Tobacco Calcium-dependent Protein Kinases Are Differentially Phosphorylated in Vivo as Part of a Kinase Cascade That Regulates Stress Response

In vivo phosphorylation sites of the tobacco calcium-dependent protein kinases NtCDPK2 and NtCDPK3 were determined in response to biotic or abiotic stress. Stress-inducible phosphorylation was exclusively located in the variable N termini, where both kinases were phosphorylated differentially despite 91% overall sequence identity. In NtCDPK2, serine 40 and threonine 65 were phosphorylated within 2 min after stress. Whereas Thr⁶⁵ is subjected to intra-molecular in vivo autophosphorylation, Ser⁴⁰ represents a target for a regulatory upstream protein kinase, and correct NtCDPK2 membrane localization is required for Ser⁴⁰ phosphorylation. NtCDPK3 is phosphorylated at least at two sites in the N terminus by upstream kinase(s) upon stress stimulus, first at Ser⁵⁴, a site not present in NtCDPK2, and also at a second undetermined site not identical to Ser⁴⁰. Domain swap experiments established that differential phosphorylation of both kinases is exclusively determined by the respective N termini. A cell death-inducing response was only observed upon expression of a truncated variant lacking the junction and calcium-binding domain of NtCDPK2 (VK2). This response required protein kinase activity and was reduced when subcellular membrane localization was disturbed by a mutation in the myristoylation and palmitoylation site. Our data indicate that CDPKs are integrated in stress-dependent protein kinase signaling cascades, and regulation of CDPK function in response to in vivo stimulation is dependent on its membrane localization.     

Density functional study of oxygen vacancy formation and spin density distribution in octahedral ceria nanoparticles

We report plane wave basis density functional theory (DFT) calculations of the oxygen vacancies formation energy in nanocrystalline CeO _2-x in comparison with corresponding results for bulk and (111) CeO₂ surface. Effects of strong electronic correlation of Ce4f states are taken into account through the use of an effective on-site Coulomb repulsive interaction within DFT+U approach. Different combinations of exchange-correlation functionals and corresponding U values reported in the literature are tested and the obtained results compared with experimental data. We found that both absolute values and trends in oxygen vacancy formation energy depend on the value of U and associated with degree of localization of Ce4f states. Effect of oxygen vacancy and geometry optimization method on spatial spin distribution in model ceria nanoparticles is also discussed.     

C-banded wheat chromosomes in wheat and triticale

Summary The C-banding patterns of wheat chromosomes in 7 hexaploid triticale and 7 wheat genotypes are described and compared. All 14 wheat chromosome pairs were individually identified in the triticales and a tetraploid wheat, and all the B and two A genome chromosome pairs in the hexaploid wheat genotypes. Little variation was found between genotypes in the distribution of C-bands but considerable variation was found in their size, total number and total length.     

Lysine provisioning by horizontally acquired genes promotes mutual dependence between whitefly and two intracellular symbionts

Horizontal gene transfer is widespread in insects bearing intracellular symbionts. Horizontally transferred genes (HTGs) are presumably involved in amino acid synthesis in sternorrhynchan insects. However, their role in insect-symbiont interactions remains largely unknown. We found symbionts Portiera, Hamiltonella and Rickettsia possess most genes involved in lysine synthesis in the whitefly Bemisia tabaci MEAM1 although their genomes are reduced. Hamiltonella maintains a nearly complete lysine synthesis pathway. In contrast, Portiera and Rickettsia require the complementation of whitefly HTGs for lysine synthesis and have lysE, encoding a lysine exporter. Furthermore, each horizontally transferred lysine gene of ten B. tabaci cryptic species shares an evolutionary origin. We demonstrated that Hamiltonella did not alter the titers of Portiera and Rickettsia or lysine gene expression of Portiera, Rickettsia and whiteflies. Hamiltonella also did not impact on lysine levels or protein localization in bacteriocytes harboring Portiera and ovaries infected with Rickettsia. Complementation with whitefly lysine synthesis HTGs rescued E. coli lysine gene knockout mutants. Silencing whitefly lysA in whiteflies harboring Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia without influencing the expression of Hamiltonella lysA. Furthermore, silencing whitefly lysA in whiteflies lacking Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia in ovarioles. Therefore, we, for the first time, demonstrated an essential amino acid lysine synthesized through HTGs is important for whitefly reproduction and fitness of both obligate and facultative symbionts, and it illustrates the mutual dependence between whitefly and its two symbionts. Collectively, this study reveals that acquisition of horizontally transferred lysine genes contributes to coadaptation and coevolution between B. tabaci and its symbionts.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

Quantitation of the niacin metabolites 1-methylnicotinamide and l-methyl-2-pyridone-5-carboxamide in random spot urine samples, by ion-pairing reverse-phase HPLC with UV detection, and the implications for the use of spot urine samples in the assessment of niacin status

