Designing accessible educational resources for people living with spinal cord injury.

Context/objective: To identify themes of interest for the production of educational resources for people with spinal cord injury (SCI).Design: A mixed-method study.Setting: Outpatient SCI community in Australia.Participants: Individuals with a SCI, or carers, family & friends of people who live with a SCI (n = 116).Interventions: Not applicable.Outcome measures: Quantify themes of interest perceived within the Australian SCI community as necessary for the development of SCI educational resources.Results: All seven individuals from the focus-group interviews suggested that educational resources on body physiology, secondary complications, injury pathophysiology, and health and wellbeing maintenance would be most pertinent for development. These themes (among others) were further explored and quantitatively evaluated via an online survey which demonstrated that interviewees ranked 'Your injury' as being of highest importance for the production of educational resources. Within each theme, the sub-categories; 'Bowel/bladder' and 'What equipment is covered in the National Disability Insurance Scheme (NDIS)' were ranked as being of highest importance for the production of educational resources.Conclusion: We have identified multiple areas of interest in the design and production of educational resources for individuals with SCI.

Does a rare mutation in PTPRA contribute to the development of Parkinson's disease in an Australian multi-incident family?

The genetic study of multi-incident families is a powerful tool to investigate genetic contributions to the development of Parkinson's disease. In this study, we identified the rare PTPRA p.R223W variant as one of three putative genetic factors potentially contributing to disease in an Australian family with incomplete penetrance. Whole exome sequencing identified these mutations in three affected cousins. The rare PTPRA missense variant was predicted to be damaging and was absent from 3,842 alleles from PD cases. Overexpression of the wild-type RPTPα and R223W mutant in HEK293T cells identified that the R223W mutation did not impair RPTPα expression levels or alter its trafficking to the plasma membrane. The R223W mutation did alter proteolytic processing of RPTPα, resulting in the accumulation of a cleavage product. The mutation also resulted in decreased activation of Src family kinases. The functional consequences of this variant, either alone or in concert with the other identified genetic variants, highlights that even minor changes in normal cellular function may increase the risk of developing PD.

Strong Predictive Algorithm of Pathogenesis-Based Biomarkers Improves Parkinson's Disease Diagnosis.

Easily accessible and accurate biomarkers can aid Parkinson's disease diagnosis. We investigated whether combining plasma levels of α-synuclein, anti-α-synuclein, and/or their ratios to amyloid beta-40 correlated with clinical diagnosis. The inclusion of amyloid beta-40 (Aß40) is novel. Plasma levels of biomarkers were quantified with ELISA. Using receiver operating characteristic (ROC) curve analysis, levels of α-synuclein, anti-α-synuclein, and their ratios with Aß40 were analyzed in an initial training set of cases and controls. Promising biomarkers were then used to build a diagnostic algorithm. Verification of the results of biomarkers and the algorithm was performed in an independent set. The training set consisted of 50 cases (age 65.2±9.3, range 44-83, female:male=21:29) with 50 age- and gender-matched controls (67.1±10.0, range 45-96 years; female:male=21:29). ROC curve analysis yielded the following area under the curve results: anti-α-synuclein=0.835, α-synuclein=0.738, anti-α-synuclein/Aß40=0.737, and α-synuclein/Aß40=0.663. A 2-step diagnostic algorithm was built: either α-synuclein or anti-α-synuclein was ≥2 times the means of controls (step-1), resulting in 74% sensitivity; and adding α-synuclein/Aß40 or anti-α-synuclein/Aß40 (step-2) yielded better sensitivity (82%) while using step-2 alone yielded good specificity in controls (98%). The results were verified in an independent sample of 46 cases and 126 controls, with sensitivity reaching 91.3% and specificity 90.5%. The algorithm was equally sensitive in Parkinson's disease of ≤5-year duration with 92.6% correctly identified in the training set and 90% in the verification set. With two independent samples totaling 272 subjects, our study showed that combination of biomarkers of α-synuclein, anti-α-synuclein, and their ratios to Aß40 showed promising sensitivity and specificity.

Draft Genome Sequence of Halomonas sp. Strain KAO, a Halophilic Mn(II)-Oxidizing Bacterium.

Halomonas sp. strain KAO is an aerobic, Mn(II)-oxidizing, halophilic bacterium. The draft genome of the isolate contains 47 contigs encompassing 3.7 Mb and a GC content of 64.22%. This sequence will provide essential information for future studies of Mn(II) oxidation, particularly under halophilic conditions.

Hunting for Familial Parkinson's Disease Mutations in the Post Genome Era.

