guided-protocol-for-fecal-microbial-characterization-16s-rrna

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

Sheba Medical Center

ABC transporters are often found to be inherently expressed in a wide variety of stem cells, where they provide improved protection from toxins. We found a subpopulation of human melanoma cells expressing multidrug-resistance gene product 1 (MDR1). This fraction co-expresses the ABC transporters, ABCB5 and ABCC2 in addition to the stem cell markers, nanog and human telomerase reverse transcriptase (hTERT). The clonogenicity and self-renewal capacity of MDR1(+) melanoma cells were investigated in single cell settings using the limiting dilution assay. We found that the MDR1(+) cells, isolated by FACS sorting, demonstrated a higher self-renewal capacity than the MDR1(-) fraction, a key stem cell feature. Moreover, MDR1(+) cells had higher ability to form spheres in low attachment conditions, a hallmark of cancer. In conclusion, these novel findings imply that the MDR1(+) cells represent melanoma stem cells and thus should be considered as a unique cellular target for future anti-melanoma therapies.

MDR1 expression identifies human melanoma stem cells.

An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel approach, m(6)A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m(6)A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks--around stop codons and within long internal exons--and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m(6)A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a fundamental role in regulation of gene expression.

Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.

Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR).

Hospitalized patients are at increased risk for acquiring healthcare-associated infections (HAIs) and inadequate nutrition. The human intestinal microbiota plays vital functions in nutrient supply and protection from pathogens, yet characterization of the microbiota of hospitalized patients is lacking. We used 16S rRNA amplicon sequencing to characterize the global pattern of microbial composition of fecal samples from 196 hospitalized patients with suspected infectious diarrhea in comparison to healthy, non-hospitalized subjects (n = 881), and to traditional culture results. We show that hospitalized patients have a significant rise in α-diversity (richness within sample) from birth to <4 years of age, which continues up to the second decade of life. Additionally, we noted a profoundly significant increase in taxa from Proteobacteria phylum in comparison to healthy subjects. Finally, although more than 60% of hospitalized samples had a greater than 10% abundance of Proteobacteria, there were only 19/196 (10%) positive cultures for Campylobacter, Salmonella, or Shigella entero-pathogens in traditional culturing methods. As hospitalized patients have increased risk for HAIs and inadequate nutrition, our data support the consideration of nutritional and/or microbial modification in this population.

Fecal microbial characterization of hospitalized patients with suspected infectious diarrhea shows significant dysbiosis.

Adenosine deaminase acting on RNA 1 (ADAR1) is the master RNA editor, catalyzing the deamination of adenosine to inosine. RNA editing is vital for preventing abnormal activation of cytosolic nucleic acid sensing pathways by self-double-stranded RNAs. Here we determine, by parallel analysis of RNA secondary structure sequencing (PARS-seq), the global RNA secondary structure changes in ADAR1 deficient cells. Surprisingly, ADAR1 silencing resulted in a lower global double-stranded to single-stranded RNA ratio, suggesting that A-to-I editing can stabilize a large subset of imperfect RNA duplexes. The duplexes destabilized by editing are composed of vastly complementary inverted Alus found in untranslated regions of genes performing vital biological processes, including housekeeping functions and type-I interferon responses. They are predominantly cytoplasmic and generally demonstrate higher ribosomal occupancy. Our findings imply that the editing effect on RNA secondary structure is context dependent and underline the intricate regulatory role of ADAR1 on global RNA secondary structure.

RNA editing by ADAR1 leads to context-dependent transcriptome-wide changes in RNA secondary structure.

Neural progenitor cells undergo somatic retrotransposition events, mainly involving L1 elements, which can be potentially deleterious. Here, we analyze the whole genomes of 20 brain samples and 80 non-brain samples, and characterized the retrotransposition landscape of patients affected by a variety of neurodevelopmental disorders including Rett syndrome, tuberous sclerosis, ataxia-telangiectasia and autism. We report that the number of retrotranspositions in brain tissues is higher than that observed in non-brain samples and even higher in pathologic vs normal brains. The majority of somatic brain retrotransposons integrate into pre-existing repetitive elements, preferentially A/T rich L1 sequences, resulting in nested insertions. Our findings document the fingerprints of encoded endonuclease independent mechanisms in the majority of L1 brain insertion events. The insertions are "non-classical" in that they are truncated at both ends, integrate in the same orientation as the host element, and their target sequences are enriched with a CCATT motif in contrast to the classical endonuclease motif of most other retrotranspositions. We show that L1Hs elements integrate preferentially into genes associated with neural functions and diseases. We propose that pre-existing retrotransposons act as "lightning rods" for novel insertions, which may give fine modulation of gene expression while safeguarding from deleterious events. Overwhelmingly uncontrolled retrotransposition may breach this safeguard mechanism and increase the risk of harmful mutagenesis in neurodevelopmental disorders.

Karen Cesarkas

Sheba Medical Center

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing