Fixation-related electrical potentials during a free visual search task reveal the timing of visual awareness.

It has been repeatedly claimed that emotional faces readily capture attention, and that they may be processed without awareness. Yet some observations cast doubt on these assertions. Part of the problem may lie in the experimental paradigms employed. Here, we used a free viewing visual search task during electroencephalographic recordings, where participants searched for either fearful or neutral facial expressions among distractor expressions. Fixation-related potentials were computed for fearful and neutral targets and the response compared for stimuli consciously reported or not. We showed that awareness was associated with an electrophysiological negativity starting at around 110 ms, while emotional expressions were distinguished on the N170 and early posterior negativity only when stimuli were consciously reported. These results suggest that during unconstrained visual search, the earliest electrical correlate of awareness may emerge as early as 110 ms, and fixating at an emotional face without reporting it may not produce any unconscious processing.

Tuning to non-veridical features in attention and perceptual decision-making: An EEG study.

When searching for a lost item, we tune attention to the known properties of the object. Previously, it was believed that attention is tuned to the veridical attributes of the search target (e.g., orange), or an attribute that is slightly shifted away from irrelevant features towards a value that can more optimally distinguish the target from the distractors (e.g., red-orange; optimal tuning). However, recent studies showed that attention is often tuned to the relative feature of the search target (e.g., redder), so that all items that match the relative features of the target equally attract attention (e.g., all redder items; relational account). Optimal tuning was shown to occur only at a later stage of identifying the target. However, the evidence for this division mainly relied on eye tracking studies that assessed the first eye movements. The present study tested whether this division can also be observed when the task is completed with covert attention and without moving the eyes. We used the N2pc in the EEG of participants to assess covert attention, and found comparable results: Attention was initially tuned to the relative colour of the target, as shown by a significantly larger N2pc to relatively matching distractors than a target-coloured distractor. However, in the response accuracies, a slightly shifted, "optimal" distractor interfered most strongly with target identification. These results confirm that early (covert) attention is tuned to the relative properties of an item, in line with the relational account, while later decision-making processes may be biased to optimal features.

Synthesis of N-aryl ß-amino acid derivatives via Cu(ii)-catalyzed asymmetric 1,4-reduction in air.

In the presence of the inexpensive and stable stoichiometric reductant polymethylhydrosiloxane (PMHS) as well as certain amounts of appropriate alcohol and base additives, the non-precious metal copper-catalyzed asymmetric 1,4-hydrosilylation of ß-aryl or ß-alkyl-substituted N-aryl ß-enamino esters was well realized to afford a diverse range of N-aryl ß-amino acid esters in high yields and excellent enantioselectivities (26 examples, 90-98% ee). This approach tolerated the handling of both catalyst and reactants in air without special precautions. The chiral products obtained have been successfully converted to the corresponding enantiomerically enriched ß-lactam and unprotected ß-amino acid ester, which highlighted the synthetic utility of the developed catalytic procedure.

Hypoxia-inducible miR-182 enhances HIF1α signaling via targeting PHD2 and FIH1 in prostate cancer.

Activation of hypoxia-inducible factor 1α (HIF1α) controls the transcription of genes governing angiogenesis under hypoxic condition during tumorigenesis. Here we show that hypoxia-responsive miR-182 is regulated by HIF1α at transcriptional level. Prolyl hydroxylase domain enzymes (PHD) and factor inhibiting HIF-1 (FIH1), negative regulators of HIF1 signaling, are direct targets of miR-182. Overexpression of miR-182 in prostate cancer cells led to a reduction of PHD2 and FIH1 expression and an increase in HIF1α level either under normoxic or hypoxic condition. Consistently, inhibition of miR-182 could increase PHD2 and FIH1 levels, thereby reducing the hypoxia-induced HIF1α expression. Matrigel plug assay showed that angiogenesis was increased by miR-182 overexpression, and vice versa. miR-182 overexpression in PC-3 prostate cancer xenografts decreased PHD2 and FIH1 expression, elevated HIF1α protein levels, and increased tumor size. Lastly, we revealed that the levels of both miR-182 and HIF1α were elevated, while the expression PHD2 and FIH1 was downregulated in a mouse model of prostate cancer. Together, our results suggest that the interplay between miR-182 and HIF1α could result in a sustained activation of HIF1α pathway, which might facilitate tumor cell adaption to hypoxic stress during prostate tumor progression.