Parkinson's disease (PD) is typically sporadic; however, multi-incident families provide a powerful platform to discover novel genetic forms of disease. Their identification supports deciphering molecular processes leading to disease and may inform of new therapeutic targets. The LRRK2 p.G2019S mutation causes PD in 42.5-68% of carriers by the age of 80 years. We hypothesise similarly intermediately penetrant mutations may present in multi-incident families with a generally strong family history of disease. We have analysed six multiplex families for missense variants using whole exome sequencing to find 32 rare heterozygous mutations shared amongst affected members. Included in these mutations was the KCNJ15 p.R28C variant, identified in five affected members of the same family, two elderly unaffected members of the same family, and two unrelated PD cases. Additionally, the SIPA1L1 p.R236Q variant was identified in three related affected members and an unrelated familial case. While the evidence presented here is not sufficient to assign causality to these rare variants, it does provide novel candidates for hypothesis testing in other modestly sized families with a strong family history. Future analysis will include characterisation of functional consequences and assessment of carriers in other familial cases.

Nursing students' perceptions of clinical relevance and engagement with bioscience education: A cross-sectional study of undergraduate and postgraduate nursing students.

BACKGROUND: Bioscience in nursing education covers a broad range of disciplinary areas (anatomy, physiology, pathophysiology and pharmacology) and underpins clinical assessment and critical thinking in nursing practice. This is imperative for patient safety and favourable patient outcomes. In nursing programs for registration, most bioscience content is taught during the early phases of the program and little incorporated into postgraduate nursing programs. OBJECTIVES: The aim of this study was to explore student's perceived relationship between clinical relevance and engagement (attention and time) with bioscience content, by surveying undergraduates and postgraduate nursing students. DESIGN: This sequential mixed methods study included two phases. METHODS: The first phase comprised of focus group interviews from one Australian University. Thematic analysis of these data, coupled with existing literature, informed the second study phase; a quantitative questionnaire. PARTICIPANTS: Focus group interviews included N = 30 students from one tertiary site; 10 from each year level. The questionnaire was administered to nursing students undertaking undergraduate nursing studies (1st, 2nd & 3rd years), and postgraduate nursing studies. RESULTS: Nursing students (n = 406) across three Australian universities (four programmes) completed the questionnaire. The clinical relevance of bioscience was widely appreciated; 91.6% of undergraduate nursing students and 98.5% of postgraduate nursing students indicated that every nurse must have a good understanding of bioscience. However, there was an inverse relationship between engagement with bioscience and timing in the curriculum, as 50% of undergraduate nursing students indicated that bioscience content took up too much of their time, compared to only 20% of postgraduate nurses (odds ratio 0.27 [0.16-0.46], p < 0.001). CONCLUSION: Nursing students' perceptions of the clinical relevance of bioscience for their career strongly corresponds with their progression through their studies. Unfortunately, as students progress to the later years of their nursing education, their engagement with formal bioscience education decreases. This poses the question 'Are we delivering bioscience content to nursing students at the appropriate time?'

Meta-analysis of genome-wide DNA methylation identifies shared associations across neurodegenerative disorders.

BACKGROUND: People with neurodegenerative disorders show diverse clinical syndromes, genetic heterogeneity, and distinct brain pathological changes, but studies report overlap between these features. DNA methylation (DNAm) provides a way to explore this overlap and heterogeneity as it is determined by the combined effects of genetic variation and the environment. In this study, we aim to identify shared blood DNAm differences between controls and people with Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. RESULTS: We use a mixed-linear model method (MOMENT) that accounts for the effect of (un)known confounders, to test for the association of each DNAm site with each disorder. While only three probes are found to be genome-wide significant in each MOMENT association analysis of amyotrophic lateral sclerosis and Parkinson's disease (and none with Alzheimer's disease), a fixed-effects meta-analysis of the three disorders results in 12 genome-wide significant differentially methylated positions. Predicted immune cell-type proportions are disrupted across all neurodegenerative disorders. Protein inflammatory markers are correlated with profile sum-scores derived from disease-associated immune cell-type proportions in a healthy aging cohort. In contrast, they are not correlated with MOMENT DNAm-derived profile sum-scores, calculated using effect sizes of the 12 differentially methylated positions as weights. CONCLUSIONS: We identify shared differentially methylated positions in whole blood between neurodegenerative disorders that point to shared pathogenic mechanisms. These shared differentially methylated positions may reflect causes or consequences of disease, but they are unlikely to reflect cell-type proportion differences.

Draft Genome Sequence of Halomonas sp. Strain ML-15, a Haloalkaliphilic, Polycyclic Aromatic Hydrocarbon-Degrading Bacterium.

Halomonas sp. strain ML-15 is an aerobic, haloalkaliphilic bacterium capable of degrading polycyclic aromatic hydrocarbons (PAHs). The draft genome sequence of the isolate contains 19 contigs encompassing 4.8 Mb and a G+C content of 65.38%. This sequence will provide essential information for future studies of PAH degradation, particularly under haloalkaliphilic conditions.

