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.

Utility of a bacterial infection model to study epithelial-mesenchymal transition, mesenchymal-epithelial transition or tumorigenesis.

DCLK1 and Lgr5 have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. Epithelial-mesenchymal transition (EMT) is a key development program that is often activated during cancer invasion and metastasis, and also imparts a self-renewal capability to disseminating cancer cells. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we observed a relative decrease in DCLK1 expression in the colonic crypts, with significant shift towards stromal staining at peak (12 days post infection) hyperplasia, whereas staining for Lgr5 and Msi-1 increased several fold. When hyperplasia was regressing (days 20-34), an expansion of DCLK1+ve cells in the CR-infected crypts compared with that seen in uninfected control was recorded. Purified colonic crypt cells exhibiting epigenetic modulation of the transforming growth factor-ß (TGFß), Wnt and Notch pathways on 12 or 34 days post infection formed monolayers in vitro, and underwent trans-differentiation into fibroblast-like cells that stained positive for vimentin, fibronectin and DCLK1. These cells when trypsinized and regrown in soft agar, formed colonospheres/organoids that developed into crypt-like structures (colonoids) in Matrigel and stained positive for DCLK1. Mice exhibiting 12 or 34 days of TMCH were given azoxymethane once for 8 h (Gp1) or weekly for 3 weeks (Gp2), and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as colonospheres in soft agar and nodules/tumors in nude mice. Crypt cells isolated from Gp2 animals failed to form the monolayers, but developed into colonospheres in soft agar and nodules/tumors in nude mice. Thus, both hyperplasia and increased presence of DCLK1+ve cells promote cellular transformation in response to a second hit. The TMCH model, therefore, provides an excellent template to study how alterations in intestinal stem cells promote trans-differentiation, crypt regeneration or colon carcinogenesis following bacterial infection.

Activation of NPY type 5 receptors induces a long-lasting increase in spontaneous GABA release from cerebellar inhibitory interneurons.

Neuropeptide Y (NPY), a widely distributed neuropeptide in the central nervous system, can transiently suppress inhibitory synaptic transmission and alter membrane excitability via Y2 and Y1 receptors (Y2rs and Y1rs), respectively. Although many GABAergic neurons express Y5rs, the functional role of these receptors in inhibitory neurons is not known. Here, we investigated whether activation of Y5rs can modulate inhibitory transmission in cerebellar slices. Unexpectedly, application of NPY triggered a long-lasting increase in the frequency of miniature inhibitory postsynaptic currents in stellate cells. NPY also induced a sustained increase in spontaneous GABA release in cultured cerebellar neurons. When cerebellar cultures were examined for Y5r immunoreactivity, the staining colocalized with that of VGAT, a presynaptic marker for GABAergic cells, suggesting that Y5rs are located in the presynaptic terminals of inhibitory neurons. RT-PCR experiments confirmed the presence of Y5r mRNA in the cerebellum. The NPY-induced potentiation of GABA release was blocked by Y5r antagonists and mimicked by application of a selective peptide agonist for Y5r. Thus Y5r activation is necessary and sufficient to trigger an increase in GABA release. Finally, the potentiation of inhibitory transmission could not be reversed by a Y5r antagonist once it was initiated, consistent with the development of a long-term potentiation. These results indicate that activation of presynaptic Y5rs induces a sustained increase in spontaneous GABA release from inhibitory neurons in contrast to the transient suppression of inhibitory transmission that is characteristic of Y1r and Y2r activation. Our findings thus reveal a novel role of presynaptic Y5rs in inhibitory interneurons in regulating GABA release and suggest that these receptors could play a role in shaping neuronal network activity in the cerebellum.

Efficacy of occupant-collected dust samples in the evaluation of residential allergen and fungal levels.

This study evaluated the ability of a resident to evaluate their home for allergens and mold using a settled dust test kit compared with evaluation and collection of settled dust by an industrial hygienist. Forty-three home residents were provided with a kit containing written instructions and a vacuum cleaner attachment for collecting a settled dust sample. Within 2 weeks of receiving the occupant-collected sample, an industrial hygienist evaluated these homes, including a visual inspection, collection of settled dust, and collection of spore trap samples. Settled dust samples were analyzed for major dog, cat, dust mite, and cockroach allergens using immunoassay methods, and for mold spore equivalents using quantitative polymerase chain reaction methods for the 13 mold species or species groups comprising the American Relative Moldiness Index (ARMI). Allergen concentrations and ARMIs were compared between the resident- and industrial hygienist-collected samples. Linear regression between the two sets of samples showed strong correlations for dog allergen (r(2) = 0.92) and cat allergen (r(2) = 0.90). Correlations for dust mite (r(2) = 0.57) and cockroach allergens (r(2) = 0.22) were lower, likely due to most samples being near the limit of detection. ARMIs were highly correlated (r(2) = 0.68) and were in categorical (high, medium, or low) agreement for 76% of residences. These results show that residents can reliably follow directions and collect settled dust samples, providing an efficient method to remotely screen homes for elevated allergen levels and to identify homes with a potential mold or moisture problem that may need further evaluation.

