Multi-ancestry Genome-Wide Association Study of Early Childhood Caries.

Early childhood caries (ECC) is the most common non-communicable childhood disease. It is an important health problem with known environmental and social/behavioral influences that lacks evidence for specific associated genetic risk loci. To address this knowledge gap, we conducted a genome-wide association study of ECC in a multi-ancestry population of U.S. preschool-age children (n=6,103) participating in a community-based epidemiologic study of early childhood oral health. Calibrated examiners used ICDAS criteria to measure ECC with the primary trait using the dmfs index with decay classified as macroscopic enamel loss (ICDAS ≥3). We estimated heritability, concordance rates, and conducted genome-wide association analyses to estimate overall genetic effects; the effects stratified by sex, household water fluoride, and dietary sugar; and leveraged the combined gene/gene-environment effects using the 2-degree-of-freedom (2df) joint test. The common genetic variants explained 24% of the phenotypic variance (heritability) of the primary ECC trait and the concordance rate was higher with a higher degree of relatedness. We identified 21 novel non-overlapping genome-wide significant loci for ECC. Two loci, namely RP11-856F16 . 2 (rs74606067) and SLC41A3 (rs71327750) showed evidence of association with dental caries in external cohorts, namely the GLIDE consortium adult cohort (n=∼487,000) and the GLIDE pediatric cohort (n=19,000), respectively. The gene-based tests identified TAAR6 as a genome-wide significant gene. Implicated genes have relevant biological functions including roles in tooth development and taste. These novel associations expand the genomics knowledge base for this common childhood disease and underscore the importance of accounting for sex and pertinent environmental exposures in genetic investigations of oral health.

Metabolomics Insights in Early Childhood Caries.

Dental caries is characterized by a dysbiotic shift at the biofilm-tooth surface interface, yet comprehensive biochemical characterizations of the biofilm are scant. We used metabolomics to identify biochemical features of the supragingival biofilm associated with early childhood caries (ECC) prevalence and severity. The study's analytical sample comprised 289 children ages 3 to 5 (51% with ECC) who attended public preschools in North Carolina and were enrolled in a community-based cross-sectional study of early childhood oral health. Clinical examinations were conducted by calibrated examiners in community locations using International Caries Detection and Classification System (ICDAS) criteria. Supragingival plaque collected from the facial/buccal surfaces of all primary teeth in the upper-left quadrant was analyzed using ultra-performance liquid chromatography-tandem mass spectrometry. Associations between individual metabolites and 18 clinical traits (based on different ECC definitions and sets of tooth surfaces) were quantified using Brownian distance correlations (dCor) and linear regression modeling of log2-transformed values, applying a false discovery rate multiple testing correction. A tree-based pipeline optimization tool (TPOT)-machine learning process was used to identify the best-fitting ECC classification metabolite model. There were 503 named metabolites identified, including microbial, host, and exogenous biochemicals. Most significant ECC-metabolite associations were positive (i.e., upregulations/enrichments). The localized ECC case definition (ICDAS ≥1 caries experience within the surfaces from which plaque was collected) had the strongest correlation with the metabolome (dCor P = 8 × 10-3). Sixteen metabolites were significantly associated with ECC after multiple testing correction, including fucose (P = 3.0 × 10-6) and N-acetylneuraminate (p = 6.8 × 10-6) with higher ECC prevalence, as well as catechin (P = 4.7 × 10-6) and epicatechin (P = 2.9 × 10-6) with lower. Catechin, epicatechin, imidazole propionate, fucose, 9,10-DiHOME, and N-acetylneuraminate were among the top 15 metabolites in terms of ECC classification importance in the automated TPOT model. These supragingival biofilm metabolite findings provide novel insights in ECC biology and can serve as the basis for the development of measures of disease activity or risk assessment.

Rapid diagnosis of Staphylococcus aureus bacteremia using S. aureus PNA FISH.

