Comparison of oral microbiome profile of polymers modified with silver and vanadium base nanomaterial by next-generation sequencing.

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.

Transcriptome and secretome analysis of Aspergillus fumigatus in the presence of sugarcane bagasse.

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-ß--xylanase (10.82 U mL-1) and endo-1,3-ß 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, ß-xylosidases, LPMOs, α-arabinofuranosidases, cellobiohydrolases, endoglucanases and ß-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.

Heterologous expression of mitochondrial nicotinamide adenine dinucleotide transporter (Ndt1) from Aspergillus fumigatus rescues impaired growth in Δndt1Δndt2 Saccharomyces cerevisiae strain.

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.

Mast cell-T cell axis alters development of colitis-dependent and colitis-independent colorectal tumours: potential for therapeutically targeting via mast cell inhibition.

BACKGROUND: Colorectal cancer (CRC) has a high mortality rate and can develop in either colitis-dependent (colitis-associated (CA)-CRC) or colitis-independent (sporadic (s)CRC) manner. There has been a significant debate about whether mast cells (MCs) promote or inhibit the development of CRC. Herein we investigated MC activity throughout the multistepped development of CRC in both human patients and animal models. METHODS: We analyzed human patient matched samples of healthy colon vs CRC tissue alongside conducting a The Cancer Genome Atlas-based immunogenomic analysis and multiple experiments employing genetically engineered mouse (GEM) models. RESULTS: Analyzing human CRC samples revealed that MCs can be active or inactive in this disease. An activated MC population decreased the number of tumor-residing CD8 T cells. In mice, MC deficiency decreased the development of CA-CRC lesions, while it increased the density of tumor-based CD8 infiltration. Furthermore, co-culture experiments revealed that tumor-primed MCs promote apoptosis in CRC cells. In MC-deficient mice, we found that MCs inhibited the development of sCRC lesions. Further exploration of this with several GEM models confirmed that different immune responses alter and are altered by MC activity, which directly alters colon tumorigenesis. Since rescuing MC activity with bone marrow transplantation in MC-deficient mice or pharmacologically inhibiting MC effects impacts the development of sCRC lesions, we explored its therapeutic potential against CRC. MC activity promoted CRC cell engraftment by inhibiting CD8+ cell infiltration in tumors, pharmacologically blocking it inhibits the ability of allograft tumors to develop. This therapeutic strategy potentiated the cytotoxic activity of fluorouracil chemotherapy. CONCLUSION: Therefore, we suggest that MCs have a dual role throughout CRC development and are potential druggable targets against this disease.

Toxicogenomic Analysis.

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.

Microbial communities of titanium versus zirconia abutments on implant-supported restorations: Biodiversity composition and its impact on clinical parameters over a 3-year longitudinal prospective study.

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.

Silencing of LRRC49 and THAP10 genes by bidirectional promoter hypermethylation is a frequent event in breast cancer.

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.

Analysis of the oral microbiome on the surface of modified dental polymers.

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.