Peter Rüthemann

Background Inhalation treatment frequently is used in dogs and cats with chronic respiratory disease. Little is known however about the performance of delivery devices and the distribution of aerosolized drugs in the lower airways. Objective To assess the performance of 3 delivery devices and the impact of variable durations of inhalation on the pulmonary and extrapulmonary deposition of nebulized ^99mtechnetium‐diethylenetriamine‐pentaacetic acid (^99mTc‐DTPA). Animals Ten university‐owned healthy Beagle dogs. Methods Prospective crossover study. Dogs inhaled the radiopharmaceutical for 5 minutes either through the Aerodawg spacer with a custom‐made nose‐muzzle mask, the Aerochamber spacer with the same mask, or the Aerodawg spacer with its original nose mask. In addition, dogs inhaled for 1 and 3 minutes through the second device. Images were obtained by 2‐dimensional planar scintigraphy. Radiopharmaceutical uptake was calculated as an absolute value and as a fraction of the registered dose in the whole body. Results Mean (±SD) lung deposition for the 3 devices was 9.2% (±5.0), 11.4% (±4.9), and 9.3% (±4.6), respectively. Differences were not statistically significant. Uptake in pulmonary and extrapulmonary tissues was significantly lower after 1‐minute nebulization, but the mean pulmonary/extrapulmonary deposition ratio (0.38 ± 0.27) was significantly higher than after 5‐minute nebulization (0.16 ± 0.1; P = .03). No significant differences were detected after 3‐ and 5‐minute nebulization. Conclusion and Clinical Importance The performance of a pediatric spacer with a custom‐made mask is comparable to that of a veterinary device. One‐minute nebulization provides lower pulmonary uptake but achieves a better pulmonary/extrapulmonary deposition ratio than does 5‐minute nebulization.

Ultraviolet (UV) light induces mutagenic cyclobutane pyrimidine dimers (CPDs) in nucleosomal DNA that is tightly wrapped around histone octamers. How global‐genome nucleotide excision repair (GG‐NER) processes CPDs despite that this chromatin arrangement is poorly understood. An increased chromatin association of CHD1 (chromodomain helicase DNA‐binding 1) upon UV irradiation indicated possible roles of this chromatin remodeler in the UV damage response. Immunoprecipitation of chromatin fragments revealed that CHD1 co‐localizes in part with GG‐NER factors. Chromatin fractionation showed that the UV‐dependent recruitment of CHD1 occurs to UV lesions in histone‐assembled nucleosomal DNA and that this CHD1 relocation requires the lesion sensor XPC (xeroderma pigmentosum group C). In situ immunofluorescence analyses further demonstrate that CHD1 facilitates substrate handover from XPC to the downstream TFIIH (transcription factor IIH). Consequently, CHD1 depletion slows down CPD excision and sensitizes cells to UV‐induced cytotoxicity. The finding of a CHD1‐driven lesion handover between sequentially acting GG‐NER factors on nucleosomal histone octamers suggests that chromatin provides a recognition scaffold enabling the detection of a subset of CPDs. Synopsis Processing of UV‐induced cyclobutane pyrimidine dimers by global‐genome nucleotide excision repair (GG‐NER) in the context of nucleosomes is facilitated by the chromatin remodeler CHD1, whose recruitment by the UV lesion sensor XPC promotes handover to downstream repair factors. UV irradiation leads to increased CHD1 association with chromatin. A subset of chromatin‐bound CHD1 co‐localizes with GG‐NER factors. CHD1 co‐localization to repair sites depends on the XPC lesion sensor. CHD1 promotes nucleosomal lesion handover from XPC to TFIIH. CHD1 depletion decreases excision repair and sensitizes to UV‐induced cytotoxicity.

Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of structurally unrelated DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) radiation or intracellular metabolic by-products. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4DDB2 and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4DDB2 or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin.

The cellular defense system known as global-genome nucleotide excision repair (GG-NER) safeguards genome stability by eliminating a plethora of structurally unrelated DNA adducts inflicted by chemical carcinogens, ultraviolet (UV) radiation or endogenous metabolic by-products. Xeroderma pigmentosum group C (XPC) protein provides the promiscuous damage sensor that initiates this versatile NER reaction through the sequential recruitment of DNA helicases and endonucleases, which in turn recognize and excise insulting base adducts. As a DNA damage sensor, XPC protein is very unique in that it (a) displays an extremely wide substrate range, (b) localizes DNA lesions by an entirely indirect readout strategy, (c) recruits not only NER factors but also multiple repair players, (d) interacts avidly with undamaged DNA, (e) also interrogates nucleosome-wrapped DNA irrespective of chromatin compaction and (f) additionally functions beyond repair as a co-activator of RNA polymerase II-mediated transcription. Many recent reports highlighted the complexity of a post-translational circuit that uses polypeptide modifiers to regulate the spatiotemporal activity of this multiuse sensor during the UV damage response in human skin. A newly emerging concept is that stringent regulation of the diverse XPC functions is needed to prioritize DNA repair while avoiding the futile processing of undamaged genes or silent genomic sequences.

DNA damage recognition subunits such as DDB2 and XPC protect the human skin from ultraviolet (UV) light-induced genome instability and cancer, as demonstrated by the devastating inherited syndrome xeroderma pigmentosum. Here we show that the beneficial DNA repair response triggered by these two genome caretakers critically depends on a dynamic spatiotemporal regulation of their homeostasis. The prolonged retention of DDB2 and XPC in chromatin, because of a failure to readily remove both recognition subunits by the ubiquitin-dependent p97/VCP/Cdc48 segregase complex, leads to impaired DNA excision repair of UV lesions. Surprisingly, the ensuing chromosomal aberrations in p97-deficient cells are alleviated by a concomitant downregulation of DDB2 or XPC. Also, genome instability resulting from an excess of DDB2 persisting in UV-irradiated cells is prevented by concurrent p97 overexpression. Our findings demonstrate that DNA damage sensors and repair initiators acquire unexpected genotoxic properties if not controlled by timely extraction from chromatin.