Lassa virus (LASV) cell entry is mediated by the interaction of the virus glycoprotein complex (GPC) with alpha-dystroglycan at the cell surface followed by binding to LAMP1 in late endosomes. However, LAMP1 is not absolutely required for LASV fusion, as this virus can infect LAMP1-deficient cells. Here, we used LASV GPC pseudoviruses, LASV virus-like particles and recombinant lymphocytic choriomeningitis virus expressing LASV GPC to investigate the role of human LAMP1 (hLAMP1) in LASV fusion with human and avian cells expressing a LAMP1 ortholog that does not support LASV entry. We employed a combination of single virus imaging and virus population-based fusion and infectivity assays to dissect the hLAMP1 requirement for initiation and completion of LASV fusion that culminates in the release of viral ribonucleoprotein into the cytoplasm. Unexpectedly, ectopic expression of hLAMP1 accelerated the kinetics of small fusion pore formation, but only modestly increased productive LASV fusion and infection of human and avian cells. To assess the effects of hLAMP1 in the absence of requisite endosomal host factors, we forced LASV fusion with the plasma membrane by applying low pH. Unlike the conventional LASV entry pathway, ectopic hLAMP1 expression dramatically promoted the initial and full dilation of pores formed through forced fusion at the plasma membrane. We further show that, while the soluble hLAMP1 ectodomain accelerates the kinetics of nascent pore formation, it fails to promote efficient pore dilation, suggesting the hLAMP1 transmembrane domain is involved in this late stage of LASV fusion. These findings reveal a previously unappreciated role of hLAMP1 in promoting dilation of LASV fusion pores, which is difficult to ascertain for endosomal fusion where several co-factors, such as bis(monoacylglycero)phosphate, likely regulate LASV entry. via fusion with acidic endosomes mediated by the viral glycoprotein complex (GPC) interaction with the intracellular receptor LAMP1. However, the requirement for LAMP1 is not absolute, as LASV can infect avian cells expressing a LAMP1 ortholog that does not interact with GPC. To delineate the role of LAMP1 in LASV entry, we developed assays to monitor the formation of nascent fusion pores, as well as their initial and complete dilation to sizes that allow productive infection of avian cells by LASV GPC pseudoviruses. This novel approach provided unprecedented details regarding the dynamics of LASV fusion pores and revealed that ectopic expression of human LAMP1 in avian cells leads to a marked acceleration of fusion but modestly increases the likelihood of complete pore dilation and infection. In contrast, human LAMP1 expression dramatically enhanced the propensity of nascent pores to fully enlarge when LASV fusion with the plasma membrane was forced by exposure to low pH. Thus, whereas the role of LAMP1 in LASV fusion is confounded by an interplay between multiple endosomal factors, the plasma membrane is a suitable target for mechanistic dissection of the roles of host factors in LASV entry.Lassa virus (LASV) enters cells  LASV is an Old World mammarenavirus that infects a broad host range of cells from different species. LASV cell entry is mediated by the viral surface glycoprotein complex (GPC), a trimeric class I fusion protein that consists of non-covalently associated surface GP1) and transmembrane GP2) subunits phosphate (BMP) . BMP speHere, we employed a combination of single LASV pseudovirus (LASVpp) tracking in live cells alongside bulk virus-cell fusion and infectivity assays to assess the effect of hLAMP1 on distinct steps of viral fusion. Pseudoviruses co-labeled with viral content marker and an internal pH sensor enable detection of single virus fusion events and monitoring the initial enlargement of fusion pores, whereas bulk fusion/infectivity assay report functional enlargement of fusion pores. We find that hLAMP1 overexpression in both human and avian cell lines moderately promotes fusion and infection through an endocytic entry pathway, whereas LASV GPC-mediated fusion at the cell surface forced by exposure to low pH was dramatically enhanced and accelerated in hLAMP1-expressing cells. Real-time imaging of forced LASVpp fusion with avian cells revealed a strong enhancement in fusion pore dilation by upon hLAMP1 expression. Our results thus provide new insights into the role of hLAMP1 in early and late stages of LASV fusion and reveal key differences in permissiveness of endosomes and the plasma membrane for LASV fusion.We used two cell lines to examine the role of LAMP1 in LASV fusion–human lung epithelial A549 cells endogenously expressing human LAMP1 (hLAMP1) and chicken DF-1 fibroblasts expressing the avian LAMP1 ortholog which does not support LASV entry , whereasConsistent with the requirement for hLAMP1, infection of DF-1 cells by HIV-1 particles pseudotyped with LASV GPC (LASVpp) measured by a single-cycle infectivity assay was ~100 fold lower than infection of A549 cells . To ruleNext, we examined the effect of ectopic hLAMP1 expression on the efficiency of LASVpp fusion with A549 and DF-1 cells using a direct virus-cell fusion assay that reports the delivery of virus-incorporated beta-lactamase (BlaM) into the cytoplasm ,15. DF-1A modest increase in LASVpp fusion and infection upon hLAMP1 expression indicates that endogenous levels of this receptor in A549 cells may be sufficient for near-optimal LASVpp entry. In contrast, the lack of a strong effect on viral fusion and infection of DF-1 cells was unexpected, suggesting that endosomal factors other than LAMP1 may facilitate LASV fusion in DF-1 cells.To probe the effects of hLAMP1 on LASVpp fusion with membranes that do not contain significant amounts of this intracellular receptor or other endosomal factors that may facilitate LASV entry and to ensure a more tractable system for viral fusion, we bypassed the internalization and endosomal trafficking steps by forcing LASVpp fusion with the plasma membrane through exposure to low pH. Ectopic expression of hLAMP1 dramatically enhanced the forced LASVpp fusion with both A549 and DF-1 cells, as measured by the BlaM assay Figs  and S2B.vs infection upon hLAMP1 expression in A549 cells might be due to less efficient post-fusion steps of infection following LASVpp entry at the plasma membrane compared to entry from endosomes in these but not DF-1 cells.Akin to the effect of hLAMP1 overexpression on the forced viral fusion , forced Given the strikingly potent enhancing effect of ectopic hLAMP1 expression on the forced LASVpp fusion, we sought to determine if such enhancement may be caused by a global, non-specific effect of hLAMP1 on the plasma membrane. Toward this goal, we measured the fusion of control pseudoviruses bearing the unrelated VSV G protein (VSVpp) with the parental and hLAMP1 expressing cells. As expected, ectopic expression of hLAMP1 did not considerably enhance VSVpp fusion through an endosomal or forced pathway . This fiIn order to validate the results obtained with HIV-1-based LASVpp, we measured the fusion of arenavirus virus-like particles (VLPs) bearing LASV GPC (referred to as LASV-VLP). VLPs were made by co-expressing the New World Junin arenavirus NP and Z proteins with LASV GPC, essentially as described in . To measFinally, we examined the effect of hLAMP1 expression on infection by recombinant LCMV viruses bearing LASV GPC (LCMV-LASV GPC) ,18. A549We next sought to determine which steps of viral fusion are facilitated by hLAMP1 expression by single virus tracking. LASVpp were labeled with the mCherry-2xCL-YFP-Vpr construct, which is incorporated into HIV-1 pseudoviruses and is cleaved by the viral protease upon virus maturation, producing free mCherry and YFP-Vpr . Loss ofUsing the aforementioned virus labeling strategy, we imaged single LASVpp fusion with DF-1 endosomes of cells expressing or lacking hLAMP1. LASVpp undergoes type II fusion with A549 cells . We alsin vitro  revealed a ~2-fold increase in the fusion rate with DF-1 cells expressing LAMP1-WT or LAMP1-d384 compared to control cells . The fasImportantly, hLAMP1 expression markedly shortened the lag between virus interior acidification (YFP quenching) and small pore formation (YFP dequenching) from ~18 min to ~8 min, on average . The larInset). Another approach to evaluate the effect of hLAMP1 on the propensity of fusion pores to enlarge is to assess the time required for nascent pore (detected by YFP dequenching) to dilate to sizes that allow mCherry release. “Instant” pore enlargement was defined as simultaneous  YFP dequenching and mCherry loss. The lag time to mCherry release was not significantly affected by hLAMP1 expression (Inset). These results show that ectopic expression of hLAMP1 does not noticeably promote the initial enlargement of fusion pores formed between LASVpp and endosomes.In DF-1 cells, we observed three single LASVpp fusion phenotypes–instant and delayed pore enlargement and stalled fusion,–irrespective of ectopic hLAMP1 expression . All thrpression , in excepression , Inset. We and others have previously shown that hLAMP1 overexpression allows LASV GPC-mediated fusion to occur at higher pH ,12. To cInset). These findings suggest that hLAMP1 promotes the initial dilation of nascent LASVpp fusion pores triggered at the cell surface to sizes that allow mCherry release. The overall fraction of single LASVpp fusion events associated with mCherry release (delayed or instantaneous) reached ~25% of all cell-bound particles for DF-1 cells expressing LAMP1-d384 .We further assessed the ability of hLAMP1 to promote fusion pore dilation by analyzing the lag time between YFP dequenching (nascent pore formation) and mCherry release as a metric for initial enlargement of fusion pores. hLAMP1 expression caused a highly significant decrease in the lag time between YFP-dequenching and mCherry release . The aveinsets to Figs The faster kinetics of fusion and significant reduction in the lag time between YFP dequenching and mCherry loss show that hLAMP1 accelerates the forced LASVpp fusion and promotes the initial enlargement of nascent pores. The hLAMP1 effect on small pore enlargement in these experiments contrasts with a non-significant effect on enlargement of LASVpp pores formed in endosomes . Note, hbona fide viral fusion events.To determine if hLAMP1 must be anchored to a cell membrane to facilitate LASVpp fusion, we expressed and purified the human LAMP1 ectodomain  in Expi293F cells  and asseSimilar to the effect of full-length hLAMP1 expression, the kinetic of forced fusion was markedly accelerated by sLAMP1 compared to the BSA control . HoweverInset). A shorter lag between YFP dequenching and mCherry release (vs ~60%) for forced fusion with sLAMP1-treated DF-1 cells compared to the full-length hLAMP1 expressing cells  indicates that the transmembrane domain of hLAMP1 may be required for efficient pore enlargement.Soluble LAMP1 did not significantly increase the overall extent of LASVpp fusion , but the release  further  release . The avells Figs , Insets The marginal effect of sLAMP1 on the initial dilation of forced fusion pores at the cell surface prompted us to test whether the soluble receptor promotes bulk LASVpp fusion, using the BlaM assay. Pseudoviruses were bound to DF-1 cells in the cold and their fusion with the plasma membrane was triggered by exposing to pH 5.0 in the presence of sLAMP1. Surprisingly, soluble LAMP1 modestly reduced the bulk fusion efficiency with mock-transduced DF-1 cells and had no effect on forced fusion with DF-1 cells expressing LAMP1-WT or LAMP1-d384 . This fiHere, we examined the role of human LAMP1 in LASV fusion with human and avian cells. A combination of single-virus tracking in live cells with functional assays monitoring productive virus fusion/infection enabled monitoring the evolution of fusion pores, from nascent pore opening to dilation to functionally relevant sizes. We have previously shown that single LASVpp undergoes type II fusion with endosomes of A549 cells, which entails viral membrane permeabilization  prior to fusion (YFP dequenching and release of the mCherry content marker) . Type IIUnexpectedly, ectopic expression of hLAMP1 has a relatively modest effect on LASV fusion/infection with endosomes across three platforms–LASVpp, LASV-VLP and recombinant LCMV/LASV-GPC viruses, even in avian cells expressing a LAMP1 ortholog that does not support LASV fusion. These results indicate that productive LASVpp fusion with endosomes may be controlled by additional endosomal host factors. Indeed, we have recently shown that the late endosome-resident lipid, bis(monoacylglycero)phosphate (BMP), greatly promotes the dilation of LASV GPC-mediated fusion pores .vs endosomes. Clearly, hLAMP1 plays a critical role in LASV fusion with the plasma membrane which likely lacks requisite endosomal co-factors that augment LASV fusion. As proposed above, the effect of hLAMP1 on LASV-endosome fusion may be masked by additional endosomal factors promoting productive LASVpp fusion and infection. Besides BMP, which we have shown to promote the enlargement of arenavirus GPC-mediated fusion pores  A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Sean P.J. WhelanAssociate EditorPLOS PathogensSusan RossSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Zhang et al. report an observational study of the role of LAMP1 binding to GPC during LASV entry, continuing an important body of work from the Melikyan laboratory. Previous work from this group and others has demonstrated that LAMP1 is not strictly required for LASV entry but enables entry earlier in the endocytic pathway. The mechanistic basis for this role of LAMP1 is accelerating LASV entry is unclear. By redirecting LASV entry to the plasma membrane through ectopic expression of LAMP1 and exposure of cells to acidic pH, they demonstrate a previously unknown role for LAMP1 binding to GPC in accelerating fusion pore expansion. Examining the role of LAMP1 during LASV entry at the plasma membrane enabled the authors to specify the contribution of LAMP1 in the absence of other endosomal factors that might impact LASV entry .The manuscript is very well written, and the data are clearly presented. The results reflect a meaningful contribution to the understanding of LASV entry and the role of the LAMP1 receptor. The manuscript also demonstrates the value of monitoring viral entry at the plasma membrane, although it might not be the natural site of entry. This approach will be of general interest to other groups engaged in the study of viruses that enter through the endosome.Reviewer #2: This excellent, very well controlled paper presents in-depth analysis of the mechanisms of membrane fusion mediated by the virus glycoprotein complex (GPC) of Lassa virus (LASV) and focuses on the role of LAMP1 that is not absolutely required for fusion and infection but rather regulates the timing and efficiency of viral entry. The authors use an exceptionally wide arsenal of state-of-the-art approaches and models to explore LAMP1 contributions in different fusion stages. Most importantly, the analysis of the single-virus fusion to endosomal and plasma membranes in live cells allows very sensitive detection of pre-fusion pores in viral envelope, nascent proton-permeable fusion pores and expanding fusion pores. Among many novel features of GPC-mediated features uncovered in this work, I am most intrigued by a much stronger LAMP1 dependency of fusion to plasma membrane vs. that to endosomal membrane and by the conclusion that LAMP1 influences more than one stage in the fusion pathway. The results of this exciting work will be of interest to researchers working on the cell entry mechanisms of diverse enveloped viruses. I have only minor suggestions/questions.Reviewer #3: In this study, Zhang et al. investigate the role of LAMP1 in supporting membrane fusion mediated by LASV GPC displayed on HIV pseudoparticles, LASV VLPs, and recombinant LASV. They employ a combination of assays measuring both bulk functional consequences of manipulations and single-particle kinetic tracking in live cells. They use two cells lines, a human cell line endogenously expressing LAMP1 that supports LASV fusion, and an avian cell line expressing a LAMP1 ortholog that does not support LASV fusion. They measure fusion in two distinct contexts, from within endosomes (endogenous pathway) or from the plasma membrane (forced pathway). To assess the contribution of LAMP1 on membrane fusion the authors overexpress WT human LAMP1 (hLAMP1) or a mutant version (d384) that preferentially traffics to the cell surface in either cell line. They discover a novel function for hLAMP1 in promoting fusion-pore dilation that might depend on the hLAMP1 transmembrane domain. This effect of hLAMP1 on LASV GPC fusion was only apparent or more pronounced on the plasma membrane .This study extends our view of viral membrane fusion dynamics and host factor roles at distinct stages of fusion. It is particularly exciting because the questions of fusion-pore expansion are difficult to dissect mechanistically - the authors overcome this hurdle by employing a very elegant assay that quantitatively reports on both small fusion-pore formation and its initial expansion, and by combining it with bulk functional assays. I look forward to seeing this work published in PloS Pathogens, but I have some major and minor points to be addressed before that point.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: I have no significant critiques to offer about this manuscript and support publication in its current form.Reviewer #2: I have only minor comments/suggestions for clarification.Reviewer #3: 1) It is not surprising that the detailed quantitative effects of different manipulations would have different outcomes for different virus systems or in different cell lines, and there are many possible reasons for that, but I find the early discussion of each of the results too speculative and distracting because it comes across inconsistent. I list some examples:a. The first sentences of the second results section give two distinct interpretations for the small effect of hLAMP1 expression on LASVpp entry in A549 vs. DF-1 cells. In the former case it is interpreted as indication of near optimal fusion, and in DF-1 cells as an indication of limited access of LASVpp to endosomal compartments. Can the efficiency of fusion for internalized particles be measured in the two contexts? Or alternatively, could the efficiency of internalization in the two cell lines be compared . Furthermore, it was not clear what was meant by the limited access until much later in the manuscript where it was explained that it refers to the efficiency of endocytosis. An alternative would be to move any speculation to the Discussion section.b. At the end of the second Results section the authors speculate on the reasons for the discordance between fold-enhancement of forced fusion vs. infection in hLAMP1 expressing A549 cells might be due to the less efficient infection following fusion at the plasma membrane. Why would the same effect not be observed for DF-1 cells?2) Most fusion and infection figures report only fold-changes. To better evaluate the data, it would be important to also include absolute values perhaps as a Supplement.3) From the presented data I am not convinced of the advantage of DF-1 cells over A549 cells for the detailed kinetic dissections of hLAMP1 effects in live-cell tracking experiments.a. The very similar relative effect of hLAMP1 expression on endosomal fusion by LASVpp in A549 and DF-1 cells is surprising even in the context of the speculated lower internalization efficiency. Even if a much smaller fraction of total particles are internalized into DF-1 vs. A549 endosomes, going from no hLAMP1 to about endogenous levels of hLAMP1 would be expected to have a much larger fold change than in the case of going from having hLAMP1 to having more of it. The relative change in the measured function should not be dependent on the efficiency of internalization (which does not change with hLAMP1 expression).b. Since there are no marked differences in the effects of hLAMP1 (over)expression in the two cell lines (and given the comment under ‘a’ above), I see no advantage of choosing DF-1 cells over the more native target. It would be important to corroborate the detailed mechanistic interpretations for the role of hLAMP1 in pore expansion in A549 cells. This would be helpful both in endosomes (same magnitude of effects in DF-1 and A549) and on the plasma membrane (since endogenous LAMP1 would not be present there just the same).4) Instant velocity plots are shown in various figures but not mentioned or described in the text.