R factors for 28 IgG1 and 2748 IgG4 structures that satisfied the disulphide connectivity in their hinges. These joint best-fit structures showed that the best-fit IgG1 models had a greater separation between the centers of their Fab regions than those for IgG4, in agreement with their hinge lengths of 15 and 12 residues, respectively. The resulting asymmetric IgG1 solution structures resembled its crystal structure. Both symmetric and asymmetric solution structures were determined for IgG4. Docking simulations with our best-fit IgG4 structures showed greater steric clashes with its receptor to explain its weaker FcγRI receptor binding compared to our best-fit IgG1 structures with fewer clashes and stronger receptor binding. Compared to earlier approaches for fitting molecular antibody structures by solution scattering, we conclude that this joint fit approach based on x-ray and neutron scattering data, combined with Monte Carlo simulations, significantly improved our understanding of antibody solution structures. The atomistic nature of the output extended our understanding of known functional differences in Fc receptor binding between IgG1 and IgG4.Small angle x-ray and neutron scattering are techniques that give solution structures for large macromolecules. The creation of physically realistic atomistic models from known high-resolution structures to determine joint x-ray and neutron scattering best-fit structures offers a, to our knowledge, new method that significantly enhances the utility of scattering. To validate this approach, we determined scattering curves for two human antibody subclasses, immunoglobulin G (IgG) 1 and IgG4, on five different x-ray and neutron instruments to show that these were reproducible, then we modeled these by Monte Carlo simulations. The two antibodies have different hinge lengths that connect their antigen-binding Fab and effector-binding Fc regions. Starting from 231,492 and 190,437 acceptable conformations for IgG1 and IgG4, respectively, joint x-ray and neutron scattering curve fits gave low goodness-of-fit  Atomistic solution structural studies from joint x-ray and neutron scattering curve fits, when combined with molecular dynamics and Monte Carlo simulations, provide a, to our knowledge, new means of determining structures. Here, this method was evaluated in detail using multiple x-ray and neutron data sets for human immunoglobulin G (IgG) 1 and IgG4 antibody subclasses. Single small families of best-fit structures were determined for both subclasses after starting from a large library of simulated structures. These structures explained the different binding modes of IgG1 and IgG4 to two different Fc receptors at a molecular level, thus illustrating the value of this new method to study antibody function. We discuss the applicability of this joint x-ray and neutron approach combined with structural simulations to other similar systems.H and VL) mediate antigen binding, whereas the Fc constant domains (CH2 and CH3) perform effector functions (H2 domains (A). Flexibility in the hinge is also relevant for function, this being exemplified in the stochastic walking of antibodies on repetitive antigenic epitopes such as on viral surfaces ), and replacing 137 mM NaCl with 50 mM NaCl or 250 mM NaCl in buffers termed PBS-50 and PBS-250. For neutron work, the light water in PBS-137 was replaced by 100% heavy water by extensive dialysis into 100% heavy water immediately before SANS experiments. By this, the H atoms in the hydration shell are replaced by D atoms, together with 90% of the protein-exchangeable H-atom content (2 (pH 7.4)).The monoclonal IgG1 6a and IgG1 19a antibodies were used here as previously described . The mon content . Additioμm). Overall, IgG1 6a and IgG1 19a were studied at eight different concentrations between 0.04 and 0.33 mg/mL and 0.12–0.96 mg/mL, respectively. Sample volumes of 50 μL were used in the BioSAXS automatic sample changer. Each sample was moved continuously in the capillary during beam exposure to reduce radiation damage. 10 time frames, each of duration 0.1 s, were acquired, alongside online checks that confirmed the absence of radiation damage during data acquisition. After this, the 10 frames were averaged. The BsxCUBE GUI interface was used for control of the automatic sample changer, and the sample settings were loaded from the ISPyB interface  at low scattering vectors, Q , give the Rg and the forward scattering at zero angle I(0)  :(1)lnI in reciprocal space into the distance distribution function P(r) in real space was carried out using the program ScÅtter (http://www.bioisis.net/users/sign_in):P(r) corresponds to the distribution of distances r between all volume elements. This yields the maximum dimension of the macromolecule L and its most commonly occurring distance vector M in real space, as well as an alternative calculation of the Rg value.This expression is valid in a ted by Q ,32:(2)lnructures . The Rg  package . For IgGectively . For IgGAtomistic scattering modeling compares theoretical scattering curves calculated from protein crystal structural models with the experimental scattering curves. For this, the antibody amino-acid residues were numbered using standard EU numbering for IgG ,34.1HZH)    to creat of IgG4 , the stand 1HZH) ,37. The 1HZH) . All disulphide bonds were retained. For this complete IgG1 starting structure, force field parameterizations were generated and hydrogen atoms added using the glycan reader component of CHARMM-GUI (A), only that at Cys226-Cys226 was present in the IgG1 b12 crystal structure. The Cys226-Cys226 conformation was retained in the initial model, with the force field parameterizations being varied to incorporate or exclude it as required below. Sequence differences between IgG1 b12, IgG1 6a, or IgG1 19a were again disregarded. Two biantennary Gal2.GlcNAc2.Man3.GlcNAc2 glycans at Asn297 were retained (A). The x-ray and neutron scattering length density of the glycans is slightly higher than that of the protein; thus, the glycan and protein components were indistinguishable by scattering  . 13 resiARMM-GUI ,38 and tARMM-GUI , 41, 42.ARMM-GUI . Of the ARMM-GUI A, only retained A. The xattering .1BBJ) (4C55) (A) were retained. The composition of the first glycan was Gal1.GlcNAc2.Man3.GlcNAc2; that for the second was a Man3.GlcNAc2.Fuc core with GlcNAc and Gal.GlcNAc branches. The 20-residue hinge 216ESKYGPPCPSCPAPEFLGGP238 and the missing C-terminal Fc residues 442SLGK445 were modeled using the PyMOL build_seq script. All the disulphide bonds within the crystal structures were retained. To complete this structure, force field parameterizations were generated and hydrogen atoms added, as for IgG1 above. Because neither the Cys226-Cys226 or Cys229-Cys229 hinge residues were positioned to form disulphide bonds in the initial structure, these bonds were not included in the force-field-parameterized model of IgG4. The initial model was energy-minimized as for IgG1.For our SASSIE modeling, the starting IgG4 model was constructed from crystal structures for Fab B72.3 (PDB: 1BBJ)  and seru) (4C55) . These h) (4C55) A were rThe hinge conformations of the new starting IgG1 and IgG4 models were rapidly sampled using dihedral Monte Carlo simulations within SASSIE  while ho220CDKTHT225 was varied in both heavy chains. The asymmetry of the starting crystal structure was retained by this simulation.Simulation 1: 100,000 models were generated, of which 68,914 (68.9%) were sterically acceptable. The lower hinge retained the Cys226-Cys226 disulphide bond, whereas the Cys229-Cys229 bond was not present. The upper-hinge peptide 215VEPKSCDKTHTCPPCPAPELLGGP238 was varied. This simulation sampled conformations with and without symmetry but resulted in a low sampling rate for conformers that showed viable disulphide bonding conformations.Simulation 2: 200,000 models were generated, of which 135,742 (67.9%) were sterically acceptable. Here, the full hinge α-carbon atoms had to be within 0.75 nm of each other to permit interchain disulphide bonding. This additional constraint removed many models.Simulation 3: 404,000 models were generated, of which 26,836 (6.6%) were sterically acceptable. The full hinge was again varied, but with the additional constraint that the Cys226-Cys226 and Cys229-Cys229 For IgG1, three Monte Carlo simulations were performed to maximize the sampling of possible conformers with plausible hinge disulphide bonding. Of a total of 704,000 randomized conformations that were generated, 231,492 structural models were sterically acceptable . The three simulations gave 27,158 models that contained both canonical disulphide bonds as follows:215VESKYGPPCPSCPAPEFLGGP238, of which 143,568 models (47.9%) were sterically acceptable. Only 110 models contained both canonical disulphide bonds.Simulation 2: As for IgG1, 300,000 trial models were generated by varying the full hinge Simulation 3: As for IgG1, 400,000 trial models were generated in which the Cys226-Cys226 and Cys229-Cys229 pairs involved in interchain disulphide bonding in IgG4 had to be within 0.75 nm of one another. From this, 46,869 models (11.7%) were acceptable. As for IgG1, this disulphide bridge constraint removed many models.For IgG4, two Monte Carlo simulations generated 700,000 IgG4 randomized conformations, from which 190,437 structural models were sterically acceptable. Simulation 1 for IgG1 was not performed for IgG4 because the Fab and Fc crystal structures used for the starting model did not contain a disulphide-bridged hinge structure. The simulations resulted in 46,979 models that contained both canonical disulphide bonds:I(Q) (I(Q) value was matched to the theoretical I(Q) value with the closest Q value. For the x-ray curves with up to 365 data points, the Q spacing is close enough for this procedure to have little effect, whereas the neutron curves have up to 45 data points and the quality of the matches is reduced  . For comI(Q) ,20. The  reduced . After tη is a scaling factor used to match the theoretical curve to the experimental I(0) value. An iterative search to minimize the R factor was used to determine η. All steps were performed in the SASSIE-web workflow (version 0.8) (https://sassie-web.chem.utk.edu/sassie2). Structures with the lowest R factors were accepted as valid models of the antibody solution structure. We note that χ2 values are often used elsewhere as a monitor of best fits to scattering curves; these, however, require errors for the experimental intensities that were not always available.B). The twofold symmetry of the antibody primary structure meant that the differentiation between Fab1 and Fab2, and consequently d2 and d3, is only there for descriptive clarity. These parameters were used previously for IgG4  and those between each Fab region with the Fc region  B. The tfor IgG4  and othefor IgG4 ,47,48. AγRI receptor were docked onto the best-fit structures for the IgG1 β and the IgG4 α and β clusters using the web server algorithm PatchDock as described previously .2O and 2H2O buffers , the corresponding previous and new Rg, Rxs1, and Rxs2 values were similar to those of IgG1 6a   . The scabaseline . This diThe atomistic solution structural modeling of IgG1 was initiated using the crystal structure of full-length IgG1 b12 . MissingR factor analysis was used to identify the errors in the experimental data sets and the R factor filter required to select best-fit models. This R factor monitored the agreement between the theoretical and experimental curves to select modeled solution structures that were consistent with the experimental