Rapid sequential clustering of NMDARs, CaMKII, and AMPARs upon activation of NMDARs at developing synapses.

Rapid, synapse-specific neurotransmission requires the precise alignment of presynaptic neurotransmitter release and postsynaptic receptors. How postsynaptic glutamate receptor accumulation is induced during maturation is not well understood. We find that in cultures of dissociated hippocampal neurons at 11 days in vitro (DIV) numerous synaptic contacts already exhibit pronounced accumulations of the pre- and postsynaptic markers synaptotagmin, synaptophysin, synapsin, bassoon, VGluT1, PSD-95, and Shank. The presence of an initial set of AMPARs and NMDARs is indicated by miniature excitatory postsynaptic currents (mEPSCs). However, AMPAR and NMDAR immunostainings reveal rather smooth distributions throughout dendrites and synaptic enrichment is not obvious. We found that brief periods of Ca2+ influx through NMDARs induced a surprisingly rapid accumulation of NMDARs within 1 min, followed by accumulation of CaMKII and then AMPARs within 2-5 min. Postsynaptic clustering of NMDARs and AMPARs was paralleled by an increase in their mEPSC amplitudes. A peptide that blocked the interaction of NMDAR subunits with PSD-95 prevented the NMDAR clustering. NMDAR clustering persisted for 3 days indicating that brief periods of elevated glutamate fosters permanent accumulation of NMDARs at postsynaptic sites in maturing synapses. These data support the model that strong glutamatergic stimulation of immature glutamatergic synapses results in a fast and substantial increase in postsynaptic NMDAR content that required NMDAR binding to PSD-95 or its homologues and is followed by recruitment of CaMKII and subsequently AMPARs.

The deubiquitinase cofactor UAF1 interacts with USP1 and plays an essential role in spermiogenesis.

Spermiogenesis defines the final phase of male germ cell differentiation. While multiple deubiquitinating enzymes have been linked to spermiogenesis, the impacts of deubiquitination on spermiogenesis remain poorly characterized. Here, we investigated the function of UAF1 in mouse spermiogenesis. We selectively deleted Uaf1 in premeiotic germ cells using the Stra8-Cre knock-in mouse strain (Uaf1 sKO), and found that Uaf1 is essential for spermiogenesis and male fertility. Further, UAF1 interacts and colocalizes with USP1 in the testes. Conditional knockout of Uaf1 in testes results in disturbed protein levels and localization of USP1, suggesting that UAF1 regulates spermiogenesis through the function of the deubiquitinating enzyme USP1. Using tandem mass tag-based proteomics, we identified that conditional knockout of Uaf1 in the testes results in reduced levels of proteins that are essential for spermiogenesis. Thus, we conclude that the UAF1/USP1 deubiquitinase complex is essential for normal spermiogenesis by regulating the levels of spermiogenesis-related proteins.

Deficiency of the Tmem232 Gene Causes Male Infertility with Morphological Abnormalities of the Sperm Flagellum in Mice.

The axoneme and accessory structures of flagella are critical for sperm motility and male fertilization. Sperm production needs precise and highly ordered gene expression to initiate and sustain the many cellular processes that result in mature spermatozoa. Here, we identified a testis enriched gene transmembrane protein 232 (Tmem232), which is essential for the structural integrity of the spermatozoa flagella axoneme. Tmem232 knockout mice were generated for in vivo analyses of its functions in spermatogenesis. Phenotypic analysis showed that deletion of Tmem232 in mice causes male-specific infertility. Transmission electron microscopy together with scanning electron microscopy were applied to analyze the spermatozoa flagella and it was observed that the lack of TMEM232 caused failure of the cytoplasm removal and the absence of the 7th outer microtubule doublet with its corresponding outer dense fiber (ODF). Co-IP assays further identified that TMEM232 interacts with ODF family protein ODF1, which is essential to maintain sperm motility. In conclusion, our findings indicate that TMEM232 is a critical protein for male fertility and sperm motility by regulating sperm cytoplasm removal and maintaining axoneme integrity.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with ß-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

The Slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice.

The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles toward nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knockout (KO) mice and HA-tagged Ssh2 knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.

