Probing lipid rafts with proximity imaging: actions of proatherogenic stimuli

Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to cluster in microdomains enriched in glycosphingolipids and cholesterol and represent a relatively selective marker of lipid rafts. In recent years, several attempts have been made to use fluorescent probes to nondisruptively label these domains in living cells. Here, we have transfected endothelial cells with a GPI-anchored thermotolerant green fluorescent protein (ttGFP) to show colocalization of this fluoroprobe with another marker of lipid rafts, urokinase-type plasminogen activator receptor-1. ttGFP was used to quantify the cell surface area occupied by lipid rafts and to examine the effect of various proatherogenic signals on lipid rafts. Exposure of endothelial cells to asymmetric dimethylarginine and oxidized LDL (oxLDL), as well as oxidant stress, reduced the cell surface area occupied by lipid rafts. Next, the property of ttGFP to undergo a shift in absorbance depending on the clustering of these molecules was utilized to perform proximity imaging (PRIM). PRIM showed that nitric oxide (NO) increased the distance between GPI-anchored ttGFP molecules clustered in lipid-rich microdomains. This "unclustering" of GPI-anchored ttGFP was not reproduced by prooxidant signals and was due to reduction in membrane-cytoskeletal constraints on the lipid rafts. These findings suggested that two fundamentally different mechanisms modulate lipid rafts: 1) substance regulation of lipid rafts involving modification of cholesterol and sphingolipids and 2) structural regulation of lipid rafts through disruption of membrane-cytoskeletal interactions, switching off the spatial confinement of lipid rafts.     

Do ants make direct comparisons?

Many individual decisions are informed by direct comparison of the alternatives. In collective decisions, however, only certain group members may have the opportunity to compare options. Emigrating ant colonies (Temnothorax albipennis) show sophisticated nest-site choice, selecting superior sites even when they are nine times further away than the alternative. How do they do this? We used radio-frequency identification-tagged ants to monitor individual behaviour. Here we show for the first time that switching between nests during the decision process can influence nest choice without requiring direct comparison of nests. Ants finding the poor nest were likely to switch and find the good nest, whereas ants finding the good nest were more likely to stay committed to that nest. When ants switched quickly between the two nests, colonies chose the good nest. Switching by ants that had the opportunity to compare nests had little effect on nest choice. We suggest a new mechanism of collective nest choice: individuals respond to nest quality by the decision either to commit or to seek alternatives. Previously proposed mechanisms, recruitment latency and nest comparison, can be explained as side effects of this simple rule. Colony-level comparison and choice can emerge, without direct comparison by individuals.     

Brain-penetrating 2-aminobenzimidazole H1-antihistamines for the treatment of insomnia

The benzimidazole core of the selective non-brain-penetrating H₁-antihistamine mizolastine was used to identify a series of brain-penetrating H₁-antihistamines for the potential treatment of insomnia. Using cassette PK studies, brain-penetrating H₁-antihistamines were identified and in vivo efficacy was demonstrated in a rat EEG/EMG model. Further optimization focused on strategies to attenuate an identified hERG liability, leading to the discovery of 4i with a promising in vitro profile.     

Skin-specific Deletion of Stearoyl-CoA Desaturase-1 Alters Skin Lipid Composition and Protects Mice from High Fat Diet-induced Obesity

Stearoyl-CoA desaturase-1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids and is an important regulator of whole body energy homeostasis. Severe cutaneous changes in mice globally deficient in SCD1 also indicate a role for SCD1 in maintaining skin lipids. We have generated mice with a skin-specific deletion of SCD1 (SKO) and report here that SKO mice display marked sebaceous gland hypoplasia and depletion of sebaceous lipids. In addition, SKO mice have significantly increased energy expenditure and are protected from high fat diet-induced obesity, thereby recapitulating the hypermetabolic phenotype of global SCD1 deficiency. Genes of fat oxidation, lipolysis, and thermogenesis, including uncoupling proteins and peroxisome proliferator-activated receptor-γ co-activator-1α, are up-regulated in peripheral tissues of SKO mice. However, unlike mice globally deficient in SCD1, SKO mice have an intact hepatic lipogenic response to acute high carbohydrate feeding. Despite increased basal thermogenesis, SKO mice display severe cold intolerance because of rapid depletion of fuel substrates, including hepatic glycogen, to maintain core body temperature. These data collectively indicate that SKO mice have increased cold perception because of loss of insulating factors in the skin. This results in up-regulation of thermogenic processes for temperature maintenance at the expense of fuel economy, illustrating cross-talk between the skin and peripheral tissues in maintaining energy homeostasis.Obesity is a multifactorial disease stemming from a combination of genetic, dietary, and lifestyle factors and the interaction between these components (1–3). The microsomal enzyme, stearoyl-CoA desaturase-1 (SCD1),³ is a critical control point in the development of metabolic diseases, including obesity and insulin resistance. SCD1 catalyzes the conversion of saturated fatty acids, such as palmitate (16:0) and stearate (18:0), into their Δ-9 monounsaturated products, palmitoleate (16:1 n-7) and oleate (18:1 n-9), respectively. Mice lacking the SCD1 enzyme because of a global deletion of the Scd1 gene (GKO) are lean and protected from diet-induced and leptin deficiency-induced obesity. These mice have a marked increase in energy expenditure and almost complete protection from high fat diet-induced weight gain and glucose intolerance (4–10).Because SCD1 is expressed in multiple tissues, including liver, brown and white adipose tissue, skeletal muscle, and skin, it has been difficult to determine the relative contributions of these tissues to the dramatically altered metabolic phenotypes of GKO mice. Studies using antisense oligonucleotide-mediated approaches to knock down Scd1 expression have reported protection from diet-induced weight gain and hepatic insulin resistance upon hepatic SCD1 inhibition (11–13). However, whereas the liver is a major target of these antisense oligonucleotides, they have also been reported to affect expression of target genes in adipose tissue (13, 14) and possibly other organs (15). Using Cre recombinase-mediated inhibition of hepatic Scd1, we recently reported that chronic deletion of SCD1 specifically in liver does not protect mice from high fat diet-induced obesity (16), suggesting that extra-hepatic tissues may play a more prominent role in the increased energy expenditure phenotype of global SCD1 deficiency (16).In addition to their hypermetabolic phenotype, global SCD1 deficiency also elicits marked cutaneous phenotypes, including dry skin, alopecia, and sebocyte hypoplasia (7, 17, 18). Given the severity of this skin phenotype in GKO mice, we sought to establish a specific role for SCD1 in the skin. In this study, we used the Cre-lox system to generate mice with a skin-specific deletion of SCD1 (SKO). We report here that SKO mice have a severe paucity of lipid-enriched sebocytes in the skin, resulting in dry skin, alopecia, and marked alterations in levels of key skin lipids. Unlike mice with global or liver-specific deletion of SCD1 (7, 16), SKO have an intact hepatic lipogenic response to dietary stimuli. However, deletion of skin SCD1 completely recapitulates the increased energy expenditure phenotype of GKO mice (7) and protects SKO mice from high fat diet-induced obesity, hepatic steatosis, and glucose intolerance. Elevation of genes encoding for cold-inducible factors, including peroxisome proliferator-activated receptor γ co-activator-1α (Pgc-1α) and uncoupling proteins (Ucps) in brown and white adipose tissue and skeletal muscle of SKO mice, suggests up-regulation of thermogenic processes for maintenance of core body temperature in SKO mice. Furthermore, the hypermetabolic phenotype of SKO mice, coupled with the loss of insulating factors in the skin, results in severe cold intolerance in SKO mice that is ameliorated by prior feeding with a high fat diet. To the best of our knowledge, this study represents the first example of skin-specific deletion of a lipogenic enzyme resulting in profound changes in systemic energy metabolism. These data elucidate an as yet under-appreciated role for skin SCD1 in triggering the altered metabolic phenotypes caused by global SCD1 deletion.     

ApoE isoform-dependent changes in hippocampal synaptic function

Accumulating evidence suggests that neurons prone to degeneration in Alzheimer's Disease (AD) exhibit evidence of re-entry into an aberrant mitotic cell cycle. Our laboratory recently demonstrated that, in a genomic amyloid precursor protein (APP) mouse model of AD (R1.40), neuronal cell cycle events (CCEs) occur in the absence of beta-amyloid (Aβ) deposition and are still dependent upon the amyloidogenic processing of the amyloid precursor protein (APP). These data suggested that soluble Aβ species might play a direct role in the induction of neuronal CCEs. Here, we show that exposure of non-transgenic primary cortical neurons to Aβ oligomers, but not monomers or fibrils, results in the retraction of neuronal processes, and induction of CCEs in a concentration dependent manner. Retraction of neuronal processes correlated with the induction of CCEs and the Aβ monomer or Aβ fibrils showed only minimal effects. In addition, we provide evidence that induction of neuronal CCEs are autonomous to primary neurons cultured from the R1.40 mice. Finally, our results also demonstrate that Aβ oligomer treated neurons exhibit elevated levels of activated Akt and mTOR (mammalian Target Of Rapamycin) and that PI3K, Akt or mTOR inhibitors blocked Aβ oligomer-induced neuronal CCEs. Taken together, these results demonstrate that Aβ oligomer-based induction of neuronal CCEs involve the PI3K-Akt-mTOR pathway.     

