Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF·c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSF_M) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSF_M variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSF_M structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSF_M and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSF_M residues critical for M-CSF·c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF·c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.     

Pollinator‐mediated selection on floral size and tube color in Linum pubescens: Can differential behavior and preference in different times of the day maintain dimorphism?

Diversity of flower traits is often proposed as the outcome of selection exerted by pollinators. Positive directional pollinator‐mediated selection on floral size has been widely shown to reduce phenotypic variance. However, the underlying mechanism of maintaining within‐population floral color polymorphism is poorly understood. Divergent selection, mediated by different pollinators or by both mutualists and antagonists, may create and maintain such polymorphism, but it has rarely been shown to result from differential behavior of one pollinator. We tested whether different behaviors of the same pollinators in morning and evening are associated with dimorphic floral trait in Linum pubescens, a Mediterranean annual plant that exhibits variable within‐population frequencies of dark‐ and light‐colored flower tubes. Usia bicolor bee‐flies, the major pollinators of L. pubescens, are mostly feeding in the flower in the morning, while in the evening they are mostly visiting the flowers for mating. In 2 years of studying L. pubescens in a single large population in the Carmel, Israel, we found in one year that dark‐centered flowers received significantly higher fraction of visits in the morning. Fitness was positively affected by number of visits, but no fitness differences were found between tube‐color morphs, suggesting that both morphs have similar pollination success. Using mediation analysis, we found that flower size was under positive directional pollinator‐mediated selection in both years, but pollinator behavior did not explain entirely this selection, which was possibly mediated also by other agents, such as florivores or a‐biotic stresses. While most pollinator‐mediated selection studies show that flower size signals food reward, in L. pubescens, it may also signal for mating place, which may drive positive selection. While flower size found to be under pollinator‐mediated selection in L. pubescens, differential behavior of the pollinators in morning and evening did not seem to explain flower color polymorphism.     

Role of the conserved carboxy-terminal α-helix of Rad6p in ubiquitination and DNA repair

RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C-terminal α-helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin-conjugating enzymes (Ubcs). This specific conservation suggests that the C-terminal a-helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error-free DNA repair in vivo. C-terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C-terminal u-helix is also essential for error-free DNA repair in vivo. Overexpression of Rad18p, a single-stranded DNA-binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C-terminal α-helix mutants to different degrees. This indicates that the C-terminal α-helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild-type capacity for error-free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.     

Late Quaternary reptile extinctions: size matters,insularity dominates

Aim A major Late Quaternary vertebrate extinction event affected mostly large‐bodied ‘megafauna’. This is well documented in both mammals and birds, but evidence of a similar trend in reptiles is scant. We assess the relationship between body size and Late Quaternary extinction in reptiles at the global level. Location Global. Methods We compile a body size database for all 82 reptile species that are known to have gone extinct during the last 50,000 years and compare them with the sizes of 10,090 extant reptile species (97% of known extant diversity). We assess the body size distributions in the major reptile groups: crocodiles, lizards, snakes and turtles, while testing and correcting for a size bias in the fossil record. We examine geographical biases in extinction by contrasting mainland and insular reptile assemblages, and testing for biases within regions and then globally by using geographically weighted models. Results Extinct reptiles were larger than extant ones, but there was considerable variation in extinction size biases among groups. Extinct lizards and turtles were large, extinct crocodiles were small and there was no trend in snakes. Lizard lineages vary in the way their extinction is related to size. Extinctions were particularly prevalent on islands, with 73 of the 82 extinct species being island endemics. Four others occurred in Australia. The fossil record is biased towards large‐bodied reptiles, but extinct lizards were larger than extant ones even after we account for this. Main conclusions Body size played a complex role in the extinction of Late Quaternary reptiles. Larger lizard and turtle species were clearly more affected by extinction mechanisms such as over exploitation and invasive species, resulting in a prevalence of large‐bodied species among extinct taxa. Insularity was by far the strongest correlate of recent reptile extinctions, suggesting that size‐biased extinction mechanisms are amplified in insular environments.     

Leishmania-sandfly interactions: an empirical field study

Phlebotomus papatasi is the sandfly vector of Leishmania major in the Jordan Valley. The objective of this study was to characterize vector-parasite relations in an active zoonotic focus. Seasonality and intensity of promastigote infection rates in female sandflies and the developmental stage of these hosts were established. On 153 trap-nights, 641 female P. papatasi were caught and examined. Of these, 48 (7.4%, range 12.9-4.8%) were infected with L. major promastigotes. Correlating the number of parasites with the gonotrophic age of the vector revealed that most infections initially are light ones, and those that survive in the vector generally prosper and proliferate. Comparing infection rates in parous and gravid flies revealed that similar proportions of gravid and parous flies are found infected. Thus, Leishmania infections do not appear to affect sandfly survival.     

Deletions in processed pseudogenes accumulate faster in rodents than in humans

Summary The relative rates of point nucleotide substitution and accumulation of gap events (deletions and insertions) were calculated for 22 human and 30 rodent processed pseudogenes. Deletion events not only outnumbered insertions (the ratio being 71 and 31 for human and rodent pseudogenes, respectively), but also the total length of deletions was greater than that of insertions. Compared with their functional homologs, human processed pseudogenes were found to be shorter by about 1.2%, and rodent pseudogenes by about 2.3%. DNA loss from processed pseudogenes through deletion is estimated to be at least seven times faster in rodents than in humans. In comparison with the rate of point substitutions, the abridgment of pseudogenes during evolutionary times is a slow process that probably does not retard the rate of growth of the genome due to the proliferation of processed pseudogenes.     

Are island‐like systems biologically similar to islands? A review of the evidence

Islands are geographically defined as land masses completely surrounded by water, and island systems have been used as models for many biogeographic, ecological, and evolutionary theories ever since Darwin's pioneering efforts. However, their biological definition is complex. Over the past few decades these theories have been applied to many study systems that only share some geographic features with island systems. These features include spatial fragmentation, limited area, spatial and temporal isolation from adjacent parts of the system, and low connectivity between different parts within the system, to mention just a few. These systems vary in their form, the matrix that surrounds them, the factors defining their borders, the extent of insularity they impose on the different taxa, and their geological similarity to different types of actual islands. Here, I seek to understand whether such island‐like systems (ILS) function biologically as true islands. In the first part, I describe the wide diversity of ILS suggested in the literature and the variation in the features that define their insularity. In the second part, I review the extent to which the main theories of island biology are applicable to these systems: species–area and species–isolation relationships, community composition, evolutionary radiations, and the extent of endemism and genetic diversity. In the third and final part, I suggest a new conceptual framework within which to classify and study the biology of ILS, as well as practical future research directions. I conclude that the term ‘biological island’ is a multi‐faceted concept, loosely related to its geographical definition. As ILS are often less isolated than true islands, and their biological patterns are only partly similar to those of true islands (and even this is true only for some ILS) the use of the term ‘island’ to describe any isolated habitat is therefore inappropriate.     

Age-dependent insemination success of sterile Mediterranean fruit flies

The Sterile Insect Technique (SIT) is used in many regions worldwide to manage wild populations of the Mediterranean fruit fly (`medfly'), an important pest species. This technique relies on released sterile males outcompeting their wild counterparts in fertilizing ova of wild females. Numerous studies have investigated the ability of sterile males to secure copulations, an essential step toward overall success. Here we progress further along the mating sequence by studying reproductive barriers that may remain ahead of sterile males that manage to secure copulations in field cage experiments and whether ability to pass these barriers is influenced by a male's age, diet and size, or the size of his mate. Amongst those virgin males that succeeded in copulating, both the number of sperm stored by mates and the chances of having any sperm stored at all decreased with age. Sperm tended to be stored asymmetrically between the females' two spermathecae, and this tendency was more apparent when few sperm were stored. In accord with effects of male age on number of sperm stored, sperm of older males were stored more asymmetrically than that of young males. We found no evidence that male size, male diet or female size influenced copula duration, number of sperm stored or allocation of sperm between the female's two spermathecae. The decline in number of sperm stored as males aged was not accompanied by age-dependent changes in copula duration, indicating that copula duration and insemination success are not deterministically linked. We discuss these results in light of their relevance to SIT and the medfly mating system.     

Systemic bud induction and retinoic acid signaling underlie whole body regeneration in the urochordate Botrylloides leachi

Regeneration in adult chordates is confined to a few model cases and terminates in restoration of restricted tissues and organs. Here, we study the unique phenomenon of whole body regeneration (WBR) in the colonial urochordate Botrylloides leachi in which an entire adult zooid is restored from a miniscule blood vessel fragment. In contrast to all other documented cases, regeneration is induced systemically in blood vessels. Multiple buds appear simultaneously in newly established regeneration niches within vasculature fragments, stemming from composites of pluripotent blood cells and terminating in one functional zooid. We found that retinoic acid (RA) regulates diverse developmental aspects in WBR. The homologue of the RA receptor and a retinaldehyde dehydrogenase-related gene were expressed specifically in blood cells within regeneration niches and throughout bud development. The addition of RA inhibitors as well as RNA interference knockdown experiments resulted in WBR arrest and bud malformations. The administration of all-trans RA to blood vessel fragments resulted in doubly accelerated regeneration and multibud formation, leading to restored colonies with multiple zooids. The Botrylloides system differs from known regeneration model systems by several fundamental criteria, including epimorphosis without the formation of blastema and the induction of a “multifocal regeneration niche” system. This is also to our knowledge the first documented case of WBR from circulating blood cells that restores not only the soma, but also the germ line. This unique Botrylloides WBR process could serve as a new in vivo model system for regeneration, suggesting that RA signaling may have had ancestral roles in body restoration events.