Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing.

Extracellular ATP controls various signaling systems including propagation of intercellular Ca ²⁺ signals (ICS). Connexin hemichannels, P2x7 receptors (P2x7Rs), pannexin channels, anion channels, vesicles, and transporters are putative conduits for ATP release, but their involvement in ICS remains controversial. We investigated ICS in cochlear organotypic cultures, in which ATP acts as an IP ₃ -generating agonist and evokes Ca ²⁺ responses that have been linked to noise-induced hearing loss and development of hair cell-afferent synapses. Focal delivery of ATP or photostimulation with caged IP ₃ elicited Ca ²⁺ responses that spread radially to several orders of unstimulated cells. Furthermore, we recorded robust Ca ²⁺ signals from an ATP biosensor apposed to supporting cells outside the photostimulated area in WT cultures. ICS propagated normally in cultures lacking either P2x7R or pannexin-1 (Px1), as well as in WT cultures exposed to blockers of anion channels. By contrast, Ca ²⁺ responses failed to propagate in cultures with defective expression of connexin 26 (Cx26) or Cx30. A companion paper demonstrates that, if expression of either Cx26 or Cx30 is blocked, expression of the other is markedly down-regulated in the outer sulcus. Lanthanum, a connexin hemichannel blocker that does not affect gap junction (GJ) channels when applied extracellularly, limited the propagation of Ca ²⁺ responses to cells adjacent to the photostimulated area. Our results demonstrate that these connexins play a dual crucial role in inner ear Ca ²⁺ signaling: as hemichannels, they promote ATP release, sustaining long-range ICS propagation; as GJ channels, they allow diffusion of Ca ²⁺ -mobilizing second messengers across coupled cells.

Significance: Accumulating evidence has underscored the role of cytosolic p53 in promoting cell death. Different reports have revealed that p53 participates in apoptosis induction by acting directly at mitochondria. However, because p53 can mediate apoptosis without its DNA-binding domain (the domain proposed to be fundamental for the targeting of p53 to mitochondria), the mitochondrial localization of p53 is likely not the only transcription-independent mechanism by which p53 promotes apoptosis. Here we demonstrate that p53 at the endoplasmic reticulum (ER) and at mitochondria-associated membranes, interacting with sarco/ER Ca ²⁺ -ATPase pumps, modulates ER–mitochondria cross-talk and, in turn, Ca ²⁺ -dependent apoptosis.

Several deficiencies affecting previous ‘‘box’’ models of the cochlea are overcome in this paper. Both mechanical and hydrodynamical aspects are treated at a level adequate to the complexity of realistic cochlear structures. The dynamics of the cochlea as a passive physical system, in the linear approximation, is described by an integral equation. It is further shown that this equation describes the properties of the working cochlea, provided a force term that accounts for hair cell motility is included. Numerical solutions for different degrees of outer hair cells activity, obtained by matrix methods in the frequency domain, are presented. Amplitudes and phases of the computer-simulated traveling waves are in fair agreement with basilar membrane responses to tones measured in various experimental conditions.