Arthrobotrys flagrans in the absence of potential fusion partners (cell monologue). They were in the same phase as growth oscillations. In contrast to the anti-phasic oscillations observed during the cell dialogue, SofT and MakB displayed synchronized oscillations in phase during the monologue. Once two fusion partners came into each other’s vicinity, their oscillation frequencies slowed down (entrainment phase) and transit into anti-phasic synchronization of the two cells’ oscillations with frequencies of 104±28 s and 117±19 s, respectively. Single-cell oscillations, transient entrainment, and anti-phasic oscillations were reproduced by a mathematical model where nearby hyphae can absorb and secrete a limited molecular signaling component into a shared extracellular space. We show that intracellular Ca2+ concentrations oscillate in two approaching hyphae, and depletion of Ca2+ from the medium affected vesicle-driven extension of the hyphal tip, abolished the cell monologue and the anti-phasic synchronization of two hyphae. Our results suggest that single hyphae engage in a ‘monologue’ that may be used for exploration of the environment and can dynamically shift their extracellular signaling systems into a ‘dialogue’ to initiate hyphal fusion.Communication is crucial for organismic interactions, from bacteria, to fungi, to humans. Humans may use the visual sense to monitor the environment before starting acoustic interactions. In comparison, fungi, lacking a visual system, rely on a cell-to-cell dialogue based on secreted signaling molecules to coordinate cell fusion and establish hyphal networks. Within this dialogue, hyphae alternate between sending and receiving signals. This pattern can be visualized via the putative signaling protein Soft (SofT), and the mitogen-activated protein kinase MAK-2 (MakB) which are recruited in an alternating oscillatory manner to the respective cytoplasmic membrane or nuclei of interacting hyphae. Here, we show that signal oscillations already occur in single hyphae of  Neurospora crassa, Fusarium oxysporum, Botrytis cinerea, and Arthrobotrys flagrans  kinase MakB which show anti-phasic, oscillatory recruitment to the plasma membrane and the nuclei of interacting cells, respectively  and observed oscillatory recruitment of SofT to single hyphal tips with a mean period of 137±17 (SD) seconds (n=164 in 18 hyphae) without other hyphae in their vicinity (n=50) hyphal tips, experiment repeated three times, (Aspergillus nidulans and raised the question whether and how these two processes might be connected in A. flagrans (A. nidulans chitin synthase B (DFL_009443) in A. flagrans, which acts as a cargo marker for intracellular transport and is crucial for polarized growth (n=50 in 7 hyphae)  , Video 3 hyphae) , Video 4 hyphae) . Indeed,e)  might transit into the anti-phasic, cell-to-cell dialogue once a partner cell appears in the vicinity, we constructed a mathematical model. Specifically, we extended an existing model of cell-to-cell communication based on anti-phasic oscillations so as to explicitly account for the uptake of signaling components from the surrounding media . This moectively . As the and Y1,2 . ChsB isIn simulations containing only a single cell, similar to the experimental results monitoring the components in a single hypha, oscillations in these different components could be observed . When plialogue) . The rolup again . This baup again . As seenup again . This ‘cup again .n=173 in 18 hyphae) was comparable to the frequency in single hyphae and was not influenced by the presence of nematodes (n=45 in 8 hyphae) in the absence of neighboring hyphae and a mean period of 117±19 (n=57 in 8 hyphae) during the cell-to-cell dialogue  but then transitioned to anti-phasic oscillations characteristic of the cell-to-cell dialogue , Video 5ematodes . As expeematodes . To furtematodes . We monidialogue , Video 6dialogue , Video 7dialogue . Additiodialogue , Video 8n=105 in 11 hyphae) and 103±27 (n=106 in 10 hyphae), respectively  seconds  was comparable to the other markers during the cell-to-cell dialogue  which is part of the low-affinity calcium uptake system in Saccharomyces cerevisiae and is essential for mating  were cultivated at 28°C on PDA . All fungal strains used in the study are listed in Escherichia coli Top10 cells were used for plasmid cloning. A. nidulans strains were cultivated at 37°C on Aspergillus minimal media and transformed as described in E. coli Top10 cells were used for plasmid cloning. A. flagrans SofT was tagged with GFP at the C-terminus and expressed under the native promoter at the sofT locus. A 1 kb region from the 3’-end of the sofT open reading frame (orf) and 0.6 kb of the terminator region were amplified by PCR from A. flagrans genomic DNA. A GFP::hph cassette was amplified from pNH57 and all three fragments were subsequently inserted into the linearized plasmid backbone pJET1.2 using Gibson assembly, resulting in plasmid pVW106. A. flagrans ChsB was tagged with either mCherry or GFP at the N-terminus and expressed under the alpha-tubulin tubA promoter. The chsB orf and the 0.9 kb 3’ region were amplified by PCR from A. flagrans gDNA and inserted into the plasmid backbone pVW92 using Gibson assembly, resulting in the tubA-mCherry-chsB plasmid pVW118. For a GFP-tagged ChsB variant, the mCherry cassette of pVW118 was replaced by GFP using Gibson assembly, resulting in pVW132.All plasmids and primers used in the study are listed in h2b promoter. The R-GECO sequence was amplified from gDNA of SNT162 -R-GECO-fragment was cloned into pVW57, resulting in pVW131.R-GECO was expressed under the histone f SNT162  and clonfigA, figB, and hamA were deleted by homologous recombination. One kb flanks homologous to the 5’ and 3’ regions of the gene of interest were amplified by PCR with 25 bp overhangs homologous to either the hygromycin-B (hph) or geneticin sulfate  resistance cassette as well as to the pJET1.2 plasmid backbone. Verification of homologous recombination was performed as described in A. flagrans RimA was tagged with mCherry at the N-terminus and expressed under the tubA promoter. The rimA orf and 3’ region were amplified by PCR from A. flagrans gDNA and assembled into the plasmid backbone pVW82 using Gibson assembly.sofT orf were amplified by PCR from A. flagrans gDNA and assembled into the plasmid backbone pJM16 using Gibson assembly for complementing the ∆sofT mutant with truncated versions of the protein. Each construct was expressed under the sofT promoter.Fragments of the A. nidulans Soft was tagged with GFP at its endogenous locus with the endogenous promoter. Fragments were amplified by PCR from A. nidulans gDNA and assembled into the Blue Heron Biotechnology pUC vector.2O, 0.4 mg MnSO4 - 4H2O, 0.88 mg ZnSO4 - 7H2O, 3 mg FeCl3 - 6H2O, 15 g agar, pH 5.5) slides supplemented with 1 µM CaCl2. For calcium chelating experiments, EGTA (stock solution 0.5 M) was added at a final concentration of 5 mM. Around 1×104A. flagrans spores were incubated on a 2×2 cm agar pad at 28°C in darkness for 12–72 hr.For microscopy, fungal strains were inoculated on thin LNA  with a 63× NA 1.4 oil objective lens (DIC M27). Time series were acquired with a gallium arsenide phosphide photomultiplier tube (GaAsP-PMT) detector, 488 or 561 nm excitation lasers were used. Live cell imaging of A. nidulans was performed using a Nikon Ti2 microscope mounted with a Yokogawa W1 spinning-disk confocal scan head and two Prime95B cameras. Images were acquired with the NIS Elements Advanced Research software (Nikon) with the 488 nm laser.Live cell imaging of Image processing and analysis were performed in FIJI  and ZEN https://zenodo.org/record/6830734#.Ys_KmS2w1TY).All raw images and data files resulting from the analyses are available at Zenodo , inhibitor (I), free vesicle (X), docked vesicle (Y), and concentration of the extracellular signaling molecule at the tip of the cell (Z). The index  indicates the specific cell. τ is a time-scaling constant to adjust the time scale without the loss of dynamics. Ω is the system size and controls the level of noise in the system. The drift function (F) readswhere G) isThe noise function , d=0 (short distance), Ω=1000. The initial conditions for the simulations were set to U(t=0) = . To minimize the effect of the initial conditions, the model was run for 200 min before any other analysis was performed.where Assuming Brownian diffusion in three dimensions, the mean square distance  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) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Naama Barkai as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Antonio Di Pietro (Reviewer #1).Thank you for submitting your article "Synchronization of oscillatory growth prepares fungal hyphae for fusion" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The reviewers' consensus is that the study's findings are valuable but that the strength of evidence provided is incomplete. It is essential that you:1) Please increase the experiments' sampling sizes – the ones used are generally small, especially considering the observed variability.2) The model was difficult to follow and added only incrementally to the model previously developed by Goryachev (Bioessays 2012). Please make its description clearer and clarify its novelty.3) The calcium experiments were incomplete and do not fully demonstrate a role of extracellular calcium in hyphal communication. Please provide additional evidence that clearly demonstrates calcium's role.4) Please improve the clarity of your figures and presented data – all reviewers had numerous constructive suggestions.Reviewer #1 (Recommendations for the authors):lines 111-139: I found the whole model section very confusing (see details below):Z1 and Z2, respectively".Lines 111-112: The term "compartments" as used here appears confusing. I suggest using the following sentence: "The concentration of signaling components in the immediate proximity of the wall of cell 1 and cell 2 is represented by 2+, H2O2, peptides, etc.). I think here they mix both concepts. I guess this paragraph should read: "As a signaling compound is taken up into a cell, activating components (or compounds?) (modeled as circles A1 and A2) become more concentrated in this cell. These activating components (compounds?), in turn, stimulate the docking of cytoplasmic signaling components (X1 and X 2) to the compartment inside of the cell membrane (Y1 and Y2). The release of membrane-docked vesicles to the extracellular space is initially blocked by high levels of activator (A1 and A2). Secretion ultimately occurs when some of the activating components are converted into auto-inhibitory components (I1 and I2), reducing the levels of activating components, thereby allowing secretion of signaling compounds into the extracellular compartments (Z1 and Z2)"?Lines 111 and following: the authors need to clearly distinguish between signalling components  or signalling compounds .Figure 3d. While the oscillations of GFP-ChsB are clearly visible in the images, those of R-GECO are not (at least to this reviewer's untrained eye). Although in the graph the R-GECO peaks are clear, to make the point the authors may want to try improving the image.Line 165: I suggest that the authors use the word "possibly" rather than "probably". While the data demonstrate fluctuations of intracellular calcium, there is no evidence shown for the direct uptake of calcium during these rapid fluctuations, and thus they cannot exclude that the calcium could originate from intracellular stores.2+-depleted conditions. The authors infer from this that "intracellular ca2+ oscillations indeed rely on extracellular ca2+". However, this conclusion is flawed because the hyphae have been depleted for calcium for several hours and therefore the intracellular stores are also likely depleted. Their data from EGTA treatment mainly confirm previous reports showing that calcium is required for hyphal fusion and also demonstrate that calcium is required for well-regulated pulsatile secretion and for the recruitment of SofT to the plasma membrane.Line 166 and subsequent: In the subsequent experiment, calcium was drastically depleted from the medium by addition of the calcium chelating agent EGTA for several hours. This led to reduced hyphal growth and abolished cell fusion. Moreover, fluorescence of R-GECO was not detectable in these caReviewer #2 (Recommendations for the authors):This paper uses live cell imaging to show the localization of a tagged protein, SofT, to hyphal tips during growth, independently of the oscillation of this protein during chemotropic interactions prior to cell fusion as shown in a published article by the authors in 2022 . In addition, the role of calcium signaling is shown by usingComments.1. It is unclear if the constructs used in this study are the same as used in the PNAS 2022 paper. If so, it should be cited and not re-described. What is SofT? And how does the SofTnprotein function on a molecular level, if known.2. SofT has been characterized in a number of fungi and oscillation of SofT during chemotropic interactions has been observed in a number of species. Is the oscillation of SoFT to hyphal tips unique in A. flagrans? and if so, why? It seems that the role of hyphae in exploring their environment is a universal feature. This aspect should be discussed.3. There have been numerous published articles on the role of calcium in oscillatory growth of hyphae, most recent in 2022 (J. Fungi), including one by the author (PNAS 2017) and which are much more rigorous in approach.4. As reported in the 2022 PNAS article, and elsewhere, cell dialog in a number of different fungi is mediated by a MAP kinase complex, which oscillates to the tip of interacting cells with opposing dynamics to SofT. This issue should be addressed, preferably by experimental data.5. Are the oscillation dynamics of SofT similar or different to that of other fungi? If different, any ideas of the biochemical/mechanistic aspects of this? Are the oscillation dynamics temperature dependent ? Could it be that this oscillation is specific for nematode-trapping fungi? Does the presence of nematodes or nematode signaling molecules affect the dynamics of SofT?6. The R-GECO oscillations are not really obvious from the panels or the movie.7. Figure 4B is not clear and could be modified.8. Is it possible that SofT is the only signaling component in this system? How would this change the model?9. What is the evidence for the role of SofT playing a role in "exploration of its environment"? What is the phenotype of the SofT mutant and how would the oscillation and phenotype support this contention?10. The role of calcium in the growth and chemotropic processes could be explored more thoroughly.Reviewer #3 (Recommendations for the authors):General points:Dedicated introduction and Discussion sections would improve the flow of the manuscript. There is a very limited discussion to contextualize this work in a larger context of cell to cell communication, membrane fusion or even the realm of coupling of biochemical oscillation. Ideally, signaling molecule(s) would be identified through experiments, but at the very least there should be a Discussion section postulating on possibilities on the possible nature of signals. Perhaps the mathematical model could be used to put limits on diffusion/secretion rates that would support the observed oscillatory behavior and could point to an ion vs macromolecule based signal?The N's are small in general (ranging from 3-12 hyphae) and it is difficult to grasp the cell to cell variability in the oscillations. With SD of 30%, it seems there is substantial variability. Experiments that show the degree to which a given cell monologue persists at a given frequency vs fluctuating would be interesting and maybe useful to compare as a function of distance to a partner to understand the transition from monologue to out of synchrony and dialogue.Figure 1:– This sentence could be more clear: "Numbers indicate the count of oscillating accumulation of each fusion protein at the hyphal times during the time course."– The peak at 60s in the ChsB histogram is perplexing because there are not clear peak-to-peak intervals of 60s. Is the plot in C not representative? Could you be more clear with how the signals are being processed and analyzed?– Is the ChsB signal plotted with fewer data points than the SofT signal? Also, the kymograph in 1B appears to be blurryFigure 2:– Could you make it more clear where (if anywhere) ChsB and SofT fit in the mathematical model?Figure 3: Could synchronization of the signals in B be quantified? It would be great to have a plot that includes data from more than just a single pair of hyphae to show that this pattern of asynchrony to synchrony is a frequent occurrence. Additionally, as noted above, it would be interesting to see how the oscillations change as a frequency of distance and starting point frequencies of the hyphae in the dialogue.Figure 4: do you have any experimental evidence for the so-called refractory period that is preventing self stimulation?If there was any way to either manipulate the oscillations to force to be in phase, potentially through local light induced recruitment of soft or in some way demonstrate that out of phase really matters or linking calcium as the direct signal, this would make all the difference in making this paper more of an advancement for the field.Could an experiment be conceptualized that somehow assesses the extracellular role of calcium as a possible signal that is conveying the antiphase information? I wonder if there are any extracellular dyes or even recombinantly supplied proteins that could be taken advantage of to image calcium, potentially in the context of small growth volumes or microfluidics? Are there ways to more transiently, locally chelate calcium between two homing tips, maybe with a photocaged chelation? Essential revisions:The reviewers' consensus is that the study's findings are valuable but that the strength of evidence provided is incomplete. It is essential that you:1) Please increase the experiments' sampling sizes – the ones used are generally small, especially considering the observed variability.We increased sample sizes and updated the respective graphs. To further validate our findings, we included measurements of the MAP kinase MakB (MAK-2) and the actin marker Lifeact 2) The model was difficult to follow and added only incrementally to the model previously developed by Goryachev (Bioessays 2012). Please make its description clearer and clarify its novelty.To clarify the novelty of our model extension, and relate our model better to the biological system, we added the following in the model section:1 and Z2 to explicitly represent the information exchange between hyphae via the shared extra-cellular medium. Among the abstract model variables, SofT as a signaling component located inside of a hyphal cell connects most closely to variables X1,2 and Y1,2. ChsB is also located inside the hyphal cells, but with localizes differentlyfrom SofT and likely does not act as a signalling component, so that ChsB can be related to the variables A1,2, and I1,2.”“The main novelty of our model is the addition of the variables ZWe further modified Figure 2A (model schematic) and revised the text on the model construction and model analysis based on the input from all three referees.3) The calcium experiments were incomplete and do not fully demonstrate a role of extracellular calcium in hyphal communication. Please provide additional evidence that clearly demonstrates calcium's role.2+ signal into hyphal growth and the cell dialogue.To provide additional evidence, we concentrated on proteins which potentially play a role in coupling the caSaccharomyces cerevisiae and is essential for mating. Interestingly, filamentous fungi like A. flagrans contain two orthologues in their genome and we identified A. flagrans FigA and FigB. Single gene deletion of figA resulted in a colony phenotype, but both gene deletion mutants still performed cell fusions and trapped nematodes . We were unable to generate a double deletion, however, this might have been since at least one protein is essential for general growth. Indeed, in a recent publication  the orthologue DhFIG_2 of Dactylellina haptotyla could only be knocked down by RNA interference. Considering that the MAP kinase MAK-2 (MakB) plays an essential role in vegetative hyphal fusion in filamentous fungi, while the MakB-orthologue Fus3 is essential for mating, we believe that a direct comparison between mating and hyphal fusion might be helpful identifying novel components of the system. We added parts of our results to the discussion and created a new figure .First, we focused on orthologues of the protein FIG1 (mating factor induced gene 1), which is part of the low-affinity calcium uptake system (LACS) in Sordaria macrospora, as published by Teichert et al., 2014. We identified HAM-10, an orthologue of Unc13/Munc13 in higher eukaryotes. Unc13/Munc13 is known to facilitate proper SNARE assembly at the vesicle release site in the synaptic cleft . The A. flagrans orthologue HamA contains a calcium dependent C2 domain, similar to orthologues in other fungi. In higher eukaryotes, the protein also contains a calmodulin-binding domain. To further investigate the role of HamA, we generated a ∆hamA deletion strain, which showed a cell fusion mutant phenotype as observed in the ∆ham-10 mutant in N. crassa  . Interestingly, in the ∆ham-10 mutant, neither Soft nor MAK-2 localized at the tip of germ tubes . We were only able to localize mCherry-ChsB in a ∆hamA mutant background and observed oscillating recruitment of the protein to the hyphal tip, indicating that HamA could play a role at the interface of combining cell dialogue signaling and growth dynamics.Interestingly, the Soft protein sequence exhibits weak homology to mammalian proteins of the aczonin/piccolo family . These proteins act as scaffold proteins in the active zone of synapses to regulate the coordinated fusion and release of vesicles and their content into the synaptic cleft. Since the neurotransmitter release is highly dependent on calcium, we screened for Soft (PRO40)-interacting proteins with calcium-dependent functions in the filamentous fungus N. crassa is involved in cell fusion, but not essential . We localized the orthologues RimA in A. flagrans which shows localization at the hyphal tip without any noticeable oscillations during our time series .One interacting protein of Munc13 is RIM1, which plays a role in linking vesicle fusion and calcium influx. We identified the orthologue in the Soft/PRO40 interacting protein dataset of Teichert et al., 2014. Similarly, the orthologous Syt1 in 2+-concentrations and how this change relates to the secretion of a putative chemo-attractive signal molecule. Our data add evidence to the hypothesis, that extracellular calcium plays a role in hyphal communication, however, we feel that deciphering the exact role during this process might be out of the scope of this short report.Additionally, calmodulin and a calcium/calmodulin-dependent protein kinase were identified as interaction partner of PRO40, indicating strong similarities between the fast and highly regulated secretion of the cell dialog in fungi and release of synaptic vesicles in neurons. Early neural cell-type can be observed in ctenophores, which are one of the earliest-branching lineages of bilaterians who share some neuronal genes . In a so-called Unc13-RIM line, the combination of voltage-gated calcium channels to the vesicle release machinery results in coupling of action potentials and calcium influx to swift vesicle release. These results further show the occurrence of pulsatile secretion across eukaryotes. In the future, it will be interesting to study how other fusion-related proteins might be involved in the interpretation of the increase in intracellular ca4) Please improve the clarity of your figures and presented data – all reviewers had numerous constructive suggestions.We improved the clarity of our figures and included additional experiments suggested by the reviewers.The following experiments were added to help strengthening our findings:We quantified the occurrence of SofT-GFP monolog on LNA and observed the same behavior on PDA. We included a new figure .sofT mutant  with truncated fragments of the protein and found that the N-terminal disordered region and the WW domain of the protein are sufficient to rescue the cell fusion deficiency of the mutant. Our results are summarized in .We performed rescue experiments of the ∆We included additional microscopy data showing that hyphae without SofT-GFP recruitment at their tips were unable to fuse, suggesting that they could not recognize each other. (new Movie S7) and new figure Figure S1.A. nidulans and observed no monolog in this species under our tested conditions , LNA,). We hypothesize that depending on the species there might be fundamental difference if a hyphal cell undergoes a signaling monologue and discuss this result in the manuscript.We localized the SofT orthologue in We localized the MAP kinase MakB and co-localized it with SofT. We discovered that both proteins show oscillations in the same phase without other hyphae in vicinity, which is the opposite of the so far observed anti-phasic oscillations observed during the cell dialogue . Additionally, we observed that decoupling of both oscillations into the anti-phasic cell dialogue occurred during the transitory phase where the growth slowed down. We feel that this observation is a strong contribution to our findings and therefore split our previous figure 3 into new figures 3 and 4, separating our transition findings  and Calcium-related  findings.We improved the visualization of R-GECO fluorescence and included previous Figure S2b into new Figure 4a for further clarification. We additionally measured actin dynamics visualized by Lifeact-GFP during the cell dialogue and included our findings in new Figure S5b.C. elegans and couldn’t observe changes in the dynamic.We quantified SofT-GFP oscillations in the presence of the nematode Reviewer #1 (Recommendations for the authors):lines 111-139: I found the whole model section very confusing (see details below):This sentence has been changed as suggested.Lines 111-112: The term "compartments" as used here appears confusing. I suggest using the following sentence: "The concentration of signaling components in the immediate proximity of the wall of cell 1 and cell 2 is represented by Z1 and Z2, respectively".2+, H2O2, peptides, etc.). I think here they mix both concepts. I guess this paragraph should read: "As a signaling compound is taken up into a cell, activating components (or compounds?) (modeled as circles A1 and A2) become more concentrated in this cell. These activating components (compounds?), in turn, stimulate the docking of cytoplasmic signaling components (X1 and X2) to the compartment inside of the cell membrane (Y1 and Y2). The release of membrane-docked vesicles to the extracellular space is initially blocked by high levels of activator (A1 and A2). Secretion ultimately occurs when some of the activating components are converted into auto-inhibitory components (I1 and I2), reducing the levels of activating components, thereby allowing secretion of signaling compounds into the extracellular compartments (Z1 and Z2)"?Lines 111 and following: the authors need to clearly distinguish between signalling components  or signalling compounds  or with a cellular component (proteins). We therefore assign the agnostic term of “component” throughout, to not give more concrete naming than would be warranted by our model.2+ from the medium….line 50. … depletion of caCorrected.Line 136: use "implies" rather than "obviate"Corrected.Line 136 and following: Please clearly explain for the non-specialist why the model implies a slowdown of the oscillation dynamics. This is not obvious.The referenced “critical slowing down” is not a property of this particular model, but rather a phenomenon that is observed for most dynamical systems that go from one dynamical behavior into another type of behavior, as one main model parameter is modified while the system keeps running. We tried to reformulate the according sentence as below. Any longer discussion would require the study of textbooks or additional articles, as the underlying mechanisms of this slowing down are well-known in the dynamical systems, but are not trivial to explain in terms of their dynamic original. We refrained from such explanations, and instead hope that the following explanation gives an intuition, and supply the according references at the end of the sentence:This “critical-slowing down” is typical in phases where a system transitions from one type of dynamic behavior to another type of dynamic behavior, due to being “caught in between” two types of behavior during the transition .Figure 3b. Please explain in the legend what T1 and T2 mean (tip 1 and tip 2?).Yes, we included the information in the figure legend.Figure 3d. While the oscillations of GFP-ChsB are clearly visible in the images, those of R-GECO are not (at least to this reviewer's untrained eye). Although in the graph the R-GECO peaks are clear, to make the point the authors may want to try improving the image.We displayed the images of 3d  in greyscale (inverted contrast) and used different frames of the time series to help with visibility. We additionally included previous Figure S2b into the new Figure 4a to further help with clarification.Line 165: I suggest that the authors use the word "possibly" rather than "probably". While the data demonstrate fluctuations of intracellular calcium, there is no evidence shown for the direct uptake of calcium during these rapid fluctuations, and thus they cannot exclude that the calcium could originate from intracellular stores.Corrected.2+-depleted conditions. The authors infer from this that "intracellular ca2+ oscillations indeed rely on extracellular ca2+". However, this conclusion is flawed because the hyphae have been depleted for calcium for several hours and therefore the intracellular stores are also likely depleted. Their data from EGTA treatment mainly confirm previous reports showing that calcium is required for hyphal fusion and also demonstrate that calcium is required for well-regulated pulsatile secretion and for the recruitment of SofT to the plasma membrane.Line 166 and subsequent: In the subsequent experiment, calcium was drastically depleted from the medium by addition of the calcium chelating agent EGTA for several hours. This led to reduced hyphal growth and abolished cell fusion. Moreover, fluorescence of R-GECO was not detectable in these caWe agree with this comment and changed our conclusion regarding the EGTA treatment.Reviewer #2 (Recommendations for the authors):This paper uses live cell imaging to show the localization of a tagged protein, SofT, to hyphal tips during growth, independently of the oscillation of this protein during chemotropic interactions prior to cell fusion as shown in a published article by the authors in 2022 . In addition, the role of calcium signaling is shown by usingComments.1. It is unclear if the constructs used in this study are the same as used in the PNAS 2022 paper. If so, it should be cited and not re-described. What is SofT? And how does the SofTnprotein function on a molecular