A score system for quality evaluation of RNA sequence tags: an improvement for gene expression profiling

Background: High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) or Sequencing-by-Synthesis (SBS) represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. Results: This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. Conclusion: These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at http://gdm.fmrp.usp.br/s3t/.S3T source code and datasets can also be downloaded from the aforementioned website.

Experimental Infection of the Opossum Didelphis aurita by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense and Amblyomma dubitatum

This work evaluated the infection of opossums (Didelphis aurita) by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and their role as amplifier hosts for horizontal transmission to Amblyomma cajennense and/or Amblyomma dubitatum ticks. Infection in D. aurita was induced by intraperitoneal inoculation with R. felis (n = 4 opossums), R. bellii (n = 4), and R. parkeri (n = 2). Another group of six opossums were inoculated intraperitoneally with Leibovitz-15 sterile culture medium, representing the uninfected groups (n = 2 opossums simultaneously to each infected group). Opossum blood samples collected during the study were used for DNA extraction, followed by real-time polymerase chain reaction targeting the rickettsial gene gltA, hematology, and detection of Rickettsia spp.-reactive antibodies by indirect immunofluorescence assay. Opossums were infested with uninfected A. cajennense and/or A. dubitatum for 30 days postinoculation (DPI). Flat ticks molted from ticks fed on opossums were allowed to feed on uninfected rabbits, which were tested for seroconversion by immunofluorescence assay. Samples of flat ticks were also tested by real-time polymerase chain reaction. Inoculated opossums showed no clinical abnormalities. Antibodies to Rickettsia spp. were first detected at the second to fourth DPI, with detectable titers until the 150th DPI. Rickettsemia was detected only in one opossum inoculated with R. parkeri, at the eighth DPI. Only one A. cajennense tick (2.0%) previously fed on a R. parkeri-inoculated opossum became infected. None of the rabbits infested with opossum-derived ticks seroconverted. The study demonstrated that R. felis, R. bellii, and R. parkeri were capable to produce antibody response in opossums, however, with undetectable rickettsemia for R. felis and R. bellii, and very low rickettsemia for R. parkeri. Further studies must be done with different strains of these rickettsiae, most importantly the strains that have never gone through in vitro passages.

Experimental Infection of Opossums Didelphis aurita by Rickettsia rickettsii and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense

The present study evaluated the infection of opossums (Didelphis aurita) by Rickettsia rickettsii and their role as amplifier hosts for horizontal transmission of R. rickettsii to Amblyomma cajennense ticks. Three groups of opossums were evaluated: on day 0, group 1 (G1) was inoculated intraperitoneally with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and group 3 (G3) was the uninfected control group. Opossum rectal temperature was measured daily. Blood samples were collected every 2 to 4 days during 30 days, and used to (1) inoculate guinea pigs intraperitoneally; (2) extract DNA followed by real-time polymerase chain reaction (PCR) targeting the rickettsial gene gltA; (3) study hematology; (4) detect R. rickettsii-reactive antibodies by indirect direct immunofluorescence assay (IFA). Blood was also collected every 10 days from days 30 to 180, to be tested by serology. Opossums were infested by uninfected A. cajennense larvae and nymphs from days 3 to 15. Engorged ticks were collected and allowed to molt in an incubator. Thereafter, the subsequent flat ticks were allowed to feed on uninfected rabbits, which were tested for seroconversion by IFA. Samples of flat ticks were also tested by real-time PCR. All G1 and G2 opossums became infected by R. rickettsii, as demonstrated by real-time PCR or/and guinea pig inoculation, but they showed no clinical abnormality. Rickettsemia was first detected at days 2 to 8, lasting intermittently till days 1 to 30. Approximately 18% and 5% of the flat ticks previously fed on G1 and G2 opossums, respectively, became infected by R. rickettsii, but only the rabbits infested with G1-derived ticks seroconverted. The study demonstrated that R. rickettsii was capable of infecting opossums without causing illness and developing rickettsemia capable of causing infection in guinea pigs and ticks, although the infection rate in ticks was low.

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.

