Microbial Evaluation Of Traumatized Teeth Treated With Triple Antibiotic Paste Or Calcium Hydroxide With 2% Chlorhexidine Gel In Pulp Revascularization.

Revascularization outcome depends on microbial elimination because apical repair will not happen in the presence of infected tissues. This study evaluated the microbial composition of traumatized immature teeth and assessed their reduction during different stages of the revascularization procedures performed with 2 intracanal medicaments. Fifteen patients (7-17 years old) with immature teeth were submitted to the revascularization procedures; they were divided into 2 groups according to the intracanal medicament used: TAP group (n = 7), medicated with a triple antibiotic paste, and CHP group (n = 8), dressed with calcium hydroxide + 2% chlorhexidine gel. Samples were taken before any treatment (S1), after irrigation with 6% NaOCl (S2), after irrigation with 2% chlorhexidine (S3), after intracanal dressing (S4), and after 17% EDTA irrigation (S5). Cultivable bacteria recovered from the 5 stages were counted and identified by means of polymerase chain reaction assay (16S rRNA). Both groups had colony-forming unit counts significantly reduced after S2 (P < .05); however, no significant difference was found between the irrigants (S2 and S3, P = .99). No difference in bacteria counts was found between the intracanal medicaments used (P = .95). The most prevalent bacteria detected were Actinomyces naeslundii (66.67%), followed by Porphyromonas endodontalis, Parvimonas micra, and Fusobacterium nucleatum, which were detected in 33.34% of the root canals. An average of 2.13 species per canal was found, and no statistical correlation was observed between bacterial species and clinical/radiographic features. The microbial profile of infected immature teeth is similar to that of primarily infected permanent teeth. The greatest bacterial reduction was promoted by the irrigation solutions. The revascularization protocols that used the tested intracanal medicaments were efficient in reducing viable bacteria in necrotic immature teeth.

Diet Carotenoid Lutein Modulates The Expression Of Genes Related To Oxygen Transporters And Decreases Dna Damage And Oxidative Stress In Mice.

Lutein (LT) is a carotenoid obtained by diet and despite its antioxidant activity had been biochemically reported, few studies are available concerning its influence on the expression of antioxidant genes. The expression of 84 genes implicated in antioxidant defense was quantified using quantitative reverse transcription polymerase chain reaction array. DNA damage was measured by comet assay and glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were quantified as biochemical parameters of oxidative stress in mouse kidney and liver. cDDP treatment reduced concentration of GSH and increased TBARS, parameters that were ameliorated in treatment associated with LT. cDDP altered the expression of 32 genes, increasing the expression of GPx2, APC, Nqo1 and CCs. LT changed the expression of 37 genes with an induction of 13 mainly oxygen transporters. In treatments associating cDDP and LT, 30 genes had their expression changed with a increase of the same genes of the cDDP treatment alone. These results suggest that LT might act scavenging reactive species and also inducing the expression of genes related to a better antioxidant response, highlighting the improvement of oxygen transport. This improved redox state of the cell through LT treatment could be related to the antigenotoxic and antioxidant effects observed.

Reduced Plasma Angiotensin Ii Levels Are Reversed By Hydroxyurea Treatment In Mice With Sickle Cell Disease.

Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin-angiotensin system (RAS) may be altered in an animal model of SCD. Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50-75mg/kg/day, 4weeks) treatment on these parameters were also determined. Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart. Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.

Proinflammatory Activity Of Primarily Infected Endodontic Content Against Macrophages After Different Phases Of The Root Canal Therapy.

This study investigated the presence of target bacterial species and the levels of endotoxins in teeth with apical periodontitis. Levels of inflammatory mediators (interleukin [IL]-1β and tumor necrosis factor [TNF]-α) were determined after macrophage stimulation with endodontic content after different phases of endodontic therapy using different irrigants. Thirty primarily infected root canals were randomly assigned into 3 groups according to the irrigant used for root canal preparation (n = 10 per group): GI: 2.5% sodium hypochlorite, GII: 2% chlorhexidine gel, and GIII (control group): saline solution. Root canal samples were taken by using paper points before (s1) and after root canal instrumentation (s2), subsequently to 17% EDTA (s3), after 30 days of intracanal medication (Ca[OH]2 + saline solution) (s4), and before root canal obturation (s5). Polymerase chain reaction (16S recombinant DNA) and limulus amebocyte lysate assay were used for bacterial and endotoxin detection, respectively. Macrophages were stimulated with the root canal contents for IL-1β/TNF-α measurement using enzyme-linked immunosorbent assay. Porphyromonas gingivalis (17/30), Porphyromonas endodontalis (15/30), and Prevotella nigrescens (11/30) were the most prevalent bacterial species. At s1, endotoxins were detected in 100% of the root canals (median = 32.43 EU/mL). In parallel, substantial amounts of IL-1β and TNF-α were produced by endodontic content-stimulated macrophages. At s2, a significant reduction in endotoxin levels was observed in all groups, with GI presenting the greatest reduction (P < .05). After a root canal rinse with EDTA (s3), intracanal medication (s4), and before root canal obturation (s5), endotoxin levels reduced without differences between groups (P < .05). IL-1β and TNF-α release decreased proportionally to the levels of residual endotoxin (P < .05). Regardless of the use of sodium hypochlorite or CHX, the greatest endotoxin reduction occurs after chemomechanical preparation. Increasing steps of root canal therapy associated with intracanal medication enhances endotoxin reduction, leading to a progressively lower activation of proinflammatory cells such as macrophages.

G-quadruplex Formation Enhances Splicing Efficiency Of Pax9 Intron 1.

G-quadruplexes are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e., interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex-forming region located near exon 1, which is present in all known sequenced placental mammals. Using circular dichroism (CD) analysis and CD melting, we showed that these sequences are able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon-intron boundary, we used a validated double-reporter splicing assay and qPCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically the splicing efficiency of human PAX9 intron 1. The less stable, rat quadruplex had a less efficient splicing when compared to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether, these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1.

The G209A mutation in the alpha-synuclein gene in Brazilian families with Parkinson's disease

A missense G209A mutation of the alpha-synuclein gene was recently described in a large Contursi kindred with Parkinson's disease (PD). The objective of this study is to determine if the mutation G209A of the alpha-synuclein gene was present in 10 Brazilian families with PD. PD patients were recruited from movement disorders clinics of Brazil. A family history with two or more affected in relatives was the inclusion criterion for this study. The alpha-synuclein G209A mutation assay was made using polymerase chain reaction and the restriction enzyme Tsp45I. Ten patients from 10 unrelated families were studied. The mean age of PD onset was 42.7 years old. We did not find the G209A mutation in our 10 families with PD. Our results suggest that alpha-synuclein mutation G209A is uncommon in Brazilian PD families.

Dna Extraction From Filter-paper Spots Of Vaginal Samples Collected After Sexual Violence.

To detect the presence of male DNA in vaginal samples collected from survivors of sexual violence and stored on filter paper. A pilot study was conducted to evaluate 10 vaginal samples spotted on sterile filter paper: 6 collected at random in April 2009 and 4 in October 2010. Time between sexual assault and sample collection was 4-48hours. After drying at room temperature, the samples were placed in a sterile envelope and stored for 2-3years until processing. DNA extraction was confirmed by polymerase chain reaction for human β-globin, and the presence of prostate-specific antigen (PSA) was quantified. The presence of the Y chromosome was detected using primers for sequences in the TSPY (Y7/Y8 and DYS14) and SRY genes. β-Globin was detected in all 10 samples, while 2 samples were positive for PSA. Half of the samples amplified the Y7/Y8 and DYS14 sequences of the TSPY gene and 30% amplified the SRY gene sequence of the Y chromosome. Four male samples and 1 female sample served as controls. Filter-paper spots stored for periods of up to 3years proved adequate for preserving genetic material from vaginal samples collected following sexual violence.

Intermittent Fasting Induces Hypothalamic Modifications Resulting In Low Feeding Efficiency, Low Body Mass And Overeating.

Intermittent fasting (IF) is an often-used intervention to decrease body mass. In male Sprague-Dawley rats, 24 hour cycles of IF result in light caloric restriction, reduced body mass gain, and significant decreases in the efficiency of energy conversion. Here, we study the metabolic effects of IF in order to uncover mechanisms involved in this lower energy conversion efficiency. After 3 weeks, IF animals displayed overeating during fed periods and lower body mass, accompanied by alterations in energy-related tissue mass. The lower efficiency of energy use was not due to uncoupling of muscle mitochondria. Enhanced lipid oxidation was observed during fasting days, whereas fed days were accompanied by higher metabolic rates. Furthermore, an increased expression of orexigenic neurotransmitters AGRP and NPY in the hypothalamus of IF animals was found, even on feeding days, which could explain the overeating pattern. Together, these effects provide a mechanistic explanation for the lower efficiency of energy conversion observed. Overall, we find that IF promotes changes in hypothalamic function that explain differences in body mass and caloric intake.

