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Alterations of the gut microbiota have been associated with stress-related disorders including depression and anxiety and irritable bowel syndrome (IBS). More recently, researchers have started investigating the implication of perturbation of the microbiota composition in neurodevelopmental disorders including autism spectrum disorders and Attention-Deficit Hypersensitivity Disorder (ADHD). In this review we will discuss how the microbiota is established and its functions in maintaining health. We also summarize both pre and post-natal factors that shape the developing neonatal microbiota and how they may impact on health outcomes with relevance to disorders of the central nervous system. Finally, we discuss potential therapeutic approaches based on the manipulation of the gut bacterial composition.

The perception of visceral pain is a complex process involving the spinal cord and higher order brain structures. Increasing evidence implicates the gut microbiota as a key regulator of brain and behavior, yet it remains to be determined if gut bacteria play a role in visceral sensitivity. We used germ-free mice (GF) to assess visceral sensitivity, spinal cord gene expression and pain-related brain structures. GF mice displayed visceral hypersensitivity accompanied by increases in Toll-like receptor and cytokine gene expression in the spinal cord, which were normalized by postnatal colonization with microbiota from conventionally colonized (CC). In GF mice, the volumes of the anterior cingulate cortex (ACC) and periaqueductal grey, areas involved in pain processing, were decreased and enlarged, respectively, and dendritic changes in the ACC were evident. These findings indicate that the gut microbiota is required for the normal visceral pain sensation. DOI: http://dx.doi.org/10.7554/eLife.25887.001, eLife digest The human gut is home to over 100 trillion microbes collectively known as the gut microbiota. These microbes help us to digest food and absorb the nutrients effectively. A diverse and stable community of gut microbes is believed to be important for good health. Recently, it has also become clear that the microbiota can also influence the brain and how we behave. For example, many studies suggest that gut microbiota can alter how an individual perceives pain, but it is not clear how this works. Rodents are often used in experiments as models of human biology. One of the most frequently used rodent models in studies of gut microbes is the “germ-free” mouse. These mice grow up in laboratory environments that are completely free of microbes, making it possible to study how having no gut microbes affects the health and behaviour of the mice. Luczynski, Tramullas et al. used germ-free mice to study how the gut microbiota influences an animal’s sensitivity to pain. The experiments show that, compared to mice with normal gut microbiota, the germ-free mice were more sensitive to pain from internal organs especially the gut. These mice also produced larger amounts of specific proteins involved in immune responses, which contributed to the animal’s increased sensitivity to pain. Allowing the germ-free mice to be colonised with gut microbes could reverse these changes. The experiments also show that the germ-free mice had changes in the size of two areas of the brain involved in sensing pain: an area called the anterior cingulate cortex was smaller, while the periaqueductal grey region was enlarged. There were also differences in individual nerve cells within the anterior cingulate cortex compared to normal mice. The findings of Luczynski, Tramullas et al. reinforce the idea that the gut microbiota is involved in the sensation of pain from internal organs, and show that hypersensitivity to this form of pain can be reversed later in life by colonising the gut with microbes. Continuing to study the impact of microbes on this type of pain could aid the development of new therapies for the treatment of pain disorders in humans. DOI: http://dx.doi.org/10.7554/eLife.25887.002

Objectives Atrial fibrillation (AF) is associated with increased morbidity and mortality in patients with hypertrophic cardiomyopathy (HCM). The primary aim of this study (HCM Risk-AF) was to determine the predictors of AF in a large multicentre cohort of patients with HCM. Exploratory analyses were performed to investigate the association between AF and survival and the efficacy of antiarrhythmic therapy in maintaining sinus rhythm (SR). Methods A retrospective, longitudinal cohort of patients recruited between 1986 and 2008 in seven centres was used to develop multivariable Cox regression models fitted with preselected predictors. HCM was defined as unexplained hypertrophy (maximum left ventricular wall thickness of >15 mm or in accordance with published criteria for the diagnosis of familial disease). 28% of patients (n=1171) had coexistent hypertension. The primary end point was paroxysmal, permanent or persistent AF detected on ECG, Holter monitoring or implantable device interrogation. Results Of the 4248 patients with HCM without preexisting AF, 740 (17.4%) reached the primary end point. Multivariable Cox regression revealed an association between AF and female sex, age, left atrial diameter, New York Heart Association (NYHA) class, hypertension and vascular disease. The proportion of patients with cardiovascular death at 10 years was 4.9% in the SR group and 10.9% in the AF group (difference in proportions=5.9%; 95% CI (4.1% to 7.8%)). The proportion of patients with non-cardiovascular death at 10 years was 3.2% in the SR group and 5.9% in the AF group (difference in proportions=2.8%; 95% CI (0.1% to 4.2%)). An intention-to-treat propensity score analysis demonstrated that β-blockers, calcium channel antagonists and disopyramide initially maintained SR during follow-up, but their protective effect diminished with time. Amiodarone therapy did not prevent AF during follow-up. Conclusion This study shows that patients with HCM who are at risk of AF development can be identified using readily available clinical parameters. The development of AF is associated with a poor prognosis but there was no evidence that antiarrhythmic therapy prevents AF in the long term.

Irritable bowel syndrome is a functional gastrointestinal disorder, with stress playing a major role in onset and exacerbation of symptoms such as abdominal pain and altered bowel movements. Stress-related disorders including anxiety and depression often precede the development of irritable bowel syndrome and vice versa. Stressor exposure during early life has the potential to increase an individual’s susceptibility to both irritable bowel syndrome and psychiatric disease indicating that there may be a common origin for these disorders. Moreover, adverse early life events significantly impact upon many of the communication pathways within the brain-gut-microbiota axis, which allows bidirectional interaction between the central nervous system and the gastrointestinal tract. This axis is proposed to be perturbed in irritable bowel syndrome and studies now indicate that dysfunction of this axis is also seen in psychiatric disease. Here we review the co-morbidity of irritable bowel syndrome and psychiatric disease with their common origin in mind in relation to the impact of early life stress on the developing brain-gut-microbiota axis. We also discuss the therapeutic potential of targeting this axis in these diseases.

UNLABELLED The infant gut microbiome is dynamic, and radical shifts in composition occur during the first 3 years of life. Disruption of these developmental patterns, and the impact of the microbial composition of our gut on brain and behaviour, has attracted much recent attention. Integrating these observations is an important new research frontier. CONCLUSION Early-life perturbations of the developing gut microbiota can impact on the central nervous system and potentially lead to adverse mental health outcomes.

Visceral hypersensitivity is a hallmark of many clinical conditions and remains an ongoing medical challenge. Although the central neural mechanisms that regulate visceral hypersensitivity are incompletely understood, it has been suggested that stress and anxiety often act as initiating or exacerbating factors. Dysfunctional corticolimbic structures have been implicated in disorders of visceral hypersensitivity such as irritable bowel syndrome (IBS). Moreover, the pattern of altered physiological responses to psychological and visceral stressors reported in IBS patients is also observed in the maternally separated (MS) rat model of IBS. However, the relative contribution of various divisions within the cortex to the altered stress responsivity of MS rats remains unknown. The aim of this study was to analyze the cellular activation pattern of the prefrontal cortex and amygdala in response to an acute psychological stressor (open field) and colorectal distension (CRD) using c-fos immunohistochemistry. Several corticoamygdalar structures were analyzed for the presence of c-fos-positive immunoreactivity including the prelimbic cortex, infralimbic cortex, the anterior cingulate cortex (both rostral and caudal) and the amygdala. Our data demonstrate distinct activation patterns within these corticoamygdalar regions including differential activation in basolateral versus central amygdala following exposure to CRD but not the open field stress. The identification of this neuronal activation pattern may provide further insight into the neurochemical pathways through which therapeutic strategies for IBS could be derived.

Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed.

A growing body of preclinical and clinical evidence supports a relationship between the complexity and diversity of the microorganisms that inhabit our gut (human gastrointestinal microbiota) and health status. Under normal homeostatic conditions this microbial population helps maintain intestinal peristalsis, mucosal integrity, pH balance, immune priming and protection against invading pathogens. Furthermore, these microbes can influence centrally regulated emotional behaviour through mechanisms including microbially derived bioactive molecules (amino acid metabolites, short-chain fatty acids, neuropeptides and neurotransmitters), mucosal immune and enteroendocrine cell activation, as well as vagal nerve stimulation.The microbiota-gut-brain axis comprises a dynamic matrix of tissues and organs including the brain, autonomic nervous system, glands, gut, immune cells and gastrointestinal microbiota that communicate in a complex multidirectional manner to maintain homeostasis and resist perturbation to the system. Changes to the microbial environment, as a consequence of illness, stress or injury, can lead to a broad spectrum of physiological and behavioural effects locally including a decrease in gut barrier integrity, altered gut motility, inflammatory mediator release as well as nociceptive and distension receptor sensitisation. Centrally mediated events including hypothalamic-pituitary-adrenal (HPA) axis, neuroinflammatory events and neurotransmitter systems are concomitantly altered. Thus, both central and peripheral pathways associated with pain manifestation and perception are altered as a consequence of the microbiota-gut-brain axis imbalance.In this chapter the involvement of the gastrointestinal microbiota in visceral pain is reviewed. We focus on the anatomical and physiological nodes whereby microbiota may be mediating pain response, and address the potential for manipulating gastrointestinal microbiota as a therapeutic target for visceral pain.

A growing body of preclinical and clinical evidence supports a relationship between the complexity and diversity of the microorganisms that inhabit our gut (human gastrointestinal microbiota) and health status. Under normal homeostatic conditions this microbial population helps maintain intestinal peristalsis, mucosal integrity, pH balance, immune priming and protection against invading pathogens. Furthermore, these microbes can influence centrally regulated emotional behaviour through mechanisms including microbially derived bioactive molecules (amino acid metabolites, short-chain fatty acids, neuropeptides and neurotransmitters), mucosal immune and enteroendocrine cell activation, as well as vagal nerve stimulation.

New Findings What is the central question of this study? Does stress sensitivity and susceptibility to inflammation innate to certain rat strains make them vulnerable to bowel dysfunction? What is the main finding and its importance? Of four different rat strains, the Lewis rat, which displays both susceptibility to gastrointestinal inflammation and sensitivity to stress, exhibits the most aberrant gastrointestinal morphology and visceral pain sensitivity. Given the similarities to human functional bowel disorders, such as irritable bowel syndrome, this may make it a good model of this disease. Irritable bowel syndrome is a common, debilitating gastrointestinal (GI) disorder characterized by episodic exacerbations of symptoms such as abdominal pain, bloating and altered bowel habit. Contributory factors for the development of irritable bowel syndrome include genetics, childhood trauma and prior GI infection leading to chronic low-grade inflammation or immune activation. Additional considerations in comprehending the chronic relapsing pattern that typifies irritable bowel syndrome symptoms are the effects of both psychosocial and infection-related stresses. Background stress and immune profiles can influence gut permeability and visceral pain sensitivity. This study examined whether innate susceptibility to inflammation and stress sensitivity in four rat strains is associated with bowel dysfunction. The pain threshold to colorectal distension was assessed in Lewis, Fischer (F344) and spontaneously hypertensive rats and compared with Sprague–Dawley control animals. Colons were subsequently excised and morphologically assessed for total length, goblet cell hyperplasia and muscle and mucosal thickness. Lewis rats displayed visceral hypersensitivity compared with other strains. At a morphological level, the gastrointestinal tract from these rats displayed mucosal goblet cell hyperplasia and alterations in muscle layer thickness. The Lewis rat strain, which is reported to have increased susceptibility to GI inflammation in addition to stress sensitivity, had the most prominent features of physiological and morphological GI dysfunction. These data support the hypothesis that background strain is a key factor in the development and exacerbation of bowel dysfunction in rodent models.

Current demands for detection of Gunshot Residue (GSR) require a reliable and rapid decentralized detection system with high sensitivity and specificity. This article reviews the use of electrochemical devices for GSR detection over the last 35 years and highlights recent advances associated with the demands of GSR field detection such as portability, speed, cost and power. Anodic stripping voltammetry (ASV) has been widely implemented for the detection of the metallic components of GSR at a variety of working electrodes. Efforts toward the detection of the organic components of GSR have also been reported using cyclic- and square-wave voltammetry. The simultaneous detection of both organic and inorganic GSR constituents has recently been examined to increase the overall information content in a single voltammetric scan. As well as this, exploitation of screen-printing fabrication allows replacement of conventional electrochemical cells with easy-to-use sensor strips Sampling methods for electrochemical GSR analysis are also advancing from acid washes or swabs to simpler abrasive methods which integrate sampling and analysis obviating the need for intermediate processing steps. The latest direction of electrochemical detection of GSR involves chemometric treatment to evaluate data allowing for more objective conclusions and increasing the automation of the system. These advances indicate great promise for investigating firearm-related crimes, and bring significant changes to the detection of GSR making electroanalysis a powerful tool for decentralized forensic analysis.

Targeting the gastrointestinal (GI) tract represents a promising strategy for local or systemic delivery of nucleic acid-based therapeutics. The development of a nano-carrier for siRNA delivery via the GI tract would enable localised therapy for a range of gastrointestinal diseases. Previously, nano-sized polymeric micelles (PM) formed by a variety of amphiphilic poly(allylamine) (PAA) derivatives have shown potential for oral delivery of hydrophobic drugs and bioactive peptides. The aim of this study was to evaluate the ability of these amphiphilic PAA-based PM for siRNA delivery via the GI tract. The physicochemical characteristics of PAA·siRNA transfection complexes and their biological efficacy in vitro were investigated. Physicochemical profiles demonstrated that PAAs and siRNA self-assembled into complexes with nano-sized diameters (150-300 nm) and cationic surface charge (+ 20 to 30 mV). The PAA·siRNA complexes were stable in the presence of salt solutions and simulated gastric/intestinal fluids. In undifferentiated Caco-2 cells, PAA·siRNA complexes achieved up to 35% cellular uptake, with successful siRNA release from endosomes/lysosomes and significant luciferase gene knockdown. These results highlight the potential of these nano-sized PM for siRNA oral delivery via the GI tract for treatment of gastrointestinal diseases.

To date, limited reports are available on the regulatory systems exerting control over bacterial synthesis of the biodegradable polyester group known as polyhydroxyalkanoates (PHAs). In this study, we performed random mini-Tn 5 mutagenesis of the Pseudomonas putida CA-3 genome and screened transconjugants on nitrogen-limited medium for reduced PHA accumulation phenotypes. Disruption of a GacS sensor kinase in one such mutant was found to eliminate medium-chain-length PHA production in Pseudomonas putida CA-3. Recombinant expression of wild-type gacS from a pBBRgacS vector fully restored PHA accumulation capacity in the mutant strain. PCR-based screening of the P. putida CA-3 genome identified gene homologues of the GacS/GacA- rsm small RNA (sRNA) regulatory cascade with 96% similarity to published P. putida genomes. However, reverse transcription-PCR (RT-PCR) analyses revealed active transcription of the rsmY and rsmZ sRNAs in gacS -disrupted P. putida CA-3, which is atypical of the commonly reported Gac/Rsm regulatory cascade. Quantitative real-time RT-PCR analyses of the phaC1 synthase responsible for polymer formation in P. putida CA-3 indicated no statistically significant difference in transcript levels between the wild-type and gacS -disrupted strains. Subsequently, SDS-PAGE protein analyses of these strains identified posttranscriptional control of phaC1 synthase as a key aspect in the regulation of PHA synthesis by P. putida CA-3.

Significant research is focused on the development of non-viral vectors for delivery of siRNA to neurons and the central nervous system. Cyclodextrins (CDs) have shown great promise as efficient and low toxicity gene delivery vectors in various cell types. Here, we investigate two CDs for siRNA delivery in a neuronal cell model. These CDs were substituted on opposite faces (primary and secondary) with amphiphilic and cationic groups. Physical properties of CD.siRNA complexes, including size, charge and stability were measured. In vitro investigations were carried out in immortalised hypothalamic neurons. Neuronal cell uptake was measured by flow cytometry and cytotoxicity was assessed by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Both CDs interacted with siRNA, yielding nanosized cationic complexes which exhibited good stability on storage. A favourable toxicity profile was demonstrated for the CD.siRNA complexes. However, only one of the two CDs mediated high levels of neuronal uptake and efficient gene silencing, equivalent to those achieved with a commercial lipid-based vector. Despite the suitability of both CDs as siRNA delivery vectors in terms of their ability to complex siRNA and the properties of the complexes yielded, only one CD achieved good transfection efficiency. This was likely due to the differences in their chemical structures. The effective CD offers great potential as a novel non-toxic vector for neuronal siRNA delivery.

Purpose: Development of RNA interference based therapeutics for neurological and neurodegenerative diseases is hindered by a lack of non-viral vectors with suitable properties for systemic administration. Amphiphilic and cationic cyclodextrins (CD) offer potential for neuronal siRNA delivery. Here, we aimed to improve our CD-based siRNA formulation through incorporation of a polyethyleneglycol (PEG) shielding layer and a cell penetrating peptide, octaarginine (R8). Methods: CD.siRNA complexes were modified by addition of an R8-PEG-lipid conjugate. Physical properties including size, charge and stability were assessed. Flow cytometry was used to determine uptake levels in a neuronal cell model. Knockdown of an exogenous gene and an endogenous housekeeping gene were used to assess gene silencing abilities. Results: CD.siRNA complexes modified with R8-PEG-lipid exhibited a lower surface charge and greater stability to a salt-containing environment. Neuronal uptake was increased and significant reductions in the levels of two target genes were achieved with the new formulation. However, the PEG layer was not sufficient to protect against serum-induced aggregation. Conclusions: The R8-PEG-lipid-CD.siRNA formulation displayed enhanced salt-stability due to the PEG component, while the R8 component facilitated transfection of neuronal cells and efficient gene silencing. Further improvements will be investigated in the future in order to optimise stability in serum and enhance neuronal specificity.

