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.

A hybrid approach to assess the structural impact of long noncoding RNA mutations uncovers key NEAT1 interactions in colorectal cancer.

Long noncoding RNAs (lncRNAs) are emerging players in cancer and they entail potential as prognostic biomarkers or therapeutic targets. Earlier studies have identified somatic mutations in lncRNAs that are associated with tumor relapse after therapy, but the underlying mechanisms behind these associations remain unknown. Given the relevance of secondary structure for the function of some lncRNAs, some of these mutations may have a functional impact through structural disturbance. Here, we examined the potential structural and functional impact of a novel A > G point mutation in NEAT1 that has been recurrently observed in tumors of colorectal cancer patients experiencing relapse after treatment. Here, we used the nextPARS structural probing approach to provide first empirical evidence that this mutation alters NEAT1 structure. We further evaluated the potential effects of this structural alteration using computational tools and found that this mutation likely alters the binding propensities of several NEAT1-interacting miRNAs. Differential expression analysis on these miRNA networks shows upregulation of Vimentin, consistent with previous findings. We propose a hybrid pipeline that can be used to explore the potential functional effects of lncRNA somatic mutations.

Discovery and Validation of Clinically Relevant Long Non-Coding RNAs in Colorectal Cancer.

Colorectal cancer (CRC) is the third most prevalent cancer worldwide, with nearly two million newly diagnosed cases each year. The survival of patients with CRC greatly depends on the cancer stage at the time of diagnosis, with worse prognosis for more advanced cases. Consequently, considerable effort has been directed towards improving population screening programs for early diagnosis and identifying prognostic markers that can better inform treatment strategies. In recent years, long non-coding RNAs (lncRNAs) have been recognized as promising molecules, with diagnostic and prognostic potential in many cancers, including CRC. Although large-scale genome and transcriptome sequencing surveys have identified many lncRNAs that are altered in CRC, most of their roles in disease onset and progression remain poorly understood. Here, we critically review the variety of detection methods and types of supporting evidence for the involvement of lncRNAs in CRC. In addition, we provide a reference catalog that features the most clinically relevant lncRNAs in CRC. These lncRNAs were selected based on recent studies sorted by stringent criteria for both supporting experimental evidence and reproducibility.

Citizen-science reveals changes in the oral microbiome in Spain through age and lifestyle factors.

The relevance of the human oral microbiome to our understanding of human health has grown in recent years as microbiome studies continue to develop. Given the links of the oral cavity with the digestive, respiratory and circulatory systems, the composition of the oral microbiome is relevant beyond just oral health, impacting systemic processes across the body. However, we still have a very limited understanding about intrinsic and extrinsic factors that shape the composition of the healthy oral microbiome. Here, we followed a citizen-science approach to assess the relative impact on the oral microbiome of selected biological, social, and lifestyle factors in 1648 Spanish individuals. We found that the oral microbiome changes across age, with middle ages showing a more homogeneous composition, and older ages showing more diverse microbiomes with increased representation of typically low abundance taxa. By measuring differences within and between groups of individuals sharing a given parameter, we were able to assess the relative impact of different factors in driving specific microbial compositions. Chronic health disorders present in the analyzed population were the most impactful factors, followed by smoking and the presence of yeasts in the oral cavity. Finally, we corroborate findings in the literature that relatives tend to have more similar oral microbiomes, and show for the first time a similar effect for classmates. Multiple intrinsic and extrinsic factors jointly shape the oral microbiome. Comparative analysis of metabarcoding data from a large sample set allows us to disentangle the individual effects.

Microbiome Profiling from Fecal Immunochemical Test Reveals Microbial Signatures with Potential for Colorectal Cancer Screening.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer deaths worldwide. Early diagnosis of CRC, which saves lives and enables better outcomes, is generally implemented through a two-step population screening approach based on the use of Fecal Immunochemical Test (FIT) followed by colonoscopy if the test is positive. However, the FIT step has a high false positive rate, and there is a need for new predictive biomarkers to better prioritize cases for colonoscopy. Here we used 16S rRNA metabarcoding from FIT positive samples to uncover microbial taxa, taxon co-occurrence and metabolic features significantly associated with different colonoscopy outcomes, underscoring a predictive potential and revealing changes along the path from healthy tissue to carcinoma. Finally, we used machine learning to develop a two-phase classifier which reduces the current false positive rate while maximizing the inclusion of CRC and clinically relevant samples.

Citizen-science based study of the oral microbiome in Cystic fibrosis and matched controls reveals major differences in diversity and abundance of bacterial and fungal species.

