Protein preference and elevated plasma FGF21 induced by dietary protein restriction is similar in both male and female mice.

Animals that are moderately protein restricted respond to this dietary stress by increasing consumption of protein-containing foods. This is true in many species, including rodents. Rodent models of protein restriction have typically relied on only male subjects, and there are plausible reasons why female rodents may respond differently to dietary protein restriction. To address this gap in knowledge, the current experiments examined protein preference after two weeks on a 5% protein diet or 20% protein control diet, in male and female mice. We found that female protein-restricted mice, like male protein-restricted mice, increase consumption of 4% casein (protein) relative to 4% maltodextrin (carbohydrate) when presented with both simultaneously. Interestingly, this increased consumption was due to more bursts in females and more licks per burst in males, indicating possible differences in mechanism by which increased intake is achieved. Stage of the estrous cycle did not affect female responses. Moreover, we measured plasma fibroblast growth factor 21 (FGF21) - a hormone induced by protein restriction and necessary for protein preference - in male and female mice. Here, we found no statistical differences between protein-restricted males, females in diestrus, or females in proestrus. In non-restricted mice FGF21 levels were low, but significantly higher in females in proestrus than females in diestrus or males. Overall, these experiments highlight the importance of including female subjects in studies of food choice and macronutrient restriction.

Sampling phasic dopamine signaling with fast-scan cyclic voltammetry in awake, behaving rats.

Fast-scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the in vivo measurement of extracellular fluctuations in multiple chemical species. The technique is frequently utilized to sample sub-second (phasic) concentration changes of the neurotransmitter dopamine in awake and behaving rats. Phasic dopamine signaling is implicated in reinforcement, goal-directed behavior, and locomotion, and FSCV has been used to investigate how rapid changes in striatal dopamine concentration contribute to these and other behaviors. This unit describes the instrumentation and construction, implantation, and use of components required to sample and analyze dopamine concentration changes in awake rats with FSCV.

Heterogeneity of dopamine neuron activity across traits and states.

Midbrain dopamine neurons fire irregularly, with interspersed clusters of high-frequency spikes, commonly called 'bursts'. In this review we examine such heterogeneity in activity, and provide insight into how it can participate in psychiatric conditions such as drug addiction. We first describe several techniques used to evaluate dopamine neuron activity, and comment on the different measures that each provides. We next describe the activity of dopamine neurons in 'basal' conditions. Specifically, we discuss how the use of anesthesia and reduced preparations may alter aspects of dopamine cell activity, and how there is heterogeneity across species and regions. We also describe how dopamine cell firing changes throughout the peri-adolescent period and how dopamine neuron activity differs across the population. In the final section, we discuss how dopamine neuron activity changes in response to life events. First, we focus attention on drugs of abuse. Drugs themselves change firing activity through a variety of mechanisms, with effects on firing while drug is present differing from those seen after drug discontinuation. We then review how stimuli that are rewarding, aversive, or salient can evoke changes in firing rate and discharge pattern of dopamine neurons, and provide behavioral relevance of dopamine signaling. Finally, we discuss how stress can modulate dopamine neuron firing and how this may contribute to the role that stressful experiences play in psychiatric disorders such as addiction and depression.

Risk of venous thromboembolism in patients treated for bacterial infection in the community with outpatient parenteral antimicrobial therapy.

BACKGROUND: It is recommended that venous thromboembolism (VTE) prophylaxis be considered for patients receiving outpatient parenteral antimicrobial therapy (OPAT), but there is no published data to quantify VTE risk in this patient group. AIM AND METHOD: The aim of this retrospective cohort study was to establish VTE incidence in patients managed through an OPAT service and assess utility of a common VTE prediction score normally used for inpatients. Consecutive episodes of OPAT between May 2009 and May 2012 were included. Patients on long-term anti-coagulants, those with an established indication for extended, outpatient VTE prophylaxis (i.e. patients referred to OPAT following hip or knee arthroplasty) were excluded. The Padua VTE Prediction Score was retrospectively applied to the cohort. The primary outcome was incidence of symptomatic VTE during or up to 90 days after completion of OPAT treatment. RESULTS: There were 780 included patient episodes; 105 (13.5%) patients had a Padua VTE risk score >3; no patients received pharmacological VTE prophylaxis during OPAT treatment. During or up to 90 days following OPAT, two proximal lower limb DVTs were diagnosed, giving VTE incidence of 2/780 (0.26%, 95% CI: 0.03-0.92%), and there were eight deaths of which none were suspected to be related to VTE. There was one intracranial haemorrhage associated death. CONCLUSION: This retrospective cohort study found a low incidence of VTE in OPAT patients, and does not support routine application of inpatient VTE prophylaxis algorithms to patients treated for infection in the community.

