Comparison between 16S rRNA and shotgun sequencing in colorectal cancer, advanced colorectal lesions, and healthy human gut microbiota.

BACKGROUND: Gut dysbiosis has been associated with colorectal cancer (CRC), the third most prevalent cancer in the world. This study compares microbiota taxonomic and abundance results obtained by 16S rRNA gene sequencing (16S) and whole shotgun metagenomic sequencing to investigate their reliability for bacteria profiling. The experimental design included 156 human stool samples from healthy controls, advanced (high-risk) colorectal lesion patients (HRL), and CRC cases, with each sample sequenced using both 16S and shotgun methods. We thoroughly compared both sequencing technologies at the species, genus, and family annotation levels, the abundance differences in these taxa, sparsity, alpha and beta diversities, ability to train prediction models, and the similarity of the microbial signature derived from these models. RESULTS: As expected, the results showed that 16S detects only part of the gut microbiota community revealed by shotgun, although some genera were only profiled by 16S. The 16S abundance data was sparser and exhibited lower alpha diversity. In lower taxonomic ranks, shotgun and 16S highly differed, partially due to a disagreement in reference databases. When considering only shared taxa, the abundance was positively correlated between the two strategies. We also found a moderate correlation between the shotgun and 16S alpha-diversity measures, as well as their PCoAs. Regarding the machine learning models, only some of the shotgun models showed some degree of predictive power in an independent test set, but we could not demonstrate a clear superiority of one technology over the other. Microbial signatures from both sequencing techniques revealed taxa previously associated with CRC development, e.g., Parvimonas micra. CONCLUSIONS: Shotgun and 16S sequencing provide two different lenses to examine microbial communities. While we have demonstrated that they can unravel common patterns (including microbial signatures), shotgun often gives a more detailed snapshot than 16S, both in depth and breadth. Instead, 16S will tend to show only part of the picture, giving greater weight to dominant bacteria in a sample. Therefore, we recommend choosing one or another sequencing technique before launching a study. Specifically, shotgun sequencing is preferred for stool microbiome samples and in-depth analyses, while 16S is more suitable for tissue samples and studies with targeted aims.

Performance of a Shotgun Prediction Model for Colorectal Cancer When Using 16S rRNA Sequencing Data.

Colorectal cancer (CRC), the third most common cancer globally, has shown links to disturbed gut microbiota. While significant efforts have been made to establish a microbial signature indicative of CRC using shotgun metagenomic sequencing, the challenge lies in validating this signature with 16S ribosomal RNA (16S) gene sequencing. The primary obstacle is reconciling the differing outputs of these two methodologies, which often lead to divergent statistical models and conclusions. In this study, we introduce an algorithm designed to bridge this gap by mapping shotgun-derived taxa to their 16S counterparts. This mapping enables us to assess the predictive performance of a shotgun-based microbiome signature using 16S data. Our results demonstrate a reduction in performance when applying the 16S-mapped taxa in the shotgun prediction model, though it retains statistical significance. This suggests that while an exact match between shotgun and 16S data may not yet be feasible, our approach provides a viable method for comparative analysis and validation in the context of CRC-associated microbiome research.

Chemopreventive effects of anthocyanins on colorectal and breast cancer: A review.

The present review has analyzed the scientific literature, available in the PubMed and Scopus databases, in order to summarize the current state of diet anthocyanin research in breast cancer (BC) and colorectal cancer (CRC) animal models but also for up-to-date human studies. For CRC, 28 preclinical and 9 clinical studies were selected in line with our search query in science databases. In relation to BC, 14 preclinical and 5 clinical studies were selected. Remarkably, all the preclinical studies, to a greater or lesser degree, suggested a chemoprevention effect of anthocyanin in BC/CRC rodent models. These encouraging results from animal models are not extrapolated to the same degree to human studies where, from the similar theoretical daily doses of anthocyanins in these studies, the opposite results were reported. Nevertheless, it is worth mentioning that the anthocyanin doses in the human studies carried out recently are low if we consider the estimated exposure to anthocyanins issued by the European Food Safety Agency (EFSA) or extremely low if we consider with caution the human equivalent dose based on body surface area from the preclinical dosage regimes used. Therefore, although some clinical data has demonstrated an inverse relation between anthocyanin consumption and BC/CRC, this could, in fact, be more relevant if we increase the daily human anthocyanin dose (as observed in animal model dose-effect studies) while new toxicological data for this flavonoid subtype are brought to light.

