Determination of testosterone esters in the hair of male greyhound dogs using liquid chromatography–high resolution mass spectrometry

The doping of greyhound dogs with testosterone is done in an attempt to improve their athletic performance, but such doping cannot easily be confirmed, especially in male dogs owing to the natural presence of endogenous testosterone. As testosterone is usually administered as its esters, their direct detection in hair would provide confirmatory evidence of the administration of a pharmaceutical product. This article demonstrates that the use of a liquid chromatography–high resolution mass spectrometry method with heated electrospray ionisation (HESI) combined with the use of amino solid‐phase extraction (SPE) cartridges for sample clean‐up, is suitable for the sensitive determination of propionate, phenyl propionate, isocaproate, decanoate, and enanthate esters of testosterone in greyhound hair. The method is linear over the range, 0.1 μg/kg–10 μg/kg, for all the testosterone esters analysed. The limits of detection (LOD) are 0.05 μg/kg for testosterone phenyl propionate, isocaproate, and decanoate, 0.025 μg/kg for testosterone propionate, and 0.25 μg/kg for testosterone enanthate. This method was applied to hair samples collected from male greyhounds before and after a single administration of a product containing several testosterone esters, each of which could be detected up to 100 days post‐administration. The study also demonstrates that tail hair is the specimen of choice for the analysis of testosterone in dog hair and that washing of dogs does not impact the analysis of testosterone esters in hair. This method may be useful in racing regulation for the detection of illegitimate use of testosterone in all species.     

P53基因在电离辐射诱导的非小细胞肺癌细胞死亡中的作用

目的研究p53基因在电离辐射诱导的非小细胞肺癌细胞死亡中的作用。方法选取H1299（p53-/-）,H1299-P53,H1299-175H细胞为研究对象。采用流式细胞术检测细胞凋亡率,WESTERN BOLT方法检测相关蛋白表达情况。结果在H1299（P53-/-）细胞中,4GY、8GY照射后细胞凋亡率分别是假照组的68.4%、50%。在H1299-P53细胞中,4GY、8GY照射后细胞凋亡率分别是假照组的1.7倍、8.1倍。在H1299-175H细胞中,4GY8、GY照射后细胞凋亡率分别是假照组的1.5倍、2倍。LC3蛋白表达在H1299呈现剂量依赖性升高,在H1299-175H呈现剂量依赖性降低,在H1299-P53中,OGY表达最高,4 GY表达最低,8 GY表达低于假照组但比4 GY高。AKT表达在H1299细胞呈剂量依赖性降低,H1299-P53在4GY表达量升高而在8GY又降低但仍高于假照组,在H1299-175H细胞中AKT表达呈剂量依赖性降低。MDM2在H1299的表达呈剂量依赖性升高,在H1299-P53细胞中呈现与AKT相同的变化趋势,在H1299-175H细胞中4GY时表达量最高8GY最低。Caspase-3在H1299细胞中随着照射剂量的增加变化不明显,在H1299-P53细胞中呈现剂量依赖性升高,在H1299-175H细胞中,0 GY表达最高,4 GY表达最低,8 GY低于假照组但高于4 GY组。结论 X射线促进P53（-/-）的H1299细胞时发生自噬,而促进H1299-P53及H1299-175H中发生凋亡。     

Nerve alterations showing autophagy in 2 patients with lichen aureus

Lichen aureus is a rare, chronic, persistent purpuric dermatosis clinically characterized by striking yellow‐ to bronze‐colored lesions. Histologically, lichen aureus differs from other pigmented purpuric dermatoses in containing dense, band‐like infiltrates closely associated with the epidermis. This report describes 2 patients with lichen aureus, a 20‐year‐old woman with a lesion on her right arm and a 51‐year‐old man with a lesion on the right side of his groin. Skin biopsy specimens revealed almost identical findings in both patients, including dense band‐like infiltrates containing lymphocytes, histiocytes with hemosiderin deposits scattered extravasated red blood cells and nerve alterations at the dermo‐epidermal interface. The nerves within the lesions were filled with granules, which stained positive with antibody to microtubule‐associated protein 1A/1B‐light chain 3, suggesting autophagy within the nerves. These altered nerves were present only in areas of band‐like dermal lymphocytic infiltration. Electron microscopy of the lesions showed the accumulation of autophagosomes in Schwann cells.     

