Project description:Exosomes are molecular entities derived from membrane vesicles of endocytic origin secreted by most cell types. These vesicles are implicated in cell-to-cell communication, deliver proteins and mRNA molecules between cells. Recent studies have shown that exosomes are found in body fluids such as saliva, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluids and breast milk. Exosomes secreted through human saliva contain mRNA may potentially be useful for diagnostic purposes. Although the exact protective mechanism of saliva RNA is a topic of debate, the consensus is that the enrichment of mRNAs in these nano-vesicles in one of the features of the biomarker discoveries. Our aim was to determine if exosomes are present in human saliva and to nano-characterize their transcriptomic content. Exosomes were purified by differential ultracentrifugation, identified by immunoelectron microscopy, flow cytometry and western blot using a CD-63 antibody. Atomic force microscopy studies revealed ultra structural analysis of both size and density of exosomes. Microarray analysis revealed the presence of 590 mRNA core transcripts are relatively stable inside the exosomes, which can be of saliva mRNA biomarkers. Exosomal mRNA stability was determined by detergent lyses with treatment of RNase. Under in vitro conditions fluorescent dye labeled saliva exosomes were able to communicate between human oral keratinocytes studied by using fluorescence microscopy. The RNA from saliva exosomes can transfer their genetic information to human oral keratinocytes and alters gene expression in the new location. Together, these results suggest that saliva is involved in mRNA trafficking via exosomes, and provides a mechanism for cargoing passenger mRNAs. Our findings are consistent with proposal that exosomes can shuttle RNAs between cells and mRNA is protected inside these vesicles may be a possible resource for biomarker discovery. Experiment Overall Design: Human saliva exosomes were purified through differential centrifugation followed by RNA extraction and hybridization on Affymetrix microarrays. We were able to obtain normal human subjects saliva which are pooled and subjected to ultracentrifugation. The protocol was approved by UCLA Institutional review board. 1 ml of saliva exosomes were used to extract RNA followed by two rounds of amplification by Actorus Amp kit. The amplified RNA was biotin labled and hybridized with Affymetrix protocol.

Project description:Background: Low birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intrauterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. In a unique cohort of 17 monozygotic (MZ) monochorionic female twins very discordant for birth weight (relative differences ranging from 21.3-35.7%), we examined if adverse prenatal growth conditions experienced by the smaller co-twins lead to systemic long-lasting DNA methylation changes. Genome-wide DNA methylation profiles were acquired from saliva DNA using the Infinium HumanMethylation450 BeadChip, targeting ~2% of all CpGs in the genome. Results: Overall, co-twins showed very similar genome-wide DNA methylation profiles. Since observed differences were almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3153 were differentially methylated between the heavy and light co-twins at nominal significance (p<0.01), of which 45 showed absolute mean β-value differences >0.05 (max=0.08). Deep bisulfite sequencing of eight such loci revealed that differences remained in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicated no significant intra-pair differences. Conclusions: Severe intrauterine growth differences observed within these MZ twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies. DNA methylation profiles of saliva from 17 Adult Female MZ MC Twins discordant for birth weight.

Project description:This is a pilot study. We are trying to detect potential salivary biomarkers in mice with a pancreatic tumor. Global gene expression profiling has shown great promise in high-throughput biomarker discovery for early disease detection in body fluids such as saliva, which is accessible, cost-effective, and non-invasive. However, this goal has not been fully realized because saliva, like many clinical samples, contains partially fragmented and degraded RNAs that are difficult to amplify and detect with prevailing technologies. Here, using nanogram scale salivary RNA as a proof-of-principle example, we describe our progress with a novel poly-A tail independent mRNA amplification strategy combined with the Affymetrix GeneChip Exon arrays. We defined a Salivary Exon Core Transcriptome (SECT) with highly similar expression profiles in healthy individuals verified by quantitative PCR. Informatics analysis of SECT provided important mechanistic insight to their potential origin and function. Finally we demonstrated the diagnostic potential of true exon level expression profiling approach with salivary exon biomarkers that accurately discriminated gender in healthy individuals. Recent studies have demonstrated that discriminatory salivary biomarkers can be readily detected upon the development of systemic diseases such as pancreatic cancer, breast cancer, lung cancer and ovarian cancer. However, the utility of salivary biomarkers for the detection of systemic diseases has been undermined due to the absence of biological and mechanistic rationale why distal diseases from the oral cavity would lead to the development of discriminatory biomarkers in saliva. Here, we examine the hypothesis that pancreatic tumor-derived exosomes are mechanistically involved in the development of pancreatic cancer-discriminatory salivary transcriptomic biomarkers. We first developed a pancreatic cancer mouse model that yielded discriminatory salivary biomarkers by implanting the mouse pancreatic cancer cell line Pan02 into the pancreas of the syngeneic host C57BL/6. The role of pancreatic cancer-derived exosomes in the development of discriminatory salivary biomarkers was then tested by engineered a Pan02 cell line that is suppressed for exosome biogenesis, implanted into the C56BL/6 mouse and examine if the discriminatory salivary biomarker profile was ablated or disrupted. Suppression of exosome biogenesis results in the ablation of discriminatory salivary biomarker development. This study supports that tumor-derived exosomes provide a mechanism in the development of discriminatory biomarkers in saliva and distal systemic diseases. We analyzed saliva from 6 healthy mice and 6 mice with a pancreatic tumor using the Affymetrix Mouse Exon Genome 430 2.0 platform. Array data was processed by dChip. No techinical replicates were performed.

Project description:Epigenetics presents a dynamic approach to assess complex individual variation in obesity susceptibility. However, few studies have examined epigenetic patterns in preschool-age children, despite the relevance of this developmental stage to trajectories of weight gain, because of difficulties obtaining blood tissue samples. This proof of principle study examined DNA methylation in 92 saliva samples, comparing Latino preschool children of normal weight mothers (Body Mass Index [BMI] <27 kg/m2 and WC <90 cm) to children of obese mothers (BMI >30 kg/m2 and WC >100 cm). We hypothesized that salivary DNA methylation patterns in Latino preschool age children born of normal weight vs obese weight mothers would be: 1) associated with maternal BMI phenotype in continuous linear regression analysis; 2) saliva could demonstrate epigenetic variation across individuals; and 3) preschool child saliva would be differentially methylated when comparing those children with obese versus normal weight mothers. One hundred and nineteen CpG sites were significantly (p-value <1.56 X 10-5, p-value adjusted <.05) associated with maternal BMI in linear regression models controlling for childâs age, gender, and BMI. Of these 119 CpG sites, 41 were found within the transcription start site, 5â UTR, 3â UTR, or another regulatory region outside of the gene body. Saliva, a practical human tissue to obtain in naturalistic settings and in pediatric populations, was confirmed to be a viable medium for genome-wide epigenetic testing with maternal weight. Although not identical to results yielded from other human tissue types (i.e., cord blood samples), saliva findings indicate potential epigenetic differences in Latino preschool children at risk for pediatric obesity. This proof of principle study examined DNA methylation in 92 saliva samples, comparing Latino preschool children of normal weight mothers (Body Mass Index [BMI] <27 kg/m2 and WC <90 cm) to children of obese mothers (BMI >30 kg/m2 and WC >100 cm). Antropometry was measured objectively according to a standardized protocol.Saliva from preschool Latino children at risk for obesity (BMI>50% < 95% participating in WIC/SNAP programs) was collected using the Oragene DNA saliva kit following a strict data collection protocol. DNA extraction was performed as per DNA Genotek's recommendations using the PrepIT L2P reagent. Extracted DNA was stored in individually barcoded cryovials at â80 degrees Fahrenheit. For children, saliva was obtained using the âbaby brushâ approach, in which small sponges attached to plastic handles are inserted between cheek and gumline to absorb saliva .Arrays were processed using standard protocol [34], with 3 samples randomly selected to serve as duplicates and 1 sample run with HapMap DNA to test functionality of reagents. Duplicates were measured for high technique consistency with Pearson correlation coefficient (>.99). Methylation data were quality controlled using Illumina GenomeStudio (V2011.1), Methylation module (V1.9.0). Samples with lower than 98% call rate (i.e. <485,000 probes) were excluded. Any non-specific cross-reacting probes, probes carrying common SNPs (MAF >1%), or any probes with p-values greater than 0.05 for more than 20% of the sample were sequentially excluded. Validation via pyrosequencing was conducted.

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Single-cell whole genome sequencing of bacteria from saliva

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