Activity-Dependent Ectopic Spiking in Parvalbumin-Expressing Interneurons of the Neocortex.

Canonically, action potentials of most mammalian neurons initiate at the axon initial segment (AIS) and propagate bidirectionally: orthodromically along the distal axon and retrogradely into the soma and dendrites. Under some circumstances, action potentials may initiate ectopically, at sites distal to the AIS, and propagate antidromically along the axon. These "ectopic action potentials" (EAPs) have been observed in experimental models of seizures and chronic pain, and more rarely in nonpathological forebrain neurons. Here we report that a large majority of parvalbumin-expressing (PV+) interneurons in the upper layers of mouse neocortex, from both orbitofrontal and primary somatosensory areas, fire EAPs after sufficient activation of their somata. Somatostatin-expressing interneurons also fire EAPs, though less robustly. Ectopic firing in PV+ cells occurs in varying temporal patterns and can persist for several seconds. PV+ cells evoke strong synaptic inhibition in pyramidal neurons and interneurons and play critical roles in cortical function. Our results suggest that ectopic spiking of PV+ interneurons is common and may contribute to both normal and pathological network functions of the neocortex.

Heat Stress and an Immune Challenge Influence Turkey Meat Quality, but Conspecific-Directed Pecking Behavior Does Not.

Heat stress (HS), immune challenges (IC) and pecking behavior are some of the many stressors poultry can experience in commercial settings that may affect bird welfare and meat quality after harvest. The first objective was to determine if HS or IC turkeys displayed greater negative effects on meat quality, and the second objective was to determine if the frequency of non-aggressive pecking behaviors among the birds was related to meat quality. Ninety-two, commercial male, beak-trimmed turkeys were used with a total of 15 rooms and 4-7 birds per room. Each treatment was applied for 1 week prior to harvest: the Control (CON) group had no stressors added, the HS group ambient temperature was approximately 29 °C for 120 min, and the IC group involved inoculating birds with a live vaccine for hemorrhagic enteritis virus. Birds were recorded and scored to quantify pecking behavior. Once harvested, carcasses were evaluated for feather retention force, pH, color, proximate analysis, fatty acid composition, shear force, and drip loss. Stress treatment resulted in HS breasts having the lowest protein content, and IC breasts having the lowest CIE L* values and the greatest shear force values. Pecking behavior had no impact on any meat quality attributes.

Development and perceptions of an academic success tool for pharmacy students.

OBJECTIVE: Some students may face challenges with graduate-level reading and writing, particularly in certain active learning pedagogies, such as team-based learning or peer instruction, which require extensive pre-reading. The objective of this study is to determine the perceived utility of an academic literacy (reading/writing) test for first professional year student pharmacists (P1s). METHODS: In a collaboration between pharmacy and linguistics faculty, an academic literacy assessment tool was developed using fall P1 course materials. After pilot testing and adjustments, the revised test was administered to all P1 students by trained facilitators, then scored. Students needing literacy support were identified, met with individually to debrief on the assessment, and offered a year-long, one-on-one tutoring program. P1 faculty participated in an end-of-semester focus group session to determine whether the assessment correctly identified students who benefited from literacy support, and to decide on the impact of subsequent support. Thematic analysis was performed on the data. RESULTS: A total of 13 students were identified as at-risk through the assessment. Since tutoring was optional, eight students met at least once, and two students met weekly during the ensuing semester. Faculty from the end-of-semester focus group 1) stated that the assessment accurately pre-identified students who struggled with literacy components of P1 coursework, and 2) expressed a wish for earlier identification of students with required instead of optional tutoring. CONCLUSIONS: Faculty perceived that the tool accurately identified students, but the timing and the volunteer nature of the follow-up tutoring limited the success of the assessment effort.

Using multiple case studies of health and justice services to inform the development of a new complex intervention for prison-leavers with common mental health problems (Engager).

