From this band, ten sequences out of 12 obtained were related to the

genus Curvibacter (class of β-proteobacteria), the two other sequences corresponding to the genus Burkholderidia (class of β-proteobacteria) (Table 5). Three other sequenced bands were visible in all treatments but they increased significantly in intensity at the end of incubation (both B3 and B4 in Vfinal of LA1, B8 in VFfinal of ARS-1620 clinical trial LB2). These three excised bands were related to the phylum PX-478 cell line Actinobacteria (with B3 affiliated to the clade acI) (Figure 4 and Table 5). Finally, the three last bands chosen to be sequenced appeared (B5 in Vfinal and VFfinal of LA2) or disappeared (both B6 and B7 in VFAfinal of LB1) at the end of incubation (Figure 4). These ones were all affiliated to the phylum Actinobacteria

(as were 85% of the sequenced DGGE bands). Note that the excised band B1 (LA1 experiment), related to the phylum Cyanobacteria (Table 5), disappeared at the end of the incubation in both VF and V treatments. Table 5 Phylogenetic information about the OTUs

corresponding Captisol concentration to the excised and sequenced DGGE bands Bands N° Number of sequenced clones OTUs Nearest uncultivated species accession no°,% similarity B1 12 Phylum: Picocyanobacteria Synechococcus sp AY224199, 98% B2 10 Class: β-proteobacteria Genus: Curvibacter EU703347, 98 EU642369, 99% B2 1 Class: β-proteobacteria Genus: Burkholderia EU642141, 98% B2 1 Class: β-proteobacteria Genus: Burkholderia EU801155, 97% EU63973669, 96% B3 9 Phylum: Actinobacteria Clade: acI FJ916243, 99% B4 11 Phylum: Actinobacteria Unidentified FN668296, 99% B5 10 Phylum: Actinobacteria Unidentified FN668268, 100% B5 1 Unclassified bacteria Metalloexopeptidase   B6 12 Phylum: Actinobacteria Unidentified FJ916291, 99% B7 11 Phylum: Actinobacteria Unidentified DQ316369, 99% B8 8 Phylum: Actinobacteria Unidentified AJ575506, 99% B8 3 Unclassified bacteria   Cluster analyses based on quantification of the band position and intensity (Figure 5) showed that, for each lake, the bacterial community structure was clearly different according to the period (early spring/summer) (Figure 5).

[32]. Although no statistical correlation was performed, it was observed that isolates belonging to the capsular type II were confined to MT1, indicating that the genetic background of this serotype may be well conserved. Higher number of isolates may corroborate these PD-1/PD-L1 assay findings. All isolates were susceptible to the antimicrobials evaluated in this study, except erythromycin and clindamycin. Although it was not an epidemiological investigation, the overall rate of erythromycin resistance among the isolates analyzed was 19.3%. Previous epidemiological and bacterial collection data from Brazilian GBS isolates showed that erythromycin resistance ranged from 4 to 14% [10–13].

A higher incidence rate LY2835219 clinical trial was observed in other regions, where erythromycin resistance up to 40% among GBS isolates was detected in Europe [15] and USA [3, 9].

In this study, resistance to both erythromycin and clindamycin was observed in GBS isolates of capsular types III and V, whereas the isolates displaying resistance only to erythromycin were exclusively found in the Ia capsular type. Similar results were previously obtained by other authors [3, 10]; however, resistant isolates for both antimicrobials were also observed among the Ib, II, IV, VI and VIII capsular serotypes [3, 34]. The mechanisms of macrolide resistance are mediated by ermA, ermB and mefA/E, and the distribution of these genes among GBS isolates in this study were in accordance with the macrolide-resistance AZD8186 cost phenotypes. These results were also observed by others [10–13]. The increasing numbers of isolates showing macrolide resistance together with the description of reduced susceptibility to penicillin emphasize the need for continued monitoring of antimicrobial susceptibility profile to identify the emergence of resistance among GBS isolates. Data of the potential virulence of GBS isolates from Brazil are limited. Three genomic islands encoding the structurally distinct types of pili (PI-1, PI-2a and PI-2b) were identified in GBS. These pili are organized

in two different loci, where PI-2a and PI-2b PLEK2 are located at the same chromosomal locus, with these being mutually exclusive [35]. To our knowledge, this is the first study describing the prevalence of the pilus island in Brazilian GBS isolates, and at least one pilus type was detected among the isolates, supporting their use as an antigen for vaccine development. The combination of PI-1 and PI-2a was the most prevalent among the GBS isolates, and this result is in agreement with previous reports [21, 36]. In addition, the presence of this combination was correlated with maternal colonization and invasive disease in adults [36]. The cyl locus of GBS consists of a cluster of twelve genes [27], and some of them can modulate cylE expression and secretion [37], which is crucial for β-H/C activity.

In order to identify an index patient, it would be helpful if risk patients were routinely swabbed upon admission. As the efficacy and cost-effectiveness

of patient screening are unproven and the quality of the evidence is poor (McGinigle et al. 2008), other deciding criteria should be established for the appraisal of MRSA infection as an AZD5363 OD in HCWs. The practice under German law is to apply the presumed causality clause in order to facilitate the recognition of OD claims in those cases where no index patient has been identified, but the infection appears to be evidently occupationally related (SGB VII, Art. 9, Para. 3). In all 17 recognized cases, it was assumed that the infected HCW had been in direct contact with AZD6244 patients likely to have proven MRSA-positive, although this could be verified in only

53% of these cases. It is apparent that the quality of evidence substantiating workplace-related infection varies. These figures show that conclusive evidence of a causal link between MRSA infection and the workplace, i.e. recorded exposure to MRSA-positive patients, was determined only in every second HCW. The procedure to adjudicate claims for recognition of MRSA infection as an OD involved both hard facts and less conclusive evidence. The strongest argument for a causal relationship was a similar Tucidinostat manufacturer genetic profile of the index patient and the HCW. The least conclusive argument was the presumption that the workplace was a healthcare setting in which MRSA was endemic. In 18% of the recognized cases, no expert appraisal was performed. This may be because many MRSA cases recovered without complications and incurred low medical costs so that an expert appraisal was deemed unwarranted. The reasons for rejecting claims for the recognition of MRSA as an OD were not analyzed in this paper. The data in the standard documentation of rejected cases are not detailed enough to allow reliable

assessment, with regard to exposure and symptoms. Furthermore, the data do not distinguish between colonization and infection. The data suggest that a large proportion of the MRSA claims were rejected by the BGW because MRSA colonization is not considered legitimate confirmation of OD. A large proportion of the rejected claims for which no specific workplace exposure Tangeritin was established were probably reported for prophylactic reasons to allow for the possibility that it should prove necessary to make an insurance claim. The German Code of Social Law (SGB VII, Art. 9, Para. 3) stipulates that sufficient probability of a workplace-related cause of disease should be established. Additional, non-occupational risks of infection were found in five cases. However, the assessors did not address risks outside the HCW’s job in their appraisal of these cases. Presumably, the assessors considered the risk of infection among HCWs to be higher than the endemic risk in the population at large.

coli conditional auxotrophs. These proteins do not bear significant sequence similarity to naturally occurring proteins, are α-helical, as per our binary code design strategy, and are extremely thermostable. Our work demonstrates that even de novo polypeptides are genuinely poised for biological action and that unevolved proteins from a binary coded combinatorial library will readily promote life. E-mail:

mafisher@princeton.​edu Origin of Plant Phenylalanine Ammonia Lyase: A Key Apoptosis inhibitor Event for Land Colonisation? Marco Fondi1, Giovanni Emiliani,2 Simonetta Gribaldo3, Renato Fani1 1Department of Evolutionary Biology, University of Florence, via Romana 19, 50125 Florence Italy; 2Department of Environmental and Forestry Technologies and Sciences, University of Florence, via S. Bonaventura 13, 50145 Florence, Italy; 3BMGE

Unit, Pasteur Institute, 75724 Paris, France Between 480 and 360 million years ago, land plants (Embryophytes) evolved, from the Charophyceae, a small group of freshwater green algae (Kenrick and Crane,1997), differentiating from simple structure (Bryophyte) to elaborate organisms showing an extraordinary array of complex organs and tissue systems (vascular plants). However, in the first stages of prototrophs terrestrialization, beneficial associations between fungi (mycorrhizal symbioses), and soil bacteria (N2 fixing), might have greatly helped early land plants to face a harsh environment characterised by important stresses including desiccation, UV radiation, and microbial attack (Selosse and Le Tacon, 1998). A key find more event for plants colonisation of land and diversification was probably represented by the molecular evolution of phenylpropanoid pathway, since these compounds are involved in many

stress response pathways (pathogens, grazing, ROS scavenging, UV screening, etc) as well as in other fundamental traits such as biosynthesis of lignin, the structural polymer able to guarantee stem rigidity and xylem (water conducting tissue) formation (Ferrer et al., and reference Amino acid therein). Despite its importance, the origin and evolution of the phenylpropanoid pathway, as well as the first advantageous physiological roles of its products are unclear. Phenylalanine Ammonia Lyase (PAL) is responsible for the first committed step of plant phenylpropanoid pathway and the complete metabolism appears to be a specific and ubiquitous feature of land plants. However, PAL SAR302503 datasheet homologues have been identified and characterized in fungi such as Aspergillus oryzae (Seshime et al., 2005). Although phenylpropanoids are largely absent in prokaryotes, PAL homologues have been recently identified in Streptomyces maritimus and Photorhabdus luminescens where they are involved in the production of antimicrobial compounds (Xiang and Moore, 2005).

The bands were visualized by using the enhanced chemiluminescence system (Pierce, Rockford, IL). To validate the reproducibility, the tests were I-BET151 mouse repeated for at least 3 times. Statistical analysis Statistical analysis was performed using the independent 2-tailed t-test. All P values were two-tailed and considered statistically significantly if less than 0.05. Means, standard errors, and P values were calculated using SPSS version 11.0 for Windows. Results Cell transformation of IEC-6 cells The method has been well established

for cell transformation of normal cells with MNNG and PMA. We treated IEC-6 cells with MNNG and PMA for 12 times. After the final treatment, we detected the colony check details formation in semisolidified agarose of normal and MNNG/PMA treated IEC-6 cells. Transformed foci of normal IEC-6 cells were 0.02% and that of MNNG/PMA treated IEC-6 cells were 0.37%. MNNG/PMA treatment markedly enhanced the production of

transformed foci (Table 3; p < 0.01). Table 3 Transformation of IEC-6 cells by MNNG and PMA1. Cell type dishes Number of clonies Clong efficiency in soft agar(%) normal 4 2 ± 0.1 0.02 MNNG/PMA 4 37 ± 0.2 0.37* * p < 0.01 compared to untreated cells. Then we detected the cell AZD3965 growth curve of normal and MNNG/PMA treated IEC-6 cells. Cell proliferation was determined by3H-TdR, which indicated the DNA synthesis. As shown in Fig. 1, cell growth of MNNG/PMA treated IEC-6 cells was significantly increased, compared with that of

normal IEC-6 cells. The increased cell growth was coincident with the property of cancer cells. To further confirm its cancerous character, MNNG/PMA treated IEC-6 cells were inoculated subcutaneously in nude mice. As expected, tumor xenografts for were detected in all animals 4 weeks later, which was coincident with the result of human cancer cell SW480. However, no tumor xenograft was visible in mice inoculated with normal IEC-6 cells even 8 weeks after inoculation. Fig. 1b showed the tumors were low- differentiated carcinomas. Histologically, the tumor cells of xenografts were arranged in flakiness and nest with round or polygon in shape. Tumor giant cells and mitotic phases could be seen. This suggested MNNG/PMA treated IEC-6 cells had been fully transformed. Figure 1 Transformation of normal ICE-6 cells. (A) Cell growth curves of normal and MNNG/PMA treated IEC-6 cells. (B) Histologically analysis of tumor xenografts inoculated with transformated IEC-6 cells. Changes of gene expression detected by microarray analysis To elucidate the molecular mechanisms involved in cell transformation of IEC-6 cells, the rat Oligo GEArray microarray was used to identify genes with altered expression level after cell tranformation, compared with its normal controls. The microarray comprised 113 genes representative of the six biological pathways involved in transformation and tumorigenesis.

4b); #selleck chemicals llc randurls[1|1|,|CHEM1|]# interestingly, these variations were less marked for dgd1 than for the WT. Interpretation of these results is beyond the scope of this study. The only aspect of the temperature dependence that we want to point out is the strong decrease of the average lifetime above 50°C (reaching 83 ps at 65°C). For dgd1 the same sharp drop in τave occurs at lower temperatures and begins at around 45°C (Fig. 4b). Fig. 4 a Chlorophyll a fluorescence decay traces

for isolated thylakoid membranes from WT (thick line) and dgd1 (dashed line), recorded by TCSPC. The presented curves are the sums of five independent measurements on different preparations. The excitation wavelength is 430 nm, and the emission is recorded at 688 nm at 25°C. The corresponding fits (fluorescence lifetimes (τ) and relative amplitudes, given in brackets) are also presented. b Temperature dependence of the average fluorescence lifetime for the WT (filled square) and dgd1 (open circle). Details about the fitting procedure are described in “Materials and methods”. The lines (solid for WT and dashed for dgd1) serve as a guide to the eye. The average lifetime values and their standard errors are determined from five independent experiments Lipid matrix: lipid packing and membrane permeability In order to study the global physical

SB-715992 clinical trial properties of the lipid matrix of thylakoids, two methods were applied: (i) time-resolved fluorescence of MC540 in thylakoid membranes, which reports on the packing of the lipid molecules; and (ii) electrochromic absorbance transients on whole leaves, which probe the energization and the permeability of thylakoid membranes. Partition of MC540 in thylakoid membranes Using the three-exponential

model for the analysis of the fluorescence decay of MC540 (see also “Materials and methods”), lifetimes of 0.19–0.23 ns (Fig. 5a), 0.66–1.08 ns (Fig. 5b), and 1.71–2.15 ns (Fig. 5c) were obtained; the lifetimes shorten with the increase of temperature. In this article, they are referred to as 200-ps, 1-ns, and 2-ns components, respectively. Fig. 5 Temperature dependencies of the parameters, obtained after the analysis of the fluorescence decays recorded for MC540 in WT and dgd1 thylakoid membranes. a–c Lifetime Tobramycin components (blue symbols) and their respective amplitudes in WT (full black symbols) and dgd1 (open black symbols). d Weighted average lifetimes of the two long-lived components for WT (filled circle) and dgd1 (open circle). The samples were thermostated for 10 min at each temperature before starting the measurements. For further details for the fitting model see also “Materials and methods” and text As shown in Fig. 5a–c, the relative amplitudes of the different lifetime components of MC540 differ for WT and dgd1.

suis in accordance to results reported for S. aureus[15]. By this we identified persister cell formation in three different S. suis strains, suggesting that this phenomenon may be a general trait among this species. Though this has to be further confirmed by testing more

S. suis strains and www.selleckchem.com/products/Tipifarnib(R115777).html antibiotics that are of higher clinical relevance to treat S. suis infections in pigs and humans, persister cells should be considered in the future in cases of ineffective antibiotic treatments or when studying antibiotic tolerance of S. suis. In line with several previous studies [3, 14, 22, 46] the number of persisters observed was higher during stationary growth of S. suis when compared to exponential grown bacteria. Type I persisters were found to be the main

source of antibiotic tolerance in our experiments. Among other stress signals, nutrient limitation in stationary growth is thought to be a trigger Fer-1 order inducing down-regulation of the metabolic activity and bacterial dormancy in energy-deprived cells which can protect the bacteria from antibiotic IKK inhibitor killing. We found some hints for involvement of the catabolic enzyme system ADS, since approximately two log-fold higher levels of persister cells were found in the exponential growth phase of an arginine deiminase knock-out strain (10ΔAD) as compared to its wild type strain. In S. suis the arginine deiminase system metabolizes arginine as a substrate to produce energy in form of ATP [38]. The diminished ATP levels

may lead to reduced general metabolic activity of strain 10ΔAD that might explain the slower growth rate (see Additional file 2: Figure S1) and enhanced number of antibiotic tolerant persister cells. Furthermore, the ccpA deficient strain exhibited lower numbers of persister cells in the stationary growth phase when compared to the wild type. This is in agreement with studies in S. gordonii showing that a ccpA knock-out resulted in an increased sensitivity of the bacteria to penicillin treatment [47]. Since CcpA is a pleiotropic regulator that is important for a balanced metabolic flux in the central carbon metabolism, the alteration of central metabolic processes may influence persister cell formation of S. suis. Accordingly, an interplay between carbohydrate consumption and formation of persisters has recently been demonstrated for E. coli[12]. Edoxaban Further studies are needed to clarify the mechanisms involved in CcpA and/or arginine deiminase dependent changes in antibiotic tolerance of S. suis. When using antibiotics with varying modes of action, resulting killing profiles were quite different, ranging from pronounced biphasic killing patterns to nearly plane curves, at least for exponential grown S. suis. These findings seem to be highly dependent on the type of antibiotic used, which is also emphasized by the fact that treatment with the β-lactam antibiotics amoxicillin and penicillin resulted in similar killing curves.

However, other studies have reported contradictory results: Merchat et al. (1996) concluded that the number of charges does not affect the activity of the PS against both bacterial Gram types [23]. Caminos et al. (2006) showed that the photodynamic activity of a tricationic CDK assay porphyrin combined with trifluoromethyl group was higher for an E. coli strain than the one observed with the corresponding tetracationic porphyrin [24]. Banfi et al. (2006) also concluded that a dicationic porphyrin was more efficient than the corresponding tetracationic derivatives against Gram (+) Staphylococcus aureus and Gram (-) E. coli and Pseudomonas aeruginosa [21]. However, our results suggest

that the number of positive charges affects the PI process. Two of the tricationic PS are the GS-7977 most efficient ones, although they have quite different partition coefficients. Comparing the photoinactivation rate of Tri-Py+-Me-PF and Tri-Py+-Me-CO2Me with the photoinactivation rate of tetracationic Tetra-Py+-Me, the results suggest that a high number of positive charges and a hydrophilic character can decrease the PI

efficiency, as shown by other studies (Jori, personal communication). On the other hand, the meso-substituent groups can play an important role on bacterial PI process. In fact, it has Fosbretabulin purchase been shown that positive charges combined with highly lipophilic groups might increase the amphiphilic character of the PS, enhancing its

affinity to bacteria [25, 27], and consequently increasing the photocytotoxic activity [24]. However, in the present study no direct correlation could be established between the PI pattern and the partition coefficients of the PS. Probably, other interactions, not accounted by log PB/W, such as the combined effect of the cationic charge and the amphiphilic character of the macrocycle is responsible for the photodynamic efficiency [19, 20, 34]. In our case, the results obtained with Tri-Py+-Me-PF and Tri-Py+-Me-CO2Me against E. coli were significantly different (p = Carbachol 0.000, ANOVA) from those obtained with the other tricationic porphyrin Tri-Py+-Me-CO2H. Tri-Py+-Me-PF, and Tri-Py+-Me-CO2Me caused a reduction below the detectable limits (~7 log) after a light dose of 21.6 J cm-2 on E. coli while Tri-Py+-Me-CO2H produced only a ~5 log survivors reduction after 64.8 J cm-2. A possible explanation for this behaviour can be the presence of the acid group in the Tri-Py+-Me-CO2H porphyrin. This acid group can be ionized when dissolved in PBS buffer and the global charge of the porphyrin decreases and, consequently, the efficiency of inactivation decreases. On the other hand, the Tri-Py+-Me-CO2Me, that has the acid group protected, shows a significantly higher (p < 0.000, ANOVA) inactivation rate for E. coli than Tri-Py+-Me-CO2H.

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neoformans for an additional 1 hr and subsequent microscopic imaging. Collection of human peripheral blood monocytes and phagocytosis Monocytes were isolated

by Ficoll-Hypaque (GE Healthcare, Piscataway, NJ) density gradient centrifugation as described previously [30]. see more Briefly, diluted venous blood from one healthy donor was diluted with Hank’s balanced salt solution (Mediatech, Herndon, Va) and was layered on top of Ficoll-Hypaque (GE Healthcare) at a 1:1 ratio and centrifuged at 2000 rpm/4°C for 15 minutes without brake. The monocyte layer was removed and red blood cells were lysed using lysing buffer (0.155 M NH4Cl pH 7.4). Cells were washed three times with Hank’s balanced salt solution and suspended in RPMI (Mediatech) media supplemented with 10% fetal calf serum (Gemini Bioproducts, West Sacramento, Ca) and cells were then plated on poly-lysine coverslip-bottom H 89 chemical structure MaTtek plates (Ashland, MA)

at a density of 2 × 105 per well in feeding media and allowed to adhere at 37°C and 10% CO2 for 6 days prior to incubation with C. neoformans, using 18B7 (10 ug/ml) or 20% human serum, for 1 hr and subsequent microscopic imaging. This study was done with the approval of our institutional review board committee at the Albert Einstein College of Medicine and prior consent was obtained from blood donors. Time-lapse imaging For live cell imaging, phagocytosis assays were done as described [9]. Briefly, 105 HPBM were plated on polylysine TPCA-1 coated coverslip bottom MatTek plates and allowed to adhere for 6 days. The media was then removed and replaced with fresh media containing C. neoformans cells (C. PRKACG neoformans to HPBM ratio of 10:1) along with monoclonal antibody (mAb) against the cryptococcal capsule (mAb 18B7, 50 μg/ml). C. neoformans

were opsonized with either mAb 18B7 or 20% guinea pig serum as indicated above. HPBMs and C. neoformans were then incubated together for 30 min at 4°C to synchronize phagocytosis, followed by 60 min incubation at 37°C to allow for completion of phagocytosis. This was followed by two washes with fresh media (1 ml each), and replenishment with 2 ml feeding media. The plates were then taken for time-lapse imaging every 4 minutes using an Axiovert 200 M inverted microscope and photographed with an AxiocamMR camera controlled by the Axio Vision 4.4 software (Carl Zeiss Micro Imaging, NY). This microscope was housed in a Plexiglas box and the temperature was stabilized at 37°C with a forced air heater system. The plate lid was kept in place to prevent evaporation, and 5% CO2 was delivered to a chamber locally at the culture dish. Quantitative analysis of phagosomal extrusion and cell to cell spread was carried out by compiling all the movies and counting the number of macrophages with internalized C.