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Roy Mwenechanya, Julie Kovářová, Nicholas J. Dickens, Manikhandan Mudaliar, Pawel Herzyk, Isabel M. Vincent, Stefan K. Weidt, Karl E. Burgess, Richard J. S. Burchmore, Andrew W. Pountain, Terry K. Smith, Darren J. Creek, Dong-Hyun Kim, Galina I. Lepesheva, Michael P. Barrett
Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana
www.plosntds.org
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0.83 0.64 0.02 0 k
(24)Tj
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1.3492 0 Td
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(development,)Tj
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(if)Tj
0.7483 0 Td
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(are)Tj
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3.0784 0 Td
(aims)Tj
2.0523 0 Td
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(the)Tj
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(disease)Tj
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2.5568 0 Td
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2.211 0 Td
(we)Tj
1.3266 0 Td
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1.4683 0 Td
(complementary)Tj
6.5083 0 Td
(high)Tj
2.0069 0 Td
(throughput)Tj
4.7906 0 Td
(data)Tj
1.9105 0 Td
(approaches)Tj
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(of)Tj
1.0148 0 Td
(metabolomics)Tj
5.8337 0 Td
(and)Tj
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(genome)Tj
3.4072 0 Td
(sequencing)Tj
4.7169 0 Td
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1.0261 0 Td
(reveal)Tj
2.5682 0 Td
(a)Tj
0.6576 0 Td
(gene)Tj
2.0806 0 Td
(whose)Tj
2.7213 0 Td
(mutation)Tj
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(causes)Tj
2.7496 0 Td
(resistance)Tj
4.1329 0 Td
(to)Tj
1.0261 0 Td
(AmB)Tj
2.3017 0 Td
(in)Tj
/F14 1 Tf
1.0375 0 Td
[()-3()2()-4()12( )]TJ
-34.9566 -1.3039 Td
[()6()16()18()4()]TJ
2.7609 0 Td
[()6()1(\n)4()7()16()6()]TJ
/F10 1 Tf
(.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 496.2897 cm
BT
/F4 1 Tf
1 TL
-0.0033 Tc
0 0 Td
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4.5071 0 Td
(and)Tj
1.989 0 Td
(methods)Tj
/F0 1 Tf
-6.4961 -1.5023 Td
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1.9134 0 Td
(culture)Tj
3.1842 0 Td
(and)Tj
1.8615 0 Td
(AmB)Tj
2.3764 0 Td
(resistance)Tj
4.7244 0 Td
(selection)Tj
ET
Q
q
1 j
1 J
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10 0 0 10 200.0125 461.2535 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
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5.8903 0 Td
(of)Tj
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[()]TJ
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0.9751 0 Td
[()6()1(\n)4()7()16()6()]TJ
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3.9912 0 Td
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(MNYC/BZ/62/M379)Tj
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(\(GIBCO\))Tj
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([)Tj
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(27)Tj
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(10%)Tj
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(bovine)Tj
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(\(FBS\))Tj
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(\(PAA)Tj
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(x)Tj
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(10)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 346.9039 439.7102 cm
BT
/F10 1 Tf
1 TL
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(5)Tj
ET
Q
q
1 j
1 J
0 w
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BT
/F10 1 Tf
1 TL
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2.8233 0 Td
[()]TJ
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(AmB)Tj
2.3017 0 Td
(\(Sigma-Aldrich\))Tj
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1.0318 0 Td
