A functional high‐content miRNA screen identifies miR‐30 family to boost recombinant protein production in CHO cells

The steady improvement of mammalian cell factories for the production of biopharmaceuticals is a key challenge for the biotechnology community. Recently, small regulatory microRNAs (miRNAs) were identified as novel targets for optimizing Chinese hamster ovary (CHO) production cells as they do not add any translational burden to the cell while being capable of regulating entire physiological pathways. The aim of the present study was to elucidate miRNA function in a recombinant CHO‐SEAP cell line by means of a genome‐wide high‐content miRNA screen. This screen revealed that out of the 1, 139 miRNAs examined, 21% of the miRNAs enhanced cell‐specific SEAP productivity mainly resulting in elevated volumetric yields, while cell proliferation was accelerated by 5% of the miRNAs. Conversely, cell death was diminished by 13% (apoptosis) or 4% (necrosis) of all transfected miRNAs. Besides these large number of identified target miRNAs, the outcome of our studies suggest that the entire miR‐30 family substantially improves bioprocess performance of CHO cells. Stable miR‐30 over expressing cells outperformed parental cells by increasing SEAP productivity or maximum cell density of approximately twofold. Our results highlight the application of miRNAs as powerful tools for CHO cell engineering, identified the miR‐30 family as a critical component of cell proliferation, and support the notion that miRNAs are powerful determinants of cell viability.     

A dynamic global vegetation model for use with climate models: concepts and description of simulated vegetation dynamics

Changes in vegetation structure and biogeography due to climate change feedback to alter climate by changing fluxes of energy, moisture, and momentum between land and atmosphere. While the current class of land process models used with climate models parameterizes these fluxes in detail, these models prescribe surface vegetation and leaf area from data sets. In this paper, we describe an approach in which ecological concepts from a global vegetation dynamics model are added to the land component of a climate model to grow plants interactively. The vegetation dynamics model is the Lund–Potsdam–Jena (LPJ) dynamic global vegetation model. The land model is the National Center for Atmospheric Research (NCAR) Land Surface Model (LSM). Vegetation is defined in terms of plant functional types. Each plant functional type is represented by an individual plant with the average biomass, crown area, height, and stem diameter (trees only) of its population, by the number of individuals in the population, and by the fractional cover in the grid cell. Three time‐scales (minutes, days, and years) govern the processes. Energy fluxes, the hydrologic cycle, and carbon assimilation, core processes in LSM, occur at a 20 min time step. Instantaneous net assimilated carbon is accumulated annually to update vegetation once a year. This is carried out with the addition of establishment, resource competition, growth, mortality, and fire parameterizations from LPJ. The leaf area index is updated daily based on prevailing environmental conditions, but the maximum value depends on the annual vegetation dynamics. The coupling approach is successful. The model simulates global biogeography, net primary production, and dynamics of tundra, boreal forest, northern hardwood forest, tropical rainforest, and savanna ecosystems, which are consistent with observations. This suggests that the model can be used with a climate model to study biogeophysical feedbacks in the climate system related to vegetation dynamics.     

Density and hydration of fresh and fixed human skeletal muscle

The maximum tetanic tension of skeletal muscle (P(0)) is often estimated based on calculation of physiological cross-sectional area (PCSA). PCSA depends on muscle volume, pennation angle, and fiber length. Studies documenting PCSA in fixed human muscles usually compute muscle volume by dividing muscle mass by density. These studies use a density value of 1.0597 g/cm(3), which was originally based on unfixed rabbit and canine muscle tissue. Due to the dehydration effects of different fixation methods, the variable hydration that occurs when fixed tissue is stored in buffered saline, and the potential for species-specific muscle density, this value may be incorrect and an accurate value for fixed human muscle density is needed. To obtain an accurate density and water content values, 4% formaldehyde-fixed (n=54) and 37% formaldehyde-fixed (n=54) cadaveric human muscle samples were divided into 6 groups (0, 6, 12, 18, 24, or 30 h) for hydration in phosphate buffered saline (PBS). Measurements of volume, water content, and mass were made enabling calculation of muscle density. Additionally, water content was measured in living muscle (n=4) to determine the appropriate hydration time in PBS. Comparisons among groups demonstrated a significant increase in muscle water content and muscle volume over time, reaching living tissue levels after 24h, but, interestingly, the hydration process did not affect muscle density. These data yield a density value (mean+/-SE) of 1.112+/-0.006 g/cm(3) in 4% formaldehyde-fixed muscle and 1.055+/-0.006 g/cm(3) in 37% formaldehyde-fixed muscle. These results indicate that the use of inappropriate hydration times or density values can produce PCSA errors of 5-10%.     

