FRETboard: Semisupervised classification of FRET traces

Förster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nanoscale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner but lack the flexibility to adapt to different experimental setups and require local installations. Here, we propose to fit models to optical signals more intuitively by adopting a semisupervised approach, in which the user interactively guides the model to fit a given data set, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios and correctly estimate parameters for up to 11 states. On in vitro data, we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis.     

Effect of gelling agent on growth and development ofCeratozamia hildae somatic embryos

Individual somatic proembryos ofCeratozamia hildae were exposed to media that differed only in gelling agent utilized. Five different gelling agents were compared in the first experiment: Bacto agar, Agargel, Gel-Gro, Phytagar, and TC agar. In addition, the effect of agar was examined at two levels. Growth, proliferation, and development were assessed. The lower level of agar did not support good somatic proembryo growth. Agargel and the high agar concentration produced cultures with good proliferation. Proembryos exposed to Phytagar, Gel-Gro, and TC agar had the highest proliferation rates. Overall, Gel-Gro was considered the best gelling agent tested. The three concentrations of Gel-Gro used in the second experiment were 2, 4, and 6 g·1⁻, with the lowest concentration representing the control, the recommended concentration. As gelling agent concentration increased, so did mortality; however, the highest Gel-Gro concentration also produced the highest numbers of good-quality, mature somatic embryos. Proliferation rate was greatest at the lowest concentration. These results suggest thatCeratozamia cultures should be exposed to different gelling agents or concentrations of gelling agents at different developmental stages in order to produce the greatest number and highest quality of somatic embryos.     

Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli.

Three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. No clear function has been assigned to the smallest of these three factors, IF1. Therefore, to investigate the role of this protein in the initiation process in Escherichia coli we have mutated the corresponding gene infA. Because IF1 is essential for cell viability and no mutant selection has so far been described, the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomal infA+ gene, followed by deletion of this infA+ gene. Using this approach, the six arginine residues of IF1 were altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. All mutants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Differences in growth phenotypes of the mutants were observed in both minimal and rich media. Some of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type infA+ allele. Several of these recombinants showed reduced growth rate and a partial cold-sensitive phenotype. This paper presents a collection of IF1 mutants designed for in vivo and in vitro studies on the function of IF1.     

Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions

To simultaneously assess the effects of exonic mutations on RNA splicing and protein functions, we report here an intron-inclusive cDNA (Intinc) expression system. As a test model, twenty-four mutations in exon 9 of the phenylalanine hydroxylase (PAH) gene were examined in an Intinc expression plasmid composed of the PAH cDNA with the exon 9 flanked by its authentic introns. When the PAH enzyme activities from the Intinc plasmid-transfected cells were compared to those of a standard cDNA expression system, five mutations resulted in significant relative differences in PAH activities attributed to altered exon 9-inclusive mRNA levels. Two of the mutations affected exon recognition probably through splice site modifications and the remaining three affected experimentally verified exon splicing enhancer (ESE) motifs. The Intinc expression system allows not only a better link between mutation genotype to disease phenotype but also contributes to further understanding of molecular mechanisms of deleterious effects of mutations.     

Synaptotagmin perturbs the structure of phospholipid bilayers

Synaptotagmin I (syt), an integral protein of the synaptic vesicle membrane, is believed to act as a Ca2+ sensor for neuronal exocytosis. Syt's cytoplasmic domain consists largely of two C2 domains, C2A and C2B. In response to Ca2+ binding, the C2 domains interact with membranes, becoming partially embedded in the lipid bilayer. We have imaged syt C2AB in association with lipid bilayers under fluid, using AFM. As expected, binding of C2AB to bilayers required both an anionic phospholipid [phosphatidylserine (PS)] and Ca2+. C2AB associated with bilayers in the form of aggregates of varying stoichiometries, and aggregate size increased with an increase in PS content. Repeated scanning of bilayers revealed that as C2AB dissociated it left behind residual indentations in the bilayer. The mean depth of these identations was 1.81 nm, indicating that they did not span the bilayer. Individual C2 domains (C2A and C2B) also formed aggregates and produced bilayer indentations. Binding of C2AB to bilayers and the formation of indentations were significantly compromised by mutations that interfere with binding of Ca2+ to syt or reduce the positive charge on the surface of C2B. We propose that bilayer perturbation by syt might be significant with respect to its ability to promote membrane fusion.     

Determining denaturation midpoints in multiprobe equilibrium protein folding experiments

Multiprobe equilibrium unfolding experiments in the downhill regime (i.e., maximal barrier < 3 RT) can resolve the folding process with atomic resolution [ Munoz ( 2002) Int. J. Quantum Chem. 90, 1522 -1528] . Such information is extracted from hundreds of heterogeneous atomic equilibrium unfolding curves, which are characterized according to their denaturation midpoint (e.g., T m for thermal denaturation). Using statistical methods, we analyze T m accuracy when determined from the extremum of the derivative of the unfolding curve and from two-state fits under different sets of simulated experimental conditions. We develop simple procedures to discriminate between real unfolding heterogeneity at the atomic level and experimental uncertainty in the single T m of conventional two-state folding. We apply these procedures to the recently published multiprobe NMR experiments of BBL [ Sadqi et al. ( 2006) Nature 442, 317 -321 ] and conclude that for the 122 single transition atomic unfolding curves reported for this protein the mean T m accuracy is better than 1.8 K for both methods, compared to the 60 K spread in T m determined experimentally. Importantly, we also find that when the pre- or posttransition baseline is incomplete, the two-state fits systematically drift the estimated T m value toward the center of the experimental range. Therefore, the reported 60 K T m spread in BBL is in fact a lower limit. The derivative method is significantly less sensitive to this problem and thus is a better choice for multiprobe experiments with a broad T m distribution. The results we obtain in this work lay the foundations for the quantitative analysis of future multiprobe unfolding experiments in fast-folding proteins.     

Production of Melanocyte‐Specific Antibodies to Human Melanosomal Proteins: Expression Patterns in Normal Human Skin and in Cutaneous Pigmented Lesions

Multiple factors affect skin pigmentation, including those that regulate melanocyte and/or keratinocyte function. Such factors, particularly those that operate at the level of the melanosome, are relatively well characterized in mice, but the expression and function of structural and enzymatic proteins in melanocytes in human skin are not as well known. Some years ago, we generated peptide‐specific antibodies to murine melanosomal proteins that proved to be instrumental in elucidating melanocyte development and differentiation in mice, but cross‐reactivity of those antibodies with the corresponding human proteins often was weak or absent. In an effort to characterize the roles of melanosomal proteins in human skin pigmentation, and to understand the underlying mechanism(s) of abnormal skin pigmentation, we have now generated polyclonal antibodies against the human melanocyte‐specific markers, tyrosinase, tyrosinase‐related protein 1 (TYRP1), Dopachrome tautomerase (DCT) and Pmel17 (SILV, also known as GP100). We used these antibodies to determine the distribution and function of melanosomal proteins in normal human skin (adult and newborn) and in various cutaneous pigmented lesions, such as intradermal nevi, lentigo simplex, solar lentigines and malignant melanomas. We also examined cytokeratin expression in these same samples to assess keratinocyte distribution and function. Immunohistochemical staining reveals distinct patterns of melanocyte distribution and function in normal skin and in various types of cutaneous pigmented lesions. Those differences in the expression patterns of melanocyte markers provide important clues to the roles of melanocytes in normal and in disrupted skin pigmentation.     

The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

Background  

Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10) or PML (promyelocytic leukemia) nuclear bodies, where it associates with TOPIIIα, and to the nucleolus.     

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Causes,historical development,effects and future challenges of a common environmental problem: eutrophication

The impact of the presence of us humans as well as our activities to nature has led to over-exploitation of natural resources and to dramatic changes in land-use including the use of artificial fertilizers contributes to the deterioration of the natural environment. The population density, industrial processes and the use of fertilizers are the main causes for the eutrophication of river systems, estuaries and seas. There are several ways to determine the actual nutrient levels back to the 1950s, back to 1900 or even back to 1800. Available data indicate that the natural background concentrations of nutrients (pristine conditions or the period prior to the widespread use of artificial fertilizers and detergents) were dramatically lower than today. Available time series on chemical and biological data collected from different parts of the world show the (sometimes tremendous) increase in nutrient levels and the related productivity. The same time series, however, also show the decreases in values when measures were taken to reduce the nutrient emissions. Investigations of different systems all over the world show that nearly every system responses differently to eutrophication. Especially physical boundary conditions play an important role in the manifestation of the ultimate effect of local eutrophication. Apart from the physical boundary conditions also the transformation and retention of nutrients in estuarine and coastal systems contribute to system specific responses. Depending on all these different conditions, site specific responses with even site specific problems may occur. The challenge of this millennium is to really reach a balance between nature and mankind including its population size. A beneficial step in this discussion is assessing criteria to reduce eutrophication back to a level acceptable to both humans and nature.