The structure of mammalian V-ATPase with mEAK-7 shows how a TLDc domain-containing protein can bind the proton pump to form an activity-sensitive interaction. V-ATPases are rotary proton pumps that serve as signaling hubs with numerous protein binding partners. CryoEM with exhaustive focused classification allowed detection of endogenous proteins associated with porcine kidney V-ATPase. An extra C subunit was found in ∼3% of complexes, whereas ∼1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 showed that mEAK-7’s TLDc domain interacts with V-ATPase’s stator, whereas its C-terminal α helix binds V-ATPase’s rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike the yeast TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that the mEAK-7:V-ATPase interaction is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. These results show that V-ATPase binding by TLDc domain proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzyme’s activity. ATP hydrolysis in the A3B3 subcomplex of the V1 region induces rotation of the rotor subcomplex, which contains subunits D, F, d, and a ring of membrane-embedded c subunits  are large, membrane-embedded protein complexes that function as proton pumps in eukaryotic cells. V-ATPase activity is essential for acidification of numerous intracellular compartments including endosomes, lysosomes, and secretory vesicles . In somesubunits . In the the ring . Rotatiothe ring , station regions , with su1 region  and subuociation .V-ATPase–driven acidification is necessary for targeting and post-translational modification of proteins in the Golgi , degradaV-ATPases interact with other proteins in the cell . In partAnalysis of V-ATPase from kidney tissue identified ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27, and nine subunits of the CCT complex as V-ATPase associated proteins . Two of 1 and VO regions .To identify low abundance complexes between mammalian V-ATPase and its binding partners, we first determined the subunit composition and structure of V-ATPase prepared from porcine kidney. We then subjected images of this complex to an exhaustive 3D classification strategy designed to detect low-population structures. This procedure revealed a small population of V-ATPase complexes with super-stoichiometric occupancy of the C subunit, and a second small population of complexes with an additional density bound to the catalytic Aignaling  and an mmEAK-7 has been proposed to activate the mTOR pathway but was not previously suggested to interact with V-ATPase , 2019. T1 region, and subunits a1, c, c″, d1, e2, RNAseK, ATP6AP1/Ac45, and ATP6AP2/PRR in the VO region Fig 1A). O region . AlthougO region . DespiteO region , the speMass spectrometry analysis of trypsin digested V-ATPase sample.Table S1 Mass spectrometry quantification of V-ATPase subunits.Table S2 left and 1 regions at 3.6–4.0 Å resolution and the VO regions at 3.7–5.8 Å resolution . This density appears to correspond to an additional copy of subunit C . This density abuts subunit B, subunit A, and peripheral stalk 3. The shape of the density does not correspond to any of the known subunits of V-ATPase. It is not clear if the fraction of particle images with this additional density represents the true fraction of V-ATPase complexes in cells with this protein bound, or if the interaction is more common but is disrupted during purification of V-ATPase. The list of proteins identified by mass spectrometry of the preparation (Table S1) was inspected to identify candidate proteins that could explain the density. After excluding V-ATPase subunits, the most abundant ∼50-kD proteins based on iBAQ scores (intensity-based absolute quantification) were the α and β subunits of mitochondrial ATP synthase followed by mEAK-7. Structures are known for both subunit α and β from ATP synthase , which regulates dauer formation in nematodes , followed by a region resembling an EF-hand domain (cyan), a TLDc domain , and a C-terminal α helix that is separated from the rest of the protein by an extended linker region (red)  . The N tleft) as well as a B1 subunit (right), facilitated mainly by hydrophobic interactions. This α helix is formed from sequence in the protein that was previously proposed to bind mTOR . Binding involving subunit A occurs through apparent ionic interactions between Thr354 and Gln317 from mEAK-7 with backbone atoms from residues Arg553 and Thr557 from subunit A, respectively, and hydrophobic interactions between Pro316 from mEAK-7 and Ala559 from subunit A (middle). Residues Gly276 and His277 from the TLDc domain of mEAK-7 interact with backbone atoms from Phe481 and Pro482 of subunit B1 (right). Together the TLDc domain and C-terminal α helix form a pincer-like grip around B1  confirmed the lysosomal localization of the protein, as shown previously (lower). Endogenous mEAK-7 expression in HEK293T cells is nearly undetectable . Whereas 3D classification identified a population comprising ∼25% of particle images where the density for mEAK-7’s N-terminal domain and TLDc domain are weak and poorly resolved, suggesting that they are loosely attached via the TLDc domain, density for mEAK-7’s C-terminal α helix remains strong and well resolved even in this 3D class . In contrast, when the C-terminal α helix is truncated most V-ATPase complexes in rotational State 2 do not bind mEAK-7 (upper middle). Only ∼22% of particle images showed any density for the N-terminal domain and TLDc domain of mEAK-7 when the C-terminal α helix was truncated, and even this density was weak and poorly resolved (lower middle). These experiments indicate that mEAK-7’s C-terminal α helix is important for the tight binding of V-ATPase by mEAK-7.To understand the relative importance of mEAK-7’s TLDc domain and C-terminal α helix for V-ATPase binding, we prepared a construct of mEAK-7 with the C-terminal α helix removed by truncation at residue Ser415. This construct was expressed heterologously in is state , left. Wupper left). In contrast, with ATP added a population of particles appeared with mEAK-7 entirely absent in rotational State 2 (upper right). In this state, even density for the C-terminal α helix is missing . Thus, it appears that rotation of the rotor, driven by ATP hydrolysis can result in the displacement of mEAK-7 from V-ATPase and disruption of the cross-link between the enzyme’s rotor and stator. Despite several biochemical experiments, we were unable to demonstrate that mEAK-7 can be fully dissociated from V-ATPase by the addition of ATP to the complex, including experiments where we washed immobilized V-ATPase:mEAK-7 extensively with a buffer containing ATP before elution of V-ATPase. These experiments suggest that mEAK-7 can remain associated to V-ATPase, either by its TLDc domain or its C-terminal α helix, during ATP hydrolysis.The observation that mEAK-7, which crosslinks the rotor and stator of V-ATPase in the structure, does not affect the enzyme’s activity in vitro or in cells suggests that the crosslink is broken during rotary catalysis. To determine the effect of ATP hydrolysis on mEAK-7 crosslinking of the rotor and the stator, V-ATPase was mixed with a ∼20× molar excess of mEAK-7, ATP was added to 10 mM and mixed, and cryoEM grids were frozen within 5 s. Given the concentration of V-ATPase (20 mg/ml) and the enzyme’s specific ATPase activity (2.9 ± 0.72 μmol ATP/min/mg), these conditions ensure that the grids were frozen before the supply of ATP was consumed. In the absence of ATP, it was impossible to find a 3D class in rotational State 2 that lacked density for mEAK-7 completely and density for mEAK-7’s C-terminal α helix was always clear , an article was published showing that the yeast TLDc protein Oxr1p binds to the yeast V1 complex, releasing subunit H and promoting dissociation of V1 from VO (1 complex (1:Oxr1p is inhibited even though subunit H is absent (left) and mEAK-7 (middle) shows notable differences in their interactions with V-ATPase: Oxr1p binds the enzyme in rotational State 1, whereas mEAK-7 binds it in rotational State 2. Furthermore, Oxr1p attaches to peripheral stalk 2, whereas mEAK-7 attaches to peripheral stalk 3. Most notably, Oxr1p binding forces the peripheral stalk 2 into a highly strained conformation (left) that is not seen in any of the peripheral stalks in V-ATPase with mEAK-7 (center). This strained peripheral stalk explains why Oxr1p binding promotes dissociation of V1 from VO, whereas mEAK-7 binding does not. Recent structures of yeast V1 and V1∆C complexes show a sharp bending of peripheral stalk 2 induced by interaction with subunit H (right). The interaction of subunit H with peripheral stalk 2 and the resulting bending of the peripheral stalk was proposed to be responsible for inhibiting ATP hydrolysis of V1, as disruption of the interaction prevents subunit H from inhibiting ATP hydrolysis  was added to the membrane fraction at 8 ml/g and resuspended with a Dounce homogenizer. Approximately 6 g of membranes were used for each preparation. The detergent glycol diosgenin (GDN) was added to 1% (wt/vol) and incubated with gentle mixing overnight. After solubilization, V-ATPase purification and assay was performed with no added lipids at 37°C as described previously . All subsequent steps were performed at 4°C. Each kidney was dissected to separate the softer cortex and medulla tissue from the more rigid pelvis tissue. The cortex and medulla tissue were blended with homogenization buffer for 1 min in a total volume of ∼500 ml for four kidneys at high speed followed by a 10-s pause and an additional 1 min of blending. Cell debris was removed by centrifugation at 800eviously , except eviously . BrieflySus scrofa (UniProt ID: A0A4X1T484) was synthesized in a pET28a(+) vector with an N-terminal 6× His tag (GenScript). The codon optimized sequence for mEAK-7 was:Recombinant mEAK-7 from GGGAATTCAAAAAGTAGGTCTGGACAAGGTCTTTGCAGCCGTTTCCTGCCGGAGGAACAAGCGGAAGTGGACGGTCTGTTCGATGCACTCAGCTCTGAGAAGCTGTCCTCACGGACCAGCCCGCGTAGCTTTAGCCTGCAAGCGCTGAAATCTCACGTAGGCGAAGCGTTGCCACCGGAGATGGTTACGCGTCTGTTCGAGGGTATGCGTCGTGCGGATCCGACAGGCAAGGCGACCGGTCCGAGCGCACGCATCAGCCAGGAGCAATTTACCTTGAGTATGAGCCATTTGCTGCGCGGCAGCAGCGAGGAAAAATCCTTGGTTATTCTGGCAATGGCTGCCGCTACCGATGGCCCAGCGGAAGCCCGTGAGGTTCTGCGCTTCACGGAAGACCTGGTGGGCAGCGTCGTGCATGTCTTACACTACCGCCAAGAGCTGCGCGGTTGGACCCAGAAACAGGCCTCTGGTTCCCCGCCTCGTGTTCAGGCGTTGGCGGCACAATTATTCTCCGAGCTGAAGCTGCAGGACGGCGAGAAGCTGCCGGGTCCGCAGCGTCTGGACTGCGATTGTGATCGTGCAGTCGTGGAAGCGTGGCTGTTCCGCGCTCCGCATGTTGCAACCTTTCTGTCCGTGGTGATTCATCAGGGGTTTCGCTTGCTGCGCTCCAGCCTGGACTTGGCGACTCTGCTGCCAGAACGTCAAGTTGACCGAGGCCGTGAATTTGCGTCGCTGCTGGACGTGCTGAGCGTTGCCTATATCAACAGTCACCTGCCGCGTGATCTCCGTCATCGTTGGCGTCTCCTTTTCGCCACGGCTCTGCACGGTCACAGCTTTGCTCAATTGTGCGGTCGTATCACCCAGCGCGGCCCGTGCGTGGTGCTGTTGGAAGATCAGGATGGCCACGTTTTTGGCGGCTTCGCGTCTTGTAGCTGGGAAGTGAAACCGCAGTTTCAGGGTGACAGCAAGTGCTTTCTTTTCTCGATCTGCCCGGCTATGGCGGTTTACACCTGCACCGGGTATAACGATCATTACATGTATCTGAATCATGGTCAGCAGACCATTCCGAATGGTCTGGGTATGGGTGGTCAACACAACTACTTCGGTCTGTGGGTTGACGTTGATTTTGGTAAAGGTCACTCCAAAGCAAAACCGACCTGTACCACGTACAGCAGCCCACAACTGTCGGCTCAAGAGGACTTCCGCTTCGAAAAGATGGAAGTTTGGGCAGTGGGCGACCCGTCTGTCACTCAACCGGCGAAAAGCAGCAAGTCCATCCTGGACGGCGACCCGGAGGCGCAAATTCTGCTTGAGGCGAGCGGCAGAAGCCGTCACTCCGAAGGTTTGCGTGCGGTGCCGGAGGACGATTAA.