Presence of Certain Foodborne Pathogens in Traditional Balearic Islands' Meat, Pastry, and Cheese Specialties Supported by European Union Quality Schemes, from 2018 to 2021.

The cuisine of the Balearic Islands (Spain, southern Europe) has several products of a great tradition, recognized worldwide and covered by European Union quality schemes, such as Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI). Among them, the most emblematic products are sobrasada de Mallorca (a type of raw curated pork meat), ensaimada de Mallorca (pastry product), and Mahón-Menorca cheese (cow's milk cheese). During 4 consecutive years (2018-2021), the presence/absence of Escherichia coli ß-glucuronidase positive (henceforth as E. coli), Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus in these products has been monitored, as well as the total yeast and mold count in ensaimada de Mallorca. The results of the microbiological analysis showed that sobrasada presented similar microbiological patterns to those of other raw curated meat products (some presence of E. coli and L. monocytogenes). Furthermore, the sobrasada de Mallorca made with white pork tended to be positive in E. coli compared to other sobrasada subtypes. In the case of ensaimada, only a reduced number of cases within filled ensaimadas (with higher moisture content) presented unacceptable mold and yeast counts (>500 colony-forming unit [CFU]/g). Finally, the Mahón-Menorca cheese presented a surprising microbiology pattern: higher E. coli contamination in the pasteurized milk cheese compared to its raw counterpart. This pattern was observed for all the years, being statistically significant in 2020. This study has detected good microbiological status in the three traditional products studied. However, worrisome issues in Good Hygienic Practices have been detected for some companies that produce pasteurized milk Mahón-Menorca cheese under the PDO quality label. The companies involved and even the competent authorities should address these problems to correct this deviation in food security.

Dietary Pattern's Role in Hepatic Epigenetic and Dietary Recommendations for the Prevention of NAFLD.

NAFLD has emerged as a significant public health concern, with its prevalence increasing globally. Emphasizing the complex relationship between dietary patterns and epigenetic modifications such as DNA methylation or miRNA expression can exert a positive impact on preventing and managing metabolic disorders, including NAFLD, within the 2030 Sustainable Development Goals. This review aims to evaluate the influence of dietary patterns on hepatic epigenetic gene modulation and provide dietary recommendations for the prevention and management of NAFLD in the general population. METHODS: Comprehensive screening and eligibility criteria identified eleven articles focusing on epigenetic changes in NAFLD patients through dietary modifications or nutrient supplementation. RESULTS AND DISCUSSION: Data were organized based on study types, categorizing them into evaluations of epigenetic changes in NAFLD patients through dietary pattern modifications or specific nutrient intake. CONCLUSIONS: The study highlights the importance of dietary interventions in managing and preventing NAFLD, emphasizing the potential of dietary patterns to influence hepatic epigenetic gene modulation. This study provides valuable insights and recommendations to mitigate the risk of developing NAFLD: (i) eat a primarily plant-based diet; (ii) increase consumption of high-fiber foods; (iii) consume more polyunsaturated and monounsaturated fatty acids; (iv) limit processed foods, soft drinks, added sugars, and salt; and (v) avoid alcohol.

Diet Impacts on Gene Expression in Healthy Colon Tissue: Insights from the BarcUVa-Seq Study.

(1) Introduction: The global rise of gastrointestinal diseases, including colorectal cancer and inflammatory bowel diseases, highlights the need to understand their causes. Diet is a common risk factor and a crucial regulator of gene expression, with alterations observed in both conditions. This study aims to elucidate the specific biological mechanisms through which diet influences the risk of bowel diseases. (2) Methods: We analyzed data from 436 participants from the BarcUVa-Seq population-based cross-sectional study utilizing gene expression profiles (RNA-Seq) from frozen colonic mucosal biopsies and dietary information from a semi-quantitative food frequency questionnaire. Dietary variables were evaluated based on two dietary patterns and as individual variables. Differential expression gene (DEG) analysis was performed for each dietary factor using edgeR. Protein-protein interaction (PPI) analysis was conducted with STRINGdb v11 for food groups with more than 10 statistically significant DEGs, followed by Reactome-based enrichment analysis for the resulting networks. (3) Results: Our findings reveal that food intake, specifically the consumption of blue fish, alcohol, and potatoes, significantly influences gene expression in the colon of individuals without tumor pathology, particularly in pathways related to DNA repair, immune system function, and protein glycosylation. (4) Discussion: These results demonstrate how these dietary components may influence human metabolic processes and affect the risk of bowel diseases.