Synthesis and structure-activity relationship of novel lactam-fused chroman derivatives having dual affinity at the 5-HT(1A) receptor and the serotonin transporter.

The structure-activity relationship (SAR) for three series of lactam-fused chroman derivatives possessing 3-amino substituents was evaluated. Many compounds exhibited affinities for both the 5-HT(1A) receptor and the 5-HT transporter. Compounds 45 and 53 demonstrated 5-HT(1A) antagonist activities in the in vitro cAMP turnover model.

In vitro studies of a prolactin antagonist, hPRL-G129R in human breast cancer cells.

Human prolactin (hPRL) has been shown to be one of the important survival/growth factors that promotes the proliferation of breast cancer cells in an autocrine/paracrine manner. In our recent studies, we demonstrated that a hPRL antagonist with a single amino acid substitution mutation (hPRL-G129R) was able to inhibit breast cancer cell proliferation via induction of apoptosis (1). In this study three independent yet related experiments were carried out regarding the effects of hPRL-G129R in breast cancer cells. We investigated the possible mechanism(s) of hPRL-G129R induced apoptosis in breast cancer cells. It is well documented that transforming growth factors (TGF) in conjunction with hormones such as estrogen and PRL play a major role in modulating the proliferation and apoptosis of mammary cells. We first investigated the relationships between hPRL/hPRL-G129R and TGFs. We show that hPRL is able to down-regulate TGF beta 1 (apoptotic factor) secretion and up-regulate TGF alpha (survival factor) secretion in a dose-dependent manner in T-47D cells. More importantly the hPRL antagonist up-regulates TGF beta 1 and down-regulates TGF alpha secretion. When hPRL-G129R was applied together with hPRL, it blocked the effects of hPRL. Secondly, we tested the possible involvement of caspases in hPRL-G129R induced apoptosis. We have shown that caspase-3 is activated by hPRL-G129R at a concentration of 250 ng/ml in T-47D breast cancer cells. Thirdly, we explored the additive effects of an anti-neoplastic drug, cisplatin, with the hPRL-G129R in T47D breast cancer cells. We show that cisplatin and hPRL-G129R when applied together resulted in about 40% growth inhibition in T-47D cells.

Inhibition of oncogene STAT3 phosphorylation by a prolactin antagonist, hPRL-G129R, in T-47D human breast cancer cells.

We have previously demonstrated that a hPRL antagonist (hPRL-G129R) was able to inhibit PRL induced breast cancer cell proliferation through induction of apoptosis. In the present study, we test the hypothesis that the inhibitory effect of hPRL-G129R in breast cancer cells occurs, at least in part, through the inhibition of oncogene STAT3 activation. We first demonstrated that STAT5 and STAT3 could be activated by either hGH or hPRL in T-47D breast cancer cells. Although the patterns of STAT5 activation by hGH and hPRL are similar, we observed a nearly 10-fold greater efficacy of hPRL in STAT3 activation as compared to that of hGH. More importantly, we have demonstrated that activation of STAT3 by hPRL could be inhibited by hPRL-G129R. Since T-47D cells coexpress GHR and PRLR, an attempt was made to dissect the molecular events mediated through hGHR or hPRLR using mouse L-cells expressing a single population of receptors (hGHR or hPRLR). To our surprise, only STAT5, not STAT3 phosphorylation was observed in these L-cells. In conclusion, our results suggest that: a) STAT3 is preferably activated through hPRLR in T-47D cells; b) hPRL-G129R is effective in inhibiting STAT3 phosphorylation; and c) the mechanism of STAT3 activation is different from that of STAT5.

A human prolactin antagonist, hPRL-G129R, inhibits breast cancer cell proliferation through induction of apoptosis.