curves. To achieve this selection, it was necessary to determine a cutoff R factor below which models were assigned as best fits depending on the experimental scattering curve, its signal/noise ratio, and its Q range. To determine this cutoff, two experimental curves for the same protein from the same instrument were used to calculate two R factors for each of the 231,492 modeled IgG1 curves. The correlation between the two R factors was assessed using both the Pearson r and Spearman rs coefficients . The final R factor cutoffs for IgG1 were determined to be 3.00, 2.00, 3.15, and 3.10% for instruments ID02, SANS2d, D11, and D22, respectively.A goodness-of-fit ficients . By gradIgG1 19a . Because 19a . Filtering for the above-determined R factor for instrument ID02 of 3.00% to give 37,412 models reduced d1 to 4.5–12 nm . The disulphide distance constraint separated the resulting 4728 models into two clusters . The 389 models with d1 of ∼6 nm and below 7 nm were denoted as Cluster α. These were Y-shaped symmetric structures because abs(d2 − d3) was low at ∼1 nm. The 4339 models with d1 of ∼9.5 nm and above 7 nm were denoted as Cluster β. Those Cluster β structures with high asymmetry and a large abs(d2 − d3) were labeled as β1, whereas those Cluster β structures showing symmetry with a low abs(d2 − d3) were labeled as β2 . These α or β clusters were only visible through the abs(d2 − d3) difference, not otherwise (A and B), hence showing the utility of the abs(d2 − d3) values to evaluate the filtered models.The structural outcome of the IgG1 6a SAXS curve fits was monitored by the d1, d2, and d3 distances between the centers of mass of the Fab and Fc regions B; , 16,46).,46.B;  curves calculated from representative Cluster α, β1, and β2 best-fit structures with the lowest R factors revealed very good visual fits (I(Q) fits were corroborated by very good visual fits with the distance distribution function P(r) for all three Clusters α, β1, and β2, especially the relative intensities of the M1 and M2 peaks . The models that passed both the R factor and disulphide distance filters showed a Fab-Fab separation d1 between 6 and 11 nm (B). The D11 and D22 fits  showed slightly reduced d1 values compared to the SANS2d fits . Unlike the SAXS modeling, no distinct α and β clusters were identified. Visual inspection of the curve fits of the theoretical scattering curves with the SANS experimental data for representative structures confirmed that both clusters provide plausible models . This range was smaller than that of 3.75–6.47 nm sampled for IgG1  was determined to give 28,084 models , with 32 (0.02% accepted) and 14,895 (32% accepted) for Simulations 2 and 3, respectively . Filtering for R factors below 3.00% limited d1 to a range between 5 and 12 nm . Interestingly, the maximum d1 value of 12 nm was similar to that for IgG1 . Application of the hinge disulphide constraint resulted in the observation of two clusters, α and β, that corresponded to d1 being below or above 7 nm, respectively . Cluster β in IgG4 showed a smaller average d1 of 8.09 nm compared to 9.34 nm in IgG1  values mostly found at 2.5 nm and labeled β1. Cluster β2 was much less populated for IgG4 compared to IgG1. When the IgG4 structures were filtered using curve fits based on the SAXS instrument BM29 with an R factor cutoff of 2.40%, very similar Clusters α, β1, and β2 were again visible , thus confirming the reproducibility of the modeling fits from two different SAXS data sets.The structural outcome of the IgG4 SAXS curve fits was monitored by the distances between the centers of mass of the Fab and Fc regions, as for IgG1 above. The 190,437 models showed a Fab separation d1 that extended to 14 nm gray, A. Filte purple, A. Inter purple, A. Applimagenta, A. Clust in IgG1  and 3. UI(Q) were seen for representative best-fit IgG4 structures for Clusters α, β1, and β2 that had the lowest R factors (P(r), including the relative intensities of the M1 and M2 peaks in the P(r) curves  and 4.78 ± 0.08, 2.58 ± 0.07, and 1.34 ± 0.11 nm (Cluster β) were in good agreement with the experimental values of 4.99 ± 0.02, 2.50 ± 0.03, and 1.37 ± 0.02 nm, respectively , 2.00% , and 2.85% , far fewer modeled structures satisfied these double x-ray and neutron filters.1)α conformations was confirmed by the exclusion of Cluster α in the double x-ray and neutron fits . There, Cluster β was centered at d1 = 9.68 ± 0.45 nm  and D22 . There, Cluster β was centered at similar d1 values of 9.06 ± 1.06 and 9.08 ± 1.06 nm α and β models passed the double x-ray and neutron filter. Both clusters showed similar populations when tested against the joint ID02-SANS2d data sets (A and B (in blue). In Clusters α and β, two of the three separations d1, d2, and d3 in IgG4 were 6.8–7.0 nm, and the third was 8.6 nm ; thus, this outcome was reproducible.For IgG4, both the Cluster ata sets . An expls 8.6 nm , unlike s 8.6 nm . Given tts blue, , C and D3)1HZH) gave separations d1 = 9.0 nm and d2 and d3 = 9.0 and 6.1 nm that resembled Cluster β (5DK3) gave separations d1 = 7.0 nm and d2 and d3 = 7.6 and 5.9 nm that were intermediate between Cluster α and β . Co. Co1HZH)The SAXS atomistic modeling analyses for IgG1 and IgG4 resulted in 4728 and 14,927 conformational models, respectively  and 3, tnstraint  and 7. Tnstraint , were viR factors for filtering based on experimental curve comparisons, and provided two different views of hydrated and unhydrated IgG structures. To model these data sets, molecular dynamics first ensured that the starting antibody protein structures for IgG1 and IgG4 based on crystal structures were physically realistic and complete. Each starting structure was then inputted into the SASSIE-web modeling workflow, in which Monte Carlo randomization of the antibody hinge structure was performed  with separations d1 of 6.0 nm and d2 and d3 = 8.8 and 7.7 nm and the other being 1103 asymmetric structures (Cluster β) with separations d1 of 8.0 nm and d2 and d3 = 8.4 and 6.0 nm . Second, these molecular structures provided new functional insights on the molecular basis for IgG1 and IgG4 complex formation with two major ligands, i.e., the globular heads of complement C1q and the high-affinity FcγRI receptor (1)β) were combined with a docking model for the interaction between human IgG1 Fc and the crystal structure of the C1q globular head, this being taken from our 2015 study . Similar results were obtained for crystal structures for the Fc-FcγRIII complexes, showing that the IgG1-FcγRIII interactions were permitted α in IgG4, the combination of the 1645 best-fit IgG4 structures with the C1q head and the FcγRI receptor showed 109 (7%) and 1340 (82%) steric overlap of the two ligands with the Fab regions, respectively (B). For Cluster β for IgG4, the combination of the 1103 best-fit IgG4 structures with the C1q head and the FcγRI receptor showed 203 (18%) and 1009 (95%) steric overlap of the two ligands with the Fab regions, respectively (C). The low overlap for the C1q-IgG4 interaction indicated that this interaction was permitted, provided that the shorter IgG4 hinge showed enough flexibility to enable the Fab regions to be displaced for complex formation. The high overlap for the FcγRI receptor indicated that IgG4 would show reduced reactivity for binding to its receptor, making this interaction unlikely. High overlap was seen also for the FcγRIII receptor, making this interaction unlikely, too.For Cluster ectively B. For Cectively C. The lNew, to our knowledge, biological insights were obtained from these atomistic IgG1 and IgG4 solution structures. First, by considering the hinge peptides as molecular structures in the fits, we have an atomistic explanation for the more elongated solution structure of IgG1 compared to that of IgG4. The atomistic modeling also showed that the IgG1 solution structure is asymmetric, this asymmetry being similar to that of the IgG1 crystal structure A. Seconreceptor . Here, areceptor ,16, provOur IgG1 and IgG4 studies complement other recent investigations on antibody solution structures. A neutron spin echo study  was condThe atomistic modeling of antibody solution structures from scattering data has evolved significantly since its original inception in 1995. Our first approach used systematic translations and rotations of separate Fab and Fc crystal structures in bovine IgG to fit SANS data, followed by the molecular modeling of the two hinge peptides into the best-fit Fab-Fc arrangement . Our secα and β solution structures. Alongside these scattering fits, the availability of high-quality scattering curves was indispensable. Here, experimental errors in these curves were considered by comparing side-by-side two independent experimental scattering curves of the same protein, then computing the R factor to show the extent of agreement between these. Our curve comparison of data from two different x-ray and three different neutron instruments showed that the lowest R factors ranged between 2.00 and 3.15%. The quality of the modeled fits surpassed this experimental limit for both IgG1 and IgG4, with R factors below 3% in the full scattering curve Q range out to 1.5 nm−1  much reduced the number of allowed structures in the ensemble. In the future, the advent of size-exclusion chromatography in SAXS and SANS data collection will improve the quality of the scattering curves by the removal of trace aggregates, leading to improved modeling outcomes.These IgG1 and IgG4 atomistic structural fits yield significantly more information from scattering experiments than previously. For this, molecular dynamics and Monte Carlo methods proved indispensable. Even though the conformational difference between IgG1 and IgG4 was evident from the x-ray and neutron scattering data , the ato1.5 nm−1  and 3, i1.5 nm−1 , not oneα and β, that gave indistinguishable scattering fits. Cluster α featured the two Fab regions ∼6 nm apart and both separated by ∼8.0–8.5 nm from the Fc region in mostly symmetric structures . The fit ambiguity resulted from the similar sizes and shapes of the Fab and Fc regions, meaning that these could be interchanged and still give good fits. For IgG1, this ambiguity was resolved by the disulphide constraint and the use of joint x-ray and neutron fits; this eliminated Cluster α to leave Cluster β as the best-fit solution structure. For IgG4, its shorter hinge meant that both Clusters α and β offered good fits, with no clear preference for either conformation. It is possible that the two clusters represent degeneracies in the modeling and not two distinct structural outcomes. Nonetheless, this result still enabled the analysis of IgG4 binding to its C1q and FcγRI ligands by docking. This outcome emphasizes the importance of combining multiple experimental data sets to understand protein solution structures, especially when combining solution data with crystal and NMR structures . A protWe have reported here in detail a new, to our knowledge, new modeling method to determine solution structures of full-length human IgG1 and IgG4 antibodies by joint SAXS and SANS studies. This opens new avenues for future structural studies of full-length antibodies. New biological insights were obtained on the IgG1 and IgG4 solution structures. The IgG1 and IgG4 atomistic structural fits have yielded significantly more information from scattering experiments than previously.D.W.W. and E.L.K.E. designed and performed the modeling analyses and wrote the manuscript. G.K.H. obtained SAXS and SANS data and analyzed these. S.J.P. conceived and coordinated the study and wrote the manuscript.


