Computing Geographical Networks Generated by Air-Mass Movement.

As air masses move within the troposphere, they transport a multitude of components including gases and particles such as pollen and microorganisms. These movements generate atmospheric highways that connect geographic areas at distant, local, and global scales that particles can ride depending on their aerodynamic properties and their reaction to environmental conditions. In this article we present an approach and an accompanying web application called tropolink for measuring the extent to which distant locations are potentially connected by air-mass movement. This approach is based on the computation of trajectories of air masses with the HYSPLIT atmospheric transport and dispersion model, and on the computation of connection frequencies, called connectivities, in the purpose of building trajectory-based geographical networks. It is illustrated for different spatial and temporal scales with three case studies related to plant epidemiology. The web application that we designed allows the user to easily perform intensive computation and mobilize massive archived gridded meteorological data to build weighted directed networks. The analysis of such networks allowed us for example, to describe the potential of invasion of a migratory pest beyond its actual distribution. Our approach could also be used to compute geographical networks generated by air-mass movement for diverse application domains, for example, to assess long-term risk of spread from persistent or recurrent sources of pollutants, including wildfire smoke.

Gait characteristics during inadvertent obstacle contacts in young, middle-aged and older adults.

BACKGROUND: When stepping over obstacles, analyses have focused on the successful trials to understand adaptive gait. However, examination of the inadvertent trips that occasionally occur in the laboratory can provide a rich source of information regarding the gait characteristics underlying trip-related falls. RESEARCH QUESTION: What gait variables during obstacle crossing are associated with inadvertent obstacle contacts, and are these variables different across the lifespan? METHODS: Three age groups included: young adults (20-35 years, N = 20), middle-aged adults (50-64 years, N = 15), and older adults (65-79 years, N = 19). A stationary, visible obstacle (26 cm tall) was placed in the middle of a walkway. Foot trajectories and head angles were compared between contact and non-contact trials. RESULTS: Twelve participants contacted the obstacle: seven young adults (3.5% of young adult trials), two middle-aged adults (1.3%), and three older adults (1.6%). Young and middle-aged adults contacted primarily with the trail limb, while older adults contacted primarily with the lead limb. Contacts occurred for different reasons: Most young adult contact trials had appropriate foot placement, but inadequate elevation; middle-aged and older adults demonstrated inappropriate foot placement before the obstacle, leading to foot contact during the swing phase. SIGNIFICANCE: Lower contact rates in the middle-aged and older adults indicates that the cautious strategies adopted during obstacle crossing are effective. Higher contact rates in young adults may indicate trial-and-error exploratory behavior. Inappropriate foot placement in the middle-aged and older adults may indicate impaired ability to gather obstacle position information during the approach phase.

Nav Channels in Damaged Membranes.

Sick excitable cells (ie, Nav channel-expressing cells injured by trauma, ischemia, inflammatory, and other conditions) typically exhibit "acquired sodium channelopathies" which, we argue, reflect bleb-damaged membranes rendering their Nav channels "leaky." The situation is excitotoxic because untreated Nav leak exacerbates bleb damage. Fast Nav inactivation (a voltage-independent process) is so tightly coupled, kinetically speaking, to the inherently voltage-dependent process of fast activation that when bleb damage accelerates and thus left-shifts macroscopic fast activation, fast inactivation accelerates to the same extent. The coupled g(V) and availability(V) processes and their window conductance regions consequently left-shift by the same number of millivolts. These damage-induced hyperpolarizing shifts, whose magnitude increases with damage intensity, are called coupled left shift (CLS). Based on past work and modeling, we discuss how to test for Nav-CLS, emphasizing the virtue of sawtooth ramp clamp. We explain that it is the inherent mechanosensitivity of Nav activation that underlies Nav-CLS. Using modeling of excitability, we show the known process of Nav-CLS is sufficient to predict a wide variety of "sick excitable cell" phenomena, from hyperexcitability through to depolarizing block. When living cells are mimicked by inclusion of pumps, mild Nav-CLS produces a wide array of burst phenomena and subthreshold oscillations. Dynamical analysis of mild damage scenarios shows how these phenomena reflect changes in spike thresholds as the pumps try to counteract the leaky Nav channels. Smart Nav inhibitors designed for sick excitable cells would target bleb-damaged membrane, buying time for cell-mediated removal or repair of Nav-bearing membrane that has become bleb-damaged (ie, detached from the cytoskeleton).

