Simultaneous Identification of Four “Legal High” Plant Species in a Multiplex PCR High‐Resolution Melt Assay,

The international prevalence of “legal high” drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real‐time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high‐resolution melt using LCGreen Plus^®. The PCR assay was evaluated based on the following: (i) specificity and selectivity—primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity—serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability—sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging “legal high” species.     

Development of a PCR High‐Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional “Unregulated Legal High” Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose),

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an “unregulated legal high” in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be “thujone‐free.” However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high‐resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three “unregulated legal high” species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.     

Social and economic impact of the commercialization of the Argus II artificial retina in the United States

Each year, the United States invests about $45 billion in research conducted by federal researchers within federal laboratories. These efforts generate extensive social benefits when results are transferred to the private sector. It is important that we effectively quantify the economic and societal impact of federal technology transfer activities to inform taxpayers and policymakers about the value of public investments in this form of research. The Argus II device, an artificial retina commercialized in the United States by Second Sight in 2013, provides a rich example of how private sector innovation can be enhanced by research collaborations with federal labs and academia. Over the 25-year journey from idea to product, Second Sight carried out research and development collaborations with six Department of Energy national laboratories and seven universities. The case of Argus II also offers valuable insight into (1) how private industry, academia, and government can work together to bring socially beneficial innovations to fruition and (2) the tradeoffs inherent in these public–private collaborations. In this paper, we use a Markov model to estimate the realized and potential future social benefits associated with Argus II. We provide an interactive tool that can be used to replicate our findings and modify assumptions using updated patient information as it becomes available. We also provide insight into the aspects of federal involvement surrounding the development of Argus II that contributed to its successful commercialization and discuss other spillover benefits from these public–private collaborations.     

Criminalization and the Collateral Consequences of Conviction

Convicted offenders face a host of so-called “collateral” consequences: formal measures such as legal restrictions on voting, employment, housing, or public assistance, as well as informal consequences such as stigma, family tensions, and financial insecurity. These consequences extend well beyond an offender’s criminal sentence itself and are frequently more burdensome than the sentence. This essay considers two respects in which collateral consequences may be relevant to the question of what the state should, or may, criminalize. First, they may be relevant according to specific accounts of criminalization, including plausible versions of the harm principle and legal moralism. Second, they may be relevant to the legitimacy of state criminalization more generally. Thus for legal theorists concerned with the issue of legitimate criminalization, normative questions raised by collateral consequences are of central importance.     

Additional predictions for forensic DNA phenotyping of externally visible characteristics using the ForenSeq and Imagen kits

Multiplex DNA typing methods using massively parallel sequencing can be used to predict externally visible characteristics (EVCs) in forensic DNA phenotyping through the analysis of single-nucleotide polymorphisms. The focus of EVC determination has focused on hair color, eye color, and skin tone as well as visible biogeographical ancestry features. In this study, we researched off-label applications beyond what is currently marketed by the manufacturer of the Verogen ForenSeq kit primer set B and Imagen primer set E SNP loci. We investigated additional EVC predictions by examining published genome wide sequencing studies and reported allele-specific gene expression and predictive values. We have identified 15 SNPs included in the ForenSeq kit panel and Imagen kits that have additional EVC prediction capabilities beyond what is published in the Verogen manuals. The additional EVCs that can be predicted include hair graying, ephelides hyperpigmented spots, dermatoheliosis, facial pigmented spots, standing height, pattern balding, helix-rolling ear morphology, hair shape, hair thickness, facial morphology, eyebrow thickness, sarcoidosis, obesity, vitiligo, and tanning propensity. The loci can be used to augment and refine phenotype predictions with software such as MetaHuman for missing persons, cold case, and historic case investigations.     

Do School Report Cards Produce Accountability Through the Ballot Box?

Public education has been transformed by the widespread adoption of accountability systems that involve the dissemination of school district performance information. Using data from Ohio, we examine if elections serve as one channel through which these accountability systems might lead to improvements in educational quality. We find little evidence that poor performance on widely disseminated state and federal indicators has an impact on school board turnover, the vote share of sitting school board members, or superintendent tenure, suggesting that the dissemination of district performance information puts little (if any) electoral pressure on elected officials to improve student achievement.     

Detection and Identification of Kratom (Mitragyna speciosa) and Marijuana (Cannabis sativa) by a Real-Time Polymerase Chain Reaction High-Resolution Melt Duplex Assay,

Mitragyna speciosa (MS), a plant commonly known as kratom, is a widely used “legal high” opiate alternative for pain relief. DNA extracted from MS and 26 additional plant species was amplified by PCR using primers targeting the strictosidine beta-D-glucosidase (SGD) and secologanin synthase 2 (SLS2) genes and detected by high-resolution melt curves using three intercalating dyes. Amplicon sizes were confirmed using agarose gel electrophoresis. The observed melt temperatures for SGD and SLS2 were 77.08 ± 0.38°C and 77.61 ± 0.46°C, respectively, using SYBR^® Green I; 80.18 ± 0.27°C and 80.59 ± 0.08°C, respectively, using Radiant^™ Green; and 82.19 ± 0.04°C and 82.62 ± 0.13°C, respectively, using the LCGreen^® PLUS dye. The SLS2 primers demonstrated higher specificity and identified MS DNA at 0.05 ng/μL. In a duplex reaction, SLS2 and tetrahydrocannabinoic acid synthase gene primers detected and differentiated MS and Cannabis sativa (CS) by melt peaks at 82.63 ± 0.35°C and 85.58 ± 0.23°C, respectively, using LCGreen^® PLUS.     

Evaluation of Two New Methods for DNA Extraction of “Legal High” Plant Species

A quick, simple, and high-yield nucleic acid isolation process is crucial for high-quality DNA analysis. The ability of the MicroGEM PDQeX phytoGEM system and Omega Bio-tek E.Z.N.A.® Plant DS Mini kit to extract PCR-ready DNA was evaluated by extracting the forensically relevant “legal high” plant species: Ipomoea purpurea, Artemisia absinthium, Mitragyna speciosa, Datura stramonium, and Papaver somniferum. The plant material was pulverized, processed using the manufacturer’s plant protocol for the PDQeX Nucleic Acid Extraction or the manufacturer’s protocol for the Omega extraction, quantified using the Invitrogen Qubit 2.0 Fluorometer, and analyzed for amplifiability by PCR using a Qiagen Rotor-Gene Q instrument and published assays. The DNA amplicons for the legal high species produced high-resolution melt curves concordant with the melts observed when DNA was isolated using the Qiagen DNeasy Plant Mini Kit in previous studies.