Crispr-Cas9 mediated complete deletion of glucagon receptor in mice display hyperglucagonemia and α-cell hyperplasia.

Glucagon receptor plays an important role in the regulation of glucose metabolism. Studies have revealed that glucagon receptor antagonism is a potential effective treatment for diabetes. However, the functions of GCGR have not been fully illustrated. Although two Gcgr truncation knockout mice models have been widely used for GCGR function studies, truncated gene may remain neomorphic and/or dominant-negative function. In this study, we took the advantages of Crispr-Cas9 technique and generated a novel allele of GCGR in the mouse that yields complete loss of GCGR protein. Our studies reveal that complete deletion of Gcgr results in hyperglucagonemia, α-cell hyperplasia, improvement of glucose tolerance. These results are similar to the Gcgr-truncated mutation in mice. Hence, we provide a novel strain of GCGR knockout mice for the GCGR function studies.

Identification of a Novel Risk Model: A Five-Gene Prognostic Signature for Pancreatic Cancer.

Objective: Biomarkers for pancreatic cancer (PCa) prognosis provide evidence for improving the survival outcome of this disease. This study aimed to identify a prognostic risk model based on gene expression profiling of microarray bioinformatics analysis. Methods: Prognostic immune genes in the TCGA-PAAD cohort were identified using the univariate Cox regression and Kaplan-Meier survival analysis. Multivariate Cox regression (stepAIC) was used to identify prognostic genes from the top 20 hub genes in the protein-protein interaction (PPI) network. A prognostic risk model was established and its performance in predicting the overall survival in PCa was validated in GSE62452. Gene mutations and infiltration immune cells in PCa tumors were analyzed using online databases. Results: Univariate Cox regression and Kaplan-Meier survival analyses identified 128 prognostic genes. Multivariate Cox regression (stepAIC) identified five prognostic genes (PLCG1, MET, TNFSF10, CXCL9, and TLR3) out of the 20 hub genes in the PPI network. A prognostic risk model was established using the signature of five genes. This model had moderate to high accuracies (AUC > 0.700) in predicting 3-year and 5-year overall survival in TCGA and GSE62452 cohorts. The Kaplan-Meier survival analysis showed that high-risk scores were correlated with poor survival outcomes in PCa (p < 0.05). Also, mutations in the five genes were related to poor survival. The five genes were related to multiple immune cells. Conclusions: The prognostic risk model was significantly correlated with the survival in PCa patients. This model modulated PCa tumor progression and prognosis by regulating immune cell infiltration.

Studying the mixture effects of brominated flame retardants and metal ions by comet assay.

This study was designed to evaluate the sensitivities of diverse cell lines on DNA damage effects and genotoxic effects of three brominated flame retardants (BFRs) and three metal ions (Cu2+, Cd2+, Hg2+) by comet assay. First, THP-1 was identified as the most sensitive cell line in terms of DNA damage among 11 kinds of cells screened. Accordingly, the THP-1 cell line was used as a model in subsequent single/combined genotoxicity tests. Single exposure tests to BFRs or metal ions revealed that the DNA damage effects increased with increasing exposure concentration. In combined exposure tests, BFRs (at concentrations of 1/2 EC50) were deployed in combination with different concentrations of Cu2+, Cd2+, or Hg2+. The results showed that the % tail DNA values were significantly increased by most mixtures. Our findings on combined toxic effects by comet assay provide valuable information for setting valid environmental safety evaluation standards.

Screening toxicological effects of different contaminants using hepatic homogenates-based ethoxyresorufin-O-deethylase in vitro.

In this paper, we demonstrated the potential of an in vitro method of liver homogenate-based ethoxyresorufin-O-deethylase (EROD) to determine the toxicological effects of multiple kinds of contaminants. We evaluated the in vitro impact of nine pharmaceutically active compounds (PhACs), 13 polycyclic aromatic hydrocarbons (PAHs), and three polychlorinated biphenyls (PCBs). There were different responses of EROD to these contaminants. The response of EROD to PhACs was quite complex, exhibiting both induction and inhibition effects. PAHs and PCBs elicited a strong inhibitory response on EROD activity at high concentrations in a dose-dependent manner. PAHs showed more inhibitory effects as the number of benzene rings increased. Our in vitro bioassay seems to be a potential method for toxicological screening of multiple types of contaminants.

A Novel c.676_677insG PHOX2B Mutation in Congenital Central Hypoventilation Syndrome.