SCN5A encodes the main subunit of the NaV1.5 channel, which mediates the fast Na+ current responsible for generating cardiac action potentials. The single nucleotide polymorphism SCN5A(R1193Q), which results in an amino acid replacement in the subunit, is common in East Asia. SCN5A(R1193Q) is often identified in patients with type 3 long QT syndrome and Brugada syndrome. However, its linkage to arrhythmic disorders is under debate. Previous electrophysiological studies performed at room temperature inconsistently reported the gain- or loss-of-function effect of SCN5A(R1193Q) on the NaV1.5 channel. More recently, it was theoretically predicted that SCN5A(R1193Q) would exert a loss-of-function effect at body temperature. Here, we experimentally assessed whether SCN5A(R1193Q) modulates the NaV1.5 channel at various temperatures including normal and febrile body temperatures. We compared voltage-gated Na+ currents in SCN5A(R1193Q)-transfected and wild-type SCN5A-transfected HEK293T cells using a whole-cell voltage-clamp technique. First, we made comparisons at constant temperatures of 25°C, 36.5°C, and 38°C, and found no difference in the conductance density, voltage dependence of gating, or time dependence of gating. This suggested that SCN5A(R1193Q) does not modulate the NaV1.5 channel regardless of temperature. Second, we made comparisons while varying the temperature from 38°C to 26°C in 3 min, and again observed no difference in the time course of the amplitude or time dependence of gating during the temperature change. This also indicated that SCN5A(R1193Q) does not modulate the NaV1.5 channel in response to an acute body temperature change. Therefore, SCN5A(R1193Q) may not be a monogenic factor that triggers arrhythmic disorders. SCN5A encodes the pore-forming subunit of the human cardiac NaV1.5 voltage-gated Na+ channel, which is responsible for generating cardiac action potentials -transfected cells  and RQ cells .To quantify the time dependence of activation, we measured the time for the current level to increase from 10% to 90% of the peak amplitude (10%–90% rise time) on the trace of f –20 mV . There wV1.5 channel availability for subsequent action potentials [SCN5A(R1193Q) altered such inactivation using a double-voltage step protocol ]. This result suggested that SCN5A(R1193Q) does not affect the inactivation limiting channel availability.In cardiomyocytes under physiological conditions, inactivation occurring at the resting state (~–80 mV) between the action potentials decreases Natentials . Therefoprotocol . As the INa amplitude against conditioning potential were similar between WT and RQ cells at all tested temperatures. Moreover, the Vhalf and slope factor of the plot did not differ significantly  between WT cells and RQ cells at any tested temperature  in the time constant estimated from the slow component between WT cells  and RQ cells  . This result suggested that SCN5A(R1193Q) does not affect recovery from inactivation.We assessed the time dependence of recovery from inactivation using a triple-voltage step protocol . INa,2/IIn the second set of experiments , we evalINa amplitude gradually decreased  in 10%–90% rise time at temperatures near 26°C, 36.5°C, and 38°C between WT cells  and RQ cells   .INa slowed down (t-test) in the time constant of the fast component at temperatures near 26°C, 36.5°C, and 38°C between WT  and RQ cells  (t-test) in the time constant of the slow component at temperatures near 26°C, 36.5°C, and 38°C between WT  and RQ cells   . Similar; n = 7) .SCN5A(R1193Q) does not reduce the cell surface expression or unitary conductance of the NaV1.5 channel. This directly indicates that SCN5A(R1193Q) does not cause the previously predicted loss of channel function that could manifest at normal or febrile body temperatures [SCN5A(R1193Q) does not cause the gating abnormalities that were previously reported to occur at room temperature [A similarity in whole-cell conductance density between WT and RQ cells at 36.5°C and 38°C  suggesteeratures . MoreoveSCN5AR93Q does perature .In this study, cells were cultured at a normal body temperature and then exposed to a test temperature during the recording session. Thus, the total time of conditioning to the febrile body temperature prior to measurement was up to ~1 h. We therefore cannot exclude the possibility that long-term  exposure to a febrile body temperature could produce a difference(s) in gating and/or membrane trafficking between WT channels and those containing SCN5A(R1193Q) subunit.SCN5A mutations were shown to cause channel malfunctions that manifest only near body temperature. For example, SCN5A(T1620M) right-shifts the voltage dependence of activation and accelerates inactivation at 32°C [SCN5A(F1344S) also right-shifts the voltage dependence of activation at 40.5°C [SCN5A splice variant lacking Q1077 showed a reduced current density at 32°C and 37°C [Some  at 32°C , while S SCN5AT16M right-sand 37°C . Such te SCN5AT16M right-sand 37°C .SCN5A(R1193Q) did not alter the voltage or time dependence of activation, inactivation, or recovery from inactivation at any tested temperature  does not modulate the NaV1.5 channel in a temperature-dependent fashion.By contrast, ure Figs –4. Theseure Figs –4. This SCN5A(R1193Q) does not modulate the NaV1.5 channel in response to an acute change of temperature. It is noteworthy that differences between the rates of activation and inactivation at body temperature and room temperature measured during a gradual temperature change  does not modulate the NaV1.5 channel at body temperature. These results together with the previously reported similarity in the MAFs of SCN5A(R1193Q) between the arrhythmic patients and the healthy controls [SCN5A(R1193Q) may not be the monogenetic cause of arrhythmic disorders.Taken together, our findings suggest that controls –10 indic


Its pronounced selectivity for the human receptor (hCXCR4), however, precludes the use of [68Ga] Pentixafor for imaging receptor expression and dynamics in CXCR4-related diseases in endogenous mouse models. To overcome this restriction, [125I]CPCR4.3, a structurally related pentapeptide ligand, has been evaluated as a preclinical tool for efficient in vitro and in vivo targeting of hCXCR4 and mCXCR4.The development and clinical translation of [68Ga] Pentixafor, [125I]CPCR4.3 showed 2.4- to 11-fold increased specific binding to human cancer cell lines with different hCXCR4 expression levels  as well as strong and highly specific binding to mCXCR4 expressing cells , which was not detectable for [68Ga]Pentixafor. This is the consequence of the equally high affinity of iodo-CPCR4 to hCXCR4 and mCXCR4  as opposed to [natGa] Pentixafor . Additionally, [125I]CPCR4.3 showed enhanced tracer internalization (factor of 1.5–2 compared to the reference). In vivo biodistribution studies in immunocompetent Black Six and immunocompromised CD-1 nude mice showed predominant hepatobiliary excretion of [125I]CPCR4.3 (logP = 0.51), leading to high activity levels in liver and intestines. However, [125I]CPCR4.3 also showed high and specific accumulation in organs with endogenous mCXCR4 expression , even at low receptor expression levels.Compared to the reference [125I]CPCR4.3 represents a sensitive and reliable tool for the species-independent quantification of CXCR4 expression. Its suboptimal clearance properties will certainly restrict its use for in vivo imaging applications using 123I (for SPECT) or 124I (for PET), but due to its high and specific accumulation in mCXCR4 expressing tissues, [125I]CPCR4.3 holds promise as a powerful preclinical tool for the investigation and quantification of CXCR4 involvement and kinetics in various murine disease models via, e.g., biodistribution and autoradiography studies.Due to its excellent hCXCR4 and mCXCR4 targeting efficiency, both in vitro and in vivo, [ To date, there is only one report on the use of [68Ga] Pentixafor for imaging of inflammation after myocardial infarction in a mouse model FBz-TN14003 Ala2-Arg3-Nal4-Gly5-), CPCR4.3, Fig. 125I]CPCR4.3 as the lead compound for this proof-of-concept study.In a different context, we recently reported on the identification of a conformationally restricted FC-131-derived cyclopentapeptide with subnanomolar affinity for hCXCR4 . Surpris125I]CPCR4.3 was investigated in a panel of human and murine cancer cell lines with different levels of endogenous CXCR4 expression as well as CHO-K1 cells transiently transfected with hCXCR4 and mCXCR4 (positive controls) and compared to the hCXCR4 selective reference compound [68Ga]Pentixafor. To evaluate the in vivo mCXCR4 targeting efficiency of [125I]CPCR4.3, comparative biodistribution studies were performed in non-tumor-bearing Black Six (immunocompetent) and CD-1 nude (immunocompromised) mice.The hCXCR4- and mCXCR4-specific binding of [CPCR4.3 was synthesized as described . The cor68Ga] Pentixafor was prepared according to a previously established protocol [125I] NaI , the reaction vessel was briefly vortexed and the labeling reaction was allowed to proceed for 15 min at RT. The peptide solution was then removed from the insoluble oxidizing agent. Separation of [125I]CPCR4.3 from unlabeled precursor was achieved using gradient RP-HPLC , gradient: 22–42% ethanol (0.5% acetic acid) in water (0.5% acetic acid) within 20 min, flow: 1 mL/min).The reference ligand [protocol . Radioio125I]CPCR4.3 was then reconstituted to an activity concentration of app. 1 MBq/100 μL using PBS and was then used for the in vivo study.For in vitro binding and uptake studies, the HPLC product fraction was used as such and diluted to the required concentration using the respective assay medium. For biodistribution experiments, excess ethanol was removed by bubbling an argon stream through the product fraction at 90 °C for 20 min. [125I]CPCR4.3 was determined via a modified shake-flask method as described previously CPCR4.3 and the reference [68Ga] Pentixafor was investigated in various cell lines with different endogenous hCXCR4  and mCXCR4  expression levels. For data validation, CHO-K1 cells transiently transfected with hCXCR4 and mCXCR4 [CXCR4-mediated cellular uptake of 15ICPCR4.35 cells in assay medium were incubated with [125I]CPCR4.3 (0.1 nM) or the reference [68Ga] Pentixafor (1 nM) at 37 °C for 60 min in the presence (non-specific binding) or absence (control) of 100 μM AMD3100 . After incubation, the tubes were centrifuged  and the supernatant was carefully removed. After washing twice with 200 μL of cold HBSS, the amount of bound radioligand in the cell pellet was quantified using a γ-counter .In the case of suspension cells , samples containing 2 × 10125I]CPCR4.3 (0.1 nM) or the reference [68Ga] Pentixafor (1 nM) at 37 °C for 60 min in the presence (non-specific binding) or absence (control) of 100 μM AMD3100 . Upon incubation, the incubation medium was removed, and cells were rinsed twice with 250 μL of HBSS and lysed using 300 μL of 1 N NaOH. The lysate was transferred to vials and combined with 250 μL of HBSS used for rinsing the wells. Quantification of the amount of free and bound activity was performed in an Automatic Gamma Counter.In the case of the adherent cell lines , app. 150,000 cells/well were seeded into PLL-coated 24-well plates  on the day prior to the experiment. On the day of the experiment, the culture medium was removed, and the cells were left to equilibrate in 200 μL of assay medium (RPMI + 5% BSA) at 37 °C for a minimum of 15 min before the experiment. Then, the cells were incubated with [50) [5 cells/sample) in Hank’s buffered salt solution (1% BSA) and [125I]CPCR4.3 as radioligand. To allow data normalization, [natGa] Pentixafor and FC-131 were included as references in this study. Experiments were performed in triplicate with n = 3 per concentration in each experiment. IC50 values were calculated using GraphPad Prism 6.01 .Affinities for human and murine CXCR4 (hCXCR4 and mCXCR4) were determined in competitive binding assays (IC50)  using ei125I]CPCR4.3 and [68Ga] Pentixafor into the human cancer cell lines HT-29, SH-5YSY, MCF-7, and LNCaP was investigated in analogy to a previously published protocol [The internalization of [protocol . Non-spe125I]CPCR4.3 (0.1 nM) at 37 °C for 30 min and washed with HBSS. In the experiment allowing ligand recycling, 250 μL of assay medium were added to the wells (n = 3). In the experiment inhibiting ligand recycling, 250 μL of assay medium containing 100 μM AMD3100 were added to the wells (n = 3). Subsequently, cells were incubated at 37 °C for 5, 15, 30, and 60 min, respectively. The supernatant was removed and combined with 250 μL of HBSS used for rinsing the cells. This fraction represents the amount of externalized ligand at the respective time point. The following lysis of the cells was performed as described for the radioligand binding study.To determine ligand washout and recycling kinetics, cells  were first incubated with [Animal experiments were performed in accordance with the current animal welfare regulations in Germany .125I]CPCR4.3, approximately 550 kBq (15 μCi) of [125I]CPCR4.3 in a total volume of 100 μL of PBS (pH 7.4) were injected intravenously (i.v.) into the tail vein of immunocompetent Black Six mice (n = 4) under isoflurane anesthesia. To determine mCXCR4 specificity of ligand accumulation, 50 μg AMD3100/mouse were coinjected (n = 4). The animals were sacrificed 60 min post injection (p.i.), and the organs of interest were dissected. The radioactivity was measured in weighted tissue samples using a γ-counter. Data are expressed in % ID/g tissue (mean ± SD). For a comparative biodistribution study in an immunodeficient mouse strain, CD-1 nu/nu mice were used. Statistical analysis (one-tailed t test) of the separate biodistribution data sets was performed using Microsoft Excel.To investigate the biodistribution of [125I]CPCR4.3 and the reference ligand [68Ga] Pentixafor to a panel of human and murine cancer cell lines with different levels of endogenous CXCR4 expression as well as to CHO-K1 cells transiently transfected with h- and mCXCR4 (positive controls) after a 60-min incubation (37 °C).Figure 125I]CPCR4.3 and [68Ga] Pentixafor follows the same pattern; both ligands exhibit highest uptake in the hCXCR4-transfected control cell line CHO-hCXCR4 and low binding to the negative control cells CHO-WT . For [125I]CPCR4.3, the level of non-specific binding was generally higher than for [68Ga]Pentixafor: in the presence of 100 μM AMD3100, 3.7 ± 0.6% of the added [125I]CPCR4.3 activity was found to be non-specifically bound to the cells after 60 min, independently from the cell line used, with only 1.3 ± 0.1% of the [68Ga] Pentixafor activity being non-specifically bound under the same conditions. This is most probably the result of the increased lipophilicity of [125I]CPCR4.3 (log P = 0.51 vs log P = − 2.9 for [68Ga]Pentixafor), favoring non-specific interactions with the cell membrane.As shown, hCXCR4-specific binding of both [125I]CPCR4.3 and the reference [68Ga] Pentixafor follow the same pattern, with the human lymphoma cell lines Jurkat and Daudi showing the highest uptake. These findings correlate well with the documented high  [125I]CPCR4.3 was found to be 2.4- (Daudi cells) to 11-fold (LNCaP cells) enhanced compared to [68Ga] Pentixafor, indicative of a higher affinity of the radioiodinated ligand to hCXCR4 and consequently a more sensitive detection of low-level CXCR4 expression.In the cell lines with endogenous hCXCR4 expression, uptake levels of both 15ICPCR4.3ference 6Ga Pentix Jurkat) ); and lo Jurkat) , LNCaP [ Jurkat) ) CXCR4 e68Ga] Pentixafor shows virtually no binding to mCXCR4 [125I]CPCR4.3 binds strongly and specifically to murine CXCR4, both in the mCXCR4-transfected CHO control cells and the murine lymphoma  and breast cancer  cell li125I]CPCR4.3 binding, radioiodinated CPCR4.3 was used as the “universal” radioligand in the subsequent determination of the hCXCR4 and mCXCR4 affinity of CPCR4.3 and its 3-iodo-Tyr- counterpart (iodo-CPCR4.3) in comparison to the commonly used reference ligand FC-131 and [natGa]pentixafor.Based on the initial ligand binding experiment demonstrating the species independence of cellular [natGa] Pentixafor shows the already documented pronounced selectivity for hCXCR4 over mCXCR4 [natGa] Pentixafor under the assay conditions used in this study.As shown in Table r mCXCR4 . The oth125I]CPCR4.3 to hCXCR4 expressing cells in comparison to [68Ga] Pentixafor observed in the initial radioligand binding study , the numerical values for the affinity of CPCR4.3 and [natGa] Pentixafor differ from previously reported data CPCR4.3 is able to target both receptors with the same affinity.As shown in our previous study on the parent peptide CPCR4.3, the successive implementation of small structural changes in the pentixafor backbone, i.e., introduction of the so-called “peptoid structure” , in whicpeptides , 20. Givproteins , alongsi125I]CPCR4.3, its internalization and externalization properties , demonstrating species independent externalization and recycling kinetics for [125I]CPCR4.3.To evaluate the extent of tracer washout and ligand recycling as well as eventual differences in ligand processing between hCXCR4 and mCXCR4 expressing cells, externalization and recycling kinetics of 15ICPCR4.3125I]CPCR4.3 under conditions allowing ligand recycling is reduced compared to the transfected cell lines (with only 50.8 ± 1.2% (HT-29) and 45.2 ± 1.9% (MCF-7) of the initial activity remaining inside the cells after 60 min), indicating a lower efficiency of ligand recycling in these cell lines. This, however, is most probably a consequence of the significantly lower hCXCR4 expression in HT-29 and MCF-7 cells compared to the transfected CHO cells, leading to less efficient tracer re-binding and reinternalization. Interestingly, a higher fraction of [125I]CPCR4.3 activity is ultimately retained in HT-29 and MCF-7 cells compared to the transfected cell lines under conditions inhibiting ligand recycling (25.1 ± 0.6% (HT-29) and 22.6 ± 1.0% (MCF-7) of the initial cellular activity after 120 min). The same effect had also been observed, when externalization kinetics of radioiodinated sst-ligands were investigated in sst2-transfected CHO cells and endogenously sst-expressing AR42J cells CPCR4.3 shows suitable cellular uptake kinetics as well as retention both in hCXCR4 and in mCXCR4 expressing cells as a prerequisite for fast and specific h/mCXCR4 targeting in vivo as well as reasonably slow washout from target tissues.Overall, [125I]CPCR4.3 to specifically target mCXCR4 expression in vivo, biodistribution studies using different mouse strains (Black Six: immunocompetent vs CD-1 nude: immunodeficient) were performed. Table To assess the potential of [125I]CPCR4.3 shows rapid clearance from the circulation and is, due to its pronounced lipophilicity (log P = 0.51), predominantly cleared via the hepatobiliary route, leading to very high accumulation in liver and intestines. The only other organs with notable tracer accumulation are stomach, kidney, and spleen. As demonstrated by coinjection of an excess of unlabeled competitor, i.e., AMD3100, the splenic uptake of [125I]CPCR4.3 is primarily CXCR4 mediated. The same applies to [125I]CPCR4.3 uptake in the other tissues with endogenous CXCR4 expression, lung and adrenals CPCR4.3 in non-target organs were observed  immune cell population in mouse spleen. In contrast, [125I]CPCR4.3 uptake levels in the mCXCR4 expressing non-lymphoid organs lung and adrenals were not statistically different in the two mouse strains. This highlights the ability of [125I]CPCR4.3 to quantitatively detect—despite clearly not optimal excretion characteristics—different levels of endogenous mCXCR4 expression in vivo. This is also supported by results from a previous study, where [125I]CPCR4.3 was able to (semi) quantitatively detect murine immune cell infiltrates in a mouse model of esophageal carcinogenesis CPCR4.3 represents a sensitive and reliable tool for the species-independent quantification of CXCR4 expression. Despite its suboptimal clearance properties due to its relatively high lipophilicity, i.e., high accumulation in the gastrointestinal system, which certainly restricts its use for in vivo imaging applications using 123I (for SPECT) or 124I (for PET), [125I]CPCR4.3 shows high and specific accumulation in mCXCR4 expressing tissues. Thus, it holds promise as a powerful preclinical tool for the investigation and quantification of CXCR4 involvement and kinetics in various murine disease models via, e.g., biodistribution and autoradiography studies. Given the successful proof-of-concept introduction of the CPCR4.3 pentapeptide scaffold as a high-affinity ligand for hCXCR4 and mCXCR4, ongoing efforts are currently directed toward the implementation of alternative radiolabeling strategies by refined chemical conjugation approaches, leading to first very promising radiometal-labeled CPCR4.3 analogs.Due to its excellent hCXCR4 and mCXCR4 targeting efficiency, both in vitro and in vivo, [


Bacillus thuringiensis insecticidal proteins (Bt toxins) have been widely used in crops for agricultural pest management and to reduce the use of chemical insecticides. Here, we have engineered Bt toxin Cry2Ab30 and bioconjugated it with 4”-O-succinyl avermectin (AVM) to synthesize Cry2Ab–AVM bioconjugate. It was found that Cry2Ab–AVM showed higher insecticidal activity against Plutella xylostella, up to 154.4 times compared to Cry2Ab30. The binding results showed that Cry2Ab–AVM binds to the cadherin-like binding protein fragments, the 10th and 11th cadherin repeat domains in the P. xylostella cadherin (PxCR10–11), with a much higher affinity (dissociation equilibrium constant DK = 3.44 nM) than Cry2Ab30 (DK = 28.7 nM). Molecular docking suggested that the macrolide lactone group of Cry2Ab–AVM ligand docking into the PxCR10–11 is a potential mechanism to enhance the binding affinity of Cry2Ab–AVM to PxCR10–11. These findings offer scope for the engineering of Bt toxins by bioconjugation for improved pest management. Bacillus thuringiensis insecticidal proteins (Bt toxins) have been shown to be highly effective in agricultural pest control +.4”-O-Succinyl avermectin was synthesized according to previously reported procedures ,43. Selecry2Ab30 was transformed into E. coli BL21 (DE3) cells. The bacterial stab was inoculated onto the LB medium containing kanamycin (35 μg/mL)  and incubated at 20 °C. The expression of Cry2Ab30 was induced with 0.2 mM isopropyl-B-D-thiogalactopyranoside (IPTG)  after the OD600nm reached 0.6. After that, Cry2Ab30 was purified using a Ni-IDA prepacked column .The production and purification of Cry2Ab30 was as described in our previous study . The plaP. xylostella cadherin (PxCR10–11) binding Cry2Ab30 were expressed and purified as described in our previous study [2HPO4, 1.8 mM KH2PO4, pH 7.4). PxCR10–11 was purified using a GST prepacked gravity column .The 10th and 11th CR domains (1258–1602 aa) in the us study . Cells w−6 mol) was firstly dissolved in 1 mL of DMF . Then, triethylamine (2 × 10−5 mol)  was added to provide protons, EDC (2 × 10−5 mol)  was added to form an O-acylisourea active intermediate, and NHS (5 × 10−5 mol)  was added to form an NHS ester intermediate; this intermediate was stirred for 0.5 h at 25 °C to activate 4”-O-succinyl avermectin. Cry2Ab30 (2 × 10−8 mol) was desalted into PBS  using a size-exclusion column , and activated 4”-O-succinyl avermectin was then added to Cry2Ab30 PBS buffer. The mixture was vortexed and allowed to react at room temperature for 2 h. After the reaction was completed, Cry2Ab–AVM was purified using a PD-10 desalting column  [4”-O-Succinyl avermectin  . Cry2Ab–NJ, USA) . The fluNJ, USA) . Further10–11 were detected by a ligand blot assay [10–11 protein in TBST buffer overnight followed by three washes of TBST buffer.The binding abilities of Cry2Ab30 and Cry2Ab–AVM to PxCRot assay . Cry2Ab310–11 were assayed on an Octet Red 96 biolayer interferometer  [10–11 was first loaded onto anti-GST biosensors  for 300 s. The binding of Cry2Ab30 and Cry2Ab–AVM to immobilized PxCR10–11 was measured with a 400 s association followed by a 400 s dissociation. The buffer was 10 mM GSH, 50 mM Tris, pH 8.0. The dissociation equilibrium constant (KD) was determined by Octet System Data Analysis software  [The binding kinetics of Cry2Ab30 or Cry2Ab–AVM to PxCRCA, USA) ,49. GST-CA, USA) ,52.P. xylostella. Different doses of Cry2Ab30 and Cry2Ab–AVM solutions were spread on the surface of the diet and allowed to dry. Samples were treated with the same volume of PBS buffer as controls. Thirty individual larvae were used for each concentration. All the bioassay experiments were repeated in triplicates. Observations were recorded at 72 h, and the mortality was determined and statistically analyzed using SPSS 17.0  [The bioassays of Cry2Ab30 and Cry2Ab–AVM were performed with second-instar IL, USA) .P. xylostella were starved for 5 h and then fed an artificial diet including Cry2Ab30 or Cry2Ab–AVM for 24 h. Larvae fed with PBS buffer  were set as negative controls. The larvae were then dissected, and the midgut tissues were then isolated and immediately fixed in 2.5% glutaraldehyde  and post-fixed in 1% OsO4 . The fixed midgut tissues were then immersed into Epon for embedding. Ultrathin sections were sliced using a Leica EM UC7 ultramicrotome , and stained with uranyl acetate and then lead citrate [Third instar larvae of  citrate . The ult10–11 was built by I-TASSER servers (http://zhanglab.ccmb.med.umich.edu/I-TASSER) and the molecular structure of 4”-O-succinyl avermectin was built using ChemBioDraw Ultra 12.0 . Docking of 4”-O-succinyl avermectin to PxCR10–11 was analyzed by Molecular Operating Environment (MOE)  with default parameters; 4”-O-succinyl avermectin was selected as the ligand [The protein structure of PxCRe ligand . All stre ligand .


