15th December 2017, Volume 130 Number 1467

Increased right cardiac sympathetic and parasympathetic nerve activity in type 2 diabetes

CT Bussey, Z Ashley, DO Schwenke, RR Lamberts

Department of Physiology, HeartOtago, School of Biomedical Sciences, University of Otago, Dunedin.

Heart function is regulated by sympathetic and parasympathetic nervous inputs, which are unbalanced in type 2 diabetes contributing to widespread cardiac dysfunction. Specifically, diabetes disturbs heart rate (HR) regulation, which is under control of the previously unstudied right cardiac sympathetic nerve activity (cSNA) and parasympathetic nerve activity (PSNA).

We measured right cSNA and PSNA in 20-week-old male Zucker type 2 diabetic fatty rats (DM, n=6–9) and non-diabetic littermates (ND, n=6–7). The right cardiac sympathetic and parasympathetic vagal nerves were placed uncut over bipolar platinum recording electrodes, during anaesthesia. Baseline cSNA and PSNA were recorded, followed by injection of β-agonist isoproterenol (1µg/kg) before and after ganglionic blockade with hexamethonium (1mg/kg). Data were expressed as mean ± standard error, with differences assessed via t–test.

Although HR was decreased, right integrated cSNA was increased in DM (ND 1.7±0.4 vs DM 6.0±2.1 µV/s, P<0.05), accompanied by increased vagal PSNA frequency (ND 2.3±1.1 vs DM 15.7±5.6 Hz, P<0.05). Diabetes reduced β- adrenergic-induced increases in HR and integrated cSNA (ND 21.0±5.9 vs DM 3.9±2.9% change, P<0.05). Disrupting transmission through nerve ganglia with hexamethonium indicated diabetes impaired β-adrenergic-induced increases in signalling frequency from the heart to the brain (cSNA: ND 35.2±11.7 vs DM -2.2±14.4 Hz; PSNA: ND 109.0±37.2 vs DM 5.7±56.5 Hz; P<0.05).

Thus right cSNA and PSNA are increased in DM, with lower HR suggesting dominant PSNA changes are an underestimated therapeutic target. Reduced β- adrenergic responsiveness of cSNA and PSNA was largely attributable to rarely studied peripheral signalling. Severe impairment of autonomic regulation is likely a key contributor to the burden of cardiac dysfunction in type 2 diabetes.

Low-intensity magnetic stimulation and excitability in the rodent neocortex as measured by local field potentials

M Sykes,1,2 A Tang,2 J Rodger,JNJ Reynolds1

1Department of Anatomy, University of Otago, Dunedin; 2Experimental and Regenerative Neuroscience, School of Animal Biology, University of Western Australia, Perth, Australia.

Repetitive transcranial magnetic stimulation is a technique used to alter cortical excitability in patients with depression or stroke, using strong (~1 tesla) magnetic pulses. Outside of the targeted, focal zone, however, are broad areas exposed to lower strength magnetic fields, below the threshold for direct depolarisation for many neurons. Increasing evidence has emerged to suggest that fields 100 milliTesla, or lower, are able to modulate cellular properties such as calcium levels and firing thresholds in vitro and excitability in vivo.

To better understand the effects of low-intensity rTMS within the cortex, we made local field potential recordings using metal wire electrodes in place of traditional, inflexible glass microelectrodes. We recorded local field potentials from layer V in the motor cortex of urethane-anaesthetised rats. Rats were treated with either one or two rounds of quadripulse (QPS-50ms or sham, followed by QPS-5ms; n=8–9 per group) or theta burst stimulation (iTBS or sham, followed by iTBS; n=3–4 per group).

No significant effects of one or multiple rounds of low-intensity stimulation were observed on the slope of evoked field potentials in animals treated with QPS (F1,14=2.79; P=0.117) or iTBS (F1,5=0.121; P=0.742) protocols. The same negative results held true for input/output curves in QPS-treated (P=0.760) and in iTBS- treated animals (P=0.396). Overall in all measured outcomes, including paired-pulse ratio and power spectral density, no significant changes were observed as a result of low intensity rTMS.

These results suggest that low intensity rTMS, at levels previously shown to modulate MEPs, does not have a substantial effect on the excitability of layer V neurons, measurable under these circumstances. However, the existence of some trends within the data suggested that potential changes are subtle, and thus require a more sensitive technique.

Supported by the Department of Anatomy Doctoral Scholarship.

Developing chlamydia-targeted protease inhibitors as a potential treatment for chlamydiosis

AA Agbowuro,1 A Springwald,J Hwang,1 JW Marsh,2 R Mazraani,LC McCaugney,3,4 MS Bogyo,WM Huston,3 AB Gamble,JDA Tyndall1

1School of Pharmacy University of Otago, Dunedin; 2Queensland University of Technology, Australia; 3University of Technology, Sydney, Australia; 4University of Oxford, United Kingdom; 5Stanford University, California, United States of America.

Chlamydia trachomatis is responsible for the most common sexually transmitted bacterial infection worldwide, and often results in complications such as ectopic pregnancy and reversible blindness. While current treatment with macrolide antibiotics is quite effective, there are growing concerns about their efficacy due to the ever-increasing prevalence of the disease. This in part can be traced to the lack of organism specificity of the drugs. We therefore aimed to develop chlamydia- specific antibacterials as a means of overcoming this drawback.

CtHtrA is a serine protease crucial for the survival and virulence of chlamydia, and a potential drug target. JO146 (Boc-Val-Pro-ValP(OPh)2) was previously identified as an inhibitor of CtHtrA (IC50=12.5µM) that is selectively toxic to the human pathogen. However, JO146 is not potent enough and may also be susceptible to enzymatic degradation, being a peptide-based compound. To improve these properties, we synthesized 24 new analogues of JO146 belonging to six different chemical classes, and optimised them for binding at the various subpockets of the enzyme. The compounds were tested in in vitro CtHtrA inhibition and C. trachomatis cell culture assays.

Non-covalent binders (N-methyl amides and valinol) were generally inactive (IC50>500µM), inferring that covalency is crucial for activity. α-Ketobenzothiazole showed comparable activity to JO146 (×0.94 relative IC50). Isoleucine and tertiary leucine were the most active residues at P1 and P3 respectively, yielding Boc-Tle-Pro-IleP(OPh)2 with an ~1,000-fold increase in cellular activity relative to JO146. A number of these inhibitors showed improved selectivity for CtHtrA over other human serine proteases.

We have successfully developed potent and selective analogues of JO146 suitable for in vivo pre-clinical studies. In addition, comprehensive structure activity relationships that would enable the design of clinically relevant inhibitors for the treatment of chlamydial infections have also been built.

Supported by a University of Otago Doctoral Scholarship.

Transport issues affect teenage wellbeing in Southland, New Zealand

AL Ward,1 R McGee,1 C Freeman,C Cameron1

1Department of Preventive and Social Medicine, Dunedin School of Medicine; 2Department Geography, University of Otago, Dunedin.

Discussions of teenage transport issues are typically framed from the perspective of crash risk and physical health. This research addresses transport in the context of wellbeing. Issues surrounding mobility affect older teenagers’ happiness and autonomy. Why teenagers choose to travel the way they do, and what impact these choices have on wellbeing, should help researchers acquire a more nuanced view of the role transportation plays in overall health.

An online survey gathered data about life satisfaction and self-perceived strengths, peer and parental attachment, transport, licensure and activities. Descriptive results were analysed and unpaired t-tests and Pearson’s chi-squared tests (χ2) performed on mean values and categorical data, to assess differences and associations by gender, and multiple linear regressions were used.

Overall response rate was 71.5% (N=786; male=49%; average age = 16.7 years). Eighty percent of respondents reported being “happy” or “very happy”; males were happier than females (t=5.12, P=0.001). There was a significant difference between peer attachment and wellbeing between genders; females showed more peer attachment (t=-5.19, P=0.001). The most common mode of transport was being a passenger in a car, followed by walking (85.1% and 69.9%, respectively). Multiple linear regressions supported the attachment and wellbeing descriptive findings. After adjusting for other variables, significant positive associations existed between having a restricted licence and life satisfaction, between cycling and life satisfaction among females, and negative associations existed between licensure and life satisfaction among males. For males driving a car and for females having a license was positively associated with self-perceived strengths.

Trends, gender differences and associations exist with regard to transport issues and wellbeing. This study provides an evidence base for future research and direction for transport-related intervention to provide transport options and infrastructure to support wellbeing among older teenagers.

Supported by a University of Otago Doctoral Scholarship.

Investigating molecular diagnostic biomarkers for myalgic encephalomyelitis/chronic fatigue syndrome

EC Sweetman,1 WP Tate,1 M Ryan2

1Department of Biochemistry; 2Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a prevalent but poorly understood illness characterised by severe debilitating fatigue, affecting approximately 1% of the global population and disproportionately affecting females. There are no agreed diagnostic markers or definitive clinical tests, and the causative agent and disease pathophysiology are ill-defined. However, immune dysfunction, chronic viral infection, and recently metabolic and mitochondrial dysregulation are proposed causes of ME/CFS. Abnormal upregulation and activation of an innate antiviral immune response protein, protein kinase R (PKR), has also been observed.

This project aimed at identifying biological pathway dysfunctions by comprehensive analysis of different classes of molecules, including micro-RNA, expressed genes (transcriptome), and cellular proteomes in a study group of 10 ME/CFS patients and 10 age-gender matched controls. For a potential diagnostic test, antibodies against (i) a non-phosphorylated PKR fragment and (ii) a phosphorylated PKR peptide were raised and purified to investigate the ratio of active:inactive PKR in ME/CFS patients.

Principal component analysis and t-tests were used to identify significant changes in the molecular data. Regulators of metabolic, immune and oxidative pathways were significantly increased. An example was Interleukin 8 (fold-change = 5.57, P=0.02).

Several key mitochondrial proteins were decreased in the ME/CFS transcriptome and proteome analyses, for example NADH dehydrogenase 1 alpha subcomplex, 5 and NADH dehydrogenase flavoprotein 1 (Fold-change <0.78, P<0.01). Possible disease biomarkers were identified, including plasma microRNAs hsa-miR-142-5p (P=0.036), hsa-let-7g(P=0.02),hsa-miR-1825 (P=0.02), and a gene transcript TNFAIP3 (P=3.23x10-21). Western immunoblots of lymphocyte and neutrophil protein extracts detected an increased ratio of active:inactive PKR in ME/CFS patients.

This study of a range of biologically important molecules within a well-characterised ME/CFS patient group identified significant dysregulation in immune inflammatory, apoptosis, oxidative stress, and metabolic pathways, and in mitochondrial functioning.

Utilisation of preventive medicines in older people approaching end-of-life

SW Narayan, PS Nishtala

School of Pharmacy, University of Otago, Dunedin.

The prevention and treatment of chronic conditions are mainly based on pharmacologic therapy; therefore having multiple chronic conditions means the use of a number of medications in individuals aged ≥65 years. Among these, preventive medicines (PMs) such as antithrombotics, cholesterol lowering and bone preserving medicines are the most commonly prescribed.

Our explorative study aimed to examine the prescribing patterns of preventive medicines in older individuals in their last year of life. This retrospective cohort study included individuals (N=99,809) aged ≥75 years who were in their last year of life. PMs examined in this study included low-dose aspirin (≤325mg/day), clopidogrel, dipyridamole, warfarin, dabigatran, statins and bisphosphonates. Multinomial and logistic regression models were used to examine the influence of age, sex, multimorbidity, socioeconomic status and presence of a life-limiting illness (cancer) on the number and type of PMs prescribed (between 2007–2012).

The number of PMs prescribed to older individuals in their last year of life was higher for males compared to females (Relative Risk Ratio 1.34, 95% CI: 1.26–1.41). The number of PMs prescribed decreased as age increased. However, the use of clopidogrel increased over three-fold from 2007–2012 (Odds Ratio (OR) 7.55, 95% CI: 6.28–9.09). In contrast, bisphosphonates use decreased significantly during the same period (OR 0.44, 95% CI: 0.40–0.49). Individuals with a diagnosis of cancer had decreased odds of PMs utilisation except for antiplatelets, aspirin monotherapy and statins, which had remarkably high odds (OR 4.07, CI: 3.84–4.31, P<0.001).

Over the last decade, a significant emphasis is being placed on the optimal use of medicines and deprescribing unnecessary medicines in individuals with multimorbidity and those approaching end-of-life. Our findings suggest that there is some evidence that the use of preventive medicines in older individuals diagnosed with cancer has declined.

Supported by a University of Otago Doctoral Scholarship.