The VP8* sequences (amino acids 46 to 231) from a human RV P[19] (GenBank accession number DQ887060 ) was expressed in E. coli BL21 and purified by glutathione S-transferase (GST) tag affinity purification, as described previously (25 (link)). Recombinant VP8* proteins of other P[II] RVs (P[4], P[6], and P[8]) and a P[10] RV were also purified using the same expression system. 15N- or 15N,13C-labeled P[19] VP8* proteins were also made for the NMR studies using the same E. coli culture procedures, with the addition of 15N-minimal growth medium (22 mM KH2PO4, 50 mM Na2HPO4, 8.5 mM NaCl, 0.1 mM CaCl2, 2 mM MgSO4, 1× BME vitamins (catalog no. B6891; Sigma), 18 mM 15NH4Cl, and 22 mM glucose) or 15N,13C-minimal growth medium supplied with 22 mM [13C]glucose (20 (link)). The GST tag of the labeled VP8* proteins was removed for the NMR study by cutting with thrombin (Sigma-Aldrich Co., St. Louis, MO), followed by fast protein liquid chromatography (FPLC) gel filtration to collect the VP8* protein fractions and concentration by ultracentrifugation. The purified VP8* proteins were stored at −80°C until needed.
Bme vitamins
Manufactured by Merck Group
Sourced in United States
BME vitamins are a line of laboratory-grade vitamins and mineral supplements produced by Merck Group. They are designed for use in scientific research and analysis applications. The core function of BME vitamins is to provide a reliable and consistent source of essential vitamins and minerals for various experimental and testing procedures.
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Lab products found in correlation
Amikin, by Bristol-Myers Squibb (5 mentions)
Medium 199, by Merck Group (5 mentions)
Foetal calf serum, by Thermo Fisher Scientific (5 mentions)
M199 medium, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Amikacin, by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
M199 media, by Merck Group (2 mentions)
15n nh4cl, by Cambridge Isotopes (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Thrombin, by Merck Group (1 mentions)
Sephacryl s 200 column, by GE Healthcare (1 mentions)
Resource s cation exchange column, by GE Healthcare (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi powder, by Thermo Fisher Scientific (1 mentions)
Medium 199, by Lonza (1 mentions)
Gentamicin sulphate, by Merck Group (1 mentions)
Schneider s drosophila medium, by Merck Group (1 mentions)
25 protocols using bme vitamins
1
Recombinant Rotavirus VP8* Protein Expression
2
Leishmania Parasite and Vector Maintenance
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Leishmania enriettii LV90 (MCAV/BR/45/LV90) and Leishmania sp. AM-2004 (MMAC/AU/2004/AM-2004; Roo1; LV756), and three human infecting Leishmania strains, L. major FVI (MHOM/IL/81/Friedlin/FVI), L. infantum CUK3 (TOB/TR/2005/CUK3) and L. donovani GR374 (MHOM/ET/2010/GR374), were used. Parasites were maintained at 23°C in M199 medium supplemented with 10% fetal calf serum (Gibco), 1% BME vitamins (Sigma), 2% sterile urine and 250 μg/ml Amikin (Amikin, Bristol-Myers Squibb), and were in culture for about 10 subpassages from an animal host before use. Before experimental feeding, parasites were washed by centrifugation and resuspended in saline solution.
Lutzomyia longipalpis (Jacobina colony) was maintained at Charles University in Prague under standard conditions [29 (link)]. Females from the colonies of Culicoides nubeculosus and C. sonorensis (both belonging to subgenus Monoculicoides) were sent to Charles University from the Pirbright Institute, UK and kept at 20°C before exposure to feeding. All insects were initially given free access to 50% sucrose supplemented with penicillin (5000 U/ml), which was replaced with sugar solution alone 3 days before experimental feeding.
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Leishmania enriettii LV90 (MCAV/BR/45/LV90) and Leishmania sp. AM-2004 (MMAC/AU/2004/AM-2004; Roo1; LV756), and three human infecting Leishmania strains, L. major FVI (MHOM/IL/81/Friedlin/FVI), L. infantum CUK3 (TOB/TR/2005/CUK3) and L. donovani GR374 (MHOM/ET/2010/GR374), were used. Parasites were maintained at 23°C in M199 medium supplemented with 10% fetal calf serum (Gibco), 1% BME vitamins (Sigma), 2% sterile urine and 250 μg/ml Amikin (Amikin, Bristol-Myers Squibb), and were in culture for about 10 subpassages from an animal host before use. Before experimental feeding, parasites were washed by centrifugation and resuspended in saline solution.
Lutzomyia longipalpis (Jacobina colony) was maintained at Charles University in Prague under standard conditions [29 (link)]. Females from the colonies of Culicoides nubeculosus and C. sonorensis (both belonging to subgenus Monoculicoides) were sent to Charles University from the Pirbright Institute, UK and kept at 20°C before exposure to feeding. All insects were initially given free access to 50% sucrose supplemented with penicillin (5000 U/ml), which was replaced with sugar solution alone 3 days before experimental feeding.
3
Establishing Lutzomyia Vector Colonies for Leishmania Research
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A colony of Lutzomyia migonei was established at Charles University in Prague from specimens captured in Baturité municipality, Ceará state, northeast Brazil (04°19′41″S, 38°53′05″W). An already-established laboratory colony of Lutzomyia longipalpis (from Jacobina, Brazil) with well-known susceptibility to L. infantum [16 (link)–18 (link)] was used as a control. Both colonies were maintained under standard conditions as previously described [19 (link)].
A viscerotropic Leishmania infantum strain (MHOM/BR/76/M4192) [20 (link)] and dermotropic L. infantum strain (ITOB/TR/2005/CUK3) [21 (link)] were maintained at 23 °C on Medium 199 (Sigma) supplemented with 10 % foetal calf serum (Gibco), 1 % BME vitamins (Sigma), 2 % human urine and 250 μg/ml Amikin (Amikin, Bristol-Myers Squibb).
A viscerotropic Leishmania infantum strain (MHOM/BR/76/M4192) [20 (link)] and dermotropic L. infantum strain (ITOB/TR/2005/CUK3) [21 (link)] were maintained at 23 °C on Medium 199 (Sigma) supplemented with 10 % foetal calf serum (Gibco), 1 % BME vitamins (Sigma), 2 % human urine and 250 μg/ml Amikin (Amikin, Bristol-Myers Squibb).
4
Maintenance of P. sergenti Sand Fly Colonies and Leishmania tropica
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Three laboratory colonies of P.sergenti were used—1) TU originating from Sanliurfa, Turkey; 2) IS originating from Amnun, Israel; and 3) ISG derived from IS females artificially infected by Ps. sergenti as described by Lantova et al. (2010) (link). Sand flies were maintained under standard conditions as previously described by Volf and Volfova (2011) . Leishmania tropica SU23 (MHOM/TR/98/HM) was maintained at 23°C on M199 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 20% foetal calf serum (Gibco, Life Technologies, Carlsbad, CA), 1% BME vitamins (Sigma-Aldrich), 2% filtered human urine, amikacin (250 mg/ml), and gentamicin (80 mg/ml).
5
Isotope-labelled PARKIN Ubl phosphorylation
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Isotope-labelled HsPARKIN Ubl domain (aa 1-72) was expressed in M9 minimal media supplemented with 4 g/L 13C-glucose, 2 g/L 15N-NH4Cl, trace elements and BME Vitamins (Sigma-Aldrich) and purified as described above. The final gel filtration was performed in NMR buffer (18 mM Na2HPO4, 7 mM NaH2PO4 pH 7.2, 150 mM NaCl, 5 mM TCEP). Isotope-labelled HsPARKIN Ubl was phosphorylated at room temperature by incubating 80 µM HsPARKIN Ubl with 2.5 µM PhPINK1, 1 mM ATP and 1x ligation buffer which was adjusted to 332.5 µl with NMR buffer, before addition of 17.5 µl D2O as lock solvent. The reaction was monitored by consecutive 1H,15N 2D BEST-TROSY (Band Selective Excitation Short Transients Transverse Relaxation Optimised Spectroscopy) experiments and quenched with apyrase.
6
Isotope-labelled PARKIN Ubl phosphorylation
7
Recombinant Histone Purification and Isotopic Labeling
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Homo sapiens histones H2A, H2B, H3, and H4 were overexpressed and purified following published protocols23 (link),61 (link). The pET-3a plasmids containing the histone sequences were transformed and overexpressed in E. coli BL21 (DE3) pLysS cells in 2 TY media. Crude WT histones were purified by 26/60 Sephacryl S-200 column (GE Healthcare) followed by the Resource S cation-exchange column (GE Healthcare). The expression of H4K20C mutant was similar to that of WT histones. H4K20C histone purified by gel-filtration (Sephacryl S-200 column) was further purified by Vydac C4 preparative HPLC chromatography (Grace).
To produce 13C-labeled,15N-labeled histones, cells were grown in 2x TY media until OD600 reached 0.5. The cells were pelleted down and resuspended in equal volume of M9 minimal media supplemented with 0.4% 13C glucose and 0.1% 15N ammonium chloride (CIL, USA), micro nutrients and BMEvitamins (Sigma-Aldrich). After 1 h acclimatization, the cells were induced with 0.4 mM IPTG at OD600 of 0.7 for overexpression for 3 h at 37 °C. The 13C-labeled,15N-labeled WT H4 and H4K20C were purified following the same procedures as the unlabeled ones.
To produce 13C-labeled,15N-labeled histones, cells were grown in 2x TY media until OD600 reached 0.5. The cells were pelleted down and resuspended in equal volume of M9 minimal media supplemented with 0.4% 13C glucose and 0.1% 15N ammonium chloride (CIL, USA), micro nutrients and BMEvitamins (Sigma-Aldrich). After 1 h acclimatization, the cells were induced with 0.4 mM IPTG at OD600 of 0.7 for overexpression for 3 h at 37 °C. The 13C-labeled,15N-labeled WT H4 and H4K20C were purified following the same procedures as the unlabeled ones.
8
Leishmania major Cultivation and Infection
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Leishmania major parasites (FV1 MHOH/IL/80/Friedlin) were cultivated in Medium199 (Sigma–Aldrich) at 23°C, supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Carlsbad, CA, United States), 1% BME vitamins (Sigma–Aldrich), 2% of sterile urine, and 250 μg/mL amikacin (Medopharm, Pozorice, Czech Republic). Adult females had access to AtbC in sucrose solution served ad libitum and changed daily for 5 days after eclosion, during the experimental infection and after infection. On day 5, females were fed through chicken skin membrane on defibrinated sheep blood (LabMediaServis, Jaromer, Czech Republic) with AtbC, seeded with 106 L. major promastigotes/mL. Blood-fed females were separated, kept under the same conditions described above, and dissected at different time intervals (indicated in figure legends). Guts (without Malpighian tubules) and carcasses (i.e., all other tissues) were dissected in sterile saline solution, collected in pools of 10, and stored at −80°C until processing.
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Leishmania major parasites (FV1 MHOH/IL/80/Friedlin) were cultivated in Medium199 (Sigma–Aldrich) at 23°C, supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Carlsbad, CA, United States), 1% BME vitamins (Sigma–Aldrich), 2% of sterile urine, and 250 μg/mL amikacin (Medopharm, Pozorice, Czech Republic). Adult females had access to AtbC in sucrose solution served ad libitum and changed daily for 5 days after eclosion, during the experimental infection and after infection. On day 5, females were fed through chicken skin membrane on defibrinated sheep blood (LabMediaServis, Jaromer, Czech Republic) with AtbC, seeded with 106 L. major promastigotes/mL. Blood-fed females were separated, kept under the same conditions described above, and dissected at different time intervals (indicated in figure legends). Guts (without Malpighian tubules) and carcasses (i.e., all other tissues) were dissected in sterile saline solution, collected in pools of 10, and stored at −80°C until processing.
9
Maintenance of Leishmania donovani GR374
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Leishmania donovani strain GR374 (MHOM/ET/2010/DM-1033) was maintained at 23°C on Medium 199 (Sigma) supplemented with 10% foetal calf serum (Gibco), 1% BME vitamins (Sigma), 2% human urine and amikin (250 μg/ml).
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Leishmania donovani strain GR374 (MHOM/ET/2010/DM-1033) was maintained at 23°C on Medium 199 (Sigma) supplemented with 10% foetal calf serum (Gibco), 1% BME vitamins (Sigma), 2% human urine and amikin (250 μg/ml).
10
Fluorescent Leishmania Parasite Growth Curves
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Established laboratory colonies of Lu. longipalpis (from Jacobina, Brazil) and Lu. migonei (from Baturité, Brazil) were used and maintained under standard conditions, as previously described19 (link). A fluorescent strains of L. (V.) braziliensis (XB29 marked with GFP) and L. (L.) infantum (OGVL marked with mCherry) by stable integration of mCherry into the 18 S rRNA locus as previously described20 (link). We have used parasites within less than 10 passages in vitro and were maintained at 23 °C on Medium 199 (Sigma-Aldrich, USA) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, USA), 1% BME vitamins (Sigma-Aldrich), 2% human urine and 250 μg/ml amikin (Bristol-Myers Squibb, USA).
For study of growth curves in-vitro, two initial doses were used: 104 promastigotes/ml and 105 promastigotes/ml. The concentration of parasites was analyzed daily for 7 days, using counting in Burker chamber. The experiments were repeated twice.
For study of growth curves in-vitro, two initial doses were used: 104 promastigotes/ml and 105 promastigotes/ml. The concentration of parasites was analyzed daily for 7 days, using counting in Burker chamber. The experiments were repeated twice.
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