In the study presented here, the performance of the S. aureus PNA FISH assay was evaluated using 285 blood cultures (from 104 patients) that had gram-positive cocci resembling staphylococci on Gram stain. The new molecular test is based on a fluorescence in situ hybridization assay using peptide nucleic acid probes targeting Staphylococcus aureus 16S rRNA and is designed for the rapid identification of Staphylococcus aureus directly from positive blood cultures. The sensitivity, specificity, and positive and negative predictive values of the S. aureus PNA FISH for the rapid identification of Staphylococcus aureus directly from positive blood culture bottles were 100, 99.4, 99.2 and 100%, respectively.

EtOH self-administration on shuttle box avoidance learning and extinction in rats.

The effects of ethanol on the acquisition and extinction of the two-way active avoidance response were examined in adult, male Wistar rats from two treatment groups, oral self-administration of alcohol solution (10% v/v ethanol and 3% w/v glucose in distilled water) and oral self-administration of control solution (3% w/v glucose in distilled water). Alcohol or control solutions were available 1 h per day during 15 days simultaneously with food, with free water for the rest of the day. Blood was drawn in the last day of this phase to evaluate blood ethanol levels (BEL). After this period, rats were tested in a two-bottle paradigm for 1 h per day and placed in a shuttle box immediately afterwards. This phase went lasted for 10 days. Subjects were trained to avoid an electric foot shock in the first 5 days (15 trials per day). Following this, half of the subjects were tested in an "easy extinction with punishment" (EEP) and the other half in a "difficult extinction with punishment" (DEP) of the avoidance response for the last 5 days. Alcohol accelerates the avoidance responding acquisition, and no significant effects of alcohol were seen in the extinction phase. Data are discussed in terms of the specificity of the effects of alcohol on learning.

Oral intake of sweetened or sweetened alcoholic beverages and open-field behavior.

The relationship between the intake of sweetened alcoholic beverages and individual differences in an open field was assessed using an oral self-administration procedure in male Wistar rats (n = 41). After four sessions in the open field, rats were gradually reduced to 80% of their ad lib body weights over a 10-day period. Rats were then allowed to drink an alcohol-containing solution (10% v/v ethanol, 3% w/v glucose) (experimental group: n = 20) or a solution of glucose (3% w/v glucose) (control group: n = 21) for 1 h/day during 9 consecutive days. Experimental rats were divided into two groups on the basis of the mean daily ethanol dose ingested (g/kg/h) in the nine sessions. The high ethanol-consuming (HEI rats), when compared with the low ethanol-consuming rats (LEI rats), only showed a tendency (p = 0.062) towards fewer global number of rearings in the open field. No relationship between open-field defecation and ethanol intake was observed. With regard to the control rats, the higher consuming also showed lower number of rearings in the open field, similarly to the experimental rats. When we divided all experimental or control rats into two subgroups on the basis of the mean daily tap-water ingested during 23 h/day, no differences in the number of rearings were found. The results suggest that rearing in a novel environment could be a predictor of susceptibility to reinforcement by sweetened or palatable beverages.

Effects of ketamine, a noncompetitive NMDA antagonist, on the acquisition of the lever-press response in rats.

We analyzed the effects of ketamine, a noncompetitive NMDA antagonist, on the acquisition of the lever-press response in the Skinner box and on motor performance both in the open field and in the inclined screen. Ninety-six adult male Wistar rats were assigned at random to eight different groups (n = 12). The first four groups received an acute intraperitoneal (IP) injection of: (a) physiological saline, (b) 4 mg/kg ketamine, (c) 8 mg/kg ketamine, or (d) 12 mg/kg ketamine, and the subjects were tested in a free lever-press response shaping in the Skinner box. The second four groups received the same substances and doses as the first four, but the subjects were tested for locomotor activity in an open field and tested immediately afterwards for motor performance in an 80 degrees inclined screen. Results showed that ketamine impaired the acquisition of the lever-press response in a dose-dependent manner, with no effects on ambulation in the open field nor on length of stay in the inclined screen. These results suggest that ketamine effects on the acquisition of the lever-press response cannot be attributed to a motor impairment, indicating a possible specific effect of ketamine on the associative learning acquisition.

Effects of ethanol, caffeine, and clorazepate on hypertonic NaCl solution intake in rats.