BACKGROUND: People in the criminal justice system have complex needs but often do not make use of services outside of prison, in many cases due to poorly joined up working between health and criminal justice services. The 'Engager' programme aimed to develop a complex collaborative care intervention for people leaving prison with common mental health problems that could support their transition into the community and facilitate joined up working between health, justice and social services. To augment our core intervention theory, we wanted to learn from innovative and forward-thinking services providing interagency support and/or treatment for people experiencing common mental health problems within the criminal justice system. We wanted to identify key elements of interagency practice to understand what was and was not effective in engaging people, maintaining their contact and improving mental health and other aspects of their lives. METHOD: We used a multiple case study design with a focused ethnographic approach in four study sites. Data came from three sources (documents, field notes and semi-structured interviews) underwent a framework analysis. RESULTS: We identified seven main themes, namely: collaboration, client engagement, client motivation, supervision, therapeutic approach, peers and preparations for ending. Engaging and motivating clients was dependent on the relationship built with the professional. This relationship was developed through building trust and rapport, which required time and respectful, open and honest communication. Professionals were often unable to build this relationship effectively if they did not work in effective interagency collaborations, particularly those which included shared practices and were supported by effective supervision. CONCLUSIONS: The multiple case study design contributed insights as to how health and justice services work together. The main themes identified are well known factors in health and justice co-working. However, the novel insights were gleaned examining interdependence and interactions in complex, multifactorial phenomena and practice, in particular the importance of shared practice and supervision models. The approach of selecting a small number of cases representing identified knowledge gaps contributed a valuable addition to the program theory and delivery for an innovative complex intervention.

Transgenerational Bisphenol A Causes Deficits in Social Recognition and Alters Postsynaptic Density Genes in Mice.

Bisphenol A (BPA) is a ubiquitous endocrine-disrupting chemical. Developmental exposure produces changes in behavior and gene expression in the brain. Here, we examined social recognition behaviors in mice from the third familial generation (F3) after exposure to gestational BPA. Second-generation mice were bred in one of four mating combinations to reveal whether characteristics in F3 were acquired via maternal or paternal exposures. After repeated habituation to the same mouse, offspring of dams from the BPA lineage failed to display increased investigation of a novel mouse. Genes involved in excitatory postsynaptic densities (PSDs) were examined in F3 brains using quantitative PCR. Differential expression of genes important for function and stability of PSDs were assessed at three developmental ages. Several related PSD genes-SH3 and multiple ankyrin repeat domains 1 (Shank1), Homer scaffolding protein 1c (Homer1c), DLG associated protein 1 (Gkap), and discs large MAGUK scaffold protein 4 (PSD95)-were differentially expressed in control- vs BPA-lineage brains. Using a second strain of F3 inbred mice exposed to BPA, we noted the same differences in Shank1 and PSD95 expression in C57BL/6J mice. In sum, transgenerational BPA exposure disrupted social interactions in mice and dysregulated normal expression of PSD genes during neural development. The fact that the same genetic effects were found in two different mouse strains and in several brain regions increased potential for translation. The genetic and functional relationship between PSD and abnormal neurobehavioral disorders is well established, and our data suggest that BPA may contribute in a transgenerational manner to neurodevelopmental diseases.

Early Life Stress Drives Sex-Selective Impairment in Reversal Learning by Affecting Parvalbumin Interneurons in Orbitofrontal Cortex of Mice.

Poverty, displacement, and parental stress represent potent sources of early life stress (ELS). Stress disproportionately affects females, who are at increased risk for stress-related pathologies associated with cognitive impairment. Mechanisms underlying stress-associated cognitive impairment and enhanced risk of females remain unknown. Here, ELS is associated with impaired rule-reversal (RR) learning in females, but not males. Impaired performance was associated with decreased expression and density of interneurons expressing parvalbumin (PV+) in orbitofrontal cortex (OFC), but not other interneuron subtypes. Optogenetic silencing of PV+ interneuron activity in OFC of control mice phenocopied RR learning deficits observed in ELS females. Localization of reversal learning deficits to PV+ interneurons in OFC was confirmed by optogenetic studies in which neurons in medial prefrontal cortex (mPFC) were silenced and associated with select deficits in rule-shift learning. Sex-, cell-, and region-specific effects show altered PV+ interneuron development can be a driver of sex differences in cognitive dysfunction.