Human breast cancer is the predominant malignancy and the leading cause of cancer death in women from Western societies. The cause of breast cancer is still unknown. Recently, the association between human prolactin (hPRL) activity and breast cancer has been reemphasized. Biologically active hPRL has been found to be produced locally by breast cancer cells that contain high levels of PRL receptor. A high incidence of mammary tumor growth has also been found in transgenic mice overexpressing lactogenic hormones. More importantly, it has been demonstrated that the receptors for sex steroids and PRL are coexpressed and cross-regulated. In this study, we report that we have designed and produced a hPRL antagonist, hPRL-G129R. By using cell proliferation assays, we have demonstrated that: (a) hPRL and E2 exhibited an additive stimulatory effect on human breast cancer cell (T-47D) proliferation; (b) hPRL-G129R possessed an inhibitory effect on T-47D cell proliferation; and (c) when antiestrogen (4-OH-tamoxifen) and anti-PRL (hPRL-G129R) agents were added together, an additive inhibitory effect was observed. We further investigated the mechanism of the inhibitory effects of hPRL-G129R in four hPRLR positive breast cancer cell lines. We report that hPRL-G129R is able to induce apoptosis in all four cell lines in a dose-dependent manner as determined by the Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The apoptosis is induced within 2 h of treatment at a dose as low as 50 ng/ml. We hope that the hPRL antagonist could be used to improve the outcome of human breast cancer therapy in the near future.

Vascular surgical society of great britain and ireland: limb salvage in the octogenarian

BACKGROUND: Critical limb ischaemia (CLI) presents a unique set of problems in the elderly patient. Foremost among these is the much greater likelihood of loss of independence and reduced quality of life if major amputation is required. For this reason it has been this unit's policy to attempt reconstructive vascular surgery in almost all cases of CLI. The outcome of this policy was examined. METHODS: All patients had surgery performed under one consultant and data were entered prospectively on to a database. RESULTS: Risk factors included diabetes (17 per cent), smoking (78 per cent) and ischaemic heart disease (31 per cent). Some 127 patients had either femoropopliteal (59), femorodistal (64) or popliteal-distal grafts (four) performed for limb-threatening ischaemia. Follow-up was performed at 3, 6 and 12 months and then at annual intervals until death. Seventeen of these patients required a subsequent major amputation, 12 at the below-knee and five at the above-knee level. Mean follow-up was 2 years. The perioperative mortality rate was 15 per cent, although eight of these patients were admitted with acute or chronic ischaemia. Cumulative graft secondary patency rate was 68 per cent at 4 years for vein grafts. Some 95 per cent of patients with patent grafts were independently mobile. CONCLUSION: Excellent results can be achieved for limb salvage with a relatively low morbidity in this group. Elderly patients with CLI do not live long and avoidance of amputation is particularly desirable in order to maximize the quality of their remaining life.

Reproducibility of the vesicular breath sounds in normal subjects.

Nonfiltered (NF) lung sounds from the apical area of the heart along with lung volumes and ECG signals were recorded from 5 normal subjects. The signals were digitized and subjected to three methods of heart sound cancellation: 75-Hz high-pass filtering (75 HF), ECG-triggered blanking (BL) and adaptive noise cancelling (AF) [IEEE Trans. Biomed. Engng 33: 1141-1148, 1986]. The sound signals were then subjected to the fast Fourier transform algorithm to obtain power spectra. Five breaths from each subject were analyzed, and their spectra were similar and slightly skewed to the right. The average values of mean, median and mode frequencies of the whole breath of 5 subjects, respectively, were for NF: 64.62 +/- 3.74, 44.57 +/- 2.06 and 36.75 +/- 1.79 Hz; for 75 HF: 150.42 +/- 17.49, 114.02 +/- 6.43 and 86.16 +/- 3.13 Hz; for BL: 81.76 +/- 6.02, 52.36 +/- 2.79, 41.10 +/- 3.15 Hz; for AF: 96.87 +/- 11.58, 68.23 +/- 10.44 and 52.25 +/- 8.97 Hz. These values showed no differences between subjects. The F values obtained by the two-way analysis of variance of all breaths of all subjects (mean, median, mode) were: NF: 0.161, 0.341, 0.089; 75 HF: 0.455, 0.042, 0.085; BL: 0.108, 0.082, 0.057; AF: 0.130, 0.204, 0.113 (all p greater than 0.1). The data revealed a remarkable lack of variation within and between subjects, suggesting similar sites and mechanisms of production and transmission.

Adaptive filtering in biological signal processing.

