Systematic Evaluation of Clinical, Nutritional, and Fecal Microbial Factors for Their Association With Colorectal Polyps.

INTRODUCTION: The identification of risk factors for precursor lesions of colorectal cancer (CRC) holds great promise in the context of prevention. With this study, we aimed to identify patient characteristics associated with colorectal polyps (CPs) and polyp features of potential malignant progression. Furthermore, a potential association with gut microbiota in this context was investigated. METHODS: In this single-center study, a total of 162 patients with CPs and 91 control patients were included. Multiple variables including information on lifestyle, diet, serum parameters, and gut microbiota, analyzed by 16S-rRNA gene amplicon sequencing and functional imputations (Picrust2), were related to different aspects of CPs. RESULTS: We observed that elevated serum alkaline phosphatase (AP) levels were significantly associated with the presence of high-grade dysplastic polyps. This association was further seen for patients with CRC. Thereby, AP correlated with other parameters of liver function. We did not observe significant changes in the gut microbiota between patients with CP and their respective controls. However, a trend toward a lower alpha-diversity was seen in patients with CRC. Interestingly, AP was identified as a possible clinical effect modifier of stool sample beta diversity. DISCUSSION: We show for the first time an increased AP in premalignant CP. Furthermore, AP showed a significant influence on the microbial composition of the intestine. Relatively elevated liver enzymes, especially AP, may contribute to the detection of precancerous dysplastic or neoplastic changes in colorectal lesions. The association between elevated AP, premalignant CP, and the microbiome merits further study.

2D and 3D Erosion Landscape Analysis of Endodontic-Treated Teeth Using EDTA and HEDP as Chelating Agents: A High-Resolution Micro-Computed Tomographic Study.

This study aimed to assess the amount of erosion during activated endodontic irrigation with either HEDP or EDTA via high-resolution micro-computed tomography. Two root canals of twenty premolars were prepared with ProTaper Next and irrigated with sodium hypochlorite. Palatal canals, which served as control groups, were sealed, while buccal canals were further irrigated with either EDTA (n = 10) or HEDP (n = 10), which served as test groups. Micro-CT was performed to measure erosion depth. For 2D and 3D measurements, non-parametric repeated ANOVA measurements and post hoc tests were performed. 2D analysis showed highly significant differences between the case groups at each position of the root (p ≤ 0.01). The cervical and apical positions showed significant differences in the EDTA group (p = 0.03). The 3D analysis also showed significant differences between both chelating agents (p < 0.01) and the case and control groups (p = 0.01). The mean erosion depths in the cervical, middle, and apical thirds of the EDTA group were 45.75, 41.79, and 32.25 µm, and for the HEDP group were 20.25, 16.40, and 15.96 µm, respectively. HEDP seems to have a significantly less erosive effect. Different irrigation protocols with harsher conditions, as might be the case during endodontic retreatment, could be assessed with micro-CT.

The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors.

BACKGROUND: Inter-individual differences in response to immune checkpoint inhibitors (ICI) remain a major challenge in cancer treatment. The composition of the gut microbiome has been associated with differential ICI outcome, but the underlying molecular mechanisms remain unclear, and therapeutic modulation challenging. METHODS: We established an in vivo model to treat C57Bl/6j mice with the type-I interferon (IFN-I)-modulating, bacterial-derived metabolite desaminotyrosine (DAT) to improve ICI therapy. Broad spectrum antibiotics were used to mimic gut microbial dysbiosis and associated ICI resistance. We utilized genetic mouse models to address the role of host IFN-I in DAT-modulated antitumour immunity. Changes in gut microbiota were assessed using 16S-rRNA sequencing analyses. FINDINGS: We found that oral supplementation of mice with the microbial metabolite DAT delays tumour growth and promotes ICI immunotherapy with anti-CTLA-4 or anti-PD-1. DAT-enhanced antitumour immunity was associated with more activated T cells and natural killer cells in the tumour microenvironment and was dependent on host IFN-I signalling. Consistent with this, DAT potently enhanced expansion of antigen-specific T cells following vaccination with an IFN-I-inducing adjuvant. DAT supplementation in mice compensated for the negative effects of broad-spectrum antibiotic-induced dysbiosis on anti-CTLA-4-mediated antitumour immunity. Oral administration of DAT altered the gut microbial composition in mice with increased abundance of bacterial taxa that are associated with beneficial response to ICI immunotherapy. INTERPRETATION: We introduce the therapeutic use of an IFN-I-modulating bacterial-derived metabolite to overcome resistance to ICI. This approach is a promising strategy particularly for patients with a history of broad-spectrum antibiotic use and associated loss of gut microbial diversity. FUNDING: Melanoma Research Alliance, Deutsche Forschungsgemeinschaft, German Cancer Aid, Wilhelm Sander Foundation, Novartis Foundation.

