Testing for midazolam and oxycodone in blood after formalin‐embalmment: About a complex medico‐legal case

The stability of compounds in formalin solution is an important factor for drug analysis in a toxicological investigation. In this article, the authors report a complex medico‐legal case involving midazolam and oxycodone. The complexity of this case comes from the fact that the body was embalmed with formalin solution before the autopsy. This technique, called thanatopraxy, allows the preservation of corpses from decomposition, the destruction of a maximal number of micro‐organisms, and the presentation of the body with a natural appearance to the family. Unfortunately, when thanatopraxy is performed before the collection of biological specimens, the toxicological results are not representative of the time of the death. In addition, the interpretation of the results is difficult, because formalin can cause oxidation of xenobiotics present in the body at the time of the death, alter the pH of the tissues and dilute the compounds. To document the chemical stability of midazolam and oxycodone in formalin solution and interpret the results, a stability study was conducted for 21 days. Blood containing midazolam and oxycodone was spiked with formalin, kept at 4°C and regularly tested for both drugs. This study showed a rapid degradation of midazolam and oxycodone (85% during the first 24 hours for oxycodone). In the peripheral blood of the victim, methanol (1.31 g/L), midazolam (74ng/mL) and oxycodone (152 ng/mL) were identified. According to the stability study, the measured concentrations in formalin fixed‐tissues are to be interpreted very carefully, knowing that significant degradation has occurred.     

Rewiring yeast sugar transporter preference through modifying a conserved protein motif

Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe³⁸Ile³⁹Met⁴⁰, Scheffersomyces stipitis rgt2 Phe³⁸ and Met⁴⁰, and Saccharomyces cerevisiae hxt7 Ile³⁹Met⁴⁰Met³⁴⁰ all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties.Molecular transporter proteins facilitate monosaccharide uptake and serve as the first step in catabolic metabolism. In this capacity, the preferences, regulation, and kinetics of these transporters ultimately dictate total carbon flux (1–3); and optimization of intracellular catabolic pathways only increases the degree to which transport exerts control over metabolic flux (4, 5). Thus, monosaccharide transport profiles and rates are important design criteria and a driving force to enable metabolic engineering advances, ultimately resulting in a biorefinery concept whereby biomass is converted via microbes into a diverse set of molecules (6–10). Among possible host organisms, Saccharomyces cerevisiae is an emerging industrial organism with well-developed genetic tools and established industrial processes and track record (11–16). However, S. cerevisiae lacks an endogenous xylose catabolic pathway and thus is unable to natively use the second most abundant sugar in lignocellulosic biomass. Decades of research have been focused on improving xylose catabolic pathways in recombinant S. cerevisiae (17–22), but less work has been focused on the first committed step of the process—xylose transport, an outstanding limitation in the efficient conversion of lignocellulosic sugars (23, 24).In S. cerevisiae, monosaccharide uptake is mediated by transporters belonging to the major facilitator superfamily (MFS) (25, 26), a ubiquitous group of proteins found across species (27). The predominant transporters in yeast are members of the HXT family (28) and are marked by efficient hexose transport (29) with lower affinities to xylose thus contributing to diauxic growth and flux limitation when attempting pentose utilization in recombinant S. cerevisiae (30). Previous efforts have attempted to identify heterologous transporters with a higher affinity for xylose over glucose (31–36). However, the vast majority of these transporters are either nonfunctional, not efficient, or not xylose specific (24, 37). Furthermore, nearly all known wild-type transporters that enable growth on xylose in yeast confer higher growth rates on glucose than on xylose (24, 37). As an alternative to bioprospecting, we have previously reported that xylose affinity and exponential growth rates on xylose can be improved via directed evolution of Candida intermedia glucose-xylose symporter 1 (GXS1) and Scheffersomyces stipitis xylose uptake 3 (XUT3) (38). These results demonstrated that mutations at specific residues (e.g., Phe⁴⁰ in C. intermedia GXS1) can have a significant impact on the carbohydrate selectivity of these MFS transporters. The fact that single amino acid substitutions can have such a significant impact on transport phenotype (38–40) indicates how simple homology based searches can be ineffective at identifying efficient xylose transporters (35, 36). However, evidence of natural xylose exclusivity is seen in the Escherichia coli xylE transporter that has recently been crystallized (41). The sequence-function flexibility of MFS transporters potentiates the capability to rewire hexose transporters from being glucose favoring, xylose permissive into being xylose-exclusive transporters.In this work, we report on the discovery of a conserved Gly³⁶-Gly³⁷-Val³⁸-Leu³⁹-Phe⁴⁰-Gly⁴¹ motif surrounding the previously identified Phe⁴⁰ residue of C. intermedia GXS1 that controls transporter efficiency and selectivity. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose, taking the general form G-G/F-XXX-G. We conduct saturation mutagenesis on Val³⁸, Leu³⁹, and Phe⁴⁰ within the variable region of this motif in C. intermedia GXS1 to demonstrate control of sugar selectivity. Next, we combine xylose-favoring mutations to create a unique mutant version of gxs1 that transports xylose, but not glucose. Finally, we demonstrate the importance of this motif in the capacity to rewire the sugar preference of other hexose transporters including S. cerevisiae hexose transporter 7 (HXT7) and S. stipitis glucose transporter/sensor (RGT2, similar to S. cerevisiae RGT2). This work serves to increase our understanding of the structure–function relationships for molecular transporter engineering and demonstrates complete rewiring of hexose transporters into transporters that prefer xylose as a substrate.     

The Effects of Race and Other Socioeconomic Factors on Health Service Use Among American Military Veterans

This study examined the extent to which racial disparities in service utilization exist in veterans (VA) and non-VA health care systems. An observational study design was used with a nationally representative sample of veterans. Logistic regression models were constructed using sociodemographic characteristics, health insurance and benefits, and health status as predictors of health service use in both VA and non-VA health care systems. A population weighted sample of 19,270 veterans from the 2001 National Survey of Veterans was used, which included 17,004 (88.24 %) White, 1,864 (9.15 %) African American, 414 (2.15 %) Native American/Alaskan Native, and 87 (0.45 %) Asian American/Pacific Islander veterans. Results showed that use of the VA health care system was not associated with race, but was associated with VA disability compensation, lack of private health insurance, and greater health care need. Contrarily, in non-VA healthcare systems, veterans who were racial minorities, less educated, and without private health insurance were less likely to use services. Together, these findings demonstrate the socioeconomic context in which health disparities exist and suggest the influence of health insurance on racial disparities in service utilization.     

The length of the Staphylococcus aureus protein A polymorphic region regulates inflammation: impact on acute and chronic infection

Staphylococcus aureus protein A (SpA) plays a critical role in the induction of inflammation. This study was aimed to determine whether the number of short sequence repeats (SSRs) present in the polymorphic region modulates the inflammatory response induced by SpA. We demonstrated that there is a dose-response effect in the activation of interferon (IFN)-β signaling in airway epithelial and immune cells, depending on the number of SSRs, which leads to differences in neutrophil recruitment. We also determined that a significant proportion of isolates from patients with chronic infections such as osteomyelitis and cystic fibrosis carry fewer SSRs than do isolates from patients with acute infections or healthy carriers and that there was an inverse correlation between the number of SSRs and the length of disease course. Given the importance of IFN signaling in eradication of S. aureus, loss of SSRs may represent an advantageous mechanism to adapt to and persist in the host.