Quantitative analysis of culturable bacteria, levels of endotoxins, inflammatory mediators and substance P in teeth with symptomatic irreversible pulpitis and in teeth with vital normal pulp tissues.

AIM: To comparatively analyse the levels of culturable bacteria, endotoxins (LPS), tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and substance P in teeth with symptomatic irreversible pulpitis (SIP) and vital normal pulp (VNP) tissues. METHODOLOGY: Thirty-two patients were included (20 teeth with SIP and 12 teeth with VNP tissues) in this cross-sectional study. Samples were collected from the full length of the root canals (microbial analysis) and periapical tissues (2 mm beyond the apex for immunological analysis), using sterile absorbent paper points. The levels of culturable bacteria (culture method), endotoxins (LAL Pyrogent 5000), TNF-α, IL-1ß and substance P (ELISA) were assessed. The Mann-Whitney test was used for comparisons between the levels of CFU/mL, LPS, TNF-α, IL-1ß and substance P in the SIP and VNP groups. The statistical analysis was performed with the significance level set at 5%. RESULTS: Culturable bacteria were recovered from all teeth with SIP. On the other hand, no positive cultures were observed in the VNP tissues group (p > .05). The levels of LPS were approximately four times higher in teeth with SIP than in teeth with VNP tissues (p < .05). Higher levels of TNF-α and substance P were detected in teeth with SIP (p < .05). On the other hand, no difference in the levels of IL-1ß was detected between the two groups (p > .05). CONCLUSION: Teeth with symptomatic irreversible pulpitis present higher levels of culturable bacteria, endotoxins, TNF-α and substance P than those with vital normal pulp tissues. On the other hand, the levels of IL-1ß were similar in teeth from both groups suggesting reduced implications of this inflammatory mediator in the early stages of infection.

Bacteria and virulence factors in periapical lesions associated with teeth following primary and secondary root canal treatment.

AIM: This cross-sectional study aimed to investigate the microbial profile and to quantify the levels of endotoxins (LPS) and lipoteichoic acid (LTA) present in periapical lesions associated with root filled teeth and those that had received root canal retreatment. It also aimed to investigate the association between microorganisms and their virulence factors with clinical and radiographic features. METHODOLOGY: Patients with periapical lesions in teeth with post-treatment endodontic disease following primary root canal treatment (n = 19) and unsuccessful root canal retreatment (n = 13) were treatment planned for endodontic microsurgery, where the periapical lesions were collected. Clinical and radiographic data were also collected. For microbiological analysis, nested polymerase chain reaction was used to detect 17 bacterial species. Levels of LPS and LTA were determined using limulus amebocyte lysate and enzyme-linked immunosorbent assays, respectively. The Student t-test or Wilcoxon-Mann-Whitney tests were applied to compare the data on LPS and LTA with clinical and radiographic features. The associations between the clinical and radiographic features and the bacterial species were analysed using the Fisher's exact test. A significance level of 5% was adopted. RESULTS: Bacterial DNA, LPS and LTA were detected in all samples. Parvimonas micra was the most commonly detected species in all groups, followed by Enterococcus faecalis, Fusobacterium nucleatum and Porphyromonas endodontalis. The type of endodontic treatment, whether a primary root canal treatment or retreatment, was not associated with the presence of any bacterial species in periapical lesions. The levels of LPS and LTA in periapical lesions of root filled teeth were not significantly different from those that had been retreated. Associations between the levels of LPS and LTA with clinical signs and symptoms were found. No association was found between specific bacteria and clinical features. CONCLUSION: Periapical lesions associated with teeth after primary root canal treatment and retreatment had similar polymicrobial composition. The levels of LPS and LTA in periapical lesions associated with teeth after primary root canal treatment and retreatment were similar, and both were associated with the same symptomatology.

Efficacy of reciprocating and ultrasonic activation of 6% sodium hypochlorite in the reduction of microbial content and virulence factors in teeth with primary endodontic infection.

AIM: To evaluate in a clinical trial the efficacy of reciprocating and ultrasonic activation of 6% sodium hypochlorite (NaOCl) in the microbial composition and reduction in microbial load as well as in levels of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in teeth with primary endodontic infections. METHODOLOGY: Samples were collected from 24 root canals with pulp necrosis and periapical lesions, before and after chemo-mechanical canal preparation. The teeth were randomly divided according to the activation protocol as follows: control group without activation (WA, n = 8), reciprocating activation group using Easy Clean tip (EC, n = 8) and ultrasonic activation group using Irrisonic insert (US, n = 8). Microbiological specimens were processed using a culture technique and microbiota composition was analysed using the checkerboard technique. The levels of LPS and LTA were quantified using limulus amebocyte lysate (LAL) and enzyme-linked immunosorbent assay (ELISA), respectively. The Fisher's exact test, Kruskal-Wallis, Dunn's and Wilcoxon's test with a significance level of P < 0.05 were used for statistical analysis. RESULTS: All initial specimens had growth of viable bacteria in fastidious anaerobe agar (FAA), with an average of 105  CFU mL-1 , whereas only one case had such growth after chemo-mechanical canal preparation. LPS and LTA were recovered in 100% of the cases. Chemo-mechanical canal preparation significantly decreased the levels of LPS and LTA (P < 0.05), but no significant differences were found between the groups (P > 0.05). Through the checkerboard technique, bacteria were found in 100% of the initial specimens with concentrations between <105 and 106 . The most frequently identified microorganisms were Prevotella nigrescens and Enterococcus hirae. After chemo-mechanical canal preparation, many species were not detected in any of the three groups tested. A significant reduction occurred in Group US, followed by Groups EC and WA. CONCLUSIONS: Activation of 6% NaOCl reduced the levels of LPS and LTA with no differences between the groups. However, ultrasonic activation was associated with a greater reduction in microbial load within root canals.

Combined use of chemical and biological compounds to control hookworm.

The aim of this study was to evaluate the combined use of different chemical (albendazole, ivermectin, glycerine and Vaseline) and biological (Monacrosporium thaumasium) compounds in the control of Ancylostoma caninum. Infective larvae of A. caninum were obtained from coprocultures of positive faeces from naturally infected dogs. We used 1% ivermectin, 1% albendazole, 100% glycerine, 100% Vaseline and an isolate of the nematophagous fungus M. thaumasium (NF34), alone or in combinations. Next, an experimental test was set up with 16 groups in microtubes, with a 24-h interaction. The groups (G1 to G15) that contained any chemical or biological compound (NF34) and/or their combined use (chemical + biological) showed a difference in relation to the control group, except G5 - Vaseline 100% without combinations. It was concluded that, even on an experimental basis, the combined use of anthelmintic drugs with biological control was efficient; however, more studies must be carried out in order to elucidate the synergistic action between chemical and biological compounds to be used in the effective control of hookworms in the future.

Clinical efficacy of EDTA ultrasonic activation in the reduction of endotoxins and cultivable bacteria.

AIM: This clinical study was conducted to investigate the influence of 17% ethylenediaminetetraacetic acid (EDTA) ultrasonic activation after chemomechanical preparation (CMP) on eliminating/reducing oral bacterial lipopolysaccharides (known as endotoxins) and cultivable bacteria in teeth with pulp necrosis and apical periodontitis. METHODOLOGY: Samples were taken from 24 root canals at several clinical periods: S1 - before CMP; S2 - after CMP; S3 - after EDTA: G1 - with ultrasonic activation (n = 12) and G2 - without ultrasonic activation (n = 12). Root canals were instrumented using Mtwo rotary files. Culture techniques were used to determine the number of colony-forming units (CFU). Limulus amebocyte lysate (LAL) was used to measure endotoxin levels. Friedman's and Wilcoxon signed-rank tests were used to compare the amount of bacteria and endotoxin levels in each period (P < 0.05). RESULTS: Endotoxins and cultivable bacteria were recovered in 100% of the initial samples (S1). CMP was effective in reducing endotoxins and bacterial load (all with P < 0.05). Higher values of endotoxin reduction were achieved with EDTA ultrasonic activation [G1, 0.02 EU mL-1 (range 0.01-0.75)] compared with the no activation group [G2, 1.13 EU mL-1 (range 0.01-8.34)] (P < 0.05). Regarding bacterial reduction, no statistically significant difference was found in S3, regardless of the group (G1, G2, P > 0.05). CONCLUSIONS: Chemomechanical preparation was effective in reducing bacteria and endotoxins, but could not completely eliminate them. The ultrasonic activation of EDTA was effective in further reducing endotoxin levels in the root canals of teeth with pulp necrosis and apical periodontitis.

