The authors evaluated a method of assisting the ventilation of adult sockeye salmon in an attempt to enhance post-release survival after fisheries capture at moderate and peak water temperatures. Though comparable recovery methods are often used by recreational anglers, the authors found this to be ineffective in enhancing post-release survival. Oncorhynchus spp.) following capture is a management tactic often used in commercial, aboriginal, and recreational fisheries. Fisheries capture and handling can be both exhausting and stressful to fish, which can limit their ability to swim and survive after release. As a result, researchers have assessed methods intended to improve post-release survival by assisting the flow of water over the gills of fish prior to release. Such approaches use recovery bags or boxes that direct water over the gills of restrained fish. This study evaluated a method of assisting ventilation that mimics one often employed by recreational anglers (i.e. holding fish facing into a current). Under laboratory conditions, wild Fraser River sockeye salmon (Oncorhynchus nerka) either received manual ventilation assistance for 1 min using a jet of water focused at the mouth or were left to recover unassisted following a capture-and-release simulation. A control group consisted of fish that were not exposed to the simulation or ventilation assistance. The experiment was conducted at 16 and 21°C, average and peak summer water temperatures for the Fraser River, and fish survival was monitored for 33 days. At 21°C, all fish perished within 3 days after treatment in all experimental groups, highlighting the consequences of handling adult sockeye salmon during elevated migration temperatures. Survival was higher at 16°C, with fish surviving on average 15–20 days after treatment. At 16°C, the capture-and-release simulation and ventilation assistance did not affect the survival of males; however, female survival was poor after the ventilation assistance compared with the unassisted and control groups. Our results suggest that the method of ventilation assistance tested in this study may not enhance the post-release survival of adult Fraser River sockeye salmon migrating in fresh water.The live release of wild adult Pacific salmon ( Understanding the physiological demands placed on a fish during fisheries capture through to recovery (assuming it is to be released) are key to evaluating conservation-driven attempts to aid recovery processes and reduce post-release mortality. Fisheries capture and handling results in a stress response and physiological exhaustion associated with both aerobic and anaerobic metabolic activity. The physiological stress response is initiated with the release of catecholamines and corticosteroids, which stimulate cardiorespiration to meet mounting tissue oxygen requirements . EncountThe duration of recovery needed for repeat burst events ranges from 40 min to several hours (reviewed by Oncorhynchus nerka) that return to the Fraser River, BC, Canada during their once-in-a-lifetime spawning migration are captured in both marine and freshwater fisheries by commercial, aboriginal, and recreational fishers. In order to protect threatened sockeye salmon stocks and to ensure spawning escapement targets are met for all stocks, managers implement catch limits and temporal fishery closures. Fraser River recreational rod-and-reel anglers will often release sockeye salmon. In 2010, the recreational fishery released one-third (∼100 000) of sockeye salmon captured in the lower Fraser River  have been evaluated. Most approaches involve ensuring adequate water flow across gill surfaces, thereby increasing the potential for more oxygen delivery during excess requirements (see Oncorhynchus kisutch) that had been caught by a marine commercial gillnet  Cultus Lake Salmon Research Laboratory (∼26 km), where passive integrated transponder tags  were inserted into the coelomic cavity for individual identification. Fish were then transferred to 10 circular 1400 l aerated tanks  supplied with filtered and UV-sterilized circulating fresh water  from Cultus Lake. The fish were held at 14°C for ≥15 h to recover from transport.−1) to 16 (five tanks) and 21°C (five tanks) to simulate the current average and record peak summer Fraser River temperatures experienced by migrating adult sockeye salmon  were used in the experiment. On 14 August 2010, the temperature was gradually increased (by ∼0.5°C h−1 (as measured at the mouth), is similar to or greater than the water speed that migrating sockeye salmon are experiencing in the Fraser River and its tributaries  control [20 males (10 at 16°C and 10 at 21°C) and 13 females (eight at 16°C and five at 21°C)]; (ii) capture-and-release simulation without assisted ventilation [18 males (10 at 16°C and eight at 21°C) and 16 females (eight at 16°C and eight at 21°C)]; and (iii) capture-and-release simulation with assisted ventilation [17 males (10 at 16°C and seven at 21°C) and 19 females (nine at 16°C and 10 at 21°C)]. The simulated fisheries capture consisted of four experimenters leaning over an ∼800 l doughnut-shaped tank  to stimulate the fish to burst swim by touching the tails of the fish or vigorously splashing behind them. After 3 min of this manual chasing, fish were immediately dip netted and exposed to air for 1 min. Similar chasing techniques have been used to stimulate exhaustive exercise in fish  with fresh flowing water at the experimental temperature. Thirty minutes later, blood was sampled (see ‘Blood sampling and laboratory assays’ below) by transferring the fish by dip net to a flow-through, foam-lined trough. Fish were finally transferred to 7000 l aerated tanks (3 m diameter) at the appropriate experimental temperature for long-term monitoring. Control fish were dip netted directly from the 1400 l tanks and sampled for blood immediately before transfer to the 7000 l tanks. All fish were monitored for survival in their respective temperatures for up to 33 days, which represents the approximate time when these wild sockeye salmon would have arrived at their natal spawning grounds .MB) and sex. Opercular tissue (7 mm diameter) was sampled for DNA stock identification , body mass (+), chloride (Cl−), potassium (K+), and osmolality] can be analysed to identify the acute response to exercise  was used on whole, well-mixed blood  , lactate and glucose , osmolality (Advanced Instruments 3320 freezing-point osmometer), chloride , and sodium and potassium , as described by Samples (∼3 ml) were obtained via caudal puncture using a heparinized vacutainer (detailed by MB) among treatment groups. No significant differences were found in either FL or MB across groups (ANOVA: P>0.05), and so groups are compared directly herein. The effects of temperature and capture-and-release treatments on physiological variables were examined using two-way ANOVA. One fish in the 21°C and assisted ventilation group died within 20 min of blood sampling, 17 h ahead of all other mortality, and exhibited anomalous blood physiological variables. This individual was subsequently removed as an outlier in all analyses of physiological variables. Plasma cortisol values were analysed separately for sex in consideration of the naturally higher plasma cortisol values of migrating female sockeye salmon in comparison to migrating male sockeye salmon, as reported in the literature .Two-way analysis of variance (ANOVA) was used to test for differences in size , glucose, osmolality, and haematocrit relative to control fish Table . Female Fish held at 21°C exhibited 100% mortality across all groups within 3 days after the simulated capture-and-release treatments . The level reached with the capture-and-release simulation (∼13 mmol l−1 with or without assisted ventilation) exceeded that proposed to be characteristic of anaerobic activity during burst swimming in sockeye salmon  exhibited rapid mortality with no significant differences among the capture-and-release treatments, including control fish. Studies have shown that water temperatures >18°C have negative survival consequences on sockeye salmon during their freshwater spawning migration  and enabled long-term monitoring, which led to the discovery of a sexual dichotomy in post-release survival. However, holding fish in tanks results in confinement stress not experienced in natural conditions .In summary, most North American recreational fisheries regulations recommend a method of manually reviving fish intended to be released after capture (


Interestingly a similar observation had previously been reported in the distantly-related Gram-positive organism Bacillus subtilis. MreB is often viewed as the bacterial actin homolog contributing to bacterial morphogenesis by coordinating peptidoglycan-synthesising complexes. In addition MreB is increasingly found to be involved in a growing number of processes including chromosome segregation and motor-driven motility. Using genetic and pharmacological approaches, we examined the possible role of MreB during natural transformation in L. pneumophila. Our data show that natural transformation does not require MreB dynamics and exclude a direct role of MreB filaments in the transport of foreign DNA and its recombination in the chromosome.Natural transformation is the process by which bacteria can actively take up and integrate exogenous DNA thereby providing a source of genetic diversity. Under specific growth conditions the coordinated expression of several genes – a situation referred to as “competence” – allows bacteria to assemble a highly processive and dedicated system that can import high molecular weight DNA. Within the cell these large imported DNA molecules are protected from degradation and brought to the chromosome for recombination. Here, we report elevated expression of  Natural transformation is a major mechanism of horizontal gene transfer (HGT) and plays a leading role in bacterial genetic diversification248recA is essential for transformation even when not part of the competence