Detection of ribonucleotides embedded in DNA by Nanopore sequencing.

Ribonucleotides represent the most common non-canonical nucleotides found in eukaryotic genomes. The sources of chromosome-embedded ribonucleotides and the mechanisms by which unrepaired rNMPs trigger genome instability and human pathologies are not fully understood. The available sequencing technologies only allow to indirectly deduce the genomic location of rNMPs. Oxford Nanopore Technologies (ONT) may overcome such limitation, revealing the sites of rNMPs incorporation in genomic DNA directly from raw sequencing signals. We synthesized two types of DNA molecules containing rNMPs at known or random positions and we developed data analysis pipelines for DNA-embedded ribonucleotides detection by ONT. We report that ONT can identify all four ribonucleotides incorporated in DNA by capturing rNMPs-specific alterations in nucleotide alignment features, current intensity, and dwell time. We propose that ONT may be successfully employed to directly map rNMPs in genomic DNA and we suggest a strategy to build an ad hoc basecaller to analyse native genomes.

Macroalgal microbiomes unveil a valuable genetic resource for halogen metabolism.

BACKGROUND: Macroalgae, especially reds (Rhodophyta Division) and browns (Phaeophyta Division), are known for producing various halogenated compounds. Yet, the reasons underlying their production and the fate of these metabolites remain largely unknown. Some theories suggest their potential antimicrobial activity and involvement in interactions between macroalgae and prokaryotes. However, detailed investigations are currently missing on how the genetic information of prokaryotic communities associated with macroalgae may influence the fate of organohalogenated molecules. RESULTS: To address this challenge, we created a specialized dataset containing 161 enzymes, each with a complete enzyme commission number, known to be involved in halogen metabolism. This dataset served as a reference to annotate the corresponding genes encoded in both the metagenomic contigs and 98 metagenome-assembled genomes (MAGs) obtained from the microbiome of 2 red (Sphaerococcus coronopifolius and Asparagopsis taxiformis) and 1 brown (Halopteris scoparia) macroalgae. We detected many dehalogenation-related genes, particularly those with hydrolytic functions, suggesting their potential involvement in the degradation of a wide spectrum of halocarbons and haloaromatic molecules, including anthropogenic compounds. We uncovered an array of degradative gene functions within MAGs, spanning various bacterial orders such as Rhodobacterales, Rhizobiales, Caulobacterales, Geminicoccales, Sphingomonadales, Granulosicoccales, Microtrichales, and Pseudomonadales. Less abundant than degradative functions, we also uncovered genes associated with the biosynthesis of halogenated antimicrobial compounds and metabolites. CONCLUSION: The functional data provided here contribute to understanding the still largely unexplored role of unknown prokaryotes. These findings support the hypothesis that macroalgae function as holobionts, where the metabolism of halogenated compounds might play a role in symbiogenesis and act as a possible defense mechanism against environmental chemical stressors. Furthermore, bacterial groups, previously never connected with organohalogen metabolism, e.g., Caulobacterales, Geminicoccales, Granulosicoccales, and Microtrichales, functionally characterized through MAGs reconstruction, revealed a biotechnologically relevant gene content, useful in synthetic biology, and bioprospecting applications. Video Abstract.

A comprehensive overview of microbiome data in the light of machine learning applications: categorization, accessibility, and future directions.

Metagenomics, Metabolomics, and Metaproteomics have significantly advanced our knowledge of microbial communities by providing culture-independent insights into their composition and functional potential. However, a critical challenge in this field is the lack of standard and comprehensive metadata associated with raw data, hindering the ability to perform robust data stratifications and consider confounding factors. In this comprehensive review, we categorize publicly available microbiome data into five types: shotgun sequencing, amplicon sequencing, metatranscriptomic, metabolomic, and metaproteomic data. We explore the importance of metadata for data reuse and address the challenges in collecting standardized metadata. We also, assess the limitations in metadata collection of existing public repositories collecting metagenomic data. This review emphasizes the vital role of metadata in interpreting and comparing datasets and highlights the need for standardized metadata protocols to fully leverage metagenomic data's potential. Furthermore, we explore future directions of implementation of Machine Learning (ML) in metadata retrieval, offering promising avenues for a deeper understanding of microbial communities and their ecological roles. Leveraging these tools will enhance our insights into microbial functional capabilities and ecological dynamics in diverse ecosystems. Finally, we emphasize the crucial metadata role in ML models development.

Unraveling C-to-U RNA editing events from direct RNA sequencing.

In mammals, RNA editing events involve the conversion of adenosine (A) in inosine (I) by ADAR enzymes or the hydrolytic deamination of cytosine (C) in uracil (U) by the APOBEC family of enzymes, mostly APOBEC1. RNA editing has a plethora of biological functions, and its deregulation has been associated with various human disorders. While the large-scale detection of A-to-I is quite straightforward using the Illumina RNAseq technology, the identification of C-to-U events is a non-trivial task. This difficulty arises from the rarity of such events in eukaryotic genomes and the challenge of distinguishing them from background noise. Direct RNA sequencing by Oxford Nanopore Technology (ONT) permits the direct detection of Us on sequenced RNA reads. Surprisingly, using ONT reads from wild-type (WT) and APOBEC1-knock-out (KO) murine cell lines as well as in vitro synthesized RNA without any modification, we identified a systematic error affecting the accuracy of the Cs call, thereby leading to incorrect identifications of C-to-U events. To overcome this issue in direct RNA reads, here we introduce a novel machine learning strategy based on the isolation Forest (iForest) algorithm in which C-to-U editing events are considered as sequencing anomalies. Using in vitro synthesized and human ONT reads, our model optimizes the signal-to-noise ratio improving the detection of C-to-U editing sites with high accuracy, over 90% in all samples tested. Our results suggest that iForest, known for its rapid implementation and minimal memory requirements, is a promising tool to denoise ONT reads and reliably identify RNA modifications.

Cyclic AMP induces reversible EPAC1 condensates that regulate histone transcription.

The second messenger cyclic AMP regulates many nuclear processes including transcription, pre-mRNA splicing and mitosis. While most functions are attributed to protein kinase A, accumulating evidence suggests that not all nuclear cyclic AMP-dependent effects are mediated by this kinase, implying that other effectors may be involved. Here we explore the nuclear roles of Exchange Protein Activated by cyclic AMP 1. We find that it enters the nucleus where forms reversible biomolecular condensates in response to cyclic AMP. This phenomenon depends on intrinsically disordered regions present at its amino-terminus and is independent of protein kinase A. Finally, we demonstrate that nuclear Exchange Protein Activated by cyclic AMP 1 condensates assemble at genomic loci on chromosome 6 in the proximity of Histone Locus Bodies and promote the transcription of a histone gene cluster. Collectively, our data reveal an unexpected mechanism through which cyclic AMP contributes to nuclear spatial compartmentalization and promotes the transcription of specific genes.

Different states of stemness of glioblastoma stem cells sustain glioblastoma subtypes indicating novel clinical biomarkers and high-efficacy customized therapies.

BACKGROUND: Glioblastoma (GBM) is the most malignant among gliomas with an inevitable lethal outcome. The elucidation of the physiology and regulation of this tumor is mandatory to unravel novel target and effective therapeutics. Emerging concepts show that the minor subset of glioblastoma stem cells (GSCs) accounts for tumorigenicity, representing the true target for innovative therapies in GBM. METHODS: Here, we isolated and established functionally stable and steadily expanding GSCs lines from a large cohort of GBM patients. The molecular, functional and antigenic landscape of GBM tissues and their derivative GSCs was highlited in a side-by-side comprehensive genomic and transcriptomic characterization by ANOVA and Fisher's exact tests. GSCs' physio-pathological hallmarks were delineated by comparing over time in vitro and in vivo their expansion, self-renewal and tumorigenic ability with hierarchical linear models for repeated measurements and Kaplan-Meier method. Candidate biomarkers performance in discriminating GBM patients' classification emerged by classification tree and patients' survival analysis. RESULTS: Here, distinct biomarker signatures together with aberrant functional programs were shown to stratify GBM patients as well as their sibling GSCs population into TCGA clusters. Of importance, GSCs cells were demonstrated to fully resemble over time the molecular features of their patient of origin. Furthermore, we pointed out the existence of distinct GSCs subsets within GBM classification, inherently endowed with different self-renewal and tumorigenic potential. Particularly, classical GSCs were identified by more undifferentiated biological hallmarks, enhanced expansion and clonal capacity as compared to the more mature, relatively slow-propagating mesenchymal and proneural cells, likely endowed with a higher potential for infiltration either ex vivo or in vivo. Importantly, the combination of DCX and EGFR markers, selectively enriched among GSCs pools, almost exactly predicted GBM patients' clusters together with their survival and drug response. CONCLUSIONS: In this study we report that an inherent enrichment of distinct GSCs pools underpin the functional inter-cluster variances displayed by GBM patients. We uncover two selectively represented novel functional biomarkers capable of discriminating GBM patients' stratification, survival and drug response, setting the stage for the determination of patient-tailored diagnostic and prognostic strategies and, mostly, for the design of appropriate, patient-selective treatment protocols.

Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors.

Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. In this work, we have analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. We highlighted the conservation of a short sequence in the genes encoding subunits of the V-ATPase complex and their assembly factors analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding our knowledge on the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization.

Amplicon-Based Microbiome Profiling: From Second- to Third-Generation Sequencing for Higher Taxonomic Resolution.

The 16S rRNA amplicon-based sequencing approach represents the most common and cost-effective strategy with great potential for microbiome profiling. The use of second-generation sequencing (NGS) technologies has led to protocols based on the amplification of one or a few hypervariable regions, impacting the outcome of the analysis. Nowadays, comparative studies are necessary to assess different amplicon-based approaches, including the full-locus sequencing currently feasible thanks to third-generation sequencing (TGS) technologies. This study compared three different methods to achieve the deepest microbiome taxonomic characterization: (a) the single-region approach, (b) the multiplex approach, covering several regions of the target gene/region, both based on NGS short reads, and (c) the full-length approach, which analyzes the whole length of the target gene thanks to TGS long reads. Analyses carried out on benchmark microbiome samples, with a known taxonomic composition, highlighted a different classification performance, strongly associated with the type of hypervariable regions and the coverage of the target gene. Indeed, the full-length approach showed the greatest discriminating power, up to species level, also on complex real samples. This study supports the transition from NGS to TGS for the study of the microbiome, even if experimental and bioinformatic improvements are still necessary.

Dysregulation of testis mRNA expression levels in hatchery-produced vs wild greater amberjack Seriola dumerili.

Reproductive dysfunctions have been recently documented in male greater amberjack Seriola dumerili caught from the wild and reared in captivity. In the present study, we compared testis transcriptome in wild fish (WILD), hatchery-produced fish with apparently normal spermatogenesis (Normal Farmed; NormalF) and hatchery-produced fish with evident reproductive dysfunction (Dysfunctional Farmed; DysF). Gene expression analysis identified 2157, 1985 and 74 differentially expressed genes (DEGs) in DysF vs WILD, NormalF vs DysF and NormalF vs WILD comparisons, respectively. In DysF, a dysregulation of several interconnected biological processes, including cell assembly, steroidogenesis and apoptosis was found. Gene enrichment of progesterone-mediated oocyte maturation, oocyte meiosis and cell cycle pathways were identified in the DysF vs NormalF comparison. Most of the DEGs involved in the enriched pathways were downregulated in DysF. The comparison of NormalF vs WILD showed that most of the DEGs were downregulated in NormalF, including a gene that encodes for a regulatory protein with a protective role in apoptosis regulation (ptpn6), indicating that spermatogenesis was dysfunctional also in the apparently "normal" hatchery-produced fish. Hence, rearing of male greater amberjack in captivity, from eggs produced by captive breeders, did not prevent the appearance of reproductive dysfunctions, and these dysfunctions involved several biological processes and metabolic pathways.

Whole-Exome and Transcriptome Sequencing Expands the Genotype of Majewski Osteodysplastic Primordial Dwarfism Type II.

Microcephalic Osteodysplastic Primordial Dwarfism type II (MOPDII) represents the most common form of primordial dwarfism. MOPD clinical features include severe prenatal and postnatal growth retardation, postnatal severe microcephaly, hypotonia, and an increased risk for cerebrovascular disease and insulin resistance. Autosomal recessive biallelic loss-of-function genomic variants in the centrosomal pericentrin (PCNT) gene on chromosome 21q22 cause MOPDII. Over the past decade, exome sequencing (ES) and massive RNA sequencing have been effectively employed for both the discovery of novel disease genes and to expand the genotypes of well-known diseases. In this paper we report the results both the RNA sequencing and ES of three patients affected by MOPDII with the aim of exploring whether differentially expressed genes and previously uncharacterized gene variants, in addition to PCNT pathogenic variants, could be associated with the complex phenotype of this disease. We discovered a downregulation of key factors involved in growth, such as IGF1R, IGF2R, and RAF1, in all three investigated patients. Moreover, ES identified a shortlist of genes associated with deleterious, rare variants in MOPDII patients. Our results suggest that Next Generation Sequencing (NGS) technologies can be successfully applied for the molecular characterization of the complex genotypic background of MOPDII.

Chitinolytic Enzymes of the Hyperparasite Fungus Aphanocladium album: Genome-Wide Survey and Characterization of A Selected Enzyme.

The filamentous fungus Aphanocladium album is known as a hyperparasite of plant pathogenic fungi; hence, it has been studied as a possible agent for plant protection. Chitinases secreted by A. album have proven to be essential for its fungicidal activity. However, no complete analysis of the A. album chitinase assortment has been carried out, nor have any of its chitinases been characterized yet. In this study, we report the first draft assembly of the genome sequence of A. album (strain MX-95). The in silico functional annotation of the genome allowed the identification of 46 genes encoding chitinolytic enzymes of the GH18 (26 genes), GH20 (8 genes), GH75 (8 genes), and GH3 (4 genes) families. The encoded proteins were investigated by comparative and phylogenetic analysis, allowing clustering in different subgroups. A. album chitinases were also characterized according to the presence of different functional protein domains (carbohydrate-binding modules and catalytic domains) providing the first complete description of the chitinase repertoire of A. album. A single chitinase gene was then selected for complete functional characterization. The encoded protein was expressed in the yeast Pichia pastoris, and its activity was assayed under different conditions of temperature and pH and with different substrates. It was found that the enzyme acts mainly as a chitobiosidase, with higher activity in the 37-50 °C range.

Lactobacillus paracasei CNCM I-5220-derived postbiotic protects from the leaky-gut.

The maintenance of intestinal barrier function is essential for preventing different pathologies, such as the leaky gut syndrome (LGS), which is characterized by the passage of harmful agents, like bacteria, toxins, and viruses, into the bloodstream. Intestinal barrier integrity is controlled by several players, including the gut microbiota. Various molecules, called postbiotics, are released during the natural metabolic activity of the microbiota. Postbiotics can regulate host-microbe interactions, epithelial homeostasis, and have overall benefits for our health. In this work, we used in vitro and in vivo systems to demonstrate the role of Lactobacillus paracasei CNCM I-5220-derived postbiotic (LP-PBF) in preserving intestinal barrier integrity. We demonstrated in vitro that LP-PBF restored the morphology of tight junctions (TJs) that were altered upon Salmonella typhimurium exposure. In vivo, LP-PBF protected the gut vascular barrier and blocked S. typhimurium dissemination into the bloodstream. Interestingly, we found that LP-PBF interacts not only with the host cells, but also directly with S. typhimurium blocking its biofilm formation, partially due to the presence of biosurfactants. This study highlights that LP-PBF is beneficial in maintaining gut homeostasis due to the synergistic effect of its different components. These results suggest that LP-PBF could be utilized in managing several pathologies displaying an impaired intestinal barrier function.

HaploCoV: unsupervised classification and rapid detection of novel emerging variants of SARS-CoV-2.

Accurate and timely monitoring of the evolution of SARS-CoV-2 is crucial for identifying and tracking potentially more transmissible/virulent viral variants, and implement mitigation strategies to limit their spread. Here we introduce HaploCoV, a novel software framework that enables the exploration of SARS-CoV-2 genomic diversity through space and time, to identify novel emerging viral variants and prioritize variants of potential epidemiological interest in a rapid and unsupervised manner. HaploCoV can integrate with any classification/nomenclature and incorporates an effective scoring system for the prioritization of SARS-CoV-2 variants. By performing retrospective analyses of more than 11.5 M genome sequences we show that HaploCoV demonstrates high levels of accuracy and reproducibility and identifies the large majority of epidemiologically relevant viral variants - as flagged by international health authorities - automatically and with rapid turn-around times.Our results highlight the importance of the application of strategies based on the systematic analysis and integration of regional data for rapid identification of novel, emerging variants of SARS-CoV-2. We believe that the approach outlined in this study will contribute to relevant advances to current and future genomic surveillance methods.

Cumulus Cell Transcriptome after Cumulus-Oocyte Complex Exposure to Nanomolar Cadmium in an In Vitro Animal Model of Prepubertal and Adult Age.

Cadmium (Cd), a highly toxic pollutant, impairs oocyte fertilization, through oxidative damage on cumulus cells (CCs). This study analysed the transcriptomic profile of CCs of cumulus-oocyte complexes (COCs) from adult and prepubertal sheep, exposed to Cd nanomolar concentration during in vitro maturation. In both age-groups, CCs of matured oocytes underwent RNA-seq, data analysis and validation. Differentially expressed genes (DEGs) were identified in adult (n = 99 DEGs) and prepubertal (n = 18 DEGs) CCs upon Cd exposure. Transcriptomes of adult CCs clustered separately between Cd-exposed and control samples, whereas prepubertal ones did not as observed by Principal Component Analysis. The transcriptomic signature of Cd-induced CC toxicity was identified by gene annotation and literature search. Genes associated with previous studies on ovarian functions and/or Cd effects were confirmed and new genes were identified, thus implementing the knowledge on their involvement in such processes. Enrichment and validation analysis showed that, in adult CCs, Cd acted as endocrine disruptor on DEGs involved in hormone biosynthesis, cumulus expansion, regulation of cell signalling, growth and differentiation and oocyte maturation, whereas in prepubertal CCs, Cd affected DEGs involved in CC development and viability and CC-oocyte communications. In conclusion, these DEGs could be used as valuable non-invasive biomarkers for oocyte competence.

UTRdb 2.0: a comprehensive, expert curated catalog of eukaryotic mRNAs untranslated regions.

