Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung

Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hyp- oxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar viru- lence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1

Clinical and Experimental Determination of Protection Afforded by BCG Vaccination against Infection with Non-Tuberculous Mycobacteria: A Role in Cystic Fibrosis?

Mycobacterium abscessus is a nontuberculous mycobacterium (NTM) of particular concern in individuals with obstructive lung diseases such as cystic fibrosis (CF). Treatment requires multiple drugs and is characterised by high rates of relapse; thus, new strategies to limit infection are urgently required. This study sought to determine how Bacille Calmette-Gu&eacute;rin (BCG) vaccination may impact NTM infection, using a murine model of Mycobacterium abscessus infection and observational data from a non-BCG vaccinated CF cohort in Sydney, Australia and a BCG-vaccinated CF cohort in Cape Town, South Africa. In mice, BCG vaccination induced multifunctional antigen-specific CD up sup T cells circulating in the blood and was protective against dissemination of bacteria to the spleen. Prior infection with M. abscessus afforded the highest level of protection against M. abscessus challenge in the lung, and immunity was characterised by a greater frequency of pulmonary cytokine-secreting CD4 T cells compared to BCG vaccination. In the clinical CF cohorts, the overall rates of NTM sampling during a three-year period were equivalent; however, rates of NTM colonisation were significantly lower in the BCG-vaccinated (Cape Town) cohort, which was most apparent for M. abscessus. This study provides evidence that routine BCG vaccination may reduce M. abscessus colonisation in individuals with CF, which correlates with the ability of BCG to induce multifunctional CD4T cells recognising M. abscessus in a murine model. Further research is needed to determine the optimal strategies for limiting NTM infections in individuals with CF

A double-blinded randomised controlled trial exploring the effect of anodal transcranial direct current stimulation and uni-lateral robot therapy for the impaired upper limb in sub-acute and chronic stroke

BACKGROUND:Neurorehabilitation technologies such as robot therapy (RT) and transcranial Direct Current Stimulation (tDCS) can promote upper limb (UL) motor recovery after stroke. OBJECTIVE:To explore the effect of anodal tDCS with uni-lateral and three-dimensional RT for the impaired UL in people with sub-acute and chronic stroke. METHODS:A pilot randomised controlled trial was conducted. Stroke participants had 18 one-hour sessions of RT (Armeo®Spring) over eight weeks during which they received 20 minutes of either real tDCS or sham tDCS during each session. The primary outcome measure was the Fugl-Meyer assessment (FMA) for UL impairments and secondary were: UL function, activities and stroke impact collected at baseline, post-intervention and three-month follow-up. RESULTS:22 participants (12 sub-acute and 10 chronic) completed the trial. No significant difference was found in FMA between the real and sham tDCS groups at post-intervention and follow-up (p = 0.123). A significant ‘time’ x ‘stage of stroke’ was found for FMA (p = 0.016). A higher percentage improvement was noted in UL function, activities and stroke impact in people with sub-acute compared to chronic stroke. CONCLUSIONS:Adding tDCS did not result in an additional effect on UL impairment in stroke. RT may be of more benefit in the sub-acute than chronic phase

Delta inulin-based adjuvants promote the generation of polyfunctional CD4+ T cell responses and protection against Mycobacterium tuberculosis infection

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.There is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis. Advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. In this report we determined if Advax displays broad applicability against important human pathogens by assessing protective immunity against infection with M. tuberculosis. The fusion protein CysVac2, comprising the M. tuberculosis antigens Ag85B (Rv1886c) and CysD (Rv1285) formulated with Advax provided significant protection in the lungs of M. tuberculosis-infected mice. Protection was associated with the generation of CysVac2-specific multifunctional CD4+ T cells (IFN-γ+TNF+IL-2+). Addition to Advax of the TLR9 agonist, CpG oligonucleotide (AdvaxCpG), improved both the immunogenicity and protective efficacy of CysVac2. Immunisation with CysVac2/AdvaxCpG resulted in heightened release of the chemoattractants, CXCL1, CCL3, and TNF, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of CysVac2-specific CD4+ T cells. As delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, CysVac2/AdvaxCpG is a strong candidate for further preclinical evaluation for progression to human trials

Measurement of motor-evoked potential resting threshold and amplitude of proximal and distal arm muscles in healthy adults. A reliability study

Purpose: Reliability of motor-evoked potential threshold and amplitude measurement of upper limb muscles is important when detecting changes in cortical excitability. The objective of this study was to investigate intra-rater, test-retest reliability and minimal detectable change of resting motor threshold and amplitude of a proximal and distal upper limb muscles, anterior deltoid and distal extensor digitorum communis in healthy adults. Method: To measure motor-evoked potential responses, transcranial magnetic stimulation was interfaced with electromyography and neuronavigation equipment. Two measurements were conducted on day 1 and a third measurement three days later. Reliability was analysed using intraclass correlation coefficients. Results: Twenty participants completed the study. Excellent intra-rater (intraclass correlation coefficient = 0.91 (extensor digitorum), 0.94 (anterior deltoid)) and good to excellent test-retest reliability (intraclass correlation coefficient = 0.69 (anterior deltoid), 0.84 (extensor digitorum)) was found for resting motor threshold. Minimal detectable change for resting motor threshold was found at 10.95% (extensor digitorum) and 16.35% (anterior deltoid) between first and third measurements. Motor-evoked potential amplitude of extensor digitorum communis had fair to good intra-rater (intraclass correlation coefficient = 0.50) and test-retest reliability (intraclass correlation coefficient = 0.65). Conclusions: Our results suggest that resting motor threshold is a reliable neurophysiological measure even for proximal shoulder muscles. Future research should further explore the reliability of motor-evoked potential amplitude before integration into neurological rehabilitation

Translation of evidence-based Assistive Technologies into stroke rehabilitation: Users' perceptions of the barriers and opportunities

Background: Assistive Technologies (ATs), defined as "electrical or mechanical devices designed to help people recover movement", demonstrate clinical benefits in upper limb stroke rehabilitation; however translation into clinical practice is poor. Uptake is dependent on a complex relationship between all stakeholders. Our aim was to understand patients', carers' (P&Cs) and healthcare professionals' (HCPs) experience and views of upper limb rehabilitation and ATs, to identify barriers and opportunities critical to the effective translation of ATs into clinical practice. This work was conducted in the UK, which has a state funded healthcare system, but the findings have relevance to all healthcare systems. Methods. Two structurally comparable questionnaires, one for P&Cs and one for HCPs, were designed, piloted and completed anonymously. Wide distribution of the questionnaires provided data from HCPs with experience of stroke rehabilitation and P&Cs who had experience of stroke. Questionnaires were designed based on themes identified from four focus groups held with HCPs and P&Cs and piloted with a sample of HCPs (N = 24) and P&Cs (N = 8). Eight of whom (four HCPs and four P&Cs) had been involved in the development. Results: 292 HCPs and 123 P&Cs questionnaires were analysed. 120 (41%) of HCP and 79 (64%) of P&C respondents had never used ATs. Most views were common to both groups, citing lack of information and access to ATs as the main reasons for not using them. Both HCPs (N = 53 [34%]) and P&C (N = 21 [47%]) cited Functional Electrical Stimulation (FES) as the most frequently used AT. Research evidence was rated by HCPs as the most important factor in the design of an ideal technology, yet ATs they used or prescribed were not supported by research evidence. P&Cs rated ease of set-up and comfort more highly. Conclusion: Key barriers to translation of ATs into clinical practice are lack of knowledge, education, awareness and access. Perceptions about arm rehabilitation post-stroke are similar between HCPs and P&Cs. Based on our findings, improvements in AT design, pragmatic clinical evaluation, better knowledge and awareness and improvement in provision of services will contribute to better and cost-effective upper limb stroke rehabilitation. © 2014 Hughes et al.; licensee BioMed Central Ltd

Non-toxic metal-cyclam complexes, a new class of compounds with potency against drug-resistant Mycobacterium tuberculosis

Tuberculosis (TB) accounted for 1.5 million deaths in 2014 and new classes of anti-TB drugs are required. We re-port a class of functionalized 1,8-disubstituted cyclam derivatives that display low micromolar activity against pathogenic myco-bacteria. These compounds inhibit intracellular growth of Mycobacterium tuberculosis, are non-toxic to human cell lines and are active against multidrug-resistant M. tuberculosis strains, indicating a distinct mode of action. These compounds warrant further appraisal as novel agents to control TB in humans.This work was supported by the National Health and Medical Research Council (NHMRC) Project APP1084266, the NHMRC Center of Research Excellence in Tuberculosis Control (APP1043225) and the University of Sydney Sydnovate Fund. We acknowledge the Centre for Drug Candidate Optimization, Monash University for the ADME studie

A destabilized bacterial luciferase for dynamic gene expression studies

Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression