Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision

An emerging DNA sequencing technique uses protein or solid-state pores to analyze individual strands as they are driven in single-file order past a nanoscale sensor. However, uncontrolled electrophoresis of DNA through these nanopores is too fast for accurate base reads. Here, we describe forward and reverse ratcheting of DNA templates through the α-hemolysin nanopore controlled by phi29 DNA polymerase without the need for active voltage control. DNA strands were ratcheted through the pore at median rates of 2.5-40 nucleotides per second and were examined at one nucleotide spatial precision in real time. Up to 500 molecules were processed at ～130 molecules per hour through one pore. The probability of a registry error (an insertion or deletion) at individual positions during one pass along the template strand ranged from 10% to 24.5% without optimization. This strategy facilitates multiple reads of individual strands and is transferable to other nanopore devices for implementation of DNA sequence analysis.     

On the vapour trail of an atmospheric imprint in insects

Terrestrial arthropods, at constant risk from desiccation, are highly sensitive to atmospheric temperature and humidity. A physiological marker of these abiotic conditions could highlight phenotypic adaptations, indicate niche partitioning, and predict responses to climate change for a group representing three-quarters of the Earth's animal species. We show that the (18)O composition of insect haemolymph is such a measure, providing a dynamic and quantitatively predictable signal for respiratory gas exchange and inputs from atmospheric humidity. Using American cockroaches (Periplaneta americana) under defined experimental conditions, we show that insects respiring at low humidity demonstrate the expected enrichment in the (18)O composition of haemolymph because of evaporation. At high humidity, however, diffusional influx of atmospheric water vapour into the animal forces haemolymph to become depleted in (18)O. Additionally, using cockroaches sampled from natural habitats, we show that the haemolymph (18)O signature is transferred to the organic material of the insect's exoskeleton. Insect cuticle, therefore, exhibits the mean atmospheric conditions surrounding the animals prior to moulting. This discovery will help to define the climatic tolerances of species and their habitat preferences, and offers a means of quantifying the balance between niche partitioning and 'neutral' processes in shaping complex tropical forest communities.     

Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism

The mouse arylamine N-acetyltransferase 2 (Nat2) and its homologue (NAT1) in humans are known to detoxify xenobiotic arylamines and are also thought to play a role in endogenous metabolism. Human NAT1 is highly over-expressed in estrogen receptor positive breast tumours and is implicated in susceptibility to neural tube defects. In vitro assays have suggested an endogenous role for human NAT1 in folate metabolism, but in vivo evidence to support this hypothesis has been lacking. Mouse Nat2 provides a good model to study human NAT1 as it shows similar expression profiles and substrate specificities. We have generated transgenic mice lacking a functional Nat2 gene and compared the urinary levels of acetylated folate metabolite para-aminobenzoylglutamate in Nat2 knockout and Nat2 wild-type mice. These results support an in vivo role for mouse Nat2/human NAT1 in folate metabolism. In addition, effects of the Nat2 deletion on sex ratios and neural tube development are described.     

Immunotherapy success in prophylaxis cannot predict therapy: prime-boost vaccination against the 5T4 oncofoetal antigen

We have investigated the tumour therapeutic efficacy of homologous and heterologous prime-boost vaccine strategies against the 5T4 oncofoetal antigen, using both replication defective adenovirus expressing human 5T4 (Ad5T4), and retrovirally transduced DC lines (DCh5T4) in a subcutaneous B16 melanoma model (B16h5T4). In naïve mice we show that all vaccine combinations tested can provide significant tumour growth delay. While DCh5T4/Adh5T4 sequence is the best prophylactic regimen (P > 0.0001), it does not demonstrate any therapeutic efficacy in mice with established tumours. In active therapy the Adh5T4/DCh5T4 vaccination sequence is the best treatment regimen (P = 0.0045). In active therapy, we demonstrate that B16h5T4 tumour growth per se induces Th2 polarising immune responses against 5T4, and the success of subsequent vaccination is dependant on altering the polarizing immune responses from Th2 to Th1. We show that the first immunization with Adh5T4 can condition the mice to induce 5T4 specific Th1 immune responses, which can be sustained and subsequently boosted with DCh5T4. In contrast immunisation with DCh5T4 augments Th2 immune responses, such that a subsequent vaccination with Adh5T4 cannot rescue tumour growth. In this case the depletion of CD25⁺ regulatory cells after tumour challenge but before immunization can restore therapeutic efficacy. This study highlights that all vaccine vectors are not equal at generating TAA immune responses; in tumour bearing mice the capability of different vaccines to activate the most appropriate anti-tumour immune responses is greatly altered compared to what is found in naïve mice.     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Proteomic analyses of intermediate filaments reveals cytokeratin8 is highly acetylated--implications for colorectal epithelial homeostasis.

Butyrate is a critical cancer-preventive element in the colon milieu whose mechanism of action is unclear, but appears to be mediated through inhibition of histone deacetylases (HDACs) and consequent alterations in global protein acetylation. Cytokeratins (CKs) have roles in cytoskeletal function as components of the intermediate filaments (IFs) and this involves CKs in the regulation of tissue homeostasis of high-turnover epithelia such as the colon. We used a 2-D gel/MS analysis to characterise the proteome of IFs, and a novel monitoring-initiated detection and sequencing (MIDAS) approach to identify acetylation sites on principal proteins. We report that CKs are highly acetylated in a colon cancer cell line, with five acetylation sites characterised on CK8 and a further one on CK18. Acetylation of CK8 is responsive to butyrate. HDAC5 is the deacetylase associated with IFs. These data indicate a novel action of butyrate as a cancer preventive agent. Acetylation of CK8 may be associated with IFs stabilisation and thereby provide a candidate mechanism for the appropriate retention or loss of epithelial cells from the flat mucosa.     

Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however, molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore, somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. Here we identify retinoic acid (RA) as this somitic signal and show that retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation, including ventral neural patterning, while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm, where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. Our data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension.     

Evaluation and mapping of the conservation significance of habitats using GIS: an example from Crete, Greece

To ensure the long-term future of NATURA 2000 sites across Europe, effective techniques are required for evaluating and monitoring their conservation significance. This paper describes a GIS-based method that uses multi-criteria evaluation (MCE) to determine the conservation significance of vegetation communities and habitats for a case study of a proposed NATURA 2000 site on the northwest coast of Crete, Greece. The method uses the most frequently used criteria for the selection of priority areas for nature conservation—species and habitat diversity, rarity of species and habitats, naturalness, threat of human disturbance and replaceability. For each community and corresponding habitat type, each criterion was scored according to field data and expert knowledge using a numerical scale. The final conservation score for each community was derived using MCE within a GIS and mapped. The results demonstrated that the method is an effective tool for evaluating and comparing conservation significance and could be applied to other sites across Europe and to monitor change.