Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). Here we report the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2Å resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions. Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

It is generally accepted that nitric oxide (NO) or its derivatives, reactive nitrogen species (RNS), are involved in the development of Parkinson's disease (PD). Recently, emerging evidence in the study of PD has indicated that protein S-nitrosylation triggers the signaling changes in neurons. In this study, SH-SY5Y cells treated with rotenone were used as a model of neuronal death in PD. The treated cells underwent significant apoptosis, which was accompanied by an increase in intracellular NO in a rotenone dose-dependent manner. The CyDye switch approach was employed to screen for changes in S-nitrosylated (SNO) proteins in response to the rotenone treatment. Seven proteins with increased S-nitrosylation were identified in the treated SH-SY5Y cells, which included proliferating cell nuclear antigen (PCNA). Although PCNA is generally located in the nucleus and participates in DNA replication and repair, significant PCNA was identified in the SH-SY5Y cytosol. Using immunoprecipitation and pull-down approaches, PCNA was found to interact with caspase-9; using mass spectrometry, the two cysteine residues PCNA-Cys81 and -Cys162 were identified as candidate S-nitrosylated residues. In addition, the evidence obtained from in vitro and the cell model studies indicated that the S-nitrosylation of PCNA-Cys81 affected the interaction between PCNA and caspase-9. Furthermore, the interaction of PCNA and caspase-9 partially blocked caspase-9 activation, indicating that the S-nitrosylation of cytosolic PCNA may be a mediator of the apoptotic pathway.

The present study was to investigate the effect of hydrogen peroxide (H2O2) on bone marrow stem cells and their endothelial differentiation and the underlying mechanisms in vitro.

Abnormal survival of retinal pigment epithelium (RPE) cells contributes to the pathogenesis of proliferative vitreoretinopathy (PVR), a sight-threatening disease. In this study, we explored the effect of the anti-rheumatic agent auranofin (AF) on RPE cell survival and studied the underlying signaling mechanisms in vitro. Our results showed that AF inhibited ARPE-19 cell survival in a dose and time-dependent manner. Application of AF induced several effects: a significant decrease in total epidermal growth factor receptor (EGFR) and an increase in phosphorylated EGFR and mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), c-Jun, mitogen activated protein kinase activated protein kinase 2(MAPKAPK2), and heat shock protein 27 (HSP27). AF also inhibited epidermal growth factor (EGF)-dependent cell proliferation and migration through affecting EGFR/MAPK signaling. The antioxidant N-acetylcysteine (NAC) blocked the AF-induced increase of reactive oxygen species (ROS) production, the reduction of total EGFR, and the phosphorylation of multiple nodes in EGFR/MAPK signaling pathway. P38MAPK inhibitor SB203580, but not inhibitors of EGFR (erlotinib), ERK (FR180204) and JNK (SP600125), suppressed AF-induced phosphorylation of EGFR/p38MAPK/MAPKAPK2/Hsp27. In conclusion, the ROS-dependent phosphorylation of EGFR/MAPK is an important signaling pathway for AF-induced inhibition of RPE cell survival, and AF may have the potential for treatment of abnormal survival of RPE cells in PVR.

Nagashima-type palmoplantar keratosis (NPPK) is an autosomal recessive form of palmoplantar keratoderma (PPK), which is caused by mutations in the SERPINB7 gene. NPPK has only been reported in Japanese and Chinese populations. The present study was conducted on 12 unrelated Chinese patients who were clinically predicted to suffer from NPPK. Mutation screening was performed by direct sequencing of the entire coding regions of SERPINB7, SLURP1, AQP5, CSTA, KRT1 and KRT9 genes. Direct sequencing of SERPINB7 revealed five homozygous founder mutations (c.796C>T) and four compound heterozygous mutations in nine patients, including one novel mutation (c.122_127delTGGTCC). Nine out of the 12 patients were diagnosed with NPPK due to SERPINB7 pathogenic mutations, and the results expanded the known mutation spectrum of NPPK. Taking the other seven reported Chinese patients, who had been definitively diagnosed with NPPK by genetic testing, into account, the present study further demonstrated that NPPK is a common entity in Mainland China, and c.796C>T is the most prevalent mutation and exerts a founder effect. Furthermore, the NPPK cases described in the current study presented a consistently mild phenotype, as compared with the degrees of phenotypic variability associated with other types of relatively severe PPK, including Mal de Meleda and Olmsted syndrome.

Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen.

Low-density lipoprotein (LDL) is non-atherogenic, while oxidized LDL (ox-LDL) is critical to atherosclerosis. N-acetylcysteine (NAC) has anti-atherosclerotic effect with largely unknown mechanisms. The present study aimed to determine if NAC could attenuate in vivo LDL oxidation and inhibit atherosclerosis. A single dose of human native LDL was injected intravenously into male C57BL/6 mice with and without NAC treatment. Serum human ox-LDL was detected 30 min after injection, reached the peak in 3 hours, and became undetectable in 12 hours. NAC treatment significantly reduced serum ox-LDL level without detectable serum ox-LDL 6 hours after LDL injection. No difference in ox-LDL clearance was observed in NAC-treated animals. NAC treatment also significantly decreased serum ox-LDL level in patients with coronary artery diseases and hyperlipidemia without effect on LDL level. Intracellular and extracellular reactive oxidative species (ROS) production was significantly increased in the animals treated with native LDL, or ox-LDL and in hyperlipidemic LDL receptor knockout (LDLR(-/-)) mice that was effectively prevented with NAC treatment. NAC also significantly reduced atherosclerotic plaque formation in hyperlipidemic LDLR(-/-) mice. NAC attenuated in vivo oxidation of native LDL and ROS formation from ox-LDL associated with decreased atherosclerotic plaque formation in hyperlipidemia.

Signal transducers and activators of transcription 3 (Stat3) has been reported to be involved in the pathogenesis of various human diseases and is constitutively active in human multiple myeloma (MM) U266 cells. The Stat3-regulated mechanisms involved in these processes, however, are not fully defined. To further understand the regulation of Stat3 activity, we performed a systematic proteomic analysis of Stat3 interacting proteins in U266 cells. This analysis, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture (SILAC), immunoprecipitation, and quantitative MS. As a result, quantitative mass spectrometry analysis allowed us to distinguish specific Stat3 interacting proteins from background proteins and led to the identification of a total of 38 proteins. Three Stat3 interacting proteins - 14-3-3ζ, PRKCB and Hsp90 - were further confirmed by reciprocal co-immunoprecipitations and surface plasmon resonance (SPR) analysis. Our results therefore not only uncover a number of Stat3 interacting proteins that possess a variety of cellular functions, but also provide new insight into the mechanisms that regulate Stat3 activity and function in MM cells.

The functional significance of NOX1/NADPH oxidase in the heart has not been explored due to its low expression relative to other NOX homologs identified so far. We aimed to clarify the role of NOX1/NADPH oxidase in the septic heart by utilizing mice deficient in the Nox1 gene (Nox1(-/Y)). Sepsis was induced by intraperitoneal administration of lipopolysaccharides (LPS: 25mg/kg) or cecal ligation and puncture (CLP) surgery. A marked elevation of NOX1 mRNA was demonstrated in cardiac tissue, which was accompanied by increased production of reactive oxygen species (ROS). In Nox1(-/Y) treated with LPS, cardiac dysfunction and survival were significantly improved compared with wild-type mice (Nox1(+/Y)) treated with LPS. Concomitantly, LPS-induced cardiomyocyte apoptosis and activation of caspase-3 were alleviated in Nox1(-/Y). The level of phosphorylated Akt in cardiac tissue was significantly lowered in Nox1(+/Y) but not in Nox1(-/Y) treated with LPS or that underwent CLP surgery. Increased oxidation of cysteine residues of Akt and enhanced interaction of Akt with protein phosphatase 2A (PP2A), a major phosphatase implicated in the dephosphorylation of Akt, were demonstrated in LPS-treated Nox1(+/Y). These responses to LPS were significantly attenuated in Nox1(-/Y). Taken together, ROS derived from NOX1/NADPH oxidase play a pivotal role in endotoxin-induced cardiomyocyte apoptosis by increasing oxidation of Akt and subsequent dephosphorylation by PP2A. Marked up-regulation of NOX1 may affect the risk of mortality under systemic inflammatory conditions.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs that regulate gene expression at the post-transcriptional levels. Recent studies show that miRNAs may function as oncogenes or tumor suppressor genes. In this study, we demonstrated that miR-518b was down-regulated in esophageal squamous cell carcinoma (ESCC) tissues and correlated with metastasis and survival. miR-518b suppressed the proliferation by inducing apoptosis and repressed the invasion in ESCC cells, but had no effect on the cell cycle. Furthermore, Rap1b was revealed to be directly regulated by miR-518b. These findings indicate that miR-518b may function as a tumor suppressor by targeting Rap1b in the development of ESCC and has important clinical and prognostic value.

Myelin transcription factor 1 (Myt1) is one of the three vertebrate C2HC-type zinc finger transcription factors that include Myt1 (Nzf1), Myt1L (Png1), and Myt3 (Nzf3, St18). All three paralogs are widely expressed in developing neuronal cells. Yet their function for mammalian development has not been investigated directly. Here we report that only Myt1 is expressed in the embryonic pancreas, in both endocrine progenitors and differentiated islet cells. Myt1(-/-) animals die postnatally, likely due to confounding effects in multiple tissues. The endocrine tissues in the embryonic Myt1(-/-) pancreas contained abnormal islet cells that expressed multiple hormones; although hormone levels were normal. We also created pancreas-specific Myt1 knockout mice. These mutant animals had no obvious physical defects from their wild-type littermates. Male mutant animals had reduced glucose-clearing abilities and abnormal multi-hormone-expressing cells present in their endocrine islets. In addition, they also had reduced Glut2 expression, and attenuated glucose-induced insulin secretion in the adult islets. Surprisingly, the expression of the Myt1 paralogs, Myt1l and Myt3, was induced in the embryonic Myt1(-/-) pancreas. The consequences of Myt1 inactivation in the developing pancreas could be masked by activation of its paralogs, Myt1l and Myt3. These findings suggest Myt1 is involved in proper endocrine differentiation and function.

Forkhead box Q1 (FOXQ1, also known as HFH1), a member of the forkhead transcription factor family, has been demonstrated to be overexpressed in multiple tumors and is thought to be an indicator of poor clinical outcomes.

The occurrence of algal blooms in drinking water sources and recreational water bodies have been increasing and causing severe environmental problems worldwide, particularly when blooms dominated by Microcystis spp. Bloom prediction and early warning mechanisms are becoming increasingly important for preventing harmful algal blooms in freshwater ecosystems. Chlorophyll fluorescence parameters (CFpars) have been widely used to evaluate growth scope and photosynthetic efficiency of phytoplankton. According to our 2-year monthly monitor datasets in Lake Erhai, a simple but convenient method was established to predict Microcystis blooms and algal cell densities based on a CFpar representing maximal photochemical quantum yield of Photosystems II (PSII) of algae. Generalized linear mixed models, used to identify the key factors related to the phytoplankton biomass in Lake Erhai, showed significant correlations between Chl a concentration and both the light attenuation coefficient and water temperature. We fitted seasonal trends of CFpars (Fv/Fm and ΔF/Fm') and algal cell densities into the trigonometric regression to predict their seasonal variations and the autocorrelation function was applied to calculate the time lag between them. We found that the time lag only existed between Fv/Fm from blue channel and algal cell densities even both Fv/Fm and ΔF/Fm' show the significant non-linear dynamics relationships with algal cell densities. The peak values of total algal cell density, cyanobacteria density and Microcystis density followed the foregoing peak value of Fv/Fm from blue channel with a time lagged around 40 days. Therefore, we could predict the possibilities of Microcystis bloom and estimate the algal cell densities in Lake Erhai ahead of 40 days based on the trends of Fv/Fm values from blue channel. The results from our study implies that the corresponding critical thresholds between Fv/Fm value and bloom occurrence, which might give new insight into prediction of cyanobacteria blooms and provide a convenient and efficient way for establishment of early warning of cyanobacteria bloom in eutrophic aquatic ecosystems.

Although recent evidence has pointed to the existence of small open reading frame (smORF)-encoded microproteins in mammals, their function remains to be determined. Skeletal muscle development requires fusion of mononuclear progenitors to form multinucleated myotubes, a critical but poorly understood process. Here we report the identification of Minion (microprotein inducer of fusion), a smORF encoding an essential skeletal muscle specific microprotein. Myogenic progenitors lacking Minion differentiate normally but fail to form syncytial myotubes, and Minion-deficient mice die perinatally and demonstrate a marked reduction in fused muscle fibres. The fusogenic activity of Minion is conserved in the human orthologue, and co-expression of Minion and the transmembrane protein Myomaker is sufficient to induce cellular fusion accompanied by rapid cytoskeletal rearrangement, even in non-muscle cells. These findings establish Minion as a novel microprotein required for muscle development, and define a two-component programme for the induction of mammalian cell fusion. Moreover, these data also significantly expand the known functions of smORF-encoded microproteins.

