Multicentric case series of scuba diving fatalities: The role of intracardiac gaseous carbon dioxide in the forensic diagnosis.

Scuba diving fatalities post-mortem diagnosis presents a higher level of forensic complexity because of their occurrence in a non-natural human life environment. Scuba divers are equipped with diving gas to breathe underwater. It is essential for them to be fully trained in order to be able to manage their dive safely despite the varying increase of ambient pressure and temperature decrease. Throughout the dive, the inhaled diving gas is dissolved in the diver's tissues during the descent and if the decompression steps are not respected during the ascent, the balance between the dissolved gas and the tissues (including blood) is disrupted, leading to a gaseous release in the organism. Depending on the magnitude of this gaseous release, free gas can occur in blood and tissue. Venous or arterial gas embolism can also occur as a consequence of decompression sickness or barotraumatism. It can also induce drowsiness that consequently leads to drowning. As a result, the occurrence of gas in dead scuba divers is very complex to interpret, as is the difficulty to distinguish it from resuscitation maneuver artifacts or body decomposition. Although the literature is scarce in this domain, significant work has been done to provide a precise intracadaveric gas sampling method to enlighten the cause and circumstances of death during the dive. The aim of this study is to obtain higher statistical significance by collecting a number of cases to confirm the gas sampling protocol and analysis and gain more information about the cause of death and the events surrounding the fatality through the establishment of clear management guidelines.

The importance of motility and chemotaxis for extra-animal survival of Salmonella enterica serovar Typhimurium and Dublin.

AIMS: This study investigated the importance of flagella and motility of Salmonella enterica serovar Typhimurium and Dublin in models of extra-animal survival. METHODS AND RESULTS: The study was performed using transposon mutants in flagella genes fliC and fljB and in chemotaxis genes cheA, cheB and cheR. Flagella and chemotaxis were found to be of minor importance for attachment to plant leaves, survival in liquid manure and interaction with the nematode C. elegans, while differences were observed between the fliC mutant and the wild-type strain of S. Dublin in interactions with amoebae. CONCLUSIONS: The study shows that flagella and chemotaxis play a minor role in extra-animal survival of these two serovars of Salmonella under the conditions tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Extra-animal survival is important in the full infection cycle for zoonotic salmonellae. Such serovars are motile. Even though the current study was only based on the characterization of two serovars, it strongly suggests that motility and chemotaxis are of minor importance during the spread of Salmonella from one animal to the next through the external environment.

Predicting soil-to-plant transfer of radionuclides with a mechanistic model (BioRUR).

BioRUR model has been developed for the simulation of radionuclide (RN) transfer through physical and biological compartments, based on the available information on the transfer of their nutrient analogues. The model assumes that radionuclides are transferred from soil to plant through the same pathways as their nutrient analogues, where K and Ca are the analogues of Cs and Sr, respectively. Basically, the transfer of radionuclide between two compartments is calculated as the transfer of nutrient multiplied by the ratio of concentrations of RN to nutrient, corrected by a selectivity coefficient. Hydroponic experiments showed the validity of this assumption for root uptake of Cs and Sr and reported a selectivity coefficient around 1.0 for both. However, the application of this approach to soil-to-plant transfer raises some questions on which are the effective concentrations of RN and nutrient detected by the plant uptake mechanism. This paper describes the evaluation of two configurations of BioRUR, one which simplifies the soil as an homogeneous pool, and the other which considers that some concentration gradients develop around roots and therefore ion concentrations at the root surface are different from those of the bulk soil. The results show a good fit between the observed Sr transfer and the mechanistic simulations, even when a homogeneous soil is considered. On the other hand, Cs transfer is overestimated by two orders of magnitude if the development of a decreasing K profile around roots is not taken into account.

An overview of BORIS: Bioavailability of Radionuclides in Soils.