Background Early-life stress and a genetic predisposition to display an anxiety- and depressive-like phenotype are associated with behavioral and gastrointestinal (GI) dysfunction. Animals exposed to early-life stress, and those genetically predisposed to display anxiety or depressive behaviors, have proven useful tools in which to study stress-related GI disorders, such as irritable bowel syndrome (IBS). IBS is a heterogeneous disorder, and likely a consequence of both genetic and environmental factors. However, the combined effects of early-life stress and a genetic predisposition to display anxiety- and depression-like behaviors on GI function have not been investigated. Methods We assessed the effect of maternal separation (MS) on behavioral and GI responses in WKY animals relative to a normo-anxious reference strain. Key Results Both non-separated (NS) WKY and WKY-MS animals displayed anxiety-like responses in the open-field test and depressive-like behaviors in the forced swim test relative to Sprague–Dawley rats. However, MS had no further influence on anxiety- and depressive-like behaviors exhibited by this stress-prone rat strain. Similarly, corticosterone levels measured after the OFT were insensitive to MS in WKY animals. However, WKY-MS displayed significantly increased colonic visceral hypersensitivity, fecal output, and altered colonic cholinergic sensitivity. Conclusions & Inferences Our data suggest that early-life stress, on the background of a genetic predisposition to display an anxiety- and depressive-like phenotype, selectively influences GI function rather than stress-related behaviors. Thus, our findings highlight the importance of genetic predisposition on the outcome of early-life adversity on GI function.

The aim of this study was to determine whether Thiel-embalmed cadavers would provide a useful anatomy teaching tool for topics that cannot be approached using formalin-fixed cadavers such as oral cavity examination and maxillary anesthesia. The suitability of Thiel-embalmed bodies for performing oral examinations was assessed by asking first-year dental and dental hygiene students at a dental school in Ireland to identify oral structures on a classmate and on a Thiel-embalmed body. The study was conducted in 2016. The ease of location was compared in the two settings, and their quality was assessed on the cadavers. The suitability of Thiel-embalmed cadavers to teach maxillary anesthesia was assessed by students' performing mock injections at five adjacent sites daily for five consecutive days, followed by inspection of the gingival surface by experienced anatomists and dentists. Data were obtained from 57 students, but only the 54 forms that were fully completed were analyzed, for an overall response rate of 85.7%. The results showed that most oral structures were more difficult to locate on cadavers. The texture and appearance of features in the cadavers were rated at a midpoint between realistic and unrealistic. The relative inexperience of the participants, the accumulation of fixative in the oral cavity, and discoloration were mentioned as potential confounding factors. Visual analysis of images obtained following repeated injections revealed no deterioration of the tissue. Importantly, the puncture marks appeared to reduce over time, suggesting that the gingival tissue maintains some elasticity following Thiel fixation. These findings suggest that Thiel-embalmed cadavers may be a useful tool to provide students more time to localize and study aspects of the oral cavity. Likewise, the recoiling capacity of gingival tissue suggests that Thiel-embalmed cadavers may provide an ideal tool for teaching injection technique of local anesthetics.

The recent literature is surveyed to explore the nature of voltammetry in room temperature ionic liquids. The extent of similarities with conventional electrochemical solvents is reported and some surprising differences are noted.

Room temperature ionic liquids (RTILs) have been applied to a microelectrode array and been demonstrated to form effective, membrane-free amperometric gas sensors. Determining the RTIL [P(6,6,6,14)][FAP] as the most appropriate choice for extended use, the amperometric quantification of oxygen has been demonstrated. The response of the sensor was quantified by both cyclic voltammetry and chronoamperometry. A range of O(2) contents (2-13% v/v) and RTIL layer thicknesses (from ca. 6 to 125 mum) have been investigated. The combination of microelectrode array and RTIL, as well as the absence of membrane and volatile solvent, results in an elegant, easy to calibrate gas sensor with potential utility in standard and nonstandard conditions.

Physicochemical changes and substantially modified electrochemical behavior have been reported when ionic liquids are degassed with nitrogen. In conventional experiments in aqueous and organic media, degassing with N(2) is commonly used to remove the electroactive dissolved oxygen. However, in hydrophilic ionic liquid media, degassing with N(2) removes not only the dissolved oxygen but also a significant amount of the adventitious water present. Given the low viscosity of water, this in turn leads to a dramatic change of the viscosity of the degassed ionic liquid and hence mass transport properties that influence voltammetric responses. In the widely used and relatively viscous room temperature ionic liquid, 1-n-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF(4)) containing the redox probe tetracyanoquinodimethane (TCNQ) and 9% (v/v) deliberately added water, 1 h degassing with very dry N(2) under benchtop conditions results in a dramatic decrease of the TCNQ reduction current obtained under steady-state conditions at a 1 mum diameter microdisc electrode. This is reflected by a change of diffusion coefficient of TCNQ (D(TCNQ)) from 2.6 x 10(-7) to 4.6 x 10(-8) cm(2) s(-1). Karl Fischer titration measurements show that almost complete removal of the deliberately added 9% water is achieved by degassing under benchtop conditions. However, displacement of oxygen by nitrogen in the ionic liquid solution results in the decrease of electrochemical reduction current by 6%, implying that dissolved gases need not be inert with respect to solvent properties. Oxygen removal by placing the BMIMBF(4) ionic liquid in a nitrogen-filled glovebox or in a vacuum cell also simultaneously leads to removal of water and alteration of voltammetric data. This study highlights that (i) important physicochemical differences may arise upon addition or removal of a solute from viscous ionic liquids; (ii) degassing with dry nitrogen removes water as well as oxygen from ionic liquids, which may have implications on the viscosity and structure of the medium; (iii) particular caution must be exercised when deoxygenation is applied in ionic liquid media as part of the protocol used in electrochemical experiments to remove oxygen; (iv) gases such as oxygen, argon, and nitrogen dissolved in ionic liquids need not be innocent with respect to the properties of an ionic liquid. The use of vacuum based techniques to eliminate all volatile solutes, including water and oxygen, is advocated.

Disruption of bacterial colonization during the early postnatal period is increasingly being linked to adverse health outcomes. Indeed, there is a growing appreciation that the gut microbiota plays a role in neurodevelopment. However, there is a paucity of information on the consequences of early-life manipulations of the gut microbiota on behavior. To this end we administered an antibiotic (vancomycin) from postnatal days 4–13 to male rat pups and assessed behavioral and physiological measures across all aspects of the brain–gut axis. In addition, we sought to confirm and expand the effects of early-life antibiotic treatment using a different antibiotic strategy (a cocktail of pimaricin, bacitracin, neomycin; orally) during the same time period in both female and male rat pups. Vancomycin significantly altered the microbiota, which was restored to control levels by 8 weeks of age. Notably, vancomycin-treated animals displayed visceral hypersensitivity in adulthood without any significant effect on anxiety responses as assessed in the elevated plus maze or open field tests. Moreover, cognitive performance in the Morris water maze was not affected by early-life dysbiosis. Immune and stress-related physiological responses were equally unaffected. The early-life antibiotic-induced visceral hypersensitivity was also observed in male rats given the antibiotic cocktail. Both treatments did not alter visceral pain perception in female rats. Changes in visceral pain perception in males were paralleled by distinct decreases in the transient receptor potential cation channel subfamily V member 1, the α-2A adrenergic receptor and cholecystokinin B receptor. In conclusion, a temporary disruption of the gut microbiota in early-life results in very specific and long-lasting changes in visceral sensitivity in male rats, a hallmark of stress-related functional disorders of the brain–gut axis such as irritable bowel disorder.

Background Breastfeeding rates in Ireland are among the lowest in Europe. Breastfeeding groups can provide support, information, and friendship for women. However, there is little research exploring community breastfeeding groups led by Public Health Nurses providing universal maternal and child care to all postnatal mothers in the community in Ireland. Aim The aim of this study was to explore breastfeeding women’s experiences of a Public Health Nurse led support group. Methods A qualitative descriptive design to explore women’s experiences of a community breastfeeding support group was conducted. Data were collected using one to one interviews with breastfeeding women (n = 7) in a primary healthcare setting. Transcripts were analysed using Burnard’s thematic content analysis. Results The overall theme identified was ‘normalising breastfeeding’ which emerged from the subthemes ‘socialising and sharing’, ‘information and support seeking’, ‘building confidence’, ‘overcoming embarrassment’, ‘negative perceptions of others’, and ‘promoting breastfeeding to others’. Discussion Women who attended the PHN led breastfeeding support group found it to be a cocoon of ‘normality’, whereas breastfeeding was considered almost something to be ashamed of in other circumstances. Many women attributed their success with breastfeeding to the support group. Conclusion Facilitating a sense of normalcy for breastfeeding women at individual, community and societal levels was essential in promoting breastfeeding. The community support group was influential in normalising breastfeeding for a sample of women, by minimising the potential for embarrassment, promoting social interaction and sharing, building confidence and knowledge. This buffered the effects of negative attitudes of others and personal feelings of shame.

Females are more likely to experience visceral pain than males, yet mechanisms underlying this sex bias are not fully elucidated. Moreover, pain sensitivity can change throughout the menstrual cycle. Alterations in the glutamatergic system have been implicated in several pain-disorders; however, whether these are sex-dependent is unclear. Thus, we aimed to investigate sex differences in the spinal cord glutamate uptake and how it varies across the estrous cycle. The activity of the glutamate transporters, excitatory amino acid transporters (EAATs) was assessed using an ex vivo aspartate radioactive uptake assay in the lumbosacral spinal cord in Sprague-Dawley male and female rats. The gene expression of EAATs, glutamate receptor subunits NR1 and NR2B and the estrogen receptors ERα & ERβ in the spinal cord were also analyzed. EAAT activity was lower in females, particularly during the estrus phase, and this was the only cycle stage that was responsive to the pharmacological effects of the EAATs activator riluzole. Interestingly, EAAT1 mRNA expression was lower in high-estrogen and high-ERα states compared to diestrus in females. We conclude that the Spinal EAAT activity in females is different to that in males, and varies across the estrous cycle. Furthermore, the expression levels of estrogen receptors also showed a cycle-dependent pattern that may affect EAATs function and expression.

Purpose– The purpose of this paper is to find out how intermediaries interpret their role working with vulnerable defendants at court.Design/methodology/approach– In this study six intermediaries who have worked with defendants were interviewed using a semi-structured interview and the interview transcripts were analysed using interpretative phenomenological analysis.Findings– Intermediaries appeared to be trying to make sense of their developing identities as professionals in the courtroom and this theme is conceptualised through social identity complexity theory.Practical implications– Health and care professionals undertaking a new function in the criminal justice sector should receive training about the psychological processes underlying developing professional identities. Such training should reduce the cognitive load when they work in the new environment and failure to undertake this training may lead to less efficient practice. Gaining an understanding of their professional positioning within the court environment may assist with retention of intermediaries in this new role.Originality/value– This is the first published study where intermediaries have been interviewed about their experiences with defendants. Recommendations are made including the requirement for additional training for intermediaries to understand the underlying psychological processes and conflicts they may experience when working with defendant cases.

RNA interference (RNAi) holds great promise as a strategy to further our understanding of gene function in the central nervous system (CNS) and as a therapeutic approach for neurological and neurodegenerative diseases. However, the potential for its use is hampered by the lack of siRNA delivery vectors, which are both safe and highly efficient. Cyclodextrins have been shown to be efficient and low toxicity gene delivery vectors in various cell types in vitro. However, to date they have not been exploited for delivery of oligonucleotides to neurons. To this end, a modified β-cyclodextrin (CD) vector was synthesised, which complexed siRNA to form cationic nanoparticles of less than 200nm in size. Furthermore, it conferred stability in serum to the siRNA cargo. The in vitro performance of the CD in both immortalised hypothalamic neurons and primary hippocampal neurons was evaluated. The CD facilitated high levels of intracellular delivery of labelled siRNA, whilst maintaining at least 80% cell viability. Significant gene knockdown was achieved, with a reduction in luciferase expression of up to 68% and a reduction in endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) expression of up to 40%. To our knowledge, this is the first time that a modified CD has been used as a safe and efficacious vector for siRNA delivery into neuronal cells.

