Toll-like receptor 4 modulates skeletal muscle substrate metabolism.

Toll-like receptor 4 (TLR4), a protein integral to innate immunity, is elevated in skeletal muscle of obese and type 2 diabetic humans and has been implicated in the development of lipid-induced insulin resistance. The purpose of this study was to examine the role of TLR4 as a modulator of basal (non-insulin-stimulated) substrate metabolism in skeletal muscle with the hypothesis that its activation would result in reduced fatty acid oxidation and increased partitioning of fatty acids toward neutral lipid storage. Human skeletal muscle, rodent skeletal muscle, and skeletal muscle cell cultures were employed to study the functional consequences of TLR4 activation on glucose and fatty acid metabolism. Herein, we demonstrate that activation of TLR4 with low (metabolic endotoxemia) and high (septic conditions) doses of LPS results in increased glucose utilization and reduced fatty acid oxidation in skeletal muscle and that these changes in metabolism in vivo occur in concert with increased circulating triglycerides. Moreover, animals with a loss of TLR4 function possess increased oxidative capacity in skeletal muscle and present with lower fasting levels of triglycerides and nonesterified free fatty acids. Evidence is also presented to suggest that these changes in substrate metabolism under metabolic endotoxemic conditions are independent of skeletal muscle-derived proinflammatory cytokine production. This report illustrates that skeletal muscle is a target for circulating endotoxin and may provide critical insight into the link between a proinflammatory state and dysregulated metabolism as observed with obesity, type 2 diabetes, and metabolic syndrome.

The cardiac neural stem cell phenotype is compromised in streptozotocin-induced diabetic cardiomyopathy.

Neural stem cells were identified in the rat heart and during scar formation and healing participated in sympathetic fiber sprouting and angiogenesis. In the setting of diabetes, impaired wound healing represents a typical pathological feature. These findings provided the impetus to test the hypothesis that experimental diabetes adversely influenced the phenotype of cardiac neural stem cells. Streptozotocin (STZ)-induced diabetic rats were associated with elevated plasma glucose levels, significant loss of body weight and left ventricular contractile dysfunction. In the heart of STZ-diabetic rats, the density of nestin immunoreactive processes emanating from cardiac neural stem cells were reduced. The latter finding was reaffirmed as nestin protein expression was significantly decreased in the heart of STZ-diabetic rats and associated with a concomitant reduction of nestin mRNA. Employing the TUNEL assay, the loss of nestin expression in STZ-diabetic rats was not attributed to widespread cardiac neural stem cell apoptosis. Insulin administration to STZ-diabetic rats with established hyperglycaemia led to a modest recovery of nestin protein expression in cardiac neural stem cells. By contrast, the administration of insulin immediately after STZ injection improved plasma glucose levels and significantly attenuated the loss of nestin protein expression. These data highlight the novel observation that nestin protein expression in cardiac neural stem cells was significantly reduced in STZ-induced type I diabetic rats. The aberrant cardiac neural stem cell phenotype may compromise their biological role and predispose the diabetic heart to maladaptive healing following ischemic injury.

Effect of chronic inhibition of nitric oxide on hypertension, insulin resistance, and cardiovascular remodeling in glucose-fed rats.

To determine the contribution of nitric oxide (NO) in cardiovascular remodeling associated to hypertension and insulin resistance, male Sprague-Dawley rats received tap water supplemented or not (control), with 10% D-glucose (G) and/or 50 mg x kg(-1) x d(-1) L-NAME to inhibit NO synthase (G-LN or LN) for 4 weeks. Systolic blood pressure increased by 12%, 26%, and 39% with G, LN, and G-LN treatments, respectively. Hyperinsulinemia and insulin resistance (homeostasis model assessment index) occurred in G-treated rats (P < 0.05) and were further increased in G-LN (P < 0.01). Plasma adrenaline concentrations were markedly increased in all treated groups, especially in G-LN (P < 0.01), whereas noradrenaline was increased in G-treated rats only. Whereas no cardiac hypertrophy or fibrosis was detected, aortic hypertrophy occurred in LN and G-LN rats (P < 0.001) without smooth muscle hyperplasia. Superoxide anion formation was increased in the aorta of all treated groups (P < 0.01) and in the heart of LN (P < 0.05), but reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity was not affected. In conclusion, the loss of the wide-range protective effects of NO, the increased vascular oxidative stress, and the sympathoadrenal hyperactivity are among the contributing factors leading to the exacerbation of hypertension and insulin resistance in G-LN. These factors were sufficient to cause vascular but not cardiac hypertrophy.

