Generation of a catalytic module on a self-folding RNA

It is theoretically possible to obtain a catalytic site of an artificial ribozyme from a random sequence consisting of a limited numbers of nucleotides. However, this strategy has been inadequately explored. Here, we report an in vitro selection technique that exploits modular construction of a structurally constrained RNA to acquire a catalytic site for RNA ligation from a short random sequence. To practice the selection, a sequence of 30 nucleotides was located close to the putative reaction site in a derivative of a naturally occurring self-folding RNA whose crystal structure is known. RNAs whose activity depended on the starting three-dimensional structure were selected with 3'-5' ligation specificity, indicating that the strategy can be used to acquire a variety of catalytic sites and other functional RNA modules.     

Mammalian variations on a theme: a Smo and Sufu surprise

Hedgehog signaling plays a critical role during development and tumorigenesis. While much mechanistic insight has come from pathway investigations in the fruit fly, recent studies suggest a distinct mammalian strategy for signaling from Smoothened to Gli through the novel protein Suppressor of Fused that may have therapeutic implications.     

Comments on a skeleton design paradigm for a demosponge

The ball-shaped marine sponge Cinachyrellalevantinensis is 3-5 cm in diameter. It filters large quantities of seawater for feeding. Sponges contain numerous, hydrated, brittle amorphous SiO? spicules of several types that form 70-80% by weight of the sponge. We performed mechanical tests to determine the functionality of the sponge skeleton. The potential effect of habitat on skeleton properties was investigated by comparing sponges from 0.5 m and 30 m depth. We determined how spicules contribute to maintaining the strength and macroscopic structural integrity of a sponge, and studied their deformation mechanisms under external loading, and their microscopic design parameters. Compression tests of cylindrical samples cut from sponges revealed their macroscopic deformation mechanisms. Experiments solely with the organic material (following spicules dissolution) revealed the contribution of the spicules to the load carrying capacity and structural integrity of the sponge. Cantilever bending tests of anchored spicules determined the strength of individual spicules, the sponge's main skeletal elements. As the strength of brittle spicules is statistical in nature, we used Weibull Statistics to define their strength and evaluate their Young's modulus. Shallow and deep-water sponges did not differ significantly neither in response to compression, nor in spicule strength under bending and tension. Spicule weight fraction within a sponge was significantly higher in shallow-water individuals. We conclude that the structural integrity and strength of this sponge's skeleton is derived from its low-strength, small spicules, produced by a cost-effective process. The operating deformation of the spicules (bending) and their design parameters make them highly efficient.     

Exclusion processes on a growing domain

A discrete model provides a useful framework for experimentalists to understand the interactions between growing tissues and other biological mechanisms. A cellular automata (CA) model with domain growth, cell motility and cell proliferation, based on cellular exclusion processes, is developed here. Average densities can be defined from the CA model and a continuum representation can be determined. The domain growth mechanism in the CA model gives rise to a Fokker-Planck equation in the corresponding continuum model, with a diffusive and a convective term. Deterministic continuum models derived from conservation laws do not include this diffusive term. The new diffusive term arises because of the stochasticity inherited from the CA mechanism for domain growth. We extend the models to multiple species and investigate the influence of the flux terms arising from the exclusion processes. The averaged CA agent densities are well approximated by the solution of nonlinear advection-diffusion equations, provided that the relative size of the proliferation processes to the diffusion processes is sufficiently small. This dual approach provides an understanding of the microscopic and macroscopic scales in a developmental process.     

The effect of a corona discharge on a lightning attachment

The interaction between the lightning leader and the space charge accumulated near the top of a ground object in the atmospheric electric field is considered using analytical and numerical models developed earlier to describe spark discharges in long laboratory gaps. The specific features of a nonstationary corona discharge that develops in the electric field of a thundercloud and a downward lightning leader are analyzed. Conditions for the development of an upward lightning discharge from a ground object and for the propagation of an upward-connecting leader from the object toward a downward lightning leader (the process determining the point of strike to the ground) are investigated. Possible mechanisms for the interaction of the corona space charge with an upward leader and prospects of using it to control downward lightning discharges are analyzed.     

CLUSTAL: a package for performing multiple sequence alignment on a microcomputer

An approach for performing multiple alignments of large numbers of amino acid or nucleotide sequences is described. The method is based on first deriving a phylogenetic tree from a matrix of all pairwise sequence similarity scores, obtained using a fast pairwise alignment algorithm. Then the multiple alignment is achieved from a series of pairwise alignments of clusters of sequences, following the order of branching in the tree. The method is sufficiently fast and economical with memory to be easily implemented on a microcomputer, and yet the results obtained are comparable to those from packages requiring mainframe computer facilities.     

Action of Patulin on a Yeast

The action of patulin on Saccharomyces cerevisiae was studied. At weak doses, the drug inhibited growth, but inhibition was transient. After 10 min, syntheses of rRNA, tRNA, and probably mRNA were blocked; this was shown by radioactive precursor incorporation assays and gel electrophoresis of RNAs. After recovery of growth, patulin disappeared from the medium. It seemed that this degradation resulted from the activity of an inducible enzymatic system. Induced cells resisted very high patulin concentrations.     

