Wounds and the origin of blood-feeding in bats

This new theory about the origin of blood-feeding in bats is based on four main premises: (1) that the diets and feeding behaviour of some bats have always varied; (2) that the Miocene mammal fauna of South America included many large (≥ 2 kg body mass) forms; (3) that wounds offered protovampaire bats the opportunity to feed on blood; and (4) that sharp, strong upper incisor teeth were a prerequisite to feeding at wounds. This theory proposes that variation in feeding behaviour led protovampire bats to feed on concentrations of insect larvae at wounds on large mammals, thence to insects and body fluids, and finally to blood. The chiropteran family Phyllostomidae is confined to the New World and its members frequently have robust upper incisor teeth and flexible foraging behaviour. Elsewhere in the world, bats with flexible foraging behaviour lack robust incisors, while those with robust incisors are more conservative in their foraging. The wound theory of the origin of blood-feeding in bats takes into account their anatomy and foraging behaviour as well as the availability of large prey. In contrast to earlier theories that blood-feeding in bats originated from fruit-eaters with robust incisors or through the gleaning of ectoparasites, the wound theory also explains why vampire bats occur only in the New World.     

A retroviral vector for siRNA expression in mammalian cells

Utilization of RNA interference (RNAi) for knockdown of gene expression has become a standard tool for the study of gene function. Short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters are widely used to achieve stable knockdown of gene expression by RNAi. We have constructed a retroviral-based shRNA expression vector, pSiRPG, as a tool for shRNA-based functional genomic studies. This vector is based on a widely used shRNA expression system and was modified to harbor an enhanced green fluorescent protein (EGFP) and a puromycin selection marker. The functionality of the elements in the pSiRPG vector was validated. The H1(TetO₂) promoter in the vector facilitates doxycycline-inducible shRNA expression, which was demonstrated in cells expressing the Tet repressor (TetR). However, we also demonstrated limited efficiency of the inhibition of shRNA expression in an uninduced TetR-expressing cell line. This observation strongly indicates that the H1(TetO₂) promoter, which is used in a wide range of vectors, is not optimal for tightly regulated shRNA expression. Stable repression of the NDRG1 protein level was observed when introducing pSiRPG constructs expressing shRNAs targeting NDRG1 into two mammary epithelial cell lines by retroviral delivery. This vector should therefore facilitate functional studies in breast cell lines that are hard to transfect with conventional plasmid-based methods.     

The origin of the tRNA molecule: implications for the origin of protein synthesis

A model for tRNA molecule origin is discussed. The model postulates that this molecule originated simply by direct duplication (and subsequent evolution) of a gene coding for an RNA hairpin structure, which can thus be hypothesized as the evolutionary precursor of the tRNA molecule. The main properties are defined for these hairpin structures and it is suggested that these structures might have housed, near their 3' end, anticodons that were transferred to the loop of the tRNA anticodon during duplication of the hairpin structures. Moreover, the main characteristics are given for the evolutionary intermediary formed by direct duplication of the hairpin structure, i.e. the double hairpin. The evolutionary stages envisaged by this model for tRNA origin seem to naturally imply some evolutionary transitions through which the origin of protein synthesis passed. Finally, some strong historical evidence is provided to corroborate the model.     

