Estimating Population-Level Coancestry Coefficients by an Admixture F Model

In this article, we develop an admixture F model (AFM) for the estimation of population-level coancestry coefficients from neutral molecular markers. In contrast to the previously published F model, the AFM enables disentangling small population size and lack of migration as causes of genetic differentiation behind a given level of F_ST. We develop a Bayesian estimation scheme for fitting the AFM to multiallelic data acquired from a number of local populations. We demonstrate the performance of the AFM, using simulated data sets and real data on ninespine sticklebacks (Pungitius pungitius) and common shrews (Sorex araneus). The results show that the parameterization of the AFM conveys more information about the evolutionary history than a simple summary parameter such as F_ST. The methods are implemented in the R package RAFM.     

Atomic Force Microscopy of Plant-Parasitic Nematodes

A simple method for atomic force microscopy (AFM) of nematode cuticle was developed to visualize the external topography of Helicotylenchus lobus, Meloidogyne javanica, M. incognita, and Xiphinema diversicaudatum. Endospores of two isolates of the nematode parasite, Pasteuria penetrans, adhering to M. incognita and X. diversicaudatum were also visualized and measured by this technique. Scanning procedures were applied to specimens killed and dehydrated in air or dehydrated and stored in glycerol. Atomic force microscopy scanning of nematodes in constant height mode yielded replicated high-resolution images of the cuticle showing anatomical details such as annulations and lateral fields. Submicrometer scale images allowed the identification of planar regions for further higher resolution scans.     

Field Efficacy of Verticillium lecanii,Sex Pheromone,and Pheromone Analogs as Potential Management Agents for Soybean Cyst Nematode

A soybean cyst nematode sex pheromone (vanillic acid), chemical analogs of the pheromone, and the fungus Verticillium lecanii were applied in alginate prills (340 kg/ha) to microplots and small-scale field plots as potential management agents for Heterodera glycines on soybean. In 1991 microplot tests, treatment with V. lecanii, vanillic acid, syringic acid plus V. lecanii, or vanillic acid plus V. lecanii lowered midseason cyst numbers compared with the untreated susceptible cultivar control, autoclaved V. lecanii treatment, or aldicarb treatment, At-harvest cyst numbers were lowest with V. lecanii and with vanillic acid treatments. Aldicarb treatment reduced midseason cyst numbers in 1992. There were no differences among seed yields either year. In the field trials, numbers of cysts were reduced one or both years with aldicarb, ferulic acid, syringic acid, vanillic acid, or 4-hydroxy-3-methoxybenzonitfile treatments, or with a resistant cultivar, compared to an untreated susceptible cultivar. Highest yields were recorded after treatment with 4-hydroxy-3-methoxybenzonitrile (1991), methyl vanillate (1992), and aldicarb (1992). These studies indicate that some chemical analogs of vanillic acid have potential for use in soybean cyst nematode management schemes.     

Evidence for a pathway that facilitates nitric oxide diffusion in the brain

Nitric oxide (NO) is a diffusible messenger that conveys information based on its concentration dynamics, which is dictated by the interplay between its synthesis, inactivation and diffusion. Here, we characterized NO diffusion in the rat brain in vivo. By direct sub-second measurement of NO, we determined the diffusion coefficient of NO in the rat brain cortex. The value of 2.2 × 10⁻⁵ cm²/s obtained in vivo was only 14% lower than that obtained in agarose gel (used to evaluate NO free diffusion). These results reinforce the view of NO as a fast diffusing messenger but, noticeably, the data indicates that neither NO diffusion through the brain extracellular space nor homogeneous diffusion in the tissue through brain cells can account for the similarity between NO free diffusion coefficient and that obtained in the brain. Overall, the results support that NO diffusion in brain tissue is heterogeneous, pointing to the existence of a pathway that facilitates NO diffusion, such as cell membranes and other hydrophobic structures.     

