Cooled feline intestine and fresh intestine did not differ in enterotomy leak pressure testing or in gross wall thickness measurement.

OBJECTIVE: To report gross anatomical gastrointestinal measurements and compare enterotomy leak pressures between fresh and cooled feline cadavers. STUDY DESIGN: Ex vivo, randomized study. ANIMALS: Fresh feline cadavers (n = 20). METHODS: Jejunal segments (8 cm) were harvested on the same day as euthanasia. From each cadaver, one segment was randomly assigned to control (C), fresh enterotomy (FE), and cooled enterotomy (CE) groups. Enterotomy construction and leak testing were performed within 12 h of euthanasia for the C and FE groups and after 17-29 h of cooling for the CE group. Initial leak pressure (ILP) and maximum intraluminal pressure (MIP) were compared. Gastrointestinal wall thickness and intraluminal diameter were measured on harvested applicable gastrointestinal divisions at up to three time points: day 1 fresh, day 2 cooled, and day 3 cooled. RESULTS: The mean (± SD) ILPs for the C, FE, and CE constructs were 600 (± 0.0), 200.3 (± 114.7), and 131.3 (± 92.6) mmHg, respectively. The C ILP was higher (p < .001) than the FE and CE ILP. The ILP (p = .11) and the MIP (p = .21) did not differ between the FE and CE constructs. Wall thickness (measured in mm) did not differ between duodenum day 1 fresh and day 2 cooled groups (p = .18) or between any jejunum day groups (p = .86). The intraluminal diameters (mean ± SD) for the duodenum, jejunum, and ileum were 5.7 (± 0.7), 5.8 (± 0.8), and 7.2 (± 2.2) mm, respectively. CONCLUSION: No difference was appreciated between FE and CE ILP and MIP. Wall thickness measurements did not differ between days for duodenum or jejunum. CLINICAL RELEVANCE: Cadaveric feline intestine cooled for up to 29 h may be used for determining intestinal leak pressures.

Use of serum osmolality to identify heart disease stage in dogs and relationship to mathematical chloride correction.

BACKGROUND: Heart failure-associated hypochloremia can be depletional from diuretics or dilutional from water retention. Serum osmolality reflects water balance but has not been evaluated in dogs with heart disease. HYPOTHESIS: To determine if serum osmolality is related to heart disease stage and amount of mathematical correction of serum chloride (Cl- ) concentrations in healthy dogs and dogs with myxomatous mitral valve degeneration (MMVD). ANIMALS: Seventy-seven dogs (20 healthy, 25 Stage B MMVD, 32 Stage C/D MMVD). METHODS: Serum Cl- concentrations were mathematically corrected. Osmolality was calculated (calOsm) and directly measured by freezing point depression (dmOsm) and compared by Bland-Altman analysis. Biochemical variables and osmolality were compared among healthy, Stage B, and Stage C/D dogs. Correlations were explored between osmolality and biochemical variables. Median and range are presented. P < .05 was considered significant. RESULTS: The calOsm was different among groups (P = .003), with Stage B (310 mOsm/kg; 306, 316) and C/D dogs (312 mOsm/kg; 308, 319) having higher calOsm than healthy dogs (305 mOsm/kg; 302, 308). Osmolality methods were moderately correlated (P < .0001, rs = .46) but with proportional bias and poor agreement. The amount of Cl- correction was negatively correlated with calOsm (P < .0001, rs = -.78) and dmOsm (P = .004, rs = -.33). Serum bicarbonate concentration was negatively correlated with Cl- (P < .0001, rs = -.67). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with Stage B and Stage C/D heart disease had higher calOsm than healthy dogs. Osmolality was inversely related to the amount of Cl- correction, which supports its use in assessing relative body water content. Poor agreement between calOsm and dmOsm prevents methodological interchange.

An LC-MS/MS method for simultaneous analysis of up to six monoamines from brain tissues.

Monoamines are a class of neuromodulators that are crucial for a variety of brain functions, including control of mood, movement, sleep and cognition. From mammals to insects, the nervous system is enriched in monoamines such as dopamine, serotonin and melatonin, analytes which range from being highly polar to non-polar. Here we developed a method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to quantify in a single run the amounts of six distinct monoamines in extracts from dissected Drosophila and mouse brain tissues. The measured monoamines were dopamine (DA), serotonin (also known as 5-hydroxytryptamine (5-HT)), octopamine (OA, an insect equivalent of norepinephrine), tyramine (TA), melatonin (MT) and N-acetylserotonin (NAS). The analytical range of these monoamines was between 0.25 and 5.0 ng/mL. This quantitative LC-MS/MS methodology has important use for simultaneous measurement of distinct neuroactive monoamines from precious biological specimens.

An LC-MS/MS Methodological Framework for Steroid Hormone Measurement from Human Serum.