−1 kanamycin (Sigma-Aldrich) and grown overnight at 37°C. A transformed colony was used to inoculate a starter culture containing 10 ml of 2xYT medium supplemented with 50 μg ml−1 kanamycin. The starter culture was grown overnight at 37°C with shaking at 240 rpm, and 10 ml of this culture was used to inoculate 1 liter of 2xTY medium supplemented with 50 μg ml−1 kanamycin. The culture was grown at 37°C with shaking at 240 rpm to an OD of ∼0.8, with the temperature then reduced to 7°C and protein expression was induced overnight by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fisher). All further steps were performed at 4°C. Cells were harvested by centrifugation at 4,000g for 15 min and the cell pellet was resuspended in PBS and centrifuged again at 4,000g for 30 min. The cell pellet was then resuspended in lysis buffer  at ∼8 ml/g of wet cell pellet. The resuspended cells were lysed by sonication (Q Sonica Q500) with 2 s on, 2 s off at 30% amplitude for 10 min, and cell debris was removed by centrifugation at 130,000g for 45 min with a Type 70 Ti Rotor (Beckman Coulter). The supernatant was filtered through a 0.22 μm filter (Sigma-Aldrich) and loaded onto a HisTrap HP 5 ml column previously equilibrated with HisTrap buffer . The column was washed with 10 column volumes (CV) of HisTrap buffer followed by elution with five CV of HisTrap buffer with imidazole added to 300 mM. Eluted protein was concentrated with a 10-kD MWCO concentrator (Amicon) to ∼500 μl before two rounds of gel filtration, first with a Superose 6 Increase 10/300 column and then a Superdex 75 10/300 column. The purified protein was pooled, flash frozen, and stored at −80°C for subsequent use.ArcticExpress competent cells (Agilent) were transformed with the plasmid and plated onto Luria broth (LB) agar supplemented with 50 μg ml2 at 50 mM (0.4 μl) was applied on the grid, and immediately before plunge freezing, 1.6 μl of V-ATPase:mEAK-7 mixture was added and mixed by pipetting for 2 s. This procedure results in a final concentration of 10 mM ATP and requires a total of 5 s from mixing to plunge freezing.Purified V-ATPase was concentrated to ∼20 mg/ml and 1.2 μl was applied onto the copper side of nanofabricated gold grids  in the e−/Å2, were collected. For V-ATPase with recombinant mEAK-7, mEAK-7ΔCterm, mEAK-7 plus ATP, and mEAK-7 plus EDTA with EGTA, cryoEM data were acquired with the same electron microscope. However, for these datasets, a lower magnification was used with a calibrated pixel size of 1.3225 Å/pixel, and 5,142, 2,013, 5,498, and 2,468 movies consisting of 29 exposure fractions each with total exposures of 36, 37, 44, and 41 e−/Å2, respectively, were collected. For the V-ATPase with mEAK-7 and calcium, a Thermo Fisher Scientific Glacios electron microscope and Falcon 4 camera with Selectris X energy filter was used. The slit width was 10 eV, and aberration-free image shift was used during data collection. The calibrated pixel size was 0.895 Å/pixel and 3,209 movies with 1,288 frames in EER mode .For V-ATPase alone, movies were aligned with UCSF MotionCor2  through elion v3  and proceference . 2D claseference . This coolishing  and tranRelion signal subtraction and focused classification strategy  and was applied to the map. Map-to-model FSC was calculated with proc3d in EMAN  (All raw (WIFF and WIFF.SCAN) files were saved in ProHits . mzXML fiz.org/) .5 cells/ml and were transfected with FuGENE 6 (Promega) transfection reagent 24 h later. RAW264.7 cells were seeded on 18 mm glass coverslips at a density of 5 × 105 cells/ml and transfected 24 h later with FuGENE HD. Cells were transfected with 3 μl of FuGENE per 1 μg of total plasmid. Both cell types were imaged 18–24 h post-transfection.The sequence for mEAK-7 was amplified from the pET28a(+) vector from GenScript for Gibson assembly (New England Biolabs), cloning into the pmCherry-N1 (Clonetech) expression vector for mammalian cells with the forward primer GTCGCCACCATGgggaattcaaaaagtaggtctggacaaggtctttgcagccgtttc and the reverse primer CTCGCCCTTGCTCACatcgtcctccggcaccgcacgcaaaccttcggagtgacggcttct, which maintains the C-terminal mCherry tag. The reverse primer CTCGCCCTTGCTCACttaatcgtcctccggcaccgcacgcaaaccttcggagtgacggct was used to create a plasmid with a stop codon before the mCherry tag for expression of mEAK-7 without the tag. HeLa and HEK293T cells were seeded on 18 mm glass coverslips at a density of 3 × 10To visualize lysosomal compartments, HeLa cells transiently expressing mEAK-7-mCherry were incubated with 250 μg/ml fluorescein isothiocyanate (FITC)-conjugated 10 kD-dextran at the time of transfection and chased with complete medium 1 h before imaging. HEK293T cells were transiently transfected with mEAK7-mCherry and LAMP1-GFP to label the lysosomes. Coverslips with cells were mounted in a Chamlide magnetic chamber, incubated at 37°C in HBSS medium, and visualized with a Zeiss Axiovert 200 M confocal microscope operated by Volocity v6.3 software. Images were acquired using a 63×/1.4 NA oil objective (Zeiss) with an additional 1.5× magnifying lens and processed using Volocity v6.3 and Adobe Illustrator.+ solutions  at different pH values , containing 10 μM nigericin and 5 μM monensin. Cells were imaged 5 min after the addition of each solution to obtain the 490 nm/440 nm fluorescence ratio corresponding to each pH standard. A calibration curve was prepared from a least-squares fit of mean background-subtracted fluorescence ratios as a function of pH. The pH measurements were compared with an unpaired two-tailed t test.To measure pH of lysosomes, HEK293T cells transiently transfected with unlabelled mEAK-7 and PLCδ-PH-RFP  were incubated with 250 μg/ml FITC-conjugated 10-kD dextran at the time of transfection and chased with complete medium 1 h before imaging to visualize lysosomal compartments. RAW264.7 cells co-transfected with unlabelled mEAK-7 and PLCδ-PH-RFP were incubated with FITC-conjugated zymosan particles, which were taken up into phagosomes, 1 h before imaging and were chased with complete medium 30 min before imaging to wash away unbound particles. Steady-state lysosomal and phagosomal pH were determined by exciting FITC labelled lysosome/phagosomes at 481 ± 15 nm and 436 ± 20 nm, respectively, and collecting emitted light at 520 ± 35 nm. The pH-dependent fluorescence intensity of FITC when excited at ∼490 nm was used as described below to determine the steady-state pH of lysosomes. The relatively pH-insensitive fluorescence intensity of FITC when excited at ∼440 nm was used to control for photobleaching during image acquisition. Multiple fields of cells were imaged for each condition, and the data were processed with Volocity v6.3. To convert fluorescence ratios to pH values, cells were sequentially subjected to isotonic KEMD-26385, EMD-26386, EMD-26387, and EMD-26388. Models have been deposited into the PDB with accession codes 7U8O, 7U8P, 7U8Q, and 7U8R. Mass spectrometry data for proteins that bind mEAK-7 were deposited into the ProteomeXchange with accession code PXD034953, through partner MassIVE with accession code MSV000089750.CryoEM maps have been deposited into the EMDB with accession codes


The second near-infrared (NIR-II) window is a fundamental modality for deep-tissue in vivo imaging. However, it is challenging to synthesize NIR-II probes with high quantum yields (QYs), good biocompatibility, satisfactory pharmacokinetics, and tunable biological properties. Conventional long-wavelength probes, such as inorganic probes  and organic dyes (which contain large π-conjugated groups), exhibit poor biosafety, low QYs, and/or uncontrollable pharmacokinetic properties. Herein, we present a bioengineering strategy that can replace the conventional chemical synthesis methods for generating NIR-II contrast agents. We use a genetic engineering technique to obtain a series of albumin fragments and recombinant proteins containing one or multiple domains that form covalent bonds with chloro-containing cyanine dyes. These albumin variants protect the inserted dyes and remarkably enhance their brightness. The albumin variants can also be genetically edited to develop size-tunable complexes with precisely tailored pharmacokinetics. The proteins can also be conjugated to biofunctional molecules without impacting the complexed dyes. This combination of albumin mutants and clinically-used cyanine dyes can help widen the clinical application prospects of NIR-II fluorophores. It is currently difficult to synthesise NIR-II probes with good quantum yields, biocompatibility and pharmacokinetics. Here the authors report a strategy to alter these properties by modifying the protein coatings with biofunctional molecules, and generate long-wavelength fluorophores for in vivo imaging. The development of high-quality NIR-II fluorophores is essential to realizing deep bioimaging11 and facilitating clinical translation. For example, clinically used cyanine dyes14 were discovered to possess a tail peak in the NIR-II window when viewed using an InGaAs camera20. However, the majority of organic NIR-II fluorophores exhibit very low fluorescence quantum yields (QYs)19 and/or poor pharmacokinetic properties. Moreover, multiple and complex synthetic steps are followed to synthesize these