Molecular shape and binding force of Mycoplasma mobile’s leg protein Gli349 revealed by an AFM study

Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349 kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the “leg” of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70 pN. These findings strongly support the idea that Gli349 is the “leg” protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.     

Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua

A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism.     

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.     

Application of a Sex Pheromone,Pheromone Analogs,and Verticillium lecanii for Management of Heterodera glycines

A mutant strain of the fungus Verticillium lecanii and selected bioregulators of Heterodera glycines were evaluated for their potential to reduce population densities of the nematode on soybean under greenhouse conditions. The bioregulators tested were the H. glycines sex pheromone vanillic acid and the pheromone analogs syringic acid, isovanillic acid, ferulic acid, 4-hydroxy-3-methoxybenzonitrile, and methyl vanillate. A V. lecanii-vanillic acid combination and a V. lecanii-syringic acid combination were also applied as treatments. Syringic acid, 4-hydroxy-3-methoxybenzonitrile, V. lecanii, V. lecanii-vanillic acid, and V. lecanii-syringic acid significantly reduced nematode population densities in the greenhouse tests. Results with vanillic acid, isovanillic acid, and ferulic acid treatments were variable. Methyl vanillate did not significantly reduce cyst nematode population densities in the greenhouse tests.     

Mortality of Pratylenchus penetrans by Volatile Fatty Acids from Liquid Hog Manure

As part of our research program assessing the use of liquid hog manure (LHM) to control root-lesion nematodes, Pratylenchus penetrans, a series of acute toxicity tests was conducted to: (i) examine if non-ionized forms of volatile fatty acids (VFA) are responsible for the mortality of P. penetrans exposed to LHM under acidic conditions, (ii) determine if Caenorhabditis elegans can be a surrogate for P. penetrans in screening tests by comparing their sensitivities to VFA, (iii) characterize the nematicidal effect of individual VFA in LHM to P. penetrans, and (iv) determine whether individual VFA in LHM interact in their toxicity to P. penetrans. LHM was significantly (P ≤ 0.05) more toxic to P. penetrans than a mixture of its main VFA components at concentrations of 5% and 10% (vol. VFA or LHM /vol. in buffer). Pratylenchus penetrans was more sensitive to acetic acid than C. elegans, whereas the sensitivity of both nematode species to n-caproic acid was similar. Individual VFA vary in their lethality to P. penetrans. n-valeric acid was the most toxic (LC₉₅= 6.8 mM), while isobutyric acid was the least toxic (LC₉₅ = 45.7 mM). Individual VFA did not interact in their toxicity to P. penetrans, and their effects were considered additive. VFA account for the majority of the lethal effect of LHM to P. penetrans under acidic conditions. Caenorhabditis elegans cannot be used as a surrogate to P. penetrans in toxicity studies using VFA. The efficacy of LHM to control P. penetrans can be evaluated by assessing its VFA content prior to application, and this evaluation is facilitated by the fact that the interaction of individual VFA appears to be simply additive.     

Evolutionary Constraints Favor a Biophysical Model Explaining Hox Gene Collinearity

The Hox gene collinearity enigma has often been approached using models based on biomolecular mechanisms. The biophysical model is an alternative approach based on the hypothesis that collinearity is caused by physical forces pulling the Hox genes from a territory where they are inactive to a distinct spatial domain where they are activated in a step by step manner. Such Hox gene translocations have recently been observed in support of the biophysical model. Genetic engineering experiments, performed on embryonic mice, gave rise to several unexpected mutant expressions that the biomolecular models cannot predict. On the contrary, the biophysical model offers convincing explanation. Evolutionary constraints consolidate the Hox clusters and as a result, denser and well organized clusters may create more efficient physical forces and a more emphatic manifestation of gene collinearity. This is demonstrated by stochastic modeling with white noise perturbing the expression of Hox genes. As study cases the genomes of mouse and amphioxus are used. The results support the working hypothesis that vertebrates have adopted their comparably more compact Hox clustering as a tool needed to develop more complex body structures. Several experiments are proposed in order to test further the physical forces hypothesis.     

Microbial biodegradation of aromatic alkanoic naphthenic acids is affected by the degree of alkyl side chain branching

Naphthenic acids (NAs) occur naturally in oil sands and enter the environment through natural and anthropogenic processes. NAs comprise toxic carboxylic acids that are difficult to degrade. Information on NA biodegradation mechanisms is limited, and there are no studies on alkyl branched aromatic alkanoic acid biodegradation, despite their contribution to NA toxicity and recalcitrance. Increased alkyl side chain branching has been proposed to explain NA recalcitrance. Using soil enrichments, we examined the biodegradation of four aromatic alkanoic acid isomers that differed in alkyl side chain branching: (4′-n-butylphenyl)-4-butanoic acid (n-BPBA, least branched); (4′-iso-butylphenyl)-4-butanoic acid (iso-BPBA); (4′-sec-butylphenyl)-4-butanoic acid (sec-BPBA) and (4′-tert-butylphenyl)-4-butanoic acid (tert-BPBA, most branched). n-BPBA was completely metabolized within 49 days. Mass spectral analysis confirmed that the more branched isomers iso-, sec- and tert-BPBA were transformed to their butylphenylethanoic acid (BPEA) counterparts at 14 days. The BPEA metabolites were generally less toxic than BPBAs as determined by Microtox assay. n-BPEA was further transformed to a diacid, showing that carboxylation of the alkyl side chain occurred. In each case, biodegradation of the carboxyl side chain proceeded through beta-oxidation, which depended on the degree of alkyl side chain branching, and a BPBA degradation pathway is proposed. Comparison of 16S rRNA gene sequences at days 0 and 49 showed an increase and high abundance at day 49 of Pseudomonas (sec-BPBA), Burkholderia (n-, iso-, tert-BPBA) and Sphingomonas (n-, sec-BPBA).     

Analysis of the formation of flower shapes in wild species and cultivars of tree peony using the MADS-box subfamily gene

Tree peony (Paeonia suffricotisa) cultivars have a unique character compared with wild species; the stamen petalody results in increased whorls of petals and generates different flower forms, which are one of the most important traits for cultivar classification. In order to investigate how petaloid stamens are formed, we obtained the coding sequence (666 bp) and genomic DNA sequence of the PsTM6 genes (belongs to B subfamily of MADS-box gene family) from 23 tree peony samples, Five introns and six exons consisted of the genomic DNA sequence. The analysis of cis-acting regulatory elements in the third and fourth intron indicated that they were highly conserved in all samples. Partial putative amino acids were analyzed and the results suggested that functional differentiation of PsTM6 paralogs apparently affected stamen petalody and flower shape formation due to due to amino acid substitution caused by differences in polarity and electronic charge. Sliding window analysis indicated that the different regions of PsTM6 were subjected to different selection forces, especially in the K domain. This is the first attempt to investigate genetic control of the stamen petalody based on the PsTM6 sequence. This will provide a basis for understanding the evolution of PsTM6 and its the function of in determining stamen morphology of tree peony.     

Nanoscale Cell Wall Deformation Impacts Long-Range Bacterial Adhesion Forces on Surfaces

