Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established.VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn VARP is bound to endosomes and functions as a protein:protein interaction platform. Here, the authors present the NMR structure of the complex between the retromer subunit VPS29 and a VARP Zn-fingernail microdomain that is structurally distinct from Zn-fingers and further show that mutations, which abolish VPS29:VARP binding, inhibit trafficking from endosomes to the cell surface. At the heart of all retromer-based coats is a 1:1:1 core heterotrimer comprised of VPS35 , VPS26 (arrestin-like fold) and VPS29 (inactive phosphatase fold), which can be identified across nearly all eukaryotic organisms5. Loss of retromer in mice is embryonically lethal7, and mutations in the core retromer components are linked to Alzheimer’s and Parkinson’s diseases (reviewed in ref. 8). In mammalian cells, retromer-based coats also contain other components including the PI3P/PI3,5P2-binding PX domain-containing sorting nexin (SNX) proteins 12 and associate with regulatory proteins/protein complexes including WASH complex14, TBC1D516, RME817, EHD1 and Rabankyrin518.Retromer complex-based coated vesicular-tubular structures mediate retrograde transport of a vast array of transmembrane proteins of widely differing function from endosomes to the cell surface or the TGN  VAMP722; is a Rab32/38 effector23; and is a GEF for Rab21/524  were created and a Surface Plasmon Resonance (SPR) assay was developed to study their binding to VPS29 under different conditions  proved unsuitable primarily due to the constructs’ rapid precipitation on stirring).The two mammalian VARP CHPLCxCxxC sequences differ only slightly in their core sequences but their 20–30 residue flanking sequences differ considerably Fig.  and are KD was measured using both equilibrium and kinetic analysis, both of which yielded a value of 2–3 µM at 12 °C.  and the reaction is exothermic. When the experiment was repeated using protein that had been dialyzed overnight against buffer lacking Zn++ but containing 10 mM EDTA in order to remove bound Zn++, and the assay carried out in buffer lacking Zn++ and containing 10 mM EDTA, the measured binding dropped ~20 fold to 45 µM . A similar weaker KD (55 µM) was also obtained in the presence of Zn++ ions when all four Cys residues, which we believe coordinate a single Zn++, were mutated to serines  terms that maintain similarity to a fixed template structure of VPS29 derived from PDB 2R17  data showed that the crystal structure of VPS29 is maintained in solution and does not change appreciably upon the formation of the complex with VARP 692–746  – Pro 713O (VARP) and Tyr 163 OH (VPS29) – Cys 711O (VARP), the latter probably being water-mediated. Simultaneous mutation of the His and Leu residues from the loop in VARP to alanines abolishes binding to wt VPS29 in our SPR assay , consistent with a block in recycling of GLUT1 from endosomal compartments to the plasma membrane. This could be rescued in transiently transfected HeLa cells expressing wtVPS29-tagRFP but not by the L26S, Y165S and L152E mutant versions of VPS29-tagRFP  on endosomes similarly to wt VPS29-TagRFP, indicating that they were correctly folded . We did not investigate further as the main focus of this work is not on TBC1D5.The lack of rescue by the L152E mutant was consistent with our previous dataDs in the 100 μM range. Notably, conformational restraining of an Asn Pro Phe motif with a non-physiological disulphide link causes a ~10 fold reduction for its cognate EH domain in KD to ~10 μM40. Similarly, removal of the Zn++ ion destabilizes the VARP Zn-finger domain, thereby reducing its affinity to VPS29. However, it is possible to regain this affinity by adding back Zn++ ions since in bio-layer interferometry it was shown that GST VARP 692–746 (fingernail 2) bound VPS29 prior to the EDTA treatment and following EDTA treatment and subsequently refolding into buffer containing Zn++ with similar affinities , the motif is actually part of a structurally defined fold (the Zn-fingernail) resulting in a decrease in entropy loss and hence relatively tight binding of 2–3 μM for the motif when compared with similar motifs such as Asp Pro Phe, Asp Pro Trp (binding AP2 α-adaptin appendage) and Asn Pro Phe (binding EH domains) all of which consist of a proline flanked by a hydrophilic and a hydrophobic residues and have K41 (PDB ID 5GTU)) were reported45. Notably, these studies used a slightly longer isoform of VPS29 that has the N-terminal sequence MAGHRLVLVL (referred to here as long VPS29) instead of MLVL; the latter was the originally reported isoform46 and is the one used in this work. Importantly, the affinities of either VPS29 isoforms for VARP were similar: KDs were 1.3 and 2.7 µM respectively, with equivalently fast on-rates  and a small peptide fragment of the endosomal Rab7 GAP TBC1D5++ cluster  due to a large total interaction interface of 850–920 Å2, consistent with its ability to outcompete VARP and TBC1D5 binding45  to the long VPS29, which is weaker than has been reported by ITC (residues 132–158: ~20 µM43). The interaction of short VPS29 lacking MAGHR was too weak to assess reliably (KD > 300 μM) likely due to factors including the interaction being mediated only by the Asn Pro Leu of TBC1D5 ; and Rab7:GTP-dependent retromer recruitment16 would be difficult as the GTP on the Rab7 would be immediately hydrolysed. Use of different relative amounts of the two versions of VPS29, for instance in different cell types, could bias the ratio of VARP to TBC1D5 in a given retromer coat, with various physiological outcomes, e.g., amount of Rab7 or VAMP7, resulting (see later).Both RidL and TBC1D5 bind to the same site on VPS29 as do the VARP Zn-fingernails using proline and leucine/isoleucine residues, but neither possesses a Cys/Znter Fig. . Bindingg45 Fig. . The MAGg45 Fig. . A shortARP Fig. ) contactChaetomium thermophilum and Chlamydomonas reinhardtii retromer coats reveals the presence of VPS35 dimers forming ~100 Å high arches extending away from the membrane with two VPS29 molecules placed at each apex47 .One Zn-fingernail can be accommodated on the surface of one Leu152-centred binding site on VPS29 Fig. . The recx47 Fig. . The exirch Fig.  would ea19). Immunoelectron microscopy, which allows short, retromer- and VARP-positive tubules to be resolved from the endosomal body from which they emanate, suggests that the retromer and VARP are enriched on the tubular processes  linkers on the Zn-fingernails. Efficient VARP recruitment into retromer-based coats is easiest to envisage occurring as the retromer coat assembles: either if the VARP is already dynamically localized to the endosome surface through transient interactions with its membrane-associated binding partners and it ‘reaches up’ to the arches or if it docks from the cytosol and ‘reaches down’ to its partners  and the ANKRD2 domains of VARP to membrane-associated VAMP7 and Rab21 will be facilitated by the unstructured   Fig. . Nonethezoa Fig. . In VPS2zoa Fig. . Finallyzoa Fig.  across e59, the former acting in late endosomal function and thus being inactivated by TBC1D5, and the latter likely acting to promote fusion of the tubule with the plasma membrane. A VARP-like protein would have acted as a GEF on Rab21, on internal membranes and the plasma membrane but likely independently from retromer60. In the opisthokont ancestor  or with both 15NH4Cl (0.5 g/L) and 13C6-glucose (2 g/L)) . Expression was carried out for 18–20 h at 28 °C following induction of expression by addition of 0.2 mM IPTG at OD600nm of 0.8.All constructs used are summarised in Supplementary Table 2H11 Tris buffer pH 7.0, 200 mM NaCl, 1 mM 2H6 DTT either in a 95:5 H2O:2H2O mixture or in 2H2O, using a VivaSpin 3000 mwco spin filter. In the latter case, buffer exchange was repeated five times with a 3–5 fold dilution so as to reduce the level of H2O from the original buffer to <0.5%. VPS29 was expressed from pGEX4T2 and purified as described above except that cleavage was carried out with thrombin.Proteins were purified in 20 mM Tris, 100 mM NaCl, pH7.4, 0.2 mM βME, 0.2 mM AEBSF on GST-Sepharose. Prescission cleavage was carried out overnight at 4 °C if needed with 0.5 mg Prescission Protease. Eluted proteins were further purified on S75 Superdex gel filtration in the same buffer. Samples for NMR were subsequently buffer exchanged into 20 mM Experiments were performed on a Biacore T200 . In the first instance, goat anti-GST polyclonal antibody was coupled to Series S CM5 Sensor Chips  by the amine coupling protocol specified in the Biacore T200 Control Software with reagents purchased from GE Healthcare Life Sciences. In brief, after the sensor surfaces were activated by applying a 1:1 mixture of 50 mM N-hydroxysuccinimide (NHS): 200 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) provided in the Biacore Amine Coupling Kit , the anti-GST antibody was diluted to 30 μg/ml in 10 mM sodium acetate, pH 5.0  and injected into all flow cells at 5 μl/min for 7 minutes, (resulting in ~10000RU (resonance units) of protein immobilized). Ethanolamine  was then injected to block remaining active sites on all four flow cells. In the next step, in order to block any high affinity sites that are present on the anti-GST pAB, 3 cycles using a 3-min injection of recombinant GST at 5 μg/ml followed by regeneration with 10 mM glycine-HCl pH 2.1 were performed.2, 0.1 mM TCEP, 0.5 μM ZnCl2, pH7.4). Two flow cells could be analysed at a time. Initially, GST-tagged proteins are injected separately into each flow cell, at 10 μg/ml at 15 μl/min for 200 s. Therefore, the blank flow cell contained GST-CLA and the second flow cell displayed GST-VARP Zn-fingernail, its mutants or GST-TBC1D5 (residues 132–156). GST-CLA is derived from the β2 adaptin subunit of human AP2 (UniProt P63010), residues 623–632, and therefore would not bind VPS29 specifically. After a stabilization period of 300 s, analytes typically between 40 and 0.3μM concentration were then injected at 10 μl/min for 300 s, followed by 300 s wash (dissociation period). Importantly, fast on and off binding events to the different GST-Zn-fingernails, as well as the GST-TBC1D5 (residues 132–156) were observed. Between each sample injection, the anti-GST antibody on the chip was regenerated by injecting 10 mM glycine-HCL pH2.1 at 30 μl/min for 60 s. At 12 °C, each flow cell could be efficiently regenerated ~50 times.The binding analysis was performed between 8 and 25 °C in VPS29 gel filtration buffer  for binding were determined from the best fit curves to the equation for a single binding site between the VARP Zn-fingernail and VPS29. On and off-rates, and resultant KD, were also calculated, using Rmax determined from the equilibrium analysis. By acquiring binding data at five different temperatures (between 4 and 25 °C), the Van ‘t Hoff equation was plotted to estimate changes in binding enthalpy and entropy. The linear form of the Van ‘t Hoff equation:Sensorgrams were processed with Biacore T200 Evaluation Software . Equilibrium dissociation constants  was used, due to the clear linearity of ln KD plotted against 1/T in that temperature range (R2 = 0.91). The equation assumes that the enthalpy and entropy are constant with temperature changes. Under standard conditions, at 25 °C, the binding is therefore characterized by favorable enthalpy and entropy changes (ΔH° ~ −20 kJ mol−1 and ΔS° ~ +36 J K−1 mol−1).(where 2) for at least 30 minutes prior to loading with GST-VARP-692-746 for 10 minutes. Every binding experiment consisted of three steps: incubation for 10 minutes in the specific assay buffer (200 mM NaCl 20 mM Tris pH 7.4 0.1 mM TCEP with either 0.5μM ZnCl2 or 10 mM EDTA) followed by 5 min incubation with VPS29 in the same buffer  (association phase), and then by 10 min incubation with the same buffer without VPS29 in order to measure VPS29 off-rate (dissociation phase). Three sequential binding experiments were therefore performed on each biosensor tip presenting GST-VARP-692-746: the first in the presence of Z++ ions, the second after removal of Zn++ ions (through the use of 10 mM EDTA), and the third in the presence of re-introduced Z++ ions. Importantly, in order to remove Zn++ ions, the biosensor-bound GST-VARP was incubated twice for 15 minutes in (200 mM NaCl 20 mM Tris pH 7.4 0.1 mM TCEP 10 mM EDTA). After binding in the presence of EDTA, the biosensor-bound GST-VARP was reloaded with Zn++ for 30 min in (200 mM NaCl 20 mM Tris pH 7.4 0.1 mM TCEP 0.5 mM ZnCl2). Data were processed with the Octet Data Acquisition 7.0 software, individually fitted using ForteBio Analysis Software 7.0, and subsequently further analysed with the PRISM software (GraphPad) in order to determine equilibrium dissociation constants (KD). Although VPS29 binding in the presence of 10 mM EDTA was observed to be non-specific and random, both binding experiments in the presence of 0.5 μM Zn++ gave similar binding profiles and equilibrium constants.Real-time kinetic measurements of VPS29 binding to immobilized GST-VARP-692-746 at 25 °C were conducted using an Octet RED96 . Samples and buffers were dispensed into 96-well microtiter plates (Greiner) at a volume of 200 µl per well. Anti-GST biosensors  were hydrated in VPS29 gel filtration buffer  and a non-native Trp residue (here numbered 747). Similarly, the VPS29 samples carried an additional non-native N-terminal sequence GSPEFGTRDR, here numbered -9-0. Samples of complex were prepared by mixing the required amounts of appropriately labelled VPS29 and VARP 692–746. NMR data were acquired using Bruker AV-I 600 and AV-III 800 spectrometers, each equipped with a cryogenically cooled triple resonance (1H/15N/13C) 5 mm probe. Experiments were conducted at 25 °C unless otherwise stated, and 1H chemical shifts were calibrated using sodium 3,3,3-trimethylsilylpropionate (TSP) as an external 1H reference; 15N and 13C chemical shifts were indirectly referenced to the 1H shifts using the ratio of gyromagnetic ratios31NMR samples of VPS29 comprised 0.3–0.6 mM 2O samples of 15N,13C-labelled free VSP29 the following datasets were acquired: 2D datasets: [15N-1H] HSQC, [13C-1H] HSQC , [13C-1H] HSQC (aromatic region in 13C), constant-time [13C-1H] HSQC , constant-time [13C-1H] HSQC (aromatic region in 13C); 3D datasets: CBCANH, CBCA(CO)NH, HNCA, HN(CO)CA, HBHANH, HBHA(CO)NH, [1H-13C-1H] HCCH-COSY, [1H-13C-1H] HCCH-TOCSY, [13C-13C-1H] HCCH-TOCSY, 15N NOESY-HSQC (τm = 150 ms), 13C NOESY-HSQC . For 2H2O samples of 15N,13C-labelled free VSP29 the following datasets were acquired: 2D datasets: [1H-1H] DQ correlation; 3D datasets: 13C NOESY-HSQC .For H2O samples of 15N,13C-labelled free VARP 692–746 the following datasets were acquired: 2D datasets: [15N-1H] HSQC, [13C-1H] HSQC , constant-time [13C-1H] HSQC , constant-time [13C-1H] HSQC (aromatic region in 13C), [1H-1H] NOESY (τm = 150 ms); 3D data sets: CBCANH, CBCA(CO)NH, HBHANH, HBHA(CO)NH, [1H-13C-1H] HCCH-COSY, [1H-13C-1H] HCCH-TOCSY, [13C-13C-1H] HCCH-TOCSY, HACAHB, HNHB, 15N NOESY-HSQC (τm = 50 ms), 15N NOESY-HSQC (τm = 150 ms), 13C NOESY-HSQC , 13C NOESY-HSQC .For H2O samples of complex containing 1:1 15N,13C-labelled VSP29 and natural abundance VARP 692–746, the following datasets were acquired: 2D datasets: [15N-1H] HSQC, [13C-1H] HSQC , [13C-1H] HSQC (aromatic region in 13C), constant-time [13C-1H] HSQC , [1H-1H] NOESY ; 3D datasets: HNCA, HN(CO)CA, CBCANH, CBCA(CO)NH, HBHA(CO)NH, [1H-13C-1H] HCCH-TOCSY, [13C-13C-1H] HCCH-TOCSY, 15N NOESY-HSQC (τm = 150 ms), 13C NOESY-HSQC , 13C NOESY-HSQC . For 2H2O samples of complex containing 1:1 15N,13C-labelled VSP29 and natural abundance VARP 692–746, the following datasets were acquired: 2D datasets: [13C-1H] HSQC , [13C-1H] HSQC (aromatic region in 13C), constant-time [13C-1H] HSQC , constant-time [13C-1H] HSQC (aromatic region in 13C), [1H-1H] NOESY ; 3D datasets: [13C-13C-1H] HCCH-TOCSY, 13C NOESY-HSQC , 13C NOESY-HSQC , 13C NOESY-HSQC , 13C NOESY-HSQC .For H2O samples of complex containing 1:1 natural abundance VSP29 and 15N,13C-labelled VARP 692–746, the following datasets were acquired: 2D datasets: [15N-1H] HSQC at 25 °C, 10 °C and 2 °C, [13C-1H] HSQC ; 3D datasets: CBCA(CO)NH, HNCA, HN(CO)CA, HBHA(CO)NH, HNHA, [1H-13C-1H] HCCH-TOCSY, [13C-13C-1H] HCCH-TOCSY, 15N NOESY-HSQC , 13C NOESY-HSQC . For 2H2O samples of complex containing 1:1 natural abundance VSP29 and 15N,13C-labelled VARP 692–746, the following datasets were acquired: 2D datasets: [13C-1H] HSQC , [13C-1H] HSQC (aromatic region in 13C), [1H-13C-1H] HCCH-COSY, [13C-13C-1H] HCCH-TOCSY, 13C NOESY-HSQC , 13C NOESY-HSQC .For H13C experiments, a period of 15N decoupling equal in length to the acquisition period was applied at the beginning of the inter-scan delay, and for 15N experiments an equivalent period of 13C decoupling was similarly applied. All spectra were processed using the program TOPSPIN versions 3.1 and 3.2  and analysed using the program CCPN analysis64.All of the NOESY datasets used for structure calculations (see below) were acquired using pulse sequences modified to ensure equal RF heating in each case, e.g., for 1H))2 + (Δδ (15N)/5.0)2).Backbone amide group chemical shift perturbations were calculated for VPS29 and for VARP 692–746 using the assignments described above using the formula Δδ =  √((Δδ(65. The conformation of the majority of the VPS29 component was restrained to a template conformation adapted from a previously published X-ray structure (see below), while the conformation of the VARP peptide and interfacial residues of VPS29 (selected on the basis of preliminary structures) were allowed to evolve under a combination of intermolecular and intra-peptide NOE-derived distance restraints, as well as limited NOE-derived restraints for the interfacial region of VPS29 and J-coupling-derived χ1 dihedral angle restraints for the interfacial region of the VARP peptide. The NOE restraints were classified into very strong (0–2.3 Å), strong (0–2.9 Å), medium (0–3.5 Å) and weak (0–5.0 Å) intensity categories. The upper distance bounds used for these categories were calibrated using assigned NOE cross-peaks in the unfiltered NOESY spectra of samples in which the Vps29 component was labelled; specifically, the intensity corresponding to the very strong category (0–2.3 Å) was set using sequential dαN peaks in regions of regular anti-parallel β-sheet, that of the strong category (0–2.9 Å) was set using sequential dNN peaks in regions of regular α-helix, that of the medium category (0–3.5 Å) was set using  dαN peaks in regions of regular α-helix, and those in the weakest category were set to be consistent with the expected approximate detection limit for NOEs, set as 5 Å. This calibration was extended for use in the various types of filtered spectra, in each case by comparing the corresponding measured intensities for particular specific NOE connections as they appeared in both the filtered and unfiltered spectra, using only signals that could be observed free of overlap in both spectra. As a check that the calibration of the intermolecular NOE-based restraints was consistent with the structure calculations and force field, test calculations were also run in which the upper bounds for just these restraints in the final set were either tightened or loosened slightly; it was found that tightening them caused a significant increase in their violations, whereas loosening had relatively little effect since violations were already very few.Structural models of the VPS29-VARP complex were generated using a hybrid NMR/X-ray crystallographic approach, using simulated annealing calculations run with the program XPLOR-NIH66, with all seleno-Met residues changed to Met and the 11–12, 40–41 and 91–92 peptide bonds set to the cis conformation; this structure was selected since it lacks the protruding conformation of helix 2 (95–108) seen in PDB 1Z2X27 that makes a crystal contact to its counterpart in a symmetry-related molecule and which is inconsistent with NOE data from the present study67. The Leu40-Cys41 peptide bond was modelled in the cis conformation because (i) in the highest resolution structure of VPS29 (pdb 5GTU) this peptide was found to be cis43, and (ii) modelling the trans conformation for Leu40-Cys41 led to persistent, severe outliers in the Ramachadran plot for this region both in pdb 2R17 and in trial calculations for our system. The protein co-ordinates from 2R17 were adapted as follows: Hydrogen atoms were first added according to standard geometries, then all atoms except for the guanidinium protons of all arginines and all atoms within 5 Å of residues 40 and 41 were fixed and the structures then subjected to Powell energy minimization (1000 steps), Langevin dynamics at 1000 K , increase of the van der Waals force constant and tilting of the NOE potential function asymptote in 2000 step cycles, switching to a square-well NOE function then cooling to 300 K in 2000 step cycles and final Powell minimization (1000 steps); this phase of the calculation allowed adaptation to the cis conformation of the 40–41 peptide bond as well as resolving atom definition issues for the arginines. All atoms except backbone amide N, C’ and O were then released and the structure subjected to 200 cycles of Powell minimization, after which all atoms were released and the structures again subjected to Powell minimization (100 steps). Only very small movements of the protein backbone occurred during this minimization; for residues 1–181) the backbone co-ordinate shift  was 0.150 Å.The VPS29 template structure was derived from residues 1–181 of the VPS29 component of the VPS29-VPS35 complex in pdb 2R1768 with a force constant of 1 kcal mol−1. Since the XPLOR-NIH calculations employed r−6 summation for all groups of equivalent protons and non-stereospecifically assigned prochiral groups, and since no stereo-assignments were made , all constraints involving protons within such groups were converted to group constraints (by using wildcards such as HB*). All lower bounds were set to zero69. Stage 1 of the two-stage protocol comprised Powell energy minimization (500 steps), Langevin dynamics at 1000 K (5000 steps), increase of the van der Waals force constant and tilting of the NOE potential function asymptote in 1000 step cycles, switching to a square-well NOE function then cooling to 300 K in 500 step cycles and final Powell minimization (500 steps). The force constants used for both the distance and