A rapid quantitative method of carisoprodol and meprobamate by liquid chromatography-tandem mass spectrometry.

The identification and quantitation of carisoprodol (Soma) and its chief metabolite meprobamate, which is also a clinically prescribed drug, remains a challenge for forensic toxicology laboratories. Carisoprodol and meprobamate are notable for their widespread use as muscle relaxants and their frequent identification in the blood of impaired drivers. Routine screening is possible in both an acidic/neutral pH screen and a traditional basic screen. An improvement in directed testing quantitations was desirable over the current options of an underivatized acidic/neutral extraction or a basic screen, neither of which used ideal internal standards. A new method was developed that utilized a simple protein precipitation, deuterated internal standards and a short 2-min isocratic liquid chromatography separation, followed by multiple reaction monitoring with tandem mass spectrometry. The linear quantitative range for carisoprodol was determined to be 1-35mg/L and for meprobamate was 0.5-50mg/L. The method was validated for specificity and selectivity, matrix effects, and accuracy and precision.

Experts and performance in histopathology--a study in breast pathology.

This study was undertaken to determine if it was possible to identify expertise within Histopathologists (trainees, district general pathologists and pathologists with a special interest in breast disease) using an objective measure of performance. The method of assessment of performance is based on the CWS (Cochran-Weiss-Shanteau) ratio formed by the individual's ability to discriminate between a spectrum of disease categories and their level of inconsistency when assessed at intervals. The slides circulated represented the spectrum of breast disease seen in routine practice. The results demonstrated the average CWS ratio to be lowest in trainees and highest in pathologists with a special interest in breast pathology although there was no statistical difference in the CWS scores obtained between the district general pathologists and pathologists with a special interest. Differences in inconsistency rather than discriminatory ability mainly accounted for the difference in the CWS ratio observed between the groups studied. The study shows that the CWS ratio is potentially a very useful tool in the assessment of pathologists with regard to assessing their progress through training.

A study of electric field standing waves on reflection microspectroscopy of polystyrene particles.

We have been investigating the mid-infrared (MIR) reflection spectrum of microparticles on mirrored substrates. Gold-coated porous alumina filters were used as a substrate to layer the particles and provide consistent reflection spectra. Polystyrene spheres with measured diameters of 0.42 microm were studied using Fourier transform infrared (FT-IR) reflection microspectroscopy, and spectra are shown for coverages in the range 0.5-6 monolayers (ML). Results show that absorption has a nonlinear, stairstep-like dependence on particle coverage and a wavelength dependence that can be explained by electric field standing waves (EFSW) caused by the mirrored substrate. The same effect is found to cause progressive weakening of the observed spectra as a function of increasing wavelength in sub-monolayer coverage measurements. Scattering effects in the spectra are consistent with surface scattering at the antinodes of the EFSW. These observations provide explanations for differences seen between optical properties of particles calculated using the specular-reflection method versus those calculated using traditional aerosol methods. A simple multilayer method for estimating particle absorption coefficients is demonstrated that compares well with values reported using ellipsometry for bulk polystyrene. Another simple method based on submonolayer coverage spectra provides spectra suitable for classification analysis but is only semi-quantitative at determining absorption coefficients.

Head injuries, pentobarbital, and the determination of death.

The Bexar County Medical Examiner's Office in San Antonio, Texas, has encountered 3 cases within a 15-month period involving decedents who were pronounced dead by brain death or cardiac death examination and who had elevated, if not toxic concentrations of pentobarbital present at the time of examination. The elevated levels of pentobarbital were discovered during an autopsy examination performed for medicolegal reasons. The diagnosis of brain death and the implications of pentobarbital intoxication during a brain death examination are discussed.

Sex differences in NeuN- and androgen receptor-positive cells in the bed nucleus of the stria terminalis are due to Bax-dependent cell death.

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is larger in males than in females of several species. We previously demonstrated that in mice lacking the pro-death gene, bax, total BNSTp cell number is increased and sex differences in cell number are eliminated. This suggests that Bax-dependent cell death underlies sexual differentiation of the BNSTp. However, it is not known what cells in the BNSTp are affected by bax deletion. Here we used immunohistochemistry and stereological techniques to quantify phenotypically-identified cells in the BNSTp of adult male and female bax -/- and bax +/+ mice. Sections were thionin-stained, or double-labeled for antigen expressed in neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) to identify mature neurons and astrocytes, respectively; an additional series was labeled for androgen receptor (AR). As previously demonstrated, sex differences in BNSTp area and overall cell number were seen in wild-type mice, but absent in bax -/- animals. In addition, sex differences (favoring males) were present in the number of NeuN+ and AR+ cells in wild-type mice. Bax gene deletion significantly increased the number of NeuN+ and AR+ cells and reduced or eliminated the sex differences in these cell types. The number of astrocytes in the BNSTp was not sexually dimorphic, nor significantly affected by bax gene status, although there was a trend for more GFAP+ cells in bax -/- mice. Overall brain weight was also greater in bax -/- animals compared with controls. We conclude that the sex differences in neuron and AR+ cell number are due at least in part to Bax-mediated cell death. Increased NeuN+ and AR+ cell number in bax -/- mice suggests that supernumerary cells in bax knockouts differentiate similarly to those in wild-type mice, and retain the capacity to respond to androgens.

Comparison of drug concentrations taken from clamped and unclamped femoral vessels.

Postmortem drug concentrations may vary depending on sampling site, volume of blood collected, and method of sampling, making it important to analyze specimens from different sites in the body to detect postmortem redistribution and avoid erroneous conclusions on cause and manner of death. Using a blind stick method to draw large amounts of blood from the femoral vessel may increase the likelihood of contamination with blood from more central sites. It has been suggested that clamping the femoral vessel before drawing the sample may eliminate possible contribution from central sites. Eight drugs from four different drug classes were evaluated to determine the difference between drug concentrations in clamped and blind stick femoral blood. Drug concentrations of three selective serotonin reuptake inhibitors, or SSRIs (sertraline, paroxetine, citalopram), two benzodiazepines (diazepam and alprazolam), two antihistamines (diphenhydramine and promethazine), and one opiate (hydrocodone) were evaluated in clamped femoral blood, blind stick femoral blood, and heart blood and compared using concentration ratios and linear regression analysis. Clamped femoral blood concentrations and blind stick femoral blood concentrations were found to have good predictability across all drug classes with ratios around 1.0, indicating good correlation between blind stick femoral and clamped femoral samples. Therefore, it can be concluded that a blind stick femoral blood sample does not have significant redistribution from central sites and is of equivalent quality to a clamped femoral sample.

Genetic background influences the behavioural and molecular consequences of neurokinin-1 receptor knockout.

Genetic background affects animal phenotype and therefore is of particular relevance to studies using genetically manipulated mice. Strain differences in hypothalamic-pituitary-adrenocortical (HPA) axis activity may contribute to background-specificity of some mutations. Here, we analysed components of the HPA axis in mice lacking a functional neurokinin-1 receptor (NK1-/-) on two backgrounds: backcrossed C57BL/6 (B6) and mixed C57BL/6 x 129/sv (129B6). We hypothesized that HPA axis activity would vary between these strains, leading to differences in the NK1-/- phenotype. We compared levels of plasma corticosterone between the groups, and found 129B6 mice exhibited elevated levels of stress-induced corticosterone compared with B6 mice, regardless of genotype. Although the level of basal corticotrophin-releasing factor and stress-induced c-fos mRNAs did not differ between the genotypes of either strain, examination of glucocorticoid receptor immunoreactivity within the hippocampus revealed that NK1-/- mice on the 129B6 background had elevated expression compared with wild-type, whilst there was no difference between genotypes in the B6 strain. Similarly, hippocampal neurogenesis in NK1-/- mice was greater than in wild-type on the 129B6 strain, and did not differ between genotypes on the B6 background. Finally, novelty- and morphine-induced locomotion were assessed. NK1-/- mice on the 129B6 background exhibited hyperlocomotion in response to novelty and greater sensitivity to the locomotor-stimulating properties of morphine than wild-type. In contrast, in B6 mice, no differences were observed between genotypes for either locomotor behaviour. In summary, we find that HPA axis activity differs between the strains and that there are profoundly background-specific effects of the NK1 receptor mutation.

Safety and effectiveness of topical dry mouth products containing olive oil, betaine, and xylitol in reducing xerostomia for polypharmacy-induced dry mouth.