Validity of Micro-Data Loggers to Determine Walking Activity of Turkeys and Effects on Turkey Gait.

Accelerometers have the potential to provide objective, non-invasive methods for detecting changes in animal behavior and health. Our objectives were to: (1) determine the effects of micro-acceleration data loggers (accelerometers) and habituation to accelerometers on turkey gait and health status, (2) determine age-related changes in gait and health status, and (3) assess the validity and reliability of the accelerometers. Thirty-six male commercial turkeys were randomly assigned to one of five groups: accelerometer and habituation period (AH), accelerometer and no habituation period (AN), VetRap bandage (no accelerometer) and habituation period (VH), bandage (no accelerometer) and no habituation period (VN), and nothing on either leg (C). Health status and body condition were assessed prior to video-recording birds as they walked across a Tekscan® pressure pad at 8, 12, and 16 weeks to determine effects of treatment on number of steps, cadence, gait time, gait distance, gait velocity, impulse, gait cycle time, maximum force, peak vertical pressure, single support time, contact time, step length, step time, step velocity, stride length, total double support time, and duty factor. Accelerometer validity and reliability were determined by comparing the number of steps detected by the accelerometer to the number of steps determined from video recordings. Several age-related changes in turkey gait were found regardless of habituation including a slower cadence at 16 weeks, shorter gait distance at 8 weeks, and slower gait velocity at 16 weeks. When comparing bandaged vs. unbandaged limbs, both treatment and age-treatment interactions were found depending on the gait parameter. Accelerometer validity and reliability were affected by both age and treatment. False discovery rate increased, while accuracy and specificity decreased with age. Validity and reliability were lowest for non-habituated birds (AN and VN). Results demonstrated that micro-data loggers do not adversely affect turkey health status, but habituation to wearing accelerometers greatly affects accelerometer reliability and validity. Accelerometer validity and turkey gait are also greatly affected by the age of the turkeys.

Working together to shape the endometrial cancer research agenda: The top ten unanswered research questions.

BACKGROUND: Endometrial cancer (EC) is the most common gynaecological cancer in developed nations and its incidence is rising. As a direct consequence, more women are dying from EC despite advances in care and improved survivorship. There is a lack of research activity and funding, as well as public awareness about EC. We sought to engage patients, carers and healthcare professionals to identify the most important unanswered research questions in EC. METHODOLOGY: The priority setting methodology was developed by the James Lind Alliance and involved four key stages: gathering research questions; checking these against existing evidence; interim prioritisation; and a final consensus meeting during which the top ten unanswered research questions were agreed using modified nominal group methodology. RESULTS: Our first online survey yielded 786 individual submissions from 413 respondents, of whom 211 were EC survivors or carers, and from which 202 unique unanswered research questions were generated. 253 individuals, including 108 EC survivors and carers, completed an online interim prioritisation survey. The resulting top 30 questions were ranked in a final consensus meeting. Our top ten spanned the breadth of patient experience of this disease and included developing personalised risk scoring, refining criteria for specialist referral, understanding the underlying biology of different types of EC, developing novel personalised treatment and prevention strategies, prognostic and predictive biomarkers, increasing public awareness and interventions for psychological issues. CONCLUSION: Having established the top ten unanswered research questions in EC, we hope this galvanises researchers, healthcare professionals and the public to collaborate, coordinate and invest in research to improve the lives of women affected by EC.

Discharges to prison from medium secure psychiatric units in England and Wales.

BACKGROUND: Early findings from a national study of discharges from 32 National Health Service medium secure units revealed that nearly twice as many patients than expected were discharged back to prison. AIMS: To compare the characteristics of those discharged back to prison with those discharged to the community, and consider the implications for ongoing care and risk. METHOD: Prospective cohort follow-up design. All forensic patients discharged from 32 medium secure units across England and Wales over a 12-month period were identified. Those discharged to prison were compared with those who were discharged to the community. RESULTS: Nearly half of the individuals discharged to prison were diagnosed with a serious mental illness and over a third with schizophrenia. They were a higher risk, more likely to have a personality disorder, more symptomatic and less motivated than those discharged to the community. CONCLUSIONS: Findings suggest that alternative models of prison mental healthcare should be considered to reduce risks to the patient and the public.

Male-male clasping may be part of an alternative reproductive tactic in Xenopus laevis.

Male Xenopus laevis frogs have been observed to clasp other males in a sustained, amplectant position, the purpose of which is unknown. We examined three possible hypotheses for this counter-intuitive behavior: 1) clasping males fail to discriminate the sex of the frogs they clasp; 2) male-male clasping is an aggressive or dominant behavior; or 3) that males clasp other males to gain proximity to breeding events and possibly engage in sperm competition. Our data, gathered through a series of behavioral experiments in the laboratory, refute the first two hypotheses. We found that males did not clasp indiscriminately, but showed a sex preference, with most males preferentially clasping a female, but a proportion preferentially clasping another male. Males that clasped another male when there was no female present were less likely to "win" reproductive access in a male-male-female triad, indicating that they did not establish dominance through clasping. However, those males did gain proximity to oviposition by continued male-male clasping in the presence of the female. Thus, our findings are consistent with, but cannot confirm, the third hypothesis of male-male clasping as an alternative reproductive tactic.

Social smokers' management of conflicted identities.

BACKGROUND: Although social smoking has increased among young adults, it remains a poorly understood behaviour. The authors explored how young adult social smokers viewed and defined smoking and the strategies they used to reconcile their conflicting smoker and non-smoker identities. The authors also examined alcohol's role in facilitating social smoking and investigated measures that would decouple drinking and smoking. METHODS: The authors conducted 13 in-depth interviews with young adult social smokers aged between 19 and 25 years and used thematic analysis to interpret the transcripts. RESULTS: The authors identified four key themes: the demarcation strategies social smokers used to avoid classifying themselves as smokers, social smoking as a tactic that ameliorates the risk of alienation, alcohol as a catalyst of social smoking and the difficulty participants experienced in reconciling their identity as non-smokers who smoke. CONCLUSIONS: Although social smokers regret smoking, their retrospective remorse was insufficient to promote behaviour change, and environmental modifications appear more likely to promote smoke-free behaviours among social smokers. Participants strongly supported extending the smoke-free areas outside bars, a measure that would help decouple their alcohol-fuelled behaviours from the identity to which they aspire.

Peptide inhibitors of the malaria surface protein, apical membrane antigen 1: identification of key binding residues.

Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5-10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to >350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C(α) H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a nonpolar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics.

Structure and inter-domain interactions of domain II from the blood-stage malarial protein, apical membrane antigen 1.

The malarial surface antigen apical membrane antigen (AMA1), from Plasmodium falciparum, is a leading candidate for inclusion in a vaccine against malaria. AMA1 is synthesised by mature blood-stages of the parasite and is located initially in the apical organelles of the merozoite. Prior to merozoite invasion of host erythrocytes, it is processed into a 66 kDa type 1 integral membrane protein on the merozoite surface. The pattern of disulphide bonds in AMA1 has been the basis for separation of the ectodomain into three domains, with three, two and three disulphide bonds, respectively. We have determined the solution structure of a 16kDa construct corresponding to the putative second domain of AMA1. While circular dichroism and hydrodynamic data were consistent with a folded structure for domain II, its NMR spectra were characterised by broad lines and significant peak overlap, more typical of a molten globule. Consistent with this, domain II bound the fluorescent dye 8-anilino-1-naphthalene sulphonate (ANS). We have nonetheless determined a structure, which defines the secondary structure elements and global fold. The two disulphide bonds link the N and C-terminal regions of the molecule, which come together to form a four-stranded beta-sheet linked to a short helix. A long loop linking the N and C-terminal regions contains four other alpha-helices, the locations of which are not fixed relative to the beta-sheet core, even though they are well-defined locally. Very recently this region of domain II has been shown to contain the epitope recognised by the invasion-inhibitory antibody 4G2, even though it does not contain any of the polymorphisms that are regarded as having arisen in response to the pressure of immune recognition.

Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.

Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

Sialic acid acts as a receptor for equine rhinitis A virus binding and infection.

Equine rhinitis A virus (ERAV) is a member of the genus Aphthovirus, family Picornaviridae, and causes respiratory disease in horses worldwide. To characterize the putative receptor molecule(s) of the ERAV isolate 393/76 (ERAV.393/76) on the surface of Vero and other cells, an assay was developed to measure the binding of purified biotinylated ERAV.393/76 virions to cells by flow cytometry. Using this assay, the level of binding to different cell types correlated with the relative infectivity of ERAV in each cell type. In particular, equine fetal kidney cells, mouse fibroblast cells, rabbit kidney-13 and Crandell feline kidney cells bound virus at high levels and produced high virus yields (> or =10(7) TCID50 ml(-1)). Madin-Darby bovine kidney and baby hamster kidney cells showed little or no binding of virus, producing yields of < or =10(1.8) TCID50 ml(-1). Treatment of Vero and other cells with sodium periodate and the metabolic inhibitors tunicamycin, benzyl N-acetyl-alpha-D-galactosamide, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and proteases indicated that part of the receptor-binding and entry complex for ERAV.393/76 is on N-linked carbohydrates and that the carbohydrate is likely to be present on a protein rather than a lipid backbone. The effect of carbohydrate-specific lectins and neuraminidases on ERAV.393/76 binding and infection of Vero and other cell types implicated alpha2,3-linked sialic acid residues on the carbohydrate complex in the binding and infection of ERAV.

Identification of a neutralizing epitope in the betaE-betaF loop of VP1 of equine rhinitis A virus, defined by a neutralization-resistant variant.

Equine rhinitis A virus strain 393/76 (ERAV.393/76) was passaged in the presence of post-infection ERAV.393/76 equine polyclonal antiserum (EPA). Viruses with increased resistance to neutralization by EPA were obtained after 15 passages. Compared with the parent virus, five plaque-purified, neutralization-resistant mutant viruses, in addition to the non-plaque-purified viruses that were examined, had a Glu-->Lys change at position 658, which is located in the predicted betaE-betaF (EF) loop of VP1. Rabbit antiserum was prepared against the isolated EF loop of ERAV.393/76 VP1 expressed as a fusion protein with glutathione S-transferase. This antiserum bound to purified ERAV.393/76 in Western blots, but not to the neutralization-resistant mutant virus or to ERAV.PERV/62, a naturally occurring ERAV strain that has a Lys residue at position 658. These results suggest that the EF loop of VP1 is involved in a neutralization epitope of ERAV.

Mapping epitopes in equine rhinitis A virus VP1 recognized by antibodies elicited in response to infection of the natural host.

Equine rhinitis A virus (ERAV) is an important respiratory pathogen of horses and is of additional interest because of its close relationship and common classification with foot-and-mouth disease virus (FMDV). As is the case with FMDV, the VP1 capsid protein of ERAV has been shown to be a target of neutralizing antibodies. In FMDV VP1, such antibodies commonly recognize linear epitopes present in the betaG-betaH loop region. To map linear B cell epitopes in ERAV VP1, overlapping fragments spanning its length were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. These fusion proteins were tested for reactivity with sera from ERAV-infected horses and with polyclonal sera from ERAV-immunized rabbits and mice. Regions at the N- and C-termini as well as the betaE-betaF and the betaG-betaH loop regions contained B cell epitopes that elicited antibodies in the natural host. GST fusion proteins of these regions also elicited antibodies following immunization of rabbits and mice, which, in general, strongly recognized native ERAV VP1 but which were non-neutralizing. It is concluded that the N-terminal region of ERAV VP1, in particular, contains strong B cell epitopes.