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The relationship between bacterial infiltration and internal conical Implant-Abutment Interfaces (IAIs) with different conicities still requires investigations that can offer valuable information in the clinical understanding of peri-implant health. The present study aimed to verify the bacterial infiltration of two internal conical connections with an angulation of 11.5° and 16° with the external hexagonal connection as a comparative after thermomechanical cycling using saliva as a contaminant. Test (n = 10) and control (n = 3) groups were set up. Evaluations were made on torque loss, Scanning Electron Microscopy (SEM), and Micro Computerized Tomography (MicroCT) after performing 2 × 106 mechanical cycles (120 N) and 600 thermal cycles (5°–55° C) with 2 mm lateral displacement. The contents of the IAI were collected for microbiological analysis. There was a difference (p < 0.05) in torque loss of the groups tested; groups from the 16° IAI obtained a lower percentage of torque loss. All groups presented contamination and the analysis of the results shows that the microbiological profile of the IAI differs qualitatively from the profile found in the saliva used for contamination. The mechanical loading affects the microbiological profile found in the IAIs (p < 0.05). In conclusion, the IAI environment may favor a microbiological profile different from that of saliva and the thermocycling condition may alter the microbial profile found in the IAI.

Following human immunodeficiency virus-1 (HIV-1) infection and antiretroviral therapy, the development of periodontal disease was shown to be favored. However, the influence of HIV-1 infection on the periodontal microbiota after non-surgical periodontal debridement (NSPD) needs a broad comprehension. This work aimed to compare the subgingival microbiological content of patients infected with HIV-1 and controls (non-infected) with periodontitis undergoing NSPD.The bacterial profile of subgingival biofilm samples of patients with HIV-1 (n = 18) and controls (n = 14) with periodontitis was assessed using 16S rRNA gene sequencing. The samples were collected at baseline, 30, and 90 days after NSPD. The taxonomic analysis of gingival microbiota was performed using a ribosomal RNA database. The microbiota content was evaluated in the light of CD4 cell count and viral load.Both HIV and control groups showed similar stages and grades of periodontitis. At baseline, the HIV group showed higher alpha diversity for both healthy and periodontal sites. Streptococcus, Fusobacterium, Veillonella and Prevotella were the predominant bacterial genera. A low abundance of periodontopathogenic bacteria was observed, and the NSPD induced shifts in the subgingival biofilm of patients with HIV-1, leading to a microbiota similar to that of controls.Different subgingival microbiota profiles were identified-a less diverse microbiota was found in patients infected with HIV-1, in contrast to a more diverse microbiota in controls. NSPD caused changes in the microbiota of both groups, with a greater impact on the HIV group, leading to a decrease in alpha diversity, and produced a positive impact on the serological immune markers in patients infected with HIV-1. Control of periodontitis should be included as part of an oral primary care, providing the oral health benefits and better control of HIV-1 infection.

The biological functions of a cell may change in response to exposure to toxic agents. Toxicogenomics employs the recent developments in genomics, transcriptomics, and proteomics to study how a chemical impacts gene/protein expression and cell functions. We describe a method for transcriptomic analysis by RNA sequencing based on Illumina HiSeq, NextSeq, or NovaSeq Systems followed by real-time qPCR validation. We also depict a method for proteomic analysis by "one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis" (1D SDS-PAGE) and a sample preparation procedure for "liquid chromatography in tandem with mass spectrometry" (LC-MS/MS), and we present some generic points to consider during LC-MS/MS.

Background Shifts in microbial communities are common over time, but they may disturb the host-microbiome homeostasis and result in inflammation of the peri-implant issues if a dysbiotic biofilm is established. Purpose Considering that different oral substrate surfaces may have a relevant impact on the microbial adhesion and colonization, the aim of this study was to investigate the microbial communities of the biofilm formed on single-implant restorations using titanium or zirconia abutments and how they correlate with clinical parameters after 3-years of implant loading. Materials and methods MiSeq sequencing of 16S rRNA amplicons was used to characterize the oral biofilms of individuals (n = 20) who were sampled longitudinally during 3 years of masticatory loading. Bioinformatics analysis and multivariate statistical analysis were used to evaluate the microbial diversity and clinical outcomes. Results Microbiomes of both abutment materials presented high alpha-diversity indices during all the experimental period, irrespective of the time of sampling. Microbial communities of titanium and zirconia were quite different over time, differing about 30% after 3 years of functional loading. Similarity of microbiomes between tested abutments and contralateral teeth was also low, ranging between 45% and 50% after 3 years of investigation. Periodontal pathogens commonly associated with peri-implantitis were found in both groups. Furthermore, both abutment materials presented strong correlations of diversity indices and microbial taxa with clinical outcomes. Conclusions The type of abutment substrate significantly influenced diversity and clustering of communities during 3 years of functional loading. The time of sampling had no effect on the variables. Large correlations were found between microbial findings and clinical outcomes.

This study aimed to evaluate two methods of the incorporation of nanostructured silver vanadate decorated with silver nanoparticles (AgVO3) into acrylic resin and characterize the profile of early and late microbial communities in class and family taxonomic level by pyrosequencing. The specimens were made by adding different concentrations of AgVO3 (1, 2.5, and 5%) to the heat-activated acrylic resin by two methods: vacuum spatulation (VS) and polymeric film (PF). A control group (0%) without AgVO3 was also obtained for both methods. After 24 h and 7 days of incubation in human saliva, biofilm samples were collected, DNA was extracted, and 16S rRNA genes were sequenced by the 454-Roche sequencing platform. Seventeen classes and 51 families of bacteria were identified. The abundance of Bacteroidia, Bacilli, Negativicutes, Fusobacteria and Betaproteobacteria classes decreased after 7 days of incubation, and Clostridia, Gammaproteobacteria, and unclassified bacteria increased. The Negativicutes and Betaproteobacteria classes were more abundant when the PF method was used, and Gammaproteobacteria was more abundant when VS was used. The incorporation of 5% AgVO3 promoted a reduction in the prevalence of Bacilli, Clostridia, Negativicutes, Betaproteobacteria, and unclassified bacteria, and increased Gammaproteobacteria. The addition of AgVO3 to acrylic resin altered the early and mature microbiome formed on the specimen surface, and the PF method presented a more favorable microbial profile than the VS method.

Breast cancer is an important public health problem, and its progression may be related to the extracellular matrix (ECM), which acts as a structural scaffold and instruction source for neoplastic cells. Laminins are ECM proteins regulating tumor biology. The laminin-derived peptide C16 regulates different properties of tumor cells. Here we analyzed C16-induced differential gene expression in MDA-MB-231 breast cancer cells. MCF-10A normal-like breast cells served as control. Among different cancer-related genes, C16 induced overexpression of GPNMB. This gene encodes a transmembrane protein GPNMB (glycoprotein non-metastatic B), involved with malignant phenotype of breast cancer cells. Immunoblot validated microarray results. To correlate gene and protein expression with cellular function, we investigated whether C16 would regulate invasion in breast cancer cells. siRNA experiments strongly suggested that C16 and GPNMB cooperate to regulate invasion of highly aggressive MDA-MB-231 cancer cells. We addressed regulatory mechanisms involved in C16-mediated increase of GPNMB protein levels in MDA-MB-231 cells, and observed that C16 stimulates β1 integrin and Src phosphorylation. Furthermore, Src inhibition decreases peptide-induced GPNMB expression levels. To contextualize in vivo our results in vitro, we addressed GPNMB immunostaining in breast cancer human tissue microarrays. Quantitative immunohistochemistry showed that GPNMB is significantly more expressed in breast cancer compared to normal tissue. We concluded that laminin-derived peptide C16 regulates gene and protein expression of GPNMB in breast cancer cells. C16 and GPNMB may cooperate to regulate invasion of highly aggressive MDA-MB-231 cells, probably through Src signaling. GPNMB presented increased expression in breast cancer in vivo compared to normal breast tissue.