level, if known.sofT-GFP constructs created in this study were designed to tag the gene in locus and transformed into A. flagrans WT. In the PNAS 2022 paper, a ∆sofT mutant strain was complemented with a GFP-sofT construct which integrated ectopically in the genome. The reasoning behind it was because of the limited number of selection markers in A. flagrans.The Neurospora crassa and Sordaria macrospora Soft-orthologues proteins SO and PRO40 are required for sexual development. The orthologue protein AoSO of Aspergillus oryzae localizes at septal pores upon damage to hyphal compartments and various stresses like temperature, pH or nutrient depletion.The Soft protein is only conserved in filamentous ascomycete fungi. The S. macrospora PRO40 acts as a scaffold protein of the cell wall integrity pathway, however its molecular function remains insufficiently understood .Soft-orthologues contain 1200-1300 amino acids with a conserved N-terminal disordered region and a central WW domain, which facilitates protein-protein interactions in signaling networks. It was shown that the A. flagrans SofT protein and performed rescue experiments of the ∆sofT mutant and included our results in Figure S2. A. flagrans SofT contains 1213 amino acids. Analysis with the NCBI domain prediction tool coupled with the iupred2a prediction tool (https://iupred2a.elte.hu) predicted an N-terminal disordered region, a WW domain (AA505-533) and a C-terminal putative phosphatase domain (AA676-1213) in the sequence . The first fragment in our rescue experiment encoded for the N-terminal region of the protein (1-504 AA) without the conserved WW domain. The second fragment encoded for the N-terminal region with the conserved WW domain (1- 540 AA). The third fragment encoded the C-terminal region of the protein containing the putative phosphatase domain (534 – 1213 AA). We individually transformed the three constructs into the ∆sofT strain and observed the growth of the mutants after genotyping. As the ∆sofT deletion shows a strong growth phenotype on solid media  with the absence of aerial mycelium, we checked the growth of the three mutants on potato dextrose agar. The ability to form aerial mycelia and perform vegetative hyphal fusion was only restored by the second fragment containing the N-terminal region with the WW domain  suggesting the importance of SofT-interacting proteins during the cell dialog.In order to further dissect its molecular role, we generated three truncated versions of the 2. SofT has been characterized in a number of fungi and oscillation of SofT during chemotropic interactions has been observed in a number of species. Is the oscillation of SoFT to hyphal tips unique in A. flagrans? and if so, why? It seems that the role of hyphae in exploring their environment is a universal feature. This aspect should be discussed.Parts of this response were included in the Results section, resulted in a new figure  and were discussed in.A. flagrans hyphae on LNA in order to characterize monolog oscillations. We found that 85% +- 4 (average +-SD) showed localization of SofT at the hyphal tip in the absence of a fusion partner. We also observed that hyphae without SofT-GFP recruitment at their tips were unable to fuse, suggesting that they could not recognize each other . However, these cells were still able to undergo cell fusion if other hyphae induced hyphal fusion. We also tested the SofT-GFP dynamics on PDA (rich nutrient media compared to low nutrient LNA) and observed recruitment to the hyphal tips and cell fusion, suggesting that nutrient availability would not be the sole cause of inducing the dynamics We quantified the presence of SofT at 3x50 hyphal tips in Aspergillus nidulans to check if oscillation of SofT at hyphal tips can be found in different fungi. We found punctate localization throughout the hyphae which resembles the punctuate localization that can be also observed in A. flagrans  but couldn’t observe any tip oscillation of A. nidulans SO during cultivation in liquid and on solid Aspergillus minimal medium as well as low nutrient agar as it is used in A. flagrans experiments. However, we also observed no hyphal fusion during these culture conditions suggesting that oscillations of Soft at hyphal tips might be related to the preparedness of hyphal fusion. Interestingly, Aspergillus species are known to rarely undergo vegetative hyphal fusion under laboratory conditions , while it can be observed more frequently in other fungi. We speculate that at least under experimental conditions there might be ways to regulate/activate the signaling monologue and it might be different in fast growing fungi (in molds like Aspergillus or Neurospora) compared to slower growing fungi which will be exciting studying in the future.We additionally identified and localized the SofT orthologue in 3. There have been numerous published articles on the role of calcium in oscillatory growth of hyphae, most recent in 2022 (J. Fungi), including one by the author (PNAS 2017) and which are much more rigorous in approach.2+ signaling during cell fusion was presented by using various ca2+ pharmacological modulators. We believe that our contribution of showing that the stepwise extension of hyphae is coordinated during the cell-to-cell dialogue and which we visualized by pulses of intracellular ca2+ accumulations include a new angle of showing the involvement of ca2+ during the process.See our general response 3 to the editor for further clarification. In the study performed by Kurian et al., (2022), evidence of ca4. As reported in the 2022 PNAS article, and elsewhere, cell dialog in a number of different fungi is mediated by a MAP kinase complex, which oscillates to the tip of interacting cells with opposing dynamics to SofT. This issue should be addressed, preferably by experimental data.A. flagrans. MakB-mCherry showed oscillatory recruitment to nuclei of growing hyphae with phases similar to the observed SofT-GFP oscillations. Interestingly, co-localization of SofT-GFP and MakB-mCherry in the same hyphae revealed that both proteins were oscillating in the same phase without other hyphae in vicinity, which is the opposite of the so far observed anti-phasic oscillations observed during the cell dialogue. Additionally, we observed that decoupling of the oscillations into the anti-phasic cell dialogue occurred during the transitory phase. We included our results in (L167) and updated the figures to create a new figure 3 and supplementary figure Figure SS.Thank you for the great suggestion. We localized the MAP kinase MakB fused to mCherry during hyphal growth of 5. Are the oscillation dynamics of SofT similar or different to that of other fungi? If different, any ideas of the biochemical/mechanistic aspects of this? Are the oscillation dynamics temperature dependent ? Could it be that this oscillation is specific for nematode-trapping fungi? Does the presence of nematodes or nematode signaling molecules affect the dynamics of SofT?Neurospora crassa, Botrytis cinerea, and A. flagrans, exhibiting minute time scales that vary across these species. Specifically, in N. crassa, the oscillation occurs approximately every 10 minutes, while in B. cinerea, the range extends from 11 to 15 minutes . It is important to note that these observations were conducted on different media, which may have an impact on the dynamics. Additionally, the growth speed and size of all tested fungi are different as well. Considering the coupling between SofT oscillation and hyphal growth, we speculate that the overall dynamics of the oscillation will be influenced by the speed of growth. We tested if SofT-GFP oscillations are modified by nematodes and could not observe any differences. We haven’t checked for changes at different temperatures but speculate that during colder incubations (when growth slows down) oscillations of SofT might slow down, too. As seen in our comment 2 we didn’t observe oscillation dynamics of Soft in A. nidulans but agree, that this observation opens new questions about whether and why some fungi show this behavior while others don’t. Finding media conditions that either repress or induce hyphal fusion and comparing different fungal species might bring new insights into the observed dynamics.So far, the oscillation dynamics of SofT/MakB have been reported in 6. The R-GECO oscillations are not really obvious from the panels or the movie.We displayed the images of 3d in greyscale (inverted contrast) and used different frames of the time series to help with visibility. We additionally included previous Figure S2b into the new Figure 4a to further clarify.7. Figure 4B is not clear and could be modified.We modified the scheme. It is now new Figure 5.8. Is it possible that SofT is the only signaling component in this system? How would this change the model?This is a justified question, which has been addressed by previous work on this type of oscillatory system . We expanded on this point in our model section, as follows:“This model requires that not one, but two species in the cell undergo a conversion, with the second species’ conversion requiring the first species’ transition to have completed to a large extent. The reason behind this is that, without this sequential conversion, there is no delay between receiving and secreting signal, and that the secreting cell would not become nonreceptive to its own signal (8).”Bioessays 34, 259-266 (2012).(8) A. B. Goryachev, A. Lichius, G. D. Wright, N. D. Read, Excitable behavior can explain the "ping-pong" mode of communication between cells using the same chemoattractant. 9. What is the evidence for the role of SofT playing a role in "exploration of its environment"? What is the phenotype of the SofT mutant and how would the oscillation and phenotype support this contention?sofT results in absence of hyphal cell fusion in the mycelia . As seen in comment 2  we observed that hyphae without SofT-GFP recruitment at their tips were unable to fuse, suggesting that they could not recognize each other.Deletion of 10. The role of calcium in the growth and chemotropic processes could be explored more thoroughly.2+ accumulations include a new angle of showing the involvement of ca2+ during the process.See our general response 3 to the editor for further clarification. We believe that our contribution of showing that the stepwise extension of hyphae is coordinated during the cell-tocell dialogue and which we visualized by pulses of intracellular caReviewer #3 (Recommendations for the authors):General points:Dedicated introduction and Discussion sections would improve the flow of the manuscript. There is a very limited discussion to contextualize this work in a larger context of cell to cell communication, membrane fusion or even the realm of coupling of biochemical oscillation. Ideally, signaling molecule(s) would be identified through experiments, but at the very least there should be a Discussion section postulating on possibilities on the possible nature of signals. Perhaps the mathematical model could be used to put limits on diffusion/secretion rates that would support the observed oscillatory behavior and could point to an ion vs macromolecule based signal?We combined results and discussion as our findings were thought to be published as short report and feel that this format gives more strength to our observations.eLife was launched meanwhile we are unsure about the format. Therefore, we updated the format.Since the new concept of N. crassa  and direct interacting protein of Soft in S. macrospora , suggesting a link between cell dialogue and the glycolytic oscillation which needs further investigation. In addition,As seen in our general response 3 to the editor we see strong similarities between the hyphal cell dialogue and neurotransmitter release in the synaptic cleft including membrane fusion and added this as additional discussion in the manuscript. Coupling of biochemical oscillations are certainly possible and indeed 6-phosphofructo-2-kinase was identified as phosphorylation target protein of MAK-2 in We added a new section "Particle size estimation for the signaling component" to the Methods. Based on this estimation, we now added the following the main manuscript text:2+ ions as well as ca2+binding proteins, but not secretory vesicles as the component that is exchanged between the hyphae.”“An estimate calculation indicates that particles of diameter of approximately 3 nm or less can traverse the distance at which two hyphae synchronize at a sufficiently short time to support synchronization . This diameter would include caThe N's are small in general (ranging from 3-12 hyphae) and it is difficult to grasp the cell to cell variability in the oscillations. With SD of 30%, it seems there is substantial variability. Experiments that show the degree to which a given cell monologue persists at a given frequency vs fluctuating would be interesting and maybe useful to compare as a function of distance to a partner to understand the transition from monologue to out of synchrony and dialogue.We increased sample sizes and updated the respective graphs. To further validate our findings, we included measurements of the MAP kinase MakB (MAK-2) and the actin marker Lifeact. .Figure 1:– This sentence could be more clear: "Numbers indicate the count of oscillating accumulation of each fusion protein at the hyphal times during the time course."Corrected.– The peak at 60s in the ChsB histogram is perplexing because there are not clear peak-to-peak intervals of 60s. Is the plot in C not representative? Could you be more clear with how the signals are being processed and analyzed?Each histogram is the result of a measurement of the fluorescent intensity with a circular selection at the hyphal tip for every given frame. The relative fluorescent intensity values are then plotted against the time as depicted in Figure 1c. The plot in C is representative for the time series shown in Figure 1b. We are not clearly sure about the first part of this comment since there is a fluorescent accumulation (peak) at 60s.– Is the ChsB signal plotted with fewer data points than the SofT signal? Also, the kymograph in 1B appears to be blurryYes, the mCherry-ChsB images were captured at reduced intervals  due to quicker photobleaching.Figure 2:– Could you make it more clear where (if anywhere) ChsB and SofT fit in the mathematical model?We added an according extension to Figure 2B, indicatint how these protein species might figure in the model. Further, we added the following to the text of our manuscript:1,2 and Y1,2 . ChsB is also located inside the hyphal cells, but localizes differently from SofT and likely does not act as a signaling component, so that ChsB can be related to the variables A1,2, and I1,2.”“Among the abstract model variables, SofT as a signaling component located inside of a hyphal cell connects most closely to variables XFigure 3: Could synchronization of the signals in B be quantified? It would be great to have a plot that includes data from more than just a single pair of hyphae to show that this pattern of asynchrony to synchrony is a frequent occurrence. Additionally, as noted above, it would be interesting to see how the oscillations change as a frequency of distance and starting point frequencies of the hyphae in the dialogue.We additionally localized the MAP kinase MakB and co-localized it with SofT. We discovered that both proteins show oscillations in the same phase without other hyphae in vicinity, which is the opposite of the so far observed anti-phasic oscillations observed during the cell dialogue . Additionally, we observed that decoupling of both oscillations into the anti-phasic cell dialogue occurred during the transitory phase and included these results in Figure 3.Figure 4: do you have any experimental evidence for the so-called refractory period that is preventing self stimulation?So far the refractory period is based on models of excitable behavior. Of note, the fusing cells are genetically identical cells and hence would produce the same putative chemoattractant molecule to recognize the partner.If there was any way to either manipulate the oscillations to force to be in phase, potentially through local light induced recruitment of soft or in some way demonstrate that out of phase really matters or linking calcium as the direct signal, this would make all the difference in making this paper more of an advancement for the field.This is an interesting thought, and we agree that decoupling components of the system will be one of the next steps forward in the field. However, we believe that our discoveries of a monologue of individual hyphae before fusion, the transition into a dialogue and the coordination of growth during the cell-to-cell dialogue provide new valuable information for the field.Could an experiment be conceptualized that somehow assesses the extracellular role of calcium as a possible signal that is conveying the antiphase information? I wonder if there are any extracellular dyes or even recombinantly supplied proteins that could be taken advantage of to image calcium, potentially in the context of small growth volumes or microfluidics? Are there ways to more transiently, locally chelate calcium between two homing tips, maybe with a photocaged chelation?Great idea! Observing cell dynamics in microfluidic chambers and manipulating will be something to consider for the future.


Tighter glycemic control can reduce the risk of microvascular complications and potentially macrovascular complications, particularly in those with a new diagnosis of DM.2In the United States, there are approximately 30 million people living with diabetes mellitus (DM), the majority (95%) of whom have type 2 DM (T2DM).3 If adequate glycemic control is not achieved with metformin, additional antihyperglycemic agents can be added.3 Additional antihyperglycemic options include sulfonylureas, thiazolidinediones, dipeptidyl peptidase-4 (DPP-4) inhibitors, sodium-glucose co-transporter 2 (SGLT-2) inhibitors, glucagon-like peptide 1 receptor agonists (GLP-1 RAs), and insulin.4 Recent evidence, largely from cardiovascular outcome trials (CVOTs), has supported the use of SGLT-2 inhibitors or GLP-1 RAs as the preferred agents to be added to metformin if additional glycemic control is needed in patients with T2DM and cardiovascular disease.6Metformin is generally the first-line medication for the treatment of T2DM.7Oral semaglutide  was approved for the treatment of T2DM in September 2019; the injectable formulation of the drug was approved in December 2017. Oral semaglutide is the first oral formulation of a GLP-1 RA approved in the United States (US). The Institute for Clinical and Economic Review (ICER) performed a systematic literature review and cost-effectiveness analysis to evaluate the health and economic outcomes of oral semaglutide for the treatment of T2DM. Here, we present the summary of our findings and highlights of the policy discussion with key stakeholders that took place at a public meeting of the New England Comparative Effectiveness Public Advisory Council (NE CEPAC) on November 14, 2019. The detailed report is available on the ICER website.We compared the clinical effectiveness of oral semaglutide with liraglutide (an injectable GLP-1 RA), empagliflozin (an SGLT-2 inhibitor), sitagliptin (a DPP-4 inhibitor), and no treatment beyond background antihyperglycemic therapy . We reviewed clinical benefits in terms of intermediate outcomes  and key benefits .19 Eight RCTs were part of PIONEER, a phase 3 clinical development program for oral semaglutide, including 1 CVOT.18 Three RCTs were CVOTs of our selected active comparator therapies.19 Finally, we included the CVOT of injectable semaglutide to inform our assessment of the key benefits of semaglutide as a molecule.11We included evidence from 12 randomized controlled trials (RCTs).Head-to-head data on intermediate outcomes were available from the PIONEER trials. Results showed that oral semaglutide reduced HbA1c more than placebo, sitagliptin, and empagliflozin and more than liraglutide at 52 weeks but not at 26 weeks. Results also showed oral semaglutide reduced body weight more than placebo, sitagliptin, and liraglutide but had similar effects compared with empagliflozin.Because the CVOTs were placebo-controlled, we did not have head-to-head data for oral semaglutide versus active comparators on key benefits. Therefore, we conducted network meta-analyses on results from the CVOTs to obtain comparative estimates on the following outcomes: 3-point MACE ; hospitalization for heart failure; and nephropathy.We found that semaglutide  reduced the risk of 3-point MACE more than sitagliptin; no significant differences were found between semaglutide and liraglutide or empagliflozin. We also found that empagliflozin reduced the risk of hospitalization for heart failure more than semaglutide. We found no differences between semaglutide and active comparators on nephropathy.Adverse events (AEs) experienced with oral semaglutide were generally mild to moderate in severity and most commonly related to gastrointestinal effects. The rates of AEs leading to discontinuation were considerably higher for oral semaglutide versus the other comparators. In the CVOTs, both formulations of semaglutide had numerically higher rates of retinopathy events compared with placebo, with a significant risk increase reported for injectable semaglutide.The comparative clinical effectiveness of oral semaglutide on key benefits was assessed through indirect comparisons that are potentially susceptible to effect modification. Additionally, by combining results from the oral and injectable semaglutide CVOTs, we assumed that the clinical benefits of these formulations are reasonably similar. In January 2020, the U.S. Food and Drug Administration expanded the indication for injectable semaglutide for MACE risk reduction, but oral semaglutide does not currently have this expanded indication. This adds additional uncertainty as to whether these agents are comparable; however, it appears that the decision may have been because the CVOT of oral semaglutide was short in duration and showed statistical noninferiority but not superiority compared with placebo. Finally, in choosing our active comparators, we hypothesized that these comparators provided estimates for the classes they represent; however, this adds additional uncertainty when assessing the comparative clinical effectiveness of oral semaglutide and other antihyperglycemic agents.20 which was based on the United Kingdom Prospective Diabetes Study (UKPDS) Outcomes Model 2.21 Oral semaglutide was compared with 4 other options: (1) ongoing background antihyperglycemic therapy, (2) sitagliptin, (3) empagliflozin, and (4) liraglutide. Outcomes included life-years (LYs) gained, quality-adjusted life-years (QALYs) gained, and total lifetime costs. The base case used a health care sector perspective and a lifetime time horizon. A cohort of US adults with self-reported diabetes and HbA1c ≥ 7 from National Health and Nutrition Examination Survey (NHANES) program 2013-14 and 2015-16 surveys (n = 362) served as the population for our microsimulations.22 The cohort had a mean age of 61.8 years, a mean duration of diabetes of 13.1 years, and a mean HbA1c of 8.7.We estimated that the lifetime cost-effectiveness of oral semaglutide added to current antihyperglycemic therapy for T2DM. We developed an adaptation of a published US-based microsimulation model,7Oral semaglutide had the highest LYs gained and QALYs gained . Oral seThe overarching limitation of our cost-effectiveness analysis is the complexity of the clinical outcomes related to T2DM. Although this complexity was accounted for in part by using a microsimulation model, individual patient outcomes are challenging to predict. Another uncertainty arises from our reliance on effect estimates obtained through indirect treatment comparisons that may be susceptible to effect modification.In the base case, we assumed that key benefits remained constant for a patient’s lifetime because of a lack of data with longer follow-up. In a scenario analysis, we observed that gradual decreases in key benefits over time led to higher costs and decreased LYs and QALYs for oral semaglutide. Another key assumption was that patients who discontinued treatment moved on to receive insulin therapy; although this allowed for direct head-to-head comparisons of treatment with an initial therapy, this treatment pathway is not likely representative of the clinical management of all patients with T2DM.The NE CEPAC is one of the independent appraisal committees composed of medical evidence experts convened by ICER to engage in the public deliberation of the evidence on clinical and cost-effectiveness of health care interventions. Their deliberation includes input from clinical experts and patient representatives specific to the condition under review, as well as formal comment from manufacturers and the public. A policy roundtable concludes each meeting during which representatives from insurers and manufacturers join clinical experts and patient representatives to discuss how best to apply the findings of the evidence to clinical practice, insurance coverage, and pricing negotiations.not adequate evidence to demonstrate that the net health benefit of adding oral semaglutide is superior to that provided by adding liraglutide. The NE CEPAC also voted 11-1 that there is not adequate evidence to distinguish the net health benefit of adding oral semaglutide from that provided by adding empagliflozin.The NE CEPAC voted 12-0 that there is adequate evidence to demonstrate that adding oral semaglutide to ongoing background therapy provides a positive net health benefit. The NE CEPAC also voted 12-0 that there is adequate evidence to demonstrate that the net health benefit of adding oral semaglutide is superior to that provided by adding sitagliptin. The NE CEPAC voted 11-1 that there is The NE CEPAC panel also voted on “other potential benefits” and “contextual considerations” as part of a process intended to signal to policymakers whether there are important considerations when making judgments about long-term value for money not adequately captured in analyses of clinical and/or cost-effectiveness. Results from votes on “other potential benefits” showed that the majority of the panel (9/12) agreed that oral semaglutide offers reduced complexity compared with liraglutide that will significantly improve patient outcomes. The results of the votes on “contextual considerations” are shown in The culminating vote of the NE CEPAC was on the overall “long-term value for money” of oral semaglutide, taking into consideration its clinical effectiveness, incremental cost-effectiveness, potential other benefits, and contextual considerations. Voting categories for long-term value for money are “low,” “intermediate,” or “high.” Of the 12 panel members, 4 voted low, 6 voted intermediate, and 2 voted high long-term value for money of oral semaglutide compared with ongoing background therapy.The policy roundtable discussion explored how best to translate the evidence and broader perspectives discussed into clinical practice and into pricing and insurance coverage policies. The key policy perspectives on coverage policy are as follows:Step therapy: We heard from payer representatives that there is very little insurer management of treatment for T2DM, but that patient resistance to injectable treatments limited the early-line use of GLP-1 therapies. This resistance will be removed with an oral GLP-1 therapy, and as a result, some payers may consider instituting step therapy. However, we heard from clinical experts that some drug classes are preferable for specific patients based on a number of interacting clinical criteria and that a routine step through SGLT-2 inhibitors or DPP-4 inhibitors would be viewed as lacking clinical nuance. We heard from payer analysts that some payers have instituted a step therapy requirement for an injectable GLP1 RA before receiving coverage for oral semaglutide. Clinical experts felt this was not clinically sensible.More broadly, clinical experts acknowledged that they will have some patients whom they believe would do equally well with an SGLT-2 inhibitor or a GLP-1 RA, and in those cases, it would be appropriate for clinicians to pick the substantially cheaper agent (currently an SGLT-2 inhibitor), given the lack of evidence demonstrating clear superiority of one therapy over the other for many patients.Payers considering step therapy with other oral agents before access to coverage for oral semaglutide should consult with patients and clinical experts to determine whether step therapy can be targeted to appropriate patients without undue administrative burden.Other coverage criteria: We heard from clinical experts that concurrent therapy with an SGLT-2 inhibitor and a GLP-1 RA is common. Some payers may wish to consider limiting an initial trial of oral therapy to 1 agent after metformin, but there may be considerable resistance from clinicians and patients, given the lack of previous experience with active management in this disease space.Prescriber criteria: Given the prevalence of T2DM and the safety profile for oral semaglutide, there appear to be no evidence-based reasons to consider restricting providers to specialists.ICER’s potential budget impact analysis suggested that only approximately 7%-14% of eligible patients with T2DM could be treated with oral semaglutide before passing a budget impact threshold of $819 million per year over 5 years. During the policy discussion, clinical experts stated their belief that, because primary care providers are often uncomfortable prescribing injectable GLP-1 RAs, oral semaglutide would be an attractive alternative for up to 50% of the eligible patient population. ICER therefore issued an “Access and Affordability Alert” as part of its final report to signal to policymakers that special measures may need to be considered to ensure that the health care system can absorb the added costs from these therapies over the short term without reducing access inappropriately or contributing to rapid growth in health care insurance costs.Clinical evidence shows that treatment with oral semaglutide resulted in better outcomes compared with ongoing background therapy and sitagliptin. Oral semaglutide provided better improvements in intermediate outcomes compared with liraglutide, although no significant differences in key benefits were found. Oral semaglutide reduced HbA1c but had similar effects on body weight compared with empagliflozin; no significant differences in 3-point MACE were found, but empagliflozin was found to reduce the risk for hospitalization for heart failure more than semaglutide.Oral semaglutide was estimated to be dominant compared with liraglutide and to have incremental cost-effectiveness ratios between $100,000 and $150,000 per QALY versus sitagliptin and background therapy alone. But oral semaglutide is unlikely to meet commonly accepted cost-effectiveness thresholds compared with empagliflozin and, with substantial uptake, could contribute to short-term budget strains in the health system. Clinicians and policymakers should work together to ensure that this important new treatment option is brought into clinical practice in a way that maximizes its health benefits without adversely affecting access and affordability.