MD Mathieu Fontaine

PharmD Alexandre Pachot

PhD Audrey Larue

PharmD Bruno Mougin

PhD Caroline Landelle

PhD Fabienne Venet

PhD Caroline Allombert

PharmD Marie-Ange ´lique Cazalis

PharmD Guillaume Monneret

PhD Alain Lepape
email: alain.lepape@chu-lyon.fr

Delayed increase of S100A9 messenger RNA predicts hospitalacquired infection after septic shock*

S

epsis is the leading cause of death among patients in noncoronary intensive care units (ICUs) and is the tenth major cause of death overall in the United States [START_REF] Martin | The epidemiology of sepsis in the United States from 1979 through 2000[END_REF]. Furthermore, the severe sepsis attack rate in French ICUs appears to have increased over the past decade [START_REF] Brun-Buisson | EPISEPSIS: A reappraisal of the epidemiology and outcome of severe sepsis in French intensive care units[END_REF]. After sep-tic shock, patients are more exposed to hospital acquired infection (HAI) as a result of the association of many risk factors such as invasive devices and a state of immunosuppression [START_REF] Landelle | Nosocomial infection after septic shock among intensive care unit patients[END_REF].

The prediction of outcome (ICUacquired infection or fatal) is of the utmost importance for selecting patients for targeted therapy aimed at restoring immune functions (in the case of associated immunosuppression) and for measures against crosstransmission. Clinical trials may have suffered over the past decades from not having been able to stratify patients owing to the clinical heterogeneity of the population [START_REF] Carlet | Sepsis: Time to reconsider the concept[END_REF].

Besides proinflammatory mediators such as cytokines and chemokines, several endogenous molecules initiate inflammatory responses by interacting with signaling receptors. Such innate signals are now generically termed alarmins because they are considered as danger-signaling molecules induced by various types of stress (e.g., trauma, infection, burns). The alarmin family includes high-mobility-group box proteins, interleukins such as interleukin-1␣, cytosolic calcium-binding proteins of the S100 family, heat-shock proteins, and nucleosomes [START_REF] Harris | Alarmin news about danger: Workshop on innate danger signals and HMGB1[END_REF][START_REF] Pugin | Dear SIRS, the concept of 'alarmins' makes a lot of sense![END_REF][START_REF] Adib-Conquy | Stress molecules in sepsis and systemic inflammatory response syndrome[END_REF]. Recent research points to prominent proinflammatory functions performed by at least three members of the S100 family, S100A8, S100A9, and S100A12, which are mainly expressed by phagocytes [START_REF] Roth | Phagocytespecific S100 proteins: A novel group of pro-inflammatory molecules[END_REF]. S100A8 and S100A9 are potent neutrophil stimulators and are involved in neutrophil migration to inflammatory sites [START_REF] Ryckman | Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion[END_REF]. S100A8 plasma levels decrease during the recovery phase from septic shock [START_REF] Payen | Gene profiling in human blood leucocytes during recovery from septic shock[END_REF]. Although S100A8 and S100A9 exist mainly as a heterodimer called calprotectin, several studies suggest that S100A9 has independent pro-and antiinflammatory functions, making it an interesting candidate biomarker in septic syndromes [START_REF] Sroussi | Oxidation of methionine 63 and 83 regulates the effect of S100A9 on the migration of neutrophils in vitro[END_REF][START_REF] Lorenzo | Macrophage suppression following phagocytosis of apoptotic neutrophils is mediated by the S100A9 calcium-binding protein[END_REF].

The aim of this study is to assess the ability of whole blood S100A9 measured at the messenger RNA (mRNA) level both in the first days after the onset of septic shock and at a later stage of the disease to predict survival and the occurrence of ICU-acquired infections.

Objective: Septic shock remains a serious disease with high mortality and increased risk of hospital-acquired infection. The prediction of outcome is of the utmost importance for selecting patients for therapeutic strategies aiming to modify the immune response. The aim of this study was to assess the capability of S100A9 messenger RNA in whole blood from patients with septic shock to predict survival and the occurrence of hospital-acquired infection.

Design: Cohort study. Setting: Two intensive care units in a university hospital.

Subjects:

The study included patients with septic shock (n ؍ 166) and healthy volunteers (n ؍ 44).

Interventions: None.

Measurements and Main Results:

For the patients with septic shock patients, overall mortality was 38% and the mean Simplified Acute Physiologic Scale II on shock onset was 52. Using quantitative reverse transcriptase-polymerase chain reactions, we found that median S100A9 messenger RNA was significantly lower in healthy volunteers than in patients with septic shock (p < .0001) between days 1 and 3 after onset of the septic shock and not significantly different between nonsurvivor and survivor patients (p ؍ .1278). However, median S100A9 messenger RNA measured on days 7-10 was significantly higher in patients who were about to contract hospital-acquired infections compared with those who were not (p ؍ .009). In the multivariate analysis, the S100A9 marker increased the probability of contracting hospital-acquired infections with an odds ratio of 1.12 per unit (p ؍ .0054).

Conclusions: S100A9 messenger RNA is increased in septic shock and its delayed overexpression is associated with the occurrence of secondary hospital-acquired infection. This biomarker may be of major interest in identifying patients with increased risk of hospital-acquired infection who could benefit from targeted therapy aimed at restoring their immune functions.

MATERIALS AND METHODS

The protocol was approved by the local ethics committee for clinical research, which waived the need for informed consent.

Patients

The first part of the cohort samples has been used in previous studies [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF]. Consecutive patients Ͼ18 yrs of age admitted for septic shock to two ICUs of a university hospital were enrolled from December 14, 2001, through May 1, 2005. The diagnosis of septic shock was based on criteria of the American College of Chest Physicians/Society of Critical Care Medicine [START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee[END_REF]. It was defined by an identifiable infection site and evidence of a systemic inflammatory response manifested by at least two of the following criteria: 1) body temperature Ͼ38°C or Ͻ36°C; 2) heart rate Ͼ90 beats/min; 3) respiratory rate Ͼ20 breaths/ min; 4) a white blood cell count Ͼ12,000/mm 3 or Ͻ4000/mm 3 ; and 5) blood pressure no higher than 90 mm Hg despite fluid resuscitation and requiring vasopressor therapy (Ͼ5 g/kg of body weight of epinephrine and/or norepinephrine). The time of onset of the state of septic shock was defined as the beginning of vasopressor therapy (day 1). All patients were treated similarly according to the standardized septic shock guidelines of our ICUs. Patients were monitored for 28 days or until death. HAI-related variables were screened during the ICU stay. A group of healthy volunteers was recruited in our hospital (occupational medicine) to act as controls.

Intervention

All blood samples were drawn by an indwelling arterial catheter. Blood samples collected between days 1 and 3 after onset of septic shock were defined as early samples and those collected between days 7 and 10 were defined as late samples. Blood samples from volunteers were collected by venous puncture. They provided only an early sample.

Data Collection

Baseline Data. Data were collected on demographic characteristics, the dates of hospital and ICU admission and discharge, admission category, the presence on admission of immune deficiency, and the presence of comorbidities. Severity was assessed at the onset of septic shock using the Simplified Acute Physiology Score II [START_REF] Gall | A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study[END_REF] and the Sepsis-related Organ Failure Assessment [START_REF] Vincent | The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/ failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine[END_REF].

Variables Related to Septic Shock. Data were collected on the date of onset of septic shock (i.e., the documentation, the origin, and identity of the causative micro-organisms; the infection site). The location of septic shock acquisition was defined as the community, if initial signs of infection were present on hospital admission, or if septic shock developed within 48 hrs of admission. Other cases were grouped in the hospital and ICU-acquired septic shock category.

Hospital-Acquired Infection-Related Variables. HAIs were defined according to the criteria of the European Network for Surveillance of nosocomial infections in ICUs [START_REF]Surveillance of Nosocomial Infections in Intensive Care Units: Protocol[END_REF], which are very close to the Centers for Disease Control and Prevention's definitions [START_REF] Horan | CDC/ NHSN surveillance definition of health careassociated infection and criteria for specific types of infections in the acute care setting[END_REF]. If a patient experienced more than one episode of the same type of HAI after septic shock, the analysis was restricted to the first episode of that type. All HAIs after septic shock were reviewed concomitantly by two investigators; in the event of dissension, agreement was reached after further discussion.

Analytical Procedures

Whole blood samples collected during the early and late sampling periods were analyzed as follows: peripheral blood mRNA expression was measured using samples collected directly in PAXGene Blood RNA tubes (PreAnalytix, Hilden, Germany). Total RNA was extracted using the PAXGene Blood RNA kit (PreAnalytix). For each RNA extraction, the residual genomic DNA was digested using the RNase-Free DNase set (Qiagen, Hilden, Germany). The integrity and quality of the total RNA was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) by means of the RNA 6000 Nano assay (Agilent).

Total RNA was reverse transcribed into cDNA using the SuperScript III reverse transcription-polymerase chain reaction (PCR) system (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. mRNA expression was quantified using quantitative real-time PCR as previously described [START_REF] Pachot | Messenger RNA expression of major histocompatibility complex class II genes in whole blood from septic shock patients[END_REF]. Briefly, PCR reactions were performed using a LightCycler instrument with the Fast-Start DNA Master SYBR Green I real-time PCR kit according to the manufacturer's instructions (Roche, Mannheim, Germany). Thermocycling was performed in a final volume of 20 L containing 3 mM MgCl 2 and 0.5 M each of the required primers. PCR was performed with an initial denaturation step of 10 mins at 95°C followed by 40 cycles of a touch-down PCR protocol (10 secs at 95°C, 10 secs annealing at 68 -58°C, and 16 secs extension at 72°C).

mRNA expression of S100A9 and the housekeeping gene peptidylpropyl isomerase B encoding for cyclophilin B were investigated using specific cDNA standards and ready-touse primer mixes obtained from Search-LC. The cDNA standard for S100A9 (accession number NM_002965.3) was prepared from purified PCR amplicons obtained with the following primer combinations: S100A9: forward primer: 5Ј-TCAAAGAGCTGGTGCGAAAA-3Ј, reverse primer: 5Ј-AACTCCTCGAAGCT-CAGCTG-3Ј, amplicon position 253 to 279, as previously described [START_REF] Pachot | Messenger RNA expression of major histocompatibility complex class II genes in whole blood from septic shock patients[END_REF].

The LightCycler 2.0 was used to determine the crossing point for individual samples. Serial dilutions of the cDNA standard were prepared in quadruplicate to generate standard curves. Relative standard curves describing the PCR efficiency of the target gene and peptidylpropyl isomerase B were created and used to perform efficiency-corrected quantification with the LightCycler software version 4 (Roche). The results were expressed as a concentration ratio between the target gene mRNA and peptidylpropyl isomerase B mRNA levels. The efficiency of peptidylpropyl isomerase B mRNA as a reference for target mRNA quantification in humans has been previously described [START_REF] Pachot | Peptidylpropyl isomerase B (PPIB): A suitable reference gene for mRNA quantification in peripheral whole blood[END_REF].

Statistical Analysis

The baseline characteristics of patients on admission to ICUs were described using frequencies with associated percentages and medians with interquartile ranges. The survivor vs. nonsurvivor groups as well as patients developing secondary HAIs or not were compared using the Mann-Whitney U test for continuous variables and the Pearson chi-squared test or Fisher's exact test, as appropriate, for categorical data. For each marker, comparisons between healthy control subjects and patients with septic shock, survivors and nonsurvivors, HAI ϩ and HAI Ϫ , were performed using the Mann-Whitney U test. A receiver operating characteristic curve for the markers was produced to evaluate the prediction between survivors vs. nonsurvivors and HAI ϩ vs. HAI Ϫ . Univariate and multivariate logistic regression was used to identify the variables associated with death or HAI with the odds ratio and their 95% confidence interval. Those variables that resulted in a p value Ͻ.15 were subjected to multivariate analysis. Backward selection was used and a p value of .05 was considered as statistically significant. Because all the quantitative variables (Simplified Acute Physiology Score II, Sepsis-related Organ Failure Assessment, age, and S100A9 values) confirmed the assumed linearity with the outcome (survivors vs. non survivors, HAI ϩ vs. HAI Ϫ ), these variables were included in the model as a continuous variable. Survival curves were built to compare the probability of death or HAIs between the patients stratified using the threshold indicated by the Youden Index calculated on the receiver operating characteristic curve. The log-rank test was used to compare survival distribution. The absolute reduction in mortality and HAIs was the difference between the highest group's event rate and the lowest group's event rate. Statistical analyses were performed with SAS version 9 (SAS Institute, Cary, NC) and Analyze-it software (Analyze-it Software, Leeds, UK).

RESULTS

One hundred sixty-seven patients were eligible for the study. Overall, 159 completed the study (Fig. 1). Forty-four healthy volunteers acted as control subjects (86% women, median age 52 yrs).

The baseline characteristics are shown in Table 1. The mean value of the Simplified Acute Physiology Score II score on shock onset was 52 and the Sepsis-related Organ Failure Assessment score on admission was 10. Patients were equally divided between medical and surgical categories. The infections were equally divided between community-acquired and hospitalacquired infections. More than 75% of all the infections involved the two usual infection sites (i.e., abdominal and pulmonary). As expected, Sepsis-related Organ Failure Assessment and Simplified Acute Physiology Score II scores were significantly higher among the nonsurvivors than the survivors (p Ͻ .0001). The overall mortality within 28 days was 38%.

Thirty-seven patients (23%) out of 159 experienced at least one HAI after septic shock irrespective of whether the septic shock followed a hospital-(HAI attack rate: 23%) or community-acquired infection (HAI attack rate: 23%). The HAI after septic shock appeared with a median delay of 15 days (interquartile range, [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF][START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee[END_REF][START_REF] Gall | A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study[END_REF][START_REF] Vincent | The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/ failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine[END_REF][START_REF]Surveillance of Nosocomial Infections in Intensive Care Units: Protocol[END_REF][START_REF] Horan | CDC/ NHSN surveillance definition of health careassociated infection and criteria for specific types of infections in the acute care setting[END_REF][START_REF] Pachot | Messenger RNA expression of major histocompatibility complex class II genes in whole blood from septic shock patients[END_REF][START_REF] Pachot | Peptidylpropyl isomerase B (PPIB): A suitable reference gene for mRNA quantification in peripheral whole blood[END_REF][START_REF] Cinel | Molecular biology of inflammation and sepsis: A primer[END_REF][START_REF] Rivers | Management of sepsis: Early resuscitation[END_REF]. To predict the risk of ICU-acquired infection, only the patients with samples obtained on both days 1-3 and 7-10 were taken into account, which added up to 93 patients. Of these 93 remaining patients, 27 had HAIs described in Table 2. A total of 45 pathogens were isolated from the 39 HAIs. Pulmonary infections were caused by Pseudomonas aeruginosa (n ϭ 6), Enterobacteriaceae (n ϭ 4), and Staphylococcus aureus (n ϭ 4). Fungi accounted for 38% of all micro-organisms from urinary tract infections. Three bloodstream infections and four catheter-related infections were caused by Gram-positive cocci. Patients who developed HAIs were more exposed (and for a longer duration) to intubation and central venous and urinary tract catheterization. The duration of exposure was censured after the onset of the secondary HAI. Furthermore, patients with HAIs stayed longer in the ICU and had a higher mortality rate.

Mortality

For predicting mortality, 148 patients with early blood sample and 93 patients with late blood sample were evaluated. The S100A9 mRNA level was significantly lower in healthy volunteers than in pa- .0931) (Fig. 2). On days 1-3, the area under the receiver operating characteristic curve was found to be 0.575 (confidence interval 0.478 -0.671; p ϭ .0644). At a cutoff of 22 chosen with the Youden index, the sensitivity of S100A9 mRNA for differentiating survivors from nonsurvivors on day 28 was 46% and its specificity 73%. However, the survival curves were significantly different because the survival rate of patients with S100A9 mRNA Ͻ22 was higher than in patients with S100A9 mRNA Ն22 (log-rank test: p ϭ .0165). On days 1-3, S100A9 mRNA was not statistically significant in the uniand multivariate analysis.

ICU-Acquired Infection

The S100A9 mRNA level at days 1-3 did not show significant differences between HAI-positive and -negative groups (median [interquartile range]: HAI ϩ : 16.390 [12.670 -25.440], HAI Ϫ : 15.960 [9.395-23.270]; p ϭ .2733). In contrast, as shown in the Figure 3, on days 7-10, the median S100A9 mRNA level was significantly higher in the HAI-positive group (10.140 [7.34 -18.08] vs. 7.160 [4.340 -11.810]; p ϭ .0090). S100A9 mRNA predicted HAI with an area under the receiver operating characteristic curve of 0.673 (confidence interval, 0.552-0.794; p ϭ .0025). At a cutoff of 6.1 chosen with the Youden index, the sensitivity of S100A9 mRNA for predicting HAI was 89% and its specificity 44%. The HAI-free distribution function is significantly different between the two groups (Fig. 4), because patients with S100A9 mRNA Ͻ6.1 had a lower rate of HAI than patients with S100A9 mRNA Ն6.1 (logrank test: p ϭ .0234). Based on this cutoff value of S100A9, the absolute reduction in HAI was 30% (relative risk, 4.2; 95% confidence interval 1.37-12.88). Multivariate analysis showed that S100A9 mRNA on days 7-10 after the onset of septic shock significantly increased the probability of HAI with an odds ratio of 1.12 per unit (95% confidence interval 1.034 -1.213; p ϭ .0054; Table 3).

DISCUSSION

Danger-associated molecular patterns are normal cell constituents that can be released into the extracellular milieu during states of cellular stress or damage and subsequently activate the immune system. S100 proteins have been defined as danger-associated molecular patterns, which might be involved in sepsis, although their precise contribution in pathophysiology remains to be elucidated [START_REF] Cinel | Molecular biology of inflammation and sepsis: A primer[END_REF]. In the present study, we showed that septic shock induced a major increase in S100A9 mRNA expression in circulating cells. Although the expression was not associated with survival, our findings suggested that an increased S100A9 mRNA level was a predictor of HAI.

