RT-qPCR for Fecal Mature MicroRNA Quantification and Validation.

By routinely and systematically being able to perform quantitative stem-loop reverse transcriptase (RT) followed by TaqMan® minor-groove binding (MGB) probe, real-time quantitative PCR analysis on exfoliated enriched colonocytes in stool, using human (Homo sapiens, hsa) micro(mi)RNAs to monitor changes of their expression at various stages of colorectal (CRC) progression, this method allows for the reliable and quantitative diagnostic screening of colon cancer (CC). Although the expression of some miRNA genes tested in tissue shows less variability in normal or cancerous patients than in stool, the noninvasive stool by itself is well suited for CC screening. An miRNA approach using stool promises to offer more sensitivity and specificity than currently used genomic, methylomic, or proteomic methods for CC screening.To present an application of employing miRNAs as diagnostic markers for CC screening, we carried out global microarray expression studies on stool colonocytes isolated by paramagnetic beads, using Affymetrix GeneChip miRNA 3.0 Array, to select a panel of miRNAs for subsequent focused semiquantitative PCR analysis studies. We then conducted a stem-loop RT-TaqMan® MGB probes, followed by a modified real-time qPCR expression study on 20 selected miRNAs for subsequent validation of the extracted immunocaptured total small RNA isolated from stool colonocytes. Results showed 12 miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p, and miR214) to have an increased expression in stool of CC patients, and that later TNM stages exhibited more increased expressions than adenomas, while 8 miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222, and miR-938) showed decreased expressions in stool of CC patients, which becomes more pronounced as the cancer progresses from early to late TNM stages (0-IV).

Role of Melt Curve Analysis in Interpretation of Nutrigenomics' MicroRNA Expression Data.

This article illustrates the importance of melt curve analysis (MCA) in interpretation of mild nutrogenomic micro(mi)RNA expression data, by measuring the magnitude of the expression of key miRNA molecules in stool of healthy human adults as molecular markers, following the intake of Pomegranate juice (PGJ), functional fermented sobya (FS), rich in potential probiotic lactobacilli, or their combination. Total small RNA was isolated from stool of 25 volunteers before and following a three-week dietary intervention trial. Expression of 88 miRNA genes was evaluated using Qiagen's 96 well plate RT2 miRNA qPCR arrays. Employing parallel coordinates plots, there was no observed significant separation for the gene expression (Cq) values, using Roche 480® PCR LightCycler instrument used in this study, and none of the miRNAs showed significant statistical expression after controlling for the false discovery rate. On the other hand, melting temperature profiles produced during PCR amplification run, found seven significant genes (miR-184, miR-203, miR-373, miR-124, miR-96, miR-373 and miR-301a), which separated candidate miRNAs that could function as novel molecular markers of relevance to oxidative stress and immunoglobulin function, for the intake of polyphenol (PP)-rich, functional fermented foods rich in lactobacilli (FS), or their combination. We elaborate on these data, and present a detailed review on use of melt curves for analyzing nutigenomic miRNA expression data, which initially appear to show no significant expressions, but are actually more subtle than this simplistic view, necessitating the understanding of the role of MCA for a comprehensive understanding of what the collective expression and MCA data collectively imply.

Feasibility of retrofitting a university library with active workstations to reduce sedentary behavior.

