Hybridization of Rous Sarcoma Virus Deoxyribonucleic Acid Polymerase Product and Ribonucleic Acids from Chicken and Rat Cells Infected with Rous Sarcoma Virus

Rous sarcoma virus (RSV)-specific ribonucleic acid (RNA) in virus-producing chicken cells and non-virus-producing rat cells infected with RSV was studied by hybridization with the endogenous deoxyribonucleic acid (DNA) product of the RSV virion DNA polymerase system. By hybridizing the total DNA product with excess virion RNA, the product DNA was separated into hybridized (“minus”) and nonhybridized (“plus”) DNA. The “minus” DNA was complementary to at least 20% of the RNA from RSV which remained of high molecular weight after denaturation. A maximum of approximately 65% hybridization was observed between “minus” DNA and RSV RNA or RSV-infected chicken cell RNA. A maximum of about 60% hybridization was observed between “minus” DNA and RSV-infected rat cell RNA. RSV-infected chicken cells contained RSV-specific RNA equivalent to about 6,000 virions per cell. RSV-infected rat cells contained RSV-specific RNA equivalent to approximately 400 virions per cell. Neither cell type contained detectable RNA complementary to virion RNA. The RSV-specific RNA in RSV-infected rat cells did not appear to be qualitatively different from that in RSV-infected chicken cells.     

A Novel Bio-Sensor Based on DNA Strand Displacement

DNA strand displacement technology performs well in sensing and programming DNA segments. In this work, we construct DNA molecular systems based on DNA strand displacement performing computation of logic gates. Specifically, a class of so-called “DNA neurons” are achieved, in which a “smart” way inspired by biological neurons encoding information is developed to encode and deliver information using DNA molecules. The “DNA neuron” is bistable, that is, it can sense DNA molecules as input signals, and release “negative” or “positive” signals DNA molecules. We design intelligent DNA molecular systems that are constructed by cascading some particularly organized “DNA neurons”, which could perform logic computation, including AND, OR, XOR logic gates, automatically. Both simulation results using visual DSD (DNA strand displacement) software and experimental results are obtained, which shows that the proposed systems can detect DNA signals with high sensitivity and accretion; moreover, the systems can process input signals automatically with complex nonlinear logic. The method proposed in this work may provide a new way to construct a sensitive molecular signal detection system with neurons spiking behavior in vitro, and can be used to develop intelligent molecular processing systems in vivo.     

Transcription of Simian Virus 40 II. Hybridization of RNA Extracted from Different Lines of Transformed Cells to the Separated Strands of Simian Virus 40 DNA

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and “revertants” isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and “revertant” cell lines. From 30 to 80% of the “early” strand and from 0 to 20% of the “late” strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.     

Patterns of mesenchymal condensation in a multiscale, discrete stochastic model

Cells of the embryonic vertebrate limb in high-density culture undergo chondrogenic pattern formation, which results in the production of regularly spaced “islands” of cartilage similar to the cartilage primordia of the developing limb skeleton. The first step in this process, in vitro and in vivo, is the generation of “cell condensations,” in which the precartilage cells become more tightly packed at the sites at which cartilage will form. In this paper we describe a discrete, stochastic model for the behavior of limb bud precartilage mesenchymal cells in vitro. The model uses a biologically motivated reaction–diffusion process and cell-matrix adhesion (haptotaxis) as the bases of chondrogenic pattern formation, whereby the biochemically distinct condensing cells, as well as the size, number, and arrangement of the multicellular condensations, are generated in a self-organizing fashion. Improving on an earlier lattice-gas representation of the same process, it is multiscale (i.e., cell and molecular dynamics occur on distinct scales), and the cells are represented as spatially extended objects that can change their shape. The authors calibrate the model using experimental data and study sensitivity to changes in key parameters. The simulations have disclosed two distinct dynamic regimes for pattern self-organization involving transient or stationary inductive patterns of morphogens. The authors discuss these modes of pattern formation in relation to available experimental evidence for the in vitro system, as well as their implications for understanding limb skeletal patterning during embryonic development.     

Molecular Threading: Mechanical Extraction,Stretching and Placement of DNA Molecules from a Liquid-Air Interface

We present “molecular threading”, a surface independent tip-based method for stretching and depositing single and double-stranded DNA molecules. DNA is stretched into air at a liquid-air interface, and can be subsequently deposited onto a dry substrate isolated from solution. The design of an apparatus used for molecular threading is presented, and fluorescence and electron microscopies are used to characterize the angular distribution, straightness, and reproducibility of stretched DNA deposited in arrays onto elastomeric surfaces and thin membranes. Molecular threading demonstrates high straightness and uniformity over length scales from nanometers to micrometers, and represents an alternative to existing DNA deposition and linearization methods. These results point towards scalable and high-throughput precision manipulation of single-molecule polymers.     

Effect of Energy Under-Reporting on Secular Trends of Dietary Patterns in a Mediterranean Population

BackgroundDiet is an important factor in the prevention of chronic diseases. Analysis of secular trends of dietary patterns can be biased by energy under-reporting. Therefore, the objective of the present study was to analyse the impact of energy under-reporting on dietary patterns and secular trends in dietary patterns defined by cluster analysis.ResultsThree clusters, “healthy”, “mixed” and “western”, were identified for both surveys. The “mixed” cluster was the predominant cluster in both surveys. Excluding EUR reduced the proportion of the “mixed” cluster up to 6.40% in the 2000 survey; this caused secular trend increase in the prevalence of the “mixed” pattern. Cross-classification analysis of all participants and PER’ data showed substantial agreement in cluster assignments: 68.7% in 2000 and 84.4% in 2005. Excluding EUR did not cause meaningful (≥15%) changes in the “healthy” pattern. It provoked changes in consumption of some food groups in the “mixed” and “western” patterns: mainly decreases of unhealthy foods within the 2000 and increases of unhealthy foods within the 2005 surveys. Secular trend effects of EUR were similar to those within the 2005 survey. Excluding EUR reversed the direction of secular trends in consumption of several food groups in PER in the “mixed” and “western” patterns.ConclusionsEUR affected distribution of participants between dietary patterns within and between surveys, secular trends in food group consumption and amount of food consumed in all, but not in the “healthy” pattern. Our findings emphasize threats from energy under-reporting in dietary data analysis.     

A Prospective Cohort Study to Examine the Association between Dietary Patterns and Depressive Symptoms in Older Chinese People in Hong Kong

Introduction

Dietary patterns are culturally specific and there is limited data on the association of dietary patterns with late-life depression in Chinese. This study examined the associations between dietary patterns and baseline and subsequent depressive symptoms in community-dwelling Chinese older people in Hong Kong.

Methods

Participants aged ≥65 year participating in a cohort study examining the risk factors for osteoporosis completed a validated food frequency questionnaire at baseline between 2001 and 2003. Factor analysis was used to identify three dietary patterns: “vegetables-fruits” pattern, “snacks-drinks-milk products” pattern, and “meat-fish” pattern. Depressive symptoms were measured at baseline and 4-year using the validated Geriatric Depression Scale. Multiple logistic regression was used for cross-sectional analysis (n = 2,902) to assess the associations between dietary patterns and the presence of depressive symptoms, and for longitudinal analysis (n = 2,211) on their associations with 4-year depressive symptoms, with adjustment for socio-demographic and lifestyle factors.

Results

The highest quartile of “vegetables-fruits” pattern score was associated with reduced likelihood of depressive symptoms [Adjusted OR = 0.55 (95% CI: 0.36–0.83), p_trend = 0.017] compared to the lowest quartile at baseline. Similar inverse trend was observed for the highest quartile of “snacks-drinks-milk products” pattern score [Adjusted OR = 0.41 (95% CI: 0.26–0.65), p_trend<0.001] compared to the lowest quartile. There was no association of “meat-fish” pattern with the presence of depressive symptoms at baseline. None of the dietary patterns were associated with subsequent depressive symptoms at 4-year.

Conclusion

Higher “vegetables-fruits” and “snacks-drinks-milk products” pattern scores were associated with reduced likelihood of baseline depressive symptoms in Chinese older people in Hong Kong. The longitudinal analyses failed to show any causal relationship between dietary patterns and depressive symptoms in this population.     

The Motility Symbiont of the Termite Gut Flagellate Caduceia versatilis Is a Member of the “Synergistes” Group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.     

Cellulose as an Architectural Element in Spatially Structured Escherichia coli Biofilms

Morphological form in multicellular aggregates emerges from the interplay of genetic constitution and environmental signals. Bacterial macrocolony biofilms, which form intricate three-dimensional structures, such as large and often radially oriented ridges, concentric rings, and elaborate wrinkles, provide a unique opportunity to understand this interplay of “nature and nurture” in morphogenesis at the molecular level. Macrocolony morphology depends on self-produced extracellular matrix components. In Escherichia coli, these are stationary phase-induced amyloid curli fibers and cellulose. While the widely used “domesticated” E. coli K-12 laboratory strains are unable to generate cellulose, we could restore cellulose production and macrocolony morphology of E. coli K-12 strain W3110 by “repairing” a single chromosomal SNP in the bcs operon. Using scanning electron and fluorescence microscopy, cellulose filaments, sheets and nanocomposites with curli fibers were localized in situ at cellular resolution within the physiologically two-layered macrocolony biofilms of this “de-domesticated” strain. As an architectural element, cellulose confers cohesion and elasticity, i.e., tissue-like properties that—together with the cell-encasing curli fiber network and geometrical constraints in a growing colony—explain the formation of long and high ridges and elaborate wrinkles of wild-type macrocolonies. In contrast, a biofilm matrix consisting of the curli fiber network only is brittle and breaks into a pattern of concentric dome-shaped rings separated by deep crevices. These studies now set the stage for clarifying how regulatory networks and in particular c-di-GMP signaling operate in the three-dimensional space of highly structured and “tissue-like” bacterial biofilms.     

The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var “Turkish Samsun” control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by “Samsun” chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.     

Bayesian Analysis of Congruence of Core Genes in Prochlorococcus and Synechococcus and Implications on Horizontal Gene Transfer

It is often suggested that horizontal gene transfer is so ubiquitous in microbes that the concept of a phylogenetic tree representing the pattern of vertical inheritance is oversimplified or even positively misleading. “Universal proteins” have been used to infer the organismal phylogeny, but have been criticized as being only the “tree of one percent.” Currently, few options exist for those wishing to rigorously assess how well a universal protein phylogeny, based on a relative handful of well-conserved genes, represents the phylogenetic histories of hundreds of genes. Here, we address this problem by proposing a visualization method and a statistical test within a Bayesian framework. We use the genomes of marine cyanobacteria, a group thought to exhibit substantial amounts of HGT, as a test case. We take 379 orthologous gene families from 28 cyanobacteria genomes and estimate the Bayesian posterior distributions of trees – a “treecloud” – for each, as well as for a concatenated dataset based on putative “universal proteins.” We then calculate the average distance between trees within and between all treeclouds on various metrics and visualize this high-dimensional space with non-metric multidimensional scaling (NMMDS). We show that the tree space is strongly clustered and that the universal protein treecloud is statistically significantly closer to the center of this tree space than any individual gene treecloud. We apply several commonly-used tests for incongruence/HGT and show that they agree HGT is rare in this dataset, but make different choices about which genes were subject to HGT. Our results show that the question of the representativeness of the “tree of one percent” is a quantitative empirical question, and that the phylogenetic central tendency is a meaningful observation even if many individual genes disagree due to the various sources of incongruence.     

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm “dynamics govern specificity” might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: A Connected Model of the Photosynthetic Unit

The concept of photosynthetic unit (PSU) is reviewed in the light of the authors' results in the fields of fluorescence and luminescence (delayed light). Models of PSU are mainly distinguished by the amount of exciton exchange which is allowed between units. The “separate” model, with its “first-order” character, is not consistent with fluorescence kinetic data. The sigmoidal rise of fluorescence under actinic light is best explained by “nonseparate” models; however, most of these models assume a delocalization of excitons or centers. The “connected” model introduced here is not subject to this criticism. It discloses a new effect (the “îlot” effect): a nonrandom grouping of fluorescent units the consequences of which are discussed. It is noted that a “two-quantum” model for the photochemical reaction gives results very similar to those of the connected model. A relation between luminescence intensity and fluorescence yield is seen as a necessary consequence of the PSU concept. Its meaning is different in separate and nonseparate models. This relation is discussed in connection with the true system II fluorescence emission.     

Developmental Trajectories of Body Mass Index Among Japanese Children and Impact of Maternal Factors during Pregnancy

Background

The aims of this study were to 1) determine the distinct patterns of body mass index (BMI) trajectories in Japanese children, and 2) elucidate the maternal factors during pregnancy, which contribute to the determination of those patterns.

Methodology/Principal Findings

All of the children (1,644 individuals) born in Koshu City, Japan, between 1991 and 1998 were followed in a longitudinal study exploring the subjects’ BMI. The BMI was calculated 11 times for each child between birth and 12 years of age. Exploratory latent class growth analyses were conducted to identify trajectory patterns of the BMI z-scores. The distribution of BMI trajectories were best characterized by a five-group model for boys and a six-group model for girls. The groups were named “stable thin,” “stable average,” “stable high average,” “progressive overweight,” and “progressive obesity” in both sexes; girls were allocated to an additional group called “progressive average.” Multinomial logistic regression found that maternal weight, smoking, and skipping breakfast during pregnancy were associated with children included in the progressive obesity pattern rather than the stable average pattern. These associations were stronger for boys than for girls.

Conclusions/Significance

Multiple developmental patterns in Japanese boys and girls were identified, some of which have not been identified in Western countries. Maternal BMI and some unfavorable behaviors during early pregnancy may impact a child’s pattern of body mass development. Further studies to explain the gender and regional differences that were identified are warranted, as these may be important for early life prevention of weight-associated health problems.     

Sperm surface galactosyltransferase activities during in vitro capacitation

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as “decapacitation factors” when added back to in vitro fertilization assays. These glycoside “decapacitation factors” inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces “decapacitation factio” competition. On the other hand “conventional” low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of “decapacitation factos” is a necessary prerequisite for sperm binding to the zona pellucida.     

Logic Gate Operation by DNA Translocation through Biological Nanopores

Logical operations using biological molecules, such as DNA computing or programmable diagnosis using DNA, have recently received attention. Challenges remain with respect to the development of such systems, including label-free output detection and the rapidity of operation. Here, we propose integration of biological nanopores with DNA molecules for development of a logical operating system. We configured outputs “1” and “0” as single-stranded DNA (ssDNA) that is or is not translocated through a nanopore; unlabeled DNA was detected electrically. A negative-AND (NAND) operation was successfully conducted within approximately 10 min, which is rapid compared with previous studies using unlabeled DNA. In addition, this operation was executed in a four-droplet network. DNA molecules and associated information were transferred among droplets via biological nanopores. This system would facilitate linking of molecules and electronic interfaces. Thus, it could be applied to molecular robotics, genetic engineering, and even medical diagnosis and treatment.