the dihedral angle restraints were 50 kcal mol−1. In stage 2, zinc ions were first defined and placed at the geometric average of the co-ordinates of the four zinc-binding cysteinyl sulfur atoms, and all necessary bond and angle terms added to the force field, following which structures were subjected to Powell energy minimization (500 steps), Langevin dynamics at 1000 K while progressively increasing the torsion angle force constant in 1000 and 2000 step cycles, switching to a square-well NOE function then cooling to 300 K in 250 step cycles and final Powell minimization (1000 steps).To calculate an ensemble of models for the complex, 50 starting structures were first created by randomizing the backbone ϕ and ψ angles of VARP 687–747 and placing the peptide at a random distance (Gaussian distribution around 150 Å) and orientation relative to a copy of the template structure for VPS29. These starting structures were then subjected to a two-stage simulated annealing protocol to generate an ensemble of conformers consistent with the NOE and dihedral angle restraints, all while applying the Ramachandran database potential of mean forceThroughout these calculations, strong NCS constraints were used to maintain the internal structure of the VPS29 component. To achieve this the VPS29 structure was duplicated, the co-ordinates of one copy shifted by 500 Å then rigidly fixed and groups of NCS constraints defined between the fixed and unfixed copies so as to maintain the VPS29 structure in the evolving co-ordinates of the unfixed copy. Once preliminary rounds of calculation had established likely regions where contacts between VPS29 and VARP might occur, the NCS terms for just these regions of VPS29 were restricted to backbone atoms and were applied with a much reduced force constant; the NCS constraints used in the final rounds of calculations are shown in Table Finally, the remaining atoms of the VPS29 N- and C-terminal tails (residues -9-0 and Ser 182) were added in a separate simulated annealing protocol. Initially all atoms of the full structure were placed at fully randomized positions within a 200 Å cube, then for those residues included in the previous stages of the calculations  the randomized co-ordinates were replaced by the previously calculated values and rigidly fixed in place. The structures were then subjected to 1000 cycles of Powell minimization, followed by 5000 steps of Langevin dynamics at 500 K, increase of the van der Waals force constant in 1000 step cycles, cooling to 300 K in 1000 step cycles and final Powell minimization (1000 steps). This protocol for adding the tails and linker was repeated independently five times for each input structure using a different randomization seed each time, and the structure with the lowest value of E retained.70 and are as follows: for VARP 710–721: most favoured 60%, additionally allowed 40%, generously allowed 0.0%, disallowed 0.0%; for VPS29 1–181 : most favoured 89.3%, additionally allowed 10.0%, generously allowed 0.0%, disallowed 0.7%. The program CLUSTERPOSE71 was used to calculate the mean rmsd of ensembles to their mean structures, and structures were visualized and mean pairwise rmsd values were calculated using the program PYMOL. Ensembles were superposed using the co-ordinates of their respective average structures; the average structures themselves are not shown.The 25 models with lowest total energy were accepted to form the final ensemble Fig. . Ramacha19 cultured under standard conditions in RPMI supplemented with 10% (vol/vol) FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. siRNA-mediated gene knock-down was performed as described before19. In short, cells were transfected (two transfections separated by 48 h) with 100 nM oligonucleotide using Oligofectamine (Life Technologies), according to the manufacturer’s protocol. On-Target Plus oligonucleotide catalogue numbers (Dharmacon/Thermo Scientific) were: non-targeting (NT) control (D-001810-01) and VPS29-1 (J-009764-09). For VPS29 ‘rescue experiments’ and colocalization studies, cells were seeded on glass coverslips and transfected with siRNA-resistant VPS29-TagRFP constructs 24 h after the second knock-down using FuGENE® 6 Transfection Reagent (Roche), according to the manufacturer’s protocol. 24 h after transfection, cells were fixed or incubated for 2 h in fresh cell culture medium prior to fixation (VPS29 ‘rescue experiments’). Knock-down of VPS29 (with actin as a control) and over-expression of VARP-GFP were assessed by immunoblotting using primary antibodies to VPS29 , actin , VARP  and secondary antibodies to IgG .HeLaM cells or HeLaM cells stably expressing VARP-GFP22. When indicated, the cytosol was extracted prior to fixation by rinsing cells briefly with PBS (with Ca2+ and Mg2+), followed by incubation for 30 s in 0.05% saponin in PBS (with Ca2+ and Mg2+), and then immediately formaldehyde fixed. Colocalization of transiently transfected VPS29-TagRFP constructs with either endogenous VPS35 or VARP-GFP was quantified using Pearson’s Correlation Coefficient, calculated using the Zeiss Zen software. Quantitative colocalization of GLUT1 with LAMP1 was performed essentially as previously described19 with cell fields randomly selected based on nuclear stain, transfected cells identified by the presence of TagRFP, and focusing using the LAMP1 signal (two independent experiments with 10 fields per condition and a minimum of 20 cells per condition). Single confocal images of cells were acquired corresponding to 1 Airy unit, and the degree of colocalization in individual cells was measured by Manders’ Colocalization (Overlap) Coefficient using Zeiss Zen software. Data are presented as box and whisker plots, in which the boxes extend from the 25th to 75th percentiles, the middle line indicates median. and whiskers represent min to max with all points shown. p values were calculated using a one-way Anova with Dunnett’s test in GraphPad Prism 5 , with the assumption that each individual cell is a biological replicate.For immunofluorescence confocal microscopy, cells were fixed with 4% paraformaldehyde in PBS for 10 min. Cells were then permeabilized by incubation with 0.1% (v/v) Triton X-100 in PBS for 5 min and incubated with blocking solution (5% BSA in PBS) for 30 min. This was followed by incubation with primary antibodies used at 1:250 dilution  and then fluorescently conjugated secondary antibodies from ThermoFisher used at 1:1000 dilution . Images were captured using a Zeiss LSM 780 confocal microscope as described elsewhereFor immunofluorescence wide field microscopy, cells were fixed, permeabilized and stained with antibodies as for confocal microscopy, except that when staining for Rab7a, 0.25% (v/v) Triton X-100 was used. Additional primary antibodies used at 1:250 dilution for immunofluorescence wide field microscopy were to VPS26 , Rab7a  and TBC1D5 . Fluorescently conjugated secondary antibodies used at 1:1000 dilution were from ThermoFisher . Cells were imaged using a Zeiss AxioPlan microscope with a X63 PlanAPO objective lens and images captured through a Hamamatsu CCD camera controlled via the manufacturer’s software. The Rab7 antibody used does not discriminate between Rab7 in the GDP- or GTP-state, but even if GDP-bound, Rab7 is unlikely removable from tubule membranes through the action of RabGDI due to steric blocking by the polymerized retromer coat.72. Sequential immunolabelling of frozen sections was performed using 1:100 rabbit anti-GFP  and 1:100 mouse anti-VPS35  as primary antibodies followed by 1:100 rabbit anti-mouse IgG  and protein A gold . EM sections were examined with a FEI Tecnai G2 Spirit BioTwin TEM .For immunoelectron microscopy, cells were fixed with 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and prepared for immunoEM of Tokuyasu frozen sections73. A hidden Markov model was generated using phylogenetically verified holozoan VARP homologues published in Herman et al.56, which was used to search predicted proteins from the genomes listed in Supplementary Table Homo sapiens genome74. A result was considered to be a positive hit if it retrieved VARP or a clear orthologue with an E-value < 0.05. A domain analysis to identify ankyrin repeats was performed using the Conserved Domain Database75, in order to provide further evidence of VARP orthology.VARP homologues were identified using HMMerE-value  < 0.05, and in a reciprocal search into the human genome, retrieved the query or a clear orthologue as a top hit, also with an E-value < 0.05. TBC1D5 proteins were identified by BLASTP using the human sequence as a query (NP_055559), with the same search parameters described for VPS29. As TBC1D5 is closely related to TBC1D13, the identity of putative TBC1D5 sequences was confirmed by phylogenetics.VPS29 homologues were identified by BLASTP searches using the human VPS29 sequence as a query (NP_476528) to search the genomes listed in Supplementary Table 76 with default parameters. All full alignments used to generate supplementary figures are available upon request.Alignments were generated for VARP, VPS29, and TBC1D5 families using MUSCLEFurther information on research design is available in the Supplementary InformationReporting Summary


Antigenic drift of influenza virus hemagglutinin (HA) is enabled by facile evolvability. However, HA antigenic site B, which has become immunodominant in recent human H3N2 influenza viruses, is also evolutionarily constrained by its involvement in receptor binding. Here, we employ deep mutational scanning to probe the local fitness landscape of HA antigenic site B in six different human H3N2 strains spanning from 1968 to 2016. We observe that the fitness landscape of HA antigenic site B can be very different between strains. Sequence variants that exhibit high fitness in one strain can be deleterious in another, indicating that the evolutionary constraints of antigenic site B have changed over time. Structural analysis suggests that the local fitness landscape of antigenic site B can be reshaped by natural mutations via modulation of the receptor-binding mode. Overall, these findings elucidate how influenza virus continues to explore new antigenic space despite strong functional constraints. Antigenic site B in influenza A virus hemagglutinin (HA) is immunodominant in circulating human H3N2 strains. Using deep mutational scanning, Wu et al. here define the local fitness landscapes of HA antigenic site B in six human H3N2 strains, providing insights into evolvability of influenza antigenicity. Throughout the past 50 years, the human H3N2 virus has acquired up to 9 new N-glycosylation sites due to constant pressure for immune evasion7. These N-glycans shield a large portion of the immunodominant HA1 globular head domain to limit antibody access to the major antigenic sites. Nonetheless, the added N-glycans can also negatively influence virus fitness when they are too close to the receptor-binding site (RBS)9. Consequently, there is a functional limit on the number of N-glycosylation sites present at any one time on the HA7. As N-glycosylation sites on the HA1 globular head domain accumulate over time and cover large regions of the HA1 surface, the more exposed membrane distal RBS in the HA head and the