Ultra-rapid cardiotoxicity of the hepatitis C virus protease inhibitor BILN 2061 in the urokinase-type plasminogen activator mouse

BACKGROUND & AIMS: Because current therapies for chronic hepatitis C virus (HCV) infections are suboptimal and associated with severe side effects, novel treatment options are needed. A small animal model has recently been developed to study HCV infections. To examine the usefulness of this human liver-urokinase-type plasminogen activator (uPA)(+/+) severe combined immune deficient (SCID) mouse for the development of HCV-targeted drugs, we evaluated the antiviral efficacy and safety of an HCV NS3-protease inhibitor, BILN 2061. METHODS: BILN 2061 was orally administered at clinical range doses for 4 days to SCID mice that differed in the presence of HCV infection, human hepatocyte grafts, and uPA zygosity. Treatment outcome was evaluated clinically, virologically, and morphologically. Using standard high-performance liquid chromatography-ultraviolet (HPLC-UV) methods and mass spectrometry, single-dose pharmacokinetics and multiple-dose drug exposures were analyzed. The (13)C-aminopyrine breath test was applied to compare in vivo liver function. RESULTS: A 4-day treatment with BILN 2061 of HCV genotype-1b infected chimeric animals reduced the viral load by >100-fold, but concomitant clinical and ultrastructural signs of cardiotoxicity appeared. BILN 2061 administration to uPA-transgenic mice induced mitochondrial swelling with aberrant cristae in cardiomyocytes, but not in skeletal muscle. Because both drug accumulation and liver function were identical in affected uPA-transgenic and nontransgenic SCID mice without cardiac involvement, the urokinase plasminogen activator transgene itself appears to be implicated. CONCLUSIONS: The human liver-uPA(+/+)SCID mouse is an interesting small animal model to evaluate the preclinical safety and efficacy of new antiviral compounds against HCV. The uPA-transgene increases the susceptibility of mice to BILN 2061-induced cardiotoxicity.     

Environmental enrichment enhances directional selectivity of primary auditory cortical neurons in rats

Environment enrichment (EE) has an important role in brain plasticity. Previous research has shown that EE increases the response strength of auditory cortical neurons, but it remains unknown whether EE can affect the directional selectivity of auditory neurons. In this study, rats were exposed to EE conditions during the developmental critical period (EE1) or after the critical period (EE2). By in vivo extracellular recording, we found that EE enhanced the directional selectivity of primary auditory cortical neurons in EE1 rats, which showed a sharper azimuth selectivity curve of auditory cortical neurons compared with normal rats. However, there was no significant difference in directional selectivity between the EE2 rats and age-matched control rats. Our findings indicate that early exposure to EE enhances the directional sensitivity of primary auditory cortical neurons. These results provide an insight into developmental plasticity in the auditory system.     

Environmental enrichment reduces mechanical hypersensitivity in neuropathic mice,but fails to abolish the phenotype of CCK2 receptor deficient mice

Genetic invalidation of CCK₂ receptors abolishes chronic constriction injury (CCI) induced mechanical hypersensitivity in mice. However, housing in environmentally enriched conditions significantly alters the phenotype of CCK₂ receptor deficient mice in all major behavioral domains. Furthermore, environmental enrichment itself has been reported to have protective effects in several rodent models of neurological diseases (brain and spinal trauma, ischemic stroke, Alzheimer's disease, etc.). In the present study we reproduced the earlier finding that mice, lacking CCK₂ receptors (−/−) are resistant to CCI-induced hypersensitivity. On the other hand, environmental enrichment substantially reduced CCI-induced mechanical hypersensitivity in wild-type (+/+) mice. Nevertheless, the phenotypic differences between wild-type (+/+) and mutant (−/−) mice in mechanical sensitivity before and after CCI-surgery were not eliminated by alternative housing conditions. These observations suggest that environmental enrichment has beneficial effects in neuropathic conditions and reinforce the causal link between CCK₂ receptors, mechanical sensitivity and the development of CCI-induced hypersensitivity.     

