ORCID ID: 0000-0002-7224-8449 (Y.Z.).Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141-and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis.

A human immunodeficiency virus type 1 variant resistant to zalcitabine (2',3'-dideoxycytidine [ddC]) was selected by sequential passage in the presence of increasing concentrations of ddC in peripheral blood mononuclear cell cultures. A mutation causing a lysine-to-arginine substitution was noted in reverse transcriptase (RT) codon 65 of this ddC-selected virus. A cloned mutant virus with this codon 65 mutation was constructed by using a novel PCR approach for site-directed mutagenesis. Characterization of this virus confirmed that the RT Lys-65--Arg substitution was necessary and sufficient for a fourfold increase in the ddC 50%N inhibitory concentration, as well as for resistance to didanosine (2',3'-dideoxyinosine [ddl]). Lys-65-*Arg and virus resistance to ddC and ddl also developed during therapy in isolates from one ddC-treated patient and two ddI-treated patients. Recombinant-expressed codon 65 mutant RT enzyme was resistant to ddCTP and ddATP ii cell-free polymerase assays. Results of mutant enzyme studies are consistent with Lys-65--*Arg leading to changes in binding of the triphosphate forms of these nucleoside analogs to the RT. These data have implications for future studies of ddC resistance, particularly those aimed at defining its clinical relevance.

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.

Abstract-Obese hypertensive patients with cardiovascular risk factor clustering and increased risk for atherosclerotic disease have increased plasma nonesterified fatty acid levels, including oleic acid (OA), and a more active renin-angiotensin-aldosterone system. Vascular smooth muscle cell (VSMC) migration and proliferation participate in the development of atherosclerotic plaque. OA and angiotensin (Ang) II induce synergistic mitogenic responses in VSMCs through sequential signaling pathways dependent on the activation of protein kinase C (PKC), oxidants (reactive oxygen species, ROS), and extracellular signal-regulated kinase (ERK) activation. We tested the hypotheses that (1) OA and Ang II have additive or synergistic effects on VSMC migration and (2) PKC, ROS, and mitogen-activated protein kinase are critical signaling molecules. OA at 100 mol/L increases VSMC migration 60Ϯ10% over control (PϽ0.001). Ang II (10 Ϫ9 mol/L) increases VSMC migration by 62Ϯ13% and 73% over control, respectively (PϽ0.01). Coincubation of cells with OA and Ang II produces a nearly additive increase in VSMC cell migration at 107Ϯ20% (PϽ0.01). Increases in VSMC migration induced by OA alone and combined with Ang II were reduced by PKC inhibition and downregulation. VSMC migration in response to OA alone and with Ang II was also inhibited by N-acetyl-cysteine, MEK inhibition, and ERK antisense. VSMC migration in response to OA alone or combined with Ang II is dependent on activation of PKC, ROS, and ERK activation, further raising the possibility that increased plasma nonesterified fatty acids and an activated renin-angiotensin-aldosterone system in subjects with the risk factor cluster contribute to accelerated atherosclerosis through a PKC, ROS, and ERK-dependent signaling pathway.