Dynamic Response Characteristics of a Sealing Airbag under Different Impact Types and Impact Pressures.

The rapid isolation of the disaster area underground coal mine can effectively prevent the spread of disaster accidents. Rapid sealing of the disaster area can be realized through sealing in the form of an inflatable capsule. The cushioning performance of the inflatable capsule to an explosion shock wave is an important factor affecting sealing reliability. The response process of a small inflatable capsule under an explosion shock wave was studied using the pipeline explosion experimental system to examine the dynamic response characteristics of the coal mine airbag under the impact load. The deformation buffering process of the inflatable and hydrogel capsules was tested by a drop hammer impact test. Results showed that under the action of three explosion impact pressures (0.3, 0.4, and 0.5 MPa), the inflatable capsule could absorb 22% of the impact energy through its deformation and reduce the maximum explosion impact pressure. Moreover, under the impact of falling weight at different heights (20, 30, 40, and 50 cm), the cushioning process of the inflatable and hydrogel capsules absorbed the impact energy through the compression deformation of the capsule, which is the loading stage. When the hammer speed decreased to zero, the deformation and absorbed energy of the capsule were at maximum. The capsule recovered its deformation and converted the absorbed energy into kinetic energy to make the hammer rebound, which is the unloading stage of the capsule. The capsule body realized the absorption and transfer of impact energy through its deformation and completed the energy buffer through the dynamic response process of multiple loading and unloading.

Moroidin, a Cyclopeptide from the Seeds of Celosia cristata That Induces Apoptosis in A549 Human Lung Cancer Cells.

Interference of microtubule dynamics with tubulin-targeted drugs is a validated approach for cancer chemotherapy. Moroidin (1) is an Urticaceae-type cyclopeptide having a potent inhibitory effect on purified tubulin polymerization. So far, moroidin has not been chemically synthesized, and its effect on cancer cells remains unknown. Herein, the cyclopeptide moroidin was isolated and identified from the seeds of Celosia cristata, and a revised assignment of its NMR data was presented. For the first time, moroidin (1) was demonstrated as having cytotoxic effects for several cancer cells, especially A549 lung cancer cells. The cellular evidence obtained showed that moroidin disrupts microtubule polymerization and decreases ß-tubulin protein levels, but is not as potent as colchicine. Molecular docking indicated that 1 has a high binding potential to the vinca alkaloid site on tubulin. Moreover, moroidin arrested A549 cells in the G2/M phase and induced cell apoptosis. The intrinsic mitochondrial pathway and AKT were involved in the moroidin-induced cell apoptosis. In addition, moroidin (1) inhibited the migration and invasion of A549 cells at sublethal concentrations.

Investigation of thermal behavior and hazards quantification in spontaneous combustion fires of coal and coal gangue.

To explore the thermal behavior and hazard during the spontaneous combustion fires (SCFs) of coal and coal gangue (CG), the characteristics of heat release and thermal transfer during the SCFs of coal and CG were tested. The results indicate that coal contains more combustibles and aromatic hydrocarbons, while CG possesses higher contents of ash and inorganic silicate. Coal has a stronger heat release capacity, while CG owns a smaller specific heat capacity, a larger thermal diffusivity and a greater thermal conductivity. Thus, CG performs better with respect to heat transfer. The apparent activation energy of coal is larger in the endothermic stage, whereas that of CG is more notable in the exothermic stage. Based on heat release and heat transfer performance, hazardous zones during the SCFs of coal and CG were identified, and the combustion growth index was established to quantify the hazard of SCF disasters. The results show that the hazard is determined by both heat release and thermal transfer capacities. Coal or CG with a combustible component of 31.3 %, which not only releases massive heat but also transfers heat quickly, corresponds to the most considerable hazard of SCF disasters.

Blended learning model via small private online course improves active learning and academic performance of embryology.

