Abstract The PowerPlex® Y23 System combines the seventeen Y-STR loci in current commercially available Y-STR kits (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, ...DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4) with six new highly discriminating Y-STR loci (DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643). These six new loci have higher gene diversities than most of the loci in other commercial Y-STR analysis kits, allowing for further distinction between unrelated male individuals. In addition, the inclusion of two rapidly mutating Y-STR loci may allow for the discrimination of related individuals. The PowerPlex® Y23 System is designed to amplify DNA from purified extracts as well as direct amplification from substrates used to collect database samples (e.g. swabs and storage cards). Validation of the PowerPlex® Y23 System includes all of the studies required by the FBI and SWGDAM. The results demonstrate that the PowerPlex® Y23 System is a robust and reliable amplification kit capable of overcoming high concentrations of commonly encountered inhibitors such as hematin, humic acid, and tannic acid. Full profiles are consistently detected with 62.5 pg of male DNA, even in the presence of excessive amounts of female DNA, establishing the PowerPlex® Y23 System as a sensitive method for Y-STR testing. Complete Y-STR profiles are detected from mixed samples with 62.5 pg of male DNA in a background of 400 ng of female DNA or 125 pg of male DNA mixed with 3000 ng of female DNA.
Highlights • The PowerQuant® System was developed to quantify human DNA and assess sample quality. • A developmental validation following SWGDAM guidelines was completed. • Degraded DNA, inhibited ...samples, and mixtures of male/female DNA were evaluated. • Results demonstrate reliability, reproducibility and suitability for forensic use. • The PowerQuant® System distinguishes degraded samples from inhibited samples.
We demonstrate the capabilities of a centrifugal polyethylene terephthalate toner (PeT) microdevice for integrated on-chip reagent mobilization, mixing, and PCR amplification for genetic analysis of ...short tandem repeats (STR). Fluid flow, including reagent mobilization and mixing, is achieved by centrifugal force, eliminating the need for bulky instrumentation. The use of a passive valve also eliminates the need for extra hardware and simplifies the chip and the device design. A custom-built system is capable of thermocycling through a dual Peltier clamping system, as well as variable rate spinning with a DC motor. A multiplex PCR amplification of alleles associated with 18 genomic loci was successfully performed on-chip, followed by capillary electrophoretic separation, which showed efficient amplification of DNA from multiple sources. The genetic profiles generated were 100% concordant with those obtained using conventional PCR methods. The resultant system represents a novel microfluidic PCR amplification platform that uses inexpensive PCR microdevices that are simple to fabricate, yet effective for complex, multiplexed PCR.
•The PowerQuant¨r) System was developed to quantify human DNA and assess sample quality.•A developmental validation following SWGDAM guidelines was completed.•Degraded DNA, inhibited samples, and ...mixtures of male/female DNA were evaluated.•Results demonstrate reliability, reproducibility and suitability for forensic use.•The PowerQuant¨r) System distinguishes degraded samples from inhibited samples.
Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant¨r) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant¨r) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay tm)s specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.
We demonstrate the capabilities of a centrifugal polyethylene terephthalate toner (PeT) microdevice for integrated on-chip reagent mobilization, mixing, and PCR amplification for genetic analysis of ...short tandem repeats (STR). Fluid flow, including reagent mobilization and mixing, is achieved by centrifugal force, eliminating the need for bulky instrumentation. The use of a passive valve also eliminates the need for extra hardware and simplifies the chip and the device design. A custom-built system is capable of thermocycling through a dual Peltier clamping system, as well as variable rate spinning with a DC motor. A multiplex PCR amplification of alleles associated with 18 genomic loci was successfully performed on-chip, followed by capillary electrophoretic separation, which showed efficient amplification of DNA from multiple sources. The genetic profiles generated were 100% concordant with those obtained using conventional PCR methods. The resultant system represents a novel microfluidic PCR amplification platform that uses inexpensive PCR microdevices that are simple to fabricate, yet effective for complex, multiplexed PCR.
We demonstrate the capabilities of a centrifugal polyethylene terephthalate toner (PeT) microdevice for genetic analysis of short tandem repeats (STR)
via
PCR amplification.
Human Cell Line and Tissue Sample Authentication Ewing, Margaret M.; McLaren, Robert S.; Hebble, Kathryn D. ...
Journal of biomolecular techniques,
05/2013, Letnik:
24, Številka:
Suppl
Journal Article
Recenzirano
Background: Short Tandem Repeat (STR) genotyping analysis is a proven technology for uniquely identifying virtually all human samples. STR genotyping was adopted as the preferred technology for ...identification of human tissue culture cell lines by the ATCC Standards Development Organization (ASN-0002: Authentication of Human Cell Lines: Standardization of STR Profiling). We developed new automation-compatible protocols/systems for generating STR profiles from human cell lines or tissue samples. Methods: We adapted the STR genotyping systems routinely used for forensic and paternity testing to better meet the needs of genomic core facilities. Modifications include balancing for higher amounts of template DNA, configuring the reagents for compatibility with high throughput robotic workstations, and supporting electrophoretic separation and analysis on a wider variety of instrument and software platforms. Results: The GenePrint® 10 and GenePrint® 21 Systems allow for multiplexed genotyping of 9 or 20 STR loci, plus the amelogenin gender marker. STR analysis with these loci uniquely identify virtually all human cell lines and tissue samples (body fluids, tissues and extracted DNA) and confirm the absence of cross-contamination or a sample switch. The GenePrint® 10 System is compatible with purified DNA or direct amplification from cells deposited on FTA® Cards (GE Whatman). Both genotyping systems meet the ASN-0002 standard and include the loci represented on the National Center for Biotechnology Information (NCBI) human cell line database:
http://www.ncbi.nlm.nih.gov/biosample?term=human%20cell%20line%20STR%20profile
Conclusions: STR genotyping analysis with the GenePrint® 10 and GenePrint® 21 Systems can establish human cell line or tissue sample identity and confirm the absence of contamination with other human cell lines or tissues. The methods are compatible with DNA concentrations, robotic protocols, instrumentation, and genotyping software typically used in genomics core facilities.
Background
Chronic pain patients increasingly seek treatment through mindfulness meditation.
Purpose
This study aims to synthesize evidence on efficacy and safety of mindfulness meditation ...interventions for the treatment of chronic pain in adults.
Method
We conducted a systematic review on randomized controlled trials (RCTs) with meta-analyses using the Hartung-Knapp-Sidik-Jonkman method for random-effects models. Quality of evidence was assessed using the GRADE approach. Outcomes included pain, depression, quality of life, and analgesic use.
Results
Thirty-eight RCTs met inclusion criteria; seven reported on safety. We found low-quality evidence that mindfulness meditation is associated with a small decrease in pain compared with all types of controls in 30 RCTs. Statistically significant effects were also found for depression symptoms and quality of life.
Conclusions
While mindfulness meditation improves pain and depression symptoms and quality of life, additional well-designed, rigorous, and large-scale RCTs are needed to decisively provide estimates of the efficacy of mindfulness meditation for chronic pain.
Preclinical studies using bone marrow derived cells to treat traumatic brain injury have demonstrated efficacy in terms of blood–brain barrier preservation, neurogenesis, and functional outcomes. ...Phase 1 clinical trials using bone marrow mononuclear cells infused intravenously in children with severe traumatic brain injury demonstrated safety and potentially a central nervous system structural preservation treatment effect. This study sought to confirm the safety, logistic feasibility, and potential treatment effect size of structural preservation/inflammatory biomarker mitigation in adults to guide Phase 2 clinical trial design. Adults with severe traumatic brain injury (Glasgow Coma Scale 5–8) and without signs of irreversible brain injury were evaluated for entry into the trial. A dose escalation format was performed in 25 patients: 5 controls, followed 5 patients in each dosing cohort (6, 9, 12 ×106 cells/kg body weight), then 5 more controls. Bone marrow harvest, cell processing to isolate the mononuclear fraction, and re‐infusion occurred within 48 hours after injury. Patients were monitored for harvest‐related hemodynamic changes, infusional toxicity, and adverse events. Outcome measures included magnetic resonance imaging‐based measurements of supratentorial and corpus callosal volumes as well as diffusion tensor imaging‐based measurements of fractional anisotropy and mean diffusivity of the corpus callosum and the corticospinal tract at the level of the brainstem at 1 month and 6 months postinjury. Functional and neurocognitive outcomes were measured and correlated