Future Physiology 2021

Atrial Compartmentalisation Proteomics: Analysis of endo-lysosomal proteins using a modified density gradient approach

Atrial Fibrillation (AF) is the most common cardiac arrhythmia. The incidence and prevalence are expected to increase in the next 30 years by 63% and 66% respectively1. As a multigenic disorder, an understanding of the molecular cascades and the cross-communication between total ion exchange and cellular stress in AF is essential for designing effective AF therapies. Mis-regulation of regulatory proteins in the endocytic pathway, including endo-lysosomes and lysosomes, has been reported in AF2. The discovery of the cardiac lysosome Ca2+ signaling3 has opened a new door to unravel the underlying molecular mechanisms of acidic organelles in AF. Organelle isolation using tissue biopsies facilitates studies investigating the roles of regulatory proteins within these pathways. We developed an endo-lysosome isolation method from tissue biopsies using Percoll and sucrose differential density gradient centrifugation. We present what we believe to be the first comprehensive dataset of C. porcellus endo-lysosomal focused organelle proteomics using label free MS/MS peptide analysis (QExec LumosTM mass spectrometer) on isolated endo-lysosomes from C. porcellus atrial tissue. Experiments performed in accordance with Home Office Guidance on the Animals (Scientific Procedures) Act (1986). Mass spectrometry data from biological replicates (n = 3) were processed and analysed using Max-quant and Perseus software (v1.5.2.4) to identify proteins. Robust criteria was used for filtration and imputation, and significantly upregulated/downregulated proteins in endo-lysosome (EL) and tissue lysate (TL) fractions were identified by volcano plot. The highest abundant EL proteins were separated using cell component filtration in Gene Ontology (GO) analysis, the molecular function was identified using the PANTHER pathway, and functional network interactions were mapped using Cytoscape analysis (v3.7.2). The presence of the EL marker proteins Lysosome associated membrane protein 2 (LAMP2), beta-Galactosidase (GLB), beta-hexosaminidase (HEXB) and the absence of mitochondrial marker Cytochrome oxidase IV (COX IV) and sarcoplasmic reticulum marker Phospholamban were confirmed by Western blot and enzyme assays. Our EL preparations demonstrate the enrichment of lysosomal markers GLB, HEXB, Ras-related protein Rab-7a and LAMP2, and identify multiple protein hits relevant to diseases that have been linked to dysfunctional lysosomal enzymes and membrane-bound proteins. These include; lysosomal α-glucosidase (GAA), a key lysosomal enzyme involved in the degradation of glycogen in lysosomes4; Cathepsin A, which serves a protective function by regulating stability and activity of beta-galactosidase, neuraminidase enzymes and plays a role in galactosialidosis5; Clusterin, identified as a potential biomarker for mucopolysaccharidosis; and Decorin, a protein that, when dysregulated, contributes to cardiac fibrosis or fibrotic stiffness. Furthermore, we identified glycogen phosphorylase, brain form (PYGB) in the EL fraction, a lysosomal enzyme that regulates glycogen mobilization, and plays a prominent role as the only marker protein elevated in the early-stage of asymptomatic patients with lysosomal storage disease Fabry disease. In addition, we identified a major complement of lysosomal cathepsins in our proteomic data that have been linked with cardiovascular diseases, such as cathepsins B, C, D and Z. The successful identification of such disease related proteins highlights the potential usage of these techniques in understanding the role of lysosomal proteins in complex cardiac diseases such as AF.

Cerebrovascular responses during voluntary breath holding are larger than rebreathing in humans

Both voluntary breath-holding and rebreathing of expired air elicit changes in respiratory gas chemostimuli (CO2 and O2) at the metabolic rate. These chemostimuli elicit increases in cerebral blood flow (CBF), proportional to the magnitude of concomitant increases in CO2 and reductions in O2. These chemostimuli also activate the sympathetic nervous system, which increases systemic blood pressure. Although blood pressure responses appear small during rebreathing, evidence from obstructive sleep apnea (OSA) patients suggests higher mean blood pressure during sleep in those with worse OSA. We aimed to assess how superimposed changes in blood gases and increases in blood pressure affect the CBF responses during breath holding vs. rebreathing. We recruited 23 healthy participants (12 females) and instrumented them with a finometer (for beat-by-beat mean arterial blood pressure; MAP), transcranial Doppler ultrasound (middle and posterior cerebral artery velocity; MCAv, PCAv) and a pneumotachometer with gas sampling via a dual gas analyzer to assess the pressure of end-tidal (PET)CO2 and O2. Participants carried out two protocols in randomized order: (a) a maximal, voluntary end-inspiratory breath hold (BH) and (b) a rebreathing (RB) test. A breath-by-breath stimulus index (SI) was calculated as PETCO2/PETO2 during rebreathing, whereas the end-tidal gas values used to calculate SI were interpolated during breath holding from initial and break point values. During both BH and RB, cerebrovascular reactivity (CVR) was calculated as the MCAv or PCAv/SI. MAP reactivity (MAPR) was calculated as the slope of the MAP/SI response. Cerebrovascular conductance (CVC; MCA or PCA/MAP) reactivity (CVCR) was calculated as the slope of the MCACVC or PCACVC/SI responses. We found that (a) CVR was larger during BH vs. RB (MCA: 146.9±69.9 vs. 67.7±29.9 cm/s/SI, P<0.0001, n=20; PCA: 78.1±30.7 vs. 45.6±21.0 cm/s/SI, P=0.0004, n=17), (b) MAPR during BH was significantly higher than during RB (116.8±67.8 vs. 35.9±26.0 mmHg/SI, P=0.0001, n=20). and (c) CVCR during BH vs. RB (MCA: 0.69±0.62 vs. 0.45±0.22 cm/s/mmHg/SI, P=0.64, n=20; PCA: 0.33±0.17 vs. 0.33±21 cm/s/SI, cm/s/mmHg/SI, P=0.9, n=17). Our data demonstrate that breath holding elicited ~3-fold increases in MAP, translating to a larger anterior and posterior CVR compared to rebreathing. These findings suggest that the sympathetic responses during voluntary breath holding were larger than those during rebreathing across similar chemostimuli, potentially due to the differential sympathetic effects of struggling against a closed glottis during breath holding. This is the first evidence that voluntary apnea has larger effects on brain blood flow beyond that elicited by blood gas stimuli alone. Our data may have implications for understanding stroke risk in clinical populations with obstructive sleep apnea, whereby patients experience intermittent breath holds throughout the night, with associated spikes in arterial blood pressure.

A dual potassium channelopathy underlies small vessel disease of the brain in a mouse model of Alzheimer’s disease

The microcirculation of the brain is increasingly viewed as a potential target for disease modifying drugs in the treatment of the patient with Alzheimer’s Dementia (AD). However, despite established changes to cerebrovascular autoregulation in patients with AD, underlying pathogenic mechanisms in isolated brain resistance arteries have not been investigated. Here we present the first detailed examination of the principal vasodilatory pathways within small cerebral arteries in an AD mouse model. For our study we used the APP23 mouse which has a seven-fold increase in amyloid precursor protein leading to neuritic plaques and also cerebrovascular accumulation of amyoid-β, similarly to patients with AD. Using a range of physiological techniques; mainly pressure myography, patch clamp electrophysiology and high-speed spinning disc confocal microscopy, we show that a global vascular dysfunction within this model is underpinned by two distinct channelopathies. Firstly we showed an attenuation of large conductance Ca2+–activated K+ (BK) channel function as a decreased contractility to the BK channel blocker, paxilline (p = <0.001, unpaired t test, n = 8-12 arteries per group). Further investigation determined that the BK channels from the APP23 vascular smooth muscle cells produced significantly less spontaneous transient outward currents (p = <0.01, two way ANOVA, n = 10-14 cells per group) and that this was correlated to a significantly reduced frequency of calcium sparks (p = <0.05, unpaired t test, n = 5-7 arteries per group). Thus, the increase in myogenic tone from these animals (p = <0.01, unpaired t test, 25-29 arteries per group) was a result of a lack of calcium spark activation of the BK channel. This observation was accompanied by a reduction in the vasodilatory capacity of the endothelial inward rectifier K+ channel, Kir2.1 (p = <0.05, unpaired t test, 10-11 arteries per group), indicating a deficit in neurovascular coupling in these animals. If present in human AD, the combination of these pathologies would account for the clinical cerebrovascular presentation seen in patients: reduced blood flow and a failure of functional hyperaemia. The data directs future research to approaches that will reverse this dual vascular channelopathy, with the ultimate aim of restoring healthy cerebral blood flow and improving clinical outcomes.

Cardiotoxicity of particulate matter air pollution in mouse models of health and disease

An estimated 4.2 million premature deaths worldwide are attributed to air pollution, primarily through increased cardiovascular (CV) morbidity and mortality. Studies have found particulate matter (PM), a subgroup of pollutants, including polyaromatic hydrocarbons (PAHs) are associated most strongly with CV disease (CVD), due to the ability of these smaller particles to enter the systemic circulation. Studies in fish have shown PAHs, such as phenanthrene (Phe), can directly alter CV electrophysiology, including prolonged action potential duration (APD) and QT interval, reduced heart rate (HR), reduced contractility and changes in underlying ionic currents. This level of detailed mechanistic investigation is lacking in mammalian models. Using the mouse model, the current study found exposure to even a single pollutant, Phe, can alter cardiac electrophysiology. Acute exposure (15 minutes) to Phe led to a significant reduction in HR and prolongation of the RR interval both ex-vivo (25uM Phe, 16% reduction in HR, P=0.022, n=5) using the isolated heart preparation and in-vivo (50ug/kg Phe, 10% reduction in HR, P=0.0043, n=7) using electrocardiographs recorded from anaesthetised mice, suggesting Phe has some direct effects on the heart, which are unaffected by peripheral factors. Wild-type mice (10-weeks) were exposed to a lower dose of Phe (30ug/kg, n=8) or vehicle only (DMSO, n=6) for a 14-day period, followed by in-vivo electrocardiograph recording and tissue collection. PQRST intervals were calculated for all animals; a significant prolongation of the QTc and P-wave duration was observed in Phe treated animals (P<0.05), suggesting the pro-arrhythmic effects of Phe previously seen in fish, may also occur in mammals. Furthermore, the weight of hearts treated with Phe was significantly higher than animals treated with vehicle only (P=0.004), this could indicate a hypertrophic response, however further molecular analysis is needed to confirm this. This study is the first to show direct effects of Phe on the CV system (CVS) of mice, both in the absence and presence of peripheral factors. This study is also the first to investigate prolonged exposure to Phe in mice, showing actions on both cardiac electrophysiology and potentially structure. More detailed electrophysiological, and long-term exposure studies combined with RNA and protein analysis of tissue are needed to investigate the full effects of Phe and reveal novel mechanisms of action. Ambient PM is now ranked 5th in the global mortality risk factor list and as the global levels of pollution continue to rise, studies like ours will help manage and mitigate this avoidable cause of disease and death.

Kinetics of synaptic vesicle pool depletion and adaptation in zebrafish lateral line hair cells

Background In auditory, vestibular and lateral line systems, ribbon synapses allow hair cells to drive high rates of sustained synaptic transmission (Goutman and Glowatzki 2007; Pichler and Lagnado 2019) and to maintain the exquisite temporal precision of sensory information. The kinetic properties of vesicle release and replenishment at these synapses have mainly been studied by measuring changes in membrane capacitance during hair cell depolarization using patch clamp, which bypasses the normal process of mechanotransduction. The zebrafish is becoming an important model for the investigation of hair cell development and function (Ricci et al. 2013; Olt et al. 2014; Pichler and Lagnado 2019), with the advantage to investigate both pre- and postsynaptic responses and the activity of afferent neurons in an intact organism in vivo. In this study, we investigated the release properties of hair cell ribbon synapses and sensory adaptation in vivo using a combination of extracellular recording and fluorescence imaging while displacing the mechanoelectrical transducer apparatus. Methods Work was licensed by the UK Home Office and approved by the University of Sheffield Ethical Review Committee. Zebrafish larvae from the following strains were used: Tg(brn3c:Gal4), Tg(UAS:iGluSnFR), Tg(UAS:GCaMP7a) and Tg(nbt:GCaMP3). Zebrafish larvae (<5.2 days post fertilisation) were anaesthetised in MS-222 (0.01%) prior to injection of α-bungarotoxin (125 μM) into the heart to suppress movement. Recordings from the ganglion cell body were performed using loose-patch voltage-clamp as previously described (Trapani et al. 2011). Mechanical displacement of the cupula was achieved by using a piezoelectric fluid jet. iGluSnFR and Ca2+ signals were recorded using a two-photon laser-scanning microscope based on a mode-locked laser system operating at 925 nm. Results Sustained displacement of the neuromast cupula elicited a transient increase in hair cell synaptic release at the stimulus onset, which quickly adapted to a lower, sustained level. The release time course was consistent with the recruitment of two pools of synaptic vesicles with different release kinetics. The time constant of the fast component, which we identify as the readily releasable pool (RRP), was 19 ms (18 neuromasts, 10 zebrafish). Short, repeated pressure stimuli lead to progressive depletion of the RRP. Recovery from depletion showed a biphasic time course, with a fast time constant of 129 ms and a slow time constant of 2.8 s (33 neuromasts, 16 zebrafish). The relatively fast adaptation of synaptic responses (21 hair cells, 15 neuromasts, 7 zebrafish) was likely due to vesicle depletion, as the hair cell calcium responses (39 hair cells, 18 neuromasts, 4 zebrafish) did not show any significant decline throughout the stimulation. Both the calcium responses in the afferent terminals (37 afferent terminals, 17 neuromasts, 5 zebrafish) and the firing rate in the afferent ganglion neurons (32 neurons, 29 zebrafish) showed adapting responses comparable to fast adaptation at the hair cell ribbon synapse. Conclusions We conclude that the hair cell synaptic release properties allow the lateral line to maintain its sensitivity in the presence of sustained stimuli, such as those produced by a constant flow of water.

Inhibition of mitochondria fission in astrocytes in the Dorsal Vagal Complex of the brain to target the metabolic profile of brown adipose tissue in high fat diet-fed rats

Background and aim: The prevalence of obesity worldwide is increasing sharply, resulting in growing costs for healthcare systems worldwide. The brown adipose tissue (BAT) is being probed as a potential target to treat obesity. BAT activation is driven by the central nervous system (CNS) and circulating glucose and fatty acids. The nucleus of the tractus solitarius (NTS) in the brainstem is an important centre that receives information regarding the nutritional status from the visceral vagal afferents and relays them to the forebrain. Astrocytes are the most abundant cells in the brain that work as energy sensor and provide the right environment for the neurones to respond to altered metabolic status. Their role in regulating systemic metabolism is of growing interest, and evidence shows their involvement in BAT thermogenesis (1). Previous studies have shown that targeting all cells or specifically astrocytes with a dominant-negative form of Drp1 (Drp1-K38A) to inhibit mitochondria fragmentation in rats NTS decreased food intake, restored insulin sensitivity in the NTS and prevented weight gain in high-fat diet (HFD)- fed-obese rats (2). Our main aim is to investigate whether targeting NTS with Drp1-K38A can affect the metabolic profile of BAT and increase adrenergic discharge to the organ and glucose uptake, which associate with enhanced metabolic demand. Materials and methods: The experimental protocol was approved by the ethical committee for the use of animals in research of the Faculty of Biological Sciences,University of Leeds and by the Home Office. 1) Rats (male, n=24) were subjected to stereotactic surgery on day 0 and received an injection of either GFAP:Drp1-K38A or GFAP:GFP in the NTS on day 1 and fed HFD diet for 15 days. On day 16 animals were sacrificed by pentobarbital overdose (60 mg/kg) and BAT dissected. 2) Rats (male, n=3) were fed with HFD for 28 days, submitted to stereotactic surgery on day 29 and injected with an adenovirus expressing CMV:Drp1K138A or CMV:GFP in the NTS on day 30. Rats where then maintained on HFD for additional 15 days and submitted to PET scan. Results: Noradrenaline precursor Tyrosine Hydroxylase (TH) levels in BAT of HFD-fed animals that received GFAP:Drp1K38-A in the NTS are increased 144% when compared to GFAP:GFP controls (p<0.001). The mRNA levels of insulin-dependent glucose transporter 4 (GLUT4) and thermogenic gene peroxisome proliferator-activated receptor gamma (PGC1A) are significantly upregulated in animals that received GFAP:Drp1K38-A in the NTS when compared to controls (p<0.0001 and p<0.05) respectively. On a protein level PGC1A is upregulated (p<0.05). Our Pilot study revealed a potential increase in glucose uptake in BAT of rat fed long-term HFD (50 days) that received CMV:Drp1K38-A when compared to control. Conclusions: Our results suggest that inhibition of mitochondria fission in NTS astrocytes restored TH reservoirs in BAT-essential for beta adrenergic-driven thermogenesis, and regulator of GLUT4 mRNA in BAT(3). Consistently with the PET scan pilot, these results may suggest that glucose uptake in BAT is favoured in animals that received either CMV:Drp1K38-A or GFAP:Drp1K38-A when compared to controls; more studies are required to test this hypothesis.

Role of CPT1C in motor function, energy state, mood, synaptic plasticity and cognition

CPT1C is an enzyme present throughout the nervous system, which is specifically expressed in neurons, showing remarkable amounts in the hypothalamus, hippocampus, cortex, cerebellum and amygdala. Unlike other CPT1 isoforms, which are involved in the transport of fatty acids inside the mitochondria for their subsequent b-oxidation, CPT1C is located in the endoplasmic reticulum (ER), where it regulates ceramide metabolism. Previous studies reported that CPT1C plays an important role in energy homeostasis and control of body weight. Although the molecular mechanisms underlying these functions remain partially unclear, CPT1C involvement in dendritic spine maturation, ER-mediated axonal transport and AMPA receptor synthesis and trafficking have been described. Due to the wide expression of this protein together with the diverse cellular mechanisms attributed, CPT1C might be involved in additional functions in different brain regions that remain unexplored. Here we carried out a systematic characterization of the role of CPT1C at different —molecular, synaptic and behavioral— levels of complexity. First, CPT1C expression pattern in different brain areas was studied by immunohistochemistry in CPT1C Knock-out (KO) mice and wild-type littermate mice. Then, the role of CPT1C in locomotor activity, energy state, anxiety- and depression-like behavior was explored. In addition, CPT1C involvement in different types of learning was investigated studying motor learning, hippocampal-dependent spatial and habituation memory, and associative instrumental learning in vivo, in behaving mice. To correlate hippocampal-dependent memory processes with synaptic plasticity changes, neural activity and dendritic spine maturation in the hippocampus were evaluated in vitro in these animals. Our data showed energy deficits and impaired locomotor activity in CPT1C KO mice. These animals also exhibited deficits in motor and instrumental learning, as well as in spatial and habituation memory. The latter effects could be attributed to an inefficient hippocampal dendritic spine maturation and long-term plasticity impairments observed at the CA3-CA1 synapse. No changes in mood state were found. Together, our results show that CPT1C is needed for learning and memory processes taking place in brain areas that underlie motor, associative, and non-associative learning, as well as confirm the role of CPT1C in hippocampal synaptic plasticity and energy homeostasis.

Sternohyoid muscle weakness in pre-symptomatic ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration and loss of motor neurons, resulting in severe muscle weakness and paralysis. The respiratory system is implicated in ALS, with patients experiencing sleep-disordered breathing and progressive respiratory muscle weakness, ultimately leading to respiratory muscle paralysis and failure. The SOD1G93A mouse model of ALS displays motor neuron degeneration and a phenotype consistent with human ALS. It remains to be determined whether muscle dysfunction in ALS is a consequence of motor unit degeneration subsequent to motor neuron loss, or whether there is a primary muscle disorder in ALS that contributes to muscle dysfunction, thereby disrupting neuromuscular junction physiology and contributing to muscle dysfunction. These views are broadly considered as “dying forward” (nerve to muscle) or “dying back” (muscle to nerve) phenomena. It is suggested that muscle impairment may exist early, before the onset of overt motor impairments in ALS mice and perhaps humans. Therefore, we hypothesize that upper airway muscle dysfunction presents early in ALS and precedes cranial motor neuron dysfunction. We aimed to examine upper airway and diaphragm muscle function in young (48-49 days) pre-symptomatic ALS mice. We examined ex vivo muscle contractile function of sternohyoid (a representative pharyngeal dilator) and diaphragm muscles for wild-type (n=12) and ALS (n=12) mice. Muscle preparations were attached to a dual-mode lever transducer system and studied in a water-jacketed muscle bath containing Krebs solution at 35oC aerated with 95%O2/5%CO2. Isometric contractions were performed to examine force-generating capacity. Isotonic contractions were performed at 0% load to examine maximum shortening (Smax) and shortening velocity (Vmax) and at 50% load to examine maximum work (Wmax) and power (Pmax) output. Values are expressed as mean ± S.D. Data were statistically compared using unpaired Student’s t test, Mann Whitney test or two-way ANOVA with Bonferroni post hoc test. P<0.05 was considered statistically significant. Sternohyoid muscle tetanic force was significantly reduced in ALS mice compared to wild-type. For the force-frequency relationship, specific force was significantly reduced at 100-150 Hz. Smax, Vmax, Wmax and Pmax were all significantly reduced in ALS sternohyoid muscle compared to wild-type. Conversely, for diaphragm muscle, there was no significant difference in force production in ALS mice compared to wild-type. Smax, Vmax, Wmax and Pmax were also equivalent for ALS and wild-type diaphragm muscle preparations. These data demonstrate sternohyoid muscle weakness (evidenced by reduced specific force and power output) in young pre-symptomatic ALS mice. Diaphragm muscle function was unaffected in pre-symptomatic ALS mice. Our study indicates that upper airway muscle dysfunction may be a prodromal signature of ALS, with potential implications for the control of upper airway patency in ALS and other neurodegenerative diseases. Current studies seek to examine brainstem and spinal cord motor neuron pools to compare motor and muscle changes in young ALS mice.

The effect of local cooling strategies on the perceptual and inflammatory response to passive heating in healthy young males

Introduction Passive elevation of body temperature can induce an acute inflammatory response that has been proposed to be beneficial for metabolic health. However, the heat strain associated with whole-body heating can be perceived as uncomfortable. While implementing local cooling strategies may partly alleviate thermal discomfort during passive heating, it is not known how such protocol adaptations impact on the acute inflammatory response. The aim of this series of studies was to investigate the effect of local cooling strategies on the acute inflammatory response to passive heating. Methods Study 1 Using a water-perfused suit, ten recreationally active males underwent three 90 min conditions: heating of the legs (50°C water) with upper-body cooling (2°C water and ice packs; LBH), whole-body heating (50°C water; WBH) and exposure to a thermoneutral temperature (36°C water; CON). Blood samples were collected before and up to 3h post-session to assess inflammatory markers. Rectal temperature, basic affect and thermal comfort were recorded at regular intervals. Study 2 Nine recreationally active male participants were immersed up to the waist for three 60 min water immersion conditions: 42°C hot water immersion (HWI), 42°C HWI with simultaneous upper-body cooling using a fan (FAN), and 36°C thermoneutral water immersion (NEU). Blood samples to determine interleukin (IL)-6 plasma concentration were collected pre- and post-water immersion; rectal temperature, basic affect and thermal comfort were assessed throughout the intervention. Results Study 1 The IL-6 incremental area under the curve (iAUC) was higher for LBH (1096±851 pg/mL*270min) and WBH (833±476 pg/mL*270min) compared with CON (565±325 pg/mL*270min; p<0.047). Rectal temperature was elevated to a larger extent in WBH (38.6±0.4°C) compared with the other trials (p<0.001), and was higher at the end of LBH (37.1±0.3°C) compared with CON (36.7±0.2°C, p=0.001). Basic affect and thermal comfort were more negative during WBH compared with LBH and CON (p<0.010). Study 2 Plasma IL-6 concentration was higher for HWI and FAN when compared with CON (p<0.001) and did not differ between HWI and FAN (p=0.221; pre to post, HWI: 1.0±0.6 to 1.5±0.7 pg/mL, FAN: 0.7±0.5 to 1.1±0.5 pg/mL, CON: 0.5±0.2 to 0.5±0.2 pg/mL). Rectal temperature was elevated to a larger extent in HWI (38.7±0.6°C) compared with FAN and NEU (p<0.001), and was higher in FAN (38.1±0.4°C) compared with NEU (37.1±0.3°C, p<0.001). At the end of immersion, basic affect was lowest for HWI (HWI: -1.8±2.0, FAN: 0.2±1.6, CON 1.0±2.1, p<0.020); thermal comfort for HWI was changed from pre into the uncomfortable range (3.0±1.0, p<0.001), whereas FAN (0.7±0.7, p=0.160) and CON (-0.2±0.7, p=0.283) remained in the comfortable range throughout. Conclusions Both aggressive upper-body cooling through a water-perfused suit and ice packs (study 1) as well as modest cooling using a fan (study 2) resulted in more positive perceptual responses compared with whole-body heating. Moreover, although LBH attenuated the response in additional cardiometabolic markers, both LBH and FAN did not attenuate the acute IL-6 response. The use of a fan may thus be considered as a practical and low-cost tool to enhance the tolerability of passive heating.

Low intensity resistance exercise with blood flow restriction induces local and systemic hypoalgesia via endogenous opioid-mediated mechanisms of pain modulation

Resistance exercise can acutely decrease pain sensitivity (hypoalgesia) both local and remote to the exercising muscle. This effect is maximised with higher intensity exercise (1) and is partly driven by endogenously produced opioid and endocannabinoid substances, which have antinociceptive effects. Resistance exercise is used to treat individuals with chronic pain, however many of these individuals are contraindicated to higher intensity exercise. It has been hypothesised that performing low intensity exercise with blood flow restriction (BFR) in the working limb could increase the analgesic effect of exercise to a similar level as high intensity exercise (2). However, the magnitude of this effect and possible mechanisms are not known. To investigate this, healthy active individuals (n=12) performed 4 trials of unilateral leg press exercise in a randomised, crossover design. The trials included: 1) low intensity exercise (30% 1RM, LIRE); 2) high intensity exercise (70% 1RM, HIRE); 3) BFR exercise (30% 1RM) with a low pressure (BFR-L) and 4) BFR exercise (30% 1RM) with a high pressure (BFR-H). Sensitivity to a pressure stimulus (pressure-pain threshold, [PPT]) measured in kilograms of force (kgf) was assessed before and 5min post-exercise using algometry, whereby an increase in PPT value represents hypoalgesia. This was performed in the exercising quadriceps muscle and three remote non-exercising muscles. Venous blood samples were taken before and 10min post-exercise. Plasma concentration of beta-endorphin and 2-arachidonoylglycerol (marker of opioid and endocannabinoid systems, respectively) were determined using enzyme linked-immunosorbence assays. Values are mean±SD and analysed with two-way repeated measures ANOVA for each outcome. Mediation analysis was performed to determine the impact of any change in plasma beta-endorphin and 2-arachidonoylglycerol concentrations on any change in PPT. There were no differences in baseline values between repeated trials for any outcome measured. In the exercising limb, at 5min post-exercise the PPT was 2.43±1.60 kgf higher, 3.72±2.24 kgf higher and 1.68±1.53 kgf higher following BFR-L, BFR-H and HIRE, respectively, compared to LIRE (all p<0.05). Following BFR-H, PPT was 1.29±1.18 kgf higher and 2.04±1.75 kgf higher compared to BFR-L and HIRE (both p<0.05). In the non-exercising remote muscles, a greater change in PPT occurred with BFR-L (11-17%), BFR-H (11-21%) and HIRE (10-18%) compared to LIRE (2-4%) (all p<0.05). At 5min post-exercise, plasma beta-endorphin concentration was greater following BFR-L (108.56±20.18 pg/ml), BFR-H (109.54±18.83 pg/ml) and HIRE (92.87±16.82 pg/ml) compared to LIRE (90.62±17.13 pg/ml) (all p<0.05). The increase in beta-endorphin was greater in BFR-L and BFR-H compared to HIRE (both p<0.05). There were no changes in plasma 2-arachidonoylglycerol concentration. The mediation model suggested that plasma beta-endorphin mediates 42% of the total effect of the exercise intervention on change in PPT. Thus, this data shows that addition of BFR to LIRE can increase the local and systemic analgesic effect of exercise (3). Importantly, the magnitude of this effect is either greater (local exercising muscle) or similar (remote non-exercising muscle) to HIRE. Moreover, analgesia with BFR exercise is partly driven by endogenous production of opioids. BFR exercise may be effective for treating individuals with chronic pain who cannot tolerate higher intensity exercise.

Different ventilatory response to hypoxia and hypercapnia in prematurely born adults

INTRODUCTION Pre-term birth, typically defined as birth occurring prior to the 36th week of gestation, has been reported to significantly hamper functional and anatomical development of the respiratory system (1). Prematurely born individuals present reduced cardiopulmonary control, as well as impaired alveologenesis and pulmonary vasculogenesis, leading to inefficient gas diffusion capacity. Either ventilation-to-perfusion mismatch and/or right heart dysfunction have been suggested as potential mechanisms responsible for alteration in respiratory function (2). Recent work from our group has also demonstrated higher oxidative stress in prematurely born adults (3), which may negatively affect both peripheral and central chemosensitivity to hypoxia and hypercapnia, respectively. The aim of this study was to investigate the resting ventilatory responses to normoxic hypercapnia and normobaric hypoxia in prematurely born, but otherwise healthy, adults. METHODS Seventeen pre-term adults (Mean±SD; age, 21±2 years; height, 177±7 cm; body mass, 68.9±7.5 kg; gestational age, 29±3 weeks; gestational weight, 1234±315 g) and fourteen age-matched full-term controls (age, 22±2 years; height, 180±5 cm; body mass, 73.5±5.6 kg; gestational age, 39±2 weeks; birth weight, 3672±499 g) underwent two 5-min resting normoxic hypercapnic periods (3% CO2 and 6% CO2, respectively), interposed by 5-min ambient air exposure (visit 1). On a second occasion, the participants underwent, again at rest, 4-min of normoxic breathing, before being exposed to 4-min of normobaric hypoxia (FIO2=0.11) (visit 2). Minute ventilation (V̇E), breathing frequency, tidal volume, and end-tidal partial pressure of CO2 (PETCO2), were continuously recorded during both sessions. RESULTS On both occasions, pre-term adults showed lower PETCO2 during rest in normocapnic normoxia than their full-term counterparts (visit 1: 36±4 vs. 39±2 mmHg, p=0.02; visit 2: 36±3 vs. 38±2 mmHg, p=0.04), while no difference was observed in V̇E. During 3% normoxic hypercapnia, PETCO2 increased to a greater extent over the first minute in pre-term, relative to full-term, adults (+11±3% vs. +16±5%, p=0.05). No difference in V̇E was observed between groups while breathing 3% CO2. During 6% hypercapnic exposure, PETCO2 increased similarly in both groups, while V̇E was higher in pre-term compared to full-term adults (30.2±7.5 vs. 23.7±4.5 L/min, p<0.0001). The relative increases in PETCO2 and V̇E during the first minute breathing 6% CO2 were higher in preterm (+31±8% and +64±35%, respectively) compared to full-term (+21±5% and +35±21%, p=0.002 and p<0.04, respectively). During normobaric hypoxia, PETCO2 and V̇E increased from normoxia to a similar extent in both groups, thus maintaining significantly lower PETCO2 in pre-term compared to full-term adults. Interestingly, exposure to normobaric hypoxia resulted in significantly higher breathing frequency among the prematurely born group, relative to their full-term counterparts. CONCLUSIONS These findings support previous evidence on prematurity-induced alterations in pulmonary function which persist into adulthood (1). Pre-term individuals presented a hypocapnic status at rest which may be related to ventilation-to-perfusion mismatch and/or cardiac dysfunction. Moreover, this was the first study showing evidence that pre-term birth is associated with increased chemosensitivity to hypercapnia, but not to hypoxia, compared to full-term birth. These novel findings might suggest that prematurity results in exaggerated central chemosensitivity despite unchanged peripheral chemosensitivity.