BACKGROUND: Libraries are an inherently sedentary environment, but are an understudied setting for sedentary behavior interventions. PURPOSE: To investigate the feasibility of incorporating portable pedal machines in a university library to reduce sedentary behaviors. METHODS: The 11-week intervention targeted students at a university library. Thirteen portable pedal machines were placed in the library. Four forms of prompts (e-mail, library website, advertisement monitors, and poster) encouraging pedal machine use were employed during the first 4 weeks. Pedal machine use was measured via automatic timers on each machine and momentary time sampling. Daily library visits were measured using a gate counter. Individualized data were measured by survey. Data were collected in fall 2012 and analyzed in 2013. RESULTS: Mean (SD) cumulative pedal time per day was 95.5 (66.1) minutes. One or more pedal machines were observed being used 15% of the time (N=589). Pedal machines were used at least once by 7% of students (n=527). Controlled for gate count, no linear change of pedal machine use across days was found (b=-0.1 minutes, p=0.75) and the presence of the prompts did not change daily pedal time (p=0.63). Seven of eight items that assessed attitudes toward the intervention supported intervention feasibility (p<0.05). CONCLUSIONS: The unique non-individualized approach of retrofitting a library with pedal machines to reduce sedentary behavior seems feasible, but improvement of its effectiveness is needed. This study could inform future studies aimed at reshaping traditionally sedentary settings to improve public health.

Diagnostic microRNA markers to screen for sporadic human colon cancer in stool: I. Proof of principle.

To present proof-of-principle application for employing micro(mi)RNAs as diagnostic markers for colon cancer, we carried out global microarray expression studies on stool samples obtained from fifteen individuals (three controls, and three each with TNM stage 0-1, stage 2, stage 3, and stage 4 colon cancer), using Affymetrix GeneChip miRNA 3.0 Array, to select for a panel of miRNA genes for subsequent focused semi-quantitative polymerase chain reaction (PCR) analysis studies. Microarray results showed 202 preferentially expressed miRNA genes that were either increased (141 miRNAs), or reduced (61 miRNAs) in expression. We then conducted a stem-loop reverse transcriptase (RT)-TaqMan® minor groove binding (MGB) probes, followed by a modified qPCR expression study on 20 selected miRNAs. Twelve of the miRNAs exhibited increased and 8 decreased expression in stool from 60 individuals (20 controls, 20 with tumor-lymph node-metastatic (TNM) stage 0-1, 10 with stage 2, five with stage 3, and 5 with stage 4 colon cancer) to quantitatively monitor miRNA changes at various TNM stages of colon cancer progression. We also used laser-capture microdissection (LCM) of colon mucosal epithelial tissue samples (three control samples, and three samples from each of the four stages of colon cancer, for a total of 15 samples) to find concordance or lack thereof with stool findings. The reference housekeeping pseudogene-free ribosomal gene (18S rRNA), which shows little variation in expression, was employed as a normalization standard for relative PCR quantification. Results of the PCR analyses confirmed that twelve miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p and miR214) had an increased expression in the stool of patients with colon cancer, and that later TNM carcinoma stages exhibited a more pronounced expression than did adenomas. On the other hand, eight miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222 and miR-938) had decreased expression in the stool of patients with colon cancer, which was also more pronounced from early to later TNM stages. Results from colon mucosal tissues were similar to those from stool samples, although with more apparent changes in expression. Cytological studies on purified stool colonocytes that employed Giemsa staining showed 80% sensitivity for detecting tumor cells in stool smears. The performance characteristics of the test confirmed that stool is a medium well-suited for colon cancer screening, and that the quantitative changes in the expression of few mature miRNA molecules in stool associated with colon cancer progression provided for more sensitive and specific non-invasive diagnostic markers than tests currently available on the market. Thus, a larger prospective and properly randomized validation study of control individuals and patients exhibiting various stages of colon cancer progression (TNM stages 0-IV) is now needed in order to standardize test conditions, and provide a means for determining the true sensitivity and specificity of a miRNA screening approach in stool for the non-invasive detection of colon cancer, particularly at an early stage (0-I). Eventually, we will develop a chip to enhance molecular screening for colon cancer, as has been accomplished for the detection of genetically-modified organisms (GMOs) in foods.

Diagnostic microRNA markers to screen for sporadic human colon cancer in blood.

