Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/αSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 can drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE that face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering. Sec17 (αSNAP) and SNAREs are receptors for the Sec18 (NSF) AAA ATPase  domains.The molecular interactions between Sec18/NSF, Sec17/αSNAP, and neuronal SNAREs were illuminated by determination of their structures when assembled without membrane anchors into the NSF/αSNAP/SNARE complex, also referred to as the 20S particle . The heahomotypic fusion and vacuole protein sorting) with multiple direct affinities. Two HOPS subunits bind Ypt7, anchored on each membrane  Fuse SNAREs . Howevere SNAREs . (3) A pe SNAREs . Reconste SNAREs . It has e SNAREs . Sec17 ae SNAREs . An intee SNAREs . Single-e SNAREs . HoweverComplete SNARE zippering is considered essential for SNARE-dependent fusion . We now Vacuole SNAREs  have an cis-SNARE complex assembly can occur spontaneously on these proteoliposomes, but assembly is stimulated by HOPS, allowing direct comparison of HOPS-dependent and HOPS-independent SNARE assembly  assay of vacuolar SNARE associations . The Qc-assembly . The aveassembly , purple,assembly , indicatassembly  but is nProteoliposomes with R, Qa, and Ypt7 (where indicated) were incubated with Qb-SNARE domain and Qc. Both SNARE domains were either fluorescently labeled at their N-termini , reducedSince this Sec17-induced SNARE conformational change is seen with C-terminal fluors but not with N-terminal fluors, requires HOPS as the SNARE assembly catalyst, and requires SNAREs that can zipper, a major part of the conformational change may be zippering itself. This might be spontaneous after Sec17-induced release of HOPS from SNAREs  or be prSec17 also interacts with partially zippered SNARE complex to promote SNARE complex stability. SNARE complex was assembled by HOPS on Ypt7/R proteoliposomes with soluble Qa, with the Qb SNARE domain labeled with a fluorophore at a cysteinyl residue upstream of the SNARE domain, and with Qc of full length (w.t.) or with the 3Δ C-terminal truncation, each bearing a fluorophore at its native cysteinyl residue upstream of the SNARE domain. When the complex of proteoliposomes with these fluorescent Qb and Qc had full-length SNARE domains, it was stable whether or not it included Sec17, as there was no loss of average FRET efficiency after addition of excess non-fluorescent Qc . In contSec17 can restore fusion when Qc has truncations at the C-terminal end of its SNARE domain , stimulaFusion assays were also performed with Ypt7/R proteoliposomes and each of the three Ypt7/single-anchored Q-SNARE proteoliposomes in the presence of HOPS and the other two soluble Q-SNAREs . With metrans-SNARE complexes, but Sec18 is not simply promoting Sec17 binding, since it is still needed for fusion when Sec17 is joined to an integral N-terminal membrane anchor , Sec18 (NSF), and SNAREs cooperate to promote membrane fusion. Sec17 has multiple functions: (a) It supports Sec18 association with SNARE complexes for their ATP-driven disassembly for subsequent rounds of fusion . Sec17/αWhile intracellular fusion reactions share many requirements, such as for SNAREs and SM protein, there are major differences as well. Synaptic vesicle fusion and other calcium-dependent secretion systems require calcium, synaptotagmin, Munc13, and complexin, none of which have their obvious counterparts in calcium-independent systems. Vacuole and endosome fusion have their SM protein as an integral subunit of the tethering complex, which is not seen in other organelles. The similarities and differences in fusion pathways at each organelle will be clarified as each is more thoroughly studied.PI3P was from Echelon , ergosterol from Sigma , fluorescent lipids from Thermo Fisher , and other lipids were from Avanti . Biobeads SM2 were from BioRad, Cy5-Streptavidin from SeraCare , biotinylated phycoerythrin from Invitrogen , and underivatized streptavidin from Thermo Fisher. Spectrapor six dialysis tubing  was from Spectrum Labs . Octyl-b-D-glucopyranoside was purchased from Anatrace .Sec17 with six acidic amino acids mutated to neutral residues, GST-Sec17 , was generated by PCR with Phusion high-fidelity DNA polymerase (NEB). The DNA fragment was cloned into BamHI- and SalI-digested pGST parallel1 vector  with an For Sec17-E34S,E35S, D38S,TCGTCGGCTGCTTCTCTTTGTGTCCAAGCAGCCACF: AGAAGCAGCCGACGAAAACTTGTATGAATCAGAACR: For Sec17 E73A, D74A, E75A,GGTAATGCAGCCGCAGCAGGAAATACCTACGTAGAF: TGCGGCTGCATTACCAGCCTTTTTCTGATAGTCAGR: GST-sQa3Δ with amino acyl residues 1–235 and GST-sQb3Δ with amino acyl residues 1–160 were generated by PCR with Phusion high-fidelity DNA polymerase (NEB). DNA fragments were cloned into BamHI- and SalI-digested pGST parallel1 vector  with an For GST-sQa3Δ,AGGGCGCCATGGATCCGATGTCCTTTTTCGACATCGAF: AGTTGAGCTCGTCGACTAGATATTCTCGTCTATGGTGGR: For GST-sQb3Δ,AGGGCGCCATGGATCCGATGAGTTCCCTATTAATAF: AGTTGAGCTCGTCGACTACAAGGTCTGTCTTGCATTTTR: To generate Qa with L238, M242, A245, L249, and A252 changed to Ala, Ser, or Gly, the pParallel1 GST vector with Qa lacking residues 228–257 was generated by inverse PCR with pParallel1 GST-Qa  using PhGACCAGCATCAGAGGGACCGF: GTCTATGGTGGTTACTTGTTR: GTAACCACCATAGACGAGAATATCTCGCATGGCCATGATAACGGCCAGAATGGCAACAAACAAGGCACCAGAGGCGACCAGCATCAGAGGSequence 1: CCTCTGATGCTGGTCGCCTCTGGTGCCTTGTTTGTTGCCATTCTGGCCGTTATCATGGCCATGCGAGATATTCTCGTCTATGGTGGTTACSequence 2: GTAACCACCATAGACGAGAATATCTCGCATAGCCATGATAACAGCCAGAATAGCAACAAACAAAGCACCAGAAGCGACCAGCATCAGAGGSequence 1: CCTCTGATGCTGGTCGCTTCTGGTGCTTTGTTTGTTGCTATTCTGGCTGTTATCATGGCTATGCGAGATATTCTCGTCTATGGTGGTTACSequence 2: GTAACCACCATAGACGAGAATATCTCGCATGCCCATGATAACGCCCAGAATGCCAACAAACAAGCCACCAGAGCCGACCAGCATCAGAGGSequence 1: CCTCTGATGCTGGTCGGCTCTGGTGGCTTGTTTGTTGGCATTCTGGGCGTTATCATGGGCATGCGAGATATTCTCGTCTATGGTGGTTACSequence 2: Vam7 mutants with cysteines inserted at the N- and C-termini of the SNARE domain were generated by inverse PCR with the Vam7 intein vector  and PhusFor Vam7-C208S,GAAAGCGATGACATTGGTACAGCAAACATAGCTCAF: CAATGTCATCGCTTTCCTTGAGCAAGGACCTCAATR: For Vam7-C208S,M250C ,GGGCAGTGTCAAATGGTGCGCGATCAAGAACAAF: CCATTTGACACTGCCCCTGTTGCAAATCGTTATR: For Vam7-C208S,A316C ,CAACAGTTGTTGAATTCTCGAGCACCACCAF: CAACAACTGTTGTTAAAATGTCTAGCCTTCTTGTTGGCR: HOPS and prenyl-Ypt7 , Ypt7-TM2 was added to 2 mM. After prewarming (27°C for 10 min) the separately GTP-exchanged proteoliposomes in a 384-well plate, fusion reactions were initiated by mixing 5 μl of each proteoliposome preparation and supplementing with other fusion factors in volumes summing to 10 μl, continuing incubation at 27°C in a Spectramax fluorescent plate reader. Fusion incubations (20 μl) in RB150  had proteoliposomes , 50 nM HOPS, the indicated concentrations of sSNAREs, 400 or 600 nM Sec17, 300 nM Sec18, 1 mM ATP or its analogs, and 3 mM MgCl2, as modified in each figure legend.Proteoliposomes were prepared by detergent dialysis from β-octylglucoside-mixed micelles for fusion assays  and SNARAssays were performed as described previously  with oneMODELLER  was usedThe MODELLER protocol consisted of an alignment step  and a modeling step (python script file modeler-input.py). The script files are shown here for synaptobrevin (nyv1):from modeller import *env = environaln = alignment(env)mdl = model)aln.append_modelaln.appendaln.salign,output='',align_block = 15, # no. of seqs. in first MSAalign_what='PROFILE',alignment_type='PAIRWISE',comparison_type='PSSM', # or 'MAT' (Caution: Method NOT benchmarked# for 'MAT')similarity_flag = True, # The score matrix is not rescaledsubstitution = True, # The BLOSUM62 substitution values are# multiplied to the corr. coef.#write_weights = True,#output_weights_file='test.mtx', # optional, to write weight matrixsmooth_prof_weight = 10.0) # For mixing data with priorsaln.editaln.writealn.writefrom modeller import *from modeller.automodel import *env = environa = automodel)a.very_fasta.starting_model = 1a.ending_model = 1a.makeThe homology models of Nyv1, Vam3, Vti1, Vam7, and Sec18, together with the crystal structure of Sec17 (PDB ID 1QQE) , were fi public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:This is a very important paper that challenges the generally accepted dogma that full zippering of SNARE complexes is essential for intracellular membrane fusion. Previous work had already shown that C-terminal truncation of one SNARE arrested liposome fusion mediated by the yeast vacuolar SNARE complex and that Sec17/Sec18 could rescued fusion, but it was argued that such rescue could arise because Sec17/Sec18 restored C-terminal zippering. This paper now shows that Sec17/Sec18 rescue fusion even when three SNAREs are crippled -by truncation or mutation- to definitively prevent zippering, thus showing that Sec17/18 have a direct, positive role in membrane fusion.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Josep Rizo as Reviewing Editor and Vivek Malhotra as the Senior Editor.Thank you for submitting your article "Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this letter to help you prepare a revised submission.Essential revisions:1. The paper is difficult to read, particularly for non-experts in the field, and is very long, presenting the most dramatic results that support the main conclusion in Figure 10. The data in this figure are minimally dependent on the 51 data panels  preceding this figure. The reviewers wonder how many readers will make it that far and believe that a short, crisp manuscript would better convey the main message of the paper. One possibility is to reformat the paper to a Short Report including a small number of experiments. If the authors feel that such format is too constraining, they could keep the paper as a full Article but should strive to shorten it considerably. Note that some of the data are not necessary for the story and/or are not very compelling. For instance the FRET data of Figure 3, the HOPS/Sec17 binding data of Figure 4 and the studies with antibodies of Figure 9 could be part of a future paper devoted to more in depth mechanistic studies, and the titrations of Figure 7 could be included as supplementary material or could also be presented in such future study. In revising the manuscript, the authors should also consider the recommendations for the authors listed below. Note that these recommendations were extracted (unedited) from the separate reviews and some of them may be useful for a future paper(s) describing data that were removed from this manuscript.2. The authors should discuss whether the mechanism underlying fusion in the in vitro assays presented in this paper could be operating in a cell under normal conditions or it only occurs when mutations are introduced into the SNAREs AND when Sec17/Sec18 are added above a threshold that might or might not be achieved in a cell (see point #1 of reviewer 3 below).Reviewer #1 (Recommendations for the authors):1. The authors should provide an explanation of why the observed FRET efficiency is so low even under the conditions under which SNARE complex assembly is most efficient and the donor and acceptor probes are expected to be very close. Is it due to low incorporation of the dyes on the proteins? It would be advisable to measure the FRET efficiency observed when the SNARE complex is assembled in solution, either with the proteins in the presence of detergent, or using only soluble SNARE domains. Does this lead to much more efficient FRET? If it does, why is SNARE complex formation on the liposomes much less efficient?2. The concept of 'entropic cage' does not seem appropriate for the model that the authors are proposing, as favorable interactions at the C-terminus of the SNARE domains should be pretty much abolished by the mutations to Ser or Gly plus the truncations of two SNAREs. It seems very likely that any proximity between the C-termini of the two membrane-anchored SNAREs would arise from steric constraints caused by the bound Sec17 molecules rather than by entropic contributions. Perhaps the term 'steric cage' or simply 'cage' seem more appropriate.3. Line 559: it seems likely that the persistence of fusion with the alanine mutations but not with the Ser mutations arises because there is still some hydrophobicity left, rather than because of higher helical propensity.4. In lines 133-134, the authors state that αSNAP stabilizes SNARE complexes, citing Ma et al. 2016, but a more balanced view would be provided if the authors also cite another single molecule study that reached the opposite conclusion .5. The label 'No Sec17, Sec18 or ATP' at the top of Figure 8 is confusing, as there is Sec17 anchored on the liposomes in some of the experiments of the top panels.6. Line 518: Qc3delta is not included in set 2 according to Figure 9.7. Line 447: mM should be μM.Reviewer #2 (Recommendations for the authors):1. In Figure 1C, I wonder what the point is of showing "sticks" in addition to "cartoon". To me this makes the images look fuzzy, without adding any usable information.2. In Figure 3, I'm not sure I understand why cis-SNARE complexes require Sec17 to complete the process of zippering? 3. In Figure 3C, I was surprised that HOPS stimulation is independent of Ypt7. What do the authors make of this?4. In Figure 7, is there evidence of cooperativity with respect to Sec17? Mightn't it be interesting to investigate this and perhaps determine a Hill coefficient?5. Also in Figure 7, why does Sec18 still have a dramatic stimulatory effect (compare 1000 nM curves in panels G and H) when Sec17 contains the LALA mutation (which ought to prevent Sec18 binding)?6. Figure 9A Set 4, indicating that Qc alone is sufficient to confer resistance to αVps33, would seem to be quite informative. Would it be interesting to repeat the experiment with Qb in place of Qc?7. What might the partial resistance to αSec18 in Figure 9, Sets 8 and 9 mean?8. The data in Figure 10 doesn't seem to agree with the data in Figure 10 —figure supplement 1.Reviewer #3 (Recommendations for the authors):1. It is striking that, in the presence of Sec17, Sec18, and HOPS, SNAREs are capable of driving efficient fusion even when several C-terminal layers are deleted. While the data are overall convincing, I am wondering if the Sec17-dependent activation is utilized in the cell with wild-type SNAREs. In other words, is such a mechanism also relevant beyond the engineered system described here? To answer this question, the authors would need to isolate Sec17 or Sec18 mutants that are only defective in stimulating fusion while remaining fully functional in cis-SNARE disassembly. Then they could test if such mutants are defective in driving fusion in vivo.2. The authors stated that the presence of SNAP-25 linker interferes with the binding with the other two α-SNAP molecules. Does this mean the mechanism proposed in this work is specific to yeast vacuole fusion rather than a general principle?eLife. Your revised article has been evaluated by Vivek Malhotra (Senior Editor), Josep Rizo as Reviewing Editor and one of the referees who reviewed the original version.Thank you for resubmitting your work entitled "Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering" for further consideration by The manuscript has been improved but there are some remaining concerns that need to be addressed, as outlined below:1. A key issue that needs to be clarified is why does the zippering of cis-SNARE complexes depend on Sec17 ? In these experiments, the R- and Qa-SNARE are embedded in the same liposomes, and the Qb- and Qc-SNAREs lack transmembrane domains. The failure of such complexes to fully zipper makes no sense unless something prevents such zippering and the natural culprit is HOPS. The authors should briefly discuss the mechanistic implications of these findings. For example, might HOPS have a proofreading function, in which it somehow prevents cis-SNARE complexes from fully zippering (but perhaps not trans-SNARE complexes)? In any case, the authors should clearly point out from the beginning of the description of the zippering experiments of Figure 2 that they involve cis-SNARE complexes. They should also explain the relevance of cis-SNARE zippering assays to a manuscript that otherwise deals with trans-SNARE zippering/fusion.2. The use of lines and sticks in Figure 1C is visually uninterpretable and therefore unhelpful. The authors are advised to remove them although this is of course up to them. Essential revisions:1. The paper is difficult to read, particularly for non-experts in the field, and is very long, presenting the most dramatic results that support the main conclusion in Figure 10. The data in this figure are minimally dependent on the 51 data panels  preceding this figure. The reviewers wonder how many readers will make it that far and believe that a short, crisp manuscript would better convey the main message of the paper. One possibility is to reformat the paper to a Short Report including a small number of experiments. If the authors feel that such format is too constraining, they could keep the paper as a full Article but should strive to shorten it considerably. Note that some of the data are not necessary for the story and/or are not very compelling. For instance the FRET data of Figure 3, the HOPS/Sec17 binding data of Figure 4 and the studies with antibodies of Figure 9 could be part of a future paper devoted to more in depth mechanistic studies, and the titrations of Figure 7 could be included as supplementary material or could also be presented in such future study. In revising the manuscript, the authors should also consider the recommendations for the authors listed below. Note that these recommendations were extracted (unedited) from the separate reviews and some of them may be useful for a future paper(s) describing data that were removed from this manuscript.We have taken this seriously, and now have 5 data figures  instead of the 9 in the original submission. For this reason, and since the Introduction and Results are more succinct, the reader won't "burn out" before getting to the "triply-crippled Q's" Figure. The 5 data figures are essential to develop the system and show its characteristics, and the supplementary figures provide depth to our consideration of the Figures. For a paper which challenges a main shibboleth of the field, we feel it's important to develop the system fully, showing the full impact and ramifications of crippling even one Q, rather than just cherry-pick one culminating figure.2. The authors should discuss whether the mechanism underlying fusion in the in vitro assays presented in this paper could be operating in a cell under normal conditions or it only occurs when mutations are introduced into the SNAREs AND when Sec17/Sec18 are added above a threshold that might or might not be achieved in a cell (see major point #1 of reviewer 3 below).Reviewer #1 (Recommendations for the authors):1. The authors should provide an explanation of why the observed FRET efficiency is so low even under the conditions under which SNARE complex assembly is most efficient and the donor and acceptor probes are expected to be very close. Is it due to low incorporation of the dyes on the proteins? It would be advisable to measure the FRET efficiency observed when the SNARE complex is assembled in solution, either with the proteins in the presence of detergent, or using only soluble SNARE domains. Does this lead to much more efficient FRET? If it does, why is SNARE complex formation on the liposomes much less efficient?We emphasize that these are ensemble FRET experiments rather than single-molecule FRET experiments. The average FRET efficiency is modest because its denominator includes all the fluorescent Qb and Qc in a bulk reaction, even though only a small per cent enter HOPS-dependent SNARE complex. We note this now directly in the text.2. The concept of 'entropic cage' does not seem appropriate for the model that the authors are proposing, as favorable interactions at the C-terminus of the SNARE domains should be pretty much abolished by the mutations to Ser or Gly plus the truncations of two SNAREs. It seems very likely that any proximity between the C-termini of the two membrane-anchored SNAREs would arise from steric constraints caused by the bound Sec17 molecules rather than by entropic contributions. Perhaps the term 'steric cage' or simply 'cage' seem more appropriate.Thank you, we have removed this term and now describe our model in terms of an "environment" that Sec17 creates.3. Line 559: it seems likely that the persistence of fusion with the alanine mutations but not with the Ser mutations arises because there is still some hydrophobicity left, rather than because of higher helical propensity.A good point, which we have now added. Thanks.4. In lines 133-134, the authors state that αSNAP stabilizes SNARE complexes, citing Ma et al. 2016, but a more balanced view would be provided if the authors also cite another single molecule study that reached the opposite conclusion .Thanks, we've now added this.5. The label 'No Sec17, Sec18 or ATP' at the top of Figure 8 is confusing, as there is Sec17 anchored on the liposomes in some of the experiments of the top panels.Thanks, our mistake! We have removed "Sec17" from these labels; as you note, what differentiates these rows of panels is the presence or absence of Sec18 and of ATP or its analogs.6. Line 518: Qc3delta is not included in set 2 according to Figure 9.Qc3delta is not included initially, but is added along with Sec17, Sec18, and ATPgammaS at 30min., 1min after the control IgG, anti-Vps33, or anti-Sec18 were added. Each of the 8 sets has all the same components, but differ in which are added from the start and which are only added after the antibodies; we've now emphasized this in the text.7. Line 447: mM should be μM.Thank you.Reviewer #2 (Recommendations for the authors):1. In Figure 1C, I wonder what the point is of showing "sticks" in addition to "cartoon". To me this makes the images look fuzzy, without adding any usable information.We included the sidechains to emphasize that these molecules are extensively interacting rather than just being near each other.2. In Figure 3, I'm not sure I understand why cis-SNARE complexes require Sec17 to complete the process of zippering? Without Sec17, these complexes apparently don't zipper all the way most of the time; they are in a metastable partially-zippered state. Fusion without Sec17 is slow because zippering is slow/infrequent; adding Sec17 enhances zippering and enhances fusion. When Qc lacks the +4 to +8 layers, it's perhaps zippered even less, and is seen to be labile to exchange.3. In Figure 3C, I was surprised that HOPS stimulation is independent of Ypt7. What do the authors make of this?As we've noted in earlier papers , HOPS has direct affinity for SNAREs, and a strict