A sensitive period for environmental regulation of eating behavior and leptin sensitivity

Western lifestyle contributes to body weight dysregulation. Leptin down-regulates food intake by modulating the activity of neural circuits in the hypothalamic arcuate nucleus (ARC), and resistance to this hormone constitutes a permissive condition for obesity. Physical exercise modulates leptin sensitivity in diet-induced obese rats. The role of other lifestyle components in modulating leptin sensitivity remains elusive. Environmentally enriched mice were used to explore the effects of lifestyle change on leptin production/action and other metabolic parameters. We analyzed adult mice exposed to environmental enrichment (EE), which showed decreased leptin, reduced adipose mass, and increased food intake. We also analyzed 50-d-old mice exposed to either EE (YEE) or physical exercise (YW) since birth, both of which showed decreased leptin. YEE mice showed no change in food intake, increased response to leptin administration, increased activation of STAT3 in the ARC. The YW leptin-induced food intake response was intermediate between young mice kept in standard conditions and YEE. YEE exhibited increased and decreased ratios of excitatory/inhibitory synapses onto α-melanocyte-stimulating hormone and agouti-related peptide neurons of the ARC, respectively. We also analyzed animals as described for YEE and then placed in standard cages for 1 mo. They showed no altered leptin production/action but demonstrated changes in excitatory/inhibitory synaptic contacts in the ARC similar to YEE. EE and physical activity resulted in improved insulin sensitivity. In conclusion, EE and physical activity had an impact on feeding behavior, leptin production/action, and insulin sensitivity, and EE affected ARC circuitry. The leptin-hypothalamic axis is maximally enhanced if environmental stimulation is applied during development.     

Synthesis of deuterium labelled diclofenac with improved isotopic enrichment

A five‐step synthesis of deuterium‐labelled diclofenac starting from 2‐phenyl[²H₅] acetic acid is described. The synthesis prevents deuterium from scrambling during the reaction. It offers the labelled compound with over 99% isotopic enrichment. It also provides a possible alternative route for the synthesis of deuterium‐labelled 4′‐hydroxydiclofenac, which is the principal human metabolite of diclofenac. Copyright © 2009 John Wiley & Sons, Ltd.     

Protein interaction landscapes revealed by advanced in vivo cross-linking–mass spectrometry

Defining protein–protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking–mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry–based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine–lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

Protein–protein interactions (PPIs) are fundamental to the formation of protein complexes and crucial for regulating cellular activities required for cell viability and homeostasis. As a result, aberrant PPIs have been implicated in a variety of human diseases, including cancers (1). Recent studies have demonstrated the potential of modulating PPIs through their interaction interfaces, representing a therapeutic paradigm for mechanism-driven drug discovery (2). Therefore, detailed analysis of PPIs at the systems level will not only advance our understanding of protein sociology and their cellular functions but also facilitate the exploration of novel interaction-based therapeutics. Affinity purification–mass spectrometry (AP-MS) has been proven powerful in profiling PPIs at the global scale due to its versatility, speed, and scope (3–6). However, PPIs can reorganize upon cell lysis, and weak/transient interactions can be lost during APs (2, 7–11), preventing the full characterization of native PPIs. Recently, AP-MS has been coupled with protein proximity labeling (i.e., APEX [ascorbate peroxidase] and BioID) to improve the detection of PPIs within vicinity (∼20 nm) (12, 13). Although successful, these approaches only provide a list of putative interactors and often result in high background due to label diffusion and/or nonspecific binding (11, 14). Additionally, cell engineering associated with AP-based methods could potentially affect protein localization, interaction, and function (2, 10, 11). Thus, reciprocal purification is often needed for further validation, and iterative tagging of a large number of baits is required to dissect proteome-wide PPI networks (3–6), leading to limited analysis throughput. Recent developments in cofractionation-based MS approaches and complex-centric data analysis have allowed global characterization of protein complexes from cell lysates with increased accuracy and throughput (15, 16). While these technologies have been effective in defining cellular PPIs and complexes, it remains challenging to differentiate between direct and indirect interactors, as well as determine PPI interfaces of proteome networks in their native environments. To advance PPI studies, cross-linking–mass spectrometry (XL-MS) has been coupled with AP to facilitate the capture and identification of interactions of protein complexes (8, 9, 17–19). The unique capability of XL-MS technologies to covalently tether proximal amino acids (AA) of interacting proteins (∼30 Å) enables preservation of native PPIs in living cells, as well as simultaneous identification of PPI identities and contact sites (20–24). Thus, XL-MS represents a high-throughput method for defining interaction network topologies at the systems level. Apart from PPI mapping, the identified cross-links can be used not only for validating and fine-tuning existing structures but also for assisting de novo structural modeling to elucidate architectures of protein complexes (19, 24–30).While in vivo PPI profiling is most attractive for unraveling protein interactions and structures in their native cellular environments, most XL-MS studies have been restricted to in vitro experiments. This is largely due to the difficulty in detecting low-abundance and heterogeneous cross-link products from complex peptide mixtures, especially at the proteome scale. Various efforts have been made to address the limitations of XL-MS analysis, resulting in technological advances in bioinformatics tools, XL reagents, and enrichment strategies (24–29). Most notably, recent innovations in MS-cleavable XL reagents have made a significant impact on XL-MS studies, enabling their applications in mapping PPIs at the systems level (21, 22, 31–34). To advance XL-MS studies, we have developed a class of sulfoxide-containing MS-cleavable XL reagents (e.g., disuccinimidyl sulfoxide [DSSO]), which allow fast and accurate identification of cross-linked peptides using multistage MS (MSⁿ) and conventional database searching tools (22, 35–38). Importantly, our MS-cleavable reagents have demonstrated effectiveness in defining PPIs and elucidating architectures of protein complexes in vitro and in vivo (22, 24, 30, 39, 40). In addition, they have been successfully employed to delineate proteome-wide interaction networks by in vitro XL of cell lysates (31, 34). While these results highlight the potential of MS-cleavable cross-linkers in PPI studies, reagents with enrichment capabilities are ultimately advantageous for enhancing MS detectability of low-abundance cross-links in complex mixtures. This has been exemplified by the success of proteome-wide in vivo XL-MS analyses using Protein Interaction Reporter, resulting in the most representative in vivo PPI network in human cells composed of 893 nodes and 1,042 edges to date (21). While impressive, the scope of the uncovered PPI networks was limited, describing only a fraction of the human interactome. To enhance in vivo XL-MS studies, we have developed two multifunctional sulfoxide-containing XL reagents, that is, Azide-A-DSBSO and Alkyne-A-DSBSO (Azide/Alkyne-tagged, acid-cleavable disuccinimidyl bissulfoxide) (22, 41), which are membrane permeable, enrichable, and MS cleavable. Both reagents carry a small bio-orthogonal handle that allows click chemistry–based biotin conjugation for enrichment of cross-linked peptides. In addition, they possess two symmetric sulfoxide-containing MS-cleavable bonds for robust cross-link separation and identification. Moreover, incorporation of an acid-cleavable site improves cross-link recovery and identification through removal of the biotin handle. Finally, the spacer length (∼14 Å) is well suited for mapping PPIs. Collectively, the combined features of Azide-A-DSBSO and Alkyne-A-DSBSO make them ideal reagents for studying PPIs in intact cells. While we have previously demonstrated the feasibility of in vivo XL-MS analysis using Azide-A-DSBSO (22), the proteome content remains underrepresented. To further in vivo XL-MS studies, we have developed an Alkyne-A-DSBSO–based in vivo XL-MS analytical platform by coupling sample preparation strategies with advanced instrumentation to maximize the yield and identification of cross-linked peptides at the global scale. These developments have enabled us to generate an expansive in vivo XL-MS dataset consisting of 13,904 unique lysine–lysine linkages (9,289 intersubunit and 4,615 intrasubunit), permitting construction of the largest in vivo human interactome to date. This network comprises 6,439 edges within 2,484 nodes, uncovering PPIs involving 795 CORUM (comprehensive resource of mammalian) protein complexes (with at least 50% subunits identified) associated with various cellular pathways. Importantly, the methodologies presented here will help facilitate future XL-MS studies of various sample origins at the systems level.     

Sex-specific behavioural effects of environmental enrichment in a transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by motor neuron degeneration, muscle wasting and paralysis. While twin studies support a role for both genetic and environmental factors in ALS, the nature of environmental modifiers is unknown. We therefore compared onset and progression of disease symptoms in female and male transgenic ALS mice (expressing the human SOD1^G93A gene mutation) and their wild-type littermates, housed in environmentally enriched versus standard conditions. Environmental enrichment significantly improved motor performance, as measured using the accelerating rotarod, in particular for female mice. This enhanced motor coordination was observed for both SOD1^G93A and wild-type mice, suggesting this effect is independent of genotype. Female SOD1^G93A mice housed with environmental enrichment were found to reach overt end-stage disease sooner than their standard-housed littermates. However, male SOD1^G93A mice did not show significantly accelerated disease progression. This evidence for environmental modulation of ALS pathogenesis in transgenic mice provides insights into activity-dependent aspects of the disease process, and may help identify molecular targets for pharmacological modulators as future therapeutics.     