Effects of thyroid hormone status on metabolic pathways of arachidonic acid in mice and humans: A targeted metabolomic approach.

Symptoms of cardiovascular diseases are frequently found in patients with hypothyroidism and hyperthyroidism. However, it is unknown whether arachidonic acid metabolites, the potent mediators in cardiovascular system, are involved in cardiovascular disorders caused by hyperthyroidism and hypothyroidism. To answer this question, serum levels of arachidonic acid metabolites in human subjects with hypothyroidism, hyperthyroidism and mice with hypothyroidism or thyroid hormone treatment were determined by a mass spectrometry-based method. Over ten arachidonic acid metabolites belonging to three catalytic pathways: cyclooxygenases, lipoxygenases, and cytochrome P450, were quantified simultaneously and displayed characteristic profiles under different thyroid hormone status. The level of 20-hydroxyeicosatetraenoic acid, a cytochrome P450 metabolite, was positively correlated with thyroid hormone level and possibly contributed to the elevated blood pressured in hyperthyroidism. The increased prostanoid (PG) I2 and decreased PGE2 levels in hypothyroid patients might serve to alleviate atherosclerosis associated with dyslipidemia. The elevated level of thromboxane (TX) A2, as indicated by TXB2, in hyperthyroid patients and mice treated with thyroid hormone might bring about pulmonary hypertension frequently found in hyperthyroid patients. In conclusion, our prospective study revealed that arachidonic acid metabolites were differentially affected by thyroid hormone status. Certain metabolites may be involved in cardiovascular disorders associated with thyroid diseases.

Hepatic p38α regulates gluconeogenesis by suppressing AMPK.

BACKGROUND & AIMS: It is proposed that p38 is involved in gluconeogenesis, however, the genetic evidence is lacking and precise mechanisms remain poorly understood. We sought to delineate the role of hepatic p38α in gluconeogenesis during fasting by applying a loss-of-function genetic approach. METHODS: We examined fasting glucose levels, performed pyruvate tolerance test, imaged G6Pase promoter activity, as well as determined the expression of gluconeogenic genes in mice with a targeted deletion of p38α in liver. Results were confirmed both in vivo and in vitro by using an adenoviral dominant-negative form of p38α (p38α-AF) and the constitutively active mitogen-activated protein kinase 6, respectively. Adenoviral dominant-negative form of AMP-activated protein kinase α (DN-AMPKα) was employed to test our proposed model. RESULTS: Mice lacking hepatic p38α exhibited reduced fasting glucose level and impaired gluconeogenesis. Interestingly, hepatic deficiency of p38α did not result in an alteration in CREB phosphorylation, but led to an increase in AMPKα phosphorylation. Adenoviral DN-AMPKα could abolish the effect of p38α-AF on gluconeogenesis. Knockdown of up-steam transforming growth factor ß-activated kinase 1 decreased the AMPKα phosphorylation induced by p38α-AF, suggesting a negative feedback loop. Consistently, inverse correlations between p38 and AMPKα phosphorylation were observed during fasting and in diabetic mouse models. Importantly, adenoviral p38α-AF treatment ameliorated hyperglycemia in diabetic mice. CONCLUSIONS: Our study provides evidence that hepatic p38α functions as a negative regulator of AMPK signaling in maintaining gluconeogenesis, dysregulation of this regulatory network contributes to unrestrained gluconeogenesis in diabetes, and hepatic p38α could be a drug target for hyperglycemia.

Thyroid hormone regulates muscle fiber type conversion via miR-133a1.

It is known that thyroid hormone (TH) is a major determinant of muscle fiber composition, but the molecular mechanism by which it does so remains unclear. Here, we demonstrated that miR-133a1 is a direct target gene of TH in muscle. Intriguingly, miR-133a, which is enriched in fast-twitch muscle, regulates slow-to-fast muscle fiber type conversion by targeting TEA domain family member 1 (TEAD1), a key regulator of slow muscle gene expression. Inhibition of miR-133a in vivo abrogated TH action on muscle fiber type conversion. Moreover, TEAD1 overexpression antagonized the effect of miR-133a as well as TH on muscle fiber type switch. Additionally, we demonstrate that TH negatively regulates the transcription of myosin heavy chain I indirectly via miR-133a/TEAD1. Collectively, we propose that TH inhibits the slow muscle phenotype through a novel epigenetic mechanism involving repression of TEAD1 expression via targeting by miR-133a1. This identification of a TH-regulated microRNA therefore sheds new light on how TH achieves its diverse biological activities.

Hepatic miR-378 targets p110α and controls glucose and lipid homeostasis by modulating hepatic insulin signalling.

Understanding the regulation of insulin signalling in tissues provides insights into carbohydrate and lipid metabolism in physiology and disease. Here we show that hepatic miR-378/378* expression changes in response to fasting and refeeding in mice. Mice overexpressing hepatic miR-378/378* exhibit pure hepatic insulin resistance. miR-378 inhibits hepatic insulin signalling through targeting p110α, a subunit of PI3K and hence a critical component of insulin signalling. Knockdown of hepatic p110α mimics the effect of miR-378, while restoration of p110α expression abolishes the action of miR-378 on insulin signalling as well as its systemic effects on glucose and lipid homeostasis. miR-378/378* knockout mice display hypoglycemia and increased hepatic triglyceride level with enhanced insulin sensitivity. Inhibition of hepatic p110α in miR-378/378* knockout mice corrects the abnormal glucose tolerance. Finally, we show that overexpression of hepatic miR-378/378* ameliorates hepatic steatosis in ob/ob mice without exacerbating hyperglycemia. Our findings establish fasting-responsive miR-378 as a critical regulator of hepatic insulin signalling.

Regulation of fatty acid composition and lipid storage by thyroid hormone in mouse liver.

BACKGROUND: Thyroid hormones (THs) are potent hormones modulating liver lipid homeostasis. The perturbation of lipid homeostasis is a hallmark of non-alcoholic fatty liver disease (NAFLD), a very common liver disorder. It was reported that NAFLD patients were associated with higher incidence of hypothyroidism. However, whether abnormal thyroid function contributes to the pathogenesis of NAFLD remains unclear. RESULTS: We used in vivo models to investigate the influence of hypothyroidism and TH on hepatic lipid homeostasis. We did not observe hepatic triglyceride accumulation in the liver of hypothyroid mice, although the liver was enlarged. We then characterized the hepatic fatty acid composition with gas chromatography-mass spectrometry in mice under different thyroid states. We found that hypothyroidism decreased saturated fatty acid (SFA) content while TH treatment restored the level of SFA. In agreement with this finding, we observed that the expression of acetyl-CoA carboxylase 1 and fatty acid synthase, the rate-limit enzymes for de novo lipogenesis (DNL), decreased in hypothyroid mice while increased after TH treatment. We also found that the ratio of C18:1n-9/C18:0 and C16:1n-7/C16:0 was decreased by TH treatment, suggesting the activity of stearoyl-CoA desaturase-1 was suppressed. This finding indicated that TH is able to suppress triglyceride accumulation by reducing fatty acid desaturation. Additionally, we found that hepatic glycogen content was substantially influenced by TH status, which was associated with glycogen synthase expression. The increased glycogen storage might explain the enlarged liver we observed in hypothyroid mice. CONCLUSIONS: Taken together, our study here suggested that hypothyroidism in mice might not lead to the development of NAFLD although the liver became enlarged. However, disturbed TH levels led to altered hepatic fatty acid composition and glycogen accumulation.

Circulating Muscle-specific miRNAs in Duchenne Muscular Dystrophy Patients.

Noninvasive biomarkers with diagnostic value and prognostic applications have long been desired to replace muscle biopsy for Duchenne muscular dystrophy (DMD) patients. Growing evidence indicates that circulating microRNAs are biomarkers to assess pathophysiological status. Here, we show that the serum levels of six muscle-specific miRNAs (miR-1/206/133/499/208a/208b, also known as myomiRs) were all elevated in DMD patients (P < 0.01). The receiver operating characteristic curves of circulating miR-206, miR-499, miR-208b, and miR-133 levels reflected strong separation between Becker's muscular dystrophy (BMD) and DMD patients (P < 0.05). miR-206, miR-499, and miR-208b levels were positively correlated with both age and type IIc muscle fiber content in DMD patients (2-6 years), indicating that they might represent the stage of disease as well as the process of regeneration. miR-499 and miR-208b levels were correlated with slow and fast fiber content and might reflect the ratio of slow to fast fibers in DMD patient (>6 years). Fibroblast growth factor, transforming growth factor-ß, and tumor necrosis factor-α could affect the secretion of myomiRs, suggesting that circulating myomiRs might reflect the effects of cytokines and growth factors on degenerating and regenerating muscles. Collectively, our data indicated that circulating myomiRs could serve as promising biomarkers for DMD diagnosis and disease progression.

Circulating miR-130b mediates metabolic crosstalk between fat and muscle in overweight/obesity.

AIMS/HYPOTHESIS: Adipose tissue is a dynamic endocrine organ that regulates whole-body energy homeostasis through the secretion of signalling molecules. Recent reports suggest that secreted microRNAs (miRNAs) may function as biologically active molecules for intercellular communication. This study aims to identify obesity-related circulating miRNA that could be secreted from adipocytes and to explore its possible role in the pathogenesis of metabolic diseases. METHODS: Real-time RT-PCR was used to evaluate the circulating level of miR-130b in mouse models of obesity as well as in humans. Luciferase assay and immunoblotting were used to verify the miRNA target. The effect of miR-130b on mouse peroxisome proliferator-activated receptor γ coactivator-1α was also investigated by electrogene transfer. RESULTS: The circulating level of miR-130b was elevated in mouse models of obesity as well as in obese Chinese individuals. More interestingly, the circulating level of miR-130b was positively correlated with BMI. Moreover, circulating miR-130b was a better predictor of the metabolic syndrome than was triacylglycerol level. Mechanistically, adipocytes secreted miR-130b during adipogenesis. TGF-ß, which is proportionately increased with obesity, stimulated miR-130b secretion from adipocytes. Furthermore, miR-130b was able to target muscle cells and reduce the expression of its direct target gene, PGC-1α (also known as PPARGC1A), which plays a key role in lipid oxidation in muscle. CONCLUSIONS/INTERPRETATION: Circulating miR-130b reflects the degree of obesity and could serve as a potential biomarker for hypertriacylglycerolaemia and metabolic syndrome. Circulating miR-130b could function as a metabolic mediator for adipose-muscle crosstalk and might be involved in the pathogenesis of obesity-associated metabolic diseases.

Attenuation of p38-mediated miR-1/133 expression facilitates myoblast proliferation during the early stage of muscle regeneration.