The ability to predict the consequences of an accidental release of radionuclides relies mainly on the level of understanding of the mechanisms involved in radionuclide interactions with different components of agricultural and natural ecosystems and their formalisation into predictive models. Numerous studies and databases on contaminated agricultural and natural areas have been obtained, but their use to enhance our prediction ability has been largely limited by their unresolved variability. Such variability seems to stem from incomplete knowledge about radionuclide interactions with the soil matrix, soil moisture, and biological elements in the soil and additional pollutants, which may be found in such soils. In the 5th European Framework Programme entitled Bioavailability of Radionuclides in Soils (BORIS), we investigated the role of the abiotic (soil components and soil structure) and biological elements (organic compounds, plants, mycorrhiza, and microbes) in radionuclide sorption/desorption in soils and radionuclide uptake/release by plants. Because of the importance of their radioisotopes, the bioavailability of three elements, caesium, strontium, and technetium has been followed. The role of one additional non-radioactive pollutant (copper) has been scrutinised in some cases. Role of microorganisms (e.g., K(d) for caesium and strontium in organic soils is much greater in the presence of microorganisms than in their absence), plant physiology (e.g., changes in plant physiology affect radionuclide uptake by plants), and the presence of mycorrhizal fungi (e.g., interferes with the uptake of radionuclides by plants) have been demonstrated. Knowledge acquired from these experiments has been incorporated into two mechanistic models CHEMFAST and BIORUR, specifically modelling radionuclide sorption/desorption from soil matrices and radionuclide uptake by/release from plants. These mechanistic models have been incorporated into an assessment model to enhance its prediction ability by introducing the concept of bioavailability factor for radionuclides.

Salmonella enterica serovar Typhimurium response involved in attenuation of pathogen intracellular proliferation.

Salmonella enterica serovar Typhimurium proliferates within cultured epithelial and macrophage cells. Intracellular bacterial proliferation is, however, restricted within normal fibroblast cells. To characterize this phenomenon in detail, we investigated the possibility that the pathogen itself might contribute to attenuating the intracellular growth rate. S. enterica serovar Typhimurium mutants were selected in normal rat kidney fibroblasts displaying an increased intracellular proliferation rate. These mutants harbored loss-of-function mutations in the virulence-related regulatory genes phoQ, rpoS, slyA, and spvR. Lack of a functional PhoP-PhoQ system caused the most dramatic change in the intracellular growth rate. phoP- and phoQ-null mutants exhibited an intracellular growth rate 20- to 30-fold higher than that of the wild-type strain. This result showed that the PhoP-PhoQ system exerts a master regulatory function for preventing bacterial overgrowth within fibroblasts. In addition, an overgrowing clone was isolated harboring a mutation in a previously unknown serovar Typhimurium open reading frame, named igaA for intracellular growth attenuator. Mutations in other serovar Typhimurium virulence genes, such as ompR, dam, crp, cya, mviA, spiR (ssrA), spiA, and rpoE, did not result in pathogen intracellular overgrowth. Nonetheless, lack of either SpiA or the alternate sigma factor RpoE led to a substantial decrease in intracellular bacterial viability. These results prove for the first time that specific serovar Typhimurium virulence regulators are involved in a response designed to attenuate the intracellular growth rate within a nonphagocytic host cell. This growth-attenuating response is accompanied by functions that ensure the viability of intracellular bacteria.

Daily time course of whole-shoot gas exchange rates in two drought-exposed Mediterranean shrubs.

Effects of drought on water relations, whole-shoot gas-exchange characteristics, and pigment and zeatin concentrations were investigated in the Mediterranean shrubs rosemary (Rosmarinus officinalis L.) and lavender (Lavandula stoechas L.). Two-year-old, greenhouse-grown plants were placed in a whole-shoot gas-exchange measurement system and subjected to 10 days of drought, resulting in severe water stress, and then re-watered for 5 days in order to study their recovery. Water stress resulted in a significant decline in maximum whole-shoot net CO2 assimilation rates (An) for both species that was associated with reductions in leaf area and stomatal conductance. Because shoot dark respiration rate (Rd) was less sensitive to water stress than An, shoot Rd/An ratio increased from about 15 to 95% during water stress. No major changes in chlorophyll and carotenoid concentrations of rosemary leaves were observed during the experiments, but chlorophyll and carotenoid concentrations fell significantly in water-stressed lavender leaves. Zeatin concentrations were higher in rosemary leaves than in lavender leaves during water stress. After re-watering, whole-shoot An and Rd rapidly recovered to their pre-drought rates.

Genetic mapping by duplication segregation in Salmonella enterica.

MudP and MudQ elements were used to induce duplications in Salmonella enterica by formation of a triple crossover between two transduced fragments and the host chromosome. The large size (36 kb) of MudP and MudQ is a favorable trait for duplication formation, probably because homology length is a limiting factor for the central crossover. Additional requirements are a multiplicity of infection of 2 or higher in the infecting phage suspensions (which reflects the need of two transduced fragments) and an exponentially growing recipient (which reflects the need of a chromosome replication fork). We describe a set of 11 strains of S. enterica, each carrying a chromosomal duplication with known endpoints. The collection covers all the Salmonella chromosome except the terminus. For mapping, a dominant marker (e.g., a transposon insertion in or near the locus to be mapped) is transduced into the 11-strain set. Several transductants from each cross are grown nonselectively, and haploid segregants are scored for the presence of the marker. If all the segregants contain the transduced marker, it maps outside the duplication interval. If the marker is found only in a fraction of the segregants, it maps within the duplicated region.

Regulation of septation: a novel role for SerC/PdxF in Salmonella?

The sfiW locus of Salmonella enterica, previously identified by mutations that suppress the cell division defect of His-constitutive (His(c)) strains, corresponds to serC, the bifunctional gene for phosphoserine-oxoglutarate aminotransferase (SerC) and 2-ketoerythroic acid 4-phosphate transaminase (PdxF). SerC- mutants form small, nearly spherical cells in a wild-type (His+) background, suggesting that the SerC/PdxF product acts as a septation antagonist. Suppression of His(c) filamentation by serC mutations may be explained by loss of the anti-septation activity of SerC/PdxF. The isolation of serC alleles that have lost their biosynthetic activities but are still able to inhibit septum formation suggests that the anti-septation activity of the SerC/PdxF product is unrelated to its known roles in serine and pyridoxine biosynthesis.

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella.

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.

DNA adenine methylase mutants of Salmonella typhimurium show defects in protein secretion, cell invasion, and M cell cytotoxicity.

Mutants of Salmonella typhimurium lacking DNA adenine methylase are attenuated for virulence in BALB/c mice. LD(50) values of a DNA adenine methylation (Dam)(-) mutant are at least 10(3)- to 10(4)-fold higher than those of the parental strain when administrated by oral or intraperitoneal routes. Dam(-) mutants are unable to proliferate in target organs but persist in low numbers in these locations. Efficient protection to challenge with the virulent parental strain is observed in mice infected with a Dam(-) mutant. Use of the ileal loop assay shows that Dam(-) mutants are less cytotoxic to M cells and fail to invade enterocytes. In the tissue culture model, lack of DNA adenine methylation causes reduced ability to invade nonphagocytic cells. In contrast, no effect is observed either in intracellular proliferation within nonphagocytic cells or in survival within macrophages. The invasion defect of Dam(-) mutants is correlated with a distinct pattern of secreted proteins, which is observed in both PhoP(+) and PhoP(-) backgrounds. Altogether, our observations suggest a multifactorial role of Dam methylation in Salmonella virulence.

Repression of IS200 transposase synthesis by RNA secondary structures.

The IS 200 transposase, a 16 kDa polypeptide encoded by the single open reading frame (ORF) of the insertion element, has been identified using an expression system based on T7 RNA polymerase. In wild-type IS 200, two sets of internal inverted repeats that generate RNA secondary structures provide two independent mechanisms for repression of transposase synthesis. The inverted repeat located near the left end of IS 200 is a transcriptional terminator that terminates read-through transcripts before they reach the IS 200 ORF. The terminator is functional in both directions and may terminate >80% of transcripts. Another control operates at the translational level: transposase synthesis is inhibited by occlusion of the ribosome-binding site (RBS) of the IS 200 ORF. The RBS (5'-AGGGG-3') is occluded by formation of a mRNA stem-loop structure whose 3' end is located only 3 nt upstream of the start codon. This mechanism reduces transposase synthesis approximately 10-fold. Primer extension experiments with AMV reverse transcriptase have provided evidence that this stem-loop RNA structure is actually formed. Tight repression of transposase synthesis, achieved through synergistic mechanisms of negative control, may explain the unusually low transposition frequency of IS 200.

Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.

DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background.

The virulence plasmids of Salmonella.

Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes. Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system. Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process. The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons. The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.

Cell division inhibition in Salmonella typhimurium histidine-constitutive strains: an ftsI-like defect in the presence of wild-type penicillin-binding protein 3 levels.