regulon, as for instance in Haemophilus influenzae10Vibrio cholerae4Streptococcus pneumoniae and Bacillus subtilis1314Integration of the internalized ssDNA into the chromosome requires a core and conserved process, homologous recombination. While RecA is primarily involved in the maintenance of chromosome integrity it is also needed to recombine the internalised DNA with the chromosome through the interaction with the transformation-specific protein DprALegionella pneumophila we stumbled upon the observation that mreB is strongly induced during competence. Our data are consistent with the induction of mreB and its paralog mbl observed during competence in the distantly-related Gram-positive Bacillus subtilis20222426272829213Pseudomonas aeruginosa inactivation of MreB leads to mislocalisation of the type IV pilus retraction ATPase PilTB. subtilismreB is induced during competence in B. subtilis and under control of the competence regulator ComK. MreB was found to interact with the ComGA ATPase of the DNA uptake system and this sequestration by ComGA was proposed to prevent cell elongation at the exit of competenceB. subtilis as an mreB deletion mutant showed no defect in transformation efficiency. However, in this species, a potential involvement of the actin-like cytoskeleton in natural transformation may be obscured by the presence of two MreB paralogs (Mbl and MreBH) that can partly maintain cell growth and morphogenesis in absence of MreBDNA uptake depends on the proton motive forceB. subtilis, examination of the L. pneumophila genome revealed a single mreB gene. We found the L. pneumophila mreB to be expressed during competence as part of an operon with comEA, a gene encoding a core component of the DNA uptake system. We investigated the mechanism of induction of mreB during competence in L. pneumophila and examined a possible role of MreB in natural transformation using genetic and pharmacological approaches. Our data suggest that the co-regulation of MreB and the DNA uptake system is coincidental and that natural transformation occurs without assistance of the MreB cytoskeleton.In contrast to L. pneumophila has previously been reported during growth under microaerophilic conditionsL. pneumophila strains AA100 and JR32, mutations in the proQ and comR genes fully de-repressed competence and resulted in high levels of transformability38proQ and comR (respectively lpp0148 and lpp2773) in the Paris strain by inserting a premature ochre codon (TAA) at position 11 . As anticipated both lpp0148TAA and lpp2773TAA mutants were highly transformable with a typical transformation frequency of 1.10−4 including under conditions where the Paris wild-type strain is not transformable (transformation frequency < 1.10−9) (data not shown). It was previously found that comEA is the most differentially expressed gene in the competent stateL. pneumophilacomEA transcript (450 nt) by Northern-blot we noticed an additional band corresponding to a larger transcript of about 1700 nucleotides (comEA in the kanamycin-resistant JR32 proQ and JR32 comR mutants (data not shown). An analysis of the comEA locus showed a small intergenic region (less than 200 bp) between the coding sequences of comEA and mreB  of ~2.5 and a similar comEA-mreB mRNA is then also detectable, thereby confirming the transcriptional readthrough between these two genes (mreB gene shows that mreB is expressed as a monocistronic mRNA of approximately 1100 nt in early growth phase (OD600 = 0.4) and is then strongly induced to form a bicistronic mRNA when comEA is expressed (OD600 = 2.5)  . We concmreB gene with a kanamycin resistance gene in the constitutively transformable lpp0148TAA mutant. Repeated attempts failed to produce kanamycin-resistant transformants. As mreB had been found to be essential in different rod-shaped bacteria such as B. subtilis, Escherichia coli and Caulobacter crescentus, we thought that it could also be the case in L. pneumophila4243mreB by replacing its promoter by a synthetic IPTG-inducible promoter  at the natural chromosomal locus of the constitutively transformable lpp0148TAA strain  and then collapsed because of cell lysis. Exactly as on solid media, concentrations of IPTG above 20 μM could support normal growth , mreB is far less expressed in the lpp0148TAAmreBindstrain than in the lpp0148TAA strain  (mreB expression levels have no dramatic consequences on the cells that show an elongated rod-shaped morphology (mreB expression in the lpp0148TAAmreBindstrain is similar to that in the Paris wild-type strain (1.08 ± 0.07). This condition effectively represents the situation where mreB is expressed at the basal level of the Paris wild-type strain independently of competence development. In this condition, the transformability of the lpp0148TAAmreBind strain is equivalent to that of the lpp0148TAA strain  the mreB transcript is below the detection level which is about 40-times below the expression level in the Paris wild-type strain. Yet this did not result in a significant difference in transformability. Collectively the data suggest that the induction of mreB during competence is coincidental and has no functional significance with respect to natural transformation.Construction of a conditional and mreB , Northerw absent . Analysitometry) . Importarphology . At 500 A strain  showing lpp0148TAAmreBindstrain can be reproducibly grown in the absence of IPTG for two generations before growth begins to slow and then stops (lpp0148TAAmreBindstrain was grown for 24 h on CYE plate with the least amount of IPTG that could support growth (20 μM). The cells were then transferred to liquid IPTG-free medium at an OD600 of 0.2 and growth was monitored along with cell morphology. In agreement with the two hours generation time of L. pneumophila, at 2 hours post-inoculation the OD600 had doubled and the bacteria retained their elongated rod shape but the mean cell diameter (0.65 μm) was significantly increased compared to their initial mean cell diameter (0.61 μm)  and their viability was strongly affected  is reduced about 6-fold when compared to that of the lpp0148TAAmreBind cells at T = 0 (3.29 × 10−6) or to the lpp0148TAA cells having competence-induced levels of MreB (3.26 × 10−6). This decreased transformability appears somehow limited given the poor state of the cells at this stage and a requirement of MreB for the functioning of the DNA uptake system can be excluded. Yet since transformation frequency is reduced a minor role cannot be ruled out. Alternatively this limited decrease in transformability of MreB-depleted cells may represent an indirect effect of the major disturbance of the cell wall that non-specifically alter the assembly and/or localisation of membrane-associated systems3345The above experiments could not rule out the possibility that the low levels of MreB required for growth could be also needed to import and/or integrate exogenous DNA. As described above, the en stops . This pr0.61 μm) . Anotheraffected . At T = affected . Beyond Caulobacter crescentus, Escherichia coli, Pseudomonas aeruginosa, Myxococcus xanthus), A22 was shown to cause a rapid and reversible disturbance of the MreB cytoskeleton thereby arresting MreB-dependent processes within one to two minutes without immediately affecting cell morphology283349L. pneumophila A22 showed antibiotic activity against this organism and cells growing in growth-limiting sub-inhibitory concentration of A22 (0.25 μg/mL) exhibit a morphology identical to MreB-depleted cells  and verified that these mutations were indeed sufficient to confer resistance to A22  and even subverted by pathogens. A similar situation exists with MreB, although its primary function is to coordinate cell wall synthesis it can also serve other active processes such as gliding motility5052mreB is up-regulated during competence in the Gram-positive B. subtilisL. pneumophila. We found that mreB was induced because it forms a bicistronic mRNA with comEA, a gene coding an essential component of the DNA uptake system. This is most likely due to inefficient transcription termination of the comEA gene and may appear as a side effect of comEA induction. Yet the co-expression of comEA and mreB as a bicistronic mRNA suggested a functional significance. For instance, during DNA uptake ComEA relocalises upon DNA binding and this process might have been dependent on MreB dynamicsmreB was important for the functioning of the DNA uptake system. By constructing an inducible allele of mreB we first found that mreB is essential in L. pneumophila, confirming it a possible target for antibiotic developmentmreB expression from that of comEA. Bacteria expressing limited amount of mreB, even below that of non-competent bacteria, remained as transformable as bacteria displaying competence-associated high levels of mreB. We could not determine whether MreB showed different localisation pattern in competent cells compared to their non-competent counterparts but large differences in expression levels of mreB seemed to have no detectable consequences on growth and morphology of the cells. Although mreB expression is required for viability, mreB levels below those found in the Paris wild-type strain were sufficient to maintain cell shape and division suggesting that normal MreB levels are not limiting during growth. Growing the mreB-inducible strain without inducer we could observe the fate of cells in which MreB levels fall below the level required to maintain the rod-shaped morphology of the bacteria. MreB-depleted bacteria rapidly change morphology and loose viability. Yet before viability is too affected these morphologically-altered cells retained the ability to take up and integrate exogenous DNA. Their transformability is relatively lower (6-fold) but the transformation system seems to remain mostly functional. Mutants of genes involved in the DNA uptake system typically completely lose transformability. For instance, deletion mutants of the comEA and comEC genes in L. pneumophila JR32 strain showed a 1000-fold decreased transformability