Species interactions, stability, and resilience of the gut microbiota - Helminth assemblage in horses.

The nature and strength of interactions entertained among helminths and their host gut microbiota remain largely unexplored. Using 40 naturally infected Welsh ponies, we tracked the gut microbiota-cyathostomin temporal dynamics and stability before and following anthelmintic treatment and the associated host blood transcriptomic response. High shedders harbored 14 species of cyathostomins, dominated by Cylicocyclus nassatus. They exhibited a highly diverse and temporal dynamic gut microbiota, with butyrate-producing Clostridia likely driving the ecosystem steadiness and host tolerance toward cyathostomins infection. However, anthelmintic administration sharply bent the microbial community. It disrupted the ecosystem stability and the time-dependent network of interactions, affecting longer term microbial resilience. These observations highlight how anthelmintic treatments alter the triangular relationship of parasite, host, and gut microbiota and open new perspectives for adding nutritional intervention to current parasite management strategies.

Improving Species Level-taxonomic Assignment from 16S rRNA Sequencing Technologies.

Analysis of the bacterial community from a 16S rRNA gene sequencing technologies requires comparing the reads to a reference database. The challenging task involved in annotation relies on the currently available tools and 16S rRNA databases: SILVA, Greengenes and RDP. A successful annotation depends on the quality of the database. For instance, Greengenes and RDP have not been updated since 2013 and 2016, respectively. In addition, the nature of 16S sequencing technologies (short reads) focuses mainly on the V3-V4 hypervariable region sequencing and hinders the species assignment, in contrast to whole shotgun metagenome sequencing. Here, we combine the results of three standard protocols for 16S rRNA amplicon annotation that utilize homology-based methods, and we propose a new re-annotation strategy to enlarge the percentage of amplicon sequence variants (ASV) classified up to the species level. Following the pattern (reference) method: DADA2 pipeline and SILVA v.138.1 reference database classification (Basic Protocol 1), our method maps the ASV sequences to custom nucleotide BLAST with the SILVA v.138.1 (Basic Protocol 2), and to the 16S database of Bacteria and Archaea of NCBI RefSeq Targeted Loci Project databases (Basic Protocol 3). This new re-annotation workflow was tested in 16S rRNA amplicon data from 156 human fecal samples. The proposed new strategy achieved an increase of nearly eight times the proportion of ASV classified at the species level in contrast to the reference method for the database used in the present research. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample inference and taxonomic profiling through DADA2 algorithm. Basic Protocol 2: Custom BLASTN database creation and ASV taxonomical assignment. Basic Protocol 3: ASV taxonomical assignment using NCBI RefSeq Targeted Loci Project database. Basic Protocol 4: Definitive selection of lineages among the three methods.

Glioblastoma Therapy: Rationale for a Mesenchymal Stem Cell-based Vehicle to Carry Recombinant Viruses.

Evasion of growth suppression is among the prominent hallmarks of cancer. Phosphatase and tensin homolog (PTEN) and p53 tumor-suppressive pathways are compromised in most human cancers, including glioblastoma (GB). Hence, these signaling pathways are an ideal point of focus for novel cancer therapeutics. Recombinant viruses can selectivity kill cancer cells and carry therapeutic genes to tumors. Specifically, oncolytic viruses (OV) have been successfully employed for gene delivery in GB animal models and showed potential to neutralize immunosuppression at the tumor site. However, the associated systemic immunogenicity, inefficient transduction of GB cells, and inadequate distribution to metastatic tumors have been the major bottlenecks in clinical studies. Mesenchymal stem cells (MSCs), with tumor-tropic properties and immune privilege, can improve OVs targeting. Remarkably, combining the two approaches can address their individual issues. Herein, we summarize findings to advocate the reactivation of tumor suppressors p53 and PTEN in GB treatment and use MSCs as a "Trojan horse" to carry oncolytic viral cargo to disseminated tumor beds. The integration of MSCs and OVs can emerge as the new paradigm in cancer treatment.

Meta-Analysis and Validation of a Colorectal Cancer Risk Prediction Model Using Deep Sequenced Fecal Metagenomes.