A set of PCRs for rapid identification and characterization of Pseudomonas syringae phylogroups.

AIMS: The aim of this study was to develop a rapid PCR-based method for the specific detection of individual phylogroups of the Pseudomonas syringae complex. METHODS AND RESULTS: Seven primer pairs were developed by analysing whole genomes of 54 Ps. syringae strains. The specificity and sensitivity of these primer pairs were assessed on 236 strains from a large and comprehensive Ps. syringae collection. The method was also validated by characterizing the phylogenetic diversity of 174 putative Ps. syringae isolates from kiwifruit and apricot orchards of southeastern France. CONCLUSION: Our PCR-based method allows for the detection and characterization of nine of the 13 Ps. syringae phylogroups (phylogroups 1, 2, 3, 4, 7, 8, 9, 10 and 13). SIGNIFICANCE AND IMPACT OF THE STUDY: To date, phylogenetic affiliation within the Ps. syringae complex was only possible by sequencing housekeeping genes. Here, we propose a rapid PCR-based method for the detection of specific phylogroups of the Ps. syringae complex. Furthermore, for the first time we reveal the presence of Ps. syringae strains belonging to phylogroups 10 and 13 as epiphytes on plants, whereas they had previously only been observed in aquatic habitats.

Inferring the evolutionary history of the plant pathogen Pseudomonas syringae from its biogeography in headwaters of rivers in North America, Europe, and New Zealand.

Nonhost environmental reservoirs of pathogens play key roles in their evolutionary ecology and in particular in the evolution of pathogenicity. In light of recent reports of the plant pathogen Pseudomonas syringae in pristine waters outside agricultural regions and its dissemination via the water cycle, we have examined the genetic and phenotypic diversity, population structure, and biogeography of P. syringae from headwaters of rivers on three continents and their phylogenetic relationship to strains from crops. A collection of 236 strains from 11 sites in the United States, in France, and in New Zealand was characterized for genetic diversity based on housekeeping gene sequences and for phenotypic diversity based on measures of pathogenicity and ice nucleation activity. Phylogenetic analyses revealed several new genetic clades from water. The genetic structure of P. syringae populations was not influenced by geographic location or water chemistry, whereas the phenotypic structure was affected by these parameters. Comparison with strains from crops revealed that the metapopulation of P. syringae is structured into three genetic ecotypes: a crop-specific type, a water-specific type, and an abundant ecotype found in both habitats. Aggressiveness of strains was significantly and positively correlated with ice nucleation activity. Furthermore, the ubiquitous genotypes were the most aggressive, on average. The abundance and diversity in water relative to crops suggest that adaptation to the freshwater habitat has played a nonnegligible role in the evolutionary history of P. syringae. We discuss how adaptation to the water cycle is linked to the epidemiological success of this plant pathogen.

Bacterial-based additives for the production of artificial snow: what are the risks to human health?

For around two decades, artificial snow has been used by numerous winter sports resorts to ensure good snow cover at low altitude areas or more generally, to lengthen the skiing season. Biological additives derived from certain bacteria are regularly used to make artificial snow. However, the use of these additives has raised doubts concerning the potential impact on human health and the environment. In this context, the French health authorities have requested the French Agency for Environmental and Occupational Health Safety (Afsset) to assess the health risks resulting from the use of such additives. The health risk assessment was based on a review of the scientific literature, supplemented by professional consultations and expertise. Biological or chemical hazards from additives derived from the ice nucleation active bacterium Pseudomonas syringae were characterised. Potential health hazards to humans were considered in terms of infectious, toxic and allergenic capacities with respect to human populations liable to be exposed and the means of possible exposure. Taking into account these data, a qualitative risk assessment was carried out, according to four exposure scenarios, involving the different populations exposed, and the conditions and routes of exposure. It was concluded that certain health risks can exist for specific categories of professional workers (mainly snowmakers during additive mixing and dilution tank cleaning steps, with risks estimated to be negligible to low if workers comply with safety precautions). P. syringae does not present any pathogenic capacity to humans and that the level of its endotoxins found in artificial snow do not represent a danger beyond that of exposure to P. syringae endotoxins naturally present in snow. However, the risk of possible allergy in some particularly sensitive individuals cannot be excluded. Another important conclusion of this study concerns use of poor microbiological water quality to make artificial snow.

Plant-pathogenic bacteria as biological weapons - real threats?

At present, much attention is being given to the potential of plant pathogens, including plant-pathogenic bacteria, as biological weapons/bioterror weapons. These two terms are sometimes used interchangeably and there is need for care in their application. It has been claimed that clandestine introduction of certain plant-pathogenic bacteria could cause such crop losses as to impact so significantly on a national economy and thus constitute a threat to national security. As a separate outcome, it is suggested that they could cause serious public alarm, perhaps constituting a source of terror. Legislation is now in place to regulate selected plant-pathogenic bacteria as potential weapons. However, we consider it highly doubtful that any plant-pathogenic bacterium has the requisite capabilities to justify such a classification. Even if they were so capable, the differentiation of pathogens into a special category with regulations that are even more restrictive than those currently applied in quarantine legislation of most jurisdictions offers no obvious benefit. Moreover, we believe that such regulations are disadvantageous insofar as they limit research on precisely those pathogens most in need of study. Whereas some human and animal pathogens may have potential as biological or bioterror weapons, we conclude that it is unlikely that any plant-pathogenic bacterium realistically falls into this category.

Traditional AMPA receptor antagonists partially block Na v1.6-mediated persistent current.

Voltage-gated Na channels and AMPA receptors play key roles in neuronal physiology. Moreover, both channels have been implicated in the pathophysiology of both grey and white matter in a variety of conditions. Dissecting out the roles of these channels requires specific pharmacological tools. In this study we examined the potential non-specific effects on Na(v)1.6 channels of five widely used AMPA receptor blockers. Using whole-cell patch clamp electrophysiology, we identified a TTX-sensitive persistent Na channel current in HEK cells stably expressing the Na(v)1.6 channel. From a holding potential of -120 mV, slow ramp depolarization to +75 mV generated an inward current that peaked at approximately -15 mV. Superfusion of purportedly specific AMPA antagonists, 1-naphthylacetyl spermine, SYM2206, CP465022, GYKI52466, blocked Na(v)1.6-mediated persistent currents in a dose-dependent manner. Each of these AMPA receptor blockers significantly inhibited (to approximately 70% of control levels) the persistent Na current at concentrations routinely used to selectively block AMPA receptors. The AMPA/kainate blocker, NBQX, did not significantly affect persistent Na channel currents. Furthermore, peak transient current was insensitive to NBQX, but was reversibly inhibited by SYM2206, CP465022 and GYKI52466. These results indicate that many commonly used AMPA receptor antagonists have modest but significant blocking effects on the persistent components of Na(v)1.6 channel activity; therefore caution should be exercised when ascribing actions to AMPA receptors based on use of these inhibitors.

Glycine betaine improves Listeria monocytogenes tolerance to desiccation on parsley leaves independent of the osmolyte transporters BetL, Gbu and OpuC.