Previous reports indicate that several anxiolytics enhance the intake of hypertonic saline in rehydrating rats. This experiment was conducted to determine the effects of repeated (5 sessions) injection (i.p.) of ethanol (0.4 or 0.8 g/kg), caffeine (20 or 40 mg/kg) or clorazepate (3 mg/kg) on the ingestion of hypertonic saline (1.8%) in water-deprived rats. Saline intake increased with the acute administration of both clorazepate and ethanol (two doses), but it decreased with caffeine (two doses). It seems that the increase or decrease of hypertonic saline ingestion following acute drug administration continues to correlate well with anxiolytic or anxiogenic actions. However, following repeated administration of caffeine and ethanol, the effects on saline intake were not maintained in a reliable manner.

The effects of caffeine in the social interaction test and on exploration in rats: comparison with ethanol and clorazepate.

The effects of caffeine (20mg/kg) in the holeboard and social interaction tests were compared with those of ethanol (0.4g and dipotassium clorazepate (3mg/kg), following acute administration in one group of rats or after five daily injections in another group. The rats were put in pairs into an unfamiliar arena with high levels of illumination (n = 80), or tested individually in the holeboard (n = 80). Acute caffeine produced no effect on the time spent in social interaction, although it enhanced the number of social contacts, and both genital and total sniffing. Following five injections, caffeine also increased the time spent in social interaction. Acute clorazepate enhanced this time but this effect showed partial tolerance after five injections. Clorazepate also enhanced the number and duration of social contacts, increasing social grooming and genital sniffing, regardless of the duration of the treatment. Ethanol increased the time spent in social interaction following five injections, and increased social grooming. In the holeboard, stimulant effects were observed for caffeine and clorazepate, showing partial tolerance and without any effect on head dipping. In the social interaction test, only a stimulant effect for caffeine was obtained. The results of this study suggest that, under some circumstances, caffeine may enhance social interaction, in a manner similar to standard anxiolytics. Such an effect is potentiated by repeated administration.

Effects of oral ethanol self-administration on the inhibition of the lever-press response in rats.

The effects of ethanol on the inhibition of a learned response were examined in adult, male Wistar rats from two treatment groups: oral self-administration of alcoholic solution (10% ethanol and 10% glucose in distilled water) and oral self-administration of sweet solution (10% glucose in distilled water). Subjects were food deprived and alcoholic or control solutions were available 1 h per day during 15 days. After this period, rats were tested in a two-bottle paradigm during 1 h per day and placed in the operant chambers immediately afterward. This phase went on for 19 days. Subjects were trained to lever press for food and were tested in a continuous reinforcement schedule, operant extinction, successive discrimination, and two-stimuli tests. Alcohol impaired the ability to inhibit previously reinforced responses but only in situations indicated by exteroceptive stimuli. Ethanol intake did not impair the lever-press behavior neither in the acquisition of the response nor in the continuous reinforcement schedule. These data suggest that the sedative effects of alcohol at this dose were not apparent in reinforcement situations, in contrast with extinction situations.

Lymphoblastic lymphoma/leukemia of T-cell origin: ultrastructural, cytochemical, and immunologic features of ten cases.

Ten cases of T-lymphoblastic lymphoma/leukemia were studied with light and electron microscopy. Cytochemical strains were performed on touch preparations, and mononuclear cell suspensions were tested for spontaneous rosette formation with sheep erythrocytes, C3 receptors, and surface immunoglobulins. The present investigation was performed to evaluate several ultrastructural parameters, mainly the nuclear shape, as diagnostic clues for this group of lymphomas. Characteristic convoluted nuclei were present in 7 to 47% of the lymphoblasts. This percentage correlated with the focal acid phosphatase reaction and E-rosette formation. Acid phosphatase was the best cytochemical marker (70-100% of the lymphoblasts showed focal reaction product). By ultrastructural cytochemistry, the reaction product was demonstrated in the Golgi cisternae and primary lysosomes. The cell suspensions obtained from different sources contained 14 to 95% E-rosette-forming cells. No specific morphologic, cytochemical, or immunologic differences were found between patients with or without mediastinal involvement.