We carried out this study to present proof-of-principal application, showing that by using a global microarray expression analysis, followed by quantitative stem-loop reverse transcriptase in conjunction with TaqMan® polymerase chain reaction (PCR) analysis of micro(mi)RNA genes, on limited number of plasma and tissue samples obtained from 20 individuals (five healthy, five TNM stage 0-1 colon cancer, five stage 2 and five stage 3), we were able to quantitatively monitor miRNA changes at the various TNM stages of colon cancer progression, particularly at the early, pre-malignant adenoma stage (e.g. polyps ≥ 1 cm with high grade dysplasia). The expression of some of the tested miRNAs showed less variability in tissue than in plasma. Nevertheless, our limited preliminary data on the plasma by itself show that plasma is well-suited for screening, and that the quantitative changes in the expression of a few cell-free circulatory mature miRNA molecules in plasma, that are associated with colon cancer progression, would provide for more sensitive and specific markers than those tests currently available on the market. In addition, analysis of miRNA molecules offers a quantitative and cost-effective non-invasive diagnostic approach for screening, than currently employed methods in a prevalent cancer that can be cured if it is detected at the early TNM stages, and that becomes deadly if not diagnosed before metastasis. Thus, a larger prospective and properly randomized clinical study using plasma derived from many control individuals and at various stages of colon cancer (TNM stages 0-IV) from patients, in order to corroborate the initial results, is now urgently needed in order to allow for a statistically valid analysis, standardizing test conditions which will provide a means for determining the true sensitivity and specificity of a miRNA-screening approach. This approach, when combined with bioinformatics analysis to correlate miRNA seed data with mRNA target data, would allow for a mechanistic understanding of how miRNAs regulate mRNA gene expression, and would offer a better comprehensive diagnostic screening test for early-detection of colon cancer non-invasively.

Diagnostic microRNA markers for screening sporadic human colon cancer and active ulcerative colitis in stool and tissue.

By routinely and systematically being able to perform quantitative stem-loop reverse transcriptase followed by TaqMan PCR expression analysis on stool and tissue samples using fifteen human (Homo sapiens, hsa) micro(mi)RNA genes selected by careful analysis of the peer-reviewed literature, we were able to monitor changes at various stages of CRC, allowing for reliable diagnostic screening of colon cancer particularly at the early, pre-malignant stages, and for difficult-to-treat active ulcerative colitis (UC). Although the expression of some of the miRNA genes tested in tissue showed less variability in CRC or UC patients than in stool, the stool by itself appears well-suited to screening. A miRNA approach using stool samples promises to offer more sensitivity and specificity than currently used screening genomic, methylomic or proteomic methods for colon cancer. Larger prospective clinical studies utilizing stool derived from many control, colon cancer or UC patients, to allow for a statistically valid analysis, are now urgently required to standardize test performance and determine the true sensitivity and specificity of the miRNA screening approach, and to provide a numerical underpinning for these diseases as a function of total RNA. Moreover, when a miRNA screening test is combined with analysis of a messenger(m)RNA expression test, which has also been considered in earlier studies to be a highly sensitive and a very specific and reliable transcriptomic approach, as outlined in this article, bioinformatics can be used to correlate microRNA seed data with mRNA target data in order to gain a mechanistic understanding of how miRNAs regulate gene expression, enabling understanding of why these miRNA genes should be informative in a screening test.

Differences in mRNA and microRNA microarray expression profiles in human colon adenocarcinoma HT-29 cells treated with either Intensity-modulated Radiation Therapy (IMRT), or Conventional Radiation Therapy (RT).