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: (No Response)Reviewer #2: 1) Can finding that hLAMP1 expression does not significantly alter the relative weights of the three types of fusion events: instant, delayed pore opening, no dilation  indicate that Lamp1 influences the stage(s) preceding the fusion pathway? For instance, can you exclude the effects of the LAMP1 expression at the surface of the cells beyond the relatively low endogenous levels of this receptor in A549 cells, Fig. 1C on cell surface binding of GPC carrying pseudoviruses?2) I am somewhat unclear on the relative effects of overexpression of WT LAMP1 that is almost absent at the plasma membrane and LAMP1 d384 that is mostly expressed at the plasma membranes. Can you please further discuss why in Fig. 2B WT LAMP1 and LAMP1 d384 similarly  promote fusion in endosomes and at the plasma membrane?3) I am intrigued by the finding that pseudoviruses exposed to acidic pH on the cell surface can still be internalized and fuse with neutral endosomes. Does this outcome (fusing in endosome rather than at the plasma membrane) correlate with how fast these virions are internalized? If fusion cannot happen faster than within certain time and if the virus happens to be internalized within this time, fusion will happen only in endosome.4) To distinguish direct effects of LAMP1 (as a receptor) from indirect effects such as domain organization in PM one may ask whether LAMP1 expression influences fusion for viral fusogens that do not utilize LAMP1 as a receptor, say for HIV Env?5) I suggest the sudden appearance of the term ‘type II fusion’ needs to be preceded by a brief explanation of the way you have classified single virus fusion events into Type 1 and 2 in Ref 20. Also is there any evidence to suggest that fusion is dependent on pre-fusion permeabilization? The authors show that LASVpp undergoes type II fusion independent of a target cell (A549 and DF-1 cells) and irrespective of whether fusion occurred in endosomes or at the plasma membrane. Can pre-fusion permeabilization be uncoupled from fusion in low pH-forced fusion model by using less acidic pH than pH 5.0.6) Fig. 2D is referred to before Fig. 2C.7) The sentence “We have previously shown that hLAMP1 overexpression allows LASV GPC-mediated fusion to occur at higher pH .” should be edited, since Ref. 10 is published by another group.Reviewer #3: 1) On page 11, second paragraph, it should be clearly stated that the single LASVpp fusion being imaged is endosomal fusion at that stage.2) Page 12, line 14, it is not clear which possibility is argued against.3) Page 14, line 13, it is commented that Fig. 6 shows efficient single LASpp fusion. It is not clear how a couple of particles report on efficiency.4) Please make clear in the text when soluble LAMP1 was added to virus particles relative to the attachment step of viruses to cells.5) Figure 2A legend, should explain that LASVpp are expressing luciferase and when luciferase signal was measured (perhaps move the intro sentences in part D to part A).6) Fig. S6 – why does LAMP1 have ladder-like appearance?7) In the discussion section, perhaps add more discussion of the endosomal factors that might be redundant to LAMP1.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at References:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 21 Jul 2022AttachmentLASV-LAMP1_PLoSPath_rebuttal(2).docxSubmitted filename: Click here for additional data file. 1 Aug 2022Dear Dr. Melikyan,We are pleased to inform you that your manuscript 'Human LAMP1 accelerates Lassa virus fusion and potently promotes fusion pore dilation upon forcing viral fusion with non-endosomal membrane' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Sean P.J. WhelanAssociate EditorPLOS PathogensSusan RossSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens <span style="font-family: "Noto Sans", sans-serif; font-size: 12px;"> orcid.org/0000-0002-7699-2064 </span> ***********************************************************Reviewer Comments : 9 Aug 2022Dear Dr. Melikyan,We are delighted to inform you that your manuscript, "Human LAMP1 accelerates Lassa virus fusion and potently promotes fusion pore dilation upon forcing viral fusion with non-endosomal membrane," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


Asthma is a chronic inflammatory disease primarily characterized by inflammation and reversible bronchoconstriction. It is currently one of the leading causes of morbidity and mortality in the world. Oxidative stress further complicates the pathology of the disease. The current treatment strategies for asthma mainly involve the use of anti-inflammatory agents and bronchodilators. However, long-term usage of such medications is associated with severe adverse effects and complications. Hence, there is an urgent need to develop newer, novel, and safe treatment modalities for the management of asthma. This has therefore prompted further investigations and detailed research to identify and develop novel therapeutic interventions from potent untapped resources. This review focuses on the significance of oxidative stressors that are primarily derived from both mitochondrial and non-mitochondrial sources in initiating the clinical features of asthma. The review also discusses the biological scavenging system of the body and factors that may lead to its malfunction which could result in altered states. Furthermore, the review provides a detailed insight into the therapeutic role of nutraceuticals as an effective strategy to attenuate the deleterious effects of oxidative stress and may be used in the mitigation of the cardinal features of bronchial asthma. Asthma is a multifaceted heterogenous chronic respiratory disease associated with various phenotypes and endotypes, sharing common characteristic features like inflammation and reversible airway obstruction  adrenergic agonists and anticholinergic agents that relax the airway smooth muscle to attenuate the bronchoconstriction, and anti-inflammatory drugs including corticosteroids and methylxanthines to suppress the airway-associated inflammation  affects every organ such as lungs and cardiovascular system , hydrogen peroxide (H2O2), and hydroxyl radicals (OH−) are mainly generated either due to mitochondria dysfunction or by the activation of the various enzyme oxidases including NADPH oxidase, P450 mono-oxygenase, cyclooxygenase, indolediamine dioxygenase, xanthine oxidase, and the Rho kinases , endogenous nitric oxide (NO), reactive nitrogen species of NO (RNS), and protection against these radicals by the endogenous biological defense system (Kirkham and Rahman 2−•) which is considered the major precursor that is mainly produced by the oxidative phosphorylation from the mitochondrial source or from the reduced nicotinamide adenine phosphate (NADPH) oxidation catalyzed by NADPH oxidases (Nox) , hydroxyl radical (HO•), singlet oxygen (1O2), peroxyl radical (HO2•), nitric oxide (•NO), peroxynitrite (ONOO-), perhydroxy radical (HO2•), hydroperoxyl radical (ROOH•), hypochlorous acid (HClO), ozone (O3), and nitric dioxide (NO2)   Barnes . TO miti) Barnes .2−) and radical hydroxylic ion (OH) due to their high instability with the presence of unpaired electrons that have the potential to initiate oxidation by several cellular substances including the lipids, proteins, and DNR to initiate the disruption of normal cell functions  and the generation of several lipid hydroperoxidase molecules in the cell membrane. LPOs generate reactive aldehydrogen formation and other bioactive molecules Fig.  Rahman . This re2 produces peroxynitrite ions (ONOO-), a highly reactive oxidant species that interacts with tyrosine to produce the stable product nitro tyrosine (R-SNO), which is known as S-nitro(syl)ation. NO2 is formed from NO, which is also reactive upon combining with HOCl (produced by MPO as described above) or with HOBr (from EPO) to produce nitrosyl chloride (NO2Cl) or nitrosyl bromide (NO2Br) which degrade into nitrite and Cl− or Br−  and NO-derived reactive nitrogen species (RNS) are another category of the free radicals which triggers the initiation of nitrosative stress in modulating the airway function in asthma pathogenesis . Despite the involvement in bioenergetics, mitochondria are also involved in maintaining the various cellular homeostasis functions including calcium regulation, alteration of redox potential, and release of protein that stimulates the caspase family of proteases  stress. Mitochondrial oxidative stress and ER stress further lead to cell apoptosis. Mitochondrial dysfunction plays a crucial role in the bioenergetics metabolism and non-energetics pathogenesis in several pulmonary diseases and the lack of mitochondrial homeostasis leads to cell injury and cell death. ROS activated by oxidative stress and inflammatory antigens are the major factors for increasing mitochondrial DNA (mtDNA) damage, dysregulation of the tricarboxylic acid (TCA) cycle, and dysregulation of the electron transport chain Reddy . FurtherAdhatoda vasica extract against hypoxia-induced mitochondrial dysfunction in acute allergic asthmatic mice. The results indicated that oral administration of Adhatoda vasica extract significantly attenuated IL-17A and hypoxia-inducible factor-1α (HIF-1α). The study showed that Adhatoda vasica extract produced anti-inflammatory effects and acted as an inflammatory marker which is responsible for mitochondrial dysfunctions  or attenuation of extracellular oxidants. It has also attenuated the higher level of lipid peroxidation induced by ONOO−. Quercetin prevented mitochondrial damage and had caused its redistribution to the cytosol by stimulation of plasma membrane oxidoreductases , while three components are present in the cytosol in a resting phase . The bonding proteins are also present in cytosol with the other components: rac1 or rac2, the two small molecular weight guanine nucleotide proteins. When cytosolic parts migrate to the cell membrane, NADPH oxidase is activated. The family NOX is gp91PHOX homologous and forms p22PHOX heterodimer except NOX5 and DOUX. As an additional EF intracellular Ca2+ binding hand domain, both NOX5 and DOUX1/DOUX2 are calcium-sensitive, whereas NOX1 to NOX3 are not directly calcium-dependent for activation -1, can be increased in normal human nasal epithelial cells  to produce superoxide , and directly cause bronchodilatation via calcium modulation . These micro- and innervated enzymes contribute to modulating the immune system and reduces the risk of various conditions in humans, including respiratory conditions , promote NO -4; Nox2B) and upregulated the antioxidant genes NADPH quinine oxidoreductase-1 (NQO1) and γ-glutamyl cysteine synthetase catalytic subunit (GCLC)   treatment to human bronchoepithelial cells where morin modulated oxidative stress responsive-MAPK pathway. The antioxidant activity of morin was shown by significant decrease in total cellular ROS and decreased phosphorylation of MAPK family protein   in BALB/c mice for the treatment of asthma. The study revealed that SKNEE attenuated the level of NF-κB and activated Nrf-2, HO-1 signaling pathways. The results indicated that SKNEE is a potential therapeutic agent for allergic airway inflammation , the subjects receiving two capsule of saffron (100 mg/day) for 8 weeks showed improvement in the frequency of clinical symptoms measured in terms of shortness of breath during the day and night time (frequency), use of standard asthma medicine , waking up at night due to asthma exacerbation, and limitation in physical activity  and 2 g/day (n = 26 patients) for 3 months. The effects were then compared with placebo control (n = 24 patients). Interestingly, forced expiratory volume (FEV) 25–75% and FEV1 (% predicted) was remarkably increased in N. sativa 2 g/day group while peak expiratory flow variability was improved in both 1 and 2 g/day groups as compared to placebo. In addition, fractional exhaled nitric oxide and (FENO) and serum IgE were reduced, and interferon gamma was increased after 3 months in both 1 and 2g/day groups. Furthermore, asthma control test score was improved drastically at 6 and 12 weeks suggesting N. sativa supplementation may improve set lung function parameters and airway inflammation in partly controlled asthma  with n = 32) were administered with a low antioxidant diet for 10 days before starting the randomized cross over trial  and tomato juice (45 mg lycopene/day) with 10 days washout period after each treatment. It was observed that low antioxidant diet was associated with worsening of asthma control score, reduction in %FEV (1) and %FVC. It also caused an increase in percent of sputum neutrophil. In contrast, both treatment groups (tomato juice and extract) decreased the influx of airway neutrophil. Furthermore, treatment with tomato extract also reduced sputum neutrophil elastase activity thus suggesting that dietary antioxidants such as lycopene may be beneficial in asthma management  and FENO decreased by 18% . This clinical trial highlights that dietary soy isoflavone supplementation is beneficial in the management of eosinophilic asthma (Kalhan et al. Another RCT studied if lycopene-rich supplement modified non-eosinophilic airway inflammation in asthma. Asthmatic subjects (It is clear from this extensive literature that oxidative stress and the imbalance between the oxidant and antioxidant systems play a major role in the initation and progression of the asthma disease. As the imbalance between the oxidant and antioxidant systems is not controlled by the endogenous antioxidant systems, it is therefore highly essential to identify the exogenous antioxidant which can restore the balance. Thus, identifying newer pharmacological agents that could restore the balance between the oxidant and antioxidant systems will become an alternate to the current therapy which are being used with limitations due to the poor response and adverse effects. Although various studies have reported on using the nutraceuticals as the alternative antioxidant therapy, which is quite promising in various in vitro and in vivo studies. Very few have been proven to be clinically effective in attenuating the disease due to poor bioavailability. Furthermore, extensive in-depth research is highly required to understand the new pathways in mediating the oxidative stress and at the same time identifying newer nutraceuticals with improved physiochemical properties which is essential in management of the asthma.


Withania somnifera, commonly known as Ashwagandha. Withaferin A has received great attention for its anticancer properties noted in cancer cells of various origins. Extracts of Withania somnifera have been used in traditional Ayurvedic and Unani Indian medicine for their various pharmacological benefits. In recent years, Withania somnifera or Ashwagandha extract has become popularized as a health supplement marketed for its stress and anxiety reducing effects. Withaferin A is one of the most studied withanolides extracted from Withania somnifera that has gained great attention for its anticancer, anti-inflammatory, metabolic, and pro-apoptotic effects. Extensive in vivo and in vitro studies have depicted Withaferin A’s interactions with key role players in cancerous activity of the cell to exert its pro-apoptotic effects. Withaferin A interactions with NF-κB, STAT, Hsp90, ER-α, p53, and TGF-β have noted inhibition in cancer cell proliferation and cell cycle arrest in G2/M stage, ultimately leading to apoptosis or cell death. This review highlights pro-apoptotic properties of Withaferin A including generation of reactive oxidative species, Par-4 activation, endoplasmic reticulum stress (ER) induction, and p53 activation. Analysis of Withaferin A’s involvement in various oncogenic pathways leading to malignant neoplasm and its pharmacologic activity in conjunction with various cancer drugs provides promising evidence in therapeutic potential of Withaferin A as a cancer treatment.Withaferin A is a C28 steroidal lactone derived from the plant  Withania somnifera has been receiving great attention due to its anticancer properties observed in various mice models and tumor cell studies. Research on the compound depicts strong potential in development of Withaferin A as a therapeutic for cancer treatment.Advances in science and medicine have dramatically impacted cancer treatments, increasing life expectancy of many individuals facing grim diagnosis of cancer. Despite medical advances, the disease still affects many individuals globally and is among the leading causes of death. Furthermore, drug resistance poses a serious problem that affects cancer cell survival and relapse. Therefore, development of potent therapeutics capable of impacting tumor progression and cancer cell proliferation is of crucial importance. Withaferin A, a phytochemical derived from plant Withania somnifera plant have been studied for therapeutic effects in cancers of various origin. The anticancer effects of the plant were determined in the study by Withania somnifera  and Withaferin A was identified as the active compound which inhibited cell proliferation , was determined as a result of computational analysis . WithafeWithania somnifera showing that 0.4585 mg/kg of Withaferin A was present  found improved bioavailability with this formulation of Withaferin A . The in aferin A . Anotheraferin A . Resultsaferin A . The same period .Withania somnifera in an in vitro absorption model system using canine kidney cell culture was performed  in Wistar rats (Withania somnifera extract (WSE) which were, control, 500, 1000, and 2000 mg/kg of body weight/day for a period of 28 days  and tumor associated macrophages (TAMs)  .in vivo studies relating Withaferin A have demonstrated that the compound suppressed growth of the cancer cells derived from human tumors and of the experimentally cancer induced rodent models (in vivo and in vitro (Withaferin A has been widely studied for its anti-cancer effects and multiple interactions contributing to its anti-cancer activity have been investigated. Many t models . Withafein vitro . Furtherin vitro . Furtherin vitro . WithafeEffects of Withaferin A treatment was studied in prostate cancer (PCA) cells utilizing SILAC-based proteomic approach where protein expression at 4 h and 24 h post exposure to Withaferin A was analyzed . UpregulEfficacy of Withaferin A has also been studied in cancer cells that are able to replicate by developing an Alternate mechanism of Lengthening of Telomeres (ALT) . Althougvia modulation of TGF-β signaling and by inhibiting TGF-β dependent Smad2 phosphorylation  is noted to contribute to Withaferin A induced apoptosis . This caoduction .Generation of ROS was noted in prostate cancer cells treated with 3-azido Withaferin A . FurtherAnticancer effects of low concentration of Withaferin A were investigated in oral cancer Ca9-22 cancer cells . Ca9-22 Studies with Withaferin A have also shown to induce apoptosis by activating the tumor suppressor protein, p53, leading to cell cycle arrest and cell death in cancer cells . ResultsWithaferin A treatment applied to different lung cancer cell lines revealed reduction in cell viability in a caspase-dependent manner and induced expression of p53 and Bax . The samvia the ATF4-ATF3-CHOP axis and initiate apoptosis  and in the androgen-insensitive prostate cancer cell lines by induction of endoplasmic reticulum (ER) stress. Withaferin A treatment of Caki cells led to induction of various ER stress markers which included phosphorylation of eukaryotic initiation factor 2a (eIF-2a), glucose-regulated protein (GRP) up regulation, and CAAT enhancer binding-protein homologous protein (CHOP) up regulation . Withafepoptosis .Induction of ER stress is noted in colorectal cancer (CRC) cells in a combination treatment of Withaferin A and 5-Fluorouracil resulting in autophagy and apoptosis was observed . Furthervia siRNAs lead to proliferation of cholangiocarcinoma cells in culture ; these pathways are often found active in human cancers . Withafevia up-regulation of Bim and Bad proteins   . Effectson (EMT) . Withafeon (EMT) .The estrogen receptor α (ER-α) is heavily studied in cancers of various origin due to its role in cell proliferation and disease progression. ER-α plays a crucial role in driving breast and uterine cancers and is expressed in greater than 70% of ER + breast tumors . In MCF-The transcription factor NF-κB plays a crucial role in controlling expression of regulatory genes for cell proliferation, cell death, and immunity . Unregulvia inhibiting activity of vascular endothelial growth factor (VEGF), a key regulator of angiogenesis. In silico molecular docking studies show favorable binding of Withaferin A with VEGF; results are comparable with Bevacizumab, an approved anti-cancer treatment  cells where the combination treatment of the two substances leads to antiproliferation of the cells and induces endoplasmic reticulum (ER) stress, resulting in apoptosis and autophagy . The antobserved . The effobserved .A recent study investigated the potential of Withaferin A as a therapeutic for cancer treatment by incorporating the compound into nanosponges (NS) which serve to transport the compound to the target cell . SRB assin vivo tumor study utilizing LWA treatment, such as, PEGylated nanoliposome encapsulating Withaferin A, revealed reduction in tumor growth and improvement in survival for Dalton Lymphoma Ascites (DLA) tumor bearing mice; it was also revealed that LWA treatment reduced tumor growth by regulating expression of Ki-67 and cyclin D1 protein (in vitro (The  protein . Furtherin vitro . This dein vitro .Structural modifications to Withaferin A resulting in various derivatives have also been assessed for cytotoxicity against various cancer cells and demonstrate significant potential in development as a therapeutic for cancer. The 3-azido analogue of Withaferin A has exhibited 35-fold increase in cytotoxicity compared to its unmodified parent molecule . In prosin vitro and in vivo investigations have depicted promising outcomes for the compound’s anticancer effects when administered alone or in combination with other available therapeutics. Studies have shown anticancer effects such as, apoptosis, ROS generation, cell cycle arrest, etc. in cancers of various origins. Despite these results, an effective minimum dosage, toxicity, and bioavailability of the compound is yet to be thoroughly evaluated. Current studies depict low bioavailability of Withaferin A which poses a barrier in determining minimal dosage to have therapeutic effect on cancerous cells. However, efforts are also being made to overcome this barrier by studying various modes of administering Withaferin A and developing transporter methods that can lead to greater bioavailability of the compound such as use of nanosponges loaded with the biomolecule or development of PEGylated nanoliposomes. Additionally, delivery of Withaferin A using novel drug delivery approaches must be considered due to its low bioavailability. There is also a need to further evaluate adverse effects of Withaferin A in various populations such as immunocompromised, pregnant, or geriatric. Structural changes to Withaferin A and development of effective analogues that produce significantly potent effects may pose another opportunity for the molecule to be developed as a therapeutic agent. Although several studies have been conducted on Withaferin A and its interactions with various role players in oncogenic activity of the cell, further evaluation of Withaferin A’s activity must be made to develop the compound as an effective therapeutic.Withaferin A is being widely studied for its numerous benefits, a major one is its potential as a therapeutic for cancer treatment. Multiple


Despite minimal differences in transmission efficiency of the sGD or fGD drive elements in single generation crosses, the reconstituted spo11 fGD cassette surprisingly exhibits slower initial drive kinetics than the unlinked sGD element in multigenerational cage studies, but then eventually catches up to achieve a similar level of final introduction. These unexpected kinetic behaviors most likely reflect differing transient fitness costs associated with individuals co-inheriting Cas9 and gRNA transgenes during the drive process.The core components of CRISPR-based gene drives, Cas9 and guide RNA (gRNA), either can be linked within a self-contained single cassette  or be provided in two separate elements , the latter offering greater control options. We previously engineered split systems that could be converted genetically into autonomous full drives. Here, we examine such dual systems inserted at the  CRISPR-based gene-drives can carry the Cas9 and guide RNA (gRNA) components in a single-linked cassette or in separate elements inserted into different genomic loci. Here the authors genetically transform and compare full versus split drives, with the former performing less efficiently than predicted. Linked gene drives  carry gene cassettes encoding the bacterial Cas9 endonuclease and a guide RNA (gRNA) sequence that directs the Cas9/gRNA ribonucleoprotein complex to cleave the genome at the site of cassette insertion5. Upon cleavage of the target site on a homologous chromosome, the gene-drive element is copied into the double stranded break (DSB) by homology-directed repair (HDR) using the drive-bearing homologous chromosome as a repair template. If this directional gene conversion process is efficient, the drive allele will be inherited in a super-Mendelian fashion (>50%). Genes encoding beneficial factors can be linked to fGD cassettes to spread these traits into susceptible populations within a few generations3, following a trajectory defined by logistical  growth kinetics4.CRISPR-based gene drives offer novel approaches for vector control by altering the genetic structures of wild insect populations7 carry beneficial cargo to reduce the vector capacity of the insect, while suppression drives9 endeavor to bias the inheritance of deleterious traits that will ultimately either kill or sterilize the insect. In addition to autonomously acting fGDs in which Cas9 and gRNA are encoded within a unitary cassette, so-called split gene drives (sGD) have been developed12 in which the copying cassette carries only the gRNA component, with Cas9 supplied from a second genomic site. In such bipartite arrangements, the gRNA-carrying cassette is copied in the presence of the Cas9 source while the Cas9-encoding element remains static . In the absence of Cas9, however, the gRNA-bearing split-drive element also is inherited at Mendelian frequencies. When at low frequencies, sGDs are predicted to spread additively through populations rather than exponentially, as do fGDs4.Gene-drive technologies can be used either to modify or suppress insect disease vector populations, depending on their design. Modification drives12. Such recoded sGDs minimize the production of nonfunctional NHEJ alleles that are resistant to Cas9 cleavage by dominantly eliminating such alleles transmitted by females through a phenomenon referred to as sterile/lethal mosaicism13. This latter process arises as a consequence of maternal inheritance of Cas9/gRNA complexes that act on the paternal allele to mutate it in a sufficient fraction of cells in progeny to either kill  or sterilize (sterile mosaicism) such offspring.Our group recently analyzed a set of sGDs inserted into essential loci carrying recoded cDNA sequences that restore functionality of the endogenous genes upon allelic cleavage and conversion12, the sGD constructs included another design feature that permits facile genetic transfiguration of the sGD into an fGD. This conversion system relies on a set of Cas9 sources that carry sGD homology arms flanking the Cas9 cassette and a gRNA (gRNAHack) that can cleave a synthetic target site placed within the sGD element. When such Mendelian Cas9 sources (inserted into AttP sites in different genomic locations) are crossed to the sGD element, the gRNAHack mediates cleavage of the sGD resulting in the insertion of the Cas9 cargo into the recipient element. Here, we provide proof-of-concept for such conversion of a recoded split drive into a full drive inserted into the spo11 locus, which is required for fertility in both sexes. This strategy for converting a sGD into a fGD follows the logic of the homology-assisted CRISPR knock-in system (or HACK)14, which efficiently replaces target loci in Drosophila. We evaluate the performance of a converted (or hacked) fGD in both single-generation crosses and cage competition experiments and compare its performance to that of the sGD from the previous experiments12. Surprisingly, instead of spreading rapidly as predicted, we find that the fGD displays slower initial drive kinetics than the sGD, although it eventually catches up and achieves a similar level of introduction into the population. The delayed drive of the fGD element may result from elevated transient fitness costs associated with co-inheritance of Cas9 and gRNA transgenes in the presence of cleavage-sensitive target alleles that exceed those produced by the sGD configuration12.Although unexploited in the previous studyspo11 sGD line12 that copied efficiently to the homologous chromosome in females (76–83%) but to a lesser degree in males (65–69%) in single-generation crosses. This spo11 sGD drove to a high level of stable introduction in multigenerational cage experiments (~85%). Because the spo11 sGD drive performed well but not perfectly, we selected it to test for conversion to an fGD by hacking -driven Cas9 transgenes were placed between a 3′ partial nonfunctional fragment of a 3xP3-tdTomato (expressed in the eye) marker and a fully active Opie2-EGFP transgene (expressed in the abdomen). These flanking sequences act as homology arms (HA) adjacent to a gRNA cleavage site located in the sGD element . The sGD carries a synthetic PAM site (hackPAM) between the two marker sequences that is unique to those constructs. One of the marker transgenes encodes an intact fluorescent protein (tdTomato) and is active in the sGD line, whereas the other marker gene (EGFP) consists only of partial 3′ sequences and is inactive. The hackPAM provides a synthetic recognition site for the hacking gRNA carried outside of the homology arms on the Cas9-donor (Cas9Hack) element  and autonomous self-mobilizing capacity as a composite fGD element. Donor Cas9Hack transgenes carrying the gRNAHack components were inserted into available fly strains containing AttP sites located either on chromosome II or chromosome III via phiC31 recombination15 . We observed comparable allelic conversion efficiencies for CCvasa using either the Mendelian sources (vCas9-III and nCas9-II) or Cas9Hack transgenes when expressed using the corresponding promoters  carried by the Cas9Hack sources had no target in these specific crosses and did not affect CCvasa copying efficiencies. As expected based on the static nature of the tested Cas9 sources, inheritance of the Cas9 transgenes in all cases approximated Mendelian frequencies  to express Cas9.We used two previously validated autosomal static Cas9 sources (nCas9-II and vCas9-III), inserted into chromosomes II and III, respectively, as reference controls for assessing the activity of Cas9Hack sources controlled by the same promoters as the Hack lines (vHack and nHack). These lines carry the same regulatory sequences