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Prior exposure to hypoxia alters DNA methylation patterns in the eastern oyster.

Environmentally induced epigenetic changes (e.g., DNA methylation) can alter genetic activity to help organisms adapt and respond to variable environments. While many studies have investigated DNA methylation as a response to a stressor at a single timepoint, less well-understood is how methylation may encode memory of past environments and influence the response to current environments (i.e., carryover effects). Oysters are an excellent natural system to study carryover effects due to their sessile nature, which may expose them to increased environmental variability. To better understand how methylation changes in response to a previous exposure of environmental stress, we conducted a fully factorial experiment exposing juvenile oysters to either control or hypoxic conditions at two timepoints separated by 60 days. After the second exposure, whole body tissue samples were collected and processed for methylRAD sequencing. Regardless of treatment, methylation was mostly found in exons. We found both the first and second exposure treatments contributed significantly to the observed variation in gene body methylation. Interestingly, oysters that were first exposed to hypoxia and later exposed to control conditions had methylation patterns that differed the most from any other condition. We found that differentially methylated genes identified in pairwise comparisons were mainly involved in the oxidative stress response, metabolism, and transcription. Together, these findings suggest that early life environments have a lasting impact on the epigenome and that the timing of stress elicits unique response strategies, which highlights potential targets of resilience for oysters.

Animals

Analgesic activity of floctafenine after cholecystectomy.

In a double-blind, crossover study involving 40 post-cholecystectomy patients, the analgesic activity of a new oral nonnarcotic agent, floctafenine, was compared with that of oral meperidine and oral propoxyphene. A behavioral approach to the measurement of pain relief, i.e., the measurement of pain on movement or coughing, was used in addition to the accepted subjective method. Floctafenine 200 mg was found to have analgesic activity intermediate between that of meperidine 75 mg and propoxyphene 65 mg. Patients reported the fewest side effects after floctafenine. Initial pain level was found to affect the final pain score. Effects on pain relief due to age, sex, time of the day, and carryover effects were also examined and not found to be significant.

Adolescent

Sleep laboratory studies of flurazepam: a model for evaluating hypnotic drugs.

The results from six separate evaluations of flurazepam 30 mg in the sleep laboratory were combined to determine the effectiveness of the drug in inducing and maintaining sleep and its effects on sleep stages in a large sample of insomniac subjects. The combined studies provide a model from which a detailed profile of the effects of a hypnotic drug over short-, intermediate-, and long-term conditions can be thoroughly evaluated. Although sleep was significantly improved on the first night of flurazepam administration, peak effectiveness of the drug did not result until the second and third consecutive drug nights. Flurazepam continued to be effective in inducing and maintaining sleep with intermediate-and long-term drug use with only a slight loss of effectiveness with long-term use. Sleep was also significantly improved on the first and second nights of drug withdrawal. Carryover effectiveness of active metabolites of flurazepam from one drug night to the next drug night and to withdrawl nights is discussed. The clinical implications are discussed with regard to the time of peak effectiveness of the drug, dosage recommendations and schedule, minimizing possible effects of the drug on daytime performance, and the rationale and method for using drug holidays in the treatment regimen. With this comprehensive profile of the drug's actions, the physician is able to more rationally and effectively utilize the drug in treating the insomniac patient. With short-term administration, flurazepam produced a slight decrease in rapid eye movement (REM) sleep and an increase in REM latency. These effects were much more pronounced with intermediate-term drug administration, again possibly due to the accumulation of active metabolites. After withdrawal there was no rebound in REM sleep. Stages 3 and 4 sleep decreased progressively through short and intermediate drug administration. With initial withdrawal, there was a slight recovery in both sleep stages.

Anti-Anxiety Agents

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of twenty-five steroids in equine serum.

Steroids are potential biomarkers for monitoring equine pregnancy. However, immunoassays currently used for their quantification suffer from cross-reactivity and limited specificity, thus requiring more accurate methods. This study reports the development and validation of a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of 25 steroids covering the main biosynthetic pathways of progestogens, corticosteroids, androgens, and estrogens. Steroids were extracted by protein precipitation followed by evaporation, derivatization, and reconstitution before LC-MS/MS analysis. A surrogate matrix was used for calibration and validation to avoid endogenous interference. Validation was performed according to and partly adapted from Clinical and Laboratory Standards Institute guidelines (CLSI), including linearity, trueness, precision, limits of detection and quantification, measurement uncertainty, recovery, matrix effects, carryover, selectivity, and stability. Calibration curves were fitted using the best-performing weighted linear or quadratic regression model, yielding excellent linearity (R2&#xa0;>&#xa0;0.990), trueness between -9.0% and 2.3%, and intra- and inter-day precision <6.3%. Lower limits of quantification ranged from 2.07 to 2250&#xa0;pg/mL depending on physiological analytes concentration. Extraction recovery averaged 24.3-114.9%, matrix effects were acceptable, and accuracy ranged from 94.4% to 98.9%. No carryover or interferences were detected. Measurement uncertainty remained <15%. This study presents the first LC-MS/MS method partially validated per CLSI criteria for the quantification of 24 steroids in equine serum. The method offers a sensitive and specific alternative to immunoassays and provides a robust tool for equine steroid profiling with potential applications in pregnancy monitoring, placentitis diagnosis, and fetal sex determination.