Adhesion of bacteria occurs on virtually all natural and synthetic surfaces and is crucial for their survival. Once they are adhering, bacteria start growing and form a biofilm, in which they are protected against environmental attacks. Bacterial adhesion to surfaces is mediated by a combination of different short- and long-range forces. Here we present a new atomic force microscopy (AFM)-based method to derive long-range bacterial adhesion forces from the dependence of bacterial adhesion forces on the loading force, as applied during the use of AFM. The long-range adhesion forces of wild-type Staphylococcus aureus parent strains (0.5 and 0.8 nN) amounted to only one-third of these forces measured for their more deformable isogenic Δpbp4 mutants that were deficient in peptidoglycan cross-linking. The measured long-range Lifshitz-Van der Waals adhesion forces matched those calculated from published Hamaker constants, provided that a 40% ellipsoidal deformation of the bacterial cell wall was assumed for the Δpbp4 mutants. Direct imaging of adhering staphylococci using the AFM peak force-quantitative nanomechanical property mapping imaging mode confirmed a height reduction due to deformation in the Δpbp4 mutants of 100 to 200 nm. Across naturally occurring bacterial strains, long-range forces do not vary to the extent observed here for the Δpbp4 mutants. Importantly, however, extrapolating from the results of this study, it can be concluded that long-range bacterial adhesion forces are determined not only by the composition and structure of the bacterial cell surface but also by a hitherto neglected, small deformation of the bacterial cell wall, facilitating an increase in contact area and, therewith, in adhesion force.     

Pipecolic acid enhances resistance to bacterial infection and primes salicylic acid and nicotine accumulation in tobacco

Distinct amino acid metabolic pathways constitute integral parts of the plant immune system. We have recently identified pipecolic acid (Pip), a lysine-derived non-protein amino acid, as a critical regulator of systemic acquired resistance (SAR) and basal immunity to bacterial infection in Arabidopsis thaliana. In Arabidopsis, Pip acts as an endogenous mediator of defense amplification and priming. For instance, Pip conditions plants for effective biosynthesis of the phenolic defense signal salicylic acid (SA), accumulation of the phytoalexin camalexin, and expression of defense-related genes. Here, we show that tobacco plants respond to leaf infection by the compatible bacterial pathogen Pseudomonas syringae pv tabaci (Pstb) with a significant accumulation of several amino acids, including Lys, branched-chain, aromatic, and amide group amino acids. Moreover, Pstb strongly triggers, alongside the biosynthesis of SA and increases in the defensive alkaloid nicotine, the production of the Lys catabolites Pip and α-aminoadipic acid. Exogenous application of Pip to tobacco plants provides significant protection to infection by adapted Pstb or by non-adapted, hypersensitive cell death-inducing P. syringae pv maculicola. Pip thereby primes tobacco for rapid and strong accumulation of SA and nicotine following bacterial infection. Thus, our study indicates that the role of Pip as an amplifier of immune responses is conserved between members of the rosid and asterid groups of eudicot plants and suggests a broad practical applicability for Pip as a natural enhancer of plant disease resistance.     

Mimicking SP-C palmitoylation on a peptoid-based SP-B analogue markedly improves surface activity

Hydrophobic lung surfactant proteins B and C (SP-B and SP-C) are critical for normal respiration in vertebrates, and each comprises specific structural attributes that enable the surface-tension-reducing ability of the lipid-protein mixture in lung surfactant. The difficulty in obtaining pure SP-B and SP-C on a large scale has hindered efforts to develop a non-animal-derived surfactant replacement therapy for respiratory distress. Although peptide-based SP-C mimics exhibit similar activity to the natural protein, helical peptide-based mimics of SP-B benefit from dimeric structures. To determine if in vitro surface activity improvements in a mixed lipid film could be garnered without creating a dimerized structural motif, a helical and cationic peptoid-based SP-B mimic was modified by SP-C-like N-terminus alkylation with octadecylamine. “Hybridized” mono- and dialkylated peptoids significantly decreased the maximum surface tension of the lipid film during cycling on the pulsating bubble surfactometer relative to the unalkylated variant. Peptoids were localized in the fluid phase of giant unilamellar vesicle lipid bilayers, as has been described for SP-B and SP-C. Using Langmuir-Wilhelmy surface balance epifluorescence imaging (FM) and atomic force microscopy (AFM), only lipid-alkylated peptoid films revealed micro- and nanostructures closely resembling films containing SP-B. AFM images of lipid-alkylated peptoid films showed gel condensed-phase domains surrounded by a distinct phase containing “nanosilo” structures believed to enhance re-spreading of submonolayer material. N-terminus alkylation may be a simple, effective method for increasing lipid affinity and surface activity of single-helix SP-B mimics.     