membrane proximal HA stem region may become increasingly prone to be targeted by antibodies10. Therefore, it is not surprising that antigenic site B, which significantly overlaps with the RBS, has become immunodominant in recent years12. While the evolution of antigenic site B is driven by immune evasion, the indispensable function of the RBS imposes a strong functional constraint on its evolution . For example, we have previously shown that such functional constraints can limit the mutational reversibility at residue 190, which is part of the antigenic site B as well as the RBS13. However, our understanding of the evolutionary constraints of antigenic site B is far from complete.H3N2 influenza virus was introduced into the human population in 1968. As the main antigen of influenza virus, the hemagglutinin (HA) from H3N2 viruses has five major antigenic sites (sites A–E)14. Deep mutational scanning, which combines saturation mutagenesis and next-generation sequencing so as to measure the phenotype of many genetic variants in parallel15, has been applied to study the fitness landscape of influenza HA18. However, most deep mutational scanning studies on influenza HA have been focused on variants that are only one mutation away from the wild-type (WT) sequence17. As a result, epistatic effects (non-additive fitness effects) between mutations, which are believed to be pervasive, are often ignored19. In addition, it is unclear to what extent epistatic effects are localized in antigenic site B and whether any substantial interaction occurs with the rest of the influenza HA sequence, which also changes during the course of evolution.A fitness landscape refers to the genotype-to-phenotype map of many related genetic variants. Characterization of the fitness landscape of the influenza HA facilitates the understanding of its evolutionary constraints and predictability of naturally occurring mutations20, and sequence variants that are previously unfit may become fit and emerge (evolutionary contingency)20. Structural analysis further reveals that the receptor-binding mode of the human H3N2 virus has itself evolved over time, providing a mechanistic basis for the evolution of the local fitness landscape of antigenic site B. This study provides insights into how antigenic site B is still able to continue evolving in the presence of the strong functional constraints that enable virus entry.In this study, we are interested in understanding how the local fitness landscape of antigenic site B in human H3N2 virus has changed over time due to epistasis when the genetic backgrounds differ for the rest of the protein. The replication fitness of 576 variants of interest in antigenic site B is measured by deep mutational scanning in each of the six different H3N2 genetic backgrounds, namely, A/Hong Kong/1/1968 (HK68), A/Bangkok/1/1979 (Bk79), A/Beijing/353/1989 (Bei89), A/Moscow/10/1999 (Mos99), A/Brisbane/10/2007 (Bris07), and A/North Dakota/26/2016 (NDako16). Our results indicate that the local fitness landscape of antigenic site B is highly dependent on the genetic background, which reveals the pervasiveness of evolutionary entrenchment and contingency during natural evolution of the human H3N2 virus. Specifically, sequence variants that are previously fit may progressively become unfit and become extinct (evolutionary entrenchment)23. We compiled an inventory of major amino acid variants that have reached high occurrence frequency (>90% in any given year) during the natural evolution of human H3N2 viruses over the past 50 years at these six residues of antigenic site B .Deep mutational scanning, which allows the phenotypes of many genetic variants to be measured in parallelUltimately, we obtained six different local fitness landscapes, each with 576 variants. Two biological replicates were performed for each experiment with a high correlation (Pearson correlation = 0.92 to 0.97) observed between replicates . In other words, we were interested in addressing whether the sequence variants at residues 156, 158, 159, 190, 193, and 196  from a naturally occurring strain that was isolated many years ago (up to five decades) would still be fit on the genetic background of a more recent strain, and vice versa. Subsequently, we plotted the preference of individual natural sequence variants  in different focal genetic backgrounds  against the year of strain isolation. This analysis tracked how natural sequence variants from a given year would fit in a given focal genetic background from a different year. A schematic diagram of this analysis is shown in Supplementary Fig. 25, which may explain why the natural sequence variants from HA with high KL divergence from Mos99 are not particularly unfit. Overall, our results suggest that evolutionary entrenchment and contingency are pervasive in the natural evolution of H3N2 HA antigenic site B  at residues 156, 158, 159, 190, 193, and 196 in a given focal genetic background change as the strains diverge from the focal genetic background. The divergences of naturally occurring strains from a given focal year were computed based on the residue-averaged Kullback–Leibler (KL) divergence (see Methods). The entire HA amino acid sequences except residues 156, 158, 159, 190, 193, and 196 were used to compute the KL divergence. A larger KL divergence indicates lower HA sequence identities between the natural variants and the focal genetic background. In most cases, the preferences of the natural sequence variants negatively correlate with KL divergence Fig. . Thus th26. However, interpreting the structure of these high-dimensional landscapes is difficult27 and highly sensitive to experimental noise. Previous work has shown that approximate fitness landscapes with additive and pairwise interactions among residues can accurately describe biophysical properties of proteins, including protein residue–residue contacts29. Additive fitness effects describe the independent contributions of each amino acid variant to fitness, whereas pairwise epistatic interaction effect describes how pairs of amino acids synergistically (positively) or antagonistically (negatively) impact the fitness31. Here we construct a statistical model to infer the additive fitness effects and pairwise epistatic interactions that underlie the six local fitness landscapes (see Methods). This model provides an interpretable description of the high-dimensional experimental landscape in the presence of the fitness measurement noise. Based on the 95% confidence, we classified each parameter into significantly positive, significantly negative, and insignificant deviation from zero . These results are shown in Supplementary Fig. Fitness landscapes of molecules are often complex with both specific and non-specific interactionsInterestingly, a significant number of parameters for additive fitness effects and also for pairwise epistatic effects had different signs in different genetic backgrounds . The crystal structure of Bris07 HA in complex with human receptor analog 6′SLNLN  was determined in our previous study. The cryved Fig. . GlcNAc-ved Fig. . For exaved Fig. . This roved Fig. . Of note13. In addition, the receptor specificity of human H3N2 viruses has been shown to be constantly evolving34.Overall, this analysis demonstrates that natural mutations in the base of RBS can influence the conformation and mode of binding of the receptor. Since antigenic site B also interacts with the receptor Fig. , changesp value = 0.007). In contrast, the similarity in the local fitness landscape of antigenic site B among different genetic backgrounds did not have significant correlation with the pairwise amino acid sequence identity of the entire HA ectodomain . This analysis substantiates the notion that the amino acid sequence at the base of the RBS can modulate the local fitness landscape of antigenic site B.To further understand how the amino acid sequence in the base of the RBS may influence the local fitness landscape of antigenic site B, we compared the pairwise amino acid identity in the base of the RBS and the pairwise correlation of the local fitness landscape of antigenic site B among different genetic backgrounds. Here the base of the RBS is defined as residues 135, 137, 145, 222, 225, and 226. The amino acid sequence of the base of the RBS for each of the strains of interest is shown in Supplementary Fig. The evolution of antigenic site B is functionally constrained owing to its significant overlap with the RBS. Subsequently, antigenic site B can only tolerate mutations that do not negatively impact receptor binding. This study demonstrates that evolutionary entrenchment and contingency are highly prevalent in antigenic site B. Such observations indicate that the evolutionary constraints of antigenic site B have been changing during the course of human H3N2 virus evolution. Concomitantly, the receptor-binding mode of human H3N2 viruses has been  evolving Fig. . With co36, it seems extremely difficult to predict the evolution of influenza virus at amino acid resolution. A previous study has shown that selection of antigenically advanced variants under antibody pressure can help predict the emerging mutations in the next antigenic drift37. However, our work here shows that, in antigenic site B, a mutation that has high fitness in one strain may be highly deleterious in another strain. The local fitness landscape of antigenic site B has been changing extensively throughout the evolution of human H3N2 viruses over the past 50 years and can be totally uncorrelated between strains that have been isolated far apart in time  coding region from A/Hong Kong/1/1968 (HK68)18. For HA, the ectodomain was from HK68, whereas the non-coding region, N-terminal secretion signal, C-terminal transmembrane domain, and cytoplasmic tail were from A/WSN/33. For NA, the entire coding region was from HK68, whereas the non-coding region of NA was from A/WSN/33. The sequences of the HA ectodomains are indicated in Supplementary Data All H3N2 viruses generated in this study were based on the influenza A/WSN/33 H1N1) eight-plasmid reverse genetics systemN1 eight-38. Primers for the insert contained the combinatorial mutations of interest and are shown in Supplementary Table Mutant libraries were generated by a ligation strategy. For each mutant library, an insert and a vector were generated by PCR using KOD DNA polymerase (EMD Millipore) according to the manufacturer’s instructions. The HA-encoding plasmid pHW2000-HA) of the influenza eight-plasmid reverse genetics system was used as the template000-HA of2) using lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. At 24 h post-transfection, cells were washed twice with phosphate-buffered saline (PBS), and cell culture medium was replaced with OPTI-MEM medium supplemented with 0.8 μg mL−1 TPCK-trypsin. Virus mutant library was harvested at 72 h post-transfection and stored at −80 °C until used. For measuring virus titer by TCID50 assay, MDCK-SIAT1 cells were washed twice with PBS prior to the addition of virus, and OPTI-MEM medium was supplemented with 0.8 μg mL−1 TPCK-trypsin.For each mutant library, transfection was performed in HEK 293T/MDCK-SIAT1 cells  co-culture (ratio of 6:1) at 60% confluence in a T75 