The comprehensive multiplatform genomics data generated by The Cancer Genome Atlas (TCGA) Research Network is an enabling resource for cancer research. It includes an unprecedented amount of microRNA ...sequence data: ~11 000 libraries across 33 cancer types. Combined with initiatives like the National Cancer Institute Genomics Cloud Pilots, such data resources will make intensive analysis of large-scale cancer genomics data widely accessible. To support such initiatives, and to enable comparison of TCGA microRNA data to data from other projects, we describe the process that we developed and used to generate the microRNA sequence data, from library construction through to submission of data to repositories. In the context of this process, we describe the computational pipeline that we used to characterize microRNA expression across large patient cohorts.
Previously we have reported that metastatic melanoma cell lines and tumor specimens have reduced expression of ADAR1 and consequently are impaired in their ability to perform A-to-I microRNA (miRNA) ...editing. The effects of A-to-I miRNAs editing on melanoma growth and metastasis are yet to be determined. Here we report that miR-378a-3p is undergoing A-to-I editing only in the non-metastatic but not in metastatic melanoma cells. The function of the edited form is different from its wild-type counterpart. The edited form of miR-378a-3p preferentially binds to the 3'-UTR of the PARVA oncogene and inhibits its expression, thus preventing the progression of melanoma towards the malignant phenotype. Indeed, edited miR-378a-3p but not its WT form inhibits melanoma metastasis in vivo. These results further emphasize the role of RNA editing in melanoma progression.
High-throughput total nucleic acid (TNA) purification methods based on solid-phase reversible immobilization (SPRI) beads produce TNA suitable for both genomic and transcriptomic applications. Even ...so, small RNA species, including miRNA, bind weakly to SPRI beads under standard TNA purification conditions, necessitating a separate workflow using column-based methods that are difficult to automate. Here, an SPRI-based high-throughput TNA purification protocol that recovers DNA, RNA and small RNA, called GSC-modified RLT+ Aline bead-based protocol (GRAB-ALL), which incorporates modifications to enhance small RNA recovery is presented. GRAB-ALL was benchmarked against existing nucleic acid purification workflows and GRAB-ALL efficiently purifies TNA, including small RNA, for next-generation sequencing applications in a plate-based format suitable for automated high-throughput sample preparation.
In forest soils, the availability of phosphate is largely dependent on phosphatase activity. We used soil imprinting to compare in situ activity and fine-scale distribution of phosphatase on soil ...profiles located across forest chronosequences of four age classes young (5–6 yrs), canopy closure (24–30 yrs), stem exclusion (61–71 yrs), and older (90–103 yrs) of mixed Douglas-fir/paper birch stands regenerated after fire or clearcutting in southern interior British Columbia, Canada. Chromatography paper treated with a mixture of substrate and colorimetric reagent was applied directly to vertical soil surfaces, accessed through root windows. Stands older than 61 years had both the highest level of in situ phosphatase activity and larger, more intense regions of activity. Bray-extractable phosphorus was negatively related to imprintable phosphatase activity. We compared the changes in phosphatase activity with differences in the ectomycorrhizal fungal (EMF) community that had been documented previously in the same stands. Of 84 ectomycorrhizal fungi found on roots in at least two of the stand-age classes, eight taxa were positively correlated and one taxon (Rhizopogon vinicolor/vesiculosus) negatively correlated with high phosphatase activity. The frequency of three taxa appeared to be positively correlated with larger areas of activity on the soil profiles. By using an imprinting approach, this study was able to demonstrate, for the first time, that in situ phosphatase activity and physical attributes of that activity (i.e., number, size, and relative rates of each area of activity) were related to concentrations of soil nutrients and with the frequency of individual ectomycorrhizal fungi.
•Phosphatase activity was imprinted along a birch – Douglas-fir chronosequence.•Older forests had larger, more frequent and more intense areas of activity.•Eight ectomycorrhizal fungal species were related with higher phosphatase.•Three ectomycorrhizal fungal taxa were related to larger areas of activity.•Soil chemistry related to amount, biological variables to area of activity.
To understand nutrient cycling in soils, soil processes and microorganisms need be better characterized. To determine whether specific trophic groups of fungi are associated with soil enzyme ...activity, we used soil imprinting to guide mm-scale sampling from microsites with high and low phosphatase activities in birch/Douglas-fir stands. Study 1 involved sampling one root window per site at 12 sites of different ages (stands); study 2 was conducted at one of the stem-exclusion stands, at which 5 root windows had been installed. Total fungal and ectomycorrhizal (EM) fungal terminal-restriction fragment length polymorphism (TRFLP) fingerprints differed between high- and low-phosphatase activity microsites at 8 of 12 root windows across 12 sites. Where differences were detected, fewer EM fungi were detected in high- than low-phosphatase activity microsites. Using 5 root windows at one site, next-generation sequencing detected similar fungal communities across microsites, but the ratio of saprotrophic to EM fungal reads was higher in high-phosphatase activity microsites in the two windows that had low EM fungal richness. In windows with differences in fungal communities, both studies indicated that EM fungi were less successful than saprotrophic fungi in colonizing fine-scale, organic matter-rich microsites. Fine-scale sampling linked with in situ detection of enzyme activity revealed relationships between soil fungal communities and phosphatase activity that could not be observed at the scales employed by conventional approaches, thereby contributing to the understanding of fine-scale phosphorus cycling in forest soils.
In a recent publication appearing in the Asian Journal of Andrology, a team of researchers investigated the relationship of early hormone exposure to adult lung function. In the publication, ＇Second ...to fourth digit ratio： a predictor of adult lung function＇, Park et al.,＇ described known sexual dimorphisms in lung function and a role for sex hormones in the development and regulation of lung function： the predominantly-male hormone, testosterone, enhances airway inflammation and the predominantly female hormone, estrogen, promotes lung development and protects against inflammation. Increasingly over the last decade and a half, digit ratio （the ratio of the lengths of the index to ring fingers） has served as a convenient biomarker for prenatal steroid exposure, and is thought to depend on exposure to testosterone relative to estrogen during development.
Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be ...used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95-100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.
Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways ...related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E-3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E-3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.
Abstract
Ectomycorrhizal fungi (EMF) provide nutrients to their hosts by means of hyphae that extend beyond nutrient-depleted rhizosphere soil. Soil bacteria may compete with EMF for nutrients or may ...act synergistically to enhance nutrient supply to hosts. To assess the interactions between hyphae and bacteria, two types of small, sand-filled mesh bags were incubated in a Pseudotsuga menziesii/Betula papyrifera forest. The bags allowed ingrowth by EMF (35-μm mesh) or excluded hyphae (0.5-μm mesh), while allowing migration of soil bacteria. After incubation, bacteria were isolated from bags using a method to enrich for Gram-positive bacteria. Isolates were assayed for phosphatase and N-acetyl glucosaminidase (NAGase) activities to assess the potential to access organic phosphorus and nitrogen. The average phosphatase activities were higher in exclusion than ingrowth bags, while NAGase activities did not differ. Streptomyces isolates, which are expected to be strong competitors and antagonists of EMF, were more prevalent in ingrowth bags and yet had lower phosphatase activities. Furthermore, there were no indications of antagonism between fungi and Streptomyces, as there were no increases in NAGase activities in ingrowth bags. We conclude that fungal hyphae can structure components of the soil bacterial community for decreased extracellular enzyme production.
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