Summary The number of strand-specific nicking endonucleases that are currently available for laboratory procedures and applications in vivo is limited, and none is sufficiently specific to nick single target sites within complex genomes. The extreme target specificity of homing endonucleases makes them attractive candidates for engineering high specificity nicking endonucleases. I-SceI is a monomeric homing enzyme that recognizes an 18 bp asymmetric target sequence, and cleaves both DNA strands to leave 3’-overhangs four base-pairs in length. In single turnover experiments using plasmid substrates, I-SceI generates transient open circle intermediates during the conversion of supercoiled to linear DNA, indicating that the enzyme sequentially cleaves the two DNA strands. A novel hairpin substrate was used to demonstrate that although wild-type I-SceI cleaves either the top or bottom DNA strand first to generate two nicked DNA intermediates, the enzyme has a preference for cleaving the bottom strand. The kinetics data are consistent with a parallel sequential reaction mechanism. Substitution of two pseudo-symmetric residues, Lys-122 and Lys-223, markedly reduces top or bottom-strand cleavage, respectively, to generate enzymes with significant strand- and sequence-specific nicking activity. The two active sites are partially interdependent since alterations to one site affect the second. The kinetics analysis is consistent with X-ray crystal structures of I-SceI/DNA complexes that reveal a role for the lysines in establishing important solvent networks that include nucleophilic water molecules thought to attack the scissile phosphodiester bonds.

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Highlights:(1) Potato extract (PE) exhibits non-toxic effects on mice.(2) Cigarette smoke (CS)–induced COPD rats exhibit significant thickened and disordered lung markings.(3) PE could improve the histopathological symptoms of lung tissue in COPD.(4) PE could increase the expression of IL-10 and reduce the expression of TNF-α and G-CSF in COPD rats.ObjectiveThis study aimed to evaluate the therapeutic effects of potato extract (PE) on cigarette smoke (CS)–induced chronic obstructive pulmonary disease (COPD).MethodsPE was first prepared by frozen centrifugation, and its amino acid composition was detected. Toxicity of PE was analyzed by changes in morphology, behavior, routine blood indexes, and biochemical criteria of mice. Then, the COPD rat model was established by CS exposure, and PE, doxofylline, and prednisolone acetate were used to treat these rats. After 45 days of treatment, the morphology and behavior of rats were recorded. In addition, the histopathology of lung tissue was evaluated by chest x-ray and hematoxylin and eosin staining. The expression of interleukine-10 (IL-10), tumor necrosis factor-α (TNF-α), and granulocyte colony-stimulating factor (G-CSF) was detected in serum and lung tissue by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively.ResultsVarious amino acids were identified in PE, and no toxicity was exhibited in mice. The CS-induced COPD rat model was successfully established, which exhibited significant thickened and disordered lung markings on 90% of the rats. After administering doxofylline and prednisolone acetate, inflammation symptoms were improved. However, side effects such as emaciation, weakness, and loosening of teeth appeared. In the PE group, obviously improved histopathology was observed in lung tissues. Meanwhile, it was revealed that PE could increase the expression of IL-10 and reduce the expression of TNF-α and G-CSF in COPD rats, and doxofylline and prednisolone acetate also elicited similar results.ConclusionOur study suggests PE might be effective in the treatment of CS-induced COPD by inhibiting inflammation.

Metabolomic analysis in mice revealed that natriuretic peptide receptor A (NPRA) is mainly involved in nucleotide biosynthesis and histidine metabolism in cardiac tissues, and in creatine metabolism, TCA cycle and pentose phosphate pathway in the plasma. Furthermore, proteomics revealed that Cox7c, Cox7b, ATP5J2, Uqcr10, and Myh7 play a vital role in the regulation of metabolic pathway. Together, deterioration of NPRA results in metabolic dysfunction involved with a protein and metaboliteinteracting pathway. Highlights• Metabolomic analysis of cardiac tissues from NPRA-deficient mice.• Metabolomic analysis of plasma from NPRA-deficient mice.• TMT-based proteomic analysis of cardiac tissues in NPRA-deficient mice.• Mechanistic insights into NPRA in mice from metabolomes and proteomes.

The constituents with hepatoprotective activity were investigated in three traditional Chinese medicine formulae for treating jaundice, namely, Zhi-Zi-Da-Huang-Tang, Yin-Chen-Hao-Tang, and Da-Huang-Xiao-Shi-Tang. By using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry, 79 chemical constituents were identified unambiguously or tentatively in three formulae based on the accurate molecular weight, mass spectrometric fragmentation behavior, and comparison with reference standards. Then the hepatoprotective activities of 27 constituents were evaluated on tert-butylhydroperoxide-injured BRL-3A cells. The results indicated that 11 constituents, including protocatechic acid (19), epijasminoside A (56), rutin (71), tetrahydropalmatine (76), rhaponticin (80), 3,4-dicaffeoylquinic acid (82), 3,5-dicaffeoylquinic acid (85), diosmetin-7-O-glucoside (90), jatrorrhizine (93), berberine (100), and daidzein (101) exerted hepatoprotective activities. Interestingly, most of the crude drugs in three formulae contained hepatoprotective active constituents, and the combinations of constituents from different crude drugs exhibited greater effects. This result provided evidence to the traditional Chinese medicine theory of combining several drugs together to exert synergistic efficacy.