flask . The extracted RNA was then reverse transcribed to cDNA using Superscript III reverse transcriptase (Life Technologies). The plasmid or the cDNA from the post-selection viral mutant libraries was amplified by PCR to add part of the adapter sequence required for Illumina sequencing using the primers listed in Supplementary Table To passage the virus mutant libraries, MDCK-SIAT1 cells at 80% confluence in a T75 flask were washed twice with PBS and infected with a multiplicity of infection (MOI) of 0.01 in OPTI-MEM medium supplemented with 0.8 μg mLA second PCR was performed to add the rest of the adapter sequence and index to the amplicon using primers: 5’-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT-3’ and 5’-CAA GCA GAA GAC GGC ATA CGA GAT XXX XXX GTG ACT GGA GTT CAG ACG TGT GCT-3’. Positions annotated by an X represented the nucleotides for the index sequence. The final PCR products were submitted for next-generation sequencing on one lane of Illumina MiSeq PE300.39. After trimming the primer sequences, both forward- and reverse-complemented reverse reads were translated into protein sequences. A paired-end read was then filtered and removed if the protein sequence translated from the forward read and that translated from the reverse-complemented reverse read did not match. Amino acids at the residues of interest were then extracted. The number of reads corresponding to each of the 576 variants was counted. For any given variant, input count represents the number of reads corresponding to the given variant in the plasmid mutant library, whereas selected count represents the number of reads corresponding to the given variant in the post-selection virus mutant library.Sequencing data were obtained in FASTQ format and were parsed using SeqIO module in BioPythonk was estimated as k and ink are selected and input (before selection) counts. A pseudocount of 1 was added to the counts to estimate the (very low) fitness of variants with selk = 0. From the replicates of selk, we computed mean and variance of each variant. We found substantial deviation from the Poisson expectation that the mean and variance are equal . We use a generalized logistic function,p1, p2, and p3 are free parameters that determine the shape. These parameters, along with the hi(a) and s(k) is sequence of variant k. We estimate the confidence interval of the estimated additive and epistatic parameters by a parametric bootstrap: bootstrapped training data are generated by a normal distribution with mean equal to the fitness and variance equal to the fitness variance, then parameters are estimated for each bootstrap and the 95% confidence interval is calculated from an ensemble of 100 bootstraps.We model fitness as a non-linear function of a sum of additive and pairwise effects. We chose a non-linear sigmoidal function43. Variant networks in Supplementary Fig. Sequence logos in Fig. http://gisaid.org)44  at TSRI. Diffraction-quality crystals were obtained from 0.1 M sodium cacodylate [pH 6.5], 6% PEG 8000, and 39% 2-methyl-2,4-pentanediol at 20 °C by sitting drop vapor diffusion method with 500 μL reservoir solution and each drop consisting 0.8 μL protein + 0.8 μL precipitant. The resulting crystals were soaked in 20 mM 6′SLNLN for 2 h, flash cooled, and stored in liquid nitrogen until data collection.The HA68 HA ectodomain, which contains HA1 residues 11–329 and HA2 residues 1–176, was fused to an N-terminal gp67 signal peptide and to a C-terminal biotinylation site, trimerization domain, and 6x His-tag. Recombinant bacmid DNA that carried the HK68 HA ectodomain construct was generated using the Bac-to-Bac system (Life Technologies). Baculovirus was generated by transfecting purified bacmid DNA into Sf9 cells  using FuGene HD (Promega). HK68 HA ectodomain was expressed by infecting suspension cultures of High Five cells  with baculovirus at an MOI of 5–10 and incubating at 28 °C shaking at 110 r.p.m. for 72 h. The supernatant was concentrated. HA0 was purified by Ni-NTA, buffer exchanged into 10 mM Tris-HCl [pH 8.0] and 50 mM NaCl, and concentrated to 10 mg mL45. The structure was solved by molecular replacement using Phaser46 with PDB: 6AOR32 as the molecular replacement model, and the structure was rebuilt using Coot47 and refined using Refmac548. Ramachandran statistics were calculated using MolProbity49.Diffraction data were collected at the APS GM/CA-CAT 23ID-B. The data were indexed and integrated and scaled using HKL2000 (HKL Research)Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3


Influenza B virus (IBV) is a respiratory pathogen that infects humans and causes seasonal influenza epidemics. However, cellular response to IBV infection in humans and mechanisms of host-mediated restriction of IBV replication are not thoroughly understood. In this study, we used next-generation sequencing (NGS) to perform transcriptome profiling of IBV-infected human lung epithelial A549 cells at 0, 6, 12, and 24 h post infection (hpi) and characterized the cellular gene expression dynamics. We observed that more than 4000 host genes were differentially regulated during the study period, which included up regulation of genes encoding proteins, having a role in the innate antiviral immune responses, immune activation, cellular metabolism, autophagy, and apoptosis, as well as down regulation of genes involved in mitosis and cell proliferation. Further analysis of RNA-Seq data coupled with RT-qPCR validation collectively showed that double-strand RNA recognition pathways, including retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3), were substantially activated following IBV infection. Taken together, these results provide important initial insights into the intimate interaction between IBV and lung epithelial cells, which can be further explored towards elucidation of the cellular mechanisms in restriction or elimination of IBV infections in humans. Influenza viruses are classified as types A, B, and C according to their distinct antigenic properties residing in two major structural proteins (matrix 1 and nucleocapsid) ,3. Among3 nucleotide substitutions per site per year for hemagglutinin (HA) and neuraminidase (NA) proteins [As with IAV, IBV contains eight segments of single-stranded negative-sense RNA. IBV evolves at an estimated rate of ~2 × 10proteins ,15,16. Tproteins ,18. IBV proteins ,20. The The global burden of annual influenza epidemics involving IBV, coupled with the increasing strong links between IBV and fatal infections in humans ,22, callIn this study, using next-generation sequencing (NGS) we investigated cellular response to IBV infection in human lung epithelial A549 cells. Significantly, we observed that the double-stranded RNA sensing pathway, consisting of RIG-I and TLR3, played an essential role in detecting IBV infection. Interestingly, in addition to the antiviral innate immune response, we also found that IBV replication resulted in profound changes in gene transcriptions involving other cellular pathways including immune activation, cellular metabolism, autophagy, apoptosis, mitosis, and cell proliferation. Taken together, these results provide important initial insights into the interactions between IBV and human lung epithelial cells, which pave the way for further investigation of cellular mechanism in restriction or elimination of IBV infections in humans.® CCL-185) cells, Madin-Darby canine kidney (MDCK) cells (ATCC® CCL-34), and human embryonic kidney cells HEK293T (ATCC® CRL-3216). Cells were maintained in Dulbecco’s minimum essential medium (DMEM) that was supplemented with 10% fetal bovine serum (FBS)  and penicillin-streptomycin  (100 U/mL). MDCK cells were used initially to propagate IBV in T75 tissue culture flasks. Briefly, MDCK cells were cultured to reach only 60% to 70% confluence at the time of infection. After infection, fresh DMEM with 0.5 μg/mL tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin  was added for additional 3-day incubation at 37 °C with 5% CO2. The infected cell cultures were then frozen and thawed. The supernatant was spun at 500× g for 10 min at 4 °C to remove the cellular debris, which was followed by traditional viral titration experiments in MDCK cells according to the method of Reed and Muench [Influenza B/Brisbane/60/2008 virus was used in this study . Cell lines acquired from the American Type Culture Collection  for this work included Human lung alveolar carcinoma epithelial cell line A549  and TPCK-treated trypsin (1 μg/mL) was added into IBV-infected cell for further cultivation. Total cellular RNA was isolated from mock-infected cells (0 hpi) and infected cells at 6, 12, and 24 hpi, respectively, with TRIZOL followed by RNA purification with ethanol. Bioanalyzer 2100  was used to assess the quality of extracted RNA. RNA that passed the quality test was further processed for cDNA library construction by using a cDNA library prep kit  according to the manufacturer’s instruction. cDNA library construction and all other procedures were conducted in Genomics Core Research Facility (GCRF) at the University of Nebraska-Lincoln (UNL). Briefly, mRNAs were purified from the total RNA using oligo(dT) magnetic beads followed by fragmentation. The resultant mRNAs were reverse transcribed to cDNAs that were subjected to an end repair process followed by ligation to the adapters. After separation in agarose gel through electrophoresis, cDNA fragments with a size of about 200 bp were excised, extracted, and amplified by PCR using two primers that match the ends of adaptors. PCR-enriched samples were then sequenced by an Illumina Genome Analyzer IIx (GA IIx) sequencer in the GCRF at UNL.Two biological replicates were sequenced for each time point . The eight samples were barcoded and sequenced in one lane. Each of the eight samples had approximately 5–7 million 100-nucleotide single-end reads. All sequencing results passed the quality control. The raw reads of the eight samples were submitted to NCBI SRA database under Biosample ID: SRS1328599.The raw read quality was checked using FastQC. The 3’ 5 nucleotides of each read, whose sequencing qualities were low, were removed using fastx_trimmer. GSNAP was then used to map raw reads to human (version: GRCh37.p13) and Influenza B virus genomes . SamtoolP value adjusted according to the Benjamini–Hochberg method to identify differentially expressed genes (P < 0.05). To further remove potential false positives, we removed differentially expressed genes with the changes of expression levels less than ±2 fold.DESeq was used to identify differentially expression human genes . Brieflyhttps://string-db.org/) to identify significantly enriched biological processes, cellular components and molecular functions, and KEGG pathways for the differentially expressed gene sets of each time point [P value of 0.05 and normalized false discovery rate of 0.25 were used to identify significantly enriched terms. A P value adjusted according to Bonferroni step down procedure was used to measure the enrichment significance of a GO term (P < 0.05) in ClueGo. Parent-child GO terms were fused with Kappa cutoff 0.4. The minimal GO level analyzed was set to 3. The enriched KEGG pathways were identified using a false discover rate of 0.05.We used GSEA database, ClueGO, and STRING database  according to the manufacturer’s instructions, which was then followed by cDNA synthesis with Oligo dT(18) primer  and Reverse Transcriptase . With Fast SYBR Green Master Mix (Thermo Fisher), qPCR reactions were conducted and read in QuantStudio 6 Flex Real-Time PCR System  according to the recommended protocol by the manufacturer. 5 TCID50/mL) and increased over a 24-h period [To investigate the host response to IBV infection, we infected human lung epithelial cells (A549) in duplicate with human IBV strain (Victoria lineage-B/Brisbane/60/2008) at a multiplicity of infection (MOI) of 1.0 . IBV reph period . The prih period .To explore cellular responses to IBV infection, we used DESeq program  to normaOverall, the number of significantly regulated genes increased over time. For all three time points post infection, a total of 4365 genes was differentially regulated. There are more up-regulated genes than down-regulated genes at each time point a. The upWe then investigated the expression dynamics of the 4365 human genes over the study period b. Most uTo better understand the innate immune response to IBV infection, we analyzed regulated genes in pathways related to viral infection including detection of infection, interferon and cytokine cascade, viral myocarditis, antigen processing and presentation, and disease tolerance . We thenPrevious studies demonstrated that influenza A virus infection is mainly detected by three classes of pattern recognition receptors: Toll-like receptors TLR3 , TLR7 and TLR8 , retinoic acid-inducible gene I (RIG-I) (5’-triphosphate RNA) and NOD-like receptor NOD-LRR- and pyrin domain-containing 3 (NLRP3). These receptors activate different downstream pathways for antiviral activities as described below. As early as 6 hpi following IBV infection, genes in the RIG-I like receptor signaling pathway and TLR3 signaling pathway were up-regulated, including DDX58 (or RIG-I), IFIH1 (or MAD5) and DHX58 (or LGP2), IL12A, IL8, IRF7, ISG15, TRIM25, and TLR3 .r = 0.85; p < 0.0001). Interestingly, we did not observe expression of TLR7 and TLR8 at all time points in A549 cells probably because both genes are not expressed in this cell line as demonstrated in a previous study [To further validate the gene expression results analyzed by next-generation sequencing, we conducted a conventional RT-qPCR experiment where we measured quantitative changes of a subset of genes in IBV-infected A549 cells as a function of time (post-infection). Selected genes are respectively involved in the innate immune pathway , autophagy (AMBRA1), and apoptosis (CASP10). As summarized in us study . Our datRIG-I and TLR7 have been reported to activate type I IFN pathway. Consistent with this, we observed that genes of the JAK-STAT signaling cascade were up regulated including IRF9, STAT1, STAT2, and JAK2, suggesting that the IFN signaling pathway was activated as early as 6 hpi. It is also surprising to observe that although TLR7 was not expressed, downstream genes of TLR7 pathway were up-regulated  , which ip < 0.01), we viewed that IRF-3 gene expression was slightly increased in A549 cells in response to IBV infection. RT-qPCR data appeared to be in agreement with the RNA-Seq result where we found that the fold-change for IRF-3 when compared to 0h hpi was 1.16 (6 hpi), 1.38 (12 hpi), and 1.14 (24 hpi), respectively (data not shown). For IFN-α and IFN-α7, similar to what was observed in RNA-Seq experiment  were up regulated at various time points examined during IBV replication .Antigen processing and presentation pathway is important for initiation of adaptive cellular and humor immune response. It has been shown that RIG-I and TLR3 can activate MHC-I system, which recruits CD8+ T cell for killing infected cells . ConsistHost resistance to viral infection or protective host response and disease tolerance play critical roles to modulate viral pathogenicity and severity of damage to host tissues and organs. Previous studies on IAV infection revealed that both processes are modulated by certain key factors . Host rel-serine, amino acid, and oxyacid) and their metabolic processes. Future study is needed to experimentally address the consequence of altered metabolic changes in cell physiology or in IBV replication and pathogenesis.The up-regulated metabolic processes are mainly involved in synthesis and metabolism of biomolecules  and regulation of protein modifications (phosphorylation and ubiquitination) . The genGenes involved in cell proliferation were dominant in down-regulated gene sets at 12 and 24 hpi . The enrThe innate immune system is the first line of host defense against invading pathogens. Analysis of changes in this intrinsic antiviral pathway in response to IBV infection is critical to elucidate IBV-host interaction. In this study, we examined IBV infection induced response in human lung epithelial A549 cells, a well characterized cell system that responds to infection robustly and has been extensively used to investigate innate immune response of pathogens, including but not limited to Ebola virus , InfluenIn addition, we observed no inducible expression of IFN-γ, representative of type II IFN response, which is in line with the fact that IFN-γ is only expressed in immune cells . Upon acFurther analysis of NGS data also suggests that IBV may somehow interfere with A549 cell proliferation, which is consistent with our in vitro proliferation data reported in our previous work . PreviouHuman A549 cell-specific transcriptome analysis also allowed us confirm the result from a previous study where the authors showed that TLR7 and TLR8 are not expressed in human epithelial A549 cells . LackingIn summary, the host transcriptomic analysis presented here sheds lights on host biological systems that are affected by IBV infection. These studies shall provide novel insights into the intricate host-pathogen interaction following IBV infection of human lung epithelial cells, which can be further explored toward the elucidation of the mechanisms by which IBV remodels the epithelial environment to promote its own replication and spread in humans.


Cudrania tricuspidata (C. tricuspidata) is widespread throughout East Asia and in China and Korea, and it is widely used as a traditional remedy against eczema, mumps, and tuberculosis. With regard to the aforementioned medical efficacy, various studies are continuously being conducted, and it has been reported that C. tricuspidata extract has various actions against inflammation, diabetes, obesity, and tumors. Therefore, we evaluated antiplatelet effects using derrone in C. tricuspidata. We examined the effect of derrone on the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and inositol 1, 4, 5-triphosphate receptor I (IP3RI), and on the dephosphorylation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinases p38 (p38MAPK), and Akt, which affects platelet function and thrombus formation. Various agonists-induced human platelets were inhibited by derrone without cytotoxicity, and it also decreased the intracellular calcium level through the signaling molecule phosphorylations. In addition, derrone inhibited glycoprotein IIb/IIIa (αIIb/β3) affinity. Thus, in the present study, derrone suppressed human platelet aggregation and thrombin-induced clot formation. In normal circulation of blood, platelets are necessary for hemostasis and thrombosis. . The carαIIb/β3 activation, but its phosphorylation suppressed its activity [3RI [2+]i mobilization i concentration i) was elevated from 100.5 ± 0.4 nM to 755.5 ± 10.2 nM by collagen (2.5 μg/mL). However, derrone (15 to 60 μM) reduced the collagen-increased [Ca2+]i levels (μM) increased inositol 1, 4, 5-triphosphate receptor type I (IP3RI) phosphorylation. This shows that derrone decreased [Ca2+]i levels through phosphorylation of IP3RI. In addition, we explored whether derrone is involved in the inhibition of dense granule secretion. Serotonin is not synthesized in megakaryocytes or platelets, but is taken up from plasma and stored in dense granules in platelets. Upon stimulation by agonists, high [Ca2+]i levels facilitate the myosin light chain and pleckstrin phosphorylation to trigger δ-granule and α-granule release, and the serotonin released by exocytosis acts as a platelet agonist on the 5-hydroxytryptamine 2 (5-HT2) receptor on platelets [δ-granule release, and, as shown in μM) dose-dependently inhibited collagen-stimulated serotonin secretion. It is known that ERK phosphorylation is involved in influx from extracellular Ca2+ [μM) inhibited collagen-induced phosphorylation of ERK2 (42 kDa).As shown in i levels . Next, wlatelets . We evallar Ca2+ ; therefoμg/mL) increased the TXA2 generation to 55.8 ± 4.8 ng/108 platelets from 1.5 ± 0.2 ng/108 platelets. However, derrone inhibited TXA2 production , were investigated. The cPLA2 has been reported to hydrolyze arachidonic acid from membrane of platelets, and p38MAPK activates cPLA2 to act as an enzyme. As shown in Figures 2 and p38MAPK phosphorylation in a dose-dependent manner.As shown in oduction . To idenαIIb/β3 affinity, the binding of fibrinogen to αIIb/β3 is increased  regulates actin, but its phosphorylation by kinases inhibits affinity , 11. As  Figures , we inve Figures . Next, wαIIb/β3 in the previous experiment, we examined whether derrone affects fibrin clot retraction using PRP. As shown in μM) effectively delayed the thrombin-stimulated fibrin clot, with inhibitory degrees of 79.8%, 59.4%, 44.5%, 42.8%, and 34.7%, respectively . These data indicate that regulation of signaling molecules such as IP3RI (Ser1756) and VASP  influences the clot retraction delay.On the platelet surface,  Figures  by upreg Figures  and elev Figures . Moreovetraction , throughC. tricuspidata, administration of extract of C. tricuspidate (50 and 100 mg/kg) reduced hyperaggregated platelet aggregation without any hepatotoxicity in high-fat diet- (HFD-) fed rats. Moreover, a decrease in thromboxane A2 production was observed in vivo [L, euchrestaflavanone A, and cudraxanthone B [L and euchrestaflavanone A increased only the cAMP levels.According to the results of studies on the antiplatelet effects of  in vivo . In addinthone B –28. Theiin vitro and did not confirm the antiplatelet effect in vivo. Although the experiment was conducted using a low concentration (15–45 μM) of derrone in vitro, it is difficult to reach this concentration in blood by ingestion. Moreover, since our study is not an in vivo study using the human body, it is difficult to prove its effect in the human body; thus, we cannot prove that these effects would be the same in people with high and low platelet counts or cardiovascular disease. However, based on the in vitro effect, we suggest that derrone has the potential to inhibit thrombosis-mediated CVDs.Our study had some limitations, in that it was conducted αIIb/β3 affinity, and clot retraction, which were achieved by the regulation of IP3RI (Ser1756), VASP (Ser157 and Ser239), cPLA2 (Ser505), and p38MAPK. In addition, derrone increases cAMP and cGMP levels in human platelets. These two cyclic nucleotides are key mediators of the antiplatelet effects. Thus, we confirmed that derrone could be a potential phytochemical for the prevention of platelet-mediated illnesses.In conclusion, we confirmed that derrone suppressed collagen-stimulated platelet aggregation through downregulation of intracellular calcium concentration,