This article describes a rapid and reliable electrochemical/enzymatic method of verifying the presence of nitroaromatic explosives. The new technique leverages both conventional voltammetric analysis and biocatalytic conversion of TNT. The simultaneous use of independent measurement schemes, based on two distinct processes, dramatically increases the information content and offers substantially improved reliability while minimizing the occurrence of false alarms. This has been accomplished by coupling direct voltammetric analysis with the biocatalytic conversion of the TNT substrate via nitroreductase (NR), which reduces a nitro group of TNT using NADH as an electron donor. This chemical reduction (30 s timescale) can then be observed using square-wave voltammetry by examination of the reductive and oxidative features. This novel protocol was found to be selective for TNT, not only when compared to DNT and NT, but also to other explosive species such as RDX and PETN. This unique dual-mode detection strategy for measuring TNT at a single device holds considerable promise for improving the probability of explosive detection and hence for diverse security screening applications.

PurposeThis paper's aim is to examine the interaction between an intermediary, a vulnerable defendant and barristers and the judge in a courtroom. The paper seeks to consider how the communication needs of vulnerable defendants, such as those with learning disabilities, should be addressed in the criminal justice system.Design/methodology/approachThe paper considers the legal landscape for dealing with vulnerable defendants. A case example and court and police interview transcripts are then used to illustrate some of the communication needs of vulnerable people in the criminal justice system.FindingsThe paper highlights the complexities of the language that is used by lawyers in the courtroom and the difficulties that this can cause for a vulnerable defendant. Additionally, this paper reveals the difficulties that the police caution can present to a vulnerable suspect in custody.Social implicationsVulnerable witnesses or defendants may be disadvantaged in understanding questions and the implications of answers that they provide in a courtroom or in attendance at a police station. The criminal justice system should support these individuals and provide guidance and training to professionals. The author identifies a need for more research in this area.Originality/valueThis is one of the first published papers to examine the interaction between an intermediary, a vulnerable defendant and barristers and the judge in the courtroom. The paper considers how the communication needs of vulnerable defendants should be addressed in the criminal justice system.

Atypical antipsychotic drugs (AAPDs) such as olanzapine have a serious side effect profile including weight gain and metabolic dysfunction, and a number of studies have suggested a role for gender in the susceptibility to these effects. In recent times, the gut microbiota has been recognised as a major contributor to the regulation of body weight and metabolism. Thus, we investigated the effects of olanzapine on body weight, behaviour, gut microbiota and inflammatory and metabolic markers in both male and female rats. Male and female rats received olanzapine (2 or 4 mg/kg/day) or vehicle for 3 weeks. Body weight, food and water intake were monitored daily. The faecal microbial content was assessed by 454 pyrosequencing. Plasma cytokines (tumour necrosis alpha, interleukin 8 (IL-8), interleuin-6 and interleukin 1-beta (IL-1β)) as well as expression of genes including sterol-regulatory element binding protein-1c and CD68 were analysed. Olanzapine induced significant body weight gain in the female rats only. Only female rats treated with olanzapine (2 mg/kg) had elevated plasma levels of IL-8 and IL-1β, while both males and females had olanzapine-induced increases in adiposity and evidence of macrophage infiltration into adipose tissue. Furthermore, an altered microbiota profile was observed following olanzapine treatment in both genders. This study furthers the theory that gender may impact on the nature of, and susceptibility to, certain side effects of antipsychotics. In addition, we demonstrate, what is to our knowledge the first time, an altered microbiota associated with chronic olanzapine treatment.

PurposeThe purpose of this paper is to examine how Registered Intermediaries are used in the England and Wales to facilitate communication between vulnerable witnesses, victims and police investigators and criminal courts.Design/methodology/approachThe paper focuses on the need for early identification of the vulnerable person so that support measures can be put in place from the outset to assist them to provide their testimony.FindingsIt is noted that real progress has been made by the introduction of legislation, specifically the Youth Justice and Criminal Evidence Act (1999), and the uptake by the police service of the subsequent special measures put in place. However, the criminal justice service cannot afford to be complacent as research demonstrates that the police and the courts need to be more effective in managing these issues.Originality/valueThe paper recommends that support measures are widened to include witnesses and suspects being interviewed by the Independent Police Complaints Commission, HM Customs and Revenue, the Department of Health and the Department of Work and Pensions.

While cathodic voltammetric measurements based on the reduction of nitro-containing explosive compounds have been well documented, little attention has been given for exploiting their anodic response, associated with the oxidation of their reaction products, for qualitative and quantitative security and forensic information. We demonstrate here that cyclic square-wave volammetry, combining the cathodic and anodic signals, offers distinct electrochemical profiles for trace nitroaromatic, nitramine and nitrate ester explosives compared to analogous cyclic-voltammetric and cathodic square-wave-voltammetric measurements. Unique electrochemical signatures are also obtained for commonly used explosive mixtures. Such distinct explosive signatures at disposable strip electrodes should facilitate rapid decentralized security screening applications.

The ability of gut microbiota to communicate with the brain and thus modulate behavior is emerging as an exciting concept in health and disease. The enteric microbiota interacts with the host to form essential relationships that govern homeostasis. Despite the unique enteric bacterial fingerprint of each individual, there appears to be a certain balance that confers health benefits. It is, therefore, reasonable to note that a decrease in the desirable gastrointestinal bacteria will lead to deterioration in gastrointestinal, neuroendocrine or immune relationships and ultimately disease. Therefore, studies focusing on the impact of enteric microbiota on the host and in particular on the central nervous system are essential to our understanding of the influence of this system. Recent studies published in this Journal demonstrate that germ-free mice display alterations in stress-responsivity, central neurochemistry and behavior indicative of a reduction in anxiety in comparison to conventional mice. Such data offer the enticing proposition that specific modulation of the enteric microbiota may be a useful strategy for stress-related disorders and for modulating the co-morbid aspects of gastrointestinal disorders such as irritable bowel syndrome and inflammatory bowel disease.

