Janet E. Barnette

PKCγ Regulates Syndecan-2 Inside-Out Signaling during Xenopus Left-Right Development

The transmembrane proteoglycan syndecan-2 cell nonautonomously regulates left-right (LR) development in migrating mesoderm by an unknown mechanism, leading to LR asymmetric gene expression and LR orientation of the heart and gut. Here, we demonstrate that protein kinase C gamma (PKCgamma) mediates phosphorylation of the cytoplasmic domain of syndecan-2 in right, but not left, animal cap ectodermal cells. Notably, both phosphorylation states of syndecan-2 are obligatory for normal LR development, with PKCgamma-dependent phosphorylated syndecan-2 in right ectodermal cells and nonphosphorylated syndecan-2 in left cells. The ectodermal cells contact migrating mesodermal cells during early gastrulation, concurrent with the transmission of LR information. This precedes the appearance of monocilia and is one of the earliest steps of LR development. These results demonstrate that PKCgamma regulates the cytoplasmic phosphorylation of syndecan-2 and, consequently, syndecan-2-mediated inside-out signaling to adjacent cells.

Regulation of casein kinase i∈ activity by Wnt signaling

The Wnt/β-catenin signaling pathway is important in both development and cancer. Casein kinase Iϵ (CKIϵ) is a positive regulator of the canonical Wnt pathway. CKIϵ itself can be regulated in vitro by inhibitory autophosphorylation, and recent data suggest that in vivo kinase activity can be regulated by extracellular stimuli. We show here that the phosphorylation state and kinase activity of CKIϵ are directly regulated by Wnt signaling. Coexpression of XWnt-8 or addition of soluble Wnt-3a ligand led to a significant and rapid increase in the activity of endogenous CKIϵ. The increase in CKIϵ activity is the result of decreased inhibitory autophosphorylation because it is abolished by preincubation of immunoprecipitated kinase with ATP. Furthermore, mutation of CKIϵ inhibitory autophosphorylation sites creates a kinase termed CKIϵ(MM2) that is significantly more active than CKIϵ and is not activated further upon Wnt stimulation. Autoinhibition of CKIϵ is biologically relevant because CKIϵ(MM2) is more effective than CKIϵ at activating transcription from a Lef1-dependent promoter. Finally, CKIϵ(MM2) expression in Xenopus embryos induces both axis duplication and additional developmental abnormalities. The data suggest that Wnt signaling activates CKIϵ by causing transient dephosphorylation of critical inhibitory sites present in the carboxyl-terminal domain of the kinase. Activation of the Wnt pathway may therefore stimulate a cellular phosphatase to dephosphorylate and activate CKIϵ

Stable Isotopes in Forensics Applications

Stable isotope analysis has a lengthy application history in the fields of biology, ecology, and geology but its application in forensic investigations is relatively new. A recent report by the National Research Council on the strength of the forensic sciences in the United States highlighted areas of weakness, including the lack of a tested scientific foundation for many of the analytical techniques used in examinations. Stable isotope analysis has a strong scientific foundation developed in the academic community and could thus play a major role in the forensic community as a powerful tool in the investigators toolbox.

Hydrogen and oxygen isotope values in hydrogen peroxide

Hydrogen peroxide (H(2)O(2)) is a widely used oxidizer with many commercial applications; unfortunately, it also has terrorist-related uses. We analyzed 97 hydrogen peroxide solutions representing four grades purchased across the United States and in Mexico. As expected, the range of hydrogen (δ(2)H, 230‰) and oxygen (δ(18)O, 24‰) isotope values of the H(2)O(2) solutions was large, reflecting the broad isotopic range of dilution waters. This resulted in predictable linear relationships of δ(2)H and δ(18)O values of H(2)O(2) solutions that were near parallel to the Meteoric Water Line (MWL), offset by the concentration of H(2)O(2) in the solution. By grade, dilute (3 to 35%) H(2)O(2) solutions were not statistically different in slope. Although the δ(2)H values of manufactured H(2)O(2) could be different from those of water, rapid H(2)O(2)-H(2)O exchange of H atoms eliminated any distinct isotope signal. We developed a method to measure the δ(18)O value of H(2)O(2) independent of dilution water by directly measuring O(2) gas generated from a catalase-induced disproportionation reaction. We predicted that the δ(18)O values of H(2)O(2) would be similar to that of atmospheric oxygen (+23.5‰), the predominant source of oxygen in the most common H(2)O(2) manufacturing process (median disproportionated δ(18)O=23.8‰). The predictable H-O relationships in H(2)O(2) solutions make it possible to distinguish commercial dilutions from clandestine concentration practices. Future applications of this work include synthesis studies that investigate the chemical link between H(2)O(2) reagents and peroxide-based explosive products, which may assist law enforcement in criminal investigations.

Radiocarbon dating of seized ivory confirms rapid decline in African elephant populations and provides insight into illegal trade

Significance C-14 dating methods can be used to determine the time of death of wildlife products. We evaluate poaching patterns of elephants in Africa by using 14C to determine lag time between elephant death and recovery of ivory by law enforcement officials. Most ivory in recent seizures has lag times of less than 3 y. Lag times for ivory originating in East Africa are shorter, on average, than the lag times for ivory originating in the Tridom region (Cameroon–Gabon–Congo). The 14C data show little or no evidence that large-scale ivory shipments contained ivory stockpiled over long time periods. Little, if any, “old” ivory (i.e., >10 y) seems to contribute to large ivory shipments. Carbon-14 measurements on 231 elephant ivory specimens from 14 large ivory seizures (≥0.5 ton) made between 2002 and 2014 show that most ivory (ca. 90%) was derived from animals that had died less than 3 y before ivory was confiscated. This indicates that the assumption of recent elephant death for mortality estimates of African elephants is correct: Very little “old” ivory is included in large ivory shipments from Africa. We found only one specimen of the 231 analyzed to have a lag time longer than 6 y. Patterns of trade differ by regions: East African ivory, based on genetic assignments of geographic origin, has a much higher fraction of “rapid” transit than ivory originating in the Tridom region of Cameroon–Gabon–Congo. Carbon-14 is an important tool in understanding patterns of movement of illegal wildlife products.