Histidine-constitutive (Hisc) strains of Salmonella typhimurium undergo cell division inhibition in the presence of high concentrations of a metabolizable carbon source. Filaments formed by Hisc strains show constrictions and contain evenly spaced nucleoids, suggesting a defect in septum formation. Inhibitors of penicillin-binding protein 3 (PBP3) induce a filamentation pattern identical to that of Hisc strains. However, the Hisc septation defect is caused neither by reduced PBP3 synthesis nor by reduced PBP3 activity. Gross modifications of peptidoglycan composition are also ruled out. D-Cycloserine, an inhibitor of the soluble pathway producing peptidoglycan precursors, causes phenotypic suppression of filamentation, suggesting that the septation defect of Hisc strains may be caused by scarcity of PBP3 substrate.

The P22 Erf protein and host RecA provide alternative functions for transductional segregation of plasmid-borne duplications.

A tandem DNA duplication carried on a ColE1-derived plasmid segregates at high frequency upon generalized transduction by phage P22 HT. Transductional segregation of the plasmid-borne duplication can be promoted either by RecA or by the Erf function of P22, indicating that transductional segregation is a consequence of the recombination events that re-circularize the plasmid in the recipient cell. RecA-mediated and Erf-mediated transduction give similar frequencies of duplication segregation and yield the same types of segregation products, indicating that two distinct recombination machineries (RecA + RecBCD and Erf + RecBCD) perform similar or identical recombination reactions on plasmid DNA substrates transduced by bacteriophage P22 HT.

The sfiX, rfe and metN genes of Salmonella typhimurium and their involvement in the His(c) pleiotropic response.

Two loci involved in the pleiotropic response of His(c) strains of Salmonella typhimurium (sfiX and sfiY) have been characterized at the molecular level. The sfiX gene (CS 44) has been identified as a homolog of the E. coli gene sanA, located downstream of the cytidine deaminase gene (cdd). The cdd-sanA (or cdd-sfiX) operon shows a highly conserved structure in E. coli and Salmonella. Like its E. coli homolog, the sfiX gene of S. typhimurium is required for vancomycin resistance at high temperature. The dual effect of sfiX mutations (induction of vancomycin sensitivity and suppression of cell division inhibition) suggests a link between SfiX function and murein synthesis. The sfiY locus (CS 85), contains two genes arranged in a single transcriptional unit. The upstream gene is a homolog of the E. coli gene rfe; mutations in this gene suppress the cell division defect of His(c) strains. The suppressor effect of rfe mutations can be reproduced by tunicamycin, suggesting that suppression of filamentation results from an increase in the intracellular concentration of UDP-N-acetyl-D-glucosamine. The gene located downstream of rfe is also found in E. coli but its function is unknown. Insertions in rfe suppress the methionine requirement of His(c) strains of S. typhimurium by a polar effect on the downstream gene, tentatively designated metN. Complementation with a rfe+ clone indicates that the rfe gene is not involved in the methionine requirement of His(c) strains. Thus metN expression appears to cause methionine auxotrophy in a His(c) background.

Underground metabolism.

All enzymes are able to use alternative substrates. When these are naturally occurring metabolites, an 'underground reaction' takes place. Examples are presented in which underground metabolism of this sort produces an observable phenotype. Although biological processes can be remarkably accurate, evolution has selected error rates far from perfect. It is suggested here that a certain level of metabolic inaccuracy, in addition to saving energy, may also confer an evolutionary advantage, for example by providing metabolic plasticity. Since underground reactions are unpredictable from DNA sequence data, caution is in order when interpreting correlations between genetic disorders and pathological syndromes.

Cloning with Mud-P22 hybrid prophages: mapping of IS200 elements on the chromosome of Salmonella typhimurium LT2.

Specific DNA fragments from the chromosome of Salmonella typhimurium LT2 were packaged in P22 capsids by induction of "locked-in" Mud-P22 hybrid prophages. High yields of the packaged DNA were obtained upon capsid disruption. DNA hybridization using a fragment of insertion sequence IS200 as probe permitted physical mapping of IS200 elements on the chromosome of S. typhimurium LT2 within +/- 1 centisome (CS). IS200 copies were found at the following locations: CS 24 (copy VI), CS 53 (copy V), CS 63 (copy I), CS 80 (copy II) and CS 93 (copy III). Copy IV, previously mapped near fliA (CS 42), was not included in our study.