Statistical Significance of Mutation Frequencies,and the Power of Statistical Testing,Using the Poisson Distribution

The Poisson distribution may be employed to test whether mutation frequencies differ from control frequencies. This paper describes how this testing procedure may be used for either one-tailed or two-tailed hypotheses. It is also shown how the power of the statistical test can be calculated, the power being the probability of correctly concluding the null hypothesis to be false.     

Neuropeptide Y and platelet aggregation by adrenaline

The effect of neuropeptide Y on platelet-rich citrated human plasma has been studied both before and after addition of adrenaline. The peptide has no aggregatory properties of its own, but in the concentrations tested it does slow and inhibit the aggregatory responses of platelets to adrenaline. This effect is dose-dependent. The implications of this finding are discussed.     

Catalytically inactive,purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA–DNA hybrids. GFP-dRNH1 binds strongly to RNA–DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA–DNA hybrids under a wide range of conditions.     

Synthesis and fluorescent labeling of beta-amyloid peptides.

Fluorescent cell analytical techniques require the incorporation of a fluorophore into the target molecule without causing a significant change in the native conformation. Many short peptides have a limited number of reactive groups that can be labeled without affecting the biological activity. In this work we present several methods for labeling beta-amyloid peptides (betaA[25-35], betaA[1-40]) and their derivatives (LPFFD, RIIGL and RVVIA) with different chromophores exclusively at the N-terminus. In the case of liquid-phase labeling, fluorescein isothiocyanate was used. The side-chain amino function of Lys, if present in the sequence, was protected with an Fmoc group, whereby the hydrophobic character of the peptide was further increased. The labeling reaction was carried out in an appropriate deaggregating solvent, DMSO. For solid-phase labeling, 5(6)-carboxyfluorescein and 7-amino-4-methyl-3-coumarinylacetic acid were applied. Several cleavage cocktails were tested for removal of the labeled amyloid peptides from the resin in order to completely suppress the oxidation of Met.     

A fluid salt-bridging cluster and the stabilization of p53

p53 is a homotetrameric tumor suppressor protein that is found to be mutated in most human cancers. Some of these mutations, particularly mutations to R337, fall in the tetramerization domain and cause defects in tetramer formation leading to loss of function. Mutation to His at this site has been found to destabilize the tetramer in a pH-dependent fashion. In structures of the tetramerization domain determined by crystallography, R337 from one monomer makes a salt bridge with D352 from another monomer, apparently helping to stabilize the tetramer. Here we present molecular dynamics simulations of wild-type p53 and the R337His mutant at several different pH and salt conditions. We find that the 337-352 salt bridge is joined by two other charged side chains, R333 and E349. These four residues do not settle into a fixed pattern of salt bridging, but continue to exchange salt-bridging partners on the nanosecond time scale throughout the simulation. This unusual system of fluid salt bridging may explain the previous finding from alanine scanning experiments that R333 contributes significantly to protein stability, even though in the crystal structure it is extended outward into solvent. This extended conformation of R333 appears to be the result of a specific crystal contact and, this contact being absent in the simulation, R333 turns inward to join its interaction partners. When R337 is mutated to His but remains positively charged, it maintains the original interaction with D352, but the newly observed interaction with E349 is weakened, accounting for the reduced stability of R337H even under mildly acidic conditions. When this His is deprotonated, the interaction with D352 is also lost, accounting for the further destabilization observed under mildly alkaline conditions. Simulations were carried out using both explicit and implicit solvent models, and both displayed similar behavior of the fluid salt-bridging cluster, suggesting that implicit solvent models can capture at least the qualitative features of this phenomenon as well as explicit solvent. Simulations under strongly acidic conditions in implicit solvent displayed the beginnings of the unfolding process, a destabilization of the hydrophobic dimer-dimer interface. Computational alanine scanning using the molecular mechanics Poisson-Boltzmann surface area method showed significant correlation to experimental unfolding data for charged and polar residues, but much weaker correlation for hydrophobic residues.     

Working with mahouts to explore the diet of work elephants in Myanmar (Burma)

At an elephant camp in central Myanmar (Burma), we interviewed mahouts and veterinarians to describe the diet of Asian elephants (Elephas maximus) in a mixed-deciduous forest. Elephants showed a broad dietary breadth (103 plant species from 42 families); consumed mostly browse (94% of plant species); and were very selective about plant parts [e.g., many trees were eaten exclusively for their bark (22%) or fruits (14%)]. The fruits from 29 plant species were recorded to be eaten by elephants. Several of these were found as fruit remains, seeds, or seedlings in elephant dung, suggesting a role of Asian elephants in seed dispersal. Work elephants and their mahouts prove to be a rich source of information to understand wild elephant ecology.     

Force field influences in beta-hairpin folding simulations

All-atom force fields are now routinely used for more detailed understanding of protein folding mechanisms. However, it has been pointed out that use of all-atom force fields does not guarantee more accurate representations of proteins; in fact, sometimes it even leads to biased structural distributions. Indeed, several issues remain to be solved in force field developments, such as accurate treatment of implicit solvation for efficient conformational sampling and proper treatment of backbone interactions for secondary structure propensities. In this study, we first investigate the quality of several recently improved backbone interaction schemes in AMBER for folding simulations of a beta-hairpin peptide, and further study their influences on the peptide's folding mechanism. Due to the significant number of simulations needed for a thorough analysis of tested force fields, the implicit Poisson-Boltzmann solvent was used in all simulations. The chosen implicit solvent was found to be reasonable for studies of secondary structures based on a set of simulations of both alpha-helical and beta-hairpin peptides with the TIP3P explicit solvent as benchmark. Replica exchange molecular dynamics was also utilized for further efficient conformational sampling. Among the tested AMBER force fields, ff03 and a revised ff99 force field were found to produce structural and thermodynamic data in comparably good agreement with the experiment. However, detailed folding pathways, such as the order of backbone hydrogen bond zipping and the existence of intermediate states, are different between the two force fields, leading to force field-dependent folding mechanisms.     

5-Keto-3-cyano-2,4-diaminothiophenes as selective maternal embryonic leucine zipper kinase inhibitors

Maternal embryonic leucine zipper kinase (MELK) is involved in several key cellular processes and displays increased levels of expression in numerous cancer classes (colon, breast, brain, ovary, prostate and lung). Although no selective MELK inhibitors have yet been approved, increasing evidence suggest that inhibition of MELK would constitute a promising approach for cancer therapy. A weak high-throughput screening hit (17, IC₅₀?≈?5?μM) with lead-like properties was optimized for MELK inhibition. The early identification of a plausible binding mode by molecular modeling offered guidance in the choice of modifications towards compound 52 which displayed a 98?nM IC₅₀. A good selectivity profile was achieved for a representative member of the series (29) in a 486 protein kinase panel. Future elaboration of 52 has the potential to deliver compounds for further development with chemotherapeutic aims.