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Background: The objective this study was to evaluate the influence of preventive remineralizing techniques on surface roughness and volume loss of dentin submitted to erosive and/or abrasive challenges., Material and Methods: One hundred and eighty specimens of bovine root dentin were made; half of each was isolated (without treatment - WT) and half was subjected to the following remineralizing techniques: fluoride varnish (FV); Regenerate Boosting Serum® (RBS); Er,Cr:YSGG laser (L); fluoride varnish+laser (FV+L); Regenerate Boosting Serum®+laser (RBS+L). The specimens were submitted to erosive, abrasive and erosive followed by abrasive challenge. Erosion was carried out for 5 minutes, twice a day for 10 days. Abrasion was performed with an electric toothbrush and slurry solution for 60 seconds. The evaluation was performed by confocal laser scanning microscopy. Analysis of variance and Tukey tests were used for surface roughness; volume loss comparison was performed using the Kruskal-Wallis test and Dunn's post-hoc ( p <0.05)., Results: There was no statistically significant difference in the surface roughness of the reference area in relation to the areas submitted to different types of treatment and challenges ( p >0.05). Regarding volume loss, the untreated group submitted to erosive/abrasive challenges showed greater percentage of volume loss compared to the other groups ( p <0.05)., Conclusions: It is concluded that preventive remineralizing techniques are effective in maintaining dentin volume after erosive/abrasive challenges. Key words: YSGG lasers, Dentin, Erosion, Tooth Abrasion., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright: © 2024 Medicina Oral S.L.)

Background: To evaluate the effectiveness of Er:YAG and Nd:YAG laser on dentin hypersensitivity prevention, associated or not to acidulated phosphate fluoride (APF) after erosive challenge., Material and Methods: 104 specimens were obtained from bovine dentine and divided into groups (n=13): G1: Er:YAG; G2: Er:YAG followed by application of APF; G3: application of APF followed by Er:YAG, simultaneously; G4: Nd:YAG; G5: Nd:YAG followed by application of APF; G6: application of APF followed by Nd:YAG, simultaneously; G7:application of APF; G8: untreated. The parameters for Er:YAG were:10s, distance of 4mm, water cooling flow of 2mL/min, 2Hz, 3.92J/cm2. For the Nd:YAG: 10s, distance of 1mm, without cooling, 10Hz, 70.7J/cm2. The erosive drink was a cola at 4°C, 3×/day for 1 minute, for 5 days. Roughness and wear analysis were done in confocal laser microscope. Data were statistically analyzed (α=0.05)., Results: As regards roughness, there was no statistically difference among the groups. The groups irradiated with Er:YAG had a volume loss lower. G6 showed higher values than the groups irradiated with Er:YAG and lower than the other groups. The other groups irradiated with Nd:YAG showed similar wear results to the control., Conclusions: The Er:YAG laser showed the lowest volume loss from wear analysis, suggesting the increased the acid resistance of dentin. Key words: Dentine sensitivity, Lasers, Sodium fluoride., Competing Interests: Conflicts of interest The authors declare that they have no conflict of interest., (Copyright: © 2024 Medicina Oral S.L.)

Obesity significantly decreases life expectancy and increases the incidence of age-related dysfunctions, including β-cell dysregulation leading to inadequate insulin secretion. Here, we show that diluted plasma from obese human donors acutely impairs β-cell integrity and insulin secretion relative to plasma from lean subjects. Similar results were observed with diluted sera from obese rats fed ad libitum, when compared to sera from lean, calorically restricted, animals. The damaging effects of obese circulating factors on β-cells occurs in the absence of nutrient overload, and mechanistically involves mitochondrial dysfunction, limiting glucose-supported oxidative phosphorylation and ATP production. We demonstrate that increased levels of adiponectin, as found in lean plasma, are the protective characteristic preserving β-cell function; indeed, sera from adiponectin knockout mice limits β-cell metabolic fluxes relative to controls. Furthermore, oxidative phosphorylation and glucose-sensitive insulin secretion, which are completely abrogated in the absence of this hormone, are restored by the presence of adiponectin alone, surprisingly even in the absence of other serological components, for both the insulin-secreting INS1 cell line and primary islets. The addition of adiponectin to cells treated with plasma from obese donors completely restored β-cell functional integrity, indicating the lack of this hormone was causative of the dysfunction. Overall, our results demonstrate that low circulating adiponectin is a key damaging element for β-cells, and suggest strong therapeutic potential for the modulation of the adiponectin signaling pathway in the prevention of age-related β-cell dysfunction., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Aim: To evaluate the presence of Streptococcus mutans (SM), main pathogen of dental caries, interleukin-6 and -10 in samples of amniotic fluid (AF) and clinical and oral data of the healthy pregnancy, to investigate the SM exposition in intrauterine life and to elucidate the presence of antibodies to SM in newborns' saliva (SA) with undetectable levels of this microorganism described in the previous study., Methods: The study involved the participation of 26 women with term deliveries and without gestational complications. General health data were collected through a questionnaire applied to the participants. The volunteers were examined orally, the caries experience (decayed, missing, and filled teeth (DMFT) scores) was calculated and unstimulated SA was collected. The acquisition of AF was performed during cesarean sections and stocked on ice. The analysis of the presence of SM-DNA was performed by real-time PCR assays with specific primers. Levels of IL-6 and IL-10 were performed by ELISA with specific kits for these interleukins in the AF samples., Results: 69.2% of the AF samples had detectable SM, and 65% of the women had the bacteria in the AF and SA at the same time. There was a positive association between the presence of SM in AF and a report of not going to the dentist frequently ( p <.05). There was no statistically significant difference between the frequency of AF with or without detection of MS and active caries ( p >.05). However, the DMFT scores of women with positive AF for SM were higher than that of women without detectable MS ( p <.05). IL-6 levels were higher than IL-10 in AF ( p <.05). There were no significant differences in the levels of interleukins between samples with detectable SM or in AF and even with clinical data analysis ( p >.05)., Conclusions: The AF samples have SM in quantifiable levels, probably associated with the greatest caries experience, and seen by the highest DMFT index. Interleukin levels were not related to the presence of MS. The presence of SM in AF may explain the mucosal immune stimulation of newborn children by the previous detection of salivary antibodies against SM.

Context: A promising option for the prevention of dental caries is the use of laser irradiation., Aims: Evaluate the effects of Er:YAG, Nd:YAG, and CO 2 laser irradiation, associated or not to 2% sodium fluoride (2% NaF), on root caries prevention., Material and Methods: One hundred and four human root dentin samples were divided in eight groups (n = 13). A 9-mm 2 -area on each dentin sample was delimited and treated as follows: G1: no treatment (control); G2: 2% NaF; G3: Er:YAG; G4: 2% NaF + Er:YAG; G5: Nd:YAG; G6: 2% NaF + Nd:YAG; G7: CO 2 ; G8: 2% NaF + CO 2 . When used, the 2% NaF was applied before irradiation for 4 min. The samples were subjected to a 2-week cariogenic challenge, consisted of daily immersion in de-remineralizing solutions for 6 h and 18 h, respectively. Knoop hardness (KHN) were evaluated (10 g and 20 s) at different depths from the dentin surface. The samples (n = 3) were prepared for scanning electron microscopy (SEM). Microhardness data were analysed by one-way analysis of variance (ANOVA) and Fisher's test (α = 5%)., Results: The Er:YAG laser group (KHN = 41.30) promoted an increase in acid resistance of the dentin (P < 0.05) when compared to all groups. There was no synergism between laser irradiation and 2% NaF application. Morphological changes were observed after irradiation with all lasers; carbonization and cracks were also observed, except when Er:YAG were used., Conclusions: Er:YAG laser irradiation can safely increase the acid resistance of the dentin surface of the root, since it promoted a significant increase in surface hardness. The application of 2% NaF did not result in a synergistic effect., Competing Interests: None

Aim: To evaluate the initial adhesion and formation of Streptococcus mutans biofilm in vitro in the presence of saliva, human colostrum and 3'-sialyllactose., Methods: Human colostrum and salivas were collected from 30 mothers and newborn postpartum. Eighteen hours culture of S. mutans was treated with colostrum or 3'-sialyllactose in three different moments: before, during, and after 24 h from the microbial inoculation. Salivas were also tested in conjunction with colostrum. The assays were realized in sterile 96-well flat-bottom microtiter plates for 24 h. The biofilms were fixed, washed, stained with crystal violet, and extracted. Absorbance was measured to evaluate biofilm growth mass., Results: Colostrum applied after and during the inoculation decreased biofilm formation when compared with the control ( p  < .05). The presence of saliva increased the biofilm biomass ( p  < .05). The application of 3'-sialyllactose reduced biofilm formation independently of moments of application ( p  < .05)., Conclusion: Saliva contributed to the proliferation of biofilm and colostrum did not prevent the initial adhesion, but interfered in the accumulation and development of microorganisms in biofilms. 3'-sialyllactose significantly decreased biofilm formation. This information expands the importance of colostrum as a potent oral antimicrobial biofluid.

Biosorbent materials are effective in the removal of spilled oil from water, but their effect on hydrocarbonoclastic bacteria is not known. Here, we show that corksorb, a cork-based biosorbent, enhances growth and alkane degradation by Rhodococcus opacus B4 (Ro) and Alcanivorax borkumensis SK2 (Ab). Ro and Ab degraded 96 ± 1% and 72 ± 2%, respectively, of a mixture of n -alkanes (2 g L -1 ) in the presence of corksorb. These values represent an increase of 6 and 24%, respectively, relative to the assays without corksorb. The biosorbent also increased the growth of Ab by 51%. However, no significant changes were detected in the expression of genes involved in alkane uptake and degradation in the presence of corksorb relative to the control without the biosorbent. Nevertheless, transcriptomics analysis revealed an increased expression of rRNA and tRNA coding genes, which confirms the higher metabolic activity of Ab in the presence of corksorb. The effect of corksorb is not related to the release of soluble stimulating compounds, but rather to the presence of the biosorbent, which was shown to be essential. Indeed, scanning electron microscopy images and downregulation of pili formation coding genes, which are involved in cell mobility, suggest that cell attachment on corksorb is a determinant for the improved activity. Furthermore, the existence of native alkane-degrading bacteria in corksorb was revealed, which may assist in situ bioremediation. Hence, the use of corksorb in marine oil spills may induce a combined effect of sorption and stimulated biodegradation, with high potential for enhancing in situ bioremediation processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martins, Freitas, Castro, Silva, Gudiña, Sequeira, Salvador, Pereira and Cavaleiro.)

This study evaluated how Er,Cr:YSGG laser, associated or not with 5% fluoride varnish, influences the surface roughness and volume loss of bovine root dentin submitted to erosive and/or abrasive wear. One hundred and twenty dentin specimens were divided into the groups: without preventive treatment (WPT), 5% fluoride varnish (FV); Er,Cr:YSGG laser irradiation (L), and varnish combined with laser (FV + L). The specimens (n = 10) were subdivided into: 1 = erosion (E); 2 = abrasion (A); and 3 = erosion followed by abrasion (E + A). The erosive solution used was a soft-drink (pH = 2.42 at 4ºC) applied in 5-min cycles twice a day for 10d. Abrasive wear involved brushing for 60s with an electric brush (1,600-oscillations/s) at a load of 2.0N. Surface roughness and volume loss were evaluated using a laser scanning confocal microscope. Roughness data were submitted to one-way ANOVA and Tukey post-hoc test. For volume loss, the Kruskal-Wallis and Dunn's post-hoc tests were used (α = 5%). The lowest values of roughness were found in the control areas of all subgroups (p > 0.05). In the experimental area, the [(WPT) + (E+A)] subgroup had a significantly higher roughness (5.712 ± 0.163 μm 2 ) than the other subgroups (p < 0.05). The L and (FV + L) groups had statistically similar roughness, regardless of the type of wear. The (FV + L) group had the lowest volume loss, regardless of the type of wear performed: [(FV + L) + (E)] = 7.5%, [(FV + L) + (A) = 7.3%, and [(FV + L) + (E + A)] = 8.1%. The subgroup [(WPT) + (E + A)] had the highest volume loss (52.3%). The proposed treatments were effective in controlling dentin roughness. Laser irradiation can be an effective method to increase root dentin resistance after challenges and limit problems related to non-carious lesions.

Primary brain tumors remain among the deadliest of all cancers. Glioma grade IV (glioblastoma), the most common and malignant type of brain cancer, is associated with a 5-year survival rate of < 5%. Melatonin has been widely reported as an anticancer molecule, and we have recently demonstrated that the ability of gliomas to synthesize and accumulate this indolamine in the surrounding microenvironment negatively correlates with tumor malignancy. However, our understanding of the specific effects mediated through the activation of melatonin membrane receptors remains limited. Thus, here we investigated the specific roles of MT1 and MT2 in gliomas and medulloblastomas. Using the MT2 antagonist DH97, we showed that MT1 activation has a negative impact on the proliferation of human glioma and medulloblastoma cell lines, while MT2 activation has an opposite effect. Accordingly, gliomas have a decreased mRNA expression of MT1 (also known as MTNR1A) and an increased mRNA expression of MT2 (also known as MTNR1B) compared to the normal brain cortex. The MT1/MT2 expression ratio negatively correlates with the expression of cell cycle-related genes and is a positive prognostic factor in gliomas. Notably, we showed that functional selective drugs that simultaneously activate MT1 and inhibit MT2 exert robust anti-tumor effects in vitro and in vivo, downregulating the expression of cell cycle and energy metabolism genes in glioma stem-like cells. Overall, we provided the first evidence regarding the differential roles of MT1 and MT2 in brain tumor progression, highlighting their relevance as druggable targets. KEY MESSAGES: • MT1 impairs while MT2 promotes the proliferation of glioma and medulloblastoma cell lines. • Gliomas have a decreased expression of MT1 and an increased expression of MT2 compared to normal brain cortex. • Tumors with a high MT1/MT2 expression ratio have significantly better survival rates. • Functional selective drugs that simultaneously activate MT1 and inhibit MT2 downregulate the expression of cell cycle and energy metabolism genes in glioma stem-like cells and exert robust anti-tumor effects in vivo.

Breast cancer is the leading cause of cancer death among women worldwide. Like other cancers, mammary carcinoma progression involves acidification of the tumor microenvironment, which is an important factor for cancer detection and treatment strategies. However, the effects of acidity on mammary carcinoma cell morphology and phenotype have not been thoroughly characterized. Here, we evaluated fundamental effects of environmental acidification on mammary carcinoma cells in standard two-dimensional cultures and three-dimensional spheroids. Acidification decreased overall mammary carcinoma cell viability, while increasing their resistance to the anthracycline doxorubicin. Environmental acidification also increased extracellular vesicle production by mammary carcinoma cells. Conditioned media containing these vesicles appeared to increase fibroblast motility. Acidification also increased mammary carcinoma cell motility when cultured with fibroblasts in spheroids. Taken together, results from this study suggest that environmental acidification induces drug resistance and extracellular vesicle production by mammary carcinoma cells that promote tumor expansion.

The recent development of IQ-CSF, the second generation of real-time quaking-induced conversion (RT-QuIC) using cerebrospinal fluid (CSF), for the diagnosis of Creutzfeldt-Jakob Disease (CJD) represents a major diagnostic advance in the field. Highly accurate results have been reported with encouraging reproducibility among different centers. However, availability is still insufficient, and only a few research centers have access to the method in developing countries. In Brazil, we have had 603 suspected cases of CJD since 2005, when surveillance started. Of these, 404 were undiagnosed. This lack of diagnosis is due, among other factors, to the lack of a reference center for the diagnosis of these diseases in Brazil, resulting in some of these samples being sent abroad for analysis. The aim of this research study is to report the pilot use of IQ-CSF in a small cohort of Brazilian patients with possible or probable CJD, implementing a reference center in the country. We stored CSF samples from patients with possible, probable or genetic CJD (one case) during the time frame of December 2016 through June 2018. All CSF samples were processed according to standardized protocols without access to the clinical data. Eight patients presented to our team with rapidly progressive dementia and typical neurological signs of CJD. We used CSF samples from seven patients with other neurological conditions as negative controls. Five out of seven suspected cases had positive tests; two cases showed inconclusive results. Among controls, there was one false-positive (a CSF sample from a 5-year-old child with leukemia under treatment). The occurrence of a false positive in one of the negative control samples raises the possibility of the presence of interfering components in the CSF sample from patients with non-neurodegenerative pathologies. Our pilot results illustrate the feasibility of having CJD CSF samples tested in Brazilian centers and highlight the importance of interinstitutional collaboration to pursue a higher diagnostic accuracy in CJD in Brazil and Latin America., (Copyright © 2020 Barbosa, Castrillo, Alvim, de Brito, Gomes, Brucki, Smid, Nitrini, Landemberger, Martins, Silva and Vieira.)

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749., (© 2019 International Society for Neurochemistry.)

PrP C is a glycoprotein capable to interact with several molecules and mediates diverse signaling pathways. Among numerous ligands, laminin (LN) is known to promote neurite outgrowth and memory consolidation, while amyloid-beta oligomers (Aβo) trigger synaptic dysfunction. In both pathways, mGluR1 is recruited as co-receptor. The involvement of PrP C /mGluR1 in these opposite functions suggests that this complex is a key element in the regulation of synaptic activity. Considering that sleep-wake cycle is important for synaptic homeostasis, we aimed to investigate how sleep deprivation affects the expression of PrP C and its ligands, laminin, Aβo, and mGluR1, a multicomplex that can interfere with neuronal plasticity. To address this question, hippocampi of control (CT) and sleep deprived (SD) C57BL/6 mice were collected at two time points of circadian period (13 hr and 21 hr). We observed that sleep deprivation reduced PrP C and mGluR1 levels with higher effect in active state (21 hr). Sleep deprivation also caused accumulation of Aβ peptides in rest period (13 hr), while laminin levels were not affected. In vitro binding assay showed that Aβo can compete with LN for PrP C binding. The influence of Aβo was also observed in neuritogenesis. LN alone promoted longer neurite outgrowth than non-treated cells in both Prnp +/+ and Prnp 0/0 genotypes. Aβo alone did not show any effects, but when added together with LN, it attenuated the effects of LN only in Prnp +/+ cells. Altogether, our findings indicate that sleep deprivation regulates the availability of PrP C and Aβ peptides, and based on our in vitro assays, these alterations induced by sleep deprivation can negatively affect LN-PrP C interaction, which is known to play roles in neuronal plasticity., (© 2020 International Society for Neurochemistry.)

The p90 ribosomal S6 kinase (RSK) family, a downstream target of Ras/extracellular signal-regulated kinase signaling, can mediate cross-talk with the mammalian target of rapamycin complex 1 pathway. As RSK connects two oncogenic pathways in gliomas, we investigated the protein levels of the RSK isoforms RSK1-4 in nontumoral brain (NB) and grade I-IV gliomas. When compared to NB or low-grade gliomas (LGG), a group of glioblastomas (GBMs) that excluded long-survivor cases expressed higher levels of RSK1 (RSK1 hi ). No difference was observed in RSK2 median-expression levels among NB and gliomas; however, high levels of RSK2 in GBM (RSK2 hi ) were associated with worse survival. RSK4 expression was not detected in any brain tissues, whereas RSK3 expression was very low, with GBM demonstrating the lowest RSK3 protein levels. RSK1 hi and, to a lesser extent, RSK2 hi GBMs showed higher levels of phosphorylated RSK, which reveals RSK activation. Transcriptome analysis indicated that most RSK1 hi GBMs belonged to the mesenchymal subtype, and RSK1 expression strongly correlated with gene expression signature of immune infiltrates, in particular of activated natural killer cells and M2 macrophages. In an independent cohort, we confirmed that RSK1 hi GBMs exclude long survivors, and RSK1 expression was associated with high protein levels of the mesenchymal subtype marker lysosomal protein transmembrane 5, as well as with high expression of CD68, which indicated the presence of infiltrating immune cells. An RSK1 signature was obtained based on differentially expressed mRNAs and validated in public glioma datasets. Enrichment of RSK1 signature followed glioma progression, recapitulating RSK1 protein expression, and was associated with worse survival not only in GBM but also in LGG. In conclusion, both RSK1 and RSK2 associate with glioma malignity, but displaying isoform-specific peculiarities. The progression-dependent expression and association with immune infiltration suggest RSK1 as a potential progression marker and therapeutic target for gliomas., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Purpose: To evaluate the influence of the Er,Cr:YSGG laser with or without the 5% fluoride varnish on the acid resistance of dentin after erosive challenge., Methods: 36 incisors were selected and sectioned, obtaining 72 specimens of 4 mm × 4 mm and randomly divided into eight groups (n = 9). In G1: application of Er,Cr:YSGG (0.1W; 5Hz, air 55%); G2: laser (0.25W; 5Hz, air 55%); G3: fluoride varnish + laser (0.1W; 5Hz, air 55%); G4: fluoride varnish + laser (0.25W, 5Hz, air 55%); G5: fluoride varnish + laser (0.1W; 5Hz, without air); G6: fluoride varnish + laser (0.25W, 5Hz, without air); G7: fluoride varnish and G8: no treatment. When used, the laser was irradiated without water cooling, scanning mode during 10 seconds. The surface roughness data were subjected to ANOVA. For wear profile, we used Kruskal-Wallis test and Dunn post-hoc, all with α= 0.05., Results: The results showed no statistically significant difference when comparing the groups as regards to the surface roughness (P> 0.05). Regarding the percentage of lost volume, the G5 and G6 groups presented the best results (G5 = 7.8% and G6 = 8.5%), with the lowest loss of dentin volume compared to other groups (P< 0.05). The G8 group (no treatment) had the highest lost volume (G8 = 39.1% followed by the G7 group (fluoride varnish), which had 25.9%., Clinical Significance: The use of Er,Cr:YSGG laser and fluoride varnish can be an effective method to increase the acid resistance of dentin after erosive challenges, and limit problems related to hypersensitivity., Competing Interests: The authors declared no conflict of interest. The study was supported by FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) (PEE-01689-16) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) (Finance Code 001)., (Copyright©American Journal of Dentistry.)

