Molecular Diversity of Fungal Phylotypes Co-Amplified Alongside Nematodes from Coastal and Deep-Sea Marine Environments

Nematodes and fungi are both ubiquitous in marine environments, yet few studies have investigated relationships between these two groups. Microbial species share many well-documented interactions with both free-living and parasitic nematode species, and limited data from previous studies have suggested ecological associations between fungi and nematodes in benthic marine habitats. This study aimed to further document the taxonomy and distribution of fungal taxa often co-amplified from nematode specimens. A total of 15 fungal 18S rRNA phylotypes were isolated from nematode specimens representing both deep-sea and shallow water habitats; all fungal isolates displayed high pairwise sequence identities with published data in Genbank (99–100%) and unpublished high-throughput 454 environmental datasets (>95%). BLAST matches indicate marine fungal sequences amplified in this study broadly represent taxa within the phyla Ascomycota and Basidiomycota, and several phylotypes showed robust groupings with known taxa in phylogenetic topologies. In addition, some fungal phylotypes appeared to be present in disparate geographic habitats, suggesting cosmopolitan distributions or closely related species complexes in at least some marine fungi. The present study was only able to isolate fungal DNA from a restricted set of nematode taxa; further work is needed to fully investigate the taxonomic scope and function of nematode-fungal interactions

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.

In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways

HOPON (Hyperbaric Oxygen for the Prevention of Osteoradionecrosis): a randomised controlled trial of hyperbaric oxygen to prevent osteoradionecrosis of the irradiated mandible: study protocol for a randomised controlled trial

Background: Osteoradionecrosis of the mandible is the most common serious complication of radiotherapy for head and neck malignancy. For decades, hyperbaric oxygen has been employed in efforts to prevent those cases of osteoradionecrosis that are precipitated by dental extractions or implant placement. The evidence for using hyperbaric oxygen remains poor and current clinical practice varies greatly. We describe a protocol for a clinical trial to assess the benefit of hyperbaric oxygen in the prevention of osteoradionecrosis during surgery on the irradiated mandible. Methods/design: The HOPON trial is a phase III, randomised controlled, multi-centre trial. It employs an unblinded trial design, but the assessment of the primary endpoint, i.e. the diagnosis of osteoradionecrosis, is assessed on anonymised clinical photographs and radiographs by a blinded expert panel. Eligibility is through the need for a high-risk dental procedure in the mandible where at least 50-Gy radiotherapy has been received. Patients are randomised 1:1 to hyperbaric oxygen arm (Marx protocol) : control arm, but both groups receive antibiotics and chlorhexidine mouthwash. The primary endpoint is the presence of osteoradionecrosis at 6 months following surgery, but secondary endpoints include other time points, acute symptoms and pain, quality of life, and where implants are placed, their successful retention. Discussion: The protocol presented has evolved through feasibility stages and through analysis of interim data. The classification of osteoradionecrosis has undergone technical refinement to ensure that robust definitions are employed. The HOPON trial is the only multi-centre RCT conducted in this clinical setting despite decades of use of hyperbaric oxygen for the prevention of osteoradionecrosis. Trial registration: European Clinical Trials Database, ID: EudraCT200700622527. First registered on 5 November 2007

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.

In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways

Distribucción de Calanus spp. determinada utilizando un sistema de identificación genética

The morphological similarity of Calanus species necessitates that the only unambiguous way of discriminating between the different species at any developmental stage is with molecular tools. We have developed a simple molecular technique to distinguish between the four species of Calanus copepods found in the North Atlantic (Calanus helgolandicus, C. finmarchicus, C. glacialis and C. hyperboreus) at any life stage. This system involves the PCR amplification of a region of the 16S rRNA gene, followed by Restriction Fragment Length Polymorphism (RFLP) analysis of the amplified product. This paper describes the application of the technique to a number of Calanus samples taken from a wide geographical range within the North Atlantic. Samples were acquired from sites ranging in latitude from the English Channel (50°N) to Tromsø (69°N), including many regions of co-occurrence. A comparison has been made between the distribution of Calanus as determined by molecular techniques and distributions established using traditional morphological identification. The molecular analysis has clearly shown extended areas of distribution and co-occurrence and has confirmed the intraspecific conservation of the restriction sites and therefore the reliability of the technique.La semejanza morfológica de las especies de Calanus obliga a que la única manera clara de discriminar entre las distintas especies en cualquier estadio de desarrollo sea mediante herramientas moleculares. Hemos desarrollado una técnica molecular simple para distinguir las cuatro especies de copépodos del género Calanus que se encuentran en el Atlántico Norte (Calanus helgolandicus, C. finmarchicus, C. glaciales and C. hyperboreus), sea cual sea el estadio de su ciclo biológico. Este sistema implica la amplificación mediante PCR de una región del gen 16S rRNA, seguida de análisis de Polimorfismo de Longitud de Fragmentos de Restricción de (RFLP) del producto amplificado. Este trabajo describe la aplicación de la técnica a varias muestras de Calanus procedentes de una amplia región geográfica en el Atlántico Norte. Se obtuvieron muestras de localidades que iban desde el canal de la Mancha (50°N) a Tromsø (69°N), incluyendo muchas regiones de presencia conjunta. Se ha hecho una comparación entre la distribución de Calanus, determinada por las técnicas moleculares, y las distribuciones sobre la base de la identificación morfológica tradicional. El análisis molecular ha demostrado claramente áreas extendidas de distribución y ocurrencia conjunta y ha confirmado la conservación intraspecífica de los lugares de restricción, con lo que se ha confirmado la fiabilidad de la técnica

Detection of fungal 18S rRNA sequences in conjunction with marine nematode 18S rRNA amplicons

Free-living nematodes constitute an important component of estuarine and marine benthic ecosystems. Some marine and soil nematodes are known to harbor microbes, including symbiotic bacteria and fungi, in their external cuticle as well as internally. While assessing diversity of marine nematodes from southwest England using molecular approaches, we found evidence of co-amplification of fungal 18S rRNA sequences in conjunction with nematode 18S rRNA sequences. Based on an 18S rRNA PCR-DGGE approach, 3 fungal clone types were detected alongside nematodes from 2 of 4 estuarine and marine sites in southwest England. At the phylogeny level, fungal clone type 1 belongs to Chaetothyriales while the other 2 clone types belong to Hypocreales. The fungal clones were co-amplified with specific marine nematode taxa indicating true ecological association rather than transient environmental contamination. The present study is the first to detect fungal 18S sequences in parallel with marine nematodes and opens up a new avenue of research for investigating ecological interactions between nematodes and fungi in the marine environment

Molecular diversity of fungal phylotypes co-amplified alongside nematodes from coastal and deep-sea marine environments.

Nematodes and fungi are both ubiquitous in marine environments, yet few studies have investigated relationships between these two groups. Microbial species share many well-documented interactions with both free-living and parasitic nematode species, and limited data from previous studies have suggested ecological associations between fungi and nematodes in benthic marine habitats. This study aimed to further document the taxonomy and distribution of fungal taxa often co-amplified from nematode specimens. A total of 15 fungal 18S rRNA phylotypes were isolated from nematode specimens representing both deep-sea and shallow water habitats; all fungal isolates displayed high pairwise sequence identities with published data in Genbank (99-100%) and unpublished high-throughput 454 environmental datasets (>95%). BLAST matches indicate marine fungal sequences amplified in this study broadly represent taxa within the phyla Ascomycota and Basidiomycota, and several phylotypes showed robust groupings with known taxa in phylogenetic topologies. In addition, some fungal phylotypes appeared to be present in disparate geographic habitats, suggesting cosmopolitan distributions or closely related species complexes in at least some marine fungi. The present study was only able to isolate fungal DNA from a restricted set of nematode taxa; further work is needed to fully investigate the taxonomic scope and function of nematode-fungal interactions