Understanding mammalian glutathione peroxidase 7 in the light of its homologs

The glutathione peroxidase homologs (GPxs) efficiently reduce hydroperoxides using electrons from glutathione (GSH), thioredoxin (Trx), or protein disulfide isomerase (PDI). Trx is preferentially used by the GPxs of the majority of bacteria, invertebrates, plants, and fungi. GSH or PDI, instead, is preferentially used by vertebrate GPxs that operate by Sec or Cys catalysis, respectively. Mammalian GPx7 and GPx8 are unique homologs that contain a peroxidatic Cys (C_P). Being reduced by PDI and located within the endoplasmic reticulum (ER), these enzymes have been involved in oxidative protein folding. Kinetic analysis indicates that oxidation of PDI by recombinant GPx7 occurs at a much faster rate than that of GSH. Nonetheless, activity measurement suggests that, at physiological concentrations, a competition between these two substrates takes place, with the rate of PDI oxidation by GPx7 controlled by the concentration of GSH, whereas the GSSG produced in the competing reaction contributes to the ER redox buffer. A mechanism has been proposed for GPx7 involving two Cys residues, in which an intramolecular disulfide of the C_P is formed with an alleged resolving Cys (C_R) located in the strongly conserved FPCNQ motif (C86 in humans), a noncanonical position in GPxs. Kinetic measurements and comparison with the other thiol peroxidases containing a functional C_R suggest that a resolving function of C86 in the catalytic cycle is very unlikely. We propose that GPx7 is catalytically active as a 1-Cys-GPx, in which C_P both reduces H₂O₂ and oxidizes PDI, and that the C_P-C86 disulfide has instead the role of stabilizing the oxidized peroxidase in the absence of the reducing substrate.     

Tree structure and cavity microclimate: implications for bats and birds

It is widely assumed that tree cavity structure and microclimate affect cavity selection and use in cavity-dwelling bats and birds. Despite the interest in tree structure and microclimate, the relationship between the two has rarely been quantified. Currently available data often comes from artificial structures that may not accurately represent conditions in natural cavities. We collected data on tree cavity structure and microclimate from 45 trees in five cypress-gum swamps in the Coastal Plain of Georgia in the United States in 2008. We used hierarchical linear models to predict cavity microclimate from tree structure and ambient temperature and humidity, and used Aikaike’s information criterion to select the most parsimonious models. We found large differences in microclimate among trees, but tree structure variables explained <28 % of the variation, while ambient conditions explained >80 % of variation common to all trees. We argue that the determinants of microclimate are complex and multidimensional, and therefore cavity microclimate cannot be deduced easily from simple tree structures. Furthermore, we found that daily fluctuations in ambient conditions strongly affect microclimate, indicating that greater weather fluctuations will cause greater differences among tree cavities.     

The impact and implications of climate change for bats

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SNAREs in mammalian sperm: possible implications for fertilization

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.     

Neotropical bats in the canopy: diversity,community structure,and implications for conservation

We compare results of parallel ground and canopy netting of bats (Microchiroptera) in three adjacent forest sites near Belém, Brazil, to document possible differences in vertical distribution of species. We caught 1871 individuals representing 49 species of three families (Emballonuridae, Phyllostomidae, Vespertilionidae). Capture effort, totaling 1955.5 mistnet hours in several cycles over a two-year period, was similar for ground and canopy nets. The canopy rigs yielded more species (n = 41) than the ground nets (n = 35), but both samples were characterized by rank abundance curves with similar shape and with a dominance of frugivores (Phyllostomidae). Nearly half (n = 24) of the species were captured in numbers too small (n < 6) to allow firm classification, but differences in capture frequencies of some of the better-sampled species in high and low nets reveal vertical stratification. Species-specific differences in diet, foraging strategies, roost sites, and sampling bias contribute to this pattern. As a result of the differential use of space among bats, alterations of forest structure are likely to result in changes in structure and function of local bat communities, but our limited knowledge of natural history and ecology of many species limits definition of changes. We see a critical need for further research into the extent to which habitat complexity influences species richness and abundance of bats. This information is especially important in view of the need to develop and apply conservation-oriented programs to maintain biodiversity. A review of recent improvements in techniques for inventorying bats shows that a combination of methods, including mistnetting and acoustic monitoring, is mandatory for such studies.     

Bacterial community dynamics on bats and the implications for pathogen resistance

The bats skin microbiota plays an important role in reducing pathogen infection, including the deadly fungal pathogen Pseudogymnoascus destructans, the causative agent of white-nose syndrome. However, the dynamic of skin bacterial communities response to environmental perturbations remains poorly described. We characterized skin bacterial community over time and space in Rhinolophus ferrumequinum, a species with high resistance to the infection with P. destructans. We collected environmental covariate data to determine what factors influenced changes in community structure. We observed significant temporal and spatial shifts in the skin bacterial community, which was mainly associated with variation in operational taxonomic units. The skin bacterial community differed by the environmental microbial reservoirs and was most influenced by host body condition, bat roosting temperature and geographic distance between sites, but was not influenced by pathogen infection. Furthermore, the skin microbiota was enriched in particular taxa with antifungal abilities, such as Enterococcus, Burkholderia, Flavobacterium, Pseudomonas, Corynebacterium and Rhodococcus. And specific strains of Pseudomonas, Corynebacterium and Rhodococcus even inhibited P. destructans growth. Our findings provide new insights in characterizing the variation in bacterial communities can inform us about the processes of driving community assembly and predict the host's ability to resist or survive pathogen infection.     

MutL homologs in restriction-modification systems and the origin of eukaryotic MORC ATPases

   

The provenance and biochemical roles of eukaryotic MORC proteins have remained poorly understood since the discovery of their prototype MORC1, which is required for meiotic nuclear division in animals. The MORC family contains a combination of a gyrase, histidine kinase, and MutL (GHKL) and S5 domains that together constitute a catalytically active ATPase module. We identify the prokaryotic MORCs and establish that the MORC family belongs to a larger radiation of several families of GHKL proteins (paraMORCs) in prokaryotes. Using contextual information from conserved gene neighborhoods we show that these proteins primarily function in restriction-modification systems, in conjunction with diverse superfamily II DNA helicases and endonucleases. The common ancestor of these GHKL proteins, MutL and topoisomerase ATPase modules appears to have catalyzed structural reorganization of protein complexes and concomitant DNA-superstructure manipulations along with fused or standalone nuclease domains. Furthermore, contextual associations of the prokaryotic MORCs and their relatives suggest that their eukaryotic counterparts are likely to carry out chromatin remodeling by DNA superstructure manipulation in response to epigenetic signals such as histone and DNA methylation.     

The ossified Meckel's cartilage and internal groove in Mesozoic mammaliaforms: implications to origin of the definitive mammalian middle ear

The ossified Meckel's cartilage is described in detail from three adult individuals of two triconodont mammals, Repenomamus and Gobiconodon , which have been discovered in the Lower Cretaceous of Liaoning, China. A possible ossified Meckel's cartilage has also been recognized in the Early Cretaceous symmetrodont Zhangheotherium from Liaoning. The rod-like ossified Meckel's cartilage in Repenomamus bridges the dentary and the ear region of the cranium. Its shape and position are similar to those of Meckel's cartilage in prenatal and in some postnatal extant mammals. The ossified Meckel's cartilage may have functioned as an attachment site for the medial pterygoid muscle. These specimens provide direct evidence for the function of the internal groove which is commonly present in the dentary of early mammals and their relatives. The evidence weakens the hypothesis of multiple origins for the definitive mammalian middle ear. It supports the assumption that a persistent or ossified Meckel's cartilage has been present in adults of the common ancestor of mammals. The new evidence of Repenomamus does not support the model in which brain expansion and negative allometry of the auditory chain are primarily responsible for the detachment of ear ossicles in mammalian ontogeny and evolution. An alternative hypothesis is proposed which does not require brain expansion as the initial factor for the detachment of ear ossicles during mammalian evolution. © 2003 The Linnean Society of London. Zoological Journal of the Linnean Society , 2003, 138 , 431–448.     

Trophic niche partitioning of cryptic species of long-eared bats in Switzerland: implications for conservation

Dietary niche partitioning is postulated to play a major role for the stable coexistence of species within a community, particularly among cryptic species. Molecular markers have recently revealed the existence of a new cryptic species of long-eared bat, Plecotus macrobullaris, in the European Alps. We studied trophic niches as well as seasonal and regional variations of diet in eight colonies of the three Plecotus species occurring in Switzerland. Faeces were collected monthly from individuals returning to roost after foraging. Twenty-one arthropod categories were recognized from the faeces. All three species fed predominantly on Lepidoptera, which made up 41%, 87% and 88% (means across colonies) of the diet composition of P. auritus, P. macrobullaris and P. austriacus, respectively. The occurrence of numerous fragments of both diurnal and flightless insects in the diet of P. auritus (but rarely in the diet of the other two species) indicates that this species mostly gleans prey from substrates. P. austriacus and P. macrobullaris are more typical aerial feeders. The latter two species have narrow trophic niches, whilst P. auritus has a much broader diet. Comparison of intraspecific and interspecific niche overlaps in P. auritus and P. macrobullaris in sympatry suggests dietary niche partitioning between these two species. In contrast, the high similarity of the trophic niches of P. austriacus and P. macrobullaris, associated with a typical parapatric distribution, indicates competitive exclusion. The best conservation measures are preservation and restoration of habitats offering a high abundance of moths, the major prey of the three Plecotus species.     

Purification of retroviral vectors for clinical application: biological implications and technological challenges

For centuries mankind led a difficult battle against viruses, the smallest infectious agents at the surface of the earth. Nowadays it is possible to use viruses for our benefit, both at a prophylactic level in the production of vaccines and at a therapeutic level in the promising field of gene therapy. Retroviruses were discovered at the end of the 19th century and constitute one of the most effective entities for gene transfer and insertion into the genome of mammalian cells. This attractive feature has intensified research in retroviral vectors development and production over the past years, mainly due to the expectations raised by the concept of gene therapy. The demand for high quality retroviral vectors that meet standard requisites from the regulatory agencies (FDA and EMEA) is therefore increasing, as the technology has moved into clinical trials. The development of safer producer cell lines that can be used in large-scale production will result in the production of large quantities of retroviral stocks. Cost-efficient and scalable purification processes are essential for production of injectable-grade preparations to achieve final implementation of these vectors as therapeutics. Several preparative purification steps already established for proteins can certainly be applied to retroviral vectors, in particular membrane filtration and chromatographic methods. Nevertheless, the special properties of these complex products require technological improvement of the existing purification steps and/or development of particular purification steps to increase productivity and throughput, while maintaining biological activity of the final product. This review focuses on downstream process development in relation to the retroviral vectors characteristics and quality assessment of retroviral stocks for intended use in gene therapy.     

Affording larger brains: testing hypotheses of mammalian brain evolution on bats

Several major hypotheses have been proposed to explain how larger brains in mammals, such as those of humans, are afforded in energetic terms. To date, these have been largely tested on primates, with some cross-mammal analysis. We use morphological, ecological, and metabolic data for 313 species of bats to examine the allometry of brain mass and to test key predictions from three of these hypotheses: the direct metabolic constraint, expensive tissue, and maternal energy hypotheses. We confirm that megachiropteran bats (entirely fruit-eating) have larger brains for their body mass than microchiropteran bats (fruit-eating and non-fruit-eating) and fruit-eating species (Megachiroptera and Microchiroptera) have larger brains than non-fruit-eating species (Microchiroptera). Although our analyses demonstrate little or no support for any of the three hypotheses, we show that 95.9% of the variance in brain mass can be explained by the independent effects of gestation length and body mass. This indicates that among bats, the duration of maternal investment plays an important role in the adult brain mass finally obtained. These analyses serve to emphasis the crucial importance of testing the general applicability of macroevolutionary hypotheses (often developed in isolation in one clade) in multiple clades with different evolutionary histories.     

Wing serial homologs and the origin and evolution of the insect wing

The origin and evolution of insect wings has been the subject of extensive debate. The issue has remained controversial largely because of the absence of definitive fossil evidence or direct developmental evidence of homology between wings and a putative wing origin. Recent identification of wing serial homologs (WSHs) has provided researchers with a potential strategy for identifying WSHs in other species. Future comparative developmental analyses between wings and WSHs may clarify the important steps underlying the evolution of insect wings.