COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ～600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.     

Surprisingly complex community discovered in the mid-Devonian fossil forest at Gilboa

The origin of trees by the mid-Devonian epoch (398-385 million years ago) signals a major change in terrestrial ecosystems with potential long-term consequences including increased weathering, drop in atmospheric CO(2), modified climate, changes in sedimentation patterns and mass extinction. However, little is known about the ecology of early forests or how changes in early terrestrial ecosystems influenced global processes. One of the most famous palaeontological records for this time is the 'oldest fossil forest' at Riverside Quarry, Gilboa, New York, USA, discovered in the 1920s. Hundreds of large Eospermatopteris sandstone casts, now thought to represent the bases of standing cladoxylopsid trees, were recovered from a horizon that was originally interpreted as a muddy swamp. After quarry operations ceased, relatively minor outcrops of similar fossils at nearby localities have provided limited opportunities to evaluate this pervasive view using modern methods. In 2010, removal of the quarry backfill enabled reappraisal of the palaeoecology of this important site. Here we describe a 1,200?m(2) map showing numerous Eospermatopteris root systems in life position within a mixed-age stand of trees. Unexpectedly, large woody rhizomes with adventitious roots and aerial branch systems identified as aneurophytalean progymnosperms run between, and probably climb into, Eospermatopteris trees. We describe the overall habit for these surprisingly large aneurophytaleans, the earliest fossil group having wood produced by a bifacial vascular cambium. The site also provides evidence for arborescence within lycopsids, extending the North American range for trees in this ecologically critical group. The rooting horizon is a dark grey sandy mudstone showing limited root penetration. Although clearly belonging to a wetland coastal plain environment, the forest was probably limited in duration and subject to periodic disturbance. These observations provide fundamental clarification of the palaeoecology of this mixed-group early forest, with important implications for interpreting coeval assemblage data worldwide.     

STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter. Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (approximately 40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED) to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.     

Early maize agriculture and interzonal interaction in southern Peru

Over the past decade, increasing attention to the recovery and identification of plant microfossil remains from archaeological sites located in lowland South America has significantly increased knowledge of pre-Columbian plant domestication and crop plant dispersals in tropical forests and other regions. Along the Andean mountain chain, however, the chronology and trajectory of plant domestication are still poorly understood for both important indigenous staple crops such as the potato (Solanum sp.) and others exogenous to the region, for example, maize (Zea mays). Here we report the analyses of plant microremains from a late preceramic house (3,431 +/- 45 to 3,745 +/- 65 14C bp or approximately 3,600 to 4,000 calibrated years bp) in the highland southern Peruvian site of Waynuna. Our results extend the record of maize by at least a millennium in the southern Andes, show on-site processing of maize into flour, provide direct evidence for the deliberate movement of plant foods by humans from the tropical forest to the highlands, and confirm the potential of plant microfossil analysis in understanding ancient plant use and migration in this region.     

Gene therapy: X-SCID transgene leukaemogenicity

Gene therapy has been remarkably effective for the immunological reconstitution of patients with severe combined immune deficiency, but the occurrence of leukaemia in a few patients has stimulated debate about the safety of the procedure and the mechanisms of leukaemogenesis. Woods et al. forced high expression of the corrective therapeutic gene IL2RG, which encodes the gamma-chain of the interleukin-2 receptor, in a mouse model of the disease and found that tumours appeared in a proportion of cases. Here we show that transgenic IL2RG does not necessarily have potent intrinsic oncogenic properties, and argue that the interpretation of this observation with respect to human trials is overstated.     

A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont

According to small subunit ribosomal RNA (ss rRNA) sequence comparisons all known Archaea belong to the phyla Crenarchaeota, Euryarchaeota, and--indicated only by environmental DNA sequences--to the 'Korarchaeota'. Here we report the cultivation of a new nanosized hyperthermophilic archaeon from a submarine hot vent. This archaeon cannot be attached to one of these groups and therefore must represent an unknown phylum which we name 'Nanoarchaeota' and species, which we name 'Nanoarchaeum equitans'. Cells of 'N. equitans' are spherical, and only about 400 nm in diameter. They grow attached to the surface of a specific archaeal host, a new member of the genus Ignicoccus. The distribution of the 'Nanoarchaeota' is so far unknown. Owing to their unusual ss rRNA sequence, members remained undetectable by commonly used ecological studies based on the polymerase chain reaction. 'N. equitans' harbours the smallest archaeal genome; it is only 0.5 megabases in size. This organism will provide insight into the evolution of thermophily, of tiny genomes and of interspecies communication.     

人Ⅱ型胶原蛋白基因的转录调控研究

利用对培养细胞的短暂染分析，对人Ⅱ型胶原蛋白基因启动子区和第一内含子区的四个可能的转录调控功能区进行了实验研究。结果显示，该基因的转显是一个强启动子驱使下，由多个抑制子或抑制子样功能区调节的。这些结果独特于已报道的大鼠相应基因的结果，但得到了大鼠相应基因研究组的证实。     

Close association of erythrocytes with surface of late mouse blastocysts: SEM analysis

Summary SEM analysis of preimplantation mouse blastocysts reveals closely associated red blood cells on involuted surfaces of trophoblasts, an indication of the capability for initial phases of phagocytosis at an early developmental stage.Acknowledgment. This study was supported by NICHD grant HD-06234 and NIH grant RR-05384. Author for reprint requests.