We demonstrate excitation of photosensitisers (PSs) by accelerated protons to produce fluorescence and singlet oxygen. Their fluorescence follows a pattern similar to the proton energy loss in ...matter, while proton-derived fluorescence spectra match the photon-induced spectra. PSs excited in dry gelatin exhibit enhanced phosphorescence, suggesting an efficient PSs triplet state population. Singlet oxygen measurements, both optically at ~1270 nm and through the photoproduct of protoporphyrin IX (PpIX), demonstrate cytotoxic singlet oxygen generation by proton excitation. The singlet oxygen-specific scavenger 1,4-diazabicyclo2.2.2octane (DABCO) abrogates the photoproduct formation under proton excitation, but cannot countermand the overall loss of PpIX fluorescence. Furthermore, in two cell lines, M059K and T98G, we observe differential cell death upon the addition of the PS cercosporin, while in U87 cells we see no effect at any proton irradiation dose. Our results pave the way for a novel treatment combining proton therapy and "proton-dynamic therapy" for more efficient tumour eradication.
Aims: The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA‐targeted species‐ and group‐specific primers ...for more accurate quantification of bacteria from faecal samples with real‐time PCR.
Methods and Results: A linear range of quantification between 0·1–10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30–4500 to 1·9 × 106–6·0 × 106 target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification.
Conclusions: Real‐time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples.
Significance and Impact of the Study: To design and optimize an extensive set of real‐time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease.
PCR primers and hybridization probes were designed for the 16S rRNA genes of six bacterial species or groups typically present in human faeces or used in the dairy industry. The primers and probes ...were applied for quantification of the target bacterial genomes added in artificial DNA mixtures or faecal DNA preparations, using dot-blot hybridization and real-time PCR with SYBR Green I and TaqMan chemistries. Dot-blot hybridization with (33)P-labelled oligonucleotide probes was shown to detect a 10 % target DNA fraction present in mixed DNA samples. Applicability of the rDNA-targeted oligonucleotide probes without pre-enrichment of the 16S gene pool by PCR was thus limited to the detection of the predominant microbial groups. Real-time PCR was performed using a 96-well format and was therefore feasible for straightforward analysis of large sample amounts. Both chemistries tested could detect and quantify a subpopulation of 0.01 % from the estimated number of total bacterial genomes present in a population sample. The linear range of amplification varied between three and five orders of magnitude for the specific target genome while the efficiency of amplification for the individual PCR assays was between 88.3 and 104 %. Use of a thermally activated polymerase was required with the SYBR Green I chemistry to obtain a similar sensitivity level to the TaqMan chemistry. In comparison to dot-blot hybridization, real-time PCR was easier and faster to perform and also proved to have a superior sensitivity. The results suggest that real-time PCR has a great potential for analysis of the faecal microflora.
Purpose
Head and neck cancer (HNC) treatment may lead to late effects and impaired health-related quality of life of survivors. Knowledge on long-term late effects after radiotherapy (RT) and ...potential underlying biological mechanisms is lacking. We assessed the prevalence of xerostomia, dysphagia, and chronic fatigue (CF) in HNC survivors ≥ 5 years post-RT, and examined associations between pro-inflammatory cytokines and late effects.
Methods
In a cross-sectional study, 263 HNC survivors treated between 2007 and 2013 were enrolled. They completed validated questionnaires assessing xerostomia and dysphagia (the EORTC QLQ-H&N35), and CF (the Fatigue Questionnaire), and underwent blood sampling and clinical examination. Pro-inflammatory cytokines were analyzed in 262 survivors and 100 healthy age- and gender-matched controls.
Results
Median time since treatment was 8.5 years. The proportions of survivors reporting xerostomia, dysphagia, and CF were 58%, 31%, and 33%, respectively, with a preponderance of females. We found no significant associations between IL-6, IL-8, IP-10, TARC, TNF, or ENA-78 and the three late effects. The odds of having elevated levels of IL-6 and IP-10 were significantly higher in the survivors compared to the controls.
Conclusions
More than one-third of long-term HNC survivors experienced xerostomia, dysphagia, and CF. Persistent inflammation, with elevated systemic cytokines, was not associated with these late effects, although HNC survivors had higher levels of some cytokines than the controls.
Implications for Cancer Survivors
This study provides new knowledge on late effects that can serve as grounds for informing patients with HNC about risk of late effects more than 5 years after RT.
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