Homologous recombination-mediated genome engineering has been broadly applied in prokaryotes with high efficiency and accuracy. However, this method is limited in realizing larger-scale genome editing with numerous genes or large DNA fragments because of the relatively complicated procedure for DNA editing template construction. Here, we describe a CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ) strategy for the rapid and efficient inactivation of bacterial gene (s) in a homologous recombination-independent manner and without the use of selective marker. Our study show that CA-NHEJ can be used to delete large chromosomal DNA fragments in a single step that does not require homologous DNA template. It is thus a novel and powerful tool for bacterial genomes reducing and possesses the potential for accelerating the genome evolution.

At appropriate concentrations, hydrogen sulfide, a well-known gasotransmitter, plays important roles in both physiology and pathophysiology. Increasing evidence suggests that modifying thiol groups of specific cysteines in target proteins via sulfhydration or persulfidation is one of the important mechanisms responsible for the biological functions of hydrogen sulfide. A variety of key proteins of different cellular pathways in mammals have been reported to be sulfhydrated by hydrogen sulfide to participate and regulate the processes of cell survival/death, cell differentiation, cell proliferation/hypertrophy, cellular metabolism, mitochondrial bioenergetics/biogenesis, endoplasmic reticulum stress, vasorelaxtion, inflammation, oxidative stress, etc. Moreover, S-sulfhydration also exerts many biological functions through the cross-talk with other post-translational modifications including phosphorylation, S-nitrosylation and tyrosine nitration. This review summarizes recent studies of hydrogen sulfide-induced sulfhydration as a posttranslational modification, an important biological function of hydrogen sulfide, and sulfhydrated proteins are introduced. Additionally, we discuss the main methods of detecting sulfhydration of proteins.

Background:The mechanisms by which H 2 S regulates inflammation remain unclear. Results: H 2 S inhibits NF-B p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to MCP-1 promoter in ox-LDL-treated macrophages by targeting the free sulfhydryl group on cysteine 38 in p65. Conclusion: H 2 S inhibits macrophage inflammation by suppressing NF-B activation. Significance: These findings reveal mechanisms for regulation of NF-B pathway by H 2 S.

Sulfur dioxide (SO2) was previously regarded as a toxic gas in atmospheric pollutants. But it has been found to be endogenously generated from metabolism of sulfur-containing amino acids in mammals through transamination by aspartate aminotransferase (AAT). SO2 could be produced in cardiovascular tissues catalyzed by its synthase AAT. In recent years, studies revealed that SO2 had physiological effects on the cardiovascular system, including vasorelaxation and cardiac function regulation. In addition, the pathophysiological effects of SO2 were also determined. For example, SO2 ameliorated systemic hypertension and pulmonary hypertension, prevented the development of atherosclerosis, and protected against myocardial ischemia-reperfusion (I/R) injury and isoproterenol-induced myocardial injury. These findings suggested that endogenous SO2 was a novel gasotransmitter in the cardiovascular system and provided a new therapy target for cardiovascular diseases.

The present study was designed to investigate the role of endogenous sulfur dioxide (SO2) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO2 inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO2 synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO2 suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO2 did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO2 dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO2 simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO2 could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in SO2-induced suppression of Erk/MAPK pathway. The present study firstly demonstrated that SO2 exerted a negative regulation of VSMC proliferation via suppressing the Erk/MAPK pathway mediated by cAMP/PKA signaling.