Root canal content from primary endodontic infection and upregulation of gelatinases in fibroblast cells.

AIM: To investigate endotoxin levels from primary endodontic infections before and after chemomechanical preparation (CMP) and to determine their antigenicity against 3T3 fibroblasts through gelatinolytic activity of matrix metalloproteinases (MMPs). METHODOLOGY: Twenty-four root canals with primary endodontic infection and apical periodontitis were selected. Samples were collected using paper points before (S1) and after chemomechanical preparation (CMP) (S2). The limulus amebocyte lysate assay was used for endotoxin measurement. Fibroblasts were stimulated with root canal contents for 24 h. Supernatants of cell cultures stimulated with root canal contents were collected after 24 h to determine the levels of MMP-2 and MMP-9 gelatinolytic activity using the zymography technique. Friedman and Wilcoxon tests were used to compare the amount of endotoxin before (S1) and after CMP (S2) (P < 0.05). Data obtained from gelatinolytic activity were analysed using anova and Tukey's tests (P < 0.05). RESULTS: Endotoxin was recovered in 100% of the samples. There was a significant reduction in endotoxin levels after CMP (P < 0.05). A correlation was found between the levels of endotoxins and MMP-2 expression (P < 0.05). Root canal contents of initial samples (S1) induced significantly greater MMP-2 expression by fibroblasts when compared to S2 and the nonstimulated group (P < 0.05). No gelatinolytic activity of MMP-9 was observed in S1, S2 and control group. CONCLUSIONS: Root canal contents from primary endodontic infections had gelatinolytic activity for MMP-2. Moreover, CMP was effective in reducing endotoxin levels and their antigenicity against fibroblasts on gelatinolytic activity.

Spectrophotometric Analysis of Apical Extrusion of Sodium Hypochlorite using Different Irrigation Protocols in an Ex Vivo Model of Immature Teeth.

OBJECTIVE: To evaluate the effect of different agitation methods on apical extrusion of 1.5% sodium hypochlorite (NaOCl) in an ex vivo model of immature teeth. METHODS: Sixty extracted human inferior incisors were prepared to simulate immature teeth and embedded in an artificial root socket made of silicone impression material. The teeth were then divided into four groups: Conventional needle irrigation (CNI) alone, CNI supplemented with Ultrasonic Irrigant Activation (UIA), EasyClean (EC), or XP-endo Finisher (XPF). Extruded NaOCl was collected, reacted with m-cresol purple, and its absorbance values were measured. The data were statistically analyzed using One-way analysis of variance with a significance level of 5%. RESULTS: All groups showed apically extruded irrigating solution, and the mean volumes of extruded NaOCl did not differ significantly between any of the test groups (p⟩0.05). CONCLUSION: The activation of 1.5% NaOCL by UIA, EC, or XPF as supplementary to CNI does not promote greater apical extrusion when compared to CNI alone in simulated immature teeth.

Impact of different methodologies on the detection of point mutations in routine air-dried fine needle aspiration (FNA) smears.

Currently the best method to select suspicious thyroid nodules for surgery is fine needle aspiration (FNA) cytology. However, FNA cytology has some inherent limitations, which can partly be overcome by molecular analysis. Therefore, molecular testing for somatic mutations has emerged as the most promising approach for molecular FNA diagnostics. The objective of this methodological study was to evaluate the feasibility of detecting BRAF, NRAS, HRAS, and KRAS mutations from routine air-dried thyroid FNA smears, and to find an optimal method for detecting these mutations in FNA samples. DNA was extracted from 110 routine air-dried FNA smears and the corresponding surgically obtained formalin-fixed paraffin-embedded tissues. The presence of BRAF, NRAS, HRAS, and KRAS mutations was assessed by real-time PCRs and high resolution melting analysis, and/or pyrosequencing in comparison to real-time PCRs using hybridization probes and fluorescence melting curve analysis. The high-resolution melting-PCRs revealed a significantly lower number of PCR failures and questionable results, and detected more mutations than the PCRs using hybridization probes. The number of PCR failures ranging from 14-16% by high-resolution melting-PCRs could be further reduced to 5-14% by adding pyrosequencing assays. Moreover, pyrosequencing increased the specificity of the assays, up to 98-100%, while the sensitivity ranged between 32-63%. In summary, the mutation detection, especially in air-dried FNA samples, improves when using PCR assays in combination with high resolution melting analysis. Additional improvement can be obtained by subsequent pyrosequencing in comparison to previously described real-time PCRs using hybridization probes and fluorescence melting curve analysis.

Microbiological profile and antimicrobial susceptibility pattern of infected root canals associated with periapical abscesses.

The aim of this investigation was to identify microorganisms from root canals with periapical abscesses and assess the susceptibility of specific anaerobic bacteria to selected antimicrobials and their ß-lactamase production. Sixty root canals were microbiologically investigated. The susceptibility of Anaerococcus prevotii, Fusobacterium necrophorum, F. nucleatum, Parvimonas micra, and Prevotella intermedia/nigrescens to antimicrobials was evaluated with the Etest, whereas ß-lactamase production was assessed with nitrocefin. A total of 287 different bacterial strains were recovered, including 201 strict anaerobes. The most frequently strict isolated anaerobes were A. prevotii, P. micra, and F. necrophorum. The selected bacteria were susceptible to all the tested antibiotics, except A. prevotii and Fusobacterium species to azithromycin and erythromycin, as well as A. prevotii and F. necrophorum to metronidazole. None of the microorganisms produced ß-lactamase. Gram-positive anaerobic bacteria predominated in the root canals with periapical abscesses. All microorganisms tested were susceptible to benzylpenicillin, amoxicillin, amoxicillin + clavulanate, cefaclor, and clindamycin, producing no ß-lactamase.

Quantification of cultivable bacteria and endotoxin in post-treatment apical periodontitis before and after chemo-mechanical preparation.

This clinical study was conducted to quantify cultivable bacteria and endotoxin in root canals with post-treatment apical periodontitis by correlating their levels with clinical features and to evaluate the effect of chemo-mechanical preparation (CMP) with 2 % chlorhexidine gel + 17 % EDTA on bacterial and endotoxin removal/elimination. Moreover, target strict Gram-negative anaerobic bacteria were investigated by polymerase chain reaction (PCR). Fifteen teeth with post-treatment apical periodontitis were sampled before (s1) and after (s2) CMP. Culture techniques determined the number of colony-forming units (CFU). PCR (16S rDNA) and limulus amebocyte lysate (LAL) assay were used for bacterial and endotoxin detection, respectively. Prevotella nigrescens (4/15), Prevotella intermedia (2/15), and Tannerella forsythia (2/15) were the most frequently detected species. Endotoxin was recovered in 100 % of the samples. At s1, bacteria and endotoxin were detected at a median value of 5.14 × 10(3) CFU/mL and 3.96 EU/mL, respectively. Higher levels of endotoxin were related to a larger size of radiolucent area (>5 mm) (p < 0.05). CMP was more effective in reducing bacteria (99.61 %) than endotoxin (60.6 %) (both p < 0.05). Our findings indicated that the levels of endotoxin found in infected root canals were related to a larger size of radiolucent area in the periapical region. Moreover, CMP was effective in reducing both bacterial and endotoxin contents in post-treatment apical periodontitis.

Evaluation of cytotoxicity and up-regulation of gelatinases in fibroblast cells by three root repair materials.

AIM: To investigate the effects of root repair materials on the cytotoxicity and gelatinolytic activity of matrix metalloproteinases (MMPs) in 3T3 fibroblasts. METHODOLOGY: Fibroblasts (3T3, 3 × 10(5) cells per well) were incubated with elutes of calcium hydroxide (Biodinâmica, Ibiporã, PR, Brazil), EndoBinder (Binderware, São Carlos, SP, Brazil) and mineral trioxide aggregate (MTA) (Angelus, Londrina, PR, Brazil) for 24 h. The cytotoxicity of all root repair materials was determined using the MTT assay. Supernatants of cell cultures incubated with materials were collected after 24 h to determine the levels of MMP-2 and MMP-9 gelatinolytic activity by gelatin zymography. Data were analysed using anova and Tukey's test. RESULTS: Cells secreted MMP-2 after 24 h with calcium hydroxide inducing significantly greater MMP-2 expression in relation to the control and the other root repair materials (P < 0.05). The cytotoxicity results revealed that there was no significant difference in the cell viability of MTA, EndoBinder and the control group. However, there was a significantly reduced cell viability of 3T3 fibroblasts in association with calcium hydroxide (P < 0.05). CONCLUSIONS: Calcium hydroxide was associated with significantly less cell viability when compared with EndoBinder and MTA. All materials had gelatinolytic activity for MMP-2 with calcium hydroxide being associated with the greatest activity.