ABSTRACT: Paired-like homeobox (PHOX)2B is considered to be the causative gene of congenital central hypoventilation syndrome (CCHS), a dominant genetic disorder that results in abnormal central respiratory control with resulting hypoventilation during sleep. In this study, we report a novel c.676_677insG (p.Ala226fs) mutation in a patient with severe CCHS, and we evaluated the function of this mutation. The mutation reduced the translation of the mutant PHOX2B protein and impaired its ability to activate the PHOX2A promoter, due to a haploinsufficiency effect. The mutant PHOX2B was able to interact with wildtype PHOX2B, resulting in retention of PHOX2B on the nuclear membrane, which may impair the normal function of the nuclear membrane, and leading to cellular morbidity. Our study provides useful information for the functional studies of PHOX2B and understanding the pathogenesis of CCHS, and thus is beneficial for the prognosis of, genetic counseling for, and development of pharmaceuticals for PHOX2B-associated diseases.

In vitro effects of brominated flame retardants, selected metals and their mixtures on ethoxyresorufin-O-deethylase activity in Mossambica tilapia liver.

The in vitro effects of individual brominated flame retardants (BFRs), selected metals, and their binary mixtures on ethoxyresorufin-O-deethylase (EROD) activity were evaluated using a plate-reader method. The BFRs, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), decabromodiphenyl oxide (BDE-209), hexabromocyclododecane (HBCD), and tetrabromobisphenol A (TBBPA), were tested at doses ranging from 0.1 ng/L to 100 µg/L. Selected metals (Cu2+, Cd2+, Hg2+, and Zn2+) were screened at doses of 0.1 mg/L to 50 mg/L. The activity of EROD was significantly induced by TBBPA, BDE-209, and Zn2+, while HBCD, Cu2+, Cd2+, and Hg2+ decreased EROD activity. Moreover, following exposure to binary mixtures of metals and BFRs, the EROD activity dose-response curves were similar to those of the metals alone, indicating that EROD activity was governed by the metals.

Chibby suppresses aerobic glycolysis and proliferation of nasopharyngeal carcinoma via the Wnt/ß-catenin-Lin28/let7-PDK1 cascade.

BACKGROUND: Great progress has been achieved in the study of the aerobic glycolysis or the so-called Warburg effect in a variety of cancers; however, the regulation of the Warburg effect in Nasopharyngeal carcinoma (NPC) has not been completely defined. METHODS: Gene expression pattern of NPC cells were used to test associations between Chibby and ß-catenin expression. Chibby siRNAs and over-expression vector were transfected into NPC cells to down-regulate or up-regulate Chibby expression. Loss- and gain-of function assays were performed to investigate the role of Chibby in NPC cells. Western blot, cell proliferation, Glucose uptake, Lactate release, ATP level, and O2 consumption assays were used to determine the mechanism of Chibby regulation of underlying targets. Finally, immunohistochemistry assay of fresh NPC and nasopharyngeal normal tissue sample were used to detect the expression of Chibby, ß-Catenin, and PDK1 by immunostaining. RESULTS: We observed that Chibby, a ß-catenin-associated antagonist, is down-regulated in nasopharyngeal carcinoma cell lines and inhibits Wnt/ß-Catenin signaling induced Warburg effect. Mechanism study revealed that Chibby regulates aerobic glycolysis in NPC cells through pyruvate dehydrogenase kinase 1(PDK1), an important enzyme involved in glucose metabolism. Moreover, Chibby suppresses aerobic glycolysis of NPC via Wnt/ß-Catenin-Lin28/let7-PDK1 cascade. Chibby and PDK1 are critical for Wnt/ß-Catenin signaling induced NPC cell proliferation both in vitro and in vivo. Finally, immunostaining assay of tissue samples provides an important clinical relevance among Chibby, Wnt/ß-Catenin signaling and PDK1. CONCLUSIONS: Our study reveals an association between Chibby expression and cancer aerobic glycolysis, which highlights the importance of Wnt/ß-catenin pathway in regulation of energy metabolism of NPC. These results indicate that Chibby and PDK1 are the potential target for NPC treatment.

Single and combined genotoxicity effects of six pollutants on THP-1 cells.

The objective of this study was to evaluate the single and combined genotoxic effects of six food pollutants (Chrysoidine G, Sudan I, acid orange II, malachite green, acrylamide, and potassium bromate) on THP-1 cells through comet assay. The results of the single tests indicated that the pollutants increased the percentage of tail DNA (% tail DNA) in a dose-dependent manner. Moreover, the % tail DNA values induced by synthetic colorants (Chrysoidine G, Sudan I, acid orange II, and malachite green) were significantly higher than those by acrylamide or potassium bromate at most concentrations. In the combined tests, Chrysoidine G (422 µmol/L) or acrylamide (400 µmol/L) was mixed with different concentrations of the other five pollutants respectively. In the first combined tests, most mixtures significantly increased the % tail DNA values with the exception of Chrysoidine G and acid orange II. In the second tests, there were no significant differences in the % tail DNA values between the single and combined tests at most cases.

