Analytical surveillance of emerging drugs of abuse and drug formulations

Uncontrolled recreational drugs are proliferating in number and variety. Effects of long-term use are unknown, and regulation is problematic, as efforts to control one chemical often lead to several other structural analogs. Advanced analytical instrumentation and methods are continuing to be developed to identify drugs, chemical constituents of products, and drug substances and metabolites in biological fluids. Several mass spectrometry based approaches appear promising, particularly those that involve high resolution chromatographic and mass spectrometric methods that allow unbiased data acquisition and sophisticated data interrogation. Several of these techniques are shown to facilitate both targeted and broad spectrum analyses, the latter of which are often of particular benefit when dealing with misleadingly labeled products or assessing a biological matrix for illicit drugs and metabolites. The development and application of novel analytical approaches such as these will help to assess the nature and degree of exposure and risk and, where necessary, inform forensics and facilitate implementation of specific regulation and control measures.     

Clutch identity and predator-induced hatching affect behavior and development in a leaf-breeding treefrog

For species with complex life cycles, transitions between life stages result in niche shifts that are often associated with evolutionary trade-offs. When conditions across life stages are unpredictable, plasticity in niche shift timing may be adaptive; however, factors associated with clutch identity (e.g., genetic or maternal) may influence the effects of such plasticity. The red-eyed treefrog (Agalychnis callidryas) is an ideal organism for investigating the effects of genetics and life stage switch point timing because embryos exhibit adaptive phenotypic plasticity in hatching time. In this study, we evaluated the effects of experimentally manipulated hatching time and clutch identity on antipredator behavior of tadpoles and on developmental traits of metamorphs, including larval period, mass, SVL, and jumping ability. We found that in the presence of dragonfly nymph predator cues at 21 days post-oviposition, tadpoles reduced both their activity level and height in the water column. Furthermore, early-hatched tadpoles were less active than late-hatched tadpoles of the same age. This difference in behavior patterns of early- and late-hatched tadpoles may represent an adaptive response due to a longer period of susceptibility to odonate predators for early-hatched tadpoles, or it may be a carry-over effect mediated by early exposure to an environmental stressor (i.e., induction of early hatching). We also found that hatching time affected both behavioral traits and developmental traits, but its effect on developmental traits varied significantly among clutches. This study shows that a single early-life event may influence a suite of factors during subsequent life stages and that some of these effects appear to be dependent on clutch identity. This interaction may represent an evolutionary response to a complex life cycle and unpredictable environments, regardless of whether the clutch differences are due to additive genetic variance or maternal effects.     

Sphingolipids: regulators of crosstalk between apoptosis and autophagy

Apoptosis and autophagy are two evolutionarily conserved processes that maintain homeostasis during stress. Although the two pathways utilize fundamentally distinct machinery, apoptosis and autophagy are highly interconnected and share many key regulators. The crosstalk between apoptosis and autophagy is complex, as autophagy can function to promote cell survival or cell death under various cellular conditions. The molecular mechanisms of crosstalk are beginning to be elucidated and have critical implications for the treatment of various diseases, such as cancer. Sphingolipids are a class of bioactive lipids that mediate many key cellular processes, including apoptosis and autophagy. By targeting several of the shared regulators, sphingolipid metabolites differentially regulate the induction of apoptosis and autophagy. Importantly, individual sphingolipid species appear to “switch” autophagy toward cell survival (e.g., sphingosine-1-phosphate) or cell death (e.g., ceramide, gangliosides). This review assesses the current understanding of sphingolipid-induced apoptosis and autophagy to address how sphingolipids mediate the “switch” between the cell survival and cell death. As sphingolipid metabolism is frequently dysregulated in cancer, sphingolipid-modulating agents, or sphingomimetics, have emerged as a novel chemotherapeutic strategy. Ultimately, a greater understanding of sphingolipid-mediated crosstalk between apoptosis and autophagy may be critical for enhancing the chemotherapeutic efficacy of these agents.     

Ion-specific Effects on Prion Nucleation and Strain Formation

Ordered, fibrous, self-seeding aggregates of misfolded proteins known as amyloids are associated with important diseases in mammals and control phenotypic traits in fungi. A given protein may adopt multiple amyloid conformations, known as variants or strains, each of which leads to a distinct disease pattern or phenotype. Here, we study the effect of Hofmeister ions on amyloid nucleation and strain generation by the prion domain-containing fragment (Sup35NM) of a yeast protein Sup35p. Strongly hydrated anions (kosmotropes) initiate nucleation quickly and cause rapid fiber elongation, whereas poorly hydrated anions (chaotropes) delay nucleation and mildly affect the elongation rate. For the first time, we demonstrate that kosmotropes favor formation of amyloid strains that are characterized by lower thermostability and higher frangibility in vitro and stronger phenotypic and proliferation patterns effectively in vivo as compared with amyloids formed in chaotropes. These phenomena point to inherent differences in the biochemistry of Hofmeister ions. Our work shows that the ionic composition of a solution not only influences the kinetics of amyloid nucleation but also determines the amyloid strain that is preferentially formed.     

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

The Soluble Interleukin-6 Receptor Is a Mediator of Hematopoietic and Skeletal Actions of Parathyroid Hormone

Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6^+/+ and IL-6^−/− mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6^−/− compared with IL-6^+/+ bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6^−/− earlier than IL-6^+/+ marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6^−/− marrow. In the skeletal system, although intermittent PTH administration to IL-6^+/+ and IL-6^−/− mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-β1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.     

Oncolytic Vesicular Stomatitis Virus in an Immunocompetent Model of MUC1-Positive or MUC1-Null Pancreatic Ductal Adenocarcinoma

Vesicular stomatitis virus (VSV) is a promising oncolytic agent against various malignancies. Here, for the first time, we tested VSV in vitro and in vivo in a clinically relevant, immunocompetent mouse model of pancreatic ductal adenocarcinoma (PDA). Our system allows the study of virotherapy against PDA in the context of overexpression (80% of PDA patients) or no expression of human mucin 1 (MUC1), a major marker for poor prognosis in patients. In vitro, we tested three VSV recombinants, wild-type VSV, VSV-green fluorescent protein (VSV-GFP), and a safe oncolytic VSV-ΔM51-GFP, against five mouse PDA cell lines that either expressed human MUC1 or were MUC1 null. All viruses demonstrated significant oncolytic abilities independent of MUC1 expression, although VSV-ΔM51-GFP was somewhat less effective in two PDA cell lines. In vivo administration of VSV-ΔM51-GFP resulted in significant reduction of tumor growth for tested mouse PDA xenografts (+MUC1 or MUC1 null), and antitumor efficacy was further improved when the virus was combined with the chemotherapeutic drug gemcitabine. The antitumor effect was transient in all tested groups. The developed system can be used to study therapies involving various oncolytic viruses and chemotherapeutics, with the goal of inducing tumor-specific immunity while preventing premature virus clearance.     

Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.