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.     

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.     

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.     

A conspicuous nickel protein in microbial mats that oxidize methane anaerobically

Anaerobic oxidation of methane (AOM) in marine sediments is an important microbial process in the global carbon cycle and in control of greenhouse gas emission. The responsible organisms supposedly reverse the reactions of methanogenesis, but cultures providing biochemical proof of this have not been isolated. Here we searched for AOM-associated cell components in microbial mats from anoxic methane seeps in the Black Sea. These mats catalyse AOM rather than carry out methanogenesis. We extracted a prominent nickel compound displaying the same absorption spectrum as the nickel cofactor F430 of methyl-coenzyme M reductase, the terminal enzyme of methanogenesis; however, the nickel compound exhibited a higher molecular mass than F430. The apparent variant of F(430) was part of an abundant protein that was purified from the mat and that consists of three different subunits. Determined amino-terminal amino acid sequences matched a gene locus cloned from the mat. Sequence analyses revealed similarities to methyl-coenzyme M reductase from methanogenic archaea. The abundance of the nickel protein (7% of extracted proteins) in the mat suggests an important role in AOM.     

Der Beweis des Hilbert-Schmidt-Theorems

Ohne Zusammenfassung Vorgelegt von H. Freudenthal Professor Hans Wußing (Leipzig) zu seinem sechzigsten Geburtstag (1987) gewidmet     

Olfaction: scent-triggered navigation in honeybees

The honeybee, Apis mellifera, navigates rapidly and accurately to food sources that are often kilometres away. They achieve this by learning visual cues, such as the location and colour of nectar-bearing flowers, and chemical cues, such as the scent and the taste of the nectar. Here we train bees to visit differently scented sugar feeders placed at specific outdoor locations and find that they can be induced to visit the same locations simply by having the corresponding scent blown into the hive, even when the destinations no longer have the food or carry the scent. A familiar nectar scent can trigger specific memories of a route and therefore expedite navigation to the food source.     

Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma

To survey hepatitis B virus (HBV) integration in liver cancer genomes, we conducted massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues. We found that HBV integration is observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%). Copy-number variations (CNVs) were significantly increased at HBV breakpoint locations where chromosomal instability was likely induced. Approximately 40% of HBV breakpoints within the HBV genome were located within a 1,800-bp region where the viral enhancer, X gene and core gene are located. We also identified recurrent HBV integration events (in ≥ 4 HCCs) that were validated by RNA sequencing (RNA-seq) and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue. We also report evidence that suggests that the number of HBV integrations is associated with patient survival.     

LI-cadherin cis-dimerizes in the plasma membrane Ca2+ independently and forms highly dynamic trans-contacts

LI-cadherin belongs to the family of 7D-cadherins that is characterized by a low sequence similarity to classical cadherins, seven extracellular cadherin repeats (ECs), and a short cytoplasmic domain. Nevertheless, LI-cadherins mediates Ca²⁺-dependent cell–cell adhesion and induces an epitheloid cellular phenotype in non-polarized CHO cells. Whereas several studies suggest that classical cadherins cis-dimerize in a Ca²⁺-dependent manner and interact in trans by strand-swapping tryptophan 2 of EC1, little is known about the molecular interactions of LI-cadherin, which lacks tryptophan 2. We thus expressed fluorescent LI-cadherin fusion proteins in HEK293 and CHO cells, analyzed their cell–cell adhesive properties and studied their cellular distribution, cis-interaction, and lateral diffusion in the presence and absence of Ca²⁺. LI-cadherin highly concentrates in cell contact areas but rapidly leaves those sites upon Ca²⁺ depletion and redistributes evenly on the cell surface, indicating that it is only kept in the contact areas by trans-interactions. Fluorescence resonance energy transfer analysis of LI-cadherin-CFP and -YFP revealed that LI-cadherin forms cis-dimers that resist Ca²⁺ depletion. As determined by fluorescence redistribution after photobleaching, LI-cadherin freely diffuses in the plasma membrane as a cis-dimer (D?=?0.42?±?0.03?μm²/s). When trapped by trans-binding in cell contact areas, its diffusion coefficient decreases only threefold to D?=?0.12?±?0.01?μm²/s, revealing that, in contrast to classical and desmosomal cadherins, trans-contacts formed by LI-cadherin are highly dynamic.