DNA Building Blocks at the Foundation of Better Survival

No abstract available.     

New Building Blocks for the Preparation of Branched Oligonucleotides

Abstract

New building blocks 2 and 3 were prepared and successfully employed for the synthesis of branched oligonucleotides 5 and 6. The structure of oligomers obtained was confirmed by electrospray ionisation mass spectrometry.     

Identifying the Evolutionary Building Blocks of the Cardiac Conduction System

The endothermic state of mammals and birds requires high heart rates to accommodate the high rates of oxygen consumption. These high heart rates are driven by very similar conduction systems consisting of an atrioventricular node that slows the electrical impulse and a His-Purkinje system that efficiently activates the ventricular chambers. While ectothermic vertebrates have similar contraction patterns, they do not possess anatomical evidence for a conduction system. This lack amongst extant ectotherms is surprising because mammals and birds evolved independently from reptile-like ancestors. Using conserved genetic markers, we found that the conduction system design of lizard (Anolis carolinensis and A. sagrei), frog (Xenopus laevis) and zebrafish (Danio rerio) adults is strikingly similar to that of embryos of mammals (mouse Mus musculus, and man) and chicken (Gallus gallus). Thus, in ectothermic adults, the slow conducting atrioventricular canal muscle is present, no fibrous insulating plane is formed, and the spongy ventricle serves the dual purpose of conduction and contraction. Optical mapping showed base-to-apex activation of the ventricles of the ectothermic animals, similar to the activation pattern of mammalian and avian embryonic ventricles and to the His-Purkinje systems of the formed hearts. Mammalian and avian ventricles uniquely develop thick compact walls and septum and, hence, form a discrete ventricular conduction system from the embryonic spongy ventricle. Our study uncovers the evolutionary building plan of heart and indicates that the building blocks of the conduction system of adult ectothermic vertebrates and embryos of endotherms are similar.     

Building Blocks of Temporal Filters in Retinal Synapses

Sensory systems must be able to extract features of a stimulus to detect and represent properties of the world. Because sensory signals are constantly changing, a critical aspect of this transformation relates to the timing of signals and the ability to filter those signals to select dynamic properties, such as visual motion. At first assessment, one might think that the primary biophysical properties that construct a temporal filter would be dynamic mechanisms such as molecular concentration or membrane electrical properties. However, in the current issue of PLOS Biology, Baden et al. identify a mechanism of temporal filtering in the zebrafish and goldfish retina that is not dynamic but is in fact a structural building block—the physical size of a synapse itself. The authors observe that small, bipolar cell synaptic terminals are fast and highly adaptive, whereas large ones are slower and adapt less. Using a computational model, they conclude that the volume of the synaptic terminal influences the calcium concentration and the number of available vesicles. These results indicate that the size of the presynaptic terminal is an independent control for the dynamics of a synapse and may reveal aspects of synaptic function that can be inferred from anatomical structure.     

Building Blocks,Hinge-Bending Motions and Protein Topology

Abstract

Here we show that the locations of molecular hinges in protein structures fall between building block elements. Building blocks are fragments of the protein chain which constitute local minima. These elements fold first. In the next step they associate through a combinatorial assembly process. While chain-linked building blocks may be expected to trial-associate first, if unstable, alternate more stable associations will take place. Hence, we would expect that molecular hinges will be at such inter-building block locations, or at the less stable, ‘unassigned’ regions.

On the other hand, hinge-bending motions are well known to be critical for protein function. Hence, protein folding and protein function are evolutionarily related. Further, the pathways through which proteins attain their three dimensional folds are determined by protein topology. However, at the same time the locations of the hinges, and hinge-bending motions are also an outcome of protein topology. Thus, protein folding and function appear coupled, and relate to protein topology. Here we provide some results illustrating such a relationship.     

Binets: Fundamental Building Blocks for Phylogenetic Networks

Phylogenetic networks are a generalization of evolutionary trees that are used by biologists to represent the evolution of organisms which have undergone reticulate evolution. Essentially, a phylogenetic network is a directed acyclic graph having a unique root in which the leaves are labelled by a given set of species. Recently, some approaches have been developed to construct phylogenetic networks from collections of networks on 2- and 3-leaved networks, which are known as binets and trinets, respectively. Here we study in more depth properties of collections of binets, one of the simplest possible types of networks into which a phylogenetic network can be decomposed. More specifically, we show that if a collection of level-1 binets is compatible with some binary network, then it is also compatible with a binary level-1 network. Our proofs are based on useful structural results concerning lowest stable ancestors in networks. In addition, we show that, although the binets do not determine the topology of the network, they do determine the number of reticulations in the network, which is one of its most important parameters. We also consider algorithmic questions concerning binets. We show that deciding whether an arbitrary set of binets is compatible with some network is at least as hard as the well-known graph isomorphism problem. However, if we restrict to level-1 binets, it is possible to decide in polynomial time whether there exists a binary network that displays all the binets. We also show that to find a network that displays a maximum number of the binets is NP-hard, but that there exists a simple polynomial-time 1/3-approximation algorithm for this problem. It is hoped that these results will eventually assist in the development of new methods for constructing phylogenetic networks from collections of smaller networks.     

Synthesis of Triamino Acid Building Blocks with Different Lipophilicities

To obtain different amino acids with varying lipophilicity and that can carry up to three positive charges we have developed a number of new triamino acid building blocks. One set of building blocks was achieved by aminoethyl extension, via reductive amination, of the side chain of ortnithine, diaminopropanoic and diaminobutanoic acid. A second set of triamino acids with the aminoethyl extension having hydrocarbon side chains was synthesized from diaminobutanoic acid. The aldehydes needed for the extension by reductive amination were synthesized from the corresponding Fmoc-L-2-amino fatty acids in two steps. Reductive amination of these compounds with Boc-L-Dab-OH gave the C4-C8 alkyl-branched triamino acids. All triamino acids were subsequently Boc-protected at the formed secondary amine to make the monomers appropriate for the N-terminus position when performing Fmoc-based solid-phase peptide synthesis.     

Swine in Biomedical Research: Creating the Building Blocks of Animal Models

Abstract

Sex of preimplantation porcine embryos was determined by DNA amplification using porcine male(Y chromosome)‐specific DNA primers in the polymerase chain reaction (PCR). In order to determine the sensitivity of this sexing method, single porcine embryos ranging from unfertilized ova to the blastocyst stage were amplified in the PCR using the Y‐specific primers, and analyzed by ethidium bromide‐staining of polyacrylamide gels. The 192 bp product which denotes the presence of the Y chromosome was seen in the embryos. The unfertilized ova which is of female origin gave no product. These results are representative of PCR analysis of a total of 34 swine embryos.

Results obtained using the PCR for sexing were validated by karyotyping and confirmed by in situ hybridization with the porcine Y‐chromosome‐specific probe. In order to confirm the sex of the embryos determined by PCR, 10 day‐old porcine preimplantation embryos were biopsied to produce a small number of cells for sex determination via PCR, while the remainder of the embryo was prepared for in situ hybridization using the biotinylated probe. In situ hybridization performed on embryos shown to be male by PCR, showed pinpoint fluorescence within the nuclei, similar to that obtained when male porcine lymphocytes were hybridized. No evidence of fluorescence was seen when in situ hybridization was performed in parallel on embryos determined to be female by the PCR.

The PCR was found to be a relatively fast, accurate and reproducible means of sex determination of swine preimplantation embryos. This capability could have significant impact on animal breeding and production programs by using PCR as a screening tool for traits of economic importance.     

Construction of Double-helical DNA Structures Based on Dinucleotide Building Blocks

Abstract

We present a new method for building full 3-D structures of DNA sequences. A database of the conformational properties of dinucleotide steps has been compiled using X-ray crystal structures of oligonucleotides. The protocol uses these dinucleotides as building blocks to generate three dimensional structures of any required sequence in any required conformation.     

Culture-Independent Characterization of Archaeal Biodiversity in Swine Confinement Building Bioaerosols

It was previously demonstrated that microbial communities of pig manure were composed of both bacteria and archaea. Recent studies have shown that bacteria are aerosolized from pig manure, but none have ever focused on the airborne archaeal burden. We sought here to develop and apply molecular ecology approaches to thoroughly characterize airborne archaea from swine confinement buildings (SCBs). Eight swine operations were visited, twice in winter and once during summer. Institute of Occupational Medicine cassettes loaded with 25-mm gelatin filters were used to capture the inhalable microbial biomass. The total genomic DNA was extracted and used as a template for PCR amplification of the archaeal 16S rRNA gene. High concentrations of archaea were found in SCB bioaerosols, being as high as 10⁸ 16S rRNA gene copies per cubic meter of air. Construction and sequencing of 16S rRNA gene libraries revealed that all sequences were closely related to methanogenic archaea, such as Methanosphaera stadtmanae (94.7% of the archaeal biodiversity). Archaeal community profiles were compared by 16S rRNA gene denaturing gradient gel electrophoresis. This analysis showed similar fingerprints in each SCB and confirmed the predominance of methanogenic archaea in the bioaerosols. This study sheds new light on the nature of bioaerosols in SCBs and suggests that archaea are also aerosolized from pig manure.Over the last 30 years, swine production in Canada evolved from small family farms to industrial facilities. Pig producers have increased animal density, building mechanization, and confinement in order to decrease working and feeding time and to optimize space, leading to an increased contamination of air by bioaerosols.Even though the swine confinement building (SCB) environment has been studied for several years, little is known about the real concentration and nature of airborne microorganisms. Moreover, increasing confinement level in modern barns has raised bioaerosol levels, modifying the health risk of exposed workers. Thus far, using culture-dependent methods was the only strategy developed and used to describe SCB bioaerosol content and levels (6, 7). However, it is well known that culture-independent approaches are more likely to reveal the presence of microorganisms never suspected in most environments (2). In aerobiology, there are only a few reports using culture-independent methods (4, 15). Nehme et al. (20) applied molecular approaches to quantify and describe the bacterial aerosols in SCB and reported as much as 10⁸ bacteria per cubic meter of air, with significantly higher concentrations during winter, when the confinement is maximal. The data obtained were also compared to recent biodiversity studies of swine manures (13, 22, 25). Anaerobic gram-positive bacteria, being the greater part of the microbiological aerosols, appeared to originate from the swine manure. Those manure biodiversity studies revealed the presence of methanogenic archaea in hog wastes (22, 25). Since bacteria observed in the aerosols seem to originate from the manure, it is plausible that archaea from pig slurries are also aerosolized.We report here the characterization of the archaeal community of SCB bioaerosols by using cultivation-independent approaches. The phylogeny of airborne archaea was assessed using 16S rRNA gene sequences. Archaeal biodiversity profiles were determined with PCR-denaturing gradient gel electrophoresis (DGGE), and the concentration of aerosolized archaea was evaluated by real-time PCR by quantifying archaeal 16S rRNA gene copies in the air samples.     

Building Blocks for Synthesis of Oligoarabinonucleotides: Preparation of Arabinonucleoside H-Phosphonates from Protected Ribonucleosides

Abstract

A straightforward and inexpensive synthesis of arabinonucleoside H-phosphonates has been developed. Arabinonucleosides were synthesised from protected ribonucleosides via 2′-keto derivatives. Reaction conditions have been optimised for compounds bearing labile N-protections. Further protecting group manipulation and phosphonylation gave the required H-phosphonate monomers.     

Biophysics and Thermodynamics: The Scientific Building Blocks of Bio-inspired Drug Delivery Nano Systems

Biophysics and thermodynamics are considered as the scientific milestones for investigating the properties of materials. The relationship between the changes of temperature with the biophysical variables of biomaterials is important in the process of the development of drug delivery systems. Biophysics is a challenge sector of physics and should be used complementary with the biochemistry in order to discover new and promising technological platforms (i.e., drug delivery systems) and to disclose the ‘silence functionality’ of bio-inspired biological and artificial membranes. Thermal analysis and biophysical approaches in pharmaceuticals present reliable and versatile tools for their characterization and for the successful development of pharmaceutical products. The metastable phases of self-assembled nanostructures such as liposomes should be taken into consideration because they represent the thermal events can affect the functionality of advanced drug delivery nano systems. In conclusion, biophysics and thermodynamics are characterized as the building blocks for design and development of bio-inspired drug delivery systems.KEY WORDS: biophysics, drug delivery nano systems, pharmaceutics, thermal analysis, thermodynamics     

Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown Function

Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diameters of 39 ± 2 nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.     

Synthesis of Modified Building Blocks Containing Amino or Thiol Moieties: Application of Modified Oligodeoxyribonucleotides

Abstract

Modified building blocks have been synthesised and used to prepare 5′- amino and 5′-mercapto-oligodeoxyribonucleotides. Subsequent labelling with fluorophores or metal cluster derivatives generates a range of very useful probes.     

Facile Synthesis of Orthogonally Protected Optically Pure Keto- and Diketopiperazine Building Blocks for Combinatorial Chemistry

A simple and convenient synthesis of orthogonally protected multi-tethered, optically pure 2-ketopiperazine and 2,5-diketopiperazine scaffolds for Fmoc and Boc combinatorial chemistry was achieved, starting from accessible chiral amino acid precursors, by sequentially utilizing reductive alkylation, dipeptide coupling and ketopiperazine ring formation as key steps. These scaffolds can introduce valuable drug-like properties in three independent directions to any medicinally relevant piperazine-based motif by “around the scaffold” drug optimization. In addition, these building blocks have a wide application scope in managing fast and efficient multi-cyclic optimization processes in the combinatorial chemistry and drug design fields.     

Total Solution Synthesis of Human Epidermal Growth Factor (h-EGF) by the Assembly of Nine Building Blocks

Human epidermal growth factor (h-EGF) composed of 53 amino acids bearing three intramolecular disulfide bridges was synthesized by the maximum protecting solution method. The synthetic h-EGF coincided with recombinant h-EGF by reverse-phase HPLC, and the sites of three intramolecular disulfide bridges were ascertained by a thermolytic digestion. The synthetic h-EGF possessed m/z 6215.7 in its FAB-MS as expected, and exhibited compatible mitogenic activity.     

Managing Potato Biodiversity to Cope with Frost Risk in the High Andes: A Modeling Perspective

Austral summer frosts in the Andean highlands are ubiquitous throughout the crop cycle, causing yield losses. In spite of the existing warming trend, climate change models forecast high variability, including freezing temperatures. As the potato center of origin, the region has a rich biodiversity which includes a set of frost resistant genotypes. Four contrasting potato genotypes –representing genetic variability- were considered in the present study: two species of frost resistant native potatoes (the bitter Solanum juzepczukii, var. Luki, and the non-bitter Solanum ajanhuiri, var. Ajanhuiri) and two commercial frost susceptible genotypes (Solanum tuberosum ssp. tuberosum var. Alpha and Solanum tuberosum ssp. andigenum var. Gendarme). The objective of the study was to conduct a comparative growth analysis of four genotypes and modeling their agronomic response under frost events. It included assessing their performance under Andean contrasting agroecological conditions. Independent subsets of data from four field experiments were used to parameterize, calibrate and validate a potato growth model. The validated model was used to ascertain the importance of biodiversity, represented by the four genotypes tested, as constituents of germplasm mixtures in single plots used by local farmers, a coping strategy in the face of climate variability. Also scenarios with a frost routine incorporated in the model were constructed. Luki and Ajanhuiri were the most frost resistant varieties whereas Alpha was the most susceptible. Luki and Ajanhuiri, as monoculture, outperformed the yield obtained with the mixtures under severe frosts. These results highlight the role played by local frost tolerant varieties, and featured the management importance –e.g. clean seed, strategic watering- to attain the yields reported in our experiments. The mixtures of local and introduced potatoes can thus not only provide the products demanded by the markets but also reduce the impact of frosts and thus the vulnerability of the system to abiotic stressors.     

Plasmid Vectors and Molecular Building Blocks for the Development of Genetic Manipulation Tools for Trypanosoma cruzi

The post genomic era revealed the need for developing better performing, easier to use and more sophisticated genetic manipulation tools for the study of Trypanosoma cruzi, the etiological agent of Chagas disease. In this work a series of plasmids that allow genetic manipulation of this protozoan parasite were developed. First of all we focused on useful tools to establish selection strategies for different strains and which can be employed as expression vectors. On the other hand molecular building blocks in the form of diverse selectable markers, modifiable fluorescent protein and epitope-tag coding sequences were produced. Both types of modules were harboured in backbone molecules conceived to offer multiple construction and sub-cloning strategies. These can be used to confer new properties to already available genetic manipulation tools or as starting points for whole novel designs. The performance of each plasmid and building block was determined independently. For illustration purposes, some simple direct practical applications were conducted.