Early identification and comprehensive resuscitation of high-risk patients are key links in the survival chain in the management of severe sepsis and septic shock [START_REF] Rivers | Management of sepsis: Early resuscitation[END_REF]. Biomarkers are needed to assess the severity of illness and to riskstratify patients to ensure proper allocation of limited resources and to develop future therapeutics [START_REF] Bochud | Pathogenesis of sepsis: New concepts and implications for future treatment[END_REF]. At the onset of septic shock, major differences in systemic S100A8 expression between non-survivors and survivors have been reported both in terms of mRNA and protein, suggesting a direct relationship between transcription and translation mechanisms [START_REF] Payen | Gene profiling in human blood leucocytes during recovery from septic shock[END_REF]. More recently, a study performed on patients with severe sepsis found no apparent correlation between serum S100A8/A9 and the severity of disease and similar levels in survivors and nonsurvivors [START_REF] Van Zoelen | Expression and role of myeloid-related protein-14 in clinical and experimental sepsis[END_REF]. The systemic expression of high-mobility-group box-1, another major alarmin involved in sepsis pathophysiology, is known to be strongly increased in severe sepsis and septic shock. Both mRNA and protein levels were shown to be significantly associated with patient outcome and the degree of organ failure [START_REF] Wang | HMGB1 as a late mediator of lethal systemic inflammation[END_REF][START_REF] Pachot | Longitudinal study of cytokine and immune transcription factor mRNA expression in septic shock[END_REF][START_REF] Sunden-Cullberg | Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock[END_REF][START_REF] Gibot | Highmobility group box 1 protein plasma concentrations during septic shock[END_REF]. In the present study, we found that the cell-circulating expression of S100A9 mRNA is strongly increased after septic shock compared with that in a healthy control population. Our data are in line with a recent proteomic study in which, of 41 serum proteins, S100A9 was found to be significantly increased in patients with severe sepsis [START_REF] Hattori | YKL-40 identified by proteomic analysis as a biomarker of sepsis[END_REF]. As described for high-mobility-group box-1, our data suggest that S100A9 could play a persistent timerelated role in sepsis pathophysiology. However, based on our cohort of patients with septic shock, whole blood S100A9 mRNA does not seem to be a relevant marker for mortality, either at the early or the late phase of the syndrome.

HAIs affect approximately 20% of patients in ICUs and are associated with substantial morbidity and mortality. The duration of exposure to invasive devices (intubation, central venous catheterization, urinary tract catheterization) before HAI onset is a classic risk factor [START_REF] Vincent | Nosocomial infections in adult intensive-care units[END_REF]. With respect to the occurrence of secondary HAIs, our data suggest that an increased S100A9 mRNA expression in whole blood is a significant predictor of HAI after septic shock when determined after the first week, but not when measured during the first 3 days of the syndrome. A persistent decrease in HLA-DR expression on circulating monocytes (mHLA-DR) has been associated with the development of septic complications after various injuries, including burns, trauma, and sepsis [START_REF] Venet | Decreased monocyte human leukocyte antigen-DR expression after severe burn injury: Correlation with severity and secondary septic shock[END_REF][START_REF] Muehlstedt | Increased IL-10 production and HLA-DR suppression in the lungs of injured patients precede the development of nosocomial pneumonia[END_REF][START_REF] Tulzo | Monocyte human leukocyte antigen-DR transcriptional downregulation by cortisol during septic shock[END_REF]. The decreased expression of mHLA-DR is probably the most established marker of immunosuppression in critically ill patients. S100A9 might indeed act, therefore, as an endogenous alarm in the early phase of septic shock, although this molecule may also contribute, directly or indirectly, to other mechanisms leading to immunosuppression and HAI [START_REF] Ehrchen | The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer[END_REF]. Whether S100A9, alone or in combination with S100A8, has more pro-or anti-inflammatory functions remains unclear. The heterodimer S100A8-A9 (also called calprotectin) is released in response to stress and has been mainly associated with proinflammatory mechanisms, neutrophil activation, and migration [START_REF] Foell | S100 proteins expressed in phagocytes: A novel group of damage-associated molecular pattern molecules[END_REF][START_REF] Steinbakk | Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin[END_REF]. These mechanisms are mediated, at least in part, by the activation of TLR4 and the receptor for advanced glycation end prod- ucts, leading to lethal endotoxin-induced shock in animal models [START_REF] Ryckman | Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion[END_REF][START_REF] Ehrchen | The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer[END_REF][START_REF] Turovskaya | RAGE, carboxylated glycans and S100A8/A9 play essential roles in colitis-associated carcinogenesis[END_REF]. However, in contrast, other studies suggest that the S100A8/A9 complex may play an important role in the suppression of acute inflammation by trapping proinflammatory cytokines in vivo [START_REF] Ikemoto | Intrinsic function of S100A8/A9 complex as an anti-inflammatory protein in liver injury induced by lipopolysaccharide in rats[END_REF][START_REF] Otsuka | Suppression of inflammation in rat autoimmune myocarditis by S100A8/A9 through modulation of the proinflammatory cytokine network[END_REF]. The anti-inflammatory effects of S100A9 itself in experimental animal models (40 -43) and in in vitro experiments [START_REF] Lorenzo | Macrophage suppression following phagocytosis of apoptotic neutrophils is mediated by the S100A9 calcium-binding protein[END_REF] have also been documented.

Our study is a new illustration that molecular biology tools and mRNA quantification in whole blood could be a reliable approach to monitoring mRNA expression levels in peripheral blood from septic patients and may contribute to staging patients with septic syndromes (19, 44 -46). However, three limitations of our study should be highlighted. First, sample standardization is difficult to obtain in a clinical context (e.g., no sampling during weekends), explaining large sampling windows. Second, the results may be influenced by competitive risk between HAI and death, because patients who died early may not have had enough time to develop an HAI. The third limitation is that the serum protein level of S100A9 was not investigated in our study.

CONCLUSIONS

Increased S100A9 mRNA measured on days 7-10 might be of interest for identifying a population of patients suscepti-ble to HAI. Further validation of this marker in multicenter studies would produce better stratification of patients [START_REF] Janes | Assessing the value of risk predictions by using risk stratification tables[END_REF]. Furthermore, this article illustrates the potential of a transcriptomic approach for identifying new candidate markers for staging septic patients.

Figure 1 .

 1 Figure 1. Flow chart of the population studied. A total of 167 patients were enrolled; one was excluded because septic shock was not confirmed, two resulting from poor-quality samples, and five as a result of missing samples. Data are expressed as counts. S, survivors; NS, nonsurvivors; HAI ϩ , hospital-acquired infection; HAI Ϫ , no HAI.

  tients with septic shock (median [interquartile range], 1.460 [1.065-1.870] vs. 16.620 [11.870 -25.835]; p Ͻ .0001). In survivors and nonsurvivors, the S100A9 mRNA level tended to decrease from days 1-3 to 7-10. However, no significant differences were observed between groups at the two time points (median [interquartile range] days 1-3, nonsurvivors: 18.070 [11.950 -29.240], survivors: 16.310 [11.950 -23.180]; p ϭ .1278; days 7-10: nonsurvivors: 10.620 [7.350 -13.870], survivors: 7.360 [4.550 -10.480]; p ϭ

Figure 2 .

 2 Figure 2. Messenger RNA (mRNA) level of S100A9 in peripheral blood of survivor (S; n ϭ 90) and nonsurvivor (NS; n ϭ 58) septic shock patients and healthy volunteers (n ϭ 44). The mRNA level was normalized to that of the housekeeping gene peptidylpropyl isomerase B (quantitative reverse transcription-polymerase chain reaction [qRT-PCR] concentration ratio). *p ϭ .0001 vs. D1-3; †p ϭ .1278 vs. NS; ‡p ϭ .0931 vs. NS, Mann-Whitney U test.

Figure 3 .

 3 Figure 3. Messenger RNA level of S100A9 in peripheral blood of patients with septic shock with hospital-acquired infection (HAI ϩ ; n ϭ 27) or without subsequent hospital-acquired infection (HAI Ϫ ; n ϭ 66) and healthy volunteers (HV; n ϭ 44). *p ϭ .2733 vs. HAI ϩ ; †p ϭ .0090 vs. HAI ϩ , Mann-Whitney U test. qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 4 .

 4 Figure 4. Kaplan-Meier analysis of patients free from hospital-acquired infection (HAI) using the threshold indicated by the Youden Index calculated on the receiver operating characteristic curve (S100A9 messenger RNA level normalized with peptidylproply isomerase B). There is a significant difference between the two curves (log-rank test, p ϭ .0234).

(Crit Care Med 2011; 39:2684 -2690) KEY WORDS: shock; septic; biologic markers; RNA; messenger; cross infection; calgranulin B

Table 1 .

 1 Patient characteristics

		Nonsurvivors	Survivors	Total
		(n ϭ 61; 38%)	(n ϭ 98; 62%)	(n ϭ 159; %)	p
	Female, no. (%)	24 (39)	33 (34)	57 (36)	.4684
	Age, median yrs (Q1-Q3)	66 (55-77)	66 (55-75)	66 (55-76)	.5492
	Sequential Organ Failure Assessment,	12 (10-14)	9 (8-11)	10 (8-12)	Ͻ.0001
	median (Q1-Q3)
	Simplified Acute Physiologic Scale	64 (56-71)	48.5 (41-54)	52 (43-63)	Ͻ.0001
	II (Q1-Q3)
	Comorbidities Ն1	38 (62)	35 (36)	73 (46)	.0011
	Chronic respiratory disease, no. (%)	15 (25)	11 (11)	26 (16)	.0267
	Suspected infection, no. (%)	1 (2)	3 (3)	4 (3)	1.0000
	Clinically documented diagnosis,	7 (11)	18 (18)	25 (16)	.2457
	no. (%)
	Microbiologically documented	53 (87)	76 (78)	129 (81)	.1435
	diagnosis, no. (%)
	Gram-negative bacilli, no. (%)	35 (57)	47 (48)	82 (52)	.2479
	Gram-positive cocci, no. (%)	27 (44)	36 (37)	63 (40)	.3453
	Fungi, no. (%)	14 (23)	17 (17)	31 (20)	.3858
	Other, no. (%)	7 (11)	10 (10)	17 (11)	.8008
	Primary site of infection
	Abdominal, no. (%)	23 (38)	40 (41)	63 (40)	.6463
	Pulmonary, no. (%)	28 (46)	38 (39)	66 (42)
	Other, no. (%)	10 (16)	20 (20)	30 (19)
	Q, quartile.

Table 2 .

 2 Characteristics and outcomes of patients with or without hospital-acquired infection (HAI)

		Patients	Patients
		With HAI	Without HAI
		(n ϭ 27; 29%)	(n ϭ 66; 71%)	p
	HAI characteristics
	Pulmonary infection	14 (51.9)
	Urinary tract infection	11 (40.7)
	Catheter-related infection	7 (25.9)
	Bloodstream infection	7 (25.9)
	Intubation
	No. (%)	27 (100)	61 (92.4)	.3165
	Duration, days	19 (14-34)	11 (7-18)	Ͻ.0001
	Central venous catheterization
	No. (%)	27 (100)	65 (98.5)	1
	Duration, days	27 (20-40)	13 (10-19)	Ͻ.0001
	Urinary tract catheterization
	No. (%)	27 (100)	61 (93.8)	.3165
	Duration, days	25 (20-40)	13 (10-21)	.0001
	Patient outcome
	Hospital stay, days	40 (29-99)	29 (21-38)	.0018
	ICU stay, days	37 (27-45)	13 (11-23)	Ͻ.0001
	ICU mortality rate, within 28 days	6 (22.2)	13 (19.7)	.784
	Crude ICU mortality rate	13 (48.2)	11 (16.7)	.0016
	Crude inhospital mortality rate	14 (51.9)	19 (28.8)	.0349
	ICU, intensive care unit.

Data represents no. of patients (%) or median value (interquartile range).

Table 3 .

 3 Uni-and multivariate analysis for hospital-acquired infection in the intensive care unit

			Univariate (n ϭ 93)			Multivariate (n ϭ 93)
		Odds	95% Confidence		Odds	95% Confidence
		Ratio	Interval	p	Ratio	Interval	p
	Gender
	Male	1	-	-
	Female	1.6	0.641-3.997	.3143
	Age, yrs	0.998	0.968-1.027	.8691
	Simplified Acute	0.999	0.961-1.037	.9381
	Physiologic
	Scale II
	Sequential Organ	1.124	0.955-1.322	.1586
	Failure Assessment
	Comorbidities
	0	1	-	-
	Ն1	1.021	0.414-2.514	.9645
	Origin of shock
	Nosocomial	1	-	-
	Community-acquired	1.144	0.467-2.803	.7682
	Infection site
	Pulmonary	1	-	-
	Abdominal	1.735	0.644-4.672	.3268
	Other	1.172	0.299-4.597	.8540
	S100A9 (messenger RNA) 1.120	1.034-1.213	.0054 1.120	1.034-1.213	.0054

Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 04/07/2023

Crit Care Med 2011 Vol. 39, No. 12 Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 04/07/2023

Mathieu Fontaine and Alexandre Pachot contributed equally to this research article.

This research was conducted with the help of logistics support (H. Thizy) from the Centre


Ethnographic studies of religion, stuck somewhere between ethnocentrism and literalism, especially in the case of Islam, have brought little distinctive anthropological analysis to bear successfully on the practical realities and political ideologies of religions. In addition, the popular assertion that certain ethnic groups that have had conflicts over centuries will continue to fight with one another is but an idea fuelled by numerous media reports and scholarly publications stressing the historical nature of conflict, much in the same way as the idea that Islam and modernity are somehow adversarial.

Frustratingly, during times of uncertainty, such essentialist explanations of difference continue to hold significant weight. As long as 'history' will function as a screen, behind which the interests of political or religious leaders are hidden, ethnic or cultural conflict will continue to appear intractable. Furthermore, there are the modern views of the nation state and all of its institutionalized mythologies, which over the last hundred years have confused, erased, desecrated and demolished our understanding of the local definitions and categories of group identity articulated in everyday life. Yet, it is only by exploring how group political identities are constructed, by disentangling the relationships between anxiety, threats and group identity responses, that the paths for conflict amelioration can be elucidated. Ger Duijzings's book tries to raise the standard of scholarship and addresses some of the issues outlined above. The introduction clearly shows a critical reading of the events and tensions that plagued Kosovo over the last hundred years. Of great interest are the details that Duijzings brings to his study of various seemingly marginal groups in Kosovo; these details shed light on important facts of identity strategies of interethnic and interdenominational relations. Duijzings reveals the paucity of Western understanding of Kosovo, which has often been presented as an area steeped in historical conflict. This conflict is generally cast as two-sided (Serb versus Albanian or Orthodox versus Muslim), with two antagonistic groups clashing and centuries-old hatreds being unleashed. The prevailing notion that 'Serbs' and 'Albanians' are distinct and largely antagonistic groups is not only a simplified version of historical and political facts, but also a nationalist distortion and represents the outcome of agitation rather than a description of root causes of the conflict.

Duijzings argues that Kosovo is not a simple case of irreconcilably distinct communities where religion is alleged to play a central role, but an example of complex, historically informed interchange between loosely defined 'communities' on the basis of loosely specified notions of religious, linguistic and/ or ethnic identity. While it has become commonplace to treat Albanian and Muslim cultural identity as interchangeable, suggesting cultural homogeneity, Duijzings documents the different facets of both Muslim and Albanian identity. By deconstructing these 'communities', he confronts the essentialist claim that Serbs and Albanians are locked in conflict due to an irreconcilable hostility between Orthodox Christianity and Islam. Actually, in the Balkans there are considerable cultural crisscrossings between Islam and Christianity; in fact, Duijzings makes it clear that the groups of the region are not historically predisposed towards conflict. Quite the contrary, the various religious and ethnic groups reflect continuous interaction with one another. It was only after the first Balkan War, in 1912, that relations between Albanians and Serbs really turned sour, much of this because the Serbian side proceeded with threats of forced assimilation, dominance and oppression. Duijzings's argument seems to be that there is after all a great deal more in common between Serbs and Albanians than one might suppose, and that the radicalization of Kosovo was not inevitable.

The dangerous politics of identity

Duijzings persuasively argues that "the formation and transformation of ethnic and religious identities is determined by wider political developments" (p. 22). Identities are political constructs in which state and religious regimes play crucial roles in their formation. Instead of regarding religion and politics as separate domains, Duijzings sees them as intimately connected formations of power. In peripheral societies like Kosovo where the official authority was often only partial, political and religious elites found particularly fertile ground for manipulating ethnic identities. In this aptly defined 'frontier society', where ethnic and religious identifications are flexible, he illustrates how group boundaries are more fluid and less institutionalized. Even though local, regional and religious identities remain very significant, the dichotomy between city and country is notable and ethnic identifications are linked to religious identity. In addition, state violence is not unrelated to processes of identity construction.

Although this is now part of history, as the processes of 'ethnic unmixing' and creation of stable one-dimensional identities through violence have eliminated much of the fluidity and interrelatedness of Kosovo society, this case is especially instructive. Duijzings asserts that "Kosovo's problems are not ethnic in origin but have been ethnicized by political and religious leaders who want easy political gain" (p. 204). If we continue to understand the conflicts in the former Yugoslavia as exclusively ethnic, we will remain largely blind to the underlying political motivations and goals set by the parties that have the most to gain from conflict. Political and religious elites attempting to hold or seize power and prestige can misdirect attention from their efforts by hiding behind the veil of mass ethnic strife.

The point is not that Duijzings is mounting a counterintuitive argument, but rather that he shows, especially in his introduction to the book, that the banal generalizations which have been endemic and so widespread in the West about the enduring character of the enmity between Serbs and Albanians (as between Christians and Muslims in the Balkans) have no basis in fact and that reality is considerably more complex. The assertions that Kosovo's population has a long history of sharing cultural traits, which includes language, religious rites and spatial identifications will go a long way in dispelling what most authors of the Balkans like to assert at best as mysteries and at worst as crude fixations. Duijzings tries to make room for a more complex understanding of regional identities, beyond the over-simplifications that often become the interpretative lens for studying the Yugoslav wars. As reviewers have noticed, 1 the very fact that Duijzings so intently tries to demonstrate crosscultural contact taking place in a number of localities is indicative of how much these fixations dominate the literature. It is time to toss these misconstructions in the theoretical and empirical garbage bin of nonsense. Unfortunately, it is only now that ethnographers and social scientists have started to produce articulate challenges to these hazardous and dangerous myths.

Though religion constitutes just one factor among many that contribute to the development of a sense of belonging to a larger group, most identity scholars consider it an identity marker that divides one group from another. The practice of religions, like any cultural marker, is a ritual linked to the historical contingencies of a particular moment. Religious practices in the Balkans have shifted over time, altering the definitions of group identity. Duijzings's (2000: 106-31) work is particularly useful in deconstructing the notion of a monolithic Islamic identity in the region. By tracing the internal divisions between the Albanian dervish orders and the Bosnian ulemas (the officially sanctioned Islamic Association) in the former Yugoslavia, he shows that what specialists of the literature call 'Balkan Islam' (Popovic 1986, Bougarel andClayer 2001) is instead manifested in a complicated variety of expressions that give rise to a number of identities. Duijzings reminds us that for Albanians "religion is almost irrelevant in official political life" (2000: 159); that Albanian nationalism is not clothed in religious terms; and that Kosovo's Muslims were never supported by Muslims from elsewhere in the former Yugoslavia or in the Balkans. This challenges the notion that knowing an individual's religious identity would facilitate placing that person within a given ethnic identification or conflict.

Duijzings draws attention to the political realities exacerbating the differences between religious groups in former Yugoslavia. First and foremost, he documents a split among Muslims in Kosovo, that is, between those who turn towards Sarajevo as the center for religious guidance, and those who do not. The former are often referred to as 'Muslim Slavs' associated with Bosnian Muslims to the north; they speak Serbian as their mother tongue and do not consider themselves to be ethnically Albanian. There is a second split among Muslims along the confessional Sunni/Shiite divide. Yet, Sunni Islam is associated with the Muslim Slavs of Sarajevo. The Islamic Association wielded all the political power from Sarajevo, while the dervish orders were largely based in underdeveloped rural areas that had fared badly at the end of the Balkan wars and conclusion of each world war. Not surprisingly, Kosovo sheiks conducted open polemics in the 1970s and resisted efforts by Sarajevo Islamic authorities to weaken the dervish orders in Kosovo. These polemics only subsided when federal state authorities intervened in 1979. The fact that Bosnian Muslims generally preferred to marry Bosnian Croats or Serbs instead of Albanian Muslims is symptomatic of the same state of affairs.

If some specialists in the area consider the opposition between Albanian popular Sufism in Kosovo and a Slavic orthodox Sunni Islam in Bosnia as a 'distorted caricature' (Clayer), the contradictions are indeed deeply rooted, going far beyond specialist conceptions caught in a mixture of literalism and a dreadful statisticizing which means absolutely nothing. These contradictions have rather a political, social and moral significance, which are better expressed in ethnic or religious discourses. As Bosnian Muslims held privileged positions regarding Islamic matters under the Yugoslav system, the proliferation of Sufism in Kosovo can be interpreted as an attempt by Albanian Muslims to reassert their autonomy in the religious sphere. In discourses related to Muslim religious revival, be it confraternal or otherwise, one recurrently finds nationalist motives, which better illustrate the necessity of considering such a powerful mobilizing force in the role that religious leaders claim they play in this field.

Yet, in the last two chapters, Duijzings attempts a comparative textual analysis of what he asserts are Albanian and Serbian examples of how religious symbolisms infiltrate 'national' imaginations. He argues that Albanian nationalists at one time might have attempted to infuse their cause with religious elements largely drawn from Bektashism. In particular, from elements drawn from a long epic poem in twenty-five cantos entitled Qerbelaja (first published in 1898), which retells the saga of Shiism by the wellknown nineteenth-century Albanian poet Naim Frashëri.

Naim Frashëri was one of the most eminent thinkers and leading figures of Albanian Bektashism, Albanian romanticism and Albanian national movement. His Bektashi epic has to be judged in the context of another monumental epic, the History of Skanderbeg, in which Frashëri narrates the exploits, successes and failures of the Albanian national hero against the Ottoman invaders, and which is held to be his most famous and greatest poetic aspiration. Even though both were published the same year and were written in the same style, one grounded on Muslim mythology and Eastern mysticism, the other on Classic mythology and Christian symbolism, it is only the latter epic that became the poem of Albanian national pride. The martyrs of Karbala, quite contrary to historical evidence and Eastern tradition, owe much to the heroism of the romantic era, including Frashëri's own poetic portray of Skanderbeg (Norris 1993: 182).

In the formulation and argumentation of nationalist ideology, Frashëri made use of both religious and realist, pantheist and gnostic conceptions, coming from both Eastern and Western traditions. Among other things, he openly suggested and theorized the reconciliation of religion with the idea of the nation. However, it is an exaggeration to argue, as Duijzings (p. 167) does, that Naim Frashëri attempt has been to bridge the religious divide of Albanians by proposing Bektashism as the national religion of Albanians. This view seems to stem from an uncritical reliance on eclectic compilations of literary histories (e.g. [START_REF] Mann | Albanian literature: An outline of prose, poetry, and drama[END_REF][START_REF] Elsie | History of Albanian literature[END_REF] found throughout Duijzings's book. The Bektashis, in spite of their concentration in Albania, were a small minority quite marginalized within Albanian society, especially before the spread of national movement. Naim Frashëri was certainly not blind to this fact. The relationship of religion and national ideas in Frashëri's conceptions has been very often uncritically investigated by scholars camped on both sides of either religious [START_REF] Qazimi | Ndikime orientale në veprën letrare të Naim Frashërit[END_REF] or nationalist [START_REF] Xholi | Naim Frashëri midis së kaluarës dhe së sotmes[END_REF] lines. There is reason to believe, however, that Naim Frashëri intended not so much to provide Albanians with a unique religion, but rather make nationalist ideas successfully acceptable to Albanians of whatever religious affiliation.

The problem, however, is not so much whether this has been, as Duijzings argues, a failed attempt to bridge the religious divide between Albanians by promoting a secular national cause that incorporates some heterodox Bektashi elements within it. Taking Naim Frashëri's epic reproduction of the Battle of Karbala and situating it on comparative grounds with such a state-and church-produced body of literature that re-creates and embellishes the Kosovo Battle mythology in Serbia, as does, is in itself a problematic issue in methodological and analytical terms. Duijzings finds both have much in common: each have been portrayed through epic song and folk verse, both record a lost battle where righteous forces were overcome by evil ones, and the main hero was sacrificed, leading to the necessity for followers to accept suffering as a step to redemption and a revolt against tyranny. Overall, Duijzings believes both lent similar weight to the growth of Albanian and Serbian nationalisms respectively during the nineteenth century.

Still, as some reviewers note, excepting the partly religious nature of both myths, they cannot be compared, either in terms of content or their elaboration and diffusion. Duijzings recognizes the lack of a deeply rooted cultural tradition for the failure of Naim Frashëri's epic to become a political symbol in Albanian nationalism. In contrast, what has made the Kosovo Battle myth so extremely powerful is its making rooted in folklorism and the church culture of Serbian nationalism. Duijzings has correctly understood it this way as suggested in his chapter on the Serbian folk-epic of . Although the Kosovo myth was created in Serbia and never resonated in Kosovo itself, throughout the twentieth century it provided for Serbs a set of roles with which to sort out into proper categories ambiguous or overlapping identities in Kosovo. The Serbian Kosovo myth and epic poetry grew out of elaboration of oral and church traditions; their religious content was closely tied to the culture harbored by the Orthodox Church, a powerful institution always influential in Serbian nationalist ideology. Actually, even after the downfall of communism and federalism in Yugoslavia, the Serbian Orthodox Church revitalized nationalism by carrying Lazar's bones around Serbia for reconsecration. Relations between church and state were enormously strengthened. Even the military murderers were transformed into heroes through epic songs, a process aptly labeled as 'gusle laundering' (p. 199), while Serb paramilitary leaders were compared to Milosh Obilic, the main hero of the Kosovo Battle.

Moreover, not only is the tendency to equate Serbian and Albanian nationalisms unjustified, it is also actually a dangerous ambush in which Serbian nationalist agitators are still trying to entrap public and scholarly opinions. The political instrumentality of the Serbian myths of the Battle of Kosovo is not exactly commensurable with Naim Frashëri's Qerbelaja, but rather with other types of political folklorism and pseudo-culturalist projects, serving either chauvinistically or ideologically charged nationalisms. Examples of this sort could be found, for instance, in the communist propaganda of Eastern and Central Europe when cultural and folkloric traditions have been instrumental for justifying and magnifying the 'authentic' foundations of the political and ideological regime, as I have shown elsewhere for the case of communist Albania [START_REF] Doja | Évolution et folklorisation des traditions culturelles[END_REF]. Other anthropologists have suggested a similar argument (e.g. [START_REF] Gossiaux | Le sens et le verbe: sur deux modes opposés d'instrumentalisation politique du folklore[END_REF]. Another parallel with the Serbian Kosovo mythologies can be found in Hitler's propaganda campaign of 1930s and early 1940s when German Volkskunde traditions have been instrumental in promoting the sinisterly reputed Nazi ideology. But to compare Frashëri's national ideology to Serbian nationalism, as Duijzings does, is like comparing Herder's romanticism to Hitler's national socialism! On the other hand, while the Battle of Kosovo was instrumental for Serbian nationalism as the mythologizing and embellishment of a local, historical and factual reality, Naim Frashëri uses the drama of Karbala as a local and historical metaphor of a mythic and ethical world view. In this sense, the relational parallel between Albanian and Serbian national ideology, if any, seems to be in difference instead of similarity. There are two different, opposed conceptualizations that give way, after instrumental transformations, to a number of single utterances, which share some similar details in style and content but are irrelevant for any relational comparison. When you compare such single utterances then, you do not compare them on a superficial, linear one-to-one basis, but only with respect to their relative setting in the whole field of constructed nationalisms, and their relative difference or similarity with all other relevant actual occurrences within the same field. Both types of transformations are variants of the same pattern, but there are differences in the form and content of their conceptualization of prerequisites and outcomes. What is lacking here is the analytical and anthropological insight to understand that the poetic and intellectual speculations of Naim Frashëri, retelling the saga of Shiism as the religion of victims of persecution, are relevant because they have been instrumental in shaping theological ideas with gnostic, dualist and pantheist conceptions that would have reasonably made it possible for the Bektashi religion to have entangled with Albanian national ideology.

Nevertheless, Duijzings's book is most welcome, especially its introduction, even though it seems not sufficiently grounded in the empirical work related to the chapters that follow. As some reviewers have remarked, while some chapters go back twelve years, the introduction was the last and most polished thing that Duijzings wrote for this collection. Though the introduction can initially excite the reader, the rest of the book can disappoint since the sophistication and intellectual maturity exhibited in the introduction is clearly lacking in much that follows. In fact, the initial excitement may encourage the reader to neglect the rich material that could have been present. The underlying problem with the entire collection is its problematic methodology, which often seems to favor a reliance on a flawed body of secondary literature rather than a combination of extensive fieldwork and a critical reading of sources. This is regretful as in the introduction Duijzings goes to great lengths to articulate a dynamic in the region that is completely at odds with what much of the literature of the past promotes.

