Switching between apparently redundant iron-uptake mechanisms benefits bacteria in changeable environments.

Bacteria often possess multiple siderophore-based iron uptake systems for scavenging this vital resource from their environment. However, some siderophores seem redundant, because they have limited iron-binding efficiency and are seldom expressed under iron limitation. Here, we investigate the conundrum of why selection does not eliminate this apparent redundancy. We focus on Pseudomonas aeruginosa, a bacterium that can produce two siderophores-the highly efficient but metabolically expensive pyoverdine, and the inefficient but metabolically cheap pyochelin. We found that the bacteria possess molecular mechanisms to phenotypically switch from mainly producing pyoverdine under severe iron limitation to mainly producing pyochelin when iron is only moderately limited. We further show that strains exclusively producing pyochelin grew significantly better than strains exclusively producing pyoverdine under moderate iron limitation, whereas the inverse was seen under severe iron limitation. This suggests that pyochelin is not redundant, but that switching between siderophore strategies might be beneficial to trade off efficiencies versus costs of siderophores. Indeed, simulations parameterized from our data confirmed that strains retaining the capacity to switch between siderophores significantly outcompeted strains defective for one or the other siderophore under fluctuating iron availabilities. Finally, we discuss how siderophore switching can be viewed as a form of collective decision-making, whereby a coordinated shift in behaviour at the group level emerges as a result of positive and negative feedback loops operating among individuals at the local scale.

H-2D ligand expression by Ly49A+ natural killer (NK) cells precludes ligand uptake from environmental cells: implications for NK cell function.

To study the adaptation of natural killer (NK) cells to their major histocompatibility complex (MHC) class I environment we have established a novel mouse model with mosaic expression of H-2D(d) using a Cre/loxP system. In these mice, we noticed that NK cells expressing the inhibitory receptor for D(d), Ly49A, were specifically underrepresented among cells with low D(d) levels. That was due to the acquisition of D(d) molecules by the Ly49A+ NK cells that have lost their D(d) transgene. The uptake of H-2D molecules via the Ly49A receptor was restricted to strong ligands of Ly49A. Surprisingly, when Ly49A+ NK cells were D(d+), uptake of the alternative ligand D(k) was not detectable. Similarly, one anti-Ly49A mAb (A1) bound inefficiently when Ly49A was expressed on D(d+) NK cells. Concomitantly, functional assays demonstrated a reduced capacity of Ly49A to inhibit H-2(b)D(d) as compared with H-2(b) NK cells, rendering Ly49A+ NK cells in D(d+) mice particularly reactive. Minor reductions of D(d) levels and/or increases of activating ligands on environmental cells may thus suffice to abrogate Ly49A-mediated NK cell inhibition. The mechanistic explanation for all these phenomena is likely the partial masking of Ly49A by D(d) on the same cell via a lateral binding site in the H-2D(d) molecule.

Trace-element and phosphorus contents in sediment associated with Cretaceous oceanic anoxic events

ABSTRACT : During my SNSF-funded Ph.D. thesis project, I studied the evolution of redox conditions and organic-carbon preservation in the western Tethyan realm during three major positive excursions in the Cretaceous δ13C record, corresponding to the Valanginian, Early Aptian and Late Cenomanian. These periods were characterized by important global environmental and climate change, which was associated with perturbations in the carbon cycle. For the period of the Valanginian δ13C excursion, total organic carbon (TOC) contents and the quality of preserved organic matter are typical of oxic pelagic settings in the western Tethys. This is confirmed by the absence of major excursions in the stratigraphic distribution of RSTE during the δ13C shift. Published TOC data from other parts of the Valanginian oceans indicate that dys- to anaerobic zones were restricted to marginal seas within the Atlantic and Southern Ocean, and to the Pacific. Phosphorus (P) and mineralogical contents suggest a stepwise climatic evolution during the Valanginian, with a humid and warm climate prior to the δ13C shift leading to an increase in continental runoff. During the δ13C shift, a decrease in detrital input and P contents suggests a change in the climate towards more and conditions. During the early Aptian oceanic anoxic event (OAE 1a), a general increase followed by a rapid decrease in P contents suggests enhanced nutrient input at the beginning of OAE 1a. The return to lower values during OAE 1 a, associated with an increase in RSTE contents, may have been related to the weakened capacity to retain P in the sedimentary reservoir due to bottom-water oxygen depletion. In basinal settings, the RSTE distribution indicates well-developed anoxic conditions during OAE la, whereas in the shallower-water environments, conditions were oxic to suboxic, rather than anoxic. Furthermore, in the deeper part of the Tethys, two distinct enrichments have been observed, indicating fluctuations in the intensity of water column anoxia during the δ73C excursion. We also studied the effect of the end-Cenomanian oceanic anoxic event (OAE 2) on an expanded section in the Chrummflueschlucht (E of Euthal, Ct Switzerland). The goal here was to identify paleoceanographic and paleoenvironmental conditions during OAE 2 in this part of the northern Tethyan margin. The results show that this section is one of the most complete sections for the Cenomanian-Turonian boundary interval known from the Helvetic realm, despite a small hiatus between sediments corresponding to peaks 1 and 2 in the δ13C record. The evolution of P contents points to an increase in the input of this nutrient at the onset of OAE 2. The trends in RSTE contents show, however, that this part of the Helvetic realm was not affected by a strong depletion in oxygen conditions during OAE 2, despite its hemipelagic position. A further goal of this project was to submit the samples to a total extraction method (a combined HF/HNO3/HCI acid digestion) and compare the results obtained by the partial HNO3 acid extraction in order to standardize the analytical prócedures in the extraction of RSTE. The obtained results for samples of OAE 1 a suggest that RSTE trends using the partial HNO3 digestion are very comparable to those obtained by the total digestion method and subsequently normalized with regards to AI contents. RÉSUMÉ : Durant ce projet de thèse, financé par le Swiss National Science Funding (SNSF), j'ai étudié l'évolution des conditions redox et de la préservation de carbone organique dans le domnaine ouesttéthysien pendant trois excursions majeures du δ13C au Crétacé correspondant au Valanginien, à l'Aptien inférieur et à la limite Cénomanien-Turonien. Ces périodes sont caractérisées par des changements climatiques et environnementaux globaux associés à des perturbations dans le cylce du carbone. Pour L'excursion positive en δ13C du Valanginien, les analyses du carbone organique total (COT) et les observations palynologiques du domaine téthysien ont présenté des indications d'environnement pélagique relativementbienoxygéné. L'absence d'enrichissements en éléments traces sensibles aux conditions redox (TE) pendant l'excursion positive en δ13C confirme ces interprétations. Les données publiées de COT dans d'autres partie du globe indiquent cependant l'existence de conditions dys- à anaérobiques dans certains bassins restreints de l'Atlantique, l'Océan Austral et du Pacifique. L'évolution du phosphore (P) et la composition minéralogique des sédiments semblent indiquer un climat relativement chaud et humide avant l'excursion en δ13C entraînant une augmentation de l'altération continentale. Pendant le shift isotopique, une diminution des apports détritiques et du P suggèrent une transition vers des conditions plus arides. À l'Aptien Inférieur, le début de l'événement anoxique (OAE 1a) est marqué par une augmentation générale du P dans les sédiments indiquant une augmentation du niveau trophique à la base de l'excursion isotopique. Durant l'événement anoxique, les sédiments sont relativement appauvris en P. Cette diminution rapide associée à des enrichissements en TE est probablement liée à une remobilisation plus importante du P lors de la mise en place de conditions anoxiques dans les eaux de fond. Dans les environnements de bassin, le comportement des TE (enrichissements bien marqués) attestent de conditions réductrices bien marquées alors que dans les environnements moins profonds, les conditions semblent plutôt oxiques à dysoxiques. De plus, deux niveaux d'enrichissement en TE ont été observés dans la partie plus profonde de la Téthys, indiquant des fluctuations assez rapides dans l'intensité de l'anoxie de la colonne d'eau. Nous avons ensuite étudié les effets de l'événement anoxique de la fin du Cenomanien (OAE 2) dans un basin marginal de la marge nord de la Téthys avec la coupe de Chrummflueschlucht (à l'est de Euthal, Ct Schwyz). Les résultats ont montré que cette coupe présente un des enregistrements sédimentaires des plus complets de l'OAE 2 dans le domaine helvétique malgré un hiatus entre le pic 1 et 2 de l'excursion en δ13C. L'évolution du P montre une augmentation au début de l'OAE 2. Cependant, la distribution des TE indique que cette région n'a pas été affectée par des conditions réductrices trop importantes. Un second aspect de ce travail a été l'étude des différentes méthodes sur l'analyse de la distribution des TE. Des échantillons de l'OAE 1a ont été soumis à deux types d'extractions, l'une dite «totale » (attaque combinée d'acides HF/HNO3/HCI) et l'autre dite partielle » (HNO3). Les résultats obtenus suggèrent que les courbes de tendances des TE acquises par extraction partielle sont semblables à celle obtenues par extraction totale et normalisées par l'AI.

Drug uptake in a rodent sarcoma model after intravenous injection or isolated lung perfusion of free/liposomal doxorubicin.

The distribution of free and liposomal doxorubicin (Liporubicin) administered by intravenous injection (IV) or isolated lung perfusion (ILP) was compared in normal and tumor tissues of sarcoma bearing rodent lungs. A single sarcomatous tumor was generated in the left lung of 35 Fischer rats, followed 10 days later by left-sided ILP (n=20) or IV drug administration (n=12), using 100 microg and 400 microg free or liposomal doxorubicin, respectively. The tumor and lung tissue drug concentration was measured by HPLC. Free doxorubicin administered by ILP resulted in a three-fold (100 microg) and 10-fold (400 microg) increase of the drug concentration in the tumor and normal lung tissue compared to IV administration. In contrast, ILP with Liporubicin resulted in a similar drug uptake in the tumor and lung tissue compared to IV injection. For both drug formulations and dosages, ILP resulted in a higher tumor to lung tissue drug ratio but also in a higher spatial heterogeneity of drug distribution within the lung compared to IV administration. ILP resulted in a higher tumor to lung tissue drug ratio and in a more heterogeneous drug distribution within the lung compared to IV drug administration.

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake.

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Galpha4 and Gbeta, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

Photodynamic induced uptake of liposomal doxorubicin to rat lung tumors parallels tumor vascular density.

BACKGROUND: Visudyne®-mediated photodynamic therapy (PDT) at low drug/light conditions has shown to selectively enhance the uptake of liposomal doxorubicin in subpleural localized sarcoma tumors grown on rodent lungs without causing morphological alterations of the lung. The present experiments explore the impact of low-dose PDT on liposomal doxorubicin (Liporubicin™) uptake to different tumor types grown on rodent lungs. MATERIAL AND METHODS: Three groups of Fischer rats underwent subpleural generation of sarcoma, mesothelioma, or adenocarcinoma tumors on the left lung. At least five animals of each group (sarcoma, n = 5; mesothelioma, n = 7; adenocarcinoma, n = 5) underwent intraoperative low-dose (10 J/cm(2) at 35 mW/cm(2) ) PDT with 0.0625 mg/kg Visudyne® of the tumor and the lower lobe. This was followed by intravenous (IV) administration of 400 µg Liporubicin™. After a circulation time of 60 min, the tumor-bearing lung was processed for HPLC analyses. At least five animals per group underwent the same procedure but without PDT (sarcoma, n = 5; mesothelioma, n = 5; adenocarcinoma, n = 6). Five untreated animals per group underwent CD31 immunostaining of their tumors with histomorphometrical assessment of the tumor vascularization. RESULTS: Low-dose PDT significantly enhanced Liporubicin™ uptake to all tumor types (sarcoma, P = 0.0007; mesothelioma, P = 0.001; adenocarcinoma, P = 0.02) but not to normal lung tissue compared to IV drug administration alone. PDT led to a significantly increased ratio of tumor to lung tissue drug uptake for all three tumor types (P < 0.05). However, the tumor drug uptake varied between tumor types and paralleled tumor vascular density. The vascular density was significantly higher in sarcoma than in adenocarcinoma (P < 0.001) and mesothelioma (P < 0.001), whereas there was no significant difference between adenocarcinoma and mesothelioma. CONCLUSION: Low-dose Visudyne®-mediated PDT selectively enhances the uptake of systemically administered liposomal doxorubicin in tumors without affecting the drug uptake to normal lung. However, drug uptake varied significantly between tumor types and paralleled tumor vascular density.

