People known to the student researcher (in Cardiff and Southampto

People known to the student researcher (in Cardiff and Southampton) who matched the criteria

were invited to take part and asked to suggest other potential participants (snowball sampling). An interview schedule was designed, based on previous qualitative studies to explore symptom experience, health-seeking behaviours and beliefs about self-medicating behaviours in relation to coughs, colds and flu(1). Following School research ethics approval, interviews were Navitoclax supplier recorded and transcribed verbatim for thematic analysis. Fifteen individuals (7 males; 8 females) took part in the research ranging in age from 18 to 75 years. Most were White Caucasian and two of Asian ethnicity. The sample consisted of students, manual and non-manual workers, professionals and retired individuals. Analysis of transcripts

yielded eleven broad themes (with a total of 35 sub-themes) to capture beliefs about self-medication for cough, colds or flu. These were: 1) Symptoms, 2) Response to symptoms, 3) Length of response, 4) Reason for response, 5) Prevention, 6) Beliefs, 7) Health-seeking behaviours, 8) Self-medication, 9) Influences, 10) Recommendations and 11) First port of call. These findings, informed the adaptation of the original SMS which was found to be relevant to coughs, colds or flu since CH5424802 the self-medicating beliefs and behaviours fitted into the three original sub-scales, which were ‘Reluctance’, ‘Don’t hesitate’ and ‘Run its course’. Statements derived from this study were added to the original SMS and existing scale items were modified for coughs, cold and flu. This provides a useful tool for pharmacists to predict how patients are likely to self manage these symptoms and understand how to optimise the advice given. Further work is needed to pilot the SMS and to test its psychometric properties for colds and flu. More qualitative research is needed to capture the views of people from a broader range of ethnic origin. 1. James DH, French DP. The development of the Self-Medicating Scale CHIR-99021 datasheet (SMS): a scale to measure people’s beliefs

about self-medication. Pharmacy World Science 2008; 30: 794–800. Wasim Baqir, Olga Crehan, Richard Murray, Richard Copeland, David Campbell Northumbria Healthcare NHS Foundation Trust, North Shields, UK This study aimed to quantify prescribing by pharmacists and determine the error rate Prevalence of prescribing and error rates measured across three district general hospitals Pharmacists prescribed for 40% of all patients across three hospitals, with an error rate of 0.3% Pharmacists can competently and safely prescribe across a number of therapeutic areas Pharmacist prescribing rapidly evolved with the introduction pharmacist independent prescribing in 2006, with pharmacists now able to prescribe all medicines.

Phyllosphere microbiota play a critical role in protecting plants

Phyllosphere microbiota play a critical role in protecting plants from diseases as well as promoting their growth by various mechanisms. There are serious gaps in our understanding of how and why microbiota composition varies across spatial and temporal scales, the ecology of leaf

surface colonizers and their interactions with their host, and the genetic adaptations that enable phyllosphere Selleck PF-562271 survival of microorganisms. These gaps are due in large part to past technical limitations, as earlier studies were restricted to the study of culturable bacteria only and used low-throughput molecular techniques to describe community structure and function. The availability of high-throughput and cost-effective molecular technologies is changing the field of phyllosphere microbiology, enabling researchers to begin to address the dynamics and composition of the phyllosphere microbiota across

a large number of samples with high, in-depth coverage. Here, we discuss and connect the most recent studies that have used next-generation molecular techniques such as metagenomics, proteogenomics, genome sequencing, and transcriptomics to gain new insights into the structure and function of phyllosphere microbiota and highlight important Doramapimod purchase challenges for future research. “
“Department of Microbiology, University College Cork, Western Road, Cork, Ireland Probiotics are live microorganisms that when administered in adequate amounts confer a health benefit on the host. They are mainly bacteria from the genera Lactobacillus and Bifidobacterium. Traditionally, functional properties of lactobacilli have been studied in more detail than those of bifidobacteria. However, many recent studies have clearly revealed that the bifidobacterial population in the human gut is far more abundant than the population of lactobacilli. Although the ‘beneficial gut microbiota’ still remains to be elucidated, it is generally believed that the presence of bifidobacteria is associated with a healthy

status of the host, and scientific evidence supports the benefits attributed to specific Bifidobacterium strains. To carry out their functional activities, Aurora Kinase bifidobacteria must be able to survive the gastrointestinal tract transit and persist, at least transiently, in the host. This is achieved using stress response mechanisms and adhesion and colonization factors, as well as by taking advantage of specific energy recruitment pathways. This review summarizes the current knowledge of the mechanisms involved in facilitating the establishment, colonization, and survival of bifidobacteria in the human gut. “
“During the course of our screening program to isolate isoprenoids from marine Actinobacteria, 523 actinobacterial strains were isolated from 18 marine sponges, a tunicate, and two marine sediments. These strains belonged to 21 different genera, but most were members of Streptomyces, Nocardia, Rhodococcus, and Micromonospora.

These data suggest that N gonorrhoeae transformation by ssDNA is

These data suggest that N. gonorrhoeae transformation by ssDNA is largely dependent on the presence of the Crick DUS12. A previous study reported efficient ssDNA transformation in N. gonorrhoeae much higher than the levels we measured

(Stein, 1991). This study did not report how much contaminating dsDNA was present in the ssDNA preparations, and therefore, those results are difficult to compare to the results obtained in this study. Our data show that there is significant dsDNA contamination of standard M13 ssDNA preparations and we added a column purification step to enrich for ssDNA molecules. It is possible that the high transformation efficiencies reported previously (Stein, 1991) were attributable to contaminating double-stranded BMS-354825 chemical structure RF DNA within the recombinant FDA approved Drug Library research buy M13 phage preparations. Our results support the observation of transformation in co-culture experiments with strains secreting ssDNA via the type IV secretion system (Dillard & Seifert, 2001). Interestingly,

Crick DUS0 ssDNA transformation was consistently, but not statistically higher than Watson DUS0 ssDNA transformation. We do not presently understand the reason why the Crick strand transforms consistently, but not statistically better without a DUS, but it could be used more efficiently during uptake or recombination into the chromosome or perhaps is more resistant to nucleases encountered during the transformation process. Although both the Watson and the Crick DUS12 sequences

enhanced transformation in both FA1090 and MS11, the magnitude of enhancement was much greater for the Crick DUS12 than the Watson DUS12 (Fig. 2). Again, these differences could be mediated at any stage in the transformation for process. The previously accepted model of dsDNA DUS12 action invokes the DUS12 sequence binding to a putative outer membrane receptor leading to increased DNA uptake into the periplasm. We have suggested that the DUS may have more complicated role during the process of transformation (Duffin & Seifert, 2010), which may include a role for the DUS beyond DNA uptake into the periplasm. Many factors are required for the complex process of transformation including DNA binding and DNA uptake into the periplasm and through the inner membrane. Prior reports have shown DUS12 dsDNA uptake is transported into the periplasm, but no reports have shown ssDNA transport. However, as all of the previous studies establish that the dsDUS mediates transport into the periplasm, we do not favor a role for the ssDUS in this step of transformation. A lack of activity in DNA uptake for ssDUS could explain the overall reduction in transformation of ssDNA compared to dsDNA.

Purified proteins were dialyzed against distilled water and then

Purified proteins were dialyzed against distilled water and then injected into a rabbit to prepare antiserum. The antisera were designated as anti-Sov32-177:2408-2499, anti-Sov178-625, anti-Sov626-1073, and anti-Kgp. A 0.3-kbp 3′-terminal region of sov was amplified from pKS32 by PCR with 5′-GGAATTCCATGGCTCCGCGTACCGGTGGG-3′ (italics: NcoI site) and 5′-GGGGTACCTAGTGATGGTGATGGTGATG-3′ (italics: KpnI site). The amplified product was digested with NcoI and KpnI and cloned into the NcoI (in the sov) and KpnI (in a pUC119 vector) sites of pKS9 (Saiki & Konishi, 2007) to create pKS36. pKS37 was constructed by ligation of a 6.2-kbp

SacI–KpnI-digested fragment from pKS25 (described below) with an annealed-oligonucleotide linker (5′-TCCATCACCATCACCATCACTAGTGGTAC-3′/5′-CACTAGTGATGGTGATGGTGATGGAAGCT-3′). pKS38 was created by ligation of a 6.2-kbp SacI–KpnI-digested fragment from BIBW2992 chemical structure pKS25 with an annealed-oligonucleotide

linker (5′-TCCGTCATCACCATCACCATCACTAGTGGTAC-3′/5′-CACTAGTGATGGTGATGGTGATGACGGAAGCT-3′). Palbociclib cell line pKS36, pKS37, and pKS38 were linearized and used to construct the P. gingivalis mutants 83K5, 83K6, and 83K7, respectively, by electroporation (Saiki & Konishi, 2007). Insertion and deletion mutations of 83K5–7 were confirmed by determining the nucleotide sequences of the DNA regions that were PCR amplified using chromosomal DNA as templates. Subcellular fractions were prepared as described in Ishiguro et al. (2009). The supernatant from a P. gingivalis cell culture (100 mL) was concentrated on an ultrafiltration membrane [10 000 Molecular weight cut off (MWCO); Sartorius Stedim Biotech] and diluted with 8 M urea (the extracellular fraction). Cell pellets were washed in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4), suspended in PBS/protease inhibitor cocktail (PIC) [PBS supplemented with a 1/100 vol. of PIC (for

use with mammalian cell and tissue extract; Sigma-Aldrich) supplemented with N-α-p-tosyl-l-lysine chloromethyl ketone hydrochloride (10 mM; Sigma-Aldrich)], sonicated (with tip #7), and ultracentrifuged at 104 000 g for 30 min at 4 °C to remove the supernatant (the cytoplasmic/periplasmic Galactosylceramidase fraction). Membrane pellets were suspended in PBS, solubilized with 2% Triton X-100 for 30 min at 4 °C, and centrifuged (104 000 g for 30 min at 4 °C) to remove the supernatant (the inner membrane fraction). Pellets were suspended in PBS (the outer membrane fraction). Inner membrane and outer membrane fractions were verified as described in Ishiguro et al. (2009) (see Supporting Information, Fig. S1). Histidine-tagged Sov in the fractions was cosedimented with Ni2+-chelated Sepharose Fast Flow resins (a histidine-tag pulldown experiment), eluted, concentrated on an ultrafiltration membrane (100 000 MWCO; Sartorius Stedim Biotech), diluted with 8 M urea, and concentrated to 50 μL.

Prior to experimental sessions, the mental capacity of subjects t

Prior to experimental sessions, the mental capacity of subjects to learn the imagery techniques was tested by the Kinesthetic and Visual Imagery Questionnaire and a chronometric test. The Kinesthetic and Visual Imagery Questionnaire is an imagery assessment tool comprised of 10 items, each scored on a five-point ordinal scale, including the image clarity (visual dimension) and the sensations intensity (kinesthetic dimension) of body movements. Each item describes an action: (i) neck flexion/extension, (ii) shoulder shrugging, (iii) forward trunk flexion, (iv) forward see more shoulder flexion, (v) elbow flexion, (vi) thumb to finger tips, (vii) knee extension, (viii) hip abduction, (ix)

foot external rotation, and (x) foot tapping. Subjects physically execute each movement and immediately afterwards imagine performing the same movement. A score of 5 corresponds

to the highest clarity/intensity, and a score of 1 corresponds to the lowest clarity/ intensity (for a review, see Malouin et al., 2007). The Kinesthetic and Visual Imagery Questionnaire scores allowed the researcher to assess each participant’s abilities and decide whether the subject was a suitable selleck kinase inhibitor candidate for MP. Comparing actual and imagined movement times, the chronometric test determined the motor imagery ability of participants. For the test, sitting on a chair with a back rest with both feet resting on the floor, the subject was asked (i) to physically write one six-letter word, and (ii) to imagine the same movement for each upper limb (dominant and non-dominant hand). Two trials were performed. The test always began with the dominant hand. A motor imagery index was calculated (imagery time/executed time) for each subject as an indicator of the temporal congruence of the imaged and physically executed task. If the duration of imagined action had a much larger variance (> 0.4) than the real movement duration, the subject was excluded. Subjects who successfully performed the chronometric test and reached high Kinesthetic and Visual Obeticholic Acid Imagery Questionnaire scores were invited

to participate in experimental sessions. For the experimental sessions, the subjects were seated in a comfortable chair, with head and arm rests. With closed eyes and through earphones, the instructions for mental activity were provided by an audiotape, recorded by a female voice. The tape lasted 13 min and consisted of three steps. Three minutes of relaxation exercises, in which the subject was instructed to imagine him/herself in a warm, relaxing place (e.g. a beach) and to contract and relax different muscle groups in the body (i.e. progressive relaxation) (Page et al., 2007). Seven minutes of mentally writing, in which the subject was instructed to imagine him/herself writing Portuguese words (a six-word set) with the non-dominant hand. Each six-word set was composed of a sequence of four/six/eight-letter words.

e nucleus raphe magnus, nucleus raphe dorsalis and locus coerule

e. nucleus raphe magnus, nucleus raphe dorsalis and locus coeruleus).

This possibility is supported by the observation that omnidirectional pause neurons (OPNs), which may modulate arousal in orienting subsystems such as the saccade generator (Optican, 2008), stop discharging during sleep (Henn et al., 1984). Further, OPN inactivation produces slower saccades (Kaneko, 1973). Consistent with this idea, increased TOT led to increased subjective selleck screening library perception of sleepiness (SSS) in the current study (Table 2). Increased air traffic density is one of the top five factors leading to poor ATC operator performance (Durso & Manning, 2008). Here we manipulated air traffic density to induce different levels of TC. Subjective and behavioral results confirmed our manipulations: higher traffic density (i.e. higher TC) led to slower RTs, more detection errors and higher levels of perceived exertion (Table 3). The above notwithstanding, increased TC did not impact (micro)saccadic or drift

dynamics our current experiment. Previous studies have found that increased TC affects saccadic dynamics (Galley & Andres, 1996; Di Stasi et al., 2010a,b, 2011) and microsaccadic rates (Pastukhov & Braun, 2010; Benedetto et al., 2011), albeit with inconsistent results. The difference between current and former

Depsipeptide ic50 results may be due partly to the presence of one or more secondary tasks (simultaneous to the participants’ primary task) in many of the previous experiments (Di Stasi et al., 2010a,b; Benedetto et al., 2011). Whatever the reason for the lack of effects of TC in our study, it is worth noting that it applied to both saccades and microsaccades, thereby lending additional support to the hypothesis that saccades and microsaccades share a common generator (Zuber et al., 1965; Otero-Millan et al., 2008, 2011; Rolfs et al., 2008; Engbert, 2012). To our knowledge, no previous research has investigated the effect of TC on drift. In our experiment, variations in TOT but not TC modulated Carnitine palmitoyltransferase II fixational and saccadic eye movement parameters. The dissociation of TOT and TC effects is important, as it satisfies several neuroergonomics criteria to establish an ideal measure of attentional state in applied settings (Parasuraman & Rizzo, 2007). Briefly, the main requirements of such an attentional measure (in our case, eye-movement based) are (Luximon & Goonetilleke, 2001): (i) sensitivity: it should detect significant variations in attentional levels; (ii) noninvasiveness: it should not interfere with the primary task; and (iii) selectivity: it should be immune to other variables.

6 and subjected to stress At different time points, samples (15

6 and subjected to stress. At different time points, samples (1.5 mL) were collected, and the cell pellets were resuspended in SDS sample buffer [60 mM Tris-HCl [pH 6.8], 30% glycerol, 2% SDS, 0.1% bromophenol blue, and 14.4 mM 2-mercaptoethanol) and boiled for 10 min to prepare the protein lysates. selleck chemical Proteins were separated by electrophoresis on a 12% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. After blocking, the blots

were probed with mouse monoclonal antibodies against the HA tag (Cell Signaling Technology, Danvers, MA) or DnaK (Stressgen, Victoria, Canada) as a loading control. Horseradish peroxidase-conjugated goat anti-mouse IgG was used as the secondary antibody. The proteins were visualized using a BM chemiluminescence blotting substrate (POD) (Roche, Mannheim, Germany). Total RNA was isolated using the RNeasy Mini kit (Qiagen) and treated with DNase I. The quantity and purity of RNA was determined

using a NanoDrop spectrophotometer (Nanodrop Tech. Inc., Wilmington, DE). cDNA was synthesized from total RNA (1 μg) using the First Strand cDNA Synthesis Kit (Roche) at the following conditions: 25 °C for 10 min, 42 °C for 60 min, 99 °C for 5 min and cooling to 4 °C. The resulting cDNA was then amplified using gene-specific primer sets. The reaction mixture was denatured (94 °C, 4 min), followed by 20 thermal cycles (94 °C for 30 s, 54 °C for 30 s, 72 °C for 50 s) and a final extension (72 °C for 10 min). The primer pair LysP-RT-F (5′-GGAAGAAGGCTTTGGTTTCG-3′) and LysP-RT-R (5′-GAGGCATACATCCCGGAGTT-3′) was used to detect the lysP transcript. The 16S rRNA gene was used DAPT datasheet as a normalization control. The amplified products were separated on a 1.5% agarose gel, stained with ethidium bromide and visualized. To identify

genes involved in the proteolytic activation of CadC, we performed a genome-wide screen to isolate mutants that prevent cadBA expression, even in the presence of the cadC gene, under acid stress conditions (pH 5.8, 10 mM lysine). The Tn10dCm transposon was used to mutagenize Salmonella strain JF3068 carrying a cadA::lacZ transcriptional fusion. Of the approximately PI-1840 30 000 random transposon insertions screened, 12 mutants were identified as white colonies on E glucose agar plates containing X-gal. The precise location of the transposon insertions was determined by sequencing of genomic DNA flanking the transposons (Welsh & McClelland, 1990). Ten insertions were mapped to the cad locus, and the remaining two insertions were located in STM4538 and yfhK, which encodes a PTS permease similar to the E. coli mannose-specific PTS enzyme IID and a putative sensor kinase, respectively. To ensure linkage of the phenotype to the transposon insertion, STM4538::Tn10dCm and yfhK::Tn10dCm were moved into the parental wild-type strain using P22-mediated transduction, and LDC assays were performed. Usually, a positive test is indicated by a purple color and a negative test by a yellow color.

Active TB needs to be excluded before considering treatment of la

Active TB needs to be excluded before considering treatment of latent infection, which is usually with isoniazid monotherapy for 6 months or isoniazid/rifampicin Dabrafenib cell line for 3 months. Starting HAART reduces the risk of reactivation of latent TB infection and is effective at reducing the incidence of new TB. We recommend that all HIV-positive patients should be offered HAART in line with the British HIV Association (BHIVA) treatment guidelines [2]. We recommend daily TB treatment whenever possible. Treatment

may be given 5 days per week, but should be intensively supervised. This option may be useful in hospital or other highly supervised settings. Three-times-per-week directly observed therapy (DOT) should only be given to patients who are stable and clinically well and where local logistics

enable this to be undertaken successfully. We do not recommend twice-weekly GSK1120212 DOT for treatment of HIV/TB coinfected patients, especially in those with CD4 counts <100 cells/μL, as it has been associated with unacceptably high rates of rifamycin resistance. In cases where multiple drug resistance is not suspected, treatment should be started with four drugs (typically rifampicin, isoniazid, pyrazinamide and ethambutol) until sensitivities are known. We recommend a 6-month treatment regimen for drug-sensitive TB outside of the central nervous system (CNS). This is usually four drugs for 2 months, followed by isoniazid and rifampicin for a further 4 months (at least 182 doses of isoniazid and rifampicin and 56 doses of pyrazinamide and ethambutol in total). In drug-sensitive TB affecting Thymidine kinase the CNS we recommend 9 months of treatment. This usually consists of four drugs for 2 months, followed by 7 months of isoniazid and rifampicin [3]. Drug-resistant disease should be treated only by specialists with experience in such cases, in line with NICE guidelines [1]. Careful attention should be paid to drug interactions between TB drugs, HAART and other therapy. Rifampicin is a powerful inducer of cytochrome 450 (CYP450)

and has effects on several metabolic pathways and P-glycoprotein (PgP). Rifampicin interacts with protease inhibitors (PIs), NNRTIs, chemokine (C-C motif) receptor 5 (CCR5) antagonists, and antimicrobials such as fluconazole. Rifabutin is a less potent inducer of CYP450 and may be used as an alternative to overcome some of these difficulties (for up-to-date drug interaction data go to Toxicity profiles of antiretrovirals and anti-tuberculosis drugs overlap and make it difficult to determine the causative drug. For example, rashes occur with NNRTIs, rifampicin and isoniazid. Isoniazid and stavudine both cause peripheral neuropathy. All patients on isoniazid should take pyridoxine to try and prevent this complication.

coli (Vine & Cole,

2011) It is currently unclear whether

coli (Vine & Cole,

2011). It is currently unclear whether this ‘activity’ is a previously unreported NO reductase or a combination of a primary chemical reaction (e.g. metal-catalyzed nitrosylation of iron-sulfur centers) followed by a reductive repair pathway. NO reacts directly with metal ions to form nitrosyl complexes. Thus, nitrosylation of iron atoms, especially iron-sulfur clusters, in enzymes such as aconitase and fumarase or in the transcription factors FNR, Fur, SoxR, OxyR, and NsrR inactivates their functions. Under oxidizing conditions, metal nitrosyl complexes can then transfer the NO moiety to –SH groups of proteins, peptides, and cysteine, or to nitrogen atoms of secondary amines. As NarG is a major catalyst for the formation of NO RG-7388 mw in the cytoplasm, protection mechanisms become essential when NarL and FNR are both active, which is during anaerobic growth in the presence of a high concentration of nitrate. Consequently, protection against damage by NO must also be activated by NarL, FNR or both. But this poses a dilemma: how can the bacteria survive when NO-induced damage is so severe that FNR can

no longer function? Enteric bacteria appear to have solved this problem by evolving multiple repair pathways, some that function when FNR (and Fur, etc.) are active, and others that deal with the greater threat when damage is so severe that FNR is itself inactivated. Mechanisms to VX-770 in vivo minimize damage caused when FNR is active include nitrite reduction by the cytoplasmic nitrite reductase, NirBD, nitrite expulsion by the nitrate and nitrite transporter, NarK (Jia et al., 2009), and possibly repair by the hybrid cluster protein, Hcp, and its reductase. Expression of the genes for all of these proteins is dependent upon FNR activation. However,

contrary to our earlier report (Filenko et al., 2007), hcp expression is not activated by NarL, but instead it is directly regulated by NsrR in response to NO (Chismon et al., 2010). Nitrosative damage to iron-sulfur centers, and possibly other iron proteins as well, is repaired in E. coli by YtfE or Fenbendazole RIC – for repair of iron centers (Justino et al., 2007; Overton et al., 2008). It is not known whether damaged iron-sulfur centers are repaired directly by the removal of the NO moiety, or whether iron is released followed by the reconstruction of the active redox center. If the former is correct, is an acceptor molecule nitrosated in the process and, if so, what is this acceptor and how is that regenerated? Synthesis of three further proteins is strongly up-regulated by nitrate-activated NarL during anaerobic growth in the presence of nitrate but is not dependent on activation by the FNR protein. These are the O6-methyl-guanine methyl transferase, Ogt; and the products of the two-gene operon, yeaR yoaG.

To normalize the number count of mitochondria and symbionts, a di

To normalize the number count of mitochondria and symbionts, a dilution curve was performed and the results obtained by Neubauer chamber counting were compared to the optical density (OD) on a wavelength of 600 nm. All the experiments were normalized to the medium efficiency by OD as 2.0 × 1010 for symbiont (OD = 0.9) and 4.5 × 108 for mithocondrion fraction (OD = 2.5). Protozoa were washed twice in PBS and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for 1 h. After being washed again in 0.1 M cacodylate buffer, pH 7.2 cells where postfixed for 1 h in 1% osmium tetroxide containing 0.8% potassium ferrocyanide, 5 mM CaCl2 in 0.1 M cacodylate buffer. Then, cells were washed, dehydrated in several crescent

concentrations of acetone and embedded in Epon: first a mix of Epon: Acetone (1 : 1) and finally pure Epon. Ultrathin sections were obtained in an Ultracut Reichert Ultramicrotome and mounted on 400 mesh copper grids, selleck products stained with uranyl acetate and lead citrate. Samples were analyzed in a Zeiss 900 transmission electron microscope. Total lipids were extracted from A. deanei metabolically labeled after growth for 24,

36, and 48 h in the presence of [32Pi]-orthophosphate or from endosymbionts and mitochondria obtained after cell fractioning of protozoa treated selleck chemicals llc or not with miltefosine for 24 h. Samples were washed with PBS, and the pellet was used for lipid extraction as described below. The lipid extraction was performed as described by Horwitz & Perlman (1987). Subsequently, the organic phase, containing the phospholipids, was solubilized with 3 mL of CHCl3 : CH3OH : HCl (200 : 100 : 0.75 v/v), and the phases were separated by centrifugation after addition of 0.3 mL of 0.6 N HCl. To purify the phospholipid Farnesyltransferase fraction, 0.5 mL of CHCl3 : CH3OH : HCl (3 : 48 : 47 v/v) was added to the organic phase and centrifuged. The pH was adjusted to 7.0 with 0.2 N NH4OH in methanol before dry under N2 gas. After lipid extraction, the protocol described by Einicker-Lamas et al. (1999) was used. Briefly, silica gel plates (Silica gel 60F254 Merck) were activated by heat, and the samples corresponding

to the lipid extracts of A. deanei, control and miltefosine-treated cells, grown in the presence of 32Pi, as well as lipid fractions derived from endosymbionts and mitochondria isolated from the host protozoan, were applied to the silica plates. The run of the samples was performed using a mobile phase (120 chloroform : 45 acetone : 39 methanol : 36 HCl : 24 H2O), as described by the method of Horwitz & Perlman (1987), for 80 min. The TLC plates were dried and exposed to develop in an iodine vapor atmosphere. Control standards (Sigma) were used to determine the phospholipids composition in each sample. When lipids were labeled by 32Pi, the TLC plate was sensibilized with 32Pi radiation, which was detected in Molecular Dynamics Storage Phosphor Screen GP after 24 h of exposure.