We carried out this in vitro molecular study to investigate the effect of two clinical X-irradiation modalities (a two-dimensional external beam radiotherapy referred to in this article as conventional RT, and a three dimensional conformal intensity-modulated radiation therapy (IMRT) on a colon adenocarcinoma HT-29 cell line. Cells were synchronized by serum deprivation 48 h before irradiation so that >90% of them were in the G(0)/G(1) phase of the cell cycle. Cells were allowed to recover 3 h after irradiation before total RNA extraction. Two types of arrays, namely Affymetrix Human HG U133A 2.0 oligonucleotide microarrays and Ambion mirVana bioarrays, were employed to study mRNA and microRNA expressions, respectively. Three flasks were used per irradiation dose, and an additional three unirradiated flasks served as control. Microarray data were validated by reverse transcriptase quantitative polymerase chain reaction, and proteins of some expressed genes were determined by Western blots. Results showed the existence of differences in expression profiles between the two irradiation modalities. IMRT appeared to influence expression of some DNA repair genes, whereas in conventional RT, some DNA repair and cell cycle-related genes that initially seemed to be preferentially expressed dwindled to normal levels. Earlier in vitro experiments using cell survival to study sublethal damage repair support our conclusions. Bioinformatic investigation revealed a correlation of gene expression with derepression effects of microRNA molecules. We have presented opinions as to how microRNAs might influence gene expression during radiation-induced stress and have suggested future avenues for research.

Robotic mitral valve repairs in 300 patients: a single-center experience.

OBJECTIVES: Mitral valve repair is the standard therapy for patients with degenerative (myxomatous) disease and severe mitral regurgitation. Robotic mitral valve repair provides the least-invasive surgical approach. We report the largest single-center robotic mitral valve repair experience. METHODS: Between May 2000 and November 2006, 300 patients underwent a robotic mitral valve repair (daVinci Surgical System; Intuitive Surgical, Inc, Sunnyvale, Calif). All operations were done with 3- to 4-cm right intercostal access, transthoracic aortic occlusion, and peripheral cardiopulmonary bypass. Repairs included 1 or a combination of trapezoidal/triangular leaflet resections, sliding plasties, chordal transfers/replacements, edge-to-edge approximations, and ring annuloplasties. Echocardiographic and survival follow-up were 93% and 100% complete, respectively. RESULTS: There were 2 (0.7%) 30-day mortalities and 6 (2.0%) late mortalities. No sternotomy conversions or mitral valve replacements were required. Immediate postrepair echocardiograms showed the following degrees of mitral regurgitation: none/trivial, 294 (98%); mild, 3 (1.0%); moderate, 3 (1.0%); and severe, 0 (0.0%). Complications included 2 (0.7%) strokes, 2 transient ischemic attacks, 3 (1.0%) myocardial infarctions, and 7 (2.3%) reoperations for bleeding. The mean hospital stay was 5.2 +/- 4.2 (standard deviation) days. Sixteen (5.3%) patients required a reoperation. Mean postoperative echocardiographic follow-up at 815 +/- 459 (standard deviation) days demonstrated the following degrees of mitral regurgitation: none/trivial, 192 (68.8%); mild, 66 (23.6%); moderate, 15 (5.4%); and severe, 6 (2.2%). Five-year Kaplan-Meier survival was 96.6% +/- 1.5%, with 93.8% +/- 1.6% freedom from reoperation. CONCLUSIONS: Robotic mitral valve repair is safe and is associated with good midterm durability. Further long-term follow-up is necessary.

The effect of frequency altered feedback on stuttering duration and type.

PURPOSE: The effect of frequency altered feedback (FAF) on stuttering type (i.e., prolongation, repetition, or silent block) and stuttering duration (i.e., average duration of stuttering event and total stuttering time) was examined. METHOD: Retrospective analyses of previously collected data from 12 adult persons who stutter who participated in an ABA time-series design while reading orally was undertaken. It was hypothesized that stuttering duration would decrease and there would be a differential reduction in the type of stuttering during FAF, concurrent with previously confirmed reduction of stuttering episodes. A total of 2,971 stuttered syllables were analyzed. RESULTS: The total stuttering duration (s/min) was significantly reduced by approximately 50% irrespective of stuttering type (p = .0014). Although significant differences in the average duration(s) of the 3 stuttering types (p = .0064) existed, FAF significantly reduced each type of stuttering by approximately 20% (p = .0055). There was no differential effect on the reduction of proportion of stuttering type during FAF (p = .36). CONCLUSIONS: FAF positively affects the speech of persons who stutter by reducing the proportion of stuttered events with a concomitant decrease in duration of residual stuttering and total stuttering time during oral reading.

