These functions position mTORC1 as a central controller of cellular homeostasis, but the behavior of this system in individual cells has not been well characterized. Here, we provide measurements necessary to refine quantitative models for mTORC1 as a metabolic controller. We developed a series of fluorescent protein-TFEB fusions and a multiplexed immunofluorescence approach to investigate how combinations of stimuli jointly regulate mTORC1 signaling at the single-cell level. Live imaging of individual MCF10A cells confirmed that mTORC1-TFEB signaling responds continuously to individual, sequential, or simultaneous treatment with amino acids and the growth factor insulin. Under physiologically relevant concentrations of amino acids, we observe correlated fluctuations in TFEB, AMPK, and AKT signaling that indicate continuous activity adjustments to nutrient availability. Using partial least squares regression modeling, we show that these continuous gradations are connected to protein synthesis rate via a distributed network of mTORC1 effectors, providing quantitative support for the qualitative model of mTORC1 as a homeostatic controller and clarifying its functional behavior within individual cells. To adapt to continuously changing microenvironments, cells must perceive the availability of multiple nutrients and adjust anabolic and catabolic processes accordingly. The kinase mTOR  is the core of a regulatory network for sensing growth factors and metabolites, including amino acids, glucose, and cellular energy charge (ATP/ADP/AMP ratios). Collectively, these factors regulate the assembly and activity of mTOR Complex 1 (mTORC1), a heteromer of mTOR, Raptor, mLST8, PRAS40, and DEPTOR . Throughin vitro conditions, where nutrients are in excess  as mTOR and its regulators are present in >103 copies per cell and can cycle rapidly between multiple states within any individual cell  or fixed single-cell snapshots (such as immunofluorescence), which can obscure the heterogeneous activity of single cells . AveragiC/N) has been frequently used as a surrogate measure of this phosphorylation, both by immunofluorescence  family of transcription factors . TFEB morescence  and by lrescence . Howeverr export . ERK andr export , with amIn this study, we set out to characterize the homeostatic control function of mTORC1 at the cellular level. We used engineered versions of TFEB and immunostaining of multiple endogenous substrates to quantify the individual and combined effect of multiple inputs on mTORC1 activity at the single-cell level. Our data show that the response to amino acids and insulin varies among mTORC1 substrates, changes continuously and dose-dependently with both stimuli, and also continues adapting on the order of hours after major stimuli. With this knowledge in hand, we were able to use multiplexed immunofluorescence data to generate quantitative models of protein synthesis as a function of mTORC1 activity. These models revealed a degree of control at the single-cell level that is consistent with a homeostatic role for mTORC1, but which is strikingly distributed across the mTORC1 signaling network.C/N . For all reporters, we quantified responses to treatment on a cell-by-cell basis using a metric, Δi, which computes the average change in reporter localization from baseline to the peak response, typically 30–60 min after treatment. Using this quantification, we observed that FL-TFEB-TRdulators , includidulators . The abidulators , was cledulators . FL-TFEBdulators . Based oC/N tracks with endogenous TFEBC/N in live cells in response to both amino acid and insulin stimuli, with minimal perturbation of endogenous mTORC1 signaling.Localization of TFEB-TR was compared to endogenous TFEB on a cell-by-cell basis using immunostaining with an antibody targeting the C-terminal region of TFEB that is absent in TFEB-TR . As withC/N, whereas mTOR inhibition only returned 3xSA-TRC/N to its baseline, indicating a reduction in its overall dynamic range . MEFs displayed a strong increase in TFEB-TRlutamine . SimilarC/N did not remain constant following each stimulus. In many cases, it showed a rebound effect following an initial peak or a gradual rise across hours  per cell was reduced under increasing extracellular amino acid concentrations, such that cells showed the largest fluctuations in TFEB-TRC/N when amino acids are limited , ULK1 (phosphorylation at Ser-757), S6K (phosphorylation at Thr-389), S6 , and TFEB (cytosolic/nuclear ratio), as well as related proteins including phosphorylated AKT, GSK3β, and ACC. Cells were cultured in minimal medium and fixed 1 hr after the introduction of stimulation conditions. We assessed protein synthesis activity in each case by pulsing cells with O-propargyl puromycin (OPP), which is incorporated into translating peptides, for 30 min prior to fixation, followed by fluorescence labeling with click chemistry. All stains were performed on the same samples using a bleach/stain cycle to yield a total of 16 measurements for each cell. We performed target staining under 16 stimulation conditions, comprising all present/absent combinations of saturating insulin, essential amino acids , glucose, and glutamine. Over 10ibutions . The meaibutions , which wibutions . The staC/N; C/N, each of which showed a pattern of intensity distinct from the pS6/p4E-BP1 axis. The maximal points for each of these stains were unique from one another and from the pS6/p4E-BP1 axis, implying that the overall phosphorylation of the mTORC1 substrate network does not occur along a singular axis.As a qualitative visualization of network activity at the single-cell level, we constructed UMAPs based on the known mTORC1 targets and their substrates . Cell-to-cell variance in protein synthesis, however, is much greater as it includes all effects, not just those directly biased by these average nutrient differences. Nevertheless, all stains together predicted 60% of the cell-to-cell variance in OPP, while the normalized targets of mTORC1 predicted 25% of the variance (C/N) each provided <5% prediction individually but afforded much higher predictivity (~25%) when combined. Such cooperativity implies that, as a controller of protein synthesis, mTORC1 activity must be viewed as a network of processes rather than a single linear pathway.We next examined the relationship of mTORC1 target phosphorylation to protein synthesis by examining OPP labeling. OPP showed an approximate correlation with pULK1, pS6, and p4-EBP1 but was not tightly associated with any one mTORC1 target . We usedC/N . This revariance , left. Tvariance . We specvariance , right. The data presented here provide two central insights that were previously unaddressed experimentally. First, live-cell imaging shows that mTORC1 activity changes within a cell are continuous, both in the sense that mTORC1 responds to various stimuli dose-dependently in continuous gradations rather than large abrupt shifts, and that mTORC1 activity can fluctuate continuously over time, changing on the order of minutes or faster. Second, immunofluorescence analysis demonstrates that different mTORC1 substrates are not co-linearly activated within individual cells. Instead, each responds with different sensitivity, and they are collectively correlated with the cellular protein synthesis rate. Together, these findings elaborate how mTORC1 operates as an analog homeostatic controller for cellular anabolic functions.Prior to this work, several observations suggested that mTORC1 activity might be discontinuous. In a number of studies, phosphorylation of mTORC1 substrates appeared to be much greater in response to combined amino acid and growth factor stimuli than in response to either stimulus alone . Furthereither the activated Rag-complex binding sites or reducing the number of TSC2 complexes on the lysosome would be sufficient to increase the number of active mTORC1 complexes  and systems-level behaviors (i.e. how the many copies of the complex behave collectively within the cell). The boolean logic of individual mTORC1 complexes does not imply strict on/off responses at the cellular level, but there are important cellular implications for this molecular behavior. Our results using sequential stimuli  suggest Our data also show that the appearance of synergistic mTORC1 activation depends on which substrate is used to track its activity, and whether phosphorylation is assessed at a single time point or over time. Notably, phosphorylation of S6K or its substrate S6 have been used in most studies that have observed a strict requirement for both amino acids and growth factors . HoweverAdditionally, it is possible that each of the nodes in the network is co-regulated by additional signals, which could explain the divergent behavior of pS6. For example, Ser 235/236 of S6 can be phosphorylated both by p70 S6K and by p90 RSK , which iin vitro studies of this model suggest that cell death results from a failure to limit energy expenditure , which in many cases were significantly different than those found in human tissues. Our findings should thus be interpreted as characterizing a set of behaviors that are possible for the mTORC1-TFEB network within a certain context. These results should be further tested under relevant conditions before concluding that they represent the behavior of the network in other cell types.Our results are based largely on experiments conducted in a single, genetically stable, immortalized cell line, and thus the relevance of our conclusions for most other cell types or for any particular tissue within the body is not known. All experiments were performed Plasmids encoding full-length human TFEB (FL-TFEB) were received as a gift from the Zoncu lab. The series of TFEB reporters, including FL-TFEB, were constructed in the pLJM1 lentiviral vector, and the coding region of TFEB was modified, as shown in Retroviral or PiggyBac systems were used to generate stably expressing reporter cell lines. Following transfection, cells were selected using puromycin (1–2 μg/mL), G418 (200 μg/mL), or geneticin (300 μg/mL). To reduce variability in reporter expression, clonal cell lines were established using limited dilution cloning. In this study, data for each reporter is representative of behaviors that were consistent across at least three single-cell clones. Reporter cell lines were confirmed mycoplasma-negative through periodic monitoring by PCR and validation with ATCC testing of selected lines.MCF10A cells clone 5E  were rouBefore imaging, cells were washed twice with their respective imaging media and then cultured in imaging experiment media at least 2 hr prior to imaging. In experiments that involved amino acid perturbation, cells were pre-incubated with ‘Imaging medium – noAA’ for at least 6 hours prior to imaging to limit residual extra amino acids from the routine cell culture media.For all imaging experiments, cells were seeded in glass-bottom 96-well plates  1 d prior to imaging. To increase the cell-to-media ratio, cells were plated in the center of the well by spotting 9000 cells on 3 uL of type I collagen (Gibco A10483-01). Following the treatment regimen, cells were fixed in 4% paraformaldehyde solution for 15 min, and then permeabilized with 100% methanol for 15 min.For immunostaining, cells were washed in PBS-T (0.1% Tween-20 in PBS) twice, blocked for 1 hr with Odyssey Blocking Buffer . A sequential staining protocol  was used2 at 37°C in an environmental chamber. Images were collected with a Nikon  20x/0.75 NA Plan Apo objective on a Nikon Eclipse Ti inverted microscope, equipped with a Lumencor SOLA or Lumencor SPECTRA X light engine. Fluorescence filters used in the experiment are DAPI , CFP , YFP , Cherry , and Cy5 . Images were acquired using Andor Zyla 5.5 sCMOS camera every 6–7 min at 2 × 2 binning. Exposure times for each channel were 25–50 ms for DAPI; 150–250 ms for CFP; 150–250 ms for YFP; 300–500 ms for Cherry; and 300–500 ms for Cy5.For live-cell microscopy experiments, cells were cultured and seeded as described in previous sections. Time-lapse wide-field microscopy was performed as detailed in Live-cell microscopy images were processed using the custom MATLAB procedure as described in our previous work . In thisThe single-cell time-series data were further filtered, requiring minimum fluorescent intensity more than two times background intensity, trace length of at least 8 hr, and no more than three contiguous missing datapoints. Missing datapoints in time series were linearly interpolated to fill the gap.For all translocation reporters , the activities of the kinases are described by the ratio of average cytosolic fluorescent intensity divided by average nuclear fluorescent intensity.For the AMPKAR2 reporter, AMPKAR2 phosphorylation status was calculated using the protocol described in For the TOP-H2B-YFP-DD reporter, the rate of intensity gain over time was measured after addition of trimethoprim (TMP), before and after a stimulus. The ratio of the rate after to that before the stimulus is used to represent the relative translation activity (RTA) induced by the stimulus.For all volatility analysis of time-series data, we used a time window between 2 and 6 hr after the final perturbation. We estimated that in this time window the kinase activities of interest have approached a quasi-steady state. Within that time window, data from every other time step were then used to calculate CV, which we used as metric of volatility. Alternating time steps were used to mitigate non-biological noise, such as imaging and segmentation noise.The displayed time series were chosen by random number generation in MATLAB with a threshold for minimum tracking time to eliminate cells in which recording was terminated prematurely due to failure of the tracking algorithm. The chosen tracks were manually verified to be representative of successfully tracked cells and consistent with the overall range of cell behaviors. Cell recordings determined by manual inspection to have poor tracking or quantification accuracy were discarded.t-test with unequal variances. A p-value of 0.05 is considered as significant for all hypothesis testing.Linear regression analysis was performed using a MATLAB implementation of the Theil-Sen estimator, which is relatively insensitive to outliers and robust to heteroskedastic data. Unless otherwise indicated, each statistical comparison was made by  In this study, Sparta et al., performed time-resolved monitoring of the mechanistic target of rapamycin complex 1 (mTORC1) response to stimuli including glucose, amino acids, and insulin at the single-cell resolution. The authors found that mTORC1 activation is graded, which supports the model of continuous adjustment of mTORC1 signaling to changes in the cellular environment rather than an "on/off" mode of mTORC1 function. Considering the pivotal role of mTORC1 in sensing a plethora of signals to orchestrate metabolic programs impacting cell fate and organismal physiology, it was thought that this study will be of interest across a broad spectrum of biomedical disciplines. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Ivan Topisirovic as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Philip Cole as the Senior Editor.Thank you for submitting your article "Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis" 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:1) It was thought that presented data support the present view of mTORC1 regulation rather than establishing a previously unappreciated model of stimuli-sensing by mTORC1. As illustrated in the individual reviews, the claim that the present view in the field is that mTORC1 functions analogous to an AND gate was found to be somewhat inaccurate. To this end, it was thought that the authors should focus on highlighting the power of their quantitative approaches that allow time-resolved measurements of mTORC1 activity at the single cell resolution that drew substantial enthusiasm from the reviewers, while deemphasizing the claim that their study challenges current paradigms of mTORC1 regulation. Moreover, it was thought that the focus should also be shifted to what were found to be novel findings .2) Some important controls were deemed to be missing. For instance, it should be verified whether manipulations in glucose, amino acid or insulin levels affect expression of TFEB reporters. In addition, the activity of PI3K/AKT signaling should be examined across the conditions as it was thought that this may be crucial to determine the extent of the effects of starvation and amino acid stimulation on mTORC1 signaling in the absence of insulin. Moreover, pharmacological and/or genetic approaches to verify whether employed stimuli are still capable to activate mTORC1 signaling when PI3K/AKT signaling is debilitated appear to be warranted. Finally, monitoring Ser235/236 phosphorylation of rpS6 may not to reflect alterations in signaling via the mTORC1/S6K axis.3) Some concerns were raised regarding the differences in cell density between baseline and treatment conditions in the examples of original imaging data that have been provided. Based on this, it was thought that more original data should be included to support the quantification plots.4) In some instances, the information provided in the methodology section lacked sufficient detail.5) The authors should acknowledge and/or experimentally address limitations of their study pertinent to relying on a single cell line and excluding physiological variables that are known to affect mTORC1 activity .Reviewer #1 (Recommendations for the authors):– The authors propose that the prevalent model of mTORC1 regulation is that analog to AND gate type of digital logic gate, whereby it is assumed mTORC1 is only active in the presence of both amino acids and growth factors under optimal cellular energy conditions. This is likely to be my subjective interpretation, but I do not think that such AND gate model of mTORC1 regulation is strongly supported by the literature. In contrary, I think that the prevailing model of mTORC1 regulation is that of a rheostat that is akin to the one proposed by the authors. With this in mind, my suggestion to the authors is not to claim the novelty of the model of mTORC1 regulation, but rather their ability to support the existing model by quantifying the effects of various stimuli on mTORC1 activity at the single-cell level, which at least I found to be quite exciting and warranted.– The major limitation of the study is that it based on a single cell line. This renders generality of the authors' conclusions somewhat questionable and should be either accompanied by testing other cell lines or commented as a limitation of the study in the manuscript. Along the same lines, the authors should consider toning down claims of how relative their findings are physiologically as although they study the effects of glucose, amino acids, and insulin on MCF10A cells, it is unlikely that their model reflects the complexity of the physiological environments which comprise fluctuations in oxygen and other factors.– Methods section regarding the exact conditions of starvation/stimulation should perhaps be further clarified. In addition, the authors should consider providing evidence that 6h starvation of MCF10A cells is sufficient to shut down PI3K/AKT signaling. Would mTORC1 be activated by amino acids in the presence of e.g., AKT inhibitor? It was also thought that it would be important to monitor potential effects of manipulations in nutrient and/or insulin on reporter expression.– Phosphorylation of S235/236 on rpS6 can be independent of the mTORC1/S6K axis and its regulation is unaltered in cells lacking S6K1 and 2 (PMID: 15060135). Ergo, using p-S235/236 on rpS6 as a readout of mTORC1 activity may be problematic.Reviewer #2 (Recommendations for the authors):1. Does the amount of nutrients/growth signals added affect total expression of TFEB fusion proteins and mutants? Is this taken into consideration in the calculation of C/N particularly during prolonged periods?2. The findings in Figures4 and 5 that TFEB-C/N responds to amino acids despite absence of insulin should be verified for the presence of insulin/growth factor signaling. According to the Methods, cells were starved for 6 hr. There could be residual insulin/PI3K signaling, thus allowing TFEB response upon amino acid addition. They should examine PI3K/Akt signaling. Would this aa response still occur upon inhibition of PI3K or Akt? What happens when starvation is prolonged to 12-16 hr before re-addition of nutrients?Reviewer #3 (Recommendations for the authors):This is an interesting manuscript from Sparta and colleagues that investigates dynamics of MTOR and TFEB signalling.eLife. For publication as a full-length paper, it would seem the narrative would need to be re-structured to highlight how findings illustrate novel insights with relevance