The behavior of ice under extreme conditions undergoes the change of intermolecular binding patterns and leads to the structural phase transitions, which are needed for modeling the convection and internal structure of the giant planets and moons of the solar system as well as H2O-rich exoplanets. Such extreme conditions limit the structural explorations in laboratory but open a door for the theoretical study. The ice phases IX and XIII are located in the high pressure and low temperature region of the phase diagram. However, to the best of our knowledge, the phase transition boundary between these two phases is still not clear. In this work, based on the second-order Møller–Plesset perturbation (MP2) theory, we theoretically investigate the ice phases IX and XIII and predict their structures, vibrational spectra and Gibbs free energies at various extreme conditions, and for the first time confirm that the phase transition from ice IX to XIII can occur around 0.30 GPa and 154 K. The proposed work, taking into account the many-body electrostatic effect and the dispersion interactions from the first principles, opens up the possibility of completing the ice phase diagram and provides an efficient method to explore new phases of molecular crystals. Water is the most studied material on earth and consists of two hydrogen atoms that are attached to a single oxygen atom. The ice phase diagram shows the preferred physical state under different thermodynamic variables, with the small changes in temperature or pressure causing the structural change from one physical state to another ,2,3. It Exploring molecular structures in extreme environments have many limitations in laboratory, such as the weak of experimental signal, the harsh conditions of high-pressure equipment, etc., but effective theoretical predictions provide an alternative approach to study the detailed crystal structures. Based on the second-order Møller–Plesset perturbation (MP2) theory, the present work proposes an ab initio method to investigate the structures, equation of state, Raman spectra and Gibbs free energies of ices IX and XIII, and predicts their phase transition at high pressure and low temperature, which provides opportunities to advance the development of high pressure structural determination through computation .With the continuous increase of computer power, molecular simulation becomes a powerful tool to predict different molecular characteristics. The ability of predictive computer simulation depends on the accuracy of the potential energy calculation . Both clsymmetry ,29,30. Tsymmetry . It is wsymmetry  using thIn this work, we demonstrate the practicability of MP2 theory by applying it to the structure simulation of ice phases IX and XIII, along with the electrostatically embedded generalized molecular fractionation (EE-GMF) method ,33. Due 3, and can be converted to ice II by warming. Ice phase IX has a similar structure with ice III, except that ice IX is a proton ordered structure while ice III is a proton disordered structure [Ice phase IX is an ultralow temperature solid structure, which is stable at temperatures below 140 K and pressures between 0.2 and 0.4 GPa. It has a square lattice with a density of 1.16 g/cmtructure ,40. Furttructure ,41. Thertructure , while ttructure .a and b, but has an error of 0.03 to 0.1 Å when predicting c due to the strong hydrogen bonding interaction along c direction. Such errors lead to the slight volume difference between the calculated results and observed data, as shown in −1. From the results of experiment and theory, the numbers of Raman peaks from theoretical simulation and experiment are the same. For the three Raman peaks below 150 cm−1, the deviations between the experimental values and theoretical results are relatively small, and the maximum deviation of the theoretical prediction is about 30 cm−1 with reference to the experimental observation for the third peak. There are three experimental Raman peaks within the frequency region between 150 and 350 cm−1. Among these three peaks, the deviations of theoretical predictions are between 50 to 70 cm−1 as compared to the experimental results. Therefore, our theoretical calculations qualitatively reproduced the experimental Raman spectrum of ice phase IX in the low frequency region. Furthermore, for the high frequency region, the predicted 2 discernible Raman bands of ice phase IX are assigned to 2 observed Raman peaks from 2800 to 3600 cm−1. −1 in the high frequency region and low frequency region, respectively. The consistency of Raman peak positions between the MP2 theory and experiment is quantitative and consistent, indicating that the MP2 method can provide accurate prediction of the ice structures at high pressures.The vibrational spectrum is regarded as a chemical recognition of a particular molecule or material, which can be used to quickly identify or distinguish structures. −1 for each band. The agreement of IR spectra between the experiment and theory further confirms the accuracy of the proposed ab initio method and provides an approach to identify the structural phases for molecular crystal. So far, no experimental IR work has been carried out and reported for ice phase XIII, and thus the present work only reports the IR comparison for ice IX. We hope to carry out further Raman and IR spectral experiments in the future to verify the proposed spectral prediction. More Raman and IR spectra analysis for ice phases IX and XIII can be found in the The proposed ab initio method allows us to further calculate the vibrational IR spectra of ice phases IX and XIII. The Gibbs free energies of ice phases IX and XIII are further calculated by the EE-GMF method. In this study, we mainly focused on the Gibbs free energy differences between Ices IX and XIII in the high pressure and low temperature region. This work demonstrates the relative stability between Ices IX and XIII in terms of Gibbs free energy. In the future study, the other phases, such as Ices II, Ih and XV, under low temperatures may also be considered and thus we could provide a more comprehensive comparison of relative stabilities of those phases in the high pressure and low temperature region. The researches along these lines are underway in our laboratory.The distance threshold was set to 5.0 Å for determining fragment pairs used in quantum mechanical calculations. The interactions beyond the distance threshold were treated by classical Coulomb interactions. All QM calculations were carried out using the Gaussian09 program .The current EE-GMF-MP2/aug-cc-pVDZ calculations may have errors arising from the incompleteness of the base set ,62. The 1212, and the ice XIII has 28 molecules in the unit cell with a symmetry of P21/a.The crystal structures, vibrational spectra, Gibbs free energies and phase transition of ice phases IX and XIII were calculated at the MP2 level with the aug-cc-pVDZ basis set ,63,64. Tkth monomer in the central unit cell (0th) is kE(0), and the energy of the dimer composed of the kth monomer in the central unit cell and the lth monomer in the ith unit cell is kEl(i)(0). The three integers of the specified cell are represented by i. The remote electrostatic energy correction is ELR , the pressure is P, and the unit cell volume is V. It should be noted that being excluded from the second sum is k = l and i = 0. Although the truncation of multibody expansion after the two-body terms (dimers) achieves an excellent cost-accuracy balance, the series can also be truncated after a three-body or higher-order term.The electronic enthalpy per unit cell  or single atomic coordinates. It should be noted that these equations ignore the derivative of the embedded electrostatic field. The geometric derivatives of dipole moment and polarizability can also be calculated using the fragmentation approach and provide information of infrared (IR) and Raman intensity.The first and second derivatives of enthalpy in Equation (1) for atomic coordinates are related to atomic force and force constant, respectively. Derivatives relating to lattice constants give lattice forces as follows,We have included the background charges in the 11 × 11 × 11 supercell. The last term of Equation (1), gives the long-range interaction in the 41 × 41 × 41 supercell through the charge-charge Coulomb interaction,Qnk is the corresponding normal mode. Only the zone-center (k = 0) vibrations have nonzero intensities and thus are IR- or Raman-active. In this study, the frequency calculated using MP2/aug-cc-pVDZ is scaled by 0.94. In addition, μEE−GMF is the dipole moment of the central unit, and T is obtained byUv is the zero-point vibration energy per unit cell at the temperature of T, and Sv is the vibration entropy per unit cell. For molecular crystals with large band gaps, only temperature effects due to phonons need to be considered. Both Uv and Sv can be derived from the partition function of phonons, vZ. In the harmonic approximation and atomic units, vZ can be written as,nk is the frequency of the phonon with the wave vector k in the nth phonon branch. The product on k must be taken over all K evenly spaced k grid points in the reciprocal unit cell. In this study, the k-grid of 21 × 21 × 21 (K = 9261) is used. According to thermodynamics, we haveωnk.By calculating the enthalpy of the per unit cell, the effect of pressure is taken into account. The following expressions can be used to calculate IR intensity and Raman intensity,F method . The GibFrom small molecules to macroscopic structures, the phase diagrams of molecular crystals have always been the hot research of scientists. The proposed fragment-based QM calculation using the MP2 method is different from the traditional methods such as the classical force field and DFT theory. The difficulty in phase diagram prediction stems from the high cost of structural screening and the low precision of the applicable method. The proposed MP2 theory combining with the fragmentation approach enables high-precision and efficient calculations on complex systems at finite pressures and temperatures, which can accurately describe all cohesive interactions and obtain reasonable results when predicting structures and energies of molecular structures and produce convinced ice phase diagram via ab initio calculation. Ice phases IX and XIII are two molecular crystals at the high pressure and low temperature, that have rarely been studied by both experiment and theory. In the present work, we quantitatively reproduce their respective structures, and vibrational spectra at the MP2/aug-cc-pVDZ level. The predicted phase transition between the phase IX and phase XIII falls within the temperature and pressure range of the experimental speculation. This work confirms for the first time the positions of phases IX and XIII in ice phase diagram at high pressures and is consistent with the experimental speculation, which can be used to assist in the high pressure exploration of molecular phases with potentially important applications.