with imaging data. Inflammatory cytokine arrays were measured in the plasma pretreatment, posttreatment, and at 1 and 6 month follow‐up. There were no serious adverse events. There was a mild pulmonary toxicity of the highest dose that was not clinically significant. Despite the treatment group having greater injury severity, there was structural preservation of critical regions of interest that correlated with functional outcomes. Key inflammatory cytokines were downregulated. Treatment of severe, adult traumatic brain injury using an intravenously delivered autologous bone marrow mononuclear cell infusion is safe and logistically feasible. There appears to be a treatment signal as evidenced by central nervous system structural preservation, consistent with previous pediatric trial data. Inflammatory biomarkers are downregulated after cell infusion. Stem Cells 2016
Video Highlight: https://youtu.be/UiCCPIe-IaQ Stem Cells 2017;35:1065–1079
Autologous bone marrow mononuclear cells (BMMNC) in escalating doses of 6, 9, 12 × 106 cells/kg were injected intravenously to treat patients with severe traumatic brain injury within 48 hours of injury and compared to concurrent, placebo‐ controlled controls in a Phase 1/2a clinical trial. Primary outcome measures included white matter volumetrics and corpus callosal (CC) fractional anisotropy (FA) as a marker of myelination/myelin loss over time. There was a strong treatment effect noted (Cohen's d) in corpus callosal FA and mean diffusivity (MD), providing powerful estimates for the follow‐on trial sample size determination. The corpus callosal fiber tractography from individual patients imaged in the early period postinjury (1 month) and at 6 months, and those images demonstrate the fiber tract drop out over time in the untreated patient that is preserved with treatment. These data are summarized in the table below.
CONTEXT Atypical antipsychotic medications are commonly used for off-label conditions such as agitation in dementia, anxiety, and obsessive-compulsive disorder. OBJECTIVE To perform a systematic ...review on the efficacy and safety of atypical antipsychotic medications for use in conditions lacking approval for labeling and marketing by the US Food and Drug Administration. DATA SOURCES AND STUDY SELECTION Relevant studies published in the English language were identified by searches of 6 databases (PubMed, EMBASE, CINAHL, PsycInfo, Cochrane DARE, and CENTRAL) from inception through May 2011. Controlled trials comparing an atypical antipsychotic medication (risperidone, olanzapine, quetiapine, aripiprazole, ziprasidone, asenapine, iloperidone, or paliperidone) with placebo, another atypical antipsychotic medication, or other pharmacotherapy for adult off-label conditions were included. Observational studies with sample sizes of greater than 1000 patients were included to assess adverse events. DATA EXTRACTION Independent article review and study quality assessment by 2 investigators. DATA SYNTHESIS Of 12 228 citations identified, 162 contributed data to the efficacy review. Among 14 placebo-controlled trials of elderly patients with dementia reporting a total global outcome score that includes symptoms such as psychosis, mood alterations, and aggression, small but statistically significant effects sizes ranging from 0.12 and 0.20 were observed for aripiprazole, olanzapine, and risperidone. For generalized anxiety disorder, a pooled analysis of 3 trials showed that quetiapine was associated with a 26% greater likelihood of a favorable response (defined as at least 50% improvement on the Hamilton Anxiety Scale) compared with placebo. For obsessive-compulsive disorder, risperidone was associated with a 3.9-fold greater likelihood of a favorable response (defined as a 25% improvement on the Yale-Brown Obsessive Compulsive Scale) compared with placebo. In elderly patients, adverse events included an increased risk of death (number needed to harm NNH = 87), stroke (NNH = 53 for risperidone), extrapyramidal symptoms (NNH = 10 for olanzapine; NNH = 20 for risperidone), and urinary tract symptoms (NNH range = 16-36). In nonelderly adults, adverse events included weight gain (particularly with olanzapine), fatigue, sedation, akathisia (for aripiprazole), and extrapyramidal symptoms. CONCLUSIONS Benefits and harms vary among atypical antipsychotic medications for off-label use. For global behavioral symptom scores associated with dementia in elderly patients, small but statistically significant benefits were observed for aripiprazole, olanzapine, and risperidone. Quetiapine was associated with benefits in the treatment of generalized anxiety disorder, and risperidone was associated with benefits in the treatment of obsessive-compulsive disorder; however, adverse events were common.
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