Tumour necrosis factor alpha (TNFα) is an important inflammatory mediator in the body andand thus, is considered an important biomarker in a host of inflammatory disorders such as ulcerative colitis. Many biosensing strategies are expensive and use complex fabrication approaches. Therefore, this study aimed to develop a simple yet robust TNFα sensor, which could conduct measurement within biological environments. 3D printing was utilised to fabricate an electrode with skyscraper structures to provide increased surface area for the sensor. The sensor was made by covalent immobilization of anti-TNFα onto carboxylic acid bearing conducting polymer thiophene-2-carboxylic (Th2CA) after electrodeposition of gold nanoparticles (AuNPs) onto the SS electrode. The development of the biosensor was characterised using field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS). The linear range for TNFα was from 160 to 1820 pg/ml with a limit of detection of 44.5pg/ml on the skyscraper (SS) immunosensor. The sensor was selective for the detection of TNFα when compared to other commonly found extracellular biological molecules. The sensor was able to monitor levels of TNFα from faecal pellets, where an increase in TNFα was observed with increasing age. These findings highlight that 3D printing could be used to make a simple yet robust immunosensor for the detection of a host of key biomolecules providing the potential for vital diagnostic tools.

BACKGROUND:Increasing age increases the incidence of chronic constipation and fecal impaction. The contribution of the natural aging process to this phenotype is unclear. This study explored the effects of age on key motility patterns in the murine colon and determined the contribution that altered neurokinin 2 (NK2) -mediated signaling made to the aging phenotype. METHODS:Mucosal reflexes, colonic migrating motor complexes (CMMCs) and colonic motility assays were explored in isolated ex vivo colons from 3, 12-14, 18- and 24-months old mice and the NK2-mediated response determined. Electrical field stimulation (EFS) or exogenous drug application were used to explore the role of the mucosa in colonic segments. KEY RESULTS:Aging reduced the force of contraction of the distal colon mucosal reflex, the frequency and force of contraction of CMMCs and the NK2-mediated component of both motility patterns. Ondansetron, a 5-HT3 receptor antagonist, blocked a component of both motility patterns in full thickness but not in mucosa-free segments of the distal colon. 5, hydroxytryptamine (5-HT) and EFS-evoked NK2-dependent contractions were reduced with increasing age. Smooth muscle sensitivity to 5-HT or neurokinin A (NKA) was not altered with age. In isolated colon motility assays application of NKA decreased transit time in 24-months colon and the NK2 antagonist GR159897 increased transit times in both 3- and 24-months old colons. CONCLUSIONS AND INFERENCES:Aging impairs key motility patterns in the murine colon. These changes involve a decrease in mucosally-evoked NK2-mediated signaling. Targeting NK2-mediated signaling may provide a novel approach to treating age-related motility disorders in the lower bowel.

Enterochromaffin (EC) cells located within the intestinal mucosal epithelium release serotonin (5-HT) to regulate motility tones, barrier function and the immune system. Electroanalytical methodologies have been able to monitor steady state basal extracellular 5-HT levels but are unable to provide insight into how these levels are influenced by key regulatory processes such as release and uptake. We established a new measurement approach, amperometry approach curve profiling, which monitors the extracellular 5-HT level at different electrode-tissue (E-T) distances. Analysis of the current profile can provide information on contributions of regulatory components on the observed extracellular 5-HT level. Measurements were conducted from ex vivo murine ileum and colon using a boron-doped diamond (BDD) microelectrode. Amperometry approach curve profiling coupled with classical pharmacology demonstrated that extracellular 5-HT levels were significantly lower in the colon when compared to the ileum. This difference was due to a greater degree of activity of the 5-HT transporter (SERT) and a reduced amount of 5-HT released from colonic EC cells. The presence of an inhibitory 5-HT4 autoreceptor was observed in the colon, where a 40% increase in extracellular 5-HT was the half maximal inhibitory concentration for activation of the autoreceptor. This novel electroanalytical approach allows estimates of release and re-uptake and their contribution to 5-HT extracellular concentration from intestinal tissue be obtained from a single series of measurements.

Serotonin, a monoamine neurotransmitter, is important in both the central nervous system (CNS) and the peripheral nervous system. Malfunction of serotonin signaling leads to various disorders. We studied serotonin signaling from serotonergic neurons inside the ventral nerve cord of Drosophila melanogaster . Serotonergic neurons and stimulated release were visualized and achieved with mCherry and channelrhodopsin-2 (an optogenetically transfected ion channel), respectively, and two electrochemical techniques quantified serotonin release and vesicular content. Mean vesicular serotonin content released during exocytosis from these neurons was 84 %, considerably higher than reported in previous studies regarding octopamine (4.5 %) and glutamate release (31 %). Serotonin content within all vesicles is uniformly changed when serotonin concentration is inhibited or enhanced. However, serotonin release exhibits two Gaussian distributions: higher frequency of small release events, and similar or slightly higher frequency of large events, resulting in differential release fractions ranging from partial (13–18 %) to full (100 %) release after treatment with agents to either enhance or diminish release. This is the first example of consistent full exocytotic release events we have observed in any system. We suggest one pool of vesicles can release significantly diverse fractions of transmitter load during exocytosis, a potentially novel pathway to regulate exocytosis and neuronal signaling.