to Cas9 as the static sources but can mediate hacking events by gRNAHack when coupled with sGD lines Fig. . We firs spo11 sGD. We crossed the hackable spo11 sGD with both vHack-A and nHack-A to obtain spo11 fGDs  using PCR amplification followed by Sanger sequencing. In contrast to recovery of these multiple independent hacking events when employing a Cas9Hack insertion near the insertion site of the spo11 sGD, an identical vHack construct inserted at a different III chromosome AttP site , which efficiently sustained hacking of the closely linked spo11 sGD, did not support conversion of the more distantly residing rab5 sGD element in preliminary experiments (2L).Having confirmed the expected performance of the Cas9Hack lines, we next used these lines to generate fGD versions of theGDs Fig. . Of the spo11 fGDs carrying either vasa-Cas9 or nos-Cas9, we next compared their allelic conversion rates to those obtained with the bipartite spo11 sGD system. We crossed G0 homozygote spo11 fGD males to homozygous WT virgin females followed by mating heterozygote F1 siblings  to their WT counterparts and then scored the fraction of fluorescently labeled F2 offspring  was greater than through males  ; nCas9-II = 53 ± 9% (F), 52 ± 6% (M)), whereas Cas9 remained integrally linked to the spo11 element in the fGD  Fig. , we dete0%) Fig.  were slispo11 fGD transgene by profiling the range of different NHEJ alleles generated in single-pair crosses  F2 offspring derived from independent crosses of an F1 trans-heterozygote female with a WT male. Following amplification and sequencing of the target site in single fGD− F2 individuals, we estimated the frequencies of NHEJ-induced alleles occurring in F1 trans-heterozygous mothers by subtracting the paternal WT allele from the sequencing reads. The analysis of fGD− flies revealed that 28% of the individuals inherited WT sequences, while 33% carried in-frame indels, and 39% had out-of-frame mutations  to 75% WT (+/+) individuals  and randomly-selected individuals were transferred at each generation for 20 generations  during which the fGD increased only gradually in frequency without achieving the higher predicted levels of final introduction. Based on the modeling summarized above, we had expected that forcing linkage between Cas9 and gRNA would lead to a heightened performance by the fGD relative to sGD in cage trials, since any fGD+ individual should be able to mediate cleavage and allelic conversion of WT alleles while the sGD+ drive would require being paired with a separate vCas9 transgene to produce drive. Moreover, the sGD when paired with Cas9 could transmit NHEJ-inducing maternal Cas9/gRNA complexes to offspring that did not inherit both the Cas9 and gRNA transgenes (75% of progeny), as compared to only 50% for a linked fGD that failed to copy. This differential maternal mutagenic effect is predicted to be further amplified due to the slower intrinsic drive kinetics of the sGD relative to the fGD. Despite the initial dip in fGD frequencies, fGD+ alleles increased to a stable plateau level in all cage replicates  despite the divergence in intermediate generations. We sequenced non-converted fGD− individuals that were present at four different timepoints: G2, G4, G8, and G20  by the fGD compared to the sGD. As mentioned above, the variety of NHEJ alleles decreased over time leveling out by G8 and remaining stable through G20 . Consistent with the disappearance of convertible WT alleles by G8, the fGD+ exhibited only a minimal increase in frequency between G8 and G20.We selected the split combination of vCas9-III + sGD for further comparison in multigenerational cage trials. We reasoned that these experiments should provide insights regarding whether forced linkage of vCas9 to the gRNA within the same fGD cassette would accelerate its drive dynamics or increase its final degree of introduction as predicted based on the different inherent drive mechanisms acting in the two systemsons Fig. . SurprisG20 Fig.  to captules Fig.  was a gr− flies .Building on the aforementioned mathematical models, we extended our analysis to the fGD by assessing two potential fitness cost implementations: (1) an active cost associated with ascending phase of the drive trajectory, where fitness costs due to Cas9/gRNA were only apparent during active cleavage events, and (2) a co-occurrence cost, where the fitness costs manifest whenever an individual has Cas9 and gRNA, regardless of active cleavage. Drive performance was estimated from respective cage trial data using a Naive–Bayes Multi-Objective Hidden Markov Model optimized with an evolutionary algorithm  and not a second class .Lastly, we assessed the capacity of predicting fGD trends based on sGD performance. To do so, we applied both our models to a range of possibilities in the two main components of the model: conversion rate (CR) and fitness costs (FC)  element targeting a locus (spo11) essential for fertility in both sexes into a full gene-drive (spo11 fGD) and compared these well-matched systems in both single-generation crosses and multigenerational cage experiments. We observed, and mathematically modeled an unexpected delay in drive of the fGD based on simple predictions derived from analysis of sGD systems and a reduced level of final introduction for the fGD that was nearly equal to that of the sGD instead of being higher as predicted by the modeling.In this study, we genetically converted a split gene-drive , Cas9 is forcibly linked to the gRNA component in the fGD and thus a higher percentage of individuals incur costs associated with co-inheritance of these CRISPR components. This effect is particularly pronounced in early generations when the great majority of non-transgenic individuals will be WT homozygous and thus have fitness advantage over the transgenic line early during the drive process. In the ascending phase of the drive, along with allelic conversion events, WT alleles can be mutated to generate transiently acting dominant mosaic phenotypes12. Fitness costs associated with co-inheritance of gRNA and Cas9 transgenes are thus confined to the active drive phase.Although the sGD and fGD performed similarly in single-generation crosses, we observed notable unexpected differences in their performance in multigenerational cage trials. The vfGD spread through a naïve population following initial introduction at low levels to ~80% of the population in 15 generations, but did so with delayed drive kinetics compared to the 3 and G7. By G8, WT alleles were no longer detected in fGD cages in contrast to the sGD, where such unmodified alleles persisted for longer. Presumably, Cas9 that is obligately produced by the fGD allele either successfully converted all WT alleles by conversion to the fGD+ state or mutated them to cleavage-resistant and largely functional NHEJ alleles, thus resulting in the fGD attaining stable levels of introduction. Despite its initial rise, the fGD did not achieve the predicted levels of introduction most likely due to the accumulation of functional NHEJ alleles. While the fGD generated NHEJ alleles  at comparable frequencies to its sGD counterpart . Thus, it is a quite an unexpected result that the recoded spo11 fGD in this study did not perform as modeled based on drive parameters extracted from the sGD in Supplementary Fig. 4. These fundamental mathematical differences in drive kinetics are well captured by our current model and predict rapid increases in the frequency of the fGD but only gradual asymptotic increases in the sGD. Moreover, as mentioned above, both can produce transgenerational lethal mosaicism even in the absence of inheritance of the CRISPR components. Therefore, the sGD is expected to generate NHEJ alleles more frequently than the fGD when only one or neither of the CRISPR components are inherited by progeny. Moreover, this greater frequency of non-productive maternal transmission of mutagenic Cas9/gRNA complexes for the sGD versus the fGD is further compounded by the inherently slower drive trajectory of the sGD relative to the fGD, which provides yet more opportunities for creating such NHEJ alleles. Thus, an sGD is predicted to generate considerably higher frequencies of NHEJ alleles than the corresponding fGD, which differs from what we observed experimentally .Importantly, as indicated in Supplementary Fig. spo11 fGD cannot be simply predicted from drive parameters extracted from the sGD. In particular, modeling suggests that the fGD incurs a substantially greater fitness cost during the drive phase when many wildtype alleles still remain in the population. These costs may be related to accumulating levels of maternal Cas9/gRNA complexes associated with maintaining fGD stocks for multiple generations or may reflect some other feature of the full versus split-drive configurations that pertains to the active drive phase. Note that the fGD remains stable in the population once attaining full introduction and thus does not appear to generate any additional generic co-occurrence fitness costs . Since the fGD increases only gradually over five generations (G4–9), one can estimate that the drive-dependent fitness cost must nearly balance the exponential drive of the element during this period. One potential contribution to this fGD lag phase could be yet greater fitness costs incurred by WT individuals inheriting Cas9/gRNA complexes from mothers homozygous for the drive element. Consistent with fGDs providing elevated levels of maternal Cas9/gRNA complexes, fGD produced a higher fraction of nonfunctional frameshift mutations than the sGD, which may reflect differing levels of Cas9 activity. While the theoretical mechanisms behind full and split drives are now fairly well understood, and the dominant interactions properly reflected in the models, higher-order impacts are not captured in these simulations. Detailed as these models are, the discrepancies suggest that yet more fine-scale models may be required to capture biochemical differences in Cas9/gRNA complexes: different categories of fitness costs (active drive versus co-occurrence) in the progeny of Cas9/gRNA-bearing males versus females or parents versus grandparents (or even great-grandparents), DNA accessibility during different developmental stages, and repair mechanisms available when different promoters are active. Modeling and analysis performed in this study, of similar or even greater complexity, is rapidly becoming