Understanding the role of objective physiological tests in assessment of survivors of severe COVID-19 pneumonia

The COVID-19 pandemic has affected millions of people worldwide. Acute COVID-19 infection can cause persistent and burdensome symptoms, impacting physiological function and quality of life (D’Cruz et al., 2021). Ideally, holistic post-COVID assessment includes physiological tests, yet infection control policies favour remote assessment of symptoms where possible. The availability of tests involving aerosol generating procedures, such as spirometry, is restricted. This study aimed to investigate the relationship between patient-reported symptoms and physiological outcomes in survivors of severe COVID-19 pneumonia, to investigate the role of in-person physiological assessment. This was a prospective single-centre observational study in the outpatient department of an urban South London teaching hospital. Survivors of severe COVID-19 pneumonia were invited for face-to-face assessment at 4-6 weeks post hospital discharge. Relationships between subjective self-reported symptoms (outcome measures listed in Table 1), 4 metre gait speed (metres/second (4MGS)) (Hirabayashi et al., 2020), and 1 minute sit to stand (repetitions/min (1STSreps) and SpO2% desaturation) (Briand et al., 2018) were assessed using correlation and multiple regression analysis. 168 patients attended clinic between June and November 2020. Patient characteristics and results of correlation analysis are shown in Table 1. Age (Beta = -0.284, p<0.001) and current mMRC dyspnoea score (Beta= -0.352, p=0.001) were independent predictors of 4MGS (overall model fit R2=0.35, p<0.001). Body mass index was the only independent predictor of 1STSreps (Beta=0.214, p=0.007, overall model fit R2=0.33, p<0.001). NRS pain (Beta=0.298, p=0.004) and cough (Beta=-0.179, p=0.041) were the only independent predictors of SpO2% nadir during 1STS (overall model fit R2=0.13, p=0.039). Respiratory symptoms were not strong predictors of 4MGS and 1STS performance, emphasising the importance of objective physiological tests in post-COVID assessments. The challenge of providing holistic care to survivors of COVID-19 has highlighted the importance of clinical respiratory physiology to the multidisciplinary team.

The influence of hyperglycaemia on the circadian clock in the TallyHO/Jng mouse model of type II diabetes

Circadian rhythms refer to endogenous biological oscillations with a 24-hour periodicity which are directly under the control of the core clock located within the hypothalamus. The disruption of said circadian rhythms via rotational or night-shift work has been linked to the development of metabolic diseases such as obesity and type 2 diabetes (T2D). The specific contributions of the core clock to this dysregulation have been elucidated following the development of tissue-specific knockout mice. For example, both skeletal muscle and hepatic-specific CLOCK and BMAL1 knockout mice display impairments in glucose control [1 2]. Despite this, evidence of circadian disruption in more physiological models of T2D is limited. In particular, although the use of diet-induced models is common, these models tend not to develop hyperglycaemia and overt T2D. Therefore, we have utilised the TallyHO/Jng mouse (JAX stock #005314) as a model of T2D. Interestingly, the development of the hyperglycaemic phenotype within this strain is not 100% penetrant which allows us the segregate the mice into two divergent strains of normoglycaemic (Tally-Low) and hyperglycaemic (Tally-High) TallyHO/Jng mice. The SWR/J mouse (JAX stock #000689) strain was utilised as the recommended healthy control. Therefore, our aim was to characterise the development of the diabetic phenotype in Tally-Low and Tally-High mice in comparison to SWR/J mice. In addition, we aimed to determine how hyperglycaemia influences the expression of circadian clock genes across the circadian cycle. Body weight and blood glucose was monitored on a weekly basis from 8 to 16 weeks of age (n=8/group). Plasma insulin was determined at 8 and 16 weeks of age (n=8/group). At 16 weeks of age, SWR/J, Tally-Low and Tally-High (n=6/group) mice were anaesthetized (50 mg pentobarbital/kg BW) and sacrificed via cervical dislocation at ZT3 (9am) and ZT15 (9 pm) and tissue samples collected. Skeletal muscle circadian clock related gene expression was assessed via RT-qPCR and protein levels via immunoblotting. Data were analysed via two-way ANOVA with Bonferroni correction and data presented as mean ± standard deviation. All procedures were approved by the Danish Animal Experiments Inspectorate and complied with European Directive 2010/63/EU for animal experiments. Tally-High mice displayed overt hyperglycaemia by 8-weeks of age (19.9 ± 5.2 mmol) in comparison to Tally-Low (8.9 ± 2.2 mmol) and SWR/J (6.7 ± 1.1 mmol) mice (P < 0.05). At 8-weeks of age, both Tally-Low (3.5 ± 1.2 ng/ml) and Tally-High (3.1 ± 1.0 ng/ml) display hyperinsulinemia in comparison to SWR/J mice (0.7 ± 0.3 ng/ml) (P < 0.05). At 16-weeks of age, skeletal muscle Bmal1 expression displayed diurnal rhythmicity between ZT3 and ZT15 in both SWR/J and Tally-Low mice (P 0.05). In conclusion, the divergent phenotype development in the TallyHO/Jng mouse represents an interesting model to study the effects of hyperglycaemia. The exacerbated hyperglycaemia in the Tally-High mice resulted in disrupted diurnal rhythmicity in Bmal1 expression in skeletal muscle. These data suggest that disruptions to the clock machinery in peripheral tissues may contribute to the divergent phenotype development in TallyHO/Jng mice.

Accentuated early postprandial appetite and appetite-related hormone responses in people with SCI versus able-bodied controls

Introduction: In persons with spinal cord injury (SCI), reduced fat-free mass and movement-related energy expenditure contribute to the increased obesity risk found in this population. Impairments in gastrointestinal function in this population are well established (Holmes & Blanke, 2019), but the regulation of appetite-related hormones, and their influence on energy intake regulation remains unknown. Additionally, low levels of total daily energy expenditure have been linked to reduced appetite sensitivity and weight gain (Long et al., 2002), potentially further impacting on energy intake regulation in persons with SCI. This study compared postprandial responses of appetite-related hormones, appetite perceptions and appetite sensitivity in persons with SCI and AB controls. Methods: In a counterbalanced order, 12 men with high-level SCI (≥T4 vertebrae) and 12 body mass and age-matched AB controls completed two trials, consuming a covert high-energy (HE; 2513 kJ) and low-energy (LE; 1008 kJ) preload on separate occasions. Subjective appetite perceptions (hunger, fullness, satisfaction and prospective future consumption [PFC]) were assessed at 30 min intervals following preload consumption (up to 150 min) and appetite sensitivity was subsequently determined from ad libitum meals. Appetite-related hormone (total PYY, GLP-1 and acylated ghrelin) responses were measured in the HE trial. Test-specific effect sizes (Cohen’s d) were calculated in adjunct to each statistical test (a large, moderate, small and trivial effect size was defined as ≥ 0.8, 0.5 < 0.8, and 0.2 < 0.5, and < 0.2, respectively) (Cohen, 1988). Results: Within the early postprandial phase (0-60 min), the SCI group displayed accentuated meal-responses for PYY (d = 0.72; p = 0.365), GLP-1 (d = 1.00; p = 0.134) and acylated ghrelin (d = 0.87; p = 0.112), though no group difference were seen from 60-150 min (d ≤ 0.59; p ≥ 0.340) (Figure 1). Moreover, subjective ratings of fullness (d = 1.09) and satisfaction (d = 0.91) were higher (P ≤ 0.028) during the first hour (Figure 2). Ad libitum energy intake was also lower in the SCI group (1086 vs. 1713 kJ, respectively, d = 1.00; P = 0.020) but no effect of trial (preload) was found (d = 0.003; p = 0.974) (Figure 3). Discussion: These findings suggest that postprandial satiety may be augmented in people with SCI. Such responses may be related to colonic dysfunction, which is believed to reduce motility in the upper gastrointestinal tract (Fynne et al., 2012). As PYY and GLP-1, and AG play a role in the inhibition and stimulation of gastrointestinal motility, respectively, our findings suggest that the accentuated postprandial gut hormone secretion may be mediated via colonic dysfunction. Additionally, reduced lean mass, commonly found in persons with SCI, may be related to the augmented satiety response (Gorgey & Dudley, 2007). In conclusion, this study shows that individuals with SCI exhibit accentuated appetite-related hormone and perceived satiety responses in the early postprandial phase, compared to age and weight-matched AB controls. These findings may reflect wider gut dysmotility in people with SCI but imply that weakened satiety may not be associated with higher rates of obesity.

Neuropeptide Y neurons within the nucleus of the solitary tract regulate feeding

Neuropeptide Y (NPY) is one of the most potent orexigenic neuropeptides in mammals. Although NPY is widely expressed within the brain, the preponderance of research investigating NPY’s effects on appetite have focussed on the hypothalamus. In the present work, we examined the distribution, neurochemical phenotype and function of NPY cells within the key homeostatic brain region, the nucleus of the solitary tract (NTS) using NPY Humanized Renilla Green Fluorescent protein (NPY-GFP; n=38) and NPY-Cre mice (n=17). All animal studies were conducted in accordance with The Animals (Scientific Procedures) Act 1986. Using in situ hybridisation histochemistry (ISSH) to label endogenous Npy mRNA and immunohistochemistry to label GFP, we observed that nearly all NPY-GFP cells co-expressed Npy mRNA (n=6). These findings validate the NPY-GFP line. Next, we assessed the distribution and neurochemical phenotype of NPY neurons within the NTS. The majority of NPY neurons are localised between -7.20 and -7.56 from bregma (n=29). As expected, the majority (63.62±4.92%) of NPY-GFP cells co-expressed paired-like homeobox 2b (Phox2b), the transcription factor essential for the development of the autonomic nervous system (n=4), and none co-expressed the neurotransmitter acetylcholine (using choline acetyltransferase), which is localised outside the NTS (n=3). Similar to the arcuate nucleus of the hypothalamus, NPY-GFP co-expression was observed with the neurotransmitter GABA (using glutamate decarboxylase67, GAD67; 22.20±2.78%; n=3). Perhaps counter-intuitively, a larger subset (54.66±7.50%; n=7) of NPY-GFP cells showed co-localisation with the anorectic protein nesfatin, although nesfatin’s function within the NTS has not been well described. Roughly a quarter (29.20±7.75%; n=7) showed co-expression for the catecholamines (using tyrosine hydroxylase) which are also classically anorectic. No NPY-GFP cells were co-expressed with the enzyme for nitric oxide, nitric oxide synthase (nNOS) (n=4). To examine the function of NTS NPY neurons, NPY-Cre mice were stereotaxically injected with the Cre-dependant designer receptor exclusively activated by designer drug (DREADD-Gq-mCherry or DREADD-Gi-mCherry) into the NTS (n=8/9 per group) under gas (2-3% isoflurane in O2) anaesthesia. We observed that activating NPY NTS neurons (DREADD-Gq-mCherry) with designer drug clozapine-N-oxide (CNO, i.p., 1 mg/kg/10 ml) significantly increased food intake whereas inhibiting NPY NTS neurons (DREADD-Gi-mCherry) significantly decreased food intake compared with saline treatment (two-tailed paired t-test or Wilcoxon sign-rank test, p<0.05). These data provide the first detailed characterisation of NPY neurons within the NTS and reveal that this population of neurons regulate appetite in mice.

The obese rat liver displays overlapping lipid signatures to the hypoxic liver, yet retains metabolic flexibility to respond to acute hypoxic stress

Hypoxia is associated with the aetiology of obesity. For instance, expanded adipose tissue experiences a lower oxygen tension and shows altered metabolism as a result [1]. However, in addition to adipose, the liver enlarges during obesity in association with ectopic fat storage [2]. Moreover, systemic inflammation leads to localised hypoxia as activated immune cells increase oxygen consumption and therefore deplete tissue oxygen tension [3]. We therefore sought to investigate the metabolic overlap between hypoxia and obesity in rat liver to determine whether hypoxia signalling plays a role in the development of obesity-related hepatic conditions, such as non-alcoholic liver disease. All experiments were carried out in accordance with the UK Home Office, Animals (Scientific Procedures) Act (1986) and were reviewed by the University of Cambridge Animal Welfare and Ethical Research Committee. Following 7-day baseline measurements in controlled environmental conditions (22°C, 55% humidity, 12h photoperiod), female Zucker obese (ZUC-Leprfa/fa) and lean (ZUC-Leprfa/+) rats were either exposed to two days of normobaric hypoxia (10.3 ± 0.2% O2; hypoxia groups) or maintained in the same environmental conditions (normoxia groups; n = 6 per group). At the end of the two-day exposure, the left lateral lobe of the liver was harvested and snap-frozen for metabolic analysis. Liquid chromatography-mass spectrometry (LC-MS) was utilised to carry out open-profile lipidomics on the liver. Using orthogonal partial least squares-discriminate analysis, the obese/normoxic lipidome was shown to differ significantly to that of the lean/normoxic lipidome (R2X(cum) = 81.4%, Q2(cum) = 84.9%, p<0.0001 ). Likewise, the lean/hypoxic lipidome differed to the lean/normoxic lipidome (R2X(cum) = 85.1%, Q2(cum) = 81.3%). These models were used to generate a shared and unique structures plot, with species within the ±0.5 region on either axis being of interest as shared or unique to obesity and/or hypoxia. Of these species of interest, a series of sphingomyelins (chain length 24:0 – 41:1) were found at lower levels in both the hypoxic and obese liver (p<0.05, one-way ANOVA with Benjamini-Hochberg false discovery rate correction, Tukey’s post hoc test for multiple comparisons). These species remained at lower levels (compared to lean/normoxic controls) when the insults were combined in the obese/hypoxic liver. Using LC-MS, the acyl-carnitine complement of the liver was also assayed. The obese/hypoxic liver had higher levels of long-chain acyl-carnitines (≥13 carbons) compared with the obese/normoxic liver, alongside lower levels of short-chain acyl-carnitines (<6 carbons). This suggests that during hypoxia, fatty acid oxidation decreases in the obese liver [4], showing that the obese liver remains metabolically flexible to respond to hypoxic conditions by promoting less oxygen-expensive pathways. Taken together, these results highlight that there are metabolic signatures common to the obese and hypoxic liver. However, the obese liver can still respond metabolically to hypoxic stimuli despite potential lipotoxicity and insulin resistance concurrent with obesity development [5].

Menstrual cycle phase but not oral contraceptive use modulates monocyte TNFα release following exposure to combined lipopolysaccharide and hyperthermia

Exercise and hyperthermia can increase intestinal permeability and cause an influx of lipopolysaccharide (LPS) into the intravascular space. LPS initiates a robust pro-inflammatory cascade which can contribute to heat-related illnesses. Fluctuations in oestrogen throughout the menstrual cycle have been shown to modulate intestinal barrier function, with indirect evidence linking high oestrogen concentrations in the luteal phase with an improved intestinal barrier function. Improved barrier function may impact pro-inflammatory responses across the menstrual cycle in response to hyperthermia-mediated LPS influx. There is little research on whether pro-inflammatory responses to LPS and/or hyperthermia differ in eumenorrheic women compared to women who take an oral contraceptive, or whether responses fluctuate across monthly hormone/pill cycles. This study aimed to quantify monocyte release of pro-inflammatory cytokine tumour necrosis factor-alpha (TNFα) following LPS-stimulation with or without hyperthermia in whole blood obtained from oral contraceptive users and eumenorrheic women throughout the pill/menstrual cycle. Volunteers using oral contraceptives (OC, n=7) were tested during the pill-free phase (PF; day 6±1), and twice during the pill phase (P1; day 14±2, and P2; 21±2 including the pill-free week). Eumenorrheic volunteers (MC, n=7) were tested during the early follicular (EF; day 6±1), ovulation (OV; day 16±2), and mid-luteal (ML; 7±1 days’ following positive ovulation test) phases of the menstrual cycle. Participants rested for 20-minutes before a 20 mL venous blood sample was drawn into a syringe and transferred into sodium-citrate tubes. Whole blood (1 mL) was stimulated with 100μg.mL-1 of LPS and one aliquot was incubated at 37°C (body temperature), and another at 40°C (simulated hyperthermia) for 6 hours. Plasma was diluted 1:40 in 1% bovine serum albuminate in phosphate-buffered saline, and TNFα concentration determined via ELISA (R&D Duoset, within-plate coefficient of variation (CV) 4%, between-plate CV 14%). Absolute TNFα concentrations (ng.mL-1) were analysed by repeated-measures ANOVA, with temperature (37°C and 40°C) and time (OC: PF, P1, P2; MC, EF, OV, ML] phase) within-subject factors and group (OC and MC) a between-subject factor. Effect sizes are expressed at partial eta2 (ηp2). TNFα concentrations were 28% greater after incubation at 40°C compared to 37°C (main effect for temperature, p<0.001, ηp2=0.610). The temperature-mediated response was similar in both groups (temperature x group interaction, p=0.784, ηp2=0.006), regardless of menstrual cycle phase or pill phase (main effect of time, p=0.212, ηp2=0.121). The TNFα response was similar between P1 vs. PF (+4%), and P1 vs. P2 (+7%) in the OC group regardless of temperature. TNFα concentrations were increased between ML vs. EF (+39%) and between ML vs. OV (+55%) regardless of temperature, however, the time x group interaction did not meet the conventional threshold for statistical significance (p=0.068, ηp2=0.200). TNFα responses were similar between the two groups when stimulated with LPS with/or without hyperthermia. A larger pro-inflammatory response occurred during the ML phase of the MC across both temperatures, despite evidence linking oestrogen with a reduction in pro-inflammatory responses. In conclusion, our data suggest that monocyte responses to immunogenic stimuli fluctuate throughout a hormone cycle in eumenorrheic women, but are more stable in those taking oral contraceptives.

Distinct roles for branched-chain amino acids and branched-chain keto acids in mediating cardiac insulin resistance in the healthy and failing heart

Perturbed branched-chain amino acids (BCAA) oxidation positively correlates with the severity of cardiac insulin resistance in heart failure 1, 2. We previously showed that cardiac specific deletion of the BCAA oxidative enzyme mitochondrial branched-chain aminotransferase (BCATm), which increases cardiac BCAA levels and decreases branched-chain keto acids (BCKA) levels, increases insulin-stimulated cardiac glucose oxidation rates 3. This enhanced cardiac insulin sensitivity is associated with an increase in the phosphorylation of protein kinase B (Akt) and activation of pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. However, whether it is the accumulation of BCAA or BCKA that is critical in mediating cardiac insulin resistance is unknown. How perturbed BCAA oxidation may mediate cardiac insulin resistance in heart failure is also unknown. To address these questions, we first examined the effects of selectively enhancing cardiac BCKA levels on cardiac insulin-stimulated glucose oxidation. We perfused isolated working mice hearts (male and female C57BL/6N, n=8-10) with high levels of BCKA (&#55349;&#57026;-keto-isocaproate 80 mM, &#55349;&#57026;-keto-&#55349;&#57027;-methylvalorate 100 mM, &#55349;&#57026;-keto-isovalorate 70 mM), levels that can be seen in diabetes and obesity 4. High levels of BCKA completely blunted insulin-stimulated glucose oxidation rates and increased fatty acid oxidation rates. We also found that BCKA abolished insulin-stimulated mitochondrial Akt, an effect that was associated with PDH deactivation. We next determined the potential protective effect of reducing cardiac BCKA levels in the failing heart. We randomized WTCre+/+ and cardiac-specific BCATm-/- mice (male, 25-30g, n=6-8) to undergo either sham surgery or transverse aortic constriction surgery to induce heart failure. Five weeks post-surgery there was a marked increase in insulin-stimulated glucose oxidation rates in the BCATm-/- failing hearts compared to the WTCre+/+ failing hearts (1.48 ± 0.2 vs 0.78 ± 0.02 µmol.g dry wt-1.min-1 respectively, p<0.05), with no significant effect on glycolysis rates. Enhanced cardiac insulin sensitivity was associated with a significant decrease in fatty acid oxidation rates in the BCATm-/- failing hearts compared to the WTCre+/+ failing hearts (0.31 ± 0.06 vs 0.67 ± 0.2 µmol.g dry wt-1.min-1 , respectively, p<0.05). This decrease in fatty acid oxidation in the BCATm-/- failing hearts was associated with a significant decrease in myocardial oxygen consumption rates. As a result, cardiac efficiency (cardiac work/myocardial oxygen consumption) was significantly increased in the BCATm-/- failing hearts compared to the WTCre+/+ failing hearts. We conclude that the accumulation of BCKA, and not BCAA, is a major contributor to cardiac insulin resistance via abrogating mitochondrial translocation of Akt. Targeting BCKA may represent a potential therapeutic approach to improve cardiac insulin-stimulated glucose oxidation in the setting of heart failure, obesity and diabetes.