Rab11-FIP1C Is a Critical Negative Regulator in ErbB2-Mediated Mammary Tumor Progression.

Rab coupling protein (FIP1C), an effector of the Rab11 GTPases, including Rab25, is amplified and overexpressed in 10% to 25% of primary breast cancers and correlates with poor clinical outcome. Rab25 is also frequently silenced in triple-negative breast cancer, suggesting its ability to function as either an oncogene or a tumor suppressor, depending on the breast cancer subtype. However, the pathobiologic role of FIP family members, such as FIP1C, in a tumor-specific setting remains elusive. In this study, we used ErbB2 mouse models of human breast cancer to investigate FIP1C function in tumorigenesis. Doxycycline-induced expression of FIP1C in the MMTV-ErbB2 mouse model resulted in delayed mammary tumor progression. Conversely, targeted deletion of FIP1C in the mammary epithelium of an ErbB2 model coexpressing Cre recombinase led to accelerated tumor onset. Genetic and biochemical characterization of these FIP1C-proficient and -deficient tumor models revealed that FIP1C regulated E-cadherin (CDH1) trafficking and ZONAB (YBX3) function in Cdk4-mediated cell-cycle progression. Furthermore, we demonstrate that FIP1C promoted lysosomal degradation of ErbB2. Consistent with our findings in the mouse, the expression of FIP1C was inversely correlated with ErbB2 levels in breast cancer patients. Taken together, our findings indicate that FIP1C acts as a tumor suppressor in the context of ErbB2-positive breast cancer and may be therapeutically exploited as an alternative strategy for targeting aberrant ErbB2 expression. Cancer Res; 76(9); 2662-74. ©2016 AACR.

Transient neonatal high oxygen exposure leads to early adult cardiac dysfunction, remodeling, and activation of the renin-angiotensin system.

Perinatal conditions (such as preterm birth) can affect adult health and disease, particularly the cardiovascular system. Transient neonatal high O(2) exposure in rat in adulthood (a model of preterm birth-related complications) leads to elevated blood pressure, vascular rigidity, and dysfunction with renin-angiotensin system activation. We postulate that neonatal hyperoxic stress also affects myocardial structure, function, and expression of renin-angiotensin system components. Sprague-Dawley pups were kept with their mother in 80% O(2) or in room air (control) from days 3 to 10 of life. Left ventricular function was assessed in 4-, 7-, 12-week-old (echocardiography) and in 16-week-old (intraventricular catheterization) male O(2)-exposed versus control rats. At 16 weeks, hearts from O(2)-exposed rats showed cardiomyocyte hypertrophy, enhanced fibrosis, and increased expression of transforming growth factor-ß1, senescence-associated proteins p53 and Rb, upregulation of angiotensin II type 1 (AT1) receptor expression (protein and AT1a/b mRNA), and downregulation of AT2 receptors. At 4 weeks (before blood pressure increase), the expression of cardiomyocyte surface area, fibrosis, p53, and AT1b was significantly increased and AT2 decreased in O(2)-exposed animals. After 4 weeks of continuous angiotensin II infusion (starting at 12 weeks), O(2)-exposed rats developed severe heart failure, with impaired myocardial mechanical properties compared with saline-infused rats. Transient neonatal O(2) exposure in rats leads to left ventricular hypertrophy, fibrosis and dysfunction, and increased susceptibility to heart failure under pressure overload. These results are relevant to the growing population of individuals born preterm who may be at higher risk of cardiac dysfunction when faced with increased peripheral resistance associated with hypertension, vascular diseases, and aging.

Building block synthesis using the polymerase chain assembly method.

De novo gene synthesis allows the creation of custom DNA molecules without the typical constraints of traditional cloning assembly: scars, restriction site incompatibility, and the quest to find all the desired parts to name a few. Moreover, with the help of computer-assisted design, the perfect DNA molecule can be created along with its matching sequence ready to download. The challenge is to build the physical DNA molecules that have been designed with the software. Although there are several DNA assembly methods, this section presents and describes a method using the polymerase chain assembly (PCA).

