two stages. The first stage extracts topics (or patterns) from text documents to focus on interesting topics. In the second stage, topics are deployed to lower level terms to address the low-frequency problem and find specific terms. The specific terms are determined based on their appearances in relevance feedback and their distribution in topics or high-level patterns. We test our proposed method with extensive experiments in the Reuters Corpus Volume 1 dataset and TREC topics. Results show that our proposed approach significantly outperforms the state-of-the-art models.

Geometric sensitivity of patient-specific finite element models of the spine to variability in user-selected anatomical landmarks

Software to create individualised finite element (FE) models of the osseoligamentous spine using pre-operative computed tomography (CT) data-sets for spinal surgery patients has recently been developed. This study presents a geometric sensitivity analysis of this software to assess the effect of intra-observer variability in user-selected anatomical landmarks. User-selected landmarks on the osseous anatomy were defined from CT data-sets for three scoliosis patients and these landmarks were used to reconstruct patient-specific anatomy of the spine and ribcage using parametric descriptions. The intra-observer errors in landmark co-ordinates for these anatomical landmarks were calculated. FE models of the spine and ribcage were created using the reconstructed anatomy for each patient and these models were analysed for a loadcase simulating clinical flexibility assessment. The intra-observer error in the anatomical measurements was low in comparison to the initial dimensions, with the exception of the angular measurements for disc wedge and zygapophyseal joint (z-joint) orientation and disc height. This variability suggested that CT resolution may influence such angular measurements, particularly for small anatomical features, such as the z-joints, and may also affect disc height. The results of the FE analysis showed low variation in the model predictions for spinal curvature with the mean intra-observer variability substantially less than the accepted error in clinical measurement. These findings demonstrate that intra-observer variability in landmark point selection has minimal effect on the subsequent FE predictions for a clinical loadcase.

Determining epithelial contribution to in vivo mesenchymal tumour expression signature using species-specific microarray profiling analysis of xenografts

Gene expression profiling using microarrays and xenograft transplants of human cancer cell lines are both popular tools to investigate human cancer. However, the undefined degree of cross hybridization between the mouse and human genomes hinders the use of microarrays to characterize gene expression of both the host and the cancer cell within the xenograft. Since an increasingly recognized aspect of cancer is the host response (or cancer-stroma interaction), we describe here a bioinformatic manipulation of the Affymetrix profiling that allows interrogation of the gene expression of both the mouse host and the human tumour. Evidence of microenvironmental regulation of epithelial mesenchymal transition of the tumour component in vivo is resolved against a background of mesenchymal gene expression. This tool could allow deeper insight to the mechanism of action of anti-cancer drugs, as typically novel drug efficacy is being tested in xenograft systems.

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.

Measurement of general and specific approaches to physical activity parenting : a systematic review

Background Parents play a significant role in shaping youth physical activity (PA). However, interventions targeting PA parenting have been ineffective. Methodological inconsistencies related to the measurement of parental influences may be a contributing factor. The purpose of this article is to review the extant peer-reviewed literature related to the measurement of general and specific parental influences on youth PA. Methods A systematic review of studies measuring constructs of PA parenting was conducted. Computerized searches were completed using PubMed, MEDLINE, Academic Search Premier, SPORTDiscus, and PsycINFO. Reference lists of the identified articles were manually reviewed as well as the authors' personal collections. Articles were selected on the basis of strict inclusion criteria and details regarding the measurement protocols were extracted. A total of 117 articles met the inclusionary criteria. Methodological articles that evaluated the validity and reliability of PA parenting measures (n=10) were reviewed separately from parental influence articles (n=107). Results A significant percentage of studies used measures with indeterminate validity and reliability. A significant percentage of articles did not provide sample items, describe the response format, or report the possible range of scores. No studies were located that evaluated sensitivity to change. Conclusion The reporting of measurement properties and the use of valid and reliable measurement scales need to be improved considerably.