A combination of ultrahigh throughput PathHunter and cytokine secretion assays to identify glucocorticoid receptor agonists

The use of ultrahigh throughput screens (uHTS) is a well-accepted mechanism to identify agonists and antagonists of target receptors. We used the Path Hunter [Path Hunter technology is a registered trademark of DiscoveRx Corporation.] technology from DiscoveRx to screen the entire Merck compound library for glucocorticoid receptor (GR) agonists in a 2.2-μl total reaction volume assayed in a 3456-well plate format. This single addition, homogenous assay which utilizes the principle of enzyme fragment complementation (EFC) to detect nuclear translocation of GR, an initial step of receptor activation, was used to successfully screen a large library of small molecules as indicated by an average signal to background ratio of approximately 4-fold and an average Z-factor value of 0.45. Hits from the HTS campaign were studied in a cytokine secretion assay in primary human monocytes to gain functional information regarding these compounds in a phenotypic and physiologically relevant setting. Our data indicate that using the PathHunter assay, we successfully identified compounds that showed agonism for the GR receptor in primary human monocytes and due to their performance in a physiologically relevant model they likely will have a better chance to evoke clinical efficacy.     

Patellofemoral interactions in walking,stair ascent,and stair descent using a virtual patella model

Restoration of normal patella kinematics is an important clinical outcome of total knee arthroplasty. Failure of the patella within total knee systems has been documented and, upon occurrence, often necessitates revision surgery. It is thus important to understand patella mechanics following implantation, subject to load states that are typically realized during walking and other gaits. Here, a computational model of the patella is developed and used to examine the effects of walking, stair ascent, and stair descent on the development of stress and contact pressure in the patella throughout the gait cycle. Motion of the patella was governed by a combination of kinematic and force control, based on knee flexion and patellofemoral joint reaction force data from the literature. Unlike most previous analyses of full gait, quasi-static equilibrium was enforced throughout the cycle. Results indicate that, though peak forces vary greatly between the three gaits, maximum contact pressure and von Mises stress are roughly equivalent. However, contact area is larger in stair ascent and descent than walking, as patellofemoral loading, implant geometry, and polyethylene yield increase conformity between the femoral component and patella. Additionally, maximum contact pressure does not coincide with maximum load except for the case of walking. Though specific to the implant design considered here, this result has important ramifications for patella testing and emphasizes the need to characterize patella mechanics throughout gait.     

Identification of EMS-Induced Mutations in Drosophila melanogaster by Whole-Genome Sequencing

Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing ~70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.     

Monoclonal Antibodies Recognize Distinct Conformational Epitopes Formed by Polyglutamine in a Mutant Huntingtin Fragment