Nanomolar Levels Of Pahs In Extracts From Urban Air Induce Mapk Signaling In Hepg2 Cells.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that occur naturally in complex mixtures. Many of the adverse health effects of PAHs including cancer are linked to the activation of intracellular stress response signaling. This study has investigated intracellular MAPK signaling in response to PAHs in extracts from urban air collected in Stockholm, Sweden and Limeira, Brazil, in comparison to BP in HepG2 cells. Nanomolar concentrations of PAHs in the extracts induced activation of MEK4 signaling with down-stream increased gene expression of several important stress response mediators. Involvement of the MEK4/JNK pathway was confirmed using siRNA and an inhibitor of JNK signaling resulting in significantly reduced MAPK signaling transactivated by the AP-1 transcription factors ATF2 and c-Jun. ATF2 was also identified as a sensitive stress responsive protein with activation observed at extract concentrations equivalent to 0.1 nM BP. We show that exposure to low levels of environmental PAH mixtures more strongly activates these signaling pathways compared to BP alone suggesting effects due to interactions. Taken together, this is the first study showing the involvement of MEK4/JNK/AP-1 pathway in regulating the intracellular stress response after exposure to nanomolar levels of PAHs in environmental mixtures.

Nat2, Xrcc1 And Hogg1 Polymorphisms, Cigarette Smoking, Alcohol Consumption And Risk Of Upper Aerodigestive Tract Cancer.

To evaluate associations between polymorphisms of the N-acetyltransferase 2 (NAT2), human 8-oxoguanine glycosylase 1 (hOGG1) and X-ray repair cross-complementing protein 1 (XRCC1) genes and risk of upper aerodigestive tract (UADT) cancer. A case-control study involving 117 cases and 224 controls was undertaken. The NAT2 gene polymorphisms were genotyped by automated sequencing and XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms were determined by Polymerase Chain Reaction followed by Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Slow metabolization phenotype was significantly associated as a risk factor for the development of UADT cancer (p=0.038). Furthermore, haplotype of slow metabolization was also associated with UADT cancer (p=0.014). The hOGG1 Ser326Cys polymorphism (CG or GG vs. CC genotypes) was shown as a protective factor against UADT cancer in moderate smokers (p=0.031). The XRCC1 Arg399Gln polymorphism (GA or AA vs. GG genotypes), in turn, was a protective factor against UADT cancer only among never-drinkers (p=0.048). Interactions involving NAT2, XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms may modulate the risk of UADT cancer in this population.

Characterization Of Microsatellite Markers Developed From Prosopis Rubriflora And Prosopis Ruscifolia (leguminosae - Mimosoideae), Legume Species That Are Used As Models For Genetic Diversity Studies In Chaquenian Areas Under Anthropization In South America.

Prosopis rubriflora and Prosopis ruscifolia are important species in the Chaquenian regions of Brazil. Because of the restriction and frequency of their physiognomy, they are excellent models for conservation genetics studies. The use of microsatellite markers (Simple Sequence Repeats, SSRs) has become increasingly important in recent years and has proven to be a powerful tool for both ecological and molecular studies. In this study, we present the development and characterization of 10 new markers for P. rubriflora and 13 new markers for P. ruscifolia. The genotyping was performed using 40 P. rubriflora samples and 48 P. ruscifolia samples from the Chaquenian remnants in Brazil. The polymorphism information content (PIC) of the P. rubriflora markers ranged from 0.073 to 0.791, and no null alleles or deviation from Hardy-Weinberg equilibrium (HW) were detected. The PIC values for the P. ruscifolia markers ranged from 0.289 to 0.883, but a departure from HW and null alleles were detected for certain loci; however, this departure may have resulted from anthropic activities, such as the presence of livestock, which is very common in the remnant areas. In this study, we describe novel SSR polymorphic markers that may be helpful in future genetic studies of P. rubriflora and P. ruscifolia.