We test here whether CDDP affects ENaC to promote renal salt and water excretion. The effects of CDDP and benzamil (BZM), a blocker of ENaC, on excretion of a sodium load were quantified. Similar to BZM, CDDP facilitated renal Na+ excretion. To directly quantify the effects on ENaC, principal cells in split-open tubules were patch clamped. CDDP, at doses comparable to those used for chemotherapy (1.5 µM), significantly decreased ENaC activity in native tubules. To further elaborate on this mechanism, the dose-dependent effects of CDDP on mouse ENaC (mENaC) heterologously expressed in Chinese Hamster Ovary (CHO) cells were tested using patch clamping. As in native tubules, CDDP significantly decreased the activity of mENaC expressed in CHO cells. Dose–response curves and competition with amiloride identified CDDP as a weak inhibitor of ENaC (apparent IC50 = 1 µM) that competes with amiloride for inhibition of the channel, weakening the inhibitory actions of the latter. Such observations are consistent with CDDP being a partial modulator of ENaC, which possibly has a binding site that overlaps with that of amiloride. These findings are consistent with inhibition of ENaC by CDDP contributing to the RSWS caused by this important chemotherapy drug.Cisplatin (CDDP) is an important anticancer drug. A common side effect of CDDP is renal salt and water-wasting syndrome (RSWS). The origin of RSWS is obscure. Emerging evidence, though, suggests that broad inhibition of sodium transport proteins by CDDP may result in decreases in tubular reabsorption, causing increases in sodium and water excretion. In this sense, CDDP would be acting like a diuretic. The effect of CDDP on the epithelial Na These drugs are powerful chemotherapy agents because of their ability to bind DNA and inhibit replication [Cisplatin  . CDDP is+/K+-ATPase [In addition to damaging cells via direct actions on DNA, CDDP, independent of the effects on DNA, also binds to and is a general inhibitor of sodium transport proteins, including Na+-ATPase ,8. Inhib+-ATPase ,10. Redu+ channel (ENaC) is a sodium-selective ion channel expressed in the luminal plasma membranes of late distal convoluted tubule, connecting tubule and collecting duct principal cells [2+, Cu2+, Hg2+, Ni2+, Pb2+ and Zn2+, are known to decrease the activity of ENaC [The epithelial Naal cells . The cooal cells . It is ual cells . This isal cells . Moreove of ENaC . It currBased on the understanding that CDDP causes RSWS, is concentrated in urine, has a general inhibitory effect on sodium transport proteins and has some chemical similarity with the guanidinium core of ENaC blockers, and that heavy metals affect the activity of ENaC, and that inhibitors of ENaC cause a natriuresis and diuresis similar to that caused by CDDP, we test here if CDDP affects ENaC activity and whether such action potentially could contribute to the RSWS caused by this widely used chemotherapy drug. Findings showed that CDDP causes a natriuresis similar to the ENaC blocker, BZM. In isolated tubules, CDDP rapidly decreases ENaC activity. In a heterologous expression system, CDDP decreases the activity of ENaC in a dose-dependent manner in a way that is competitive with the effects of amiloride. Together, these results are consistent with CDDP being a partial inhibitor of ENaC, with inhibition of this key renal ion channel possibly contributing to the RSWS caused by CDDP.+ load  plus treatment with CDDP, BZM or a vehicle. Excretion of this sodium load was then followed over the next four hours. + load at 2 and 4 h following the injection of 100 µL of a 0.9% NaCl solution with BZM , CDDP  and a vehicle. At 2 h, the BZM group had significantly greater Na+ excretion compared with the saline-injected control group. At the 4 h time point, Na+ excretion was significantly greater in the CDDP and BZM groups compared with the control, with CDDP and BZM not being different. As expected, these results are consistent with CDDP, like BZM, facilitating Na+ excretion.Male mice were housed socially at three mice/cage with free access to water and food. After 2 days, mice received an Nao, 1.098 ± 0.19 vs. 0.3725 * ± 0.0917, before and after CDDP treatment, respectively, * p < 0.05 vs. Before). Split-open tubules isolated from the kidneys were prepared for single-channel patch clamp electrophysiology in the cell-attached configuration. 50 of 0.46 ± 0.10 µM and 1.0 ± 0.18 µM for amiloride and CDDP, respectively. Maximal responses on mENaC activity, calculated as an inhibition percentage (Emax), were 91.9 ± 0.80 vs. 56.6 ± 4.2% for amiloride and CDDP, respectively, with the latter being significantly lower than the former. These results demonstrate that CDDP is a partial inhibitor of ENaC.Chinese Hamster Ovary (CHO) cells expressing mENaC were patch clamped in the whole-cell configuration under voltage clamp conditions in the presence and absence of amiloride. Similar to the ENaC blocker amiloride, CDDP reduced mENaC activity in CHO cells in a dose-dependent manner. As reported in 50 = 499 ± 0.02 nM and 1.59 ± 0.19 µM for amiloride in the absence and presence of CDDP, where * p < 0.05 vs. amiloride) and a reduced Emax for amiloride .We noticed in patch clamp experiments that CDDP seemed to interfere with the amiloride block of the channel. To further explore this, we tested using patch clamp electrophysiology if CDDP competed with amiloride for a block of mENaC in an overexpression system using CHO cells. This was done by generating amiloride inhibition curves for mENaC in the presence of 10 µM of CDDP. Our findings confirm that CDDP caused an expected natriuresis. In isolated tubules, CDDP rapidly decreased ENaC activity. In a heterologous expression system, CDDP decreased the activity of mENaC in a dose-dependent manner. The inhibition curve for CDDP on ENaC showed this drug to be a partial inhibitor of the channel. CDDP competed with amiloride for the inhibition of ENaC, reducing the effects of this more powerful inhibitor, which suggested that CDDP and amiloride may have overlapping binding sites on the channel.+ excretion as a consequence of inhibiting ENaC [Cisplatin is an important chemotherapy drug. Possible side effects of CDDP include RSWS ,17,18,19ing ENaC .Exploring the mechanism underlying this CDDP-dependent natriuresis more deeply, we found, as supported by the results presented in CDDP was shown to be a partial inhibitor of ENaC in the current study, as supported by the results in A close inspection of the molecular structure of CDDP shows that this molecule shares some similarity with the pyrazinoyl guanidine group present in ENaC blockers, such as BZM and amiloride. Perhaps this explains how both could potentially be open channel blockers of ENaC that compete for a common or overlapping binding site. However, such contemplations are tempered by the need for further and more precise investigation of this possibility, including a precise mapping of both the amiloride and CDDP binding sites within ENaC.2+ and Hg2+, decrease ENaC activity [2+, Ni2+ and Cu2+ increase the activity of ENaC [+/K+-ATPase or the mineralocorticoid receptor. Inhibitory effects of CDDP on both of these proteins have been reported before [An alternative explanation of a mechanism consistent with the observation that CDDP is a weak inhibitor of ENaC that competes with pore blockers of this ion channel is that CDDP contains platinum at its core, and ENaC is particularly susceptible to the effects of heavy metals. Many heavy metals, including Cdactivity . However of ENaC . It is ud before ,22.+/K+-ATPase and the mineralocorticoid receptor, CDDP also likely has a more direct action on ENaC. Consequently, both the direct and secondary actions of CDDP on ENaC cause the RSWS associated with this important anticancer drug. We make this argument because CDDP affected ENaC in the current study in a heterologous system that did not include a mineralocorticoid receptor. In addition, the spatiotemporal effects of CDDP on ENaC in the current patch clamp study were rapid and localized to the plasma membrane. The effects of the mineralocorticoid receptor on ENaC are via transactivation localized to the nucleus, which manifests in minutes to hours. In the current study, CDDP rapidly reduced ENaC activity at the plasma membrane within seconds. Moreover, the competitive nature of CDDP and amiloride on ENaC are most consistent with, at least, some direct effect on the channel that is independent of secondary actions mediated by inhibition of the pump.While the current findings, due to limitations in methodology, cannot discount possible contribution from secondary effects of CDDP on ENaC, we believe the simplest interpretation of the current results is that, in addition to possible secondary effects mediated by inhibition of the NaAll animal use and welfare adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Protocols were reviewed and approved by the Institutional Animal care and Use Committee of the University of Texas Health Science Center at San Antonio. Mice were housed and cared for in the Laboratory Animal Resources Facility at the University of Texas Health Science Center at San Antonio, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and licensed by the United States Department of Agriculture.+ load  plus treatment with CDDP , BZM  or the vehicle. Urine volume and urinary Na+ concentration were evaluated every 2 h over a 4 h period. Urinary Na+ was quantified with a flame photometer . Animals were observed during the whole time of the experimental period for clinical signs of pain or altered behavior. None were noticed.Metabolic cage experiments followed previously published protocols with minor modifications . Briefly2, 2 MgCl2, 5 glucose and 10 HEPES (pH 7.4) and the pipette solution contained (in mM) 140 LiCl, 2 MgCl2 and 10 HEPES (pH 7.4). Channel activity  was calculated as previously described [Split-open tubules amenable to patch clamp analysis were prepared as previously described . Brieflyescribed . After r2-terminal eCFP using a Polyfect reagent  as described previously [+ current with a bath solution of (in mM) 150 NaCl, 1 CaCl2, 2 MgCl2 and 10 HEPES (pH 7.4) and a pipette solution of (in mM) 120 CsCl, 5 NaCl, 2 MgCl2, 5 EGTA, 10 HEPES, 2 ATP and 0.1 GTP (pH 7.4). Current recordings were acquired with an Axopatch 200 B (Axon Instruments) patch clamp amplifier interfaced via a Digidata 1550 B (Axon Instruments) to a PC running the pClamp 11 suite of software (Axon Instruments). All currents were filtered at 1 kHz. Cells were clamped to a 40 mV holding potential with voltage ramps (500 ms) from 60 down to −100 mV, used to elicit current. ENaC activity is in the amiloride-sensitive current density at −80 mV. Whole-cell capacitance, on average 8–10 pF, was compensated. Series resistance, on average 3–6 megaohms, were also compensated. Cells expressing mENaC were evaluated by quantifying the macroscopic current in the presence and absence of amiloride. Dose–response curves for amiloride and CDDP were collected. In another set of experiments, cells were exposed to CDDP (10 µM) and a dose–response curve for amiloride was then collected. The EC50 and maximal response (Emax), expressed as inhibition (% inhibition) of channel activity, then were calculated. As stated above, this dose and the time course of CDDP showed no toxic effects for cell capacitance, and input resistances remained constant over the course of these experiments.CHO cells were maintained in a culture with Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin using standard methods . Cells weviously ,27. In beviously ,27,28. Ip ≤ 0.05 was considered significant.Values were reported as mean ± standard error of the mean (SEM). Data were compared by a Student-t-test and a One-way ANOVA, followed by Bonferroni’s multiple comparisons test when appropriate and a +/K+-ATPase.In conclusion, we demonstrated here that CDDP decreases ENaC activity, likely via a binding site on the channel that, at least partially, overlaps with that of amiloride. We also confirmed here that CDDP causes a natriuresis that is similar to that caused by ENaC blockers. ENaC blockers work as diuretics because they increase sodium excretion. We concluded that the direct inhibitory effect of CDDP on ENaC contributes to the RSWS caused by this important chemotherapy drug. This action of CDDP appears to be in addition to and separate from the effects of this drug on DNA replication, the mineralocorticoid receptor and Na