The 5' and 3' untranslated regions of eukaryotic mRNAs (UTRs) play crucial roles in the post-transcriptional regulation of gene expression through the modulation of nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization, and message stability. Since 1996, we have developed and maintained UTRdb, a specialized database of UTR sequences. Here we present UTRdb 2.0, a major update of UTRdb featuring an extensive collection of eukaryotic 5' and 3' UTR sequences, including over 26 million entries from over 6 million genes and 573 species, enriched with a curated set of functional annotations. Annotations include CAGE tags and polyA signals to label the completeness of 5' and 3'UTRs, respectively. In addition, uORFs and IRES are annotated in 5'UTRs as well as experimentally validated miRNA targets in 3'UTRs. Further annotations include evolutionarily conserved blocks, Rfam motifs, ADAR-mediated RNA editing events, and m6A modifications. A web interface allowing a flexible selection and retrieval of specific subsets of UTRs, selected according to a combination of criteria, has been implemented which also provides comprehensive download facilities. UTRdb 2.0 is accessible at http://utrdb.cloud.ba.infn.it/utrdb/.

Profiling RNA Editing in Single Cells.

RNA editing is a widespread molecular phenomenon occurring in a variety of organisms. In humans, it mainly involves the deamination of adenosine to inosine (A-to-I) in double-stranded RNAs by ADAR enzymes. A-to-I RNA editing has been investigated in different tissues as well as in diverse experimental and pathological conditions. By contrast, its biological role in single cells has not been explored in depth. Recent methodologies for cell sorting in combination with deep sequencing technologies have enabled the study of eukaryotic transcriptomes at single cell resolution, paving the way to the profiling of their epitranscriptomic dynamics.Here we describe a step-by-step protocol to detect and characterize A-to-I events occurring in publicly available single-cell RNAseq experiments from human alpha and beta pancreatic cells.

The Underestimated Role of the p53 Pathway in Renal Cancer.

The TP53 tumor suppressor gene is known as the guardian of the genome, playing a pivotal role in controlling genome integrity, and its functions are lost in more than 50% of human tumors due to somatic mutations. This percentage rises to 90% if mutations and alterations in the genes that code for regulators of p53 stability and activity are taken into account. Renal cell carcinoma (RCC) is a clear example of cancer that despite having a wild-type p53 shows poor prognosis because of the high rate of resistance to radiotherapy or chemotherapy, which leads to recurrence, metastasis and death. Remarkably, the fact that p53 is poorly mutated does not mean that it is functionally active, and increasing experimental evidences have demonstrated this. Therefore, RCC represents an extraordinary example of the importance of p53 pathway alterations in therapy resistance. The search for novel molecular biomarkers involved in the pathways that regulate altered p53 in RCC is mandatory for improving early diagnosis, evaluating the prognosis and developing novel potential therapeutic targets for better RCC treatment.

Extension of the shelf-life of fresh pasta using modified atmosphere packaging and bioprotective cultures.

Microbial stability of fresh pasta depends on heat treatment, storage temperature, proper preservatives, and atmosphere packaging. This study aimed at improving the microbial quality, safety, and shelf life of fresh pasta using modified atmosphere composition and packaging with or without the addition of bioprotective cultures (Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium spp., and Bacillus coagulans) into semolina. Three fresh pasta variants were made using (i) the traditional protocol (control), MAP (20:80 CO2:N2), and barrier packaging, (ii) the experimental MAP (40:60 CO2:N2) and barrier packaging, and (iii) the experimental MAP, barrier packaging, and bioprotective cultures. Their effects on physicochemical properties (i.e., content on macro elements, water activity, headspace O2, CO2 concentrations, and mycotoxins), microbiological patterns, protein, and volatile organic compounds (VOC) were investigated at the beginning and the end of the actual or extended shelf-life through traditional and multi-omics approaches. We showed that the gas composition and properties of the packaging material tested in the experimental MAP system, with or without bioprotective cultures, positively affect features of fresh pasta avoiding changes in their main chemical properties, allowing for a storage longer than 120 days under refrigerated conditions. These results support that, although bioprotective cultures were not all able to grow in tested conditions, they can control the spoilage and the associated food-borne microbiota in fresh pasta during storage by their antimicrobials and/or fermentation products synergically. The VOC profiling, based on gas-chromatography mass-spectrometry (GC-MS), highlighted significant differences affected by the different manufacturing and packaging of samples. Therefore, the use of the proposed MAP system and the addition of bioprotective cultures can be considered an industrial helpful strategy to reduce the quality loss during refrigerated storage and to increase the shelf life of fresh pasta for additional 30 days by allowing the economic and environmental benefits spurring innovation in existing production models.