Evaluation of cytotoxicity and up-regulation of gelatinases in human fibroblast cells by four root canal sealers.

AIM: To investigate the effects of root canal sealers on the cytotoxicity and gelatinolytic activity of matrix metalloproteinases (MMPs) in human fibroblasts. METHODOLOGY: Human fibroblasts (MRC5, 3×10(5) cells per well) were incubated directly or indirectly with AH Plus, Endomethasone N, Pulp Canal Sealer EWT or Sealapex for 30 min, 1, 4 or 24 h (time-points). The cytotoxicity of all root canal sealers was determined by counting viable cells using the trypan blue exclusion assay. Supernatants of cell cultures incubated with root sealers directly or indirectly were collected after each time-point to determine the levels of MMP-2 and MMP-9 gelatinolytic activity by gelatin zymography. Data were analysed using anova and the Tukey's tests. RESULTS: Cells secreted MMP-2 after periods of 4 and 24 h; however, there were no significant differences between the sealers. Secretion of gelatinases was elevated by root canal sealers in direct contact with the cell monolayer when compared to indirect contact (P < 0.05). At the time-points tested, no gelatinolytic activity could be detected in the control group without the sealers. The cytotoxicity results revealed that all sealers were cytotoxic in both contact forms. Sealapex had the lowest cytotoxicity and AH Plus the most cytotoxicity. CONCLUSIONS: All root canal sealers induced the expression of MMP-2 in MRC5 fibroblasts. AH Plus had the highest cytotoxicity amongst the tested sealers, but all were associated with cytotoxic effects.

Effect of root canal sealers on bond strength of fibreglass posts cemented with self-adhesive resin cements.

AIM: This study evaluated the effects of different root canal sealers on the bond strength of a fibreglass post cemented with self-adhesive resin cements. METHODOLOGY: The root canals of 50 extracted maxillary single-rooted canine teeth were prepared with the crown-down technique and randomly divided into five groups according to the sealer used: group 1: control group, gutta-percha points only (no sealer); group 2: AH Plus (resin-based sealer); group 3: self-etch Epiphany (resin-based sealer); group 4: Sealer 26 (calcium hydroxide-based sealer); and group 5: Endomethasone (zinc oxide eugenol-based sealer). The root canals were filled with gutta-percha, the cold lateral compaction technique, except for group 3 where Resilon was used. Post spaces were prepared, and fibreglass posts were cemented with the self-adhesive cement RelyX Unicem. Bonded specimens were sectioned into 1-mm-thick slabs, and a push-out test was performed in a universal machine. Failure modes were observed and classified into five types: (i) adhesive between the post and resin cement; (ii) mixed, with resin cement covering 0-50% of the post diameter; (iii) mixed, with resin cement covering 50-100% of the post surface; (iv) adhesive between resin cement and root canal; and (v) cohesive in dentine. Data of bond strength were submitted to anova and Tukey test (α = 0.05). RESULTS: No significant difference was detected between control group, AH Plus, Epiphany and Sealer 26 (P > 0.05). The Endomethasone group had significantly lower bond strength values than the other sealers (P < 0.05). The prevalence of mixed fractures and adhesive cement-dentine failure was verified in the eugenol-containing sealer group; in the control group, the resin-based and calcium hydroxide-based sealer groups, the predominant mode of failure was the mixed type. CONCLUSION: Endomethasone interfered negatively with the bond to root dentine; however, AH Plus, Epiphany and Sealer 26 did not interfere in the bond strength of a fibreglass post cemented with self-adhesive resin cements.

Long-term sealing ability of resin-based root canal fillings.

