Mobile and Connected Health Technology Needs for Older Adults Aging in Place: Cross-Sectional Survey Study.

BACKGROUND: An increasing number of mobile and wearable devices are available in the market. However, the extent to which these devices can be used to assist older adults to age in place remains unclear. OBJECTIVE: This study aimed to assess older adults' perceptions of using mobile and connected health technologies. METHODS: Using a cross-sectional design, a total of 51 participants were recruited from a senior community center. Demographics and usage of mobile or wearable devices and online health communities were collected using a survey questionnaire. Descriptive statistics assessed usage of devices and online health communities. The Fisher exact test was used to examine the relationship between technology usage and having access to a smartphone. RESULTS: The sample was primarily comprised non-Hispanic white (35/51, 69%), educated (39/51, 76% any college), and female (36/51, 71%) participants, with an average age of 70 (SD 8) years. All participants were insured and nearly all lived at home (49/51, 94%). A total of 86% (44/51) of the participants had heard of wearable health devices, but only 18 out of 51 (35%) had ever used them. Over 80% (42/51) expressed interest in using such devices and were interested in tracking exercise and physical activity (46/51, 90%), sleep (38/51, 75%), blood pressure (34/51, 67%), diet (31/51, 61%), blood sugar (28/51, 55%), weight (26/51, 51%), and fall risk (23/51, 45%). The greatest concerns about using wearable devices were cost (31/51, 61%), safety (14/51, 28%), and privacy (13/51, 26%); one-fourth (12/51) reported having no concerns. They were mostly interested in sharing data from mobile and connected devices with their health care providers followed by family, online communities, friends, and no one. About 41% (21/51) of the older adults surveyed reported having ever heard of an online health community, and roughly 40% (20/51) of the participants reported being interested in joining such a community. Most participants reported having access to a smartphone (38/51, 74%), and those with such access were significantly more likely to show interest in using a wearable health device (P<.001) and joining an online health community (P=.05). CONCLUSIONS: Our findings suggest that, although few older adults are currently using mobile and wearable devices and connected health technologies for managing health, they are open to this idea and are mostly interested in sharing such data with their health care providers. Further studies are warranted to explore strategies to balance the data sharing preference of older adults and how to best integrate mobile and wearable device data with clinical workflow for health care providers to promote healthy aging in place.

Proximity Ligation Real-Time PCR: A protein-based confirmatory method for the identification of semen and sperm cells from sexual assault evidence.

The positive identification of seminal fluids in sexual assault crimes is considered crucial evidence to determine whether a sexual act occurred or not. However, current presumptive methods lack specificity and sensitivity. Confirmation of semen by microscopic examination of spermatozoa is laborious, time consuming, and can sometimes lead to negative or inconclusive results. Here we report the use of the Proximity Ligation Real-Time PCR (PLiRT-PCR) assay as an attractive and promising confirmatory method for the identification of semen and sperm proteins using two polyclonal antibodies, Prostate Specific Antigen (PSA) and Sperm-Specific Protein (SP10), respectively. PLiRT-PCR, relies on protein recognition by pairs of proximity probes (antibody-DNA conjugates) that give rise to a ligated DNA strand. The ligated DNA strand is then amplified and detected by qPCR.

Bioinformatic removal of NUMT-associated variants in mitotiling next-generation sequencing data from whole blood samples.

Nuclear mitochondrial DNA segments (NUMTs) have arisen because of the transposition of segments of the mitochondrial DNA genome (mitogenome) into the nuclear genome. When using a "mitotiling" strategy, NUMTs may be more readily amplified when targeting the entire mitogenome compared to the control region, as hundreds of primers are required for complete sequencing coverage. In samples with a high percentage of nuclear DNA copies per cell, such as whole blood, NUMT coenrichment may be exacerbated. The present study examined bioinformatic approaches for removing NUMTs and NUMT-associated variants (NAVs) from next-generation sequence data generated using two mitotiling kits (Precision ID and QIAseq). Across 16 samples with low mtDNA copy number, NUMT coenrichment produced 890 NAVs with >5% variant frequency. The use of the consensus sequence to eliminate NUMT reads proved to be effective for QIAseq data, and resulted in >85% NAV removal in Precision ID data. This method was bolstered by NAV filtering in Precision ID analysis. Alternative high stringency mapping to the revised Cambridge Reference Sequence (rCRS) and the human genome reference GRCh38 for the QIAseq data caused a reduction in mitogenome coverage without complete NUMT removal. These bioinformatic solutions facilitate mitotiling sequence data analysis for low-level variant detection.