A simple ion-pairing reverse-phase HPLC method, with UV diode array detection, was developed and validated for quantitation of the urinary niacin metabolites 1-methylnicotinamide and l-methyl-2-pyridone-5-carboxamide in a single run. Urine samples were purified using a polymer-based mixed mode anion exchange reverse-phase cartridge. Analysis was performed on a reverse-phase C18 column, using a methanol gradient elution system, containing phosphate buffer pH 7.0, 1-heptanesulphonic acid as the ion-pairing agent and trimethylamine as a modifier. The assay was applied to the measurement of the niacin status of two subjects using spot urine samples. The samples were collected over 4 consecutive days and at four time points during 1 day. Status, expressed as the concentration ratios (2-PYR or 1-MN)/creatinine and 2-PYR/l-MN, varied within and between days and was least for fasting samples. This work illustrates the potential of spot urine sampling for niacin status assessment, but highlights the need for further validation prior to its use in field nutritional surveys.     

Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.     

Identification and analysis of products formed from phospholipids in the free radical oxidation of human low density lipoproteins

Phospholipids reside in the surface layer of LDLs and constitute approximately 20-25% of the particle by weight. We report a study of the primary products generated from the most abundant molecular species of phosphatidylcholines present in LDL during in vitro free radical oxidations. The 13-hydroperoxides of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) and 1-stearoyl-2-linoleoyl-sn-glycero-phosphocholine (SLPC) and the 15-hydroperoxides of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphocholine (SAPC) were found to increase in a time-dependent manner and in significant amounts even in the presence of alpha-tocopherol. Phospholipid alcohols also formed during the course of the oxidations. Early in the LDL oxidations, while alpha-tocopherol was still present, the thermodynamically favored trans,trans products of PLPC and SLPC were found to form in significantly larger quantities than those formed from cholesteryl linoleate. Additionally, quantities of PAPC 11-hydroperoxide (11-OOH) decreased over time relative to PAPC 15-OOH, even while alpha-tocopherol was still present in the oxidation, presumably as a result of further oxidation of PAPC 11-OOH to form cyclic peroxide oxidation products. These results suggest that alpha-tocopherol is more closely associated with the inner cholesteryl ester-rich hydrophobic core of an LDL particle and is not as effective as an antioxidant in the outer phospholipid layer as it is in the lipid core.     

Agent-based dynamic knowledge representation of Pseudomonas aeruginosa virulence activation in the stressed gut: Towards characterizing host-pathogen interactions in gut-derived sepsis

Background

There is a growing realization that alterations in host-pathogen interactions (HPI) can generate disease phenotypes without pathogen invasion. The gut represents a prime region where such HPI can arise and manifest. Under normal conditions intestinal microbial communities maintain a stable, mutually beneficial ecosystem. However, host stress can lead to changes in environmental conditions that shift the nature of the host-microbe dialogue, resulting in escalation of virulence expression, immune activation and ultimately systemic disease. Effective modulation of these dynamics requires the ability to characterize the complexity of the HPI, and dynamic computational modeling can aid in this task. Agent-based modeling is a computational method that is suited to representing spatially diverse, dynamical systems. We propose that dynamic knowledge representation of gut HPI with agent-based modeling will aid in the investigation of the pathogenesis of gut-derived sepsis.

Methodology/Principal Findings

An agent-based model (ABM) of virulence regulation in Pseudomonas aeruginosa was developed by translating bacterial and host cell sense-and-response mechanisms into behavioral rules for computational agents and integrated into a virtual environment representing the host-microbe interface in the gut. The resulting gut milieu ABM (GMABM) was used to: 1) investigate a potential clinically relevant laboratory experimental condition not yet developed - i.e. non-lethal transient segmental intestinal ischemia, 2) examine the sufficiency of existing hypotheses to explain experimental data - i.e. lethality in a model of major surgical insult and stress, and 3) produce behavior to potentially guide future experimental design - i.e. suggested sample points for a potential laboratory model of non-lethal transient intestinal ischemia. Furthermore, hypotheses were generated to explain certain discrepancies between the behaviors of the GMABM and biological experiments, and new investigatory avenues proposed to test those hypotheses.

Conclusions/Significance

Agent-based modeling can account for the spatio-temporal dynamics of an HPI, and, even when carried out with a relatively high degree of abstraction, can be useful in the investigation of system-level consequences of putative mechanisms operating at the individual agent level. We suggest that an integrated and iterative heuristic relationship between computational modeling and more traditional laboratory and clinical investigations, with a focus on identifying useful and sufficient degrees of abstraction, will enhance the efficiency and translational productivity of biomedical research.