Perhaps this is due to the fact that this is not a book in the classical sense; as Duijzings states in the preface, "this book is not the one I initially intended to write" (p. ix). In the early 1990s his research project considered what seemed to be a stable community, Letnica, a Catholic, ethnic-Croat enclave in Kosovo near Macedonia. The discussion about Letnica, however, is restricted to just the first chapter of his book (pp. 37-64). His ambitious project probably suffered from the accelerated events in Kosovo during the Milosevic era, when nearly the entire Croat population of Letnica became refugees in Croatia. Thus, as it has been suggested, the uneven quality of presentation and argument among the case studies discussed in the book may be largely due to the fact that Duijzings was only able to do extensive fieldwork in just this one case. Certainly, the unfortunate realities of the 1990s linked to the nature of identity politics might have changed dramatically the project of many anthropologists, though not necessarily for all of them.

Sifting through a body of secondary sources Duijzings forcibly tries to fit the material into what he correctly knows about the nature of identity. Unfortunately, his over-reliance on the work of both respected Balkan commentators such as [START_REF] Hasluck | Christianity and Islam under the Sultans[END_REF] and [START_REF] Malcolm | Kosovo: a short history[END_REF] is unhealthy. Both produced some of the best professional secondary resources you could hope to use for Kosovo. However, to assume authority while making unsubstantiated statements is a dangerous trap that Duijzings might have clearly understood. They cannot provide us the analytical foundation for what has been, ultimately in their case, an attempt to historicize a phenomenon that today is rather difficult to monitor anthropologically. In addition, while some reviewers have noted that his analysis seems to be problematic as far it is based not on field observations, but on an old secondary literature which does not give sufficient details and makes the comparison difficult (Clayer), others point that it is remarkable how many authoritative sources are totally ignored in parts of Duijzings's book (Norris).

However, Duijzings's scholarship runs especially into trouble when he relies heavily on local publications, from newspapers to post-1986 Serbian 'social science', which makes the reading of his case studies terribly frustrating, as he uses too often these politically charged and blatant propaganda sources to substantiate realities that are central to his argument. This is especially evident in his brief discussion of 'the making of Egyptians' in . Even though some reviewers believe that "this short chapter alone conveys and explains the transience and political function of identity in the Balkans better than most of the voluminous tomes on the subject" (Bellamy), others aptly point out that Duijzings relies excessively on the nationalist discourse by journalists and scholars, which "reflects a dangerous process of legitimizing 'points of view' when they are in fact scandalously false and contrived" (Blumi). More importantly, some classic and very dangerous nationalist mythologies (especially those that are all too often referenced with no qualification in Duijzings's book, such as the Serbian fascist propaganda about Albanian efforts to force assimilation) promulgate accusations that have never been substantiated with documentary evidence and fly in the face of history. As with much of this type of literature, these assertions are typically made by people whose authority stems from their political allegiance to nationalist projects. Duijzings himself is far too careful to outwardly subscribe to the premise of theories of this kind. Nevertheless, by incorporating this material so heavily into the content, the impact on his analysis is blurred and confused. The danger of adopting this flawed literature while attempting to contribute to the study of shifting identity is that it implicitly accepts the foundation -if not the content -of highly distorted suppositions.

In that context, instead of subscribing to charges of Albanian 'hegemony' and 'genocide' (!) there are far more productive and helpful ways of exploring individual motivations and cultural consequences of adopting one particular identity or another. A related problem, I suspect, might be this regrettable tendency of British and American publishers to tolerate a good deal of literature written by authors who are not fluent in the necessary languages, but who nonetheless seem to think that by dressing their stuff up as 'political science' they will somehow attain greater wisdom. What is clear, however, is that Duijzings is at his best when he does not depend on secondary literature but is informed by his own fieldwork and an intimate knowledge of the situation on the ground. A focus on marginal activities at the grassroots level helps Duijzings make important observations about the nature of identity and the role of religion in conflict and cohesion in Kosovo. By considering examples of identity development in Kosovo that most Western scholars overlook, several of the studies -in one way or another -demonstrate the incomplete nature of identity conversion.

For instance, he examines ethnically mixed pilgrimages and ambiguous sanctuaries as a laboratory of identity, where you can see both fission and fusion at work. In such religious events Catholics, Orthodox and Muslims come together at each other's sanctuaries and shrines, and rituals are shared and group identities are blurred. While showing that Catholic and Muslim Albanians have enjoyed largely unproblematic relations, he describes the history of crypto-Catholicism in Kosovo and explains how it united Albanians, making Islam and Christianity both part of their common identity. He can also talk knowledgeably about Albanian dervishes, the revival of popular Sufism and the Bektashis. The latter, a religious brotherhood that is dominant in Albania, is also present in Kosovo and Western Macedonia.

Finally, all of these cases together with its introduction make of this book an original contribution to the literature on identity politics in the Balkans, making it possible to elucidate contemporary problems of nationalism. Rather than casting ethnic conflict as a retreat to 'ancient hatreds', this book views these violent moments as social rupture, thus providing insight into the motivations and political formations that rush into the vacuums accompanying social dislocations. Ideas about ethnic separation were transmitted by religious/political elites and reproduced in the form of group identity formation. The divides between Serb and Albanian as between Christian and Muslim were contingent upon the immediate nature of social threats and the political-social dislocations that accompanied them.

  Religion and the politics of identity in Kosovo, London: Hurst, 2000, xv + 238 pp., ISBN 0231120990.

	Albert Doja

Focaal -European Journal of Anthropology 43 (

2004

): 149-156

Ger Duijzings,

  Duijzings knows Serbo-Croat (he teaches Serbo-Croat Studies in London), but probably not Albanian as he himself suggests while acknowledging the translation of some original texts by one of his Albanian students (p. 173). Certainly, you can negotiate your own way through Kosovo speaking Serbo-Croat, but if you also lack extensive fieldwork, this becomes a considerable shortcoming as anyone listening to an Albanian muhabet in Kosovo's cafés will have a better chance of knowing what is truly going on.


Pierre Tapie

Alexis M Prevost

Lorraine Montel

Léa-Laetitia Pontani
email: lea-laetitia.pontani@sorbonne-universite.fr

Elie Wandersman
email: elie.wandersman@sorbonne-universite.fr

A simple method to make, trap and deform a vesicle in a gel

We present a simple method to produce giant lipid pseudo-vesicles (vesicles with an oily cap on the top), trapped in an agarose gel. The method can be implemented using only a regular micropipette and relies on the formation of a water/oil/water double droplet in liquid agarose. We characterize the produced vesicle with fluorescence imaging and establish the presence and integrity of the lipid bilayer by the successful insertion of α-Hemolysin transmembrane proteins. Finally, we show that the vesicle can be easily mechanically deformed, non-intrusively, by indenting the surface of the gel.

INTRODUCTION

Vesicles have been widely used to mimic cellular compartmentalization and reproduce in vitro specific biological functions with bottom-up approaches [1,2]. In many recent studies, the focus was put on the encapsulation of complex biological reactions inside the vesicles, in order to express proteins [3] or genes [4], or to reconstitute and study the protein filaments of the cytoskeleton, such as the actin cortex [5] or microtubule asters [6]. Many other works aim to mimic the cell membrane properties, as for instance membrane fusion [7][8][9], or cell communication, via the insertion of transmembrane proteins in liposomes. In particular, mechanosensitive channels [10,11] have been inserted in the membrane of liposomes, allowing in turn to measure their conductance under mechanical stress. The production of vesicles usually relies on either hydration methods or inverted emulsion templates. Hydration methods rely on the swelling of dried lipid films in an aqueous buffer [12,13]. They are relatively simple to operate but yield polydisperse vesicle sizes and a relatively non homogeneous encapsulation efficiency [14][15][16]. The production rate of unilamellar vesicles can be improved by using electric fields (electroformation methods [17]) but fragile proteins can be damaged by the applied fields [18,19]and the technique is also limited to buffers with low ionic concentrations. Inverted emulsion templates, on the other hand, are based on the forced passage of a water-in-oil emulsion droplets through a water/oil interface [5,20]. This method can be developed in microfluidic chips [21][22][23][24], yielding monodisperse vesicle sizes, that are limited by the microfluidic channel dimensions. In both methods (hydration or emulsions), the resulting vesicles are dispersed in the outer medium, which requires additional steps to handle or transfer them in different environments (micropipettes [25], optical trapping [26], etc). These extra steps, relying on specific technical skills, make it difficult to replicate many experiments and collect large statistics on the systems proper-ties. In particular, in order to study mechanotransduction processes, it is required to mechanically stimulate the vesicles. In practice, this has typically been achieved with local membrane deformations (pipette suction [27], fluid flows [28,29], AFM [30,31]). However, these methods do not reproduce faithfully the nature of mechanical perturbations in tissues. In addition, they overlook the mechanical coupling between cells and their biological visco-elastic environment. We propose here a new technique, that provides a versatile platform for the straightforward production of biomimetic pseudo-vesicles (a vesicle with an oily cap on top), but also allows for their trapping and non-intrusive mechanical excitation.

PSEUDO-VESICLE FORMATION

The vesicles are produced from a water in lipidcontaining oil droplet configuration, that is formed in a liquid, warm, agarose solution (details on the chemicals can be found in the Methods Section). We first draw a small volume (about 500 nL) of the internal aqueous phase (Figure 1a), using a 2.5 µL micropipette (Eppendorf). Second, we move the pipette into the oil/lipids container and suck about 50 nL of it (Figure 1b), by turning the adjustment wheel of the micropipette while the tip is immersed in the oil. The pipette tip thus contains an oil/water sandwich. Last, we move the pipette into the warm agarose solution (temperature T ≈ 38 • C, see details in the Methods Section) where we expel the oil/water sandwich by turning backward the adjustment wheel of the micropipette. During this expulsion phase, the oil phase first grows into a droplet in the liquid agarose (Figure 1c), followed by the aqueous phase that grows inside of it (Figure 1d,e). Finally, we move up the pipette rapidly in the air, which detaches the double droplet from the tip due to viscous friction from the agarose solution (Figure 1f). Immediately after its formation, the inner water droplet is thus surrounded by an oil layer containing phospholipids. These lipids therefore redistribute at both oil wa- ter interfaces, with their hydrophilic heads turned towards the inner water phase on one side and towards the agarose solution on the other side. While the agarose solution cools down, the double droplet slowly sediments in the agarose solution and eventually gets trapped in the formed gel. During this cooling process, the oil layer of the double droplet creams under gravity to form an oil cap, while the lipids stabilizing the former oil/water interfaces zip up a lipid bilayer on the lower part of the droplet (Figure 1g). The double droplet becomes a pseudo-vesicle. This simple micro-pipette method yields pseudo-vesicles with a diameter of 601 ±58µm (N=98). Smaller sizes can be obtained using smaller tips and a micro-injector (see Methods Section). The production rate of the method is around 5 pseudo-vesicles per minute and the rate of success is overall about 30%. Once trapped in the gel, the pseudo-vesicle is stable for several hours and can be kept overnight if the gel-containing cuvette is sealed to prevent evaporation. Moreover, being trapped in the gel, pseudo-vesicles can easily be observed with a microscope, as in [32].

MEMBRANE CHARACTERISATION AND FUNCTIONALIZATION Fluorescence imaging

To characterize the pseudo-vesicle and probe the existence of a lipid bilayer, we first used fluorescent markers dispersed in both the internal aqueous phase (carboxyfluorescein, emission wavelength λ c = 525 nm, green, see Methods Section) and the oil phase (Nile red, emission wavelength λ n = 636 nm, red). The pseudo-vesicle is produced in the agarose gel as described above, and fur-ther imaged using a confocal microscope, with a 4x objective. The fluorescence images from both the green and red channels are recorded (see Figure 2a), showing that the fluorescent water phase is efficiently encapsulated in the pseudo-vesicle. Secondly, we added green fluorescent lipids (NBD-PC, 1wt%) to the lipid mixture present in the oil phase labelled with Nile Red. A composite image obtained by confocal microscopy is shown on Figure 2b. On Figure 2c, we plot for each channel the normalized radial intensity profiles ((I(r) -I(0))/(I cap + I(0)), where I cap is the intensity in the oil cap in a given channel. These profiles reveal a significant increase of the fluorescent signal arising from the lipids at the boundary between the inner aqueous phase and the outer agarose gel. On the contrary there is no detectable fluorescence signal arising from the oil phase, indicating that there is not a measurable layer of oil in the bilayer, at the confocal microscope spatial resolution and in the limit of our fluorescence imaging sensitivity. Altogether, our results confirm that the inner phase is efficiently encapsulated in a lipid bilayer devoid of significant amounts of oil. This doesn't rule out the presence of oil traces at submicrometer length scales in the bilayer, as evidenced in emulsion template formed vesicles [33]. However, it was shown in [34] that such oil residues in the membrane did not impair significantly the mechanical properties nor the functionality of the membrane. The membrane tension γ b can be estimated from the contact angles of the oil cap with the aqueous and agar phases (see Supplementary Information) and yields γ b = 4.15 ± 0.33 mN/m obtained from 13 images on N=8 pseudo-vesicles), which is much larger than values usually reported in electroformed lipid vesicles [35,36], γ b ∼ 10 -3 mN/m. On the contrary, our membrane tension value compares well with those obtained in Droplet Interface Bilayers (DIBs) which are planar lipid bilayers obtained by putting in contact two aqueous droplets bathing in an oil+lipid mixture (see for instance [37][38][39]). In DIBs, there is also water/oil interfaces surrounding the lipid bilayer. The high γ b value we measure in our pseudo-vesicle is thus, as in DIBs, presumably due to the presence of the oil cap which pins and stretches the bilayer on both sides.

Nanopores insertion

To further probe the presence of a lipid bilayer and its functionality, we inserted α-Hemolysin (αHL) transmembrane proteins in the lipid bilayer. αHL is an heptameric nanopore [40] through which carboxyfluorescein molecules can diffuse [41]. Practically, we found that the direct dissolution of αHL monomers in the internal aqueous phase was decreasing the pseudo-vesicle stability, due to a nanopore induced modification of the oil/water surface tension (See Supplementary Information). We therefore used Small Unilamellar Vesicles (SUV) containing αHL nanopores, primarily prepared (see Methods Section) and dispersed in the fluorescent aqueous internal phase loaded with carboxyfluorescein. The pseudovesicle is then formed as described above, allowing for the SUVs contained in the inner phase to fuse with the bilayer, as previously shown in [42]. This fusion allows in turn the insertion of functional transmembrane channels into the lipid bilayer [43,44], αHL in our case (Figure 2d). Under an epifluorescence macroscope, we imaged the carboxyfluorescein leakage across the pseudo-vesicle by acquiring one image every 4 minutes, for two hours. We performed control experiments using on the one hand SUVs devoid of αHL nanopores, and on the other hand pseudo-vesicles prepared without SUVs nor αHL. Using image analysis, we measured the average intensity inside (resp. outside) the pseudo-vesicle I in (resp. I out ) from which we determine the fluorescence intensity contrast Γ = (I in -I out )/(I in + I out ). The time variation of the normalized contrast Γ(t)/Γ(t = 0) is presented on Figure 2e. Clearly, the normalized contrast decreases over time for pseudo-vesicles containing αHL whereas it remains constant for αHL-free pseudo-vesicles. This establishes that the lipid bilayer can be functionalized with a transmembrane protein. More quantitatively, fitting the normalized contrast with an exponential decay (solid line on Fig. 2e), we obtain a characteristic release time of 5.9 ± 0.3 .10 4 s. This timescale is expected to scale with the volume of the vesicle [45]. Taking into account our large vesicle size, our results are in good agreement with values reported in the literature [34,45,46]. Error bars are SE of data. R = 316±24 µm is the average pseudo-vesicle size. d) Sketch of αHL insertion in the pseudo-vesicle membrane, using αHL loaded SUV. e) Time evolution of the normalized contrast Γ(t)/Γ(t = 0), for αHL loaded pseudo-vesicles (red disks, N=5) and control experiments without αHL (black squares, N=5). Error bars are SD of data. The solid line is an exponential fit of the data Γ(t)/Γ(t = 0) = e -t/τ ≈ 1 -t/τ , with τ = 5.9 ± 0.3 .10 4 s.

MECHANICAL EXCITATION

Since the pseudo-vesicle is trapped within the bulk of the agarose gel, it can be deformed by indenting the surface of the gel (Figure 3a). We compress the surface of the gel with a square piston (surface S=1 cm 2 ) mounted of a motorized z-translation stage (indentation amplitude, ∆z = 1 mm, Figure 3b-e), while measuring the applied normal force F (Methods Section). With our fluorescence macroscope, we image a pseudo-vesicle containing carboxyfluorescein, as the pseudo-vesicle is cyclically deformed (Figure 3b-d). Using image analysis, we fit an ellipse to the pseudo-vesicle shape (excluding the oil cap from the analysis, Figure 3c). The time variation of the long a and short b axis of the ellipse are plotted on Figure 3f, from which we compute the ellipse's eccentricity e = 1 -b 2 /a 2 . The variation of eccentricity, ∆e = e(F ̸ = 0) -e(F = 0) can be used as a proxy for strain. On Figure 3g, we plot the compressive stress σ = F/S as a function of e. A linear relationship is observed, σ = K e ∆e, with an effective compression modulus of the pseudo-vesicle K e = 12.1 ± 0.6 kPa. The value of K e is well distinct from the gel's compression modulus K gel ≈ 85 kPa (see inset of Figure 3g). In addition, we performed complementary experiments (see Supplementary Information) showing that the presence of the oil cap was not affecting the pseudo-vesicle deformation. Our method thus offer a easy-to-use platform to study lipid bilayer mechanics or mechanotransduction processes.

CONCLUSION

Overall our approach constitutes a very simple method to produce and trap pseudo-vesicles within a gel, which is easy to set up and inexpensive. On the one hand, the method only requires small volumes of encapsulated phase (∼ 1 µL of sample), in contrast with usual microfluidic techniques [21] which usually require hundreds of microliters of solutions in order to obtain stable flows. On the other hand, it is based on gentle manipulation, thus avoiding any protein denaturation which can be caused by the application of electric fields [18,19] as in electroformation techniques. It makes it of special interest for the use of valuable and delicate biological samples. Note, however, that the estimated value of the membrane tension in our system is much larger than values usually reported on electroformed vesicles, presumably due to the presence of the oil cap which pins and stretches the bilayer on both sides. This effect should be taken into consideration for further applications of the method. Last, because the pseudo-vesicles are trapped in a gel, they are easily localized and do not require post-production handling, in contrast with emulsion template methods. The pseudo-vesicles can also be easily deformed by indenting the gel surface, allowing to finely tune both the stress amplitude and frequency. Finally, the pseudo-vesicles are embedded in a viscoelastic gel which better mimics the mechanical properties of biological tissues.

METHODS SECTION

Chemicals

Unless specified, all chemicals were purchased from Sigma Aldrich, Merk inc.

Internal/external aqueous phase

The internal aqueous phase of the double droplet contains 10 mM Tris buffer (pH=7.5), 400 mM sucrose, and 100 mg/mL Dextran (M w =40.10 3 g/mol,). For fluorescent pseudo-vesicles, we added 20 µM of carboxyfluorescein to this buffer. The osmolarity of the internal phase, measured with a Löser TYP6 osmometer is about 600 mOsm. The external phase is made of a 3%wt low gelling temperature Agarose, dissolved in a buffer containing 10 mM Tris (pH=7.5) and 200 mM potassium chloride. Prior to agarose addition we adjust the osmolarity to 380 mosm. Agarose is then dissolved in the buffer on a hot plate and the resulting solution is maintained in a liquid form at 85 • C.

Oil phase containing the phospholipids

The oil phase is a 50/50 wt/wt mixture of Hexadecan and Silicon oil AR20 in which dried lipids are dissolved. The lipid mixture is the one described in [45] to mimic the bacterial membrane composition and maximize its stability in droplet interface bilayer geometries. It consists of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 81.1 wt%), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, 10.8 wt%), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DOPG, 5.4 wt%) and cholesterol (2.7 wt%). Note that such a high concentration of DOPC prevents the formation of lipid domains both in the bilayer [47,48] and at the oil/water interface [49]. In practice, we prepare a solution of 7.4 mg of this lipid mixture dissolved in chloroform. The lipids are then dried under nitrogen and kept under inert atmosphere in a 2 mL vial at -20 • C for several weeks. Immediately before use, we add 2 mL of the oil phase to yield a total lipid concentration of 3.7 mg/mL, and place the vial in an ultrasonic bath for 30 minutes at 30 • C. This oil/lipid mixture can then be used for a few days.

For confocal imaging, we labelled separately the oil phase and the phospholipids. The oil phase is labelled with Nile Red with the following procedure: a small amount of solid Nile Red (typically the tip of a spatula) is dissolved in 300 µL of acetone. 5 mL of silicone oil is then poured onto the acetone and stirred overnight at room temperature in order to transfer the Nile Red dye into the oil phase while evaporating all traces of solvent.

For the labelling of the lipid bilayer, fluorescent lipids were incorporated in the lipid mix following the above-mentioned procedure. We use 1-Myristoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-Glycero-3-Phosphocholine or NBD-PC lipids that are excited at 480 nm, allowing us to image simultaneously the oil phase labelled with Nile Red and the lipid bilayer labelled with NBD-PC. In that case the final lipid concentrations in the mix yield: 80.3wt% DOPC, 10.7wt% DPhPC, 2.7wt% Cholesterol, 5.4wt% DOPG, 1wt% NBD-PC.

SUV preparation

The above-mentioned lipid mixture (total lipid mass 1.25 mg) is dried under nitrogen in a test tube and left to dry further in a vacuum chamber overnight. The next day, 1 mL of the internal aqueous phase is added to the lipid film and the solution is placed in a probe sonicator for 30 minutes using on/off cycles of 15/5 seconds, respectively.

Preparation of αHL solutions and integration in SUV αHL monomers are prepared in the internal aqueous buffer at a concentration of 250 µg/mL. In order to obtain SUV decorated with αHL nanopores, 30 µL of this αHL solution was added to 120 µL of the SUV solution, yielding a final pore monomer concentration of 50 µg/mL. The resulting mix is incubated at room temperature for about an hour and used as the inner phase of the pseudovesicles.

Vesicle formation and mechanical excitation

The oil/aqueous phase sandwich is made following the steps described in the main text. The warm agarose solution is poured into a spectrophotometer cuvette (dimensions 10x10x40 mm 3 ).

Temperature of the agarose is measured with a temperature thermocouple (USB-TC01, National Instrument). When the temperature reaches about 38 • C, the double droplet is formed. The system is left to cool down to room temperature for about 15 minutes, so that the agarose gels around the pseudo-vesicle.

Note that the method can in principle be used in liquid (agarose-free) environments, which would allow to use oil-cap removal techniques as described in [22][23][24]. In our first trials, however, the high sedimentation velocities of the produced droplets (that were large, and significantly denser than the outer phase) were destabilizing the pseudo-vesicles during their formation. Reducing the pseudo-vesicle size (using a smaller injection tip diameter), adjusting the density of the inner phase or increasing the external phase viscosity will lower the sedimentation speed of the produced double droplet and sounds like a promising route to obtain stable pseudo-vesicles in liquid environments.

For mechanical excitation experiments in agarose, a thin rectangular sheet of Plexiglas (width 1 mm) is added on one side of the cuvette to cover its wall, prior to liquid agarose pouring. As the agarose is gelling, this sheet is carefully removed, leaving an empty space between the agarose gel and the cuvette wall. Indeed, due to Poisson effect, a lateral expansion of the gel occurs as it is vertically compressed. This empty space is required to allow for this lateral expansion and a proper elastic deformation of the gel. The cuvette is placed and fixed on the device displayed in Figure 3a. The piston has been 3D printed to fit the cuvette size and a thin Plexiglas sheet is glued to the piston face indenting the agarose surface. The piston is mounted on a translation stage controlled by a Newport LTA-HL linear actuator (Z resolution of 1 µm). As the piston indents the gel, it deflects the set of two planar cantilevers attached to the base of the sample holder. A capacitive sensor measures the deflection of the can-tilevers. Knowing the stiffness of these cantilever (from previous calibration) we deduce the applied normal force F (the measurement noise on F is 1 mN). The first step of the indentation experiments consists in determining the gel surface position. To this end, the Z position of the piston is lowered by steps of 0.1 mm, while measuring the normal force. As the force reaches 5 mN the ramp is stopped and this Z position is taken as the origin for Z coordinates. Subsequently, we impose a cyclic deformation of the gels, by indenting the gel, from this surface position, by a value ∆Z, by steps of 100 µm. At each step, the force is measured. The pseudo-vesicle is imaged at each indention step (using a pulse of blue light, duration 500 ms) with a Leica Macroscope and a Pointgrey camera (BFLY-U3-23S6C-C).

Smaller pseudo-vesicle production

To reach smaller pseudo-vesicle sizes, we use a smaller injection tip. In practice we use a Polyurethan tubing (Phymep) with an outer diameter of 240 µm and an inner diameter of 130 µm. The tubing is connected to a 50 µL syringe (Hamilton), mounted on a home-made microinjector. The pseudo-vesicles are produced in agarose gel with the fluorescent inner buffer and imaged through epifluorescence. We determine their sizes using image analysis and find an average diameter D = 410 ± 45µm (N=6). In principle, smaller sizes could be obtained by using a smaller injection tip. The only experimental difficulty will consist in forming the oil/water sandwich. The use of a commercial micro-injector will be required.

  FIG.1. a-f) (upper row) Sketch of the double emulsion production method. Green/orange/blue colors stand for internal/oil/external phases, respectively. (bottom row) Bright field images. Scale bars = 500 µm. The produced double emulsion droplet in f) sediments in the liquid agarose and eventually gets trapped as the agarose gels. Simultaneously, the surrounding oil shell creams at the top of the droplet and a lipid bilayer is zipped on the lower part. A few minutes later, the pseudo-vesicle has been formed and is trapped in the gel, as sketched in g). Bottom panel: Fluorescence macroscope image of a pseudo-vesicle loaded with carboxyfluorescein trapped in an agarose gel. Scale bar = 200 µm.

FIG. 2

 2 FIG.2. a) Composite confocal microscopy image of a pseudovesicle trapped in an agarose gel. The internal phase contains carboxyfluorescein (green), while the oil phase contains Nile Red (red). b) Composite image obtained by adding 1wt% fluorescent NBD-PC in the lipid mixture (green) and an oil containing Nile Red (red). Yellow color = red + green channels. For clarity, the image has been smoothed with a 2 pixel radius Gaussian filter. Scale bars = 200 µm. c) Normalized radial fluorescence intensity profile, averaged over N=8 pseudo-vesicles labelled as in b). The green triangles (resp. red disks) show the fluorescent lipids channel (resp. fluorescent oil channel). Error bars are SE of data. R = 316±24 µm is the average pseudo-vesicle size. d) Sketch of αHL insertion in the pseudo-vesicle membrane, using αHL loaded SUV. e) Time evolution of the normalized contrast Γ(t)/Γ(t = 0), for αHL loaded pseudo-vesicles (red disks, N=5) and control experiments without αHL (black squares, N=5). Error bars are SD of data. The solid line is an exponential fit of the data Γ(t)/Γ(t = 0) = e -t/τ ≈ 1 -t/τ , with τ = 5.9 ± 0.3 .10 4 s.

FIG. 3

 3 FIG. 3. a) Sketch of the mechanical excitation setup. bd) Macroscope fluorescence images of pseudo-vesicles either relaxed ((b) Z=0) or deformed (Z=0.5 and 1 mm in (c,d), respectively). In (c) the red line is the fit of the contour of the lower part of the pseudo-vesicle with an ellipse. Scale bar = 400 µm. e) Piston position Z(t). f) Major (red) and minor (blue) axes of the fitted ellipse as a function of time, for a 1000 s long cyclic indentation. g) Compressive stress σ as a function of the ellipse's eccentricity e. Grey crosses correspond to all data points from f), black circles are averages within bins of eccentricity δe= 0.01. Error bars are SD of the data. The red doted line is a linear fit to the data. Inset: Normal force as a function of the piston indentation. The line is a linear fit F = κz, from which the gel compression modulus can be deduced, K gel = κH/S, with H the height of the gel.

Acknowledgements

The authors thank Mathieu Letrou and Sophie Cribier for their valuable help with SUV preparation. EW acknowledges financial support from Emergence Sorbonne University, LLP acknowledges support from Agence Nationale de la Recherche (BOAT, ANR-17-CE30-0001) and from Emergence(s) Ville de Paris.

DATA AVAILABILITY

All data generated or analyzed during this study are included in this published article and its supplementary information files. Correspondence and requests for raw data and materials should be addressed to E.W. or L.-L.P.