The effects of oral contraceptives on exercise performance in women: A systematic review and meta-analysis

Background: Oral contraceptive pills (OCPs) are double agents, which downregulate endogenous concentrations of oestradiol and progesterone whilst simultaneously providing daily supplementation of exogenous oestrogen and progestin during the OCP-taking days. This altered hormonal milieu differs significantly from that of eumenorrheic women and might impact exercise performance, due to changes in ovarian hormone-mediated physiological processes. Objective: To explore the effects of OCPs on exercise performance in women and to provide evidence-based performance recommendations to users. Methods: This review complied with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. A between-group analysis was performed, wherein performance of OCP users was compared with naturally menstruating women, and a within-group analysis was conducted, wherein performance during OCP consumption was compared with OCP withdrawal. For the between-group analysis, women were phase matched in two ways: (1) OCP withdrawal versus the early follicular phase of the menstrual cycle and (2) OCP consumption versus all phases of the menstrual cycle except for the early follicular phase. Study quality was assessed using a modified Downs and Black Checklist and a strategy based on the recommendations of the Grading of Recommendations Assessment Development and Evaluation working group. All meta-analyses were conducted within a Bayesian framework to facilitate probabilistic interpretations. Results: 42 studies and 590 participants were included. Most studies (83%) were graded as moderate, low or very low quality, with 17% achieving high quality. For the between-group meta-analysis comparing OCP users with naturally menstruating women, posterior estimates of the pooled effect were used to calculate the probability of at least a small effect (d ≥ 0.2). Across the two between-group comparison methods, the probability of a small effect on performance favouring habitual OCP users was effectually zero (p < 0.001). In contrast, the probability of a small effect on performance favouring naturally menstruating women was moderate under comparison method (1) (d ≥ 0.2; p = 0.40) and small under comparison method (2) (d ≥ 0.2; p = 0.19). Relatively large between-study variance was identified for both between-group comparisons ( !0.5 = 0.16 [95% credible interval (CrI) 0.01–0.44] and !0.5 = 0.22 [95% CrI 0.06–0.45]). For the within-group analysis comparing OCP consumption with withdrawal, posterior estimates of the pooled effect size identified almost zero probability of a small effect on performance in either direction (d ≥ 0.2; p ≤ 0.001). Conclusions: OCP use might result in slightly inferior exercise performance on average when compared to naturally menstruating women, although any group-level effect is most likely to be trivial. Practically, as effects tended to be trivial and variable across studies, the current evidence does not warrant general guidance on OCP use compared with non-use. Therefore, when exercise performance is a priority, an individualised approach might be more appropriate. The analysis also indicated that exercise performance was consistent across the OCP cycle.

The atypical antipsychotic drugs aripiprazole and clozapine have direct effects to increase beta-cell proliferation and protect against apoptosis

Motivation: Atypical antipsychotic drugs (AAPs) are prescribed to control various psychiatric conditions, such as schizophrenia, bipolar disorder, or they are used as an add-on therapy for major depressive disorder. The prevalence of diabetes is 3-fold higher in people suffering from mental illnesses [1] and some antipsychotic drugs have been linked to increased risk of hyperglycaemia and type 2 diabetes (T2D) [2]. However, AAPs vary in side effects related to T2D and the mechanisms of action of individual AAPs at islet beta-cells are not fully understood. Therefore, this project aimed to investigate the direct effects of therapeutic concentrations of two widely used AAPs, aripiprazole and clozapine, on mouse beta-cell function. Methods: Beta-cell proliferation was measured by quantifying BrdU incorporation into MIN6 beta-cell DNA after 48h exposure to 1µM aripiprazole or 2µM clozapine using a BrdU ELISA, and viability was measured by Trypan blue staining. ATP generation and apoptosis of MIN6 cells and mouse islets were quantified following a 48h incubation in the absence or presence of AAPs using CellTiter-Glo 3D and Caspase-Glo assays, respectively. Chronic effects of AAPs on glucose-stimulated insulin secretion from MIN6 cells and mouse islets were determined by static incubation experiments and secreted insulin was measured by radioimmunoassay. Results: Aripiprazole and clozapine significantly increased MIN6 beta-cell proliferation (OD 450nm, control: 0.59±0.02; +1µM aripiprazole: 0.65±0.01; +2µM clozapine: 0.67±0.02, n=8, P0.1). These AAPs had a protective effect against palmitate-induced apoptosis of MIN6 cells and mouse islets (MIN6 cells: luminescence units, control: 653,970±28,403; +1µM aripiprazole: 541,061±13,281; +2µM clozapine: 555,632±16,741, n=8, P<0.001; mouse islets: control: 62,424± 7,921; +1µM aripiprazole: 40,799±4,841; +2µM clozapine: 41,627±3,857, n=8, P0.1; control: 0.92±0.09; +2µM clozapine: 1.18±0.26, n=7, P>0.1) or mouse islets (ng/islet/h, control: 2.77±0.37; +1µM aripiprazole: 3.97±0.67, n=7, P>0.1; control: 1.70±0.74; +2µM clozapine: 2.15±0.79, n=7, P>0.1). Conclusion: Therapeutically relevant concentrations of aripiprazole and clozapine are well-tolerated by beta-cells, with no impairment of viability. They induce beta-cell proliferation and reduce apoptosis, which is of benefit to restore functional beta-cell mass. Our data support these AAPs being recommended for treating psychiatric conditions in patients with glucose dysregulation.

A comprehensive analysis of gene expression in the kidney of the one-humped Arabian camel (Camelus dromedarius) reveals a role for cholesterol in water conservation during dehydration

The one-humped Arabian camel is the most important livestock animal in arid and semi-arid regions and continues to provide basic necessities to millions of people in areas where food production is problematic. In the current context of global warming, there is renewed interest in the adaptive mechanisms that enable camelids to survive in arid conditions. Extensive evidence shows the impressive set of adaptations that allows dromedaries to thrive in such environment and recent investigations described genomic signatures that revealed evolutionary adaptations to desert environments (1-2). We now present a comprehensive catalogue of the transcriptomes of the male dromedary kidney. We performed RNAseq in samples from 15 adult dromedaries (5 controls, 5 dehydrated and 5 rehydrated). Using DESeq2, we identified 985 DEG in the kidney (cortex and medulla) of dehydrated camels. Gene Ontology (GO) analyses suggested an enrichment of the cholesterol biosynthetic pathway and an overrepresentation of categories related to “ion transmembrane transport”. The RNAseq data confirmed that 13 key genes involved in the cholesterol biosynthesis pathway were significantly (padj < 0.05) downregulated in the kidney medulla during dehydration. These data were validated by RT-qPCR (log2FC ranged 0.34 – 0.87). Further, we recently reconstructed the transcriptional regulatory network of the camel kidney during chronic dehydration. We identified 49 differentially expressed transcription factors and 10 enriched regulons. Among those, SREBF1 and SREBF2 were significantly downregulated in dehydration compared to control (padj < 0.05). They play a key role in the regulation of cholesterol synthesis and GO analyses of their significantly changed targets confirmed an enrichment of that pathway. Based on our hypothesis of a role for cholesterol during dehydration, we identified DEGs with known roles in the countercurrent multiplication process which are affected by changes in the level of cholesterol. Cholesterol changes the state of K+ inward rectifiers (KCNJ8), by directly binding onto it, to “silent”, it also inhibits Na+ leakage and indirectly decreases Na+-K+-ATPase activity. Moreover, cholesterol depletion results in the accumulation of AQP2 in the plasma membrane. KCNJ8 (padj < 0.01) and ATP1B3 (non-significant) showed a ~0.5-2 fold increase in gene expression during dehydration in the medulla, whilst SLC9A7 expression level trended towards a smaller increase (non-significant). Relative expression of AQP2 showed a 2-fold increase (padj < 0.001). Our datasets suggest that the suppression of cholesterol biosynthesis may facilitate water retention in the kidney of the dromedary by indirectly enhancing the osmotic gradient along the medullary interstitium that is required for the subsequent AQP-2-mediated water reabsorption. Ethical statement and statistics Samples were shipped to the University of Bristol under the auspices of a DEFRA Import Licence (TARP/2016/063). This study was approved by the Animal Ethics Committee of the United Arab Emirates University (approval ID: AE/15/38) and the University of Bristol Animal Welfare and Ethical Review Board. Benjamini-Hochberg was used to calculate significance levels in differential gene expression analysis and One-way ANOVA to determined differences between RT-qPCR experimental groups (n=5). Alternatively, we used Kruskal-Wallis test combined with Benjamini-Hochberg method for groups which followed non-normal distribution.