that can be appreciated by a broad audience. The paper should be revised to focus on a smaller number of specific biological questions that could be addressed by the single cell signalling data.While this reviewer appreciates the elegance and power of the experimental system, and the substantial amount of data in the manuscript, we did not find that the data provide enough clear insight into the mechanisms of MTOR regulation or signalling to justify publication in For example, the authors were able to demonstrate that the truncated TFEB-TR reporter behaves similarly to full length, but that a 3xSA-TFEB-TR lacking GSK3beta modification sites was poorly responsive, but these are not significant enough advances for the field.The authors aimed to use cell based reporters to investigate digital vs gradual modes of MTOR signalling, an interesting concept for signalling systems biology. Data in Figure 4 support the notion of incremental responses to amino acids and also to insulin signalling. The study proceeds with investigation of multiple signalling inputs, developing highly granular analysis of sequential additions. The biological mechanistic insight from these experiments remains unclear. Additional information may be gained from analysis of different combinations of simultaneous stimulation eg AA+insulin, as explored initially in the manuscript. Linking further with the initial part of the paper, it is interesting that TFEB responses differed with other MTOR downstream signalling, most notably with P-S6, but also with P-ULK1-S757. It would be interesting if further mechanistic insight could be demonstrated by modulation of responses by gene silencing of essential components of AA sensing, insulin signalling or TFEB signalling pathway. In addition, this paper would be greatly strengthened if some of the cell-based imaging readouts can be complemented by cell population immuno-blot analysis for key experiments. Essential revisions:1) It was thought that presented data support the present view of mTORC1 regulation rather than establishing a previously unappreciated model of stimuli-sensing by mTORC1. As illustrated in the individual reviews, the claim that the present view in the field is that mTORC1 functions analogous to an AND gate was found to be somewhat inaccurate. To this end, it was thought that the authors should focus on highlighting the power of their quantitative approaches that allow time-resolved measurements of mTORC1 activity at the single cell resolution that drew substantial enthusiasm from the reviewers, while deemphasizing the claim that their study challenges current paradigms of mTORC1 regulation. Moreover, it was thought that the focus should also be shifted to what were found to be novel findings .We agree that this shift in perspective is helpful, and we have refocused the work on the quantitative gains of our live-cell measurements and sequential immunofluorescent stains. We also now provide more formal quantitative modeling to address how mTORC1 activity relates quantitatively to protein synthesis rates. The manuscript text has been edited and reorganized according to this newly refined focus. To provide a complete discussion of the existing literature, we have kept a short introduction to the AND gate concept, but we no longer claim that it is the predominant view of the field or that the novelty of our study lies in the refutation of this concept.2) Some important controls were deemed to be missing. For instance, it should be verified whether manipulations in glucose, amino acid or insulin levels affect expression of TFEB reporters.A control for total expression of TFEB reporter (TFEB C+N) in response to amino acid stimulus is now provided as Figure 1—figure supplement 1F.In addition, the activity of PI3K/AKT signaling should be examined across the conditions as it was thought that this may be crucial to determine the extent of the effects of starvation and amino acid stimulation on mTORC1 signaling in the absence of insulin.AKT activity in live cells is reported now in Figure 4, across serial stimulation experiments including:starvation, glucose/glutamine re-feeding, amino acid re-feeding, and insulin treatments.Moreover, pharmacological and/or genetic approaches to verify whether employed stimuli are still capable to activate mTORC1 signaling when PI3K/AKT signaling is debilitated appear to be warranted.Pharmacological inhibition of AKT is reported in Figure 3—figure supplement 1C (using MK2206). In this panel, cells received full amino acids; note that upon MK-2206 treatment, TFEB-TR localization returns to the C/N level for amino-acid stimulated cells (about 1.0), not to the amino acid starved baseline level (about 0.8) as observed in Figure 3B. If amino acids were unable to activate mTORC1 signaling in the presence of the inhibitor, we would expect the TFEB C/N ratio to drop to the starved baseline of 0.8.Finally, monitoring Ser235/236 phosphorylation of rpS6 may not to reflect alterations in signaling via the mTORC1/S6K axis.We agree that the interpretation of data from this phospho-epitope needs to take into account the fact that it can be phosphorylated by both p70 S6K and p90 RSK. This convergence is one mode by which alternate regulation can come into play. In the revised manuscript, measurements of substrate phosphorylation are now provided all together in Figure 6, so that the signaling targets may be evaluated in a broader context. We have also now directly commented on this pathway convergence in the results and discussion.3) Some concerns were raised regarding the differences in cell density between baseline and treatment conditions in the examples of original imaging data that have been provided. Based on this, it was thought that more original data should be included to support the quantification plots.Example images are now found in Figure 1 and Figure 6—figure supplement 1. The image data used in all of these experiments include much larger regions of cells than can be reasonably visualized in a figure panel, and each dataset is composed of multiple experiments. Image panels shown were chosen essentially at random and were cropped to a scale that would allow the viewer to assess the level of cellular detail present in the dataset, so the apparent density in these cases is arbitrary. We did not observe systematic differences in cell density across treatments, and we have now indicated this in the respective figure legend. We have revised the former Figure S1  to include larger fields of cells with more similar densities across conditions, but please note that these images still represent only a small fraction of the data , all of which are represented in the summary plots and model and are included in the supplemental data file for Figure 6. Regarding the trends visible in the former Figure 2B,D , we agree that the visible trend is subtle, but it is consistent with the trends shown in the quantification plots, which best represent the full dataset.4) In some instances, the information provided in the methodology section lacked sufficient detail.We have reviewed and revised the methodology to ensure complete detail. We have included a description of and reference regarding the sequential staining technique employed to collect many immunofluorescent measurements in the same cells. We also include the customized media formulations in the materials tables. The base medium for all starvation conditions was lacking both glutamine and glucose; where noted (“noAA” formulation) the base medium was also lacking all amino acids.5) The authors should acknowledge and/or experimentally address limitations of their study pertinent to relying on a single cell line and excluding physiological variables that are known to affect mTORC1 activity .We have included text specifically discussing the limitations that must be considered when interpreting our experiments. However, to mitigate the limited interpretability of data from a single cell line, we have also added experiments employing mouse embryonic fibroblast (MEF) cells, a very different context from the MCF10As used previously. These experiments are presented in Figure 4G.Reviewer #1 (Recommendations for the authors):– The authors propose that the prevalent model of mTORC1 regulation is that analog to AND gate type of digital logic gate, whereby it is assumed mTORC1 is only active in the presence of both amino acids and growth factors under optimal cellular energy conditions. This is likely to be my subjective interpretation, but I do not think that such AND gate model of mTORC1 regulation is strongly supported by the literature. In contrary, I think that the prevailing model of mTORC1 regulation is that of a rheostat that is akin to the one proposed by the authors. With this in mind, my suggestion to the authors is not to claim the novelty of the model of mTORC1 regulation, but rather their ability to support the existing model by quantifying the effects of various stimuli on mTORC1 activity at the single-cell level, which at least I found to be quite exciting and warranted.We agree with this perspective and have shifted our focus accordingly; please see the response in the essential revisions section for more details.– The major limitation of the study is that it based on a single cell line. This renders generality of the authors' conclusions somewhat questionable and should be either accompanied by testing other cell lines or commented as a limitation of the study in the manuscript. Along the same lines, the authors should consider toning down claims of how relative their findings are physiologically as although they study the effects of glucose, amino acids, and insulin on MCF10A cells, it is unlikely that their model reflects the complexity of the physiological environments which comprise fluctuations in oxygen and other factors.We agree and have added these limitations as the last section in the Discussion, as well as added data from another cell line (MEFs). Please see the response in the essential revisions section for more details.– Methods section regarding the exact conditions of starvation/stimulation should perhaps be further clarified. In addition, the authors should consider providing evidence that 6h starvation of MCF10A cells is sufficient to shut down PI3K/AKT signaling. Would mTORC1 be activated by amino acids in the presence of e.g., AKT inhibitor? It was also thought that it would be important to monitor potential effects of manipulations in nutrient and/or insulin on reporter expression.Data addressing this point can be found in Figure 3B and Figure 3 supplement 1 – C. Please see our response to this comment under point 2 of the essential revisions section for more details.– Phosphorylation of S235/236 on rpS6 can be independent of the mTORC1/S6K axis and its regulation is unaltered in cells lacking S6K1 and 2 (PMID: 15060135). Ergo, using p-S235/236 on rpS6 as a readout of mTORC1 activity may be problematic.We agree that there is redundancy with other kinases in the regulation of this site, and we have moderated our discussion of this readout to account for this.Reviewer #2 (Recommendations for the authors):1. Does the amount of nutrients/growth signals added affect total expression of TFEB fusion proteins and mutants? Is this taken into consideration in the calculation of C/N particularly during prolonged periods?We show a plot of the total amount of TFEB reporter  in Figure S1F-G. In general, changes in reporter expression are on a much more gradual time scale (6 or more hours) and do not have a significant impact on the ratio of cytosolic to nuclear intensity (C/N), which as a proportion inherently adjusts for the total amount of reporter as long as there are no saturation effects. We also observe no evidence that the reporter is saturating within any of the conditions we have tested.2. The findings in Figures 4 and 5 that TFEB-C/N responds to amino acids despite absence of insulin should be verified for the presence of insulin/growth factor signaling. According to the Methods, cells were starved for 6 hr. There could be residual insulin/PI3K signaling, thus allowing TFEB response upon amino acid addition. They should examine PI3K/Akt signaling. Would this aa response still occur upon inhibition of PI3K or Akt? What happens when starvation is prolonged to 12-16 hr before re-addition of nutrients?Data addressing this point can be found in Figure 3B and Figure 3 supplement 1 – C. Please see our response to this comment under point 2 of the essential revisions section for more details.Reviewer #3 (Recommendations for the authors):This is an interesting manuscript from Sparta and colleagues that investigates dynamics of MTOR and TFEB signalling.While this reviewer appreciates the elegance and power of the experimental system, and the substantial amount of data in the manuscript, we did not find that the data provide enough clear insight into the mechanisms of MTOR regulation or signalling to justify publication in eLife. For publication as a full-length paper, it would seem the narrative would need to be re-structured to highlight how findings illustrate novel insights with relevance that can be appreciated by a broad audience. The paper should be revised to focus on a smaller number of specific biological questions that could be addressed by the single cell signalling data.For example, the authors were able to demonstrate that the truncated TFEB-TR reporter behaves similarly to full length, but that a 3xSA-TFEB-TR lacking GSK3beta modification sites was poorly responsive, but these are not significant enough advances for the field.We thank the reviewer for their careful assessment of the paper and the recognition of the paper’s strengths. We agree with this criticism of the original manuscript, where we did not fully articulate the new insights for a general signal transduction audience. We have now restructured the manuscript and rewritten the