AIM: To evaluate the ability of two resin-based filling materials to provide immediate and long-term sealing of the root canal. METHODOLOGY: A total of eighty-two human roots were instrumented and filled with AH Plus/gutta-percha or Epiphany/Resilon. Root filled teeth were sealed coronally either with Coltosol or Clearfil SE Bond/Filtek Z250 or were left unsealed. The quality of root canal sealing was assessed by a fluid filtration method performed at immediate and 180-day time intervals. Mean fluid filtration rates were analyzed by three-way repeated measures anova and Tukey post hoc test. RESULTS: Specimens filled with Epiphany/Resilon exhibited higher leakage than specimens filled with AH Plus/gutta-percha (P < 0.05), regardless of the coronal sealing condition and period of evaluation. No difference was detected between coronal restorative materials (P > 0.05), whilst leakage in teeth without any coronal restoration was significantly higher (P < 0.05). After storage, a significant decrease in leakage (P < 0.05) was observed in all experimental groups. CONCLUSIONS: AH Plus/gutta-percha provided superior root canal sealing at both immediate and 180-day time periods. The presence of a coronal seal reduced leakage significantly. Storage of root filled specimens did not disturb the sealing ability of the tested materials.

Thickness of dentine in mesial roots of mandibular molars with different lengths.

AIM: To measure the minimum thickness of the distal (furcal) root dentine associated with the buccal and lingual canals of the mesial roots of mandibular first molars with different lengths. METHODOLOGY: The mesial roots of 285 mandibular first molars were allocated into three groups according to their length: group I - long (24.14 mm +/- 0.85), group II - medium (22.10 mm +/- 0.65) and group III - short (19.97 mm +/- 0.75). The minimum thickness of the distal (furcal) root dentine associated with the buccal and lingual canals of the mesial roots 2 mm below the furcation was measured. The distance between the buccal and lingual canals, and the depth of concavity in the distal surface of the mesial roots were also measured. anova and Tukey-Kramer were used to test for significant differences among the groups. RESULTS: The minimum thickness of the distal wall of the mesiobuccal canal was significantly different (P < 0.05) between group I (long) and III (short), with long teeth having the smallest mean values. No significant difference was found in the thickness of the distal wall of the mesiolingual canal among the groups studied (P > 0.05). The shortest distance between the mesiobuccal and the mesiolingual canals was observed in group III (P < 0.05). The distal (furcal) concavity was deeper in group I (P < 0.05) when compared with the other groups. CONCLUSION: There was a significant difference in the minimum thickness of the distal (furcal) root wall of the mesiobuccal canal of mandibular first molars 2 mm below the furcation between group I (long) and group III (short) teeth. The thinnest walls were found in the longest teeth. The deepest concavities in the distal (furcal) walls of the mesial roots were found in the longest roots.

A cutoff thyroglobulin value suggestive of distant metastases in differentiated thyroid cancer patients.

Serum thyroglobulin is used as part of the early postoperative assessment of differentiated thyroid cancer (DTC) since there is a clear relationship between an increased risk of recurrence and persistent disease after initial treatment and high postoperative stimulated thyroglobulin (ps-Tg) values. Thus, although ps-Tg above 10-30 ng/mL is considered an independent predictor of worse prognosis, the value that is associated with distant metastases is not defined. Thus, this was our objective. We selected 655 DTC patients from a nuclear medicine department database (Irmandade Santa Casa de Misericórdia de São Paulo, Brazil). All patients had received total thyroidectomy and radioactive iodine (RAI) therapy and had ps-Tg values higher than 10 ng/mL with negative anti-thyroglobulin antibodies. Then, we selected patients who presented post-therapy whole-body scan with pulmonary and/or bone uptake but with no mediastinum or cervical uptake. Patients with negative findings on functional imaging or any doubt on lung/bone uptake were submitted to additional exams to exclude another non-thyroid tumor. Of the 655 patients, 14.3% had pulmonary and 4.4% bone metastases. There was a significant difference in ps-Tg levels between patients with and without metastases (P<0.001). The cutoff value of ps-Tg was 117.5 ng/mL (sensitivity: 70.2%; specificity: 71.7%) for those with lung metastasis, and 150.5 ng/mL (sensitivity: 79.3%; specificity: 85%) for those with bone metastasis. The cutoff value for patients with eitherpulmonary or bone metastasis was 117.5 ng/mL (sensitivity: 70.2%; specificity: 83.7%). Our findings demonstrated that ps-Tg could predict distant metastasis in DTC patients. We identified a cutoff of 117.5 ng/mL with a high negative predictive value of 93.7%.

From sequence to chromosome: the tip of the X chromosome of D. melanogaster.

