Differentiation potential of intestinal mesenchyme and its interaction with epithelial cells: a study using beta-galactosidase-expressing fibroblast lines

Rat intestinal fibroblast lines (F1:G9 and A1:F1) differing in their potential to support intestinal mucosal development were marked with reporter genes to investigate their differentiation potential. The fibroblasts were transfected with plasmids expressing either beta-galactosidase (with or without a nuclear localisation signal) or green fluorescent protein (GFP). Transfection using Tfx50 or Fugene was more efficient than electroporation. The expression of beta-galactosidase was more stable and stronger than GFP. Cells were optimally labelled using the plasmid pL27B-GAL, and sub-clones with a strong and uniform nuclear expression of beta-galactosidase were isolated. These clones expressed beta-galactosidase even after prolonged passage in the absence of selection. The beta-galactosidase tagged lines (F1:G9gal and A1:F1gal) retained the morphological characteristics, viability and differentiation properties of the parental non-transfected lines. In co-culture with a colorectal tumour cell line Caco-2, the F1:G9gal and A1:F1gal cells differed in their morphological organisation but this did not change their expression of smooth muscle alpha-actin.     

Using remote sensing image texture to study habitat use patterns: a case study using the polymorphic white-throated sparrow (Zonotrichia albicollis)

Aim Ecological factors that vary along spatial scales can greatly influence the outcome of evolution. However, often it is difficult for researchers to identify significant ecological variables that might be associated with the geographical distribution of phenotypes and genotypes in nature. In this paper, we use remote sensing image texture analyses to investigate breeding territories of the white‐throated sparrow (Zonotrichia albicollis), a polymorphic passerine species that has been shown to segregate spatially according to morph. Our aim was to examine how an integrative measure of landscape feature, such as image texture, could be used to distinguish subtle differences in habitat use and, presumably, habitat selection. Location A population of white‐throated sparrows located 1.5 miles east of the Cranberry Lake Biological Station in the Adirondack Mountains of St Lawrence County, New York, was used in this study; the study site itself was approximately 1 km². Methods We analysed 27 territories (16 white males; 11 tan males) to determine if image texture can be used to distinguish differences in vegetation and structure in territories of the two colour morphs. First (variance)‐ and second (homogeneity)‐order image texture measurements with three different window sizes were derived using the green, red, and near infrared bands of Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) data. Texture was then placed into t‐tests to determine statistical differences between the territories of the two male morphs. Results Significant differences between the territories of the two morphs were found in both variance and homogeneity texture measures in the red and green ASTER bands in all three window sizes. There were no differences with any near infrared texture measures. Main conclusions Examination of first‐ and second‐order statistical measures indicated that the territories of tan males have significantly more spectral variance than the territories of white males, while white males’ territories are significantly more homogeneous. These findings are consistent with the previous finding of the morphs settling in ‘high’ and ‘low’ neighbour density habitats that differ with respect to the amount of useable area on their borders. Previous study has also suggested that white and tan male territories differ according to overall landscape structure, not vegetation composition. Therefore, we suggest that red and green spectra may be more useful when examining differences in territory structure and possibly territory quality of similar edge‐type species. Our results suggest that image texture is a useful tool for ecological and evolutionary modelling of the distributions of phenotypes/genotypes across landscapes.     

Physical interaction between the strawberry allergen Fra a 1 and an associated partner FaAP: Interaction of Fra a 1 proteins and FaAP

The strawberry fruit allergens Fra a 1.01E, Fra a 1.02 and Fra a 1.03 belong to the group of pathogenesis‐related 10 (PR‐10) proteins and are homologs of the major birch pollen Bet v 1 and apple allergen Mal d 1. Bet v 1 related proteins are the most extensively studied allergens but their physiological function in planta remains elusive. Since Mal d 1‐Associated Protein has been previously identified as interaction partner of Mal d 1 we studied the binding of the orthologous Fra a 1‐Associated Protein (FaAP) to Fra a 1.01E/1.02/1.03. As the C‐terminal sequence of FaAP showed strong auto‐activation activity in yeast 2‐hybrid analysis a novel time resolved DNA‐switching system was successfully applied. Fra a 1.01E, Fra a 1.02, and Fra a 1.03 bind to FaAP with K_D of 4.5 ± 1.1, 15 ± 3, and 11 ± 2 nM, respectively. Fra a 1.01E forms a dimer, whereas Fra a 1.02 and Fra a 1.03 bind as monomer. The results imply that PR‐10 proteins might be integrated into a protein‐interaction network and FaAP binding appears to be essential for the physiological function of the Fra a 1 proteins.     

Further studies related to the scale-up of high cell density Escherichia coli fed-batch fermentations: the additional effect of a changing microenvironment when using aqueous ammonia to control pH

In this work, we report on the further development of the scale-down, two-compartment (STR + PFR) experimental simulation model. For the first time, the effect on high cell density Escherichia coli fed-batch fermentations of a changing microenvironment with respect to all three of the major spatial heterogeneities that may be associated with large-scale processing (pH, glucose, and dissolved oxygen concentration) were studied simultaneously. To achieve this, we used traditional microbiological analyses as well as multiparameter flow cytometry to monitor cell physiological response at the individual cell level. It was demonstrated that for E. coli W3110 under such conditions in a 20 m(3) industrial fed-batch fermentation, the biomass yield is lower and final cell viability is higher than those found in the equivalent well-mixed, 5L laboratory scale case. However, by using a combination of the well-mixed 5L stirred tank reactor (STR) with a suitable plug flow reactor (PFR) to mimic the changing microenvironment at the large scale, very similar results to those in the 20 m(3) reactor may be obtained. The similarity is greatest when the PFR is operated with a mean residence time of 50 sec with a low level of dO(2) and a high glucose concentration with either a pH of 7 throughout the two reactors or with pH controlled at 7 in the STR by addition into the PFR where the pH is > 7.     

Nutrigerontology: why we need a new scientific discipline to develop diets and guidelines to reduce the risk of aging‐related diseases

Many diets and nutritional advice are circulating, often based on short‐ or medium‐term clinical trials and primary outcomes, like changes in LDL cholesterol or weight. It remains difficult to assess which dietary interventions can be effective in the long term to reduce the risk of aging‐related disease and increase the (healthy) lifespan. At the same time, the scientific discipline that studies the aging process has identified some important nutrient‐sensing pathways that modulate the aging process, such as the mTOR and the insulin/insulin‐like growth factor signaling pathway. A thorough understanding of the aging process can help assessing the efficacy of dietary interventions aimed at reducing the risk of aging‐related diseases. To come to these insights, a synthesis of biogerontological, nutritional, and medical knowledge is needed, which can be framed in a new discipline called ‘nutrigerontology’.     

Comparison of the anatomical morphology of cervical vertebrae between humans and macaques: related to a spinal cord injury model

Non-human primates are most suitable for generating cervical experimental models, and it is necessary to study the anatomy of the cervical spine in non-human primates when generating the models. The purpose of this study was to provide the anatomical parameters of the cervical spine and spinal cord in long-tailed macaques (Macaca fascicularis) as a basis for cervical spine-related experimental studies. Cervical spine specimens from 8 male adult subjects were scanned by micro-computed tomography, and an additional 10 live male subjects were scanned by magnetic resonance imaging. The measurements and parameters from them were compared to those of 12 male adult human subjects. Additionally, 10 live male subjects were scanned by magnetic resonance imaging, and the width and depth of the spinal cord and spinal canal and the thickness of the anterior and posterior cerebrospinal fluid were measured and compared to the relevant parameters of 10 male adult human subjects. The tendency of cervical parameters to change with segmental changes was similar between species. The vertebral body, spinal canal, and spinal cord were significantly flatter in the human subjects than in the long-tailed macaques. The cerebrospinal fluid space in the long-tailed macaques was smaller than that in the human subjects. The anatomical features of the cervical vertebrae of long-tailed macaques provide a reference for establishing a preclinical model of cervical spinal cord injury.     

An approach to the production of helminth antigens in vitro: The formation of hybrid cells between Fasciola hepatic a and a rat fibroblast cell line

Using polyethylene glycol, hybrid cells were formed between rat fibroblasts lacking the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT, B.C. 2.4.2.8) and cells of the liver fluke Fasciola hepatica. The hybrid cells survived in a medium containing hypoxanthine, aminopterin and thymidine (HAT) indicating that the enzyme deficiency of the parental rat cells had been corrected. Isoelectric focusing in agarose gels showed that the HGPRT activity in the hybrids was of F. hepatica rather than rat origin. F. hepatica chromosomes could not be identified with certainty in hybrids; and fluke antigens, other than HGPRT, could not be detected in them or in culture medium in which they had grown.     

Simulating the effects of climate change on the distribution of an invasive plant,using a high resolution,local scale,mechanistic approach: challenges and insights

The growing economic and ecological damage associated with biological invasions, which will likely be exacerbated by climate change, necessitates improved projections of invasive spread. Generally, potential changes in species distribution are investigated using climate envelope models; however, the reliability of such models has been questioned and they are not suitable for use at local scales. At this scale, mechanistic models are more appropriate. This paper discusses some key requirements for mechanistic models and utilises a newly developed model (PSS[gt]) that incorporates the influence of habitat type and related features (e.g., roads and rivers), as well as demographic processes and propagule dispersal dynamics, to model climate induced changes in the distribution of an invasive plant (Gunnera tinctoria) at a local scale. A new methodology is introduced, dynamic baseline benchmarking, which distinguishes climate‐induced alterations in species distributions from other potential drivers of change. Using this approach, it was concluded that climate change, based on IPCC and C4i projections, has the potential to increase the spread‐rate and intensity of G. tinctoria invasions. Increases in the number of individuals were primarily due to intensification of invasion in areas already invaded or in areas projected to be invaded in the dynamic baseline scenario. Temperature had the largest influence on changes in plant distributions. Water availability also had a large influence and introduced the most uncertainty in the projections. Additionally, due to the difficulties of parameterising models such as this, the process has been streamlined by utilising methods for estimating unknown variables and selecting only essential parameters.     

A novel yeast two-hybrid approach to identify CDPK substrates: characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein

Calcium-dependent protein kinases (CDPKs) are sensor-transducer proteins capable of decoding calcium signals in diverse phosphorylation-dependent calcium signaling networks in plants and some protists. Using a novel yeast two-hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11-interacting protein. AtDi19 is a member of a small family of stress-induced genes. The interaction was confirmed using pull-down assays with in vitro translated AtCPK11 and GST-AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca(2+)-dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase-substrate interactions without affecting specificity.     

Simulation of greenhouse gases following land‐use change to bioenergy crops using the ECOSSE model: a comparison between site measurements and model predictions

This article evaluates the suitability of the ECOSSE model to estimate soil greenhouse gas (GHG) fluxes from short rotation coppice willow (SRC‐Willow), short rotation forestry (SRF‐Scots Pine) and Miscanthus after land‐use change from conventional systems (grassland and arable). We simulate heterotrophic respiration (R_h), nitrous oxide (N₂O) and methane (CH₄) fluxes at four paired sites in the UK and compare them to estimates of R_h derived from the ecosystem respiration estimated from eddy covariance (EC) and R_h estimated from chamber (IRGA) measurements, as well as direct measurements of N₂O and CH₄ fluxes. Significant association between modelled and EC‐derived R_h was found under Miscanthus, with correlation coefficient (r) ranging between 0.54 and 0.70. Association between IRGA‐derived R_h and modelled outputs was statistically significant at the Aberystwyth site (r = 0.64), but not significant at the Lincolnshire site (r = 0.29). At all SRC‐Willow sites, significant association was found between modelled and measurement‐derived R_h (0.44 ≤ r ≤ 0.77); significant error was found only for the EC‐derived R_h at the Lincolnshire site. Significant association and no significant error were also found for SRF‐Scots Pine and perennial grass. For the arable fields, the modelled CO₂ correlated well just with the IRGA‐derived R_h at one site (r = 0.75). No bias in the model was found at any site, regardless of the measurement type used for the model evaluation. Across all land uses, fluxes of CH₄ and N₂O were shown to represent a small proportion of the total GHG balance; these fluxes have been modelled adequately on a monthly time‐step. This study provides confidence in using ECOSSE for predicting the impacts of future land use on GHG balance, at site level as well as at national level.     

Using molecular markers with high mutation rates to obtain estimates of relative population size and to distinguish the effects of gene flow and mutation: a demonstration using data from endemic Mauritian skinks

We propose a method of analysing genetic data to obtain separate estimates of the size (N(p)) and migration rate (m(p)) for the sampled populations, without precise prior knowledge of mutation rates at each locus ( micro(L)). The effects of migration and mutation can be distinguished because high migration has the effect of reducing genetic differentiation across all loci, whereas a high mutation rate will only affect the locus in question. The method also takes account of any differences between the spectra of immigrant alleles and of new mutant alleles. If the genetic data come from a range of population sizes, and the loci have a range of mutation rates, it is possible to estimate the relative sizes of the different N(p) values, and likewise the m(p) and the micro(L). Microsatellite loci may also be particularly appropriate because loci with a high mutation rate can reach mutation-drift-migration equilibrium more quickly, and because the spectra of mutants arriving in a population can be particularly distinct from the immigrants. We demonstrate this principle using a microsatellite data set from Mauritian skinks. The method identifies low gene flow between a putative new species and populations of its sister species, whereas the differentiation of two other populations is attributed to small population size. These distinct interpretations were not readily apparent from conventional measures of genetic differentiation and gene diversity. When the method is evaluated using simulated data sets, it correctly distinguishes low gene flow from small population size. Loci that are not at mutation-migration-drift equilibrium can distort the parameter estimates slightly. We discuss strategies for detecting and overcoming this effect.     

With eyes wide open: a revision of species within and closely related to the Pocillopora damicornis species complex (Scleractinia; Pocilloporidae) using morphology and genetics

Molecular studies have been instrumental for refining species boundaries in the coral genus Pocillopora and revealing hidden species diversity within the extensively studied global species Pocillopora damicornis. Here we formally revise the taxonomic status of species closely related to and within the P. damicornis species complex, taking into account both genetic evidence and new data on morphometrics, including fine‐scale corallite and coenosteum structure. We found that mitochondrial molecular phylogenies are congruent with groups based on gross‐morphology, therefore reflecting species‐level differentiation. However, high levels of gross morphological plasticity and shared morphological characteristics mask clear separation for some groups. Fine‐scale morphological variation, particularly the shape and type of columella, was useful for differentiating between clades and provides an excellent signature of the evolutionary relationships among genetic lineages. As introgressive hybridization and incomplete lineage sorting complicate the delineation of species within the genus on the basis of a single species concept, the Unified Species Concept may represent a suitable approach in revising Pocillopora taxonomy. Eight species are herein described (P. damicornis, P. acuta, P. aliciae, P. verrucosa, P. meandrina, P. eydouxi, P. cf. brevicornis), including a novel taxon – P ocillopora bairdi sp. nov. (Schmidt‐Roach, this study). Citation synonyms and type materials are presented. © 2014 The Linnean Society of London     

The easy road to genome‐wide medium density SNP screening in a non‐model species: development and application of a 10 K SNP‐chip for the house sparrow (Passer domesticus)

With the advent of next generation sequencing, new avenues have opened to study genomics in wild populations of non‐model species. Here, we describe a successful approach to a genome‐wide medium density Single Nucleotide Polymorphism (SNP) panel in a non‐model species, the house sparrow (Passer domesticus), through the development of a 10 K Illumina iSelect HD BeadChip. Genomic DNA and cDNA derived from six individuals were sequenced on a 454 GS FLX system and generated a total of 1.2 million sequences, in which SNPs were detected. As no reference genome exists for the house sparrow, we used the zebra finch (Taeniopygia guttata) reference genome to determine the most likely position of each SNP. The 10 000 SNPs on the SNP‐chip were selected to be distributed evenly across 31 chromosomes, giving on average one SNP per 100 000 bp. The SNP‐chip was screened across 1968 individual house sparrows from four island populations. Of the original 10 000 SNPs, 7413 were found to be variable, and 99% of these SNPs were successfully called in at least 93% of all individuals. We used the SNP‐chip to demonstrate the ability of such genome‐wide marker data to detect population sub‐division, and compared these results to similar analyses using microsatellites. The SNP‐chip will be used to map Quantitative Trait Loci (QTL) for fitness‐related phenotypic traits in natural populations.     

Reversible binding interactions between the tryptophan enantiomers and albumins of different animal species as determined by novel high performance liquid chromatographic methods: an attempt to localize the D- and L-tryptophan binding sites on the human serum albumin polypeptide chain by using protein fragments

The stereoselectivity of the reversible binding interactions between the D- and L-tryptophan enantiomers and serum albumins of different animal species and fragments of human serum albumin (HSA) was investigated by applying three novel high performance liquid chromatographic (HPLC) arrangements. The separations were performed by means of (1) an achiral (diol-bond), (2) a chiral (bovine serum albumin-bond) silica gel sorbent, and (3) a column switching technique which uses both the diol- and HSA-bond HPLC stationary phases. A polarimetric detector and/or an ultraviolet (UV) spectrophotometer were used to monitor the separation process. HPLC arrangement 3 allowed the evaluation of enantioselective binding for D- and L-tryptophan to different albumins and albumin fragments. At present, column switching can be considered the technique of the broadest applicability for investigating the reversible binding interactions between a protein and drug enantiomers. Chirality 9:373–379, 1997. © 1997 Wiley-Liss, Inc.