Upregulation of YKL-40 Promotes Metastatic Phenotype and Correlates with Poor Prognosis and Therapy Response in Patients with Colorectal Cancer.

YKL-40 is a heparin- and chitin-binding glycoprotein that belongs to the family of glycosyl hydrolases but lacks enzymatic properties. It affects different (patho)physiological processes, including cancer. In different tumors, YKL-40 gene overexpression has been linked to higher cell proliferation, angiogenesis, and vasculogenic mimicry, migration, and invasion. Because, in colorectal cancer (CRC), the serological YKL-40 level may serve as a risk predictor and prognostic biomarker, we investigated the underlying mechanisms by which it may contribute to tumor progression and the clinical significance of its tissue expression in metastatic CRC. We demonstrated that high-YKL-40-expressing HCT116 and Caco2 cells showed increased motility, invasion, and proliferation. YKL-40 upregulation was associated with EMT signaling activation. In the AOM/DSS mouse model, as well as in tumors and sera from CRC patients, elevated YKL-40 levels correlated with high-grade tumors. In retrospective analyses of six independent cohorts of CRC patients, elevated YKL-40 expression correlated with shorter survival in patients with advanced CRC. Strikingly, high YKL-40 tissue levels showed a predictive value for a better response to cetuximab, even in patients with stage IV CRC and mutant KRAS, and worse sensitivity to oxaliplatin. Taken together, our findings establish that tissue YKL-40 overexpression enhances CRC metastatic potential, highlighting this gene as a novel prognostic candidate, a predictive biomarker for therapy response, and an attractive target for future therapy in CRC.

Natural and after colon washing fecal samples: the two sides of the coin for investigating the human gut microbiome.

To date several studies address the important role of gut microbiome and its interplay with the human host in the health and disease status. However, the selection of a universal sampling matrix representative of the microbial biodiversity associated with the gastrointestinal (GI) tract, is still challenging. Here we present a study in which, through a deep metabarcoding analysis of the 16S rRNA gene, we compared two sampling matrices, feces (F) and colon washing feces (CWF), in order to evaluate their relative effectiveness and accuracy in representing the complexity of the human gut microbiome. A cohort of 30 volunteers was recruited and paired F and CWF samples were collected from each subject. Alpha diversity analysis confirmed a slightly higher biodiversity of CWF compared to F matched samples. Likewise, beta diversity analysis proved that paired F and CWF microbiomes were quite similar in the same individual, but remarkable inter-individual variability occurred among the microbiomes of all participants. Taxonomic analysis in matched samples was carried out to investigate the intra and inter individual/s variability. Firmicutes, Bacteroidota, Proteobacteria and Actinobacteriota were the main phyla in both F and CWF samples. At genus level, Bacteirodetes was the most abundant in F and CWF samples, followed by Faecalibacterium, Blautia and Escherichia-Shigella. Our study highlights an inter-individual variability greater than intra-individual variability for paired F and CWF samples. Indeed, an overall higher similarity was observed across matched F and CWF samples, suggesting, as expected, a remarkable overlap between the microbiomes inferred using the matched F and CWF samples. Notably, absolute quantification of total 16S rDNA by droplet digital PCR (ddPCR) revealed comparable overall microbial load between paired F and CWF samples. We report here the first comparative study on fecal and colon washing fecal samples for investigating the human gut microbiome and show that both types of samples may be used equally for the study of the gut microbiome. The presented results suggest that the combined use of both types of sampling matrices could represent a suitable choice to obtain a more complete overview of the human gut microbiota for addressing different biological and clinical questions.