requirement for Ypt7 is only seen at low SNARE levels . The FRET signals of SNARE complexes, by the assay in Figure 2, would have been too low had we employed the very low SNARE levels which confer strict Ypt7-dependence.4. In Figure 7, is there evidence of cooperativity with respect to Sec17? Mightn't it be interesting to investigate this and perhaps determine a Hill coefficient?We agree, but a more detailed study will be needed to determine a Hill coefficient.5. Also in Figure 7, why does Sec18 still have a dramatic stimulatory effect (compare 1000 nM curves in panels G and H) when Sec17 contains the LALA mutation (which ought to prevent Sec18 binding)?The LALA mutant blocks Sec17 stimulation of Sec18-mediated 4-SNARE complex disassembly, but hasn't been shown to block all Sec17:Sec18 binding.6. Figure 9A Set 4, indicating that Qc alone is sufficient to confer resistance to αVps33, would seem to be quite informative. Would it be interesting to repeat the experiment with Qb in place of Qc?This is just what was done in Set 2.7. What might the partial resistance to αSec18 in Figure 9, Sets 8 and 9 mean?We can only speculate about the reasons. For example, the partial resistance would be consistent with HOPS:4-SNARE assembly creating a very favorable binding site for Sec18, so that the SNAREs binding Sec18 yields an antibody-resistant complex about as fast as other Sec18s are captured in solution and inactivated by their antibody. Perhaps when Sec18 is added from the start without Sec17, as in Set 6, it binds to non-functional SNARE subcomplexes.8. The data in Figure 10 doesn't seem to agree with the data in Figure 10 —figure supplement 1.We made a labeling error and have fixed it.Reviewer #3 (Recommendations for the authors):1. It is striking that, in the presence of Sec17, Sec18, and HOPS, SNAREs are capable of driving efficient fusion even when several C-terminal layers are deleted. While the data are overall convincing, I am wondering if the Sec17-dependent activation is utilized in the cell with wild-type SNAREs. In other words, is such a mechanism also relevant beyond the engineered system described here? To answer this question, the authors would need to isolate Sec17 or Sec18 mutants that are only defective in stimulating fusion while remaining fully functional in cis-SNARE disassembly. Then they could test if such mutants are defective in driving fusion in vivo.This is one of our long-term plans! For now though, we do point out that Schwartz et al., (2017) showed that the vacuole fragmentation phenotype from Qc3delta is cured by Sec17 overexpression. We've also added to the text that our concentrations of Sec17 and Sec18 are in the same range as published in vivo concentrations; the intracellular concentrations of Sec17 and Sec18 are 150 to 1100nM and 250-760nM, respectively .2. The authors stated that the presence of SNAP-25 linker interferes with the binding with the other two α-SNAP molecules. Does this mean the mechanism proposed in this work is specific to yeast vacuole fusion rather than a general principle?A more thorough investigation of other fusion systems will be needed to evaluate the generality of Sec17/Sec18 direct promotion of fusion. Many other Qb and Qc SNAREs are not joined as in SNAP-25, but we don't know whether or not this will correlate with whether Sec17/Sec18 stimulate fusion.We note that our model doesn't depend on the precise stoichiometry of Sec17:SNAREs. CryoEM structures of NSF/SNAP complexes suggest 2:1, 3:1, or 4: stoichiometries.The manuscript has been improved but there are some remaining concerns that need to be addressed, as outlined below:1. A key issue that needs to be clarified is why does the zippering of cis-SNARE complexes depend on Sec17 ? In these experiments, the R- and Qa-SNARE are embedded in the same liposomes, and the Qb- and Qc-SNAREs lack transmembrane domains. The failure of such complexes to fully zipper makes no sense unless something prevents such zippering and the natural culprit is HOPS. The authors should briefly discuss the mechanistic implications of these findings. For example, might HOPS have a proofreading function, in which it somehow prevents cis-SNARE complexes from fully zippering (but perhaps not trans-SNARE complexes)? In any case, the authors should clearly point out from the beginning of the description of the zippering experiments of Figure 2 that they involve cis-SNARE complexes. They should also explain the relevance of cis-SNARE zippering assays to a manuscript that otherwise deals with trans-SNARE zippering/fusion.Why does zippering need Sec17? We agree that "...the natural culprit is HOPS", and have added emphasis to this for clarity, now explicitly noting that "… the enhanced FRET between the SNAREs in the presence of HOPS and Sec17  is not seen without HOPS (bar 8) or Sec17 (bar 10)." We hadn't stated this clearly enough before.cis-SNARE complexes. We also more clearly cast the study in Figure 2 as one way to examine the interactions of HOPS, Sec17, and the SNAREs, a preamble to functional studies presented in the later figures. This is now pointed out both right before Figure 2 and in the Discussion.We now point out explicitly that the model we're examining in Figure 2 is of We now more cautiously refer throughout to Sec17/HOPS induced conformational change in the SNAREs, reflected by altered FRET, rather than just "zippering". We discuss why zippering is a reasonable, even likely, explanation for this conformational change.We too yearn for a physical assay of the completion of zippering of SNAREs in trans, but this is not yet feasible: trans-SNARE complexes are far less abundant than cis-complexes, engaging no more than a few per cent of the SNAREs . Furthermore, introducing two somewhat bulky fluorophores in the crowded environment at the C-termini of SNARE domains of trans-complex with HOPS, Sec17, and two tightly apposed membranes could well induce artifacts.2. The use of lines and sticks in Figure 1C is visually uninterpretable and therefore unhelpful. The authors are advised to remove them although this is of course up to them.We're removed the "lines and sticks" from Figure 1C, as requested.


Chronic total occlusion (CTO) of coronary arteries is a common finding in patients with known or suspected coronary artery disease (CAD). Although tremendous advances have been made in the interventional treatment of CTOs over the past decade, correct patient selection remains an important parameter for achieving optimal results. Non-invasive imaging can make a valuable contribution. Ischemia and viability, two major factors in this regard, can be displayed using echocardiography, single-photon emission tomography, positron emission tomography, computed tomography, and cardiac magnetic resonance imaging. Each has its own strengths and weaknesses. Although most have been studied in patients with CAD in general, there is an increasing number of studies with positive preselectional factors for patients with CTOs. The aim of this review is to provide a structured overview of the current state of pre-interventional imaging for CTOs. Chronic total occlusion (CTO) of coronary arteries is a common finding in coronary angiograms of patients with known or suspected coronary artery disease (CAD). Despite their frequency, CTOs are the most reliable predictor of an incomplete revascularization. This is the result of two major factors: (a) a lack of data from randomized controlled trials regarding a benefit on mortality and (b) the lower success rate accompanied by a higher complication rate of an interventional revascularization. Large registry studies have shown that CTO percutaneous coronary interventions (PCI) can reduce mortality –4. The ECurrent guidelines and position papers therefore recommend CTO PCI in the case of ongoing symptoms and viable myocardia in the CTO territory –10. HoweViability is by definition present in segments with preserved systolic function. In contrast, dysfunctional segments are not a priori avital. The term “hibernation” was introduced to explain a potentially dysfunctional myocardium resulting in ischemia. Information is gained from an initial coronary angiogram where well-developed collaterals correlate with less extensive scarring, indicating a more viable myocardium , 17. A nFor the detection of a hibernating myocardium, a broad range of non-invasive imaging modalities is available. An overview is presented in Echocardiography is inexpensive and available nearly everywhere. It is the standard modality for the evaluation of global and regional cardiac function. Besides pre-interventional examinations, the bedside use of echocardiography in the catheter laboratory provides the opportunity to detect complications during interventions, such as pericardial effusions or intramural hematomas.However, the value and validity of echocardiography depend on the investigator's experience. In CAD patients, an increase in regional contractility under dobutamine stress (“contractile reserve”) can show viability and improvement of regional function after revascularization –29. The In addition to the examination under dobutamine stress, echocardiography at rest can predict viability under certain circumstances. An end-diastolic wall thickness (EDWT) of >0.6 cm in dysfunctional segments is a marker of hibernation. In a trial involving 45 patients undergoing 2D echocardiography, DSE, and rest-redistribution thallium-201 (Tl-201) tomography before revascularization, an EDWT of >0.6 cm had 94% sensitivity and 50% specificity with a ROC curve similar to the maximum Tl-201 uptake, while DSE increased specificity to 88% . More reMyocardial perfusion scintigraphy (MPS) is a nuclear medicine technique that is used for around 50 years to assess regional left ventricular myocardial perfusion, diastolic and systolic function as well as to differentiate hibernating myocardium from transmural or non-transmural infarct. Three dimension images of myocardium are acquired after injecting radiopharmaceuticals with high first pass extraction by the myocardium using the single emission computed tomography (SPECT) technique. In majority of the centers the images are additionally corrected for attenuation correction using low dose CT images acquired simultaneously with SPECT images. As radioisotopes preferentially Technetium-99m (Tc-99m), specially in Europe and rarely Thallium-201 (Tl-201) are used . Tl-201 The rarely used Tl-201 is a potassium analog and uses the Na/K ATPase system of viable myocardial cells. Its initial myocardial uptake is proportional to blood flow and it is rapidly cleared from the blood. After that up to 4 h a re-equilibration takes place when Tl-201 concentration levels are lower in the blood. This is also called redistribution and the process is directly proportional to blood flow to the area and viable myocardial cells. If the stress and rest images show matched homogeneous normal tracer uptake there is no sign of ischemia or infarction. If the tracer uptake during stress is abnormal but with normal uptake during rest / redistribution that is a sign for ischemia. Perfusion defects on both stress and rest images usually means there is a scar. To check that area for hibernating myocardium a reinjection of Tl-201 and another image acquisition after 18–24 h can be carried out. If there is Tl-201 uptake, there is hibernating myocardium in this area. If there is a scar no tracer uptake