Combination of Cobe AutoPBSC and Gambro Elutra as a platform for monocyte enrichment in dendritic cell (DC) therapy: clinical study

Monocytes are a common source for generating dendritic cells (DCs). The aim of the present study was to evaluate the efficiency of a platform for monocyte collection and enrichment in a clinical setting. The platform was based on the combination of two semiautomated devices; the Cobe Spectra Auto PBSC for mononuclear cells (MNC) collection followed by counterflow elutriation for monocyte enrichment (Gambro BCT Elutra). Twenty-four patients with various types of epithelial cancer participated in the study. MNC collections were first performed as large volume leukapheresis (LVL). Subsequently, MNC products were processed with an elutriation system for monocyte isolation. LVL resulted in the collection of MNC at a median of 8.1 x 10(9) cells, containing of 31.4% monocytes. A similar efficacy was also shown in patients with lower peripheral blood counts. Elutriation of the MNC product with the Cobe Elutra device resulted in the enrichment of monocytes at a median of 2.7 x 10(9) cells, with a recovery of 80.2% and a purity of 90.7%. These monocytes were then successfully developed into DCs for clinical therapy after in vitro manipulation. These data suggest that the combination of the Cobe Spectra Auto PBSC and the Gambro BCT Elutra is an effective platform for monocyte enrichment in clinical practice according to GCP standards and GMP guidelines, and can be easily implemented in the clinical routine under current DC protocols.     

Effect of environmental enrichment on fearful behavior and gastrin-releasing peptide receptor expression in the amygdala of prenatal stressed rats

Prenatal stressed offspring exhibit more fearful behavior in behavioral tests, which can be reversed by environmental enrichment (EE). However, the physiological basis of these phenomena remains unclear. Previous studies revealed that abnormal fearful behavior of prenatally stressed offspring may be a consequence of increased activities of CRFergic systems (corticotropin-releasing factor and its receptors) in the amygdala. Gastrin-releasing peptide receptors (GRPR) also have an important role in regulating amygdala-dependent, fear-related learning. The aim of this study was to examine weather prenatal stress and EE can affect the expression of GRPR in the amygdala. We reported here that prenatal chronic stress (subjected to immobilization and bright light stress for 45 min three times per day) caused increased fearfulness in defensive withdrawal test but had no effect on the expression of GRPR in the amygdala. However, enriched environment housing treatment on postnatal days 21-60 can dramatically increase the expression of GRPR in amygdala and reduce fearfulness in the defensive withdrawal test. Our results demonstrate for the first time that EE can modify the expression of GRPR in the amygdala, which might contribute to our understanding of the physiological effects of environmental enrichment.     

基于生物信息学筛选脓毒症中性粒细胞活化的关键基因

目的基于生物信息学筛选脓毒症中性粒细胞活化的关键基因。 方法从GEO数据库中下载4个成人脓毒症芯片数据集(GSE69528、GSE28750、GSE57065、GSE95233),进行差异表达基因(DEG)筛选。基因本体论(GO)分析DEG,STRING数据库构建蛋白互作网络,Cytoscape软件提取关键基因,CIBERSORT算法估算数据集中的免疫细胞表达水平,ROC曲线评价关键基因对脓毒症患者的诊断价值。取儿童脓毒症相关芯片数据集(GSE66099)进行验证。 结果4个成人脓毒症数据集筛选出299个重叠DEG,其中112个基因上调,187个基因下调。GO主要富集于中性粒细胞活化、中性粒细胞脱颗粒、肽酶调节活性、糖胺聚糖结合、特殊颗粒、囊泡腔等。筛选出脓毒症中性粒细胞活化的关键基因10个,在脓毒症中表达水平均上调,ROC曲线下面积均大于0.7,具有较好的诊断价值。儿童脓毒症数据集验证结果与成人脓毒症数据集结果一致。 结论基于生物信息学方法筛选出脓毒症中性粒细胞活化相关的10个关键基因,为脓毒症的早期诊断、预后判断及治疗提供了潜在新靶点。