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(pressure)Tj
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1.1849 0 Td
(limiting)Tj
3.3959 0 Td
(dilution)Tj
3.4129 0 Td
(to)Tj
1.0261 0 Td
(1)Tj
0.6917 0 Td
(cell/ml)Tj
2.9593 0 Td
(in)Tj
1.0318 0 Td
(20)Tj
1.1736 0 Td
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(out)Tj
1.5534 0 Td
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(96-well)Tj
3.1238 0 Td
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ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 341.1212 cm
BT
/F0 1 Tf
1 TL
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(sensitivity)Tj
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Q
q
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1 J
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BT
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1 TL
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(adaptation)Tj
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(of)Tj
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(assay)Tj
2.2336 0 Td
([)Tj
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(28)Tj
0 g
(].)Tj
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(A)Tj
0.8958 0 Td
(starting)Tj
3.2201 0 Td
(density)Tj
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(1)Tj
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0.6917 0 Td
[()]TJ
/F10 1 Tf
0.7823 0 Td
(10)Tj
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Q
q
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1 J
0 w
7.4999 0 0 6.6659 368.4472 315.5527 cm
BT
/F10 1 Tf
1 TL
-0.0133 Tc
0 0 Td
(6)Tj
ET
Q
q
1 j
1 J
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BT
/F10 1 Tf
1 TL
-0.01 Tc
0 0 Td
(cells/ml)Tj
3.2711 0 Td
(were)Tj
2.0976 0 Td
(incubated)Tj
4.1216 0 Td
(at)Tj
0.9411 0 Td
(27C)Tj
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(in)Tj
1.0205 0 Td
(the)Tj
1.4513 0 Td
(presence)Tj
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1.0034 0 Td
(various)Tj
3.1011 0 Td
(drug)Tj
2.0863 0 Td
(concentrations)Tj
6.0944 0 Td
(for)Tj
1.372 0 Td
(72)Tj
1.1565 0 Td
(hours)Tj
2.4832 0 Td
(in)Tj
1.026 0 Td
(a)Tj
0.6463 0 Td
(96-well)Tj
3.0841 0 Td
(microtiter)Tj
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(plate.)Tj
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4.1385 0 Td
(\(Sigma-)Tj
-32.7228 -1.3039 Td
(Aldrich\))Tj
3.5603 0 Td
(in)Tj
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(1)Tj
/F16 1 Tf
[()]TJ
/F10 1 Tf
1.2529 0 Td
(phosphate-buffered)Tj
7.937 0 Td
(saline)Tj
2.4604 0 Td
(\(PBS\))Tj
2.4887 0 Td
(\(Sigma-Aldrich\))Tj
6.6671 0 Td
(pH)Tj
1.4967 0 Td
(7.4)Tj
1.3776 0 Td
(solution)Tj
3.4356 0 Td
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(added)Tj
2.6305 0 Td
(to)Tj
1.0148 0 Td
(a)Tj
-37.0201 -1.2983 Td
(concentration)Tj
5.7429 0 Td
(of)Tj
0.9978 0 Td
(44.6)Tj
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[()]TJ
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(and)Tj
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(cells)Tj
1.8935 0 Td
(incubated)Tj
4.1215 0 Td
(for)Tj
1.3662 0 Td
(a)Tj
0.6463 0 Td
(further)Tj
2.9877 0 Td
(48)Tj
1.1622 0 Td
(hours.)Tj
2.7043 0 Td
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1.7631 0 Td
(fluorescence)Tj
5.1137 0 Td
(of)Tj
1.0034 0 Td
(the)Tj
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3.0784 0 Td
(dye)Tj
1.61 0 Td
(was)Tj
1.6781 0 Td
(measured)Tj
4.0592 0 Td
(on)Tj
1.2643 0 Td
(a)Tj
0.6519 0 Td
(FLUOstar)Tj
4.1839 0 Td
(OPTIMA)Tj
4.0194 0 Td
(\(BMG)Tj
2.7213 0 Td
(LabTech,)Tj
3.8608 0 Td
(Germany\))Tj
4.2802 0 Td
(spectrometer)Tj
-31.4075 -1.2982 Td
(set)Tj
1.2812 0 Td
(at)Tj
0.9411 0 Td
(excitation)Tj
4.0932 0 Td
(and)Tj
1.7121 0 Td
(emission)Tj
3.7304 0 Td
(wavelengths)Tj
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1.0034 0 Td
(530)Tj
1.6327 0 Td
(nm)Tj
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(and)Tj
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(590)Tj
1.627 0 Td
(nm,)Tj
1.7972 0 Td
(respectively.)Tj
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1.7688 0 Td
(drugs)Tj
2.4435 0 Td
(used)Tj
-35.3647 -1.3039 Td
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1.0204 0 Td
(the)Tj
1.4514 0 Td
(susceptibility)Tj
5.3574 0 Td
(assays,)Tj
2.812 0 Td
(unless)Tj
2.6475 0 Td
(stated)Tj
2.5285 0 Td
(otherwise,)Tj
4.2518 0 Td
(were)Tj
2.0977 0 Td
(bought)Tj
3.016 0 Td
(from)Tj
2.1827 0 Td
(Sigma-Aldrich.)Tj
6.2191 0 Td
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1.3266 0 Td
(assess)Tj
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(the)Tj
1.4513 0 Td
(sensitivity)Tj
4.1556 0 Td
(to)Tj
1.0148 0 Td
(H)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 273.8267 218.4944 cm
BT
/F10 1 Tf
1 TL
-0.0133 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 277.3417 220.1385 cm
BT
/F10 1 Tf
1 TL
-0.01 Tc
0 0 Td
(O)Tj
ET
Q
q
1 j
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(33)Tj
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1.7291 0 Td
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([)Tj
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(34)Tj
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4.0422 0 Td
(and)Tj
-33.5109 -1.2982 Td
(washed)Tj
3.1748 0 Td
(then)Tj
2.0183 0 Td
(re-suspended)Tj
5.5842 0 Td
(in)Tj
1.0375 0 Td
(100)Tj
/F16 1 Tf
1.6441 0 Td
[()]TJ
/F10 1 Tf
(l)Tj
0.9751 0 Td
(transfection)Tj
4.9889 0 Td
(buffer)Tj
2.6249 0 Td
(\(90)Tj
1.5137 0 Td
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1.9331 0 Td
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Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 479.6787 577.2472 cm
BT
/F10 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(4)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 485.4614 578.948 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(pH7.3;)Tj
2.9083 0 Td
(5)Tj
0.6916 0 Td
(mM)Tj
1.9332 0 Td
(KCl;)Tj
-34.0778 -1.3039 Td
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1.1622 0 Td
(mM)Tj
1.9219 0 Td
(HEPES)Tj
3.1578 0 Td
(pH7.3;)Tj
2.897 0 Td
(0.15)Tj
1.8652 0 Td
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1.9219 0 Td
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ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 349.3417 564.2645 cm
BT
/F10 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 352.9133 565.9086 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(\))Tj
0.5499 0 Td
(and)Tj
1.7234 0 Td
(added)Tj
2.6532 0 Td
(to)Tj
1.0148 0 Td
(10)Tj
/F16 1 Tf
1.1622 0 Td
[()]TJ
/F10 1 Tf
(g)Tj
1.1849 0 Td
(of)Tj
1.0091 0 Td
(plasmid)Tj
3.3846 0 Td
(DNA)Tj
2.3697 0 Td
(in)Tj
1.0262 0 Td
(a)Tj
0.6463 0 Td
(cuvette)Tj
3.0614 0 Td
(before)Tj
-35.0756 -1.2982 Td
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4.9889 0 Td
(with)Tj
1.9956 0 Td
(an)Tj
1.2019 0 Td
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3.0557 0 Td
(biosystems)Tj
4.5807 0 Td
(NucleofectorII)Tj
6.0774 0 Td
(\(Lonza\))Tj
3.3392 0 Td
(using)Tj
2.3924 0 Td
(program)Tj
3.702 0 Td
(U-033.)Tj
2.9594 0 Td
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-34.2932 -1.2983 Td
(cells)Tj
1.9218 0 Td
(were)Tj
2.1203 0 Td
(incubated)Tj
4.1783 0 Td
(on)Tj
1.2756 0 Td
(ice)Tj
1.3322 0 Td
(for)Tj
1.389 0 Td
(10)Tj
1.1679 0 Td
(minutes)Tj
3.4695 0 Td
(and)Tj
1.7291 0 Td
(then)Tj
2.0182 0 Td
(transferred)Tj
4.6375 0 Td
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1.8425 0 Td
(pre-warmed)Tj
5.142 0 Td
(10)Tj
1.1679 0 Td
(ml)Tj
1.2869 0 Td
(of)Tj
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3.5546 0 Td
(supplemented)Tj
5.8563 0 Td
(with)Tj
1.9956 0 Td
(10%)Tj
1.9276 0 Td
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4.1498 0 Td
(and)Tj
1.7347 0 Td
(left)Tj
1.4741 0 Td
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1.0261 0 Td
(recover)Tj
3.1918 0 Td
(for)Tj
1.3889 0 Td
(18)Tj
1.1679 0 Td
(hours)Tj
2.5172 0 Td
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(27C.)Tj
2.4548 0 Td
(G418)Tj
2.3584 0 Td
(dis-)Tj
-35.7502 -1.304 Td
(ulfate)Tj
2.4377 0 Td
(salt)Tj
1.5591 0 Td
(\(Sigma-Aldrich\))Tj
6.7577 0 Td
(at)Tj
0.9525 0 Td
(50)Tj
/F16 1 Tf
1.1735 0 Td
[()]TJ
/F10 1 Tf
(g/ml)Tj
2.5851 0 Td
(was)Tj
1.7008 0 Td
(added)Tj
2.6589 0 Td
(to)Tj
1.0261 0 Td
(select)Tj
2.3981 0 Td
(cells)Tj
1.9162 0 Td
(carrying)Tj
3.5603 0 Td
(the)Tj
1.4683 0 Td
(plasmids.)Tj
3.9798 0 Td
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-34.1741 -1.2982 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(selected)Tj
3.3449 0 Td
(cells)Tj
1.9218 0 Td
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(obtained)Tj
3.7304 0 Td
(in)Tj
1.0375 0 Td
(the)Tj
1.4682 0 Td
(presence)Tj
3.7134 0 Td
(of)Tj
1.0148 0 Td
(50)Tj
/F16 1 Tf
1.1678 0 Td
[()]TJ
/F10 1 Tf
(g/ml)Tj
2.5909 0 Td
(G418)Tj
2.3584 0 Td
(in)Tj
1.0375 0 Td
(growth)Tj
3.0727 0 Td
(medium)Tj
3.6056 0 Td
(by)Tj
1.1849 0 Td
(lim-)Tj
-35.8579 -1.2983 Td
(iting)Tj
2.0636 0 Td
(dilution.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 454.4503 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Subcellular)Tj
5.0929 0 Td
(localisation)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 437.4425 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Immunofluorescence)Tj
8.7476 0 Td
(microscopy)Tj
4.8756 0 Td
(was)Tj
1.6951 0 Td
(performed)Tj
4.473 0 Td
(with)Tj
1.9956 0 Td
(WT)Tj
1.7972 0 Td
(and)Tj
1.7291 0 Td
(AmB)Tj
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(resistant)Tj
3.583 0 Td
(cell)Tj
1.559 0 Td
(lines)Tj
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(GFP-CYP51)Tj
5.176 0 Td
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1.0998 0 Td
(tagged)Tj
2.8347 0 Td
(sterol)Tj
2.4264 0 Td
(reductase)Tj
4.0138 0 Td
(\(GFP-SR\))Tj
4.1499 0 Td
(from)Tj
2.2054 0 Td
(episomal)Tj
3.7927 0 Td
(vectors.)Tj
3.2882 0 Td
(200)Tj
/F16 1 Tf
1.6441 0 Td
[()]TJ
/F10 1 Tf
(l)Tj
0.9751 0 Td
(of)Tj
-36.0281 -1.2983 Td
(mid-log)Tj
3.4015 0 Td
(phase)Tj
2.4831 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(collected)Tj
3.7417 0 Td
(and)Tj
1.7291 0 Td
(washed)Tj
3.1805 0 Td
(two)Tj
1.7064 0 Td
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2.3925 0 Td
(with)Tj
1.9956 0 Td
(PBS,)Tj
2.0579 0 Td
(fixed)Tj
2.194 0 Td
(in)Tj
1.0375 0 Td
(1%)Tj
1.4513 0 Td
(formaldehyde)Tj
-31.4133 -1.2982 Td
(\(methanol-free,)Tj
6.4459 0 Td
(Thermo)Tj
3.4809 0 Td
(Scientific\))Tj
4.2406 0 Td
(for)Tj
1.389 0 Td
(30)Tj
1.1679 0 Td
(minutes.)Tj
3.6906 0 Td
(Triton)Tj
2.8233 0 Td
(X-100)Tj
2.6532 0 Td
(\(Sigma-Aldrich\))Tj
6.7578 0 Td
(was)Tj
1.7008 0 Td
(added)Tj
-34.35 -1.304 Td
(up)Tj
1.2642 0 Td
(to)Tj
1.0261 0 Td
(final)Tj
2.0013 0 Td
(concentration)Tj
5.8223 0 Td
(0.1%)Tj
2.1543 0 Td
(and)Tj
1.7291 0 Td
(incubated)Tj
4.1783 0 Td
(for)Tj
1.3833 0 Td
(10)Tj
1.1735 0 Td
(minutes,)Tj
3.6907 0 Td
(afterwards,)Tj
4.6375 0 Td
(glycine)Tj
3.05 0 Td
(was)Tj
1.7008 0 Td
(added)Tj
2.6589 0 Td
(to)Tj
-36.4703 -1.2982 Td
(the)Tj
1.4683 0 Td
(final)Tj
2.0012 0 Td
(concentration)Tj
5.8224 0 Td
(of)Tj
1.0148 0 Td
(0.1)Tj
1.3946 0 Td
(M)Tj
1.1168 0 Td
(and)Tj
1.7292 0 Td
(incubated)Tj
4.1782 0 Td
(for)Tj
1.3833 0 Td
(an)Tj
1.2019 0 Td
(additional)Tj
4.2803 0 Td
(10)Tj
1.1678 0 Td
(minutes.)Tj
3.6907 0 Td
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2.16 0 Td
(were)Tj
2.126 0 Td
(centri-)Tj
-34.7355 -1.2983 Td
(fuged,)Tj
2.6758 0 Td
(resuspended)Tj
5.2328 0 Td
(in)Tj
1.0374 0 Td
(PBS,)Tj
2.058 0 Td
(spread)Tj
2.8573 0 Td
(on)Tj
1.2756 0 Td
(microscopy)Tj
4.8699 0 Td
(slides)Tj
2.4037 0 Td
(and)Tj
1.7348 0 Td
(left)Tj
1.474 0 Td
(to)Tj
1.0262 0 Td
(dry.)Tj
1.7971 0 Td
(Slides)Tj
2.5228 0 Td
(were)Tj
2.1203 0 Td
(washed)Tj
-33.0857 -1.3039 Td
(with)Tj
1.9842 0 Td
(PBS)Tj
1.8255 0 Td
(and)Tj
1.7234 0 Td
(blocked)Tj
3.3392 0 Td
(with)Tj
1.9899 0 Td
(PBS,)Tj
2.0523 0 Td
(0.1%)Tj
2.143 0 Td
(Triton)Tj
2.8176 0 Td
(X-100,)Tj
2.863 0 Td
(0.1%)Tj
2.143 0 Td
(BSA)Tj
1.9502 0 Td
(\(Sigma-Aldrich\))Tj
6.7521 0 Td
(for)Tj
1.3776 0 Td
(10)Tj
1.1622 0 Td
(minutes.)Tj
-34.1232 -1.2983 Td
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3.4979 0 Td
(antibody)Tj
3.7644 0 Td
(against)Tj
3.0273 0 Td
(the)Tj
1.4684 0 Td
(ER)Tj
1.4116 0 Td
(specific)Tj
3.1918 0 Td
(chaperone)Tj
4.3937 0 Td
(BiP)Tj
1.6271 0 Td
([)Tj
0.83 0.64 0.02 0 k
(37)Tj
0 g
(],)Tj
2.0749 0 Td
(a)Tj
0.6577 0 Td
(gift)Tj
1.5363 0 Td
(from)Tj
2.2054 0 Td
(Professor)Tj
3.9684 0 Td
(J.)Tj
0.7767 0 Td
(Bangs)Tj
-33.6016 -1.2982 Td
(\(University)Tj
4.7394 0 Td
(of)Tj
1.0148 0 Td
(Buffalo,)Tj
3.3166 0 Td
(New)Tj
2.0636 0 Td
(York\),)Tj
2.7949 0 Td
(was)Tj
1.7008 0 Td
(applied)Tj
3.1521 0 Td
(in)Tj
1.0318 0 Td
(dilution)Tj
3.4129 0 Td
(of)Tj
1.0148 0 Td
(1:5000,)Tj
3.05 0 Td
(overnight,)Tj
4.3087 0 Td
(at)Tj
0.9524 0 Td
(4C.)Tj
1.9729 0 Td
(Subse-)Tj
-34.5257 -1.304 Td
(quently,)Tj
3.4525 0 Td
(slides)Tj
2.4038 0 Td
(were)Tj
2.1203 0 Td
(washed)Tj
3.1804 0 Td
(three)Tj
2.2621 0 Td
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(PBS)Tj
1.8368 0 Td
(and)Tj
1.7291 0 Td
(incubated)Tj
4.1783 0 Td
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(anti-rabbit)Tj
-31.8215 -1.2982 Td
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2.4717 0 Td
(Fluor)Tj
2.3981 0 Td
(antibody)Tj
3.7644 0 Td
(\(Molecular)Tj
4.6318 0 Td
(Probes\).)Tj
3.5149 0 Td
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4.218 0 Td
(1)Tj
0.6973 0 Td
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(incubation,)Tj
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(slides)Tj
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-33.1538 -1.2983 Td
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2.262 0 Td
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(PBS,)Tj
2.0579 0 Td
(dried)Tj
2.3414 0 Td
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1.7292 0 Td
(mounted)Tj
3.8721 0 Td
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1.9955 0 Td
(5)Tj
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0.6974 0 Td
[()]TJ
/F10 1 Tf
(M)Tj
1.6214 0 Td
(4',6-diamidino-2-phenylindol)Tj
11.8997 0 Td
(e)Tj
0.6407 0 Td
(\(DAPI\).)Tj
-33.5053 -1.3039 Td
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4.9435 0 Td
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3.617 0 Td
(software)Tj
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4.1386 0 Td
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5.964 0 Td
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3.634 0 Td
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1.9219 0 Td
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2.126 0 Td
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2.0013 0 Td
(100)Tj
1.6441 0 Td
(nM)Tj
1.6611 0 Td
(MitoTracker)Tj
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4.6317 0 Td
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3.2882 0 Td
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0.9524 0 Td
(25C)Tj
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1.3833 0 Td
(30)Tj
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3.6907 0 Td
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5.7429 0 Td
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1.9219 0 Td
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1.0148 0 Td
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-34.3953 -1.2983 Td
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1.7291 0 Td
(mounted)Tj
3.8721 0 Td
(with)Tj
1.9956 0 Td
(DAPI.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 195.9874 cm
BT
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1 TL
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(Results)Tj
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0 -1.4976 Td
(Selection)Tj
4.2803 0 Td
(for)Tj
1.3748 0 Td
(AmB)Tj
2.3764 0 Td
(resistant)Tj
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3.9544 0 Td
(L)Tj
/F0 1 Tf
(.)Tj
/F9 1 Tf
1.0488 0 Td
(mexicana)Tj
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4.4504 0 Td
(and)Tj
1.8615 0 Td
(its)Tj
1.2094 0 Td
(characterisation)Tj
ET
Q
q
1 j
1 J
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10 0 0 10 200.0125 161.0078 cm
BT
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1 TL
-0.004 Tc
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(AmB)Tj
2.3017 0 Td
(resistant)Tj
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3.5829 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
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0.9751 0 Td
[()6()1(\n)4()7()16()6()]TJ
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3.9912 0 Td
(promastigotes)Tj
5.8507 0 Td
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2.1259 0 Td
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3.5603 0 Td
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(drug)Tj
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(concen-)Tj
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2.9423 0 Td
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1.0375 0 Td
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3.0387 0 Td
(medium)Tj
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1.9729 0 Td
(18)Tj
1.1736 0 Td
(passages)Tj
3.5773 0 Td
(stretching)Tj
4.2066 0 Td
(over)Tj
1.9729 0 Td
(six)Tj
1.3152 0 Td
(months.)Tj
3.5093 0 Td
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3.1351 0 Td
(this)Tj
1.6724 0 Td
(period,)Tj
3.0614 0 Td
(a)Tj
-36.2208 -1.3039 Td
(23-fold)Tj
3.0897 0 Td
(increase)Tj
3.4696 0 Td
(\(P)Tj
1.1225 0 Td
(=)Tj
0.7994 0 Td
(0.0007\))Tj
3.1634 0 Td
(in)Tj
1.0375 0 Td
(EC)Tj
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Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 339.137 133.3417 cm
BT
/F10 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(50)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 348.4346 134.9858 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
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(above)Tj
2.5455 0 Td
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1.7802 0 Td
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1.0148 0 Td
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3.9912 0 Td
(\(WT\))Tj
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-35.9203 -1.2982 Td
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1.7348 0 Td
(S1)Tj
1.1678 0 Td
(Fig)Tj
0 g
(\).)Tj
2.0466 0 Td
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4.0819 0 Td
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3.0784 0 Td
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(a)Tj
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(drug)Tj
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(concentration)Tj
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(above)Tj
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(0.27)Tj
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1.8709 0 Td
[()]TJ
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1.6214 0 Td
(could)Tj
2.4491 0 Td
(not)Tj
1.5761 0 Td
(be)Tj
1.1508 0 Td
(achieved.)Tj
-33.6356 -1.2983 Td
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1.7802 0 Td
(acquired)Tj
3.702 0 Td
(AmB)Tj
2.3017 0 Td
(resistance)Tj
4.1329 0 Td
(was)Tj
1.7008 0 Td
(stable)Tj
2.4887 0 Td
(over)Tj
1.9786 0 Td
(at)Tj
0.9524 0 Td
(least)Tj
1.9842 0 Td
(15)Tj
1.1679 0 Td
(passages)Tj
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(in)Tj
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(drug)Tj
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(free)Tj
1.7292 0 Td
(medium.)Tj
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2.5795 0 Td
(was)Tj
1.7007 0 Td
(no)Tj
1.2756 0 Td
(appreciable)Tj
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