and no detectable transformability, respectivelyIn eukaryotes actin filament structures that control the shape of the cell are also used in other non-essential cellular processes -2-aminoethanesulfonic acid]-buffered yeast extract (AYE) or on solid media ACES-buffered charcoal yeast extract (CYE) plates. Liquid cultures were performed in 13-mL tube containing 3 mL of medium in shaking incubator at 200 rpm. Alternatively, growth was monitored in 100 μL in 96-well plates placed inside a temperature-controlled plate reader. The plate was submitted to intermittent shaking  and growth was monitored by measuring absorbance at 600 nm every 15 min. When appropriate kanamycin and gentamicin were used respectively at 15 μg/mL and 10 μg/mL. Sucrose counter-selection was done on CYE plate containing 5% (w/vol) sucrose. The MreB inhibitor A22 was purchased either from Sigma-Aldrich or Calbiochem, solubilised in dimethyl sulfoxide, aliquoted and stored at −80 °C.L. pneumophila Paris clinical isolate (Outbreak isolate CIP107629). Plasmid sequences are available upon request. Oligonucleotides used in this study are listed in The strains in this study are derived from the lpp0148 and lpp2773 genes were constructed in two steps. First a “kan-sacB” cassette carrying a kanamycin resistance gene and the counter-selectable sacB gene were inserted in the lpp0148 and lpp2773 genes. To do so the upstream and downstream regions (2 kb each) of lpp0148 and lpp2773 were amplified with primers carrying 30-nucleotide sequences complementary to ends of the kan-sacB cassette. The upstream and downstream region were assembled to the kan-sacB cassette by PCR overlap extension  and used to transform the Paris wild-type strain by natural transformation followed by selection on kanamycinlpp0148 and lpp2773 genes were then replaced by alleles in which an ochre stop codon (TAA) was introduced between the eleventh and twelfth codons. The mutated alleles were synthesised by overlapping PCR and introduced in the strains by natural transformationlpp0148TAA and lpp2773TAA containing the premature stop codon.Markerless mutants of the mreB was obtained by replacing its promoter by a synthetic IPTG-inducible promoter at the natural chromosomal locus in the lpp0148TAA mutant strain. A synthetic cassette was constructed to carry the lacIq gene, followed by a gentamicin resistance gene and a synthetic promoter with a –35 box (TTGCTT) and a –10 box (TATAAT) each followed by a LacO operator site (ATTGTGAGCGGATAACAATT). The cassette was integrated between the comEA (lpp0872) and mreB (lpp0873) gene by natural transformation as described above. Transformants were selected on CYE plates containing gentamicin and IPTG (0.5 mM). The resulting lpp0148TAAmreBind strain requires IPTG for growth in liquid and solid media.The inducible mutant of mreB were transferred to a new genetic background by natural transformation of the PCR-amplified mreB alleles in the lpp0148TAAmreBind strain. Colonies that could grow in the absence of IPTG had acquired the A22-resistant mreB allele and lost the lacIq and gentamicin resistance cassette.The A22 resistant alleles of ihfB interrupted by a kanamycin resistance geneThe pGEM-ihfB::Kan plasmid used in natural transformation experiments was constructed by cloning a 5 kb fragment containing Bacteria were grown in AYE to the desired optical density and cells were fixed by adding 1 mL of culture to a 2-mL tube on ice containing an equal volume of methanol. The mixed suspension was kept on ice until the bacterial cells were collected by centrifugation  and then stored at −80 °C. Thawed bacterial pellets were resuspended in 50 μL of RNAsnap buffer comEA (comEA2-NB) or mreB (mreB-NB) in ULTRAhyb Ultrasensitive Hybridisation Buffer . Following overnight hybridisation the membranes were washed twice in 2X SSC buffer containing 0.1% SDS at 65 °C according to the ULTRAhyb manufacturer instructions. Probed membranes were revealed using horseradish peroxidase-conjugated streptavidin and enhanced luminol substrate . Luminescence signals were acquired using an imaging workstation equipped with a charge-coupled device camera (Thermo).Northern-blot analysis were performed as previously describedL. pneumophila Paris wild-type strain at different time points during growth was determined as follows. The strain was streaked on CYE solid medium and plates were incubated overnight at 37 °C. Bacteria were then resuspended in AYE and this suspension was used to inoculate 50 mL AYE at a starting OD600 of 0.05 in 500-mL Erlenmeyer flasks. Cultures were incubated under constant shaking at 30 °C and, when an OD600 of interest was reached, the volume containing 1.109 cells was collected, centrifuged for 3 minutes at 5000 g and pellets were resuspended in 200 μL AYE. This cell suspension was mixed with 3 μg of pGEM-ihfB::Kan and incubated 30 min at 30 °C without shaking . 20 U of DNase I (Sigma) was added and the volume was adjusted to 1 mL with AYE. The cells were incubated 2 h at 37 °C shaking and ten-fold serial dilutions were then plated on non-selective medium (CYE) and selective medium (CYE + Kanamycin 15 μg/mL). Plates were incubated for 72 h at 37 °C and colony-forming units (CFU) counting was performed. Transformation frequency is the ratio of the number of CFUs counted on selective medium divided by the number of CFUs counted on non-selective medium.The transformation ability of lpp0148TAAmreBind strain compared to the lpp0148TAA strain was determined as follows. The lpp0148TAAmreBind strain was streaked on CYE solid medium containing IPTG at 20 μM. The lpp0148TAA strain was streaked on CYE solid medium without IPTG. Plates were incubated overnight at 37 °C, and bacteria were resuspended at an OD600 = 0.2 in 2 mL AYE, containing 20 or 500 μM IPTG. Tubes were incubated under constant shaking at 37 °C. When an OD600 of approx. 1 was reached, 1 mL of culture was centrifuged for 3 min at 5000 g in a table-top microcentrifuge, and pellets were resuspended in 200 μL of distilled water containing 2 μg of pGEM-ihfB::Kan. After 20 minutes of incubation at 37 °C without shaking, tubes were centrifuged 3 minutes at 5000 g and pellets were washed twice in AYE. Pellets were finally resuspended in 200 μL AYE. Ten-fold serial dilutions were then plated on non-selective medium  and selective medium . Plates were incubated for 72 h at 37 °C and transformation frequencies were determined as previously described. The effect of A22 inhibitor on transformation was determined by adding A22 at the indicated concentration along with the transforming DNA.The transformation ability of the lpp0148TAAmreBind strain in the absence of IPTG was determined similarly except that centrifugation steps were avoided to preserve bacterial morphology. Transformation experiments were done using 1 mL of culture and 5 μg pGEM-ihfB::Kan. The lpp0148TAAmreBind strain was streaked on CYE medium containing 20 μM IPTG and incubated overnight at 37 °C. The strain was then resuspended in AYE media at an OD600 = 0.2 and incubated under constant shaking at 37 °C. After 0, 2, 4, 6 and 8 hours, 1050 μl of culture was taken and put into contact with 5 μg of pGEM-ihfB::Kan for 20 minutes at 37 °C. After 20 min of incubation at 37 °C, 50 μL of the suspension were used to perform ten-fold serial-dilutions which were then plated on selective and non-selective medium  and the remaining culture (~1 mL) was used to flood a plate of selective medium. Plates were incubated 72 h at 37 °C and transformation frequencies were determined as previously described. At each tested time, OD600 of the culture was measured, and 1 mL of culture was used to perform microscopic observation of the bacteria as described further below.The transformation ability of the lpp0148TAA strain on CYE medium containing A22 at 25, 50 or 100 μg/mL. After 72 h of incubation at 37 °C, single colonies were picked and streaked on CYE medium. After 48 h of incubation at 37 °C, mutants were collected and stored in AYE medium with 15% glycerol at −80 °C. We analysed 4, 8 and 2 mutants obtained on plate containing A22 at 25, 50 and 100 μg/mL, respectively. Sequencing confirmed that the selected mutants each harboured mutations in the mreB gene  was then placed at the centre of each plates. Plates were incubated 3 days at 37 °C before reading.Strains of interest were resuspended at an ODlpp0148TAAmreBind strain when grown in the absence of IPTG were quantified by measuring the width of bacterial cells at mid-cell. The measurement of bacterial cells width was performed on pictures from the microscopy experiments, using the KLONK Image Measurement software. At least one thousand bacterial cells were measured for each condition. A Shapiro-Wilk test confirmed the Normal distribution of the measured parameter for each condition (p < 0.01). Significance testing was performed using a Welsh t-tests and two samples with a P value < 0.01 were considered significantly different.Bacteria were collected from 1 mL of culture by centrifugation 3 min at 5000 g and pellets were resuspended in 300 μL PBS Formaldehyde 3.7% and incubated at room temperature for 30 min. Acid-washed (ethanol/HCl 1 M) glass coverslips were coated with poly-L-lysine by immersion in a poly-L-lysine 0.01% solution in distilled water (Sigma-Aldrich) for 5 min. Fixed bacteria in PBS Formaldehyde 3.7% were pipetted on the air-dried coverslips and let to settle and stick to the coverslips for about 30 min. Coverslips were then washed twice with PBS, and DNA was labelled using Hoechst 33288 (12 μg/mL in PBS) for 1 h at room temperature. Coverslips were washed twice in PBS and mounted using 8 μL of mounting solution (DAPCO). After an overnight incubation at 4 °C, slides were observed and imaged with an epifluorescence microscope (Zeiss Axioplan 2). Morphological changes observed in the How to cite this article: Juan, P.-A. et al. Natural transformation occurs independently of the essential actin-like MreB cytoskeleton in Legionella pneumophila. Sci. Rep.5, 16033; doi: 10.1038/srep16033 (2015).


One pathway through which pandemic influenza strains might emerge is reassortment from coinfection of different influenza A viruses. Seasonal influenza vaccines are designed to target the circulating strains, which intuitively decreases the prevalence of coinfection and the chance of pandemic emergence due to reassortment. However, individual-based analyses on 2009 pandemic influenza show that the previous seasonal vaccination may increase the risk of pandemic A(H1N1) pdm09 infection. In view of pandemic influenza preparedness, it is essential to understand the overall effect of seasonal vaccination on pandemic emergence via reassortment.In a previous study we applied a population dynamics approach to investigate the effect of infection-induced cross-immunity on reducing such a pandemic risk. Here the model was extended by incorporating vaccination for seasonal influenza to assess its potential role on the pandemic emergence via reassortment and its effect in protecting humans if a pandemic does emerge. The vaccination is assumed to protect against the target strains but only partially against other strains. We find that a universal seasonal vaccine that provides full-spectrum cross-immunity substantially reduces the opportunity of pandemic emergence. However, our results show that such effectiveness depends on the strength of infection-induced cross-immunity against any novel reassortant strain. If it is weak, the vaccine that induces cross-immunity strongly against non-target resident strains but weakly against novel reassortant strains, can further depress the pandemic emergence; if it is very strong, the same kind of vaccine increases the probability of pandemic emergence.Two types of vaccines are available: inactivated and live attenuated, only live attenuated vaccines can induce heterosubtypic immunity. Current vaccines are effective in controlling circulating strains; they cannot always help restrain pandemic emergence because of the uncertainty of the oncoming reassortant strains, however. This urges the development of universal vaccines for prevention of pandemic influenza. Mutations, which cause relatively small but frequent changes in antigenicity of virus strains, are responsible for the seasonal influenza epidemics. Vaccines used to control seasonal flu must be reviewed twice each year in anticipation of the upcoming winter influenza season In some instances, a reassortant virus can have high pathogenicity in animals and humans. The exchange of genes between pairs of influenza A virus subtypes increased virulence in animal models, including reassortment between subtypes H9N2 and H1N1, between H5N1 and H1N1, and between H3N2 and H5N1 Since pandemic influenza is caused by a novel strain, a delay of around 6 months is to be expected until vaccine starts to become available due to the time taken for the various manufacturing steps e.g. . SeasonaStudies from animal models show that infection with influenza A viruses can induce partial heterosubtypic immunity and empirical analyses also indicate that immunity acquired from natural infection in humans can also partially protect the patients against other strains Two different types of seasonal influenza vaccines are available: inactivated and live attenuated. Both are safe and effective in inducing protective antibody responses against matching seasonal strains of influenza, but only live attenuated vaccines can induce heterosubtypic immunity There is a strong case to be made for the development of cross-reactive vaccines that induce immunity against different subtypes of influenza, different strains of the same subtypes (broad-spectrum protection) to control seasonal and pandemic influenza To answer the above questions, in this study we extend our previous population dynamics model of pandemic emergence via reassortment To study the potential role of vaccination for seasonal influenza in constraining the emergence and spread of novel strains due to reassortment between two co-circulating strains of influenza A virus, we consider a human population which is infected by two strains. Strain 1 is resident, there is a vaccination programme against it, and then strain 2 which is supposed as a minor strain and not included in the vaccine is introduced. Co-circulation of two strains can cause coinfection which then provides chance for reassortment. Empirical studies suggest that two influenza A virus strains of co-infection can simultaneously transmit from person-to-person e.g., . We assuSpecifically, the following assumptions apply.µ so the total population size N remains unchanged. Homogeneous mixing of the population is assumed, for simplicity. Hence we ignore age and spatial heterogeneity.Neglecting the additional mortality caused by the virulence of infections, births and deaths are assumed to occur at the same rate i =  {1, 2} does not affect susceptibility to subsequent infection by the other virus strain 3-iI1) can be further infected by strain 2 as easily as those uninfected with strain 1, and vice versa.During the infectious period, the presence of a strain immediate interference, reducing the transmissibility of strain i from dually-infected persons by factor iφ to iβiφ with 0<iφ<1, where iβ, i =  {1, 2}, is the transmission rate of strain i from singly-infected persons to uninfected persons; and b) post-recovery cross-immunity, reducing the susceptibility to strain 3-i of individuals that have been infected with strain i =  {1, 2} but now recovered by factor φ with 0<φ <1 due to partial cross-immunity.The interaction between resident strains occurs in two distinct forms: a) βd with a constraint φ1β1+φ2β2+βd≤ min, which ensures the low transmissibility of double infection compared to that of single infections.Simultaneous transmission of two strains from dually-infected individuals occurs at a rate ψ with 0<ψ <1.A reassortant strain generated from coinfection is distinct from the two resident strains and their coinfections by having higher transmissibility. However, people who recovered from previous single or/and dual infections with resident strains possess some residual cross-immunity against the reassortant strain and reduce their susceptibility by factor φV and 1-ψV, respectively. Because the vaccine efficacy cannot be 100%, the proportion p which is protected is the product of coverage and efficacy  is far smaller than those who are susceptible (S) and, generally, those with protective immunity  at any particular time point for influenza diseases, the contacts of individuals infectious with reassortant strain (Ir) with those that are infectious with resident strains are ignored, hence there is no transition from those in I1, I2, JV2 or Id to infections with the reassortant strain.Since the number of people that are infected with resident strains , the assumption here is a simple way to model it within a population ignoring age structure cf., . FurtherThe deterministic version of the model can be specified by a set of differential equations:i =  {1, 2} areIn the above equations, the force of infection of strain Ω. Given initial sizes of compartments, the programme first determines the time of the next event, which follows an exponential distribution with mean 1/Ω. The nature of the next event is chosen at random, with each of the 41 events having a probability equal to its own rate divided by Ω. For example, the probability PE for an event E to occur during the time interval  isSurveillance shows that influenza epidemics in any given year are mostly dominated by a single virus A strain t and Δt ∼ exp(1/Ω). After each occurrence, the sizes of the compartments are updated according to the picked events. The simulation programme was coded in Visual c++.Here rateE is the rate for event E see  at time R0), defined as the average number of secondary infections that result from the introduction of a single infectious individual into an entirely susceptible population, is an important parameter used to describe the transmissibility of pathogens. R0 for the influenza A virus is typically in the range 1.2–2.4 while the mean infectious period appears to be shorter than 4 days βd = 0.20×β1 = 0.1). By assuming the same infectious period, the baseline values of transmission coefficient are chosen (see R0r)  = 1.6, is slightly higher than that of endemic strains (R01 = R02 = 1.5). The assumption of a higher R0 for the emerging pandemic strain than that of resident strains might be indirectly supported by the observation that the new pandemic strain quickly replaces previously circulating subtypes −5 per coinfection per day We first show the basic characteristics of the infectious processes and then explore the different estimates of vaccination efficacy. The basic reproductive number  administered intramuscularly or live attenuated influenza vaccine (LAIV and Q/LAIV) administered intranasally. Vaccinations are targeted at young children and at-risk groups. Many countries are shifting from targeting vaccination at high-risk groups to universal policies with the aim of reducing transmission and thus providing indirect benefits to the population. Influenza vaccines can provide moderate protection against virologically confirmed influenza Vaccine effectiveness in preventing laboratory-confirmed influenza illness when the vaccine strains are well matched to circulating strains is 70–90% in randomized, placebo-controlled trials conducted among children and young healthy adults; it is lower for the strains that are less matched The systematic reviews and meta-analyses The duration of vaccine-induced immunity appears to be shorter than that induced by natural infection In order to investigate the impact of vaccine for seasonal influenza on the emergence probability and the attack rate once it emerges, we assume the following definition for a pandemic emerged from reassortant strains: the total proportion infected with reassortant strain during the one year period since the introduction of the second resident strain  must be greater than 5% in view of the observation that the attack rate of seasonal influenza is about 5–10% R01 ≡ β1/(γ1+µ) is the basic reproductive number for strain 1. One single infection seed with strain 2 was then introduced from outside of the population. Ten million stochastic realisations were used to obtain the emergence probability of pandemic via reassortment.Here Upon the introduction of strain 2, three possible consequences come even under the same values of model parameters: no reassortant strain emerges; reassortant strains are generated but do not persist; reassortant strains emerge and develop into a pandemic  becomes very weak , its enhanced level against non-target resident strains actually increases the pandemic risk and attack rate once a pandemic emerges.Under the realistic circumstance, cross-immunity generated by vaccination and via infection more likely differs. If the cross-immunity induced by vaccination is weaker, then the probability of pandemic emergence will increase  and 3. FψV = φV = 0.5. When the durations of both immunities are 2 years, the probability of pandemic emergence is 2.1% per year; while if the duration of both immunities increases to 10 years, the probability decreases to 0.12% per year. That is, the emergence probability is inversely proportional to the duration of vaccine-derived immunity. Further, ψ<φ). To illustrate the possible patterns, we assume a fairly strong cross-immunity against endemic strain of φ = 80% (ψV≥ψ and φV = φ). Further, if ψV = ψ, the average size of pandemic remains the same once it begins to emerge. While if ψV>ψ, then the average size of pandemic will be reduced. This is obvious: given the same level of cross-immunity against endemic strain, stronger cross-immunity against reassortant strain due to vaccination will more reduce the activity of reassortant strain once it emerges; therefore the vaccination decreases both the emergence probability and the size of pandemic.Next we scrutinize how the structure of cross-immunity influences the outcome once vaccine was in use. First consider a situation where the primary infection induces much weaker cross-immunity against a reassortant strain than against an endemic strain (i.e.  