Introducing the concept of biocatalysis in the classroom: The conversion of cholesterol to provitamin D 3

Biocatalysis is a fundamental concept in biotechnology. The topic integrates knowledge of several disciplines; therefore, it was included in the course “design and optimization of biological systems” which is offered in the biochemistry curricula. We selected the ciliate tetrahymena as an example of a eukaryotic system with potential for the biotransformation of sterol metabolites of industrial interest; in particular, we focused on the conversion of cholesterol to provitamin D3. The students work with wild type and recombinant strains and learn how sterol pathways could be modified to obtain diverse sterol moieties. During the course the students identify and measure the concentration of sterols. They also search for related genes by bioinformatic analysis. Additionally, the students compare biotransformation rates, growing the ciliate in plate and in a bioreactor. Finally, they use fluorescence microscopy to localize an enzyme involved in biotransformation. The last day each team makes an oral presentation, explaining the results obtained and responds to a series of key questions posed by the teachers, which determine the final mark. In our experience, this course enables undergraduate students to become acquainted with the principles of biocatalysis as well as with standard and modern techniques, through a simple and robust laboratory exercise, using a biological system for the conversion of valuable pharmaceutical moieties.Fil: De Luca, Belén M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gonzalez, Rodrigo Horacio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Nusblat, Alejandro David. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

"Going back to our roots": second generation biocomputing

Researchers in the field of biocomputing have, for many years, successfully "harvested and exploited" the natural world for inspiration in developing systems that are robust, adaptable and capable of generating novel and even "creative" solutions to human-defined problems. However, in this position paper we argue that the time has now come for a reassessment of how we exploit biology to generate new computational systems. Previous solutions (the "first generation" of biocomputing techniques), whilst reasonably effective, are crude analogues of actual biological systems. We believe that a new, inherently inter-disciplinary approach is needed for the development of the emerging "second generation" of bio-inspired methods. This new modus operandi will require much closer interaction between the engineering and life sciences communities, as well as a bidirectional flow of concepts, applications and expertise. We support our argument by examining, in this new light, three existing areas of biocomputing (genetic programming, artificial immune systems and evolvable hardware), as well as an emerging area (natural genetic engineering) which may provide useful pointers as to the way forward.Comment: Submitted to the International Journal of Unconventional Computin

Microzooplankton community structure investigated with imaging flow cytometry and automated live-cell staining

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Marine Ecology Progress Series 550 (2016): 65-81, doi:10.3354/meps11687.Protozoa play important roles in grazing and nutrient recycling, but quantifying these roles has been hindered by difficulties in collecting, culturing, and observing these often-delicate cells. During long-term deployments at the Martha’s Vineyard Coastal Observatory (Massachusetts, USA), Imaging FlowCytobot (IFCB) has been shown to be useful for studying live cells in situ without the need to culture or preserve. IFCB records images of cells with chlorophyll fluorescence above a trigger threshold, so to date taxonomically resolved analysis of protozoa has presumably been limited to mixotrophs and herbivores which have eaten recently. To overcome this limitation, we have coupled a broad-application ‘live cell’ fluorescent stain with a modified IFCB so that protozoa which do not contain chlorophyll (such as consumers of unpigmented bacteria and other heterotrophs) can also be recorded. Staining IFCB (IFCB-S) revealed higher abundances of grazers than the original IFCB, as well as some cell types not previously detected. Feeding habits of certain morphotypes could be inferred from their fluorescence properties: grazers with stain fluorescence but without chlorophyll cannot be mixotrophs, but could be either starving or feeding on heterotrophs. Comparisons between cell counts for IFCB-S and manual light microscopy of Lugol’s stained samples showed consistently similar or higher counts from IFCB-S. We show how automated classification through the extraction of image features and application of a machine-learning algorithm can be used to evaluate the large high-resolution data sets collected by IFCBs; the results reveal varying seasonal patterns in abundance among groups of protists.This research was supported in part by NSF (grants OCE-1130140, OCE-1434440), NASA (grants NNX11AF07G and NNX13AC98G), the Gordon and Betty Moore Foundation (grants 934 and 2649), and the Woods Hole Oceanographic Institution’s Innovative Technology Program

Further Open Problems in Membrane Computing

A series of open problems and research topics in membrane com- puting are pointed out, most of them suggested by recent developments in this area. Many of these problems have several facets and branchings, and further facets and branchings can surely be found after addressing them in a more careful manner

Two Refinements of the Template-Guided DNA Recombination Model of Ciliate Computing

To solve the mystery of the intricate gene unscrambling mechanism in ciliates, various theoretical models for this process have been proposed from the point of view of computation. Two main models are the reversible guided recombination system by Kari and Landweber and the template-guided recombination (TGR) system by Prescott, Ehrenfeucht and Rozenberg, based on two categories of DNA recombination: the pointer guided and the template directed recombination respectively. The latter model has been generalized by Daley and McQuillan. In this thesis, we propose a new approach to generate regular languages using the iterated TGR system with a finite initial language and a finite set of templates, that reduces the size of the template language and the alphabet compared to that of the Daley-McQuillan model. To achieve computational completeness using only finite components we also propose an extension of the contextual template-guided recombination system (CTGR system) by Daley and McQuillan, by adding an extra control called permitting contexts on the usage of templates. Then we prove that our proposed system, the CTGR system using permitting contexts, has the capability to characterize the family of recursively enumerable languages using a finite initial language and a finite set of templates. Lastly, we present a comparison and analysis of the computational power of the reversible guided recombination system and the TGR system. Keywords: ciliates, gene unscrambling, in vivo computing, DNA computing, cellular computing, reversible guided recombination, template-guided recombination

Evolutionary distances in the twilight zone -- a rational kernel approach

Phylogenetic tree reconstruction is traditionally based on multiple sequence alignments (MSAs) and heavily depends on the validity of this information bottleneck. With increasing sequence divergence, the quality of MSAs decays quickly. Alignment-free methods, on the other hand, are based on abstract string comparisons and avoid potential alignment problems. However, in general they are not biologically motivated and ignore our knowledge about the evolution of sequences. Thus, it is still a major open question how to define an evolutionary distance metric between divergent sequences that makes use of indel information and known substitution models without the need for a multiple alignment. Here we propose a new evolutionary distance metric to close this gap. It uses finite-state transducers to create a biologically motivated similarity score which models substitutions and indels, and does not depend on a multiple sequence alignment. The sequence similarity score is defined in analogy to pairwise alignments and additionally has the positive semi-definite property. We describe its derivation and show in simulation studies and real-world examples that it is more accurate in reconstructing phylogenies than competing methods. The result is a new and accurate way of determining evolutionary distances in and beyond the twilight zone of sequence alignments that is suitable for large datasets.Comment: to appear in PLoS ON

Non-enclosure methods for non-suspended microalgae cultivation: literature review and research needs

Microalgae are getting more interests from industry and science communities. Applications of these small, unicellular microorganisms are countless: from fourth generation biofuels, through fish feed to pharmaceuticals. Ordinary methods of cultivation may be associated with many problems such as high costs, high energy consumption, and low product yield. It is difficult to control contaminations in open ponds while photobioreactors are mainly at laboratory scale and expensive to scale-up. Scientists are investigating various methods of microalgae cultivation and processing to overcome those problems. One of the novel approaches is the non-suspended method for microalgae culturing, where microalgae are grown on attached surfaces. Growing microalgae on surfaces is an attractive option and showing promising results. In comparison with ordinary suspended photobioreactors, the attached systems offer higher biomass yields, easy to scale-up with better light distribution within the reactor and better control of contamination. Moreover, the consumption of water can be drastically reduced. So far, there is not enough research for this method. Limited studies have been reported on enclosure mode of this approach with algae encapsulation into matrix. It is found that this mode would be difficult to scale up due to high costs of the enclosure material and difficulty of separating microalgae from matrix. Non-enclosure mode is more promising way of non-suspended cultivation. So far, no work has been carried out to conduct non-suspended culturing with the use of aeroterrestrial microalgae. They are species growing on the surfaces at highly humid environments. Using them in attached cultivation systems could potentially lower the water consumption to minimum. Studies have shown that the biomass of lower water content can be produced if compared to non-suspended cultivation methods. In addition, mechanization of the cultivation and harvesting processes would be less complex, as the product will not be immersed in the liquid. There would be no need for glass reactors, as lights can be placed in the spaces between surfaces. The light distribution is predicted to be the highest among all existing methods, as there would be no free floating particles absorbing and reflecting light. It will only need humid conditions, rich in CO2 between attachment surfaces. To evaluate potential advantages for non-suspended culturing of aeroterrestrial microalgae in non-enclosure way, proper experiments need to be conducted. In this review, basic concepts of attached cultivation system are discussed, focusing on the studies of biofilm formation including factors affecting deposition and systems. The detailed description of aeroterrestrial microalgae is included to give insight into potential applications of the species into attached cultivation systems