(−)Epigallocatechin-3-gallate inhibits the spontaneous firing of rat locus coeruleus neuron

(−)Epigallocatechin-3-gallate (EGCG), a tea catechin, has been known to cause many biological actions, such as anxiolytic and hypotensive effects in behavioral studies. However, to date, few reports investigate its neuronal modulation. In this study, intracellular recording was used to test the neuronal modulation of different catechins on locus coeruleus (LC) neuron, which has been demonstrated to be affected by cardiovascular function regulation and stressful events. Several catechins (1–1000 μM) were tested, including: (−)catechin (C), (−)catechingallate (CG), (−)epicatechin (EC), (−)epicatechin-3-gallate (ECG), (−)epigallocatechin (EGC) and EGCG. The results showed that catechins EC, ECG, EGC and EGCG could inhibit the spontaneous firing of the LC neurons; furthermore, these catechins show potency and efficacy in the order of EGCG > ECG > EC ≈ EGC. Among the tested catechins, EGCG was the most potent in inhibiting LC's spontaneous firing with IC₅₀ of 20.5 μM. This caused us to further examine the EGCG's desensitization and tolerance properties. When continuously administering EGCG at 1–300 μM for 20 min, no acute desensitization appeared. However, repeated applications of 300 μM EGCG at 5 min each time showed different results. The second and third applications induced less responses compared to that of the first application, suggesting a development of tolerance towards EGCG in inhibiting LC neuronal activity. Our data suggest that EGCG can inhibit LC neuron's spontaneous firing in a dose-dependent manner, with developed tolerance only when high concentration of EGCG is repeatedly applied.     

Simultaneous quantification of isoniazid,rifampicin, ethambutol and pyrazinamide by liquid chromatography/tandem mass spectrometry

A remediable cause of poor treatment response in drug‐susceptible tuberculosis (TB) patients may be low plasma levels of one or more of the first‐line anti‐TB drugs. The aim of this work was to develop an accurate and precise LC‐MS/MS method for simultaneous quantification of all four first‐line anti‐TB drugs in plasma suitable for therapeutic drug monitoring (TDM). To adjust for degradation and losses during sample preparation, isotopically labeled compounds were used as internal standards. Plasma samples spiked with internal standards were extracted using protein precipitation with methanol and acetonitrile. Simultaneous separation of all four drugs was accomplished with a Chromolith Reversed‐Phase column and mobile phases consisting of water, methanol, ammonium acetate and formic acid with subsequent mass spectrometric quantification. The linear range of the calibration curve for isoniazid was 0.5–10 mg/L, for rifampicin 0.75–30 mg/L, for ethambutol 0.25–10 mg/L and for pyrazinamide 4–80 mg/L. The lower limit of quantification was 0.5 mg/L, 0.75 mg/L, 0.25 mg/L and 4.0 mg/L, respectively. Precision estimated by the coefficient of variation was <15% for all four drugs. The LC‐MS/MS method can readily be used for simultaneous quantification of first‐line anti‐TB drugs in plasma and is well suited for TDM.     

Studies on the hippocampal formation: From basic development to clinical applications: Studies on schizophrenia

The hippocampal formation plays a critical role in cognitive function. The developmental events that shape the hippocampal formation are continuing to be elucidated and their implications for brain function are emerging as well as applying those advances to interventions that have important possibilities for the treatment of brain dysfunction. The story told in this chapter is about the use of the in oculo transplant method to illuminate intrinsic and extrinsic features that underlie the development of the dentate gyrus and adjacent hippocampus and the role of one molecule in the hippocampus and schizophrenia. Schizophrenia, originally conceptualized as a dysfunction in dopaminergic neurotransmission, is now known to involve multiple neuronal systems. Dysfunction of hippocampal neurons is emerging as one of its signature pathological features. Basic insights into the development and function of hippocampal interneurons form the basis of a new treatment initiative for this illness. Evidence for the role of the alpha 7-nicotinic acetylcholine receptor in the development and function of these neurons in rodents has led to human trials of nicotinic agonists for cognitive dysfunction in schizophrenia and the possibility of improving hippocampal development in children at risk for schizophrenia by perinatal supplementation with choline, which can act as an alpha 7-nicotinic acetylcholine receptor agonist.     

Autophagy: assays and artifacts

Autophagy is a fundamental and phylogenetically conserved self‐degradation process that is characterized by the formation of double‐layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation. The increasing significance attached to autophagy in development and disease in higher eukaryotes has placed greater importance on the validation of reliable, meaningful and quantitative assays to monitor autophagy in live cells and in vivo in the animal. To date, the detection of processed LC3B‐II by western blot or fluorescence studies, together with electron microscopy for autophagosome formation, have been the mainstays for autophagy detection. However, LC3 expression levels can vary markedly between different cell types and in response to different stresses, and there is also concern that over‐expression of tagged versions of LC3 to facilitate imaging and detection of autophagy interferes with the process itself. In addition, the realization that it is not sufficient to monitor static levels of autophagy but to measure ‘autophagic flux’ has driven the development of new or modified approaches to detecting autophagy. Here, we present a critical overview of current methodologies to measure autophagy in cells and in animals. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Retino-hypothalamic-pineal hypothesis in the pathophysiology of primary headaches

Primary headaches include migraine, tension, cluster headaches, paroxysmal hemicrania and miscellaneous headaches unassociated with structural lesions. A putative role of the retino-hypothalamic-pineal (RHP) axis in the pathophysiology of primary headaches is reviewed in terms of (1) retinal dysfunction, (2) hypothalamic dysfunction and human circadian desynchrony, (3) pineal melatonin dysfunction and (4) rostral limbic dysfunction mediating the human stress response. Unified RHP hypothesis is proposed, suggesting that an acute, periodic or chronic, circadian desynchrony and dysfunction of the whole or part of the RHP axis is implicated in the pathophysiology of primary headaches. Supportive evidence for the RHP hypothesis, including recent PET studies showing changes in dorsal pons, hypothalamus and rostral limbic structures, is presented.     

Different metabolic profiles of K1 serotype and non-serotype K1 and K2 Klebsiella pneumoniae isolates in oral infection mice model

K1 or K2 serotype Klebsiella pneumoniae isolate caused clinical pyogenic liver abscess (KLA) infection is prevalent in many areas. It has been identified that K1 or K2 serotype K. pneumoniae isolates caused KLA infection in mice by oral inoculation. In our study, K1 serotype K. pneumoniae isolate Kp1002 with hypermucoviscosity (HV)-positive phenotype caused KLA infection in C57BL/6 mice by oral inoculation. Simultaneously, non-serotype K1 and K2 isolate Kp1014 with HV-negative phenotype failed to cause KLA infection in the same manner. It seems that gastrointestinal tract translocation is the pathway by which K1 or K2 serotype K. pneumoniae caused KLA infection. Liquid chromatography-tandem mass spectrometry was used to further analyze metabolic profile changes in mice with KLA infection. Data showed that after Kp1002 or Kp1014 oral inoculation, serum Phosphatidylcholine (PC) and Lysophosphatidylcholine (LPC) levels significantly changed in mice. Some PC and LPC molecules showed changes both in the Kp1002 KLA group and the Kp1014 no-KLA group compared with the control group. The level of 18:1/18:2-PC significantly changed in the Kp1002 KLA group compared with the control group, but showed no change between the Kp1014 no-KLA group and the control group. The level of 18:1/18:2-PC might have been particularly affected by KLA infection caused by K1 serotype K. pneumoniae Kp1002. It may be a potential biomarker for KLA infection.     

Programmed cell death 1 (PD-1) regulates the effector function of CD8 T cells via PD-L1 expressed on target keratinocytes

Programmed cell death 1 (PD-1) is an inhibitory molecule expressed by activated T cells. Its ligands (PD-L1 and -L2; PD-Ls) are expressed not only by a variety of leukocytes but also by stromal cells. To assess the role of PD-1 in CD8 T cell-mediated diseases, we used PD-1-knockout (KO) OVA-specific T cell-receptor transgenic (Tg) CD8 T cells (OT-I cells) in a murine model of mucocutaneous graft-versus-host disease (GVHD). We found that mice expressing OVA on epidermal keratinocytes (K14-mOVA mice) developed markedly enhanced GVHD-like disease after transfer of PD-1-KO OT-I cells as compared to those mice transferred with wild-type OT-I cells. In addition, K14-mOVA × OT-I double Tg (DTg) mice do not develop GVHD-like disease after adoptive transfer of OT-I cells, while transfer of PD-1-KO OT-I cells caused GVHD-like disease in a Fas/Fas-L independent manner. These results suggest that PD-1/PD-Ls-interactions have stronger inhibitory effects on pathogenic CD8 T cells than does Fas/Fas-L-interactions. Keratinocytes from K14-mOVA mice with GVHD-like skin lesions express PD-L1, while those from mice without the disease do not. These findings reflect the fact that primary keratinocytes express PD-L1 when stimulated by interferon-γ in vitro. When co-cultured with K14-mOVA keratinocytes for 2 days, PD-1-KO OT-I cells exhibited enhanced proliferation and activation compared to wild-type OT-I cells. In addition, knockdown of 50% PD-L1 expression on the keratinocytes with transfection of PD-L1-siRNA enhanced OT-I cell proliferation. In aggregate, our data strongly suggest that PD-L1, expressed on activated target keratinocytes presenting autoantigens, regulates autoaggressive CD8 T cells, and inhibits the development of mucocutaneous autoimmune diseases.