The oral microbiome–organisms residing in the oral cavity and their collective genome–are critical components of health and disease. The fungal component of the oral microbiota has not been characterized. In this study, we used a novel multitag pyrosequencing approach to characterize fungi present in the oral cavity of 20 healthy individuals, using the pan-fungal internal transcribed spacer (ITS) primers. Our results revealed the “basal” oral mycobiome profile of the enrolled individuals, and showed that across all the samples studied, the oral cavity contained 74 culturable and 11 non-culturable fungal genera. Among these genera, 39 were present in only one person, 16 genera were present in two participants, and 5 genera were present in three people, while 15 genera (including non-culturable organisms) were present in ≥4 (20%) participants. Candida species were the most frequent (isolated from 75% of participants), followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). Four of these predominant genera are known to be pathogenic in humans. The low-abundance genera may represent environmental fungi present in the oral cavity and could simply be spores inhaled from the air or material ingested with food. Among the culturable genera, 61 were represented by one species each, while 13 genera comprised between 2 and 6 different species; the total number of species identified were 101. The number of species in the oral cavity of each individual ranged between 9 and 23. Principal component (PCO) analysis of the obtained data set followed by sample clustering and UniFrac analysis revealed that White males and Asian males clustered differently from each other, whereas both Asian and White females clustered together. This is the first study that identified the “basal mycobiome” of healthy individuals, and provides the basis for a detailed characterization of the oral mycobiome in health and disease.

Several studies indicate the importance of colonic microbiota in metabolic and inflammatory disorders and importance of diet on microbiota composition. The effects of alcohol, one of the prominent components of diet, on colonic bacterial composition is largely unknown. Mounting evidence suggests that gut-derived bacterial endotoxins are cofactors for alcohol-induced tissue injury and organ failure like alcoholic liver disease (ALD) that only occur in a subset of alcoholics. We hypothesized that chronic alcohol consumption results in alterations of the gut microbiome in a subgroup of alcoholics, and this may be responsible for the observed inflammatory state and endotoxemia in alcoholics. Thus we interrogated the mucosa-associated colonic microbiome in 48 alcoholics with and without ALD as well as 18 healthy subjects. Colonic biopsy samples from subjects were analyzed for microbiota composition using length heterogeneity PCR fingerprinting and multitag pyrosequencing. A subgroup of alcoholics have an altered colonic microbiome (dysbiosis). The alcoholics with dysbiosis had lower median abundances of Bacteroidetes and higher ones of Proteobacteria. The observed alterations appear to correlate with high levels of serum endotoxin in a subset of the samples. Network topology analysis indicated that alcohol use is correlated with decreased connectivity of the microbial network, and this alteration is seen even after an extended period of sobriety. We show that the colonic mucosa-associated bacterial microbiome is altered in a subset of alcoholics. The altered microbiota composition is persistent and correlates with endotoxemia in a subgroup of alcoholics.

Since the initial annotation of miRNAs from cloned short RNAs by the Ambros, Tuschl, and Bartel groups in 2001, more than a hundred studies have sought to identify additional miRNAs in various species. We report here a meta-analysis of short RNA data from Drosophila melanogaster, aggregating published libraries with 76 data sets that we generated for the modENCODE project. In total, we began with more than 1 billion raw reads from 187 libraries comprising diverse developmental stages, specific tissue- and cell-types, mutant conditions, and/or Argonaute immunoprecipitations. We elucidated several features of known miRNA loci, including multiple phased byproducts of cropping and dicing, abundant alternative 5′ termini of certain miRNAs, frequent 3′ untemplated additions, and potential editing events. We also identified 49 novel genomic locations of miRNA production, and 61 additional candidate loci with limited evidence for miRNA biogenesis. Although these loci broaden the Drosophila miRNA catalog, this work supports the notion that a restricted set of cellular transcripts is competent to be specifically processed by the Drosha/Dicer-1 pathway. Unexpectedly, we detected miRNA production from coding and untranslated regions of mRNAs and found the phenomenon of miRNA production from the antisense strand of known loci to be common. Altogether, this study lays a comprehensive foundation for the study of miRNA diversity and evolution in a complex animal model.

Summary microRNAs are endogenous non-coding small RNAs with important roles in many biological pathways; their generation and activity are under precise regulation [1–3]. Emerging evidence suggests that miRNA pathways are precisely modulated with controls at the level of transcription [4–8], processing [9–11] and stability [12,13], with miRNA deregulation linked with diseases [14] and neurodegenerative disorders [15]. In the Drosophila miRNA biogenesis pathway, long primary miRNA transcripts undergo sequential cleavage [16–18] to release the embedded miRNAs. Mature miRNAs are then loaded into Argonaute 1 (Ago1) within the RNA-induced silencing complex (RISC) [19,20]. Intriguingly, we found that Drosophila miR-34 displays multiple isoforms that differ at the 3'end, suggesting a novel biogenesis mechanism involving 3'end processing. To define the cellular factors responsible, we performed an RNAi screen and identified a putative 3'→5' exoribonuclease CG9247/nibbler essential for the generation of the smaller isoforms of miR-34. Nibbler (Nbr) interacts with Ago1 and processes miR-34 within RISC. Deep sequencing analysis revealed a larger set of multi-isoform miRNAs that are controlled by nibbler. These findings suggest that Nbr-mediated 3' end processing represents a critical step in miRNA maturation that impacts miRNA diversity.

Members of Msi family of RNA binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here we demonstrate that in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.