Residual axillary lymph node involvement after neoadjuvant systemic therapy (NST) is the determining factor for postmastectomy radiation therapy (PMRT). Preoperative identification of patients needing PMRT is essential to enable shared decision-making when choosing the optimal timing of breast reconstruction. We determined the risk of positive sentinel lymph node (SLN) after NST in clinically node-negative (cN0) breast cancer.All cT1-3N0 patients treated with NST followed by mastectomy and SLNB between 2010 and 2016 were identified from the Netherlands Cancer Registry. Rate of positive SLN for different breast cancer subtypes was determined. Logistic regression analysis was performed to determine correlated clinicopathological variables with positive SLN.In total 788 patients were included, of whom 25.0% (197/788) had positive SLN. cT1-3N0 ER+HER2+, cT1-3N0 ER−HER2+ , and cT1-2N0 triple-negative patients had the lowest rate of positive SLN: 7.2–11.5%, 0–6.3%, and 2.9–6.2%, respectively. cT1-3N0 ER+HER2− and cT3N0 triple-negative patients had the highest rate of positive SLN: 23.8–41.7% and 30.4%, respectively. Multivariable regression analysis showed that cT2 , cT3 , grade 3 , and ER+HER2− subtype  were correlated with positive SLN.In cT1-3N0 ER+HER2+, cT1-3N0 ER−HER2+, and cT1-2N0 triple-negative patients treated with NST, immediate reconstruction can be considered an acceptable option due to low risk of positive SLN. In cT1-3N0 ER+HER2− and cT3N0 triple-negative patients treated with NST, risks and benefits of immediate reconstruction should be discussed with patients due to the relatively high risk of positive SLN. Rates of reconstruction at the time of mastectomy  have increased considerably over the past decades.11Therefore, the purpose of this study was to determine the overall risk of a positive sentinel lymph node (SLN) after NST in cT1-3N0 breast cancer patients and for the different breast cancer subtypes to support preoperative shared decision-making.This study was approved by the Privacy Review Board of the Netherlands Cancer Registry (NCR), managed by the Netherlands Comprehensive Cancer Organization (IKNL). All cT1-3N0 breast cancer patients who had undergone NST (chemotherapy with or without trastuzumab) with subsequent mastectomy and sentinel lymph node biopsy (SLNB), from January 2010 through December 2016, were identified from the NCR.On-site trained registrars of NCR collect data from patients’ medical records from all hospitals in the Netherlands. Data were collected on age, tumor characteristics , and treatment regimens . The axillary nodal status was determined before NST administration by ultrasound. Patients were considered cN0 if ultrasound showed no suspicious lymph nodes or in the case of negative tissue sampling. The pathologic examination of the SLN was performed according to the national guidelines.24The ER (Estrogen Receptor) and HER2  status were determined by immunohistochemistry. Immunohistochemistry testing of ER and HER2 was in accordance with the Dutch national guidelines.According to the Dutch national guidelines of 2008 and 2012, the indication for systemic therapy was based on age, tumor size, tumor grade, and receptor status.p value < 0.05 was considered statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences .Descriptive analyses were performed to evaluate the overall rate of a positive SLN and for the different breast cancer subtypes for cT1-3N0 patients. Patients were stratified into four subtypes with ER-positive(+)HER2+, ER-negative(−)HER2+, ER+HER2−, and triple-negative patients. The PR (Progesterone Receptor) was not included in the determination of the ER+HER2+, ER−HER2+, and ER+HER2− subtypes. Univariable logistic regression analysis was applied to determine the association of patient and/or tumor characteristics with a positive SLN. Multivariable logistic regression analysis was used to adjust for potential confounders. Odds ratios (ORs) with 95% confidence interval (CI) were presented. A two-sided n = 782). Other exclusion criteria were the unknown date of SLNB or NST (n = 259), unknown SLNB outcome (n = 33), neoadjuvant endocrine therapy (n = 30), unknown breast cancer subtype (n = 13), and distant metastases at primary breast cancer diagnosis or within 91 days after surgery (n = 9). A total of 788 patients  were included for final analyses, of whom 106 (13.5%) ER+HER2+, 54 (6.9%) ER−HER2+, 474 (60.1%) ER+HER2−, and 154 (19.5%) triple-negative patients. Immediate breast reconstruction was performed in 378 (48.0%) patients, and PMRT was applied to 288 (36.5%) patients. In patients with immediate breast reconstruction, 150 (39.7%) received PMRT. PMRT was delivered in 156 (79.2%) patients with a positive SLN. A complete overview of all patient, tumor, and treatment characteristics is shown in Table A total of 1914 patients were diagnosed with cT1-3N0 breast cancer between January 2010 and December 2016 in the Netherlands and treated with NST followed by mastectomy and SLNB. Patients were excluded if the SLNB had been performed before NST  had a negative SLN, 69 (8.8%) had micrometastases, and 128 (16.2%) had macrometastases. The overall rate of a positive SLN for cT1-3N0 patients was 25.0% (197/788). The rate of a positive SLN per subtype was 10.4% (11/106) for the cT1-3N0 ER+HER2+, 3.7% (2/54) for the cT1-3N0 ER−HER2+, 35.9% (170/474) for the cT1-3N0 ER+HER−, and 9.1% (14/154) for the cT1-3N0 triple-negative patients. Table In the ER+HER2+ subgroup, 7.2% (2/28) of the cT1N0 patients had a positive SLN, 11.5% of the cT2N0 (6/52), and 11.5% (3/26) of the cT3N0 patients. Of these patients with a positive SLN, 27.3% (3/11) had micrometastases, and 72.7% (8/11) had macrometastases. In these ER+HER2+ patients, 86.8% (92/106) had received neoadjuvant trastuzumab.In the ER−HER2+ subgroup, the cT1N0 and cT3N0 patients had no positive SLN, and 6.3% (2/32) of the cT2N0 patients had a positive SLN. Both cT2N0 patients with a positive SLN had micrometastases. Neoadjuvant trastuzumab was administered in 92.6% (50/54) of these ER−HER2+ patients.In the ER+HER2− subgroup, 23.8% (20/84) of the cT1N0 patients had a positive SLN, 36.6% (90/246) of the cT2N0, and 41.7% (60/144) of the cT3N0 patients. Of these patients with a positive SLN, 34.7% (59/170) had micrometastases and 65.3% (111/170) had macrometastases.In the triple-negative subgroup, the rate of a positive SLN for cT1N0 and cT2N0 patients was respectively 2.9% (1/34) and 6.2% (6/97). This increased up to 30.4% (7/23) for the cT3N0 patients. Micrometastases were found in 35.7% (5/14) of the SLN positive patients and 64.3% (9/14) had macrometastases.Clinical T1-3N0 ER+HER2+, cT1-3N0 ER−HER2+, and cT1-2N0 triple-negative patients had the lowest rate of a positive SLN: 7.2–11.5%, 0–6.3%, and 2.9–6.2%, respectively. Clinical T1-3N0 ER+HER2− and cT3N0 triple-negative patients had the highest rate of a positive SLN: 23.8–41.7% and 30.4%, respectively (Table p = 0.022), cT2 stage , cT3 stage , lobular histology , and ER+HER2− subtype  were associated with higher odds of a positive SLN. Tumor grade 2  and tumor grade 3  were associated with lower odds of a positive SLN. After adjustment for confounders, the multivariable analysis showed that cT2 stage , cT3 stage , and ER+HER2− subtype  were correlated with higher odds of a positive SLN. Grade 3  remained correlated with lower odds of a positive SLN. Table The univariable analysis showed that age ≥ 40 years . We found that cT2 stage, cT3 stage, and ER+HER2− subtype were correlated with higher odds of a positive SLN after NST. Grade 3 was correlated with lower odds of a positive SLN after NST. The lowest risk of a positive SLN was in the ER−HER2+ subtype (3.7%), whereas the highest risk of a positive SLN was in the ER+HER2− subtype (35.7%).–30–33At present, there are no results of randomized trials addressing the role of PMRT following NST. The pooled analysis of National Surgical Breast and Bowel Project (NSABP) B-18 and B-27, and several retrospective studies have addressed the advantages of PMRT after NST.27–41–The increasing use of NST and the inability to predict the axillary lymph node status after NST have raised questions regarding the patients who do need PMRT and increased the complexity of immediate breast reconstruction planning. Immediate breast reconstruction reduces the number of surgical procedures and has a better aesthetic outcome due to decreased scarring and preservation of the breast skin envelope.34Clinical decision-making regarding the need for PMRT and immediate breast reconstruction requires a multidisciplinary approach and careful patient counseling. The results in this study indicate that cT1-3N0 ER+HER2− and cT3N0 triple-negative patients treated with NST have a high-risk of a positive SLN. For these patients, SLNB as a separate procedure preceding to reconstruction can be considered to provide information about the pathologic axillary lymph nodes prior to definitive surgery. If the SLN is positive, the breast reconstruction can then be delayed, or a tissue expander can be placed to preserve the breast skin envelope.It has not been fully elucidated whether micrometastases in the axillary lymph nodes after NST should be counted as an indicator for treatment with PMRT. Limited data are available on micrometastases and the LRR rate. In the Mamtani et al.A strength of this study is the nationwide character of the data including general, academic, and cancer centers, and thus all patients in the Netherlands. Also, the large patient population gave us the opportunity to divide patients into subgroups and therefore the ability to determine the risk of a positive SLN for the different breast cancer subtypes. This study also has certain limitations, such as its retrospective design which made it impossible to retrieve missing data on tumor grade or subtype, date of SLNB or NST, and SLNB outcome. Furthermore, based on the available data it was not possible to determine whether a full course of chemotherapy regimen was completed. Lastly, the lymphovascular invasion was not available in the NCR database and could therefore not be included in the analyses.In conclusion, these results showed that in cT1-3N0 ER+HER2+, cT1-3N0 ER−HER2+, and cT1-2N0 triple-negative patients treated with NST, immediate breast reconstruction can be considered an acceptable option due to the low risk of a positive SLN and therefore a decreased likelihood of PMRT. However, in cT1-3N0 ER+HER2− and cT3N0 triple-negative patients treated with NST, the risks and benefits of immediate breast reconstruction should be discussed with these patients due to the relatively high risk of a positive SLN and therefore an increased likelihood of PMRT. In these high-risk patients with a desire for immediate breast reconstruction, an SLNB after NST can be performed before breast surgery to determine the need for PMRT and discuss the potential complications of PMRT thoroughly with the patient in the case of a positive SLN prior to the reconstruction. For both situations, this study provides data for adequate shared decision-making.


