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.

Comparison of the coat protein, movement protein and RNA polymerase gene sequences of Australian, Chinese, and American isolates of barley yellow dwarf virus transmitted by Rhopalosiphum padi

Barley yellow dwarf luteovirus-GPV (BYDV-GPV) is a common problem in Chinese wheat crops but is unrecorded elsewhere. A defining characteristic of GPV is its capacity to be transmitted efficiently by both Schizaphis graminum and Rhopaloshiphum padi. This dual aphid species transmission contrasts with those of BYDV-RPV and BYDV-SGV, globally distributed viruses, which are efficiently transmitted only by Rhopaloshiphum padi and Schizaphis graminum respectively. The viral RNA sequences encoding the coat protein (22K) gene, the movement protein (17K) gene, the region surrounding the conserved GDD motif of the polymerase gene and the intergenic sequences between these genes were determined for GPV and an Australian isolate of BYDV-RPV (RPVa). In all three genes, the sequences of GPV and RPVa were more similar to those of an American isolate of BYDV-RPV (RPVu) than to any other luteovirus for which there is data available. RPVa and RPVu were very similar, especially their coat proteins which had 97% identity at the amino acid level. The coat protein of GPV had 76% and 78% amino acid identity with RPVa and RPVu respectively. The data suggest that RPVu and RPVa are correctly named as strains of the same serotype and that GPV is sufficiently different from either RPV strain to be considered a distinct BYDV type. The coat protein and movement protein genes of GPV are very dissimilar to SGV. The polymerase sequences of RPVu, RPVa and GPV show close affinities with those of the sobemo-like luteoviruses and little similarity with those of the carmo-like luteoviruses. The sequences of the coat proteins, movement proteins and the polymerase segments of BYDV serotypes, other than RPV and GPV, form a cluster that is separate from their counterpart sequences from dicot-infecting luteoviruses. The RPV and GPV isolates consistently fall within a dicot-infecting cluster. This suggests that RPV and GPV evolved from within this group of viruses. Since these other viruses all infect dicots it seems likely that their common ancestor infected a dicot and that RPV and GPV evolved from a virus that switched hosts from a dicot to a monocot.

The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67 348 polypeptide encoded by genome segment 8

The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSV S8 is 1 914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1 810 which is capable of encoding a protein of M(r) 67 348. The N-terminal amino acid sequence of a ~43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto- catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a ~43K major capsid protein.

Developing an evidence-based clinical pathway for the assessment, diagnosis and management of acute Charcot Neuro-Arthropathy : a systematic review

Background: Charcot Neuro-Arthropathy (CN) is one of the more devastating complications of diabetes. To the best of the authors' knowledge, it appears that no clinical tools based on a systematic review of existing literature have been developed to manage acute CN. Thus, the aim of this paper was to systematically review existing literature and develop an evidence-based clinical pathway for the assessment, diagnosis and management of acute CN in patients with diabetes. Methods: Electronic databases (Medline, PubMed, CINAHL, Embase and Cochrane Library), reference lists, and relevant key websites were systematically searched for literature discussing the assessment, diagnosis and/or management of acute CN published between 2002-2012. At least two independent investigators then quality rated and graded the evidence of each included paper. Consistent recommendations emanating from the included papers were then fashioned in a clinical pathway. Results: The systematic search identified 267 manuscripts, of which 117 (44%) met the inclusion criteria for this study. Most manuscripts discussing the assessment, diagnosis and/or management of acute CN constituted level IV (case series) or EO (expert opinion) evidence. The included literature was used to develop an evidence-based clinical pathway for the assessment, investigations, diagnosis and management of acute CN. Conclusions: This research has assisted in developing a comprehensive, evidence-based clinical pathway to promote consistent and optimal practice in the assessment, diagnosis and management of acute CN. The pathway aims to support health professionals in making early diagnosis and providing appropriate immediate management of acute CN, ultimately reducing its associated complications such as amputations and hospitalisations.

Update on the inaugural Sydney Diabetic Foot Conference 2013

The first International Diabetic Foot Conference in Australia was hosted at Liverpool Hospital in Sydney during May 30-31, 2013. In response to the growing diabetes epidemic globally and more locally to Australia, the conference provided the perfect bridge for interaction between the multidisciplinary team members involved in diabetes care and the opportunity to assimilate the most up-to-date evidence-based medicine from some of the most respected researchers in the field.

Queensland’s high risk foot database : tracking the length and width of Queensland’s foot ulcers

Background Foot ulcers are a leading cause of avoidable hospital admissions and lower extremity amputations. However, large clinical studies describing foot ulcer presentations in the ambulatory setting are limited. The aim of this descriptive observational paper is to report the characteristics of ambulatory foot ulcer patients managed across 13 of 17 Queensland Health & Hospital Services. Methods Data on all foot ulcer patients registered with a Queensland High Risk Foot Form (QHRFF) was collected at their first consult in 2012. Data is automatically extracted from each QHRFF into a Queensland high risk foot database. Descriptive statistics display age, sex, ulcer types and co-morbidities. Statewide clinical indicators of foot ulcer management are also reported. Results Overall, 2,034 people presented with a foot ulcer in 2012. Mean age was 63(±14) years and 67.8% were male. Co-morbidities included 85% had diabetes, 49.7% hypertension, 39.2% dyslipidaemia, 25.6% cardiovascular disease, 13.7% kidney disease and 12.2% smoking. Foot ulcer types included 51.6% neuropathic, 17.8% neuro-ischaemic, 7.2% ischaemic, 6.6% post-surgical and 16.8% other; whilst 31% were infected. Clinical indicator results revealed 98% had their wound categorised, 51% received non-removable offloading, median ulcer healing time was 6-weeks and 37% had ulcer recurrence. Conclusion This paper details the largest foot ulcer database reported in Australia. People presenting with foot ulcers appear predominantly older, male with several co-morbidities. Encouragingly it appears most patients are receiving best practice care. These results may be a factor in the significant reduction of Queensland diabetes foot-related hospitalisations and amputations recently reported.

Reduction in the incidence of diabetes lower extremity amputations in Queensland : 2005-2010

Background Lower extremity amputation is a common end stage complication among people with diabetes. Since 2006, the Queensland Diabetes Clinical Network has implemented programs aimed at reducing diabetes-related amputations. The aim of this retrospective observational study was to determine the incidence of diabetes lower extremity amputations in Queensland from 2005 to 2010. Methods Data on all Queensland diabetes-related lower extremity amputation admissions from 2005-2010 was obtained using diabetes amputation-related ICD-10-AM (hospital discharge) codes. Queensland diabetes amputation incidences were calculated for both general and diabetes populations using population data from the Australian Bureau of Statistics and National Diabetes Services Scheme respectively. Chi-squared tests were used to assess changes in amputation incidence over time. Results Overall, 4,443 admissions for diabetes-related amputation occurred; 32% (1,434) were major amputations. The diabetes-related amputation incidence among the general population (per 100,000) reduced by 18% (18.2 in 2005, to 15.0 in 2010, p < 0.001); major amputations decreased by 24% (6.6 to 4.7, p < 0.01). The incidence among the diabetes population (per 1,000) reduced by 40% (6.7 in 2005, to 4.0 in 2010, p < 0.001); major amputations decreased by 45% (2.3 to 1.2, p < 0.001). Conclusion This paper appears to be the first to report a significant reduction in diabetes amputation incidence in an Australian state. This decrease has coincided with the implementation of several diabetes foot clinical programs throughout Queensland. Whilst these results are encouraging in the Australian context, further efforts are required to decrease to levels reported internationally.

Is the clinical Queensland High Risk Foot Form valid or reliable for research?