Transtinib, a potent tyrosine kinase inhibitor inhibits L858R/T790M mutant NSCLC cell lines and xenografts.

Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations initially respond well to the EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. However, clinical efficacy is limited by the development of resistance. In most cases, this resistance is in the form of the T790M mutation. Here, we report the design, synthesis and biochemical evaluation of a novel series of irreversible EGFR tyrosine kinase inhibitors (EGFR-TKIs) that are derived from the anilinoquinazoline scaffold. Guided by molecular modeling, this series of analogs was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and to achieve high levels of anti-tumor activity in cell cultures and in xenografts. The most promising compound 13c ((E) -N - (4 - (4 - (3-fluorobenzyloxy) -3- chlorophenylamino) -7-ethoxyquinazolin-6-yl) -3- ((S) -pyrrolidin-2-yl)acrylamide, which we named Transtinib) displayed strong anti-proliferative activity against the H1975 and A431 cell lines with IC50 values of 34 nM and 62 nM, respectively. In xenograft models, Transtinib significantly decreases tumor size for a prolonged period of time. These results suggest that Transtinib is a potential cancer therapeutic drug lead for the inhibition of mutant EGFR to overcome the development of resistance.

[Applications of habitat equivalency analysis in ecological damage assessment of oil spill incident].

Habitat equivalency analysis (HEA) is one of the methods commonly used by U.S. National Oceanic and Atmospheric Administration in natural resources damage assessment, but rarely applied in China. Based on the theory of HEA and the assessment practices of domestic oil spill incidents, a modification on the HEA was made in this paper, and applied to calculate the habitat value in oil spill incidents. According to the data collected from an oil spill incident in China, the modified HEA was applied in a case study to scale the compensatory-restoration. By introducing the ecological service equivalent factor to transfer various habitats, it was achieved to value of the injured habitats in ecological damage assessment of oil spill incident.

Anaerobic utilization of phenanthrene by Rhodopseudomonas palustris.

A phenanthrene-utilizing bacterium was anaerobically isolated and identified as Rhodopseudomonas palustris. It tolerated up to 100 mg phenanthrene l(-1) and degraded 50% of 50 mg phenanthrene l(-1) over 10 days. The presence of phenanthrene caused a prolonged lag phase (2-3 days) in cell growth and affected the photopigments biosynthesis, while DMSO (the solvent for phenanthrene) had no impact on cell growth. The cell surface hydrophobicity of the isolate was also increased.

An isotope (18O, 15N, and 2H) technique to investigate the metal ion interactions between the phosphoryl group and amino acid side chains by electrospray ionization mass spectrometry.

Cationic metal ion-coordinated N-diisopropyloxyphosphoryl dipeptides (DIPP-dipeptides) were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). Two novel rearrangement reactions with hydroxyl oxygen or carbonyl oxygen migrations were observed in ESI-MS/MS of the metallic adducts of DIPP-dipeptides, but not for the corresponding protonated DIPP-dipeptides. The possible oxygen migration mechanisms were elucidated through a combination of MS/MS experiments, isotope ((18)O, (15)N, and (2)H) labeling, accurate mass measurements, and density functional theory (DFT) calculations at the B3LYP/6-31 G(d) level. It was found that lithium and sodium cations catalyze the carbonyl oxygen migration more efficiently than does potassium and participation through a cyclic phosphoryl intermediate. In addition, dipeptides having a C-terminal hydroxyl or aromatic amino acid residue show a more favorable rearrangement through carbonyl oxygen migration, which may be due to metal cation stabilization by the donation of lone pair of the hydroxyl oxygen or aromatic π-electrons of the C-terminal amino acid residue, respectively. It was further shown that the metal ions, namely lithium, sodium, and potassium cations, could play a novel directing role for the migration of hydroxyl or carbonyl oxygen in the gas phase. This discovery suggests that interactions between phosphorylated biomolecules and proteins might involve the assistance of metal ions to coordinate the phosphoryl oxygen and protein side chains to achieve molecular recognition.

Genome wide exploration of the origin and evolution of amino acids.

BACKGROUND: Even after years of exploration, the terrestrial origin of bio-molecules remains unsolved and controversial. Today, observation of amino acid composition in proteins has become an alternative way for a global understanding of the mystery encoded in whole genomes and seeking clues for the origin of amino acids. RESULTS: In this study, we statistically monitored the frequencies of 20 alpha-amino acids in 549 taxa from three kingdoms of life: archaebacteria, eubacteria, and eukaryotes. We found that the amino acids evolved independently in these three kingdoms; but, conserved linkages were observed in two groups of amino acids, (A, G, H, L, P, Q, R, and W) and (F, I, K, N, S, and Y). Moreover, the amino acids encoded by GC-poor codons (F, Y, N, K, I, and M) were found to "lose" their usage in the development from single cell eukaryotic organisms like S. cerevisiae to H. sapiens, while the amino acids encoded by GC-rich codons (P, A, G, and W) were found to gain usage. These findings further support the co-evolution hypothesis of amino acids and genetic codes. CONCLUSION: We proposed a new chronological order of the appearance of amino acids (L, A, V/E/G, S, I, K, T, R/D, P, N, F, Q, Y, M, H, W, C). Two conserved evolutionary paths of amino acids were also suggested: A-->G-->R-->P and K-->Y.

N-Benzyl-2-propynamide.

Pale-yellow crystals of the title compound, C(10)H(9)NO, have been obtained by the reaction of benzyl-amine and methyl propiolate. Weak inter-molecular hydrogen bonding is observed between acetyl-enic H and carbonyl O atoms. The crystal packing is stabilized by these C-H⋯O and by N-H⋯O inter-molecular hydrogen-bonding inter-actions.

Molecular modeling of zinc and copper binding with Alzheimer's amyloid beta-peptide.

Aggregation of the amyloid beta-peptide (Abeta) into insoluble fibrils is a key pathological event in Alzheimer's disease. Cu(II) and Zn(II) ions were reported to be able to induce Abeta aggregation at nearly physiological concentrations in vitro. In this study, the binding modes of Cu(II) and Zn(II) in this process were explored by molecular modeling. In the pre-associated Abeta, Ntau atom of imidazole ring of His14, O atom of carbonyl of main-chain and two O atoms of water occupied the four ligand positions of the complex. While in the aggregated form of Abeta, the His13(N)-Metals-His14(N) bridges were formed through metal cross-linking action. These results would be helpful to put insight on revealing the formation mechanism of pathogenic Abeta aggregates in brain.

A new theoretical model for the origin of amino acid homochirality.

Amino acid homochirality, as a unique behavior of life, could have originated synchronously with the genetic code. In this paper, phosphoryl amino-acid-5'-nucleosides with P-N bond are postulated to be a chiral origin model in prebiotic molecular evolution. The enthalpy change in the intramolecular interaction between the nucleotide base and the amino-acid side-chain determines the stability of the particular complex, resulting in a preferred conformation associated with the chirality of amino acids. Based on the theoretical model, our experiments and calculations show that the chiral selection of the earliest amino acids for L-enantiomers seems to be a strict stereochemical/physicochemical determinism. As other amino acids developed biosynthetically from the earliest amino acids, we infer that the chirality of the later amino acids was inherited from the precursor amino acids. This idea probably goes far back in history, but it is hoped that it will be a guide for further experiments in this area.

Molecular modeling of the intercalation of porphyrins into alpha-zirconium phosphate.

In this work, n-alkylamines (number of carbon atoms ranging from 3 to 10) were investigated in detail by molecular modeling as spacers for intercalating porphyrins into alpha-zirconium phosphate (alpha-ZrP). Pre-intercalated n-alkylamines can form either a flat monolayer or a canted bilayer in the gallery of alpha-ZrP. Based on the interlayer state and intercalative potential of the two modes in alpha-ZrP, it is suggested that the flat monolayer is a better spacer than the bilayer and that n-propylamine (PA) and n-butylamine (BA) in mobile monolayers are the best spacers among the n-alkylamines studied, as is also found experimentally. The intercalation behavior of TMPyP [5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin] and several other porphyrins was investigated by calculating the intercalative potential. The calculated results showed that the porphyrins were densely packed in a canted monolayer model, and an increase of polarity of the substituent would facilitate the intercalation of the porphyrins.

Molecular modeling of B-DNA site recognition by Ru intercalators: molecular shape selection.

In this work, molecular modeling methods have been applied to the interaction characterization of polypyridyl transitional-metal complexes with the oligonucleotide (B-DNA fragment). In order to explore the factors governing the groove recognition and intercalative depth, we establish a simple and practical docking method (step-by-step docking operation) to obtain potential curves while making complexes inset into B-DNA along an assigned path. Energy values in the potential curve are obtained from energy minimization of binding geometries. Modeling results clearly show that the optimum binding conformation corresponding to the global minimum in the potential curve for each complex is found to correlate well with the experimental results. Our results also confirm that minor changes of the ligand structure can lead to profound influences on binding geometries, so the molecular shape of the complexes is a predominant factor in governing the binding mode. Moreover, we find that the vdW force and "water molecular effect" are strongly associated with molecular-shape selection in our model. These results complement and extend the knowledge of the nature of these complexes binding to B-DNA.