While blended learning has been growing in popularity in recent years, the effectiveness of this procedure remains controversial. In this report, we assess the effectiveness of blended learning of embryology within international medical students. The participants were international medical students taking embryology in the Bachelor of Medicine and Bachelor of Surgery program. The blended learning group (BLG) consisted of students (n = 43) in the 2018-2019 academic year, taught with blended learning model via a customized small private online course (SPOC). The control traditional teaching group (TTG) consisted students (n = 48) in the 2017-2018 academic year, taught with traditional teaching model. Academic performance, including mean scores and passing ratios on the final exam of two groups were compared and analyzed with a t-test. In addition, a questionnaire directed toward evaluating student's perceptions with the blended learning was administered to students in BLG. The majority of students in BLG actively participated in online self-study activities and discussion in face-to-face class sessions. The mean score and passing ratio were significantly greater than those of students in TTG (p < 0.01). Results from the questionnaire revealed that the majority of BLG students felt that this method was beneficial for their learning of human embryology. The blended learning model, that integrates SPOC with face-to-face class lectures proved a more effective means for the teaching of embryology than the traditional lecture-based teaching model. This blended learning method may serve as a feasible model that can be readily applied for use in other medical courses.

TIGAR mitigates atherosclerosis by promoting cholesterol efflux from macrophages.

BACKGROUND AND AIMS: TP53-induced glycolysis and apoptosis regulator (TIGAR) is now characterized as a fructose-2,6-bisphosphatase to reduce glycolysis and protect against oxidative stress. Recent studies have demonstrated that TIGAR is associated with cardiovascular disease. However, little is known about its role in atherosclerogenesis. In this study, we aimed to investigate the effect of TIGAR on atherosclerosis and explore the underlying molecular mechanism. METHODS: The Gene Expression Omnibus (GEO) datasets were used to analyze the differential expression of relative proteins. THP-1-derived macrophages were used as an in vitro model and apolipoprotein E-deficient (Apoe-/-) mice were used as an in vivo model. [3H] labeled cholesterol was used to assess the capacity of cholesterol efflux and reverse cholesterol transport (RCT). Both qPCR and Western blot were used to evaluate the mRNA and protein expression, respectively. Lentiviral vectors were used to disturb the expression of TIGAR in vitro and in vivo. Oil Red O, hematoxylin-eosin, and Masson staining were performed to evaluate atherosclerotic plaques in Apoe-/- mice fed a Western diet. Conventional assay kits were used to measure the levels of reactive oxygen species (ROS), plasma lipid profiles and 27-hydroxycholesterol (27-HC). RESULTS: Our results showed that TIGAR is increased upon the formation of macrophage foam cells and atherosclerosis. TIGAR knockdown markedly promoted lipid accumulation in macrophages. Silencing of TIGAR impaired cholesterol efflux and down-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 by interfering with liver X receptor α (LXRα) expression and activity, but did not influence cholesterol uptake by macrophages. Additionally, this inhibitory effect of TIGAR deficiency on cholesterol metabolism was mediated through the ROS/CYP27A1 pathway. In vivo experiments revealed that TIGAR deficiency decreased the levels of ABCA1 and ABCG1 in plaques and aorta and impaired the capacity of RCT, thereby leading to the progression of atherosclerosis in Apoe-/- mice. CONCLUSIONS: TIGAR mitigates the development of atherosclerosis by up-regulating ABCA1 and ABCG1 expression via the ROS/CYP27A1/LXRα pathway.

Smooth muscle-specific HuR knockout induces defective autophagy and atherosclerosis.

Human antigen R (HuR) is a widespread RNA-binding protein involved in homeostatic regulation and pathological processes in many diseases. Atherosclerosis is the leading cause of cardiovascular disease and acute cardiovascular events. However, the role of HuR in atherosclerosis remains unknown. In this study, mice with smooth muscle-specific HuR knockout (HuRSMKO) were generated to investigate the role of HuR in atherosclerosis. HuR expression was reduced in atherosclerotic plaques. As compared with controls, HuRSMKO mice showed increased plaque burden in the atherosclerotic model. Mechanically, HuR could bind to the mRNAs of adenosine 5'-monophosphate-activated protein kinase (AMPK) α1 and AMPKα2, thus increasing their stability and translation. HuR deficiency reduced p-AMPK and LC3II levels and increased p62 level, thereby resulting in defective autophagy. Finally, pharmacological AMPK activation induced autophagy and suppressed atherosclerosis in HuRSMKO mice. Our findings suggest that smooth muscle HuR has a protective effect against atherosclerosis by increasing AMPK-mediated autophagy.