Composite electrodes are an effective and cheap way to utilise a wide range of carbon materials to make electrodes. More recently thermoplastics have been widely used as the binder to make carbon composite electrodes, as varying fabrication approaches, such as 3D printing, can make highly reproducible electrodes. However, there is a clear need to understand how the electrochemical performance of different carbon allotrope materials varies when made into sensors. We accessed polylactic acid (PLA) thermoplastic filaments containing carbon black, graphite, graphene, multiwall carbon nanotube (MWCNT) and carbon fiber using various electrochemical techniques. Graphite/PLA and graphene/PLA electrodes showed the best electron transfer kinetics. Graphene/PLA electrodes had the greatest sensitivity and lowest limit of detection for the measurement of serotonin. CB/PLA was least prone to electrode fouling from oxidative by-product generated from the oxidation of serotonin. 3D printing was used to make various carbon allotrope materials into complex shapes to evaluate the batch uniformity of the printed parts. Of all the materials explored, CB/PLA had the best resolution and batch uniformity when compared to PLA. Overall, our study highlights that the type of the carbon allotrope plays as much influence as amount of carbon on the electrochemical performance of carbon thermoplastic electrodes. These findings will provide significant guidance on the appropriate choice of carbon thermoplastic composite materials when designing electrodes for a wide range of applications.

Serotonin or 5-hydroxytryptamine (5-HT) is an important neurotransmitter in the central nervous system and the periphery. Most 5-HT (~99%) is found in the periphery where it regulates the function of the gastrointestinal (GI) tract and is an important regulator of platelet aggregation. However, the remaining 1% that is found in the central nervous system (CNS) can regulate a range of physiological processes such as learning and memory formation, mood, food intake, sleep, temperature and pain perception. More recent work on the CNS of invertebrate model systems has shown that 5-HT can directly regulate lifespan. This chapter will focus on detailing how CNS 5-HT signalling is altered with increasing age and the potential consequences this has on its ability to regulate lifespan.

The response to psychological stress can differ depending on the type and duration of the stressor. Acute stress can facilitate a "fight or flight response" and aid survival, whereas chronic long-term stress with the persistent release of stress hormones such as cortisol has been shown to be detrimental to health. We are now beginning to understand how this stress hormone response impacts important processes such as DNA repair and cell proliferation processes in breast cancer. However, it is not known what epigenetic changes stress hormones induce in breast cancer. Epigenetic mechanisms include modification of DNA and histones within chromatin that may be involved in governing the transcriptional processes in cancer cells in response to changes by endogenous stress hormones. The contribution of endogenous acute or long-term exposure of glucocorticoid stress hormones, and exogenous glucocorticoids to methylation patterns in breast cancer tissues with different aetiologies remains to be evaluated. In vitro and in vivo models were developed to investigate the epigenetic modifications and their contribution to breast cancer progression and aetiology. A panel of triple negative breast cancer cell lines were treated with the glucocorticoid, cortisol which resulted in epigenetic alteration characterised by loss of methylation on promoter regions of tumour suppressor genes including ESR1, and loss of methylation on LINE-1 repetitive element used as a surrogate marker for global methylation. This was verified in vivo in MDA-MB-231 xenografts; the model verified the loss of methylation on ESR1 promoter, and subsequent increase in ESR1 expression in primary tumours in mice subjected to restraint stress. Our study highlights that DNA methylation landscape in breast cancer can be altered in response to stress and glucocorticoid treatment.

Introduction Serotonin (5-HT) is a key signalling molecule within the mucosal epithelium of the intestinal wall and has been shown to be an important modulator of motility. At present, no single approach has been established for simultaneous dual measurement of 5-HT overflow and circular muscle contraction. Methods We developed a 3D-printed carbon black/polylactic acid (PLA) electrochemical sensor, which had a geometry suitable for ex vivo measurement in the guinea pig anorectum. Phasic changes in the current were used to track contractility, whilst basal changes were used to track changes in mucosal 5-HT signalling. Using amperometric detection, the sensitivity and stability of the device for 5-HT measurements was assessed. The device was compared with an isometric force transducer for tracking of anorectal contractions. Results The 3D-printed electrochemical sensor had a linear range in physiological concentrations of 5-HT (1−10 μM) present within the intestinal tract and a limit of detection of 540 nM. There was a significant correlation in the amplitude and duration of individual contractions when comparing the measurements using an isometric force transducer and 3D-printed electrochemical sensor (p<0.001, n=7). Finally, in the presence of 1 μM fluoxetine, the sensor was able to monitor a reduction in contractility (p<0.001, n=7) as well as an increase in 5-HT overflow (p<0.001, n=7). The sensor was stable for 5-HT measurement following ex vivo tissue measurements. Conclusions The 3D-printed sensor can simultaneously measure 5-HT overflow and contractility in the anorectum. This single device will have significant potential for clinical measurements of anorectum function and signalling that can direct therapeutic management of patients with lower bowel disorders.

