Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold‐based and scaffold‐free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS—five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long‐term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick‐freeze/deep‐etch electron microscopy. Both the scaffold‐free and the collagen‐based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell‐derived matrix in the scaffold‐based constructs was significantly lower than that produced by scaffold‐free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell‐derived matrix. The production of highly organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of controlling fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response. Biotechnol. Bioeng. 2012; 109: 2683–2698. © 2012 Wiley Periodicals, Inc.     

Transformation of the oilseed crop Camelina sativa by Agrobacterium-mediated floral dip and simple large-scale screening of transformants

Camelina sativa is a promising under-exploited oilseed crop with potential to become a biofuel feedstock. The ability to transform C. sativa would allow for the rapid introduction of novel traits into this emerging crop. We report the development of an Agrobacterium-based floral dip transformation method, requiring no vacuum-infiltration step, with transformation efficiencies up to 0.8%. C. sativa cultivars Ames 26665, ??Calena?? A3U7761, Ames 1043, and ??Celine?? were tested using Agrobacterium tumefaciens strains GV3101, EHA105, and At503. Use of all strains and cultivars resulted in transformed plants; however, GV3101 was the only Agrobacterium strain and Ames 1043 the only C. sativa cultivar to yield transformed plants under all conditions tested. Progeny analysis revealed that in approximately 78% of the transformed plants, the transgene segregated as a single locus. Furthermore, a high-throughput, filter paper-based PCR method was developed to screen marker-free transformed plants. Together, these methods will allow for easier introduction of new genes into this promising oilseed crop.     

Total evidence, consensus, and bat phylogeny: A distance-based approach

Resolution of the total evidence (i.e., character congruence) versus consensus (i.e., taxonomic congruence) debate has been impeded by (1) a failure to employ validation methods consistently across both tree-building and consensus analyses, (2) the incomparability of methods for constructing as opposed to those for combining trees, and (3) indifference to aspects of trees other than their topologies. We demonstrate a uniform, distance-based approach which allows for comparability among the results of character- and taxonomic-congruence studies, whether or not an identical suite of taxa has been included in all contributing data sets. Our results indicate that total-evidence and consensus trees differ little in topology if branch lengths are taken into account when combining two or more trees. In addition, when character-state data are converted to distances, our method permits their combination with information produced by techniques which generate distances directly. Moreover, treating all data sets or trees as distance matrices avoids the problem that different numbers of characters in contributing studies may confound the conclusions of a total-evidence or consensus analysis. Our protocol is illustrated with an example involving bats, in which the three component studies based on serology, DNA hybridization, and anatomy imply distinct phylogenies. However, the total-evidence and consensus trees support a fourth, somewhat different, topology resolved at all but one node and which conforms closely to the currently accepted higher category classification of Chiroptera.     

Quantifying how acquired interactions with native and invasive insects influence population growth rates of a non-indigenous plant

Non-native species often acquire novel interspecific interactions, which are central to several hypotheses of invasion success, including biotic resistance and invasional meltdown. However, the outcome of these interactions is not often linked with the demographic evidence based on the full life cycle of the species. The Philippine Ground Orchid (Spathoglottis plicata) has invaded Puerto Rico and has acquired both negative and positive interspecific interactions involving the native weevil Stethobaris polita and the invasive red fire ant Solenopsis invicta, respectively. We studied a population in the Rio Abajo Forest, and asked how these interactions affect population demography by using a combination of field, experimental and modelling approaches. Stage-structured matrix population models based on four years of field observations showed that the population of S. plicata is growing at a rate (λ) of 1.05 under natural conditions. When we modified fecundity values based on experimental exclusion of weevils and ants, the control treatment showed a similar λ. Excluding weevils increased λ to 1.20, whereas the exclusion of ants decreased λ to 1.03. When we incorporate demographic and environmental stochasticity in our models, exclusion of invasive red fire ants significantly reduces the orchid abundance over time. Although weevils offer some biotic resistance to S. plicata, these effects do not prevent orchid population growth and expansion. On the other hand, invasive red fire ants have a positive effect on the invasive orchid’s λ, partially supporting the invasional meltdown hypothesis. This study presents a method that allows one to combine opposing mechanisms of species interactions within the same quantitative framework, and the results highlight the importance of considering acquired plant–animal interactions and stochastic processes when evaluating the population growth rates and dynamics of invasive plants.     

Are Megabats Big?

Traditionally, bats (Order Chiroptera) are divided into two suborders, Megachiroptera (“megabats”) and Microchiroptera, and this nomenclature suggests a consistent difference in body size. To test whether megabats are, in fact, significantly larger than other bats, we compared them with respect to average body mass (log transformed), using both conventional and phylogenetic statistics. Because bat phylogeny is controversial, including the position of megabats, we employed several analyses. First, we derived two generic-level topologies for 101 genera, one with megabats as the sister of all other bats (“morphological” tree), the other with megabats as the sister of one specific group of microbats, the Rhinolophoidea (“molecular” tree). Second, we used a recently published “supertree” that allowed us to analyze body mass data for 656 species. In addition, because the way body mass has evolved is generally unknown, we employed several sets of arbitrary branch lengths on both topologies, as well as transformations of the branches intended to mimic particular models of character evolution. Irrespective of the topology or branch lengths used, log body mass showed highly significant phylogenetic signal for both generic and species-level analyses (all P≤ 0.001). Conventional statistics indicated that megabats were indeed larger than other bats (P ? 0.001). Phylogenetic analyses supported this difference only when performed with certain branch lengths, thus demonstrating that careful consideration of the branch lengths used in a comparative analysis can enhance statistical power. A conventional Levene's test indicated that log body mass was more variable in megabats as compared with other bats (P=0.075 for generic-level data set, P ? 0.001 for species-level). A phylogenetic equivalent, which gauges the amount of morphospace occupied (or average minimum rate of evolution) relative to topology and branch lengths specified, indicated no significant difference for the generic analyses, but did indicate a difference for some of the species-level analyses. The ancestral bat is estimated to have been approximately 20–23 g in body mass (95% confidence interval approximately 9–51 g).     