The gut microbiome is a potential modifiable risk factor for colorectal cancer (CRC). We re-analyzed all eight previously published stool sequencing data and conducted an MWAS meta-analysis. We used cross-validated LASSO predictive models to identify a microbiome signature for predicting the risk of CRC and precancerous lesions. These models were validated in a new study, Colorectal Cancer Screening (COLSCREEN), including 156 participants that were recruited in a CRC screening context. The MWAS meta-analysis identified 95 bacterial species that were statistically significantly associated with CRC (FDR < 0.05). The LASSO CRC predictive model obtained an area under the receiver operating characteristic curve (aROC) of 0.81 (95%CI: 0.78−0.83) and the validation in the COLSCREEN dataset was 0.75 (95%CI: 0.66−0.84). This model selected a total of 32 species. The aROC of this CRC-trained model to predict precancerous lesions was 0.52 (95%CI: 0.41−0.63). We have identified a signature of 32 bacterial species that have a good predictive accuracy to identify CRC but not precancerous lesions, suggesting that the identified microbes that were enriched or depleted in CRC are merely a consequence of the tumor. Further studies should focus on CRC as well as precancerous lesions with the intent to implement a microbiome signature in CRC screening programs.

Gut microbiota resilience in horse athletes following holidays out to pasture.

Elite horse athletes that live in individual boxes and train and compete for hours experience long-term physical and mental stress that compromises animal welfare and alters the gut microbiota. We therefore assessed if a temporary period out to pasture with conspecifics could improve animal welfare and in turn, favorably affect intestinal microbiota composition. A total of 27 athletes were monitored before and after a period of 1.5 months out to pasture, and their fecal microbiota and behavior profiles were compared to those of 18 horses kept in individual boxes. The overall diversity and microbiota composition of pasture and control individuals were temporally similar, suggesting resilience to environmental challenges. However, pasture exposure induced an increase in Ruminococcus and Coprococcus that lasted 1-month after the return to individual boxes, which may have promoted beneficial effects on health and welfare. Associations between the gut microbiota composition and behavior indicating poor welfare were established. Furthermore, withdrawn behavior was associated with the relative abundances of Lachnospiraceae AC2044 group and Clostridiales family XIII. Both accommodate a large part of butyrate-producing bacterial genera. While we cannot infer causality within this study, arguably, these findings suggest that management practices maintained over a longer period of time may moderate the behavior link to the gut ecosystem beyond its resilience potential.

Consumption evaluation of one apple flesh a day in the initial phases prior to adenoma/adenocarcinoma in an azoxymethane rat colon carcinogenesis model.

Colorectal cancer (CRC) is the fourth cancer with the most new cases reported in 2018 worldwide. Consumption of fruit and vegetables is a protective factor against the risk of CRC. Beyond this, flavonoids could orchestrate these healthy effects. Apart from containing the typical apple flavonoids, red-fleshed apples also contain anthocyanins, mainly cyanidin-3-O-galactoside (Cy3Gal). Through an azoxymethane rat carcinogenesis model, a study was carried out in order to assess the possible protective effects of apple polyphenols, with special attention to anthocyanins. In addition, apart from negative and positive controls, a group with chemotherapy with 5-fluorouracil (5FU) was included to compare their performance against the output collected from the animal treatments with white-fleshed apple (WF), red-fleshed apple (RF) and Cy3Gal (AE). Although the 5FU group presented the best performance towards aberrant crypt foci (ACF) inhibition (70.1%), rats fed with white-fleshed apples ('Golden Smoothee') were able to achieve 41.3% ACF inhibition, while none of the challenged treatments (WF, RF and AE) suffered mucin depletion in their colonocytes. Expression changes of 17 genes related to CRC were assessed. In detail, the ACF inhibition phenotype detected in 5FU and WF groups could be explained through the expression changes detected in the apoptosis-related genes of Aurka, p53 and Cox2. Moreover, in the apple consumption groups (WF and RF), a reduced protein expression of matrix metalloproteinases with gelatinase activity (MMP-2 and 9) was detected. Overall, our study suggests an effect of apple polyphenols and apple anthocyanin Cy3Gal against colon carcinogenesis, retarding/diminishing the appearance of the precancerous markers studied.

Priming for welfare: gut microbiota is associated with equitation conditions and behavior in horse athletes.

We simultaneously measured the fecal microbiota and multiple environmental and host-related variables in a cohort of 185 healthy horses reared in similar conditions during a period of eight months. The pattern of rare bacteria varied from host to host and was largely different between two time points. Among a suite of variables examined, equitation factors were highly associated with the gut microbiota variability, evoking a relationship between gut microbiota and high levels of physical and mental stressors. Behavioral indicators that pointed toward a compromised welfare state (e.g. stereotypies, hypervigilance and aggressiveness) were also associated with the gut microbiota, reinforcing the notion for the existence of the microbiota-gut-brain axis. These observations were consistent with the microbiability of behaviour traits (> 15%), illustrating the importance of gut microbial composition to animal behaviour. As more elite athletes suffer from stress, targeting the microbiota offers a new opportunity to investigate the bidirectional interactions within the brain gut microbiota axis.

Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model.

Colorectal cancer (CRC), also known as colon cancer, is the third most common form of cancer worldwide in men and the second in women and is characterized by several genetic alterations, among them the expression of several genes. 1,2-dimethylhydrazine (DMH) and its metabolite azoxymethane (AOM) are procarcinogens commonly used to induce colon cancer in rats (DMH/AOM rat model). This rat model has been used to study changes in mRNA expression in genes involved in this pathological condition. However, a lack of proper detailed PCR primer design in the literature limits the reproducibility of the published data. The present study aims to design, optimize and validate the qPCR, in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, for seventeen genes commonly used in the DMH/AOM rat model of CRC (Apc, Aurka, Bax, Bcl2, ß-catenin, Ccnd1, Cdkn1a, Cox2, Gsk3beta, IL-33, iNOs, Nrf2, p53, RelA, Smad4, Tnfα and Vegfa) and two reference genes (Actb or ß-actin and B2m). The specificity of all primer pairs was empirically validated on agarose gel, and furthermore, the melting curve inspection was checked as was their efficiency (%) ranging from 90 to 110 with a correlation coefficient of r 2 > 0.980. Finally, a pilot study was performed to compare the robustness of two candidate reference genes.

Seasonal Variability of the Phytochemical Composition of New Red-Fleshed Apple Varieties Compared with Traditional and New White-Fleshed Varieties.

The main objective of this study was to evaluate the impact of the season on the apple phytochemical composition (phenolic compounds, triterpenes, and organic and ascorbic acids). For this proposal, four red-fleshed and five white-fleshed apple varieties from two consecutive seasons (2015 and 2016) were studied. A significant interaction with the season in some compounds was observed. The total phenolic content in the apple flesh from 2015 was higher than that from 2016 probably related with the lower rainfall during the harvest period in 2015 that could have favored hydric stress in the apple trees. The impact of the season on the apple skin was different. The 2016 season was characterized by higher maximum and minimum temperatures resulting in a higher content of flavonols, triterpenes, and organic acids. Anthocyanin concentration in both the flesh and skin of the red-fleshed apples showed no clear relationship to the season, and each variety showed an individual pattern.

Impact of dietary supplementation with olive and thyme phenols on alpha-tocopherol concentration in the muscle and liver of adult Wistar rats.

A preliminary study to evaluate the effect of dietary supplementation with olive phenols (oleuropein, hydroxytyrosol and secoiridoids), thyme phenols and a combination of these (5 mg per kg rat weight per day) on the α-tocopherol concentrations in the muscle and liver of healthy adult Wistar rats over 21 days was conducted. In addition, the excretion of α-tocopherol through the faeces was examined. The results demonstrated that the diet supplemented with some phenolic compounds of olive and thyme increased α-tocopherol (P < 0.05) in the liver of female rats, although the α-tocopherol content in the diet of all groups was identical. In addition, a synergic effect between the olive phenols and thyme was observed. Therefore, our study indicates a protective effect of olive and thyme phenols supplemented in the diet on α-tocopherol, resulting in a higher concentration of endogenous α-tocopherol in the rat liver.

Phytochemical Profiles of New Red-Fleshed Apple Varieties Compared with Traditional and New White-Fleshed Varieties.

This study is an exhaustive chemical characterization of the phenolic compounds, triterpenes, and organic and ascorbic acids in red-fleshed apple varieties obtained by different breeding programs and using five traditional and new white-fleshed apple cultivars as reference. To carry out these analyses, solid-liquid extraction (SLE) and ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) were used. The results showed that the red-fleshed apples contained, in either the flesh or peel, higher amounts of phenolic acids (chlorogenic acid), anthocyanins (cyanidin-3-O-galactoside), dihydrochalcones (phloretin xylosyl glucoside), and organic acids (malic acid) but a lower amount of flavan-3-ols than the white-fleshed apples. These quantitative differences could be related to an up-regulation of anthocyanins, dihydrochalcones, and malic acid and a down-regulation of flavan-3-ols (anthocyanin precursors) in both the flesh and peel of the red-fleshed apple varieties. The reported results should be considered preliminary because the complete phytochemical characterization of the red-fleshed apple cultivars will be extended to consecutive harvest seasons.