Evaluation of Laser Phototherapy in the Inflammatory Process of the Rat's TMJ Induced by Carrageenan

Aim: The aim of this study was to evaluate, by light microscopy, the effects of laser phototherapy (LPT) at 780nm or a combination of 660 and 790 nm, on the inflammatory process of the rat temporomandibular joint (TMJ) induced by carrageen. Background: Temporomandibular disorders (TMDs) are frequent in the population and generally present an inflammatory component. Previous studies have evidenced positive effects of laser phototherapy on TMDs. However, its mechanism of action on the inflammation of the TMJ is not known yet. Materials and Methods: Eighty-five Wistar rats were divided into 9 groups: G1, Saline; G2, Saline + LPT IR; G3, Saline + LPT IR + R; G4, Carrageenan; G5, Carrageenan + LPT IR; G6, Carrageenan + LPT IR + R; G7, previous LPT + Carrageenan; G8, previous LPT + carrageenan + LPT IR; and G9, previous LPT + carrageenan + LPT IR + R, and then subdivided in subgroups of 3 and 7 days. After animal death, specimens were taken, routinely cut and stained with HE, Sirius Red, and Toluidine Blue. Descriptive analysis of components of the TMJ was done. The synovial cell layers were counted. Results: Injection of saline did not produced inflammatory reaction and the irradiated groups did not present differences compared to non-irradiated ones. After carrageenan injection, intense inflammatory infiltration and synovial cell layers proliferation were observed. The infrared irradiated group presented less inflammation and less synovial cell layers number compared to other groups. Previous laser irradiation did not improve the results. Conclusion: It was concluded that the LPT presented positive effects on inflammatory infiltration reduction and accelerated the inflammation process, mainly with IR laser irradiation. The number of synovial cell layers was reduced on irradiated group.

LED Phototherapy to Prevent Mucositis: A Case Report

Objective: The purpose of this case report was to evaluate the efficacy of phototherapy using light-emitting diodes (LEDs) to prevent oral mucositis in a Hodgkin's disease patient treated with the ABVD ( doxorubicin [Adriamycin], bleomycin, vinblastine, and dacarbazine) chemotherapy regimen. Background Data: Mucositis is a common dose-limiting complication of cancer treatment, and if severe it can lead to alterations in treatment planning or suspension of cancer therapy, with serious consequences for tumor response and survival. Therefore, low-power lasers and more recently LEDs, have been used for oral mucositis prevention and management, with good results. Materials and Methods: In this study, a 34-year-old man received intraoral irradiation with an infrared LED array (880 nm, 3.6 J/cm(2), 74 mW) for five consecutive days, starting on chemotherapy day 1. In each chemotherapy cycle, he received the ABVD protocol on days 1 and 15, and received LED treatment for 5 d during each cycle. To analyze the results, the World Health Organization (WHO) scale was used to grade his mucositis, and a visual analogue scale (VAS) was used for pain evaluation, on days 1, 3, 7, 10, and 13 post-chemotherapy. Results: The results showed that the patient did not develop oral mucositis during the five chemotherapy cycles, and he had no pain symptoms. Conclusion: LED therapy was a safe and effective method for preventing oral mucositis in this case report. However, further randomized studies with more patients are needed to prove the efficacy of this method.

Evaluation of the Bond Strength Between a Composite Resin and Enamel Submitted to Bleaching Treatment and Etched with Er:YAG Laser

Background: The use of laser irradiation for dental surface treatment may increase tooth-composite bond strength. Its use on bleached teeth may decrease the waiting time between bleaching and restorative procedures. Objective: This study aimed to evaluate the bond strength between a composite resin and bovine enamel bleached with 35% hydrogen peroxide and etched with Er:YAG laser. Materials and Methods: Thirty bovine teeth were randomly divided into six groups (n = 5): G1, unbleached and restored 24 h after storage in artificial saliva, etching with 35% phosphoric acid (PA) (control); G2, unbleached and restored 24 h after storage in artificial saliva, etching with Er:YAG laser and 35% PA; G3, bleached and restored immediately afterward, etching with 35% PA; G4, bleached and restored 24 h after bleaching, etching with 35% PA; G5, bleached and restored immediately afterward, etching with Er:YAG and 35% PA laser; G6, bleached and restored 24 h after bleaching, etching with Er:YAG laser and 35% PA. Bond strength was quantitatively evaluated by microtensile test (1.0 mm/min). Data were submitted to statistical analysis using ANOVA and Tukey tests (alpha - 0.05). Results: Bond strength values (MPa) were G1, 26.17 +/- 4.44; G2, 28.87 +/- 3.94; G3, 17.25 +/- 4.58; G4, 21.93 +/- 5.02; G5, 16.69 +/- 2.31; and G6, 29.06 +/- 8.31. There was no statistically significant difference among groups G1, G2, and G6 (p - 0.119), which presented higher bond strength than group G4, followed by groups G3 and G5. Conclusion: Er:YAG irradiation of bleached surfaces may favor bonding procedures when performed 24 h after bleaching.