One of the rewards of having a Drosophila melanogaster whole-genome sequence will be the potential to understand the molecular bases for structural features of chromosomes that have been a long-standing puzzle. Analysis of 2.6 megabases of sequence from the tip of the X chromosome of Drosophila identifies 273 genes. Cloned DNAs from the characteristic bulbous structure at the tip of the X chromosome in the region of the broad complex display an unusual pattern of in situ hybridization. Sequence analysis revealed that this region comprises 154 kilobases of DNA flanked by 1.2-kilobases of inverted repeats, each composed of a 350-base pair satellite related element. Thus, some aspects of chromosome structure appear to be revealed directly within the DNA sequence itself.

Ex vivo antimicrobial activity of several bleaching agents used during the walking bleach technique.

AIM: To investigate ex vivo the antimicrobial activity of a paste of sodium perborate associated with various vehicles comparing it with 37% carbamide peroxide and 35% hydrogen peroxide. METHODOLOGY: The antimicrobial activity of these agents was evaluated against three microorganisms: Enterococcus faecalis, Streptococcus mutans and Candida albicans. One millilitre of each tested substance was placed on the bottom of wells of 24-well cell culture plates. Six wells were used for each time period and group. Two millilitres of the microbial suspension was ultrasonically mixed for 10 s with the bleaching pastes and placed in contact with them for 10, 30, 45 s; 1, 3, 5, 10, 20, 30 min; and 1 and 2 h. After each period of time, 1 mL from each well was transferred to tubes containing 2 mL of freshly prepared brain heart infusion agar + neutralizers. Agar plates were inoculated in appropriate gaseous conditions. Data were analysed statistically by the Kruskal-Wallis test with the level of significance set at P < 0.05. RESULTS: In all groups containing chlorhexidine (groups 3, 5 and 7), the antimicrobial activity of the bleaching paste was significantly increased when compared with groups with other kinds of vehicle (groups 1, 2, 4, 6 and 8). For all tested groups, the most resistant microorganism was E. faecalis. CONCLUSIONS: Chlorhexidine when used as a vehicle for sodium perborate enhanced its antimicrobial activity.

Fat-soluble vitamins in human lactation.

Lactation has three distinct phases: colostrum, transition, and mature, with the quantity of fat-soluble vitamins and carotenoids tending to decline while the total fat content increases. The number of deliveries seems to be directly related to higher concentrations of beta-carotene and vitamin E in colostrum. Little is known about vitamin quantities during the other phases. In the present study, vitamin A and E concentrations during different phases of lactation were measured in primiparous and multiparous mothers to analyze the variation and suitability for the diet of a breastfed child. Phase of lactation and number of deliveries were highly significant for log mean vitamin A while only one phase showed significance for log mean vitamin E. There was a sharp decline in the levels of vitamin A and E in the course of the initial phase of lactation. The variability between measurements declined as colostrum transitioned to mature milk. There were significant measurable inter-group differences in retinol levels in milk obtained during early lactation. Vitamin A and E content can reach 2.5 and 1.4 times of levels recommended intake, respectively.

Asthma prevalence and risk factors in early childhood at Northern Portugal.

Asthma is the commonest and most important chronic non-infectious disease in childhood and it has become more prevalent in recent years. There is a shortage of studies in relation to early childhood and so, as part of the INAIRCHILD project, this cross-sectional study aimed to assess the prevalence of asthma and its associated risk factors, namely demographic, environmental, psychosocial and clinical factors for infants and preschoolers living in Northern Portugal. Data concerning asthma prevalence were collected through questionnaires based on those from the International Study of Asthma and Allergies in Childhood (ISAAC-derived), the questionnaires were distributed to 1042 children attending the 17 nurseries involved in the INAIRCHILD project (10 in urban and suburban context, and 7 in rural context). The response rate was 48%. Prevalence of asthma based on symptomatology and odds ratio was calculated. Around 52% of the studied children presented at least one of the respiratory symptoms investigated (wheeze, dyspnea and cough) in the absence of upper respiratory infections. The prevalence of asthma was 10.7%, comparable to the figures for Portuguese schoolchildren (6-7 years old) reported by the national Directorate-General of Health, thus showing that an early diagnosis might be possible and helpful for the mitigation of childhood asthma. Environmental context (urban, suburban or rural), gender and family asthma history showed clear associations with asthma prevalence, namely non-rural location, male gender, and having an asthmatic parent were found to be risk factors.