Highlights d e-CHACRs can efficiently copy and inactivate Cas9 activity (99%) d e-CHACRs spread to 100% prevalence in cage trials and eliminate Cas9 activity d ERACRs often copy but can also damage the target chromosome d ERACRs can efficiently delete and completely replace a gene drive in population cages

Articular chondrocytes are known to synthesize large amounts of nitric oxide in response to exposure to interleukin-1, but the role of this radical in proteoglycan turnover remains controversial. In this study, we used two different inhibitors of nitric oxide synthase, NG-methyl-L-arginine and thiocitrulline, to study the effects of nitric oxide on the synthesis and breakdown of proteoglycan in rabbit articular cartilage. Synthesis of nitric oxide by cartilage slices in response to treatment with interleukin-1 and a partially purified mixture of synovial cytokines known as chondrocyte-activating factors peaked during the first 2 days of culture and then fell to low levels, despite daily replenishment with fresh medium and cytokines to the cultures. The production of nitric oxide was completely inhibited by NG-methyl-L-arginine and thiocitrulline. Interleukin-1 and the chondrocyte-activating factors inhibited proteoglycan synthesis and accelerated proteoglycan breakdown in the slices of cartilage. Both nitric oxide synthase inhibitors substantially counteracted the suppression of proteoglycan synthesis but exacerbated proteoglycan catabolism occurring in response to interleukin-1 and the chondrocyte-activating factors. The accelerated catabolism was associated with increased levels of matrix metalloproteinases in the conditioned medium. This dual effect of nitric oxide complicates decision making with regard to the possible clinical applications of nitric oxide agonists or antagonists in diseases of cartilage.

Chondrocytes in arthritic cartilage respond poorly to insulin-like growth factor I (IGF-I). Studies with inducible nitric oxide synthase (iNOS) knockout mice suggest that NO is responsible for part of the cartilage insensitivity to IGF-I. These studies characterize the relationship between NO and chondrocyte responses to IGF-I in vitro, and define a mechanism by which NO decreases IGF-I stimulation of chondrocyte proteoglycan synthesis. Lapine cartilage slices, chondrocytes, and cartilage from osteoarthritic (OA) human knees were exposed to NO from the donors S-nitroso-N-acetylpenicillamine (SNAP) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NO synthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of (35)SO(4). IGF-I receptor phosphorylation was evaluated with Western analysis. SNAP, DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells, or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis in cartilage and monolayer cultures of chondrocytes. OA cartilage responded poorly to IGF-I; however, the response to IGF-I was restored by culture with N(G)-monomethyl-L-arginine (L-NMA). IGF-I receptor phosphotyrosine was diminished in chondrocytes exposed to NO. These studies show that NO is responsible for part of arthritic cartilage/chondrocyte insensitivity to anabolic actions of IGF-I; inhibition of receptor autophosphorylation is potentially responsible for this effect.

The polypterids (bichirs and ropefish) are extant basal actinopterygian (ray-finned) fishes that breathe air and share similarities with extant lobe-finned sarcopterygians (lungfishes and tetrapods) in lung structure. They are also similar to some fossil sarcopterygians, including stem tetrapods, in having large paired openings (spiracles) on top of their head. The role of spiracles in polypterid respiration has been unclear, with early reports suggesting that polypterids could inhale air through the spiracles, while later reports have largely dismissed such observations. Here we resolve the 100-year-old mystery by presenting structural, behavioural, video, kinematic and pressure data that show spiracle-mediated aspiration accounts for up to 93% of all air breaths in four species of Polypterus. Similarity in the size and position of polypterid spiracles with those of some stem tetrapods suggests that spiracular air breathing may have been an important respiratory strategy during the fish-tetrapod transition from water to land.