AIMS: To investigate the effect of glycine betaine (GB) on the survival of Listeria monocytogenes on leaf surfaces under low relative humidity (RH). METHODS AND RESULTS: The addition of GB (> or = 25 mmol l(-1)) improved the survival of L. monocytogenes under low RH on parsley leaves, thus suggesting that GB can improve the tolerance of L. monocytogenes to desiccation. Ten times less GB was needed to improve L. monocytogenes survival under low RH on nonbiological surfaces compared with parsley leaves, suggesting that, on the leaf surface, L. monocytogenes may have to compete for the available GB with autochthonous bacteria and/or the plant itself. Wild type and mutants carrying deletions in the three GB uptake systems, BetL, Gbu and OpuC, behaved similarly with and without added GB on parsley leaves (P > 0.05). In addition, preaccumulation of GB, triggered by osmotic stress prior to inoculation, failed to improve survival under low RH compared with osmotic stress without GB accumulation. CONCLUSIONS: Exogenous GB had a protective effect on L. monocytogenes cells from desiccation during survival on parsley leaves. This effect was independent of intracellular GB accumulation by the known uptake systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Presence of GB could improve the survival of L. monocytogenes to desiccation on leaf surfaces and nonbiological surfaces.

Fate of Listeria spp. on parsley leaves grown in laboratory and field cultures.

AIMS: To investigate the population dynamics of Listeria monocytogenes and Listeria innocua on the aerial surfaces of parsley. METHODS AND RESULTS: Under 100% relative humidity (RH) in laboratory and regardless of the inoculum tested (10(3)-10(8) CFU per leaf), counts of L. monocytogenes EGDe, LO28, LmP60 and L. innocua CIP 80-12 tended towards approx. 10(5) CFU per leaf. Under low RH, Listeria spp. populations declined regardless to the inoculum size (10(4)-10(8) CFU per leaf). L. innocua CIP 80-12 survived slightly better than L. monocytogenes in the laboratory and was used in field cultures. Under field cultures, counts of L. innocua decreased more rapidly than in the laboratory, representing a decrease of 9 log(10) in 2 days in field conditions compared to a decrease of 4.5 log(10) in 8 days in the laboratory. Counts of L. innocua on tunnel parsley cultures were always higher (at least by 100 times) than those on unprotected parsley culture. CONCLUSIONS: Even with a high inoculum and under protected conditions (i.e. plastic tunnels), population of L. monocytogenes on the surface of parsley on the field would decrease by several log(10) scales within 2 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Direct contamination of aerial surfaces of parsley with L. monocytogenes (i.e. through contaminated irrigation water) will not lead to contaminated produce unless it occurs very shortly before harvest.

Viable but non-culturable Listeria monocytogenes on parsley leaves and absence of recovery to a culturable state.

AIMS: To investigate the presence of viable but non-culturable Listeria monocytogenes during survival on parsley leaves under low relative humidity (RH) and to evaluate the ability of L. monocytogenes to recover from VBNC to culturable state under satured humidity. METHODS AND RESULTS: Under low RH (47-69%) on parsley leaves, the initial number of L. monocytogenes populations counted on non selective media (10(9) L. monocytogenes per leaf on TSA) was reduced by 6 log10 scales in 15 days, whereas number of viable L. monocytogenes counted under the microscope was reduced by 3-4 log10 scales, indicating the presence of VBNC cells. This was demonstrated on three L. monocytogenes strains (EGDe, Bug 1995 and LmP60). Changing from low to 100% RH permitted an increase of the culturable counts of L. monocytogenes and this growth was observed only when residual culturable cells were present. Moreover, VBNC L. monocytogenes inoculated on parsley leaves did not become culturable after incubation under 100% RH. CONCLUSIONS: Dry conditions induced VBNC L. monocytogenes on parsley leaves but these VBNC were likely unable to recover culturability after transfer to satured humidity. SIGNIFICANCE AND IMPACT OF STUDY: Enumeration on culture media presumably under-estimates the number of viable L. monocytogenes on fresh produce after exposure to low RH.

Comparison of the phenotypes and genotypes of biofilm and solitary epiphytic bacterial populations on broad-leaved endive.

The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety. Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor. However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies. Broad-leaved endive (Cichorium endivia var. latifolia) harbors biofilms containing fluorescent pseudomonads. These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads. To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive. Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation. The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains. However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms. We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states. This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.