Nutraceutical targeting of the bile acid receptor, farnesoid X receptor, for intestinal disease

Introduction: Bile acids, classically known for their roles in dietary fat absorption are now recognised as hormones critical to regulating intestinal and metabolic function, including mucosal immune responses, epithelial proliferation/apoptosis, and transepithelial transport and barrier function. Downregulation of the nuclear bile acid receptor, farnesoid X receptor (FXR), in intestinal epithelium occurs in inflammatory bowel disease, colorectal cancer, obesity and diabetes, whereas FXR activation prevents disease progression in pre-clinical models. Thus, strategies to upregulate epithelial FXR expression have been proposed to treat such conditions. Previous studies suggest that a particular class of plant-derived phytochemicals, denoted KFS1, modulates FXR activity. Plant sources have been reported to contain these phytochemicals, such as KFS1, which are proposed to have FXR modulatory activity. Aim: The aim of the current study was to investigate the potential for developing plant extracts, rich in phytochemicals, such as KFS1, as a new novel nutraceutical-based approach to treat and prevent intestinal and metabolic diseases. Methods: T84 colonic epithelial cells and primary cultures of murine epithelial colonic enteroids were employed to examine the effects of a common-dietary phytochemcial, KFS1, and a KFS1-rich plant extract on FXR expression and FXR signaling in vitro. Ex vivo studies were carried out on human colonic tissue obtained during endoscopy with ethical approval from Beaumont Hospital. In vivo studies in C57BL/6 mice were carried out and proximal colonic tissue were examined with ethical approval from the HPRA. Expression of FXR and FGF19/15, an established marker of FXR activation, was assessed by qRT-PCR, ELISA and immunoblotting. Data are expressed as mean ± SEM for a series of n experiments. Results: KFS1 treatment (5 µM; 24hrs) increased FXR mRNA and protein expression in T84 cells by 4.2 ± 0.4 (p<0.05; n = 4) and 1.7 ± 0.1 fold (p<0.05; n = 7), respectively. KFS1 also enhanced FGF-19 protein expression, an established marker of FXR activity, in response to the FXR agonist, GW4064 (5 µM), by 4.1 ± 0.5 fold (p<0.001; n = 7). Similarly, KFS1 significantly upregulated FXR expression by 2.1 ± 0.4 fold in human biopsies (p<0.05; n = 6). Oral KFS1 administration to mice had a tendency to upregulate colonic epithelial FXR signalling in vivo and increased FXR expression and GW4064-induced FGF15 mRNA by 2.3 ± 0.2 (p<0.01; n = 4) and 2.2 ± 0.1 fold (p<0.01; n = 4) in murine epithelial colonic enteroids. Finally, a methanolic KFS1-rich plant extract, denoted QE1, verified by LC/MS to contain KFS1, increased FXR protein expression in T84 cells by 1.8 ± 0.2 fold (p<0.05; n = 5) and enhanced GW4064-induced FGF19 protein release by 1.9 ± 0.1 fold (p<0.01; n = 9). Conclusion: In conclusion, our data demonstrate that KFS1-containing plant extracts modulate expression and activity of the nuclear bile acid receptor, FXR, in the intestinal epithelium. Given the critical role of FXR in maintenance of gut health and metabolic function, such extracts have significant therapeutic and commercial potential to be developed as a novel first-in-class “FXR-targeted nutraceutical” for treatment and prevention of common intestinal and metabolic disorders.

Modulation of cerebrospinal fluid production by the Transient Receptor Potential Vanilloid 4 channel: implications for hydrocephalus and other disorders of brain fluid volume

Cerebrospinal fluid (CSF) secretion into the brain ventricular system is an active process via the choroid plexus (CP) – a specialized, convoluted epithelial sheet surrounding a fenestrated capillary bed which is fed from the cerebral arterioles. The final CSF composition differs from a simple plasma filtrate implicating the CP epithelial cells (CPe) as an important regulatory site controlling CSF composition and volume. Solute and water transport across the blood-CSF barrier are tightly controlled by ion and water transport proteins. Transient Receptor Potential Vanilloid 4 (TRPV4) is a polymodal, non-selective cation channel which functions as a regulatory hub protein in epithelia, including the CP. Previous studies from our group demonstrated d that TRPV4-selective antagonists were effective at reducing ventriculomegaly in a genetic rat model of severe postnatal hydrocephalus, a condition resulting from excess CSF (Hochstetler and Smith et al., 2020). In CPe, it was found that activation of TRPV4 caused the influx of both Ca2+ and Na+ ions, and entry of both of these ions was selectively blocked by a TRPV4-selective antagonist. Systemic treatment with TRPV4 antagonists reduced TRPV4 mRNA expression in the CP in hydrocephalic rats, but not in normal rats, indicating a role for TRPV4 as part of a regulatory pathway in CSF production under pathological conditions. To study the mechanism of CSF secretion by the CP, we utilized two continuous, high resistance CP cell lines: the porcine choroid plexus – Reims (PCP-R) and the human choroid plexus papilloma (HIBCPP) cells. The current experiments utilize Ussing chamber electrophysiological techniques, western blotting, RT-PCR, and fluorescent immunolabeling. Immunolabeling and mRNA expression demonstrate a robust abundance for TRPV4 and other well-characterized membrane proteins in both cell lines. In the lines, TRPV4 activation stimulates a large change in short circuit current (ISC), a measure of net electrogenic ion flux. This change in ISC is coupled with a substantial change in transepithelial membrane conductance, a measure of cellular permeability. The transepithelial membrane transport changes due to TRPV4 activation for human and porcine cells are similar in overall effect, but differ in baseline, duration, and magnitude, indicating the possibility of species-specific CP transport mechanisms. TRPV4 activation can be inhibited by pre- incubation with TRPV4 antagonists, and the changes in ion flux and conductance can be reversed by addition of TRPV4 antagonists after initiation of the response, these studies substantiate the specificity of the mediator-induced changes and provide an in vitro model for testing potential drug actions. Furthermore, in both the porcine and human cells, the TRPV4 response is dependent upon K+ secretion, Cl- absorption, and Na+ secretion. These data confirm a role for TRPV4 in regulating ionic gradients, thereby modulating both electrogenic and electroneutral transporters, and controlling CSF production. The promising results of these studies, coupled with published data from other investigators suggesting a role for TRPV4 in blood-brain-barrier permeability and astrocyte volume regulation, shows the importance of TRPV4 as a regulatory hub protein in maintaining brain fluid volume.

Acidic pH affects the expression of ASIC1 and is associated with hypomethylation of ASIC1 promoter region in pancreatic ductal adenocarcinoma

The development and progression of pancreatic ductal adenocarcinoma (PDAC) are linked to the physiology and microenvironment of the exocrine pancreas. The temporally highly variable extracellular pH landscape in pancreatic ducts challenges epithelial cells in maintaining a constant intracellular pH [1]. We hypothesize that changes in pH affect cellular processes leading to an altered expression of ion channels. This in turn helps driving transformation from usual to malignant cell behaviour. We investigate if the already known dysregulation of the acid sensing ion channel 1 (ASIC1) [2] is regulated by DNA methylation. To investigate if a prolonged acidic pH treatment has an effect on DNA methylation and thereby the expression of ASIC1, PDAC cell lines Panc-1 and MiaPaca-2 were incubated in medium with a pH of 7.4 or 6.5 over 30 days. A ductal-like control cell line hTERT-HPNE was grown in pH 7.4. Under these conditions, mRNA expression of ASIC1 was obtained by qPCR, and DNA methylation levels were obtained by sodium bisulfite-treated genomic DNA sequencing. Human PDAC tissue samples from non-tumour and tumour tissue were used to acquire the ASIC1 expression by classical immunohistochemical analysis (IHC). Our preliminary IHC results showed that ASIC1 tends to be upregulated in tumour tissue compared to normal tissue (n=10, intensity score in tumour =2, vs. 0 in non-tumour tissue). ASIC1 was significantly upregulated in Panc-1 and MiaPaca-2 cell lines, compared to the control hTERT-HPNE (n=3, Welch Correction test, p< 0.0001 and p<0.01, respectively). The acidic pH treatment (pH 6.5) resulted in further ASIC1 upregulation, when compared to cells treated with pH 7.4, both in Panc-1 and MiaPaca-2 cells (n=3, Welch Correction test, p< 0.001 and p< 0.01, respectively). To test our hypothesis that ASIC1 gene expression is regulated by DNA methylation, we performed bisulfite sequencing interpreted by a semi-quantitative analysis, where the ratio between the peak of thymines and cytosines was measured. Different CpG islands of the ASIC1 promoter region were to some extent methylated in DNA from MiaPaca-2 cells treated with pH 7.4 (n=2), sum of methylation in 26 CpG islands was Σ95% and Σ28%, respectively. In contrast, CpG islands of the ASIC1 promoter region from MiaPaca-2 treated with pH 6.5, had lower DNA methylation levels (n=2), sum of methylation in 26 CpG islands was Σ1.35% and Σ1.18%, respectively. Collectively, these results show that ASIC1 is upregulated in human tissue, and is significantly upregulated in PDAC cell lines Panc-1 and Miapaca-2. Prolonged exposure to acidic pH, results in an upregulation of ASIC1 in both cell lines. This upregulation might be governed by DNA methylation of specific ASIC1 CpG sites, suggesting that the ASIC1 gene promoter region is hypomethylated in cells exposed to prolonged acidic treatments. Further studies should assess ASIC1 expression and DNA methylation status in human tissue. Furthermore, a quantitative analysis and method to show that DNA methylation levels are reversible can reveal that the upregulation of ASIC1 in PDAC is governed by DNA methylation and affected by the temporally highly variable pH landscape in pancreatic ducts.

Students’ perspectives of physiology e-learning during the COVID-19 pandemic in Nigerian universities

During the COVID-19 pandemic, several institutions and medical schools globally transited to online learning, as evident in few Nigerian Universities (1). This study explores the perspectives of medical students regarding Physiology e-learning amid the COVID-19 lockdown. An online descriptive, cross-sectional study was conducted between July and September, 2020. Current undergraduate medical students aged 17 years or older across five private-owned Universities who transited to virtual learning constituted the study population. Using Google forms, a web-based questionnaire was administered to participants via WhatsApp messenger. The questionnaire was developed using validated questions from previously published studies. Overall, 200 participants responded (response rate = 65%). Of the 190 valid responses, 48 (25.3%) were males, and 157 (82.6%) were in the age bracket of 17-20 years. Of the 190 participants who engaged in learning Physiology online during the lockdown, 78 (41.1%) were pursuing the Bachelor of Medicine and Bachelor of Surgery (MBBS) program, 69 (36.3%) Bachelor of Pharmacy (B.Pharm), while 16 (8.4%) were studying Bachelor of Science in Physiology (Majors; B.Sc.). More than half (n = 122, 64.2%) of the study participants agree to actively participate in online classes, 68 (35.8%) had a suitable home environment for online learning, 32 (16.8%) agree that the quality of online teaching equals classroom teaching, while 36 (19%) students are satisfied with learning Physiology online. Furthermore, 135 (71.1%) participants prefer mornings as their learning periods compared to afternoons, 149 (78.4%) supplement the online lectures with further private study, while only 46 (24.2%) students agree that online lectures only will sufficiently equip them with required Physiology knowledge. The evaluation of the experiences and perspectives of students learning Physiology online during the COVID-19 pandemic in Nigeria is imperative due to the novelty of the learning mode. Thus, there is a need to encourage the consideration of adopting and integrating blended (virtual) learning in Physiology education in Nigeria.

