Single‐dose administration of clenbuterol is detectable in dried blood spots

Clenbuterol is a β₂‐agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time‐consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 μg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post‐ingestion, with additional urine collections on days 7 and 10. Using LC–MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post‐ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7–10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 μg.     

Effects of childhood adversity on the volumes of the amygdala subnuclei and hippocampal subfields in individuals with major depressive disorder

BackgroundReductions in total hippocampus volume have frequently been reported in MRI studies in major depressive disorder (MDD), but reports of differences in total amygdala volume have been inconsistent. Childhood maltreatment is an important risk factor for MDD in adulthood and may affect the volume of the hippocampus and amygdala. In the present study, we examined associations between the volumes of the amygdala subnuclei and hippocampal subfields and history of childhood maltreatment in participants with MDD.MethodsWe recruited 35 patients who met the DSM-IV criteria for MDD and 35 healthy controls. We acquired MRI data sets on a 4.7 T Varian Inova scanner. We manually delineated the amygdala subnuclei (lateral, basal and accessory basal nuclei, and the cortical and centromedial groups) and hippocampal subfields (cornu ammonis, subiculum and dentate gyrus) using reliable volumetric methods. We assessed childhood maltreatment using the Childhood Trauma Questionnaire in participants with MDD.ResultsIn participants with MDD, a history of childhood maltreatment had significant negative associations with volume in the right amygdala, anterior hippocampus and total cornu ammonis subfield bilaterally. For volumes of the amygdala subnuclei, such effects were limited to the basal, accessory basal and cortical subnuclei in the right hemisphere, but they did not survive correction for multiple comparisons. We did not find significant effects of MDD or antidepressant treatment on volumes of the amygdala subnuclei.LimitationsOur study was a cross-sectional study.ConclusionOur results provide evidence of negative associations between history of childhood maltreatment and volumes of medial temporal lobe structures in participants with MDD. This may help to identify potential mechanisms by which maltreatment leads to clinical impacts.     

