To signal a conjunction of many inputs negative regulation is likely

Cells make many transitions from an old to a new phase of activity - between inactive and active states of an enzyme, or between phases of the cell cycle. If a cell is to survive, molecular prerequisites for functioning in the new phase should be available before a transition occurs. The cell’s survival is more likely if a regulatory network gates the transition, preventing its occurrence until the prerequisites are available. Suppose a specific conjunction of inputs is required for a network, from which a single output governs the transition. Then we suggest that cells are likely to use negative regulation - a gating network based on a logical disjunction of signals for the absence of prerequisites - rather than positive regulation - a logical conjunction of signals for their presence. That is, if a logical conjunction of n prerequisites A1 ANDA2 AND … ANDAn is needed in the new phase, a negative regulatory network is likely to enforce the corresponding logical disjunction, NOT (NOTA1 ORNOTA2 OR … ORNOTAn). Five examples illustrate this conclusion. Arguments based on performance criteria support the hypothesis: negative regulation is more economical than positive regulation, because networks for computing OR can use fewer and simpler parts than those for computing AND. Negative regulation can increase reliability, because a mechanism that uses fewer, simpler parts is less likely to fail. And, a negative regulatory network can be more robust - less susceptible to errors resulting from noisy input.     

A linear function for the melting behavior of lipids

The melting behavior of a variety of saturated long chain compounds is shown to be related to hydrocarbon chain length by the equation TN = C₀ + T_∞N where T is the absolute melting temperature, and N is the number of long chain carbon atoms. The constants C₀ and T_∞ are determined graphically or analytically from TN vs. N data. The linear relationship, derived from fundamental thermodynamic principles, is empirically satisfied. For each homologous series considered, coefficients of the equation provide a rational means for correlation and comparison with other polymorphs and indicate the relative importance of chain length, chain parity (even or odd), and headgroup polarity to melting behavior.     

Behaviour of a glycosphingolipid with unsaturated fatty acid in phosphatidylcholine bilayers

N-(Oleoyl)galactosylceramide with perdeuterated acyl chain was prepared by partial synthesis, and studied by wide line ²H-NMR in phospholipid liposomes. Spectra were obtained for low glycolipid concentrations in bilayers of dimyristoyl-, distearoyl-, and 1-palmitoyl-2-oleoylphosphatidylcholines. In an attempt to isolate the effects of glycosphingolipid fatty acid cis unsaturation on glycolipid behaviour in membranes, spectral findings related to the above species were compared to literature NMR data for pure 1-palmitoyl-2-oleoylphosphatidylcholine bilayers in which the oleoyl chain of the phospholipid had been deuterated, and to analogously deuterated glycerol based lipids in Acholeplasma laidlawii membranes. The results for N-(oleoyl-d₃₃)galactosylceramide proved to be qualitatively and quantitatively very similar to published data dealing with glycerol based lipids at comparable temperatures. In addition, the results were strikingly similar for glycolipids dispersed in saturated and unsaturated phospholipid host matrices. It would appear that the primary effects of cis 9,10 fatty acid unsaturation in glycosphingolipids (at low concentration in fluid phospholipid membranes) are the same as those of fatty acid cis unsaturation in glycerolipids. It further appears that the overall dynamic behaviour of N-(oleoyl)galactosylceramide in fluid phospholipid membranes is very similar to that of glycerolipids with comparable acyl chains.     

Equilibria and stability of a class of positive feedback loops

Positive feedback loops are common regulatory elements in metabolic and protein signalling pathways. The length of such feedback loops determines stability and sensitivity to network perturbations. Here we provide a mathematical analysis of arbitrary length positive feedback loops with protein production and degradation. These loops serve as an abstraction of typical regulation patterns in protein signalling pathways. We first perform a steady state analysis and, independently of the chain length, identify exactly two steady states that represent either biological activity or inactivity. We thereby provide two formulas for the steady state protein concentrations as a function of feedback length, strength of feedback, as well as protein production and degradation rates. Using a control theory approach, analysing the frequency response of the linearisation of the system and exploiting the Small Gain Theorem, we provide conditions for local stability for both steady states. Our results demonstrate that, under some parameter relationships, once a biological meaningful on steady state arises, it is stable, while the off steady state, where all proteins are inactive, becomes unstable. We apply our results to a three-tier feedback of caspase activation in apoptosis and demonstrate how an intermediary protein in such a loop may be used as a signal amplifier within the cascade. Our results provide a rigorous mathematical analysis of positive feedback chains of arbitrary length, thereby relating pathway structure and stability.     

