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PMC:7795856 / 23329-34206 JSONTXT

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LitCovid-PubTator

Id Subject Object Predicate Lexical cue tao:has_database_id
413 132-135 Gene denotes Tα1 Gene:134864
414 400-403 Gene denotes Tα1 Gene:134864
415 1132-1135 Gene denotes Tα1 Gene:134864
416 1251-1254 Gene denotes Tα1 Gene:134864
417 1294-1297 Gene denotes Tα1 Gene:134864
418 126-131 Species denotes human Tax:9606
419 176-183 Species denotes E. coli Tax:562
420 394-399 Species denotes human Tax:9606
421 442-449 Species denotes E. coli Tax:562
422 1270-1284 Species denotes synthetic gene Tax:2005392
423 1288-1293 Species denotes human Tax:9606
424 558-565 Chemical denotes alanine MESH:D000409
425 838-850 Chemical denotes tetracycline MESH:D013752
431 1874-1877 Gene denotes Tα1 Gene:134864
432 1712-1719 Species denotes E. coli Tax:562
433 1952-1962 Chemical denotes ampicillin MESH:D000667
434 1964-1969 Chemical denotes TBamp MESH:C064881
435 2244-2249 Chemical denotes TBamp MESH:C064881
465 3319-3322 Gene denotes Tα1 Gene:134864
466 3334-3337 Gene denotes Tα1 Gene:134864
467 3509-3512 Gene denotes Tα1 Gene:134864
468 3763-3766 Gene denotes Tα1 Gene:134864
469 4032-4035 Gene denotes Tα1 Gene:134864
470 4142-4145 Gene denotes Tα1 Gene:134864
471 2576-2587 Chemical denotes citric acid MESH:D019343
472 2851-2867 Chemical denotes ammonium sulfate MESH:D000645
473 3006-3016 Chemical denotes AEX buffer
474 3018-3022 Chemical denotes sAEX
475 3030-3038 Chemical denotes Tris/HCl
476 3045-3049 Chemical denotes EDTA MESH:D004492
477 3127-3131 Chemical denotes sAEX
478 3454-3465 Chemical denotes sAEX buffer
479 3579-3589 Chemical denotes CEX buffer
480 3597-3607 Chemical denotes Na-citrate
481 3676-3693 Chemical denotes TOYOPEARL Sulfate
482 3747-3750 Chemical denotes CEX
483 3842-3846 Chemical denotes NaCl MESH:D012965
484 4098-4102 Chemical denotes NaCl MESH:D012965
485 4208-4218 Chemical denotes AEX buffer
486 4226-4238 Chemical denotes ethanolamine MESH:D019856
487 4239-4242 Chemical denotes HCl MESH:D006851
488 4387-4397 Chemical denotes AEX buffer
489 4460-4464 Chemical denotes NaCl MESH:D012965
490 4564-4572 Chemical denotes peptides MESH:D010455
491 4624-4629 Chemical denotes water MESH:D014867
492 2597-2610 Disease denotes EmulsiFlex C5 MESH:C537005
493 4277-4299 Disease denotes mL Fractogel® EMD TMAE MESH:C562593
501 4974-4977 Gene denotes Tα1 Gene:134864
502 4985-4988 Gene denotes Tα1 Gene:134864
503 5211-5222 Gene denotes apoferritin Gene:2495
504 5269-5280 Gene denotes transferrin Gene:7018
505 5185-5198 Gene denotes thyroglobulin Gene:7038
506 5160-5173 Gene denotes thyroglobulin Gene:7038
507 4771-4779 Chemical denotes Superose
510 5463-5468 Chemical denotes water MESH:D014867
511 5470-5495 Disease denotes Veolia Water Technologies MESH:D000069578
522 6365-6385 Gene denotes alkaline phosphatase Gene:16870
523 6565-6585 Gene denotes alkaline phosphatase Gene:16870
524 6387-6389 Gene denotes AP Gene:16870
525 6341-6346 Species denotes mouse Tax:10090
526 5841-5849 Chemical denotes Bis–Tris MESH:C026272
527 5901-5937 Chemical denotes 3-(N-morpholino)propanesulfonic acid MESH:C008550
528 5939-5943 Chemical denotes MOPS MESH:C008550
529 6146-6154 Chemical denotes Tween 20 MESH:D011136
530 6034-6053 Disease denotes dry blotting system MESH:D034721
531 6283-6288 Disease denotes PBS/T MESH:D011535
541 6774-6777 Gene denotes Tα1 Gene:134864
542 6785-6788 Gene denotes Tα1 Gene:134864
543 6873-6878 Gene denotes RPC 1 Gene:11128
544 6812-6824 Chemical denotes acetonitrile MESH:C032159
545 6835-6846 Chemical denotes formic acid MESH:C030544
546 6930-6942 Chemical denotes acetonitrile MESH:C032159
547 6953-6964 Chemical denotes formic acid MESH:C030544
548 7037-7049 Chemical denotes acetonitrile MESH:C032159
549 7060-7071 Chemical denotes formic acid MESH:C030544
553 7787-7790 Gene denotes Tα1 Gene:50719
554 7502-7513 Species denotes Wistar rats Tax:10116
555 7871-7874 Species denotes rat Tax:10116
557 8507-8510 Gene denotes Tα1 Gene:134864
563 8569-8572 Gene denotes Tα1 Gene:134864
564 8754-8757 Gene denotes Tα1 Gene:134864
565 8869-8872 Gene denotes Tα1 Gene:134864
566 8783-8788 Chemical denotes water MESH:D014867
567 8836-8841 Chemical denotes water MESH:D014867
580 9694-9707 Gene denotes serum albumin Gene:213
581 10059-10062 Gene denotes Tα1 Gene:50719
582 10210-10213 Gene denotes Tα1 Gene:50719
583 10335-10355 Gene denotes alkaline phosphatase Gene:16870
584 10381-10401 Gene denotes alkaline phosphatase Gene:16870
585 10778-10781 Gene denotes Tα1 Gene:50719
586 9790-9793 Species denotes rat Tax:10116
587 9949-9952 Species denotes rat Tax:10116
588 10085-10088 Species denotes rat Tax:10116
589 10804-10807 Species denotes rat Tax:10116
590 10550-10573 Chemical denotes p-nitrophenyl phosphate MESH:C008644
591 10269-10275 Disease denotes mL PBS MESH:D011535

LitCovid-sentences

Id Subject Object Predicate Lexical cue
T131 0-2 Sentence denotes 4.
T132 3-24 Sentence denotes Materials and Methods
T133 26-30 Sentence denotes 4.1.
T134 31-69 Sentence denotes Construction of the Expression Plasmid
T135 70-357 Sentence denotes A bicistronic operon for the simultaneous expression of human Tα1 (UniProt P06454, residues 2–29) and the E. coli N-acetyltransferase RimJ (UniProt P0A948) flanked by the restriction sites NdeI and HindIII was prepared using gene synthesis (Thermofisher Scientific, Regensburg, Germany).
T136 358-698 Sentence denotes To this end, the coding sequence of human Tα1 was codon-optimized for expression in E. coli and linked to the RimJ structural gene via the nucleotide sequence GCCTGAAGAGCAGAAAATAAA comprising a “GCC” alanine codon, the opal stop codon “TGA”, a SapI restriction site for insertion of the PAS gene cassette, and a ribosome-binding site (RBS).
T137 699-878 Sentence denotes The entire gene fragment was subcloned via NdeI and HindIII on a derivative of pASK75 [36] for cytoplasmic expression under control of the tetracycline promoter/operator (tetp/o).
T138 879-1169 Sentence denotes Subsequently, a substantially non-repetitive sequence-verified PAS gene cassette [34] encoding a PAS#1 polypeptide of 601 amino acids was inserted via the SapI restriction site to create the expression vector for the C-terminally PASylated thymosin α1 (Tα1-PAS), pASK75-Tα1-PAS#1(600)/RimJ.
T139 1170-1432 Sentence denotes To construct a plasmid for the bacterial production of an N-terminally PASylated Tα1 (PAS-Tα1), the synthetic gene of human Tα1 was amplified via PCR using the primers THY-For (5’-AGCTCTTCTGCCAGTGATGCAGCAGTTGATACC) and THY-Rev (5’-GCTCAAGCTTAGTTCTCGGCTTCTTCCAC).
T140 1433-1635 Sentence denotes The PCR product was digested with SapI and HindIII and inserted via these restriction sites downstream of the PAS#1(600) sequence preceded by Met and Pro encoded on a suitable pASK75 plasmid derivative.
T141 1637-1641 Sentence denotes 4.2.
T142 1642-1691 Sentence denotes Bacterial Production of Recombinant PASylated Tα1
T143 1692-1889 Sentence denotes The BL21 derivative E. coli strain NEBexpress® (New England Biolabs, Frankfurt am Main, Germany) was transformed with the plasmids pASK75-Tα1-PAS#1(600)/RimJ or pASK75-MP-PAS#1(600)-Tα1 from above.
T144 1890-2045 Sentence denotes Two milliliters of Terrific Broth supplemented with 100 µg/mL ampicillin (TBamp) was inoculated with a single colony and grown overnight at 37 °C, 170 rpm.
T145 2046-2183 Sentence denotes This overnight culture was used to inoculate a 50 mL preculture, which was grown under the same conditions until OD550 = 1.1 was reached.
T146 2184-2308 Sentence denotes Subsequently, the 50 mL preculture was transferred into 2 L TBamp and incubated in a 5 L baffled flask at 37 °C and 100 rpm.
T147 2309-2457 Sentence denotes After reaching OD550 = 0.6, the temperature was decreased to 26 °C and the cells were subsequently induced at OD550 = 1 with 200 µg/mL aTc for 15 h.
T148 2459-2463 Sentence denotes 4.3.
T149 2464-2493 Sentence denotes Purification of PASylated Tα1
T150 2494-2657 Sentence denotes Bacteria were harvested by centrifugation and disrupted in the presence of 100 mM citric acid using an EmulsiFlex C5 cell homogenizer (Avestin, Mannheim, Germany).
T151 2658-2813 Sentence denotes After centrifugation for 20 min (39,200× g) at 16 °C, the supernatant was filter-sterilized using a 0.45 µm syringe filter (Sartorius, Göttingen, Germany).
T152 2814-2958 Sentence denotes The clear filtrate was adjusted with ammonium sulfate to 30% saturation, stirred for 30 min at RT, and centrifuged for 30 min (39,200× g) at RT.
T153 2959-3147 Sentence denotes The precipitate was solubilized in subtractive AEX buffer (sAEX; 20 mM Tris/HCl, 1 mM EDTA, pH 8.5), filter-sterilized, and dialyzed twice against a 100-fold volume of sAEX buffer at 4 °C.
T154 3148-3297 Sentence denotes Subsequent chromatography steps were performed on an ÄKTA Explorer 100 system (GE Healthcare, Freiburg, Germany) operated at a flow rate of 5 mL/min.
T155 3298-3466 Sentence denotes The dialyzed sample (Tα1-PAS or PAS-Tα1) was first applied to a 5 mL TOYOPEARL NH2-750F column (Tosoh Bioscience, Griesheim, Germany) equilibrated with the sAEX buffer.
T156 3467-3625 Sentence denotes The flow-through containing the PASylated Tα1 was collected and dialyzed twice against a 100-fold volume of the CEX buffer (20 mM Na-citrate, pH 3.0) at 4 °C.
T157 3626-3758 Sentence denotes The protein solution was then applied to an 85 mL TOYOPEARL Sulfate-650F column (Tosoh Bioscience) equilibrated with the CEX buffer.
T158 3759-3903 Sentence denotes PAS-Tα1 quantitatively bound to the column and the pure protein was eluted using a NaCl concentration gradient (0–250 mM over 2 column volumes).
T159 3904-4126 Sentence denotes In the case of Tα1-PAS, the CEX flow-through fraction contained the N-terminally acetylated protein, whereas the non-acetylated Tα1-PAS stayed bound to the column and could again be eluted in a NaCl concentration gradient.
T160 4127-4398 Sentence denotes The acetylated Tα1-PAS fraction from the flow-through was dialyzed twice against AEX buffer (20 mM ethanolamine/HCl, pH 10) and then applied to a 100 mL Fractogel® EMD TMAE (M)strong anion exchanger (Merck Millipore, Burlington, MA, USA) equilibrated with the AEX buffer.
T161 4399-4539 Sentence denotes This time, highly pure acetylated Tα1-PAS was eluted using a NaCl concentration gradient (0–200 mM over 2 column volumes) in the AEX buffer.
T162 4540-4683 Sentence denotes Both purified PASylated peptides were dialyzed 5 times against a 200-fold volume of water, lyophilized, and stored at −20 °C until further use.
T163 4685-4689 Sentence denotes 4.4.
T164 4690-4730 Sentence denotes Analytical Size Exclusion Chromatography
T165 4731-4918 Sentence denotes Analytical SEC was performed on a 24 mL Superose® 6 10/300 GL column (GE Healthcare) using an ÄKTA Explorer 10 system operated at a flow rate of 0.5 mL/min with PBS as the running buffer.
T166 4919-5373 Sentence denotes To determine the apparent molecular size, purified PAS-Tα1 or PAS-Tα1 (100 µL) was applied to the column and the elution volume was used for linear interpolation from a half-logarithmic calibration line obtained using the reference proteins thyroglobulin (octamer), thyroglobulin (tetramer), apoferritin, β-amylase, alcohol dehydrogenase (tetramer), transferrin, ovalbumin, and carboanhydrase as well as blue dextran (all from Merck, Darmstadt, Germany).
T167 5375-5379 Sentence denotes 4.5.
T168 5380-5404 Sentence denotes Endotoxin Quantification
T169 5405-5748 Sentence denotes Tα1-PAS samples were diluted to 1 mg/mL in endotoxin-free water (Veolia Water Technologies, Celle, Germany), heated to 90 °C for 5 min, and, after cooling down to RT, bacterial endotoxins were quantified using an Endosafe® system (Charles River Laboratories, Wilmington, MA, USA) with an FDA-licensed PTS™ cartridge (10–0.1 EU/mL sensitivity).
T170 5750-5754 Sentence denotes 4.6.
T171 5755-5776 Sentence denotes Western Blot Analysis
T172 5777-6087 Sentence denotes The whole cell protein extract was applied to a 4–12% SurePAGE™ Bis–Tris gradient gel (Genscript, Piscataway, NJ, USA) with 3-(N-morpholino)propanesulfonic acid (MOPS) as running buffer and electrotransferred onto a nitrocellulose membrane using an iBlot 1 dry blotting system (ThermoFisher, Waltham, MA, USA).
T173 6088-6245 Sentence denotes After washing 3 times with PBS supplemented with 0.1% v/v Tween 20 (PBS/T), the membrane was incubated with 1 µg/mL anti-PAS antibody in PBS/T for 1 h at RT.
T174 6246-6417 Sentence denotes The membrane was washed 3 times with PBS/T and incubated with a 1:5000 dilution of a goat anti-mouse IgG (Fc-specific) alkaline phosphatase (AP) conjugate (Merck) for 1 h.
T175 6418-6644 Sentence denotes After washing twice with PBS/T and twice with PBS, the blot was developed by a chromogenic reaction using BCIP (37.5 µg/mL) and NBT (150 µg/mL) in alkaline phosphatase buffer (100 mM Tris/HCl, pH 8.8, 100 mM NaCl, 5 mM MgCl2).
T176 6646-6650 Sentence denotes 4.7.
T177 6651-6711 Sentence denotes Reverse-Phase Chromatography (RPC) and ESI Mass Spectrometry
T178 6712-6965 Sentence denotes Directly after the CEX chromatography, a 200 µl sample of PAS-Tα1 or PAS-Tα1 was adjusted to 2% v/v acetonitrile, 0.1% v/v formic acid and applied to a Resource RPC 1 mL column (GE Healthcare) equilibrated with 2% v/v acetonitrile, 0.1% v/v formic acid.
T179 6966-7188 Sentence denotes The protein was eluted using a concentration gradient of up to 80% v/v acetonitrile, 0.1% v/v formic acid over 20 column volumes at a flow rate of 2 mL/min while spectrophotometrically monitoring protein elution at 225 nm.
T180 7189-7367 Sentence denotes The eluted protein fraction was directly injected into a maXis quadrupole time of flight (Q-TOF) instrument (Bruker Daltonics, Bremen, Germany) operated in the positive ion mode.
T181 7368-7441 Sentence denotes Raw data was deconvoluted using the MaxEntX algorithm (Bruker Daltonics).
T182 7443-7447 Sentence denotes 4.8.
T183 7448-7480 Sentence denotes Pharmacokinetic Analysis in Rats
T184 7481-7620 Sentence denotes A PK study in female Wistar rats at 8–9 weeks of age was conducted at the Aurigon Toxicological Research Center (ATRC, Dunakeszi, Hungary).
T185 7621-7762 Sentence denotes Up to 3 animals per cage were housed in a controlled environment at 22 ± 3 °C with a relative humidity of 50 ± 20%, 12 h light and 12 h dark.
T186 7763-7887 Sentence denotes Endotoxin-free purified Tα1-PAS (3.4 mg/kg) was administered subcutaneously via a single injection into the rat dorsal area.
T187 7888-7965 Sentence denotes Blood samples (100 µL) were taken from 5 animals each at various time points.
T188 7966-8225 Sentence denotes Following collection in tubes containing tri-potassium ethylenediaminetetraacetic acid (K3-EDTA) (Greiner Bio-One, Frickenhausen, Germany), samples were centrifuged at room temperature for 10 min (3000× g) and the resulting plasma was stored at −15 to −30 °C.
T189 8226-8473 Sentence denotes Tα1-PAS in these samples was quantified using sandwich ELISA (see below) and the data were analyzed using the Phoenix WinNonlin 6.3 software (Certara, Princeton, NJ, USA) using a one-compartment model assuming 1st order absorption and elimination.
T190 8475-8479 Sentence denotes 4.9.
T191 8480-8514 Sentence denotes Quantification of Purified Tα1-PAS
T192 8515-8605 Sentence denotes Due to the absence of aromatic side chains, PASylated Tα1 does not absorb light at 280 nm.
T193 8606-8728 Sentence denotes Thus, for reliable quantification via the peptide backbone absorption, an extinction coefficient at 225 nm was determined.
T194 8729-8842 Sentence denotes To this end, highly pure Tα1-PAS was lyophilized from water, weighed, and dissolved in a defined volume of water.
T195 8843-9077 Sentence denotes The concentration of this Tα1-PAS solution was additionally quantified by measuring the backbone peptide groups using UV absorption at 205 nm using a generic calculated extinction coefficient [75], which led to a fair agreement (±6%).
T196 9078-9231 Sentence denotes The absorbance at 225 nm was measured for various dilutions of the gravimetrically prepared peptide stock solution and plotted against the concentration.
T197 9232-9339 Sentence denotes The linear range was fitted by a straight line, resulting in an extinction coefficient of 253,057 M−1⋅cm−1.
T198 9340-9444 Sentence denotes While most buffers strongly absorb light at 205 nm, such a spectral overlap is usually absent at 225 nm.
T199 9446-9451 Sentence denotes 4.10.
T200 9452-9489 Sentence denotes ELISA Quantification of PASylated Tα1
T201 9490-9610 Sentence denotes A 96-well Nunc Maxisorb ELISA plate (ThermoFisher) was coated with 100 µg/mL anti-PAS antibody in PBS at 4 °C overnight.
T202 9611-9751 Sentence denotes After washing twice with PBS/T, free binding sites were blocked with 3% w/v bovine serum albumin (BSA) in PBS/T at room temperature for 1 h.
T203 9752-9974 Sentence denotes After washing 3 times with PBS/T, the rat plasma samples were applied in dilution series in PBS/T supplemented with 0.5% (v/v) plasma from an untreated animal (to maintain a constant proportion of rat plasma constituents).
T204 9975-10123 Sentence denotes In the same manner, a standard curve was prepared using dilution series of purified Tα1-PAS (used for spiking rat plasma) at defined concentrations.
T205 10124-10193 Sentence denotes After incubation for 1 h at RT, wells were washed 3 times with PBS/T.
T206 10194-10456 Sentence denotes To detect bound Tα1-PAS, wells were incubated for 1 h with 50 µl of a 1 µg/mL PBS/T solution of the second anti-PAS antibody conjugated with alkaline phosphatase using the Lightning-Link alkaline phosphatase antibody labeling kit (BioTechne, Wiesbaden, Germany).
T207 10457-10586 Sentence denotes After washing twice with PBS/T and twice with PBS, the enzymatic activity was detected using p-nitrophenyl phosphate (0.5 mg/mL).
T208 10587-10877 Sentence denotes To this end, the plate was incubated for 20 min at 30 °C, the absorbance was measured at 405 nm using a SpectraMax M5e microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA) and the Tα1-PAS concentrations in rat plasma samples were quantified by comparison with the standard curve.