Unified Workflow for the Rapid and In-Depth Characterization of Bacterial Proteomes.

Bacteria are the most abundant and diverse organisms among the kingdoms of life. Due to this excessive variance, finding a unified, comprehensive, and safe workflow for quantitative bacterial proteomics is challenging. In this study, we have systematically evaluated and optimized sample preparation, mass spectrometric data acquisition, and data analysis strategies in bacterial proteomics. We investigated workflow performances on six representative species with highly different physiologic properties to mimic bacterial diversity. The best sample preparation strategy was a cell lysis protocol in 100% trifluoroacetic acid followed by an in-solution digest. Peptides were separated on a 30-min linear microflow liquid chromatography gradient and analyzed in data-independent acquisition mode. Data analysis was performed with DIA-NN using a predicted spectral library. Performance was evaluated according to the number of identified proteins, quantitative precision, throughput, costs, and biological safety. With this rapid workflow, over 40% of all encoded genes were detected per bacterial species. We demonstrated the general applicability of our workflow on a set of 23 taxonomically and physiologically diverse bacterial species. We could confidently identify over 45,000 proteins in the combined dataset, of which 30,000 have not been experimentally validated before. Our work thereby provides a valuable resource for the microbial scientific community. Finally, we grew Escherichia coli and Bacillus cereus in replicates under 12 different cultivation conditions to demonstrate the high-throughput suitability of the workflow. The proteomic workflow we present in this manuscript does not require any specialized equipment or commercial software and can be easily applied by other laboratories to support and accelerate the proteomic exploration of the bacterial kingdom.

Genome-wide association study in 8,956 German individuals identifies influence of ABO histo-blood groups on gut microbiome.

The intestinal microbiome is implicated as an important modulating factor in multiple inflammatory1,2, neurologic3 and neoplastic diseases4. Recent genome-wide association studies yielded inconsistent, underpowered and rarely replicated results such that the role of human host genetics as a contributing factor to microbiome assembly and structure remains uncertain5-11. Nevertheless, twin studies clearly suggest host genetics as a driver of microbiome composition11. In a genome-wide association analysis of 8,956 German individuals, we identified 38 genetic loci to be associated with single bacteria and overall microbiome composition. Further analyses confirm the identified associations of ABO histo-blood groups and FUT2 secretor status with Bacteroides and Faecalibacterium spp. Mendelian randomization analysis suggests causative and protective effects of gut microbes, with clade-specific effects on inflammatory bowel disease. This holistic investigative approach of the host, its genetics and its associated microbial communities as a 'metaorganism' broaden our understanding of disease etiology, and emphasize the potential for implementing microbiota in disease treatment and management.

In Vitro Effect of Er:YAG Laser on Different Single and Mixed Microorganisms Being Associated with Endodontic Infections.

Objective: The purpose of this in vitro study was to evaluate the antimicrobial effect of activated irrigation with different modes of erbium-doped yttrium aluminum garnet (Er:YAG) laser application on microorganisms related to secondary endodontic infection. Background: Er:YAG laser has been recommended as an adjuvant tool for root canal disinfection during endodontic treatment. Materials and methods: Laser-activated irrigation (LAI) with 300 or 600 µm tips were tested with or without intermittent irrigation with 0.9% sodium chloride (NaCl) solution against different microorganisms (five single strains and dual species (Streptococcus gordonii combined with Actinomyces oris or Fusobacterium nucleatum) in root canals after 3 days of incubation. In a 21-day infection model, LAI was used together with intermittent rinsing with sodium hypochlorite (NaOCl) against the dual-species mixtures; here the incidence of microbial regrowth after up to 7 days was monitored. Results: In the 3-day root infection model, LAI protocols did not show any significant reduction of the microbial load when compared with manual irrigation with saline solution. In the 21-day infection, S. gordonii combined with A. oris were not detectable anymore after applying the LAI protocol with a 600 µm tip (30 mJ/10 pps) up to 7 days after treatment. Conclusions: Application of LAI with a 600 µm tip by using an Er:YAG laser might be advantageous in treatment of endodontic infections.