Animals

Alternating treatments design: one strategy for comparing the effects of two treatments in a single subject.

A little used and often confused design, capable of comparing two treatments within a single subject, has been termed, variously, a multielement baseline design, a multiple schedule design, and a randomization design. The background of these terms is reviewed, and a new, more descriptive term, Alternating Treatments Design, is proposed. Critical differences between this design and a Simultaneous Treatment Design are outlined, and experimental questions answerable by each design are noted. Potential problems with multiple treatment interference in this procedure are divided into sequential confounding, carryover effects, and alternation effects and the importance of these issues vis-a-vis other single-case experimental designs is considered. Methods of minimizing multiple treatment interference as well as methods of studying these effects are outlined. Finally, appropriate uses of Alternating Treatments Designs are described and discussed in the context of recent examples.

Behavior Therapy

Clinical evaluation of a quaternary ammonium-containing mouthrinse.

This was a double-blind, radomized, crossover study of adult subjects to evaluate the effect of a test mouthrinse (Cepacol) on plaque accumulation. The study was divided into two parts, four weeks each, one in which only a mouthwash was used (part I) and the other in which a mouthwash and toothbrushing were used (part II). 1. The test mouthrinse produced a satistically significant reduction in dental plaque when compared to a placebo rinse. 2. The GI averaged approximately 1.0 throughout the study regardless of which mouthrinse was used. 3. A possible carryover effect of the test mouthrinse was noted. 4. A lower plaque score was seen in 67 to 75% of all patients during the period in which the test mouthrinse was used as compared to the placebo. 5. Ten subjects reported a burning sensation of the tongue with the test mouthrinse. No objective adverse effects were seen.

Adult

Astragalus membranaceus therapy in patients with spinocerebellar ataxia type 3.

BACKGROUND AND AIM: Spinocerebellar ataxia type 3 (SCA3) is the most common form of hereditary cerebellar ataxia. The insulin/insulin-like growth factor 1 (IGF1) system (IIS) has been proposed as a potential target for disease-modifying therapy. Astragalus membranaceus (AM) may modulate the IIS pathway and reduce neurodegeneration, as indicated by plasma neurofilament light chain (NfL), a biomarker of disease progression. EXPERIMENTAL PROCEDURE: A randomized, triple-blind, placebo-controlled crossover trial was conducted in 32 patients with SCA3. Participants received AM or placebo for three months, followed by a one-month washout, then crossed over to the alternate treatment. Plasma NfL and IIS-related markers were measured. Twenty-three participants completed the trial. RESULTS AND CONCLUSION: Intention-to-treat analysis revealed a modest within-treatment reduction in NfL levels (27.6&#xa0;&#xb1;&#xa0;10.7 vs. 25.6&#xa0;&#xb1;&#xa0;9.8&#xa0;pg/mL, P&#xa0;=&#xa0;0.040) and increased insulin (12.5&#xa0;&#xb1;&#xa0;15.2 vs. 21.1&#xa0;&#xb1;&#xa0;15.5 &#x3bc;IU/mL, P&#xa0;=&#xa0;0.004) after AM. However, the adjusted regression model showed no significant between-treatment difference in NfL (P&#xa0;=&#xa0;0.127), and changes in NfL were not correlated with insulin. No significant changes were observed in IGF1 or IGF-binding proteins. Exploratory subgroup analyses suggested larger biomarker changes in later-stage patients. No carryover effects or demographic differences were noted. Overall, AM administration was associated with modest within-treatment biomarker changes in NfL and insulin, although the adjusted between-treatment difference in NfL was not statistically significant. These findings should be interpreted cautiously as preliminary and hypothesis-generating observations rather than confirmatory evidence of therapeutic efficacy.

Astragalus membranaceus

On assessment of bioequivalence under a higher-order crossover design.

In bioavailability studies of two formulations of a drug, the standard two-sequence, two-period crossover design is usually considered to assess bioequivalence. The standard two-sequence, two-period crossover design, however, may not be useful when differential carryover effects are present. In addition, it does not provide independent estimates of intrasubject variabilities for the two formulations. To overcome these problems, alternatively, a higher-order crossover design may be considered. In this paper, we derive statistical methods based on Schuirmann's two one-sided tests procedure for assessing bioequivalence for some commonly used higher-order crossover designs. Four designs, including Balaam's design, the two-sequence dual design, and two four-period designs (with two and four sequences), are considered. The relative merits of these designs as compared to the standard two-sequence, two-period design are discussed. Two examples concerning bioequivalence are used to illustrate the use of these methods.

Biological Availability

Therapeutic antagonism between anticholinergics and neuroleptics: possible involvement of cholinergic mechanisms in schizophrenia.

We have answered technical criticisms of our work in which anticholinergic agents were added to ongoing neuroleptic treatment in an ABA' research design. The suggested analysis of variance for repeated measures of the three periods is inappropriate because of the expected carryover effects from continuous neuroleptic treatment. The multivariate analysis of various parameters seems unsuitable because homogeneity of covariance cannot be ensured due to the heterogeneity of schizophrenia and the diverse factors represented in the psychopathology measures. We have summarized the results of recent parametric and nonparametric analyses of combined data from our three studies to show that the significant effects clearly pointed to therapeutic antagonism between anticholinergic agents and neuroleptics. We suggest that cholinergic neurons may be part of some crucial discriminative control mechanisms in the brain organization that are ineffective in schizophrenia and lead to a relative overactivity of the opposing catecholaminergic neurons in the midbrain-limbic circuitry which promote repetition of behaviors in goal-directed activity.

Antipsychotic Agents

[Treatment of organic brain psychosyndrom in the old age. A double blind study with Hydergin (author's transl)].

The efficacy of Hydergin was compared against placebo in a 15 weeks cross-over trial in 51 patients with cerebral insufficiency. The daily dosage was 3 mg of Hydergin. Criteria of evaluation consisted of clinical rating and electroencephalographic registrations, which were evaluated visually and partly automatically. The clinical symptoms as well as the electroencephalographic criteria (base line activity, theta- and delta activity) were both positively influenced by Hydergin. The base-line activity was stabilised and the alpha activity of the power spectrum increased. The most impressive result was the carryover effect of Hydergin, which could still be demonstrated in the post trial period. In case of "dizziness" the good results were limited to the period of active treatment. The relations between symptomatic and more basic therapy of cerebral insufficiency will be discussed.

Aged

In-vitro methods for determining minimal lethal concentrations of antimicrobial agents.

To determine the minimal lethal concentration of an antimicrobial agent, broth dilution tests may be performed and then a sample from each tube showing no visible growth may be subcultured onto a drug-free agar medium. By counting the number of viable cells recovered from each tube, the minimal concentration of drug required to kill 99.9% of the cells in the initial inoculum can be determined. Studies were undertaken to determine the circumstances under which false-negative tests could occur as a result of continued inhibition of growth by the antimicrobic carried over in the sample. Drug carryover did significantly reduce the number of viable cells recovered from broth containing relatively high concentrations of antimicrobic: the larger the sample, the greater the effect of drug carryover. The effect was minimal with samples of 10 microliter or less. Furthermore, the effect of drug carryover was reduced by spreading the sample over the surface of an agar medium. The relative precisions of four methods for performing subcultures with such small-volume samples were determined; coefficients of variation were 16-25%. Sampling errors inherent in the procedure for subculturing broth tubes should be taken into consideration when interpreting results of individual tests, and replicate subcultures will improve the reliability of the colony counts.

Enterococcus faecalis

Effect of cage size on patterns of activity and health of beagle dogs.

Thirty-two male beagle dogs were assigned at random, 16 to standard size cages (30 X 30 X 30 inches) and 16 to large cages (90 X 30 X 30 inches) for a period of 13 weeks, and then were "crossed-over" for an additional period of 13 weeks. The dogs were observed daily and weighed weekly. Electrocardiographic and ophthalmologic examinations were made once and hematologic and biochemical measurements were made twice during the control period and at monthly intervals during the study. Photographs were taken of each dog every 60 seconds, 7 days a week, for about 8.5 hours each day. The film was processed and analyzed for the acf standing sitting, lying, or sleeping. In addition, dogs in the large cages were scored for the region (front, middle, or back) occupied. No statistically significant differences were found between dogs in the standard or large cages with respect to weight gain, percent of time standing, and percent of time sleeping. Statistically significant (p less than 0.05) differences were found for percent of time sitting (standard cages 12.7%; large cages 9.4%) and percent of time lying (standard cages 6.6%; large cages 8.3%); however, the differences were not large enough to be of any practical concern. Transient patterns of response over 13-week periods of the study were essentially the same (statistically verified) for dogs in either size cage. Also statistical results showed that there was no significant carryover (residual) effect associated with any of the parameters measured. No beneficial or adverse effects were noted that could be related to the size of the two cages. The size of the standard cage appeared adequate for laboratory beagle dogs and no advantage was found when the dogs were in larger cages with respect to behavior, patterns of activity, or health.

Animals

Automated fluorometric analysis of epinephrine in lidocaine hydrochloride injection.

A rapid automated method for determining epinephrine in lidocaine hydrochloride injection is presented. The method is an adaptation of the official USP XIX fluorometric procedure and yields equivalent results. The linearity, accuracy, precision, excipient/background effect, and carryover characteristics of the automated system are described. A recovery study indicated that intact epinephrine is measured accurately in the presence of its degradation products. Analysis of several commercial preparations is reported.

Autoanalysis

The influence of males and females on the psychophysical judgments of females.

A transfer paradigm was employed to test the hypothesis that females influenced by males on one task would show more carryover of the influence effect to other tasks than would females influenced by other females. Thirty female college students made judgments on three cross-modal matching tasks. Experimental subjects gave their judgments on one task along with two male or two female confederates, who appeared to give discrepant judgments. They then made another series of private judgments on the other two tasks. Comparison to a control group and to an original series of provate judgments revealed a significant direct influence effect, but minimal transfer to the other two tasks. There was a nonsignificant tendency for the subjects to be more directly influenced by other females than by males, but this influence by the other females was accompanied by a decline in task confidence. Influence by males on females did not result in lowered confidence. This finding may be restricted to quantitative tasks and to American college students.

Adolescent

Epidemiological studies on Nematodirus species in sheep.

Faecal egg count and pasture larval studies on N battus and N filicollis in lambs showed distinct differences in epidemiology, the infective larvae of N battus hatching over a short restricted period in spring, whereas those of N filicollis show an extended period of hatching, beginning in autumn and increasing steadily through the winter to reach a spring peak. This difference was considered to be responsible for the much slower annual rate of increase recorded for N filicollis. Pasture infection with both species survived ploughing and reseeding, though in very small numbers, and one year's rest from sheep by alternate grazing with cattle proved equally effective in reducing the carryover of infection, and offered a practical method of controlling these species.

Animals

The recovery of mice from influenza virus infection: adoptive transfer of immunity with immune T lymphocytes.

Transfer of primary or secondary influenza-immune spleen cells to mice infected intranasally with influenza virus resulted in a significant clearance of virus from the lungs and the protection of the recipients from death. The antiviral activity was associated only with intact, viable cells and was not due to carryover of virus. The effector cell population responsible for the antiviral effect was shown to be T cells. Thus, the removal of adherent, phagocytic and Ig+ cells did not affect the antiviral activity, whereas it was destroyed with antitheta serum and complement. Antiviral activity was specific and was best expressed if the virus used to infect the recipients and to generate immune cells was the same strain. Further work will be necessary to define rigorously the role of different viral antigens in cell-mediated immune response to influenza virus infection.

Animals

Functional differentiation of T cell precursors. I. Parameters of carrier-specific tolerance in murine helper T cell precursors.

Irradiated mice reconstituted with bone marrow from sheep gamma-globulin- (SGG) tolerant syngeneic donors display reduced IgG responsiveness to challenge with trinitrophenylated (TNP)-SGG compared with recipients of normal marrow. This effect is SGG-specific and is due neither to suppressor T cells nor to antigen carryover. "Helper T cell precursor tolerance" can be induced with as little as 40 micrograms tolerogen (SGG). Unlike mature helper T cells, these precursors show both a rapid induction and rapid waning patterns, suggesting a high rate of turnover. Our results imply that marrow helper T cell precursors bear antigen-specific receptors and that the T cell repertoire must be at least partially generated before residence in the thymus.

Animals

Effect of carrier-specific tolerance on the generation of helper cell function.

The effect of carrier-specific tolerance on the development of helper cell function was studied in irradiated rats reconstituted with normal or carrier-tolerant bone marrow. Bone marrow (BM) from normal rats or rats tolerant to sheep IgG (SGG) was transferred to lethally irradiated syngeneic recipients, which were challenged 3 to 5 weeks later when immunocompetence to T-dependent antigens was shown to have recovered. When recipients were challenged with the 2,4,6-trinitrophenyl (TNP) hapten coupled to SGG in adjuvant, groups which received SGG-tolerant BM were 70-80% unresponsive in terms of anti-TNP plaque-forming cells compared to recipients of normal BM. This effect was carrier (SGG)-specific and was reversed when normal thymocytes were transferred with tolerant BM. Moreover, neither tolerant BM nor tolerant thymocytes were able to suppress the responsiveness of normal BM at a 1:1 ratio. These cell-mixing experiments imply that the reduction in helper cell function is due neither to suppressor cells nor antigen carryover in the tolerant BM. It is suggested that a BM precursor of the helper T cell may be rendered tolerant and therefore already possesses antigen specificity prior to thymic migration.

Animals