Decoupling of Massive Right-handed Neutrinos

We investigate the effect of B+L - violating anomalous generation of massive right-handed neutrinos on their decoupling, when the right-handed neutrino mass is considerably greater than the right-handed gauge boson masses. Considering normal annihilation channels, the Lee-Weinberg type of calculation, in this case, gives an upper bound of about 700 Gev, which casts doubt on the existence of such a right-handed neutrino mass greater than right-handed gauge boson masses. We examine the possibility that a consideration of anomalous effects related to the SU(2)_R gauge group may turn this into a lower bound of the order of 100 Tev.Comment: 28 Pages, Latex, 2 figure

Increased arterial pressure in mice with overexpression of the ADHD candidate gene calcyon in forebrain.

The link between blood pressure (BP) and cerebral function is well established. However, it is not clear whether a common mechanism could underlie the relationship between elevated BP and cognitive deficits. The expression of calcyon, a gene abundant in catecholaminergic and hypothalamic nuclei along with other forebrain regions, is increased in the brain of the spontaneously hypertensive rat (SHR) which is a widely accepted animal model of essential hypertension and attention deficit hyperactivity disorder (ADHD). Previous studies demonstrated that mice with up-regulation of calcyon in forebrain (CalOE) exhibit deficits in working memory. To date, there is no evidence directly connecting calcyon to BP regulation. Here, we investigated whether forebrain up-regulation of calcyon alters BP using radiotelemetry. We found that CalOE mice exhibited higher mean arterial pressure (MAP) compared to tTA controls. Plasma norepinephrine levels were significantly higher in CalOE mice compared to tTA controls. Silencing the transgene with doxycycline normalized BP in CalOE mice, whereas challenging the mice with 4% high salt diet for 12 days exacerbated the MAP differences between CalOE and tTA mice. High salt diet challenge also increased proteinuria and urinary thiobarbituric acid reactive substances (TBARs) in tTA and CalOE; and the increases were more prominent in CalOE mice. Taken together, our data suggest that upregulation of calcyon in forebrain could increase BP via alterations in noradrenergic transmission and increased oxidative stress during high salt challenge. Overall, this study reveals that calcyon could be a novel neural regulator of BP raising the possibility that it could play a role in the development of vascular abnormalities

Genetic Dissection of the Impact of miR-33a and miR-33b during the Progression of Atherosclerosis

Summary: As an important regulator of macrophage cholesterol efflux and HDL biogenesis, miR-33 is a promising target for treatment of atherosclerosis, and numerous studies demonstrate that inhibition of miR-33 increases HDL levels and reduces plaque burden. However, important questions remain about how miR-33 impacts atherogenesis, including whether this protection is primarily due to direct effects on plaque macrophages or regulation of lipid metabolism in the liver. We demonstrate that miR-33 deficiency in Ldlr−/− mice promotes obesity, insulin resistance, and hyperlipidemia but does not impact plaque development. We further assess how loss of miR-33 or addition of miR-33b in macrophages and other hematopoietic cells impact atherogenesis. Macrophage-specific loss of miR-33 decreases lipid accumulation and inflammation under hyperlipidemic conditions, leading to reduced plaque burden. Therefore, the pro-atherogenic effects observed in miR-33-deficient mice are likely counterbalanced by protective effects in macrophages, which may be the primary mechanism through which anti-miR-33 therapies reduce atherosclerosis. : miR-33a and miR-33b, the miR-33 family of miRNAs, are important regulators of reverse cholesterol transport and atherosclerosis. Price et al. have developed genetic models to explore the specific roles of miR-33a and miR-33b in atherosclerotic plaque formation. Their findings highlight both the utility and potential issues involved in anti-miR-33 therapies. Keywords: Atherosclerosis, miR-33, HDL-C, metabolism, cholestero

Endotoxin Disrupts Circadian Rhythms in Macrophages via Reactive Oxygen Species.

The circadian clock is a transcriptional network that functions to regulate the expression of genes important in the anticipation of changes in cellular and organ function. Recent studies have revealed that the recognition of pathogens and subsequent initiation of inflammatory responses are strongly regulated by a macrophage-intrinsic circadian clock. We hypothesized that the circadian pattern of gene expression might be influenced by inflammatory stimuli and that loss of circadian function in immune cells can promote pro-inflammatory behavior. To investigate circadian rhythms in inflammatory cells, peritoneal macrophages were isolated from mPer2luciferase transgenic mice and circadian oscillations were studied in response to stimuli. Using Cosinor analysis, we found that LPS significantly altered the circadian period in peritoneal macrophages from mPer2luciferase mice while qPCR data suggested that the pattern of expression of the core circadian gene (Bmal1) was disrupted. Inhibition of TLR4 offered protection from the LPS-induced impairment in rhythm, suggesting a role for toll-like receptor signaling. To explore the mechanisms involved, we inhibited LPS-stimulated NO and superoxide. Inhibition of NO synthesis with L-NAME had no effect on circadian rhythms. In contrast, inhibition of superoxide with Tempol or PEG-SOD ameliorated the LPS-induced changes in circadian periodicity. In gain of function experiments, we found that overexpression of NOX5, a source of ROS, could significantly disrupt circadian function in a circadian reporter cell line (U2OS) whereas iNOS overexpression, a source of NO, was ineffective. To assess whether alteration of circadian rhythms influences macrophage function, peritoneal macrophages were isolated from Bmal1-KO and Per-TKO mice. Compared to WT macrophages, macrophages from circadian knockout mice exhibited altered balance between NO and ROS release, increased uptake of oxLDL and increased adhesion and migration. These results suggest that pro-inflammatory stimuli can disrupt circadian rhythms in macrophages and that impaired circadian rhythms may contribute to cardiovascular diseases by altering macrophage behavior

Circadian clock control of Nox4 and reactive oxygen species in the vasculature.