Fractalkine (cx3cl1) Is Involved In The Early Activation Of Hypothalamic Inflammation In Experimental Obesity.

Hypothalamic inflammation is a common feature of experimental obesity. Dietary fats are important triggers of this process, inducing the activation of toll-like receptor-4 (TLR4) signaling and endoplasmic reticulum stress. Microglia cells, which are the cellular components of the innate immune system in the brain, are expected to play a role in the early activation of diet-induced hypothalamic inflammation. Here, we use bone marrow transplants to generate mice chimeras that express a functional TLR4 in the entire body except in bone marrow-derived cells or only in bone marrow-derived cells. We show that a functional TLR4 in bone marrow-derived cells is required for the complete expression of the diet-induced obese phenotype and for the perpetuation of inflammation in the hypothalamus. In an obesity-prone mouse strain, the chemokine CX3CL1 (fractalkine) is rapidly induced in the neurons of the hypothalamus after the introduction of a high-fat diet. The inhibition of hypothalamic fractalkine reduces diet-induced hypothalamic inflammation and the recruitment of bone marrow-derived monocytic cells to the hypothalamus; in addition, this inhibition reduces obesity and protects against diet-induced glucose intolerance. Thus, fractalkine is an important player in the early induction of diet-induced hypothalamic inflammation, and its inhibition impairs the induction of the obese and glucose intolerance phenotypes.

Evidences Of Endocytosis Via Caveolae Following Blood-brain Barrier Breakdown By Phoneutria Nigriventer Spider Venom.

Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood-brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

Development And Characterization Of Microsatellite Loci For Tabebuia Cassinoides (bignoniaceae).

Tabebuia cassinoides (Lam.) DC., popularly known as caxeta, is a tree species that belongs to the plant family Bignoniaceae. This species is endemic to the Brazilian Atlantic Forest and is widely exploited commercially. To date, little is known about its genetic structure, preventing the establishment of adequate management plans for this taxon. The objective of this study was to construct a microsatellite-enriched genomic library for T. cassinoides to select polymorphic loci, and standardize polymerase chain reaction amplification conditions. Of the 15 loci examined, 5 were polymorphic. The number of alleles per locus ranged from 2 to 8, with a mean of 4.4. The microsatellite loci described here represent the basis for detailed population genetic studies of this species, which will greatly contribute for the development of better conservation strategies for this taxon.

Two-component System Vicrk Regulates Functions Associated With Establishment Of Streptococcus Sanguinis In Biofilms.

Streptococcus sanguinis is a commensal pioneer colonizer of teeth and an opportunistic pathogen of infectious endocarditis. The establishment of S. sanguinis in host sites likely requires dynamic fitting of the cell wall in response to local stimuli. In this study, we investigated the two-component system (TCS) VicRK in S. sanguinis (VicRKSs), which regulates genes of cell wall biogenesis, biofilm formation, and virulence in opportunistic pathogens. A vicK knockout mutant obtained from strain SK36 (SKvic) showed slight reductions in aerobic growth and resistance to oxidative stress but an impaired ability to form biofilms, a phenotype restored in the complemented mutant. The biofilm-defective phenotype was associated with reduced amounts of extracellular DNA during aerobic growth, with reduced production of H2O2, a metabolic product associated with DNA release, and with inhibitory capacity of S. sanguinis competitor species. No changes in autolysis or cell surface hydrophobicity were detected in SKvic. Reverse transcription-quantitative PCR (RT-qPCR), electrophoretic mobility shift assays (EMSA), and promoter sequence analyses revealed that VicR directly regulates genes encoding murein hydrolases (SSA_0094, cwdP, and gbpB) and spxB, which encodes pyruvate oxidase for H2O2 production. Genes previously associated with spxB expression (spxR, ccpA, ackA, and tpK) were not transcriptionally affected in SKvic. RT-qPCR analyses of S. sanguinis biofilm cells further showed upregulation of VicRK targets (spxB, gbpB, and SSA_0094) and other genes for biofilm formation (gtfP and comE) compared to expression in planktonic cells. This study provides evidence that VicRKSs regulates functions crucial for S. sanguinis establishment in biofilms and identifies novel VicRK targets potentially involved in hydrolytic activities of the cell wall required for these functions.