Background High-risk foot complications such as neuropathy, ischaemia, deformity, infections, ulcers and amputations consume considerable health care resources and typically result from chronic diseases. This study aimed to develop and test the validity and reliability of a Queensland High Risk Foot Form (QHRFF) tool. Methods Phase one involved developing a QHRFF using an existing diabetes high-risk foot tool, literature search, expert panel and several state-wide stakeholder groups. Phase two tested the criterion-related validity along with inter- and intra-rater reliability of the final QHRFF. Three cohorts of patients (n = 94) and four clinicians, representing different levels of expertise, were recruited. Validity was determined by calculating sensitivity, specificity and positive predictive values (PPV). Kappa and intra-class correlation (ICC) statistics were used to establish reliability. Results A QHRFF tool containing 46-items across seven domains was developed and endorsed. The majority of QHRFF items achieved moderate-to-perfect validity (PPV = 0.71 – 1) and reliability (Kappa/ICC = 0.41 – 1). Items with weak validity and/or reliability included those identifying health professionals previously attending the patient, other (non-listed) co-morbidity, previous foot ulcer, foot deformity, optimum offloading and optimum footwear. Conclusions The QHRFF had moderate-to-perfect validity and reliability across the majority of items, particularly identifying individual co-morbidities and foot complications. Items with weak validity or reliability need to be re-defined or removed. Overall, the QHRFF appears to be a valid and reliable tool to assess, collect and measure clinical data pertaining to high-risk foot complications for clinical or research purposes.

Biomechanical characteristics of peripheral diabetic neuropathy : a systematic review and meta-analysis of findings from the gait cycle, muscle activity and dynamic barefoot plantar pressure

Background: Diabetic peripheral neuropathy is an important cause of foot ulceration and limb loss. This systematic review and meta-analysis investigated the effect of diabetic peripheral neuropathy on gait, dynamic electromyography and dynamic plantar pressures. Methods: Electronic databases were searched systematically for articles reporting the effect of diabetic peripheral neuropathy on gait, dynamic electromyography and plantar pressures. Searches were restricted to articles published between January 2000 and April 2012. Outcome measures assessed included spatiotemporal parameters, lower limb kinematics, kinetics, muscle activation and plantar pressure. Meta-analyses were carried out on all outcome measures reported by ≥3 studies. Findings: Sixteen studies were included consisting of 382 neuropathy participants, 216 diabetes controls without neuropathy and 207 healthy controls. Meta-analysis was performed on 11 gait variables. A high level of heterogeneity was noted between studies. Meta-analysis results suggested a longer stance time and moderately higher plantar pressures in diabetic peripheral neuropathy patients at the rearfoot, midfoot and forefoot compared to controls. Systematic review of studies suggested potential differences in the biomechanical characteristics (kinematics, kinetics, EMG) of diabetic neuropathy patients. However these findings were inconsistent and limited by small sample sizes.; Interpretation: Current evidence suggests that patients with diabetic peripheral neuropathy have elevated plantar pressures and occupy a longer duration of time in the stance-phase during gait. Firm conclusions are hampered by the heterogeneity and small sample sizes of available studies. Interpretation: Current evidence suggests that patients with diabetic peripheral neuropathy have elevated plantar pressures and occupy a longer duration of time in the stance-phase during gait. Firm conclusions are hampered by the heterogeneity and small sample sizes of available studies.

Evaluating the impact of high risk foot training on undergraduate podiatry students

Background Diabetes is the leading cause of high risk foot (HRF) complications, admissions and lower limb amputation. Best practice training of podiatrists is known to have a beneficial impact on such outcomes; however, there has been a paucity of studies into undergraduate diabetes podiatry training. The primary aim of this paper was to investigate the changes in final year podiatry students’ confidence, knowledge and clinical practice in the management of HRF complications. Methods This was a prospective longitudinal study of final year podiatry students (n=25) at the Queensland University of Technology. All participants throughout 2011 undertook an intervention of a series of “hands on” HRF workshops, on-campus clinics and external clinical rotations. Outcome measures included customised confidence and knowledge surveys in HRF management across four time points. A timed simulated case scenario was used to evaluate changes in clinical practice at two time points. Friedman and Wilcoxon Signed Rank Tests were used to calculate differences between time points Results Overall improvements between the first and last time points were demonstrated in 20/21 confidence items (p<0.001), 12/27 clinical practice items (p<0.05) and 3/12 knowledge items (p<0.001). Although 8/12 knowledge items recorded high baseline scores of over 80%. Conclusions Overall, it appears student confidence and clinical practice improved with the introduction of designated HRF activities, whilst knowledge remained high. This suggests “hands on” practice and not didactic lectures improve students’ clinical confidence and practice. Results from the 2012 student cohort will also be presented at this conference.

Australian Diabetes Foot Network : practical guideline on the provision of footwear for people with diabetes

Trauma, in the form of pressure and/or friction from footwear, is a common cause of foot ulceration in people with diabetes. These practical recommendations regarding the provision of footwear for people with diabetes were agreed upon following review of existing position statements and clinical guidelines. The aim of this process was not to re-invent existing guidelines but to provide practical guidance for health professionals on how they can best deliver these recommendations within the Australian health system. Where information was lacking or inconsistent, a consensus was reached following discussion by all authors. Appropriately prescribed footwear, used alone or in conjunction with custom-made foot orthoses, can reduce pedal pressures and reduce the risk of foot ulceration. It is important for all health professionals involved in the care of people with diabetes to both assess and make recommendations on the footwear needs of their clients or to refer to health professionals with such skills and knowledge. Individuals with more complex footwear needs (for example those who require custom-made medical grade footwear and orthoses) should be referred to health professionals with experience in the prescription of these modalities and who are able to provide appropriate and timely follow-up. Where financial disadvantage is a barrier to individuals acquiring appropriate footwear, health care professionals should be aware of state and territory based equipment funding schemes that can provide financial assistance. Aboriginal and Torres Strait Islanders and people living in rural and remote areas are likely to have limited access to a broad range of footwear. Provision of appropriate footwear to people with diabetes in these communities needs be addressed as part of a comprehensive national strategy to reduce the burden of diabetes and its complications on the health system.

The genome organization and affinities of an Australian isolate of carrot mottle umbravirus

The genomic sequence of an Australian isolate of carrot mottle umbravirus (CMoV-A) was determined from cDNA generated from dsRNA. This provides the first data on the genome organization and phylogeny of an umbravirus. The 4201-nucleotide genome contains four major open reading frames (ORFs). Analysis suggests that ORF2 encodes an RNA-dependent RNA polymerase, that ORF4 encodes a movement protein, and that the virus has no coat protein gene. The functions of ORFs 1 and 3 remain unknown. ORF2 is probably translated following ribosomal frameshifting. ORFs 3 and 4 are probably translated from a subgenomic mRNA. Sequence comparisons showed CMoV-A to be closely related to pea enation mosaic RNA2 NA2), but also to have affinities with the Bromoviridae. These findings shed light on the relationships between the luteoviruses, PEMV, and the umbraviruses and on the relationships between the carmo-like viruses and the Bromoviridae.

Soybean dwarf luteovirus contains the third variant genome type in the luteovirus group

Complementary DNAs covering the entire RNA genome of soybean dwarf luteovirus (SDV) were cloned and sequenced. Computer analysis of the 5861 nucleotide sequence revealed five major open reading frames (ORFs) possessing conservation of sequence and organisation with known luteovirus sequences. Comparative analyses of the genome structure show that SDV shares sequence homology and features of gene organisation with barley yellow dwarf virus (PAV isolate) in the 5' half of the genome, yet is more closely related to potato leafroll virus in its 3' coding regions. In addition, SDV differs from other known luteoviruses in possessing an exceptionally long 3' terminal sequence with no apparent coding capacity. We conclude from these data that the SDV genome represents a third variant genome type in the luteovirus group.

Characterisation of the subgenomic RNAs of an Australian isolate of barley yellow dwarf luteovirus

We have characterised the subgenomic RNAs of an Australian isolate of BYDV-PAV. Northern blot analyses of infected plants and protoplasts have shown that this isolate synthesises three subgenomic RNAs. Precise mapping of the transcription start sites of all three subgenomic RNAs and translational analyses of subgenomic RNA 2 and 3 have revealed a number of features. First, the transcription start site of subgenomic RNA 1 in this isolate differs markedly from the start site determined for an Illinois isolate of BYDV-PAV. Second, the start sites of subgenomic RNA 1 and 2 occur at a sequence that closely resembles the 5' end sequence of the genomic RNA (5'AGUGAAGA). Third, subgenomic RNA 2 appears to express ORF 6 of BYDV-PAV but the gene product is truncated due to the appearance of a new stop codon in the sequence. Last, subgenomic RNA 3, which is abundantly transcribed and encapsidated by the virus particle, appears to have no coding ability. We postulate that this novel subgenomic RNA has a regulatory function.