Characterizing Microbial Signatures on Sculptures and Paintings of Similar Provenance.

The preservation of artwork challenges museums, collectors, and art enthusiasts. Currently, reducing moisture, adjusting the type of lighting, and preventing the formation of mold are primary methods to preserving and preventing deterioration. Other methods such as ones based in detailed knowledge of molecular biology such as microbial community characterization using polymerase chain reaction (PCR) and sequencing have yet to be explored. Such molecular biology approaches are essential to explore as some environmental bacteria are capable of oxidizing nonpolar chemical substances rich in hydrocarbons such as oil-based paints. Using 16S rDNA Illumina Sequencing, we demonstrate a novel finding that there are differing bacterial communities for artwork from roughly the same era when comparing paintings on wood, paintings on canvases, and sculptures made of stone and marble. We also demonstrate that there are specific genera such as Aeromonas known for having oxidase positive strains, present on paintings on wood and paintings on canvas that could potentially be responsible for deterioration and fading as such organisms produce water or hydrogen peroxide as a byproduct of cytochrome c oxidase activity. The advantages of these genomics-based approaches to characterizing the microbial population on deteriorating artwork provides immense potential by identifying potentially damaging species that may not be detected using conventional methods in addition to addressing challenges to identification, restoration, and preservation efforts.

Identification of individuals by trait prediction using whole-genome sequencing data.

Prediction of human physical traits and demographic information from genomic data challenges privacy and data deidentification in personalized medicine. To explore the current capabilities of phenotype-based genomic identification, we applied whole-genome sequencing, detailed phenotyping, and statistical modeling to predict biometric traits in a cohort of 1,061 participants of diverse ancestry. Individually, for a large fraction of the traits, their predictive accuracy beyond ancestry and demographic information is limited. However, we have developed a maximum entropy algorithm that integrates multiple predictions to determine which genomic samples and phenotype measurements originate from the same person. Using this algorithm, we have reidentified an average of >8 of 10 held-out individuals in an ethnically mixed cohort and an average of 5 of either 10 African Americans or 10 Europeans. This work challenges current conceptions of personal privacy and may have far-reaching ethical and legal implications.

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.

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

BACKGROUND: MicroRNAs (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. METHODS: The 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. RESULTS: A 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). CONCLUSIONS: These 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 Alzheimer's 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.

Seventy-five years of service: an overview of the College of American Pathologists' proficiency testing program in histocompatibility and identity testing.

The Histocompatibility and Identity Testing Committee offers an overview of the College of American Pathologists' (CAP) Proficiency Testing (PT) program, commemorating its significant 75th anniversary in 2024. The CAP PT program has undergone significant growth and evolution over the years, ultimately achieving Centers for Medicare and Medicaid Services approval. In 1979, CAP's partnership with the American Association for Clinical Histocompatibility Testing marked a pivotal moment, leading to the creation of the first proficiency testing survey in 1980. This laid the foundation for various PT programs managed by the CAP Histocompatibility and Identity Testing Committee, including HLA antibody testing, HLA molecular typing, engraftment monitoring, parentage/relationship testing, HLA disease associations and drug risk, and HLA-B27 typing. Each program's distinctive considerations, grading methodologies, and future prospects are detailed here, highlighting the continual evolution of histocompatibility and identity testing PT to support emerging technologies and evolving laboratory practices in the field.

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.

Allele frequencies of 19 STR loci in a Philippine population generated using AmpFlSTR multiplex and ALF singleplex systems.

Allele frequencies for the 19 short tandem repeat (STR) loci CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S306, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, DHFRP2 (FOLP23), F13A01, FES/FPS, FGA, TH01, TPOX, and vWA were obtained from a sample of 106 unrelated Filipinos from different regions of the Philippine archipelago.

The effects of metal ion PCR inhibitors on results obtained with the Quantifiler(®) Human DNA Quantification Kit.

Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025-18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler(®) Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler(®) Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler(®) Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that targets human DNA in the Quantifiler(®) kit. Evidence of inhibition was observed for the human-specific assay at a lower metal concentration than detected by the IPC, for all metals examined except calcium. These results strongly suggest that determination of a "true negative" sample should not be based solely on the failure of the IPC to indicate the presence of a PCR inhibitor and indicate that amplification of all samples should be attempted, regardless of the quantification results.

Identification of a D8S1179 primer binding site mutation and the validation of a primer designed to recover null alleles.

A population study of Chamorros and Filipinos using short tandem repeat (STR) loci amplified with the AmpFlSTR Profiler Plus PCR amplification kit demonstrated an excess of observed homozygosity at the D8S1179 locus. Use of a different set of D8S1179 primers to type the same samples did not demonstrate an excess of homozygosity and showed discordant genotypes at the D8S1179 locus. A single point mutation, G-to-A transition, 16 nucleotides from the 3' end of the reverse primer, was identified to cause allele dropout when using the AmpFlSTR Profiler Plus primer set. An additional D8S1179 reverse primer specific for the variant was constructed resulting in the recovery of the null allele. The primer was included in the newly developed AmpFlSTR Identifiler PCR amplification kit. No deleterious effects or non-specific peaks were observed in validation experiments evaluating primer concentration, Mg2+ concentration, annealing temperature and population samples.

Developmental validation of a single-tube amplification of the 13 CODIS STR loci, D2S1338, D19S433, and amelogenin: the AmpFlSTR Identifiler PCR Amplification Kit.

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433). By incorporating five-dye fragment analysis technology and non-nucleotide linkers, previously optimized AmpFlSTR kit primer sequences have been maintained. This kit has been developed in accordance with the standards of the forensic community as defined by the DNA Advisory Board. Validation studies were performed to include developmental validation, and the results support the use of the AmpFlSTR Identifiler PCR Amplification Kit for human identity and parentage testing.

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 Alzheimer's 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.

Internal validation of human mitochondrial DNA quantification using real-time PCR.

The quantity of mitochondrial DNA (mtDNA) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data. Validation of a human mtDNA real-time quantitative PCR (qPCR) assay demonstrated its high degree of reproducibility and precision as well as an extremely sensitive threshold of detection (0.0001 pg/µL or approximately six human mtDNA copies/µL). A study of 35 nonprobative bone and teeth evidence samples revealed that 20 pg of mtDNA template is recommended for successful HV1 and HV2 sequence analysis; however, as little as 0.013 pg can generate a full mtDNA profile when using enhanced amplification reactions. The assay can also detect PCR inhibition and is useful for identifying samples that may benefit from re-purification. Overall, the assay is an excellent method to quantify mtDNA and is useful for determining the best analytical approach for successful sequencing.

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.

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.

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.