IntroductionMelatonin is synthesized from 5-HT by the enzyme hydroxyindole-O-methyltransferase and the EC cells maybe a site of synthesis and release of mucosal melatonin. Although the presence of melatonin in the gastrointestinal tract is not disputed its role in regulating gastrointestinal motility and its mechanism of action are still debated.MethodsWe used electrochemical and chromatographic methods to detect the regulation of mucosal melatonin release from intact segments of 3 month old C57BL/6 murine colon. Colonic migratory motor complexes (CMMC) were recorded in the presence of melatonin, MT2 receptor antagonist 4-P-PDOT and MT1/2 receptor antagonist luzindole. Functional bioassays were carried out to study how varying concentrations of melatonin influenced electrical field stimulated (EFS) colonic contractions. The influence of melatonin on feacal pellet was explored.ResultsMelatonin release was shown to 2-fold greater than serotonin, when released from the colon (n=6). Melatonin release occurred on demand during mechanical stimulation but was not released by a chemical stimulus, the bile salt deoxycholic acid. EFS of isolated colon segments caused contraction at lower frequencies but relaxation at higher frequencies. In the proximal colon, 5 µM melatonin facilitated contraction at all EFS frequencies (p<0.05, n=6), however this was not altered in the distal colon. In the presence of tetrodotoxin (TTX), melatonin did not alter KCl stimulated muscle contraction. Melatonin caused a reduction in CMMC amplitude in the proximal colon (p<0.01, n=5) but did not influence the distal colon. Melatonin did not influence the velocity of CMMCs (n=5). Melatonin significantly decreased colonic transit times of an artifical faecal pellet (p<0.001, n=5), however luzindole significantly increased colonic transit times (p<0.01, n=5).ConclusionsOur findings highlight that melatonin is present and released from the colonic mucosa and has an important functional role in influencing muscle contraction. Therefore, melatonin signalling pathways may serve to be important targets to direct therapeutic development.

Social isolation (SI) is a major health risk in older people leading to cognitive decline. This study examined how SI and age influence performance in the novel object recognition (NOR) and elevated plus maze (EPM) tasks in C57BL/6 mice aged 3 or 24 months. Mice were group-housed (groups of 2-3) or isolated for 2 weeks prior to experimentation. Following NOR and EPM testing hippocampal norepinephrine (NE), 5, hydroxytryptamine (5-HT), 5, hydroxyindole acetic acid (5-HIAA), corticosterone (CORT) and interleukin-6 (IL-6) were determined and serum collected for basal CORT analysis. A separate set of mice were exposed to the forced swim test (FST), sacrificed immediately and serum CORT determined. SI impaired performance in the NOR and the FST, reduced hippocampal 5-HT, increased hippocampal IL-6 and increased serum CORT post-FST in young mice. Aged mice either failed to respond significantly to SI (NOR, FST, hippocampal 5-HT, serum CORT post FST) or SI had synergistic effects with age (hippocampal NE, 5-HIAA:5-HT). In conclusion, the lack of response to SI in the aged mice may affect health by preventing them adapting to new stressors, while the synergistic effects of SI with age would increase allostatic load and enhance the deleterious effects of the ageing process.

Ageing is associated in many organisms with a reduction in motor movements. We have previously shown that the rate of feeding movements of the pond snail, Lymnaea, decreased with age but the underlying cause is not fully understood. Here, we show that dopamine in the cerebro‐buccal complex is an important signalling molecule regulating feeding frequency in Lymnaea and that ageing is associated with a decrease in CNS dopamine. A proteomic screen of young and old CNSs highlighted a group of proteins that regulate stress responses. One of the proteins identified was 14‐3‐3, which can enhance the synthesis of dopamine. We show that the Lymnaea 14‐3‐3 family exists as three distinct isoforms. The expression of the 29 kDa isoform (14‐3‐3Lym3) in the cerebro‐buccal complex decreased with age and correlated with feeding rate. Using a 14‐3‐3 antagonist (R18) we were able to reduce the synthesis of L‐DOPA and dopamine in ex vivo cerebro‐buccal complexes. Together these data suggest that an age‐related reduction in 14‐3‐3 can decrease CNS dopamine leading to a consequential reduction in feeding rate.

Background Epigenetic changes are highly responsive to environmental changes, including stress. The release of stress hormones; such as glucocorticoids, in response to stress have been shown to induce epigenetic modifications in neuronal cells. However, the role of glucocorticoids on epigenetic changes underlying important processes such as cell cycle regulation, apoptosis, and proliferation in breast cancer are not yet established. Furthermore, it is not known if cortisol can induce irreversible epigenetic changes on these cellular processes and whether these changes relate to the duration of the stress response. In this study, cortisol-induced epigenetic changes and the potential involvement of DNA methylation was assessed. Methods We analyzed the expression levels of maintenance DNA methyltrasferase (DNMT1) in MDA-MB-231, Hs-578T, MCF7, and T47D breast cancer cell lines by real-time PCR. We also used Qiagen Epitect Methyl ll Complete PCR array for Tumour Suppressor genes to analyse the level of methylation in 94 tumour suppressor genes. The methylation level on the Long Interspersed Nuclear Element (LINE-1) was used as surrogate marker for global DNA methylation. Results Our results show that cortisol significantly decreased the expression of DNMT1 in the triple negative cells lines MDA-MB-231 (p Conclusions Taken together, cortisol induced aberrant methylation patterns which may have important implications on progression of the disease. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Propofol is the most widely used intravenous anaesthetic agent for maintenance of anaesthesia and sedation. Studies in varying regions of the bowel have shown conflicting differences on the effects of propofol on motility. There the aim of this study was to understand the influence of propofol on colonic function and explore by which mechanism any changes occur. Functional studies were conducted using isolated colonic tissue from C57BL6 mice which were exposed to 5 μM propofol. Faecal pellet motility, colonic migratory motor complexes (CMMCs) and functional bioassays were utilised to monitor colonic function and nitric oxide production was monitored by amperometry. There was a signficant reduction in amplitude of CMMCs in the distal colon in the presence of 5 μM propofol, however no difference was observed in the proximal colon. A signficant increase in the 5-HT evoked contractions were observed in distal colon in the presence of 5 μM propofol. Additionally, a reduction in the NO production in the presence of 5 μM propofol was only observed in the distal colon. As a result, in the presence of 5 μM propofol, faecal pellet transit was increased, and velocity was reduced. At clinically relevant doses, propofol was shown to reduce colonic motility by inhibiting nitric oxide synthase in only the distal region of the colon. Our findings indicate that propofol has a considerable influence on colonic signalling mechanisms and impairs colonic motility, which may have implications in its clinical use especially for maintenance.

Serotonin (5-HT) is a key signalling molecule within the mucosal epithelium of the intestinal wall and has been shown to be an important modulator of motility. At present no single approach has been established for simultaneous dual measurement of 5-HT overflow and circular muscle contraction. We have developed a 3D printed carbon black / polylactic acid (PLA) electrochemical sensor, which had a geometry suitable for ex vivo measurement in the anorectum. The device was characterised for sensitivity and stability for 5-HT measurements as well as suitability for accurate tracking of anorectal contractions. The 3D printed electrochemical sensor had a linear range in physiological concentrations of 5-HT (1 - 10 µM) present within the intestinal tract and a limit of detection of 540 nM. The sensor was stable for 5-HT measurement following ex vivo tissue measurements. There was a signficant correlation in the amplitude and duration of individual contractions when comparing the measurements using an isometric force transducer and 3D printed electrochemical sensor. Finally, in the presence of 1 µM fluoxetine, the sensor was able to monitor a reduction in contractility as well as an increase in 5-HT overflow as predicted. Overall the 3D printed sensor has the ability to conduct dual simultaneous measurements of 5-HT overflow and contractility. This single device will have significant potential for clinical measurements of anorectum function and signalling, that can direct therapeutic management of patients with bowel disorders.

Serotonin and kynurenine are formed following metabolism of the essential amino acid tryptophan. Both molecules play important biological roles and the balance of how tryptophan metabolism varies to either the serotonin or kynurenine pathway may provide key insight into the inflammatory status of the biological region. At present complex chromatographic methods are utilised which predominately focus on either monitoring analytes in the serotonin or kynurenine pathway rather than both. Our study develops a simple yet robust methodology for the monitoring of tryptophan metabolism. We utilised isocratic reverse phase high-performance liquid chromatography with simultaneously dual electrochemical detection. This approach allowed for separation of co-eluted analytes and identification of analytes from both pathways within 14 minutes. For all analytes, limits of detection were <35 nM. No crosstalk was observed when dual simultaneous detection was conducted in a radial flow cell. Responses from the hippocampus, blood and ileum mucosa highlighted that each region had a varying ratio of serotonin to kynurenine pathway, indicating varied approaches to tryptophan metabolism. The developed method can monitor how the metabolism of tryptophan varies between the two pathways which can provide insight into the inflammatory state of reach region with age and disease.