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) domain in the N-terminal region of huntingtin (htt). PolyQ expansion above 35–40 results in disease associated with htt aggregation into inclusion bodies. It has been hypothesized that expanded polyQ domains adopt multiple potentially toxic conformations that belong to different aggregation pathways. Here, we used atomic force microscopy to analyze the effect of a panel of anti-htt antibodies (MW1–MW5, MW7, MW8, and 3B5H10) on aggregate formation and the stability of a mutant htt-exon1 fragment. Two antibodies, MW7 (polyproline-specific) and 3B5H10 (polyQ-specific), completely inhibited fibril formation and disaggregated preformed fibrils, whereas other polyQ-specific antibodies had widely varying effects on aggregation. These results suggest that expanded polyQ domains adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies, which has important implications for understanding the structural basis for polyQ toxicity and the development of intrabody-based therapeutics for HD.Huntington disease (HD)⁵ is a fatal neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the protein huntingtin (htt), which leads to its aggregation into fibrils (1). HD is part of a growing group of diseases that are classified as “conformational diseases,” which include Alzheimer disease (AD), Parkinson disease (PD), the prion encephalopathies, and many more (2–4). The length of polyQ expansion in HD is tightly correlated with disease onset, and a critical threshold of 35–40 glutamine residues is required for disease manifestation (5). Biochemical and electron microscopic studies with htt fragments demonstrated that expanded polyQ repeats (>39) form detergent-insoluble aggregates that share characteristics with amyloid fibrils (6–8), and the formation of amyloid-like fibrils by polyQ was confirmed by studies with synthetic polyQ peptides (9). Collectively, these studies demonstrated a correlation between polyQ length and the kinetics of aggregation. This phenomenon has been recapitulated in cell-culture models that express htt fragments (10–12). Although it is clear that proteins with expanded polyQ repeats assemble into fibrils in vitro, recent studies have reported that htt fragments can also assemble into spherical and annular oligomeric structures (13–16) similar to those formed by Aβ and α-synuclein, which are implicated in AD and PD, respectively.While the major hallmark of HD is the formation of intranuclear and cytoplasmic inclusion bodies of aggregated htt (17), the role of these structures in the etiology of HD remains controversial. For instance, the onset of symptoms in a transgenic mouse model of HD follows the appearance of inclusion bodies (18), while other studies indicate that inclusion body formation may protect against toxicity by sequestering diffuse, soluble forms of htt (10, 19, 20). Based on the direct correlation between polyQ length, htt aggregation propensity, and toxicity (6), it has been hypothesized that the aggregation of htt may mediate neurodegeneration in HD. However, there is no clear consensus on the aggregate form(s) that underlie toxicity, and there likely exist bioactive, oligomeric aggregates undetectable by traditional biochemical and electron microscopic approaches whose formation precedes disease symptoms. Although identification of the one or more toxic species of htt that trigger neurodegeneration in HD remains elusive, such species might exist in a diffuse, mobile fraction rather than in inclusion bodies (19). A thioredoxin-polyQ fusion protein was recently reported to exhibit toxicity in a meta-stable, β-sheet-rich, monomeric conformation (21), suggesting that polyQ can adopt multiple monomeric conformations, only some of which may be toxic. Consistent with such a scenario, molecular dynamic simulations and fluorescence correlation spectroscopy experiments with synthetic polyQ peptides indicate that polyQ domains can adopt a heterogeneous collection of collapsed conformations that are in equilibrium before aggregation (22–25).Although biochemical, biophysical, and computational approaches have yielded insight into the structures formed by polyQ in vitro, whether such structures form in vivo remains largely unknown. Indeed, determining the conformational state of any misfolded/aggregated protein in situ and/or in vivo remains a major technical challenge.Toward this goal, antibodies have been explored as a potentially powerful tool for detecting specific conformations or multimeric states of aggregated proteins in situ. Antibodies specific for amyloid fibrils often do not react with natively folded globular proteins from which they are derived, suggesting that such antibodies recognize a conformational epitope (26, 27). Several antibodies display conformation-dependent interactions with amyloids, aggregation intermediates, or natively folded precursor proteins. For example, there are antibodies specific for paired helical filaments of Tau (28–31), of aggregated forms of Aβ ranging from dimers to fibrils (32–34), and of native (35) or disease-related (36) forms of the prion protein. Antibodies have also been developed that are specific for common structural motifs associated with amyloid diseases, such as oligomers (37) and fibrils (38), independent of the peptide sequence of the amyloid forming protein from which they are derived, suggesting the potential for a common mechanism of aggregation and toxicity for these diseases.With regard to htt, several antibodies (MW1, MW2, MW3, MW4, MW5, IC2, and IF8), which are specific for polyQ repeats, stain Western blots of htt with expanded polyQ repeats much more strongly than htt with normal polyQ length (39, 40), suggesting that these antibodies may recognize abnormal polyQ conformations. Furthermore, these polyQ-specific antibodies have distinct staining patterns in immunohistochemical studies of brain tissue sections (39). In one study, the affinity and stoichiometry of MW1 binding to htt increased with polyQ length, suggesting a “linear lattice” model for polyQ (41). This model is supported by a crystal structure of polyQ bound to MW1, which showed that polyQ can adopt an extended, coil-like structure (42). However, an independent structural study showed that the anti-polyQ antibody 3B5H10 binds to a compact β-sheet-like structure of polyQ in a monomeric htt fragment.⁶ These results clearly indicate that polyQ domains can fold into at least two unique, stable, monomeric conformations and suggest that the “linear lattice” model is not generally applicable to all polyQ structures.Not only are antibodies useful for understanding what polyQ structures exist in situ, especially in the diffuse htt fraction of neurons, but antibodies and/or intrabodies may also have potential as therapeutic agents. For example, several studies showed that intrabodies reduce htt toxicity in cellular models (44–49). Moreover, one intrabody (C4) slows htt aggregation and prolongs lifespan in a Drosophila model of HD (50, 51), while another (mEM48) ameliorates neurological symptoms in a mouse model of HD (48).Three of the antibodies examined in this study (MW1, MW2, and MW7) modulate htt-induced cell death when co-transfected as single-chain variable region fragment antibodies (scFvs) in 293 cells with htt exon 1 containing an expanded polyQ domain (46). In these studies MW1 and MW2, which bind to the polyQ repeat in htt, increased htt-induced toxicity and aggregation (46). Conversely, MW7, which binds to the polyproline (polyP) regions adjacent to the polyQ repeat in htt, decreased its aggregation and toxicity (46). Interestingly, MW7 has also been shown to increase the turnover of mutant htt in cultured cells and reduce its toxicity in corticostriatal brain slice explants (49).Given the difficulty in understanding which specie(s) of htt exist and mediate pathogenesis in the putative toxic diffuse fraction of neurons, we sought to rigorously characterize the conformational specificity of a panel of anti-htt antibodies, the best in situ probes currently available for distinguishing specie(s) of htt. We reasoned that if htt can adopt multiple conformations that mediate different aggregation pathways, then anti-htt antibodies should differentially alter htt aggregation pathways by stabilizing or sequestering the specific conformers or aggregates they recognize. We therefore examined the effects of various antibodies on mutant htt fragment fibril formation and stability by atomic force microscopy (AFM). Our results are consistent with the hypothesis that monoclonal antibodies recognize distinct conformational epitopes formed by polyQ in a mutant htt fragment.     

