Tumour biology: senescence in premalignant tumours

Oncogene-induced senescence is a cellular response that may be crucial for protection against cancer development, but its investigation has so far been restricted to cultured cells that have been manipulated to overexpress an oncogene. Here we analyse tumours initiated by an endogenous oncogene, ras, and show that senescent cells exist in premalignant tumours but not in malignant ones. Senescence is therefore a defining feature of premalignant tumours that could prove valuable in the diagnosis and prognosis of cancer.     

The global distribution of clinical episodes of Plasmodium falciparum malaria

Interest in mapping the global distribution of malaria is motivated by a need to define populations at risk for appropriate resource allocation and to provide a robust framework for evaluating its global economic impact. Comparison of older and more recent malaria maps shows how the disease has been geographically restricted, but it remains entrenched in poor areas of the world with climates suitable for transmission. Here we provide an empirical approach to estimating the number of clinical events caused by Plasmodium falciparum worldwide, by using a combination of epidemiological, geographical and demographic data. We estimate that there were 515 (range 300-660) million episodes of clinical P. falciparum malaria in 2002. These global estimates are up to 50% higher than those reported by the World Health Organization (WHO) and 200% higher for areas outside Africa, reflecting the WHO's reliance upon passive national reporting for these countries. Without an informed understanding of the cartography of malaria risk, the global extent of clinical disease caused by P. falciparum will continue to be underestimated.     

Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.     

The Amazon basin in transition

Agricultural expansion and climate variability have become important agents of disturbance in the Amazon basin. Recent studies have demonstrated considerable resilience of Amazonian forests to moderate annual drought, but they also show that interactions between deforestation, fire and drought potentially lead to losses of carbon storage and changes in regional precipitation patterns and river discharge. Although the basin-wide impacts of land use and drought may not yet surpass the magnitude of natural variability of hydrologic and biogeochemical cycles, there are some signs of a transition to a disturbance-dominated regime. These signs include changing energy and water cycles in the southern and eastern portions of the Amazon basin.     

Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR

Inhibition of the BRAF(V600E) oncoprotein by the small-molecule drug PLX4032 (vemurafenib) is highly effective in the treatment of melanoma. However, colon cancer patients harbouring the same BRAF(V600E) oncogenic lesion have poor prognosis and show only a very limited response to this drug. To investigate the cause of the limited therapeutic effect of PLX4032 in BRAF(V600E) mutant colon tumours, here we performed an RNA-interference-based genetic screen in human cells to search for kinases whose knockdown synergizes with BRAF(V600E) inhibition. We report that blockade of the epidermal growth factor receptor (EGFR) shows strong synergy with BRAF(V600E) inhibition. We find in multiple BRAF(V600E) mutant colon cancers that inhibition of EGFR by the antibody drug cetuximab or the small-molecule drugs gefitinib or erlotinib is strongly synergistic with BRAF(V600E) inhibition, both in vitro and in vivo. Mechanistically, we find that BRAF(V600E) inhibition causes a rapid feedback activation of EGFR, which supports continued proliferation in the presence of BRAF(V600E) inhibition. Melanoma cells express low levels of EGFR and are therefore not subject to this feedback activation. Consistent with this, we find that ectopic expression of EGFR in melanoma cells is sufficient to cause resistance to PLX4032. Our data suggest that BRAF(V600E) mutant colon cancers (approximately 8-10% of all colon cancers), for which there are currently no targeted treatment options available, might benefit from combination therapy consisting of BRAF and EGFR inhibitors.     

Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut

The largest mucosal surface in the body is in the gastrointestinal tract, a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T-cell help, undergo class switch recombination of their immunoglobulin receptor to IgA, and differentiate to become plasma cells. However, IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA(+) plasma cells also produce the antimicrobial mediators tumour-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA(+) plasma cells can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-α and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B-lineage cells.     

Computational redesign of endonuclease DNA binding and cleavage specificity

The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site approximately 10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.     

Disruption of extended defects in solid oxide fuel cell anodes for methane oxidation

Point defects largely govern the electrochemical properties of oxides: at low defect concentrations, conductivity increases with concentration; however, at higher concentrations, defect-defect interactions start to dominate. Thus, in searching for electrochemically active materials for fuel cell anodes, high defect concentration is generally avoided. Here we describe an oxide anode formed from lanthanum-substituted strontium titanate (La-SrTiO3) in which we control the oxygen stoichiometry in order to break down the extended defect intergrowth regions and create phases with considerable disordered oxygen defects. We substitute Ti in these phases with Ga and Mn to induce redox activity and allow more flexible coordination. The material demonstrates impressive fuel cell performance using wet hydrogen at 950 degrees C. It is also important for fuel cell technology to achieve efficient electrode operation with different hydrocarbon fuels, although such fuels are more demanding than pure hydrogen. The best anode materials to date--Ni-YSZ (yttria-stabilized zirconia) cermets--suffer some disadvantages related to low tolerance to sulphur, carbon build-up when using hydrocarbon fuels (though device modifications and lower temperature operation can avoid this) and volume instability on redox cycling. Our anode material is very active for methane oxidation at high temperatures, with open circuit voltages in excess of 1.2 V. The materials design concept that we use here could lead to devices that enable more-efficient energy extraction from fossil fuels and carbon-neutral fuels.     

Mechanochemical analysis of DNA gyrase using rotor bead tracking

DNA gyrase is a molecular machine that uses the energy of ATP hydrolysis to introduce essential negative supercoils into DNA. The directionality of supercoiling is ensured by chiral wrapping of the DNA around a specialized domain of the enzyme before strand passage. Here we observe the activity of gyrase in real time by tracking the rotation of a submicrometre bead attached to the side of a stretched DNA molecule. In the presence of gyrase and ATP, we observe bursts of rotation corresponding to the processive, stepwise introduction of negative supercoils in strict multiples of two. Changes in DNA tension have no detectable effect on supercoiling velocity, but the enzyme becomes markedly less processive as tension is increased over a range of only a few tenths of piconewtons. This behaviour is quantitatively explained by a simple mechanochemical model in which processivity depends on a kinetic competition between dissociation and rapid, tension-sensitive DNA wrapping. In a high-resolution variant of our assay, we directly detect rotational pauses corresponding to two kinetic substeps: an ATP-independent step at the end of the reaction cycle, and an ATP-binding step in the middle of the cycle, subsequent to DNA wrapping.     

RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP

Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication, and therefore a potentially important drug target. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 +/- 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 +/- 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors.