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Shanna K. Williams, Zachary P. Weiner, Robert D. Gilmore
Human neuroglial cells internalize Borrelia burgdorferi by coiling phagocytosis mediated by Daam1
www.plosone.org
www.plosone.org
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[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6034 0 Td
(B31-A3)Tj
3.2655 0 Td
(wild)Tj
1.9446 0 Td
(type)Tj
-33.7037 -1.2982 Td
(\(WT\))Tj
2.4774 0 Td
(and)Tj
1.7291 0 Td
(the)Tj
1.4684 0 Td
(B31-A3)Tj
3.2598 0 Td
(mutant)Tj
3.1464 0 Td
(strain)Tj
2.5002 0 Td
(with)Tj
2.0012 0 Td
(inactivated)Tj
/F9 1 Tf
4.5978 0 Td
[()13()4()]TJ
/F10 1 Tf
2.5852 0 Td
([)Tj
0.83 0.64 0.02 0 k
(26)Tj
0 g
(])Tj
1.8538 0 Td
(expressing)Tj
4.422 0 Td
(green)Tj
2.4492 0 Td
(fluorescent)Tj
-32.4905 -1.3039 Td
(protein)Tj
3.1521 0 Td
(\(GFP\))Tj
2.7042 0 Td
(were)Tj
2.1203 0 Td
(generated)Tj
4.1385 0 Td
(by)Tj
1.1793 0 Td
(introducing)Tj
4.9719 0 Td
(the)Tj
/F9 1 Tf
1.4683 0 Td
[(\n)-68()]TJ
/F10 1 Tf
(-containing)Tj
6.0378 0 Td
(plasmid,)Tj
3.617 0 Td
(pMC2498)Tj
4.1952 0 Td
([)Tj
0.83 0.64 0.02 0 k
(27)Tj
0 g
(])Tj
1.8482 0 Td
(\(pro-)Tj
-35.4328 -1.2983 Td
(vided)Tj
2.4207 0 Td
(by)Tj
1.1792 0 Td
(Melissa)Tj
3.2088 0 Td
(Caimano,)Tj
4.1159 0 Td
(University)Tj
4.405 0 Td
(of)Tj
1.0148 0 Td
(Connecticut\),)Tj
5.7146 0 Td
(into)Tj
1.8369 0 Td
(the)Tj
1.474 0 Td
(cells)Tj
1.9162 0 Td
(by)Tj
1.1849 0 Td
(electroporation)Tj
6.3609 0 Td
(as)Tj
-34.8319 -1.2983 Td
(described)Tj
4.0535 0 Td
([)Tj
0.83 0.64 0.02 0 k
(28)Tj
0 g
(].)Tj
2.0749 0 Td
(B31-5A4NP1)Tj
5.5049 0 Td
(strain)Tj
2.5001 0 Td
([)Tj
0.83 0.64 0.02 0 k
(29)Tj
0 g
(])Tj
1.8539 0 Td
(was)Tj
1.7007 0 Td
(engineered)Tj
4.6431 0 Td
(to)Tj
1.0205 0 Td
(produce)Tj
3.5206 0 Td
(BBA66)Tj
3.0161 0 Td
(by)Tj
1.1792 0 Td
(fusing)Tj
2.6815 0 Td
(the)Tj
1.4684 0 Td
(con-)Tj
-35.2174 -1.3039 Td
(stitutive)Tj
/F9 1 Tf
3.4128 0 Td
[()-36()4()]TJ
/F10 1 Tf
1.7802 0 Td
(gene)Tj
2.0806 0 Td
(promoter)Tj
4.0365 0 Td
(to)Tj
1.0262 0 Td
(the)Tj
/F9 1 Tf
1.4683 0 Td
[()13()4()]TJ
/F10 1 Tf
2.5852 0 Td
(coding)Tj
2.9593 0 Td
(sequence)Tj
3.8608 0 Td
(in)Tj
1.0375 0 Td
(pMC2498)Tj
4.1952 0 Td
(by)Tj
1.1792 0 Td
(replacing)Tj
3.9175 0 Td
(the)Tj
/F9 1 Tf
1.4683 0 Td
[()-36()4()]TJ
/F10 1 Tf
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(driven-)Tj
/F9 1 Tf
[(\n)-68()]TJ
/F10 1 Tf
4.3199 0 Td
(coding)Tj
2.9594 0 Td
(sequence)Tj
3.8551 0 Td
(with)Tj
1.9955 0 Td
(the)Tj
/F9 1 Tf
1.4741 0 Td
[()13()4()]TJ
/F10 1 Tf
2.5851 0 Td
(coding)Tj
2.9594 0 Td
(sequence)Tj
3.8607 0 Td
(\(produced)Tj
4.3824 0 Td
(by)Tj
1.1849 0 Td
(PCR)Tj
2.0636 0 Td
(amplification\))Tj
-31.6401 -1.2982 Td
(by)Tj
1.1792 0 Td
(EcoRI-BamHI)Tj
5.9754 0 Td
(digestion)Tj
3.8834 0 Td
(sites)Tj
1.9332 0 Td
(present)Tj
3.1635 0 Td
(downstream)Tj
5.193 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
/F9 1 Tf
1.4683 0 Td
[()-36()4()]TJ
/F10 1 Tf
1.7802 0 Td
(promoter.)Tj
4.2633 0 Td
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5.4538 0 Td
(the)Tj
/F9 1 Tf
-35.3081 -1.3039 Td
[()-36()4()13()4()]TJ
/F10 1 Tf
4.473 0 Td
(cassette)Tj
3.2428 0 Td
(from)Tj
2.2053 0 Td
(pMC2498)Tj
4.1953 0 Td
(was)Tj
1.7008 0 Td
(excised)Tj
3.1124 0 Td
(by)Tj
1.1792 0 Td
(XbaI-BamHI)Tj
5.4482 0 Td
(digestion)Tj
3.8834 0 Td
(and)Tj
1.7291 0 Td
(subcloned)Tj
4.2973 0 Td
(into)Tj
-35.4668 -1.2983 Td
(the)Tj
1.4683 0 Td
(multiple)Tj
3.5659 0 Td
(cloning)Tj
3.2259 0 Td
(site)Tj
1.5703 0 Td
(of)Tj
1.0148 0 Td
(pBSV2G,)Tj
3.9062 0 Td
(a)Tj
0.6519 0 Td
(vector)Tj
2.6986 0 Td
(used)Tj
2.0579 0 Td
(for)Tj
1.389 0 Td
(transforming)Tj
/F9 1 Tf
5.4992 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
1.0148 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6091 0 Td
([)Tj
0.83 0.64 0.02 0 k
(30)Tj
0 g
(,)Tj
0.83 0.64 0.02 0 k
1.7348 0 Td
(31)Tj
0 g
(].)Tj
-34.4067 -1.2983 Td
(The)Tj
1.7801 0 Td
(two)Tj
/F9 1 Tf
1.7121 0 Td
[()-36()4()13()4()]TJ
/F10 1 Tf
4.4731 0 Td
(cassette)Tj
3.2428 0 Td
(plasmid)Tj
3.3959 0 Td
(constructs)Tj
4.3369 0 Td
(were)Tj
2.126 0 Td
(separately)Tj
4.1839 0 Td
(electroporated)Tj
5.9924 0 Td
(into)Tj
1.8369 0 Td
(B31-)Tj
-33.0801 -1.2982 Td
(5A4NP1)Tj
3.6226 0 Td
(resulting)Tj
3.7304 0 Td
(in)Tj
1.0374 0 Td
(both)Tj
2.058 0 Td
(cis-)Tj
1.6214 0 Td
(and)Tj
1.7348 0 Td
(trans-expressed)Tj
/F9 1 Tf
6.4573 0 Td
[()13()4()]TJ
/F10 1 Tf
2.5852 0 Td
(strains,)Tj
3.084 0 Td
(B31:pMC2498-BBA)Tj
7.9313 0 Td
(66)Tj
1.1679 0 Td
(and)Tj
-35.0303 -1.3039 Td
(B31:pBSV2G-BBA66.)Tj
8.8043 0 Td
(The)Tj
1.7802 0 Td
(strains)Tj
2.8629 0 Td
(were)Tj
2.126 0 Td
(induced)Tj
3.4639 0 Td
(for)Tj
1.3833 0 Td
(BBA66)Tj
3.0161 0 Td
(production)Tj
4.7168 0 Td
(by)Tj
1.1792 0 Td
(growth)Tj
3.0784 0 Td
(in)Tj
1.0318 0 Td
(BSK-II)Tj
-33.4429 -1.2983 Td
(pH)Tj
1.4966 0 Td
(7.5)Tj
1.3833 0 Td
(at)Tj
0.9411 0 Td
(34C)Tj
2.2167 0 Td
(in)Tj
1.0261 0 Td
(capped)Tj
3.0558 0 Td
(tubes)Tj
2.33 0 Td
(to)Tj
1.0148 0 Td
(a)Tj
0.6407 0 Td
(density)Tj
3.0954 0 Td
(of)Tj
1.0034 0 Td
(5)Tj
0.686 0 Td
(x)Tj
0.6747 0 Td
(10)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 405.1842 454.507 cm
BT
/F10 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(7)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 410.8535 450.085 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(cells)Tj
1.9105 0 Td
(per)Tj
1.5193 0 Td
(ml.)Tj
1.5024 0 Td
(1)Tj
0.686 0 Td
(ml)Tj
1.2756 0 Td
(of)Tj
1.0034 0 Td
(each)Tj
2.0239 0 Td
(mid-logarithmic)Tj
-31.005 -1.2982 Td
(cultures)Tj
3.3902 0 Td
(was)Tj
1.6894 0 Td
(pelleted)Tj
3.3222 0 Td
(and)Tj
1.7235 0 Td
(resuspended)Tj
5.2214 0 Td
(in)Tj
1.0318 0 Td
(4)Tj
0.6859 0 Td
(ml)Tj
1.2813 0 Td
(of)Tj
1.0035 0 Td
(BSK-II)Tj
2.9705 0 Td
(media)Tj
2.6873 0 Td
(pH)Tj
1.4967 0 Td
(6.8,)Tj
1.6157 0 Td
(and)Tj
1.7234 0 Td
(incubated)Tj
4.167 0 Td
(at)Tj
0.9467 0 Td
(an)Tj
1.1962 0 Td
(ele-)Tj
-36.1527 -1.3039 Td
(vated)Tj
2.3584 0 Td
(temperature)Tj
5.125 0 Td
(\(37C\))Tj
2.9083 0 Td
(for)Tj
1.389 0 Td
(24)Tj
1.1679 0 Td
(hrs.)Tj
1.7064 0 Td
(Cultures)Tj
3.6397 0 Td
(were)Tj
2.1203 0 Td
(grown)Tj
2.8005 0 Td
(under)Tj
2.6192 0 Td
(kanamycin)Tj
4.6431 0 Td
(and)Tj
1.7291 0 Td
(gentamicin)Tj
-32.2069 -1.2983 Td
(selection.)Tj
3.9401 0 Td
(Multiplex)Tj
4.1046 0 Td
(PCR)Tj
2.0636 0 Td
(established)Tj
4.5977 0 Td
(that)Tj
1.7802 0 Td
(each)Tj
2.0296 0 Td
(BBA66-expressing)Tj
/F9 1 Tf
7.5683 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
1.0148 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6091 0 Td
(strain)Tj
2.5002 0 Td
(con-)Tj
-34.2082 -1.2983 Td
(tained)Tj
2.7156 0 Td
(all)Tj
1.1508 0 Td
(plasmids)Tj
3.7531 0 Td
(as)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(parental)Tj
3.4923 0 Td
(B31-5A4NP1.)Tj
-12.3987 -1.3039 Td
(Human)Tj
3.3222 0 Td
(neuroglioma)Tj
5.3348 0 Td
(cell)Tj
1.559 0 Td
(lines,)Tj
2.2904 0 Td
(H4)Tj
1.4626 0 Td
(and)Tj
1.7348 0 Td
(HS683,)Tj
3.1125 0 Td
(were)Tj
2.1258 0 Td
(purchased)Tj
4.3313 0 Td
(from)Tj
2.2054 0 Td
(the)Tj
1.4683 0 Td
(American)Tj
4.1953 0 Td
(Type)Tj
-34.3386 -1.2982 Td
(Culture)Tj
3.2768 0 Td
(Collection)Tj
4.3483 0 Td
(\(ATCC,)Tj
3.4073 0 Td
(Manassas,)Tj
4.2746 0 Td
(Virginia\),)Tj
4.0989 0 Td
(maintained)Tj
4.7904 0 Td
(in)Tj
1.0318 0 Td
(25)Tj
1.1735 0 Td
(ml)Tj
1.2869 0 Td
(vented)Tj
2.897 0 Td
(cell)Tj
1.5591 0 Td
(culture)Tj
3.0387 0 Td
(flasks)Tj
-35.1833 -1.2983 Td
(\(Corning,)Tj
4.1556 0 Td
(Corning,)Tj
3.8154 0 Td
(NY\))Tj
1.9275 0 Td
(and)Tj
1.7291 0 Td
(grown)Tj
2.8006 0 Td
(in)Tj
1.0318 0 Td
(Dulbecco's)Tj
4.5921 0 Td
(modified)Tj
3.8494 0 Td
(Eagle's)Tj
2.931 0 Td
(medium)Tj
3.6056 0 Td
(\(DMEM\))Tj
3.9912 0 Td
(with)Tj
2.0012 0 Td
(4)Tj
-36.4305 -1.3039 Td
(mM)Tj
1.9332 0 Td
(l-glutamine)Tj
4.8472 0 Td
(containing)Tj
4.5241 0 Td
(4.5)Tj
1.4003 0 Td
(g/l)Tj
1.2586 0 Td
(glucose,)Tj
3.4015 0 Td
(1.5)Tj
1.3947 0 Td
(g/l)Tj
1.2585 0 Td
(sodium)Tj
3.2314 0 Td
(bicarbonate,)Tj
5.1704 0 Td
(and)Tj
1.7291 0 Td
(supplemented)Tj
-30.149 -1.2983 Td
(with)Tj
1.9956 0 Td
(10%)Tj
1.9275 0 Td
(fetal)Tj
1.9106 0 Td
(bovine)Tj
2.931 0 Td
(serum.)Tj
2.948 0 Td
(All)Tj
1.3946 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(grown)Tj
2.7949 0 Td
(at)Tj
0.9525 0 Td
(37C)Tj
2.2279 0 Td
(with)Tj
1.9956 0 Td
(5%)Tj
1.457 0 Td
(CO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 479.8488 331.4267 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 10 485.6314 333.0708 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(in)Tj
1.0374 0 Td
(a)Tj
0.652 0 Td
(humidified)Tj
-30.2511 -1.2982 Td
(incubator.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 281.9905 cm
BT
/F16 1 Tf
1 TL
-0.0033 Tc
0 0 Td
[()]TJ
/F14 1 Tf
(.)Tj
/F16 1 Tf
1.0771 0 Td
[()4()5()-1()1()-1()4()-34()8( )-7()-12(\n)]TJ
/F14 1 Tf
4.8426 0 Td
(cell)Tj
1.6346 0 Td
(infections)Tj
4.3418 0 Td
(and)Tj
1.7906 0 Td
(immunofluorescent)Tj
8.4567 0 Td
(staining)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 264.9826 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Cell)Tj
1.7971 0 Td
(coincubations)Tj
5.8393 0 Td
(with)Tj
/F9 1 Tf
2.0013 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
1.0148 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6034 0 Td
(followed)Tj
3.651 0 Td
(by)Tj
1.1792 0 Td
(immunofluorescent)Tj
8.1297 0 Td
(staining)Tj
3.4129 0 Td
(were)Tj
2.1203 0 Td
(per-)Tj
-33.749 -1.2982 Td
(formed)Tj
3.1577 0 Td
(as)Tj
1.0148 0 Td
(follows)Tj
3.0558 0 Td
(for)Tj
1.3889 0 Td
(all)Tj
1.1452 0 Td
(assays:)Tj
-8.5662 -1.2983 Td
(H4)Tj
1.4683 0 Td
(and)Tj
1.7291 0 Td
(HS683)Tj
2.8913 0 Td
(cells)Tj
1.9163 0 Td
(were)Tj
2.1259 0 Td
(grown)Tj
2.795 0 Td
(to)Tj
1.0261 0 Td
(confluency,)Tj
4.8359 0 Td
(harvested)Tj
4.0422 0 Td
(using)Tj
2.3867 0 Td
(0.05%)Tj
2.6305 0 Td
(trypsin)Tj
3.0388 0 Td
(with)Tj
1.9955 0 Td
(10)Tj
1.1736 0 Td
(mM)Tj
-35.2514 -1.3039 Td
(EDTA,)Tj
3.0443 0 Td
(and)Tj
1.7292 0 Td
(enumerated)Tj
5.0173 0 Td
(using)Tj
2.3924 0 Td
(a)Tj
0.6519 0 Td
(Petroff-Hausser)Tj
6.5537 0 Td
(counting)Tj
3.8041 0 Td
(chamber.)Tj
3.9514 0 Td
(Cells)Tj
2.16 0 Td
(were)Tj
2.126 0 Td
(seeded)Tj
2.9083 0 Td
(at)Tj
0.9468 0 Td
(a)Tj
-35.2854 -1.2983 Td
(density)Tj
3.101 0 Td
(of)Tj
1.0148 0 Td
(1)Tj
/F15 1 Tf
0.6917 0 Td
[()]TJ
/F10 1 Tf
0.7937 0 Td
(10)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 265.5496 217.4173 cm
BT
/F10 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(6)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 271.2755 212.9952 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(cells)Tj
1.9218 0 Td
(/)Tj
0.5499 0 Td
(well)Tj
1.8199 0 Td
(in)Tj
1.0318 0 Td
(cell)Tj
1.5647 0 Td
(culture)Tj
3.0387 0 Td
(plates)Tj
2.5002 0 Td
(containing)Tj
4.524 0 Td
(coverslips)Tj
4.1556 0 Td
(\(Corning\).)Tj
4.4957 0 Td
(Cells)Tj
2.16 0 Td
(were)Tj
-34.8885 -1.2982 Td
(allowed)Tj
3.2882 0 Td
(to)Tj
1.0261 0 Td
(attach)Tj
2.6362 0 Td
(to)Tj
1.0204 0 Td
(the)Tj
1.474 0 Td
(coverslips)Tj
4.1499 0 Td
(for)Tj
1.3889 0 Td
(24)Tj
1.1679 0 Td
(hr)Tj
1.1225 0 Td
(and)Tj
1.7291 0 Td
(cell)Tj
1.5591 0 Td
(culture)Tj
3.0387 0 Td
(medium)Tj
3.6057 0 Td
(was)Tj
1.7007 0 Td
(replaced)Tj
3.583 0 Td
(with)Tj
2.0012 0 Td
(fresh)Tj
-34.4916 -1.3039 Td
(media)Tj
2.6929 0 Td
(prior)Tj
2.2507 0 Td
(to)Tj
1.0261 0 Td
(addition)Tj
3.5999 0 Td
(of)Tj
/F9 1 Tf
1.0148 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
1.0148 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6034 0 Td
(culture.)Tj
/F9 1 Tf
3.2655 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
1.0148 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6091 0 Td
(were)Tj
2.1203 0 Td
(added)Tj
2.6589 0 Td
(to)Tj
1.0261 0 Td
(the)Tj
1.4684 0 Td
(cells,)Tj
2.1486 0 Td
(and)Tj
-34.5143 -1.2983 Td
(incubated)Tj
4.1782 0 Td
(at)Tj
0.9525 0 Td
(37C)Tj
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(cells)Tj
1.9218 0 Td
(per)Tj
1.5307 0 Td
(coverslip.)Tj
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Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 612.2834 cm
BT
/F14 1 Tf
1 TL
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1.8803 0 Td
(infections)Tj
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(and)Tj
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(immunofluorescence)Tj
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(labeling)Tj
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(of)Tj
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(BBA66-expressin)Tj
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(g)Tj
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0.7087 0 Td
[()]TJ
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(.)Tj
/F16 1 Tf
-29.4994 -1.1669 Td
[()4()5()-1()1()-1()4()-34()8( )-7()-12(\n)]TJ
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(and)Tj
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(human)Tj
3.1796 0 Td
(Daam1)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 581.2724 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
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(Cell)Tj
1.7971 0 Td
(infection)Tj
3.7927 0 Td
(assays)Tj
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(and)Tj
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(immunofluorescent)Tj
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(stainings)Tj
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(were)Tj
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(performed)Tj
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(duplicate)Tj
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(each)Tj
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(line,)Tj
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[()]TJ
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(.)Tj
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[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
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(and)Tj
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(timepoint)Tj
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(with)Tj
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(the)Tj
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(following)Tj
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2.0013 0 Td
[()]TJ
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(.)Tj
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[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
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4.6034 0 Td
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(strains)Tj
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(fixation)Tj
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(-20C)Tj
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(20)Tj
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(min)Tj
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(2)Tj
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(hr)Tj
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(coverslips)Tj
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(were)Tj
2.1259 0 Td
(blocked,)Tj
3.5773 0 Td
(cells)Tj
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(were)Tj
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(incubated)Tj
4.1782 0 Td
(with)Tj
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(rabbit)Tj
2.6022 0 Td
(anti-Daam1)Tj
5.0173 0 Td
(antibody)Tj
3.7644 0 Td
(\(Protein-)Tj
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(tech\))Tj
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(followed)Tj
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(goat)Tj
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(anti-rabbit)Tj
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(IgG)Tj
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(\(Molecular)Tj
4.6318 0 Td
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(by)Tj
1.1849 0 Td
(Life)Tj
1.7348 0 Td
(Technologies\))Tj
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(\(1:100\))Tj
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(for)Tj
1.3833 0 Td
(1)Tj
0.6973 0 Td
(hr)Tj
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(at)Tj
0.9524 0 Td
(37C)Tj
2.228 0 Td
(in)Tj
1.0375 0 Td
(a)Tj
0.652 0 Td
(humidified)Tj
4.6658 0 Td
(chamber.)Tj
3.9515 0 Td
(Cells)Tj
2.1599 0 Td
(were)Tj
2.1203 0 Td
(costained)Tj
4.0139 0 Td
(for)Tj
1.3833 0 Td
(BBA66)Tj
3.016 0 Td
(\(as)Tj
1.355 0 Td
(described)Tj
-33.7604 -1.2983 Td
(above\))Tj
2.8856 0 Td
(and)Tj
1.7291 0 Td
(the)Tj
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1.4684 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
1.0148 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6091 0 Td
(strains)Tj
2.8629 0 Td
(B31:pBSV2G-BBA66)Tj
8.5776 0 Td
(and)Tj
1.7291 0 Td
(B31:pMC2498-BBA)Tj
7.9313 0 Td
(66)Tj
1.1679 0 Td
(were)Tj
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(stained)Tj
3.0784 0 Td
(with)Tj
1.9955 0 Td
(fluorescein-labelled)Tj
8.0164 0 Td
(goat)Tj
1.9218 0 Td
(anti-)Tj
/F9 1 Tf
[()-2()11()-9()20()15()12()4()]TJ
5.2554 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6091 0 Td
(IgG)Tj
1.7405 0 Td
(\(1:200\))Tj
3.0274 0 Td
(\(Kirkegaard)Tj
5.0229 0 Td
(and)Tj
-34.6674 -1.2983 Td
(Perry)Tj
2.398 0 Td
(Laboratory)Tj
4.6432 0 Td
(Inc.\))Tj
2.0976 0 Td
(for)Tj
1.389 0 Td
(1)Tj
0.6916 0 Td
(hr)Tj
1.1225 0 Td
(at)Tj
0.9525 0 Td
(37C)Tj
2.2223 0 Td
(in)Tj
1.0375 0 Td
(a)Tj
0.6519 0 Td
(humidified)Tj
4.6658 0 Td
(chamber.)Tj
3.9515 0 Td
(Colocalization)Tj
5.9811 0 Td
(calculations)Tj
-31.8045 -1.2983 Td
(were)Tj
2.1203 0 Td
(performed)Tj
4.473 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
4.0478 0 Td
(above.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 421.1149 cm
BT
/F14 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Cell)Tj
1.8803 0 Td
(infections)Tj
4.3417 0 Td
(comparing)Tj
4.7481 0 Td
(coiling)Tj
3.0567 0 Td
(events)Tj
2.8347 0 Td
(between)Tj
3.6284 0 Td
(BBA66-)Tj
3.5244 0 Td
(expressing)Tj
/F16 1 Tf
4.6063 0 Td
[()]TJ
/F14 1 Tf
(.)Tj
/F16 1 Tf
-28.6206 -1.1669 Td
[()4()5()-1()1()-1()4()-34()8( )-7()-12(\n)]TJ
/F14 1 Tf
4.8425 0 Td
(and)Tj
1.7953 0 Td
(BBA66-deficient)Tj
/F16 1 Tf
7.1623 0 Td
[()]TJ
/F14 1 Tf
(.)Tj
/F16 1 Tf
1.0771 0 Td
[()4()5()-1()1()-1()4()-34()8( )-7()-12(\n)]TJ
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 390.1606 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Cell)Tj
1.7971 0 Td
(infection)Tj
3.7927 0 Td
(assays)Tj
2.6249 0 Td
(and)Tj
1.7291 0 Td
(immunofluorescent)Tj
8.1297 0 Td
(stainings)Tj
3.7701 0 Td
(were)Tj
2.126 0 Td
(performed)Tj
4.4673 0 Td
(in)Tj
1.0375 0 Td
(duplicate)Tj
3.8835 0 Td
(for)Tj
1.3832 0 Td
(each)Tj
-34.7411 -1.3039 Td
(cell)Tj
1.559 0 Td
(line,)Tj
/F9 1 Tf
1.9275 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
1.0148 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6091 0 Td
(strain,)Tj
2.727 0 Td
(and)Tj
1.7291 0 Td
(timepoint)Tj
4.1669 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
4.0535 0 Td
(above)Tj
2.5455 0 Td
(with)Tj
1.9956 0 Td
(the)Tj
1.4683 0 Td
(following)Tj
3.9798 0 Td
(modifica-)Tj
-32.7909 -1.2982 Td
(tions.)Tj
2.4264 0 Td
(H4)Tj
1.4627 0 Td
(and)Tj
1.7291 0 Td
(HS683)Tj
2.8913 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(grown)Tj
2.7949 0 Td
(and)Tj
1.7348 0 Td
(seeded)Tj
2.9027 0 Td
(on)Tj
1.2756 0 Td
(poly-L-lysine)Tj
5.4878 0 Td
(coverslips)Tj
4.1499 0 Td
(for)Tj
1.389 0 Td
(24)Tj
1.1678 0 Td
(hr.)Tj
/F9 1 Tf
1.3493 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
-34.8035 -1.2983 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6034 0 Td
(B31:pMC2498-BBA)Tj
7.9313 0 Td
(66)Tj
1.1678 0 Td
(and)Tj
1.7292 0 Td
(B31:pBSV2G-BBA66)Tj
8.5832 0 Td
(were)Tj
2.1203 0 Td
(added)Tj
2.6646 0 Td
(to)Tj
1.0261 0 Td
(the)Tj
1.4683 0 Td
(cells)Tj
1.9162 0 Td
(at)Tj
0.9525 0 Td
(a)Tj
0.6576 0 Td
(MOI)Tj
-34.8205 -1.3039 Td
(of)Tj
1.0147 0 Td
(100.)Tj
1.8709 0 Td
(Cells)Tj
2.16 0 Td
(were)Tj
2.1203 0 Td
(fixed)Tj
2.194 0 Td
(with)Tj
1.9956 0 Td
(methanol)Tj
4.0251 0 Td
(at)Tj
0.9525 0 Td
(-20C)Tj
2.5795 0 Td
(for)Tj
1.3833 0 Td
(20)Tj
1.1735 0 Td
(min)Tj
1.8539 0 Td
(after)Tj
2.0352 0 Td
(1)Tj
0.6973 0 Td
(and)Tj
1.7292 0 Td
(2)Tj
0.6973 0 Td
(hr)Tj
1.1168 0 Td
(post-infection.)Tj
6.0548 0 Td
(Cells)Tj
-35.6539 -1.2983 Td
(were)Tj
2.1203 0 Td
(stained)Tj
3.0784 0 Td
(for)Tj
1.3889 0 Td
(Daam1)Tj
3.1181 0 Td
(and)Tj
/F9 1 Tf
1.7291 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
1.0148 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6091 0 Td
(strains)Tj
2.863 0 Td
(B31:pBSV2G-BBA66)Tj
8.5776 0 Td
(and)Tj
1.7348 0 Td
(B31:pMC2498-)Tj
-30.2341 -1.2982 Td
(BBA66)Tj
3.016 0 Td
(were)Tj
2.1203 0 Td
(stained)Tj
3.0784 0 Td
(with)Tj
2.0012 0 Td
(fluorescein-labelled)Tj
8.0107 0 Td
(goat)Tj
1.9219 0 Td
(anti-)Tj
/F9 1 Tf
[()-2()11()-9()20()15()12()4()]TJ
5.2554 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6091 0 Td
(IgG)Tj
1.7405 0 Td
(\(1:200\))Tj
3.0273 0 Td
(\(Kir-)Tj
-34.7808 -1.304 Td
(kegaard)Tj
3.3732 0 Td
(and)Tj
1.7291 0 Td
(Perry)Tj
2.3981 0 Td
(Laboratory)Tj
4.6431 0 Td
(Inc.\))Tj
2.0976 0 Td
(for)Tj
1.389 0 Td
(1)Tj
0.6916 0 Td
(hr)Tj
1.1225 0 Td
(at)Tj
0.9525 0 Td
(37C.)Tj
2.4491 0 Td
(Coverslips)Tj
4.3937 0 Td
(were)Tj
2.1203 0 Td
(mounted)Tj
3.8721 0 Td
(with)Tj
2.0012 0 Td
(ProLong)Tj
-33.2331 -1.2982 Td
(Diamond)Tj
4.0591 0 Td
(Antifade)Tj
3.6907 0 Td
(with)Tj
1.9956 0 Td
(DAPI)Tj
2.5398 0 Td
(\(Molecular)Tj
4.6318 0 Td
(Probes)Tj
2.948 0 Td
(by)Tj
1.1849 0 Td
(Life)Tj
1.7348 0 Td
(Technologies\))Tj
5.8733 0 Td
(and)Tj
1.7292 0 Td
(viewed)Tj
2.999 0 Td
(using)Tj
2.3924 0 Td
(the)Tj
-35.7786 -1.2983 Td
(Zeiss)Tj
2.2336 0 Td
(LSM)Tj
2.126 0 Td
(800)Tj
1.6441 0 Td
(confocal)Tj
3.6 0 Td
(laser)Tj
2.0522 0 Td
(scanning)Tj
3.8211 0 Td
(microscope)Tj
4.8416 0 Td
(to)Tj
1.0204 0 Td
(enumerate)Tj
4.4901 0 Td
(the)Tj
1.474 0 Td
(number)Tj
3.4129 0 Td
(of)Tj
1.0148 0 Td
(coiling)Tj
2.948 0 Td
(events)Tj
-34.6788 -1.3039 Td
(\(Daam-1)Tj
3.8097 0 Td
(pseudopods)Tj
4.9946 0 Td
(enwrapping)Tj
5.0003 0 Td
(a)Tj
0.652 0 Td
(spirochete\))Tj
4.6828 0 Td
(identified)Tj
4.0535 0 Td
(in)Tj
1.0374 0 Td
(1000)Tj
2.126 0 Td
(total)Tj
2.0013 0 Td
(neuroglial)Tj
4.2576 0 Td
(cells.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 230.0031 cm
BT
/F14 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Detection)Tj
4.3086 0 Td
(of)Tj
/F16 1 Tf
1.0678 0 Td
[()]TJ
/F14 1 Tf
(.)Tj
/F16 1 Tf
1.0771 0 Td
[()4()5()-1()1()-1()4()-34()8( )-7()-12(\n)]TJ
/F14 1 Tf
4.8473 0 Td
(BBA66)Tj
3.1796 0 Td
(by)Tj
1.2142 0 Td
(immunofluorese)Tj
6.9118 0 Td
(nt)Tj
1.1197 0 Td
(staining)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 212.9952 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Induced)Tj
/F9 1 Tf
3.5376 0 Td
[()]TJ
/F10 1 Tf
(.)Tj
/F9 1 Tf
1.0148 0 Td
[()11( )12()6(\n)2()4()11()-23()9()2()-9()]TJ
/F10 1 Tf
4.6091 0 Td
(B31:pMC2498-BBA66)Tj
9.0935 0 Td
(and)Tj
1.7347 0 Td
(B31:pBSV2G-BBA66)Tj
8.5776 0 Td
(were)Tj
2.126 0 Td
(grown)Tj
2.7949 0 Td
(to)Tj
1.0262 0 Td
(a)Tj
0.6519 0 Td
(den-)Tj
-35.1663 -1.2982 Td
(sity)Tj
1.5987 0 Td
(of)Tj
1.0148 0 Td
(~1)Tj
1.2756 0 Td
(x)Tj
0.6859 0 Td
(10)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 255.2881 204.4346 cm
BT
/F10 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(8)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 261.0141 200.0125 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(cells)Tj
1.9218 0 Td
(/)Tj
0.5499 0 Td
(ml)Tj
1.287 0 Td
(and)Tj
1.7291 0 Td
(1)Tj
0.6973 0 Td
(ml)Tj
1.2869 0 Td
(of)Tj
1.0148 0 Td
(each)Tj
2.0239 0 Td
(strain)Tj
2.5059 0 Td
(was)Tj
1.6951 0 Td
(collected,)Tj
3.9684 0 Td
(centrifuged,)Tj
5.006 0 Td
(and)Tj
1.7291 0 Td
(washed)Tj
3.1805 0 Td
(\(3X)Tj
-34.6958 -1.3039 Td
(HBSS\).)Tj
3.0841 0 Td
(Washed)Tj
3.4582 0 Td
(cells)Tj
1.9218 0 Td
(were)Tj
2.1203 0 Td
(resuspended)Tj
5.2328 0 Td
(in)Tj
1.0374 0 Td
(100)Tj
/F15 1 Tf
1.6441 0 Td
[()]TJ
/F10 1 Tf
(l)Tj
0.9751 0 Td
(of)Tj
1.0148 0 Td
(dH)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 417.8267 185.3291 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 10 421.3984 186.9732 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
(O)Tj
0.9694 0 Td
(and)Tj
1.7291 0 Td
(spotted)Tj
3.1521 0 Td
(onto)Tj
2.0806 0 Td
(glass)Tj
2.0863 0 Td
(slides)Tj
2.4095 0 Td
(and)Tj
-34.5654 -1.2982 Td
(allowed)Tj
3.2882 0 Td
(to)Tj
1.0261 0 Td
(air)Tj
1.2926 0 Td
(dry.)Tj
1.7972 0 Td
(Dried)Tj
2.5455 0 Td
(cells)Tj
1.9218 0 Td
(were)Tj
2.1203 0 Td
(fixed)Tj
2.194 0 Td
(using)Tj
2.3868 0 Td
(methanol)Tj
4.0251 0 Td
(at)Tj
0.9524 0 Td
(-20C)Tj
2.5795 0 Td
(for)Tj
1.3833 0 Td
(20)Tj
1.1735 0 Td
(min)Tj
1.8539 0 Td
(followed)Tj
3.651 0 Td
(by)Tj
1.1792 0 Td
(3X)Tj
-35.3704 -1.2983 Td
(HBSS)Tj
2.5172 0 Td
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