Insights into cerebrovascular complications and Alzheimer disease through the selective loss of GRK2 regulation

Alzheimer disease (AD) and stroke are two leading causes of age-associated dementia. Increasing evidence points to vascular damage as an early contributor to the development of AD and AD-like pathology. In this review, we discuss the role of G protein-coupled receptor kinase 2 (GRK2) as it relates to individuals affected by AD and how the cardiovasculature plays a role in AD pathogenesis. The possible involvement of GRKs in AD pathogenesis is an interesting notion, which may help bridge the gap in our understanding of the heart–brain connection in relation to neurovisceral damage and vascular complications in AD, since kinases of this family are known to regulate numerous receptor functions both in the brain, myocardium, and elsewhere. The aim of this review is to discuss our findings of overexpression of GRK2 in the context of the early pathogenesis of AD, because increased levels of GRK2 immunoreactivity were found in vulnerable neurons of AD patients as well as in a two-vessel occlusion (2-VO) mammalian model of ischaemia. Also, we consider the consequences for this overexpression as a loss of G-protein coupled receptor (GPCR) regulation, as well as suggest a potential role for GPCRs and GRKs in a unifying theory of AD pathogenesis, particularly in the context of cerebrovascular disease. We synthesize this newer information and attempt to put it into context with GRKs as regulators of diverse physiological cellular functions that could be appropriate targets for future pharmacological intervention.     

Neuronal mitochondrial amelioration by feeding acetyl-L-carnitine and lipoic acid to aged rats

Brain function declines with age and is associated with diminishing mitochondrial integrity. The neuronal mitochondrial ultrastructural changes of young (4 months) and old (21 months) F344 rats supplemented with two mitochondrial metabolites, acetyl-L-carnitine (ALCAR, 0.2%[wt/vol] in the drinking water) and R-α-lipoic acid (LA, 0.1%[wt/wt] in the chow), were analysed using qualitative and quantitative electron microscopy techniques. Two independent morphologists blinded to sample identity examined and scored all electron micrographs. Mitochondria were examined in each micrograph, and each structure was scored according to the degree of injury. Controls displayed an age-associated significant decrease in the number of intact mitochondria (P = 0.026) as well as an increase in mitochondria with broken cristae (P < 0.001) in the hippocampus as demonstrated by electron microscopic observations. Neuronal mitochondrial damage was associated with damage in vessel wall cells, especially vascular endothelial cells. Dietary supplementation of young and aged animals increased the proliferation of intact mitochondria and reduced the density of mitochondria associated with vacuoles and lipofuscin. Feeding old rats ALCAR and LA significantly reduced the number of severely damaged mitochondria (P = 0.02) and increased the number of intact mitochondria (P < 0.001) in the hippocampus. These results suggest that feeding ALCAR with LA may ameliorate age-associated mitochondrial ultrastructural decay and are consistent with previous studies showing improved brain function.     

Epidemiology of Invasive Klebsiella pneumoniae with Hypermucoviscosity Phenotype in a Research Colony of Nonhuman Primates

Invasive Klebsiella pneumoniae with hypermucoviscosity phenotype (HMV K. pneumoniae) is an emerging human pathogen that, over the past 20 y, has resulted in a distinct clinical syndrome characterized by pyogenic liver abscesses sometimes complicated by bacteremia, meningitis, and endophthalmitis. Infections occur predominantly in Taiwan and other Asian countries, but HMV K. pneumoniae is considered an emerging infectious disease in the United States and other Western countries. In 2005, fatal multisystemic disease was attributed to HMV K. pneumoniae in African green monkeys (AGM) at our institution. After identification of a cluster of subclinically infected macaques in March and April 2008, screening of all colony nonhuman primates by oropharyngeal and rectal culture revealed 19 subclinically infected rhesus and cynomolgus macaques. PCR testing for 2 genes associated with HMV K. pneumoniae, rmpA and magA, suggested genetic variability in the samples. Random amplified polymorphic DNA analysis on a subset of clinical isolates confirmed a high degree of genetic diversity between the samples. Environmental testing did not reveal evidence of aerosol or droplet transmission of the organism in housing areas. Further research is needed to characterize HMV K. pneumoniae, particularly with regard to genetic differences among bacterial strains and their relationship to human disease and to the apparent susceptibility of AGM to this organism.Abbreviations: AGM, African green monkey; HMV K. pneumoniae, invasive Klebsiella pneumoniae with hypermucoviscosity phenotype; NHP, nonhuman primate; RAPD, random amplification of polymorphic DNAKlebsiella pneumoniae is an enteric, gram-negative, lactose-fermenting bacillus with a prominent capsule. This bacterium has been associated with peritonitis, septicemia, pneumonia, and meningitis in both Old and New World primates,^10,13,29 although it also is reported to constitute normal fecal and oral flora in many nonhuman primates (NHP).¹² Pathogenic strains associated with the upper respiratory tract typically are heavily encapsulated.¹² Over the past several decades, human medical literature indicates the emergence of an invasive K. pneumoniae disease in Taiwan and other Asian countries, in which community-acquired pyogenic liver abscesses have been attributed to strains of invasive K. pneumoniae with a unique hypermucoviscous phenotype (HMV K. pneumoniae).^{6,17-19,21,26,34} The hypermucoviscous phenotype has also been associated with other serious complications, including bacteremia, meningitis, and endophthalmitis. This strain of Klebsiella has become an emerging cause of pyogenic liver abscesses in some nonAsian countries, including the United States.^16,20,36,39 The majority of clinical cases of HMV K. pneumoniae are in the Asian population, particularly in patients with diabetes mellitus.^3,4,33 Determination of the HMV phenotype typically is based on a positive string test.^8,35,39Several virulence factors have been associated with HMV K. pneumoniae. Klebsiella spp. generally develop prominent polysaccharide capsules which increase virulence by protecting the bacteria from phagocytosis and preventing destruction by bactericidal serum factors. Capsular serotypes K1 or K2 have been reported as the major virulence determinants for human HMV K. pneumoniae liver abscesses.^5,8,37,38 In addition, the mucoviscosity-associated gene magA, which encodes a structural outer membrane protein of the K1 serotype, and rmpA (regulator of the mucoid phenotype gene; located on a plasmid) have been proposed as virulence factors.^{9,27,31,40,41} Recently, it was suggested that 2 clones, CC23 ^K1 and CC82^K1, are strongly associated with primary liver abscess and respiratory infection, respectively.²Over a period of several months in 2005 to 2006, 7 African green monkeys (AGM; Chlorocebus aethiops) in the US Army Medical Research Institute of Infectious Diseases research colony developed abscesses in multiple locations and either died or were euthanized when the abscesses were determined to be nonresectable.³⁵ HMV K. pneumoniae of the K2 serotype and carrying rmpA was determined to be the cause of the infection in 1 case, and the 6 other cases had similar clinical and pathologic features. This report³⁵ is the only documentation, to our knowledge, of natural infection with HMV K. pneumoniae in NHP. As a result of these cases, the US Army Medical Research Institute of Infectious Diseases instituted policies to exclude HMV K. pneumoniae from the colony. The organism was included as a specific pathogen-free requirement for vendors, and K. pneumoniae culture results were reported during quarantine periods and on routine semiannual examination for all colony NHP.