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.

AQME: A forensic mitochondrial DNA analysis tool for next-generation sequencing data.

The feasibility of generating mitochondrial DNA (mtDNA) data has expanded considerably with the advent of next-generation sequencing (NGS), specifically in the generation of entire mtDNA genome (mitogenome) sequences. However, the analysis of these data has emerged as the greatest challenge to implementation in forensics. To address this need, a custom toolkit for use in the CLC Genomics Workbench (QIAGEN, Hilden, Germany) was developed through a collaborative effort between the Armed Forces Medical Examiner System - Armed Forces DNA Identification Laboratory (AFMES-AFDIL) and QIAGEN Bioinformatics. The AFDIL-QIAGEN mtDNA Expert, or AQME, generates an editable mtDNA profile that employs forensic conventions and includes the interpretation range required for mtDNA data reporting. AQME also integrates an mtDNA haplogroup estimate into the analysis workflow, which provides the analyst with phylogenetic nomenclature guidance and a profile quality check without the use of an external tool. Supplemental AQME outputs such as nucleotide-per-position metrics, configurable export files, and an audit trail are produced to assist the analyst during review. AQME is applied to standard CLC outputs and thus can be incorporated into any mtDNA bioinformatics pipeline within CLC regardless of sample type, library preparation or NGS platform. An evaluation of AQME was performed to demonstrate its functionality and reliability for the analysis of mitogenome NGS data. The study analyzed Illumina mitogenome data from 21 samples (including associated controls) of varying quality and sample preparations with the AQME toolkit. A total of 211 tool edits were automatically applied to 130 of the 698 total variants reported in an effort to adhere to forensic nomenclature. Although additional manual edits were required for three samples, supplemental tools such as mtDNA haplogroup estimation assisted in identifying and guiding these necessary modifications to the AQME-generated profile. Along with profile generation, AQME reported accurate haplogroups for 18 of the 19 samples analyzed. The single errant haplogroup assignment, although phylogenetically close, identified a bug that only affects partial mitogenome data. Future adjustments to AQME's haplogrouping tool will address this bug as well as enhance the overall scoring strategy to better refine and automate haplogroup assignments. As NGS enables broader use of the mtDNA locus in forensics, the availability of AQME and other forensic-focused mtDNA analysis tools will ease the transition and further support mitogenome analysis within routine casework. Toward this end, the AFMES-AFDIL has utilized the AQME toolbox in conjunction with the CLC Genomics Workbench to successfully validate and implement two NGS mitogenome methods.

A performance evaluation of Nextera XT and KAPA HyperPlus for rapid Illumina library preparation of long-range mitogenome amplicons.

Next-generation sequencing (NGS) facilitates the rapid and high-throughput generation of human mitochondrial genome (mitogenome) data to build population and reference databases for forensic comparisons. To this end, long-range amplification provides an effective method of target enrichment that is amenable to library preparation assays employing DNA fragmentation. This study compared the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and the KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, Wilmington, MA) for enzymatic fragmentation and indexing of ∼8500bp mitogenome amplicons for Illumina sequencing. The Nextera XT libraries produced low-coverage regions that were consistent across all samples, while the HyperPlus libraries resulted in uniformly high coverage across the mitogenome, even with reduced-volume reaction conditions. The balanced coverage observed from KAPA HyperPlus libraries enables not only low-level variant calling across the mitogenome but also increased sample multiplexing for greater processing efficiency.

Concordance and reproducibility of a next generation mtGenome sequencing method for high-quality samples using the Illumina MiSeq.

Sanger-type sequencing (STS) of mitochondrial DNA (mtDNA), specifically the control region (CR), is routinely employed in forensics in human identification and missing persons scenarios. Yet next-generation sequencing (NGS) has the potential to overcome some of the major limitations of STS processing, permitting reasonable paths forward for full mitochondrial genome (mtGenome) sequencing, while also offering higher-throughput and higher sensitivity capabilities. To establish the accuracy and reproducibility of NGS for the development of mtDNA data, 90 DNA extracts that were previously used to generate forensic quality full mtGenomes using STS were sequenced using Nextera XT library preparation and the Illumina MiSeq. Using the same amplicon product, replicate library sets were generated and sequenced at different laboratories, and analysis was performed in replicate using the CLC Genomics Workbench. Both sequencing sets resulted in 99.998% of positions with greater than 10X coverage when 96 samples (including controls) were multiplexed. Overall, 99.9996% concordance was observed between the NGS data and the STS data for the full mtGenome. The only "discordant" calls involved low level point heteroplasmies, with the differences resulting from stochastic variation and/or the increased sensitivity of NGS. Higher sensitivity also allowed for the detection of a mixed sample previously not detected with STS. Additionally, variant calls were reproducible between sequencing sets and between software analysis versions with the variant frequency only differing by 0.23% and 0.01%, respectively. Further validation studies and specialized software functionality tailored to forensic practice should facilitate the incorporation of NGS processing into standard casework applications. The data herein comprise the largest, and likely most thoroughly examined, complete mtGenome STS-NGS concordance dataset available.

Mobile and Wearable Technology Needs for Aging in Place: Perspectives from Older Adults and Their Caregivers and Providers.

There is an increasing number of wearable trackers and mobile devices in the burgeoning world of digital health, the purpose of the study is to explore the role of these mobile and wearable tools among older adults aging in place. We conducted a cross sectional study using individual interviews with older adults and surveys with their caregivers or providers. We interviewed 29 residents living in a retirement community, and surveyed 6 caregivers or providers. The older adults had an average age of 88 years, most did not express interests on technology and heavily relied on providers for health tracking, while their professional caregivers or providers saw a great need to access older adults' health information collected from these mobile and wearable tools. Educating the older old on the benefits of mobile and wearable tools may address such discrepancy on needs of adopting mobile and wearable tools for aging in place.

T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA.

Cancer cells often arise progressively from "normal" to "pre-cancer" to "transformed" to "local metastasis" to "metastatic disease" to "aggressive metastatic disease". Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic "punctuated evolution" (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6-10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7-10 days. T1-A, T-2A, T3-HA and T4-PA cells when injected i.v. into the tail produced local metastasis in non-nude or nude NIH/Swiss mice. T4-PA cells were more widely metastatic than T3-HA cells when injected i.v. into nude mice. Evaluation of the injected mice indicated a general increase in metastatic potential of each cell line in the progression as compared to the GhrasT-NIH/3T3 transformed cells. A new photomicrographic technique to follow growth rates within six preselected 2×2mm(2) grids per plate is described. Average doubling times of the transformed cells GhrasT-NIH/3T3 (17h), T1A (17.5h), T2A (15.5h), T3-HA (17.5h) and T4-PA (18.5h) (average 17.2h) were significantly faster (P=0.006) than NIH Swiss primary embryonic cells and NIH/3T3 cells (22 h each). This cell series is currently used in this lab for studies of cancer cell inhibitors, mitochondrial biogenesis and gene expression and is available for further study by other investigators for intra- and inter-laboratory comparisons of WGS, transcriptome sequencing, SKY and other analyses. The genome rearrangements in these cells together with their phenotypic properties may help provide more insights into how one tumorigenic progression occurred to produce the various cell lines that led to the highly metastatic T4-PA cell line.

Full mtGenome reference data: development and characterization of 588 forensic-quality haplotypes representing three U.S. populations.

Though investigations into the use of massively parallel sequencing technologies for the generation of complete mitochondrial genome (mtGenome) profiles from difficult forensic specimens are well underway in multiple laboratories, the high quality population reference data necessary to support full mtGenome typing in the forensic context are lacking. To address this deficiency, we have developed 588 complete mtGenome haplotypes, spanning three U.S. population groups (African American, Caucasian and Hispanic) from anonymized, randomly-sampled specimens. Data production utilized an 8-amplicon, 135 sequencing reaction Sanger-based protocol, performed in semi-automated fashion on robotic instrumentation. Data review followed an intensive multi-step strategy that included a minimum of three independent reviews of the raw data at two laboratories; repeat screenings of all insertions, deletions, heteroplasmies, transversions and any additional private mutations; and a check for phylogenetic feasibility. For all three populations, nearly complete resolution of the haplotypes was achieved with full mtGenome sequences: 90.3-98.8% of haplotypes were unique per population, an improvement of 7.7-29.2% over control region sequencing alone, and zero haplotypes overlapped between populations. Inferred maternal biogeographic ancestry frequencies for each population and heteroplasmy rates in the control region were generally consistent with published datasets. In the coding region, nearly 90% of individuals exhibited length heteroplasmy in the 12418-12425 adenine homopolymer; and despite a relatively high rate of point heteroplasmy (23.8% of individuals across the entire molecule), coding region point heteroplasmies shared by more than one individual were notably absent, and transversion-type heteroplasmies were extremely rare. The ratio of nonsynonymous to synonymous changes among point heteroplasmies in the protein-coding genes (1:1.3) and average pathogenicity scores in comparison to data reported for complete substitutions in previous studies seem to provide some additional support for the role of purifying selection in the evolution of the human mtGenome. Overall, these thoroughly vetted full mtGenome population reference data can serve as a standard against which the quality and features of future mtGenome datasets (especially those developed via massively parallel sequencing) may be evaluated, and will provide a solid foundation for the generation of complete mtGenome haplotype frequency estimates for forensic applications.

Description of a teaching method for research education for palliative care healthcare professionals.

OBJECTIVE: Despite the rapidly growing availability of palliative care services, there is still much to be done in order to better support clinicians who are starting research programs. Among the barriers identified in the literature, methodological issues and lack of research training programs are often reported. Our aim was to describe an educational research method for healthcare professionals working in palliative care and to report the result of a survey conducted among a three-year sample of students. METHOD: The course was provided for a multidisciplinary group and was open to all healthcare professionals involved in palliative care. It took place over a single session during a full day. We used a 20-question e-survey to assess student outcomes (e.g., satisfaction, current status of their project). RESULTS: We received answers from 83 of the 119 students (70%) who took the course. The majority were physicians (n = 62, 75%), followed by nurses (n = 17, 21%). During the class, students assessed the role of the teacher as an information provider (n = 51, 61%), role model (n = 36, 43%), and facilitator (n = 33, 40%), and considered all of these roles as suitable, with a score of 3.9-4.7 out of 5. Participants reported a high level of support from the teacher, with a mean score of 8.2 (SD, 1.7) out of 10, and good overall satisfaction with a mean score of 7.6 (1.8). Finally, 51 participants (77%) were able to start their research project after the class, 27 (41%) to complete it, and 8 (12%) to submit their research to a journal or conference. SIGNIFICANCE OF RESULTS: Our results suggest that newer teaching methods such as roleplay, group work, and target acquisition are feasible and effective in a palliative research curriculum. Additional studies are needed to confirm the objective outputs of educational interventions, including research outputs.

Development of forensic-quality full mtGenome haplotypes: success rates with low template specimens.