Exploiting Natural Variation of Secondary Metabolism Identifies a Gene Controlling the Glycosylation Diversity of Dihydroxybenzoic Acids in Arabidopsis thaliana

Plant secondary metabolism is an active research area because of the unique and important roles the specialized metabolites have in the interaction of plants with their biotic and abiotic environment, the diversity and complexity of the compounds and their importance to human medicine. Thousands of natural accessions of Arabidopsis thaliana characterized with increasing genomic precision are available, providing new opportunities to explore the biochemical and genetic mechanisms affecting variation in secondary metabolism within this model species. In this study, we focused on four aromatic metabolites that were differentially accumulated among 96 Arabidopsis natural accessions as revealed by leaf metabolic profiling. Using UV, mass spectrometry, and NMR data, we identified these four compounds as different dihydroxybenzoic acid (DHBA) glycosides, namely 2,5-dihydroxybenzoic acid (gentisic acid) 5-O-β-D-glucoside, 2,3-dihydroxybenzoic acid 3-O-β-D-glucoside, 2,5-dihydroxybenzoic acid 5-O-β-D-xyloside, and 2,3-dihydroxybenzoic acid 3-O-β-D-xyloside. Quantitative trait locus (QTL) mapping using recombinant inbred lines generated from C24 and Col-0 revealed a major-effect QTL controlling the relative proportion of xylosides vs. glucosides. Association mapping identified markers linked to a gene encoding a UDP glycosyltransferase gene. Analysis of Transfer DNA (T-DNA) knockout lines verified that this gene is required for DHBA xylosylation in planta and recombinant protein was able to xylosylate DHBA in vitro. This study demonstrates that exploiting natural variation of secondary metabolism is a powerful approach for gene function discovery.     

Role of Capsular Colanic Acid in Adhesion of Uropathogenic Escherichia coli

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.     

Effects of Inducers of Systemic Acquired Resistance on Reproduction of Meloidogyne javanica and Rotylenchulus reniformis in Pineapple

The potency of the inducers of systemic acquired resistance (SAR), acibenzolar-s-methyl, DL-α-amino-n-butyric acid (AABA), DL-β-amino-n-butyric acid (BABA), γ-amino-n-butyric acid (GABA), p-aminobenzoic acid (PABA), riboflavin, and salicylic acid (SA), in reducing reproduction of Meloidogyne javanica and Rotylenchulus reniformis in pineapple was investigated. All inducers were applied as foliar sprays to 1-mon-old pineapple plants (20 ml/plant) grown in 22-cm-diam. pots in the greenhouse. Two days after application, 10,000 eggs of M. javanica or R. reniformis were inoculated onto the plants. Six months after inoculation, nematode reproduction was measured. Acibenzolar decreased R. reniformis egg production by 58% compared to the nontreated control (P ≤ 0.05). Acibenzolar, BABA, and riboflavin reduced M. javanica egg production by 60% to 64% compared to the nontreated control (P ≤ 0.05). The point in the pineapple SAR pathway that each compound activates may explain the differing results between M. javanica and its giant cells and R. reniformis and its syncytia. Foliar application of acibenzolar at 100 and 200 mg/liter decreased by 30% and 60%, respectively, the number of M. javanica eggs as compared to the nontreated control. Fresh shoot weight of pineapple treated with 50, 100, 200, and 400 mg/liter acibenzolar was reduced by 1.2%, 3.3%, 9.9%, and 33% compared to the nontreated pineapple, respectively (P ≤ 0.05). Foliar application of acibenzolar may activate intrinsic resistance of pineapple to M. javanica and R. reniformis and may have a role in the sustainable management of nematodes in pineapple.