The invagination of excess surface area by shrinking neurons.

Over most of their surface, neurons are surrounded by a narrow extracellular gap across which they make adhesive cell-cell contacts. Thus constrained, how do they regulate their geometry when osmotically perturbed? Specifically, are there any interesting consequences of local osmosis in such conditions? Using confocal imaging of shrinking neurons in culture, we observe water exiting into the cell-substratum gap. This water efflux generates a hydrostatic pressure that, at discrete (low adhesion) sites, causes the neuron's excess plasma membrane to invaginate, thus compensating for shrinkage with a pseudo-intracellular volume. To identify the minimal requirements of the process, a compartment/flux model was constructed. It comprises, essentially, a large liposome adhering in a labyrinthine fashion to a substratum. The model predicts that invaginations form at the cell-substratum interface under the influence of local osmosis, provided that adhesion across the gap is neither too tight nor too loose. Local osmosis in the central nervous system, in contrast to epithelia, is usually considered a mishap, not a physiological opportunity. We postulate, however, that local osmotic forces acting in conjunction with confined extracellular spaces could be harnessed in service of surface area, shape, and volume regulation when intense neural activity alters a neuron's osmotic balance.

Mechanoprotection of the plasma membrane in neurons and other non-erythroid cells by the spectrin-based membrane skeleton.

Though the cytomechanics of spectrin have been explored only for erythrocytes, it is thought that the spectrin skeleton acts universally to support the otherwise mechanically vulnerable cell surface bilayer. Evidence for this role is beginning to accumulate and is reviewed here. Compared to that for erythrocytes, cells whose simplicity facilitates biophysical approaches, the evidence is indirect. One way that membrane skeleton/bilayer interactions have been probed is via the behavior of mechanosusceptible ion channels - channel whose gating is perturbed by abnormally high bilayer tension. These initially unresponsive channels become progressively more mechanoresponsive as stretch and chemical reagents damage the membrane skeleton. The straightforward implication is that the intact membrane skeleton is mechanoprotective. In non-erythroid cells there is continual trafficking of bilayer to and from the plasma membrane. Some of the traffic involves spectrin-lined vacuolar membrane. Several lines of evidence suggest that when neurons elongate and remodel their neurites, membrane skeleton-based mechanoprotection allows the dynamic vacuoles and the plasma membrane to participate in mechanosensitive surface area expansion and retrieval.

Stretch-activation and stretch-inactivation of Shaker-IR, a voltage-gated K+ channel.

Mechanosensitive (MS) ion channels are ubiquitous in eukaryotic cell types but baffling because of their contentious physiologies and diverse molecular identities. In some cellular contexts mechanically responsive ion channels are undoubtedly mechanosensory transducers, but it does not follow that all MS channels are mechanotransducers. Here we demonstrate, for an archetypical voltage-gated channel (Shaker-IR; inactivation-removed), robust MS channel behavior. In oocyte patches subjected to stretch, Shaker-IR exhibits both stretch-activation (SA) and stretch-inactivation (SI). SA is seen when prestretch P(open) (set by voltage) is low, and SI is seen when it is high. The stretch effects occur in cell-attached and excised patches at both macroscopic and single-channel levels. Were one ignorant of this particular MS channel's identity, one might propose it had been designed as a sophisticated reporter of bilayer tension. Knowing Shaker-IR's provenance and biology, however, such a suggestion would be absurd. We argue that the MS responses of Shaker-IR reflect not overlooked "mechano-gating" specializations of Shaker, but a common property of multiconformation membrane proteins: inherent susceptibility to bilayer tension. The molecular diversity of MS channels indicates that susceptibility to bilayer tension is hard to design out of dynamic membrane proteins. Presumably the cost of being insusceptible to bilayer tension often outweighs the benefits, especially where the in situ milieu of channels can provide mechanoprotection.

Molecular cloning and functional expression of Xenopus laevis oocyte ATP-activated P2X4 channels.

All cells contain mechanosensitive ion channels, yet the molecular identities of most are unknown. The purpose of our study was to determine what encodes the Xenopus oocyte's mechanosensitive cation channel. Based on the idea that homologues to known channels might contribute to the stretch channels, we screened a Xenopus oocyte cDNA library with cation channel probes. Whereas other screens were negative, P2X probes identified six isoforms of the P2X4 subtype of ATP-gated channels. From RNase protection assays and RT-PCR, we demonstrated that Xenopus oocytes express P2X4 mRNA. In expression studies, four isoforms produced functional ATP-gated ion channels; however, one, xP2X4c, had a conserved cysteine replaced by a tyrosine and failed to give rise to functional channels. By changing the tyrosine to a cysteine, we showed that this cysteine was crucial for function. We raised antibodies against a Xenopus P2X4 C-terminal peptide to investigate xP2X4 protein expression. This affinity purified anti-xP2X4 antibody recognized a 56 kDa glycosylated Xenopus P2X4 protein expressed in stably transfected HEK-293 cells and in P2X4 cDNA injected oocytes overexpressing the cloned P2X4 channels; however, it failed to recognize proteins in control, uninjected oocytes. This suggests that P2X4 channels and mechanosensitive cation channels are not linked. Instead, oocyte P2X4 mRNA may be part of the stored pool of stable maternal mRNA that remains untranslated until later developmental stages.

Cell surface area regulation and membrane tension.

The beautifully orchestrated regulation of cell shape and volume are central themes in cell biology and physiology. Though it is less well recognized, cell surface area regulation also constitutes a distinct task for cells. Maintaining an appropriate surface area is no automatic side effect of volume regulation or shape change. The issue of surface area regulation (SAR) would be moot if all cells resembled mammalian erythrocytes in being constrained to change shape and volume using existing surface membrane. But these enucleate cells are anomalies, possessing no endomembrane. Most cells use endomembrane to continually rework their plasma membrane, even while maintaining a given size or shape. This membrane traffic is intensively studied, generally with the emphasis on targeting and turnover of proteins and delivery of vesicle contents. But surface area (SA) homeostasis, including the controlled increase or decrease of SA, is another of the outcomes of trafficking. Our principal aims, then, are to highlight SAR as a discrete cellular task and to survey evidence for the idea that membrane tension is central to the task. Cells cannot directly "measure" their volume or SA, yet must regulate both. We posit that a homeostatic relationship exists between plasma membrane tension and plasma membrane area, which implies that cells detect and respond to deviations around a membrane tension set point. Maintenance of membrane strength during membrane turnover, a seldom-addressed aspect of SA dynamics, we examine in the context of SAR. SAR occurs in both animal and plant cells. The review shows the latter to be a continuing source of groundbreaking work on tension-sensitive SAR, but is principally slanted to animal cells.

A New Semi-Selective Medium for Xanthomonas campestris pv. vitians, the Causal Agent of Bacterial Leaf Spot of Lettuce.

A semi-selective medium containing maltose, methyl green, and antibiotics (MMG) was developed for the isolation of Xanthomonas campestris pv. vitians. The semi-selective medium was evaluated based on plating efficiency of X. campestris pv. vitians in cell suspensions of pure cultures from leaves and soil. MMG medium allowed recovery of 5.7 to 30.6% of the X. campestris pv. vitians colonies recovered on nutrient agar and 0.1 to 8.4% of those recovered on 1/10-strength tryptic soy agar. MMG inhibited growth of most background bacteria and allowed reliable identification of X. campestris pv. vitians. The semi-selective medium contained (per liter) maltose (10 g), tryptone (5 g), K2HPO4 (3.5 g), KH2PO4 (2.75 g), trace elements (0.02 to 1.0 mg), methyl green (2 ml of a 1% aqueous solution), amoxicillin (32 mg), cephalothin (32 mg), cycloheximide (50 mg), and agar (15 g).