Production methods for pre-clinical studies in animal models, and ultimately for human clinical trials, must be capable of producing large quantities of high-quality lentiviral vector in an efficient and cost-effective manner. We report here a medium-scale method (from 1.5 L to 6 L of vector supernatant) for lentiviral vector production in adherent cell cultures using the NUNC™ EasyFill™ Cell Factory™ from Thermo Fisher Scientific. Downstream purification uses a Mustang Q XT5 anion exchange capsule from Pall, and an ultracentrifugation step to concentrate the vector. This method is capable of producing lentiviral vector with concentrated titres of 10 Gene-addition therapy is a promising therapeutic approach with the potential to treat or cure a range of genetic and acquired diseases by introducing a working copy of the appropriate gene into affected target cells. Lentiviral (LV) vectors are advantageous for therapeutic development because they transduce dividing and non-dividing cells, can be pseudotyped to target specific cell types by altering surface receptor recognition elements, and show high levels of gene transfer efficiency .Phase II trials for metachromatic leukodystrophy , WiskottProducing sufficient quantities of LV vector for use in pre-clinical animal studies and clinical trials has proven challenging. Typically, LVs are produced using either stable packaging cell lines or by the transient transfection of adherent human embryonic kidney (HEK) 293T cells . UltimatCurrent methods for transient LV production are based on the multi-plasmid transfection of adherent HEK 293T cells in single monolayer flasks. However, up-scaling LV production using monolayer flasks is time-consuming, labour intensive, and requires large work spaces for cell cultivation ,14,15. Rvia a Mustang Q XT5 anion exchange step followed by ultracentrifugation . Our results show that concentrating the supernatant up to 3000 times enables us to achieve volumes of 500 µl/cell factory at a titer of 108–109 TU/ml (determined by RT-PCR). Although the initial expansion of cells to seed a cell factory remains time consuming, gains are made using multi-layer cell factories, as they avoid the use of multiple monolayer flasks, where one 10-layer cell factory is equivalent to 36 T175 flasks. The use of cell factories increases the surface area available for cell cultivation, thereby improving vector yields, while also decreasing the transfection and post-transfection media-change times, and reducing labor costs. Although this method describes use of a second-generation system, it is applicable to production of first, second or third generation LV vectors by choosing the appropriate plasmid ratios. It can also be used for producing LV vectors with alternative pseudotypes  with or GP64 .✓ HEK 293T cells ✓ Dulbecco’s Modified Eagle’s Medium (DMEM) ✓ Fetal calf serum (FCS)  ✓ Penicillin-streptomycin  2 cell culture flasks, red filter screw cap (T75) ✓ 75 cm✓ Corning tissue-culture treated culture dishes 150 mm × 25 mm ✓ Phosphate buffered saline (PBS) ✓ TrypLE express cell dissociation reagent ✓ NUNC™ EasyFill™ Cell Factory™ System 2HPO4 ✓ Na✓ HEPES ✓ NaCl 2·2H2O ✓ CaCle.g., for our second generation, 5-plasmid system we use: pTat, pRev, pGag-Pol, pVSV-G and an expression plasmid. See Table 1 for details). Users should produce the plasmids for their system in the appropriate quantity.✓ Lentiviral packaging, envelope, and expression plasmids ✓ L-Glutamine ✓ OptiPRO™ SFM ✓ Penicillin-streptomycin  ✓ Fetal calf serum (FCS)  ✓ Bovine serum albumin (BSA) ✓ NaCl ✓ 0.9% saline for injection e.g., mouse serum if the vector is to be delivered to mice) to minimize potential immune reaction.✓ Heat-inactivated serum from the species that the vector preparation is intended for ✓ 50 mm Polydisc AS 0.45 µm Whatman filter ® Q XT5 capsule ✓ Mustang✓ Masterflex peroxide-cured silicone tubing L/S 16 ✓ Male luer lock with 3.2 mm (1/8”) hose barb connector ✓ Female luer lock with 3.2 mm (1/8”) hose barb connector ✓ 14 ml thin wall polyallomer ultracentrifuge tubes ✓ NaOH ✓ NaCl ® L/S® pump ✓ Cole Parmer Masterflex™ L-100 XP Ultracentrifuge with a SW40 Ti rotor ✓ Optima® J-E Centrifuge with a JA-10 rotor ✓ Avanti✓ HEK 293T cell growth media: DMEM, 10% FCS, 10 units/ml Penicillin, 10 µg/ml Streptomycin2HPO4, 50 mM HEPES, 0.28 M NaCl in water, adjust pH to 7.08 and filter-sterilise✓ 2× HeBS buffer: 1.5 mM Na2·2H2O in water and filter-sterilise✓ 2.5 M CaCl✓ Harvest media: OptiPRO™ SFM, 10 units/ml Penicillin, 10 µg/ml Streptomycin, 4 mM L-glutamine2O and incubate at 65°C for 30 min✓ 2% heat inactivated serum: use serum from the species that the vector preparation is intended for to minimize potential immune reaction. Dilute serum in H✓ 200 mM L-Glutamine in water and filter-sterilise✓ 2.5% (w/v) BSA in PBS✓ 1.5 M NaCl in water and filter sterilise✓ Mustang Q XT5 column equilibration buffer: 1 M NaOH in water✓ Mustang Q XT5 column storage buffer: 0.1 M NaOH and 1 M NaCl in waterCulture of sufficient HEK 293T cells1.1. Pre-warm PBS, DMEM/10%FCS/1%Pen-Strep, and 10% TrypLE-Express in PBS to 37°C.1.2. Thaw a vial of frozen HEK 293T cells rapidly in a 37°C water bath and transfer to a 50 ml conical tube and make up to 50 ml with PBS. Centrifuge at 450× g for 5 min at 4°C. Aspirate the supernatant, re-suspend the cell pellet in DMEM/10% FCS/1% Pen-Strep and transfer to a T75 flask. Incubate at 37°C in 5% CO2.1.3. When the cells are 90% confluent, split cells (1:4) by aspirating media and washing cells with 4 ml PBS. Aspirate PBS and trypsinize cells using 4 ml of 10% TrypLE-Express in PBS, incubate for 5 min at room temperature, or until cells start to detach. Add 4 ml of media, spritz cells and transfer 2 ml to 3 × T75 flasks containing 12 ml of media.NOTE: It takes 20–24 h for HEK 293T cells to double so ensure that splitting the cells (1:2 or 1:4) is performed at the same time each morning.1.4. When cells are 90% confluent (approximately 48 h after seeding), repeat step two to achieve a total of 11 × T75 flasks.1.5. When the 11 × T75 flasks are 90% confluent, harvest cells as previously and transfer one T75 flask into one 150 mm round culture dish with a final volume of 32 ml.Cell factory seeding2.1. Pre-warm PBS, DMEM/10%FCS/1%Pen-Strep, and 10% TrypLE-Express in PBS to 37°C.2.2. When the HEK 293T cells in the 150 mm round culture dishes are 90% confluent, harvest by first aspirating the media and washing cells with 6 ml of PBS. Aspirate PBS and detach cells by incubating them with 6 ml of 10% TrypLE-Express for 5 min. Add 6 ml of media to neutralize TrypLE-Express, spritz cells and transfer to a 1 L bottle.TIP: Using a left-over 1 L media bottle is a useful alternative to using multiple tubes or containers.2.3. Rinse each round culture dish with fresh media and add to the 1 L bottle containing the cell suspension.2.4. Perform a viable cell count on the cell suspension and determine the volume of cells required to achieve a final concentration of 3.476 × 105 cells/ml in a total of 1500 ml. Transfer this volume to a sterile 2 L bottle and bring up to a total of 1500 ml with media.CRITICAL STEP: Using a cell density of 1.65 × 105 cells /cm2 will ensure that the cells will reach 70%–90% confluency at 24 h post-seeding.2.5. Pour the cell suspension into a NUNC™ EasyFill™ Cell Factory™ System and equilibrate the chambers as per the manufacturer’s instructions.TIP: Pour the cell suspension into the large port and ensure that media enters all the layers during pouring. This will minimize the formation of bubbles within the large port that can make it difficult to transfer the full 1500 ml.2.6. Seed a T75 flask at a density of 6.14 × 106 cells/ml to use as a reference of confluency for the cell factory. The reference flask is used to assess cell confluency because the contents of the cell factory cannot be observed under the microscope.TIP: This number of cells in a T75 ensures that the density is 1.65 × 105 cells/cm2, which is the same as the cell factory seeding density.Plasmid transfection3.1. We advise that users optimize the plasmid ratios for their system using small-scale preparations. Here we use example ratios for a second-generation, 5-plasmid LV vector system.3.2. Prepare plasmid mix  in a 50 ml conical tube as per Table 1.3.3. Add the plasmid mix to a 50 ml conical tube containing 4.5 ml of 2.5 M CaCl2 and make up to 45 ml with sterile water.CRITICAL STEP: Ensure the pH of the 2× HeBS is exactly 7.08 as this is critical for effective DNA calcium-phosphate co-precipitation.3.4. Aliquot 22.5 ml of room temperature 2× HeBS into two 225 ml conical