Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%; P < 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P < 0.05) and improved the sensitivity of pathogen detection (P < 0.01), with a 20-to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples. Clinical microbiology is one of the most rapidly changing areas of laboratory medicine today due to the introduction of new technologies and automation (1). Molecular testing, such as PCR, is becoming the de facto gold standard for the detection of pathogens that are difficult to culture by offering high sensitivity and specificity and a rapid turnaround time (2). Syndrome-based multiplex molecular assays can detect up to 30 of the most common pathogens associated with respiratory infections, gastroenteritis, and central nervous system (CNS) infections (3-6). However, the complete list of infectious agents associated with these infections greatly exceeds the capabilities of even the best multiplex assays. These less common organisms, for which tests are not readily available, are likely responsible for many cases of undiagnosed illness, particularly in critically ill patients and those with compromised immunity (7,8). Therefore, there is increasing interest in the application of novel technologies, such as next-generation sequencing (NGS), for unbiased detection of pathogens in clinical samples.Among the various challenges with the implementation of NGS for routine pathogen detection using metagenomics, the presence of an overwhelming amount of host DNA is one of the most important problems to be addressed. A previous metagenomic study on nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections revealed that up to ϳ95% of raw NGS reads were of human DNA (9). The su...

Vitamin D deficiency affects approximately 80% of individuals in some countries and has been linked with gut dysbiosis and inflammation. While the benefits of vitamin D supplementation on the gut microbiota have been studied in patients with chronic diseases, its effects on the microbiota of otherwise healthy individuals is unclear. Moreover, whether effects on the microbiota can explain some of the marked inter-individual variation in responsiveness to vitamin D supplementation is unknown. Here, we administered vitamin D to 80 otherwise healthy vitamin D-deficient women, measuring serum 25(OH) D levels in blood and characterizing their gut microbiota pre- and post- supplementation using 16S rRNA gene sequencing. Vitamin D supplementation significantly increased gut microbial diversity. Specifically, the Bacteroidetes to Firmicutes ratio increased, along with the abundance of the health-promoting probiotic taxa Akkermansia and Bifidobacterium. Significant variations in the two-dominant genera, Bacteroides and Prevotella, indicated a variation in enterotypes following supplementation. Comparing supplementation responders and non-responders we found more pronounced changes in abundance of major phyla in responders, and a significant decrease in Bacteroides acidifaciens in non-responders. Altogether, our study highlights the positive impact of vitamin D supplementation on the gut microbiota and the potential for the microbial gut signature to affect vitamin D response.

Plant genomes contain genetically encoded isoforms of most nucleotide sugar interconversion enzymes. Here we show that Arabidopsis thaliana has five genes encoding functional UDP-Dglucose/UDP-D-galactose 4-epimerase (named UGE1 to UGE5). All A. thaliana UDP-D-glucose 4-epimerase isoforms are dimeric in solution, maximally active in vitro at 30 -40°C, and show good activity between pH 7 and pH 9. In vitro, UGE1, -3, and -5 act independently of externally added NAD ؉ , whereas cofactor addition stimulates the activity of UGE2 and is particularly important for UGE4 activity. UGE1 and UGE3 are most efficiently inhibited by UDP. The five isoforms display k cat UDP-Gal values between 23 and 128 s ؊1 and K m UDP-Gal values between 0.1 and 0.3 mM. This results in enzymatic efficiencies ranging between 97 and 890 mM ؊1 s ؊1 for UGE4 ‫؍‬ UGE1 < UGE3 < UGE5 < UGE2.The K m UDP-Glc values, derived from the Haldane relationship, were 0.76 mM for UGE1, 0.56 mM for UGE4, and between 0.13 and 0.23 mM for UGE2, -3, and -5. The expression of UGE isoforms is ubiquitous and displays developmental and cell type-dependent variations. UGE1 and -3 expression patterns globally resemble enzymes involved in carbohydrate catabolism, and UGE2, -4, and -5 expression is more related to carbohydrate biosynthesis. UGE1, -2, and -4 are present in the cytoplasm, whereasUGE4 is additionally enriched close to Golgi stacks. All UGE genes tested complement the UGE4 rhd1 phenotype, confer increased galactose tolerance in planta, and complement the galactose metabolization deficiency in the Saccharomyces cerevisiae gal10 mutant. We suggest that plant UGE isoforms function in different metabolic situations and that enzymatic properties, gene expression pattern, and subcellular localization contribute to the differentiation of isoform function.The biosynthesis of plant carbohydrates requires specific glycosyltransferases that act on activated sugars, typically uridine diphosphate, adenosine diphosphate, and guanosine diphosphate hexoses and pentoses. In addition to acting as biosynthetic substrates, nucleotide sugars are modified at their glycosyl moieties by nucleotide sugar interconversion enzymes to generate different sugars and are intermediates in the uptake of the free sugars released from the breakdown of nutritional or storage carbohydrates and other sources. The biochemistry and reaction mechanism of many nucleotide sugar interconversion pathways have been reviewed previously (1). cDNAs coding for nucleotide sugar interconverting enzymes have been cloned and recombinant proteins purified, leading the way to x-ray crystallography (2-5). The complete sequencing of entire genomes revealed a surprising over-representation of genes encoding putative isoforms of nucleotide sugar interconversion enzymes in plant genomes (6, 7), but the functional significance of this apparent genetic redundancy remains to be established.One of the best characterized nucleotide sugar interconversion enzymes is UDP-glucose 4-epimerase (EC 5.1.3.2; UGE), 4 which interconve...

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The lack of multi-omics cancer datasets with extensive follow-up information hinders the identification of accurate biomarkers of clinical outcome. In this cohort study, we performed comprehensive genomic analyses on fresh-frozen samples from 348 patients affected by primary colon cancer, encompassing RNA, whole-exome, deep T cell receptor and 16S bacterial rRNA gene sequencing on tumor and matched healthy colon tissue, complemented with tumor whole-genome sequencing for further microbiome characterization. A type 1 helper T cell, cytotoxic, gene expression signature, called Immunologic Constant of Rejection, captured the presence of clonally expanded, tumor-enriched T cell clones and outperformed conventional prognostic molecular biomarkers, such as the consensus molecular subtype and the microsatellite instability classifications. Quantification of genetic immunoediting, defined as a lower number of neoantigens than expected, further refined its prognostic value. We identified a microbiome signature, driven by Ruminococcus bromii, associated with a favorable outcome. By combining microbiome signature and Immunologic Constant of Rejection, we developed and validated a composite score (mICRoScore), which identifies a group of patients with excellent survival probability. The publicly available multi-omics dataset provides a resource for better understanding colon cancer biology that could facilitate the discovery of personalized therapeutic approaches.