Vermont State Hospital

The following report summarizes a study performed on seized drug exhibits collected in two U.S. states to evaluate the presence and identification of cutting agents. Aliquots of seized drug materials from Kentucky (n = 200) and Vermont (n = 315) were prepared using a dilute-and-shoot procedure. Initial analysis was performed using gas chromatography-mass spectrometry (GC-MS) followed by analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF). Active compounds detected overall included caffeine (31.0%), quinine/quinidine (24.7%), levamisole (11.6%), acetaminophen, (8.2%) and procaine (8.2%). These compounds were found with several drugs of abuse, such as heroin, fentanyl, methamphetamine, and cocaine. This novel information about cutting agents used to dilute or alter drugs of abuse is important to criminal investigations and in the management of acute intoxications at health centers. However, common methodologies for analysis and standard reporting practices frequently do not include cutting agents, resulting in lacking or inadequate information regarding prevalence of these substances.

Cyclic‐AMP‐dependent protein kinase A (PKA) is a serine threonine kinase that is involved in regulating cellular processes such as migration, proliferation, metabolism and survival in response to a variety of environmental cues. PKA exists in cells as an inactive tetrameric holoenzyme, comprised of two regulatory (R) and two catalytic (C) subunits. Activation of PKA's enzymatic activity typically occurs in response to stimuli that induce increases in intracellular cyclic adenosine monophosphate (cAMP). In conducting a proteomics screen to identify novel binding partners of PKA, we found that the C subunit of PKA was consistently associated with the La‐related binding protein 4 (LARP4) in HEK293 cells. LARP4 is a member of the evolutionarily conserved family of La‐related proteins yet the functions of LARP4 are largely unknown. Therefore, we sought to further investigate this novel signaling complex. The binding of LARP4 to PKA was validated through co‐immunoprecipitation experiments. Results revealed that LARP4 was present in immunoprecipitates of the C and RII subunits, indicating that LARP4 interacts with PKA holoenzymes. Moreover, LARP4 immunoprecipitates demonstrated significant PKA kinase activity even in the absence of cAMP, suggesting that the PKA associated with LARP4 is active. Preliminary experiments conducted with mouse embryo fibroblasts (MEFs) from wildtype litter control or LARP4 knockout mice revealed differential phosphorylation of endogenous PKA substrates as measured by western blot analysis using a phospho‐PKA substrate antibody. This result suggests that the interaction of LARP4 with PKA may regulate downstream signaling targets. Current experiments are focused on characterizing the biophysical interaction between LARP4 and PKA and determining how this molecular complex influences cellular function.Support or Funding InformationThis work was supported by a Scientist Development Grant from the AHA (to PBD) and an Endowed Professorship (College of Nursing and Health Sciences, University of Vermont).

This review focuses upon a critical step in forensic biology: detection and quantification of human DNA from biological samples. Determination of the quantity and quality of human DNA extracted from biological evidence is important for several reasons. Firstly, depending on the source and extraction method, the quality (purity and length), and quantity of the resultant DNA extract can vary greatly. This affects the downstream method as the quantity of input DNA and its relative length can determine which genotyping procedure to use-standard short-tandem repeat (STR) typing, mini-STR typing or mitochondrial DNA sequencing. Secondly, because it is important in forensic analysis to preserve as much of the evidence as possible for retesting, it is important to determine the total DNA amount available prior to utilizing any destructive analytical method. Lastly, results from initial quantitative and qualitative evaluations permit a more informed interpretation of downstream analytical results. Newer quantitative techniques involving real-time PCR can reveal the presence of degraded DNA and PCR inhibitors, that provide potential reasons for poor genotyping results and may indicate methods to use for downstream typing success. In general, the more information available, the easier it is to interpret and process the sample resulting in a higher likelihood of successful DNA typing. The history of the development of quantitative methods has involved two main goals-improving precision of the analysis and increasing the information content of the result. This review covers advances in forensic DNA quantification methods and recent developments in RNA quantification.

ABSTRACT Interest in non-timber forest products (NTFPs) and forest certification burgeoned in the late 1980s in response, partly, to concern over destruction of tropical rainforests. Eventually the two concepts were merged and proposed as a marketing option for NTFP producers in both temperate and tropical countries. This paper examines the experiences of NTFP certification within the Forest Stewardship Council over the past eight years. We describe some of the impediments that NTFP producers have encountered in obtaining certification, as well as successes. We find that certification and NTFPs are often incompatible. For those cases where NTFP certification is warranted, we offer several suggestions for improving the likelihood of success. The triumph of neoliberalism in international trade and aid circles all but assures continued commercialization of NTFPs. Certification may help ensure the environmental sustainability of such initiatives but it must not become a tool to exclude gatherers and or threaten local livelihoods.

A single duplex assay to determine both the amount of total human DNA and the amount of male DNA in a forensic sample has been developed. This assay is based on TaqMan technology and uses the multicopy Alu sequence to quantitate total human DNA and the multicopy DYZ5 sequence to quantitate Y chromosomal (male) DNA. The assay accepts a wide concentration range of input DNA (2 muL of 64 ng/microL to 0.5 pg/microL), and also allows detection of PCR failure. The PCR product sizes Alu (127 bp) and DYZ5 (137bp) approximate that of the smaller short tandem repeats (STRs) which should make the assay predictive of STR success with degraded DNA. The assay was optimized for probe/primer concentrations and BSA addition and validated on its reproducibility, on its human specificity, on its nonethnic variability, for artificial mixtures and adjudicated casework, for the effect of inhibitors and for state of DNA degradation. This assay should prove very usual in forensic analyses because knowing the relative amounts of male versus female DNA can allow the examiner to decide which samples may yield the most probative value in a case or direct the samples to methods that would yield the greatest information.

Prior to forensic implementation, a profiling system requires validation following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). In this work two such systems, AmpFlSTR Profiler Plus and AmpFfSTR COfiler have been validated according to the guidelines provided by TWGDAM. Profiler Plus and COfiler simultaneously amplify nine and six STR loci respectively; both also amplify a portion of the amelogenin gene. Performance of the two STR multiplex systems under conditions set forth by TWGDAM was robust and reproducible, indicating that these systems are suitable for use in forensic analysis. Additionally, specific sections of the TWGDAM validation guidelines are especially valuable in terms of familiarizing users with particular limitations of the systems prior to taking on casework.