Bayesian Geostatistical Analysis and Ecoclimatic Determinants of Corynebacterium pseudotuberculosis Infection among Horses

Kansas witnessed an unprecedented outbreak in Corynebacterium pseudotuberculosis infection among horses, a disease commonly referred to as pigeon fever during fall 2012. Bayesian geostatistical models were developed to identify key environmental and climatic risk factors associated with C. pseudotuberculosis infection in horses. Positive infection status among horses (cases) was determined by positive test results for characteristic abscess formation, positive bacterial culture on purulent material obtained from a lanced abscess (n = 82), or positive serologic evidence of exposure to organism (≥1:512)(n = 11). Horses negative for these tests (n = 172)(controls) were considered free of infection. Information pertaining to horse demographics and stabled location were obtained through review of medical records and/or contact with horse owners via telephone. Covariate information for environmental and climatic determinants were obtained from USDA (soil attributes), USGS (land use/land cover), and NASA MODIS and NASA Prediction of Worldwide Renewable Resources (climate). Candidate covariates were screened using univariate regression models followed by Bayesian geostatistical models with and without covariates. The best performing model indicated a protective effect for higher soil moisture content (OR = 0.53, 95% CrI = 0.25, 0.71), and detrimental effects for higher land surface temperature (≥35°C) (OR = 2.81, 95% CrI = 2.21, 3.85) and habitat fragmentation (OR = 1.31, 95% CrI = 1.27, 2.22) for C. pseudotuberculosis infection status in horses, while age, gender and breed had no effect. Preventative and ecoclimatic significance of these findings are discussed.     

A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast

Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5′ and 3′ gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ~0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of ~40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.     

Synthesis, calorimetric studies, and crystal structures of N, O-diacylethanolamines with matched chains

Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of N-acylethanolamines (NAEs) under physiological conditions. Because the content of NAEs in a variety of organisms increases in response to stress, it is likely that DAEs may also be present in biomembranes. In view of this, a homologous series of DAEs with matched acyl chains (n = 10–20) have been synthesized and characterized. Transition enthalpies and entropies obtained from differential scanning calorimetry show that dry DAEs with even and odd acyl chains independently exhibit linear dependence on the chainlength. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-, O-Didecanoylethanolamine (DDEA), N-, O-dilauroylethanolamine (DLEA), and N-, O-dimyristoylethanolamine (DMEA) crystallized in the orthorhombic space group Pbc₂₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that DDEA, DLEA, and DMEA are isostructural and adopt an L-shaped structure with the N-acyl chain and the central ethanolamine moiety being essentially identical to the structure of N-acylethanolamines, whereas the O-acyl chain is linear with all-trans conformation. In all three DAEs, the lipid molecules are organized in a bilayer fashion wherein the N-acyl and O-acyl chains from adjacent layers oppose each other.     

Prenatal polymetallic exposure and small for gestational age: A case-control study in Taiyuan,China

BackgroundStudies focused on independent effects of metals on small for gestational age, failing to account for potential interdependence among metals.MethodsIn this case-control study, we selected 187 pregnant women and 187 matched controls from the First Hospital of Shanxi Medical University. Determination of 12 elements in the venous blood of pregnant women before delivery by ICP-MS. Logistic regression, weighted quantile sum regression (WQSR) and Bayesian kernel machine regression (BKMR) were used to estimate the overall effect and identify important mixture components that drive the associations with SGA.ResultsAn increased risk of SGA was associated with As (OR= 1.06,95%CI: 1.01,1.12), Cd (OR= 1.24,95%CI: 1.04,1.47) and Pb (OR= 1.05,95%CI: 1.02,1.08), while Zn (OR= 0.58,95%CI: 0.45,0.76) and Mn (OR= 0.97,95%CI: 0.94,0.99) were protective factors for SGA. In the WQSR positive model, the mixture of heavy metals has a positive combined effect on SGA (OR= 1.74,95%CI: 1.15, 2.62), with Sb and Cd having the highest weights. The BKMR models confirmed that the metal mixture was associated with decreased risk of SGA when the concentration of 12 metals was between the 30th percentile and the 65th percentile, and Zn and Cd had the greatest independent effect. Zn and SGA may not be linearly correlated, higher Zn level may reduce the effect of Cd on the risk of SGA.ConclusionsOur study suggested that exposure to multiple metals was associated with risk of SGA, and the observed association with multiple metals was dominated by Zn, Cd. Sb exposure during pregnancy may also increase the risk of SGA.     

Balance of Interactions Determines Optimal Survival in Multi-Species Communities

We consider a multi-species community modelled as a complex network of populations, where the links are given by a random asymmetric connectivity matrix J, with fraction 1 − C of zero entries, where C reflects the over-all connectivity of the system. The non-zero elements of J are drawn from a Gaussian distribution with mean μ and standard deviation σ. The signs of the elements J _ij reflect the nature of density-dependent interactions, such as predatory-prey, mutualism or competition, and their magnitudes reflect the strength of the interaction. In this study we try to uncover the broad features of the inter-species interactions that determine the global robustness of this network, as indicated by the average number of active nodes (i.e. non-extinct species) in the network, and the total population, reflecting the biomass yield. We find that the network transitions from a completely extinct system to one where all nodes are active, as the mean interaction strength goes from negative to positive, with the transition getting sharper for increasing C and decreasing σ. We also find that the total population, displays distinct non-monotonic scaling behaviour with respect to the product μC, implying that survival is dependent not merely on the number of links, but rather on the combination of the sparseness of the connectivity matrix and the net interaction strength. Interestingly, in an intermediate window of positive μC, the total population is maximal, indicating that too little or too much positive interactions is detrimental to survival. Rather, the total population levels are optimal when the network has intermediate net positive connection strengths. At the local level we observe marked qualitative changes in dynamical patterns, ranging from anti-phase clusters of period 2 cycles and chaotic bands, to fixed points, under the variation of mean μ of the interaction strengths. We also study the correlation between synchronization and survival, and find that synchronization does not necessarily lead to extinction. Lastly, we propose an effective low dimensional map to capture the behavior of the entire network, and this provides a broad understanding of the interplay of the local dynamical patterns and the global robustness trends in the network.     

Hydrolysis of polynucleotides and the characterization of their secondary structure. A theoretical study

1. Single-stranded RNA may be regarded as an assembly of L hairpin loops each stabilized by N base pairs and each containing b unpaired residues; one loop is connected to another by c residues. 2. A theory based on the statistics of the random degradation of linear polymers was developed to relate N, b and c with the probability, p, of hydrolysing a diesterified phosphate bond. 3. The number of residues per hairpin loop, which is 2N+b, is related to the fraction, f, of the original loops remaining intact by the equation: 2N+b=logf/log(1–p). 4. The theory was extended to show that the number of residues per loop may be evaluated by fractionating the RNA after hydrolysis and examining the secondary structure of each fraction. Fragments that are shorter than the hairpin loop cannot reproduce the original secondary structure. The probability that a fragment will form an intact loop increases most rapidly for fragments of between 2N+b and 2(2N+b)+c residues. 5. The probability of producing a fragment capable of forming one, and only one, hairpin loop was related to N, b and c.     

Genes for fibrogenesis in the determination of susceptibility to myocardial infarction

A group of patients with ischemic heart disease and myocardial infarction (N = 156) and a reference population sample (N = 300) were genotyped for 58 single nucleotide polymorphisms (SNPs) in the genes involved in extracellular matrix function and collagen metabolism or associated with cardiovascular diseases and atherosclerotic plaque stability. Genotyping was performed by mass-spectrometry with two multiplex sets of 27 and 31 SNPs. The study revealed different genetic composition of predisposition to cardiovascular disease continuum (CVDC) syntropy (patients with concomitant conditions: hypercholesterolemia, hypertension, and type-II diabetes mellitus, N = 96) and to isolated myocardial infarction (without these conditions, N = 60). Only the KIAA1462 gene (rs3739998) showed associations with both CVDC syntropy (OR = 1.71; 95% CI 1.19–2.45; р = 0.003) and isolated infarction (OR = 1.58; 95% CI 1.05–2.40; р = 0.028). Isolated myocardial infarction was also associated with LIG1 (rs20579) (OR = 2.08; 95% CI 1.06–4.17; р = 0.028) and ADAMDEC1 (rs3765124) (OR = 1.63; 95% CI 1.07–2.50; р = 0.020). CVDC syntropy was associated with CDKN2BAS1 (rs1333049) (OR = 1.48; 95% CI 1.03–2.12; р = 0.029) and APOA2 (rs5082) (OR = 1.47; 95% CI 1.02–2.11; р = 0.035). So, genes involved in fibrogenesis contribute to predisposition to the myocardial infarction as well. Isolated myocardial infarction and CVDC syntropy can be considered as pathogenetically different cardiovascular conditions, with different genes that contribute to the susceptibility.     

Loop energy in DNA

The loop function ?(N), representing the statistical weight of N complementary residues in a closed ring, has been determined by analysis of high-resolution melting curves of a series of recombinant homopoly(A · T)_N inserts in pBR322 DNA, where 150 > N > 50 base pairs (bp). Loops are found more stable and therefore presumably less elastic than expected for an ideal, freely jointed chain. A value of 97 ± 2 bp is obtained for the empirical orientation-stiffness parameter D in the expression for nonideal chains, ?(N) = (N + D)^?1.7. The 10% increase in apparent stiffness over that of an ideal chain closely coincides with the extent of residual stacking in the loop. It is thus concluded that the more favorable loop energy, such as expected of smaller loops, is due to the incipient helical orientation of some residues, predisposing the loop to reclosure. A quantitative loop function is essential for the prediction and assignment of domains in DNA.     

Improving melon and cucumber photosynthetic activity, mineral composition, and growth performance under salinity stress by grafting onto Cucurbita hybrid rootstocks

The aim of the current work was to determine whether grafting could improve salinity tolerance of melon and cucumber, and whether possible induction of tolerance to salt stress was associated with the protection of the photosynthetic apparatus. Two greenhouse experiments were carried out to determine gas exchange, mineral composition, growth and yield of melon (Cucumis melo L. cv. Cyrano) and cucumber (Cucumis sativus L. cv. Akito) plants, either ungrafted or grafted onto the Cucurbita hybrid rootstocks (Cucurbita maxima Duch. × Cucurbita moschata Duch.), ??P360??, and ??PS1313??, respectively. Plants were grown hydroponically and supplied with two nutrient solutions ?? a nonsalinized control and a salinized solution which contained 40 mmol L^?1 of NaCl. Salinity induced a smaller decrease in leaf area index (LAI), in grafted-compared to ungrafted plants. Similarly, the P _N and g _s reduction in NaCl treatment compared to control were significantly lower in grafted plants (34% and 34%, respectively, for melon and 14% and 15.5%, respectively, for cucumber) compared to ungrafted plants (42% and 40%, respectively, for melon and 30% and 21%, respectively, for cucumber). In all grafting combinations, negative correlations were recorded between Na⁺ and Cl^? in the leaf tissue and P _N. Grafting reduced concentrations of sodium, but not chloride, in leaves. Under saline conditions a smaller reduction in melon and cucumber shoot biomass dry mass and fruit yield were recorded, with positive correlations between shoot biomass, yield and P _N. These results suggest that the use of salt tolerant Cucurbita rootstock can improve melon and cucumber photosynthetic capacity under salt stress and consequently crop performance.     

Boolean analysis of cell regulation networks

A comparison is made between the predictions of the Boolean and continuous analysis of a regulation model when the formation of two mediators interacting by cross-inhibition is stimulated by one or two specific signals. For such a system, the Boolean analysis reproduces the characteristics of behaviour previously predicted by continuous analysis (multiple stable states of opposite type, discontinuous transition, and associated hysteresis phenomenon). The qualitative agreement between the two methods allows a qualitative but rigorous treatment of regulation systems in which the Boolean analysis is applicable. From a general schematic representation of interaction in bidirectional control systems, we analyse by the Boolean method a large range of possible systems of increasing complexities which could theoretically apply. Previously unforeseen consequences of some systems are described. After that, we give a logical analysis of a well-known system (negative loop grafted with additional external controls) and discuss the application of such a system to explain certain oscillatory phenomena in the cell, showing the disrupting role of an additional control on the expected behaviour. Thus, when the analysis of a model including a negative loop does not indicate the possibility of experimentally suggested oscillations, we propose other simple logical structures which can predict this behaviour. Finally, we show a logical analysis of an opposite type of example of cell regulation where the biochemical observations can be accounted for simply by a negative loop grafted with one input variable.     

Solution of Levinthal’s paradox is possible at the level of the formation and assembly of protein secondary structures

The complete volume of a protein’s conformation space is smaller by many orders of magnitude at the level of secondary-structure elements as compared with the conformation of amino-acid residues. According to Levinthal’s estimate, the latter is ~10^2L, with L being the number of residues in the chain, while the former, at the level of secondary structures, increases no faster than ~L^N with N being the number of the secondary-structure elements. N is approximately L/15 according to the statistics of protein structures. This drastic decrease in the exponent (L/15 instead of 2L) considerably reduces the sampling space and explains the reason that sampling of the conformation space at the level of secondary-structure elements does not prevent the protein chain from finding its most stable structure.     

An efficient in planta transformation of Jatropha curcas (L.) and multiplication of transformed plants through in vivo grafting

An efficient and reproducible Agrobacterium-mediated in planta transformation was developed in Jatropha curcas. The various factors affecting J. curcas in planta transformation were optimized, including decapitation, Agrobacterium strain, pin-pricking, vacuum infiltration duration and vacuum pressure. Simple vegetative in vivo cleft grafting method was adopted in the multiplication of transformants without the aid of tissue culture. Among the various parameters evaluated, decapitated plants on pin-pricking and vacuum infiltrated at 250 mmHg for 3 min with the Agrobacterium strain EHA 105 harbouring the binary vector pGA 492 was proved to be efficient in all terms with a transformation efficiency of 62.66 %. Transgene integration was evinced by the GUS histochemical analysis, and the GUS positive plants were subjected to grafting. Putatively transformed J. curcas served as "Scion" and the wild type J. curcas plant severed as "Stock". There was no occurrence of graft rejection and the plants were then confirmed by GUS histochemical analysis, polymerase chain reaction (PCR) and Southern hybridization. Genetic stability of the grafted plants was evaluated by using randomly amplified polymorphic DNA (RAPD), marker which showed 100 % genetic stability between mother and grafted plants. Thus, an efficient in planta transformation and grafting based multiplication of J. curcas was established.     

Cyclic Negative Feedback Systems: What is the Chance of Oscillation?

Many biological oscillators have a cyclic structure consisting of negative feedback loops. In this paper, we analyze the impact that the addition of a positive or a negative self-feedback loop has on the oscillatory behavior of the three negative feedback oscillators proposed by Tsai et al. (Science 231:126–129, 2008) where, in contrast with numerous oscillator models, the interactions between elements occur via the modulation of the degradation rates. Through analytical and computational studies we show that an additional self-feedback affects the oscillatory behavior. In the high-cooperativity limit, i.e., for large Hill coefficients, we derive exact analytical conditions for oscillations and show that the relative location between the dissociation constants of the Hill functions and the ratio of kinetic parameters determines the possibility of oscillatory activities. We compute analytically the probability of oscillations for the three models and show that the smallest domain of periodic behavior is obtained for the negative-plus-negative feedback system whereas the additional positive self-feedback loop does not modify significantly the chance to oscillate. We numerically investigate to what extent the properties obtained in the sharp situation applied in the smooth case. Results suggest that a switch-like coupling behavior, a time-scale separation, and a repressilator-type architecture with an even number of elements facilitate the emergence of sustained oscillations in biological systems. An additional positive self-feedback loop produces robustness and adaptability whereas an additional negative self-feedback loop reduces the chance to oscillate.     

Conformational aspects of N-glycosylation of proteins. Studies with linear and cyclic peptides as probes

Conformational aspects of N-glycosylation of glycoproteins have been studied by using a series of peptides which contained, in addition to the `marker sequence' Asn-Gly-Thr, two cysteine residues in various positions of the peptide chain. The presence of two cysteines permitted a partial fixation of the above triplet sequence in cyclic structures of various size by intramolecular disulphide bond formation. Comparison of the glycosyl acceptor properties of the linear peptides and their corresponding cyclic analogues allows the following statements. The considerably lower acceptor capabilities of the cyclic derivatives indicate that the restriction of rotational degrees of freedom imposed by disulphide bonding results in a conformation which hinders a favourable interaction of the peptide substrate with the N-glycosyltransferase. On the other hand, the glycosylation rate of linear peptides increases with increasing chain length, suggesting that the amino acids on both the N- and C-terminal side of the `marker sequence' may contribute to a considerable extent to the induction of an `active' conformation. Realization of a potential sugar attachment site requires a hydrogen bond interaction within the `marker sequence' between the oxygen of threonine (serine) as the hydrogen bond acceptor and the β-amide of asparagine as the donor [Bause & Legler (1981) Biochem. J. 195, 639–644]. This interaction is obviously facilitated when the peptide chain can adopt a conformation which resembles a β-turn or other loop structure. The available experimental and statistical data are discussed in terms of possible structural features for N-glycosylation, with the aid of space-filling models.