Systematic discovery of in vivo phosphorylation networks

Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, respectively. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.     

Molecular architecture of a miRNA-regulated 3' UTR

Animal genomes contain hundreds of microRNAs (miRNAs), small regulatory RNAs that control gene expression by binding to complementary sites in target mRNAs. Some rules that govern miRNA/target interaction have been elucidated but their general applicability awaits further experimentation on a case-by-case basis. We use here an assay system in transgenic nematodes to analyze the interaction of the Caenorhabditis elegans lsy-6 miRNA with 3' UTR sequences. In contrast to many previously described assay systems used to analyze miRNA/target interactions, our assay system operates within the cellular context in which lsy-6 normally functions, a single neuron in the nervous system of C. elegans. Through extensive mutational analysis, we define features in the known and experimentally validated target of lsy-6, the 3' UTR of the cog-1 homeobox gene, that are required for a functional miRNA/target interaction. We describe that both in the context of the cog-1 3' UTR and in the context of heterologous 3' UTRs, one or more seed matches are not a reliable predictor for a functional miRNA/target interaction. We rather find that two nonsequence specific contextual features beyond miRNA target sites are critical determinants of miRNA-mediated 3' UTR regulation. The contextual features reside 3' of lsy-6 binding sites in the 3' UTR and act in a combinatorial manner; mutation of each results in limited defects in 3' UTR regulation, but a combinatorial deletion results in complete loss of 3' UTR regulation. Together with two lsy-6 sites, these two contextual features are capable of imparting regulation on a heterologous 3' UTR. Moreover, the contextual features need to be present in a specific configuration relative to miRNA binding sites and could either represent protein binding sites or provide an appropriate structural context. We conclude that a given target site resides in a 3' UTR context that evolved beyond target site complementarity to support regulation by a specific miRNA. The large number of 3' UTRs that we analyzed in this study will also be useful to computational biologists in designing the next generation of miRNA/target prediction algorithms.     

Functional specificity lies within the properties and evolutionary changes of amino acids

The rapid increase in the amount of protein sequence data has created a need for automated identification of sites that determine functional specificity among related subfamilies of proteins. A significant fraction of subfamily specific sites are only marginally conserved, which makes it extremely challenging to detect those amino acid changes that lead to functional diversification. To address this critical problem we developed a method named SPEER (specificity prediction using amino acids' properties, entropy and evolution rate) to distinguish specificity determining sites from others. SPEER encodes the conservation patterns of amino acid types using their physico-chemical properties and the heterogeneity of evolutionary changes between and within the subfamilies. To test the method, we compiled a test set containing 13 protein families with known specificity determining sites. Extensive benchmarking by comparing the performance of SPEER with other specificity site prediction algorithms has shown that it performs better in predicting several categories of subfamily specific sites.     

Monuments, power and personhood in the British Neolithic

Recent research on British Neolithic monuments describes how the ordering of space within these sites contributed to the production and maintenance of dominant discourses. This article argues that aspects of this work are implicitly built on conceptions of personhood specific to post-Enlightenment thought, resulting in a somewhat static and one-dimensional conception of power relations during the period. One way out of this problem is provided by anthropological and feminist literature in which an alternative characterization of the self as inherently fluid and relational has been outlined. This facilitates a shifting and contextual conception of power relations which can be reconciled more easily with the evidence from Neolithic monuments for the continuous creation and reinterpretation of spatial meanings.     

Strengthening relationships: amyloids create adhesion nanodomains in yeasts

Budding yeasts adhere to biotic or abiotic surfaces and aggregate to form biofilms, using wall-anchored glycoprotein adhesins. The process is paradoxical: adhesins often show weak binding to specific ligands, yet mediate remarkably strong adherence. Single-molecule atomic force microscopy (AFM), genomics, biochemistry and cell biology have recently explained the puzzle, with Candida albicans Als adhesins as the paradigm. The strength of adhesion results partly from force-activated amyloid-like clustering of hundreds of adhesin molecules to form arrays of ordered multimeric binding sites. The various protein domains of eukaryotic adhesins cooperate to facilitate this fascinating new mechanism of activation.     

A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry

The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.     

The DNA binding of insect Fork head factors is strongly influenced by the negative cooperation of neighbouring bases

The Drosophila melanogaster Fork head and Bombyx mori SGF1/Fork head proteins are key regulators of tissue specific gene expression in the modified larval labial glands. Here we use the competitive electrophoretic mobility shift assay to create a detailed Fork head binding matrix and we investigate some unusual features of the Fork head interaction with DNA. We found that the Fork head-DNA interaction is context dependent--the binding specificity of the protein is partly determined by specific combinations of neighbouring bases. Although the total number of the sub-optimal dinucleotide steps is not high, the negative cooperation of neighbouring bases significantly contributes to the overall binding site specificity. Our results allow efficient recognition of insect Fork head binding sites and we show that the putative Fork head cognate elements preferentially accumulate in the near upstream region of genes abundantly expressed in the labial gland.     

In praise of dyestuff histochemistry

The merits of low molecular weight reagents (mini-stains) for localising tissue components, are compared with those of high molecular weight (macro-stains), with respect to stoichiometry, ease of tissue penetration, and particularly specificity. Macro-stains, which are based mainly on antibodies, are likely to be more specific, but this will not always be the case. The concept of specificity in the design of mini-stains is examined, and its application to the delivery of e.g. Cupromeronic blue to particular substrates in the "critical electrolyte concentration" system, is discussed. The much greater resolving power of mini-stains in electron histochemistry has permitted the elucidation of four specific binding sites for proteoglycans along the collagen fibrils, validating the 'one proteoglycan: one binding site' hypothesis.     

Rationale for an integrated approach to genetic epidemiology

Conclusion: Genetic knowledge is now in the public domain and its interpretation by the media and the citizens brings the issues into the public forum of discussion for the necessary ethical, legal and socio-cultural evaluation of its application. Science is being perceived by some as dangerous and as requiring international regulation. Others feel that genetic knowledge will be the breakthrough that will permit medical progress and individual autonomy with regards to personal health and lifestyle choices. The mapping of the human genome has already yielded valuable information on an increasing number of diseases and their variants. Prevailing popular and journalistic archetypes ("imaginaires") used in the media are perceived by the producers as slowing down the possible application of genetic knowledge. The answers to these dilemmas are not readily apparent nor are they prescribed by classical philosophy of medicine. Since genetic knowledge eventually resides with the individual who carries the genes of disease and/or susceptibility, a logical approach to integration of this knowledge at a societal level would seem to reside with individual education and decision-making. The politics of the ensuing social debate could transform the current social contract since an individual's interests need to be balanced against those of his or her immediate family in the sharing of information. The ethical foundations of such a contract requires the genetic education of "Everyone" as a matter of urgent priority. Genetic education should not serve ideological power struggles between the medical establishment and the ethical-legal alliance. Instead, it should ensure the transfer of knowledge to physicians, to patients, to users, to planners, to social science and humanities researchers and to politicians, so that they may make "informed" and free decisions....     

Two conflicting needs affecting predator mobbing by great tits,Parus major

Predator mobbing by great tits, Parus major, was studied by manipulating two contextual variables: the proximity of danger to the nest hole (close versus far), and breeding condition (non-breeding versus nest building versus care of nestlings). In five experiments pitting these contextual variables against each other, great tits were presented with a multi-species mobbing chorus signalling danger to which they responded by typical predator harassment. Based on the ‘move on’ hypothesis of harassment and on context manipulation, harassment levels specific for each of the five experiments could be predictably ordered; the ensuing rank order of scores reflected, by its nature, a series of four strong inference tests. Whereas two latency measures supported the move on hypothesis, two distance-from-danger measures suggested nest hole concealment to be an opposing need; a fifth measure of harassment, comprising both latency and distance, held an intermediate position, thus tying in with the compromise nature of harassment. Considering the ‘phantom’ nature of the unseen danger stimulus applied, the defence levels observed must be regarded as moderately conservative. There are natural danger stimuli (e.g. cats) that more effectively prevent great tits displaying predator harassment.     

Dissection of the locus control function located on the chicken lysozyme gene domain in transgenic mice.

The entire chicken lysozyme gene locus including all known cis-regulatory sequences and the 5' and 3' matrix attachment sites defining the borders of the DNase I sensitive chromatin domain, is expressed at a high level and independent of its chromosomal position in macrophages of transgenic mice. It was concluded that the lysozyme gene locus carries a locus control function. We analysed several cis-regulatory deletion mutants to investigate their influence on tissue specificity and level of expression. Position independent expression of the gene is lost whenever one of the upstream tissue specific enhancer regions is deleted, although tissue specific expression is usually retained. Deletion of the domain border fragments has no influence on copy number dependency of expression. However, without these regions an increased incidence of ectopic expression is observed. This suggests that the domain border fragments may help to suppress transgene expression in inappropriate tissues. We conclude, that position independent expression of the lysozyme gene is not controlled by a single specific region of the locus but is the result of the concerted action of several tissue specific upstream regulatory DNA elements with the promoter.     

Host specificity among Unionicola spp. (Acari: Unionicolidae) parasitizing freshwater mussels

Water mites of Unionicola spp. are common parasites of freshwater mussels as adults, living on the gills, or mantle and foot of their hosts and using these tissues as sites of oviposition. The present study addresses specialization among North American Unionicola mussel-mites using 2 measures of host specificity: (1) the number of host species used by a species of mite; and (2) a measure that considers the taxonomic distinctness of the hosts utilized by mites, weighted for their prevalence in the different hosts. Results of this study indicate the Unionicola spp. mussel-mites are highly host specific, with most species occurring in association with 1 or 2 species of hosts. If 2 or more host species are utilized, they are typically members of the same genus. These data are consistent with studies examining the dispersal abilities and host recognition behavior for members of the group. When the average values of host specificity for Unionicola subgenera were mapped on a phylogenetic tree for these taxa, a clade comprised of gill mites appeared to be more host specific than a clade consisting of mantle mites. There were, however, no apparent patterns of host specificity within each of the clades. Differences in specificity between the 2 lineages may reflect either a long evolutionary history that gill mites have had with host mussels or the intense competition among gill mites for oviposition sites within unionid mussels, leading to increased host specialization.     

Space competition in the acorn woodpecker: Power struggles in a cooperative breeder

During a 5-year study of the cooperatively living acorn woodpecker Melanerpes formicivorus, I observed 26 irruptions of agonistic behaviour involving large numbers of intruders invading a territory. Evidence from several sources suggests that these invasions are usually contests among potential immigrants for the opportunity to replace a missing group member (hence, ‘power struggles’). Thus, power struggles indicate intense competition among individuals to transfer out of their natal group. Such dispersal is not necessarily excluded by the hypothesis that individuals gain directly by living in cooperative groups through either increased foraging efficiency or better predator defence. However, birds involved in power struggles expend considerable energy and incur considerable risks. These costs are likely to be an acceptable part of dispersal only if cooperative living is a result of birds being forced to remain in groups as a result of resource localization.     

Paradoxical relationships between active transport and global protein distributions in neurons

Neural function depends on continual synthesis and targeted trafficking of intracellular components, including ion channel proteins. Many kinds of ion channels are trafficked over long distances to specific cellular compartments. This raises the question of whether cargo is directed with high specificity during transit or whether cargo is distributed widely and sequestered at specific sites. We addressed this question by experimentally measuring transport and expression densities of Kv4.2, a voltage-gated transient potassium channel that exhibits a specific dendritic expression that increases with distance from the soma and little or no functional expression in axons. In over 500 h of quantitative live imaging, we found substantially higher densities of actively transported Kv4.2 subunits in axons as opposed to dendrites. This paradoxical relationship between functional expression and traffic density supports a model—commonly known as the sushi belt model—in which trafficking specificity is relatively low and active sequestration occurs in compartments where cargo is expressed. In further support of this model, we find that kinetics of active transport differs qualitatively between axons and dendrites, with axons exhibiting strong superdiffusivity, whereas dendritic transport resembles a weakly directed random walk, promoting mixing and opportunity for sequestration. Finally, we use our data to constrain a compartmental reaction-diffusion model that can recapitulate the known Kv4.2 density profile. Together, our results show how nontrivial expression patterns can be maintained over long distances with a relatively simple trafficking mechanism and how the hallmarks of a global trafficking mechanism can be revealed in the kinetics and density of cargo.     

Chaetal arrangement provides no support for a close relationship of Sabellidae and Sabellariidae (Annelida)

Sabellid and sabellariid polychaetes are regarded as sister groups in a number of recent phylogenetic analyses. This is based mainly on a shared specific arrangement of chaetae referred to as chaetal inversion. Remarkably, the uncini have a notopodial position in the abdomen, whereas capillary chaetae occur in the neuropodia in both taxa in contrast to the situation in putative relatives. However, in sabellids uncini and capillary chaetae change their position completely at the border between thorax and abdomen, whereas uncini are missing in the parathorax of Sabellariidae. Due to this difference the significance of the chaetal inversion for systematics has been subject to discussion for years. Serial semithin sections of parapodia of the Sabellidae Sabella pavonina, Branchiomma bombyx, Fabricia stellaris, and of the Sabellariidae Sabellaria alveolata were studied in order to obtain detailed information on their chaetal arrangement and sites of chaetal origin. SEM investigations and computer-aided 3D-reconstructions provide deep insight into the spatial organization of the rami. Though differing externally, the principal chaetal arrangement and the location of the formative sites turned out to be almost identical within the species of Sabellidae. Most chaetae are aligned in straight transverse rows with a dorsal site of origin within neuropodia and a ventral one in notopodia as is common in sedentary polychaetes. Semicircular and spiral arrangements are revealed to be modified transverse rows. Only in thoracic notopodia does an additional dorsocaudal formative site form distinct rows. The chaetal inversion in Sabellidae is additionally characterized by an abrupt change of capillary chaetae and uncini along with a sudden change of the parapodial morphology at the border between thorax and abdomen. All chaetae of S. alveolata are aligned in transverse rows with the same location of the formative sites as in sabellids and other sedentary polychaetes. However, in contrast to sabellids the chaetae are not inverted across a parathoracic abdominal border. Moreover, there is no inversion of the parapodial structure from parathorax to abdomen and the neuropodial chaetal composition changes gradually from parathorax to abdomen. The chaetal arrangement in Sabellariidae thus cannot be described as inverted along the body-axis as in Sabellidae. Evolutionary steps implied by the assumption of an inverted chaetal pattern in a supposed common ancestor are discussed. It is concluded that the specific chaetal arrangement of Sabellidae and Sabellariidae arose independently and therefore provides no support for a sistergroup relationship of sabellids and sabellariids.     

How important are the common factors in psychotherapy? An update

The common factors have a long history in the field of psychotherapy theory, research and practice. To understand the evidence supporting them as important therapeutic elements, the contextual model of psychotherapy is outlined. Then the evidence, primarily from meta‐analyses, is presented for particular common factors, including alliance, empathy, expectations, cultural adaptation, and therapist differences. Then the evidence for four factors related to specificity, including treatment differences, specific ingredients, adherence, and competence, is presented. The evidence supports the conclusion that the common factors are important for producing the benefits of psychotherapy.