Estrogens and androgens are important regulators of sexual development and physiological processes in men and women, acting on numerous organs throughout the body. Moreover, they can contribute to a variety of pathologies, including osteoporosis, cancer, and cardiovascular and neurologic diseases. Analysis of estrogens and androgens in biological samples has been commonly performed using immunoassays for many years. However, these assays are suboptimal, as there is cross-reactivity with similar analytes, and they have moderate specificity and sensitivity. Thus, there is a clinical need to develop highly sensitive and specific methods for the accurate measurement of estrogen and androgen concentrations. Herein, we describe the development of three liquid chromatography coupled tandem mass spectrometry-based methods that incorporate the use of a Triple Quadrupole Mass Spectrometer for quantitative measurement of endogenous concentrations of various steroid hormones in human serum samples: (1) the simultaneous measurement of testosterone, androstenedione, and cortisol, (2) dehydroepiandrosterone (DHEA), and (3) 17ß-estradiol (E2). The use of derivatizing reagents, Girard's reagent P and dansyl chloride, allowed for significant gains in sensitivity in the analysis of DHEA and E2, respectively, relative to the underivatized analyte. These procedures proved efficient and adequately sensitive for steroid hormone analysis in extracted patient sera samples from older men and postmenopausal women, providing reliable data down to low nanogram/ml and sub-nanogram/ml levels. Moreover, utilizing the combination of highly specific mass transitions associated with these analytes and their respective internal deuterated standards provided a high degree of specificity to the identity of these hormones.

Dynamic Remodeling of Membranes and Their Lipids during Acute Hormone-Induced Steroidogenesis in MA-10 Mouse Leydig Tumor Cells.

Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM-ER-mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling.

Selecting effective intervention strategies for escape-maintained academic-performance problems: Consider giving 'em a break!

This study compared consequence-and antecedent-based strategies to determine which treatments or combination of treatments produced the strongest improvements in math computation fluency with four elementary-aged students whose math computation was under the control of an escape contingency. Functional analyses were conducted to identify elementary-school students whose academic responding was under a negative-reinforcement contingency. A multielement design was then used to examine the impact of four treatments (DNRA, DRA, task choice, and task choice plus DRA) on each student's rate of correct digits per min. All four treatments increased rate of responding. Differentiated results were obtained for all participants, indicating a reliable effect. Yet, participants responded differently to the treatments, illustrating the need to investigate and adapt interventions for escape-motivated behavior on a case-by-case basis. Results are also discussed in terms of the effectiveness of choice relative to reinforcement procedures, whether there were additional benefits to combining treatments, and which type of reinforcement procedures (DRA or DNRA) appears to be more effective for students whose behavior is under the control of an escape contingency.

TSPO mutations in rats and a human polymorphism impair the rate of steroid synthesis.

The 18 kDa translocator protein (TSPO) is a ubiquitous conserved outer mitochondrial membrane protein implicated in numerous cell and tissue functions, including steroid hormone biosynthesis, respiration, cell proliferation, and apoptosis. TSPO binds with high affinity to cholesterol and numerous compounds, is expressed at high levels in steroid-synthesizing tissues, and mediates cholesterol import into mitochondria, which is the rate-limiting step in steroid formation. In humans, the rs6971 polymorphism on the TSPO gene leads to an amino acid substitution in the fifth transmembrane loop of the protein, which is where the cholesterol-binding domain of TSPO is located, and this polymorphism has been associated with anxiety-related disorders. However, recent knockout mouse models have provided inconsistent conclusions of whether TSPO is directly involved in steroid synthesis. In this report, we show that TSPO deletion mutations in rat and its corresponding rs6971 polymorphism in humans alter adrenocorticotropic hormone-induced plasma corticosteroid concentrations. Rat tissues examined show increased cholesteryl ester accumulation, and neurosteroid formation was undetectable in homozygous rats. These results also support a role for TSPO ligands in diseases with steroid-dependent stress and anxiety elements.

ATRX is a novel progesterone-regulated protein and biomarker of low developmental potential in mammalian oocytes.

A multi-species meta-analysis of published transcriptomic data from models of oocyte competence identified the chromatin remodelling factor ATRX as a putative biomarker of oocyte competence. The objective of the current study was to test the hypothesis that ATRX protein expression by cumulus-oocyte complexes (COCs) reflects their intrinsic quality and developmental potential. In excess of 10,000 bovine COCs were utilised to test our hypothesis. COCs were in vitro matured (IVM) under conditions associated with reduced developmental potential: IVM in the presence or absence of (1) progesterone synthesis inhibitor (Trilostane); (2) nuclear progesterone receptor inhibitor (Aglepristone) or (3) an inducer of DNA damage (Staurosporine). ATRX protein expression and localisation were determined using immunocytochemistry and Western blot analysis. A proportion of COCs matured in the presence or absence of Trilostane was in vitro fertilised and cultured, and subsequent embryo development characteristics were analysed. In addition, ATRX expression was investigated in 40 human germinal vesicle-stage COCs. Our results showed that ATRX is expressed in human and bovine germinal vesicle oocytes and cumulus cells. In bovine, expression decreases after IVM. However, this decline is not observed in COCs matured under sub-optimal conditions. Blastocyst development rate and cell number are decreased, whereas the incidence of abnormal metaphase phase spindle and chromosome alignment are increased, after IVM in the presence of Trilostane (P < 0.05). In conclusion, localisation of ATRX to the cumulus cell nuclei and oocyte chromatin, after IVM, is associated with poor oocyte quality and low developmental potential. Furthermore, ATRX is dynamically regulated in response to progesterone signalling.

Identifying controlling variables for math computation fluency through experimental analysis: the interaction of stimulus control and reinforcing consequences.

This study investigated a method for conducting experimental analyses of academic responding. In the experimental analyses, academic responding (math computation), rather than problem behavior, was reinforced across conditions. Two separate experimental analyses (one with fluent math computation problems and one with non-fluent math computation problems) were conducted with three elementary school children using identical contingencies while math computation rate was measured. Results indicate that the experimental analysis with non-fluent problems produced undifferentiated responding across participants; however, differentiated responding was achieved for all participants in the experimental analysis with fluent problems. A subsequent comparison of the single-most effective condition from the experimental analyses replicated the findings with novel computation problems. Results are discussed in terms of the critical role of stimulus control in identifying controlling consequences for academic deficits, and recommendations for future research refining and extending experimental analysis to academic responding are made.

Negative energy balance affects imprint stability in oocytes recovered from postpartum dairy cows.

Ovarian follicle development in post-partum, high-producing dairy cows, occurs in a compromised endogenous metabolic environment (referred to as negative energy balance, NEB). Key events that occur during oocyte/follicle growth, such as the vital process of genomic imprinting, may be detrimentally affected by this altered ovarian environment. Imprinting is crucial for placental function and regulation of fetal growth, therefore failure to establish and maintain imprints during oocyte growth may contribute to early embryonic loss. Using ovum pick-up (OPU), oocytes and follicular fluid samples were recovered from cows between days 20 and 115 post-calving, encompassing the NEB period. In a complimentary study, cumulus oocyte complexes were in vitro matured under high non-esterified fatty acid (NEFA) concentrations and in the presence of the methyl-donor S-adenosylmethionine (SAM). Pyrosequencing revealed the loss of methylation at several imprinted loci in the OPU derived oocytes. The loss of DNA methylation was observed at the PLAGL1 locus in oocytes, following in vitro maturation (IVM) in the presence of elevated NEFAs and SAM. Finally, metabolomic analysis of postpartum follicular fluid samples revealed significant differences in several branched chain amino acids, with fatty acid profiles bearing similarities to those characteristic of lactating dairy cows. These results provide the first evidence that (1) the postpartum ovarian environment may affect maternal imprint acquisition and (2) elevated NEFAs during IVM can lead to the loss of imprinted gene methylation in bovine oocytes.

De novo synthesis of steroids and oxysterols in adipocytes.

Local production and action of cholesterol metabolites such as steroids or oxysterols within endocrine tissues are currently recognized as an important principle in the cell type- and tissue-specific regulation of hormone effects. In adipocytes, one of the most abundant endocrine cells in the human body, the de novo production of steroids or oxysterols from cholesterol has not been examined. Here, we demonstrate that essential components of cholesterol transport and metabolism machinery in the initial steps of steroid and/or oxysterol biosynthesis pathways are present and active in adipocytes. The ability of adipocyte CYP11A1 in producing pregnenolone is demonstrated for the first time, rendering adipocyte a steroidogenic cell. The oxysterol 27-hydroxycholesterol (27HC), synthesized by the mitochondrial enzyme CYP27A1, was identified as one of the major de novo adipocyte products from cholesterol and its precursor mevalonate. Inhibition of CYP27A1 activity or knockdown and deletion of the Cyp27a1 gene induced adipocyte differentiation, suggesting a paracrine or autocrine biological significance for the adipocyte-derived 27HC. These findings suggest that the presence of the 27HC biosynthesis pathway in adipocytes may represent a defense mechanism to prevent the formation of new fat cells upon overfeeding with dietary cholesterol.

Improving oral reading fluency with a peer-mediated intervention.

This study examined the effects of an experimentally derived, peer-delivered reading intervention on the oral reading fluency of a first-grade student who had been referred for poor reading fluency. Same-grade peers were trained to lead the target student through a structured intervention protocol based on the results of a brief experimental analysis. Results indicated that reading improvements were obtained and are discussed in terms of selecting efficient interventions for use by peers.

A comparison of two flash-card methods for improving sight-word reading.

Flash cards have been shown to be useful for teaching sight-word reading. To date, the most effective flash-card instruction method is incremental rehearsal (IR). This method involves the instructor interspersing unknown stimulus items into the presentation of known stimulus items. In this study, we compared IR to a modified IR procedure-strategic incremental rehearsal (SIR)-to determine whether the effects of IR might be improved by incorporating variables likely to increase word acquisition. These included increased opportunities to respond to unknown stimuli, using learner responding as a basis for changing instructional items, and systematic prompting methods. An A-B-A-B design was used to compare the effects of IR and SIR for increasing sight-word reading with 4 elementary school students. Results indicated that students read more words correctly with SIR than with IR. In addition, similar patterns of responding were seen at a 2-week follow-up.

Evaluation of the multiple-stimulus without replacement preference assessment method using activities as stimuli.

The current study examined the accuracy of the multiple-stimulus without replacement (MSWO) preference assessment for identifying preferred common classroom activities as reinforcers with children with behavioral disorders. The accuracy of predictions from the MSWO regarding high, medium, and low stimulus preference was tested by providing contingent access to activities for completing math problems within an independent seatwork format. Overall, there was an interaction effect between preference ranking (high, medium, or low) and number of problems completed. The results confirm and extend previous findings regarding the accuracy of predictions with the MSWO. The findings also reveal, however, some individual differences that may account for instances in which student behavior did not conform to predictions of stimulus preference assessments.

Improving reading outcomes for children with learning disabilities: using brief experimental analysis to develop parent-tutoring interventions.

The present study assessed the effects of summer parent tutoring on 3 children with learning disabilities using empirically derived reading interventions. Brief experimental analyses were used to identify customized reading fluency interventions. Parents were trained to use the intervention strategies with their children. Parents implemented the procedures during parent-tutoring sessions at home and results were measured continuously in high-word-overlap and low-word-overlap passages to determine whether generalization occurred. Parent and child satisfaction with the procedures was assessed. Results demonstrated generalized increases in reading fluency in both high-word-overlap and low-word-overlap passages as a function of parent tutoring. Also, acceptability ratings by children and their parents indicated that they viewed the interventions as acceptable and effective. Results are discussed in terms of structuring reading fluency interventions that promote generalization and maintenance of treatment effects.

Refining the experimental analysis of academic skills deficits: part II. Use of brief experimental analysis to evaluate reading fluency treatments.

The technology of brief experimental analysis is just beginning to be used for identification of effective treatments for individual students who experience difficulty with oral reading fluency. In this study, the effect of a reading fluency treatment package was examined on easy and hard passages, and generalization was assessed on passages with high content overlap. The results suggest that the treatment package increased reading fluency for all 3 students. Effects were moderated by difficulty level for all 3 students. Results are discussed in terms of future refinements to the procedures, validation of the methods, and potential applications in clinical and school settings.

Refining the experimental analysis of academic skills deficits: Part I. An investigation of variables that affect generalized oral reading performance.

Experimental analyses for improving reading fluency deficits have rarely targeted generalized increases in academic responding. As a consequence, the variables that may help students to generalize newly learned forms of academic responding like reading are not well understood. Furthermore, experimental analyses of reading fluency interventions have not systematically examined difficulty level as a variable that may affect instructional outcomes. The experiment reported in this paper expands (a) the measurement of the dependent variables to include generalized increases across tasks (reading passages) and (b) the combination of independent variables used to produce measurable generalized increases. The results demonstrate the importance of combining reward and instructional variables (including difficulty level) to produce generalized increases and how those variables can be meaningfully investigated prior to making treatment recommendations.

An experimental analysis of reading interventions: generalization across instructional strategies, time, and passages.

This study examined the generalized effects of three treatment conditions (performance based, skill based, and a combination of the two) on oral reading fluency by an elementary school student. Results indicated equal effectiveness of all treatments, maintenance, and possible evidence of generalization across passages.

Improving oral reading fluency: a brief experimental analysis of combining an antecedent intervention with consequences.

A brief experimental analysis was used to evaluate the relative effectiveness of combining two consequences (contingent reinforcement or performance feedback) with an antecedent intervention (listening passage preview and repeated readings) on the oral reading fluency of 6 elementary students. The antecedent intervention increased the number of correctly read words per minute for all 6 students. For 4 of the students, pairing the antecedent intervention with either of the consequences resulted in higher reading rates over the antecedent intervention alone. Undifferentiated results were obtained for the remaining 2 participants. These results suggest that combining an antecedent intervention with consequences may enhance the oral reading fluency of students with reading problems. However, individual responsiveness to the different intervention components indicates that brief experimental analyses are warranted to identify the most effective intervention.