Evidence of a Recessively Inherited CCN3 Mutation as a Rare Cause of Early-Onset Parkinsonism.

The study of consanguineous families has provided novel insights into genetic causes of monogenic parkinsonism. Here, we present a family from the rural Khyber Pakhtunkhwa province, Pakistan, where three siblings were diagnosed with early-onset parkinsonism. Homozygosity mapping of two affected siblings and three unaffected family members identified two candidate autozygous loci segregating with disease, 8q24.12-8q24.13 and 9q31.2-q33.1. Whole-exome sequence analysis identified a single rare homozygous missense sequence variant within this region, CCN3 p.D82G. Although unaffected family members were heterozygous for this putative causal mutation, it was absent in 3,222 non-Parkinson's disease (PD) subjects of Pakistani heritage. Screening of 353 Australian PD cases, including 104 early-onset cases and 57 probands from multi-incident families, also did not identify additional carriers. Overexpression of wild-type and the variant CCN3 constructs in HEK293T cells identified an impaired section of the variant protein, alluding to potential mechanisms for disease. Further, qPCR analysis complemented previous microarray data suggesting mRNA expression of CCN3 was downregulated in unrelated sporadic PD cases when compared to unaffected subjects. These data indicate a role for CCN3 in parkinsonism, both in this family as well as sporadic PD cases; however, the specific mechanisms require further investigation. Additionally, further screening of the rural community where the family resided is warranted to assess the local frequency of the variant. Overall, this study highlights the value of investigating underrepresented and isolated affected families for novel putative parkinsonism genes.

Analysis of DNA methylation associates the cystine-glutamate antiporter SLC7A11 with risk of Parkinson's disease.

An improved understanding of etiological mechanisms in Parkinson's disease (PD) is urgently needed because the number of affected individuals is projected to increase rapidly as populations age. We present results from a blood-based methylome-wide association study of PD involving meta-analysis of 229 K CpG probes in 1,132 cases and 999 controls from two independent cohorts. We identify two previously unreported epigenome-wide significant associations with PD, including cg06690548 on chromosome 4. We demonstrate that cg06690548 hypermethylation in PD is associated with down-regulation of the SLC7A11 gene and show this is consistent with an environmental exposure, as opposed to medications or genetic factors with effects on DNA methylation or gene expression. These findings are notable because SLC7A11 codes for a cysteine-glutamate anti-porter regulating levels of the antioxidant glutathione, and it is a known target of the environmental neurotoxin ß-methylamino-L-alanine (BMAA). Our study identifies the SLC7A11 gene as a plausible biological target in PD.

Enhancement of artemisinin concentration and yield in response to optimization of nitrogen and potassium supply to Artemisia annua.

BACKGROUND AND AIMS: The resurgence of malaria, particularly in the developing world, is considerable and exacerbated by the development of single-gene multi-drug resistances to chemicals such as chloroquinone. Drug therapies, as recommended by the World Health Organization, now include the use of antimalarial compounds derived from Artemisia annua--in particular, the use of artemisinin-based ingredients. Despite our limited knowledge of its mode of action or biosynthesis there is a need to secure a supply and enhance yields of artemisinin. The present study aims to determine how plant biomass can be enhanced while maximizing artemisinin concentration by understanding the plant's nutritional requirements for nitrogen and potassium. METHODS: Experiments were carried out, the first with differing concentrations of nitrogen, at 6, 31, 56, 106, 206 or 306 mg L(-1) being applied, while the other differing in potassium concentration (51, 153 or 301 mg L(-1)). Nutrients were supplied in irrigation water to plants in pots and after a growth period biomass production and leaf artemisinin concentration were measured. These data were used to determine optimal nutrient requirements for artemisinin yield. KEY RESULTS: Nitrogen nutrition enhanced plant nitrogen concentration and biomass production successively up to 106 mg N L(-1) for biomass and 206 mg N L(-1) for leaf nitrogen; further increases in nitrogen had no influence. Artemisinin concentration in dried leaf material, measured by HPLC mass spectroscopy, was maximal at a nitrogen application of 106 mg L(-1), but declined at higher concentrations. Increasing potassium application from 51 to 153 mg L(-1) increased total plant biomass, but not at higher applications. Potassium application enhanced leaf potassium concentration, but there was no effect on leaf artemisinin concentration or leaf artemisinin yield. CONCLUSIONS: Artemisinin concentration declined beyond an optimal point with increasing plant nitrogen concentration. Maximization of artemisinin yield (amount per plant) requires optimization of plant biomass via control of nitrogen nutrition.

Pipeline to gene discovery - Analysing familial Parkinsonism in the Queensland Parkinson's Project.