Apical Leakage of Three Resin-Based Endodontic Sealers after 810-nm-Diode Laser Irradiation

Objective: To evaluate the influence of 810-nm-diode laser irradiation, applied before root canal filling, on apical sealing ability of three different resin-based sealers (AH Plus, EndoRez, and RealSeal). Background: Lasers have been widely used in endodontics. The dentin wall changes caused by laser irradiation could improve the sealing ability of endodontic cements. Methods: Sixty single-rooted human teeth were divided into six groups, according to the endodontic sealer used and previous 810-nm-diode laser irradiation. The protocol for laser irradiation was 2.5W in a continuous wave, in scanning mode, with four irradiations per tooth. After sample preparation, they were analyzed according to apical leakage with silver nitrate impregnation. Results: The RealSeal sealer achieved minimum leakage rates (1.24 mm), with significant differences at the 1% level (Tukey's test, p < 0.01) from AH Plus (1.84 mm) in nonirradiated groups. When the laser was used, there were also significant differences at the 5% level (p < 0.05) between irradiated groups (1.31 and 1.78 mm, respectively). Conclusion: The 810-nm-diode laser irradiation did not promote significant differences in apical leakage.

Severity of Oral Mucositis in Patients Undergoing Hematopoietic Cell Transplantation and an Oral Laser Phototherapy Protocol: A Survey of 30 Patients

Background Data and Objective: Oral mucositis (OM) is one of the worst cytotoxic effects of chemotherapy and radiotherapy in patients undergoing hematopoietic cell transplantation (HCT), and it causes severe morbidity. Laser phototherapy has been considered as an alternative therapy for prevention and treatment of OM. The aim of this study was to describe the incidence and severity of OM in HCT patients subjected to laser phototherapy, and to discuss its effect on the oral mucosa. Patients and Methods: Information concerning patient age and gender, type of basic disease, conditioning regimen, type of transplant, absence or presence of pain related to the oral cavity, OM grade, and adverse reactions or unusual events were collected from 30 patients undergoing HCT (allogeneic or autologous). These patients were given oral laser phototherapy with a InGaAIP laser (660 nm and 40 mW) daily. The data were tabulated and their frequency expressed as percentages. Results: In the analysis of those with OM, it was observed that 33.4% exhibited grade I, 40% grade II, 23.3% grade III, and 3.3% grade IV disease. On the most critical post-HCT days (D+5 and D+8), it was observed that 63.3% of patients had grade I and 33.3% had grade II disease; no patients had grade III or IV disease in this period. This severity of OM was similar to that seen in other studies of laser phototherapy and OM. Conclusion: The low grades of OM observed in this survey show the beneficial effects of laser phototherapy, but randomized clinical trials are necessary to confirm these findings.

In Vitro Evaluation of Enamel Erosion After Nd:YAG Laser Irradiation and Fluoride Application

Objective: Previous investigations have demonstrated improved enamel demineralization resistance after laser irradiation. Due to the possibility of a synergistic effect between laser and fluoride, this study investigated the effect of fluoridated agents and Nd:YAG irradiation separately and in combination on enamel resistance to erosion. Methods: One hundred bovine enamel blocks were randomly divided into 10 groups: G1, untreated (control); G2, acidic phosphate fluoride (APF) (1.23% F) for 4 min; G3, fluoride varnish for 6 h (NaF, 2.26%); G4, 0.5 W Nd: YAG laser (250 mm pulse width, 10 Hz, 35 J/cm(2), with uniform velocity for 30 sec in each application); G5, 0.75 W Nd:YAG laser (52.5 J/cm(2)); G6, 1.0 W Nd:YAG laser (70 J/cm(2)); G7, APF + 0.75 W Nd:YAG laser; G8, 0.75 W Nd:YAG laser + APF; G9, fluoride varnish + 0.75 W Nd:YAG laser; and G10, 0.75 W Nd:YAG laser + fluoride varnish. During 10 d the erosive cycle was conducted by immersion of the blocks in Sprite light for 1 min, followed by immersion in artificial saliva for 59 min. This procedure was consecutively repeated four times per day. In each day, during the remaining 20 h, the blocks were maintained in artificial saliva. The wear was evaluated by profilometry (days 5 and 10). Data were tested by two-way ANOVA and Bonferroni's tests (p < 0.05). Results: The mean wear at days 5 and 10 was, respectively: G1, 1.83 and 2.67 mu m; G2, 1.04 and 2.60 mu m; G3, 1.03 and 2.48 mu m; G4, 1.13 and 2.47 mu m; G5, 1.07 and 2.44 mu m; G6, 1.0 and 2.35 mu m; G7, 0.75 and 2.27 mu m; G8, 0.80 and 2.12 mu m; G9, 0.76 and 2.47 mu m; and G10, 1.09 and 2.46 mu m. At day 5, all the experimental groups presented significant lesser wear when compared to control group. However, at 10 d, only G7 and G8 were still different from control. Conclusions: The association between APF application and laser irradiation seems to be an alternative preventive measure against dental erosion.

PAR evaluation of treated Class I extraction patients

Objective: To evaluate treatment changes and quality of finishing occlusion in Class I patients treated with four premolar extractions. Material and Methods: Dental casts of 94 subjects (50 males and 44 females) were evaluated. Mean pretreatment age was 13.46 years, and mean treatment time was 2.09 years. The peer assessment rating (PAR) index was obtained from pretreatment and posttreatment dental casts. Results: The mean pretreatment PAR index of 29.46 was reduced to 6.32 at posttreatment stage, achieving a reduction of 78.54% with treatment. There was correlation between the initial PAR and correction during treatment, that is, the more severe the malocclusion the greater the treatment changes. Conclusion: The cases evaluated showed a high-standard orthodontic finishing.

Application of Low-Level Laser Irradiation (LLLI) and rhBMP-2 in Critical Bone Defect of Ovariectomized Rats: Histomorphometric Evaluation

Objectives: The aim of this study was to evaluate the osteogenic potential of recombinant human bone morphogenetic protein-2 (rhBMP-2) and low-level laser irradiation (LLLI), isolated or combined in critical bone defects (5mm) in parietal bone using ovariectomized female rats as an experimental animal model. Materials and Methods: Forty-nine female Wistar rats, bilaterally ovariectomized (OVX), were divided into seven treatment groups of seven animals each: (I) laser in a single application, (II) 7 mu g of pure rhBMP-2, (III) laser and 7 mu g of pure rhBMP-2, (IV) 7 mu g of rhBMP-2/monoolein gel, (V) laser and 7 mu g of rhBMP-2/monoolein gel, (VI) laser and pure monoolein gel, and (VII) critical bone defect controls. The low-level laser source used was a gallium aluminum arsenide semiconductor diode laser device (lambda = 780 nm, D = 120 J/cm(2)). Results: Groups II and III presented higher levels of newly formed bone than all other groups with levels of 40.57% and 40.39%, respectively (p < 0.05). The levels of newly formed bone of groups I, IV, V, and VI were similar with levels of 29.67%, 25.75%, 27.75%, and 30.64%, respectively (p > 0.05). The area of new bone formation in group VII was 20.96%, which is significantly lower than groups I, II, III, and VI. Conclusions: It was concluded that pure rhBMP-2 and a single dose of laser application stimulated new bone formation, but the new bone formation area was significantly increased when only rhBMP-2 was used. Additionally, the laser application in combination with other treatments did not influence the bone formation area.

Evaluation of Protective Effect of a Water-In-Oil Microemulsion Incorporating Quercetin Against UVB-Induced Damage in Hairless Mice Skin

Purpose. Histological aspects were considered in order to evaluate the in vivo photoprotective effect of a w/o microemulsion containing quercetin against UVB irradiation-induced dermal damages. The toxicity in cell culture and the potential skin irritation resulting from topical application of this formulation were investigated. Methods. Mouse dorsal surfaces were treated topically with 300 mg of the unloaded and quercetin-loaded (0.3%, w/w) microemulsions before and after exposure to UVB (2.87 J/cm(2)) irradiation. The untreated control groups irradiated and non-irradiated were also evaluated. UVB-induced histopathological changes as well as the photoprotective effect of this formulation were evaluated considering the parameters of infiltration of inflammatory cells, epidermis thickening (basale and spinosum layers) and collagen and elastic fiber contents. The cytotoxicity of the reported formulation was evaluated in L929 mice fibroblasts by MTT assay and the skin irritation was investigated after topical application of both unloaded and quercetin-loaded microemulsions once a day for 15 days. Results. The results demonstrated that the w/o microemulsion containing quercetin reduced the incidence of histological skin alterations, mainly the connective-tissue damage, induced by exposure to UVB irradiation. This suggests that protective effects of this formulation against UV-induced responses are not secondary to the interference of UV transmission (i.e., blocking the UVB radiation from being absorbed by the skin), as is usually implied with UVB absorbers and sunscreens, but is instead due to different biological effects of this flavonoid. Furthermore, by evaluating the cytotoxic effect on L929 cells and histological aspects such as infiltration of inflammatory cells and epidermis thickness of hairless mice, the present study also demonstrated the lack of toxicity of the proposed system. Conclusion. Based on these mice models, a detailed characterization of the w/o microemulsion incorporating quercetin effects as a photochemoprotective agent on human skin is presented.

Bone Marrow Concentrate and Bovine Bone Mineral for Sinus Floor Augmentation: A Controlled, Randomized, Single-Blinded Clinical and Histological Trial-Per-Protocol Analysis

Purpose: The purpose of this work was to evaluate the potential of substituting autogenous bone (AB) by bone marrow aspirate concentrate (BMAC). Both AB and BMAC were tested in combination with a bovine bone mineral (BBM) for their ability of new bone formation (NBF) in a multicentric, randomized, controlled, clinical and histological noninferiority trial. Materials and Methods: Forty-five severely atrophied maxillary sinus from 26 patients were evaluated in a partial cross-over design. As test arm, 34 sinus of 25 patients were augmented with BBM and BMAC containing mesenchymal stem cells. Eleven control sinus from 11 patients were augmented with a mixture of 70% BBM and 30% AB. Biopsies were obtained after a 3-4-month healing period at time of implant placement and histomorphometrically analyzed for NBF. Results: NBF was 14.3%+/- 1.8% for the control and nonsignificantly lower (12.6%+/- 1.7%) for the test (90% confidence interval: -4.6 to 1.2). Values for BBM (31.3%+/- 2.7%) were significantly higher for the test compared with control (19.3%+/- 2.5%) (p < 0.0001). Nonmineralized tissue was lower by 3.3% in the test compared with control (57.6%; p = 0.137). Conclusions: NBF after 3-4 months is equivalent in sinus, augmented with BMAC and BBM or a mixture of AB and BBM. This technique could be an alternative for using autografts to stimulate bone formation.

The Coupled Aerosol and Tracer Transport model to the Brazilian developments on the Regional Atmospheric Modeling System (CATT-BRAMS) - Part 1: Model description and evaluation

We introduce the Coupled Aerosol and Tracer Transport model to the Brazilian developments on the Regional Atmospheric Modeling System (CATT-BRAMS). CATT-BRAMS is an on-line transport model fully consistent with the simulated atmospheric dynamics. Emission sources from biomass burning and urban-industrial-vehicular activities for trace gases and from biomass burning aerosol particles are obtained from several published datasets and remote sensing information. The tracer and aerosol mass concentration prognostics include the effects of sub-grid scale turbulence in the planetary boundary layer, convective transport by shallow and deep moist convection, wet and dry deposition, and plume rise associated with vegetation fires in addition to the grid scale transport. The radiation parameterization takes into account the interaction between the simulated biomass burning aerosol particles and short and long wave radiation. The atmospheric model BRAMS is based on the Regional Atmospheric Modeling System (RAMS), with several improvements associated with cumulus convection representation, soil moisture initialization and surface scheme tuned for the tropics, among others. In this paper the CATT-BRAMS model is used to simulate carbon monoxide and particulate material (PM(2.5)) surface fluxes and atmospheric transport during the 2002 LBA field campaigns, conducted during the transition from the dry to wet season in the southwest Amazon Basin. Model evaluation is addressed with comparisons between model results and near surface, radiosondes and airborne measurements performed during the field campaign, as well as remote sensing derived products. We show the matching of emissions strengths to observed carbon monoxide in the LBA campaign. A relatively good comparison to the MOPITT data, in spite of the fact that MOPITT a priori assumptions imply several difficulties, is also obtained.