This in vitro study evaluated the influence of the Er,Cr:YSGG laser, associated or not to desensitizing agents, in the prevention of acid erosion in bovine root dentin. Eighty dentin specimens were selected and divided into eight groups (n = 10): G1: negative control; G2: positive control (5% fluoride varnish-FV); G3: Er,Cr:YSGG laser; G4: FV + laser; G5: 3% potassium oxalate; G6: 3% potassium oxalate + laser; G7: biphasic calcium silicate/phosphate gel (gel); G8: gel + laser. Laser parameters: 0.5 W, 6.25 J/cm 2 at 1-mm distance. The erosive drink used was a cola soft-drink (pH = 2.42 at 4 °C), lasting 5 min, twice a day, with 6-h intervals between the challenges, during 14 days. Kolmogorov-Smirnov and Levene's tests were satisfied. The surface roughness data were submitted to ANOVA and Tukey post hoc tests. For the wear profile, Kruskal-Wallis and Dunn post hoc tests were used. Afterwards, the Spearman correlation test was performed. All statistical tests assumed a significance level of 5% (α = 0.05). G1 presented the highest surface roughness value after the erosive challenge (3.586 μm 2  ± 0.205 μm 2 ) and the G7 presented the lowest surface roughness value after the erosive challenge (1.071 μm 2  ± 0.180 μm 2 ). For the lost volume, G4 presented the lowest percentage (9.7% ± 0.9%), while G1 had the highest percentage (41.8% ± 2.5%), both with p < 0.05. There was a weak correlation between the response variables (ρ = 0.33). All groups presented lower values of surface roughness and loss of volume when compared to the negative control group. For the surface roughness, the biphasic calcium silicate/phosphate gel presented the best result. For volume loss, the 5% fluoride varnish + Er,Cr:YSGG laser showed the best results compared to the other groups.

Rectal cancer represents one third of the colorectal cancers that are diagnosed. Neoadjuvant chemoradiation is a well-established protocol for rectal cancer treatment reducing the risk of local recurrence. However, a pathologic complete response is only achieved in 10-40% of cases and the mechanisms associated with resistance are poorly understood. To identify potential targets for preventing therapy resistance, a proteomic analysis of biopsy specimens collected from stage II and III rectal adenocarcinoma patients before neoadjuvant treatment was performed and compared with residual tumor tissues removed by surgery after neoadjuvant therapy. Three proteins, Ku70, Ku80, and Rab5C, exhibited a significant increase in expression after chemoradiation. To better understand the role of these proteins in therapy resistance, a rectal adenocarcinoma cell line was irradiated to generate a radiotherapy-resistant lineage. These cells overexpressed the same three proteins identified in the tissue samples. Furthermore, radiotherapy resistance in this in vitro model was found to involve modulation of epidermal growth factor receptor (EGFR) internalization by Rab5C in response to irradiation, affecting expression of the DNA repair proteins, Ku70 and Ku80, and cell resistance. Taken together, these findings indicate that EGFR and Rab5C are potential targets for the sensitization of rectal cancer cells and they should be further investigated. KEY MESSAGES: • Rab5C orchestrates a mechanism of radioresistance in rectal adenocarcinoma cell. • Rab5C modulates EGFR internalization and its relocalization to the nucleus. • In the nucleus, EGFR can modulate the expression of the DNA repair proteins, Ku70 and Ku80. • Rab5C, Ku70, and Ku80 are overexpressed in tumor tissues that contain tumor cells that are resistant to chemoradiation treatment.

Glioblastoma (GBM) is one of the most aggressive cancers, with median survival of less than 2 years. Despite of considerable advance in molecular classification of GBMs, no improvements in therapy have been described. The scenario is further complicated by tumor heterogeneity and the relationship among genetic, transcriptional and functional findings. Classically, gene expression has been evaluated by steady-state mRNA, however, this does not take translational control into consideration, which contributes considerably to the composition of the proteome. In this study, we evaluated the transcriptomic and translatomic signature of a GBM obtained from a single patient focusing in tumor heterogeneity. In a sampling of eight fragments, we investigated the translation rates, mTORC1 and ERK1/2 pathways and identified both total and polysome associated mRNAs. An increased translation rate was observed in fragments with high-grade histological features. High-grade histology was also associated with the expression of genes related to extracellular matrix (ECM) and angiogenesis, in both transcriptomes and translatomes. However, genes associated with epithelial to mesenchymal transition and stress response, were observed only in translatomes from high-grade fragments. Overall, our results demonstrate that isolation of translated mRNA can be used to identify biomarkers and reveal previously unrecognized determinants of heterogeneity in GBMs.

Objective: To detect Streptococcus mutans in colostrum and saliva of neonates and compare with its detection in saliva of mothers., Methods: Forty-three healthy women, full-term gestations with no complications, submitted to elective Cesarean section, and their newborns were included in the study. Samples were investigated by polymerase chain reaction to detect S. mutans in genetic material from the samples., Results: Approximately 16% of colostrum samples showed S. mutans , but not correlated with the presence of the bacteria in both samples of saliva. S. mutans was detected in 49 and 30% of saliva samples of mothers and neonates, respectively. There was a positive correlation in S. mutans detection between types of saliva. The number of maternal samples of saliva with detectable S. mutans was smaller in women receiving dental treatment during pregnancy. Tooth brushing, three times a day, influenced the detection of S. mutans in both the saliva and the colostrum., Conclusion: Although maternal saliva may present S. mutans , few samples of colostrum present the bacteria. The presence of bacteria in neonate saliva may be related to contact before birth. Dental treatment and hygiene habits seem to influence the detection of S. mutans in samples of maternal saliva and colostrum.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