Hepatic HuR protects against the pathogenesis of non-alcoholic fatty liver disease by targeting PTEN.

The liver plays an important role in lipid and glucose metabolism. Here, we show the role of human antigen R (HuR), an RNA regulator protein, in hepatocyte steatosis and glucose metabolism. We investigated the level of HuR in the liver of mice fed a normal chow diet (NCD) and a high-fat diet (HFD). HuR was downregulated in the livers of HFD-fed mice. Liver-specific HuR knockout (HuRLKO) mice showed exacerbated HFD-induced hepatic steatosis along with enhanced glucose tolerance as compared with control mice. Mechanistically, HuR could bind to the adenylate uridylate-rich elements of phosphatase and tensin homolog deleted on the chromosome 10 (PTEN) mRNA 3' untranslated region, resulting in the increased stability of Pten mRNA; genetic knockdown of HuR decreased the expression of PTEN. Finally, lentiviral overexpression of PTEN alleviated the development of hepatic steatosis in HuRLKO mice in vivo. Overall, HuR regulates lipid and glucose metabolism by targeting PTEN.

Myristica fragrans promotes ABCA1 expression and cholesterol efflux in THP-1-derived macrophages.

Myristica fragrans is a traditional herbal medicine and has been shown to alleviate the development of atherosclerosis. However, the anti-atherogenic mechanisms of M. fragrans are still to be addressed. In this study, we explored the effect of M. fragrans on lipid metabolism and inflammation and its mechanisms in THP-1-derived macrophages. The quantitative polymerase chain reaction and western blot analysis results showed that M. fragrans promotes cholesterol efflux from THP-1-derived macrophages and reduces intracellular total cholesterol, cholesterol ester, and free cholesterol contents in a dose- and a time-dependent manner. Further study found that liver X receptor alpha (LXRα) antagonist GGPP significantly blocked the upregulation of ABCA1 expression with M. fragrans treatment. In addition, chromatin immunoprecipitation assay confirmed that GATA binding protein 3 (GATA3) can bind to the LXRα promoter, and inhibition of GATA3 led to the downregulation of LXRα and ATP-binding cassette subfamily A member 1 expression. Furthermore, M. fragrans reduced lipid accumulation, followed by decreasing tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß and increasing IL-10 produced by THP-1-derived macrophages. Therefore, M. fragrans is identified as a valuable therapeutic medicine for atherosclerotic cardiovascular disease.

Coronavirus disease 2019 (COVID-19): current status and future perspectives.

Coronavirus disease 2019 (COVID-19) originated in the city of Wuhan, Hubei Province, Central China, and has spread quickly to 72 countries to date. COVID-19 is caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [previously provisionally known as 2019 novel coronavirus (2019-nCoV)]. At present, the newly identified SARS-CoV-2 has caused a large number of deaths with tens of thousands of confirmed cases worldwide, posing a serious threat to public health. However, there are no clinically approved vaccines or specific therapeutic drugs available for COVID-19. Intensive research on the newly emerged SARS-CoV-2 is urgently needed to elucidate the pathogenic mechanisms and epidemiological characteristics and to identify potential drug targets, which will contribute to the development of effective prevention and treatment strategies. Hence, this review will focus on recent progress regarding the structure of SARS-CoV-2 and the characteristics of COVID-19, such as the aetiology, pathogenesis and epidemiological characteristics.

Beta-hydroxybutyric acid attenuates neuronal damage in epileptic mice.

ß-Hydroxybutyric acid (BHBA) reportedly has neuroprotective and anti-oxidation properties. The present study aimed to investigate the protective effects of BHBA against epilepsy. C57BL/6 J mice were exposed to lithium chloride and pilocarpine to induce epilepsy and then were administrated with 300 mg/kg/day BHBA for 30 days. The learning impairment was evaluated via Morris Water Maze. Neuron loss and cell apoptosis were detected through Nissl staining and TUNEL staining. The levels of oxidative stress-related factors were determined by commercial kits. The protein expression levels of AMP-activated protein kinase (AMPK), p-AMPK, peroxisome proliferator-activated receptor alpha (PPARα), anti-apoptotic Bcl-2, and pro-apoptotic Bax were measured through Western blots. It was found BHBA improved epilepsy- caused learning deficiency and attenuated epilepsy-mediated neuron loss and cell apoptosis in the hippocampus. BHBA ameliorated oxidative stress via decreasing the levels of reactive oxygen species and malondialdehyde plus strengthening the activities of glutathione peroxidase and superoxide dismutase. BHBA also promoted the phosphorylation of AMPK and upregulated PPARα in the epileptic hippocampus. In conclusion, BHBA attenuates neuronal damage in epileptic mice, which is associated with its anti-apoptotic and anti-oxidative effects as well as the activation of AMPK and PPARα.

Maternal hyperglycemia disturbs neocortical neurogenesis via epigenetic regulation in C57BL/6J mice.

Offspring of mothers with hyperglycemia during pregnancy have a higher incidence of long-term neuropsychiatric disorders than offspring from a normal pregnancy, indicating that neocortical neurogenesis might be affected by maternal hyperglycemia. A paucity of study evaluating the effects of hyperglycemia on neocortical neurogenetic differentiation of neural stem cells, and the mechanism remains unclear. We sought to investigate the the roles and possible molecular mechanism of maternal hyperglycemia on neocortical neurogenetic differentiation of neural stem cells. We established a mouse model of a hyperglycemic pregnancy to study effects of intrauterine exposure to maternal hyperglycemia on neocortical neurogenesis. We observed morphological changes in the neocortex and detected the neurogenetic differentiation of neural stem cells in offspring affected by high glucose levels. We investigated the regulatory network between epigenetic modification and transcription factors in differentiated neural stem cells under hyperglycemic conditions. Maternal hyperglycemia disturbs neocortical lamination in some non-malformed offspring. Our results suggested that hyperglycemia altered the early-born neuron fate and the distribution of newborn neurons in deep layers by promoting the earlier differentiation of neural stem cells. Altered histone acetylation and its regulation on the transcription of proneural genes might be correlated to the disrupted differentiation of neural stem cells and altered distribution of newborn projection neurons in the neocortex. Our data raised the possibility that maternal hyperglycemia in pregnancy disturbs the laminar distribution of neocortical projection neurons in some non-malformed offspring via epigenetic regulation on neural stem cell differentiation and the birthdate of neocortical neurons.

Melatonin Enhances Proliferation and Modulates Differentiation of Neural Stem Cells Via Autophagy in Hyperglycemia.

Dysfunction of neural stem cells (NSCs) has been linked to fetal neuropathy, one of the most devastating complications of gestational diabetes. Several studies have demonstrated that melatonin (Mel) exerted neuroprotective actions in various stresses. However, the role of autophagy and the involvement of Mel in NSCs in hyperglycemia (HG) have not yet been fully established. Here, we found that HG increased autophagy and autophagic flux of NSCs as evidenced by increasing LC3B II/I ratio, Beclin-1 expression, and autophagosomes. Moreover, Mel enhanced NSCs proliferation and self-renewal in HG with decreasing autophagy and activated mTOR signaling. Consistently, inhibition of autophagy by 3-Methyladenine (3-Ma) could assist Mel effects above, and induction of autophagy by Rapamycin (Rapa) could diminish Mel effects. Remarkably, HG induced premature differentiation of NSCs into neurons (Map2 positive cells) and astrocytes (GFAP positive cells). Furthermore, Mel diminished HG-induced premature differentiation and assisted NSCs in HG differentiation as that in normal condition. Coincidentally, inhibiting of NSCs autophagy by 3-Ma assisted Mel to modulate differentiation. However, increasing NSCs autophagy by Rapa disturbed the Mel effects and retarded NSCs differentiation. These findings suggested that Mel supplementation could contribute to mimicking normal NSCs proliferation and differentiation in fetal central nervous system by inhibiting autophagy in the context of gestational diabetes. Stem Cells 2019;37:504-515.

Pregnancy-Associated Plasma Protein-A Accelerates Atherosclerosis by Regulating Reverse Cholesterol Transport and Inflammation.