tubes.3.5. Begin vortexing the first 225 ml conical tube, and using a 25 ml serological pipette add 22.5 ml of plasmid solution dropwise over 30–40 s. Continue to vortex for a further 20–30 s and set aside at room temperature.CRITICAL STEP: Smaller tubes/containers will not support vortexing this large volume at a high speed without spillage. Ensure the plasmid mix is added in a dropwise fashion to maximize complex formation between plasmids and calcium phosphate.3.6. After a total of 90 s has elapsed from initial vortexing of the first 2× HeBS in the 225 ml conical tube, begin vortexing the second 225 ml conical tube containing 2× HeBS and again add 22.5 ml of plasmid solution dropwise over 30–40 s. Continue to vortex for a further 20–30 s and set aside.3.7. Retrieve the NUNC™ EasyFill™ Cell Factory™ System from the incubator, remove both caps and gently pour ~400 ml of media into a 1 L bottle via the small port, ensuring that cell disturbance is minimized. After four minutes has elapsed from initial vortexing of the first 225 ml conical tube, add the DNA complexes from the first 225 ml conical tube to the ~400 ml of media. After a total of five and a half minutes has elapsed, add the second 225 ml conical tube.CRITICAL STEP: This incubation period is important to aid in the plasmid-calcium phosphate complex formation.3.8. With care to minimize cell disruption, gently pour the media containing the plasmid solution back into the 10-layer cell factory via the large port, equilibrate again as per the manufacturer’s instructions, and place back in the 37°C incubator with 5% CO2 for eight hours.3.9. During the eight-hour incubation, pre-warm 1.5 L of OptiPRO SFM supplemented with 4 mM L-glutamine and 1% Pen-Strep to 37°C.3.10. At eight hours post-transfection, perform a media change by pouring out all of the media from the NUNC™ EasyFill™ Cell Factory™ System via the small port (remove as much excess media as possible) and subsequently add 1.5 L of pre-warmed OptiPRO™ SFM into the large port. Pour gently to ensure minimal disturbance to adhered cells and equilibrate media amongst the layers.TIP: Eliminating FCS from the final vector product is recommended to minimize the risk of a potential immune response to in vivo.3.11. Incubate at 37°C and 5% CO2 for a further 40 h.LV vector harvest4.1. Pre-weigh 2 × 1 L bottles.TIP: Use the OptiPRO™ SFM bottles from the previous day.4.2. Remove the NUNC™ EasyFill™ Cell Factory™ System from the incubator and carefully pour the viral supernatant into the pre-weighed 1 L bottles.4.3. Add 200 ml of PBS to the large port of the NUNC™ EasyFill™ Cell Factory™ System and equilibrate to rinse any remaining viral supernatant. Pour the PBS rinse into the pre-weighed 1 L bottles.4.4. Weigh the bottles again to determine the volume of supernatant collected and add BSA (stored on ice) to achieve a final concentration of 0.1% BSA to the supernatant.HINT: Divide the volume of the supernatant (assuming 1 g = 1 ml) by 24 and then add that volume of 2.5% BSA (w/v) in PBS to make a final BSA concentration of 0.1%.4.5. Transfer the supernatant to 500 ml centrifuge bottles. Rinse each 1 L bottle with 20 ml PBS and add to 500 ml centrifuge bottles.4.6. Centrifuge supernatant at 400× g for five minutes at 4°C to remove cellular debris. Pour supernatant back into the 1 L bottles and keep refrigerated at 4°C until required.Lentiviral purification and concentration5.1. To set up the Mustang Q XT5 column purification system , connect a 70 cm piece of MasterFlex tubing to the Watson Marlow 323 pump. Pump PBS through the tubing at a rate of 10 ml/min, ensuring no bubbles are present within the tubing.CRITICAL STEP: It is very important to ensure no air bubbles enter the system as the presence of bubbles can cause damage to the lentivirus particles and can significantly reduce the final yield.5.2. Attach a 50 mm Polydisc AS 0.45 µm Whatman filter at the end of the tubing, secure with a cable-tie, and continue to flush PBS until the filter is saturated.TIP: Ensure that the inlet side of the 0.45 µm filter is facing downward so that gravity forces the supernatant to pass through the filter across the entire bed.5.3. Attach a 10 cm length of MasterFlex tubing with a tubing clamp to the outlet end of the 50 mm Polydisc AS 0.45 µm Whatman filter, secure with a cable-tie, and flush with PBS, ensuring no bubbles are present within the tubing.5.4. Attach a male M6 thread with a hose barb connector to the Mustang Q XT5 column and connect the inlet port to the 10 cm piece of MasterFlex tubing, securing with a cable-tie. Connect a 40 cm piece of MasterFlex tubing to the outlet port with a male M6 thread with a hose barb connector, securing with a cable-tie, and place the Mustang Q XT5 column in a retort stand with the outlet facing upwards. Place the end of the tubing into a large container to collect the waste.TIP: Like the filter, ensure that the outlet port of the Mustang Q column is facing up so that gravity forces the supernatant to pass through the entire surface area of the column.5.5. Continue to pump PBS through the system at 10 ml/min to flush out the buffer that the column is stored in. Once the pH is ~7, the system is ready to use (this can be assessed by using pH indicator strips or a pH meter).CAUTION: A pH of ~7.20 must be used for all solutions post-harvest as this is the optimal pH environment for the vector.5.6. Place the 70 cm length of tubing into a 1 L bottle of vector supernatant that has been stored at 4°C. Pump the supernatant through at 10 ml/min until system has been completely flushed with supernatant and then increase the pump rate to 30 ml/min.5.7. Once all but a few ml of the vector supernatant has been pumped through the system, rinse 1 L bottles with 150 ml of PBS containing 0.1% BSA and pump through the system.5.8. Turn off the pump before the 10 cm length of tubing has air introduced into it to ensure air bubbles do not enter the Mustang Q XT5 column. Close the tubing clamp and cut off the lower 7 cm of tubing. Attach a female luer fitting with a hose-barb connector to the end of the tubing (that is attached to the column), and secure with a cable-tie.CAUTION: Clamping of the tubing is important to prevent supernatant leaking from the system due to gravity.5.9. Using a 50 ml male luer lock syringe, flush the column with 60 ml of PBS.5.10. Cut off all but the last 10 cm of the 40 cm piece of tubing connected to the Mustang Q XT5 outlet port. Using a 50 ml syringe, elute the vector with 25 ml of 1.5 M NaCl (stored on ice), and capture into 25 ml of 2% heat inactivated animal serum (stored on ice).5.11. Transfer the elution equally into 4 × 14 ml thin wall polyallomer tubes and place into chilled SW 40 Ti buckets. Centrifuge at 20000 rpm for 90 min at 4°C.5.12. Following ultracentrifugation, gently pour off the supernatant, ensuring that the vector pellet is not disturbed, and blot tubes on tissue paper to remove as much residual liquid as possible.5.13. Resuspend the vector pellets in 0.9% saline (stored on ice) by pipetting up and down, while ensuring that no bubbles form and clumps are no longer visible. Rinse tubes to maximize vector recovery. Once resuspended, add 0.1% (v/v) heat inactivated animal serum and keep on ice when not re-suspending.TIP: To maximize vector recovery, initially resuspend vector pellets in less than a quarter of the desired final volume. Transfer this volume between each ultracentrifuge tube and finally transfer into a screw-cap 1.5 ml microcentrifuge tube. Then rinse the centrifuge tubes with a further quarter of the final resuspension volume, pool into the same 1.5 ml screw-top microcentrifuge tube and repeat with a third wash with a quarter of the final resuspension volume. Top up to the desired final volume.5.14. Aliquot vector into screw cap tubes and store at −80°C. Avoid freeze-thawing as this can decrease titers up to 10-fold /cycle.5.15. Test virus functionality using an appropriate method based on the expression plasmid used .Mustang Q XT5 column equilibration and storage6.1. After elution, equilibrate the column by pumping 25 ml of equilibration buffer at room temperature through the column at 10 ml/min. Stop the pump, close the tubing clamp, and allow to rest for 30 min.6.2. Pump through 25 ml of Mustang Q XT5 column storage buffer at room temperature at 10 ml/min, stop the pump, disconnect the column, and cap the inlet and outlet ports. Store at room temperature until next use.NOTE: This allows for long term usage of the column and will limit bacterial growth.Table 2.Possible problems and their troubleshooting solutions are listed in