requisite in the gene-drive toolbox, both for the analysis of experimental data22 and simulation of potential results to inform conservation23 and disease-elimination campaigns24. In particular, we demonstrate the application of a Naive–Bayes multi-observation HMM that optimizes the regular HMM in a parallel framework by incorporating phenotypic and genotypic data.Mathematical modeling confirmed that many of the inferred drive parameters are similar but not identical for the sGD and fGD population cage experiments. Modest deviations between the data and model regarding the degree of final sGD versus fGD introduction may reflect assumptions that the NHEJ allelic profile and the dynamics of their elimination were the same in both fGD and sGD scenarios. However, as mentioned above, in early generations of fGD cages and corresponding to the period of drive delay, nonfunctional NHEJ alleles were simplified and cleared more quickly than for the sGD. Also, WT alleles persisted longer in the sGD cages, perhaps providing less time to winnow deleterious alleles from the population Fig. . Thus, aspo11 alleles remain.In summary, this study provides a direct comparison between split and full-drive configurations in a defined chromosomal locus and validation of a general method for efficiently converting one to the other by genetic crossing. We also provide proof-of-principle for a flexible system to seamlessly convert sGDs into linked fGDs inserted into exactly the same chromosomal location using the same molecular components. Only modest differences in performance were observed between sGD and fGD in single-generation crosses. Given that the fGD is always linked to Cas9, we had expected based on modeling that it should drive more rapidly and completely than the sGD, since the element does not drive when separated from Cas9. Surprisingly, however, we observed the opposite result in multigenerational crosses where fGD drive initially exhibited a transient delay relative to the sGD before achieving comparable levels of final introduction. Our analysis suggests that this unexpected early stalling of the fGD reflects a transiently acting dominant process of sterile mosaicism in which individuals inheriting the drive element and a WT allele or strong sterile phenotypes incur a fitness cost while WT alleles are still present in the population. This temporary penalty resulting from obligate co-inheritance of Cas9 and gRNA transgenes does not prevent the fGD from reaching comparable stable levels of final introgression and vanishes once the drive has attained final levels of introduction and no WT or nonfunctional Both split and full-drive systems offer potential advantages for field applications. The split system provides potential benefits for more localized and controllable spread of the CRISPR components, while full gene drives could be implemented in contexts where long-lasting protection conferred by associated effector genes and conversion of potential incoming WT alleles may be desired. The efficient and versatile hacking strategy we describe here can be employed to optimize the performance of key components in split configurations and then convert such systems into full drives. An important lesson of this study, however, is that the final performance of the fGD needs to be empirically determined, as biochemical differences limit the predictive value of sGD models applied to an fGD linked configuration. The seamless mobilization of Cas9 into the sGD provides a range of experimental advantages, in particular for species where transgenesis is extremely laborious and time intensive.We have complied with all relevant ethical regulations for animal testing and research and conformed to the UCSD Biosafety committee-approved biological use authorization.E. coli , isolated and sequenced. Primer sequences used for the creation of the different Cas9Hack plasmids can be found in the Supplementary File. spo11 and rab5 sGD plasmid construction has been described previously12. Recoded cDNA fragments were designed by using non-sub-optimal alternative codons from CRISPR cut site to gene’s stop codon and synthesized as gBlocks™ (Integrated DNA Technologies). Codon usage frequencies were kept as similar as possible to that of the endogenous sequence.All plasmids were cloned using standard recombinant DNA techniques. Plasmid and genomic DNA sequences were amplified using Q5 Hotstart Master Mix  and Gibson assembled with NEBuilder HiFi DNA Assembly Master Mix . The resulting plasmids were transformed into NEB DH5-alpha chemically competent http://www.rainbowgene.com). Each Cas9Hack construct was injected into AttP-harboring lines expressing integrase in the X chromosome (Bloomington #R8621 and #R8622). Injected embryos were received as G0 larvae, and allowed to emerge and 3–4 females were intercrossed to 3–4 males. G1 progeny were screened for positive transgene marker (green body). All transgenic flies that displayed the marker were then balanced using Sco/CyO  or TM3/TM6  and kept on a w1118 background. Homozygous stocks were maintained in absence of any balancer alleles or markers associated to the initial inserted line. Correct and complete transgene insertions in homozygous stocks were validated through PCR amplification and Sanger sequencing.Plasmids were purified using the PureLink Fast Low-endotoxin Maxi Plasmid Purification kit . All plasmids were sequenced prior to injection. Embryo injections were carried out at Rainbow Transgenic Flies, Inc.  and Cas9 (~50%) were calculated using the resulting F2 progeny by scoring the phenotypic markers associated to each transgenic cassette. Hacking efficiency experiments were performed by crossing sGD/Cas9 heterozygotes to the opposite gender carrying a balancer and detecting their progeny displaying double-marked phenotype over that balancer  flies were carried out to obtain F1 fGD+/fGD− heterozygotes, which were used to seed the initial generation. Wildtype or heterozygous males and virgin females were collected and separately matured for 3–5 days. Cages were seeded at a phenotypic frequency of 25% fGD+ heterozygotes  to 75% fGD− . Upon transfer into fresh bottles for each generation, flies were allowed to mate and lay eggs for 3 days, then were removed from the cage (Gn) and bottles were kept for 10 days to allow for the subsequent generation to develop to adulthood. Adult progeny (Gn+1) was randomly separated into two pools and scored; one pool collected for sequencing analyses while the other was used to seed the following generation. If the two pools differed much phenotypically, frequencies were averaged in order to reduce variability and stochastic extremes. Continuous sampling and passage were carried out for 20 generations.All population cage experiments were conducted at 25 °C with a 12-h day–night cycle using 250 ml bottles containing standard cornmeal medium. Crosses between homozygous fGD2, G4, G8, and G20). Target sequences were amplified using specific gene primers that also contained adapter sequences. Non-fragmented amplicons were sequenced using Illumina-based technology , with final data being delivered as FASTQ reads and aligned to a reference sequence for each gene of interest to detect indel formation. Primer sequences used for either single fly or cage trial deep-sequencing analyses can be found in the Supplementary File.To extract fly genomic DNA for single fly resistant allele sequence analysis, single flies were squashed in lysis solution , incubated at 37 °C for 30 min and deactivated at 95 °C for 2 min. After extraction, each sample was diluted in water and stored at −20 °C if needed. 1–2 µl of each diluted DNA extraction was used as a template for a 25 µl PCR reaction that covered the flanking regions of the gRNA cut site, which were used to sequence the alleles. Sanger sequencing in individual non-fluorescent flies was performed at Genewiz, Inc. in San Diego, CA to obtain the single-cross NHEJ data. NHEJ allele sequences were obtained from Sanger chromatographs by isolating the WT sequence first and then annotating the remaining allelic sequence. For cage trials, 20–25 non-fluorescent flies were pooled and their DNA extracted at each sampling generation  design allowed active cleavage of the target chromosome and the possibility of super-Mendelian inheritance. When cleavage occurred, a fraction of the cut alleles could be properly repaired via HDR, and the remaining cut alleles underwent NHEJ repair, generating in or out-of-frame resistant alleles. The effects of shadow-drive17, in which Cas9 protein is deposited in the embryo of a female individual who does not carry the Cas9 allele, but whose own mother does, were also accommodated. Fitness costs were implemented as fractional reductions in male and female fecundity, consistent with spo11 activity, and tested under active cleavage conditions as well as co-occurrence of Cas9 and gRNAs. Additional details on the model implementation and likelihood function used in sGD and fGD model fitting can be found in the Supplementary Information. Phenotype and genotype mappings are provided in Supplementary Tables Model fitting was performed using a discrete-generation adaptation of the Mosquito Gene Drive Explorer (MGDrivE)26.Simulated model trajectories for Fig. Initial graphs were generated using Prism 9  and modified using Adobe Illustrator  to visually fit the rest of the non-data figures featured in the paper. Supplementary Figs. spo11 fGD lines) was conducted in plastic disposable vials in an ACL-2 facility, in accordance with the Institutional Biosafety Committee-approved protocol from the University of California San Diego. All vials were frozen for 48 h prior to autoclaving and discarding the flies. ACL-2 samples were also frozen for at least 48 h before their removal from the contained facility.All research involving non-hackable gRNA cassettes/split gene drives was performed in glass vials in an ACL-1 facility, whereas research involving full gene drives (Further information on research design is available in the Supplementary Information FileReporting Summary