Bloodmeal microfilariae density and the uptake and establishment of Wuchereria bancrofti infections in Culex quinquefasciatus and Aedes aegypti

The relationship between ingestion of microfilariae (mf), production of infective larvae (L3) and mf density in human blood has been suggested as an important determinant in the transmission dynamics of lymphatic filariasis. Here we assess the role of these factors in determining the competence of a natural vector Culex quinquefasciatus and a non vector Aedes aegypti to transmit Wuchereria bancrofti. Mosquitoes were infected via a membrane feeding procedure. Both mosquito species ingested more than the expected number of microfilariae (concentrating factor was 1.28 and 1.81 for Cx. quinquefasciatus and Ae. aegypti, respectively) but Cx. quinquefasciatus ingested around twice as many mf as Ae. aegypti because its larger blood meal size. Ae. aegypti showed a faster mf migration capacity compared to Cx. quinquefasciatus but did not allow parasite maturation under our experimental conditions. Similar proportions of melanized parasites were observed in Ae. aegypti (2.4%) and Cx. quinquefasciatus (2.1%). However, no relationship between rate of infection and melanization was observed. We conclude that in these conditions physiological factors governing parasite development in the thorax may be more important in limiting vectorial competence than the density of mf ingested.

Are oxygen uptake kinetics modified when using a respiratory snorkel?

PURPOSE: The aim of this study was to compare VO2 kinetics during constant power cycle exercise measured using a conventional facemask (CM) or a respiratory snorkel (RS) designed for breath-by-breath analysis in swimming. METHODS: VO2 kinetics parameters-obtained using CM or RS, in randomized counterbalanced order-were compared in 10 trained triathletes performing two submaximal heavy-intensity cycling square-wave transitions. These VO2 kinetics parameters (ie, time delay: td1, td2; time constant: τ1, τ2; amplitude: A1, A2, for the primary phase and slow component, respectively) were modeled using a double exponential function. In the case of the RS data, this model incorporated an individually determined snorkel delay (ISD). RESULTS: Only td1 (8.9 ± 3.0 vs 13.8 ± 1.8 s, P < .01) differed between CM and RS, whereas all other parameters were not different (τ1 = 24.7 ± 7.6 vs 21.1 ± 6.3 s; A1 = 39.4 ± 5.3 vs 36.8 ± 5.1 mL x min(-1) x kg(-1); td2 = 107.5 ± 87.4 vs 183.5 ± 75.9 s; A2' (relevant slow component amplitude) = 2.6 ± 2.4 vs 3.1 ± 2.6 mL x min(-1) x kg(-1) for CM and RS, respectively). CONCLUSIONS: Although there can be a small mixture of breaths allowed by the volume of the snorkel in the transition to exercise, this does not appear to significantly influence the results. Therefore, given the use of an ISD, the RS is a valid instrument for the determination of VO2 kinetics within submaximal exercise.

Oxygen uptake during early cardiogenesis of the chick.

Oxidative metabolism of the isolated embryonic heart of the chick has been determined using a spectrophotometric technique allowing global as well as localized micromeasurements of the O2 uptake. Entire hearts, excised from embryos of 10 somites (primordia fused, stage 10 HH) and 40 somites (S shaped, stage 20 HH) were placed in a special chamber under controlled metabolic conditions where they continued to beat spontaneously and regularly. During the 32 h of development, the O2 consumption of the whole heart increased from 0.9 +/- 0.1 to 5.3 +/- 0.8 nmol O2/h. These values corrected for protein content were, however, comparable (0.45 nmol O2.h-1.micrograms-1). At stage 10-12, the O2 uptake varied along the cardiac tube (from 0.74 to 1.0 nmol O2.h-1.mm-2). From stage 10 to 20, the O2 uptake per unit area of ventricle wall increased from 0.7 +/- 0.2 to 1.8 +/- 0.2 nmol O2.h-1.mm-2, and the O2 uptake per myocardial volume during one cardiac cycle varied from 7 to 2.5 nmol O2/cm3. These results indicate that, despite an intense morphogenesis, the cardiac tissue has a rather low and stable oxidative metabolism, although the O2 requirement of the whole heart increases significantly. Moreover, the normalized suprabasal aerobic energy expenditure decreases throughout early cardiogenesis. The functional integrity of the isolated embryonic heart combined with the experimental possibilities of the microtechnique make the preparation appropriate for studying the changes in cardiac metabolism during development.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Smoking and Public Health: A Review of Reviews of Interventions to Increase Smoking Cessation, Reduce Smoking Initiation and Prevent Further Uptake of Smoking - Evidence Briefing

This evidence briefing collates review-level evidence of interventions including those targeted at pregnant women and evidence for tackling health inequalities in smoking.

Smoking and Public Health: A Review of Reviews of Interventions to Increase Smoking Cessation, Reduce Smoking Initiation and Prevent Further Uptake of Smoking - Summary

The briefing identifies gaps in primary and review-level research and makes a number of recommendations, including further measures to address health inequalities.

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.

Hyperthermia and maximal oxygen uptake in men and women.

To compare the effect of hyperthermia on maximal oxygen uptake (VO2max) in men and women, VO2max was measured in 11 male and 11 female runners under seven conditions involving various ambient temperatures (Ta at 50% RH) and preheating designed to manipulate the esophageal (Tes) and mean skin (Tsk) temperatures at VO2max. The conditions were: 25 degrees C, no preheating (control); 25, 35, 40, and 45 degrees C, with exercise-induced preheating by a 20-min walk at approximately 33% of control VO2max; 45 degrees C, no preheating; and 45 degrees C, with passive preheating during which Tes and Tsk were increased to the same degree as at the end of the 20-min walk at 45 degrees C. Compared to VO2max (l x min(-1)) in the control condition (4.52+/-0.46 in men, 3.01+/-0.45 in women), VO2max in men and women was reduced with exercise-induced or passive preheating and increased Ta, approximately 4% at 35 degrees C, approximately 9% at 40 degrees C and approximately 18% at 45 degrees C. Percentage reductions (7-36%) in physical performance (treadmill test time to exhaustion) were strongly related to reductions in VO2max (r=0.82-0.84). The effects of hyperthermia on VO2max and physical performance in men and women were almost identical. We conclude that men and women do not differ in their thermal responses to maximal exercise, or in the relationship of hyperthermia to reductions in VO2max and physical performance at high temperature. Data are reported as mean (SD) unless otherwise stated.

Dynamic responses of oxygen uptake at the onset and end of moderate and heavy exercise in trained subjects.

Inconsistencies about dynamic asymmetry between the on- and off-transient responses in VO2 are found in the literature. Therefore the purpose of this study was to examine VO2 on- and off-transients during moderate- and heavy-intensity cycling exercise in trained subjects. Ten men underwent an initial incremental test for the estimation of ventilatory threshold (VT) and, on different days, two bouts of square-wave exercise at moderate (<VT) and heavy (>VT) intensities. VO2 kinetics in exercise and recovery were better described by a single exponential model (<VT), or by a double exponential with two time delays (>VT). For moderate exercise, we found a symmetry of VO2 kinetics between the on- and off-transients (i.e., fundamental component), consistent with a system manifesting linear control dynamics. For heavy exercise, a slow component superimposed on the fundamental phase was expressed in both the exercise and recovery, with similar parameter estimates. But the on-transient values of the time constant were appreciably faster than the associated off-transient, and independent of the work rate imposed (<VT and >VT). Our results do not support a dynamically linear system model of VO2 during cycling exercise in the heavy-intensity domain.