Metatranscriptome and resistome of the endodontic microbiome.

INTRODUCTION: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance (AR) gene expression in endodontic infections. METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/ secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochaetes, and Synergistetes were among the non-dominant phyla. The top ten species were mainly represented by obligate (or quasi-obligate) anaerobes, including Gram-negative (e.g., Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia and Tannerella sp. oral taxon HOT-286) and Gram-positive species (e.g., Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (e.g., glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/ protection were the main resistance mechanisms. CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in antimicrobial resistance.

Enhancing Antibiotic Efficacy in Regenerative Endodontics by Improving Biofilm Susceptibility.

INTRODUCTION: Various strategies have been researched to enhance the susceptibility of biofilms, given their tolerance to antibiotics. This study evaluated the effect of the anti-microbial peptide nisin in association with antibiotics used in regenerative endodontics, exploring different treatment times and biofilm growth conditions. METHODS: A mixture of 10 bacterial species was cultivated on dentin specimens anaerobically for 21 days. Biofilms were treated with 1 mL of high-purity nisin Z (nisin ZP, 200 µg/mL) and a triple antibiotic mixture (TAP: ciprofloxacin + metronidazole + minocycline, 5 mg/mL), alone or in combination. The effectiveness of antimicrobial agents was assessed after 1 and 7 days. During the 7-day period, biofilms were treated under 2 conditions: a single dose in a nutrient-depleted setting (ie, no replenishment of growth medium) and multiple doses in a nutrient-rich environment (ie, renewal of medium and antimicrobial agents every 48 h). After treatments, biofilm cells were dispersed, and total colony-forming units were counted. RESULTS: After 1 d-treatment, nisin ZP + TAP resulted in 2-log cell reduction compared to TAP alone (P < .05). After 7 d-treatment with a single dose, nisin ZP + TAP and TAP reduced bacteria to nonculturable levels (P < .05), whereas repeated antimicrobial doses did not eliminate bacteria in a nutrient-rich environment. No bacterial reduction was observed with nisin ZP alone in any treatment time. CONCLUSIONS: The additional use of nisin improved the TAP activity only after a short exposure time. Longer exposure to TAP or nisin + TAP in a nutrient-deprived environment effectively eliminated biofilms.

Antibiofilm Activity of LL-37 Peptide and D-Amino Acids Associated with Antibiotics Used in Regenerative Endodontics on an Ex Vivo Multispecies Biofilm Model.

The antimicrobial peptide LL-37 and D-amino acids (D-AAs) have been proposed as antibiofilm agents. Therefore, this study aimed to test the antimicrobial effect of antibiofilm agents associated with antibiotics used in regenerative endodontic procedures (the triple antibiotic paste­TAP: ciprofloxacin + metronidazole + minocycline). An endodontic-like biofilm model grown on bovine dentin discs was used in this study. After 21-day growth, the biofilms were treated with 1 mg/mL TAP, 10 µM LL-37, an association of LL-37 + TAP, 40 mM D-AAs solution, an association of D-AAs + TAP, and phosphate-buffered saline (negative control). Colony forming unit (CFU) data were analyzed by two-way ANOVA and Tukey's multiple comparison test (p < 0.05). LL-37 + TAP showed the best antibacterial activity (7-log10 CFU/mL ± 0.5), reaching a 1 log reduction of cells in relation to the negative control (8-log10 CFU/mL ± 0.7) (p < 0.05). In turn, no significant reduction in bacterial cells was observed with TAP, LL-37, D-AAs, and D-AAs + TAP compared to the negative control. In conclusion, the combination of antibiotics and LL-37 peptide showed mild antibacterial activity, while the combination of antibiotics and D-AAs showed no activity against complex biofilms.

Genomics and epidemiology for gastric adenocarcinomas (GE4GAC): a Brazilian initiative to study gastric cancer

Gastric cancer (GC) is the fifth most common type of cancer worldwide with high incidences in Asia, Central, and South American countries. This patchy distribution means that GC studies are neglected by large research centers from developed countries. The need for further understanding of this complex disease, including the local importance of epidemiological factors and the rich ancestral admixture found in Brazil, stimulated the implementation of the GE4GAC project. GE4GAC aims to embrace epidemiological, clinical, molecular and microbiological data from Brazilian controls and patients with malignant and pre-malignant gastric disease. In this letter, we summarize the main goals of the project, including subject and sample accrual and current findings

Differential expression of Zif268 and c-Fos in the primary visual cortex and lateral geniculate nucleus of normal Cebus monkeys and after monocular lesions.

The transcription factors c-Fos and Zif268 have been used as markers of neuronal activity, and they also have been implicated in neuronal plasticity. In this study, we investigated the expression of c-Fos and Zif268 proteins in the lateral geniculate nucleus (LGN) and in the cortical primary visual area (V1) of normal adult Cebus apella monkeys and in animals with monocular lesions. In the LGN, the reaction for c-Fos showed immunopositive cells in both magnocellular (M) and parvocellular (P) layers; however, the label was heavier in P layers. In animals that suffered monocular lesions, the immunocytochemistry for c-Fos showed more labeling in layers related to the normal eye compared with those of the lesioned eye. No specific label was observed after the reaction for Zif268 in the LGN. In V1, the reaction for both Zif268 and c-Fos showed a pattern of lamination in which heavier labeling was found in layers 2/3, 4A, 4C, and 6. After monocular lesions, we observed a clear pattern of ocular dominance columns in V1 for both c-Fos and Zif268, in which the columns related to the normal eye are more heavily labeled than those related to the lesioned eye. This pattern is more evident in layer 4C after c-Fos reaction, whereas, after Zif268, it is more clearly observed in layers 2/3. These results suggest that, in addition to be regulated by functional activity, these transcription factors are involved in different processes during cortical reorganization.