can be detected , 38.The Tc-99m-labeled radiotracers are monovalent cations that enter cells through their lipophilic characteristics. Their uptake is also dependent on blood flow but as well as on electrochemical gradients of the plasma- and mitochondrial-membranes, the cellular pH and intact energy pathways. In the myocytes they are trapped mainly in the mitochondria with minimal washout and no redistribution in the blood. After the intravenous injection these tracers are first cleared by the liver and excreted through the bile. This makes it impossible to assess the myocardial uptake of the inferior wall right after injection and leads to a delay of 15–45 min before the start of the imaging acquisition, especially for Sestamibi. Tetrofosmin allows earlier imaging acquisition because of lower hepatic uptake. Stress and rest images can be acquired in 2-day and 1-day protocols that show no significant differences . For theThe accurate interpretation of the myocardial perfusion imaging with SPECT is crucial. Absolute quantification of perfusion is more common with PET than with SPECT. According to the literature the sensitivity and the specificity of gated myocardial SPECT studies to diagnose clinically significant CAD with Tc-99m tracers is 88.3 and 75.8%, respectively . Cutoff Visual interpretation of three vessels CAD is a challenge for nuclear cardiology field. However, with the development and standardization of protocols as well as advances in the scanner types  there is a remarkable improvement in the quantification of myocardial wall thickening, contraction, and dilatation as well as in measurement of ejection fraction. Left ventricular dilatation in stress in comparison to rest can be quantified as transient ischemic dilatation (TID). A TID value above 1.3 is generally considered to be significant and can be used for assessment of three vessels disease .However, the comparatively low spatial resolution of SPECT images compared to that of other imaging modalities is a potential disadvantage, as it can lead to overdiagnosis of subendocardial infarctions. Nevertheless, this is not a real disadvantage in the examination of patients with CTOs since transmurality is the main question in this case. There are new predefined criteria for differentiating between transmural and non-transmural infarction in SPECT images .Overall, SPECT appears to be a useful examination modality for patients with CTOs. A radiation dose of 6–24 mSv, depending on the protocol and tracer used (lower radiation doses for Tc-99m and higher radiation doses for Tl-201) , as wellPositron emission tomography (PET) is another nuclear medicine imaging technique that is in use for around 40 years in some specialized centers with a broader use during the last years. The main advantages of PET over SPECT are higher-quality images because of high-energy emitted photons (511 keV) with improved spatial and temporal resolution and shorter half-lives of the radioisotopes (mostly some minutes) as well as less radiation exposure , 46. By For the imaging with PET positron emitting radionuclides like Rubidium-82 (Rb-82), Oxygen-15 (O-15), Nitrogen-13 (N-13), and Flouride-18 (F-18) are used and incorporated into biochemical molecules. Rb-82 is a generator product, the other named radioisotopes are cyclotron products and for O-15 and N-13 with very short half-lives of 2 and 10 min, respectively, making an onsite cyclotron necessary. For perfusion imaging O-15 water, N-13 Ammonia, Rb-82 and F-18 Flurpiridaz are used with stress and rest imaging as in MPS. O-15 water is ideal for quantification of MBF in absolute terms. It can be seen as a one-compartment tracer kinetic model as it has no barrier effect form cellular membranes. Because of its very short half-life and poor contrast images between blood pool and the myocardium its use is limited. N-13 Ammonia is cationic and its first-pass extraction is related to blood flow for low flow rates. It reaches the cytosols via passive diffusion or active transport and is incorporated into the amino acid pool or diffuses back into the blood. Rb-82 is a potassium analog with a very short half-life of 75 s. During first-pass its extraction is not very high and builds a plateau with higher blood flow rates. F-18 Flurpiridaz is a novel PET mitochondrial complex-1 inhibitor in preparation with a half-life of 120 min. It has a high extraction rate with high flow and therefore makes absolute quantification of blood flow possible . As for For the differentiation of a perfusion defect in both stress and rest between a scar region and ischemic but viable region F-18-Fluorodeoxyglucose (FDG) is used. F-18-FDG competes with glucose for transport as well as phosphorylation by hexokinase. Once it is in the cytosol and phosphorylated it cannot be further processes by glycogen synthesis and is therefore trapped in the cell , 48. ItsIn CAD patients in general, an ischemia-driven revascularization approach guided by PET could result in a reduction in angina severity and a slight improvement in the left ventricular ejection fraction . StulijfComputed tomography (CT) angiography has traditionally been used for anatomical evaluations of the coronary tree. With the advent of new-generation scanners with dual-source imaging, 64-row scanners, and modern software techniques, plaque morphology and regional calcium burden evaluations and functional assessments have also become possible , 52.Since CTOs are found in many patients with known or suspected CAD, CT examinations can also contribute to their diagnosis. However, the distinction between a “true” CTO and subtotal stenosis constitutes a diagnostic difficulty. The distal lumen is often contrasted via collaterals, and the spatial resolution of CT is relatively low in comparison to classical angiography. Diagnostic markers such as a lesion length over 9 mm and the so-called reverse attenuation gradient sign have proven to be helpful, enabling a clear diagnosis in most cases –55. AparA key task of the pre-interventional examination of CTO patients is the abovementioned viability test. While cardiac CT was previously used for purely anatomical assessments of the coronary tree, it can also be used for hibernation assessments. These are based on classic measures, such as global and regional function, wall thickness, and wall thickening in systole, while assessment methods derived from CMR have not yet found their way into clinical routine. Reduced perfusion can be indicated by reduced contrast during the arterial phase. Similar to the flooding behavior of gadolinium-containing magnetic resonance imaging (MRI) contrast agents, delayed (late) enhancement with iodine-containing contrast media indicates myocardial scarring. A mismatch between underperfusion  and late enhancement indicates a potential intervention target . An addiFor the prediction of interventional recanalization success, the Computed Tomography Registry of Chronic Total Occlusion Revascularization (CT-RECTOR) score was established by analogy with angiography-based scores, such as the Japanese Multicenter CTO Registry (J-CTO) and Prospective Global Registry for the Study of Chronic Total Occlusion Intervention (PROGRESS-CTO) scores . It is cThe actual procedure can also be planned using CT angiography. Depending on factors such as good collateralization or the presence of an intensively calcified proximal cap, the access route (antegrade or retrograde) can be determined, or in the case of severe calcifications over short distances, an early switch to a stiffer wire can be decided. The anticipation of a need for rotablation or debulking devices is a potentially time-saving and patient-friendly factor . In a siCT angiography can also be used to detect in-stent restenosis in follow-up investigations. However, as it has shown a high negative predictive value but a low positive predictive value –68, its In summary, pre-interventional multi-slice CT is a useful additional pre-interventional diagnostic tool. If necessary, it should be combined with other examination modalities to detect hibernation. Among all diagnostic modalities, CT is the closest to integrating diagnosis, risk calculation, intervention planning, and follow-up care in one modality.Cardiovascular magnetic resonance (CMR) is the gold standard for the evaluation of the left and right ventricular volumes and function, as well as ischemia and viability. A major advantage of CMR over nuclear and CT imaging is the lack of a need for ionizing radiation and the better tolerance of gadolinium-containing contrast media compared to iodine-containing contrast media. Its spatial and temporal resolutions are good thanks to modern sequences, such as balanced steady-state free precision imaging, ECG-gating, and breath holding techniques . Image qIschemia can be visualized using adenosine perfusion CMR. Less contrasted myocardial areas under vasodilator stress indicate a myocardium at risk . In CAD Two modalities have been developed for viability assessment: low-dose dobutamine CMR (LDD-CMR) and LGE. LDD-CMR is the older method, but it has shown excellent results in predicting functional recovery after revascularization, even in direct comparison with other imaging techniques , 81. In LGE imaging is a valuable tool for distinguishing a hibernating myocardium from post-infarction scarring. Five to 15 min after the administration of a gadolinium-based contrast agent, focal deposits of gadolinium in the myocardium show a widening of the extracellular volume, indicating fibrosis and scarring, because of a delayed washout. LGE has shown a good correlation with PET examinations and excellent inter- and intra-observer agreement . In stabIn a trial involving 59 patients with successful CTO PCIs, a pre-interventional LGE extent of <50% was associated with functional recovery of the related segment . In anotFor even better workups, new techniques, such as parametric mapping, are currently under investigation. In a first study, the extracellular volume was found to be superior to LGE for functional recovery assessments .Using CMR, patients have already been examined after complex PCI in order to investigate the extent and clinical impact of a peri-interventional infarction. This revealed a relevant proportion of patients with new LGE after PCI, which in turn had a negative impact on the clinical outcome . TechniqA central point of future developments, besides a further increase in diagnostic accuracy, will be a reduction in patient exposure to contrast media or radiation. Since CMR typically works without radiation, a further improvement of CMR coronary angiography would be an obvious direction in the quest to overcome the disadvantages of CT in CTO intervention planning. CMR coronary angiography is already feasible for the evaluation of plaque morphology and composition –93. At tAnother goal for further improvement of interventional outcomes is the integration of imaging modalities into the actual intervention. Opolski et al. demonstrated the feasibility and safety of an augmented-reality glass that provides the interventional cardiologist with additional information from coronary CT angiography . This isAs already discussed, each modality has its own strengths and weaknesses. Therefore, fusion imaging, such as PET/CT or PET/MRI, could improve the pre-interventional workup of CTO patients. For example, a combination of morphological assessments using CT with metabolic assessments using PET could improve patient selection . In a fiIn summary, the use of non-invasive imaging for ischemia and viability assessments before interventional recanalization of a CTO is desirable. Although some imaging techniques have clear advantages over others, the selection depends mainly on regional availability and expertise. Better patient selection and prediction of interventional success should be the target of future prospective studies.JK and DB: concept and writing of the manuscript. NE and VP: writing of the SPECT and PET section, proofreading, and image examples. MK, SM, JM, NN, TS, MT, and TT: interpretation of the sources, proofreading, and image example. WR: supervision, concept, and proofreading. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Orthotopic heart transplantation (HTX) is the gold standard to treat end-stage heart failure. Numerous risk stratification tools have been developed in the past years. However, their clinical utility is limited by their poor discriminative ability. High sensitivity troponin T (hsTnT) is the most specific biomarker to detect myocardial cell injury. However, its prognostic relevance after HTX is not fully elucidated. Thus, this study evaluated the predictive value of postoperative hsTnT for 1-year survival and days alive and out of hospital (DAOH) after HTX.This retrospective cohort study included patients who underwent HTX at the University Hospital Duesseldorf, Germany between 2011 and 2021. The main exposure was hsTnT concentration at 48 h after HTX. The primary endpoints were mortality and DAOH within 1 year after surgery. Receiver operating characteristic (ROC) curve analysis, logistic regression model and linear regression with adjustment for risk index for mortality prediction after cardiac transplantation (IMPACT) were performed.Out of 231 patients screened, 212 were included into analysis . One-year mortality was 19.7% (40 patients) and median DAOH was 298 days (229–322). ROC analysis revealed strongest discrimination for mortality by hsTnT at 48 h after HTX [AUC = 0.79 95% CI 0.71–0.87]. According to Youden Index, the cutoff for hsTnT at 48 h and mortality was 1640 ng/l. After adjustment for IMPACT score multivariate logistic and linear regression showed independent associations between hsTnT and mortality/DAOH with odds ratio of 8.10 [95%CI 2.99–21.89] and unstandardized regression coefficient of −1.54 [95%CI −2.02 to −1.06], respectively.Postoperative hsTnT might be suitable as an early prognostic marker after HTX and is independently associated with 1-year mortality and poor DAOH.The online version contains supplementary material available at 10.1186/s40001-022-00978-4. Numerous risk stratification tools have been developed in the past years, but their clinical use is often limited by insufficient predictive values . Recently, Devereaux et al. investigated the prognostic value of high-sensitivity troponin I (hsTnI) in patients undergoing cardiac surgery and showed that levels of hsTnI were independently associated with mortality . The hsTnT levels at 48 h after HTX showed numerically strongest discrimination for 1-year mortality regarding the AUC. Youden Index determined a cutoff of 1640 ng/L for hsTnT at 48 h after HTX  for non-events and 27.5% (95% CI 14.6–43.9) for events. Regarding NARI, the model was able to identify 114/1000 patients more at risk for 1-year mortality. Corresponding reclassification tables were added to the supplements. In ROC analysis the AUC for the model including troponin was significantly higher as compared to the model only including IMPACT score . The net benefit curve suggested highest net benefit for the combined use of IMPACT and hsTnT  days vs. above cutoff 278 (14–308) days, p ≤ 0.0001]. Results for other hsTnT sampling timepoints are presented in the supplements. In a multivariable linear regression model, association of continuous hsTnT elevation (per 100 ng/L) and DAOH remained significant when adjusted for points on the IMPACT score   which is supposed to optimize the allocation of the limited donor organs. In the lung transplantation setting, a similar score already exists ) which is also used to prioritize waiting list candidates 12 years and older based on a combination of waitlist urgency and post-transplant survival. Based on the data of this study, postoperative hsTnT may also be included into such a score for early re-estimation of postoperative risk and prognosis. Further studies should focus on potential interventions depending on hsTnT values that might be able to prevent complications and finally to reduce mortality after HTX. These may include intensified monitoring or standardized protocols for strict follow-up of these patients.Strengths of the presented data include first, standardized troponin measurement at multiple time points with high data completeness; second, complete 12-month follow-up. Further, we did not only address mortality but also DOAH, a more patient-centered endpoint to quantify life impact . We are The generalizability of these findings may be hampered by the single-center design. However, characteristics such as 1-year mortality were in line with the current literature.Early hsTnT levels after HTX surgery are independently associated with poor 1-year survival and reduced DAOH. Therefore, early hsTnT concentrations might be useful for early risk reassessment to tailor postoperative therapy or decision-making in the intensive care unit.Additional file 1: Figure S1. Study Flowchart. Figure S2. Association of hsTnT levels at different timepoints above cutoff and days alive and out of hospital. Table S1. Reclassification tables of a 1-year mortality prediction model using IMPACT compared to a model using IMPACT and hsTnT.


Pseudo-progression and flare-up phenomena constitute a novel diagnostic challenge in the follow-up of patients treated with immune-oncology drugs. We present a case study on pulmonary flare-up after Idecabtagen Vicleucel (Ide-cel), a BCMA targeting CAR T-cell therapy, and used single-cell RNA-seq (scRNA-seq) to identify a Th17.1 driven autoimmune mechanism as the biological underpinning of this phenomenon. By integrating datasets of various lung pathological conditions, we revealed transcriptomic similarities between post CAR T pulmonary lesions and sarcoidosis. Furthermore, we explored a noninvasive PET based diagnostic approach and showed that tracers binding to CXCR4 complement FDG PET imaging in this setting, allowing discrimination between immune-mediated changes and true relapse after CAR T-cell treatment. In conclusion, our study highlights a Th17.1 driven autoimmune phenomenon after CAR T, which may be misinterpreted as disease relapse, and that imaging with multiple PET tracers and scRNA-seq could help in this diagnostic dilemma. CAR T-cell therapy is establishing itself as a new standard of care in relapsed and refractory multiple myeloma (RRMM), and the field is moving towards use of this modality in early treatment lines . For exa18F-Fluorodeoxyglucose (FDG) PET/CT imaging is increasingly used in addition to serological M-protein testing and bone marrow aspirates for staging prior to and after CAR T infusion. Whether FDG is the best tracer in this setting has yet to be determined, as the technique is being based not only on the quantification of increased glucose uptake by tumor cells but also by others such as inflammatory cells [Appreciating the high incidence of extramedullary lesions and patchy disease patterns seen in the RRMM setting, whole body ry cells . Given try cells , 5. Pseury cells , 7. OtheHere, we report on a sarcoidosis-like flare-up after anti-BCMA CAR T-cell product Ide-cel and present our strategy to discriminate between autoimmune phenomena and true relapse using multiple PET tracers along with single-cell RNA-sequencing (scRNA-seq).6 CAR T cells was infused. The patient experienced rapid-onset cytokine release syndrome (CRS) grade 1, which was successfully treated with repetitive doses of tocilizumab. Respiratory symptoms fully resolved during bridging therapy but came back 10 days after CAR T infusion. Disease assessment was performed at 3 months of follow-up revealing undetectable MRD in a bone marrow aspirate and full resolution of focal bone lesions but still disseminated FDG uptake at the lung and mediastinal lymph nodes. To narrow the differential diagnoses down, we performed a PET using 68Ga-DOTA(0)-Phe(1)-Tyr(3)-octreotide (68Ga-DOTATOC) that usually detects sarcoidosis with high sensitivity and specificity [68Ga-Pentixafor (binds to CXCR4), which is an alternative tracer to FDG in the detection of malignant plasma cells [A 61-year-old Caucasian male was diagnosed with IgA kappa MM with osteolytic bone disease, anemia, and 80% bone marrow infiltration by malignant plasma cells. In addition, biopsy-proven extramedullary disease (EMD) was found located to both lungs, explaining months of violent coughing prior to presentation. Fluorescent in situ hybridization revealed high-risk cytogenetics including t, amp(1q), and del(1p). The patient responded to induction therapy using bortezomib, cyclophosphamide, dexamethasone, but stem cell harvest was skipped due to the COVID-19 pandemic. A bortezomib based bridging therapy failed, M-protein levels increased and salvage therapy was administered using daratumumab, pomalidomide, dexamethasone to which the patient was refractory. Given the limited options at this stage, the patient was treated with the anti-BCMA CAR T-cell product ide-cel. PET imaging using multiple tracers was conducted at baseline, 3-month follow-up, and 6-month follow-up Fig. . Baselinma cells   and genes associated with Th17 T-cells , indicative for a Th1-polarized Th17 phenotype , and IL17F were not expressed . Consequently, we termed this cluster CD8 Th17.1-like. Also CD8-positive cells with Th17-like features and plasticity towards IFNG expression have been linked to autoimmune diseases [IFNG, TNF, CSF2 (GM-CSF), CCL20, and IL26, highlighting their polyfunctional nature [IFNG, TNF, TBX21, CXCR3) and four Th17-associated  genes in individual cells. They were considered co-expressing when at least one Th1- and one Th17-associated gene was detected in a single cell, and we confirmed co-expression in the majority of Th17.1 and CD8 Th17.1-like cells. , of which none was identified as plasma cell Fig. . Qualityand CSF2 –14, whilsed Fig. . Of noteig. Fig. . Consequl nature , 24 , expressing high levels of SPP1, recently identified in severe COVID-19 ARDS and IPF (idiopathic pulmonary fibrosis), was absent from CART-BAL, sarcoidosis and HC BALs [While we observed aberrant T-cells in the CART-BAL dataset, the macrophage compartment in CART-BAL, sarcoidosis, and HC was mainly comprised of unremarkable tissue resident alveolar macrophages. A pro-fibrotic macrophage phenotype  and CCND2 expression of which a considerable amount was in a proliferative state , followed by high-dose melphalan and autotransplant two weeks after ERT [At 6 month follow-up, the patient was still coughing. CAR T-cell numbers in the peripheral blood declined from initially 10% to 0.05% of PBMCs, and humoral immunity started to recover. FDG PET showed the known sarcoidosis-like pulmonary changes, but also revealed a number of new soft-tissue lesions located to the musculature of both lower limbs, suspicious for extramedullary relapse. In contrast to the previous scans, these lesions were CXCR4 positive Fig. . We biopate Fig. . CXCR4 eion Fig. . EPCAM eion Fig. . We compion Fig. . Whetherfter ERT . The patWe hypothesize that CRS after CAR T-cell infusion triggered expansion of pathogenic Th17.1 T-cells in the lung leading to sarcoidosis-like manifestations. Th1-polarized Th17 cells are key effectors of autoimmune inflammation  includinBCMA-loss [PET imaging using multiple tracers is a noninvasive approach to decode tumor heterogeneity in MM . Using tCMA-loss , 43.Finally, we report the case of a BCMA positive relapse after anti-BCMA CAR T. While antigen loss is a rare tumor-intrinsic mechanism of resistance , 43, theIn conclusion, we demonstrated a clinically relevant autoimmune phenomenon after CAR T-cell therapy that can be delineated using scRNA-seq and PET imaging.68Ga-Pentixafor, as described previously [18F-FDG and 68Ga-DOTATOC were prepared as described previously [18F-FDG, patients fasted for at least 6 h with blood glucose levels below 160 mg/dl at time of scan. For 68Ga-Pentixafor and 68Ga-DOTATOC PET/CT, fasting was not required. Spiral CT with or without intravenous contrast was conducted and included a field of view from the base of the skull to the proximal thighs. PET emission data were acquired in three-dimensional mode . After decay and scatter correction, PET data were then reconstructed iteratively using the algorithm provided by Siemens Esoft . Image interpretation was conducted on a visual basis and carried out by an expert reader (RAW).We used a fully automated synthesis module  to synthesize eviously . 