BACKGROUND: Recent studies have suggested that pregnancy-associated plasma protein-A (PAPP-A) is involved in the pathogenesis of atherosclerosis. This study aim is to investigate the role and mechanisms of PAPP-A in reverse cholesterol transport (RCT) and inflammation during the development of atherosclerosis. Methods and Results: PAPP-A was silenced in apolipoprotein E (apoE-/-) mice with administration of PAPP-A shRNA. Oil Red O staining of the whole aorta root revealed that PAPP-A knockdown reduced lipid accumulation in aortas. Oil Red O, hematoxylin and eosin (HE) and Masson staining of aortic sinus further showed that PAPP-A knockdown alleviated the formation of atherosclerotic lesions. It was found that PAPP-A knockdown reduced the insulin-like growth factor 1 (IGF-1) levels and repressed the PI3K/Akt pathway in both aorta and peritoneal macrophages. The expression levels of LXRα, ABCA1, ABCG1, and SR-B1 were increased in the aorta and peritoneal macrophages from apoE-/-mice administered with PAPP-A shRNA. Furthermore, PAPP-A knockdown promoted RCT from macrophages to plasma, the liver, and feces in apoE-/-mice. In addition, PAPP-A knockdown elevated the expression and secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), tumor necrosis factor-α, and interleukin-1ß through the nuclear factor kappa-B (NF-κB) pathway. CONCLUSIONS: The present study results suggest that PAPP-A promotes the development of atherosclerosis in apoE-/-mice through reducing RCT capacity and activating an inflammatory response.

Melatonin enhances atherosclerotic plaque stability by inducing prolyl-4-hydroxylase α1 expression.

OBJECTIVE: Melatonin, an endogenous neurohormone secreted predominately by the pineal gland, has a variety of physiological functions. However, its protective role in atherosclerosis is not clear. In this study, we sought to investigate the potential effects of melatonin in modulating atherosclerotic plaque stability in apolipoprotein E knockout (ApoE) mice. METHOD AND RESULTS: Smooth muscle cells were treated with melatonin, which significantly increased mRNA and protein levels of a key intracellular enzyme essential for collagen maturation and secretion, prolyl-4-hydroxylase α1 (P4Hα1). Mechanistically, melatonin increased Akt phosphorylation and transcriptional activation of specificity protein 1 (Sp1), which bound with the P4Hα1 promoter and then induced P4Hα1 expression. Pretreatment with either Akt inhibitor LY294002 or Sp1 inhibitor mithramycin A (MTM) could inhibit melatonin-induced P4Hα1 expression. Finally, atherosclerotic lesions were induced by placing a perivascular collar on the right common carotid artery of ApoE mice, which were received with or without different doses of melatonin or MTM. High-dose melatonin enhanced atherosclerotic plaque stability in ApoE mice in vivo by inducing the expression of P4Hα1, which was reversed by MTM. CONCLUSION: We propose that melatonin supplementation may provide a novel and promising approach to atherosclerosis treatment.

Nicotine induces endothelial dysfunction and promotes atherosclerosis via GTPCH1.

Smoking is a major preventable risk factor for atherosclerosis. However, the causative link between cigarette smoke and atherosclerosis remains to be established. The objective of this study is to characterize the role of GTP cyclohydrolase 1 (GTPCH1), the rate-limiting enzyme for de novo tetrahydrobiopterin (BH4) synthesis, in the smoking-accelerated atherosclerosis and the mechanism involved. In vitro, human umbilical vein endothelial cells were treated with nicotine, a major component of cigarette smoke, which reduced the mRNA and protein levels of GTPCH1 and led to endothelial dysfunction. GTPCH1 overexpression or sepiapterin could attenuate nicotine-reduced nitric oxide and -increased reactive oxygen species levels. Mechanistically, human antigen R (HuR) bound with the adenylateuridylate-rich elements of the GTPCH1 3' untranslated region and increased its stability; nicotine inhibited HuR translocation from the nucleus to cytosol, which downregulated GTPCH1. In vivo, nicotine induced endothelial dysfunction and promoted atherosclerosis in ApoE-/- mice, which were attenuated by GTPCH1 overexpression or BH4 supplement. Our findings may provide a novel and promising approach to atherosclerosis treatment.