An examination of the flexion-relaxation phenomenon in the cervical spine in lumbo-pelvic sitting

The flexion-relaxation phenomenon (FRP) is well documented at end-range lumbar spine flexion in both standing and sitting however, the FRP has been insufficiently investigated in cervico-thoracic musculature. The aim of this study was to determine whether the FRP occurs during forward flexion of the neck, in lumbo-pelvic sitting, amongst a pain-free population. Surface electromyography (EMG) was used to measure muscle activation in 20 (10 men, 10 women) asymptomatic subjects in selected cervico-thoracic muscles during four, 5-s phases (upright posture, forward flexion, full flexion and return to upright) while subjects were positioned in lumbo-pelvic sitting. Spinal kinematics were simultaneously measured using an electromagnetic motion tracking device. No FRP was observed in upper trapezius or thoracic erector spinae (T4). When using visual methods to determine the presence/absence of the FRP, five subjects were believed to show evidence of the FRP in the cervical erector spinae. However, when using various non-visual criteria to determine the existence of the FRP, substantial variations (0–13 subjects) were evident. We recommend that criteria based upon relatively large differences in muscle activation should be considered when defining the FRP. These findings are of significance for future investigations examining specific cervical pain disorders.     

Biomarker evaluation and comparison using the controls as a reference population

The classification accuracy of a continuous marker is typically evaluated with the receiver operating characteristic (ROC) curve. In this paper, we study an alternative conceptual framework, the "percentile value." In this framework, the controls only provide a reference distribution to standardize the marker. The analysis proceeds by analyzing the standardized marker in cases. The approach is shown to be equivalent to ROC analysis. Advantages are that it provides a framework familiar to a broad spectrum of biostatisticians and it opens up avenues for new statistical techniques in biomarker evaluation. We develop several new procedures based on this framework for comparing biomarkers and biomarker performance in different populations. We develop methods that adjust such comparisons for covariates. The methods are illustrated on data from 2 cancer biomarker studies.     

Norbornyllactone-substituted xanthines as adenosine A(1) receptor antagonists

During the search for second-generation adenosine A(1) receptor antagonist alternatives to the clinical candidate 8-(3-oxa-tricyclo[3.2.1.0(2,4)]oct-6-yl)-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (BG9719), we developed a series of novel xanthines substituted with norbornyl-lactones that possessed high binding affinities for adenosine A(1) receptors and in vivo activity.     

Analysis of nutrient concentrations in the diet,serum, and urine of giraffe from surveyed North American zoological institutions

The objectives of the present research were to conduct a survey to investigate the health history and feeding practices of giraffe in captivity in North America and to obtain samples of hay, concentrate, browse, urine, and serum to compare across zoos, possible factors relating to the development of urolithiasis. Forty‐one out of 98 institutions contacted responded, representing 218 giraffe. All responding zoos fed concentrate and alfalfa hay was the primary forage. Sixty‐five percent of zoos fed browse and 43 different species of browse were listed. Six zoos reported a history of urolithiasis, seven reported wasting syndrome, and 10 reported sudden death. The median daily amount (as fed) of concentrate and hay offered were 5.45 kg (range of 2.73–9.55 kg) and 6.82 kg (range of 2.53–12.50 kg), respectively. The concentrate:hay ratio of the offered diet ranged from 0.22 to 3.47 with a median value of 0.79. Forty‐three percent of the institutions offered a ratio greater than 1:1. Samples of concentrate and hay (six zoos), serum (five zoos), and urine (seven zoos) were obtained for chemical analyses. Analyzed nutrient content of the consumed diet, measured by weighing feed and orts for three consecutive days, met recommendations for giraffe, but was excessive for crude protein and P. Concentrate:hay and serum P were positively correlated (r=0.72; P<0.05). High dietary P content and a high level of concentrate relative to hay may be contributing factors to urolith formation and warrant further investigation. Zoo Biol 29:457–469, 2010. © 2009 Wiley‐Liss, Inc.