18F-FDGeviously , 46. PETCART-BAL sample was obtained at 3-month follow-up, diluted in PBS and filtered through a 40 μm mesh resulting in a single cell suspension. EMD sample was obtained at 6-month follow-up by ultrasound guided push biopsy. The biopsy was filtered with a 100 μm strainer followed by tissue digestion using Collagenase A (10 mg/mL), Collagenase D (10 mg/mL), DNAse I (2 mg/mL). Digestion was stopped with EDTA (100 mM) followed by a second filtering through a 70 μm mesh resulting in a single cell suspension.Obtained single cell suspensions were adjusted to a concentration of 400 cells/μl and 1000 cells/μl for CART-BAL and EMD, respectively, and loaded into the 10x Chromium controller for scRNA-seq. Following the detailed protocol provided by 10x genomics, single cell 3’ reagent kit v3.1 was used for reverse transcription, cDNA amplification and library construction. Libraries were quantified using QubitTM 2.0 Fluorometer (ThermoFischer) and quality was checked by 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). S2 flow cells were used for sequencing on a NovaSeq 6000 sequencer (Illumina). CART-BAL has been run in duplicate to obtain technical replicates.EPCAM) were removed. Quality filtering thresholds for the number of detected genes, number of detected unique RNA molecules (UMIs) and fraction of mitochondrial UMI-counts are available in Supplementary Table Raw sequencing data was demultiplexed and quality-checked using the CellRanger v.3.1.0) ‘mkfastq’ script. Alignment and transcript quantification was performed using the CellRanger ‘count’ script against the GRCh38 (Ensembl93) human genome assembly. Ambient RNA signals were removed using SoupX to mitigate potential effects of RNA released from dead or ruptured cells . The obt.0 ‘mkfasFor analysis of CART-BAL T-cells respective clusters  were isolated from the dataset using the subset function and the subset was reanalysed repeating basic Seurat steps (described above) based on 1000 highly variable features, the first 15 PCA-dimensions, and clustering resolution parameter set to 0.7.BAL Single-cell transcriptomes generated in this study (CART-BAL) and three previous studies, including BAL samples from four pulmonary sarcoidosis patients , three hOf note, dataset HC4 from the Liao M. et al. paper was excluded due to patient privacy restrictions. In the original study alignment was performed using a different CellRanger version and reference as desired for the integration pipeline in the present study .T-cells and monocyte/macrophage (Mono/Macro) cells were separated from the integrated dataset and reanalysed, repeating integration as described above. For T-cells and monocyte/macrophage subsets 3000 and 1000 highly variable genes, 55 and 25 first PCA-dimensions, 55 and 25 first MNN-dimensions, and clustering resolutions of 1.5 and 0.4 were used, respectively, for dimensional reduction and Louvain clustering.T-cells of a publicly available dataset including three patients suffering from cutaneous sarcoidosis were extracted after preprocessing using the common CellRanger version v3.1.0) and reference  as well as quality filtering   and fraction of mitochondrial UMI-counts are available . T-cell subpopulations were annotated upon careful assessment of expressed genes. particularly, expression of canonical lineage and cell state markers was interrogated : CCR7, TCF7, S1PR1, IL7R, SELL; naive T-cells: CCR7, TCF7, LEF1; MAIT: SLC4A10, ME1; cytotoxic lymphocytes (CTL): Granzymes, PRF1; CD4 CTL: CD4, CCL4, CCL5, PRF1; IFN-responsive: ISG15, MX1, RSAD2). Macrophage subpopulations were annotated in an analogous fashion .Major cell types were annotated based on expression of canonical marker genes  based on their average expression in all cells. Expression of genes from a given signature was calculated and subtracted by the aggregated expression of a control feature set ctrl = 100) selected randomly from the bins the analysed signature genes were part of. Statistical significance was assessed by pairwise, one-sided  Wilcoxon rank-sum test (R function wilcox.test) of each group compared to the average. Signatures were retrieved from the literature. Signatures and respective sources are listed in Supplementary table 00 selectDifferential expression analysis was performed using the Wilcoxon rank-sum test implementation of the Seurat function FindAllMarkers (only.pos = TRUE). Differential expression analysis results are available in Supplementary Table EMD cells were thawed in RPMI supplemented with 10% FCS. Staining was performed in PBS at 4 °C with the following antibodies: CD184/CXCR4 – PerCP/eFluor710 , CD326/EpCAM – APC , CD269/BCMA – PE , CD38- FITC , CD138 – BV510 , CD45 – BV605 , CD3 – AF700 . For dead cell exclusion, Fixable Viability Dye eFluor ™ 780  was used. A FcGr blocking step was not performed. Events were acquired on the Attune NxT (Thermofisher). Data was analyzed with FlowJo version 10.8.0.p-value <0.05 was considered as statistically significant.The non-parametric two-sided Wilcoxon rank-sum test was used to compare gene expression values between groups of cells. A Supplementary InformationSupplementary Table 1Supplementary Table 2


Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV’s human cell entry receptor (AAVR) contains polycystic kidney disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA (enzyme-linked immunosorbent assay) studies, it is shown that AAVGo.1 binds to human AAVR more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of AAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure. There are only minor conformational adaptations in AAVR, but there is a near-rigid rotation of PKD1 with maximal displacement of the receptor domain by ~1 Å compared to PKD1 bound to AAV5. AAVGo.1 joins AAV5 as the second member of an emerging class of AAVs whose mode of receptor-binding is completely different from other AAVs, typified by AAV2.IMPORTANCE Adeno-associated virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy, with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor AAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors. Primateectively . The Capectively , 5.motif at N-terminus with eight cysteines (MANEC), while the central extracellular portion contains five polycystic kidney disease (PKD) domains numbered 1 to 5 from N terminus to C terminus  . AAVR isterminus . The C tterminus . Experimterminus .For human AAVR, a few structural complexes with AAV are known to14. TDomain-swap mutants indicate a secondary role for PKD1 with AAV2 and other serotypes , but mulAAV gene therapy utilizes primate serotypes of which AAV5 is currently the only known representative with primarily PKD1 interactions. AAV5 was initially isolated from a human penile lesion  and is tHere, binding of AAVGo.1, AAV5, and AAV2 to domain combinations of AAVR is compared by ELISA (enzyme-linked immunosorbent assay). Then, high-resolution structures are determined by cryo-EM of AAVGo.1 alone, and in complex with a PKD12 fragment of AAVR. These are compared to corresponding structures of AAV5 to understand the diversity of interactions that are compatible with productive receptor-binding.AAVGo.1 virus-like particles  were produced using Sf9 cells and examined via transmission electron microscope TEM;  and B. ACryo-EM single particle analysis (SPA) yielded a reconstruction at 2.9 Å resolution for AAVGo.1 . SharpenThe AAVGo.1 capsid has a similar protein fold and surface topology to AAV5 structures , 11, 22.AAVGo.1 and AAV5 capsid proteins have high homology (94%). All 42 amino acid differences occur in the VP3 region , and the majority of these differences are located on the exterior of the capsid . Some ofMinor structural differences are observed between AAV5 and AAVGo.1. The AAVGo.1 DE loop (VR-II), which forms the outer edge of the pore-like structure of the 5-fold axis, protrudes an additional 1.5 Å compared to AAV5  to D. A y axis of AAVGo.1, similar to previous cryo-EM studies of either PKD12 or PKD1-5 bound to AAV5 .Cryo-EM single particle analysis (SPA) yielded reconstructions at 2.4 Å for the PKD12 complex  that wer353 seen in both AAV5 and AAVGo.1 complexes suggests the potential for (rigid) pivoting around the binding site. Receptor pivoting has not been observed in individual AAV5 or AAVGo.1 complexes, but when comparing PKD1 as bound to AAVGo.1 or AAV5, we see an overall rotation in the receptor position  and the AAVGo.1 VR-IV loop. Contact has been observed between PKD1 Lys399 and VR-IV residues Asn442 and Asn443 of AAV5 , and the Ile349 interaction is absent in AAVGo.1  for AAVGo.1 Asp547 also affects PKD1 Arg353 binding subsite electrostatic interactions G478. AAVGo.1 residues Thr477 to Ser482 are of below-average order in the sharpened maps of AAVGo.1 alone. In the AAVR complex, the AAVGo.1 map here is somewhat more ordered. Together, these indicate that the loop is intrinsically dynamic. The double serine insertion is within the epitope of AAV5-neutralizing monoclonal antibody MAb HL2476 , and if Prior to this work, all complexes between AAV and AAVR were fundamentally similar to the AAV2 complex , 9, 12, Within what we might now characterize as a PKD1-binding clade of AAVs, structural differences in the AAVR complexes of AAV5 and AAVGo.1 are minimal. There is an overall rotation of the PKD1 domain, but otherwise differences are very subtle, consistent with modest differences in transduction efficiencies. It is plausible that the structural differences observed could account for different transduction efficiencies, but explicit associations would be somewhat speculative at this time. Many factors beyond avidity affect transduction. Furthermore, avidity is the result not only of the interactions seen, but from free energy changes in protein order or solvation upon receptor binding that are not quantifiable by cryo-EM.At a more qualitative level, it is an intriguing possibility that the avidity of the complex between human AAV5 and AAVR is not fully optimized, as suggested by tighter binding of a domesticated goat AAV. Is this due to compromise between receptor binding and immune neutralization escape? Or is evolutionary selection of high avidity limited by another uncharacterized step in the virus’s life cycle where dissociation from AAVR is needed?6PKD1, His6PKD12, and His6PKD15) were expressed and purified as previously described (AAV2 (0.9 mg/mL) and AAV5 (7.6 mg/mL) virus-like particles (VLPs) were produced using Sf9 cells and purified as previously described , 11. AAVescribed .6PKD1, His6PKD12, or His6PKD15) and detected with anti-6× His tag horseradish peroxidase (HRP) (Abcam number ab1187) antibody. 3,3′5,5′-Tetramethylbenzidine (TMB) ELISA Substrate (Abcam number ab171523) was added to each well, and development was stopped using 1 M hydrochloric acid. The optical density of plates was evaluated at 450 nm using a microplate reader (BioTek Synergy H1 Hybrid Multi-Mode Reader). Curves were fitted via nonlinear regression, and the 50% effective concentrations (EC50) were calculated using GraphPad Prism 8, using the 95% confidence limits as error estimates.For the PKD1 and PKD12 binding assays, ELISAs were carried out in duplicate using a modified direct ELISA protocol , 11. AAVFor the antibody binding assays, AAV5 and AAVGo.1 (50 μL at 3 μL/mL in 100 mM sodium bicarbonate buffer at pH 9.6) were incubated in multiple wells of a 96-well plate  overnight on a rocking platform. The wells were then washed three times with Tween 20 Tris-buffered saline  followed by blocking with bovine serum albumin  and incubated for 1 h. Unbound BSA was removed via three additional washes with TTBS. One hundred microliters of antibodies, diluted in TTBS, were added and incubated for 1 h at the following final concentrations: 2 μg/mL of HLA2476 , 100 μg/mL of ADK5a , and 4 μg/mL of ADK5b . After 1 h, unbound antibodies were removed and the wells were washed three times with TTBS. One hundred microliters of a secondary HRP-conjugated, goat-derived, anti-mouse antibody  were then added at 4 μg/mL and incubated for 1 h. Unbound secondary antibody was removed via washing three times with TTBS. Ninety microliters of TMB ELISA substrate were then added for 5 to 10 min. The reaction was quenched with 80 μL of 1 M HCL, and the absorbance was measured at 450 nm using a plate reader.AAVGo.1 and AAVGo.1-PKD12 complexes were prepared and imaged as previously described for AAV2 and AAV5 , 12. BriCryo-EM grids were screened using an FEI Tecnai F30 Twin 300 kV TEM in preparation for single-particle Cryo-EM. AAVGo.1, and AAVGo.1-PKD12 complexes were imaged using an FEI Titan Krios equipped with a Gatan K3 digital camera in superresolution mode at a dose rate of 40 frames per 3.12 s exposure. Automat0.143) (Relion 3.1.0 was used to process AAVGo.1 . Seven h0.143) .0.143; Relion 3.1.1 was used for the complex of PKD12 with AAVGo.1 . A total7kp3) in Coot (7kpn). The models and imaging parameters were refined using RSRef 0.5.6 (A preliminary model for AAVGo.1 was generated using the AAVGo.1 VP3 protein sequence threaded into the 2.1 Å AAV5 structure (PDB ID:  in Coot . The staef 0.5.6  with a f7TI4 for AAVGo.1 and ID 7TI5 for the complex) and EMDB (ID EMD-25909 for AAVGo.1 and ID EMD-25910 for the complex).Atomic models and cryo-EM maps are available at the PDB (ID


The majority of adult neural stem cells (aNSCs) are in a distinct metabolic state of reversible cell cycle exit also known as quiescence. The rate of aNSC activation determines the number of new neurons generated and directly influences the long-term maintenance of neurogenesis. Despite its relevance, it is still unclear how aNSC quiescence is regulated. Many factors contribute to this, like aNSC heterogeneity, the lack of reliable quiescence markers, the complexity of the neurogenic niches or the intricacy of the transcriptional and post-transcriptional mechanisms involved. In this perspective article I discuss possible solutions to these problems. But, first and foremost, I believe we require a model that goes beyond a simple transition toward activation. Instead, we must acknowledge the full complexity of aNSC states, which include not only activation but also differentiation and survival as behavioural outcomes. I propose a model where aNSCs dynamically transition through a cloud of highly interlinked cellular states driven by intrinsic and extrinsic cues. I also show how a new perspective enables us to integrate current results into a coherent framework leading to the formulation of new testable hypothesis. This model, like all others, is still far from perfect and will be reshaped by future findings. I believe that having a more complete view of aNSC transitions and embracing their complexity will bring us closer to understand how aNSC activity and neurogenesis are controlled throughout life. Neurogenesis is preserved in specific regions or neurogenic niches in the adult brains of most mammals . The sub-We still ignore how signal integration works in aNSCs. A variety of different signals have been involved in balancing quiescence and activation in these cells. For instance, NOTCH and BMP are pro-quiescence signals while WNT and SHH are considered pro-activation ones . Metabol-Transcriptional profiling is not an accurate readout of the position of NSCs along the quiescence to activation transition. Recent data has pointed out that unlike other cell types, the correlation between mRNA and protein content in quiescent NSCs is very poor . In addi-Quiescence-specific markers do not exist for aNSCs. Despite many genes being enriched in quiescent NSCs compared with active NSCs, none of them has been proven to be a reliable quiescence-specific marker. The main reason for this is the huge overlap in expression profile between astrocytes and aNSCs  . This pr-Adult neural stem cells are heterogeneous. SEZ NSCs are spatially heterogeneous, as they retain the regional identity that was instructed to them during development . Spatial-in vivo. The maintenance of quiescence depends on intrinsic as well as niche signals. The mere fact of dissociating the tissue for subsequent sorting and sequencing is enough to induce the activation of satellite cells, the resident quiescent cells of the muscle , we must embrace their complexity in full. Therefore, instead of the classical view of a linear transition from quiescence to activation, I propose a model where adult NSCs exist in a vast matrix of cellular states . These sin vivo (At one specific time each NSC cell occupies a defined position in the states matrix. But if we were able to follow that same NSC over time we would be able to observe it travelling (or reversibly transitioning) through different positions . NSCs inin vivo , 2022. Tin vivo  and Cellin vivo  on scRNAin vivo, would be fully possible to achieve thanks to the in vitro system. In vitro aNSC states exist in a fully defined and stable environment and are thus unlikely to be common (or even exist) in vivo. Direct manipulation of signalling and metabolism in aNSC in vitro will also allow evaluating the effects of single and combined cues on the transition of aNSCs between states. In addition, the in vitro system will enable us to determine the degree of intrinsic heterogeneity of adult NSCs in different conditions. Intrinsic heterogeneity can be driven, for instance, by oscillations in the levels of key transcription factors  and thenic NSCs .The new framework immediately prompts us to think of the heterogeneity of stem cells in a different way and allows the integration of clonal and label-retention data without the need of invoking the existence of distinct stem cell types. The clonal approaches typically used for the study of adult neurogenesis label those cells with the highest activity of a particular promoter at the time of tamoxifen administration. They will therefore label the positions in the matrix where that promoter is most active. But labelled cells might not constitute a separate lineage, as they do not necessarily need to be restricted to one single area of the matrix and could partially overlap with the high activity of other promoters also used for clonal analysis. For instance, high activity of the Gli1 promoter could happen anywhere where SHH signalling is high and is likely to overlap, at least partially, with high expression of unrelated genes such as Nestin or Ascl1. It is important to note that promoter activity and gene function do not always come hand in hand. Even less in adult NSCs, where mRNA and protein are poorly correlated. Nevertheless, the observed behavioural biases could still be linked to the function of the gene used for labelling the cells. For instance, high Ascl1 mRNA expression, although does not always lead to activation due to post-transcriptional regulation, could increase the probability of NSCs to activate. On the other hand, it is easy to imagine that resting NSCs, which recently proliferated, are likely to remain in a pro-activation zone after division. This alone will make them more likely to become activated again than dormant NSCs, which did not activate during the labelling period. Finally, cells which are very distant in the matrix might have very distinct signal transduction capabilities, both due to differential expression of signalling pathway components and different metabolic characteristics. This readily explains how NSCs respond differently to the same stimuli depending on their state .The model makes it clear that the likeliness of a NSC becoming activated depends on two things: their current state , and their signalling environment . But what I personally find most exciting about this framework is that it allows us to generate hypothesis and design specific experiments to test them. Two particularly tantalising ones are:One still unanswered question in the field is how interventions that affect neurogenesis impact the maintenance of aNSCs over long periods of time. Stimuli such as exercise, dietary changes or an enriched environment affect adult neurogenesis . Howeverin vivo is associated with the loss of their self-renewing ability  are highest in the most quiescent cells and repeated activation of DG NSCs  ability . This su ability . Such a The way we think about a subject often affects the questions we ask and due to the oversimplification associated with models, significant features could be overlooked. I believe it is time to incorporate the intricacy of stem cell behaviour into our working models, even if we still do not have enough data to fully support them. In fact, this, as any model is only meant to serve as a starting point, since it will continuously evolve through the addition of new data. Experiments, on the other hand, are independent of the model and will remain significant regardless of it. Although my focus is on aNSCs, the proposed framework will be useful for other adult and embryonic stem cell transitions, reprogramming strategies and cancer research. One interesting avenue would be to further explore the continuum of states that exist between glia and neural stem cells. Glial cells respond to injury and have the potential to generate new neurons in the adult brain. By investigating the aNSC to glia transition as well as the reactivation path of glial cells, we could identify important roadblocks for glia-to-neuron transitions . Other sThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.NU conceived and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JE-P declared a past co-authorship with the author NU to the handling editor.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


