Background: Sampling nasal lining fluid (NLF) via nasosorption is minimally invasive and well tolerated, but the feasibility of assessing the nasal microbiome using these samples is unknown. However, low biomass makes airway samples particularly susceptible to issues related to contaminant DNA. In this study, we evaluated the suitability of DNA isolated using methodologies for low-biomass respiratory samples and assessed how well lining fluid collected via nasosorption captures the nasal microbial diversity and composition compared to the traditional swab sampling approach. Methods: Nasal swabs and NLF were collected from adult volunteers. DNA was extracted from a mock microbial community and NLF using a column-based kit (ZymoBIOMICS), a precipitation-based kit (Qiagen), or a previously published precipitation-based method. Quality and quantity of DNA was assessed and short-read 16s rRNA sequencing performed to assess feasibility and extraction bias. An optimised extraction methodology was then used to extract DNA from NLF and nasal swabs, and long-read 16s rRNA sequencing performed to compare microbial profiles between NLF and nasal swabs. Taxonomy was assigned using the nf-core/ampliseq pipeline, the PacificBiosciences/pb-16S-nf pipeline, or the software Emu, and downstream analyses were performed using R packages phyloseq and mixOmics. Results: All extraction methods recovered DNA from the mock community, but only precipitation-based methods yielded sufficient DNA from NLF. Extraction methodologies significantly affected microbial profiles, with mechanical lysis needed to minimize bias against resilient genera. Profiles obtained from NLF and swabs were comparable with long-read sequencing. Conclusions: Our findings demonstrate the feasibility of profiling the nasal microbiome using NLF collected via nasosorption and validated two extraction methodologies as suitable for full-length 16s rRNA sequencing of low-biomass respiratory samples. Our data demonstrate the importance of unbiased DNA extraction methodologies in low-biomass respiratory samples, and the subsequent impact of DNA extraction on observed microbial profiles. Additionally, we demonstrated NLF may be an appropriate surrogate samples for nasal swabs to assess the nasal microbiome using 16s rRNA sequencing.
Historical studies, using Northern blot hybridization, RT-PCR and cDNA library construction have demonstrated the presence of a variety of mRNA molecules in platelets. The development of microarray technology has allowed further characterization of the transcripts represented in the platelet transcriptome. In this review, these studies will be summarized and their findings in relation to the study of platelet function and the identification of disease risk genes discussed.
Clostridium difficile infection (CDI) is a global health threat associated with high rates of morbidity and mortality. Conventional antibiotic CDI therapy can result in treatment failure and recurrent infection. C. difficile produces biofilms which contribute to its virulence and impair antimicrobial activity. Some bacteriophages (phages) can penetrate biofilms and thus could be developed to either replace or supplement antibiotics. Here, we determined the impact of a previously optimized 4-phage cocktail on C. difficile ribotype 014/020 biofilms, and additionally as adjunct to vancomycin treatment in Galleria mellonella larva CDI model. The phages were applied before or after biofilm establishment in vitro, and the impact was analyzed according to turbidity, viability counts and topography as observed using scanning electron and confocal microscopy. The infectivity profiles and efficacies of orally administered phages and/or vancomycin were ascertained by monitoring colonization levels and larval survival rates. Phages prevented biofilm formation, and penetrated established biofilms. A single phage application reduced colonization causing extended longevity in the remedial treatment and prevented disease in the prophylaxis group. Multiple phage doses significantly improved the larval remedial regimen, and this treatment is comparable to vancomycin and the combined treatments. Taken together, our data suggest that the phages significantly reduce C. difficile biofilms, and prevent colonization in the G. mellonella model when used alone or in combination with vancomycin. The phages appear to be highly promising therapeutics in the targeted eradication of CDI and the use of these models has revealed that prophylactic use could be a propitious therapeutic option.
A diagnosis of ovarian cancer has an impact on many areas of a woman's life which define her identity and life story. Women are forced to reconsider the future ‘scenes’ of their life scripts as they deal with the loss of fertility, a challenge to plans and existential threat. This paper considers this experience within the theoretical framework of the Self-Memory System. Alongside this theory, the author shares her use of dramatherapy tools applied to weekend residential courses run across the UK on four occasions to support women affected by ovarian cancer.
Highlights•RQ is present in mitochondria isolated from certain mouse and human tissues•RQ carries electrons to fumarate as the electron acceptor, independently of O2 levels•The ETC can be reprogrammed to the RQ/fumarate pathway using genetic and pharmacologic tools•Reprogramming the ETC mitigates hypoxia-induced damage in vitro and in vivoSummaryUbiquinone (UQ), the only known electron carrier in the mammalian electron transport chain (ETC), preferentially delivers electrons to the terminal electron acceptor oxygen (O2). In hypoxia, ubiquinol (UQH2) diverts these electrons onto fumarate instead. Here, we identify rhodoquinone (RQ), an electron carrier detected in mitochondria purified from certain mouse and human tissues that preferentially delivers electrons to fumarate through the reversal of succinate dehydrogenase, independent of environmental O2 levels. The RQ/fumarate ETC is strictly present in vivo and is undetectable in cultured mammalian cells. Using genetic and pharmacologic tools that reprogram the ETC from the UQ/O2 to the RQ/fumarate pathway, we establish that these distinct ETCs support unique programs of mitochondrial function and that RQ confers protection upon hypoxia exposure in vitro and in vivo. Thus, in discovering the presence of RQ in mammals, we unveil a tractable therapeutic strategy that exploits flexibility in the ETC to ameliorate hypoxia-related conditions.Graphical abstract
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