Myoblast proliferation following myotrauma is regulated by multiple factors including growth factors, signal pathways, transcription factors, and miRNAs. However, the molecular mechanisms underlying the orchestration of these regulatory factors remain unclear. Here we show that p38 signaling is required for miR-1/133a clusters transcription and both p38 activity and miR-1/133 expression are attenuated during the early stage of muscle regeneration in various animal models. Additionally, we show that both miR-1 and miR-133 reduce Cyclin D1 expression and repress myoblast proliferation by inducing G1 phase arrest. Furthermore, we demonstrate that miR-133 inhibits mitotic progression by targeting Sp1, which mediates Cyclin D1 transcription, while miR-1 suppresses G1/S phase transition by targeting Cyclin D1. Finally, we reveal that proproliferative FGF2, which is elevated during muscle regeneration, attenuates p38 signaling and miR-1/133 expression. Taken together, our results suggest that downregulation of p38-mediated miR-1/133 expression by FGF2 and subsequent upregulation of Sp1/Cyclin D1 contribute to the increased myoblast proliferation during the early stage of muscle regeneration.

Ligand-dependent corepressor acts as a novel corepressor of thyroid hormone receptor and represses hepatic lipogenesis in mice.

BACKGROUND & AIMS: Transcriptional co-regulators assist nuclear receptors to control the transcription and maintain the metabolic homeostasis. Ligand-dependent corepressor (LCOR) was reported to function as a transcriptional corepressor in vitro. We found LCOR expression decreased in fatty livers of leptin-deficient (ob/ob) mice, diet-induced obese mice, as well as patients, suggesting LCOR may play a role in lipid homeostasis. We sought to investigate the physiological role of LCOR in vivo and elucidate the underlining molecular mechanisms. METHODS: The effect of LCOR on hepatic lipid accumulation and thyroid hormone receptor (TR) mediated expression of lipogenic genes was studied in vitro and in vivo. RESULTS: Ectopic expression of LCOR via intravenous infection with LCOR adenovirus decreased the hepatic triglyceride level in wild type, ob/ob, and diet-induced obese mice. Interestingly, overexpression of LCOR repressed the thyroid hormone induced expression of lipogenic genes and non-lipogenic genes, and ameliorated hepatic steatosis in obese mice, suggesting that LCOR might regulate lipogenesis as a novel TR corepressor. Furthermore, our study revealed that LCOR could interact with TRß1 in the presence of the ligand, which resulted in competitive binding and reduced recruitment of steroid receptor coactivator-1/3 (SRC-1/3) to the promoter region of TR target genes. CONCLUSIONS: Our data suggest that LCOR is likely to suppress TRß1-mediated hepatic lipogenesis by decreasing binding and recruitment of SRCs to TRß1. Our study reveals the physiological function of hepatic LCOR in lipid metabolism and the mechanism by which LCOR regulates lipogenesis. Hepatic LCOR may be a potential target for treating hepatic steatosis.

Nucleolin links to arsenic-induced stabilization of GADD45alpha mRNA.

The present study shows that arsenic induces GADD45alpha (growth arrest and DNA damage inducible gene 45alpha) mainly through post-transcriptional mechanism. Treatment of the human bronchial epithelial cell line, BEAS-2B, with arsenic(III) chloride (As3+) resulted in a significant increase in GADD45alpha protein and mRNA. However, As3+ only exhibited a marginal effect on the transcription of the GADD45alpha gene. The accumulation of GADD45alpha mRNA is largely achieved by the stabilization of GADD45alpha mRNA in the cellular response to As3+. As3+ is able to induce binding of mRNA stabilizing proteins, nucleolin and less potently, HuR, to the GADD45alpha mRNA. Although As3+ was unable to affect the expression of nucleolin, treatment of the cells with As3+ resulted in re-distribution of nucleolin from nucleoli to nucleoplasm. Silencing of the nucleolin mRNA by RNA interference reversed As3+-induced stabilization of the GADD45alpha mRNA and accumulation of the GADD45alpha protein. Stabilization of GADD45alpha mRNA, thus, represents a novel mechanism contributing to the production of GADD45alpha and cell cycle arrest in response to As3+.