Rhonda K. Roby

Anti-cancer effects of novel flavonoid vicenin-2 as a single agent and in synergistic combination with docetaxel in prostate cancer.

The present study was conducted to determine the efficacy of novel flavonoid vicenin-2 (VCN-2), an active constituent of the medicinal herb Ocimum Sanctum Linn or Tulsi, as a single agent and in combination with docetaxel (DTL) in carcinoma of prostate (CaP). VCN-2 effectively induced anti-proliferative, anti-angiogenic and pro-apoptotic effect in CaP cells (PC-3, DU-145 and LNCaP) irrespective of their androgen responsiveness or p53 status. VCN-2 inhibited EGFR/Akt/mTOR/p70S6K pathway along with decreasing c-Myc, cyclin D1, cyclin B1, CDK4, PCNA and hTERT in vitro. VCN-2 reached a level of 2.6±0.3μmol/l in serum after oral administration in mice which reflected that VCN-2 is orally absorbed. The i.v. administration of docetaxel (DTL), current drug of choice in androgen-independent CaP, is associated with dose-limiting toxicities like febrile neutropenia which has lead to characterization of alternate routes of administration and potential combinatorial regimens. In this regard, VCN-2 in combination with DTL synergistically inhibited the growth of prostate tumors in vivo with a greater decrease in the levels of AR, pIGF1R, pAkt, PCNA, cyclin D1, Ki67, CD31, and increase in E-cadherin. VCN-2 has been investigated for radioprotection and anti-inflammatory properties. This is the first study on the anti-cancer effects of VCN-2. In conclusion, our investigations collectively provide strong evidence that VCN-2 is effective against CaP progression along with indicating that VCN-2 and DTL co-administration is more effective than either of the single agents in androgen-independent prostate cancer.

Simultaneous quantification of mitochondrial DNA copy number and deletion ratio: A multiplex real-time PCR assay

Mitochondrial dysfunction is implicated in a vast array of diseases and conditions, such as Alzheimers disease, cancer, and aging. Alterations in mitochondrial DNA (mtDNA) may provide insight into the processes that either initiate or propagate this dysfunction. Here, we describe a unique multiplex assay which simultaneously provides assessments of mtDNA copy number and the proportion of genomes with common large deletions by targeting two mitochondrial sites and one nuclear locus. This probe-based, single-tube multiplex provides high specificity while eliminating well-to-well variability that results from assaying nuclear and mitochondrial targets individually.

MicroRNA-940 suppresses prostate cancer migration and invasion by regulating MIEN1

BackgroundMicroRNAs (miRNAs) are crucial molecules that regulate gene expression and hence pathways that are key to prostate cancer progression. These non-coding RNAs are highly deregulated in prostate cancer thus facilitating progression of the disease. Among the many genes that have gained importance in this disease, Migration and invasion enhancer 1 (MIEN1), a novel gene located next to HER2/neu in the 17q12 amplicon of the human chromosome, has been shown to enhance prostate cancer cell migration and invasion, two key processes in cancer progression. MIEN1 is differentially expressed between normal and cancer cells and tissues. Understanding the regulation of MIEN1 by microRNA may enable development of better targeting strategies.MethodsThe miRNAs that could target MIEN1 were predicted by in silico algorithms and microarray analysis. The validation for miRNA expression was performed by qPCR and northern blotting in cells and by in situ hybridization in tissues. MIEN1 and levels of other molecules upon miRNA regulation was determined by Western blotting, qPCR, and immunofluorescence. The functional effects of miRNA on cells were determined by wound healing cell migration, Boyden chamber cell invasion, clonal and colony formation assays. For knockdown or overexpression of the miRNA or overexpression of MIEN1 3′UTR, cells were transfected with the oligomiRs and plasmids, respectively.ResultsA novel miRNA, hsa-miR-940 (miR-940), identified and validated to be highly expressed in immortalized normal cells compared to cancer cells, is a regulator of MIEN1. Analysis of human prostate tumors and their matched normal tissues confirmed that miR-940 is highly expressed in the normal tissues compared to its low to negligible expression in the tumors. While MIEN1 is a direct target of miR-940, miR-940 alters MIEN1 RNA, in a quantity as well as cell dependent context, along with altering its downstream effectors. The miR-940 inhibited migratory and invasive potential of cells, attenuated their anchorage-independent growth ability, and increased E-cadherin expression, implicating its role in mesenchymal-to-epithelial transition (MET).ConclusionsThese results, for the first time, implicate miR-940, a regulator of MIEN1, as a promising novel diagnostic and prognostic tool for prostate cancer.

Mitochondrial DNA deletions in Alzheimer's brains: a review.

Mitochondrial dysfunction and increased oxidative stress have been associated with normal aging and are possibly implicated in the etiology of late‐onset Alzheimers disease (AD). DNA deletions, as well as other alterations, can result from oxidative damage to nucleic acids. Many studies during the past two decades have investigated the incidence of mitochondrial DNA deletions in postmortem brain tissues of late‐onset AD patients compared with age‐matched normal control subjects. Published studies are not entirely concordant, but their differences might shed light on the heterogeneity of AD itself. Our understanding of the role that mitochondrial DNA deletions play in disease progression may provide valuable information that could someday lead to a treatment.

Genetic analysis of 7 medieval skeletons from the Aragonese Pyrenees.

Aim To perform a genetic characterization of 7 skeletons from medieval age found in a burial site in the Aragonese Pyrenees. Methods Allele frequencies of autosomal short tandem repeats (STR) loci were determined by 3 different STR systems. Mitochondrial DNA (mtDNA) and Y-chromosome haplogroups were determined by sequencing of the hypervariable segment 1 of mtDNA and typing of phylogenetic Y chromosome single nucleotide polymorphisms (Y-SNP) markers, respectively. Possible familial relationships were also investigated. Results Complete or partial STR profiles were obtained in 3 of the 7 samples. Mitochondrial DNA haplogroup was determined in 6 samples, with 5 of them corresponding to the haplogroup H and 1 to the haplogroup U5a. Y-chromosome haplogroup was determined in 2 samples, corresponding to the haplogroup R. In one of them, the sub-branch R1b1b2 was determined. mtDNA sequences indicated that some of the individuals could be maternally related, while STR profiles indicated no direct family relationships. Conclusions Despite the antiquity of the samples and great difficulty that genetic analyses entail, the combined use of autosomal STR markers, Y-chromosome informative SNPs, and mtDNA sequences allowed us to genotype a group of skeletons from the medieval age.

Role of genomics in eliminating health disparities.

The Texas Center for Health Disparities, a National Institute on Minority Health and Health Disparities Center of Excellence, presents an annual conference to discuss prevention, awareness education, and ongoing research about health disparities both in Texas and among the national population. The 2014 Annual Texas Conference on Health Disparities brought together experts in research, patient care, and community outreach on the “Role of Genomics in Eliminating Health Disparities.” Rapid advances in genomics and pharmacogenomics are leading the field of medicine to use genetics and genetic risk to build personalized or individualized medicine strategies. We are at a critical juncture of ensuring such rapid advances benefit diverse populations. Relatively few forums have been organized around the theme of the role of genomics in eliminating health disparities. The conference consisted of three sessions addressing “Gene-Environment Interactions and Health Disparities,” “Personalized Medicine and Elimination of Health Disparities,” and “Ethics and Public Policy in the Genomic Era.” This article summarizes the basic science, clinical correlates, and public health data presented by the speakers.

Optimization of primer specific filter metrics for the assessment of mitochondrial DNA sequence data

Filter metrics are used as a quick assessment of sequence trace files in order to sort data into different categories (i.e. high quality, review, and low quality) without human intervention. The filter metrics consist of two numerical parameters for sequence quality assessment: trace score (TS) and contiguous read length (CRL). Primer-specific settings for the TS and CRL were established using a calibration dataset of 2817 traces and validated using a concordance dataset of 5617 traces. Prior to optimization, 57% of the traces required manual review before import into a sequence analysis program, whereas after optimization only 28% of the traces required manual review. After optimization of primer-specific filter metrics for mitochondrial DNA sequence data, an overall reduction of review of trace files translates into increased throughput of data analysis and decreased time required for manual review.

Grading of qualitative and quantitative responses in the PI proficiency survey of the College of American Pathologists for mailings in 1997–2000

Abstract The PI Survey offered jointly by the American Association of Blood Banks (AABB) and the College of American Pathologists (CAP) provides biological samples for proficiency testing that mirror paternity casework samples. A grading scheme providing for three levels of performance (good, acceptable, and unacceptable) has been developed for responses provided by subscribers. Reported here is a review of the performance of participants using RFLP and PCR/STR methods for the 1997–2000 period. In general, laboratories reporting phenotypes and paternity index values using RFLP methods exhibited a fairly constant rate of unacceptable responses over the 4-year period. In contrast, laboratories using PCR/STR systems improved dramatically in the frequency of unacceptable responses (an average of 3.5% for mailings in 1997 vs. 0.7% for mailings in 2000). A comparison of unacceptable responses for RFLP and PCR/STR methods over the 4-year period shows that whereas phenotype designations for PCR/STR systems is better than for RFLP systems, the opposite is true for reported paternity index values.

Proficiency testing programs for DNA typing laboratories offered by the College of American Pathologists

Abstract The College of American Pathologists (CAP) has several proficiency testing programs targeted to laboratories that perform DNA typing of STR loci. Two well-known programs for parentage testing and forensic labs have been available since the early 1990s and are used by labs worldwide. New proficiency surveys introduced by CAP within the last 2 years may also be of interest to DNA typing labs interested in STR typing for new sources of revenue. Recently added surveys include one for labs monitoring engraftment in bone marrow transplant patients, a second survey for labs that perform phenotyping of convicted offenders for databasing programs, and a survey for labs that perform mitochondrial DNA sequencing. Presented here are brief introductions to the proficiency surveys offered by CAP that are of potential interest to DNA typing labs.

Mitochondrial Genome Interrogation for Forensic Casework and Research Studies

This unit describes methods used in the analysis of mitochondrial DNA (mtDNA) for forensic and research applications. UNIT describes procedures specifically for forensic casework where the DNA from evidentiary material is often degraded or inhibited. In this unit, protocols are described for quantification of mtDNA before amplification; amplification of the entire control region from high‐quality samples as well as procedures for interrogating the whole mitochondrial genome (mtGenome); quantification of mtDNA post‐amplification; and, post‐PCR cleanup and sequencing. The protocols for amplification were developed for high‐throughput databasing applications for forensic DNA testing such as reference samples and population studies. However, these same protocols can be applied to biomedical research such as age‐related disease and health disparities research. Curr. Protoc. Hum. Genet. 81:14.9.1‐14.9.23.