The objective of this experiment was to determine the effect of high versus low progesterone (P4) during the pre-dominance or dominance phase (or both) of ovulatory follicle development on follicular dynamics and fertility of lactating dairy cows. Progesterone (P4) was manipulated to reach high (H) or low (L) serum concentrations during the pre-dominance phase (d 0 to 4 of the wave) and dominance phase (d 5 to 7 of the wave) of a second follicular wave ovulatory follicle, creating 4 treatments: H/H, H/L, L/H, and L/L. Luteolysis was induced with PGF 2α on d 7 of the wave and ovulation was induced with GnRH 56 h after PGF 2α . Cows (n = 558) received artificial insemination (AI) 16 h following GnRH. Pregnancy was determined at 6 intervals during gestation and at calving to quantify pregnancy loss beginning at d 23 post-AI utilizing pregnancy-specific protein B (PSPB) in novel within-cow comparisons. Cows with single ovulations assigned to the L/L treatment had greater pre-ovulatory follicle diameter compared with cows assigned to the L/H or H/L treatments. Cows with single ovulations had greater pre-ovulatory follicle diameter compared with cows with double ovulations. Low P4 in H/L, L/H, and L/L increased double ovulation rate compared with H/H. Cows with double ovulations had greater pregnancies per AI (P/AI) on d 23 post-AI compared with cows with single ovulations but had greater losses if ovulations were unilateral. Cows with low P4 during the entire period of the ovulatory follicle development also had greater P/AI on d 23 post-AI compared with cows with high P4 during both phases. However, full-term P/AI was not different between treatments. This was a result of the greater incidence of pregnancy losses between d 35 and 56 of gestation for cows with unilateral double ovulations compared with bilateral double ovulations and single ovulatory cows. Cows with single ovulation and low circulating P4 during the dominance period of follicle development had increased pregnancy losses between d 35 and 56 of gestation compared with cows with single ovulations and high P4. The PSPB measurements on d 16 and 23 post-AI were highly accurate in the prediction of pregnancy at d 28. The PSPB differed on d 23 and 28 between cows that had versus cows that did not have pregnancy losses between d 28 and 35 of gestation. In summary, circulating concentrations of P4 during ovulatory follicle development affected numbers of follicles ovulated and timing of subsequent pregnancy losses., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The aims of this study were to analyze the presence of Streptococcus mutans (SM)-DNA in cord blood (CB), maternal peripheral blood (PB), and maternal saliva (SA) and compare with data collected in health surveys. Sixty-four healthy women with pregnancies to term and without complications attending for elective cesarean section in the Clinical Hospital of Ribeirao Preto, Sao Paulo were included. Samples of PB and unstimulated SA were obtained on the day of hospitalization and samples of CB were collected after the delivery section. Samples were investigated using polymerase chain reaction for the presence of SM-DNA using specific primers. The results show over 50% of the sample of PB and CB showed SM-DNA detectable. There was a positive correlation between the SM detection in PB/CB and SA (P < 0.05). Pregnant women, who reported tooth brushing more than three times a day, often showed detectable SM-DNA in PB and CB (P < 0.05). In conclusion, the majority of children can have contact with SM-DNA during the intrauterine life by the CB. SM probably transferred from salivary habitat to PB and CB. The tooth brushing can be associated to S. mutans detection in blood samples.

Objectives: This study evaluated the effects of a bleaching agent on the composition, mechanical properties, and surface topography of 6 conventional glass-ionomer cements (GICs) and one resin-modified GIC., Materials and Methods: For 3 days, the specimens were subjected to three 20-minute applications of a 37% H 2 O 2 -based bleaching agent and evaluated for water uptake (WTK), weight loss (WL), compressive strength (CS), and Knoop hardness number (KHN). Changes in surface topography and chemical element distribution were also analyzed by energy-dispersive X-ray spectroscopy and scanning electron microscopy. For statistical evaluation, the Kruskal-Wallis and Wilcoxon paired tests ( α = 0.05) were used to evaluate WTK and WL. CS specimens were subjected to 2-way analysis of variance (ANOVA) and the Tukey post hoc test ( α = 0.05), and KH was evaluated by one-way ANOVA, the Holm-Sidak post hoc test ( α = 0.05), and the t -test for independent samples ( α = 0.05)., Results: The bleaching agent increased the WTK of Maxxion R, but did not affect the WL of any GICs. It had various effects on the CS, KHN, surface topography, and the chemical element distribution of the GICs., Conclusions: The bleaching agent with 37% H 2 O 2 affected the mechanical and surface properties of GICs. The extent of the changes seemed to be dependent on exposure time and cement composition., Competing Interests: Conflict of Interest: No potential conflict of interest relevant to this article was reported.

Cellular prion protein (PrP C ) is widely expressed and displays a variety of well-described functions in the central nervous system (CNS). Mutations of the PRNP gene are known to promote genetic human spongiform encephalopathies, but the components of gain- or loss-of-function mutations to PrP C remain a matter for debate. Among the proteins described to interact with PrP C is Stress-inducible protein 1 (STI1), a co-chaperonin that is secreted from astrocytes and triggers neuroprotection and neuritogenesis through its interaction with PrP C . In this work, we evaluated the impact of different PrP C pathogenic point mutations on signaling pathways induced by the STI1-PrP C interaction. We found that some of the pathogenic mutations evaluated herein induce partial or total disruption of neuritogenesis and neuroprotection mediated by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase A (PKA) signaling triggered by STI1-PrP C engagement. A pathogenic mutant PrP C that lacked both neuroprotection and neuritogenesis activities fail to promote negative dominance upon wild-type PrP C . Also, a STI1-α7-nicotinic acetylcholine receptor-dependent cellular signaling was present in a PrP C mutant that maintained both neuroprotection and neuritogenesis activities similar to what has been previously observed by wild-type PrP C . These results point to a loss-of-function mechanism underlying the pathogenicity of PrP C mutations., (© 2018 International Society for Neurochemistry.)

PI3K/Akt/mTOR pathway activation is a hallmark of high-grade gliomas, which prompted clinical trials for the use of PI3K and mTOR inhibitors. However, the poor results in the original trials suggested that better patient profiling was needed for such drugs. Thus, accurate and reproducible monitoring of mTOR complexes can lead to improved therapeutic strategies. In this work, we evaluated the expression and phosphorylation of mTOR, RAPTOR, and rpS6 in 195 human astrocytomas and 30 normal brain tissue samples. The expression of mTOR increased in glioblastomas, whereas mTOR phosphorylation, expression of RAPTOR, and expression and phosphorylation of rpS6 were similar between grades. Interestingly, the overexpression of total and phosphorylated mTOR as well as phosphorylated rpS6 (residues 240-244) were associated with wild-type IDH1 only glioblastomas. The expression and phosphorylation of mTOR and phosphorylation of rpS6 at residues 240-244 were associated with a worse prognosis in glioblastomas. Our results suggest that mTOR and rpS6 could be used as markers of overactivation of the PI3K-mTOR pathway and are predictive factors for overall survival in glioblastomas. Our study thus suggests that patients who harbor IDH1 wild-type glioblastomas might have increased benefit from targeted therapy against mTOR.