Oscillatory dynamics of cell cycle proteins in single yeast cells analyzed by imaging cytometry.

Progression through the cell division cycle is orchestrated by a complex network of interacting genes and proteins. Some of these proteins are known to fluctuate periodically during the cell cycle, but a systematic study of the fluctuations of a broad sample of cell-cycle proteins has not been made until now. Using time-lapse fluorescence microscopy, we profiled 16 strains of budding yeast, each containing GFP fused to a single gene involved in cell cycle regulation. The dynamics of protein abundance and localization were characterized by extracting the amplitude, period, and other indicators from a series of images. Oscillations of protein abundance could clearly be identified for Cdc15, Clb2, Cln1, Cln2, Mcm1, Net1, Sic1, and Whi5. The period of oscillation of the fluorescently tagged proteins is generally in good agreement with the inter-bud time. The very strong oscillations of Net1 and Mcm1 expression are remarkable since little is known about the temporal expression of these genes. By collecting data from large samples of single cells, we quantified some aspects of cell-to-cell variability due presumably to intrinsic and extrinsic noise affecting the cell cycle.

Does atorvastatin induce aortic smooth muscle cell apoptosis in vivo?

It has been reported that HMG-CoA reductase inhibitors such as atorvastatin induce vascular smooth muscle cell (SMC) apoptosis in vitro. However, this effect remains to be demonstrated in vivo. The present studies were designed to test the ability of atorvastatin to induce SMC apoptosis in vivo, using the spontaneously hypertensive rat (SHR) as a well-known reference model of SMC apoptosis induction in vivo by cardiovascular drugs including the calcium channel blocker amlodipine. Atorvastatin was administered to SHR for 3 or 6 weeks either alone or together with amlodipine, a drug combination clinically available to patients. Primary endpoints included aortic medial hypertrophy and aortic SMC hyperplasia, internucleosomal DNA fragmentation and expression of the apoptosis regulatory proteins Bax and Bcl-2. The SHR aorta showed no evidence of SMC apoptosis induction by atorvastatin, even at the high dose of 50 mg kg(-1) day(-1), although the statin significantly reduced oxidative stress after 3 weeks and blood pressure after 6 weeks of administration. Amlodipine-induced regression of aortic hypertophy and aortic SMC hyperplasia were dose- and time-dependent, but there was no interaction between atorvastatin and amlodipine in modulating the primary endpoints. These results do not support the notion that atorvastatin induces SMC apoptosis in the aortic media in vivo.

Orbital complications of sinusitis in children.

BACKGROUND: Orbital complications of sinusitis are uncommon but can result in significant morbidity if not appropriately managed. OBJECTIVE: This study was conducted to evaluate the clinical presentation, diagnosis, management, and outcome of orbital complications of sinusitis in children treated at our institution over a 10-year period. METHODS: The study retrospectively reviewed cases of 139 children with evidence of orbital complications of sinusitis admitted to the Montreal Children's Hospital between January 1990 and March 2000. Factors assessed included the clinical presentation, radiologic findings, management, and outcome (length of admission, complications). Complications were classified as preseptal if they did not penetrate the periorbita. Postseptal complications were defined as those penetrating the periorbita and were further subdivided into cellulitis and abscess categories. RESULTS: Seventy-two percent of patients presented with preseptal cellulitis, 19% with orbital cellulitis, and 9% with subperiosteal abscess. Ophthalmoplegia and proptosis at presentation were found to be predictors of postseptal disease, although computed tomography (CT) was necessary to differentiate between cellulitis and abscess. Preseptal disease resolved with antibiotics in all cases. Postseptal disease was treated medically and in some cases surgically, although surgery did not affect outcome. CONCLUSION: Preseptal complications of sinusitis can be diagnosed clinically without a CT scan and should be treated with an appropriate course of intravenous antibiotics. Postseptal complications of sinusitis can be diagnosed by the presence of ophthalmoplegia or proptosis and mandate a CT scan to differentiate abscess from orbital cellulitis. Management of these patients should include intravenous antibiotics, reserving surgery for selected cases.