Detection of DNA-Psoralen Photoadducts in Mammalian Skin

An immunofluorescence (IF) method for the detection of 8-methoxypsoralen (8-MOP) photoadducts to DNA has been developed to assess nuclear damage in keratinocytes and melanocytes after psoralen plus UVA (PUVA) treatment, both under in vitro and in vivo conditions. Cryostat sections of the albino and pigmented guinea pig and human skin were used for in vitro studies to establish minimal and maximal drug concentration and UVA dosimetry for the detection of DNA-8-MOP photoadducts. Limits of detection were as low as 10 ng/cm2 8-MOP and 1 J/cm2 UVA for skin sections and sodium bromide-split epidermal sheets. Guinea pigs treated with topical PUVA revealed positive IF stain in epidermal cell nuclei at a threshold dose of 100 μg/cm2: 8-MOP and 13 J/cm2 UVA. Pretreatments of cryostat cuts with ethanol and alkali before IF test enhanced the sensitivity of detection in vivo about 10-fold and enabled us to follow the repair of DNA damage after treating normal guinea pig skin with a dose of 50 μg/cm2 8-MOP plus 6 J/cm2 UVA. The most interesting findings were as follows: (1) A sensitive method to detect PUVA-induced nuclear damage in epidermal and dermal cells was developed. (2) PUVA treatment induced nuclear DNA damage to melanocytes as well as to adjacent keratinocytes, and melanocytes appeared to be 10 times less vulnerable to photo-damage than keratinocytes. (3) There was a greater propensity for the proliferative cells to be damaged by PUVA. (4) PUVA induced nuclear damage up to 700 μm depth in the dermis. (5) The usefulness of the IF test in detecting DNA damage in μg and ng amounts in vivo and in following the repair of damaged DNA induced by PUVA

Identification of Papillomaviruses in Butchers' Warts

We have studied the papillomaviruses found in the hand warts of 60 butchers, most of them from 2 distant slaughtershouses. Warts differing in morphology and location were studied separately. The viruses were identified by molecular hybridization, restriction enzyme analysis and immunoflurescence. Four known human papillomaviruses (HPV-1, HPV-2, HPV-3, HPV-4) were detected and one hitherto unknown papillomavirus was identified in 9 butchers. The DNA of the latter virus did not anneal with any of the RNAs complementary to either HPV-1 to HPV-5 or bovine papillomavirus type 1 (BPV-1) DNAs, and showed a Hind II+III restriction enzyme cleavage pattern distinct from those of known HPVs and BPVs. This virus showed distinct antigenic properties, as shown by immunofluorescence, using HPV-1, -2, -3, -5, and BVP-1 antisera. It may represent a new type of human papillomarvirus(HPV-7) or a yet unidentified animal papillomavirus. In addition, 6 butchers were found to be infected with a papillomavirus, distinct from the known skin HPVs and from BPV-1, which could not be characterized by restriction enzyme analysis. Eleven butchers were found to be infected by 2 viruses.A characteristic histological pattern was found to be associated with the different papillomaviruses

Massive-Scale RNA-Seq Analysis of Non Ribosomal Transcriptome in Human Trisomy 21

Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenilated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes—possibly novel miRNA targets or regulatory sites for gene transcription—were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders