DOI: 10.5061/dryad.dv41ns2bb
title: Data from: Unraveling the biochemical aspects of the interaction between ticks and <em>Leishmania</em> using a tick cell line

description: Leishmaniasis comprises a group of vector-borne neglected tropical
 diseases caused by species of the obligatory intracellular parasite
 Leishmania, transmitted by the bite of dipteran sand flies. Infected dogs
 serve as the primary domestic reservoir of Leishmania parasites and are
 often found in close association with various arthropods, such as fleas
 and ticks. There have been recent reports of Leishmania infections
 occurring in areas non-endemic for sand fly species, leading to
 reconsideration of the hypothesis that other arthropods, such as ticks,
 may also play a significant role in the natural history and epidemiology
 of leishmaniasis. Here, we used a tick cell line as a tool to study
 Leishmania infantum and tick interaction. The results showed that L.
 infantum can bind and proliferate inside Ixodes scapularis IDE8 tick
 cells. The infection reduced tick cell viability and induced ROS
 production. Lipid profile analysis showed that the presence of L. infantum
 increased oxysterol in tick cells and influenced tick cell lipid
 biosynthesis, since an increase in glycerolipids and esterified
 cholesterol was observed in infected cells at 48. Further experiments are
 necessary to elucidate whether Leishmania can overcome the various
 biochemical and tissue barriers within ticks and be transmitted to the
 host.
description: <strong>Parasite</strong>
 <em>Leishmania</em> <em>infantum</em>
 (MCAN/BR/2008/1112), originally isolated in 2008 from a dog in Brazil, was
 maintained in Schneider’s insect medium supplemented with 10% fetal bovine
 serum (FBS) and 50 µg/mL gentamicin (Sigma) at 26 °C or Schneider’s insect
 medium supplemented with 10% FBS, 2% human urine, and 50 µg /mL gentamicin
 (Sigma) at 26 °C, in sealed T25 tissue culture flasks with weekly
 subculture. <strong>Tick Cell
 Line</strong> The <em>I.
 scapularis</em> tick cell line IDE8 was cultured in sealed T25
 flasks in 5 mL of complete L15B medium as described previously (Marotta et
 al., 2018), at 32 °C.
 <em><strong>Leishmania</strong></em>** -
 Tick Cells Interaction In Vitro** IDE8 tick cells (2 x
 10<sup>5</sup> per well) were seeded onto round glass
 coverslips in 24-well plates and maintained at 34 °C overnight. After this
 period, the cells were incubated with <em>L. infantum</em>
 promastigotes at a multiplicity of infection of 5 parasites to 1 cell (MOI
 5) for 2 h. Following the incubation, free parasites were removed by
 washing with PBS, and the interaction was assessed at 2 h, 24 h, and 48 h.
 At the end of each time period, the coverslips were fixed in methanol and
 stained with Giemsa or Diff-Quick. The association index (% infected cells
 x number of parasites/cell) was determined by counting at least 200 cells
 per coverslip. At 48h post, *Leishmania *infected-IDE8
 tick cells were incubated with Schneider´s insect medium and incubated to
 27 ºC for more 48h, to allow viable parasites inside the cells to grow.
 Then, the number of promastigotes recovered was counted in Neubauer
 chamber. <strong>Lactate Dehydrogenase (LDH)
 Activity</strong> To evaluate tick cell viability
 during <em>Leishmania</em> interaction, IDE8 tick cells (2 x
 10<sup>5</sup>/ well of a 24-well plate) were incubated with
 or without <em>L. infantum</em> promastigotes (MOI 5) for 2 h,
 24 h or 48 h at 34 °C, as described above. Then, 50 µL of supernatant was
 used to evaluate LDH activity using a CytoTox96 Non-Radioactive
 Cytotoxicity Assay kit (Promega). LDH activity was read at 490 nm at 23°C
 using a SpectraMax spectrophotometer (Molecular Devices).
 <strong>Reactive Oxygen Species (ROS) Production by Tick
 Cells</strong> To measure ROS production of tick
 cells during <em>Leishmania</em> interaction, IDE8 tick cells
 (2 x 10<sup>5</sup> per well of a 24-well plate) were
 incubated with or without <em>L. infantum</em> promastigotes
 (MOI 5) at 34 °C for 2 h, non-adherent parasites were removed by washing
 and the cells were incubated at 34°C. After 48 h, tick cells were washed
 with PBS, counted, and assayed for ROS production. Extracellular hydrogen
 peroxide (H<sub>2</sub>O<sub>2</sub>) production
 was quantified by the Amplex Red oxidation method (Invitrogen^®^). Tick
 cells were added to a reaction medium containing PBS, 10 µM Amplex Red,
 and 0.1 U/mL horseradish peroxidase (HRP) in a final volume of 0.2 mL at
 room temperature. Reactions without cells were considered as blanks, and
 uninfected cells were used as controls. After 1 h of reaction, resorufin
 formation was measured by the change in absorbance at 540 nm
 (Rocco-Machado et al., 2019). <strong>Lipid
 Extraction</strong> IDE8 tick cells
 (1x10<sup>6</sup>/mL in T25 flasks) were incubated with or
 without <em>L. infantum</em> promastigotes (MOI 5) for 48 h at
 34 °C, as described above. After this period, tick cells were washed three
 times with PBS and used for lipid extraction, which was performed
 according to Bligh and Dyer (1959). A mixture of
 methanol:chloroform:H<sub>2</sub>O (2:1:0.8 v/v) was added to
 the samples. After intermittent agitation for 2 h, the solution was
 centrifuged for 20 min at 3300 x g in a clinical centrifuge and the
 supernatant was collected. The precipitate was subjected to a second
 extraction with the same mixture, followed by intermittent agitation for 1
 h, and centrifugation for 20 min at 2000 x g. The supernatants were
 pooled, and 1.0 ml of water and 1.0 ml of chloroform were added thereto.
 After stirring and verifying the presence of two phases, the material was
 centrifuged again for 30 min at 3300 x g. The organic phase (lower),
 containing the lipids, was then removed with the aid of a Pasteur pipette
 and stored at -4<sup>o</sup>C.
 <strong>High Performance Thin Layer Chromatography
 (HPTLC)</strong> Extracted lipids were analyzed
 by HPTLC, as described previously for neutral lipids (Kawooya et al.,
 1988) and phospholipids (Horwitz and Perlman, 1987). Each lipid spot was
 identified by comparison with lipid standards run in parallel. Aliquots of
 5 μg each of 1-oleoyl-rac-glycerol (MG), 1,3-diolein (DAG), glycerol
 trioleate (TAG), cholesterol (CHO), cholesteryl palmitate (CHOE), oleic
 acid (FA), phosphatidylcholine (PC), phosphatidylethanolamine (PE),
 phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI)
 and lysophosphatidylcholine (LPC) were used as the lipid standards,
 purchased from Sigma-Aldrich^®^ (St Louis, Missouri, USA). To visualize
 the lipids, plates were immersed in a carbonization solution consisting of
 8% CuSO<sub>4</sub> and 10%
 H<sub>3</sub>PO<sub>4</sub> for 10 s and heated at
 110 °C for 20 min. Plates were analyzed by densitometry using ImageMaster
 TotalLab sofware (TotalLab, Newcastle, UK).
 <strong>Measurement of uptake of
 <sup>3</sup>H-Palmitic Acid Precursor</strong>
 IDE8 tick cells (1x10<sup>6</sup>/mL in T25 flasks)
 were incubated with or without <em>L. infantum</em>
 promastigotes (MOI 5) at 34 °C as described above. Cells were incubated
 with 100 µCi of ³H-palmitate (³H-palmitic acid 16:0 [9.10-3H(N)]
 (PerkinElmer, Boston, MA) complexed with 0.01 g fatty acid-free albumin
 (BSA-FFA, Sigma-Aldrich^®^, St Louis, Missouri, USA). After 48 h of
 interaction cells were subjected to lipid extraction and HPTLC. The lipid
 spots were scraped off the silica sheet, and radioactivity associated with
 each lipid was determined by scintillation counting using a PerkinElmer
 TriCarb scintillator. <strong>Statistical
 Analysis</strong> The data were analyzed using
 Student’s t-test to compare two groups and ANOVA for more than two groups.
 Analyses were performed using GraphPad Prism 8.0 software. Statistical
 differences were considered significant when *p *≤ 0.05.
description: # Data from: Unraveling the biochemical aspects of the interaction between
 ticks and *Leishmania* using a tick cell line Dataset DOI:
 [10.5061/dryad.dv41ns2bb](10.5061/dryad.dv41ns2bb) ## Description of the
 data and file structure This dataset contains experimental data from
 Filgueiras et al (2025), testing the hypothesis that tick cell could
 interact with the *Leishmania* parasite and that the presence of the
 parasite cause biochemical alterations in tick cells. Each file
 corresponds to each figure of the paper, containing : (1) tick cell and
 *Leishmania* interaction in different time points, Figures 2 (A, B ,C),
 (2) Biochemical results, Figure 3 (A-C), (3) Lipid metabolism (Figures 4
 A-G) ### Files and variables #### File: Figure_2_A.xlsx
 **Description:** Tick cells were incubated with *Leishmania* parasite. The
 percentage of infected- tick cell was determined at 2, 24 and 48 hours by
 counting at least 200 fixed and stained cells under an optical microscope
 ##### Variables * The blank cells are not applicable  #### File:
 Figure_2_B.xlsx **Description:** Tick cells were incubated with
 *Leishmania* parasite. The number of parasite per tick cells was
 determined at 2, 24 and 48 hours by counting at least 200 fixed and
 stained cells under an optical microscope ##### Variables * The blank
 cells are not applicable #### File: Figure_2_C.xlsx **Description:** Tick
 cells were incubated with *Leishmania* parasite. The Association Index
 (AI) was determined at 2, 24 and 48 hours. The AI was determined by
 counting at least 200 fixed and stained cells under an optical microscope.
 IA is calculated multiplying the % infected cells x number of
 parasites/cell ##### Variables * The blank cells are not applicable  ####
 File: Figure_3A.xlsx **Description:** Tick cell viability was analysed
 through LDH activity using a CytoTox96 Non-Radioactive Cytotoxicity Assay
 kit (Promega) at 48 hours. Tick cells were incubated with *Leishmania*
 parasite and the supernatant was collected, and lactate dehydrogenase
 (LDH) activity read using a spectrophotometer.  ##### Variables * The
 blank cells are not applicable  * The percentage of viable cells were
 calculated considering optical density of uninfected cells as control
 (100%) #### File: Figure_3B.xlsx **Description:** ROS production was
 determined through extracellular hydrogen peroxide (H~2~O~2~) that was
 quantified by the Amplex Red oxidation method (Invitrogen). Reactions
 without cells were considered as blanks, and uninfected cells were used as
 controls. ##### Variables * The blank cells are not applicable  * pico mol
 H~2~O~2~ x 2 x 105 cells: (numeric) #### File: Figure_3_C.xlsx
 **Description:** This experiment was used to obtain a semi-quantitative
 quantification of the relative abundance of different lipid classes
 present in the cell. The objective was to evaluate the steady-state lipid
 profile by quantifying changes in the relative abundance of neutral lipid
 classes in IDE8 cells at a specific time point during infection (48 hours)
 with L. infantum. This approach quantifies the relative mass of neutral
 lipids accumulated in the cells. The samples obtained in lipid composition
 assays (without lipid precursors) had their lipids extracted and
 resuspended. They were analyzed by high-performance thin layer
 chromatography (HPTLC) on a 20 x 10 cm silica plate with an aluminum
 support. For the analysis of neutral lipid profiles, a solvent system
 composed was used (VOGEL, ZIEVE and CARLETON, 1962). The lipids were
 identified by comparison with commercial standards , such as:
 1-oleoyl-rac-glycerol (monoacylglycerol - MAG); 1,2-dioleoyl-sn-glycerol
 (diacylglycerol 1,2 - DAG 1,2); 1,3-diolein (diacylglycerol 1,3 - DAG
 1,3); glycerol trioleate (triacylglycerol - TAG); 3β-cholest-5-en-3-ol
 (cholesterol - CHO); cholesteryl palmitate (esterified cholesterol -
 CHOE); 24-hydroxycholesterol (oxysterol - OXI); and oleic acid (free fatty
 acid - FA). 15 μL of each of these standards was used on each silica
 plate. After drying, the silica plates were sprayed with Charring's
 reagent (RUIZ and OCHOA, 1997). The HPTLCs were dried, and digitized
 (scanned) and the pixel densitometry associated with each lipid band was
 analyzed in the Image Master Total Lab version 1.11 program and the pixel
 volume or arbitrary units (A.U.) were used to compose the graphs, which
 represents the pixel intensity relative to each lipid band. This value
 allows a comparison of the relative abundance of a lipid between different
 samples ##### Variables * The blank cells are not applicable  * The data
 refer to arbitrary number determined by densitometry associated with each
 lipid  * The variables are IDE8 alone and IDE8 incubated with *Leishmania*
 #### File: Figure_4_C.xlsx Description: The methodology used above refered
 to Figure 4 (A, B, C, D, E, F, G). This experiment was designed to
 quantitatively measure metabolic activity, specifically the conversion of
 palmitate into more complex lipids. to quantify the activity of lipid
 synthesis pathways that use palmitate as a substrate. This was achieved by
 measuring the incorporation of the radioactive precursor, ³H-palmitate,
 into the different classes of lipids over time (24 and 48 hours). To
 determine fatty acid incorporation, at the time of infection, 5μCi of the
 ³H-FA-BSA complex was incubated individually/well in 500 μL of culture
 medium. Samples were collected after 24 and 48 hours of parasite
 interaction. After incubation, the cells were washed twice and Lipids were
 extracted as previously described. To characterize neutral lipid
 metabolism, the same solvent system described previously (for the lipid
 profile) was used. After the run, the dried plates were stained with
 iodine vapor, and the selected spots were marked with a pencil. The iodine
 was evaporated from the HPTLC in a fume hood, and the previously marked
 spots were scraped off. The silica was collected, allocated into vials,
 and the radioactivity associated with each spot was determined by liquid
 scintillation. The liquid scintillation counter quantifies radiation by
 estimating how much the sample scintillates compared to its internal
 scintillation standards—whether for alpha, beta, or alpha and beta
 radiation—emitted by isotopes such as carbon-14 (14C), phosphorus-32
 (³²P), and the one used in this work, tritium (³H). Therefore, it
 generates the result in Counts per Minute (CPMA) of radiation that
 composes a given sample. Since the amount of CPMA depends on the precision
 of the device used, a conversion to a universal radiation unit is
 necessary. Therefore, the results in CPMA were converted to
 Disintegrations per Minute (DPM) with the aid of an external counting
 standard, the Quench Curve for Tritium (Packard/PerkinElmer). With the
 CPMA results in hand, the transformed Spectral Index of the External
 Standard (tSIE) column of the samples was interpolated to the tSIE of the
 Quench Curve, which was paired with the curve's efficiency
 percentage, using GraphPad Prism (v. 6.1, GraphPad Inc., CA, USA). The
 program generated the sample's efficiency percentage (%Efficiency).
 The sample was then
 corrected:![](data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAVAAAABFCAYAAAAchL5FAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAAFxEAABcRAcom8z8AAC/GSURBVHhe7Z13dBTV98A/u+k9kARCCL136S300IuAdEWRpqAgVaqAYEEQASsdpAlIJyIQeu+dQGghpJBAei+7e39/LCTZyYaifEX8zeecOZzz7n3DZGfmznv33nefRkQEFRUVFZUXRqtsUFFRUVF5PlQDqqKiovIXUQ2oioqKyl9ENaAqKioqfxHVgKqoqKj8RVQDqqKiovIX0ahpTCr/NXQpiew7sI8/du8HjTV1fJrRtEENCnl6orXQoHmsJ/pMwiMiQGuJhUZDeloK6Rk6BA0OTvko7OmO5omyEjHw6GEEaTrBysICg8GAq2s+7O3tlJpZGHQZPIqKxiCQlpyMe6FCODk4KNXMoGfTonlcjEhj2JhPKWBvpVRQeUWoBlTlP0N6SgK/zP2SuYs3U8CrIFEPwsCgJzoqGlc3dxq/9R4LvpuO42OjKBlJrFzwHV9/t5Q0gQJFSlOsUH4wZHL/7i0iYnX06jeET0d8gJur0jAKR3euZ/joKUSnpCMCY6fOZNiA3gq9x0gGU0cO5detezEgVKzdnJ9mf0mp4l5KzVzcOruXSrVbkomGY8GxNCjqolRReVWIisp/gOSYUOncvIEAMmjSXHmQkP5YopdZw/oKIL0HfKzoJfLo+mUpDQI28v2m06LX60WfkSzH9vwudapWFEC6D/5SUjKVPUVEp5M2Rb0EEEA+mDxLqZHF1YO/i5u1UQ/s5HBAiFIlD1Klf8f6AojWuYCsv/BAqaDyClF9oCqvPRnJSQx671227j/O2HlrWfTFCDydrB9LtYwYP5R8ri7kL+6r6AkpWiuiNJC/aAnaNn8DrVaL1sqeBi27sXXlLAq5WPD7qkXcD32o7AoWGoIzNfTs2gSvgk7cufeIDKUOgCQyavx0XEtWwM5SQ5GmPalVxlupZZYdq5dx4NIDhvX1xZCUQFhYrFJF5RWiGlCV155ty75h7Y791Gz9Lp8NzT2FtvKqws9LlzJ4QEuliOSEIOIEChUsTClXU99ioWoNcC3kCanBZCTFm8gADHE3CE/TUL5ia/I72XPldhDxqQalGsvmz+RytDXt6tdHrxN6dWiAnaVSKzcZKbFMmjSd/mMm06B+dTCkkRb8SKmm8gpRDajKa01s5F0mfDkXNHZMmToJJ7PxFQd6dX2LqoXslQLO/bEZgGLV2mYFl7KID0OXlAw4gsWTEW02Z/x2YLDQUqReC+zQEnnjLqnxqSY6MWGBTJj0LaPGjsbOMZ0MoEJhTxOdvFj0+Wge2pdg1EfvYjAYQxWpiRFKNZVXiGpAVV5r/Jb/wp3IZKq2eItO9csqxc9k4/4rAHTp3lAp4s/NfxIWGodHpfrk8yyoFON3KhA7ezua1K+Mg4MNxN1Gk5FkojN9/Ficq7fm4w7Vubp/N9h6UahEFRMdc0QGXeDLxZv4ZPzn2GutSBHjkDUh7YFSVeUVohpQldeYdDbsOQDA+/17KYXPJiOa0KhwwJHa3oVMZSkRzF3wKynAJx/1pXA+W1M5Bu6FheCavyCl89vj7pIPSCBO0rM0Lh3YxMJ1f/LD/LncC32I/7UoylSvRLVqxU3OZI55X03BpkBdhvdpBYDG1hWAxOQEhabKq0Q1oCqvLWGnDnP90lWwzE+1EiWV4mcSeu0MYffCKFClCc4FPTEYBDEYOOPvR5tW7fA/fY2uQyYybHDfXNP79Oh73LtxhcKlmwBQv1xhAAJjdUaF1Ed8OnocNXqOpU3NUty9e41MoLS7J4WUtljB1UNbmL3En7kLZ+Hw2CUhGqP7QRefZqqs8kpRDajKa8uxgDvciUnHq4YPRcuXU4qfyfHjATxMMfDwuj+1KxShgIcb+T0K0X3wOMITNXy3cA2rv/8SZwtlT7h9M5TLNyJp2aM9AOXqVgNg34kQANYtm8+JYB1LvpkIwPVThwDwrGHUz5O0RCZN/hyf3h/SpckbWc2OtsbcT116Sg5llVeNakBVXltiY0MBqFOxICVcX/xRvhMVggHwbdWOT0ePZszYMYwZ8ylL1m7m8qVzjBzcJ89o+aO4IBLEgg41jVN/+6KVAIg+dxN9UggjJ87lw0+/oEJhR/S6THbvOQ5o6N6lruJMpuzbvJjtRy9RvFQJ1q9fx5o1a9iwYQNHDh4FIEP/bAN67MgB9p24aD6lSuWl8uJPnYrKvwIhPtI42nO3tnrhB1mfGkXQMT/Q2tJz4FQ+HTeO8eMnMmnCaHzrP3s0e8lvHVbeDXB1LgCAhdbDKEi6wKdjJ+BYth6fDzf6ZXVpD7l4PwqtnSdVvYy+THMYUiIZO/UbipYoS+yFXSxZspQVK1awdOlSTp0wGtDEzDRyJ0pl8yjwDH3fakfH1n0IuKFG7P/XvOhzp6LyL8FAekIcAFoxGrG8yb1aOSI8ii37buNU0JMaDSsqxc/AwGq/8zRuUZsC7kaHZnFHa4rawP49q1i5/g9mzpqN3ePha/CRLSSmpOJdviHOjk6Kc2Uz66vpXIm1ZvvBQ2zz+xN/f3/8/f3ZvXs3i2ZOwhJ4lJSBaZzflO07t6ErUIzUxOscuHxJKVZ5yagGVOU1xQJHD+P0WWthPrCSFveA+fO+4/zdaKWImEdBRBnA08WLGgVy53g+DV1iKHfjUqhbrhg2j9sKlfSgQpl8RMcm4fP2CN5qViNLf73fBdL10KSND462ZhyqwIPAs8yas5A+A8dRrWjuPFFrJ1vsgcTEDFLzmJunxgSzZo0fk8ZOpYijJatX/K5UUXnJqAZU5bXFu6QxnzIo5oGZMSYs+/Fbxk+eio1j7gT6a8f3A+D+Rhul6JkEnj5MUpoe76Lls9qs3IuSv2hp3AuX5ssJw3No67kdbczd9CnvkUd1J2HWtCnE6m34dPjbSiEA1tZW2GkhPDKOqLjHkX4FezeuJNaxJD3f7EC7VnW5emQnt6IzlWoqLxHVgKq8tnRo14pmlT3ZvWEzK7caDSKAPj2ZTat/YuTnc3Fxf4MKBXIb0HXrdwFQqPyzczKV7Ni6i4yMNDw9Smc3ahyYteB3Th07QEWvfFnNCaG3OHfU6L90tsluz8nxHcv5Yd2f2Dk6U7yA+Sl+1KNYEgyQHHiJoMAgpRjIZNEKPxo09MXV1Yl6Pi3ISHrAqpU7lIoqLxNldREVldeJ6yf8xadaSXF295aeb/eVAQMGSNuWzcRCi2g0Wuk7cYWJflL0I/n6k37ikc9BrKyspFi1xrJwzVZJM9HKm0WzZ0iFEh5iZWUt7bq8K+fuRipVsgi7ekre69ZOtFoLsbKyEt+2nWXTqVvZCpkZsub7WVKqSEHRWliKnYOz9Br2hdwIT815Gtm/abU0a1xHLK2sxMrSQhq0fUuOBoSZ6JzfuU4qlqss91IeN8QGSilPJ6nf+T1JMpioqrxE1HqgKq89yfFRnD1/ga3b/EjLMFC6XFWaNW+Am7MjHoULY2+ZnYukS0/n5o0b2Dg5Y2mhJSM1GWycKFWiyHNNx24FXMdgaY2tjQWJiYl4FS9DfkfzmfGp8VHcDXmAg6MzGg0kJiXjXLAIRd0fjzLFQNDNQDI0ltjaWCMGA3HJmZQsVQpnu2xfacT9IKIS03BytAeE5KQUPLyL4+HyeGQtBiYM7MYPO2+y+qdppGfqsbC2YPLHHxIYa8uVK2eoXEqx0krlpZCHARVSkpLRWlljaZH9WAkarCzNO8HNkZqUCJbWufpoLSzQmncGZZGenIQOLTY21sYoqtYKy2c94QYDOoMBxEBqRiaO9g55+JxUVP47xD+4RdWKtenQ7wOKe7k/btUQdvMM85ds4McN+/ioe3NFL5WXgXkDakhn48I5fLl4Axq0pCcnkpiSSprOihp1mjO4Xwe8i5WlTg1j8nBe7F02h6m/rCZFryEzLZXEpGQydZk4uxWg+9v9aVynBo2aNMJcYPLiH5uYPHMmoUl6RJ+OY0Fftm+Yg1s+85nNKQ+D6dO1D3cSEsnI1NOocz9+mjEGG0vVgqr8t1n11cdM23GHs8f+JF+OQcbD4Kt4F69C6SZ9CTi4MmcXlZeFck7/hLSkaGlVvZwAUsq3t8ydO1fmzp0rUyaOkvxONmJh7SzDxn8pSTplz2x0aUnyXs+2Akjhqg3k68fn+O672VK3WnkBpH3/cRKbZv4kC+dNyar2rdUWlvAHMUqVLOaOH5ilW6VOR7l1P0KpoqLy3yMpXMq5O0v/IaOVEpHER9K6mINY2bnJ4Vt5vzsqf508DaiISN9WzQSQZXtOmLQHB16RRlVKCiB9hkyW1HTzBlBE5PPJwwSQkdO+MWlPjA6VasUKCSDzfz9pInvC/p2rBK2NuFkgNvZ2EhhmfjuDe5f2i6etteTXImht5MffjytVVFT+cyRG3Ze+nYzvaMGSVWTVzhOSHS/KkLWLvpX8jwcVleu2kpthj0z6q/x9nuJVTCU08RFgTWkH0zSQomUrs2zlIgq5O7D2l+84fiHQRJ6FIYVHNy8CFni4mU73HfMXpkfjqgD4bzSmlCi5ffoIXmXr0btBGdLTMgmKMZNBLOmMGzsB91rtyJ/PBRs7G5o1rKDUUlH5z6HPTKNWi84sXLSIqaM/JCMlZ9V8AxqnAnyxYBGLFi3ig7fbkZpu5v1R+XsoLeoTEq4fkLIFrMWuWD25FpqoFIuISLt6NQSQoXPWKEUiIhITEijVCtqJbaHScuh+7kSRuf3bCSAde3ymFInoM6VLnVLSaeA4WfG1cXo+e9MZpZbsXD1XnJ28Zc2SX8TFxU4cC9SV+KQMpZqKiorKSyfPEejuo1e5/TCDBnUrU8rLUSkGoE5ZY8Tv4PFrShEAoZHRXIlMpaSnJ42KPFn0lo3/pXsAlKqXu0KNTpfO8cvB1KtRjGI16wEQeu22iU5C2F2GfTKd9yfPwDYzivj4VGp27oadrdl9Hf42ep2OlJSUv3ToDU8rAaGiovI6kqcBDY0OxgCULeiJTR6BbNeCxnw2XVSyUgRAQsRZDIB7iZq5CtLeuXiIE7fuYOFYkPe711ZIISnkOI/S9BRyKoa9aykAHkSaugp+mT2NWMfiTB/dj4MnLgPwjm8ZrMxE9V8Gf/62gqLeRfD29n7Boyjbz5gafxUVldcf8wY0PZFbB/cANrhXy3utcHSY0efiUiZ38QOAPcvXA9CxRzeT9ruXz9Oz8zvEJqQzasrXVPbOXU1nx+INiKMXRes1xTnDuJ43OjQsSx56cS8z5q9j2uyfcLZI415wAGCPR76iOc7ycrGxtcMjnwtu7h54eDzf4e7uhpuHBzbPYdX1ej06nQ69Xq8e6qEer/B48h4+C7N5oDEPgqlZoTQPLd04fvM+1fKbr1bTtn4ddp08w4jvNzB3WHeFNJOq5Spw5eYdxkz/imoliiLo8Nv8O5u2/IFrgcIMGf0Zn48dbDap/u0u7Tl5+xG3rpzmweWjvFGjEWUadeT4ge2gS6FHm8bcloqc9V/Jw0sHqN+sBTH563D06H6qeOVe+/wy0GVmkJqSipi53mdh5+CIVY5FCUqioqIYNWoUN2/exDLHyhkVFZV/Hr1eT5EiRVi0aBGurnnXcDVrQIOun6Rkxfp4lahL6N2TuabfACSHUr1OAy4GhLB1/2nebGY6DU8OOUfVek25+8CJnr3b4+5qSXqGDlc3d4oVK0HTlq2pXLqYSZ8sUqNp3dyHOOd6nNq9nPTIQBrVqUF0wVrcPn2Io9t+oVX3Sew6cZkmNb3x27Cajj370vTN7uzZuoE8PaCiIyk5HXtHhzyG3q+O8PBw2rRpw7Vr5v3JKioq/ywlSpTgxIkTeHg8LpZtBrMG9MCKz2n+/jQa9pnAkTVfmTWgB1b/SIf3hqEtWpPzR3dTprCbifzQbwtp3edDir45jNNbvsfV3Eny4P7l4zTwacabk1fx06c9QBdH52b12R3mwr4/1/Ghbx3q953Mwq+MZcN+nTOKfmPm0uv9Cfy27Cvl6QAwGHRM7N+RZX9eZMovm/m4a32lyivFYDAQGxuLTme+VJmKiso/i4WFBfnz50erzXu4ZdaADunSmgVb9/DV0i1M6N9ZKQYy6P+mL8u3H+GD8fP4+etPco3o5syYxJgpXzFu+mxmfjZGIX06B3dto0Xbzmw/d5P2NcoAMLBna5ZuC6Ra1dLoM1PYvWcPXh6OGDLT6NuyJmsP3eCL384wqVd2IduchFw7RvVaPkSnwVvvj2DjsrlKlWdy9oA/85b+ipmf7BloGDZ1FvXKeikFKioqrzOKtCYRSZOmjd4QQHYeyGOF0MblAoh7yTfk1oMEpVhEMmTskE4CGvlu4Tal8JlsXDJJwFGCQrJLhX046D0BBDSyZEf2dSXHRUqx/PZiae0ogY/istqVTBzWT3oNHCrtmtUW16LVJDA0WanyTH77ftbja3jxY8WBy8rTqaiovObkGoHGXj9Kw8a+XDdU5NSJfdQpa1oE9uaZg7Ru7Mu9dAeWbtxG/65NTeQAyQ/u0KFWVQ4l5uOPg6doW8O4Z/ZzIcKAhiXwM7Qg4MBi3OyMY9vZQ97n0wUraNpjGPvWf5814o28eZjilZpi6VWf8IADODnkDnglBF+jxZs9+WLdIeL9ZtBz7HxW7jhM3w6NlKpPJTLkHucuXMKgtXzuKk8iAhotNRo0xiuf+XxaFRWV1xSlRfVbt1Q0IL59h5m0Z8RFyfpf5kpRG8TB3VuWrvMzkefk+tkDAohXuTckJPcCpKeTFiUullp5e/oPJs03d6+Url37yIEz903a13w3TgCp3m20ZJpIsln5w+fiXbm5iIhEB50TS5DG/SYp1VRUVFReCBMDei/wqrSuU1YAcS/gIW3btJbWrVtLu45vSrN6tcVOo5GWHbrKqcu3c3YzISY6QgZ0bWqcutrll7V7z0lqerpSzSwpiXEyd4qx+EilDgPkVkjexQ8Mer3cOndIKnu7CSAuZZtIwN2QHMUUjOgzU6Rt7XIye83urLb3WlQVW9eiEpGUl8n992BQ/kGvCVGRD+TK5Yty4+YtCQ15IMlJL/olfbmEBV2Tnr16yYwlO5QilX8pev2LPfw63Yu/z3pd3oWQngeTKfxP385k/9HjaK1sSE9LIynZuMLIsXBpmtSqSds2rShbpjiW2rznr36blrN8zQ60FhYY0ODo6MRn02dQusizAygBZ/by+exF6A2CWFjh49OWkcP6KtUAyExL5euJY7kSHAZaS/SioXaj5oz65MOsnRIBbh9ZR5kWwxg5fhxVS7qDBnZvWc26bfv4ecMJhnQ3LhP9dyFs3/gb6zdsIiFNR8euPejd522ccnsnsti3YzNJ1gXo2MqHp9wesyTGRLDFbw+2dvZoNZCakoJOr0ej0SAiaDQabGxtsbK0pECxMvjUrpYraJhFejzzZn3F0k37qVO9Agf8/yQ8xYN12/3p7JPtyjEYdPy5cyepaWm0bN8JFzvzVd2fl7MnjnIvLIJaPj4U91Qu7EhncGdfFm87Cjau7D1+hhY1cuxn9AqQ5FgOH9xHRIojjZo1xevx9sgqEHr3BktXrKTLwFFULfqkQHQeGDL5c8N69h3axfFLd2jSog2+bTvQokFNpaYJgedOsHPfHrb7+eNVqjxtOnSiXZt2uDm8YA620qL+1xjcxVc8S1WW/v3ekx49ekjvPm9Lrzdbi60G6fTe6Fwj1leOQS/fTRsj+QqVksXLV8ivvy6UEp4u0rHfcEnQ6ZXaIiJydf9vAsjQKfPlBT/aIiISHx0usyd+LFaPA14NmzSVPm+/LT169JBevXtL9y4dxdnWQgDpPXSMmL8KEdEnycSBHQUrV/l931kREZk7ordUql1fghUD0LiwK2IJAtZyJzzKVPjCZEjzuhUFkA07DyqFsub7CWLn4i4r1q2TRuWLSol6b0rs3xt4/G2uHt0tliA2XtXlkmIPpP+v6NOTZfH8mVK+lLeAlRy9+1CpYkKmPlXGDu4lgHwydposXbpUmlQvJ2jt5dMvluX5nG5bs0i8ne2lQbO3ZOnSpTJmaF8BpHaznnInLF6p/lT+0wb0/oUjUsLTXRZtN77MWRjipVH1smJZuJJcCnmxH+x/zc2Te8VVi4xa4p/V5vfzJAFk9dZ9JroiIumJ0VK/YhEpUqeDRL/4DCYLfehFqVDQRsBNTl4IUopl2cwJAsi02eYrb4mI7Nv4swDyzuTF2Y2Z6ZKcmPs3PrR2jgDi0XSwxKX9Baufg/ig01LEAcGmpJxUFA4OOLJdXBwdZfrSP0REJOROgHgWdJPh038y0fun+f3XHwWQ5u16K0X/Lzm6Z7O0bPiG2NtZGl1yBb3kYtjTikDr5JspIwSQ6T+sz2qNCguSOmUKCDjKij9OmfQQEbkXcExcLJFqTXtIZo6P6NyJgwSQNgMmvdCg6j9tQH+aMVzsrN2UzSIiMnNoDwFkwfpdStErZdWPUwQ0cj00++sbefukaECGTP/eRFdEZM2Pxqr9Gw/+vTSpS/7rxcUSKVTrLQlOyP0IhR5dId7FSsiyw+FKURat61YQ0MipgGClKBdBNy7J+g0b5Mz1u0rRC5MYFSpbN6wTv33HJVUx7Phz5UwZNO47kwDjBf+V0r33UMljI4R/hOA712XDhg1y8sRVpehfSXpKslw6ntsgmUOXGCVBt29JRh4zppxkpibK1BHvS6nK1eXn1etlj982cddoxD6/x1MNaFRYkHg7Wolb0RryZCPSJyz95jMBpEm3oZKgGFR80rWVALLG37Q05v3Lp8XLArFxKCD+p57/mfxvGlBdvGxcNjdrSjr757USHJ09h7xwcp90qFlaAHErWl12Hzhq0v3VYZDpAzsI5Jfg0OxpbXxkkLhYIm36T5Gc7/zD2xcknxXS5u3JL/TVNMfy+d8KIH2HjVeKREQkNSZE9h30l/g83on4wKPi6mgtTsUaSeyLp9j+z9Dp87KShtc2QPcqOLjhJ9FqreWDKc8YuWckyvC+7cXSspAE5niG88KQkSy7/tgi5+8at+DRxydIGa1GbPK5P9WAnt42TwBp2m2sUiS3jm4WTwcLwaGkXLmZvYuFLiFIShR0FHCRG4GhJn0kI1YGvVlbAJk533Qr7KeRZyzgdcfRrRAr1v7G+vXrcDGz7Wy3EdPYsGED878ckXdA5B9HiEpIAGxBk31VFhZW2Gs0pKZlIE8aDQamTx5Pol1xvp85xuxy2+dHeBh1HYBihYylA5+g1+uISUzDNp83zZv44qz4sRJiojl/7CAffzSauKQM7Ozh519+5NvZs9nit89UGbh86QJzvvmaTz8ZwqBRkwmKy3vp6qOIYH6a8yVdWvnSslVrho+ayJqNO9AZjL9CRHgYi3+ax4zPxvP2+4PwO59dresJFloLYiLv8c0Xk/H19aVVqzdZsX4HaXpNrlzeg7u20u3NjnwycQZx6Y+vSzLZvHY5rdu2Y/gn4wgKe2TaSUHk/TvM/XoGHVq1pGXrNnw65XOWbTpA+uNysCHXTzFv3hzGjPyE9wZ+QnxKuvIUWdy+fp6RHw3C19eX7u99xNGz14iNjmDh3B+4GR6rVP+fUqpGE9o3rMDC6R8x9usFSrERSWLCsH58v+oPPvzkI4q4Oys1cqGxsqd1u85UL1EQgMSkxOxn/CmsWroVgLLV31CKKFGxKm6FPCD5LlEJEVntFw4fIiomCYo0QRwVgUYrV5xLG1cxBkU+MJU9DaVFVXmVGGT8274CLnIvNDuFK/FRiLhbaqTNwOwR6PENCwWQyd9k+3/+Kmkx4dKliptg7Sq/+d/JIcmUfj3ayuer83ZzHNmxWQYNGCDly5YQQKpXbyg9e/WUHt27y5zvNyvVJSkhTka/300AKV+jniRkmhkGGjJkw5JZ4uzsLA2atJA2LVtKI596AkjR6h0lOd04DE5PT5e1v8wUQNBay9mQ3CvRtv+2TDwcbKR2wybSsmVL8W3aRNydbaVWoy5y9/EquozkGPn2i0lSo15TqVWllADy6YKDkhoVKmM+6COtO3eT6pWM6X11mnc1v5GiPl2Wz/pUXF2cpFHzVtK6ZUvxqV1FACni+7HEPo4TpSVEyIC2xjS/AlW7SYLS5yAikpksU0Z9LBqttdRu2FRatmwpbdt3kJKly0gBr+KSz72IhET/88P8xPDb0qtJBQHko88VI9GMeBkzsIsAMmriV6ayFyAuLExKP8cItH69OgLIjNU7lSIRfYLUrmy8Xz/vzN7P7df5MwQQrzdHSIiZeMHkkSMFkMbvjJFkM4+lOVQD+i9jwYyPBbQSEJbtA40JuyYWIOO++UVERPTJMeJTpoCUrdNJnmxeEhsVKYGBgXLnbpCkpJt5Op5CUOAVsQBx9iwkuy9cl5uBgRIYcFGGDzJGOI+cfbafbtT77QWNk/y6P+8c4Sfs27ZIAGnbaZxSJCIiK2aNEkC6DvlcEtIfP8m6JClogTTrP83EXREVYvxt3L18JENhjM8d2S52WqRJy24Sl8NQTR9tDBj0HTNbDAaRoNN+0qnPYAl6ECe3A44IIM36fiNThvSWz775XlJFJCX8ppQvaCfkLyF77ySZ/D8iIrNG9BdA3pn8g6Q8+a/SHwkgQz//1kT3h7HDBZDhc5fmdr0YDDJp5AcCyMhxX0pKDoWx3dsLIBXqvyO6XB3/GeIiQ6Rn02rG65/xxIgmyYQPjR/F4eO+VPR4MZ7PgKaIT33jNfy4aa9SKCJp0vgNY1bG0IXZH//5k42Lbuq8N1Fyf2pF5o8yGtACzd+VcKVjNQ9UA/ov4/ifa0UDMnVhdhT+0IqZgsZWNh+8JCIiy7/4SNDayY7jV0RE5M9NK6VR7XoyZMgQ6dahldRt0kbOBuSOpOfFhYMbBBB7l9IyaPAHMmjwB/L+O91EA2Jftpncefj00U5S2E2pXdhR3IpXkbuJZkZUChZ+NkAA+XxD7j2uLhzZKbYWSIUGnSUxx/oLfXqKTB/7gWzceyynulzat1YAafHBTJO0lfSkJHm3+RuClb3svWi6m2tK8Ekp62EnNvkrSkBQlGSkpUjG487nj2wQjQap1LiFfDYv2xdmiA2VtpU8xLJgGTkZZpp2dNJ/i9iC1Gj9jiTluIiMzDR5992B4u9/OKe6DBto/DCtXJt7Nd/RbcY6E9Ua9hTl0oNlH7wjgAx6SibEP0FixF3p17amADJi4gwZP7SPAPLJ5C+Uqi/McxlQ3UPxqWnccn2FX+60NRG9tKxjHP2/96Mx+0JE5IvxYwWQdh9Py/Xbiois+sxoQB18ekuI+W3gcqEa0H8ZusQYGdSlldg5FJPt+0/K+eN7pWapItKt/2eSJCIhAWfE2Rrp/OF0MYhIwPlj4mJrIVN/NE6X0+IeSoXCHlLBp4s8yjl8eQoLPzNu2vfm6CU5WjOkSZlC0qTz+yaBK3NcPH1ULEFq+XRUinKjy5Ru9coL2Mj1EGPgIJt06dvBRwBZtt18IRslP48zjvx+ViwtDr59VSxAPLxLyp3IGImOjs464uNDpGY147bc+08YP0pPWDKhnwBSvn5nicuxN2HojUvibWchpas1MHn5dOmx0qNFDUFjL6v+yP1ByIU+Ubo0rSI4eYv/RdO/PzM9Vd5qXFHASjb45x71D+vVWQDZvMuc0cgmISFBElLNmYiXhyE5SgZ3aSI8DtSOmDRdqfKXeC4DaogWn9pGV8KCLblT+0T04vvYgI5anj0QmTnBaEAbDZgs5uzjqslGA+rdqr9EPmdq7r8nfqICgIVjPhas38gXozqxde0yFq5YR69Pv2Td4uk4IHw1eTRJtiX59vMRaDCwd8dS4tOtGPp+ewBsXDwY0qUF14/u4OzxC8rTm2XPkasA9G5bPkerFR8OHE7/Hh141mYkcWGn0AFFKjVQinKRqUvk+M0Q7ApVwc3BNMgQfPEU+w4fxauSDy19nr6S5Al7LtwELCnlZVqwJjMpFNHAo9C7VClhukdVoULlOHfpLgDxCYkm/bYfvgjAlzOn4ZKjMndEXAShqXqKlq1jstIt4PJltu4/T7k3atC2Va0cEvM8DLzK+QtXKV+5MnWqGQMnT4h5cJ2thwPIX7QGHZubbgMOOm6F3UHjWBxPr7y37dYnx/Bmy/rUa9mXkJg0pfilobFzwsU1x/2zN/1b/qdo7MHSuCwv/kmwzwQdGenG7TjKexn3bQNwsDQ+yRGJaSSb2eMxNsX4bylPd5xy74FpFtWA/gvRWjkxavr3LF28kAULFzNmcF8stHBkwwJ+2XyYGV/NoVQBJzIT4tmzaB32ThVxkOyQcjPfWoCOsMTcUencPCIkNgKwp2g+02VzvT4dz7u9u5q0mWP74o0ANGvXXCnKxaMLO4lNSOaNps1xcrEzkV2+fY/wBKhRtQLe+Z5jSV30TcLvXcXCuxaeJcuZiHSJMYiAZ+EyLFu2jOXLl2cdy5YtY/PmzWzZsoW61bM/GoaMSG6FhGJZsCo1y5tmI9zwXwuAb6cOJu3h926QIeBdtgFuz3HJl+6EERwvlC5WAmWMOnD/RvRA9eatsVa8mRG3znP5fCBV6tWicqXce4g94eifmzl2/hoBR3/n4hVjZsVLJzOe8YN7Mnv5DkaNnciovq2ZN+kDRs74Qan5P8IWgxgza0KCYpRC0KeRrDfuo1Yqx3ZErh4OANw9c4+4yNyG90nsvZSXC7bPmdaiGtDXhOToCEZMnE6Zmh0ZPthY5DopPZMjD1Kw0npjMGS/vfm8jRvrRSRIVlteBO3fS8jte7iUrY/H436m6PDbtYsrdyOVAiOSyq4boYAFjSvk/WI/Yf1vB0nVQav6JbFVPH1pCcZRoYdbCVNBHpw+do4rN+No0rAc5QqbGmMLp/wIYOdkR8+ePXMdXbp0oXPnzhTyyN5J4c7hPYSER1G3USMKuOcsPWhgwZpDgJaWNU2vLeL2JQDK1a5j0p4XMSGnAChZNXcZyL27AwCoUrVUrhdz66qVhCdnUK1MSZyUwsdIeirr1/9O3+FjsNXAyh2508j+LrrESD4e8A7fLNnKmGnfMWfWl8z+cTkD29Rg3pThDJn6zxjRBp7G+337hulOvQCZMQ9ITXwEVgXJ75D9TFYuXRxnK9AHnYTEKJM+ABli3CSzpKfHc6cF5nErVP5trJw3jfN3Ypj//WwcH08t9UC6VpvrZj8xm1bPMSLaff4eD5KhadNKFCtgaoQATv/xG716vUNQrPl8xdg7F4mNjkBbxAcXl2dP4y7FPAK0FHXObawl1TidtrV2UYrMcj3iAalAqfwlTabVAHauxbEHomNiuP7I/LUr8T8ZSJIO6lb05nEZWgDSom4SHB2JZbGmuLmZugrSkhIAcM+nHE/mRjLS2LN2G2gsaN4ut7sjNMV459ycTe9D6I2TzP7lV0BLiXJ5F7+5fd6fvVfjGdZ3IC2qFObApjW81El8ykNGDOrNT6v8GPnZHGZPHQmA1rkQP67aQu+WdVkwfTgjZ/yo7PncaNAYn2cNaJ9SFafXh28DEHjhsFJEwKVLBIfE8UaLVhQvlV3EqGqT5hR0dwVCuRcXbdLHkBJF8NmDoHGhco3nrxOsGtDXgPCAY4z/eiE9PvqMtg2yp6pOlhqq24OOYCy0xikLQHqCcVrj5ZDn9npZRIYZ/Z8VC1bItRmfPi2JiV9+i3M+T1rVzG3wAE4cOcmD+Ew6dGqMlxkDbIIuhrSwG2BdDIfqxhHY4b27CXkYB4BlfuO0OTrunkk3MLD215/Zsu+kSWtc2E0A7Ks3AyDgwkkuXL8NgJenF+939SHhYSg/L15j0g8gJOAEP/04j9ikjMctOkIjrgOWFCxkmpx96/w5Hj5KpWm7JpQoYENSVAi79h0BoGApo6/y5pUrJn0gnSXzZ7HtgNGnCpCWmcbuGw+xtLKkcTmju2T/rt2EPDR+OBqWNvrrAu9kj/YfBgXy1azFVG3TEbCidEMfAE4e2MutoPAsPYAF85ZQqVZDqlUrR4O27Yi+d52N/jdMdP4qCY9CGPT2W/y0/gATpv/Ed9NHmcht3IuyfPVa+vpWZd6UYXw8bT6pmc/eFliJpYMNcRrQZwpJGXmbpzK12lPV04Xg2+fwO2WcuTzh981bSTZA//f64GqdbYS19l68294XgJUr/XL0gMvnTrDxyF0avPkWHRrljAU8A2VUSeXfhcGgk/c7NxBbr4pyM1KRvabLkBkDfAVLewlNyI66/jbpPQFX2Xcy75xMgy5TAk4dkmK2xijqsNlb5fr161nH4f27pGWDGgKITxfz+ZoiItMnGiOXM2Ybc1SfSma0dGpQRtDYy/yVfvLTzFFSr3l3uRthLDYScfuSVCmeT7C0lZ+WrZWw0BDZtWW1tGxSTwqWqCd/HLxgcro5kwcLIP3HfSd+636RGjUbyY7D2VH1WxcOSvnCLgIa6dZ/rFy6fk+Cg+7K5xNGSKWKFWXAuPkSn2zMlYoNvy0VXRFs3OTgHdMkwAt/rBZAyrbpK3t27pTOPnVk9LdLRUQk+OppKe5sIWAjv6zeLGGhobL790XSqlFtKV+nlfjluAcpyXFSqqCTgIX4HTwqE4Z/IM1835LQKGNe6f2re8USxNqlmOw6dlYO/PG71GnoKyvW75AV3xhzQ6f+vE0WzpkodRu3ldOB2VH8hPuXpUalCrJ673UREbnkv0bstUjvIVOydP4OmxYZFyyMnmpa6FxJ4sN70qluWcHeXS4F513PNydxcXFZz92k4caMEEA6vfeJXHvcHhWfO/d2x5JvxArE1bumnLpwXcLDw2T7yjliD9Kiz1iJN5M+khB5SxpVLSKA/LB0nYSHh8u9wLNSt6K32LuVk4NXFEs8n4FqQP/lbF32nQDy1aLsfLacnPDfLJYgE3/YICIiGfGR8kZJL2nadaikPiWLKTX6gXw98gNp3bq1tO/QSbr16iU9evTIOrp27SLNmjWT9u3by5qD15TdjWTGy0c9agvYyOLfjyilZlm9aL44WiJuBQrLgKGjJSA41kR+YMd6aduigQBiYecgTVu1lUFDx0tQWO69t84dPyhli3qI1sJaOnXrI9v3mxpYEZGAY7vk7a7tpbCHswDiWbKitO3YRX7+zXR1Vdi9W9KxdWsZ9NHwXGlbmUmR0q+bsQhFzTo+MvvHlSY5pzs3r5ZWjY15kRbWdtKyZWv5eOxUCc+ZByUiInrZ/MNnYqdFnDzKSr+Px0toVM6/yyBL50yRAk4WgtZG2nfuJiu2GNNwTh7aJYUdbcXK2lV69hskZ26aFnVZ8MVYKVWxSY6WDGlUqajkK1VP7kU/Z1b4U4h+GCE7zFQDM8eDW1fkwMEDkv6cxYoPHTog3bp1kx49ekjb1q2kffv20r59O2nTpo306NFDunfvLpuOXFR2ExGRzWsWSavmTcTKUitgIVVrNJARE2ZKdGLeD3/o9VMy+O2uUqxgPgGkcJHS0rHLO3LwhPHj8yLk2hNJ5d9DemoCnX0bEedYk4O7l+Xy8wEg6Sz+ZjLfr9hJtZpvkBofTbyNF7/Mn0MZb9P9rHKi1+nQiWBjZZy4G/R6DDkeBY1Gg4XF0xOYwq5foH7tGiQ6l+HQ2atU9XpKxeccnD1+CL1tPurWqKoUGTGkcmD/QdLEmnoNG5PPXulcyOb+ravcDouhUdPGuVwQOQm+dYWLAXcpWKws9d7InQZk3Lvqsf/NDLqUWPbvP0alWvUo7GmmyG9GAgcPHSVFb0Wjxk1wss/7tzh99DCZ1h40rJP7OgBuXD7NvcgkmjRvjl2OWxB49hQPkoWmTUz9oJkpiXRoWI2bGQVo1bAKBgGNxoIz+//g4p1Qdp+4RKt6efzW/wJEDGRm6tBqtVhY5NhvTASdXo+IYGFhmbdP1JDBvn37SEyGGnXrU7SQq1LDLLeunOXanVC8ipShTk1l2tjzoRrQfzFiMBCXkIiTswuWebuDAIgKv8+toBDsnD2oVLksVnk8ay+TU/s3Ua9FN6q37sP5Xbn9jCr/DJf8V9PwnS/w89tIWa98j4OIGm5fOUzTtr3p/skcNswz9VmqvBxUA6ryl5nYvy1fL9/Fqt2XeKfVv3eE81/nLZ8qaMs14velP5u0p0aFUq90Ce7ZlebO/eu4P22IrvKXeMa4RkVFiZ4DB/by4+xJzFq+iw79PqNnC9V4vhoM7Px1DpuPXSUy+BHRCU8yCowkxUZgZ2MgIeIG075ZQrpeHSu9bFQDqvJCGJIe8OXUiUz9ZhlTvl3Cyh+nYvV0V6nK/4i48Bus2LKXhj6N0GXGs2Ddjhy1NNNZtWU7FK9L48aNCDi8mYt3nmdlmsqLoE7hVV4Mg57k1FQsLKyxtc07UKLyv8egyyBdJ9hYW2HQ60jL0OHoYP9YKiSnpGJjY4NWo0GXmQFaS6yfZ3WFynOjGlAVFRWVv4g6hVdRUVH5i6gGVEVFReUvohpQFRUVlb/I/wHgnsKHN0uMHAAAAABJRU5ErkJggg==) The unit used is Disintegrations per Minute (DPM), as DPM represents an absolute and quantitative measure of radioactive activity, corrected for detector efficiency. Unlike raw counts (CPM - Counts per Minute), which can vary depending on the equipment, DPM provides a true and standardized value of the amount of precursor incorporated. The use of DPM is the gold standard for metabolic flux studies, as it allows for direct and rigorous comparison of biosynthetic activity across different conditions, reflecting metabolic flux accurately and quantitatively. ##### Variables * The blank cells are not applicable  * The variables are IDE8 alone and IDE8 infected with *Leishmania* at 24 and 48 h  * The data (number) refer to desintegration per minutes  #### File: Figure_4_A.xlsx **Description:** Phospholipid  ##### Variables * The blank cells are not applicable  * The variables are IDE8 alone and IDE8 infected with *Leishmania* at 24 and 48 h  * The data (number) refer to desintegration per minutes  #### File: Figure_4_B.xlsx **Description:** 1.2 DAG  ##### Variables * The blank cells are not applicable  * The variables are IDE8 alone and IDE8 infected with *Leishmania* at 24 and 48 h  * The data (number) refer to desintegration per minutes  #### File: Figure_4_F.xlsx **Description:** CHOE ##### Variables * The blank cells are not applicable  * The variables are IDE8 alone and IDE8 infected with *Leishmania* at 24 and 48 h  * The data (number) refer to desintegration per minutes  #### File: Figure_4_E.xlsx **Description:** TAG ##### Variables * The blank cells are not applicable  * The variables are IDE8 alone and IDE8 infected with *Leishmania* at 24 and 48 h  * The data (number) refer to desintegration per minutes  #### File: Figure_4_G.xlsx **Description:** FA  ##### Variables * The blank cells are not applicable  * The variables are IDE8 alone and IDE8 infected with *Leishmania* at 24 and 48 h  * The data (number) refer to desintegration per minutes  #### File: Figure_4_D.xlsx **Description:** 1.3 DAG ##### Variables * The blank cells are not applicable  * The variables are IDE8 alone and IDE8 infected with *Leishmania* at 24 and 48 h  * The data (number) refer to desintegration per minutes