An intein with genetically selectable markers provides a new approach to internally label proteins with GFP

<p>Abstract</p> <p>Background</p> <p>Inteins are proteins that catalyze their own removal from within larger precursor proteins. In the process they splice the flanking protein sequences, termed the N-and C-terminal exteins. Large inteins frequently have a homing endonuclease that is involved in maintaining the intein in the host. Splicing and nuclease activity are independent and distinct domains in the folded structure. We show here that other biochemical activities can be incorporated into an intein in place of the endonuclease without affecting splicing and that these activities can provide genetic selection for the intein. We have coupled such a genetically marked intein with GFP as the N-terminal extein to create a cassette to introduce GFP within the interior of a targeted protein.</p> <p>Results</p> <p>The <it>Pch </it>PRP8 mini-intein of <it>Penicillium chrysogenum </it>was modified to include: 1) aminoglycoside phosphotransferase; 2) imidazoleglycerol-phosphate dehydratase, His5 from <it>S. pombe </it>; 3) hygromycin B phosphotransferase; and 4) the transcriptional activator LexA-VP16. The proteins were inserted at the site of the lost endonuclease. When expressed in <it>E. coli</it>, all of the modified inteins spliced at high efficiency. Splicing efficiency was also greater than 96% when expressed from a plasmid in <it>S. cerevisiae</it>. In addition the inteins conferred either G418 or hygromycin resistance, or histidine or leucine prototropy, depending on the inserted marker and the yeast genetic background. DNA encoding the marked inteins coupled to GFP as the N-terminal extein was PCR amplified with ends homologous to an internal site in the yeast calmodulin gene <it>CMD1</it>. The DNA was transformed into yeast and integrants obtained by direct selection for the intein's marker. The His5-marked intein yielded a fully functional calmodulin that was tagged with GFP within its central linker.</p> <p>Conclusions</p> <p>Inteins continue to show their flexibility as tools in molecular biology. The <it>Pch </it>PRP8 intein can successfully tolerate a variety of genetic markers and still retain high splicing efficiency. We have shown that a genetically marked intein can be used to insert GFP in one-step within a target protein <it>in vivo</it>.</p

Collaborative care : primary health workforce and service delivery in Western New South Wales - a case study

Objective: To explore how four small towns in rural New South Wales known as the 4Ts are addressing challenges accessing quality care and sustainable health services through a collaborative approach to workforce planning using the collaborative care framework. Design: Descriptive case study approach. Setting: The collaborative care project was developed as a result of ongoing partnerships between 2 rural Local Health Districts, 2 Primary Health Networks and a non-governmental health workforce organisation. The collaboration works with 5 subregions each comprising 2 or more rural communities. This paper focuses on the 4Ts subregion. Participants: Stakeholders of the collaborative design including organisations and the community. Intervention: A place-based approach to co-designing health services with community in one sub-region of Western New South Wales. Main outcome measures: A synthesis of field observations and experiences of community and jurisdictional partners in implementation of the 4Ts subregional model. Mapping of implementation processes against the collaborative care framework. Results: The collaborative care framework is a useful planning and community engagement tool to build health workforce literacy and to impact on system change at the local level. We identify key elements of effectiveness in establishing the 4Ts model, including the need for coordinated health system planning, better integrating existing resources to deliver services, community engagement, building health workforce literacy and town-based planning. Conclusion: This study adds to the body of knowledge about how to successfully develop a collaborative primary health care workforce model in practice. The findings demonstrate that the implementation of a collaborative primary health care workforce model using the collaborative care framework can improve service access and quality, which in turn might facilitate workforce sustainability

High expression of nicotinamide N-methyltransferase in patients with sporadic Alzheimer’s disease

We have previously shown that the expression of nicotinamide N-methyltransferase (NNMT) is significantly increased in the brains of patients who have died of Parkinson’s disease (PD). In this study, we have compared the expression of NNMT in post-mortem medial temporal lobe, hippocampus and cerebellum of 10 Alzheimer’s disease (AD) and 9 non-disease control subjects using a combination of quantitative Western blotting, immunohistochemistry and dual-label confocal microscopy coupled with quantitative analysis of colocalisation. NNMT was detected as a single protein of 29 kDa in both AD and non-disease control brains, which was significantly increased in AD medial temporal lobe compared to non-disease controls (7.5-fold, P < 0.026). There was no significant difference in expression in the cerebellum (P = 0.91). NNMT expression in AD medial temporal lobe and hippocampus was present in cholinergic neurones with no glial localisation. Cell-type expression was identical in both non-disease control and AD tissues. These results are the first to show, in a proof-of-concept study using a small patient cohort, that NNMT protein expression is increased in the AD brain and is present in neurones which degenerate in AD. These results suggest that the elevation of NNMT may be a common feature of many neurodegenerative diseases. Confirmation of this overexpression using a larger AD patient cohort will drive the future development of NNMT-targetting therapeutics which may slow or stop the disease pathogenesis, in contrast to current therapies which solely address AD symptoms

Alcohol prevention for school students: Results from a 1-year follow up of a cluster-randomised controlled trial of harm minimisation school drug education

Aims: The Drug Education in Victorian Schools (DEVS) programme taught about licit and illicit drugs over two years (2010–2011), with follow up in the third year (2012). It focussed on minimising harm and employed participatory, critical-thinking and skill-focussed pedagogy. This study evaluated the programme’s residual effectiveness at follow up in reducing alcohol-related risk and harm. Methods: A cluster-randomised, controlled trial was conducted with a student cohort during years eight (13–14 years old), nine (14–15 years old) and 10 (15–16 years old). Schools were randomly allocated to the DEVS programme (14 schools, n = 1163), or their usual drug education (7 schools, n = 589). Multi-level models were fitted to the data, which were analysed on an intent-to-treat basis. Statistically significant findings: Over the 3 years, there was a greater increase in intervention students’ knowledge about drugs, including alcohol. Their alcohol consumption did not increase as much as controls. Their alcohol-related harms decreased, while increasing for controls. There were fewer intervention group risky drinkers, and they reduced their consumption compared to controls. Similarly, harms decreased for intervention group risky drinkers, while increasing for controls. Conclusions: Skill-focussed, harm minimisation drug education can remain effective, subsequent to programme completion, in reducing students’ alcohol consumption and harm, even with risky drinkers

Nicotinamide N-methyltransferase catalyses the N-methylation of the endogenous ß-carboline norharman: evidence for a novel detoxification pathway

Nicotinamide N-methyltransferase (NNMT) is responsible for the N-methylation of nicotinamide to 1-methylnicotinamide. Our recent studies have demonstrated that NNMT regulates cellular processes fundamental to the correct functioning and survival of the cell. It has been proposed that NNMT may possess β-carboline (BC) N-methyltransferase activity, endogenously and exogenously produced pyridine-containing compounds which, when N-methylated, are potent inhibitors of Complex I and have been proposed to have a role in the pathogenesis of Parkinson's disease. We have investigated the ability of recombinant NNMT to N-methylate norharman (NH) to 2-N-methylnorharman (MeNH). In addition, we have investigated the toxicity of the BC NH, its precursor 1,2,3,4-tetrahydronorharman (THNH) and its N-methylated metabolite MeNH, using our in vitro SH-SY5Y NNMT expression model. Recombinant NNMT demonstrated NH 2N-methyltransferase activity, with a Km of 90 ± 20 µM, a kcat of 3 × 10(-4) ± 2 × 10(-5) s(-1) and a specificity constant (kcat/Km) of 3 ± 1 s(-1) M(-1) THNH was the least toxic of all three compounds investigated, whereas NH demonstrated the greatest, with no difference observed in terms of cell viability and cell death between NNMT-expressing and non-expressing cells. In NNMT-expressing cells, MeNH increased cell viability and cellular ATP concentration in a dose-dependent manner after 72 and 120 h incubation, an effect that was not observed after 24 h incubation or in non-NNNT-expressing cells at any time point. Taken together, these results suggest that NNMT may be a detoxification pathway for BCs such as NH

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tile only ( 1 85-110 dB HL, 250-500 Hz). In the remaining 7 cases, residual hearing was maintained up to at least 6 months after operation with minor changes. Insertion depth angles in these cases ranged from 285 to 420°. For these subjects, the mean preoperative score for words presented at 65 dB SPL was 22%. Mean postoperative scores were 56% for CI alone, and 68% for CI plus ipsilateral hearing aid (p ! 0.05, paired t). For sentences presented in multitalker babble noise at 5 dB SNR, mean scores were 61% CI alone, and 75% CI+IpsiHA (p ! 0.01, paired t). Conclusions: Hearing was conserved during surgery and over time in 70% of conventional candidates implanted with the Nucleus 24 Contour Advance CI who had significant levels of preoperative low-frequency residual hearing ( ^ 60 dB HL). These conventional candidates for CI also benefited from improved speech recognition in noise when using combined ipsilateral electrical and acoustic stimulation

Taking a positive spin: preserved initiative and performance of everyday activities across mild Alzheimer’s, vascular, and mixed dementia

Objectives: The literature commonly evaluates those daily activities which are impaired in dementia. However, in the mild stages, people with dementia (PwD) are still able to initiate and perform many of those tasks. With a lack of research exploring variations between different dementia diagnoses, this study sought to investigate those daily activities with modest impairments in the mild stages and how these compare between Alzheimer's disease (AD), vascular dementia (VaD) and mixed dementia. Methods: Staff from memory assessment services from nine National Health Service trusts across England identified and approached informal carers of people with mild dementia. Carers completed the newly revised Interview for Deteriorations in Daily Living Activities in Dementia 2 assessing the PwD's initiative and performance of instrumental activities of daily living (IADLs). Data were analysed using analysis of variance and Chi-square tests to compare the maintenance of IADL functioning across AD, VaD, and mixed dementia. Results: A total of 160 carers returned the Interview for Deteriorations in Daily Living Activities in Dementia 2, of which 109, 21, and 30 cared for someone with AD, VaD, and mixed dementia, respectively. There were significant variations across subtypes, with AD showing better preserved initiative and performance than VaD for several IADLs. Overall, PwD showed greater preservation of performance than initiative, with tasks such as preparing a hot drink and dressing being best maintained. Conclusion: Findings can help classify dementia better into subtypes in order to receive bespoke support. It suggests that interventions should primarily address initiative to improve overall functioning