The treatments for dentin hypersensitivity (DH) may change the surface roughness of the root dentin, which can lead to biofilm accumulation, increasing the risk of root caries. The aim was to compare the surface roughness of root dentin after different treatments of DH and the biofilm formation on those surfaces. After initial surface roughness (Sa) assessment, 50 bovine root fragments received the following treatments (n = 10): G 1-no treatment; G2-5% sodium fluoride varnish; G3-professional application of a desensitizing dentifrice; G4-toothbrushing with a desensitizing dentifrice; and G5-diode laser application (908 nm; 1.5 W, 20 s). The Sa was reevaluated after treatments. Afterward, all samples were incubated in a suspension of Streptococcus mutans at 37 °C for 24 h. The colony-forming units (CFU) were counted using a stereoscope, and the results were expressed in CFU/mL. The one-way ANOVA and the Tukey's tests compared the roughness data and the results obtained on the bacterial adhesion test (α = 5%). G2 (2.3 ± 1.67%) showed similar Sa variation than G1 (0.25 ± 0.41%) and G5 (5.69 ± 0.99%), but different from group G3 (9.05 ± 2.39%). Group 4 showed the highest Sa variation (30.02 ± 3.83%; p < 0.05). Bacterial adhesion was higher in G4 (2208 ± 211.9), suggesting that bacterial growth is greater on rougher surfaces. The diode laser and the conventional treatments for DH may change the surface roughness of the root dentin, but only brushing with desensitizing dentifrice induced a higher bacteria accumulation on root dentin surface.

The control of pre-analytical-factors in human biospecimens collected for health research is currently required. Only two previous reports using post-mortem brain samples have tried to address the impact of cold-ischemia on tissue pH. Here we report pH variations according to time (third-order polynomial model) in mice for liver, kidney and lung samples. Tissue alkalosis in cold-ischemia time may be an underlying mechanism of gene expression changes. Therefore, tissue-pH regulation after organ removal may minimize biological stress in human tissue samples.

Prion protein (PrP C ) was initially described due to its involvement in transmissible spongiform encephalopathies. It was subsequently demonstrated to be a cell surface molecule involved in many physiological processes, such as vesicle trafficking. Here, we investigated the roles of PrP C in the response to insulin and obesity development. Two independent PrP C knockout (KO) and one PrP C overexpressing (TG20) mouse models were fed high-fat diets, and the development of insulin resistance and obesity was monitored. PrP C KO mice fed high-fat diets presented all of the symptoms associated with the development of insulin resistance: hyperglycemia, hyperinsulinemia, and obesity. Conversely, TG20 animals fed high-fat diets showed reduced weight and insulin resistance. Accordingly, the expression of peroxisome proliferator-activated receptor gamma (PPARγ) was reduced in PrP C KO mice and increased in TG20 animals. PrP C KO cells also presented reduced glucose uptake upon insulin stimulation, due to reduced translocation of the glucose transporter Glut4. Thus, our results suggest that PrP C reflects susceptibility to the development of insulin resistance and metabolic syndrome., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Objective: To describe e compare the specificity of IgA antibodies against bacteria extract of Klebsiella pneumoniae , Staphylococcus aureus , Escherichia coli , and Salmonella enteritidis ., Methods: Colostrum samples were aseptically collected in the first 12 hours after C-section delivery. The specificity of IgA against bacteria extracts was analyzed by the Western blot., Results: The findings showed proteins of high molecular weight frequently detectable in the samples. S. aureus was the most frequently found bacterium in the samples (p<0.05). Approximately 93.8, 56.3, 62.5 and 60.4% of samples presented IgA reactive to S. aureus , K. pneumoniae , S. enteritidis, and E. coli, respectively. Roughly 40% of samples showed no IgA reactive to K. pneumoniae, S. enteritidis and E. coli ., Conclusion: Clinical evidence of the importance of breastfeeding for the immune protection of neonates was consistent with the observed immunological findings, since most samples showed IgA reactive against the species tested. The application and development of immunotherapies during pregnancy, focused on frequently detected antigens, could be an important tool to enhance the presence of IgA in colostrum.

Gerstmann-Sträussler-Scheinker is a genetic prion disease and the most common mutation is p.Pro102Leu. We report clinical, molecular and neuropathological data of seven individuals, belonging to two unrelated Brazilian kindreds, carrying the p.Pro102Leu. Marked differences among patients were observed regarding age at onset, disease duration and clinical presentation. In the first kindred, two patients had rapidly progressive dementia and three exhibited predominantly ataxic phenotypes with variable ages of onset and disease duration. In this family, age at disease onset in the mother and daughter differed by 39 years. In the second kindred, different phenotypes were also reported and earlier ages of onset were associated with 129 heterozygosis. No differences were associated with apoE genotype. In these kindreds, the codon 129 polymorphism could not explain the clinical variability and 129 heterozygosis was associated with earlier disease onset. Neuropathological examination in two patients confirmed the presence of typical plaques and PrPsc immunopositivity.

Iron is an essential micronutrient for several physiological functions, including the regulation of dopaminergic neurotransmission. On the other hand, both iron, and dopamine can affect the folding and aggregation of proteins related with neurodegenerative diseases, such as cellular prion protein (PrP C ) and α-synuclein, suggesting that deregulation of iron homeostasis and the consequential disturbance of dopamine metabolism can be a risk factor for conformational diseases. These proteins, in turn, are known to participate in the regulation of iron and dopamine metabolism. In this study, we evaluated the effects of dietary iron restriction on brain ferritin levels, dopamine metabolism, and the expression levels of PrP C and α-synuclein. To achieve this goal, C57BL/6 mice were fed with iron restricted diet (IR) or with normal diet (CTL) for 1 month. IR reduced iron and ferritin levels in liver. Ferritin reduction was also observed in the hippocampus. However, in the striatum of IR group, ferritin level was increased, suggesting that under iron-deficient condition, each brain area might acquire distinct capacity to store iron. Increased lipid peroxidation was observed only in hippocampus of IR group, where ferritin level was reduced. IR also generated discrete results regarding dopamine metabolism of distinct brain regions: in striatum, the level of dopamine metabolites (DOPAC and HVA) was reduced; in prefrontal cortex, only HVA was increased along with the enhanced MAO-A activity; in hippocampus, no alterations were observed. PrP C levels were increased only in the striatum of IR group, where ferritin level was also increased. PrP C is known to play roles in iron uptake. Thus, the increase of PrP C in striatum of IR group might be related to the increased ferritin level. α-synuclein was not altered in any regions. Abnormal accumulation of ferritin, increased MAO-A activity or lipid peroxidation are molecular features observed in several neurological disorders. Our findings show that nutritional iron deficiency produces these molecular alterations in a region-specific manner and provide new insight into the variety of molecular pathways that can lead to distinct neurological symptoms upon iron deficiency. Thus, adequate iron supplementation is essential for brain health and prevention of neurological diseases.

In most mammalian brains, the subventricular zone (SVZ) is a germinative layer that maintains neurogenic activity throughout adulthood. Neuronal precursors arising from this region migrate through the rostral migratory stream (RMS) and reach the olfactory bulbs where they differentiate and integrate into the local circuitry. Recently, studies have shown that heat shock proteins have an important role in cancer cell migration and blocking Hsp90 function was shown to hinder cell migration in the developing cerebellum. In this work, we hypothesize that chaperone complexes may have an important function regulating migration of neuronal precursors from the subventricular zone. Proteins from the Hsp90 complex are present in the postnatal SVZ as well as in the RMS. Using an in vitro SVZ explant model, we have demonstrated the expression of Hsp90 and Hop/STI1 by migrating neuroblasts. Treatment with antibodies against Hsp90 and co-chaperone Hop/STI1, as well as Hsp90 and Hsp70 inhibitors hinder neuroblast chain migration. Time-lapse videomicroscopy analysis revealed that cell motility and average migratory speed was decreased after exposure to both antibodies and inhibitors. Antibodies recognizing Hsp90, Hsp70, and Hop/STI1 were found bound to the membranes of cells from primary SVZ cultures and biotinylation assays demonstrated that Hsp70 and Hop/STI1 could be found on the external leaflet of neuroblast membranes. The latter could also be detected in conditioned medium samples obtained from cultivated SVZ cells. Our results suggest that chaperones Hsp90, Hsp70, and co-chaperone Hop/STI1, components of the Hsp90 complex, regulate SVZ neuroblast migration in a concerted manner through an extracellular mechanism.

The present study compared IgA specificity against oral streptococci in colostrum and saliva samples. Sixty-two mother-and-child pairs were included; samples of colostrum (C) and saliva (MS) were collected from the mothers and saliva samples were collected from babies (BS). The specificity of IgA against Streptococcus mutans and S. mitis were analyzed by western blot. Only 30% of babies' samples presented IgA reactivity to S. mutans, while 74 and 80% of MS and C, respectively, presented this response. IgA reactivity to S. mutans virulence antigens (Ag I/II, Gtf and GbpB) in positive samples showed differences between samples for Gtf and especially for GbpB (p < 0.05), but responses to Ag I/II were similar (p > 0.05). The positive response of Gtf-reactive IgA was different between C (90%) and MS (58%) samples (p < 0.05), but did not differ from BS (p > 0.05). GbpB was the least detected, with 48 and 26% of C and MS, and only 5% of BS samples presenting reactivity (p > 0.05). Eight percent of MS and C samples presented identical bands to SM in the same time-point. In conclusion, the differences of IgA response found between C and MS can be due to the different ways of stimulation, proliferation and transportation of IgA in those secretions. The colostrum has high levels of IgA against S. mutans virulence antigens, which could affect the installation and accumulation process of S. mutans, mainly by supplying anti-GbpB IgA to the neonate.

Heat shock proteins (HSPs) are abundant cellular proteins involved with protein homeostasis. They have both constitutive and inducible isoforms, whose expression levels are further increased by stress conditions, such as temperature elevation, reduced oxygen levels, infection, inflammation and exposure to toxic substances. In these situations, HSPs exert a pivotal role in offering protection, preventing cell death and promoting cell recovery. Although the majority of HSPs functions are exerted in the cytoplasm and organelles, several lines of evidence reveal that HSPs are able to induce cell responses in the extracellular milieu. HSPs do not possess secretion signal peptides, and their secretion was subject to widespread skepticism until the demonstration of the role of unconventional secretion forms such as exosomes. Secretion of HSPs may confer immune system modulation and be a cell-to-cell mediated form of increasing stress resistance. Thus, there is a wide potential for secreted HSPs in resistance of cancer therapy and in the development new therapeutic strategies.

Background: Glioblastoma (GBM), a highly aggressive brain tumor, contains a subpopulation of glioblastoma stem-like cells (GSCs) that play roles in tumor maintenance, invasion, and therapeutic resistance. GSCs are therefore a promising target for GBM treatment. Our group identified the cellular prion protein (PrP C ) and its partner, the co-chaperone Hsp70/90 organizing protein (HOP), as potential target candidates due to their role in GBM tumorigenesis and in neural stem cell maintenance., Methods: GSCs expressing different levels of PrP C were cultured as neurospheres with growth factors, and characterized with stem cells markers and adhesion molecules markers through immunofluorescence and flow cytometry. We than evaluated GSC self-renewal and proliferation by clonal density assays and BrdU incorporation, respectively, in front of recombinant HOP treatment, combined or not with a HOP peptide which mimics the PrP C binding site. Stable silencing of HOP was also performed in parental and/or PrP C -depleted cell populations, and proliferation in vitro and tumor growth in vivo were evaluated. Migration assays were performed on laminin-1 pre-coated glass., Results: We observed that, when GBM cells are cultured as neurospheres, they express specific stemness markers such as CD133, CD15, Oct4, and SOX2; PrP C is upregulated compared to monolayer culture and co-localizes with CD133. PrP C silencing downregulates the expression of molecules associated with cancer stem cells, upregulates markers of cell differentiation and affects GSC self-renewal, pointing to a pivotal role for PrP C in the maintenance of GSCs. Exogenous HOP treatment increases proliferation and self-renewal of GSCs in a PrP C -dependent manner while HOP knockdown disturbs the proliferation process. In vivo, PrP C and/or HOP knockdown potently inhibits the growth of subcutaneously implanted glioblastoma cells. In addition, disruption of the PrP C -HOP complex by a HOP peptide, which mimics the PrP C binding site, affects GSC self-renewal and proliferation indicating that the HOP-PrP C complex is required for GSC stemness. Furthermore, PrP C -depleted GSCs downregulate cell adhesion-related proteins and impair cell migration indicating a putative role for PrP C in the cell surface stability of cell adhesion molecules and GBM cell invasiveness, respectively., Conclusions: In conclusion, our results show that the modulation of HOP-PrP C engagement or the decrease of PrP C and HOP expression may represent a potential therapeutic intervention in GBM, regulating glioblastoma stem-like cell self-renewal, proliferation, and migration.

The mammalian target of rapamycin (mTOR) binds to several protein partners and forms two complexes, termed mTOR complexes 1 and 2 (mTORC1/2), that differ in components, substrates, and regulation. mTORC2 contains the protein Rapamycin-insensitive companion of mTOR (RICTOR); phosphorylates kinases of the AGC family, such as Akt; and controls the cytoskeleton. Even though the regulation of mTORC2 activity remains poorly understood, the hyperactivation of this signaling pathway has been shown to contribute to the oncogenic properties of gliomas in experimental models. In this work, we evaluated expression and phosphorylation of Akt, and expression of RICTOR and Ki-67 in 195 human astrocytomas of different grades (38 cases of grade I, 49 grade II, 15 grade III, and 93 grade IV) and 30 normal brains. Expression and phosphorylation of Akt increased with histological grade and correlated with a worse overall survival in glioblastomas (GBMs). RICTOR was overexpressed in grade I and II astrocytomas and demonstrated a shift to nuclear localization in GBMs. Nuclear RICTOR was associated to increased proliferation in GBMs. Our results point to an increase in total and phosphorylated Akt in high-grade gliomas and to a possible role of RICTOR in proliferations of high-grade GBM cells., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The aim was to evaluate the surface roughness and bacterial adhesion on enamel irradiated with high intensity lasers, associated or not to a fluoride varnish. Eighty fragments of bovine enamel were equally divided in 8 groups (n=10). Group 1 was not treated and Group 2 received only a 5% fluoride varnish application. The other groups were irradiated with an Er:Cr:YSGG (8.92 J/cm2), an Nd:YAG (84.9 J/cm2) and a diode laser (199.04 J/cm2), associated or not to a 5% fluoride varnish. The surface roughness was measured before and after treatments. Afterward, all samples were incubated in a suspension of S. mutans at 37 °C for 24 h. The colony-forming units (CFU) were counted by a stereoscope and the results were expressed in CFU/mm2. One-way ANOVA and the Tukey´s test compared the roughness data and the Student´s test compared the results obtained in the bacterial adhesion test (a=5%). The results showed that the irradiated samples without varnish presented the same roughness and the same bacterial adhesion that the non-irradiated samples. However, samples irradiated in the presence of fluoride varnish showed higher surface roughness and higher bacterial adhesion than the non-irradiated samples and those irradiated without varnish. Presence of pigments in the varnish increased the lasers' action on the enamel surface, which produced ablation in this hard tissue and significantly increased its surface roughness. For this reason, the enamel's susceptibility to bacterial adhesion was higher when the irradiation of the samples was made in presence of fluoride varnish.

Prion protein modulates many cellular functions including the secretion of trophic factors by astrocytes. Some of these factors are found in exosomes, which are formed within multivesicular bodies (MVBs) and secreted into the extracellular space to modulate cell-cell communication. The mechanisms underlying exosome biogenesis were not completely deciphered. Here, we demonstrate that primary cultures of astrocytes and fibroblasts from prnp-null mice secreted lower levels of exosomes than wild-type cells. Furthermore, prnp-null astrocytes exhibited reduced MVB formation and increased autophagosome formation. The reconstitution of PRNP expression at the cell membrane restored exosome secretion in PRNP-deficient astrocytes, whereas macroautophagy/autophagy inhibition via BECN1 depletion reestablished exosome release in these cells. Moreover, the PRNP octapeptide repeat domain was necessary to promote exosome secretion and to impair the formation of the CAV1-dependent ATG12-ATG5 cytoplasmic complex that drives autophagosome formation. Accordingly, higher levels of CAV1 were found in lipid raft domains instead of in the cytoplasm in prnp-null cells. Collectively, these findings demonstrate that PRNP supports CAV1-suppressed autophagy to protect MVBs from sequestration into phagophores, thus facilitating exosome secretion.

The prion protein (PrP C ) has been suggested to operate as a scaffold/receptor protein in neurons, participating in both physiological and pathological associated events. PrP C , laminin, and metabotropic glutamate receptor 5 (mGluR5) form a protein complex on the plasma membrane that can trigger signaling pathways involved in neuronal differentiation. PrP C and mGluR5 are co-receptors also for β-amyloid oligomers (AβOs) and have been shown to modulate toxicity and neuronal death in Alzheimer's disease. In the present work, we addressed the potential crosstalk between these two signaling pathways, laminin-PrP C -mGluR5 or AβO-PrP C -mGluR5, as well as their interplay. Herein, we demonstrated that an existing complex containing PrP C -mGluR5 has an important role in AβO binding and activity in neurons. A peptide mimicking the binding site of laminin onto PrP C (Ln-γ1) binds to PrP C and induces intracellular Ca 2+ increase in neurons via the complex PrP C -mGluR5. Ln-γ1 promotes internalization of PrP C and mGluR5 and transiently decreases AβO biding to neurons; however, the peptide does not impact AβO toxicity. Given that mGluR5 is critical for toxic signaling by AβOs and in prion diseases, we tested whether mGlur5 knock-out mice would be susceptible to prion infection. Our results show mild, but significant, effects on disease progression, without affecting survival of mice after infection. These results suggest that PrP C -mGluR5 form a functional response unit by which multiple ligands can trigger signaling. We propose that trafficking of PrP C -mGluR5 may modulate signaling intensity by different PrP C ligands., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The objective of this study was to evaluate the effects of diode laser and a desensitizing dentifrice on dentin permeability. Fifty-two root dentin fragments were obtained (5 × 5mm) and treated with 24% EDTA gel. The samples were divided into 4 groups (n = 13): G1, control (no treatment); G2, diode laser (λ = 908 nm, 1.5 W, continuous mode, 20s); G3, application of abrasive dentifrice for 1 minute (Elmex Sensitive Professional (International Gaba); and G4, application of abrasive dentifrice for 1 minute followed by irradiation with diode laser. Ten samples per group were immersed in 2% methylene blue solution for 4h. The specimens were washed, longitudinally sectioned, observed under optical microscopy, photographed and assessed based on the degree of dye leakage. The remaining samples were observed under scanning electron microscopy (SEM). The leakage data were subjected to ANOVA test, followed by Tukey's t-test (α = 5%). Groups 2, 3 and 4 showed less dye penetration than the control group (p < 0.05), but were similar among each other. SEM images showed that dentinal tubules were open in G1, and fused and occluded in G2. Group 3 showed dentinal tubules that were occluded by the metal ions from the toothpaste. G4 presented similar characteristics to G3, and the presence of fused dentin. The diode laser and the dentifrice were effective in reducing dentinal permeability, and the combination of the two treatments did not show better results than either one used alone.

The A33 protein, expressed in colorectal tumors, is a target for improving treatment of patients with colorectal cancer. Over the last decade, studies have tested anti-A33 antibody as a therapeutic agent for these patients. Preclinical results were promising, but clinical trials did not confirm positive results. Here, immunohistochemistry in colorectal cancer tissue showed that samples from well-differentiated tumors presented a strong A33 membrane staining, whereas poorly differentiated tumors and mucinous adenocarcinomas showed weak cytoplasmic and nuclear staining. Moderately differentiated tumors presented variable staining. We suggest that in future clinical trials, patients should be selected on the basis of membrane expression of A33.

Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

Glioblastoma (GBM) is currently the most aggressive form of brain tumor identified, and STAT3 is known to play an important role in gliomagenesis. Moreover, while several studies have used pharmacological approaches to modulate STAT3 activity, the results have been contradictory. In this study, expressions of STAT3, pSTAT3 (Y705), and pSTAT3 (S727) were evaluated using immunohistochemistry assays of tissue microarrays containing non-neoplastic tissue (NN, n=12), grade II astrocytomas (n=33), grade III astrocytomas (n=12), and GBM (n=85) specimens. In GBM specimens, STAT3 was overexpressed and exhibited greater nuclear localization compared with lower grade astrocytomas and NN. Conversely, nuclear localization of pSTAT3 (Y705) and pSTAT3 (S727) exhibited a similar phenotype in both GBMs and NNs. MET was also detected as a non-canonical pathway marker for STAT3. For tumors with higher levels of STAT3 nuclear localization, and not pSTAT3 (Y705) and pSTAT3 (S727), these specimens exhibited increased levels of MET expression. Thus, a non-canonical pathway may mediate a proportion of the STAT3 that translocates to the nucleus. Moreover, tumors which exhibited greater nuclear localization of STAT3 corresponded with patients that presented with lower rates of recurrence-free survival and overall survival. In contrast, the phosphorylated forms of STAT3 did not correlate with patient survival. These findings suggest that phosphorylation-independent mechanisms may mediate the nuclear translocation and activation of STAT3. Further studies are needed to identify the mechanisms involved, especially those that provide targets to achieve efficient inhibition and control of GBM progression., (Copyright © 2016 Elsevier GmbH. All rights reserved.)