Ancient DNA Methods Improve Forensic DNA Profiling of Korean War and World War II Unknowns.

The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.

Next generation sequencing of STR artifacts produced from historical bone samples.

STR artifacts are commonly observed in electrophoretic data and can complicate interpretation of the profiles produced. Even when a consensus approach is applied, reproducible artifacts have the potential to convolute the analysis. DNA obtained from historical bone samples is often heavily degraded and damaged, requiring the use of more sensitive procedures to increase allele recovery. Additionally, skeletal remains exposed to environmental conditions may be afflicted with microbial DNA contamination that cross-reacts with the primers during short tandem repeat (STR) multiplex amplification. STR artifacts manifested as a result of these circumstances can be sourced and characterized using new sequencing technologies to potentially ease the analysis burden. For this study, PCR product from 17 low-quality bone samples exhibiting reproducible autosomal and Y-chromosomal STR (Y-STR) artifacts in capillary electrophoresis (CE) data were sequenced with next generation sequencing (NGS). Sequenced reads were bioinformatically sorted using STRait Razor to determine the authenticity of alleles and confirm the profile generated by CE. Sequence data from the PCR products and a subset of the associated extracts were further analyzed with Kaiju to classify the microbial species present and identify potential sources of artifact peaks. A suspected Y-STR artifact was similar in sequence to Pseudomonas sp. BAY1663, a species ubiquitously found in soil. Regions of homology were observed between the Pseudomonas genome and the presumed primer binding locations for Y-STRs included in the AmpFlSTR Y-Filer STR kit. Characterization of such supposed artifact peaks may aid in interpretation of CE data and ultimately lead to increased confidence in the reported results.

Mitochondrial DNA haplogrouping to assist with the identification of unknown service members from the World War II Battle of Tarawa.

The World War II Battle of Tarawa, 1943, was a devastating conflict that resulted in losses of more than 1100 American and 4690 Japanese troops. The United States government aims to identify and repatriate the remains of all missing American service members through the Defense Prisoner of War/Missing in Action (POW/MIA) Accounting Agency (DPAA) and its partners such as the Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory (AFMES-AFDIL). Remains associated with the Battle of Tarawa have been recovered from field excavations conducted by History Flight, a DPAA strategic partner, as well as from the National Memorial Cemetery of the Pacific (NMCP) in Hawaii where unknowns have been disinterred for identification. DNA testing at the AFMES-AFDIL has produced mitochondrial DNA (mtDNA) sequences from 1027 case samples to date. Haplogroup assignments indicate that more than one third (36.2 %) of field-collected samples are likely of Asian maternal ancestry. Therefore the field collections from the Tarawa battlefield comprise the remains of American service members but also those of foreign nationals from Asia. The mtDNA of the NMCP unknowns is similar in ancestry proportion to the family reference sample distribution. The DPAA uses the ancestry information gleaned from mtDNA sequence data in conjunction with anthropological evidence to make foreign national determinations. In this way, mtDNA haplogrouping is used to sort the commingled and fragmentary remains recovered from Tarawa between Americans and foreign nationals, which are then repatriated to their country of origin.

Developmental validation of a Nextera XT mitogenome Illumina MiSeq sequencing method for high-quality samples.

Generating mitochondrial genome (mitogenome) data from reference samples in a rapid and efficient manner is critical to harnessing the greater power of discrimination of the entire mitochondrial DNA (mtDNA) marker. The method of long-range target enrichment, Nextera XT library preparation, and Illumina sequencing on the MiSeq is a well-established technique for generating mitogenome data from high-quality samples. To this end, a validation was conducted for this mitogenome method processing up to 24 samples simultaneously along with analysis in the CLC Genomics Workbench and utilizing the AQME (AFDIL-QIAGEN mtDNA Expert) tool to generate forensic profiles. This validation followed the Federal Bureau of Investigation's Quality Assurance Standards (QAS) for forensic DNA testing laboratories and the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines. The evaluation of control DNA, non-probative samples, blank controls, mixtures, and nonhuman samples demonstrated the validity of this method. Specifically, the sensitivity was established at ≥25 pg of nuclear DNA input for accurate mitogenome profile generation. Unreproducible low-level variants were observed in samples with low amplicon yields. Further, variant quality was shown to be a useful metric for identifying sequencing error and crosstalk. Success of this method was demonstrated with a variety of reference sample substrates and extract types. These studies further demonstrate the advantages of using NGS techniques by highlighting the quantitative nature of heteroplasmy detection. The results presented herein from more than 175 samples processed in ten sequencing runs, show this mitogenome sequencing method and analysis strategy to be valid for the generation of reference data.

Performance evaluation of a mitogenome capture and Illumina sequencing protocol using non-probative, case-type skeletal samples: Implications for the use of a positive control in a next-generation sequencing procedure.

Next-generation ancient DNA technologies have the potential to assist in the analysis of degraded DNA extracted from forensic specimens. Mitochondrial genome (mitogenome) sequencing, specifically, may be of benefit to samples that fail to yield forensically relevant genetic information using conventional PCR-based techniques. This report summarizes the Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory's (AFMES-AFDIL) performance evaluation of a Next-Generation Sequencing protocol for degraded and chemically treated past accounting samples. The procedure involves hybridization capture for targeted enrichment of mitochondrial DNA, massively parallel sequencing using Illumina chemistry, and an automated bioinformatic pipeline for forensic mtDNA profile generation. A total of 22 non-probative samples and associated controls were processed in the present study, spanning a range of DNA quantity and quality. Data were generated from over 100 DNA libraries by ten DNA analysts over the course of five months. The results show that the mitogenome sequencing procedure is reliable and robust, sensitive to low template (one ng control DNA) as well as degraded DNA, and specific to the analysis of the human mitogenome. Haplotypes were overall concordant between NGS replicates and with previously generated Sanger control region data. Due to the inherent risk for contamination when working with low-template, degraded DNA, a contamination assessment was performed. The consumables were shown to be void of human DNA contaminants and suitable for forensic use. Reagent blanks and negative controls were analyzed to determine the background signal of the procedure. This background signal was then used to set analytical and reporting thresholds, which were designated at 4.0X (limit of detection) and 10.0X (limit of quantiation) average coverage across the mitogenome, respectively. Nearly all human samples exceeded the reporting threshold, although coverage was reduced in chemically treated samples resulting in a ∼58% passing rate for these poor-quality samples. A concordance assessment demonstrated the reliability of the NGS data when compared to known Sanger profiles. One case sample was shown to be mixed with a co-processed sample and two reagent blanks indicated the presence of DNA above the analytical threshold. This contamination was attributed to sequencing crosstalk from simultaneously sequenced high-quality samples to include the positive control. Overall this study demonstrated that hybridization capture and Illumina sequencing provide a viable method for mitogenome sequencing of degraded and chemically treated skeletal DNA samples, yet may require alternative measures of quality control.

Determining the impact of establishing a psychogeriatric outreach team network in long-term care.

OBJECTIVES: The primary objective of this study was to describe a model for specialized psychogeriatric consultation to long-term care homes in a large metropolitan Canadian city and to provide an overview of the diagnostic and demographic data of patients referred for assessment. METHODS: Forty long-term care homes and 13 geriatric mental health outreach teams were surveyed and provided feedback on the model. A retrospective chart review (N=88) was also conducted to confirm the survey results and to provide an overview of the types of patients being seen. RESULTS: Team data indicated that 96% of the homes they served (N=81) were using their services, that all referrals were appropriate, and that their recommendations were implemented in over 50% of cases. Referred patients tended to be older (41% age 85 or older); were referred mainly for agitation, aggression, or depressed mood (over 90%); and mainly had a mood or cognitive disorder (over 90%). CONCLUSIONS: These preliminary data suggest that the implementation of specialized psychogeriatric consultation to long-term care may be beneficial, but future studies are required to clarify its usefulness.

hivstigma.com, an innovative web-supported stigma reduction intervention for gay and bisexual men.

An intervention to address stigma directed toward HIV-positive men and to enhance the sexual health of gay and bisexual men was developed through a community-based process involving HIV prevention workers, public health, government and researchers. The intervention aimed to diminish stigma, create greater support for HIV-positive men, make disclosure safer and easier, discourage reliance on disclosure to prevent transmission and encourage testing. The question, 'If you were rejected every time you disclosed, would you?' was widely disseminated in the gay community and supported by the Web site, hivstigma.com, to encourage participation in blog-based discussions. Eight bloggers moderated lively discussions over 5 months. There were 20 844 unique visitors to the site averaging more than 5 min each; 4384 visitors returned more than 10 times. About 1,942 men answered a pre-test survey on a popular gay dating site and 1791, a post-test evaluation. Results show a statistically significant shift among those aware of the intervention toward reduced stigma-related attitudes and behaviors and toward recognition that HIV-positive gay men face stigma in the gay community and that stigma reduces the likelihood of HIV disclosure.

Partnership experiences: involving decision-makers in the research process.

OBJECTIVES: To describe researchers' experiences with involving health system managers and public policy-makers (i.e. decision-makers) in the research process, and decision-makers' experiences with the research process, including their assessments of the benefits and costs of the involvement, and their recommendations for facilitating it. METHODS: We conducted semi-structured interviews with principal investigators and research staff for the seven research programmes funded by the Canadian Health Services Research Foundation in the 1999 and 2000 competition years, and with the decision-makers they involved in the research programmes. RESULTS: We identify three models of decision-maker involvement--formal supporter, responsive audience, and integral partner--each of which yielded important contributions to the research process. Four factors--the stage of the research process, time commitment required, alignment between decision-maker expertise and programme needs, and an existing relationship between the researcher and decision-maker--influenced the role played by decision-makers. CONCLUSIONS: While on balance a beneficial experience, the further promotion of decision-maker involvement in the research process should involve helping researchers and decision-makers identify strategic opportunities for decision-maker involvement and support the costs associated with the involvement. Consideration should also be given to undertaking and evaluating interactions between researchers and decision-makers outside of the research process.

The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides.

The majority of cytosolic proteins in eukaryotes contain a covalently linked acetyl moiety at their very N terminus. The mechanism by which the acetyl moiety is efficiently transferred to a large variety of nascent polypeptides is currently only poorly understood. Yeast N(alpha)-acetyltransferase NatA, consisting of the known subunits Nat1p and the catalytically active Ard1p, recognizes a wide range of sequences and is thought to act cotranslationally. We found that NatA was quantitatively bound to ribosomes via Nat1p and contained a previously unrecognized third subunit, the N(alpha)-acetyltransferase homologue Nat5p. Nat1p not only anchored Ard1p and Nat5p to the ribosome but also was in close proximity to nascent polypeptides, independent of whether they were substrates for N(alpha)-acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associated complex) and the Hsp70 homologue Ssb1/2p interact with a variety of nascent polypeptides on the yeast ribosome. A direct comparison revealed that Nat1p required longer nascent polypeptides for interaction than NAC and Ssb1/2p. Delta nat1 or Delta ard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while Delta nat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by Delta nat1 or Delta ard1 were partially suppressed by overexpression of SSB1. The combination of data suggests that Nat1p presents the N termini of nascent polypeptides for acetylation and might serve additional roles during protein synthesis.

Measuring the impact of health research.

Measuring the decision-making impact of applied health research should constitute a core function for many research funders and research organizations. Different target audiences warrant different measures of impact. The target audiences for applied health research include the general public, patients (and their families), clinicians, managers (in hospitals, regional health authorities and health plans), research and development officers (in biotechnology firms) and public policy-makers (i.e. elected officials, political staff and civil servants). Making meaningful assessments within peer groups that fund or produce similar types of research knowledge for similar types of target audiences makes more sense than a one-size-fits-all approach to impact assessment. User-pull and interactive measures of impact (i.e. measures of cultural shifts that would facilitate the on-going use of research knowledge to inform decision-making) can supplement more traditional producer-push measures that assess researchers' active efforts to inform decision-making and the outcome of these efforts. Cultural shifts may include the creation of a research-attuned culture among decision-makers and a decision-relevant culture among researchers. Moving beyond whether research was used to examine how it was used is also important. Research knowledge may be used in instrumental, conceptual or symbolic ways. These actions, coupled with on-going refinements to the proposed assessment tool as research evidence evolves, would take us a long way towards assessment and accountability in the health sector.

Do Canadian civil servants care about the health of populations?

OBJECTIVES: This article describes Canadian civil servants' awareness of, attitudes toward, and self-reported use of ideas about the determinants of health. METHODS: Federal and provincial civil servants in departments of finance, labor, social services, and health were surveyed. RESULTS: With civil servants in finance departments a notable exception, most Canadian civil servants see the health of populations as a relevant outcome for their sectors. Many (65%) report that ideas about the determinants of health have already influenced policymaking in their sector, but most (83%) say they need more information about the health consequences of the policy alternatives their departments face. CONCLUSIONS: Civil servants should consider developing accountability structures for health and researchers should consider producing and transferring more policy-relevant research.

A functional chaperone triad on the yeast ribosome.

The chaperones RAC (ribosome-associated complex), consisting of Ssz1p and zuotin, and Ssb1/2p are associated with ribosomes of yeast. Ssb1/2p was previously shown to form a crosslink product to polypeptides trapped in ribosome-nascent chain complexes (RNCs) in vitro. Here we show that an efficient crosslink of the nascent chain to Ssb1/2p depends on the presence of functional RAC. The crosslink to Ssb1/2p was significantly diminished if (i) RAC was removed from RNCs: a process reversed by addition of purified RAC; (ii) RAC carried a mutation in the J-domain of zuotin, leading to its inactivation in vivo; (iii) RAC's Ssz1p subunit was absent because RNCs were generated in a Deltassz1-derived translation extract. In vivo the same specific set of growth defects caused by the absence of any of the three chaperones was also displayed by a Deltassb1/2Deltassz1Deltazuo1 strain. The combination of in vitro and in vivo data supports a model in which Ssb1/2p, Ssz1p, and zuotin act in concert on nascent chains while they are being synthesized.

Examining the role of health services research in public policymaking.

Conceptual, methodological, and practical issues await those who seek to understand how to make better use of health services research in developing public policy. Some policies and some policymaking processes may lend themselves particularly well to being informed by research. Different conclusions about the extent to which policymaking is informed by research may arise from different views about what constitutes health services research (is it citable research or any professional social inquiry that can aid in problem solving?) or different views about what constitutes research use (is it explicit uses of research only, or does it also include tacit knowledge or the positions of stakeholders when they are informed by research and are influential in the policymaking process?). Some conditions may favor the use of research in policymaking, like sustained interactions between researchers and policymakers. Results from an exploratory study on the use of health services research by Canadian provincial policymakers illustrate these issues.