INTRODUCTION: Family based study designs provide an informative resource to identify disease-causing mutations. The Queensland Parkinson's Project (QPP) has been involved in numerous genetic screening studies; however, details of the families enrolled into the register have not been comprehensively reported. This article characterises the families enrolled in the QPP and summarises monogenic forms of hereditary Parkinsonism found in the register. METHOD: The presence of pathogenic point mutations and copy number variations (CNVs) were, generally, screened in a sample of over 1000 PD patients from the total of 1725. Whole exome sequencing (WES) was performed on eighteen probands from multiplex families. RESULTS: The QPP contains seventeen incidences of confirmed monogenic forms of PD, including LRRK2 p.G2019S, VPS35 p.D620N, SNCA duplications and PARK2 p.G430D (hom) & exon 4 deletion (hom). Of these seventeen, five belong to multi-incident families, while another eight have a family history of at least one other case of PD. In additional families, WES did not identify known forms of monogenic Parkinsonism; however, three heterozygous mutations in PARK2, p.R275W, p.Q34fs, and a 40bp deletion in exon 3 were identified. Of these three mutations, only the 40bp deletion segregated with disease in a dominant inheritance pattern. CONCLUSION: Eighteen probands have screened negative for known CNVs and mutations that cause clear monogenic forms of PD. Each family is a candidate for further genetic analysis to identify genetic variants segregating with disease. The families enrolled in the QPP provide a useful resource to aid in identifying novel forms of monogenic PD.

Continuous flow in open microfluidics using controlled evaporation.

This paper presents a method for programming the flow rate of liquids inside open microfluidic networks (MFNs). A MFN comprises a number of independent flow paths, each of which starts with an open filling port, has a sealed microchannel in which assays can be performed, and an open capillary pump (CP). The MFN is placed over Peltier elements and its flow paths initially fill owing to capillary forces when liquids are added to the filling ports. A cooling Peltier element underneath the filling ports dynamically prevents evaporation in all filling ports using the ambient temperature and relative humidity as inputs. Another Peltier element underneath the CPs heats the pumps thereby inducing evaporation in the CPs and setting the flow rate in the microchannels. This method achieves flow rates in the microchannels ranging from approximately 1.2 nL s(-1) to approximately 30 pL s(-1), and is able to keep 90% of a 0.6 microL solution placed in an open filling port for 60 min. This simple and efficient method should be applicable to numerous assays or chemical reactions that require small and precise flow of liquids and reagents inside microfluidics.

Comprehensive assessment of genetic sequence variants in the antioxidant 'master regulator' NRF2 in idiopathic Parkinson's disease.

Parkinson's disease (PD) is a complex neurodegenerative disorder influenced by a combination of genetic and environmental factors. The molecular mechanisms that underlie PD are unknown; however, oxidative stress and impairment of antioxidant defence mechanisms have been implicated as major contributors to disease pathogenesis. Previously, we have reported a PD patient-derived cellular model generated from biopsies of the olfactory mucosa, termed hONS cells, in which the NRF2-mediated antioxidant response pathway genes were among the most differentially-expressed. To date, few studies have examined the role of the NRF2 encoding gene, NFE2L2, and PD. In this study, we comprehensibly assessed whether rare and common NFE2L2 genetic variations modify susceptibility to PD using a large Australian case-control sample (PD=1338, controls=1379). We employed a haplotype-tagging approach that identified an association with the tagging SNP rs2364725 and PD (OR = 0.849 (0.760-0.948), P = 0.004). Further genetic screening in hONS cell lines produced no obvious pathogenic variants in the coding regions of NFE2L2. Finally, we investigated the relationship between xenobiotic exposures and NRF2 function, through gene-environment interactions, between NFE2L2 SNPs and smoking or pesticide exposure. Our results demonstrated a significant interaction between rs2706110 and pesticide exposure (OR = 0.597 (0.393-0.900), P = 0.014). In addition, we were able to identify some age-at-onset modifying SNPs and replicate an 'early-onset' haplotype that contains a previously identified 'functional promoter' SNP (rs6721961). Our results suggest a role of NFE2L2 genetic variants in modifying PD susceptibility and onset. Our findings also support the utility of testing gene-environment interactions in genetic studies of PD.

Rare POLG1 CAG variants do not influence Parkinson's disease or polymerase gamma function.

A recent meta-analysis suggested that rare CAG repeat variants in the gene that encodes polymerase gamma (POLG1) predispose individuals to develop Parkinson's disease (PD); alternative alleles were proposed to increase risk by 27%. In the current case-control study of 2255 Australians, we observed no statistical association between individuals possessing rare CAG repeat genotypes and PD (p=0.178); a subsequent meta-analysis of 2852 PD cases and 2833 controls was also non-significant (OR=1.085, p=0.124). Moreover, mitochondrial DNA synthesis (p=0.427) or Complex I activity (p=0.639) were not different in cells derived from individuals with different POLG1 genotypes. These data provide no evidence to suggest CAG repeat length in POLG1 affects PD susceptibility.