Forensic mitochondrial DNA (mtDNA) testing requires appropriate, high quality reference population data for estimating the rarity of questioned haplotypes and, in turn, the strength of the mtDNA evidence. Available reference databases (SWGDAM, EMPOP) currently include information from the mtDNA control region; however, novel methods that quickly and easily recover mtDNA coding region data are becoming increasingly available. Though these assays promise to both facilitate the acquisition of mitochondrial genome (mtGenome) data and maximize the general utility of mtDNA testing in forensics, the appropriate reference data and database tools required for their routine application in forensic casework are lacking. To address this deficiency, we have undertaken an effort to: (1) increase the large-scale availability of high-quality entire mtGenome reference population data, and (2) improve the information technology infrastructure required to access/search mtGenome data and employ them in forensic casework. Here, we describe the application of a data generation and analysis workflow to the development of more than 400 complete, forensic-quality mtGenomes from low DNA quantity blood serum specimens as part of a U.S. National Institute of Justice funded reference population databasing initiative. We discuss the minor modifications made to a published mtGenome Sanger sequencing protocol to maintain a high rate of throughput while minimizing manual reprocessing with these low template samples. The successful use of this semi-automated strategy on forensic-like samples provides practical insight into the feasibility of producing complete mtGenome data in a routine casework environment, and demonstrates that large (>2kb) mtDNA fragments can regularly be recovered from high quality but very low DNA quantity specimens. Further, the detailed empirical data we provide on the amplification success rates across a range of DNA input quantities will be useful moving forward as PCR-based strategies for mtDNA enrichment are considered for targeted next-generation sequencing workflows.

A 50-SNP assay for biogeographic ancestry and phenotype prediction in the U.S. population.

When an STR DNA profile obtained from crime scene evidence does not match identified suspects or profiles from available databases, further DNA analyses targeted at inferring the possible ancestral origin and phenotypic characteristics of the perpetrator could yield valuable information. Single Nucleotide Polymorphisms (SNPs), the most common form of genetic polymorphisms, have alleles associated with specific populations and/or correlated to physical characteristics. We have used single base primer extension (SBE) technology to develop a 50 SNP assay (composed of three multiplexes) designed to predict ancestry among the primary U.S. populations (African American, East Asian, European American, and Hispanic American/Native American), as well as pigmentation phenotype (eye, hair, and skin color) among European American. We have optimized this assay to a sensitivity level comparable to current forensic DNA analyses, and shown robust performance on forensic-type samples. In addition, we developed a prediction model for ancestry in the U.S. population, based on the random match probability and likelihood ratio formulas already used in forensic laboratories. Lastly, we evaluated the biogeographic ancestry prediction model using a test set, and we evaluated an existing model for eye color with our U.S. sample set. Using these models with recommended thresholds, the 50 SNP assay provided accurate ancestry information in 98.6% of the test set samples, and provided accurate eye color information in 61% of the European samples tested (25% were inconclusive and 14% were incorrect). This method, which uses equipment already available in forensic DNA laboratories, is recommended for use in U.S. forensic casework to provide additional information about the donor of a DNA sample when the STR profile has not been linked to an individual.

Directed evolution of a fluorogen-activating single chain antibody for function and enhanced brightness in the cytoplasm.

Directed evolution is an exceptionally powerful tool that uses random mutant library generation and screening techniques to engineer or optimize functions of proteins. One class of proteins for which this process is particularly effective is antibodies, where properties such as antigen specificity and affinity can be selected to yield molecules with improved efficacy as molecular labels or in potential therapeutics. Typical antibody structure includes disulfide bonds that are required for stability and proper folding of the domains. However, these bonds are unable to form in the reducing environment of the cytoplasm, stymieing the effectiveness of optimized antibodies in many research applications. We have removed disulfide-forming cysteine residues in a single chain antibody fluorogen-activating protein (FAP), HL4, and employed directed evolution to select a derivative that is capable of activity in the cytoplasm. A subsequent round of directed evolution was targeted at increasing the overall brightness of the fluoromodule (FAP-fluorogen complex). Ultimately, this approach produced a novel FAP that exhibits strong activation of its cognate fluorogen in the reducing environment of the cytoplasm, significantly expanding the range of applications for which fluoromodule technology can be utilized.