The high dependence of conventional optimal filtering methods on the a priori knowledge of the signal and noise statistics render them ineffective in dealing with signals whose statistics cannot be predetermined accurately. Adaptive filtering methods offer a better alternative, since the a priori knowledge of statistics is less critical, real time processing is possible, and the computations are less expensive for this approach. Adaptive filtering methods compute the filter coefficients "on-line", converging to the optimal values in the least-mean square (LMS) error sense. Adaptive filtering is therefore apt for dealing with the "unknown" statistics situation and has been applied extensively in areas like communication, speech, radar, sonar, seismology, and biological signal processing and analysis for channel equalization, interference and echo canceling, line enhancement, signal detection, system identification, spectral analysis, beamforming, modeling, control, etc. In this review article adaptive filtering in the context of biological signals is reviewed. An intuitive approach to the underlying theory of adaptive filters and its applicability are presented. Applications of the principles in biological signal processing are discussed in a manner that brings out the key ideas involved. Current and potential future directions in adaptive biological signal processing are also discussed.

Inspiratory and expiratory vesicular breath sounds.

Unfiltered breath sounds (NF) from the apical area of the heart, lung volume and ECG signals were recorded in 5 normal subjects. The signals were digitized and subjected to three methods of heart sound cancellation: 75-Hz high-pass filtering (75 HF), ECG-triggered blanking (BL) and adaptive filtering (AF). The sound signals were then subjected to the fast Fourier transform algorithm to obtain power spectra. Inspiratory and expiratory phase sounds of five breaths of each subject were analyzed separately. The inspiratory and expiratory sound power spectra were very similar and skewed slightly to the right, and therefore characterized by median frequencies. The differences between inspiratory and expiratory median frequencies were insignificant for NF: 42.90 +/- 2.03 (mean +/- SD) vs. 46.64 +/- 2.53 Hz (p greater than 0.1); for 75 HF: 106.43 +/- 10.27 vs. 118.22 +/- 6.30 Hz (p greater than 0.5); for BL: 44.46 +/- 3.33 vs. 66.73 +/- 2.93 Hz (p greater than 0.1), for AF: 49.72 +/- 5.68 vs. 79.20 +/- 13.07 Hz (p greater than 0.1). We conclude that the lack of significant differences suggests similar mechanisms and sites of production of inspiratory and expiratory vesicular breath sounds.

Autoregressive modeling of lung sounds: characterization of source and transmission.

In this communication, we discuss the application of autoregressive modeling to lung sounds analysis. The lung sounds source in the airway is modeled as a white noise source, consisting of one or a combination of the following sources: random white noise sequence, periodic train of impulses, and impulsive bursts of energy. The acoustic transmission through the lung parenchyma and chest wall is modeled as an all-pole filter. Using this method, the source and transmission characteristics of lung sounds are estimated separately, based on the lung sounds at the chest wall. To illustrate the potential validity of the model, lung sound segments in known disease conditions were selected from teaching tapes and the source and transmission characteristics were estimated by applying the model. The estimated characteristics were found to be consistent with current knowledge of the generation and transmission of lung sounds in the known conditions.

Characteristics of normal lung sounds after adaptive filtering.

Lung sounds were recorded from five normal male subjects during tidal breathing. Simultaneous electrocardiograms were recorded and used as index signals to generate simulated heart sounds for digital subtraction from recorded lung sounds to obtain purer lung sounds. Five random breaths from each subject were analyzed. Sound signals were band-pass filtered 25 to 1,000 Hz (antialiasing), digitized at 3,000 Hz, and then subjected to (1) direct fast Fourier transform (FFT) without filtering (NF); (2) digital high-pass filtering at 75 Hz and subsequent FFT (75 HzF); (3) adaptive filtering and subsequent FFT (AF). The FFT algorithms of all lung sounds were characterized by mean, median, and mode frequencies. The mean, median, and mode of NF were lower than those of 75 HzF (64.98 +/- 4.04 versus 150.42 +/- 17.49, mean +/- SE, p less than 0.003; 44.57 +/- 2.06 versus 111.81.5.78, p less than 0.0003; 36.81 +/- 1.77 versus 86.16 +/- 3.13, p less than 0.0001) and those of AF (64.98 +/- 4.04 versus 96.87 +/- 11.58, p less than 0.01; 44.57 +/- 2.06 versus 68.23 +/- 10.44, p less than 0.05; 36.81 +/- 1.78 versus 52.24 +/- 8.97, p less than 0.06). The mean, median, and mode of AF were lower than those of 75 HzF (96.87 +/- 11.58 versus 150.42 +/- 17.49, p less than 0.02; 68.23 +/- 10.44 versus 111.81 +/- 5.77, p less than 0.007; 52.24 +/- 8.97 versus 86.16 +/- 3.73, p less than 0.01). The results indicated that by filtering out low frequency heart sounds, the frequency spectrum of lung sounds was moved upward.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of heart sounds interference with lung sounds.

An index to quantify the contamination of lung sounds by heart sounds is described. Using the index, the efficacy of high pass filtering and adaptive filtering methods for the reduction of heart sounds is evaluated.