Effective Educational Strategies Combining Emerging Genetic Science and Native American Cultural Issues

The purpose of this paper is to describe how new topics in genetic science are implemented and evaluated within Genetic Education for Native Americans (GENA®) workshops. These workshops are typically implemented during professional conferences, training programs, Native American meetings and at tribal colleges. As genetic science evolves, public health educators are more and more likely to need to understand increasingly complex components within genetic research. These research discoveries are likely to impact cardiovascular health, cancer treatments, prevention and control of diabetes. The public and patients want to understand health information that affects them personally, as well as their communities. The focus of this paper is on GENA® objective 14 (emerging genetic science: microRNA), but the three 2006 3-hour workshops also addressed excerpts of GENA® objectives 5 and 29. at three meetings: the 2006 summer and fall Disparities Training Programs held in Houston, Texas and the 2006 Society for the Advancement of Chicanos and Native Americans in the Sciences (SACNAS) conference (October, Tampa, Florida). The emphasis on objective 14 is because it was updated during spring 2006 (initial focus was on stem cell research) and selected components of objectives 5 and 20 have been published elsewhere. The paper briefly describes the content, interactive learning opportunity and the evaluation from the three 2006 workshops. The overall findings verify the effectiveness (p value of less than .01) of GENA® to significantly increase knowledge level of workshop participants about emerging genetic science breakthroughs

Interactive Lesson on the Cell

The purpose of this article is to explain how a basic hands-on lesson about the human cell was developed, implemented and evaluated through Genetic Education for Native Americans (GENA®) workshops

MicroRNA-26a Is Strongly Downregulated in Melanoma and Induces Cell Death through Repression of Silencer of Death Domains (SODD)

Melanoma is an aggressive cancer that metastasizes rapidly and is refractory to conventional chemotherapies. Identifying microRNAs (miRNAs) that are responsible for this pathogenesis is therefore a promising means of developing new therapies. We identified miR-26a through microarray and quantitative reverse-transcription–PCR (qRT-PCR) experiments as an miRNA that is strongly downregulated in melanoma cell lines as compared with primary melanocytes. Treatment of cell lines with miR-26a mimic caused significant and rapid cell death compared with a negative control in most melanoma cell lines tested. In surveying targets of miR-26a, we found that protein levels of SMAD1 (mothers against decapentaplegic homolog 1) and BAG-4/SODD were strongly decreased in sensitive cells treated with miR-26a mimic as compared with the control. The luciferase reporter assays further demonstrated that miR-26a can repress gene expression through the binding site in the 3′ untranslated region (3′UTR) of SODD (silencer of death domains). Knockdown of these proteins with small interfering RNA (siRNA) showed that SODD has an important role in protecting melanoma cells from apoptosis in most cell lines sensitive to miR-26a, whereas SMAD1 may have a minor role. Furthermore, transfecting cells with a miR-26a inhibitor increased SODD expression. Our findings indicate that miR-26a replacement is a potential therapeutic strategy for metastatic melanoma, and that SODD, in particular, is a potentially useful therapeutic target

Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments

Beyond the Ribosome: Extra-translational Functions of tRNA Fragments

High-throughput sequencing studies of small RNAs reveal a complex milieu of noncoding RNAs in biological samples. Early data analysis was often limited to microRNAs due to their regulatory nature and potential as biomarkers; however, many more classes of noncoding RNAs are now being recognized. A class of fragments initially excluded from analysis were those derived from transfer RNAs (tRNAs) because they were thought to be degradation products. More recently, critical cellular function has been attributed to tRNA fragments (tRFs), and their conservation across all domains of life has propelled them into an emerging area of scientific study. The biogenesis of tRFs is currently being elucidated, and initial studies show that a diverse array of tRFs are genera ted from all parts of a tRNA molecule. The goal of this review was to present what is currently known about tRFs and their potential as biomarkers for the earlier detection of disease

Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity

Mycobacterium tuberculosis releases membrane vesicles (MV) that modulate host immune responses and aid in iron acquisition, although they may have additional unappreciated functions. MV production appears to be a regulated process, but virR remains the only characterized genetic regulator of vesiculogenesis. Here, we present data supporting a role for the M. tuberculosis Pst/SenX3-RegX3 signal transduction system in regulating MV production. Deletion of pstA1, which encodes a transmembrane component of the phosphate-specific transport (Pst) system, causes constitutive activation of the SenX3-RegX3 two-component system, leading to increased protein secretion via the specialized ESX-5 type VII secretion system. Using proteomic mass spectrometry, we identified several additional proteins hyper-secreted by the ΔpstA1 mutant, including LpqH, an MV-associated lipoprotein. Nanoparticle tracking analysis revealed a 15-fold increase in MV production by the ΔpstA1 mutant. Both hyper-secretion of LpqH and increased MV release required RegX3 but were independent of VirR, suggesting that Pst/SenX3-RegX3 controls MV release by a novel mechanism. Prior proteomic analysis identified ESX-5 substrates associated with MV. We therefore hypothesized that MV release requires ESX-5 activity. We constructed strains that conditionally express eccD5, which encodes the predicted ESX-5 transmembrane channel. Upon EccD5 depletion, we observed reduced secretion of the ESX-5 substrates EsxN and PPE41, but MV release was unaffected. Our data suggest that ESX-5 does not affect vesicle production and imply that further characterization of the Pst/SenX3-RegX3 regulon might reveal novel mechanisms of M. tuberculosis vesicle biogenesis.In Gram-negative bacteria, MV derived from the outer membrane have diverse functions in bacterial physiology and pathogenesis, and several factors regulating their production have been identified. Though Gram-positive bacteria and mycobacteria that lack an outer membrane also produce vesicles with described roles in pathogenesis, the mechanisms of MV biogenesis in these organisms remain poorly characterized. Defining mechanisms of MV biogenesis might yield significant insights into the importance of MV production during infection. In M. tuberculosis, only a single genetic element, virR, is known to regulate MV production. Our work reveals that the Pst/SenX3-RegX3 signal transduction system is a novel regulator of MV biogenesis that controls MV production by a mechanism that is independent of both VirR and activation of the specialized ESX-5 protein secretion system. Understanding which genes in the RegX3 regulon cause increased MV production might reveal novel molecular mechanisms of MV release

MicroRNA-137 targets microphthalmia-associated transcription factor in melanoma cell lines

Micropthalmia-associated transcription factor (MITF) is the master regulator of melanocyte development, survival, and function. Frequent alteration in the expression of MITF is detected in melanoma, but the mechanism(s) underlying the alteration in expression have not been completely determined. In these studies, we have identified microRNA-137 (miR-137) as a regulator of MITF expression. The genomic locus of miR-137 at chromosome 1p22 places it in a region of the human genome previously determined to harbor an allele for melanoma susceptibility. Here, we show that expression of mature miR-137 in melanoma cell lines down-regulates MITF expression. Further, we have identified a 15-bp variable nucleotide tandem repeat located just 5' to the pre-miR-137 sequence, which alters the processing and function of miR-137 in melanoma cell lines

Methods for Extracting and Characterizing RNA from Urine: for downstream PCR and RNAseq Analysis

Readily accessible samples such as urine or blood are seemingly ideal for differentiating and stratifying patients, however, it has proven a daunting task to identify reliable biomarkers in such samples. Noncoding RNA holds great promise as a source of biomarkers distinguishing physiologic wellbeing or illness