Urban water - a new frontier in isotope hydrology.

ABSTRACT Isotope hydrology has focused largely on landscapes away from densely inhabited regions. In coming decades, it will become increasingly more important to focus on water supplies and dynamics within urban systems. Stable isotope analyses provide important information to water managers within large cities, particularly in arid regions where evaporative histories of water sources, vulnerabilities, and reliabilities of the water supplies can be major issues. Here the spatial and vertical understanding of water supporting urban systems that comes from stable isotope analyses can serve as a useful management tool. We explore this research frontier using the coupled natural–human landscape of the Salt Lake Valley, USA, with its greater than one million inhabitants. We first provide data on the stable isotope ratios of the hydrologic system’s primary components: precipitation, incoming surface waters, and terminus waters in this closed basin. We then explore the spatial and temporal patterns of drinking waters within the urban landscape and the new opportunities to better link isotope ratio data with short- and long-term management interests of water managers.

14C Calibration Curves for Modern Plant Material from Tropical Regions of South America

Two Δ14C calibration curves have been produced that allow determination of the statistical average age of coca leaf and cocaine base specimens produced for the time period 1979–2009. These calibration curves are based on field collections of specimens in Bolivia, Colombia, Ecuador, and Peru. The coca leaf F14C and Δ14C calibration curves can be used to predict the ages of botanical tissues collected in tropical South America and possibly extended to other tropical locations. The cocaine F14C and Δ14C calibration curves can be used to predict the ages of seized cocaine specimens. Because the Δ14C of the atmosphere is diminishing, the precision of this approach for age determinations will continue to get less precise over time as atmospheric 14C content continues to decline.

Accuracy and precision of a laser-spectroscopy approach to the analysis of δ2H and δ18O in human urine

The isotope ratio analysis of body water often involves large sample numbers and lengthy sample processing. Here we demonstrate the ability of isotope ratio infrared spectroscopy (IRIS) to rapidly and accurately analyse the isotope ratios of water in urine. We analysed water extracted from human urine using traditional isotope ratio mass spectrometry (IRMS) and compared those values with IRIS-analysed extracted water and un-extracted urine. Regression analyses for δ2H and δ18O values between (1) extracted water analysed via IRMS and IRIS and (2) urine and extracted water analysed via IRIS were significant (R 2=0.99). These results indicate that cryogenic distillation of urine was not required for an accurate estimate of the isotopic composition of urine when using IRIS.

The influences of cultivation setting on inflorescence lipid distributions, concentrations, and carbon isotope ratios of Cannabis sp.

While much is known about how the growth environment influences many aspects of floral morphology and physiology, little is known about how the growth setting influences floral lipid composition. We explored variations in paraffin wax composition in Cannabis sp., a cash crop grown both indoors and outdoors across the United States today. Given an increased focus on regulation of this crop, there are additional incentives to certify the setting of Cannabis cultivation. To understand the impacts of the growth environment, we studied distributions, concentrations, and carbon isotope ratios of n-alkanes isolated from Cannabis sp. inflorescences to assess if variations within these lipid parameters were related to known growth settings of specimens seized by federal agents. We found that Cannabis plants cultivated under open-field settings had increased inflorescence paraffin wax abundances and greater production of lower molecular weight n-alkanes relative to plants grown in enclosed environments. Further, the carbon isotope ratios of n-C29 from Cannabis plants grown in enclosed environments had relatively lower carbon isotope (δ(13)C) values compared to plants from open-field environments. While this set of observations on seized plant specimens cannot address the particular driver behind these observations, we posit that (a) variations in irradiance and/or photoperiod may influence the distribution and concentration of inflorescence lipids, and (b) the δ(13)C value of source CO2 and lipid concentration regulates the δ(13)C values of inflorescence n-C29 and bulk Cannabis plant materials. Nonetheless, by using a cultivation model based on δ(13)C values of n-C29, the model correctly identified the growth environment 90% of time. We suggest that these lipid markers may be used to trace cultivation methods of Cannabis sp. now and become a more powerful marker in the future, once the mechanism(s) behind these patterns is uncovered.

The potential for application of ink stable isotope analysis in questioned document examination

We investigated a novel application of stable isotope abundance analysis of nitrogen (15N), carbon (13C), hydrogen (2H), and oxygen (18O) to characterize pen ink. We focused on both ballpoint and gel pen inks. We found that the isotope ratios of ink from pens purchased together in a package were similar and within-package stable isotope ratio variability was not significantly larger than the variability of isotope reference materials used during analysis. In contrast, the isotope ratios of ink from pens of the same brand purchased in three states of the continental USA were significantly different from each other and there was isotope ratio variation among pens of the same brand but different, unknown production periods. The stable isotope ratios of inked paper were statistically distinguishable using measured δ15N values. Paper inked with different gel pens was statistically distinguishable using measured δ2H values. The capacity of stable isotope ratios to differentiate among ballpoint inks as well as gel inks shows that stable isotope analysis may be a useful and quantifiable investigative technique for questioned document examination, although current sample size requirements limit its utility. Application of the technique in casework will require the development of micro-scale sampling and analysis methods.