Introduction: Cystic fibrosis (CF) is an autosomal genetic disease, associated with the production of excessively thick mucosa and with life-threatening chronic lung infections. The microbiota of the oral cavity can act as a reservoir or as a barrier for infectious microorganisms that can colonize the lungs. However, the specific composition of the oral microbiome in CF is poorly understood.Methods: In collaboration with CF associations in Spain, we collected oral rinse samples from 31 CF persons (age range 7-47) and matched controls, and then performed 16S rRNA metabarcoding and high-throughput sequencing, combined with culture and proteomics-based identification of fungi to survey the bacterial and fungal oral microbiome.Results: We found that CF is associated with less diverse oral microbiomes, which were characterized by higher prevalence of Candida albicans and differential abundances of a number of bacterial taxa that have implications in both the connection to lung infections in CF, as well as potential oral health concerns, particularly periodontitis and dental caries.Conclusion: Overall, our study provides a first global snapshot of the oral microbiome in CF. Future studies are required to establish the relationships between the composition of the oral and lung microbiomes in CF.

Target Enrichment Enables the Discovery of lncRNAs with Somatic Mutations or Altered Expression in Paraffin-Embedded Colorectal Cancer Samples.

Long non-coding RNAs (lncRNAs) play important roles in cancer and are potential new biomarkers or targets for therapy. However, given the low and tissue-specific expression of lncRNAs, linking these molecules to particular cancer types and processes through transcriptional profiling is challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues are abundant resources for research but are prone to nucleic acid degradation, thereby complicating the study of lncRNAs. Here, we designed and validated a probe-based enrichment strategy to efficiently profile lncRNA expression in FFPE samples, and we applied it for the detection of lncRNAs associated with colorectal cancer (CRC). Our approach efficiently enriched targeted lncRNAs from FFPE samples, while preserving their relative abundance, and enabled the detection of tumor-specific mutations. We identified 379 lncRNAs differentially expressed between CRC tumors and matched healthy tissues and found tumor-specific lncRNA variants. Our results show that numerous lncRNAs are differentially expressed and/or accumulate variants in CRC tumors, thereby suggesting a role in CRC progression. More generally, our approach unlocks the study of lncRNAs in FFPE samples, thus enabling the retrospective use of abundant, well documented material available in hospital biobanks.

Pochonia chlamydosporia Induces Plant-Dependent Systemic Resistance to Meloidogyne incognita.

Meloidogyne spp. are the most damaging plant parasitic nematodes for horticultural crops worldwide. Pochonia chlamydosporia is a fungal egg parasite of root-knot and cyst nematodes able to colonize the roots of several plant species and shown to induce plant defense mechanisms in fungal-plant interaction studies, and local resistance in fungal-nematode-plant interactions. This work demonstrates the differential ability of two out of five P. chlamydosporia isolates, M10.43.21 and M10.55.6, to induce systemic resistance against M. incognita in tomato but not in cucumber in split-root experiments. The M10.43.21 isolate reduced infection (32-43%), reproduction (44-59%), and female fecundity (14.7-27.6%), while the isolate M10.55.6 only reduced consistently nematode reproduction (35-47.5%) in the two experiments carried out. The isolate M10.43.21 induced the expression of the salicylic acid pathway (PR-1 gene) in tomato roots 7 days after being inoculated with the fungal isolate and just after nematode inoculation, and at 7 and 42 days after nematode inoculation too. The jasmonate signaling pathway (Lox D gene) was also upregulated at 7 days after nematode inoculation. Thus, some isolates of P. chlamydosporia can induce systemic resistance against root-knot nematodes but this is plant species dependent.

Microbiome and colorectal cancer: Roles in carcinogenesis and clinical potential.

The gastrointestinal tract harbors most of the microbiota associated with humans. In recent years, there has been a surge of interest in assessing the relationships between the gut microbiota and several gut alterations, including colorectal cancer. Changes in the gut microbiota in patients suffering colorectal cancer suggest a possible role of host-microbe interactions in the origin and development of this malignancy and, at the same time, open the door for novel ways of preventing, diagnosing, or treating this disease. In this review we survey current knowledge on the healthy microbiome of the gut and how it is altered in colorectal cancer and other related disease conditions. In describing past studies we will critically assess technical limitations of different approaches and point to existing challenges in microbiome research. We will have a special focus on host-microbiome interaction mechanisms that may be important to explain how dysbiosis can lead to chronic inflammation and drive processes that influence carcinogenesis and tumor progression in colon cancer. Finally, we will discuss the potential of recent developments of novel microbiota-based therapeutics and diagnostic tools for colorectal cancer.

Long Non-Coding RNAs As Potential Novel Prognostic Biomarkers in Colorectal Cancer.

Colorectal cancer (CRC) is the fourth most common cause of death worldwide. Surgery is usually the first line of treatment for patients with CRC but many tumors with similar histopathological features show significantly different clinical outcomes. The discovery of robust prognostic biomarkers in patients with CRC is imperative to achieve more effective treatment strategies and improve patient's care. Recent progress in next generation sequencing methods and transcriptome analysis has revealed that a much larger part of the genome is transcribed into RNA than previously assumed. Collectively referred to as non-coding RNAs (ncRNAs), some of these RNA molecules such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been shown to be altered and to play critical roles in tumor biology. This discovery leads to exciting possibilities for personalized cancer diagnosis, and therapy. Many lncRNAs are tissue and cancer-type specific and have already revealed to be useful as prognostic markers. In this review, we focus on recent findings concerning aberrant expression of lncRNAs in CRC tumors and emphasize their prognostic potential in CRC. Further studies focused on the mechanisms of action of lncRNAs will contribute to the development of novel biomarkers for diagnosis and disease progression.

Role of the neurotrophin network in eating disorders' subphenotypes: body mass index and age at onset of the disease.

Eating disorders (ED) are severe psychiatric diseases that most likely result from, and are sustained by socio-cultural, psychological and biological factors. We explored whether members of the neurotrophin family are disease-modifying factors of quantitative traits, potentially contributing to the outcome or prognosis of the disease. We studied lifetime minimum and maximum body mass index (minBMI and maxBMI) and age at onset of the disease in a sample of 991 ED patients from France, Germany, Italy and Spain and analysed 183 genetic variants located in 10 candidate genes encoding different neurotrophins and their receptors. We used a hierarchical model approach to include prior genetic knowledge of the specific and found that variants in CNTF, in its receptor CNTFR, and in NTRK2 were significantly associated with a lower age at onset of the ED. In addition, one variant in NTRK1 was associated with a higher minBMI. The results suggest that for these two subphenotypes, CNTF, CNTFR, NTRK1 and NTRK2 might act as disease-modifying factors and add preliminary evidence to the global hypothesis that EDs are the result of complex interactions and reciprocal controls between the immune, endocrine and central nervous systems.

Association of NTRK3 and its interaction with NGF suggest an altered cross-regulation of the neurotrophin signaling pathway in eating disorders.

Eating disorders (EDs) are complex psychiatric diseases that include anorexia nervosa and bulimia nervosa, and have higher than 50% heritability. Previous studies have found association of BDNF and NTRK2 to ED, while animal models suggest that other neurotrophin genes might also be involved in eating behavior. We have performed a family-based association study with 151 TagSNPs covering 10 neurotrophin signaling genes: NGFB, BDNF, NTRK1, NGFR/p75, NTF4/5, NTRK2, NTF3, NTRK3, CNTF and CNTFR in 371 ED trios of Spanish, French and German origin. Besides several nominal associations, we found a strong significant association after correcting for multiple testing (P = 1.04 x 10(-4)) between ED and rs7180942, located in the NTRK3 gene, which followed an overdominant model of inheritance. Interestingly, HapMap unrelated individuals carrying the rs7180942 risk genotypes for ED showed higher levels of expression of NTRK3 in lymphoblastoid cell lines. Furthermore, higher expression of the orthologous murine Ntrk3 gene was also detected in the hypothalamus of the anx/anx mouse model of anorexia. Finally, variants in NGFB gene appear to modify the risk conferred by the NTRK3 rs7180942 risk genotypes (P = 4.0 x 10(-5)) showing a synergistic epistatic interaction. The reported data, in addition to the previous reported findings for BDNF and NTRK2, point neurotrophin signaling genes as key regulators of eating behavior and their altered cross-regulation as susceptibility factors for EDs.

Characterisation of novel splicing variants of the tyrosine hydroxylase C-terminal domain in human neuroblastic tumours.

Alternative splicing of human tyrosine hydroxylase (hTH) transcripts appears to occur mainly in the N-terminal domain, giving rise to at least eight different isoforms. We recently reported the existence of hTH transcript variants resulting from splicing of exons 8 and 9, within a region previously thought to be constant. The mRNA distribution of these novel hTH isoforms in neuroblastic tumours and in foetal adrenal glands was analysed by conventional and real-time RT-PCR. The presence of the target protein was determined by Western blotting, immunoprecipitation and protein analysis. Transcripts lacking exons 8 and 9 were widely distributed in the tissues analysed. Characterisation of full-length mRNA revealed that splicing of exons 8 and 9 was always associated with splicing of exons 2 (hTH-Delta2,8,9) or 1b and 2 (hTH-Delta1b,2,8,9). In addition, one variant detected on Western blots in several tumours fits the predicted size (58 kDa) of the isoforms lacking exons 8 and 9. In conclusion, the two novel isoforms reported here (hTH-Delta2,8,9 and hTH-Delta1b,2,8,9) represent the first full-length isoforms with alternative splicing in the hTH C-terminal domain. These results demonstrate for the first time the existence of hTH isoforms Delta2,8,9 and Delta1b,2,8,9. Their general distribution in neuroblastoma and adrenal glands and translation into protein suggest a significant functional role for these novel hTH isoforms, which merit further study.