Interactive quizzing in online classes: The suitability of Kahoot! for face-to-face and online delivery in health sciences and medical education

Recent events have fast-tracked the transition of university teaching from face-to-face to online, remote delivery. The disruption to the delivery of standard classes, as a result of the COVID-19 pandemic, affected students across all levels of education.1 In order to continue teaching and learning, the delivery of content was replaced with live online modalities. This placed a significant challenge on tertiary educators to find learning activities that are transferrable between face-to-face and online teaching whilst promoting equal engagement.2 One such method, interactive quizzing, is a tool commonly used in face-to-face classes that could enhance engagement and enjoyability.3 The primary outcome of this study was to evaluate the suitability of using the interactive quizzing platform, Kahoot!, as a teaching tool that can be effective between face-to-face and online classes. In order to achieve this outcome, this study aimed to compare learner perceptions of the interactive quizzing platform Kahoot! when used in either a face-to-face or online setting within a health sciences and medical course. A total of 174 first-year health sciences and medical students from an Australian university enrolled in this study. Two study groups were formed based on whether the participants were currently enrolled in a face-to-face (n = 72) or online (n = 102) provision of their subject. Participants attended a one-hour physiology lecture, either in a face-to-face class or online during live sessions, then completed a 10-item Kahoot! interactive quiz based on the session content. Following the provision of the quiz, participants completed a four-question Likert scale survey related to their experiences and provided written responses to three open-ended questions regarding their perceptions of using the interactive quizzing platform. Overall, participants in both the face-to-face and online learning groups highly rated their learning experience using interactive quizzing. There were no significant differences (Student’s two-tailed t-test) between experiences from using Kahoot! during a face-to-face or online session. In particular, responses from the face-to-face and online participants on the Likert scale (1 = strongly disagree, 5 = strongly agree) for the statement “I enjoyed using Kahoot!” were 4.71 ± 0.54 and 4.81 ± 0.68, respectively (p = 0.3). Three overall themes emerged from qualitative analysis of student perceptions that were comparable between the two groups. These themes were (1) interactive quizzing is enjoyable, (2) interactive quizzing is engaging, and (3) interactive quizzing helps my learning. Participants utilising interactive quizzing in an online setting reported increased engagement whilst learning due to the fun, attractive environment and interactive nature of the platform, despite being isolated in a passive learning environment. This study identifies Kahoot! as a teaching tool that is equally effective across both face-to-face and online teaching sessions, and thus presents interactive quizzing as a variable instrument for assessment and revision that can be utilised in health sciences and medical courses. As many tertiary institutions are currently split between online, face-to-face or mixed-mode curricula, it is increasingly important to highlight technology that can be rapidly and easily transferred between various modes of delivery.

Using 3D holographic technology (HoloLens) for asthma education in health sciences and medicine

Medical and health sciences education requires constant updating and refining to best prepare students for the expectations of working in the modern healthcare professional environment. Learning about a range of diseases is a core component within these courses, and a fundamental part of many curricula and assessment-focussed outcomes. One common example is asthma, a widespread and prevalent respiratory condition estimated to affect up to 339 million people globally [1]. However, learning about the management of asthma requires students to integrate knowledge of physiology, anatomy, pathology, immunology, pharmacology and more. It also necessitates a need for an understanding of the lungs and its associated structures in 3D space. This can be difficult when studying from a textbook or lecture notes alone. In recent years, there has been a shift towards technology-enhanced learning to deliver content in an engaging manner [2]. Emerging technology, such as the Microsoft HoloLens, is of great interest as it can provide 3D representations of the human body, while also encouraging interactivity with any presented organs or systems [3]. Though never employed for the specific use of teaching asthma, the HoloLens shows potential as a way to effectively explain the mechanisms underlying asthma, and its associated multidisciplinary concepts. The aim of this honours research project will be to assess whether a textbook-style written delivery, or a three-dimensional (3D) augmented reality HoloLens resource, is more effective for learning. This will be a randomised-control trial utilising pre- and post-testing with first year health sciences and medical students. Lessons will be set up with an instructional module explaining the epidemiology, anatomy, physiology, pathophysiology, immunology and pharmacology of the respiratory system and asthma. The control group are to be provided with a printed textbook-style version of the lesson, with 2-dimensional diagrams, while the HoloLens intervention group viewing the models in 3D, with the text read out as an audio transcript. Though data collection will commence shortly, it is hypothesised that learning through augmented reality using the HoloLens device will provide a better overall learning experience and improve test performance for health sciences and medical students.

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Neurophysiological Bases of Human Movement 2025

Monday 19 April - Thursday 22 April 2021

Future Physiology 2021

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10:00 SA01 Physical activity and cardiovascular fitness

10:30 OC01 Atrial compartmentalisation proteomics: Analysis of endo-lysosomal proteins using a modified density gradient approach

10:45 OC02 Cerebrovascular responses during voluntary breath holding are larger than rebreathing in humans

11:00 OC03 A dual potassium channelopathy underlies small vessel disease of the brain in a mouse model of Alzheimer’s disease

11:15 OC04 Cardiotoxicity of particulate matter air pollution in mouse models of health and disease

11:30 SA02 Glucocorticoids and the renal-control of circadian blood pressure

14:00 SA03 Improving sleep and rehabilitation after stroke

14:30 OC05 Kinetics of synaptic vesicle pool depletion and adaptation in zebrafish lateral line hair cells

14:45 OC06 Inhibition of mitochondria fission in astrocytes in the dorsal vagal complex of the brain to target the metabolic profile of brown adipose tissue in high fat diet-fed rats

15:00 OC07 Role of CPT1C in motor function, energy state, mood, synaptic plasticity and cognition

15:15 OC08 Sternohyoid muscle weakness in pre-symptomatic ALS mice

15:30 SA04 Bright daytime light enhances circadian amplitude in a diurnal mammal

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10:00 SA05 Metabolic implications of the timing of nutrition and exercise – could the pandemic realign our rhythms?

10:30 OC09 The effect of local cooling strategies on the perceptual and inflammatory response to passive heating in healthy young males

10:45 OC10 Low intensity resistance exercise with blood flow restriction induces local and systemic hypoalgesia via endogenous opioid-mediated mechanisms of pain modulation

11:00 OC11 Different ventilatory response to hypoxia and hypercapnia in prematurely born adults

11:15 OC12 Understanding the role of objective physiological tests in assessment of survivors of severe COVID-19 pneumonia

11:30 SA06 How can exercise physiologists and the exercise sciences come together to support the post-COVID pandemic response?

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14:00 SA07 The impact of short-term muscle disuse on muscle mass regulation and insulin resistance

14:30 OC13 The influence of hyperglycaemia on the circadian clock in the TallyHO/Jng mouse model of type II diabetes

14:45 OC14 Accentuated early postprandial appetite and appetite-related hormone responses in people with SCI versus able-bodied controls

15:00 OC15 Neuropeptide Y neurons within the nucleus of the solitary tract regulate feeding

15:15 OC16 The obese rat liver displays overlapping lipid signatures to the hypoxic liver, yet retains metabolic flexibility to respond to acute hypoxic stress

15:30 SA08 Inactivity and skeletal muscle metabolism: Effects, counter-interventions and novel methodologies in a pandemic-effected society

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10:00 SA09 Islet glucagon secretion in response to high fat diet

10:30 OC17 Menstrual cycle phase but not oral contraceptive use modulates monocyte TNFα release following exposure to combined lipopolysaccharide and hyperthermia

10:45 OC18 Distinct roles for branched-chain amino acids and branched-chain keto acids in mediating cardiac insulin resistance in the healthy and failing heart

11:00 OC19 The effects of oral contraceptives on exercise performance in women: A systematic review and meta-analysis

11:15 OC20 The atypical antipsychotic drugs aripiprazole and clozapine have direct effects to increase beta-cell proliferation and protect against apoptosis

11:30 SA10 A time for growth: A circadian transcription factor regulating metabolic phenotype through the growth axis

14:00 SA11 Virtual Gut Feelings: intestinal organoid applications in metabolic and inflammatory diseases

14:30 OC21 A comprehensive analysis of gene expression in the kidney of the one-humped Arabian camel (Camelus dromedarius) reveals a role for cholesterol in water conservation during dehydration

14:45 OC22 Nutraceutical targeting of the bile acid receptor, farnesoid X receptor, for intestinal disease

15:00 OC23 Modulation of cerebrospinal fluid production by the transient receptor potential vanilloid 4 channel: Implications for hydrocephalus and other disorders of brain fluid volume

15:15 OC24 Acidic pH affects the expression of ASIC1 and is associated with hypomethylation of ASIC1 promoter region in pancreatic ductal adenocarcinoma

15:30 SA12 The hidden metabotropic life of nicotinic acetylcholine receptor subunits α7, α9 and α10 in monocytes

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10:00 SA13 Black Academia and ME: My experience of the ever-evolving research world

10:30 OC25 Students’ perspectives of physiology e-learning during the COVID-19 pandemic in Nigerian universities

10:45 OC26 Interactive quizzing in online classes: The suitability of Kahoot! for face-to-face and online delivery in health sciences and medical education

11:00 OC27 Using 3D holographic technology (HoloLens) for asthma education in health sciences and medicine

11:15 SA14 Breaking down the barriers to engagement: Has COVID 19 stopped us reaching out?

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10:30 OC01 Atrial compartmentalisation proteomics: Analysis of endo-lysosomal proteins using a modified density gradient approach

11:15 OC04 Cardiotoxicity of particulate matter air pollution in mouse models of health and disease

11:15 OC20 The atypical antipsychotic drugs aripiprazole and clozapine have direct effects to increase beta-cell proliferation and protect against apoptosis

10:45 OC26 Interactive quizzing in online classes: The suitability of Kahoot! for face-to-face and online delivery in health sciences and medical education 