Recent studies have shown that circadian clock disruption is associated with pathological remodeling in the arterial structure and vascular stiffness. Moreover, chronic circadian disruption is associated with dysfunction in endothelial responses and signaling. Reactive oxygen species have emerged as key regulators in vascular pathology. Previously, we have demonstrated that circadian clock dysfunction exacerbates superoxide production through eNOS uncoupling. To date, the impact of circadian clock mutation on vascular NADPH oxidase expression and function is not known. The goal in the current study was to determine if the circadian clock controls vascular Nox4 expression and hydrogen peroxide formation in arteries, particularly in endothelial and vascular smooth muscle cells. In aorta, there was an increase in hydrogen peroxide and Nox4 expression in mice with a dysfunctional circadian rhythm (Bmal1-KO mice). In addition, the Nox4 gene promoter is activated by the core circadian transcription factors. Lastly, in synchronized cultured human endothelial cells, Nox4 gene expression exhibited rhythmic oscillations. These data reveal that the circadian clock plays an important role in the control of Nox4 and disruption of the clock leads to subsequent production of reaction oxygen species

LPS promotes a circadian phase-shift at low concentrations.

<p>Varying doses of LPS (0, 5, 20 and 100ng/ml) were added into the luminescence buffer at Time 0 after serum shock. <b>(A)</b> <i>Left panel</i>: Bioluminescence were recorded every 2h continuing for 68 hours. <i>Right panel</i>: Relative cell numbers were measured via a cell viability assay at the end of the luminescence measurements (mean ± SEM, n = 4, one-way ANOVA, ns versus Control). <b>(B)</b> Oscillation curves were analyzed by cosinor and acrophase compared by one-way Anova with Bonferroni post hoc correction (mean ± SEM, n = 8, *<i>p</i><0.05, <i>versus</i> control, # <i>p</i><0.05, <i>versus</i> LPS 5ng/ml).</p

LPS stimulates ROS release from peritoneal macrophage and elevated ROS impairs the function of circadian transcription factors.

<p><b>(A)</b> Peritoneal macrophages were isolated from <i>mPer2</i>^luciferase transgenic mice and subjected to different treatments over 24h (LPS 20ng/ml, LPS-RS 5μg/ml, gp91 ds-tat 1μM). Unstimulated or basal superoxide release was monitored using L-012 chemiluminescence (mean ± SEM, n = 5, one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus Control). <b>(B)</b> Peritoneal macrophages from <i>mPer2</i>^luciferase mice were subjected to different treatments (LPS 20ng/ml, PEG-SOD, 100U/ml, Tempol 0.4mM) and bioluminescence recorded every 2h for 68 hours (mean ± SEM, n = 5, acrophase were compared via one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus control. # p<0.05, versus LPS). <b>(C)</b> U2OS <i>Bma1</i>^luciferase cells were transduced with active or inactive <i>Nox5</i> adenovirus (15 MOI) and the oscillation of expressed luciferase activity recorded every 2h after serum shock. Oscillation curves were analyzed by cosinor and acrophase compared by one-way Anova with Bonferroni post hoc correction (mean ± SEM, n = 6, *p<0.05, versus Control, # p<0.05, versus Nox5 active). <b>(D)</b> <i>Per1</i> promoter transactivation was assessed by a dual luciferase assay in transfected COS cells expressing BMAL1, BMAL1+CLOCK in the presence or absence of the ROS generator NOX5 or an inactive NOX5 enzyme (H268Q), (mean ± SEM, n = 5, one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus <i>Bmal1</i> alone. # p<0.05, versus Nox5 active). <b>(E)</b> SOD-sensitive superoxide production was monitored by L-012 chemiluminescence. Results are presented as mean ± SEM, n = 6, one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus <i>Bmal1</i> alone.</p

Circadian clock disruption increases LDL uptake by macrophages.

<p>Peritoneal macrophages were isolated from WT, <i>Bmal1</i> KO or <i>Per</i>-TKO mice and cells exposed to oxLDL (50μg/ml) for 72h. <b>(A)</b> Oil red O staining for lipid uptake in macrophages. The extent of staining was quantitated by measurement of fluorescent intensity. (mean ± SEM n = 4, t-test, *p<0.05 versus WT). <b>(B)</b> Measurement of cholesterol ester and free cholesterol content from macrophages. (mean ± SEM n = 4, t-test, *p<0.05 versus WT</p