Camping in a Different Tree: Results of Molecular Systematic Studies of Bats Using DNA–DNA Hybridization

We examined taxa from 13 of the 17 chiropteran families, using single-copy DNA hybridization. Five taxa that either represented points of controversy in systematics or were members of problematic families were included in the experiment. The resulting data were used to build phylogenetic trees of 14 and 19 taxa, and by combining this study's data with those from two previous studies, a supertree of 36 taxa was constructed. The trees based on the three different matrices are compared and contrasted, and a phylogenetic hypothesis supporting the association of the rhinolophoid and the pteropodid groups of bats is presented. On the basis of this study, we conclude that the phylogenetically correct placement of the family Nycteridae is in a clade that does not include their putative relatives, the Rhinolophoidea. Our results suggest that the Emballonuridae, while a monophyletic group, are well embedded within the Yangochiroptera, and do not comprise the sister taxon to all other microbats. This study supports earlier DNA-hybridization results with respect to the placement of Mystacinidae within the Noctilionoidea, replicating those earlier findings. Finally, we determine that Miniopterus may well warrant recognition as a family distinct from the Vespertilionidae in which it is usually placed.     

Studies on bacteria-like particles sampled from the stratosphere

Bacteria-like particles recovered from the stratosphere and deposited on cellulose acetate membranes have been analysed to confirm their bacterial nature. One particle appeared to be attached to an inorganic particle apparently by mucoid material typically produced by bacteria. A filamentous structure, morphologically similar to a fungal hypha, was also observed. EDS analysis showed that the particles were all non-mineral and therefore could be biological in nature. However, the composition of several clumps of nanobacteria-sized particles were found, by SIMS analysis, to be inconsistent with that of bacteria. The results show that it is dangerous to assume that bacteria-like particles seen under scanning electron microscopy are necessarily bacteria.     

Base-compositional biases and the bat problem. III. The questions of microchiropteran monophyly.

Using single-copy DNA hybridization, we carried out a whole genome study of 16 bats (from ten families) and five outgroups (two primates and one each dermopteran, scandentian, and marsupial). Three of the bat species represented as many families of Rhinolophoidea, and these always associated with the two representatives of Pteropodidae. All other microchiropterans, however, formed a monophyletic unit displaying interrelationships largely in accord with current opinion. Thus noctilionoids comprised one clade, while vespertilionids, emballonurids, and molossids comprised three others, successively more closely related in that sequence. The unexpected position of rhinolophoids may be due either to the high AT bias they share with pteropodids, or it may be phylogenetically authentic. Reanalysis of the data with varying combinations of the five outgroups does not indicate a rooting problem, and the inclusion of many bat lineages divided at varying levels similarly discounts long branch attraction as an explanation for the pteropodid-rhinolophoid association. If rhinolophoids are indeed specially related to pteropodids, many synapomorphies of Microchiroptera are called into question, not least the unitary evolution of echolocation (although this feature may simply have been lost in pteropodids). Further, a rhinolophoid-pteropodid relationship--if true--has serious implications for the classification of bats. Finally, among the outgroups, an apparent sister-group relation of Dermoptera and Primates suggests that flying lemurs do not represent the ancestors of some or all bats; yet, insofar as gliding of the type implemented in dermopterans is an appropriate model for the evolution of powered mammalian flying, the position of Cynocephalus in our tree indirectly strengthens the argument that true flight could have evolved more than once among bats.     

Isolation and identification of mucinolytic actinomycetes

Biochemical and physiological tests, and 16S rRNA gene sequences, were used to classify nine Actinomycete strains isolated from soil and sand samples in Thailand. These strains were isolated based on their ability to readily degrade mucin glycoproteins. A turbidometric based mucinolytic assay was developed to confirm this. In addition all strains showed significant production of proteases. Phylogenetic analysis of the strains revealed that from the nine isolated Actinomycete strains eight were closely related to Streptomyces species and one was identified as belonging to the genus Kitasatospora. The biochemical and physiological tests performed identified two strain pairs that were similar (with only 3.9% difference observed) and this was in accordance with the phylogenetic results obtained. The remaining strains were distinct from each other, with the soil-isolated strains forming a separate clade to the sand-isolated strains in the inferred phylogenetic trees. The isolated mucinolytic Actinomycete strains will be the subject of further investigations into their proteolytic and glycosidic activity. Mucin degrading enzymes such as these are studied for their potential to be used for the development of a drug delivery system.