Robotic mitral valve repair for anterior leaflet and bileaflet prolapse.

BACKGROUND: Centers have expanded indications for robotic mitral valve repairs to include complex pathologic features. We studied our results after robotic mitral valve repair for anterior leaflet or bileaflet prolapse. METHODS: Data were collected contemporaneously on 289 patients operated on from May 2000 to September 2006. Every patient underwent preoperative transesophageal echocardiography. Follow-up consisted of serial echocardiograms, clinic visits, and phone conversations with patients and their physicians. RESULTS: A total of 66 patients (anterior leaflet, n = 14; and bileaflet, n = 52) were identified. Mean age was 52.6 +/- 7.1 years, and 57 (86%) patients had New York Heart Association functional class II or III symptoms. Cardiopulmonary bypass and cross-clamp times were 171 +/- 52 and 132 +/- 39 minutes, respectively. The 30-day and late mortality rates were 3% (n = 2) for each time point. There were no device-related or perfusion-related complications or sternotomy conversions. Complications included 2 strokes (3%), 2 bleeding reexplorations (3%), and 10 pleural effusions requiring intervention (15%). The length of hospital stay for surviving patients was 5 +/- 3 days, and time to extubation averaged 9.5 +/- 13 hours. A total of 6 (9%) patients required valve reoperation. Mean follow-up was 795 +/- 495 days, and echocardiographic mitral regurgitation (n = 60) was none or trace (n = 35, 58.3%), mild (n = 19, 31.6%), moderate (n = 2, 3.3%), and severe (n = 4, 6.7%). CONCLUSIONS: Robotic mitral valve repair for anterior leaflet and bileaflet prolapse is feasible and safe. Outcomes and degree of late mitral regurgitation are similar to series using conventional techniques. Long-term follow-up is required to formally address the efficacy of robotic repair techniques.

Modeling survival in colon cancer: a methodological review.

The Cox proportional hazards model is the most widely used model for survival analysis because of its simplicity. The fundamental assumption in this model is the proportionality of the hazard function. When this condition is not met, other modifications or other models must be used for analysis of survival data. We illustrate in this review several methodological approaches to deal with the violation of the proportionality assumption, using survival in colon cancer as an illustrative example.

Standardization for transcriptomic molecular markers to screen human colon cancer.

Establishing test performance criteria for a transcriptomic colon cancer marker approach must be carried out in a standardized fashion in order tso ensure that the test will perform the same way in any laboratory, anywhere. Condition of sample preservation and shipping prior to total RNA extraction is critical, and we recommend preserving stool samples in an appropriate preservative and shipping them in cold packs so as to keep stools at 4 degrees C. It is not necessary to isolate colonocytes to obtain adequate RNA for testing. It is, however, important to obtain samples from both mucin-rich and non-mucin rich to have a good representation of both left- and right-side colon cancers. Employing a commercial total RNA extraction kit that contains an RLT buffer from Qiagen Corporation (Valencia, CA, USA) removes bacterial RNA from stool preparations and results in a high yield of undegraded RNA of human origin. Genes selected based on the enormous resources of NCI's Cancer Genome Anatomy project give good results. Primers for PCR should span more than one exon. Use of semiquantitative PCR, preferably with several reference housekeeping genes of various copy numbers, depending on tested genes, should enhance confidence in the quantitative results. Having standardized the testing conditions in our ongoing work, it is now imperative that a larger prospective randomized clinical study utilizing stool and tissue samples derived from several control and colon cancer patients, to allow for statistically valid analyses, be conducted in order to determine the true sensitivity and specificity of the transcriptomic screening approach for this cancer whose incidence is on the rise worldwide.