Polypharmacy is a common cause of salivary hypofunction, producing symptoms of dry mouth or xerostomia, especially among older populations. As the number of older people continues to increase, polypharmacy-induced salivary hypofunction is becoming an increasing problem. Many over-the-counter products are available for relieving symptoms of dry mouth, but few have been tested in controlled clinical investigations. The purpose of this investigation was to evaluate the safety and efficacy of a group of topical dry mouth products (toothpaste, mouth rinse, mouth spray and gel) containing olive oil, betaine and xylitol. Forty adults were entered into this single-blinded, open-label, cross-over clinical study and 39 completed all the visits. Subjects were randomly assigned at baseline to using the novel topical dry mouth products daily for 1 week, or to maintain their normal dry mouth routine care. After 1 week, they were crossed over to the other dry mouth regimen. The results demonstrated that the use of the novel topical dry mouth products increased significantly unstimulated whole salivary flow rates, reduced complaints of xerostomia and improved xerostomia-associated quality of life. No clinically significant adverse events were observed. These data suggest that the daily use of topical dry mouth products containing olive oil, betaine and xylitol is safe and effective in relieving symptoms of dry mouth in a population with polypharmacy-induced xerostomia.

The role of endocrine disrupters in water recycling: risk or mania?

The widespread occurrence of endocrine disrupting chemicals (EDCs), such as steroid hormones, in secondary wastewater effluents has become a major concern in the water recycling practice. This paper investigates the risk of steroid hormone breakthrough during nanofiltration membrane filtration in water recycling applications. The results indicate a dynamic equilibrium between adsorption and desorption of steroid hormone with regard to the membrane. This equilibrium can be pH dependent and there is a possibility for release of steroid hormones at high pH during membrane cleaning procedures or erratic pH variations. Increase in water recovery can severely increase the hormone breakthrough concentration. The results also indicate a possibility of accumulation of steroid hormones in the NF membrane, followed by subsequent release.

Screening postmortem whole blood for oxycodone by ELISA response ratios.

The objective of this study was to investigate the accuracy of screening postmortem whole blood for oxycodone using the ratio of the oxycodone immunoassay response to the response for the specimen obtained with a general opiate-class immunoassay. Fifty eight specimens which were negative for opiates and 158 postmortem whole blood specimens positive for opiates including 66 specimens known to contain oxycodone were assayed. Specimens were diluted 1:5 with assay buffer and analyzed by both the Neogen Oxymorphone/Oxycodone ELISA and the Neogen Opiate Group ELISA (Neogen Corporation, Lexington KY). The oxycodone equivalents in ng/mL from the Oxymorphone/Oxycodone ELISA were divided by the morphine equivalents in ng/mL from the Opiates ELISA to obtain an Oxycodone/Opiates Response Ratio. This ratio was compared with the GC/MS data for all specimens and for opiate positive specimens. Receiver Operating Characteristic (ROC) analysis suggested that optimum relative response ratio was 2.0. The sensitivity of the ELISA response ratio for the presence of oxycodone at a response ratio cutoff of 2.0 was 89.4% +/- 3.8% and the specificity was 88.1% +/- 3.2%. Specimens with a ratio of 2.0 or higher had a greater than 50% probability (positive predictive value) of containing oxycodone in a population with a greater than 15% prevalence of oxycodone.

Identification of gammadeltaT lymphocytes in human periapical lesions.

Endodontic (root canal) therapy is required when the pulp of a tooth becomes necrotic due to a bacterial infection or trauma. A proportion of patients who receive endodontic therapy subsequently have periapical (around the tooth root) lesions detected by radiolucency. Currently, there are no means to identify susceptible patients. Although tissue from periapical lesions has been described as inflammatory, inflammatory cell types and their functions have been poorly characterized. For example, T lymphocytes were identified using pan specific anti-CD3 mAb, which recognizes both alphabeta and gammadeltaT cells. Using the current model of gammadeltaT cells as immunoregulatory cells; gammadeltaT cells can mediate protective or destructive milieus. We postulated that patients who have a periapical lesion, as identified by radiographic bone loss, mount a gammadeltaT cell response. We collected specimens removed by surgery from both periapical lesions and other oral tissues, generated total RNA and performed reverse-transcriptase polymerase chain reaction to identify rearranged delta genes. Results were confirmed with semi-nested polymerase chain reaction. In addition, we demonstrate that these lesions contain a population of CD3+ cells that are alphabetaT cell receptor negative, implying that these cells are gammadeltaT cells. Here we show that 36/37 of periapical lesions and only 2/11 of other lesions contain gammadeltaT cells (P<0.0001). Vdelta2+ T cells were the most common subtype identified (30/36) in these samples. This is the first report in the literature of the presence of gammadeltaT cells in human periapical lesions.

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: purification, crystallization and structure determination.

We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 A resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 A resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.

Suicide gene therapy for premalignant disease: a new strategy for the treatment of intraepithelial neoplasia.

The potential of gene therapy to treat premalignant disease or recurrent cancer has not been investigated. The goal of the present investigation was to explore the efficacy of pro-drug-mediated, suicide gene therapy as a strategy to treat incipient neoplasia in stratified squamous epithelium. To test this strategy, a tissue model of premalignancy was generated by mixing normal human keratinocytes (NHK) that express the bacterial cytosine deaminase gene (CD) with premalignant keratinocytes which have been genetically marked with the bacterial gene for beta-galactosidase (II-4-beta-gal) in skin-like organotypic cultures. Preliminary studies in monolayer cultures demonstrated that CD-transduced NHK (NHK/CD) efficiently expressed the transgene and deaminated the pro-drug 5-fluorocytosine (5FC) to the toxic product 5-fluorouracil (5FU). The capacity of NHK/CD to kill II-4-beta-gal cells through bystander effect was assayed in both submerged culture and in the organotypic model of premalignancy. In submerged cultures, it was found that CD-mediated killing of II-4-beta-gal cells did not require cell-cell contact and that the LD(50) of 5FC for efficient bystander killing of II-4-beta-gal was 0.5 mM. When this concentration of pro-drug was used in organotypic cultures, a significant number of dysplastic II-4-beta-gal cells were eliminated from the tissue. Bystander killing of II-4-beta-gal cells was related to the number of NHK/CD present. These findings demonstrated that potentially malignant keratinocytes could be eliminated from a dysplastic tissue through activation of pro-drug and killing of adjacent cells through the bystander effect. By establishing an in vitro model to eliminate premalignant cells using suicide gene therapy, these studies provide a new approach for the treatment of incipient cancer as it develops, thereby preventing invasive disease.

Adverse events, including death, associated with the use of 1,4-butanediol.

BACKGROUND: 1,4-Butanediol is an industrial solvent that, when ingested, is converted to gamma-hydroxybutyrate, a drug of abuse with depressant effects, primarily on the central nervous system. After reports of toxic effects of gamma-hydroxybutyrate and its resultant regulation by the federal government, 1,4-butanediol and gamma-butyrolactone, another precursor of gamma-hydroxybutyrate and an industrial solvent, began to be marketed as dietary supplements. We investigated reports of toxic effects due to the ingestion of 1,4-butanediol and reviewed the related health risks. METHODS: From June 1999 through December 1999, we identified cases of toxic effects of 1,4-butanediol involving patients who presented to our emergency departments with a clinical syndrome suggesting toxic effects of gamma-hydroxybutyrate and a history of ingesting 1,4-butanediol and patients discovered through public health officials and family members. We used gas chromatography-mass spectrometry to measure 1,4-butanediol or its metabolite, gamma-hydroxybutyrate, in urine, serum, or blood. RESULTS: We identified nine episodes of toxic effects in eight patients who had ingested 1,4-butanediol recreationally, to enhance bodybuilding, or to treat depression or insomnia. One patient presented twice with toxic effects and had withdrawal symptoms after her second presentation. Clinical findings and adverse events included vomiting, urinary and fecal incontinence, agitation, combativeness, a labile level of consciousness, respiratory depression, and death. No additional intoxicants were identified in six patients, including the two who died. The doses of 1,4-butanediol ingested ranged from 5.4 to 20 g in the patients who died and ranged from 1 to 14 g in the nonfatal cases. CONCLUSIONS: The health risks of 1,4-butanediol are similar to those of its counterparts, gamma-hydroxybutyrate and gamma-butyrolactone. These include acute toxic effects, which may be fatal, and addiction and withdrawal.

What should we do? Papillary thyroid carcinoma in a lymph node but normal thyroid tissue--how should we proceed?

Papillary cell carcinoma of the thyroid is a relatively indolent disease, usually presenting as an asymptomatic mass in the thyroid gland that is either noted by the patient or diagnosed at routine clinical examination. Although the prognosis is generally good there are a number of controversies in the management of this condition. The significance of age at presentation, size of the tumour and the presence of lymph node metastasis are still disputed. We present the unusual case of a woman found to have papillary cell thyroid carcinoma within a lymph node during a cosmetic thyroid lobectomy. The thyroid tissue removed was not found to contain tumour. We describe the evidence behind the decision to proceed to total thyroidectomy and the finding of a tiny focus of papillary cell thyroid carcinoma within the contralateral lobe.