For advanced cancer, surgery may not be possible at the site of lymph node metastasis, such as para-aortic lymph node metastasis. Systemic administration of anticancer drugs has been performed in these cases, but treatment results are still inadequate. This study investigated the efficiency of drug delivery to intra-abdominal lymph nodes by administering an anticancer drug retrogradely to lymphatic vessels in order to deliver the drug directly to the metastatic lymph nodes. Thoracic duct infusion resulted in the same concentration of paclitaxel in abdominal lymph nodes as via systemic administration, but the serum concentration was lower. The results show that thoracic infusion may achieve higher paclitaxel doses than systemic administration. Infusion of anti-cancer drugs into the thoracic duct may yield clinical benefits for patients with extensive lymphatic metastases in abdominal malignancies.Gastrointestinal cancer with massive nodal metastases is a lethal disease. In this study, using a porcine model, we infused the anti-cancer drug Paclitaxel (PTX) into thoracic ducts to examine the efficiency of drug delivery to intra-abdominal lymph nodes. We established a technical method to catheterize the thoracic duct in the necks of pigs. We then compared the pharmacokinetics of PTX administered intrathoracically with those of systemic (intravenous) infusion. Serum, liver, and spleen concentrations of PTX were significantly lower following thoracic duct (IT) infusion than after intravenous (IV) administration approximately 1–8 h post-infusion. However, PTX levels in abdominal lymph nodes were maintained at relatively high levels up to 24 h after IT infusion compared to after IV infusion. Concentrations of PTX in urine were much higher after IT administration than after IV administration. After IT infusion, the same concentration of PTX was obtained in abdominal lymph nodes, but the serum concentration was lower than after systemic infusion. Therefore, IT infusion may be able to achieve higher PTX doses than IV infusion. IT delivery of anti-cancer drugs into the thoracic duct may yield clinical benefits for patients with extensive lymphatic metastases in abdominal malignancies. Lymphatic metastasis usually occurs in the direction of the lymph flow. In abdominal malignancies, such as gastrointestinal or ovarian cancer, metastatic tumor cells in regional lymph nodes move to the retroperitoneal para-aortic nodes and then spread to the left neck lymph nodes (Virchow’s nodes) through the thoracic duct ,2. ExtenRecently, new routes of administration have been developed for the administration of anticancer drugs. The intraperitoneal administration of Paclitaxel (PTX) has been performed for peritoneal dissemination in gastric cancer cases, producing excellent clinical results. ,12 ThereThe thoracic duct, the body’s central lymphatic vessel, originates in the cisterna chyli in the retroperitoneum, ascends between the esophagus and the descending aorta in the mediastinum, and flows into the left venous angle in humans ,17. We hPTX (Taxol) was purchased from Bristol-Myers Squibb Japan . The PTX used is a commercial product, with 100 mg of PTX dissolved in 8.35 mL of polyoxyethylene castor oil and 8.35 mL of ethanol. Porcine models, comprising 4 female pigs with an average body weight of 28.2 kg (range: 20.8–36.0 kg), were purchased from Sanesu Breeding Co., Ltd.  and were housed individually at the Center for Development of Advanced Medical Technology (CDAMTech), Jichi Medical University. All animal handling procedures in this study complied with the Jichi Medical University Guide for Laboratory Animals, the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health , and the ARRIVE guidelines . The InsAfter the pigs were placed in a supine position under inhalation anesthesia with sevoflurane, a 15 cm longitudinal incision was made 1 cm to the left of the midline at the 1/3 level on the cranial crest, caudal side. The left anterior cervical muscle was dissected, the left external jugular vein and the left subclavian vein were ligated and dissected, and then the first rib was dissected and removed 2 cm from the sternum attachment. We then performed a laparotomy, and 2 mL of Patent Blue  were injected into the mesenteric lymph nodes of the small intestine using a fine needle. Cervical lymphatic vessels could be identified in the remaining left internal carotid artery and left subclavian artery bifurcation a few minutes after the dye injection. Discharge of the lymph was confirmed after cannulation with a 24G SURFLO needle was performed A,B. The After intravenous administration of 100 mg of hydrocorton , 30 mg of PTX  were administrated from the cannulated thoracic duct, as described above. Pigs were systematically cannulated in the left internal jugular vein with a 24G Surflo needle, into which PTX was then intravenously infused. At 1, 3, 8, 12, and 24 h post-infusion, blood and urine samples were collected and centrifuged at 2,000 rpm for 10 min at 4 °C. Supernatants were then collected, placed in cryotubes, and stored at −80 °C. After thawing the samples, PTX concentrations were measured with high-performance liquid chromatography–mass spectrometry (LC-MS/MS).Fifty µL of each liquid sample , to which 200 µL of methanol and 20 µL of internal standard reagent  had been added, were vortexed for 30 s and then centrifuged . Supernatants were then subjected to spin filtering. Filtrates were analyzed by LC-MS/MS . Tissue samples (10 mg), to which 20 µL of internal standard reagent  and 1 mL of 0.1% formic acid–MeOH had been added, were homogenized and centrifuged . Supernatants (500 µL) were transferred to other tubes, to which 200 µL of water had been added. Samples were then vortexed and centrifuged , and supernatants were analyzed.18 analytical column  was used, and the column oven and autosampler were set to 40 °C and 4 °C, respectively. Mobile phase A was 0.1% formic acid-water, and mobile phase B was acetonitrile. The flow rate was set to 0.5 mL/min, and the injection volume was 3 μL. The gradient was 40% B in 2.7 min, ramping up to 100% by 4 min, remaining at 100% until 6 min, and then decreasing over 0.5 min, followed by an equilibration step at 40% B for 1.5 min.For LC analyses, a YMC-Triart CPTX and PTX-d5 were detected in ESI positive mode. MS/MS conditions were as follows: Nebulizer gas flow (3 L/min), heating gas flow (10 L/min), interface temperature (300 °C), desolvation temperature (526 °C), heat block temperature (400 °C), and drying gas flow (10 L/min). Collision energies were 24 V, 22 V, and 21 V for PTX, and 24 V, 25 V, and 21 V for PTX-d5. PTX, and PTX-d5 were observed at m/z 854 > 286 and m/z 859 > 291, respectively. PTX concentrations of the biological samples were quantified by the area ratio with the internal standard reagent (PTX-d5) added to the samples.PTX was added to the serum at a concentration of 0.005–5 µg/mL to prepare an 8-point calibration curve. The accuracy and linearity of the calibration curve, carryover, diurnal variation, inter-day variation, and sample stability were confirmed. Recovery tests of PTX added to porcine serum  were performed, and accuracies were 110% for porcine serum . For uriConcentrations in various organs were measured in the early and late phases, since frequent tissue collection with such an invasive procedure could seriously affect pharmacokinetics. In late phase analysis, some organs were taken at 8, 12, and 24 h after PTX infusion. Para-aortic lymph nodes were additionally taken only at the end of the experiment. Immediately after collection, each organ was stored in its collected state in a cryotube at −80 °C. Later, 10 mg samples of these organs were homogenized, and PTX concentrations were measured, as described above.t-tests, and p values < 0.05 were considered statistically significant.Results were analyzed using Student’s Immediately after PTX was administrated through the intrathoracic duct (IT), considerable amounts of PTX were detected in sera. However, concentrations of PTX 1 to 12 h after administration were significantly lower than those after intravenous administration (IV). The PTX concentration decreased with time, ultimately disappearing from the serum after 24 h with both IT and IV administration A. In conIn the first set of experiments, PTX accumulation in various organs was examined at 1 and 3 h post-infusion. In the lungs, liver, and spleen, PTX concentrations after IV administration were more than twice as high as after IT administration. However, there were no significant differences between the two infusion methods in terms of PTX concentrations in mesenteric lymph nodes .In the next set of experiments, we compared PTX concentrations 8, 12, and 24 h after IV and IT infusion. Although PTX concentrations were still high in the liver and spleen at 8 h, there were no differences in PTX levels in any organs between IV and IT administration after 12 h. However, 24 h after IT administration, relatively high PTX concentrations were still present in mesenteric and para-aortic lymph nodes compared to those following IV administration A,B.The thoracic duct is the main collecting vessel of the lymphatic system, and it drains lymph from the abdomen and lower extremities into the venous blood stream . Thus, tWe succeeded in cannulating the terminal thoracic duct in pig necks, and attempted to achieve the selective retrograde administration of PTX to the lymphatic system. We selected PTX because the concentration of PTX in the lymph in the thoracic duct is maintained at high levels after intraperitoneal infusion, probably due to its high molecular weight and hydrophobic properties . Using tOn the other hand, PTX concentrations in urine were considerably higher with IT than IV administration, especially at early time points. This suggests that most IT-infused PTX was retrogradely transported to the renal cortex and excreted from the kidneys, since the renal lymphatic outflow is connected to the para-aortic lymph nodes through the thoracic duct . HoweverIn summary, we established an excellent experimental model to assess the pharmacodynamics of IT-administrated drugs. Using this system, we examined the drug delivery efficiency of the IT infusion of PTX for extensive metastatic lymph nodes in the abdomen. After IT infusion, the same concentrations of PTX were obtained in abdominal lymph nodes, but with lower serum concentrations than after IV infusion. Thus, it may be possible to achieve higher PTX doses with IT than with IV administration. However, the amount of urinary excretion may present a risk of renal toxicity and requires further study. When anti-cancer drugs suitable for IT administration are found, this method may be ideal for the treatment of patients with extensive para-aortic lymph node metastases without distant metastases to other organs.Using a novel pig model, we found that the IT administration of anti-cancer drugs may offer a significant pharmacokinetic advantage and has clinical merit for patients with extensive lymph node metastases (ELM), including para-aortic lymph node metastases from abdominal malignancies.