The neurochemical basis of depression focuses on alterations in the monoaminergic and amino acid neurotransmitter systems. Moreover, decreases in serum levels of the neurotrophin brain-derived neurotrophic factor (BDNF) have led to the more recent neurotrophic hypothesis of depression. Chronic stress is one of the major predisposing factors to developing the disorder and thus we investigated the impact of chronic restraint stress on the levels of several neurotransmitters and their metabolites in a genetic animal model of depression, the Wistar Kyoto (WKY) rat. Behavioural analysis of WKY rats indicated both a depressive and anxiety-like phenotype compared to their Sprague Dawley (SD) controls. WKY animals showed similar stress-induced decreases in hippocampal GABA, noradrenaline and dopamine as their SD counterparts while exhibiting a divergent decrease in 5-HT, 5-HIAA and DOPAC. WKY rats also showed a stress-dependent increase in GABA concentrations in the amygdala compared to the SD animals. Moreover, WKY but not SD rats had a chronic stress-induced decrease in serum BDNF levels. Together these data show that there are specific strain-dependent changes in neurotransmitter and neurotrophin levels in response to chronic stress which may predispose WKY animals to a depressive-like phenotype.

The electrochemistry of various gases, including oxygen, carbon dioxide, hydrogen, ammonia, hydrogen sulfi de, sulfur dioxide and nitrogen dioxide, in room temperature ionic liquids (RTILs) is reviewed. The application of RTILs to gas sensor design and development is highlighted.

Accessible summary • Many people with a learning disability are frightened when they are interviewed by the police. • They are worried that the police will not understand their story and sometimes keep quiet because they do not understand what is happening. • It can also be very worrying for a person with a learning disability if they have to attend court and give evidence. • The law allows for vulnerable witnesses to have access to support from a professional known as an intermediary. • The law does not yet support vulnerable offenders in this way, but It may now be possible to get professional support from an intermediary if you are an offender. • The intermediary might be able to help you communicate with the police and with people at court if you are a witness or an offender. Summary Being interviewed by the police as a witness or as a suspect can be a frightening experience for many people; these feelings can be multiplied for many people who have a learning disability and who do not understand the legal proceedings. Intermediaries were introduced by the Youth Justice and Criminal Evidence Act 1999 as one of the special measures available to help facilitate communication between the police, the courts and the vulnerable witness. Research has shown that this measure has been valuable in assisting the vulnerable witness to testify. This study was an exploratory examination of the recent use of intermediaries with defendants. The defendant does not by law have the right to an intermediary; however the Coroner’s and Justice Bill (2008–09), is attempting to amend this situation. Meanwhile judges are starting to request an intermediary for vulnerable defendants, in the interests of justice. Using a mixed methods approach this study found that intermediaries were very positive about the need for vulnerable defendants to have support. The intermediaries also highlighted that they required additional training and they raised concerns about how their role was viewed by the courts when they were assisting defendants.

The detection of H2S was studied using several mediators in room temperature ionic liquids (RTILs) at a microelectrode. Each mediator was characterised voltammetrically, and H2S was added to observe if any mediation took place. A successful mediator was 3,5-tert-butyl-obenzoquinone. Cyclic voltammetry was carried out in [C2mim]-[NTf2]. A reductive wave was observed and attributed to the two-electron reduction of the mediator. No oxidative signal was observed. H2S was flowed through the system. Cyclic voltammograms showed a decrease in the reductive wave of the mediator and the onset of an oxidative signal due to the reaction between the mediator and H2S to form an adduct. This reaction is reversible and on purging the system with N2, the original reductive signal of the mediator was recovered. © 2010 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim.

Do gastrointestinal symptoms in patients with inflammatory bowel disease (IBD) in apparent remission reflect the coexistence of irritable bowel syndrome (IBS) or subclinical inflammation? The aims of this study were as follows: (i) to prospectively determine the prevalence of IBS symptoms in IBD patients in remission; and (ii) to determine whether IBS symptoms correlate with levels of fecal calprotectin.Remission was defined by physician assessment: Crohn's disease (CD) activity indexor=150 and ulcerative colitis disease activity indexor=3, and serum C-reactive protein10, while off corticosteroids or biologics. Quality of life (QOL) (by inflammatory bowel disease questionnaire), the hospital anxiety and depression scale (HAD), and fecal calprotectin were measured.Rome II criteria for IBS were fulfilled in 37/62 (59.7%) of CD patients and by 17/44 (38.6%) of those with ulcerative colitis (UC). However, fecal calprotectin was significantly elevated above the upper limit of normal in both IBD patient groups, indicating the presence of occult inflammation. Furthermore, calprotectin levels were significantly higher in CD and UC patients with criteria for IBS than in those without IBS-type symptoms. QOL scores were lower and HAD scores higher among UC patients with IBS symptoms in comparison to those who did not have IBS symptoms.IBS-like symptoms are common in patients with IBD who are thought to be in clinical remission, but abnormal calprotectin levels suggest that the mechanism in most cases is likely to be occult inflammation rather than coexistent IBS.

Theory of chronoamperometry on ring-recessed microelectrodes and their arrays is presented. Analysis of the current transients measured at thin rings and their arrays shows that chronoamperometry at such devices is an excellent method of simultaneous measurement of the diffusion coefficient D and the product n[A], where n is the number of electrons transferred and [A] is the concentration of electroactive analyte. A generic, accurate and easy to use method of experimental chronoamperometric data analysis is proposed. It is shown that the method can be applied to the simultaneous measurement of D and n[A] in solution. The method is shown to be applicable to an analysis of data recorded at single disk and ring electrodes as well as arrays of these electrodes even in the case of diffusional non-independence of electrodes in the array. © 2010 Elsevier B.V. All rights reserved.

Aim To establish any differences between patients with and without a diagnosis of personality disorders, being treated in a secure inpatient service for offenders with intellectual disability (ID) in the UK. Method A cohort study involving a selected population of people with ID and offending behaviours. Results The study included a total of 138 patients, treated over a 6 year period – 77 with a dissocial or emotionally unstable personality disorder and 61 without. Women were more likely to be in the personality disorder group. Both groups had high prevalence of abuse with no significant differences. Depressive disorders and substance abuse were more common in the personality disorder group, while epilepsy and autistic spectrum disorders were more common in the non-personality disorder group. Rather than differences, what was more striking was the rate and range of these comorbidities across both groups. Although past histories of violence and institutional aggression were no different, compulsory detention under criminal sections and restriction orders were more common in the personality disorder group. There were no differences in treatment outcomes. Conclusions Although about half of patients detained in secure units for offenders with ID have a personality disorder, there were more similarities than differences between this group and the rest. While good treatment outcomes supported the case for specialised secure treatment units for people with ID, the case for establishing a more specialised ID–personality disorder unit was less convincing. There is also a need to explore whether there are alternative diagnostic models that can delineate better the group with personality difficulties in this population.