Action of patulin on a yeast

The action of patulin on Saccharomyces cerevisiae was studied. At weak doses, the drug inhibited growth, but inhibition was transient. After 10 min, syntheses of rRNA, tRNA, and probably mRNA were blocked; this was shown by radioactive precursor incorporation assays and gel electrophoresis of RNAs. After recovery of growth, patulin disappeared from the medium. It seemed that this degradation resulted from the activity of an inducible enzymatic system. Induced cells resisted very high patulin concentrations.     

Positional recognition of a tRNA determinant dependent on a peptide insertion

The crystal structure of a catalytic fragment of Aquifex aeolicus AlaRS and additional data suggest how the critical G3:U70 identity element of its cognate tRNA acceptor stem is recognized. Though this identity element is conserved from bacteria to the cytoplasm of eukaryotes, Drosophila melanogaster mitochondrial (Dm mt) tRNA(Ala) contains a G:U base pair that has been translocated to the adjacent 2:71 position. This G2:U71 is the major determinant for identity of Dm mt tRNA(Ala). Sequence alignments showed that Dm mt AlaRS is differentiated from G3:U70-recognizing AlaRSs by an insertion of 27 amino acids in the region of the protein that contacts the acceptor stem. Precise deletion of this insertion from Dm mt AlaRS gave preferential recognition to a G3:U70-containing substrate. Larger or smaller deletions were ineffective. The crystal structure of the orthologous A. aeolicus protein places this insertion on the surface, where it can act as a hinge that provides positional switching of G:U recognition.     

Design constraints on a synthetic metabolism

A metabolism is a complex network of chemical reactions that converts sources of energy and chemical elements into biomass and other molecules. To design a metabolism from scratch and to implement it in a synthetic genome is almost within technological reach. Ideally, a synthetic metabolism should be able to synthesize a desired spectrum of molecules at a high rate, from multiple different nutrients, while using few chemical reactions, and producing little or no waste. Not all of these properties are achievable simultaneously. We here use a recently developed technique to create random metabolic networks with pre-specified properties to quantify trade-offs between these and other properties. We find that for every additional molecule to be synthesized a network needs on average three additional reactions. For every additional carbon source to be utilized, it needs on average two additional reactions. Networks able to synthesize 20 biomass molecules from each of 20 alternative sole carbon sources need to have at least 260 reactions. This number increases to 518 reactions for networks that can synthesize more than 60 molecules from each of 80 carbon sources. The maximally achievable rate of biosynthesis decreases by approximately 5 percent for every additional molecule to be synthesized. Biochemically related molecules can be synthesized at higher rates, because their synthesis produces less waste. Overall, the variables we study can explain 87 percent of variation in network size and 84 percent of the variation in synthesis rate. The constraints we identify prescribe broad boundary conditions that can help to guide synthetic metabolism design.     

Magnetic resonance imaging of a microvascular-interstitium model on a microfluidic device

We report a microvascular-interstitium model on microfluidic devices to study leakage of drugs from blood vessels under in vivo-like flow conditions. We employed magnetic resonance imaging to demonstrate the compatibility of the model for experimental animals and humans. We observed transport of two types of different molecular-weight contrast agents into the model interstitium. The ratio of the transport rates of agents agreed with the ratio calculated from diffusion coefficients of the agents. We expect that the model will be useful for the estimation and evaluation of leakage of many kinds of agents in vivo.     

Square-cut: a segmentation algorithm on the basis of a rectangle shape

We present a rectangle-based segmentation algorithm that sets up a graph and performs a graph cut to separate an object from the background. However, graph-based algorithms distribute the graph's nodes uniformly and equidistantly on the image. Then, a smoothness term is added to force the cut to prefer a particular shape. This strategy does not allow the cut to prefer a certain structure, especially when areas of the object are indistinguishable from the background. We solve this problem by referring to a rectangle shape of the object when sampling the graph nodes, i.e., the nodes are distributed non-uniformly and non-equidistantly on the image. This strategy can be useful, when areas of the object are indistinguishable from the background. For evaluation, we focus on vertebrae images from Magnetic Resonance Imaging (MRI) datasets to support the time consuming manual slice-by-slice segmentation performed by physicians. The ground truth of the vertebrae boundaries were manually extracted by two clinical experts (neurological surgeons) with several years of experience in spine surgery and afterwards compared with the automatic segmentation results of the proposed scheme yielding an average Dice Similarity Coefficient (DSC) of 90.97±2.2%.     

Demographic effects of prolonged drought on a nascent introduction of a semi-aquatic snake

Faced with a nascent introduction of a non-native species, conservationists need to quickly determine how a population performs in its new environment. Although correlative models can predict environmental suitability at a coarse scale, they often neglect short-term climatic variability, instead relying on long-term averages. Accurately projecting the fate of any particular introduction requires demographic data on how a population responds to a novel environment. The recent introduction of watersnakes (genus Nerodia) poses a risk to California’s already imperiled aquatic vertebrate fauna. Despite inhabiting a seemingly suitable climate, a non-native Nerodia sipedon population in central California is likely to have been affected by a prolonged extreme drought from 2012 to 2015. We studied the only known population of N. sipedon in California for 3 years from 2013 to 2015, and estimated its abundance and annual survival. Its abundance declined from a peak of 218 (95% Credible Interval 149–313) in August 2013 to 97 (80–119) in July 2015. Annual survival of N. sipedon from 2013 to 2014 (0.23, 0.13–0.39) and from 2014 to 2015 (0.29, 0.18–0.41) was lower than survival estimates reported from native populations. Snake body condition, the abundance of large adult females, and prey availability all declined throughout the study. We conclude that the population of N. sipedon declined from 2013 to 2015, likely due to decreasing habitat and prey availability from the prolonged regional drought. This study highlights the importance of the effect of climatic extremes on the trajectory of introduced populations in a novel environment.     

Asymmetric amyloid fibril elongation: a new perspective on a symmetric world

Amyloids are insoluble, fibrous proteins formed through the aggregation of misfolded proteins. They accumulate in the tissue of individuals with degenerative diseases, such as Parkinson's and Alzheimer's. The purpose of this study was to determine whether fibril growth from an initial model fibril seed is unidirectional or bidirectional. The prevailing theory on amyloid formation is that a symmetric fibril elongates equally from both ends. This study provides evidence to the contrary; the process occurs predominately unidirectionally, demonstrating that amyloid fibrils may be asymmetric and propagate mostly in one direction. Alexa Fluor 568 labeled insulin fibrils were seeded into a native insulin solution and allowed to elongate at 65°C while the kinetics of fibril growth was monitored. The resulting elongated fibrils were labeled with thioflavin-T, and the fluorescent images of the fibrils show that a majority of the elongated fibrils propagated along only one end of the seed, with the remaining labeled fibrils having bidirectional elongation or no elongation. Using a crystallographic model, we offer a structural explanation for asymmetric growth of the insulin fibrils. Thus, instead of the current view that fibrils grow symmetrically from both ends of the fibril, this is the first evidence that insulin amyloid fibrils formed in solution are asymmetric and appear to grow from only one end.     

Replication-Defective Vector Based on a Chimpanzee Adenovirus

An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.     

Effect of a foreign male's age on acceptance into a conspecific colony in a Neotropical wasp

We conducted field experiments to examine whether young males of different age cohorts were accepted from alien colonies in the swarm‐founding wasp Polybia paulista. We showed, as for young females in other social wasp species, that newly emerged males were frequently accepted from alien colonies. Our study suggests that young males of P. paulista acquire colony‐specific chemical odors shortly after emergence.     

Hare on potentiality: a rejoinder

Richard Hare's comment on an article by Lockwood in Bioethics (article and comment appeared in 1988 Jul; 2(3): 187-213, 214-226) pinpoints their crucial disagreement over the extent to which one is entitled to sacrifice the potential for worthwhile life of individuals whose actuality is already an established fact. Lockwood argues that, even though we can all feel gratitude toward our parents for our existence, the obligation one has towards an actual individual not to prevent his or her own potentiality for human life from being realized is far stronger than any obligation one has to bring into existence, or not to prevent from coming into existence, an otherwise merely possible individual with a similar potentiality.     

The effect of a constant efflux on solute movement to a root

This paper presents two analytical solutions to solute uptake and release by a root (the root exhibits efflux) when both mass and diffusive flows are considered and the water content of the soil is maintained constant. The first solution is for a constant solute concentration at some finite distance from the root, while the second is when there is no solute recharge from the soil outside a cylinder coaxle with the root.Several examples are presented to show the effect of the effluxive term. It is shown that the point where the ratio of efflux to initial ion concentration in solution is equal to the difference between root absorbing power and water flux at the root, is critical to nutrient depletion or buildup at the root surface. It is also shown for the case of no recharge that the concentration profiles when efflux is considered are markedly different than where efflux is not considered.Contribution from the Purdue Agric. Exp. Stn., West Lafayette, IN, 47907. Journal Paper Number7458.     

Stereochemical studies on a plasmid-coded fluoroacetate halidohydrolase

The stereochemical course of action of haloacetate halidohydrolase H-1 from Pseudomonas sp., strain A, which catalyzes the dehalogenation of fluoroacetate to glycolate, has been determined by enzymatic analysis of products from incubations with both enantiomers of 20-fluoropropionate, and by ¹H NMR analysis of the ester of (?)-α-methoxy-α-(trifluoromethyl)phenylacetic acid with phenacyl [2-²H₁]glycolate derived from the product of incubation with the (S)-monodeuterofluoroacetate. The results support a direct displacement mechanism for this enzyme, since they indicate that the reaction is catalyzed with inversion of configuration.