φ = 80% . As showψV = φV = 0), the probability of pandemic emergence will be substantially increased, compared to the situation of no such vaccination (more than 5 times in φV>0 and ψV>0), the emergence probability will decrease with both φV and ψV , some interesting phenomenon comes out. Given a level of vaccine-induced cross-immunity against novel reassortant strains (i.e. fixing ψV), increasing the level of vaccine-induced cross-immunity against endemic strains will decrease the emergence probability but increase the 1-year attack rate of pandemic   it always helps reduce the pandemic risk; however, the influence of cross-immunity against endemic trains (φV) is conditional on the strength of infection-induced cross-immunity against oncoming reassortant strain (ψ). When ψ is weak, φV decreases both the emergence probability and attack rate of pandemic; if ψ becomes strong but still is weaker than φ, φV decreases the emergence probability but increases the 1-year attack rate of pandemic. Finally, when ψ increases to the level of φ, φV increases both the emergence probability and the 1-year attack rate of pandemic. Note that as ψ increases from 0.0 in These results suggest that compared to the special situation where vaccination does not induce any cross-immunity, where vaccine-induced cross-immunity against novel reassortant strains occurs (Λr(1-ψV)  and Λr(1-ψ) for the 1st and 3rd steps, respectively; the green path). Therefore, given all other conditions remaining unchanged, increasing the vaccine-induced cross-immunity against novel reassortant strains (ψV) will decrease the contribution from path 1 and thus reduce the pandemic risk. In contrast, the effect of φV, which controls the contribution of path 2, depends on the levels of ψ. In the situation where there is much weaker infection-induced cross-immunity against reassortant strain (ψ), increasing φV will reduce the contribution through path 2, which decrease the effective reproductive number of reassortant strain and the probability for pandemic emergence. Once emerged, the spread of reassortant strain will be constrained by cross-immunity against both reassortant strains (ψV) and resident strains (φV), and so will be the size of pandemic (ψ) becomes strong enough to match or exceed the level of φ so that the contribution from path 2 becomes very weak, increasing φV will hardly change the contribution of path 2 but reduce the chance for people vaccinated to get infected with non-target resident strain (i.e. path 2) and allow people vaccinated to stay in class V longer. This then increases the contribution of path 1: more people vaccinated to become directly infected with reassortant strain, which hence indirectly increases the overall probability of pandemic emergence (ψ is strong but still weaker than φ), there is somewhat weak but yet strong enough contribution from path 2. Increasing φV will directly reduce this bit contribution, which consequently decreases the emergence probability; at the same time it increases the direct contribution from path 1 which results in an increase in 1-year attack rate of pandemic  are at least of the similar strengths to those naturally generated (φ and ψ), vaccination can reduce the probability of pandemic emergence (If all cross-immunity components generated from vaccination (mergence . The promergence , which imergence . With seOwing to the striking diversity in genetic and antigenic properties of reassortant strains, cross-immunity induced by primary infection might not match well to novel reassortant strains. With low levels of infection-induced cross-immunity, the emergence probability of pandemics via reassortment can be high . Our simA universal vaccine would reduce the risk of pandemic emergence via reassortment. Currently, LAIVs may produce some heterosubtypic immunity but TIVs cannot. A vaccine for seasonal influenza that offers little protection against a reassortant strain might increase the risk of pandemic emergence cf., . If heteR0 of the oncoming reassortant strain is lower than that of the endemic strains, the probability for the reassortant strain to develop into a pandemic will be reduced substantially (R0 could be made effectively more transmissible under vaccination for seasonal influenza (In this theoretical study, we show that the effect of seasonal vaccination depends on the interaction between cross-immunity induced by vaccination and that acquired through natural infection. As we try to explore the impact of vaccine for seasonal influenza on emergence probability of pandemic via reassortment, we focused on a simple situation: the epidemiological properties of the invader strain are identical to that of the prevailing strain and the oncoming reassortant strain is slightly better transmissible. If the invader strain is different from the prevailing one, the coexistence and coinfection will be reduced, and so does reassortment antially  and the nfluenza . That isAs the name 'pandemic' suggests, pandemic influenza must take place globally. Though we consider a population of the UK size, the qualitative conclusion obtained should be readily applied to the whole world. Given all other conditions being the same, the overall probability of pandemic emergence via reassortment will increase with the population size. Under the circumstance where all cross-immunity has a duration of 10 years and strength 20%, for example, the probability of pandemic emergence is 0.13 per century for a population size of one million (Table 4 of Generation and emergence of pandemic strains is a mysterious process, and we do not have decisive evidence about whether reassortment events leading to historical pandemics occurred in humans or other influenza hosts. For simplicity, we only model the scenario where reassortment occurs among humans, which might apply to pandemic strains in 1957 and 1968 e.g., . To mode


Leishmaniaspp,Migonemyia migonei, and theEvandromyiacortelezzii-sallesi complex south of its known distribution in thecentral temperate region of Argentina, in the province of Córdoba. The persistence ofthis phlebotomine in the northern border of the province, its association with a caseof cutaneous leishmaniasis, and the new record in the outskirts of the city ofCórdoba, the second most populated in the country, strengthens the need for regularvector surveillance and a case detection-sensitive health system in vulnerableregions, even in temperate climates.We report the presence of the competent vector for  The province of Córdoba is located in the geographical centre of Argentina. The area of theprovince sampled in this report has a climate gradient from subtropical with a dry seasonin the northeast corner to temperate in the centre-west up to the foothills, with a meantemperature of 16-17ºC and 750-800 mm of annual precipitation . This biThe northern border of the province is close to the southernmost known reports of vectorialtransmission of American cutaneous leishmaniasis (ACL) in the central region of Argentina,in the province of Santiago del Estero. Despite the fact that Córdoba was monitored forculicid-transmitted arbovirus for more than 40 years, the first reported captures ofphlebotomine sandflies in the province were in Altos de Chipión, in 2004/2005, 236 km fromthe southern record in Santiago del Estero (Los Telares 28º 58’47”S 63º27’04”W), and in LasJarillas, within the dry Chaco region Figure,  Salomón. FurtherThis paper presents three new records of phlebotomine sandflies in the province of Córdoba,their persistence in the northeast humid region and in the Espinal region captured close toCórdoba without previous records, the second most populated city in Argentina , and captures related to the ACL case.2 were placed in four sites (3 with sandflies) between December 2011-May2012, every 15 days. The sites are humid areas with vegetated or rural landscapes with cowsand wild foxes in the surroundings; (ii) the city of Córdoba, where the traps were locatedin three sites: the Zoo (31º25’34’’S 64º10’33’’W), the Botanical Garden (31º23’13’’S64º14’58’’W), and the “Bajo Grande” sewage treatment plant (31º23’38’’S 64º04’36’’W), thelast being the only one with phlebotomines. “Bajo Grande” is located in the outskirts ofthe city 10 km from downtown in a nonurbanised area, where four CDC traps baited withCO2 and separated from each other by 500 m were working seasonally for twonights from 2013-2015. The environment is close to the Suquía River border and has treesand grass, a house 100 m from the traps with roaming dogs and chickens, and wild animalssuch as foxes, hares, rats, and other rodents; (iii) the city of Unquillo, with six trapslocated in henhouses, pigsties, and horse stables. The single trap with sandflies was fromthe henhouse in the yard of the ACL case. The former two trappings were made duringarbovirus surveillance study designs, the last one for the focus study of the ACL case.Further captures were made in the course of surveillance for the spread of leishmaniasisvectors in urban and periurban environments during February 2013  at 10 sites at Altos de Chipión and seven sites at Dean Funes (30º26’S64º21’W), but no phlebotomines were captured. Species were identified with a microscopeusing Galati’s key .Evandromyia cortelezzii andEvandromyia sallesifemales were identified as belonging to thecortelezzii complex  because the females areindistinguishable by morphological characteristics.The captures were made in (i) La Para, 64 km from former captures at Altos de Chipión andsharing the basin of Mar Chiquita Lagoon . CDC miniature light traps baitedwith COMigonemyia migonei was reported previously ACLoutbreaks  in the eastern humid Chaco and gallery forestsof rivers associated with Nyssomyia nei- vai dominance  time-space scattering of ACL cases (sporadic transmission) in thewestern dry Chaco, with ominance .Mg. migonei was also reported (31º35’S60º17’W), while in the residual woods of the dry Chaco far from the river (28º38’S59º51’W), N. neivai, Ev. cortelezzii, and Mg.migonei were found in very low abundance with no dominance between the species.Therefore, the results presented here show the same species pattern in the Espinal regionas in the dry Chaco region with sporadic transmission. The Espinal region reaches theParaná River in the west, where Mg. migonei is aknown vector of Leishmania braziliensis intensified awareness and “looking for” sandflies in areas where their abundance is usuallylow and their presence sporadic, (ii) residual populations from a more extended historicalarea that is currently fragmented, (ii) sporadic extended colonisation during optimalclimate periods but subject to extinctions, or (iv) actual spread through “least-cost”environmental paths .In conclusion, although the low abundance of vectors could imply a low probability oftransmission in peridomestic environments, sporadic cases or an emergent trend could occur,or even visceral cases, due to increased parasite circulation. Surveillance inleishmaniasis-vulnerable contiguous areas and receptive areas due to the reported presenceof vectors should be intensified, even in temperate climate regions. The spread andcolonisation trends of vectors should be monitored, the Health System should be sensitisedand enabled to provide early detection and proper treatment of the cases, and eachconfirmed case will require a study of the focus to assess autochthony.


Restorations involved the bulk fill placement of conventional microhybrid resin composite  (Group 1) or flowable “low-shrinkage” resin composite  (Group 2). Two FBG sensors were used per restoration for real-time measurement of cuspal linear deformation and temperature variation. Group comparisons were determined using ANCOVA (α = 0.05) considering temperature as the covariate. A statistically significant correlation between cuspal deflection, time, and material was observed (p < 0.01). Cuspal deflection reached 8.8 μm (0.23%) and 7.8 μm (0.20%) in Groups 1 and 2, respectively. When used with bulk fill technique, flowable resin composite SDR™ induced significantly less cuspal deflection than the conventional resin composite Esthet•X® HD (p = 0.015) and presented a smoother curve slope during the polymerization. FBG sensors appear to be a valid tool for accurate real-time monitoring of cuspal deformation.Polymerization shrinkage is a major concern to the clinical success of direct composite resin restorations. The aim of this study was to compare the effect of polymerization shrinkage strain of two resin composites on cuspal movement based on the use of fiber Bragg grating (FBG) sensors. Twenty standardized Class II cavities prepared in upper third molars were allocated into two groups ( Volumetric shrinkage remains a major drawback to the clinical performance of the resin composite restorations. Shrinkage leads to deformation of the resin composites and generates stress due to the confinement of the resin to the cavity walls generated by the bonding procedure. This shrinkage stress is transferred to the tooth and may lead to cuspal deflection or enamel microcracks, whereas stress at the tooth-composite interface increases the likelihood of interfacial adhesive failures .μm [Cuspal deflection occurs due to the interaction between the polymerization shrinkage stress of the resin composite, the adhesive interface, and the compliance of the cavity wall . Compliaμm , 6, 8, 9μm , 3, 10; μm , 4, 5, 9μm ; and theμm , 12. In μm , 12. ConAdditionally, the development of inward cuspal deflection can also be related to the strategies employed for managing shrinkage stress of resin composites . These cMany methods have been used to evaluate cuspal deflection, involving technologies that go from linear variable differential transformers (LVDT) , 4, 11, ™) and a conventional microhybrid resin composite (Esthet•X® HD) using fiber Bragg grating (FBG) sensors. The null hypothesis stated that there are no significant differences in cuspal displacement generated by the two resin composites.The aim of this study was to compare the cuspal displacement induced by the polymerization shrinkage of a bulk fill resin composite . The teeth were cleaned and visually inspected to guarantee absence of hypoplastic defects, fractures, or cracks. Teeth were then stored at room temperature in a 10% buffered formalin solution (pH 7.0) for up to 3 months after extraction.n = 10) ensuring a variance of the mean BPW between groups lower than 5%.To standardize the dimensions of the molars, the teeth were selected based on the maximum buccal-palatal width (BPW), varying between 9.5 mm and 10.6 mm, and on the mesiodistal distance, varying between 8.1 mm and 9.7 mm, measured with a digital micrometer gauge . Teeth were then randomly distributed into two groups  2 mm apical from the cement-enamel junction (CEJ), with the long axis vertically oriented. Standardized large mesio-occluso-distal (MOD) cavities were prepared in each molar using a tungsten carbide round-ended bur in a high-speed handpiece  with copious water irrigation followed by a cone shaped bur of large diameter  mounted in a low-speed handpiece with water coolant indicated for inlay cavity preparations. The cusps were minimally frayed and all internal angles rounded. The width of the proximal boxes was approximately two-thirds the BPW . The occ™, Dentsply DeTrey, Konstanz, Germany) according to manufacturer instructions followed by a 10-second light-curing exposure with a LED light-curing unit  in its “low mode” program emitting 650 mW/cm2  used as control  with a UV light (248 nm) from a KrF excimer laser, using the phase mask technique.με), allowing the FBG sensor to detect not only the increasing of the distance between the cusps but also its approximation. For this, one side of the fiber was glued  to a controllable translation stage and the other one to the cusp farthest from it. After the glue drying time (15 min), the fiber was tensioned and bonded to the other cusp (nearest of the controllable translation stage). A new waiting period of 15 min was performed. The second FBG was placed parallel to the first one, but not bonded nor pretensioned, being only sensitive to temperature variations. The gratings' wavelength was measured using a sm 125–500 interrogation system  with a measurement range of 1510–1590 nm, wavelength resolution of 1 pm, and an acquisition frequency of 2 Hz. One drawback of the FBG based sensors is their cross-sensitivity to both strain and temperature. For that, in this study two FBGs were used to measure the setting cuspal deformation and the temperature variation. One of them, sensitive to strain and temperature variations, was placed perpendicular to the buccal cusp. This grating was previously tensioned (about 500 ™ or Esthet•X® HD) and the same LED light-curing unit was used for polymerization in the soft-start mode, running at 650 mW/cm2 for the first 5 seconds and at 1200 mW/cm2 in the following period. Light-curing tip was placed 1 mm above the samples, with an incidence angle near 90°. The samples were initially irradiated for 30 seconds and after a 5-minute break a second light-curing period of 30 seconds was applied. In all tests, data were continuously acquired for 10 minutes from the beginning of the polymerization.After the setup, the cavities were gently bulk filled with resin composite according to two groups .α = 0.05.Statistical analysis was performed using SPSS 20.0® . Cuspal deflection variation between groups was determined with ANCOVA, considering temperature as a covariate. Repeated measures ANOVA considering Greenhouse correction was used to analyze cuspal deflection variation within time for each group. Significance level was set at Temperature rise induced by the irradiation with the LED light-curing unit is represented in ™, a temperature variation of 38.2°C and 30.8°C was obtained for the same periods.The mean temperature rise registered from all tests during light curing of the restorations is shown in The average cuspal deformation, resulting from the ten tests of each resin, during the 10 minutes of monitoring is represented in Descriptive statistics of cuspal deflection in each period is shown in F = 245.37, p < 0.01, partial η2 = 0.935), as expressed in p < 0.01 for all comparisons between 0.5 minutes and 5.5, 6, and 10 minutes within each group). A similar behavior was found after the second polymerization period (6 to 10 minutes) but with lower deformation (p < 0.01). Only SDR presented no differences in cuspal deformation after the postpolymerization clearance periods (p = 0.051 for the 5.5- and 10-minute comparison).Over the time of polymerization, the two resin composites presented a significant variation in material behavior  = 52.69, p < 0.01, partial η2 = 0.756). t-test for independent samples equality of means revealed deformation values statistically significantly higher in Esthet•X® HD compared to SDR™  (In the mean time until the second polymerization (0.5 to 5.5 minutes), Esthet•X® HD curve deflection decreased continuously until 2 minutes pass. From this moment, values remained constant until the terminus of this light-curing free period, reaching a mean of −2296.9 074.1 με . There w= 0.016) .με of cuspal deflection and SDR™  −1166.3 με  = 11.81, p = 0.006, partial η2 = 0.542)  , meaning™, at 10 minutes, were −2277.2 με and −2001.2 με, respectively (p = 0.015)  .με) was converted to micrometers (μm), according to the following equation:In order to determine the shortening distance between the cusps , deformation expressed in microstrain (μm) for the more relevant periods of 0.5 and 10 min were 5.0 μm (0.13%) and 8.8 μm (0.23%) in the case of the Esthet•X® HD and 2.5 μm (0.06%) and 7.8 μm (0.20%) for the SDR™ resin, respectively.The average results obtained in micrometers  of each group reported high variability among samples, which could be due to the employment of natural teeth with nonhomogenous morphological and structural characteristics as previously described for other experimental models using natural teeth. In fact, it was not possible to determine the residual thickness of both dentin and enamel, which in conjunction with slight differences in the amount of the resin used to fill the tooth could compromise the compliance behavior and consequently induce different cuspal displacements. Notwithstanding this, molar teeth choice together with the large Class II MOD cavity design used in the present study has the advantage of wider surface area for fiber bonding, contrarily to premolars mostly used in other studies , 6, 7 whFBG sensors methodology allowed a real-time monitoring of the deformations occurring during resin composite curing as well as the thermal behavior during this procedure , 20. For™ with a conventional microhybrid RBC Esthet•X® HD assessing their performance in bulk fill adhesive MOD molar restorations by measuring cuspal deformation with FBG sensors during a two-step curing protocol in order to deliver a reliable energy density to the restoration. Although conventional microhybrid RBCs have restricted indications for bulk fill placement technique and SDR™ application advocates the use of a microhybrid resin composite for the final covering layer, the purpose was to equalize the restorative protocol in order to isolate and evaluate the individual biomechanical behavior of both materials in similar one-increment situations related to the potential advantage evocated for the “low-shrinkage” one. Significant differences were found between the two materials; therefore the null hypothesis was rejected.The present study compared the marketed low-shrinkage flowable RBC SDR™, which could enhance faster and higher stress development at the tooth/restoration adhesive interface. At the end of the subsequent five-minute pause period both RBCs reached higher shrinkage values, although the polymerization kinetics of SDR™ seems to develop more gradually than that achieved by Esthet•X® HD. This may be related to the functionality of the polymerization modulator incorporated in SDR™ resin matrix. In theory, when this modulator interacts with the photoinitiator (camphorquinone) the polymerization kinetics can be controlled by delaying the gel point, by slowing the rate of polymerization and elastic modulus development, and by reducing polymer cross-linking and, consequently, shrinkage stress [The greatest difference was detected at the end of the first 30 seconds curing period. At this point, Esthet•X® HD induced significantly more deformation  than SDRe stress , 24.™ than Esthet•X® HD. This can be further explained by the lower filler content exhibited by SDR™, as an inverse linear relationship between coefficient of linear thermal expansion and the filler volume fraction of the resin composite has been observed by different researchers [Cuspal deformation curves showed three expansion peaks during the experimental curing period for both resin composites. As soon as the first light irradiation period started, a discrete expansion and transitory peak were detected. However, at the beginning of the second curing light exposure, an expressive and prolonged expansion peak could be observed during all the 30-second irradiation time. These events can be interpreted as the thermal expansion effect caused either by the heat from the curing light or by the exothermic nature of the free radical polymerization of dimethacrylate monomers, as pointed out by other authors , 19, 20.earchers . Anotherμm (0.23%) and 7.8 μm (0.20%) for the maxillary molar teeth restored with Esthet•X® HD and SDR™ resins, respectively. SDR™ presented significantly less final cuspal deflection than Esthet•X® HD. Indeed, in the few studies available concerning SDR™, polymerization stress was reported to be considerably lower than that of conventional flowable resin composites, being comparable to other marketed low shrinking resin composites [™ [™ and x-tra base) have significantly reduced cuspal deflection during light irradiation when compared to a conventional RBC (GrandioSO), reporting a total mean cuspal deflection of 4.63 μm (1.19), 4.73 μm (0.99), and 11.26 μm (2.56) for SDR™, x-tra base, and GrandioSO, respectively. Other recent studies conducted by Tauböck et al. [™ generated significantly lower shrinkage forces compared with the microhybrid Esthet•X® HD when irradiation was performed at continuous high irradiance, even though axial polymerization shrinkage of the SDR composite exceeded that of the microhybrid. Nevertheless, the authors revealed no benefit of SDR regarding shrinkage force generation when modulated curing protocols with low initial irradiance were applied, such as in the soft-start mode, arguing a low responsiveness of SDR to modulated photoactivation due to the predominant effect of the polymerization modulator on reaction kinetics and stress development. In the present study, to control shrinkage-induced tensions, both resin composites were polymerized using a soft-start curing mode, with the expectation that this approach could reduce cuspal movement and improvement of restoration interfacial integrity. One can speculate that if a continuous high level curing irradiance had been used, higher cuspal displacement could have taken place, particularly in the microhybrid Esthet•X® HD samples.In the last measurement (10 minutes), the mean total cuspal deflection was 8.8 mposites  and margmposites  and arousites [™ . Differek et al.  reportedμε can be noticed. Considering the initial distance between the cusps of 3885 μm, it corresponds to an absolute resolution of 0.003 μm. During the last few years, research work devoted to studying and exploring the potential application of fiber optic technology in biomedicine has increased significantly [Some drawbacks can be pointed out to the methodology employed in this study concerning FBG sensors. Cross-sensitivity to both strain and temperature requires specific techniques to compensate for the thermal influence, which was dealt with as an additional Bragg grating. The information obtained is wavelength encoded and extracting real information from the wavelength shift involves the development of particular applications. Other disadvantages consist on the inherent fiber fragility, which makes the manipulation and the bonding of the fiber to the teeth difficult . Severalficantly .To the extent of the knowledge of the authors concerning the scientific published literature, this is the first known study to measure tooth cuspal deflection with fiber Bragg grating sensors. Fiber Bragg sensors can be extensively applied in future studies, comparing different protocols or clinical modified variables useful for shrinkage stress management in clinical practice.™ polymerization kinetics induced less stress to dental structure than Esthet•X® HD, as the mean cuspal deflection values were statistically different between the two resin composites.Within the conditions of this research protocol, SDRDespite the limitations of this in vitro study the optical FBG sensors seem to be a suitable measurement method to evaluate tooth dimensional changes related to cuspal deflection induced by resin composite polymerization shrinkage, with some advantages over other techniques, namely, continuous real-time assessment of tooth biomechanical behavior.