Introduction section to better emphasize what quantitative data were lacking in the mTOR field and how these data would be necessary to understand mTOR’s function as a regulator of homeostasis. We have also performed a substantial in-depth analysis of our multiplexed dataset  in which we ask how mTORC1 activity relate to protein synthesis. In this analysis, we show that while mTORC1 readouts are sufficient to almost completely explain protein synthesis activity on average, these readouts only partially predict protein synthesis rate within individual cells, implying the existence of additional regulators that correlate with mTORC1 on the bulk level but not within single cells. We also show that normalized mTORC1 targets are poor individual predictors of protein synthesis within single cells but are highly synergistic, implying that control of protein synthesis by mTORC1 is achieved by regulation of a network of factors rather than any individual downstream effector. Both of these insights are important for fully understanding how mTORC1 acts as a homeostatic regulator of cellular anabolic activity, and we think they will be essential for future efforts to quantitatively model cellular homeostasis.


This paper introduces an enhanced energy auditing process including a ring-fencing mechanism for the selection of energy efficiency measures considering a multi-stage planning of the energy retrofitting project. The proposed ring-fencing approach enables SMEs to overcome the barrier of lack of capital for the implementation of energy efficiency measures by implementing first no-cost measures and only after that energy savings are accumulated considering the installation of low-cost, medium-cost and finally high-cost measures. The advantages of the proposed methodology are illustrated by means of three case studies, where a variety of energy efficiency measures were first identified throughout the auditing process involving three different SMEs, then most effective measures were selected and scheduled to be implemented according to a multi-annual plan while considering budget and operational constraints. The results of the pilot studies show that the business owners have improved their decision-making with respect to energy efficiency upgrades by engaging in the auditing process and accepting the recommendations about the suggested interventions to maximize financial  benefits. IEA, 2017). SMEs of European Member States would play a vital role towards achieving combined 32.5% improvement in energy efficiency by 2030 as outlined in Energy Efficiency Directive targets. Small and Medium Enterprises (SMEs) have enormous potential to save energy as they account for 99.98% of European enterprises  to date, resulting in harnessing only a small energy saving potential of SMEs. The elimination of the costs associated with energy auditing and the availability of a professional auditor going on-site have been considered fundamental to increase the uptake of energy efficiency measures from SMEs . As the result of an online survey, the project team identified a list of barriers/challenges being faced by SMEs that are preventing them to implement energy-efficiency upgrades.D2.3) of the project.One of the European Commission’s Horizon 2020 projects,From the above figure, it is clear that a considerable percentage of SMEs agreed that lack of finance is one of the major barriers for them to implement suggested EEMs.When possible, building owners would prefer self-financing to avoid the financing costs. Furthermore, many small energy efficiency projects  are difficult to finance because of lack of interest of ESCOs and the high underwriting costs for lenders with respect to the capital investment . The SPEEDIER project has highlighted that this barrier also exists in the industrial sector and among various types of SMEs.In the residential sector, the lack of funds for the upfront cost of an energy efficiency retrofit project is one of the key barriers for owners and occupiers in Ireland , then big investment (medium-cost EEMs) and finally bigger investment (high-cost EEMs). To summarize, this funding mechanism recommends starting from implementation of no-cost EEMs first and then ring fence the saving achieved from each stage of ECM implementation for higher investment grade EEMs implementation. In this iterative cycle, cost savings are determined against agreed baseline for each stage of implementation. The achieved savings are reinvested for implementation of additional measures.In  , even though other ranking criteria could also be considered within the considered cost groups (sub-ranking criteria), such as the payback period, the COD5.2) of the SPEEDIER project.The ring-fencing mechanism is being implemented within the business through introducing SPEEDIER Service, which has defined several stages of SPEEDIER Service implementation: Engage, Identify, Implement, Review and Repeat.2 emissions), and the budget constraints as the factors determining the choices of the decision maker  and building 1 has 3,779 m² of floor area, building 2 consists of 1,350 m² of floor area and building 3 (purchased in 2021) consists of 2,790 m² of floor area. The total floor area on site is 5,129 m² (2020) and 7,919 m² (2021).The purpose of energy billing analysis is to help understand the site’s yearly usage and how seasonal changes can affect energy usage. There are two main headings: electrical usage and fossil fuel usage. Electrical usage is taken from the on-site Electricity Supply Board (ESB) main incomer and appears on each monthly or bi-monthly billing.Electrical usage. The final electrical consumption (TEFC) for 2020 is calculated to be 528,410 kWh , which is equivalent to 171 tCO₂ emission as shown inThermal usage. Natural gas is used as the fossil fuel on site. The monthly billing for period January 2020 to December 2020 was analysed, and the estimated monthly usage was obtained. Natural gas usage accounts for 100% of total thermal final consumption (TTFC) on site and was calculated to be 713,989 kWh FY2019/20,  which is equivalent to 146 tCO2 emission as shown inTotal energy breakdown. Combining fossil fuel and electrical consumption a breakdown of cost, carbon emissions and total primary energy required. Total Primary Energy Requirement (TPER) is a measure of energy consumption that also accounts for the energy that is consumed and/or lost beyond the boundary of the considered organisation, notably in generating and distributing the electricity that is used by the organization .Business-1 has two electricity meters installed. Electricity is responsible for 43% of total site consumption (TSC), with 57% associated consumption to fossil fuels . Total primary energy demand is split 56:44 with electricity predominantly. This is due to the conversion factor ) is 1.89 for electricity, due to transmission losses on the grid, and most fossil fuels having a TPER to TFC conversion factor of 1.1. Cost is broken down 58% electrical, 42% fossil fuel  and carbon emissions are split 54:46 electricity to fossil fuels Electrical consumption accounts for the largest portion of energy consumption and so can yield the highest savings.Energy consuming equipment. Building 1 is heated by two gas fired 80 kW boilers (one on standby). Building 1 has a mixture of office sections and manufacturing sections. For the office sections heat is suppliedvia radiators in older section and radiant panels in the newer section. There is a thermostat for the old section of offices and for the new section of offices there are five different thermostats.All buildings are on a time schedule of 07:00–17:00 h Monday to Friday and 07:00–12:00 h on Saturdays.Within the office areas of building 1 there are two air handling units (AHU) that ventilate air throughout these spaces. Within the manufacturing areas of building 1, 2 and 3 there are mechanical fan units that circulate air. There are also mechanical fans throughout the toilet facilities. All are operating on a time schedule pre-determined by the BMS system on site.There are four split air conditioning units that service building 1, three single outdoor units (ODU) provide cooling for the older part of building 1 and one double ODU providing cooling for the new section of building 1. There are other air conditioning split units and chiller units on site but are only used for manufacturing purposes.Lighting in building 1 consists of 60% fluorescent fittings consisting of 4×22-W FL tubes in office areas and twin 58-W FL tubes in manufacturing areas. The remaining 40% lighting of building 1 consists of 32-W panel LED in office areas and 53-W mega man LED fittings in manufacturing areas.Building 2 consists of 100% LED in manufacturing areas (same as in building 1) and fluorescent lighting in office areas (same as building 1). Building 3 is 90% fluorescent in manufacturing areas and 100% fluorescent in office areas. All lighting is controlled locally.There is a large amount of equipment on site, primarily used in manufacturing processes. These other pieces of equipment will not be part of energy measure consideration as the bespoke nature of the business and equipment rules them out. There is a lot of office equipment used on site, including printers, monitors and PCs. These pieces of equipment will be the main focus for a behavioural change energy measureThe SPEEDIER expert suggested a suitable list of energy saving measures to business-1 and presented a five-year implementation plan for same. Some of the EEMs were not selected and agreed by business-1 for the implementation plan. Below is the detailed list of suggested and agreed EEMs along with reason of non-agreement for some of the non-selected EEMs.No-cost EEMs. Five no-cost EEMs were identified, and business-1 agreed to implement three of them. The EEMs which were not selected are change the electricity supplier and increase the maximum import capacity (MIC) by 30 kVA. Since business-1 had changed their supplier just before the energy assessment so changing electricity supplier would have not been possible. Indeed, the literature has described relational switching costs as the costs which are mainly associated with the loss of personal relationships and emotional bonds with employees of the current energy supplier. In fact, when a relationship with an energy supplier ceases to exist, although no cash goes lost, the outcome of such investments such as time and expenses which have already spent while developing the relationship, are lost  and future expected energy consumption, the financial controller of the business-1 did not agree to change the MIC. However, the agreed EEMs selected to implement were related to building heating system. The list of selected EEMs is shown below inFrom Year 1 over €5,327 can be saved and used to implement low to mid cost measures.Reduce thermostat set pointBERIreland.ie. As the gas is only being used for heating in the manufacturing floorvia the two blower units in buildings 1 and 2 and also the office areasvia radiators the total thermal consumption will be reduced.Small changes can start the road to a sustainable future. Reducing the thermostat by 1°C during the heating season within the office and manufacturing areas is estimated to reduce heating costs by 2% as a low estimate, it is stated to reduce energy costs by up to 10% according toReduce time schedule of heatingAnother quick and easy savings option is the reduce the number of hours of heating on site. Turning off the heating an hour before close or an hour before that can yield significant savings. A behavioural change campaign to wear jumpers and jacket days accompanies this measure well, as telling staff the reasoning behind the changes, the understanding can be used in their own homes and also encourage people to be more energy efficient.Boiler set point optimisationIf operating temperatures are reduced, typically to 50/30°C, or the temperature differentials can be widened to offer a lower return temperature (80/50°C) then there are significant efficiency gains to be had. A lower return temperature provides greater opportunities for condensing boilers to actually condense. A condensing boiler needs the return temperature to be as low as possible, and at 30°C a condensing boiler would theoretically obtain 95% efficiency. Turning down the boiler set point to 50°C, and reducing the flow rate of the main header pump will optimise the condensing gas boiler.Low–mid-cost EEMs. Low–mid-cost is any measure under EUR 1000. One low–mid-cost ECM was identified shown below inTotal costs for low-cost measures amounts to EUR 300 with yearly savings of EUR 118,  and can be used with no-cost measures to springboard the implementation of high-cost measures.Reduce fan speed of air handling unitAir handling units are normally designed and sized for maximum occupancy of the area they are ventilating. Occupancy has a huge effect on the amount air changes per hour required, CIBSE guide A: Environmental Design states that 10 L/s per person is the suggested and tried and tested requirement for air handling units. Occupancy-driven air handling units are more energy efficient, less likely to breakdown and provide better control for human comfort levels.High-cost EEMs. Business-1 was suggested five high-cost EEMs. After a series of conversation and exploring various procurement options they agreed to implement two of them. EEMs which were not agreed are installation of renewable energy generation , installation of electric vehicle charger and electrification of heating system . Regarding all of these EEMs, business-1 found none of the procurement options suitable for them. So, business-1 agreed to consider implementation of these EEMs in future and will keep exploring and find a suitable procurement options. High cost is any measure over EUR 1000. The list of selected and agreed to implement EEMs are presented inThe discounted payback period of this measure