Background Sugarcane bagasse has been proposed as a lignocellulosic residue for second-generation ethanol (2G) produced by breaking down biomass into fermentable sugars. The enzymatic cocktails for biomass degradation are mostly produced by fungi, but low cost and high efficiency can consolidate 2G technologies. A. fumigatus plays an important role in plant biomass degradation capabilities and recycling. To gain more insight into the divergence in gene expression during steam-exploded bagasse (SEB) breakdown, this study profiled the transcriptome of A. fumigatus by RNA sequencing to compare transcriptional profiles of A. fumigatus grown on media containing SEB or fructose as the sole carbon source. Secretome analysis was also performed using SDS-PAGE and LC-MS/MS. Results The maximum activities of cellulases (0.032 U mL-1), endo-1,4-β--xylanase (10.82 U mL-1) and endo-1,3-β glucanases (0.77 U mL-1) showed that functional CAZymes (carbohydrate-active enzymes) were secreted in the SEB culture conditions. Correlations between transcriptome and secretome data identified several CAZymes in A. fumigatus. Particular attention was given to CAZymes related to lignocellulose degradation and sugar transporters. Genes encoding glycoside hydrolase classes commonly expressed during the breakdown of cellulose, such as GH-5, 6, 7, 43, 45, and hemicellulose, such as GH-2, 10, 11, 30, 43, were found to be highly expressed in SEB conditions. Lytic polysaccharide monooxygenases (LPMO) classified as auxiliary activity families AA9 (GH61), CE (1, 4, 8, 15, 16), PL (1, 3, 4, 20) and GT (1, 2, 4, 8, 20, 35, 48) were also differentially expressed in this condition. Similarly, the most important enzymes related to biomass degradation, including endoxylanases, xyloglucanases, β-xylosidases, LPMOs, α-arabinofuranosidases, cellobiohydrolases, endoglucanases and β-glucosidases, were also identified in the secretome. Conclusions This is the first report of a transcriptome and secretome experiment of Aspergillus fumigatus in the degradation of pretreated sugarcane bagasse. The results suggest that this strain employs important strategies for this complex degradation process. It was possible to identify a set of genes and proteins that might be applied in several biotechnology fields. This knowledge can be exploited for the improvement of 2G ethanol production by the rational design of enzymatic cocktails. Electronic supplementary material The online version of this article (10.1186/s12864-018-4627-8) contains supplementary material, which is available to authorized users.

Objectives This study characterized the microbial diversity of formed biofilm on the surface of acrylic resins modified with nanostructured silver vanadate decorated with silver nanoparticles (AgVO3) after incubation in human saliva. Design Resin specimens prepared with AgVO3 at concentrations 0%, 1%, 2.5%, and 5% by either vacuum mixing or polymer solubilization were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). After 24 h and 7 days of saliva incubation, biofilm samples were collected from the surface of the specimens. The 16S rDNA genes were amplified, sequenced with the 454-Roche next-generation sequencing platform and analyzed to identify the Operational Taxonomic Units at the genus or higher level. Results Significant differences in the dispersion pattern of the nanoparticles were observed among the two different methods of AgVO3 incorporation. In the microbiological analysis, a total of 103 genera and 7 more inclusive taxa, representing the phyla Bacteroidetes, Firmicutes and Proteobacteria were identified colonizing resin surfaces. The incorporation method of the AgVO3 had little to no significant effect on the microbiota of samples. Significant time and concentration-dependent responses to AgVO3 caused changes in the taxonomic profile at the phylum and genus level. Conclusions The results show differences in relation to the microbial diversity of modified resins during the initial phase of biofilm maturation. The incorporation of AgVO3 seems to significantly affect the colonizing microbiota.

Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Aspergillus fumigatus remains poor. Thus, we investigated whether Ndt1A could alter fungi survival. To this end, we engineered the expression of an Ndt1A-encoding region in a Δndt1Δndt2 yeast strain. The resulting cloned Ndt1A protein promoted the mitochondrial uptake of nicotinamide adenine dinucleotide (NAD+), generating a large mitochondrial membrane potential. The NAD+ carrier utilized the electrochemical proton gradient to drive NAD+ entrance into mitochondria when the mitochondrial membrane potential was sustained by succinate. Its uptake has no impact on oxidative stress, and Ndt1A expression improved growth and survival of the Δndt1Δndt2 Saccharomyces cerevisiae strain.

Objectives This study employed culture-independent molecular techniques to extend the characterization of the microbial diversity of biofilm associated with either titanium or zirconia implant-abutments, including not-yet-cultivated bacteria species, and to identify and quantify species recovered from peri-implantar/periodontal sulci, supragingival biofilm and the internal parts of implants. Probing depth, clinical attachment level, bleeding on probing, and marginal bone level were also evaluated over time and correlated with biofilm formation. Methods Twenty healthy participants were analyzed. DNA-Checkerboard and 16S-rDNA-Pyrosequencing were used to quantify and determine species identity. Results 161 bacterial taxa representing 12 different phylotypes were found, of which 25% were non-cultivable. Species common to all sites belonged to genera Fusobacterium, Prevotella, Actinomyces, Porphyromonas, Veillonella and Streptococcus. While some species were subject-specific and detected in most sites, other species were site-specific. Moderate to higher levels of unclassified species were found colonizing titanium-related sites. Pathogenic and non-pathogenic species were detected colonizing oral sites in both materials. Titanium-related sites presented the highest total microbial count and higher counts of pathogenic species. Conclusions Our results revealed differences regarding microbial diversity and microorganisms counts in oral biofilm associated with titanium or zirconia. The obtained data suggests a possible relation between microbiological findings and clinical outcomes. Significance Next-generation methods of detection have provided new insights on complex microbiota colonizing different sites of oral cavity. The present study demonstrates relevant differences in the communities and microbial counts colonizing different tested substrates with consequent significant differences in the clinical-outcomes, suggesting a probably different mechanism for specific bacterial adhesion.

O câncer de mama representa importante problema de saúde pública. O microambiente tumoral tem participação no câncer. Peptídeos derivados da laminina estão envolvidos com progressão tumoral. O peptídeo C16 (KAFDITYVRLKF), da cadeia gama-1 da laminina, estimula adesão, migração e invasão celular, metástase e angiogênese. Para avaliar o efeito deste peptídeo na expressão gênica de células de câncer de mama (MDA-MB-231), células foram tratadas com C16 ou peptídeo controle (FKLRVYTIDFAK) por 24 horas. Após tratamento, a expressão gênica foi avaliada por microarray. C16 regulou a expressão de 80 genes em células MDA-MB-231, incluindo genes associados ao câncer. Os genes FGFR3, GPNMB e SPOCK1 tiveram suas expressões aumentadas após tratamento com C16, como confirmado por PCR quantitativo. Ensaios de invasão em câmaras de Boyden também indicam que C16 estimula invasão de células MDA-MB-231, porém C16 não mostrou efeito sobre proliferação e apoptose. Nossos dados sugerem que o peptídeo C16 regula expressão gênica e estimula invasão de células de câncer de mama MDA-MB-231. Human breast cancer constitutes a worldwide health care problem. During cancer progression, tumor cells are engaged in an interplay with their microenvironment. Studies have shown that peptides derived from laminin are involved in tumor progression. Among them, C16 (KAFDITYVRLKF), derived from laminin gamma-1 chain, is a cell-adhesive peptide that increases cell migration, invasion, metastasis and angiogenesis. This prompted us to analyze whether C16 would regulate gene expression in human breast cancer cells (MDA-MB-231). Cells were treated with C16 or scrambled peptide control (FKLRVYTIDFAK) for 24 hours and gene expression was analyzed by microarray. Eighty genes were regulated by C16, including genes associated with cancer. Among them, FGFR3, GPNMB and SPOCK1 expression was increased by C16, as confirmed by qPCR. C16 also increased MDA-MB-231 cell invasion. However, no effect on cell proliferation and apoptosis was observed. Concluding, our data suggest that C16 regulates gene expression and enhances invasion of metastatic MDA-MB-231 breast cancer cells.

Previously we found that levels of LRRC49 (leucine rich repeat containing 49; FLJ20156) transcripts were elevated in ER-positive breast tumors compared with ER-negative breast tumors. The LRRC49 gene is located on chromosome 15q23 in close proximity to the THAP10 (THAP domain containing 10) gene. These two genes have a bidirectional organization being arranged head-to-head on opposite strands, possibly sharing the same promoter region. Analysis of the promoter region of this gene pair revealed the presence of potential estrogen response elements (EREs), suggesting the potential of this promoter to be under the control of estrogen. We used quantitative real-time PCR (qPCR) to evaluate the expression of LRRC49 and THAP10 in a series of 72 primary breast tumors, and found reduced LRRC49 and THAP10 expression in 61 and 46% of the primary breast tumors analyzed, respectively. In addition, the occurrence of LRRC49/THAP10 promoter hypermethylation was examined by methylation specific PCR (MSP) in a sub-group of the breast tumors. Hypermethylation was observed in 57.5% of the breast tumors analyzed, and the levels of mRNA expression of both genes were inversely correlated with promoter hypermethylation. We investigated the effects of 17beta-estradiol on LRRC49 and THAP10 expression in MCF-7 breast cancer cells and found both transcripts to be up-regulated 2- to 3-fold upon 17beta-estradiol treatment. Our results show that the transcripts of LRRC49/THAP10 bidirectional gene pair are co-regulated by estrogen and that hypermethylation of the bidirectional promoter region simultaneously silences both genes. Further studies will be necessary to elucidate the role of LRRC49/THAP10 down-regulation in breast cancer.

Background ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we investigated mRNA and protein levels of ADAMTS-1 in normal and neoplastic tissues using qPCR, immunohistochemistry and immunoblot analyses, and we addressed the role of ADAMTS-1 in regulating migration, invasion and invadopodia formation in breast tumor cell lines. Results In a series of primary breast tumors, we observed variable levels of ADAMTS-1 mRNA expression but lower levels of ADAMTS-1 protein expression in human breast cancers as compared to normal tissue, with a striking decrease observed in high-malignancy cases (triple-negative for estrogen, progesterone and Her-2). This result prompted us to analyze the effect of ADAMTS-1 knockdown in breast cancer cells in vitro. MDA-MB-231 cells with depleted ADAMTS-1 expression demonstrated increased migration, invasion and invadopodia formation. The regulatory mechanisms underlying the effects of ADAMTS-1 may be related to VEGF, a growth factor involved in migration and invasion. MDA-MB-231 cells with depleted ADAMTS-1 showed increased VEGF concentrations in conditioned medium capable of inducing human endothelial cells (HUVEC) tubulogenesis. Furthermore, expression of the VEGF receptor (VEGFR2) was increased in MDA-MB-231 cells as compared to MCF7 cells. To further determine the relationship between ADAMTS-1 and VEGF regulating breast cancer cells, MDA-MB-231 cells with reduced expression of ADAMTS-1 were pretreated with a function-blocking antibody against VEGF and then tested in migration and invasion assays; both were partially rescued to control levels. Conclusions ADAMTS-1 expression was decreased in human breast tumors, and ADAMTS-1 knockdown stimulated migration, invasion and invadopodia formation in breast cancer cells in vitro. Therefore, this series of experiments suggests that VEGF is involved in the effects mediated by ADAMTS-1 in breast cancer cells.