The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.     

A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion

The activation of heterodimeric (α/β) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes, including blood coagulation, tissue remodeling, and cancer metastasis. This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin β cytoplasmic tail, but how these binding events are spatiotemporally regulated remains obscure. Here we report the crystal structure of a dormant talin, revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface. Unexpectedly, the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane. Such a dual inhibitory topology for talin is consistent with the biochemical and functional data, but differs significantly from a previous model. We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) – a known talin activator, membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS. Such an electrostatic “pull-push” process promotes the relief of the dual inhibition of talin-FERM, which differs from the classic “steric clash” model for conventional PIP2-induced FERM domain activation. These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.     

The potential therapeutic use of stem cells in cartilage repair

As our population demographics change, osteoarthritis and cartilage defects are becoming more prevalent. The discovery of stems cells and their ability for indefinite regeneration has revolutionised the way cartilage problems are viewed. Tissue engineering has been shown to be the ideal way of repairing articular cartilage lesions, i.e. back to native tissue. Cartilage is an ideal tissue engineering target as it is avascular, aneural and alymphatic. The two main types of stem cells being investigated in chondrogenesis are embryological and mesenchymal stem cells. Research into embryological stem cells has been surrounded by controversy because of ethical, religious and social concerns. We discuss the use of embryological and mesenchymal stem cells in cartilage repair and the various factors involved in the differentiation into chondrocytes. We also discuss commonly used mesenchymal stem cell markers and their limitations.     

An empirical test of the F2 screen for detection of Bacillus thuringiensis-resistance alleles in tobacco budworm (Lepidoptera: Noctuidae)

Insects exposed to genetically modified crops expressing Bacillus thuringiensis (Bt) toxins are under intense selection pressure that could result on widespread Bt resistance. Screening for early indications of Bt resistance developing in targeted Lepidoptera is conducted in many of the regions where genetically modified cotton and corn have been commercialized. Heliothis virescens (F.) (Lepidoptera: Noctuidae) has been selected in the laboratory to have a gene for resistance to Cry1Ac. We used this laboratory line to test the assumptions and theoretical predictions related to detection of recessive Bt-resistant alleles in field populations based on a second generation (F2) screen. By creating single-pair families from mating a heterozygous Cry1Ac-resistant moth with a Cry1Ac-susceptible moth, we simulated the most common genotype when Bt-resistance alleles are at low frequency in the field. The second generation (F2) neonates of single-pair families were screened daily with diagnostic concentration bioassays. Cry1Ac-resistant homozygous larvae were detected, but the proportion of resistant larvae was generally below the theoretical expectation of 6.25% and was influenced by the moth F1 sib-mating density and by the day of oviposition of F2 eggs. Logistical considerations such as F1 sib-mating density and F2 neonate screening are important for the successful implementation of a reliable method.     

Rapid learning of shelter position in an intertidal fish, the shanny Lipophrys pholis L.

The homing ability of an intertidal fish, the shanny Lipophrys pholis , was investigated using two experiments that were based on the shanny's natural propensity to home to a refuge. A displacement experiment demonstrated that the fish were able to accurately locate the previous position of a refuge once the shelter itself had been removed so that it could not be used as a cue to directly signal the goal location. This shows that the shanny can encode information about its familiar surroundings into a spatial map and use this information to home. A second experiment in which the cues internal and external to the experimental tank were put in conflict with one another suggested that the shanny can encode cues that are both intra- and external-tank cues in its representation of space, but that there is individual variation in the type of cues that are used, or memorized.     

Structural basis of membrane invagination by F-BAR domains

BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.     

Quantification of isotope encoded proteins in 2-D gels using surface enhanced resonance Raman

A strategy for quantification of multiple protein isoforms from a complex sample background is demonstrated, combining isotopomeric rhodamine 6G (R6G) labels and surface-enhanced Raman in polyacrylamide matrix. The procedure involves isotope-encoding by lysine-labeling with (R6G) active ester reagents, isoform separation by 2-DGE, fluorescence quantification using internal standardization to water, and silver nanoparticle deposition followed by surface-enhanced Raman detection. R6G sample encoding and standardization enabled the determination of total protein concentration and the distribution of specific isoforms using the combined detection approach of water-referenced fluorescence spectral imaging and ratiometric quantification. A detection limit of approximately 13.5 picomolar R6G-labeled protein was determined for the surface-enhanced Raman in a gel matrix (15-fold lower than fluorescence). High quantification accuracies for small differences in protein populations at low nanogram abundance were demonstrated for human GMP synthetase (hGMPS) either as purified protein samples in a single-point determination mode (3% relative standard deviation, RSD%) or as HCT116 human cancer cellular lysate in an imaging application (with 16% RSD%). These results represent a prototype for future applications of isotopic surface-enhanced resonance Raman scatter to quantification of protein distributions.     

Catalytic mechanism of human DNA polymerase lambda with Mg2+ and Mn2+ from ab initio quantum mechanical/molecular mechanical studies

DNA polymerases play a crucial role in the cell cycle due to their involvement in genome replication and repair. Understanding the reaction mechanism by which these polymerases carry out their function can provide insights into these processes. Recently, the crystal structures of human DNA polymerase lambda (Pollambda) have been reported both for pre- and post-catalytic complexes [García-Díaz et al., DNA Repair 3 (2007), 1333]. Here we employ the pre-catalytic complex as a starting structure for the determination of the catalytic mechanism of Pollambda using ab initio quantum mechanical/molecular mechanical methods. The reaction path has been calculated using Mg(2+) and Mn(2+) as the catalytic metals. In both cases the reaction proceeds through a two-step mechanism where the 3'-OH of the primer sugar ring is deprotonated by one of the conserved Asp residues (D490) in the active site before the incorporation of the nucleotide to the nascent DNA chain. A significant charge transfer is observed between both metals and some residues in the active site as the reaction proceeds. The optimized reactant and product structures agree with the reported crystal structures. In addition, the calculated reaction barriers for both metals are close to experimentally estimated barriers. Energy decomposition analysis to explain individual residue contributions suggests that several amino acids surrounding the active site are important for catalysis. Some of these residues, including R420, R488 and E529, have been implicated in catalysis by previous mutagenesis experiments on the homologous residues on Polbeta. Furthermore, Pollambda residues R420 and E529 found to be important from the energy decomposition analysis, are homologous to residues R183 and E295 in Polbeta, both of which are linked to cancer. In addition, residues R386, E391, K422 and K472 appear to have an important role in catalysis and could be a potential target for mutagenesis experiments. There is partial conservation of these residues across the Pol X family of DNA polymerases.     

Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase Cgamma1, Grb2, and Src family kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.     

Combining simple blood tests to identify primary care patients with unexpected weight loss for cancer investigation: Clinical risk score development,internal validation,and net benefit analysis

BackgroundUnexpected weight loss (UWL) is a presenting feature of cancer in primary care. Existing research proposes simple combinations of clinical features (risk factors, symptoms, signs, and blood test data) that, when present, warrant cancer investigation. More complex combinations may modify cancer risk to sufficiently rule-out the need for investigation. We aimed to identify which clinical features can be used together to stratify patients with UWL based on their risk of cancer.Methods and findingsWe used data from 63,973 adults (age: mean 59 years, standard deviation 21 years; 42% male) to predict cancer in patients with UWL recorded in a large representative United Kingdom primary care electronic health record between January 1, 2000 and December 31, 2012. We derived 3 clinical prediction models using logistic regression and backwards stepwise covariate selection: Sm, symptoms-only model; STm, symptoms and tests model; Tm, tests-only model. Fifty imputations replaced missing data. Estimates of discrimination and calibration were derived using 10-fold internal cross-validation. Simple clinical risk scores are presented for models with the greatest clinical utility in decision curve analysis. The STm and Tm showed improved discrimination (area under the curve ≥ 0.91), calibration, and greater clinical utility than the Sm. The Tm was simplest including age-group, sex, albumin, alkaline phosphatase, liver enzymes, C-reactive protein, haemoglobin, platelets, and total white cell count. A Tm score of 5 balanced ruling-in (sensitivity 84.0%, positive likelihood ratio 5.36) and ruling-out (specificity 84.3%, negative likelihood ratio 0.19) further cancer investigation. A Tm score of 1 prioritised ruling-out (sensitivity 97.5%). At this threshold, 35 people presenting with UWL in primary care would be referred for investigation for each person with cancer referred, and 1,730 people would be spared referral for each person with cancer not referred. Study limitations include using a retrospective routinely collected dataset, a reliance on coding to identify UWL, and missing data for some predictors.ConclusionsOur findings suggest that combinations of simple blood test abnormalities could be used to identify patients with UWL who warrant referral for investigation, while people with combinations of normal results could be exempted from referral.

Dr. Brian D Nicholson and colleagues investigate whether combinations of routine blood tests could be used to stratify patients in UK with unexpected weight loss based on their risk of cancer.