Accumulating evidence has shown that altered microRNA (miR) modulation is implicated in the pathologies of ischemic stroke. However, it is unclear whether and how hsa-miR-19a-3p mediates cerebral ischemic injury. Herein, we investigated the functional role of miR-19a-3p in cerebral ischemic injury and explored its underlying regulatory mechanism.

As an active component in wolfberry, lycium barbarum polysaccharides (LBP) are capable of protecting retinal neurons in several animal disease models. Here, we asked whether LBP rescues the retinal morphology and function in rd1 mouse, a photoreceptor fast-degenerating animal model of retinitis pigmentosa, and in particular focused on LBP's effects on the function of retinal ganglion cells (RGCs) during photoreceptor degeneration.

Endoplasmic reticulum (ER) stress results in the activation of the unfolded protein response (UPR), a process that is involved in the pathogenesis of many inflammatory diseases. However, the role of ER stress in chronic rhinosinusitis with nasal polyps (CRSwNP) has yet to be elucidated. In this study, we found that the protein expression levels of a range of ER stress regulators, including p-PERK, ATF4, ATF6 and XBP1s, were significantly increased in CRSwNP compared to controls. Importantly, the expression of ATF4 and XBP1s was positively correlated with heightened inflammation in CRSwNP. In human nasal epithelial cells, the ER stress inducer tunicamycin (TM) could potentiate Toll-like receptors (TLRs) induced proinflammatory cytokines production. Furthermore, we found that the silencing of XBP1, but not ATF4 or ATF6, abrogated the proinflammatory effect of TM. Mechanistically, ER stress did not affect the NF-κB, MAPK or IRF3 signaling pathways. However, the ER stress regulator XBP1s was able to bind directly to the promoter region of inflammatory genes to modulate gene transcription. Besides, the commensal bacteria Staphylococcus aureus and several inflammatory factors, such as IL4, IL13, IL17 and IFNγ, could induce ER stress in epithelial cells. Collectively, ER stress plays a crucial role in facilitating TLR-induced inflammation. Targeting XBP1 can inhibit the inflammatory response, thus offering a potential approach to treat CRSwNP.

Metabolic syndrome (MS) is a complex multisystem disease. Traditional Chinese medicine (TCM) is effective in preventing and treating MS. Syndrome differentiation is the basis of TCM treatment, which is composed of location and/or nature syndrome elements. At present, there are still some problems for objective and comprehensive syndrome differentiation in MS. This study mainly proposes a solution to two problems. Firstly, TCM syndromes are concurrent, that is, multiple TCM syndromes may develop in the same patient. Secondly, there is a lack of holistic exploration of the relationship between microscopic indexes, and TCM syndromes. In regard to these two problems, multilabel learning (MLL) method in machine learning can be used to solve them, and a microcosmic syndrome differentiation model can also be built innovatively, which can provide a foundation for the establishment of the next model of multidimensional syndrome differentiation in MS.

It is well recognized that ventromedial hypothalamus (VMH) serves as a satiety center in the brain. However, the feeding circuit for the VMH regulation of food intake remains to be defined. Here, we combine fiber photometry, chemo/optogenetics, virus-assisted retrograde tracing, ChR2-assisted circuit mapping and behavioral assays to show that selective activation of VMH neurons expressing steroidogenic factor 1 (SF1) rapidly inhibits food intake, VMH SF1 neurons project dense fibers to the paraventricular thalamus (PVT), selective chemo/optogenetic stimulation of the PVT-projecting SF1 neurons or their projections to the PVT inhibits food intake, and chemical genetic inactivation of PVT neurons diminishes SF1 neural inhibition of feeding. We also find that activation of SF1 neurons or their projections to the PVT elicits a flavor aversive effect, and selective optogenetic stimulation of ChR2-expressing SF1 projections to the PVT elicits direct excitatory postsynaptic currents. Together, our data reveal a neural circuit from VMH to PVT that inhibits food intake.

Diabetes may be one of the risk factors of cognitive impairment. In this study, we aimed to investigate the relationship between diabetes status and cognitive impairment among the middle-aged and elderly population (≥40 years) in Chinese rural communities.