fluorophores22. The photoluminescence of small molecules is controlled by their energy gap, which depends on their chemical structure through π-conjugated groups23. Hence, long-wavelength fluorophores bear large π-conjugated groups 26. However, fluorophores with large π-conjugated groups usually have inherently low QYs and poor pharmacokinetics18.Near-infrared II (NIR-II) fluorescence imaging is preferrable to near-infrared I (NIR-I) imaging as this window provides reduced tissue scattering and autofluorescence, thus achieving deeper tissue penetration with superior contrast and resolution3. Additionally, large conjugated fluorophores are usually hydrophobic and do not exhibit satisfying pharmacokinetic properties22. In recent years, efforts have been made to synthesize rigid structures bearing electron-withdrawing groups at the center of the scaffold and/or to introduce shielding groups to reduce collisional quenching29. For example, rigid cyclohexanol structures have been introduced at the center of cyanine/polymethine dyes to produce improved dye variants. Researchers introduced a rigid alkyl thiophene moiety to increase the dihedral angle in a donor-acceptor-donor (D-A-D) dye to improve QYs19. Shielding units containing long polyethylene glycol (PEG) groups have also been used to develop good-performance D-A-D dyes and reduce collisional quenching. Some researchers have introduced dialkoxy-substituted benzene- and/or fluorene-based groups as the shielding units  to develop S-D-A-D-S fluorophores that exhibit relatively high QYs. By modifying the PEG and S groups, the dispersion of these molecules in solution can be improved and nonradiative decay caused by collisional quenching/π-π stacking can be reduced31.Large π-bridges can potentially induce nonradiative decay through vibrational relaxation, collisional quenching, and/or π-π stacking-induced quenching. Molecular simulation studies revealed that the low QYs exhibited by fluorophores containing large π-bridges could be attributed to the properties of the excited states of the π-bridges. The excited states can be directly attacked and subsequently quenched by water molecules and they are also prone to oxidation7 that can potentially influence the QY of the dyes. Moreover, some cyanine/polymethine dyes, such as the clinically used indocyanine green (ICG), do not possess modifiable units in their scaffolds and so cannot be easily conjugated to other moieties. Most importantly, achieving a good-performance fluorophore with high QY, biosafety, clinical translation potential, and satisfying pharmacokinetic properties as well as terrific biofunction is extremely challenging. We have previously reported that cyanine dyes can “hitchhike” with serum albumin in vivo to produce ultra-bright NIR-II fluorophores with improved pharmacokinetics16. The cyanine dyes inserted into a hydrophobic pocket of albumin, which stabilized the dyes and so reduced nonradiative decay and synergistically increased the extent of π-conjugation. The protein shell also protected the dyes from collisional quenching and provided biocompatibility. Inspired by this work, here we screen high-affinity recombinant subunits of albumin that could be used to generate a series of editable protein shells to chaperone dyes. Using yeast to express fragments of albumin, we successfully identify the exact binding domain for cyanine dyes. This allows us to re-engineer a series of minimal subunit-incorporating albumin variants. We use this genetic engineering technique to tune the size and brightness of cyanine/polymethine dye@protein complexes  to identify the exact binding domain for cyanine dyes. HSA consists of three independent subdomains that contain highly homologous sequences: domain I (DI), domain II (DII), and domain III (DIII)Kd = 0.57 nM) than HSA (Kd = 1.7 nM)  and slower dissociation (Koff = 0.1 s−1) with DIII compared to HSA   Fig. . IR-783 PBS Fig. . We furtPBS Fig. . The resThe binding behavior and loading capacity of cyanine dyes and albumin were further studied by ultra-high performance liquid chromatography–mass spectrometry (UHPLC-MS). Each DIII molecule bound only one IR-783 molecule . The results indicated that the dye-labeled peptide sequence was C(dye)C(carbamidomethylation)TESLVNR. The b and y ion series were further analyzed to identify the exact binding residue. The full mass (MS1) of the peptide was calculated to be 1770.749 m/z (measurement accuracy < 5 ppm). The measurement accuracy for the fragments was < 10 ppm . The results revealed that the Cys476 residue was the potential binding site  and Cl–C groups and to understand the reaction mechanism , prostate-specific membrane antigen-617 (PSMA-617) and cyclic Arg-Gly-Asp (cRGD), which specifically bind to somatostatin receptor (SSTR), PSMA and integrin αVβ3, respectively. SH-modified TATE, PSMA-617 and cRGD were first reacted with maleimide-NHS ester. Following this, the purified products were conjugated to DIII Fig. . The reaPichia pastoris with their 3D structures retained  in DIII that binds cyanine dyes, resulting in increased brightness. Using this information, the independent DIIIa and DIIIb subunits were expressed by recombinant ned Fig. 41–43. Thnts Fig. . To idennts Fig. . The brints Fig. . Thus, tSince DIIIa is a fragment of DIII with approximately half its molecule weight, we hypothesized that the rate of renal excretion of IR-783@DIIIa would be higher than that of IR-783@DIII. The pharmacokinetic properties of IR-783@DIIIa were evaluated in healthy mice Fig. . As expe32. The three domains of albumin  are homologous  of endogenous albumin if the DI, DII, or DIII units are substituted with each other. Hence, we re-engineered a recombinant HSA plasmid with 3 copies of the DIII sequence (triple DIII or TDIII)  and (glycine-glycine-glycine-glycine-serine)2 (-[Gly-Gly-Gly-Gly-Ser]2-). After the recombinant Pichia pastoris clones were identified by polymerase chain reaction (PCR), TDIII was expressed in Pichia pastoris and verified by SDS-PAGE  without altering the inserted cyanine dyes. However, the ideal pharmacokinetics of a fluorophore depend on the imaging application. For example, renally cleared probes are preferrable for molecular imaging due to their safety, while long-circulating probes are useful for vascular or lymphatic imaging. Long-circulating probes are also preferred for imaging-guided surgery. As dye@DIII/DIIIa are rapidly cleared through the renal system, they cannot function as long-circulating probes. IR-783@albumin was shown to be a long-circulating probe with a circulation time of 19 days, comparable to that of endogenous albuminII) Fig. . To retaAGE Fig. . And alsAGE Fig. .Fig. 6Dyn = 5). The dye-to-protein molar ratios investigated were 0.25:1, 0.5:1, 0.75:1, 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1 and 16:1. The samples were then exposed to a xenon lamp (780 nm) and the fluorescence intensity of each sample was detected by NIR-I (700–900 nm) and NIR-II (1100–1300 nm) detectors  were mixed with various molar concentrations of IR-783  NIR-II imaging was performed in mice with shaved heads, and vessels underneath the skull and skin were imaged with high resolution  and imaged lymphatic vessels. The ultra-bright IR-783@TDIII was able to illuminate the tiny lymphatic vessels with a resolution of 87.8 μm per pixel Fig. . NIR-II 16. Thus, dye@TDIII could be excellent candidates for imaging-guided surgery with translational potential.Intraoperative navigation technique is widely used in the field of medicine. Currently, various cyanine dyes are used in the clinic, but these exhibit rapid photobleaching and clearance. We hypothesized that dye@TDIII could address the existing problems in the field. We first compared IR-783@TDIII to the clinically used ICG to study the optical properties of the probes  for binding cyanine dyes. To our surprise, we observed that Cl-containing cyanine dyes formed covalent bonds with endogenous or mutant albumin, while Cl-free dyes formed non-covalent bonds. Some researchers have reported that Cys34 on albumin could covalently bind with IR-78351. However, the binding site was speculated on Cys34 since it is the only free cystine on albumin. In this study, we systematically studied and discovered a binding site (Cys476) on subdomain IIIa which could also covalently bond with Cl-containing cyanine dyes, and this finding has never been reported before. To confirm our finding, we used domain III or subdomain IIIa (without Cys34) instead of full albumin to react with Cl-containing dyes, and systematically studied the nucleophilic substitution of Cys476 and Cl-containing dyes. Based on this finding, we designed a series of recombinant albumin variants to chaperone Cl-containing dyes for in vivo NIR-II bioimaging. Furthermore, we determined that the interaction between cyanine dyes and albumin occurs in two independent stages and studied the mechanism of brightness enhancement. In the first stage, dyes bind to albumin or albumin mutants through non-covalent bonds, resulting in increased brightness though strengthened TICT. In the second stage, a “clasp” formed by the SH– group of Cys476 and the Cl–C group of Cl-containing dyes stabilizes the dyes through nucleophilic substitution. One group has observed some absorption spectra shifts  but with no explanation52, while, according to this phenomenon, another group provided with a possible explanation that a noncovalent adduct would be slowly transformed covalent51. However, they did not provide with more evidence for the speculation.The discovery of an albumin chaperone that functions as a brightness amplifierThis discovery provides a platform for the development of dyes that can attach themselves to serum albumin for improved pharmacokinetics. Cyanine dyes protected with a shell of albumin mutant or variant were shown to have enhanced optical properties. We also showed that the shells can be modified by fusion with a biofunctional peptide or conjugation with a small molecule. Additionally, the sizes of the bioengineered shells can be tuned to tailor the pharmacokinetic properties and clearance pathway. These excellent properties make the molecules suitable candidates for use in the fields of molecular imaging and imaging navigated surgery. In this study, we presented several applications in biofunctional imaging and imaging-guided surgery using complexes composed of bioengineered albumin fragments of various sizes with tailored pharmacokinetics and clearance pathways. We also designed an albumin mimic composed of three tandem DIII, which showed the highest brightness enhancement and generated dye complexes with albumin-like in vivo behavior.In conclusion, we demonstrated an alternative strategy to conventional chemical modification techniques to develop targeted NIR-II probes. We developed a facile strategy to design bright and editable NIR-II fluorophores that exhibit tailored pharmacokinetic properties to address the problems of existing NIR-II probes. Our innovation uses existing cyanine dyes with peak emission at ~800 nm, which could achieve full color imaging in both the NIR-I and NIR-II windows to provide accurate diagnosis and guide surgery. We believe that development of cyanine dyes with peak emission at 1000–1500 nm will further promote our genetic engineering strategy. More importantly, this genetic engineering strategy would provide us with a platform for modifying NIR-II dyes and exhibit an additional perspective to design dyes. Our strategy can help the design and development of NIR-II probes with clinical translation potential in the near future.The investigators adhered fully to the “Guide for the Care and Use of Animals” by the NIH Clinical Center Animal Care and Use Committee (NIHACUC), and all animal work was conducted in compliance with protocols approved by the NIHACUC.The aimed sequences of DIIIa and DIIIb were designed as follows. To make sure the connection part of DIIIa and DIIIb including, we designed an overlay segment with several amino acid residues (highlighted by blue color) on each of them.The amino acid sequence of DIIIa (381–494):LEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSThe amino acid sequence of DIIIb (490–585):KTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKATo make sure the subdomains were successfully expressed and purified , we used two strategies to achieve the small proteins: His tag and GST tag. Typically, a thrombin cleavage site  was insert between tag and the targeting protein, respectively.GST tag sequence:MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPHRDHence, the GST-LVPRGS-DIIIa/DIIIb:MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPHRDLVPRGSLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVS*MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPHRDLVPRGSKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA*After achieved the above corresponding pPIC9K recombinant plasmid, we transformed them into GS115 strain. Specifically, the pPIC9K-DIIIa/DIIIb plasmid was linearized with XhoI and EcoR1, respectively. Then, GS115 strain was transformed by electroporation. Recombinant Pichia clones were isolated on RD plate. After the clones appeared in the plate, washed the clones with sterile water, and put them onto YPD + GS115 (1000 µg/ml) plate. Recombinant Pichia clones were identified by PCR.PCR primes, suggest by Invitrogen yeast protocol, are documented as below:5′AOX: 5′ ctgctgatagcctaacgttc 3′3′AOX: 5′ gctgatcaggagcaagctcg 3′−1 in 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05 % (w / v) sodium azide, pH 7.0. The column was TSK G3000SWXL (Tosoh Bioscience), and the eluted proteins were detected by UV at 280 nm compared to an HSA standard. Finally, proteins were analyzed using SDS-PAGE in PhastGelTM electrophoresis system and appropriate media.Then, yeast (1 mL) was inoculated into 10 ml BMMD medium (2 % w / v glucose), and grown for 48 h on a rotary shaker of 250 r.p.m. at 30 °C. After that, 4 ml of each culture was inoculated in 2 × 200 ml BMMD media to grow for 120 h on a rotary shaker of 250 r.p.m at 30 °C. Proteins produced by recombinant Pichia were purified using the AlbuPure matrix (ProMetic BioSciences). Briefly, the supernatant was collected after centrifugation and filtered with 0.2 µm vacuum filter membranes (Millipore). Subsequently, a Pall Filtron LV system equipped with an Omega 10 kDa filter  was employed to further concentrate the filtered supernatant. The column was equilibrated with 50 mM sodium acetate pH 5.3 and loaded with the concentrated supernatant. After that, the column was washed with 10 column volume (CV) of equilibration buffer and 50 mM ammonium acetate pH 8.0 (10 CV) to elute the unbound fraction. The bound protein was eluted with a series of buffers, 50 mM ammonium acetate, 10 mM octanoate pH 8.0, 50 mM ammonium acetate, 30 mM sodium octanoate pH 8.0 or 200 mM potassium thiocyanate, respectively. Then, the eluted protein was concentrated and filtered against 10 CV of 50 mM NaCl by Vivaspin20 10 kDa PES (Sartorius). GP- HPLC was performed to quantify the HSA variants. Samples were chromatographed at a flow rate of 1 ml minThe DNA sequence of TDIII:ATGAAGTGTTGTGCTGCTGCTGACCCACACGAATGTTACGCTAAGGTTTTCGACGAGTTCAAGCCATTGGTTGAGGAACCACAGAACCTGATCAAGCAGAACTGTGAGTTGTTCGAGCAGCTGGGTGAGTACAAGTTCCAGAACGCTTTGTTGGTCAGATACACCAAGAAGGTCCCACAGGTTTCCACTCCAACCTTGGTTGAAGTCTCCAGAAACCTTGGTAAGGTCGGTTCCAAGTGTTGTAAGCACCCTGAGGCTAAGAGAATGCCATGTGCTGAAGATTACTTGTCCGTCGTCTTGAACCAGTTGTGCGTCTTGCACGAAAAGACTCCAGTTTCCGACAGAGTTACCAAGTGCTGTACTGAGTCCTTGGTCAACAGACGTCCATGTTTCTCTGCTTTGGAGGTCGACGAAACCTACGTGCCAAAAGAGTTCAACGCTGAGACTTTCACTTTCCACGCTGACATCTGTACCCTGTCCGAAAAAGAGAGACAGATCAAGAAGCAGACTGCCTTGGTCGAGTTGGTTAAGCACAAGCCAAAGGCTACCAAAGAGCAGTTGAAGGCTGTTATGGATGACTTCGCTGCCTTCGTTGAGAAGTGTTGCAAGGCTTTGGAAAAGTGTTGCGCAGCTGCAGATCCTCATGAGTGTTACGCCAAAGTCTTTGATGAGTTTAAGCCCCTGGTCGAAGAACCCCAAAACTTGATTAAGCAAAACTGCGAACTGTTTGAGCAATTGGGCGAGTACAAATTTCAAAACGCCCTGCTGGTTAGGTACACTAAGAAAGTTCCTCAGGTGTCTACCCCAACTTTGGTCGAGGTTTCTAGGAACTTGGGTAAAGTGGGTTCTAAGTGCTGCAAACATCCAGAGGCCAAAAGAATGCCTTGCGCAGAGGACTACTTGTCTGTTGTTCTGAACCAGCTTTGTGTGCTGCACGAGAAAACCCCAGTCTCTGATAGAGTCACCAAATGTTGCACCGAGTCTCTGGTTAACCGTAGACCATGTTTTTCCGCCTTGGAAGTGGATGAGACTTACGTCCCTAAAGAGTTTAACGCCGAAACCTTTACCTTTCACGCCGATATCTGCACTTTGTCTGAGAAAGAGCGTCAGATTAAGAAACAAACCGCTCTGGTCGAACTTGTCAAGCACAAACCTAAAGCCACAAAAGAACAACTGAAGGCCGTCATGGACGATTTTGCCGCTTTTGTTGAGAAATGCTGTAAGGCCCTTGAAAAGTGCTGTGCCGCAGCCGATCCACATGAATGCTATGCTAAAGTGTTCGATGAGTTCAAACCACTTGTGGAAGAACCTCAGAATCTTATCAAACAAAATTGCGAGCTTTTCGAACAGTTGGGAGAGTATAAGTTTCAAAATGCCTTGTTGGTGCGTTACACAAAAAAGGTGCCTCAAGTCTCCACTCCTACTCTGGTTGAGGTTTCCCGTAACCTGGGAAAAGTTGGTAGCAAATGCTGCAAGCACCCCGAAGCTAAACGTATGCCTTGTGCCGAGGATTATCTGAGCGTTGTCTTGAATCAGCTGTGTGTCCTTCATGAGAAAACTCCCGTTTCTGACCGTGTCACTAAGTGTTGTACCGAAAGCTTGGTGAACAGAAGGCCTTGCTTTTCTGCTCTGGAAGTTGACGAGACATATGTTCCCAAAGAGTTCAACGCAGAAACATTCACATTTCATGCAGACATCTGCACACTTAGCGAGAAAGAAAGGCAAATCAAAAAGCAAACAGCCCTGGTTGAGCTGGTCAAACATAAGCCCAAGGCCACAAAAGAGCAGCTTAAAGCAGTAATGGACGATTTCGCTGCATTTGTCGAAAAGTGTTGTAAAGCCTAAGCGGCCGCThe amino acid sequence of TDIII MKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA*The His tag was used in this sequence design since the TDIII is a big protein with molecule weight similarly equal to wild type albumin.The amino acid sequence fused with cleavage site for thrombin and His tag (TDIII-LVPRGS-His tag).MKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALVPRGSHHHHHH.Most of the cyanine dyes were purchased from Sigma-Aldrich. IR-12N3 was purchased from Nirmidas Biotech Co. Indocyanine Green  for human injection was purchased from Dandong Yichuang Pharmaceutical Co., LTD. PBS was purchased from HyClone. Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) were purchased from Sigma-Aldrich. Fetal Bovine Serum (FBS) was purchased from Neuromics. Sucrose was purchased from Sigma-Aldrich.Binding affinity of cyanine dyes with albumin variants was determined by biolayer interferometry (BLI) by interaction using an Octet Red96 system (fortéBio), respectively. Because of the same protocol, binding affinity of IR-783 with HSA was taken as an example. IR-783 powder was freshly dissolved in anhydrous DMSO (26.7 mM). Then, IR-783 is serially diluted into different concentrations  by PBS. The assay protocol was briefly described as follows. After washing with 1 × PBS for 60 s, biotinylated HSA (1 μg/mL) was loaded to the biosensor for 600 s, and after another 60 s washing, quenching with biocytin was performed for 180 s. Followed by another 60 s washing, association for 600 s and dissociation for another 600 s were performed in turn. Data was calculated and analyzed using Octet Analysis software v 7.0.Synthesis of dye@HSA and dye@HSA-domains/variants complex shared the same protocol and IR-783-DIII complex was taken as an example. DIII powder was dissolved in 1XPBS with a concentration of 20 mg/mL (855 µM); IR-783 powder was freshly dissolved in anhydrous DMSO (26.7 mM). 500 µL DIII PBS solution was first added in 500 µL PBS under slight vortexing. Then, 8, 16, 32, 48, 96 μL of the IR-783 (26.7 mM) was added into the DIII solutions, respectively . Finally, the suspension was vortexed for 30 s and heated at 60 °C for 10 min.Since we do not know if the conjugation of TATE/PSMA-617 would impact the binding of IR-783, we attempted to add IR-783 before or after the conjugation of TATE/PSMA. Results indicated that both routes did not lead to obvious fluorescence diminishing; hence, we conclude that tailoring DIII does not impact the in vitro and in vivo behavior of the fluorophores. IR-783@DIII or DIIIa (TDIII) was reacted with maleimide-PEG-NHS ester (Sigma) at RT. for 4 h under shaker. Then products  were filtered with 10 k filter against PBS for 5 times to move unreacted maleimide-PEG-NHS ester. The maleimide-PEG-NHS labeled compounds were incubated with PSMA-SH and TATE-SH peptides (molar ratio of protein and peptide was 1:5), as described in our previous paper. After that, the conjugates were washed with 10 K filter against PBS for 5 times.UV-Vis-NIR spectrophotometer (Cary 6000i) with background correction was employed to measure the optical absorption spectrum. Fluorescence spectrophotometry was carried out on a Hitachi F-7000 fluorescence spectrophotometer. Fluorescence microscopy was performed on an Olympus fluorescence microscope.DIII-cRGD was mixed with HCCA (α-cyano-4-hydroxycinnamic acid) in a 1:1 ratio, 2 μL of sample and 2 μL of HCCA. Sample matrix was mixed and loaded onto a MALDI target plate (MTP) 384 by direct droplet method. It was allowed to dry for a few minutes. MALDI-TOF mass spectrometry was performed on Autoflex maX (Bruker) instrument. Finally, the data was plotted using Graphpad 8.0.Mice were shaved using Nair hair removal cream and anesthetized using isoflurane before placing them for injection of imaging agents. For each imaging experiment, at least 3 mice were used as a parallel group cohort. All NIR-II images were collected on a Princeton InGaAs array. The excitation laser was an 808 nm laser set-up. Emission was typically collected with different long pass filters. A lens set was used for obtaining tunable magnifications, ranging from 1× (whole body) to 2.5× (high magnification) by changing the relative position of two NIR achromats . NIR-II microscopy was built based on the previous report, and the excitation laser was a 785 nm laser set-up.2, 37 °C). All cells were tested to be free of mycoplasma.AR42J (from ATCC) were cultured in F-12k medium with 20% heat-inactivated FBS and 1% penicillin and streptomycin. PC3 (from ATCC), PC3-PIP (from NIBIB at NIH) and 4T1-fluc cell lines (from ATCC) were cultured in RPMI-1640 medium with 10% heat-inactivated FBS and 1% penicillin and streptomycin. Cells were grown in a humidified atmosphere (5% CO6) were inoculated to nude mice to grow subcutaneous tumor, respectively. 4T1-fluc cells (1.0 × 105) were inoculated in the mammary fat pad to establish the orthotopic mammary tumor model/metastatic tumor model.Nude mice , C57BL/6j mice  and balb/c mice  were purchased from Jackson’s Laboratory . Bedding, nesting material, food, and water were provided ad libitum. Ambient temperature was controlled at 20 to 22 °C with 12 h light/12 h dark cycles. AR42J or PC3 cells  were intravenously injected with PBS, DIII, IR-783@DIII, IR-783@HSA, IR-783@DIIIa, and IR-783@TDIII, respectively. Peripheral blood was collected to analyze subtypes of lymphatic cells. Briefly, blood was collected from the treated mice on day 3, day 7 and day 14. Blood cells were enriched by centrifugation. Red blood cells were lysed using ACK lysis buffer for 10 min at room temperature. Then, cells were washed twice in PBS and stained with the according antibodies for 15 min. After that, cells were washed with FCS buffer (PBS buffer with 0.1% FBS), and resuspended for flow cytometric analysis. Flow cytometry was conducted on a Beckman CytoFlex S flow cytometer. Data were analyzed using FlowJo V10 version.The ground state (S0) geometries of IR-783 and ICG molecules were optimized using the B3LYP-GD3GJ/6-31 G(d) method dispersion correction. The excited state (S1) properties were calculated using the time-dependent (TD) LC-BLYP*/6-31 G(d) method with the polarizable continuum model (PCM). The HOMO and LUMO distributions were displayed using VMD code. All the (TD)DFT calculations were performed using Gaussian 16 software.Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSuppl Movie 1Suppl Movie 2Suppl Movie 3


Caenorhabditis elegans. We observed that both the catalytic (KIN-3::V5) and regulatory (KIN-10::2xMyc) subunits of the Casein Kinase II (CK2) holoenzyme complex are associated with meiotic DNA, enriched in the midvalent rings during meiotic divisions in fertilizedC. elegans oocytes.By using CRISPR/Cas9 genome-editing, we have generated epitope-tagged KIN-3 and KIN-10 expressing strains at the endogenous C-terminal loci in C. eleganscatalytic and regulatory subunits are encoded bykin-3andkin-10, respectively . We used CRISPR/Cas9 genome-editing to generate epitope-tagged KIN-3::V5 and KIN-10::2xMyc expressing strains at the endogenous C-terminal loci. By staining early embryos with commercially available epitope antibodies, we observed the localization of KIN-3::V5 and KIN-10::2xMyc in fertilizedC. elegansoocytes. During meiosis in fertilizedC. elegansoocytes, both the catalytic (KIN-3) and regulatory (KIN-10) subunits of the CK2 holoenzyme are associated with meiotic DNA, enriched around the center of the bivalent, referred to as the ring complex . It has been shown that AIR-2/Aurora B and KLP-19 localize to the ring complexes associated with meiosis I bivalent and meiosis II chromosomes. Both AIR-2 and KLP-19 are required for proper chromosome segregation duringC. elegansmeiosis . The close association of KIN-3 and KIN-10 with meiotic DNA suggests that CK2 kinase activity might influence chromosome organization and segregation during meiotic divisions in theC. elegans oocyte. In support of this, previous work has reported that depletion of CK2 results in polar body extrusion failure and extra DNA, likely due to meiotic errors in fertilizedC. elegansoocytes . A study in porcine oocytes has also shown that CK2 localizes to meiotic chromosomes and that CK2 activity is required for normal meiotic progression . Thus, CK2 function during meiotic division appears to be evolutionarily conserved.The kinase Casein Kinase II (CK2), a tetrameric holoenzyme, consists of two catalytic (CK2α) and two regulatory (CK2β) subunits . TheC. elegansCulture:All strains were derived from the wild-type Bristol N2 strain and maintained on MYOB plates seeded withEscherichia coliOP50 at 20°C.Immunostaining and Confocal Microscopy: Immunofluorescence and confocal microscopy were performed as described . For immunostaining, the following primary and secondary antibodies were used at 1:3000 dilutions: α-Myc , α-V5 , and Alexa Fluor 488 and 568 secondary antibodies . Confocal microscopy was performed using a Nikon Eclipse Ti-U microscope equipped with a Plan Apo 60×1.4 NA lens, a Spinning Disk Confocal (CSU X1), and a Photometrics Evolve 512 camera. MetaMorph software  was used for image acquisition and Adobe Photoshop/Illustrator 2022 for image processing.CRISPR/Cas9 Genome Editing: For genome editing, we used the co-CRISPR technique described previously . To design crRNA, we used the CRISPOR webserver . Animals were microinjected with a mixture of commercially available SpCas9  and custom-designed oligonucleotides  including crRNAs at 0.4–0.8 µg/ml  tracrRNA at 12 µg/ml, and single-stranded DNA oligonucleotides at 25–100 ng/ml. After injection, we screened fordpy-10(cn64) II/+rollers in F1 progeny and genotyped F2 for the epitope-tag insertion. The genome editing was verified by Sanger Sequencing . All theC. elegansstrains generated in this study produce nearly 100% viable progeny.Single-stranded DNA oligonucleotides homologous repair templates  for genome editing were as follows.GGTAAGCCTATCCCAAATCCTTTGTTGGGTCTGGACTCCACGTAAAATTTCTTTCTATTTTTTTTTTAATTTTCCTGKIN-3::V5 tag at the C-terminus (5'-3'): CATCGAATTCCGCTTCTTCTCAATCCTCCGATGCTAAAATTGACGGCGCTGGAGGTTCCGGTGGTTCTGGTGGATCCGAACAAAAACTGATATCTGAAGAAGACCTTGAGCAGAAGTTGATTAGTGAGGAGGATCTTTGAGCCACTTTCTTCCTTATTTTTGTTTTGATTTCKIN-10::2xMyc tag at the C-terminus (5'-3'): CAAAACAACACGACTCCAGCCGGGCAACAATCTGGCGGCCAGTTCAACAACTATGGTCTCGGTGGCTCTGGTGGAAGTGGAGGCTCAN2: wild-type (CGC), MTU137:kin-3(mhs464[KIN-3::V5]) I (This study), MTU598:kin-10(mhs688[KIN-10::2xMyc]) I (This study)


S-adenosyl-L-methionine (SAM) L-tyrosine lyase and catalyzes the L-tyrosine Cα–Cβ bond break to produce dehydroglycine and p-cresol while the radical SAM L-tryptophan lyase NosL cleaves the L-tryptophan Cα–C bond to produce 3-methylindole-2-carboxylic acid. It has been difficult to understand the features that condition one C–C bond break over the other one because the two enzymes display significant primary structure similarities and presumably similar substrate-binding modes. Here, we report the crystal structure of L-tyrosine bound ThiH from Thermosinus carboxydivorans revealing an unusual protonation state of L-tyrosine upon binding. Structural comparison of ThiH with NosL and computational studies of the respective reactions they catalyze show that substrate activation is eased by tunneling effect and that subtle structural changes between the two enzymes affect, in particular, the hydrogen-atom abstraction by the 5´-deoxyadenosyl radical species, driving the difference in reaction specificity.2-iminoacetate synthase ThiH is a radical  ThiH is a radical SAM L-tyrosine lyase involved in the biosynthesis of the thiazole ring of vitamin B1. Here, the authors report the crystal structure of ThiH in complex with its L-tyrosine substrate, revealing an unexpected protonation state and tunneling effect that lowers the reaction energy barrier. It enables, for example, the formation of acetyl coenzyme A by pyruvate dehydrogenase3 and pyruvate: ferredoxin oxidoreductase4 as well as the transfer of a glycoaldehyde from a ketosugar to an aldosugar by transketolase5. Dehydroglycine (DHG), also termed 2-iminoacetate, is a key precursor in the bacterial biosynthesis of the thiazole ring that constitutes the core of TPP. In aerobic prokaryotes, such as the extensively studied Bacillus subtilis, DHG is produced by the oxygen-dependent glycine oxidase7. Such glycine oxidation is not possible under anaerobic conditions and anaerobes, including the facultative bacterium Escherichia coli, use instead the 2-iminoacetate synthase ThiH that catalyzes DHG formation from L-tyrosine processing , derived from vitamin B1, is a key ubiquitous cofactorS-adenosyl-L-methionine (SAM) superfamily13 and contains one [Fe4S4] cluster that reductively cleaves SAM to form both methionine and a highly reactive 5´-deoxyadenosyl radical (5´-dA•) species. Subsequently, 5´-dA• abstracts from L-tyrosine, a hydrogen atom, long proposed to be the phenolic hydrogen atom10. By analogy, the same reaction step would be catalyzed by the radical SAM tyrosine lyase HydG involved in the assembly of the [FeFe]-hydrogenase active site15. ThiH and HydG are closely related members of the radical SAM superfamily sharing 27% sequence identity16. Both enzymes catalyze the cleavage of L-tyrosine Cα–Cβ bond to generate p-cresol16 and DHG17. The latter is used by HydG at a second active site of the enzyme to synthesize the CO and CN– diatomic ligands20 that are part of the so-called [FeFe]-hydrogenase H-cluster22. Conversely, ThiH transfers DHG to ThiG, where it is further processed to produce the thiazole ring of thiamine11.ThiH is a member of the radical 23, involved in the synthesis of the thiopeptide antibiotic nosiheptide24. NosL shares about 23% primary structure identity with them25. But, whereas ThiH and HydG cleave the L-tyrosine Cα–Cβ bond17, NosL cleaves the L-tryptophan Cα-C bond instead and produces a 3-methylindole-2-carboxylic acid (MIA)27 , an observation supported by a subsequent biochemical study28. In addition, structure-based sequence comparisons of NosL with tyrosine lyases ThiH and HydG show that, in the latter enzymes, the corresponding [L-Tyr-NH•] would be formed25. Using electron paramagnetic resonance spectroscopy (EPR), a methylene-centered p-cresyl radical intermediate (p-cresyl•) was trapped during L-tyrosine scission by HydG15 and, more recently, the 5´-dA• was trapped using p-coumaric acid instead of L-tyrosine29.These two tyrosine lyases are often compared to the radical SAM tryptophan lyase NosLThermosinus carboxydivorans (Tc) in complex with its L-tyrosine substrate. In this study, an unexpected substrate protonation state, supported by its interactions in our X-ray model, has been confirmed by molecular dynamics (MD) simulations. More specifically, deprotonation of the L-tyrosine hydroxyl moiety is in agreement with the very recent report that, in HydG, the ensuing p-cresyl• intermediate is most probably deprotonated as well, and corresponds to a 4-oxidobenzyl radical instead30. The description of the mechanism of direct hydrogen atom abstraction by 5´-dA• at the amino nitrogen position of L-tyrosine, obtained by using hybrid quantum mechanical / molecular mechanical (QM/MM) methods, supports a highly controlled and tight local environment, inducing a tunneling effect during hydrogen transfer that we have estimated analytically to be about 50% of the reaction barrier. Additional calculations performed on both ThiH and NosL show that this step corresponding to the radical formation [L-Tyr-NH•] ([L-Trp-NH•] in NosL) is crucial for the orientation of the subsequent bond cleavage step . In addition, comparison of ThiH with HydG and NosL has revealed unique structural motions, most likely responsible for specific substrate access and product release sites, showing that these enzymes work as assembly lines. Thus, by combining X-ray crystallography and computational chemistry, we provide a rarely achieved mechanistic insight into the hydrogen-atom abstraction step catalyzed by radical SAM enzymes and underscore how subtle changes at the active site afford both substrate selectivity and chemical regiospecificity.Here, we report the crystal structure of ThiH from TcThiH was incubated with its substrate L-tyrosine, 5´-deoxyadenosine (5´-dA) and methionine as mimics of the SAM cleavage products prior to crystallization. Brownish prism-shaped crystals that diffracted to 1.27 Å resolution were obtained. In this crystal form, two independent ThiH molecules termed chains A and B were present in the asymmetric unit. Surface interaction calculations using the PDBePISA server31 supported the fact that the protein does not form quaternary structures in the crystals and would likely correspond to monomers in solution, in agreement with our previous observation when performing size exclusion chromatography. The refined model . We have also performed a 250-ns molecular dynamics (MD) simulation of L-Tyr bound TcThiH (COO−)  Fig. .TcThiH numbering), all the key residues involved in L-tyrosine binding are strictly conserved in both proteins  and 0.25 Å (molecule B), in the same range derived from comparing molecules A and B of the same crystal (see above). The Fo-Fc residual electron density map at the substrate-binding site indicates features that do not correspond to L-tyrosine, best modeled as two water molecules and a sulfate ion from the crystallization condition 25, in order to identify what makes such difference in substrate specificity. TcThiH and SaNosL share about 23% identity mainly located at key positions either to stabilize the tridimensional architecture or because they are important for their function. Yet, the two structures are very similar and display a RMSD of 2.1 Å for 281 Cα-atoms superimposed. The main structural differences lay in the N-terminal α-domain. As can be seen in Fig. TcThiH and SaNosL, respectively). Their respective dihedral angles are also the same. Strikingly, the main significant difference corresponds to a one-residue insertion at the C-terminal end of strand β8  and the L-tryptophan bound SaNosL structure we had previously solved25, both crystallized with 5´-dA, we first focused on the 5´-dA• attack on their respective substrates. Since the attack occurs for both models at the substrates’ amino group  for [5´-dA• + L-Tyr]-TcThiH and [5´-dA• + L-Trp]-SaNosL, respectively  at the start of the scan. By contrast, for NosL, the C5´ and adenine parts are energetically crossing, i.e. the characters of LUMO and LUMO + 1 are exchanged and their corresponding energies remain close within ~0.2 eV range. This means that 5´-dA• is influenced by subtle changes in its environment. This results in an inflection of the energy increase  in addition to the first lowest point (Φ = −47.9°) mirroring that of ThiH. As it turns out, this energy scan inflection for NosL opens an alternative path leading ultimately to the Cα–C bond scission whereas the absolute minimum common to both ThiH and NosL would lead for NosL to the non-productive Cα–Cβ bond scission . Hence, we decided to further investigate the impact of a hydrogen tunneling effect for the reaction to proceed  and 25 °C (SaNosL), corresponding to the optimal growth of T. carboxidivorans and S. actuosus, respectively.The transition state geometries for these reactions were validated by frequency calculations. Strikingly, the unique imaginary frequency exhibits a large absolute value greater than 2000 cmect Fig. 38. In adp-cresyl• and DHG  to extract a sampling of active site conformations corresponding to normal structural “breathing” in order to determine whether some of them could separate the products further. As a reference, we first scanned the Cα–Cβ bond break using a reduced quantum model consisting of about 300 atoms extracted from our QM/MM model of [L-Tyr-NH•], and observed that the energy reaches a minimum when Cα and Cβ are about 2.7 Å apart but goes back up at greater scanned distances  are necessary for product release.However, a product escape route is observed through a gate defined by residues Y76 and Y181 Fig. , left anydG Fig. , center;osL Fig. , right. TcThiH has shown that the substrate acquires an unusual protonation state upon its binding at the active site. Indeed, the phenol functional group of L-tyrosine is deprotonated and the resulting phenolate stabilized by four anion-dipole interactions with strictly conserved residues in ThiH but also in the tyrosine lyase HydG  between the NcoI and BamHI restriction sites, leading to a protein containing an N-ter Strep-tag and corresponding to the pTcThiH construct. The corresponding DNA sequence is as follow:The synthetic gene of ThiH from CCATGGGACATCATCATCATCATCACTCCGGCACGTTCTATGATGTGATTGAAGATTACCGCCACTTTGATTTCGCAGCGTATTTCGCGAAAGTTACCGATTCTGACGTCCGTCGCATCCTGCGTCAGGATCGCCTGTCAGCCCTGGACTTTCTGACGCTGCTGTCGCCGCAAGCGGAAGCCTATCTGGAAGAAATGGCACAGAAAGCTCATCGTCTGACCGTTCAACACTTTGGCCGCACCATGCTGCTGTATACGCCGCTGTACCTGGCGAACTATTGCGTGAATCAGTGCGTTTACTGTGGTTTCCAACTGAAAAACAAACTGGAACGTAAAAAACTGACCCTGGCGGAAGTGGAACAGGAAGCCCAACTGATTGCGGCCACCGGCCTGAAACATATTCTGATCCTGACGGGTGAAAGTCGCCAGCACTCACCGGTCTCGTATATCAAAGATTGTGTGAACATCCTGAAAAAATACTTTAGCTCTATCAGCATCGAAATTTATCCGCTGACCCAGGAAGAATACGCAGAACTGATCGGCGCTGGTGTTGACGGCCTGACGATTTACCAGGAAGTGTATAACGAAGAAGTTTATGCGGAAATGCATCCGGCCGGCCCGAAACGTAATTACCGTTTCCGCCTGGAAGCACCGGAACGTGCATGCCAGGCCGGTATGCGTACCGTGAACATCGGCGCTCTGCTGGGTCTGAATGATTGGCGCCAGGAAGCATTTTTCACGGGTCTGCATGCTGATTATCTGCAACGTCGCTTTCCGGACGTGGAAGTTAGTATTTCCCCGCCGCGTATGCGTCCGCACCTGGGCGGTTTCCCGCCGCGTGTGGTTGTCAGCGATCAGAACCTGGTCCAATATGTGCTGGCATTTCGTCTGTTCATGCCGCGTAGTGGTATCACCCTGTCCACGCGTGAAAATGGTCGTCTGCGTGACGCGATGGTCCGTCTGGGCGTGACCAAAATGAGCGCAGGTTCTTGTACGGCTGTTGGCGGTCGTAGCGATCAGGAAGCCGTCGGCCAGTTCCAAATTTCTGACGAACGCACCGTTGCGGAAGTCGCAGCTATGCTGTACGCCCAGGGTTATCAACCGGTGTACAAAGATTGGCAGGCACTGAGGATCCwhich leads to the following protein sequence:MWSHPQFEKASGTFYDVIEDYRHFDFAAYFAKVTDSDVRRILRQDRLSALDFLTLLSPQAEAYLEEMAQKAHRLTVQHFGRTMLLYTPLYLANYCVNQCVYCGFQLKNKLERKKLTLAEVEQEAQLIAATGLKHILILTGESRQHSPVSYIKDCVNILKKYFSSISIEIYPLTQEEYAELIGAGVDGLTIYQEVYNEEVYAEMHPAGPKRNYRFRLEAPERACQAGMRTVNIGALLGLNDWRQEAFFTGLHADYLQRRFPDVEVSISPPRMRPHLGGFPPRVVVSDQNLVQYVLAFRLFMPRSGITLSTRENGRLRDAMVRLGVTKMSAGSCTAVGGRSDQEAVGQFQISDERTVAEVAAMLYAQGYQPVYKDWQAL.thiH gene was coexpressed with the isc operon and the SAM synthase gene coding metK from E. coli using an E. coli BL21(DE3) strain containing both pTcThiH and our home-made pRSF-ISC-MetK plasmids25. Cells were grown aerobically at 37 °C under agitation in 4 L of TB medium supplemented with 50 mM MOPS/KOH, pH 7.1, 0.5% D-glucose, ampicillin (100 µg.ml−1) and kanamycine (50 µg.ml−1) up to an OD600 of about 0.6. They were subsequently transferred in 2 L sealed bottles into a glove box with an anaerobic atmosphere containing less than 5 ppm O2 under constant stirring at 20 °C. After about 15 min, the bottles were opened to equilibrate the medium with the atmosphere of the glove box and the medium was supplemented with 10 mL MEM Vitamin solution 100X (SIGMA M6895); 4 g fumarate; 500 µL 0.5 M cysteine and 750 µL 0.2 M L-methionine per L of culture. Fifteen more minutes later, the medium was enriched with 250 µL 1 M ammonium iron(III) citrate. Protein expression was induced when the OD600 reached 1 with addition of 1 mM final concentration isopropyl-β-D-1-thiogalactopyranoside and cells were further grown overnight before being harvested and frozen in liquid nitrogen. All the purification steps were performed under anaerobic conditions. Cell pellets were resuspended in the glove box in buffer A  supplemented with EDTA-free protease inhibitor cocktail. They were subsequently disrupted by sonication and the crude-extract was cleared by centrifugation in sealed tubes at 15,000 × rpm during 30 min at 4 °C. The clarified supernatant was filtered, and loaded onto a streptavidin-agarose column (Iba®) equilibrated with anaerobic cleared lysate was injected into a Strep-tactin column equilibrated with buffer A. After an extensive wash step, the protein was eluted with a 1 mM desthiobiotin solution in buffer A. The iron-sulfur cluster was subsequently reconstituted. The protein was incubated 15 min with 5 mM DTT prior to the addition of 200 nM NifS from Azotobacter vinelandii. 5 excess FeCl3 and L-cysteine were then added and the solution was kept under mild stirring overnight at 20 °C. The protein was further purified to homogeneity by gel filtration using a GE-healthcare highload 16/600 S200 prep-grade column equilibrated with buffer B (50 mM Tris pH 8 and 100 mM NaCl). The protein eluted as two peaks corresponding to a dimer and a monomer, respectively. Only the fractions corresponding to the monomer (major peak) were pooled, concentrated to 10 mg/mL and stored in liquid nitrogen. Iron content was determined using the method of Fish48 and indicated over 3.9 iron equivalents per ThiH molecule.The recombinant 4)2SO4, 100 mM MES buffer pH 6.5, 15% dioxane and 10 mg/mL TcThiH. The protein was preincubated with 1 mM 5´-dA and 1 mM L-methionine. When required, 5-excess L-tyrosine was added to the crystallization drop. Crystals were cryoprotected using 25% glycerol in addition to the crystallization condition and were subsequently mounted in cryoloops before flash cooling in liquid propane in the glove box49. Data were collected at SOLEIL at beamlines PROXIMA-1 and PROXIMA-2A and were processed using the XDS package50  as a search model. The initial model was manually corrected using COOT52 and refined using PHENIX53 to an Rwork = 0.153 and Rfree = 0.174 with good geometry and over 98% residues in the most allowed region of the Ramachandran plot54. This model was subsequently used to solve the ligand-free TcThiH crystal structure following the same procedure, leading to a model with good geometry. The corresponding statistics are reported in Supplementary Table Crystals were obtained using the vapor diffusion method. Initial conditions were determined by screening 1248 conditions using a Gryphon robot  setup in a glove box and were subsequently manually optimized. Crystals suitable for X-ray diffraction experiments were obtained with 1.4–1.6 M (NH55.Details of all calculations are given in the Supplementary Information. All MD simulations, QM and QM/MM calculations were performed within the Schrödinger suiteFurther information on research design is available in the Supplementary information filePeer Review FileReporting Summary


We found that LTBR and CREB1 exhibited a significant upregulation in lungs of mouse model of BPD. LTBR and CREB1 expression were also increased by hyperoxia in A549 and ATII cells. According to results of cell counting kit-8 assay and flow cytometry analysis, silencing of LTBR rescued the suppressive effect of hyperoxia on cell viability and its promotive effect on cell apoptosis of A549 and ATII cells. Bioinformatics revealed CREB1 as a transcriptional factor for LTBR, and the luciferase reporter assay and ChIP assay subsequently confirmed it. The NF-κB pathway was regulated by LTBR. CREB1 induced LTBR expression at the transcriptional level to regulate NF-κB pathway and further modulate A549 and ATII cells viability and apoptosis. In conclusion, this study revealed the CREB1/LTBR/NF-κB pathway in BPD and supported the beneficial role of LTBR silence in BPD by promoting viability and decreasing apoptosis of lung epithelial cells.Bronchopulmonary dysplasia (BPD) is a prevalent chronic pediatric lung disease. Aberrant proliferation and apoptosis of lung epithelial cells are important in the pathogenesis of BPD. Lymphotoxin beta receptor (LTBR) is expressed in lung epithelial cells. Blocking LTBR induces regeneration of lung tissue and reverts airway fibrosis in young and aged mice. This study is aimed at revealing the role of LTBR in BPD. A mouse model of BPD and two  A review research concludes that BPD may be caused by various factors like premature birth, fetal growth restriction, mechanical ventilation, oxygen toxicity, inflammation, and genetic susceptibility [Bronchopulmonary dysplasia (BPD), initially described by Northway et al., is caused by oxygen supply and mechanical ventilation in premature infants with severe respiratory distress syndrome . Accorditibility . Despitetibility , pulmonatibility  were devtibility . Thus, iBPD is a chronic disease featured by disrupting the alveolar together with microvascular development of the peripheral lung. The regeneration or induced growth of type II alveolar epithelial (ATII) cells has been the focus feature of lung regeneration research . Thus, eβ receptor (LTBR) signaling in ATII cells induces kinase NIK through nonclassical nuclear factor-kappaB (NF-κB) signaling. Blocking LTBR signaling limits broncho-associated lymphoid tissue formation and reduces alveolar epithelial cell apoptosis, thereby driving alveolar regeneration [Activation of the LTneration . LTBR sineration . LTBR mRneration . LTBR exneration , a contiTranscription factors respond to stimuli of the external environment or signals at different stages of development, activate or inhibit gene transcription, and thus control the expression of different genes. It is very important to identify key transcription factors in BPD. cAMP responsive element binding protein 1 (CREB1) regulates late-stage lung development in mammals . CREB1 iκB signaling plays a crucial role in cell survival, differentiation, and innate immunity [κB dimers are not active in the cytoplasm of cells related to a suppressive protein, IκB. Under the conditions of cytokines, growth factors, or bacterial products, IκB kinases IKK-α and -β activate NF-κB, leading to IκB phosphorylation and degradation. Clearance of IκB allows translocation of NF-κB complexes into the nucleus and modulates a variety of downstream targets that affect inflammation, cell adhesion, and cell survival [κB-mediated gene modulation is implicated in the pathogenesis of BPD [The NF-immunity . NF-κB dsurvival . Many sts of BPD .in vivo and its role in the apoptosis of lung epithelial cells in vitro. Hyperoxia-stimulated A549 and ATII cells were used as the in vitro model of BPD [κB pathway was assessed, providing new ideas for understanding the pathogenesis of BPD.This study is aimed at evaluating the expression of LTBR in the BPD lung injury mouse model l of BPD , 19. Then = 8) or the control group (n = 8). To establish the BPD model, mice were exposed to 100% O2 in a medium-sized airtight polypropylene chamber on postnatal days 1-4, followed by recovering for 10 days [Neonatal C57BL/6 mice of both sexes  were used for establishing the model of BPD. Animal studies were conducted in compliance with the ARRIVE guidelines under the approval of the animal care and ethic committee of First Affiliated Hospital of Nanjing Medical University. Mice were housed under controlled standard conditions . Neonatal mice in the normoxia group were exposed to room air and fed by lactating mice. Mice were randomly divided into the BPD model group  and dissolved in RNase-free water. Reverse transcription of total RNA into cDNA was performed using a High-Capacity cDNA Reverse Transcription Kit . Next, the QuantiFast SYBR® Green PCR Kit (QIAGEN) was used for a rapid and specific quantitative detection of target cDNA. PCR analysis was performed on a LightCycer®480II Instrument, 96-well block . LTBR or CREB1 expression was calculated using the 2t method  and was Mouse LTBR, forward, 5′-TGAACACTGGAACCATCTC-3′; reverse, 5′-ACACTCATTGTCCAGATACAC-3′.Human LTBR, forward, 5′-GAAGGGTAACAACCACTGC-3′; reverse, 5′-CTTGGTTCTCACACCTGGT-3′.Mouse CREB1, forward, 5′-GAAGAGGAGACTTCAGCCC-3′; reverse, 5′-TAATGGCAATGTACTGCCCA-3′.Human CREB1, forward, 5′-ATGCAGCTGTAACAGAAGC-3′; reverse, 5′-CATAGATACCTGGGCTAATGTG-3′.Mouse GAPDH, forward, 5′-ACTCTTCCACCTTCGATGC-3′; reverse, 5′-CCGTATTCATTGTCATACCAGG-3′.Human GAPDH, forward, 5′-TCAAGATCATCAGCAATGCC-3′; reverse, 5′-CGATACCAAAGTTGTCATGGA-3′.The radial alveolar count (RAC) determines alveolar septation and is a test of alveologenesis. Through images at 100× magnification, a perpendicular line was made from a respiratory bronchiole to the nearest pleural edge or fibrovascular septum. Airspaces or saccules through this line were counted . Scion I2. A549 and ATII cells were exposed to different concentrations of O2  in a standard modular chamber  at 37°C to assess the effects of hyperoxia on LTBR and CREB1 expression. The normoxia condition contains 21% O2. Based on the results of the preliminary assays, A549 and ATII cells were exposed to 85% O2 for 48 h to mimic the in vitro model of BPD. Si-NC, si-LTBR, si-CREB1, empty pcDNA 3.1, pcDNA 3.1-LTBR, and pcDNA 3.1-CREB1 were commercially provided by GenePharma . These oligonucleotides or vectors were transfected into A549 and ATII cells using Lipofectamine 3000  for 24 h at 37°C. The transfection sequences used were as follows: si-NC: 5′-AACCATCACTTACAAGAAACC-3′; si-CREB1: 5′-GCTCGATAAATCTAACAGTTA-3′; si-LTBR: 5′-CCATCCATACTTCCCTGACTT-3′; pcDNA3.1: 5′-CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCAT-3′; pcDNA 3.1-CREB1: 5′- ATGACCATGGAATCTGGAGCCGAGAACCAGCAGAGTGGAGATGCAGCTGTAACAGAAGCTGAAAACCAACAAATGACAGTTCAAGCCCAGCCACAGATTGCCACATTAGCCCAGGTATCTATGCCAGCAGCTCATGCAACATCATCTGCTCCCACCGTAACTCTAGTACAGCTGCCCAATGGGCAGACAGTTCAAGTCCATGGAGTCATTCAGGCGGCCCAGCCATCAGTTATTCAGTCTCCACAAGTCCAAACAGTTCAGATTTCAACTATTGCAGAAAGTGAAGATTCACAGGAGTCAGTGGATAGTGTAACTGATTCCCAAAAGCGAAGGGAAATTCTTTCAAGGAGGCCTTCCTACAGGAAAATTTTGAATGACTTATCTTCTGATGCACCAGGAGTGCCAAGGATTGAAGAAGAGAAGTCTGAAGAGGAGACTTCAGCACCTGCCATCACCACTGTAACGGTGCCAACTCCAATTTACCAAACTAGCAGTGGACAGTATATTGCCATTACCCAGGGAGGAGCAATACAGCTGGCTAACAATGGTACCGATGGGGTACAGGGCCTGCAAACATTAACCATGACCAATGCAGCAGCCACTCAGCCGGGTACTACCATTCTACAGTATGCACAGACCACTGATGGACAGCAGATCTTAGTGCCCAGCAACCAAGTTGTTGTTCAAGCTGCCTCTGGAGACGTACAAACATACCAGATTCGCACAGCACCCACTAGCACTATTGCCCCTGGAGTTGTTATGGCATCCTCCCCAGCACTTCCTACACAGCCTGCTGAAGAAGCAGCACGAAAGAGAGAGGTCCGTCTAATGAAGAACAGGGAAGCAGCTCGAGAGTGTCGTAGAAAGAAGAAAGAATATGTGAAATGTTTAGAAAACAGAGTGGCAGTGCTTGAAAATCAAAACAAGACATTGATTGAGGAGCTAAAAGCACTTAAGGACCTTTACTGCCACAAATCAGAT-3′ and pcDNA 3.1-LTBR: 5′-ATGGAAGCGACAGGAATCTCATTAGCATCTCAATTAAAGGTGCCTCCATATGCGTCGGAGAACCAGACCTGCAGGGACCAGGAAAAGGAATACTATGAGCCCCAGCACCGCATCTGCTGCTCCCGCTGCCCGCCAGGCACCTATGTCTCAGCTAAATGTAGCCGCATCCGGGACACAGTTTGTGCCACATGTGCCGAGAATTCCTACAACGAGCACTGGAACTACCTGACCATCTGCCAGCTGTGCCGCCCCTGTGACCCAGTGATGGGCCTCGAGGAGATTGCCCCCTGCACAAGCAAACGGAAGACCCAGTGCCGCTGCCAGCCGGGAATGTTCTGTGCTGCCTGGGCCCTCGAGTGTACACACTGCGAGCTACTTTCTGACTGCCCGCCTGGCACTGAAGCCGAGCTCAAAGATGAAGTTGGGAAGGGTAACAACCACTGCGTCCCCTGCAAGGCCGGGCACTTCCAGAATACCTCCTCCCCCAGCGCCCGCTGCCAGCCCCACACCAGGTGTGAGAACCAAGGTCTGGTGGAGGCAGCTCCAGGCACTGCCCAGTCCGACACAACCTGCAAAAATCCATTAGAGCCACTGCCCCCAGAGATGTCAGGAACCATGCTGATGCTGGCCGTTCTGCTGCCACTGGCCTTCTTTCTGCTCCTTGCCACCGTCTTCTCCTGCATCTGGAAGAGCCACCCTTCTCTCTGCAGGAAACTGGGATCGCTGCTCAAGAGGCGTCCGCAGGGAGAGGGACCCAATCCTGTAGCTGGAAGCTGGGAGCCTCCGAAGGCCCATCCATACTTCCCTGACTTGGTACAGCCACTGCTACCCATTTCTGGAGATGTTTCCCCAGTATCCACTGGGCTCCCCGCAGCCCCAGTTTTGGAGGCAGGGGTGCCGCAACAGCAGAGTCCTCTGGACCTGACCAGGGAGCCGCAGTTGGAACCCGGGGAGCAGAGCCAGGTGGCCCACGGTACCAATGGCATTCATGTCACCGGCGGGTCTATGACTATCACTGGCAACATCTACATCTACAATGGACCAGTACTGGGGGGACCACCGGGTCCTGGAGACCTCCCAGCTACCCCCGAACCTCCATACCCCATTCCCGAAGAGGGGGACCCTGGCCCTCCCGGGCTCTCTACACCCCACCAGGAAGATGGCAAGGCTTGGCACCTAGCGGAGACAGAGCACTGTGGTGCCACACCCTCTAACAGGGGCCCAAGGAACCAATTTATCACCCATGACTGA-3′.Lung epithelial A549 cell line was commercially provided by ATCC  and was cultured in F-12 K Medium  supplemented with 10% FBS. Human ATII cells and HEK-293 T cells were purchased from Procell  and cultured in human ATII cell complete culture medium and DMEM, respectively. Cells were maintained in a 37°C incubator in the atmosphere of 95% and 5% CO2. Next, cells were added with 10 μL of silencing or overexpressing plasmids. Ten μL of CCK-8 solution was added, and then cells were cultured in the incubator for 2 h. OD value at 450 nm was read using a SpectraMax iD5 microplate reader .A CCK-8 kit  was applied to detect A549 and ATII cell viability. In brief, transfected cells were plated in 96-well plate and precultured in an incubator at 37°C and 5% COμg/lane) was separated by 11% SDS-PAGE at 110 V for 2 h and transferred to 0.22 μm PVDF membranes . The membranes were incubated with LTBR antibody , CREB1 antibody , IKKα (phospho T23) antibody , IKKα antibody , p52 antibody , RELB antibody , or GAPDH antibody  at 4°C overnight. After washing with PBS, membranes were incubated with the secondary antibody IgG  for 2 h at room temperature. Proteins were detected by enhanced chemiluminescence  based on the manufacturer's instructions. Protein band grey intensity was quantified using the Quantity One software .Total protein was extracted from lung tissues and A549 and ATII cells using RIPA lysis buffer . Protein concentration was measured with a BCA protein assay kit . Total protein  for 15 min at room temperature in dark. Finally, the cells were resuspended in 400 μL 1 × Binding Buffer, mixed on ice, and analyzed by FACS using flow cytometry  within one hour. Cells were gated into four groups: Annexin V−/PI−, Annexin V+/PI+, Annexin V+/PI−, and Annexin V−/PI+. In this study, cell apoptosis rate was defined as the percentage of Annexin V+/PI+ cells (in late apoptosis stage).A549 and ATII cells were harvested, washed with PBS, and fixed with 1% paraformaldehyde overnight at 4°C. Next, cells were washed again, resuspended in 100 μg, Abcam) or anti-IgG  for one night. After washing and elution, samples were treated with 5 M NaCl for heating, and then incubated with proteinase K for 1 h. The bound DNA fragments were purified via DNA Extraction Kit  and analyzed by real-time PCR.This assay was performed with a ChIP assay kit  following the manufacturer's instructions. Briefly, A549 and ATII cells were cross-linked with 1% formaldehyde and sonicated to shear DNA to lengths between 200~1000 base pairs. Cell lysates were incubated at 4°C with protein A/G beads coated with the anti-CREB1 antibody .Wild type (WT) LTBR gene promoter at different sites and the mutated (wt) sites were subcloned into the pGL3 luciferase reporter vector (GeneCopoeia). Mutation was performed using a QuickMutation™ Site-Directed Mutagenesis Kit . HEK-293 T tool cells at the concentration of 5 × 10t-test and one-way ANOVA with Dunnett's or Tukey's post hoc test. A p < 0.05 was deemed as statistically significant. For in vitro studies, three biological replicates × three technical replicates were applied.Data are presented as mean ± SD. For expression and luciferase activity values, data are represented as fold changes to mean control. For cell viability values, data are represented as percentage (%) of mean control. GraphPad Prism 8  was used to perform statistical tests including unpaired two-tailed Student's 2 in A549 cells  expression data of LTBR in clinical lung tissues of BPD are lacked; (2) there is a close association of LTBR and WNT/ignaling , while tκB pathway and further modulated lung epithelial cell viability and apoptosis. This study may aid in understating the pathogenesis and treatment of BPD.In conclusion, this work innovatively demonstrated the upregulation of LTBR and CREB1 in BPD. LTBR and CREB1 are positive regulators of cell apoptosis and negative regulators of cell viability of A549 and ATII cells under hyperoxia. CREB1 induced LTBR expression at the transcriptional level to regulate NF-


Synema Simon, 1864 is a relatively large genus of family Thomisidae Sundevall, 1833 and currently includes 124 species distributed worldwide, except for the Polar Regions. However, Synema can be regarded as being poorly represented in China, with only seven species, three of which are endemic.Synema from Guiyang City in China, is described under the name of S.guiyang J. Zhang, Q. Lu & H. Yu, sp. nov. Detailed descriptions and photographs of the new species are provided. DNA barcodes  of the species were obtained to confirm matching of the sexes and for future use in molecular studies.A new spider species of the genus  Thomisidae is one of the largest spider families, with 171 genera and 2159 valid species distributed worldwide, 51 genera and 306 species of which are recorded from China , Tmarus Simon, 1875 (226 species) and Thomisus Walckenaer, 1805 (143 species) . Currently, only seven Synema species are known from China, amongst them, three are endemic . Despite being common, the genus remains inadequately studied because: more than half of the species are known from a single sex or juveniles  ; original descriptions are rather brief and lack illustrations or illustrations are inadequate  . CurrentSynema specimens of crab spiders in the same location, which are with similar habitus, markings, leg spination and other characters .A DNA barcode was also obtained for the species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for 21 specimens, using the primers LCOI1490 (5’-GGTCAACAAATCATAAAGATATTG-3’) and HCOI2198 (5’-TAAACTTCAGGGTGACCAAAAAAT-3’). For additional information on extraction, amplification and sequencing procedures, see All measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters were taken at the widest point. The total body length does not include the length of the chelicerae or spinnerets. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follow All specimens are deposited at the Museum of Guizhou Education University, Guiyang, Guizhou, China .J. Zhang, Q. Lu & H. Yusp. n.BB6AFCEC-3FC5-5F68-A0A0-CFB80E085396C659D2A5-4612-4DCA-9EA3-1A43DBB0A5DCType status:Holotype. Occurrence: recordedBy: Hao Yu; individualID: YHTHO002; individualCount: 1; sex: male; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: GenBank: ON435709; Taxon: order: Araneae; family: Thomisidae; genus: Synema; specificEpithet: guiyang; scientificNameAuthorship: J. Zhang, Q. Lu & H. Yu,; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Guiyang City; locality: Guiyang Forest Park; decimalLatitude: 26.55540319; decimalLongitude: 106.75898910; Identification: identifiedBy: Hao Yu; dateIdentified: 2021-11; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2021; month: 8; day: 10; Record Level: institutionID: MGEU; basisOfRecord: PreservedSpecimenType status:Paratype. Occurrence: recordedBy: Hao Yu; individualID: YHTHO003; individualCount: 1; sex: female; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: GenBank: ON435708; Taxon: order: Araneae; family: Thomisidae; genus: Synema; specificEpithet: guiyang; scientificNameAuthorship: J. Zhang, Q. Lu & H. Yu,; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Guiyang City; locality: Guiyang Forest Park; decimalLatitude: 26.55540319; decimalLongitude: 106.75898910; Identification: identifiedBy: Hao Yu; dateIdentified: 2021-11; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2021; month: 8; day: 10; Record Level: institutionID: MGEU; basisOfRecord: PreservedSpecimenMale (holotype)  Fig. A–C. TotaCarapace Fig. A, C yellAbdomen Fig. A–C elongLegs basically yellowish-brown Fig. E, all lePalp Fig. A–D. TibiFemale .DNAbarcode: 5'TATTTGGAGCTTGATCTGCTATAGTAGGGACGGCTATAAGAGTGTTAATTCGTATGGAATTAGGA AGATCTGGAAGATTATTAGGAAATGATCATCTTTATAATGTAATTGTTACCGCTCATGCTTTTGTCATGATTTTTTTTATA GTAATACCTATTTTAATTGGGGGTTTTGGAAATTGATTAGTACCTTTAATGTTAGGGGCTCCTGATATATCTTTCCCTCG GATAAATAATTTATCTTTTTGATTATTACCCCCTTCATTATTTTTACTATTTATATCTTCTATAGTAGAGGTAGGTGTGGGG GCAGGATGAACTGTTTATCCTCCTCTAGCTTCTAGAGTTGGGCATATAGGAGGATCTATAGATTTTGCTATTTTTTCTTT ACATTTAGCTGGGGCTTCTTCTATTATAGGGGCGGTTAATTTTATTTCTACTATTATTAATATACGAACTAGAGGTATAAG AATAGAAAAGGTTCCTTTGTTTGTATGATCTGTATTAATTACAGCTATTTTACTTCTTTTGTCTTTACCTGTATTAGCAGG TGCTATTACTATATTATTAACTGATCGTAATTTTAACACTTCTTTTTTTGATCCTGCAGGGGGAGGGGATCCAATTTTATT TCAACATTTGTTTTGATTTTT3' .S.albomaculatum and S.chikunii in having the similar habitus  ; anterior metatarsi and tibiae with dense hairs (vs. few hairs in Spilosynema); PER slightly recurved (vs. distinctly recurved in Spilosynema). Strictly based on this diagnosis, it seems that the new species should be assigned to the genus Spilosynema . Therefore, it is very strange (or interesting) that S.guiyang sp. nov. exhibits typical somatic features of Spilosynema and genitalic features of Synema. In view of the fact that somatic characters are either poorly marked or variable, they are thus not sufficient for distinguishing the Spilosynema and Synema. Consequently, S.guiyang sp. nov. is assigned tentatively to the genus Synema in the present paper for the lack of a better solution.However, some genitalic characteristics are not mentioned in the diagnosis of ned Fig. B, D. In