Incited by public fascination and engineering application, water-skipping of rigid stones and spheres has received considerable study. While these objects can be coaxed to ricochet, elastic spheres demonstrate superior water-skipping ability, but little is known about the effect of large material compliance on water impact physics. Here we show that upon water impact, very compliant spheres naturally assume a disk-like geometry and dynamic orientation that are favourable for water-skipping. Experiments and numerical modelling reveal that the initial spherical shape evolves as elastic waves propagate through the material. We find that the skipping dynamics are governed by the wave propagation speed and by the ratio of material shear modulus to hydrodynamic pressure. With these insights, we explain why softer spheres skip more easily than stiffer ones. Our results advance understanding of fluid-elastic body interaction during water impact, which could benefit inflatable craft modelling and, more playfully, design of elastic aquatic toys. Stone skipping on water is a very classical phenomenon, but the relevant fluid dynamics is more complicated than what is normally expected. Here, Belden et al. study the effect of elasticity on the skipping dynamics and show why soft spheres can skip much easier than solid spheres. We observe that elastic spheres can skip for impact angles nearly three times larger than predicted for rigid spheres. Experiments and numerical modelling show that the spheres deform throughout impact in response to elastic waves propagating in the material. In some cases these elastic waves actually interact with the air–water interface to create nested cavities. We determine how the deformed geometry scales with material properties and initial impact kinematics. Using an analytical model to relate deformation to the hydrodynamic lift force, we identify the mechanisms by which elastic spheres skip so readily on water. Furthermore, we compute the normal and tangential restitution coefficients and find, surprisingly, that they display analogous behaviour to liquid droplets bouncing on inclined liquid filmsρs) will skip on water (density ρw)7810βo, raising the question of how the elastic response enables this enhanced skipping behaviour. To investigate the mechanisms underlying elastic sphere skipping, we film the water impact of custom-made elastomeric spheres with a high-speed camera viewing from the side. Rigid and elastic spheres having nearly the same radius R and density ρs are shown experimentally impacting the water in Uo and impact angle βo, but the elastic sphere has a shear modulus G that is four orders of magnitude smaller than that of the rigid sphere material. Within a few milliseconds after impact, the elastic sphere deforms dramatically and rides along the front of a cavity on the air–water interface before lifting off the surface. By the time the elastic sphere is two diameters above the surface (t≈25 ms), the rigid sphere has plunged nearly the same distance below it. The elastic sphere evidently experiences a larger upward vertical force from the water.Prior research has reported an upper bound on the impact angle of α that, unlike for skipping stonesThe extreme sphere deformation is more evident in R, G and ρs) and impact conditions (Uo and βo) as the experimental test shown in −. In some cases this elastic wave impacts the air–water interface at time tw, thus initiating a matryoshka cavity  to the water surface 2. The equation for the vertical motion of the COM is thenwhere d2 is the vertical coordinate of the COM and g is gravity. We can now predict how the hypothesized mechanisms relate to skipping. First, a larger area Sw increases the magnitude of the hydrodynamic force term in Sw increases with decreasing shear modulus G for a given applied compressive stressα thereby increasing the vertical force component that lifts the sphere off the surface. We note that the governing equations for the sphere deformation predict a steady-state solution in which the attack angle evolves as − (see α to be governed by the speed of elastic waves propagating in the sphere through the characteristic distance R such that G) will assume a smaller rate of change of α, thereby increasing the upward vertical force that enables skipping.where e 1− see . While tG. Measurements from the Abaqus results show that the rate of change of the attack angle scales as − deformations , we recover the rigid skipping regime, in which Umin is independent of shear modulus but is very sensitive to βo. For rigid spheres impacting above ρ*=ρs/ρw, prior research suggests spheres may broach , but not skipUmin decreases monotonically with decreasing shear modulus. Our analytical model accurately predicts the experimental and numerical results in this regime. The minimum speed is also much less sensitive to βo in the elastic skipping regime and as a result we observe skipping at impact angles nearly three times larger than predicted for rigid spheres . For threshold skipping cases, we expect the characteristic acceleration G limit our numerical modelling shows that Umin, we find Umin approaches the G1/5 relation in the limit of small G, indicating that the only mechanism by which reducing shear modulus enhances skipping in this limit is through the increased wetted area. However, for larger G (>≈10 kPa), Umin deviates from the G1/5 relation as the coupling between shear modulus and α becomes important (Sw and α) become negligible and the sphere is effectively rigid  and steep impact angles, making use of the relationship between λmax and βo limit, tc/tw≈1 occurs at values of λmax→1  and mode 1− transitional (tc/tw>1) regimes , we believe the physics underlying the elastic sphere impact are common to the large deformation hydroelastic response of surface-riding and skipping compliant bodies. Models of these structures, such as inflatable boats, typically ignore extreme elastic deformation even though it is known to affect drag, stability and slamming loadsR=25.8±1 mm was fabricated from Nylon DuraForm PA using selective laser sintering. The uncertainty on each sphere radius represents the 95% confidence interval based on several independent measurements. A thin Lycra casing was loosely fitted around each sphere in order to prevent undesired particles from adhering to the surface and to reduce the friction between the sphere and the launching mechanism from which it was fired.In order to control material properties, custom elastomeric spheres were fabricated from a high performance platinum-cure silicone rubber called Dragon Skin produced by Smooth-On, Inc., which consists of two liquid constituent parts. Once the two constituents are mixed, the material sets without requiring heat treatment. The shear modulus was varied by adding a silicone thinner to the mixture before setting, which reduces the material shear modulus by decreasing the polymer cross-linking density. Sphere materials with three different shear moduli were fabricated by adding 0, 1/3 and 1/2 parts thinner by mass ratio. Before setting, the liquid mixture was placed in a vacuum chamber to remove any entrained air. For our experiments, spheres were fabricated by curing the liquid mixture in smooth, machined aluminium moulds to produce spheres with three different radii: 20.1±0.8 mm; 26.2±0.8 mm; and 48.8±0.9 mm. A rigid sphere with GG to find the value that produced the best fit between the numerically simulated and experimentally measured force-displacement curves. The elastomeric spheres used in our experiments had shear moduli of 97.2, 28.5 and 12.3 kPa corresponding to 0, 1/3 and 1/2 parts thinner, respectively. According to the manufacturer of the rigid sphere (3D Systems—Quickparts Solutions), the elastic modulus of the selective laser sintering Nylon DuraForm PA material is 1.59 × 106 kPa, which—assuming a Poisson's ratio of 0.4—gives a shear modulus of G=5.66 × 105 kPa.The silicone rubber was so compliant that traditional uniaxial ‘dogbone' testing on our Instron machine was not feasible as the forces generated were too small to be reliably measured. To overcome this, we performed a test in which the spheres were compressed on the Instron to generate a quasi-static force-displacement curve. This test set-up was then numerically modelled in Abaqus with the sphere material described by a neo-Hookean hyperelastic constitutive model, parameterized by the shear modulus Uo and angle βo. Impact events were illuminated with diffuse white back lighting and filmed with either NAC GX-3 or Photron SA3 high-speed cameras acquiring at 1,000–2,000 frames per second (fps).Spheres were launched at the water surface from a variable-angle, pressure-driven cannon consisting of a pressure chamber for compressed air, a sliding cylindrical piston and interchangeable barrels. Sliding the cylindrical piston allowed air to flow from the pressure chamber into the barrel, thus forcing the sphere to accelerate out of the barrel and strike the water surface with impact speed Uo and angle βo were measured from images of the sphere before water impact using a cross-correlation algorithm. The mean uncertainties on Uo and βo are ±1.09 m s−1 and ±1.75°, respectively (computed at 95% confidence level). The same algorithm was used to measure the exit speed and angle of the sphere after lifting off the surface. Also, the rigid body rotation of the sphere was estimated after skipping by tracking reference markers on the exterior of the Lycra casing. It was found for mode 1− skip types that the rotational kinetic energy was typically <8% of the translational kinetic energy after water exit. Furthermore, for these impacts the wave time tw was typically <30% of the period of rigid body rotation measured after skipping. The collision time tc, wave time tw and vibration mode classification were all determined from manual inspection of the high-speed images. The minimum skipping speed Umin was found experimentally by performing successive experiments with identical conditions but with increasing speed until the sphere skipped.The impact speed R, density ρs and shear modulus G, as well as the impact speed Uo and angle βo were set to match experimental values. The three-dimensional computational domain consists of a water tank  with a symmetry plane coinciding with the plane of motion. For the numerical results presented in Umin is the average of the impact speeds for a skipping case and the non-skipping case with the nearest Uo. The numerical marker error bars reported in Details of the Abaqus numerical model of the elastic sphere impact are contained in referencee1, e2, e3}  and after deformation (y) are related by y=d+Vx. The velocity and acceleration fields follow as V and its time derivatives λ1, λ2 and λ3=1/λ1λ2, which are aligned with the body-fixed {m1, m2, m3} coordinates, respectively 31An approximate analytical approach to modelling the impact between a compliant elastomeric sphere and a fluid surface is outlined 2=π(λ1R)(R/λ1λ2), which gives Sw, UB and βB can be written in terms of the generalized coordinates describing the sphere deformation 2 as the characteristic wetted area and Using tc≈O(10−1)s, which gives a characteristic acceleration of −2 (with R≈O(10−2)m). Therefore, in the case of the minimum impact speed, we expect gravity to be an order of magnitude larger than the sphere acceleration such thatFor cases that barely skip, we have observed G, simulations show that sin (α+βB) cos α is of order 1 s for our experiments. We find the characteristic acceleration −2 and thus can neglect gravity. Replacing Umin with Uo in tc gives To determine how er 1 see . Thus, tHow to cite this article: Belden, J. et al. Elastic spheres can walk on water. Nat. Commun. 7:10551 doi: 10.1038/ncomms10551 (2016).Supplementary Figures 1-3, Supplementary Notes 1-3 and Supplementary ReferencesElastic spheres deform dramatically upon water impact. An elastic sphere impacting the water surface assumes a favorably oriented disk-like shape that promotes skipping. Large amplitude elastic oscillations initiated upon impact persist after the sphere lifts off the water surface. This video demonstrates vibration mode 2 in which the sphere assumes an ellipsoidal shape with oscillating major and minor axes.c is longer than the elastic wave propagation time tw.Sphere vibrations induce a matryoshka cavity. In what we refer to as the transitional impact regime, elastic spheres carve nested disturbances on the surface forming a matryoshka cavity. When viewed from the side (left hand video frame), the disturbances appear to result from rigid body rotation of the sphere. However, when viewed in a time-synced top view (right hand video frame), the stitching on the sphere casing indicates minimal rigid rotation. Rather, the video reveals an elastic wave propagating around the circumference of the sphere as the source of the nested cavities. A matryoshka cavity forms when the collision time to decreases with each skip, which prolongs skipping despite Uo diminishing. This video qualitatively demonstrates this behaviour as an elastic sphere skips across a lake.Elasticity allows spheres to walk on water. The lift-enhancing dynamics of elastic spheres enable multiple skips over long trajectories. Modeling and laboratory observations suggest that the impact angle β