RIG-I–like receptor LGP2 protects tumor cells from ionizing radiation

An siRNA screen targeting 89 IFN stimulated genes in 14 different cancer cell lines pointed to the RIG-I (retinoic acid inducible gene I)–like receptor Laboratory of Genetics and Physiology 2 (LGP2) as playing a key role in conferring tumor cell survival following cytotoxic stress induced by ionizing radiation (IR). Studies on the role of LGP2 revealed the following: (i) Depletion of LGP2 in three cancer cell lines resulted in a significant increase in cell death following IR, (ii) ectopic expression of LGP2 in cells increased resistance to IR, and (iii) IR enhanced LGP2 expression in three cell lines tested. Studies designed to define the mechanism by which LGP2 acts point to its role in regulation of IFNβ. Specifically (i) suppression of LGP2 leads to enhanced IFNβ, (ii) cytotoxic effects following IR correlated with expression of IFNβ inasmuch as inhibition of IFNβ by neutralizing antibody conferred resistance to cell death, and (iii) mouse embryonic fibroblasts from IFN receptor 1 knockout mice are radioresistant compared with wild-type mouse embryonic fibroblasts. The role of LGP2 in cancer may be inferred from cumulative data showing elevated levels of LGP2 in cancer cells are associated with more adverse clinical outcomes. Our results indicate that cytotoxic stress exemplified by IR induces IFNβ and enhances the expression of LGP2. Enhanced expression of LGP2 suppresses the IFN stimulated genes associated with cytotoxic stress by turning off the expression of IFNβ.Several studies have shown that the response of tumor cells to ionizing radiation (IR) is associated with IFN-mediated signaling (1–6). IFN signaling leads to the induction of multiple IFN stimulated genes (ISGs) (7, 8) and activates growth arrest and cell death in exposed cell populations (9). The precise mechanism of IR-mediated induction of IFN signaling is unknown. Tumor cell clones that survive an initial cytotoxic insult are subsequently resistant to exposure to both IR and prodeath components of IFN signaling (10). These clones express IFN-dependent enhanced levels of constitutively expressed ISGs, which overlap in part with ISGs initially induced by cytotoxic stress. Many of these constitutively expressed ISGs have been characterized as antiviral genes (11). Recently, enhanced levels of constitutively expressed ISGs have been reported in advanced cancers and were often associated with a poor prognosis related to aggressive tumor growth, metastatic spread, resistance to IR/chemotherapy, or combinations of these factors (11–18). The studies presented in this report are based on the hypothesis that a specific set of constitutively expressed ISGs, whose enhanced expression is by cytotoxic stress, confers a selective advantage to individual tumor clones (5, 9, 10, 13, 19).To test this hypothesis, we designed a targeted siRNA screen against 89 ISGs selected from two sources. The first included ISGs identified in our earlier screen and designated the IFN-Related DNA Damage Signature (IRDS) (1, 13). The second set included related ISG signatures that have been reported in the literature (as described in Methods and Dataset S1). The 89 genes were individually targeted in 14 tumor cell lines derived from malignant gliomas, lung, breast, colon, head and neck, prostate, and bladder cancers.The most significant finding from this screen was that the RNA helicase Laboratory of Genetics and Physiology 2 (LGP2) encoded by DHX58 [DEXH (Asp-Glu-X-His) box polypeptide 58] confers survival and mediates the response to IR of multiple tumor cell lines. LGP2 acts as a suppressor of the RNA-activated cytoplasmic RIG-I RIG-I (retinoic acid inducible gene I)–like receptor’s pathway (20, 21). This pathway is a subtype of pattern recognition receptors responsible for primary recognition of pathogen and host-associated molecular patterns and the subsequent activation of type I IFN production that orchestrates an innate immune response (22–24). In addition to its role in inhibiting IFNβ expression, Suthar et al. recently demonstrated that LGP2 governs CD8+ T-cell fitness and survival by inhibiting death-receptor signaling (25). Here we demonstrate that suppression of LGP2 leads to an enhanced IFNβ expression and increased killing of tumor cells. Our results thereby provide a mechanistic connection between IR-induced cytotoxic response in tumor cells and the LGP2–IFNβ pathway.     

Species‐specific differences in the medial prefrontal projections to the pons between rat and rabbit

The medial prefrontal cortex (mPFC) of both rats and rabbits has been shown to support trace eyeblink conditioning, presumably by providing an input to the cerebellum via the pons that bridges the temporal gap between conditioning stimuli. The pons of rats and rabbits, however, shows divergence in gross anatomical organization, leaving open the question of whether the topography of prefrontal inputs to the pons is similar in rats and rabbits. To investigate this question, we injected anterograde tracer into the mPFC of rats and rabbits to visualize and map in 3D the distribution of labeled terminals in the pons. Effective mPFC injections showed labeled axons in the ipsilateral descending pyramidal tract in both species. In rats, discrete clusters of densely labeled terminals were observed primarily in the rostromedial pons. Clusters of labeled terminals were also observed contralateral to mPFC injection sites in rats, appearing as a less dense "mirror‐image" of ipsilateral labeling. In rabbits, mPFC labeled corticopontine terminals were absent in the rostral pons, and instead were restricted to the intermediate pons. The densest terminal fields were typically observed in association with the ipsilateral pyramidal tract as it descended ventromedially through the rabbit pons. No contralateral terminal labeling was observed for any injections made in the rabbit mPFC. The results suggest the possibility that mPFC inputs to the pons may be integrated with different sources of cortical inputs between rats and rabbits. The resulting implications for mPFC or pons manipulations for studies of trace eyeblink in each species are discussed. J. Comp. Neurol. 522:3052–3074, 2014. © 2014 Wiley Periodicals, Inc.     

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane‐dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel‐filtered platelets, intact or loaded with calcium‐sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine‐positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine‐negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha‐granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine‐positive platelets formation several‐fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate‐limiting, for subpopulation segregation. Inhibition of mitogen‐activated protein kinase kinase (U0126) and glycoprotein IIb‐IIIa (Monafram^®) was without effect, whereas inhibitors of phosphatidylinositol 3‐kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.