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Salinity is an important environmental factor that affects the yield and quality of large yellow croaker (Larimichthys crocea) during aquaculture. Here, whole-genome bisulfite sequencing (WGBS), RNA-seq, bisulfite sequencing PCR (BSP), quantitative real-time PCR (qPCR), and dual luciferase reporter gene detection technologies were used to analyze the DNA methylation characteristics and patterns of the liver genome, the expression and methylation levels of important immune genes in large yellow croaker in response to salinity stress. The results of WGBS showed that the cytosine methylation of CG type was dominant, CpGIsland and repeat regions were important regions where DNA methylation occurred, and the DNA methylation in upstream 2k (2000bp upstream of the promoter) and repeat regions had different changes in the liver tissue of large yellow croaker in the response to the 12‰, 24‰, 36‰ salinity stress of 4 w (weeks). In the combined analysis of WGBS and transcriptome, the complement and coagulation cascade pathways were significantly enriched, in which the complement-related genes C7, C3, C5, C4, C1R, MASP1, and CD59 were mainly changed in response to salinity stress. In the studied area of MASP1 gene promoter, the methylation levels of many CpG sites as well as total cytosine were strongly negatively correlated with mRNA expression level. Methylation function analysis of MASP1 promoter further proved that DNA methylation could inhibit the activity of MASP1 promoter, indicating that salinity may affect the expressions of complement-related genes by DNA methylation of gene promoter region.

Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous cells derived from immature myeloid cells (IMCs). MDSCs are known to play important roles in tumor immune evasion. While we know that there are a large number of circulating and tumor-infiltrating MDSCs existing in gastric cancer (GC) patients, the phenotypic characteristics and arginase 1 (ARG1) expression levels of these MDSCs remain very unclear. In our study, flow cytometric analysis of circulating MDSCs from 20 gastric adenocarcinoma (GAC) patients found that ≥80% ARG1-expressing MDSCs were mainly early-stage MDSCs (HLA-DR−CD33+CD14−CD15−MDSCs). In addition, our investigation showed that tumor-infiltrating MDSCs from 6 GAC patients consisted of >35% ARG1-expressing naïve MDSCs (HLA-DR−CD33−CD11b−CD14−CD15−MDSCs), >15% early-stage MDSCs and >40% monocytic MDSCs (HLA-DR−CD14+MDSCs). This preliminary study describes the phenotypic characteristics and ARG1 expression levels of MDSCs from GAC patients and shows that circulating and tumor-infiltrating ARG1-expressing cells were mainly immature and monocytic MDSCs, which provides information to better understand the mechanisms that allow gastric cancer cells to evade the immune system.

GNAO1 encodes Gαo, a heterotrimeric G protein alpha subunit in the Gi/o family. In this report, we used a Gnao1 mouse model “G203R” previously described as a “gain-of-function” Gnao1 mutant with movement abnormalities and enhanced seizure susceptibility. Here, we report an unexpected second mutation resulting in a loss-of-function Gαo protein and describe alterations in central synaptic transmission.Whole cell patch clamp recordings from Purkinje cells (PCs) in acute cerebellar slices from Gnao1 mutant mice showed significantly lower frequencies of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) compared to WT mice. There was no significant change in sEPSCs or mEPSCs. Whereas mIPSC frequency was reduced, mIPSC amplitudes were not affected, suggesting a presynaptic mechanism of action. A modest decrease in the number of molecular layer interneurons was insufficient to explain the magnitude of IPSC suppression. Paradoxically, Gi/o inhibitors (pertussis toxin), enhanced the mutant-suppressed mIPSC frequency and eliminated the difference between WT and Gnao1 mice. While GABAB receptor regulates mIPSCs, neither agonists nor antagonists of this receptor altered function in the mutant mouse PCs. This study is the first electrophysiological investigation of the role of Gi/o protein in cerebellar synaptic transmission using an animal model with a loss-of-function Gi/o protein.Significance StatementThis is the first report on the electrophysiological mechanisms of a movement disorder animal model with monoallelic Gnao1 loss. This study illustrates the role of Gαo protein in regulating GABA release in mouse cerebellum. This study could also facilitate the discovery of new drugs or drug repurposing for GNAO1-associated disorders. Moreover, since GNAO1 shares pathways with other genes related to movement disorders, developing drugs for the treatment of GNAO1-associated movement disorders could further the pharmacological intervention for other monogenic movement disorders.

Background: Myeloid-derived suppressor cells (MDSCs) promote immunosuppression in the tumor microenvironment, support tumor growth and survival, and may contribute to immunotherapy resistance. Recent studies showed that tumor-derived exosomes (TDEs) can induce MDSCs accumulation and expansion, the mechanisms of which are largely unknown. Methods: The morphologies and sizes of the exosomes was observed by using a JEM-1400 transmission electron microscope. MicroRNA(miR)-107 and ARG1, DICER1, PTEN, PI3K, AKT, mTOR, and NF-kB mRNAs were quantified by quantitative reverse tanscription PCR. Dual-Luciferase Reports Assay were used to examine the expression of genes which was targeted by miR-107. The expression of proteins were analyzed by using western blot. Results: MiR-107 was not only overexpressed in gastric cancer cells but also enriched in their secreted TDEs. Also, these miR-107 enriched TDEs could be taken up by HLA-DR-CD33+MDSCs, where miR-107 was able to target and suppress expression of DICER1 and PTEN genes. Dampened DICER1 expression supported expansion of MDSCs , while decreased PTEN led to activation of the PI3K pathway, resulting in increased ARG1 expression. Furthemore, gastric cancer-derived miR-107 TDEs, when dosed intravenously into mice, were also capable of inducing expansion of CD11b+Gr1+/high MDSCs in mouse peripheral blood and altering expression of DICER1, PTEN, ARG1, and NOS2 in the MDSCs. Conclusions: Our findings demonstrate for the first time that gastric cancer-secreted exosomes are able to deliver miR-107 to the host MDSCs where they induce their expansion and activition by targeting DICER1 and PTEN genes, thereby may provide novel cancer therapeutics target for gastric cancer.

MicroRNAs (miRNAs) play an important role in regulating gene expression, and myostatin (MSTN) has been widely recognized as a key gene for muscle growth and development. Through high-throughput sequencing to study the effects of starvation on miRNA transcriptomes in Larimichthys crocea muscle tissue, we found that the expression of miR-2014, miR-1231 and miR-1470 were significantly different between fasting and normal feeding Larimichthys crocea. Bioinformatics analysis predicted that miR-2014, miR-1231 and miR-1470 target MSTN mRNA 3'UTR. To verify the accuracy of predictions, we constructed double luciferase plasmids containing MSTN 3'UTR and confirmed that miR-2014-5p and miR-1231-5p can inhibit MSTN expression by targeting MSTN 3'UTR using double luciferase experiments, while miR-1470 is not involved in regulation. Subsequent site-directed mutation experiments reflected the specificity of the target sequence. In addition, quantitative PCR experiments revealed that miR-2014-5p and miR-1231-5p may participate in the regulation of MSTN expression in fasting and refeeding period, respectively. These results implied that miRNA may take part in muscle growth regulation during starvation. It provides some insights into the molecular regulation mechanism of MSTN in response to starvation stress in fish.

Recently smoothing deep neural network based classifiers via isotropic Gaussian perturbation is shown to be an effective and scalable way to provide state-of-the-art probabilistic robustness guarantee against $\ell_2$ norm bounded adversarial perturbations. However, how to train a good base classifier that is accurate and robust when smoothed has not been fully investigated. In this work, we derive a new regularized risk, in which the regularizer can adaptively encourage the accuracy and robustness of the smoothed counterpart when training the base classifier. It is computationally efficient and can be implemented in parallel with other empirical defense methods. We discuss how to implement it under both standard (non-adversarial) and adversarial training scheme. At the same time, we also design a new certification algorithm, which can leverage the regularization effect to provide tighter robustness lower bound that holds with high probability. Our extensive experimentation demonstrates the effectiveness of the proposed training and certification approaches on CIFAR-10 and ImageNet datasets., Comment: AAAI2020

Growth and meat quality are concerned issues in aquaculture. The inhibition of MSTN can lead to significant muscle proliferation in mammals, as well as a decrease in muscle fat content. In fish, the effect of MSTN inhibition on muscle fat content is ambiguous. In this study, we constructed transgenic red carp with Larimichthys crocea MSTN1 propeptide (LcMSTN1 propeptide) to study how the inhibition of MSTN1 affects the growth and muscle fat content in fish. Southern Blotting showed that LcMSTN1 propeptide was integrated into the red carp genome. In muscle tissue, the expression of MSTN1 propeptide in transgenic red carp was significantly higher than that of non-transgenic red carp (P

Objective:To define molecular mechanisms underlying the clinical spectrum of epilepsy and movement disorder in individuals with de novo mutations in the GNAO1 gene.Methods:We identified all GNAO1 mutations reported in individuals with epilepsy (early infantile epileptiform encephalopathy 17) or movement disorders through April 2016; 15 de novo mutant alleles from 25 individuals were introduced into the Gαo subunit by site-directed mutagenesis in a mammalian expression plasmid. We assessed protein expression and function in vitro in HEK-293T cells by Western blot and determined functional Gαo-dependent cyclic adenosine monophosphate (cAMP) inhibition with a coexpressed α2A adrenergic receptor.Results:Of the 15 clinical GNAO1 mutations studied, 9 show reduced expression and loss of function (LOF; 50 values for α2A adrenergic receptor–mediated inhibition of cAMP. The GNAO1 LOF mutations are associated with epileptic encephalopathy while GOF mutants (such as G42R, G203R, and E246K) or normally functioning mutants (R209) were found in patients with movement disorders with or without seizures.Conclusions:Both LOF and GOF mutations in Gαo (encoded by GNAO1) are associated with neurologic pathophysiology. There appears to be a strong predictive correlation between the in vitro biochemical phenotype and the clinical pattern of epilepsy vs movement disorder.

Neurodevelopmental disorder with involuntary movements (Online Mendelian Inheritance in Man: 617493) is a severe, early onset neurologic condition characterized by a delay in psychomotor development, hypotonia, and hyperkinetic involuntary movements. Heterozygous de novo mutations in the

Neurodevelopmental disorder with involuntary movements (NEDIM, OMIM: 617493) is a severe, early onset neurological condition characterized by a delay in psychomotor development, hypotonia, and hyperkinetic involuntary movements. Heterozygous de novo mutations in the GNAO1 gene cause NEDIM. Gαo, the gene product of GNAO1, is the alpha subunit of Go, a member of the heterotrimeric Gi/o family of G-proteins. Go is found abundantly throughout the brain but the pathophysiological mechanisms linking Gαo functions to clinical manifestations of GNAO1-related disorders are still poorly understood. One of the most common mutant alleles among the GNAO1 encephalopathies is the c.626G>A or p.Arg209His (R209H) mutation. We developed heterozygous knock-in Gnao1+/R209H mutant mice using CRISPR/Cas9 methodology to assess whether a mouse model could replicate aspects of the NEDIM clinical pattern. Mice carrying the R209H mutation exhibited increased locomotor activity and a modest gait abnormality at 8-12 weeks. In contrast to mice carrying other mutations in Gnao1, the Gnao1+/R209H mice did not show enhanced seizure susceptibility. Levels of protein expression in multiple brain regions were unchanged from WT mice but the nucleotide exchange rate of mutant R209H Gαo was 9 times faster than WT. The atypical neuroleptic risperidone has shown efficacy in a patient with the R209H mutation. It also alleviated the hyperlocomotion phenotype observed in our mouse model but suppressed locomotion in WT mice as well. In this study, we show that Gnao1+/R209H mice mirror elements of the patient phenotype and respond to an approved pharmacological agent.

BackgroundInfants and children with dominantde novomutations inGNAO1exhibit movement disorders, epilepsy, or both. Children with loss-of-function (LOF) mutations exhibit Epileptiform Encephalopathy 17 (EIEE17). Gain-of-function (GOF) mutations or those with normal function are found in patients with Neurodevelopmental Disorder with Involuntary Movements (NEDIM). There is no animal model with a human mutantGNAO1allele.ObjectivesHere we develop a mouse model carrying a humanGNAO1mutation and determine whether clinical features of theGNAO1mutation including movement disorder would be evident in the mouse model.MethodsA mouseGnao1knock-in GOF mutation (G203R) was created by CRISPR/Cas9 methods. The resulting offspring and littermate controls were subjected to a battery of behavioral tests. A previously reported GOF mutant mouse knock-in (Gnao1+/G184S) was also studied for comparison.ResultsGnao1+/G203Rmutant mice are viable and gain weight comparably to controls. Homozygotes are non-viable. Grip strength was decreased in both males and females. MaleGnao1+/G203Rmice were strongly affected in movement assays (RotaRod and DigiGait) while females were not. MaleGnao1+/G203Rmice also showed enhanced seizure propensity in the pentylenetetrazole kindling test. Mice with a G184S GOF knock-in also showed movement-related behavioral phenotypes but females were more strongly affected than males.ConclusionsGnao1+/G203Rmice phenocopy children with heterozygousGNAO1G203R mutations, showing both movement disorder and a relatively mild epilepsy pattern. This mouse model should be useful in mechanistic and preclinical studies ofGNAO1-related movement disorders.

Given a large number of covariates $Z$, we consider the estimation of a high-dimensional parameter $\theta$ in an individualized linear threshold $\theta^T Z$ for a continuous variable $X$, which minimizes the disagreement between $\text{sign}(X-\theta^TZ)$ and a binary response $Y$. While the problem can be formulated into the M-estimation framework, minimizing the corresponding empirical risk function is computationally intractable due to discontinuity of the sign function. Moreover, estimating $\theta$ even in the fixed-dimensional setting is known as a nonregular problem leading to nonstandard asymptotic theory. To tackle the computational and theoretical challenges in the estimation of the high-dimensional parameter $\theta$, we propose an empirical risk minimization approach based on a regularized smoothed loss function. The statistical and computational trade-off of the algorithm is investigated. Statistically, we show that the finite sample error bound for estimating $\theta$ in $\ell_2$ norm is $(s\log d/n)^{\beta/(2\beta+1)}$, where $d$ is the dimension of $\theta$, $s$ is the sparsity level, $n$ is the sample size and $\beta$ is the smoothness of the conditional density of $X$ given the response $Y$ and the covariates $Z$. The convergence rate is nonstandard and slower than that in the classical Lasso problems. Furthermore, we prove that the resulting estimator is minimax rate optimal up to a logarithmic factor. The Lepski's method is developed to achieve the adaption to the unknown sparsity $s$ and smoothness $\beta$. Computationally, an efficient path-following algorithm is proposed to compute the solution path. We show that this algorithm achieves geometric rate of convergence for computing the whole path. Finally, we evaluate the finite sample performance of the proposed estimator in simulation studies and a real data analysis.

Oriented assembly of low-dimensional building blocks into their higher order three-dimensional (3D) multifunctional architectures is a fascinating technique to improve the electrochemical performance of random low-dimensional materials. Here, we have successfully realized two 3D CuO ordered nanostructures (CONs) assembled by 1D and 2D building blocks via a facile solvothermal method with mixed solutions of deionized water and ethylene glycol (EG). The synergistic effect of EG and n-butylamine on the crystal nucleation and growth process dominates the fabrication of various 3D architectures. Compared with disorganized 2D nanoflakes, 3D CONs exhibit higher specific surface areas, more convenient electron and ion mobility, and greater structural stability, which contribute to the rapid and reversible redox reaction in pseudocapacitors. Impressively, the electrochemical characteristics are greatly improved by 3D CON electrodes, showing high specific capacitance (541 and 585 F g−1 at 1 A g−1), good rate capability (retaining 81% and 79% at 20 A g−1), and stable cycle life (85.3% and 86.8% capacitance retention after 8000 cycles). More importantly, the asymmetric supercapacitor based on 3D CONs expresses excellent cycling stability (85.3% capacitance retention after 10 000 cycles) and high energy density (31.47 W h kg−1 at a power density of 892 W kg−1). The design of the 3D porous ordered nanostructures would provide a novel and ideal approach for enhancing comprehensive performance of other electrode materials in energy conversion and storage fields.

Mutations in the GNAO1 gene cause a complex constellation of neurological disorders including epilepsy, developmental delay, and movement disorders. GNAO1 encodes Gαo, the α subunit of Go, a member of the Gi/o family of heterotrimeric G protein signal transducers. Go is the most abundant membrane protein in the mammalian central nervous system and plays major roles in synaptic neurotransmission and neurodevelopment. GNAO1 mutations were first reported in early infantile epileptic encephalopathy 17 (EIEE17) but are also associated with a more common syndrome termed neurodevelopmental disorder with involuntary movements (NEDIM). Here we review a mechanistic model in which loss-of-function (LOF) GNAO1 alleles cause epilepsy and gain-of-function (GOF) alleles are primarily associated with movement disorders. We also develop a signaling framework related to cyclic AMP (cAMP), synaptic vesicle release, and neural development and discuss gene mutations perturbing those mechanisms in a range of genetic movement disorders. Finally, we analyze clinical reports of patients carrying GNAO1 mutations with respect to their symptom onset and discuss pharmacological/surgical treatments in the context of our mechanistic model.

An efficient one-pot N-alkylation of benzimidazole and benzotriazole from carbonyl compounds and tosylhydrazide has been accomplished via copper powder-catalyzed N—H bond insertion affording N-alkylated products in good yields. The reaction can tolerate a wide range of carbonyl compounds, such as aryl, alkyl, heterocyclic and α,β-unsaturated ketones, and aldehydes.

It is meaningful to exploit copper sulfide materials with desired structure as well as potential application due to their cheapness and low toxicity. A low-temperature and facile solvothermal method for preparing three-dimensional (3D) hierarchical covellite (CuS) microspheres from an ionic liquid precursor [Bmim]2Cu2Cl6 (Bmim = 1-butyl-3-methylimidazolium) is reported. The formation of CuS nanostructures was achieved by decomposition of intermediate complex Cu(Tu)3Cl (thiourea = Tu), which produced CuS microspheres with diameters of 2.5-4 μm assembled by nanosheets with thicknesses of 10-15 nm. The ionic liquid, as an "all-in-one" medium, played a key role for the fabrication and self-assembly of CuS nanosheets. The alkylimidazolium rings ([Bmim](+)) were found to adsorb onto the (001) facets of CuS crystals, which inhibited the crystal growth along the [001] direction, while the alkyl chain had influence on the assembly of CuS nanosheets. The CuS microspheres showed enhanced electrochemical performance and high stability for the application in supercapacitors due to intriguing structural design and large specific surface area. When this well-defined CuS electrode was assembled into an asymmetric supercapacitor (ASC) with an activated carbon (AC) electrode, the CuS//AC-ASC demonstrated good cycle performance (∼88% capacitance after 4000 cycles) and high energy density (15.06 W h kg(-1) at a power density of 392.9 W kg(-1)). This work provides new insights into the use of copper sulfide electrode materials for asymmetric supercapacitors and other electrochemical devices.

To explore whether there was tissue remodeling in the nasal mucosa of allergic rhinitis (AR) patients and detect the protein expressions of transforming growth factor beta1 (TGF-beta1), matrix metalloproteinases 9 (MMP-9) and tissue inhibitors of matrix metalloproteinases 1 (TIMP-1) in the nasal mucosa of these patients.Pathologic staining was used to explore the mucosa of the middle turbinate tissues from 16 patients with mild AR, from 12 patients with severe AR, and from 15 non-AR, respect. The infiltrating of eosinophils and damage of epithelium were examined by the hematoxylin-eosin staining; goblet cells were counted by the alcian blue-periodic acid-schiff staining; the percentage area of extracellular matrix was determined by the MT; the protein expressions were measured by ELISA of TGF-beta1, MMP-9 and TIMP-1 in the middle turbinate tissues.Compared with the control group, significant eosinophil infiltration and goblet cells were observed in both AR groups (P0.05). Evident epithelial damage and extracellular matrix deposition were observed in severe-AR group (P0.05). The expressions of TGF-beta1, MMP-9 and TIMP-1 in AR tissues were significant increased (P0.05).The nasal mucosa remodeling was observed in AR groups. The characteristics were as follows: eosinophils infiltration, epithelial damage, goblet cells hyperplasia and extracellular matrix deposition. TGF-beta1, MMP-9, TIMP-1 may play a role in the tissue remodeling processes.

The immortalized human bronchial epithelial cell line, BEP2D, can become malignantly transformed following its irradiation with alpha particles. Exposed cells progress through a latent period of eight to ten weeks prior to exhibiting malignant properties. The molecular basis for this delay is unclear, however it is thought to involve a manifestation of genomic instability brought on by ionizing radiation. In this study we addressed whether the microsatellite mutator phenotype which arises in cells that lack mismatch repair function could have played a role in the radiation-induced transformation of BEP2D cells. Three cell lines, including BEP2D and two transformed clonal derivatives, H2BT2L and R30T1L, were examined for the presence of the microsatellite mutator phenotype using a selectable reporter system developed in our laboratory. This reporter system is based on the ability of stably transduced cells to restore the reading frame of a hygromycin B phosphotransferase transferase gene rendered out of frame by the insertion of a (CA)(13) repeat tract immediately downstream of the ATG start codon. The parental BEP2D cell line had a relatively low hygromycin B resistant colony formation frequency of 2.36 x 10(-4). This value was similar to the frequencies of two malignantly transformed derivatives H2BT2L (0.996 x 10(-4)) and R30T1L (1.87 x 10(-4)). We therefore conclude that the H2BT2L and R30T1L cell lines are not deficient in their hMSH2, hMLH1, hPMS2 or hMSH3 mismatch repair gene functions, and that other factors orchestrated the alpha particle induced malignant transformation of H2BT2L and R30T1L cells.