There is increasing evidence to suggest that metabotropic glutamate (mGlu) receptors including mGlu(7) receptor are important in the pathophysiology of stress-related psychiatric disorders such as anxiety and major depression. mGlu(7) receptor is highly expressed in the hippocampus, a key region involved in the modulation of depression-related behaviour. Moreover, mice deficient in mGlu(7) receptor have an antidepressant-like behaviour and altered stress response. To our knowledge there is little information as to whether stressful phenotypes can influence hippocampal mGlu(7) receptor levels. To this end, we examined hippocampal mGlu(7) receptor mRNA expression in two models of depression, the stress-sensitive Wistar Kyoto (WKY) and the maternally separated model of early-life stress. In situ hybridization analysis revealed that the WKY, but not the maternally separated (MS) rats displayed selective increases in mGlu(7) receptor mRNA expression in subregions of the hippocampus compared to relevant controls. These data suggest that higher levels of this receptor could affect the behaviour in response to stressful conditions and may play a role in WKY animal's susceptibility to stress-related disorders. However, the data in maternally separated animals confirm that whilst hippocampal mGlu(7) receptors maybe involved in certain aspects of stress biology, an increased expression is not necessary for the manifestation of depression-related phenotype per se.

The voltammetry of N,N-dimethyl-p-phenylenediamine, DMPD, in the room temperature ionic liquid (RTIL) 1-butyl-3-methylimidazolium tetrafluoroborate, [C4mim][BF4] is studied by means of microdisc voltammetry over a wide range of voltage scan rates incorporating the transition from linear to convergent diffusion. Two voltammetric waves were recorded corresponding to the formation of the radical cation DMPD+ and dication DMPD 2+; both are stable on the voltammetric time scale. Double potential step chronoamperometry was used to measure the diffusion coefficients of DMPD and DMPD+. These values, in conjunction with simulations of the cyclic voltammetry, permit the inference of the equilibrium constant and kinetics for the comproportionation of DMPD and DMPD2+ forming DMPD+ to be studied. © 2009 American Chemical Society.

Microelectrode arrays have unique electrochemical properties such as small capacitive-charging currents, reduced iR drop, and steady-state diffusion currents. These properties enable the use of microelectrode arrays and have captured much interest in the field of electrochemistry. Techniques for the fabrication of such arrays are reviewed. The relative features and merits of different techniques are also discussed.

Background Adverse early life events are associated with a maladaptive stress response system and might increase the vulnerability to disease in later life. Several disorders have been associated with early life stress, ranging from depression to irritable bowel syndrome. This makes the identification of the neurobiological substrates that are affected by adverse experiences in early life invaluable. Methods The purpose of this study was to assess the effect of early life stress on the brain-gut axis. Male rat pups were stressed by separating them from their mothers for 3 hours daily between postnatal days 2–12. The control group was left undisturbed with their mothers. Behavior, immune response, stress sensitivity, visceral sensation, and fecal microbiota were analyzed. Results The early life stress increased the number of fecal boli in response to a novel stress. Plasma corticosterone was increased in the maternally separated animals. An increase in the systemic immune response was noted in the stressed animals after an in vitro lipopolysaccharide challenge. Increased visceral sensation was seen in the stressed group. There was an alteration of the fecal microbiota when compared with the control group. Conclusions These results show that this form of early life stress results in an altered brain-gut axis and is therefore an important model for investigating potential mechanistic insights into stress-related disorders including depression and IBS.

The effect of water content on room-temperature ionic liquids (RTILs) was studied by Karl Fischer titration and cyclic voltammetry in the following ionic liquids: tris(P-hexyl)tetradecylphosphonium trifluorotris(pentafluoroethyl) phosphate [P14,6,6,6][NTf2], N-butyl-N-methyl- pyrrolidiniumbis(trifluoromethylsulfonyl)imide [C4mpyrr][NTf 2], 1-hexyl-3-methylimidazolium tris(perfluoroethyl) trifluorophosphate [C6mim][FAP], 1-butyl3-methylimidazolium bis(trifluoromethylsulfonyl)imide [C4mim] [NTf2], 1-butyl-3-methylimidazoliium bis(trifluoromethylsulfonyl)imide [C 4dmim] [NTf2], N-hexyltriethylammonium bis(trifluoromethylsolfonyl)imide [N6,2,2,2][NTf2], 1-butyl-3-methylimidazolium hexafluorophosphate [C4mim][PF 6], l-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [C2mim][NTf2], 1-butyl-3-methylimidazolium tetrafluoroborate [C4mim][BF4], 1-hexyl-3- methylimidazolium iodide [C4mim][I], 1-butyl-3-methylimidazolium trifluoromethylsulfonate [C4mim][OTf], and 1-hexyl-3- methylimidazolium chloride [C6mim][Cl]. In addition, electrochemically relevant properties such as viscosity, conductivity, density, and melting point of RTILs are summarized from previous literature and are discussed. Karl Fisher titrations were carried out to determine the water content of RTILs for vacuum-dried, atmospheric, and wet samples. The anion in particular was found to affect the level of water uptake. The hydrophobicity of the anions adhered to the following trend: [FAP]- > [NTf 2]- > [PF6]- > [BF 4]- > halides. Cyclic voltammetry shows that an increase in water content significantly narrows the electrochemical window of each ionic liquid. The electrochemical window decreases in the following order: vacuum-dried > atmospheric > wet at 298 K > 318 K > 338 K. The anodic and cathodic potentials vs ferrocene internal reference are also listed under vacuum-dried and atmospheric conditions. The data obtained may aid the selection of a RTIL for use as a solvent in electrochemical applications. © 2008 American Chemical Society.