Molecular Weight Dependence of Exciton Diffusion in Poly(3‐hexylthiophene)

A joint experimental and theoretical study of singlet exciton diffusion in spin‐coated poly(3‐hexylthiophene) (P3HT) films and its dependence on molecular weight is presented. The results show that exciton diffusion is fast along the co‐facial π–π aggregates of polymer chromophores and about 100 times slower in the lateral direction between aggregates. Exciton hopping between aggregates is found to show a subtle dependence on interchain coupling, aggregate size, and Boltzmann statistics. Additionally, a clear correlation is observed between the effective exciton diffusion coefficient, the degree of aggregation of chromophores, and exciton delocalization along the polymer chain, which suggests that exciton diffusion length can be enhanced by tailored synthesis and processing conditions.     

Assessing SSU rDNA Barcodes in Foraminifera: A Case Study using Bolivina quadrata

The Small Subunit Ribosomal RNA gene (SSU rDNA) is a widely used tool to reconstruct phylogenetic relationships among foraminiferal species. Recently, the highly variable regions of this gene have been proposed as DNA barcodes to identify foraminiferal species. However, the resolution of these barcodes has not been well established, yet. In this study, we evaluate four SSU rDNA hypervariable regions (37/f, 41/f, 43/e, and 45/e) as DNA barcodes to distinguish among species of the genus Bolivina, with particular emphasis on Bolivina quadrata for which ten new sequences ( KY468817 – KY468826 ) were obtained during this study. Our analyses show that a single SSU rDNA hypervariable sequence is insufficient to resolve all Bolivina species and that some regions (37/f and 41/f) are more useful than others (43/e and 45/e) to distinguish among closely related species. In addition, polymorphism analyses reveal a high degree of variability. In the context of barcoding studies, these results emphasize the need to assess the range of intraspecific variability of DNA barcodes prior to their application to identify foraminiferal species in environmental samples; our results also highlight the possibility that a longer SSU rDNA region might be required to distinguish among species belonging to the same taxonomic group (i.e. genus).     

Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes.     

The earthworm genus Pleionogaster (Clitellata: Megascolecidae) in southern Luzon, Philippines

An earthworm biodiversity survey of the Philippines has yielded 14 new species of the perichaetine megascolecid genus Pleionogaster, previously known from only a few species from scattered Philippine locations. Bicol, the southern peninsula of Luzon, has intact forests on several isolated volcanic peaks and other remote areas. Collections made in these forests yielded the following new species, here presented by type location: Mt. Malinao, Pleionogaster albayensis, P. bicolensis, P. castilloi, P. malinaoensis, P. tiwiensis; Mt. Isarog, P. ffitchae, P. isarogensis; Mt. Bulusan, P. bulusanensis, P. hongi, P. sorsogonensis; Catanduanes Island, P. nautsae, P. viracensis; Caramoan Peninsula, P. caramoanensis, P. nillosae. Most of the species were found only in the neighborhood of the type locality, but P. bicolensis occurs in two locations in northern Bicol. Intraspecific variation in P. castilloi was observed between northern and southern flanks of Mt. Malinao. The importance of several previously overlooked Pleionogaster traits is demonstrated by their homogeneity within species reported here.Full article published online at http://www.senckenberg.de/odes/06-08.htm.     

Using ecological theory to plan restorations of the endangered Beach jacquemontia (Convolvulaceae) in fragmented habitats

Population viability and metapopulation theory and models are heuristic tools that can be used to plan restorations and assess their success. Using examples from South Florida, USA, we review background information and ongoing reintroduction experiments with the federally endangered coastal perennial vine, beach jacquemontia, Jacquemontia reclinata (Convolvulaceae). All known wild populations are declining in isolated habitat fragments varying in size, occupied area, and degree of isolation. Eleven reintroduction sites ranging in size from 422 to 4800 m² within the extant species’ range have been identified that have characteristics suitable for J. reclinata introductions and have land managers amenable to restoration efforts. Previous RAPD analysis indicated that genetic diversity of natural populations was positively correlated with population size; the two largest populations had the highest genetic diversity and the smallest populations had relatively low genetic diversity. Despite habitat fragmentation and large distances between some populations, migration rates were very high among populations (m=4.05). Experimental crosses indicated the species has a mixed mating system. From 2001 to 2005, we have introduced 935 J. reclinata in seven experiments in five locations using plants propagated ex situ at Fairchild Tropical Botanic Garden. Reintroductions have dramatically increased the number of plants in the wild by 72%. Survival from the time of transplant to 2005 ranged from 2% to 97%, was not significantly correlated with metapopulation parameters, such as, founding population size, patch size, or connectivity to extant populations. Reintroduced plants are contributing seed and pollen to the wild populations, but no recruited seedlings have yet been observed. Although it may take decades before we can consider the reintroduced populations to be self-sustainable, we argue that planning restorations for rare species based on predictions from ecological theory is advisable to allow a higher probability of success.     

First experiences with free‐ranging giant day geckos (Phelsuma madagascariensis grandis,Gray 1870) in the masoala rainforest exhibit in Zurich Zoo,Switzerland

Sixty giant day geckos (Phelsuma madagascariensis grandis) have been introduced into the Masoala rainforest exhibit of Zurich Zoo. Eighteen geckos were monitored during a 3‐month study using the telemetry method. In general, the geckos were able to adapt well under these semi‐natural conditions of the exhibit. The geckos accessed to nearly all locations in the exhibit; some of the animals remained stationary, whereas others covered dozens of meters within a week. They preferred four species of plants as well as the artificial structures supporting the walls and roof of the exhibit, and displayed natural behaviors. Animals were observed feeding on all kind of arthropods and nectar substances, and also on food items dropped from feeding stations for other species. Deliberate feeding targeted at the geckos was finally reduced to a calcium‐rich squash along the visitor's main path. Reproduction was observed frequently within the exhibit. These findings suggest that the geckos will be able to build up a self‐sustaining population and contribute to a nearly‐authentic experience for zoo visitors. Zoo Biol 0:1–7, 2006. © 2006 Wiley‐Liss, Inc.     

Campylobacter infection of broiler chickens in a free-range environment

Campylobacter jejuni is the most common cause of bacterial gastroenteritis worldwide, with contaminated chicken meat considered to represent a major source of human infection. Biosecurity measures can reduce C. jejuni shedding rates of housed chickens, but the increasing popularity of free-range and organic meat raises the question of whether the welfare benefits of extensive production are compatible with food safety. The widespread assumption that the free-range environment contaminates extensively reared chickens has not been rigorously tested. A year-long survey of 64 free-range broiler flocks reared on two sites in Oxfordshire, UK, combining high-resolution genotyping with behavioural and environmental observations revealed: (i) no evidence of colonization of succeeding flocks by the C. jejuni genotypes shed by preceding flocks, (ii) a high degree of similarity between C. jejuni genotypes from both farm sites, (iii) no association of ranging behaviour with likelihood of Campylobacter shedding, and (iv) higher genetic differentiation between C. jejuni populations from chickens and wild birds on the same farm than between the chicken samples, human disease isolates from the same region and national samples of C. jejuni from chicken meat.