The insertion of a ureteral access sheath (UAS) is a frequent procedure during flexible ureteroscopy (fURS) to facilitate kidney stone treatment. The aim of this study was to investigate the influence of 12/14 French (F) UAS on fURS outcomes.We performed a retrospective monocentric analysis of fURS procedures conducted at the Department of Urology  for kidney stone treatment via lithotripsy or basket stone retrieval between September 2013 and June 2017. Uni- and multivariate analyses were done with the help of RStudio (Version 1.0.136) software.p < 0.05). UAS usage correlated with elevated radiation exposure in seconds , prolonged operation time in minutes , length of hospital stay over 48 h , more frequent postoperative systemic inflammatory response syndrome  and lower postoperative stone-free rates . Moreover, we conducted uni- and multivariate subgroup analysis for cases with multiple kidney stones (≥ 2) and comparable stone burden; UAS was inserted in 48.3% of these cases (71/147). On multivariate logistic regression, UAS insertion was statistically associated with prolonged operation time in minutes . No statistical differences regarding radiation exposure, stone-free rates, postoperative SIRS rates or LOS were noted between UAS and non-UAS patients with multiple kidney stones of similar size (p > 0.05).In total, 283 consecutive fURS were analyzed. UAS was applied in 98 cases (34.63%). The insertion of UAS was preferred in cases with multiple kidney stones and larger median maximal stone diameter (12/14F UAS does not seem to improve overall outcomes in fURS for kidney stones. In patients with multiple kidney stones it may be associated with elevated operation time without a clear benefit in terms of improved stone-free status or reduced perioperative complication rate. Further prospective randomized studies to specify the indications for UAS usage are urgently needed. The insertion of a ureteral access sheath (UAS) is a frequent procedure during flexible ureteroscopy (fURS) for kidney stone treatment . DecreasA retrospective monocentric analysis of fURS procedures was performed, conducted at the Department of Urology  for kidney stone treatment via lithotripsy or basket stone retrieval between September 2013 and June 2017.The details of the fURS procedure have already been described in our previous manuscripts , 14. TheHolmium laserlithotripsy (Lumenis VersaPulse® PowerSuite™ 100 W) was performed with a reusable SlimLine™ 200 μm fiber (Boston Scientific). Total laser energy was defined as the cumulative lithotripsy energy via the semirigid and flexible ureteroscope. Calculi extraction during fURS was conducted with Stonizer® tipless  or NGage®  nitinol baskets. A ureteral double-J stent was reinserted depending on the complexity of fURS and significance of ureteral lesions.Stone-free status (SFS) was determined intraoperatively by the endourologist with the assistance of intraoperative fluoroscopy. Radiological postoperative re-evaluation with computed tomography (CT) or delayed kidney, ureter and bladder (KUB) radiography was always performed in case of uncertainty regarding SFS. The length of hospital stay (LOS) for a routine postoperative course was limited to 48 postoperative hours. Every patient with at least one stone episode in the past was categorized as a recurrent stone former.The determination of the infundibulopelvic angle (IPA) was based on preoperative native CT images in the coronal plane and intraoperative retrograde pyelography (RPG) images. The IPA was measured in accordance with the El-Bahnasy definition, as the angle between the ureteropelvic axis and the central axis of the lower pole of the infundibulum . CT-base3) were fulfilled in patients assigned to postoperative SIRS group  and larger median maximal stone diameter . Detailed results of univariate testing are presented in Table p < 0.0001], prolonged operation time , length of hospital stay , more frequent postoperative SIRS  and lower rate of postoperative SFS . Reported relevant complications were defined as at least Clavien-Dindo Grade 2 (CD ≥ 2) events. In the UAS group, these included one intraoperative bleeding with postoperative SIRS, one ureteral perforation with postoperative SIRS, and eleven postoperative SIRS without intraoperative complications. In the non-UAS group, we recorded two intraoperative perforations of the calyceal system, one ureteral perforation, eight postoperative SIRS without intraoperative complications, one relevant postoperative gross hematuria, and one aspiration pneumonia.In total, 283 consecutive fURS for stone surgery performed between September 2013 and June 2017 were analyzed. 12/14F UAS was applied in 98 cases (34.63%). On univariate analysis, the insertion of 12/14F UAS was preferred in cases with multiple kidney stones . No differences regarding radiation exposure, SFR, postoperative SIRS rates and LOS were noted between UAS and non-UAS cases with multiple kidney stones of similar size (p > 0.05). The results of the multivariate binary logistic regression for this subgroup are presented in Table In order to reduce bias due to heterogenic cohorts demonstrated in aforementioned overall analysis, we conducted uni- and multivariate subgroup analysis for cases with multiple kidney stones ≥ 2) that are primarily considered for 12/14F UAS usage in our department. In this subanalysis stone characteristics were comparable between the cohorts. 12/14F UAS was inserted in 48.3% of cases (71/147), irrespective of median maximal stone diameter, median stone density, presence and size of lower pole stones (p > 0.05). Full results of the univariate testing for this subgroup are presented in Table  that areOur study presents the retrospective outcomes of 12/14F UAS usage during fURS. Overall analysis as well as subanalysis for multiple stones of similar burden confirmed no clear benefit of 12/14F UAS. Due to the heterogeneity of stone characteristics demonstrated in overall analysis we suggest a special focus on the subgroup analysis of statistically comparable cases with multiple kidney stones (≥ 2) and similar stone size.The reduction of bacterial backflow to the venous system due to lowered intrarenal pressure during ureteroscopy has been proposed as a rationale for UAS usage in sepsis prevention , 19. AdvMoreover, we were unable to prove the benefit of 12/14F UAS usage on SFS, fluoroscopy and operating time neither in the overall cohort, nor in subgroup analysis of cases with multiple calculi.The analyzed cases operated with UAS were correlated in uni- and multivariate analysis with a prolonged operating time. We should discuss the possible reasons of these unexpected results. A relevant percentage (ca. 40%) of fURS is being performed at our department by residents in training . fURS wiThe presented results put into question the rationale for 12/14F UAS usage, but however should be cautiously discussed, taking into consideration the limitations of the study. Firstly, the usage of UAS was allowed in every case, depended on subjective surgeon preference. Despite the similar parameters characterizing both groups (UAS and non-UAS) with multiple calculi, we are aware that endourologists at our department tend to use UAS in patients with a more demanding anatomy or stone burden. Easier cases, even with multiple kidney stones, are often treated by fURS without UAS usage. This must be considered as a bias, diminishing the accuracy of the presented comparison.2O both under passive and active irrigation.Secondly, we acknowledge some deficits in gathered data due to the retrospective design of our study. The surgeries were performed mostly as “basketing” fURS for smaller stones without the need of lithotripsy, or “dusting plus basketing” versus “dusting plus active hand irrigation” fURS for larger stone burden. The exact type of the stone removal following the lithotripsy ( “dusting plus basketing” vs. “dusting plus active hand irrigation”) was not recorded in the study; hence our retrospective analysis does not provide data regarding how many UAS cases were conducted with active calyceal irrigation with a manual syringe pump. In our practice, some unexperienced surgeons may tend to irrigate more vigorously during fURS to obtain a clear view, which may possibly lead to infectious complications. Here UAS size plays a crucial role in lowering the pressure, especially during active irrigation, which may provide benefit especially in terms of preventing SIRS development. Noureldin et al. showed in a porcine in vivo model that 12/14 F UAS provided a sufficient, 12-fold reduction in intrarenal pressure during fURS with gravity irrigation, but only a fivefold reduction under active pump irrigation [Thirdly, we were unable to record the detailed status of intraoperative ureteral lesions during fURS. The rate of ureteral perforations was low (one case for the UAS group and one case for the non-UAS group). The high rates of postoperative stenting in the analyzed cohort have to be interpreted as a preventive measure. Placement of a mono-J catheter or double-J catheter on the string should be discussed as an alternative for postoperatively stone-free patients to spare them additional cystoscopy for double-J stent extraction .Despite the mentioned drawbacks, mostly due to the retrospective character, our study enriches the limited and ambiguous evidence on UAS application. Only one randomized controlled trial has been conducted so far on UAS insertion for kidney stone treatment. Kourambas et al. found no influence of 12/14F UAS usage regarding SFS and complications, but UAS application allowed for a significant reduction in operating time .Traxer et al. confirmed in a multicentric prospective non-randomized study on a large number  of cases no influence of UAS on SFS as well as parallel significant reduction of infectious complications . No subgp = 0.042).One of the few studies demonstrating an actual advantage of UAS usage in terms of higher rates of SFS was published by L’esperance et al. . The autThe literature describing differences between various UAS sizes is scarce. A broad majority of the cited publications analyzed the application of universal 12/14F UAS. 12/14F UAS is considered an “all-rounder”, eligible both for a stented and non-stented ureter . Taking Based on our retrospective results, the usage of 12/14F UAS does not seem to improve outcomes in fURS for kidney stones. In patients with multiple kidney stones it may be associated with elevated operation time without a clear benefit in terms of improved SFS or reduced perioperative complication rate. However, relevant limitations of retrospective study design have to be considered. Prospective randomized studies are urgently needed to define the conditions of effective, safe and cost-effective UAS usage.