Differentiation of ncRNAs from small mRNAs in Escherichia coli O157:H7 EDL933 (EHEC) by combined RNAseq and RIBOseq - ryhB encodes the regulatory RNA RyhB and a peptide, RyhP.

BACKGROUND: While NGS allows rapid global detection of transcripts, it remains difficult to distinguish ncRNAs from short mRNAs. To detect potentially translated RNAs, we developed an improved protocol for bacterial ribosomal footprinting (RIBOseq). This allowed distinguishing ncRNA from mRNA in EHEC. A high ratio of ribosomal footprints per transcript (ribosomal coverage value, RCV) is expected to indicate a translated RNA, while a low RCV should point to a non-translated RNA. RESULTS: Based on their low RCV, 150 novel non-translated EHEC transcripts were identified as putative ncRNAs, representing both antisense and intergenic transcripts, 74 of which had expressed homologs in E. coli MG1655. Bioinformatics analysis predicted statistically significant target regulons for 15 of the intergenic transcripts; experimental analysis revealed 4-fold or higher differential expression of 46 novel ncRNA in different growth media. Out of 329 annotated EHEC ncRNAs, 52 showed an RCV similar to protein-coding genes, of those, 16 had RIBOseq patterns matching annotated genes in other enterobacteriaceae, and 11 seem to possess a Shine-Dalgarno sequence, suggesting that such ncRNAs may encode small proteins instead of being solely non-coding. To support that the RIBOseq signals are reflecting translation, we tested the ribosomal-footprint covered ORF of ryhB and found a phenotype for the encoded peptide in iron-limiting condition. CONCLUSION: Determination of the RCV is a useful approach for a rapid first-step differentiation between bacterial ncRNAs and small mRNAs. Further, many known ncRNAs may encode proteins as well.

Citrullination in the periodontium--a possible link between periodontitis and rheumatoid arthritis.

OBJECTIVES: The aim of the present study was to assess human and bacterial peptidylarginine deiminase (PAD) activity in the gingival crevicular fluid (GCF) in the context of serum levels of antibodies against citrullinated epitopes in rheumatoid arthritis and periodontitis. MATERIALS AND METHODS: Human PAD and Porphyromonas gingivalis-derived enzyme (PPAD) activities were measured in the GCF of 52 rheumatoid arthritis (RA) patients (48 with periodontitis and 4 without) and 44 non-RA controls (28 with periodontitis and 16 without). Serum antibodies against citrullinated epitopes were measured by ELISA. Bacteria being associated with periodontitis were determined by nucleic-acid-based methods. RESULTS: Citrullination was present in 26 (50%) RA patients and 23 (48%) controls. PAD and PPAD activities were detected in 36 (69%) and 30 (58%) RA patients, respectively, and in 30 (68%) and 21 (50%) controls, respectively. PPAD activity was higher in RA and non-RA patients with periodontitis than in those without (p = 0.038; p = 0.004), and was detected in 35 of 59 P. gingivalis-positive samples, and in 16 of 37 P. gingivalis-negative samples in association with high antibody levels against that species. CONCLUSIONS: PAD and PPAD activities within the periodontium are elevated in RA and non-RA patients with periodontitis. PPAD secreted by P. gingivalis residing in epithelial cells may exert its citrullinating activity in distant regions of the periodontium or even distant tissues. CLINICAL RELEVANCE: In periodontitis, the citrullination of proteins/peptides by human and bacterial peptidylarginine deiminases may generate antibodies after breaching immunotolerance in susceptible individuals.

Acid shock of Listeria monocytogenes at low environmental temperatures induces prfA, epithelial cell invasion, and lethality towards Caenorhabditis elegans.

BACKGROUND: The saprophytic pathogen Listeria monocytogenes has to cope with a variety of acidic habitats during its life cycle. The impact of low-temperature coupled with pH decrease for global gene expression and subsequent virulence properties, however, has not been elucidated. RESULTS: qRT-PCR revealed for the first time a transient, acid triggered prfA induction of approximately 4-fold, 5.7-fold, 7-fold and 9.3-fold 60 to 90 min after acid shock of L. monocytogenes at 37°C, 25°C, 18°C, and 10°C, respectively. Comparable data were obtained for seven different L. monocytogenes strains, demonstrating that prfA induction under these conditions is a general response of L. monocytogenes. Transcriptome analysis revealed that the in vivo-relevant genes bsh, clpP, glpD, hfq, inlA, inlB, inlE, lisR, and lplA1 as well as many other genes with a putative role during infection are transiently induced upon acid shock conducted at 25°C and 37°C. Twenty-five genes repressed upon acid shock are known to be down regulated during intracellular growth or by virulence regulators. These data were confirmed by qRT-PCR of twelve differentially regulated genes and by the identification of acid shock-induced genes influenced by σB. To test if up regulation of virulence genes at temperatures below 37°C correlates with pathogenicity, the capacity of L. monocytogenes to invade epithelial cells after acid shock at 25°C was measured. A 12-fold increased number of intracellular bacteria was observed (acid shock, t = 60 min) that was reduced after adaptation to the level of the unshocked control. This increased invasiveness was shown to be in line with the induction of inlAB. Using a nematode infection assay, we demonstrated that Caenorhabditis elegans fed with acid-shocked L. monocytogenes exhibits a shorter time to death of 50% (TD50) of the worms (6.4 days) compared to infection with unshocked bacteria (TD50 = 10.2 days). CONCLUSIONS: PrfA and other listerial virulence genes are induced by an inorganic acid in a temperature-dependent manner. The data presented here suggest that low pH serves as a trigger for listerial pathogenicity at environmental temperatures.

FTIR-based polyphasic identification of lactic acid bacteria isolated from traditional Greek Graviera cheese.

This study used a combination of phenotypic, physical (Fourier Transformed Infra-Red [FTIR] spectroscopy) and molecular (RFLP and SSCP analysis of 16S rRNA genes) methods to identify the lactic acid bacteria (LAB) flora present in traditional Greek Graviera cheese after five weeks of ripening. A total of 300 isolates collected from high dilution plates of TSAYE (incubated at 30 °C), M-17 (22 °C) and M-17 (42 °C) agar media were clustered by FTIR and then representative strains of each cluster were cross-identified blindly by all methods. Based on their FTIR spectra, 282 isolates were LAB grouped in 28 clusters. The LAB species identified and their prevalence in the cheese samples were: Lactobacillus casei/paracasei (68.8%), Lactobacillus plantarum (19.5%), Streptococcus thermophilus (8.9%), Enterococcus faecium (2.1%), and Lactococcus lactis (0.7%). Also, Staphylococcus equorum (11 isolates), Corynebacterium sp. (5 isolates) and Brevibacterium sp. (1 isolate) were recovered from TSAYE. Comparative identification results showed that phenotypic and molecular methods were in mutual agreement as regards the LAB species identified. The present polyphasic identification approach based on rapid FTIR screening of 10-fold more isolates than a previous classical identification approach allowed or improved detection of few sub-dominant species; however the predominant LAB species in the cheese samples were the same with both approaches.

Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo.

We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage PhiX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy.

Teeth: malignant neoplasms in the dental pulp?

Common pathologies of the dental pulp differentiate between acute and chronic inflammatory states caused by caries or dental trauma. Inflammations of the dental pulp as a result of neoplastic alterations are generally considered non-existent. In fact, using the search phrase "dental pulp" combined with "sarcoma", "carcinoma", or "neoplasms" in PubMed when using the MeSH search mode yielded no reports on primary malignant neoplasms. However, a hand search yields clinical reports on pulpal tumours that were published over a century ago. In this Essay, the results of a hand search in historic published work are presented. Furthermore, deductive reflections are done on general tumour pathogenesis with respect to specific anatomical prerequisites of the dental pulp. Because of the restricted space in a tooth, tumour expansion will probably lead to the formation of irritation dentine by secondary odontoblasts and, subsequently, to a haemorrhage infarct of the pulp. One hypothesis states that a purported neoplasm of the dental pulp leads to a chronic appositive pulpitis and-sooner or later-will be treated likewise by root-canal treatment or extraction. Further research, including stem-cell studies, is recommended.