was calculated to be 4 years.SEAI, 2022) are available for the measures listed which will reduce the payback period.High-cost measures are more focused on carbon abatement rather than cost savings. SEAI grants . Monitoring and targeting have been found to have multiple benefits for industry, such as:Energy cost savings, typically 2–5% due to the ability to identify and rectify excessive consumption as it occurs;Improved data for justifying capital investment;Improve operations and maintenance (O&M)Improved budgeting due to better prediction of future energy consumption;Waste avoidance, not just energy, also water and materials;Ability to benchmark against competitors and sister companies;Measurement and verification of savings from capital investment activities; andCompliance with ISO 50001 requirements.A facility’s energy performance can be better understood by comparing it against similar facilities of the same typology, performing a detailed analysis to identify high intensity of energy consumption, high baseload consumption, energy consumption when the facility is unoccupied on night-time on weekdays, energy consumption when the facility is unoccupied on night-time on weekends, and energy consumption when the facility is unoccupied on weekends. Such a detailed approach supports a better decision-making for energy efficiency investments than the traditional analyses based on aggregate monthly or annual data .Lighting upgradeMost areas at business-1 have LED lighting already installed in buildings 1 and 2, but there are some lighting fixtures that are still fluorescent. The acquisition of building 3 has brought an opportunity to carry out a mass change over measure. With building 3 having a good quantity of FL light fixtures. From site visits there was 60% of building 1 with FL fixtures, and 90% of building 3 having FL fixtures.There is assumed to be 270 fixtures ready to change, with a nominal fixture being the twin 58-W T5. The replacement fixture 53-W LED has a lot of savings attached. An assumed cost per fixture and installation cost brings the project to EUR 20,000–25,000. The yearly savings were calculated at EUR 5,958.21 per year or alternatively 17 tonnes of carbon per year. The discounted payback period of this measure is calculated to be 4.5 years.If all measures were introduced the site would perform:14% more energy efficient year on year,carbon saved will amount to 13% of site carbon emissions,and a 13% reduction in energy costs.Implementation planThe yearly implementation plan for the next 5 years suggested by the SPEEDIER expert for business 1 is depicted in belowSPEEDIER expert suggested to implement no-cost measures (reduce thermostat set point by 1°C and reduce heating time by 1 hour) in year 1, whereas business-1 agreed to invest their own capital and implement monitoring and targeting system in year 1 as M&T implementation will yield more savings in coming years. Set-point temperature of thermostat has a significant impact on heating (and cooling) energy consumption . In yearBusiness-1 wanted to reduce the boiler set point temperature by 1°C after getting a full understanding of energy usage and pattern through installed monitoring and targeting system in year-1. In year 3, business-1 agreed to implement lighting measure and change the fluorescent bulbs with LEDs of same lux level.Business-1 also agreed to installation of renewable energy generation , installation of electric vehicle charger and electrification of heating system  in years 4 and 5. But they didn’t find any suitable quotation and procurement options at this point of time to provide commitment for ECM implementation, indeed they agreed keep exploring a suitable quotation and implementation of these EEMs in future. Business-1 faced the barrier of hidden costs (lack of time and/or management costs) associated with the procurement of PV-panel and heat pump measures . So,FigBelowThe second case study is a business based in Ireland and will be called business-2, here after. Business-2 is a goat’s cheese manufacturing small enterprise based in Ireland, which has been producing cheese since 1999 from their manufacturing plant. There are five full-time staff; and during the summertime the staff can increase to 15. Manufacturing is the main type of end use on site. In 2019, business-2 produced 35 tonnes of product, from an average herd of 300 goats.The purpose of energy billing analysis is to help understand the site’s yearly usage and how seasonal changes can affect energy usage. An electricity meter, 20 solar PV panels and fossil fuels are used to satisfy the energy needs of business-2. Electrical usage. Business-2 has an annual total electrical final consumption (TEFC) of 72,505 kWh accounting for 64% of total site final consumption (TSFC). The solar panels produce 3.6% of imported electricity with scope for further increasing the PV array. Electricity for 2019 was calculated to be 69,967 kWh , which is equivalent to 23 tCO2 emission as shown inThermal usage. Fossil fuels are provided by kerosene and LPG boilers with an annual total thermal final consumption (TTFC) of 40,822 kWh (36% of TSFC). It is noted that on-site there is a 1000-L LPG bulk tank unconnected to the LPG boiler, hence why there are bottled LPG deliveries throughout the year. Kerosene usage on site accounts for 96% of total fossil fuel usage on site for year 2019, with 4% accounting for LPG.Total energy breakdown. Combining fossil fuel and electrical consumption (with supplementary PV array) a breakdown of cost, carbon emissions and total primary energy required as shown inElectricity is responsible for 63% of total site consumption (TSC), with 37% associated consumption to fossil fuels . Total primary energy requires is split 75:25 for predominantly electricity. This is due to the conversion factor (TPER to TFC) is 1.89, due to transmission losses on the grid, and most fossil fuels having a TPER to TFC conversion factor of 1.1. Cost is broken down 82% electrical, 18% fossil fuel  and carbon emissions are split 68:32 electricity to fossil fuels  as shown inElectrical consumption accounts for the largest portion of energy consumption and so can yield the highest savings.Energy consuming equipment. The main building is heated by a single fire bird kerosene boiler, estimated to be 26 kW. Some of the spaces are heatedvia underfloor heating at a set point of approximately 20°C. Hot water is heatedvia an ACV cylinder with approximately 500-L capacity at a set point of 60°C.via under floor heating (60 m²). This system is temperature controlled and time controlled. It was noted that the thermostats for space heating were faulty or in need of repair. There was a substantial insulation project undertaken within the last 3 years that focused primarily on cavity insulation and attic insulation.Space heating is providedThere are seven domestic hot water outlets that see sporadic use throughout a working week. The time schedule for hot water is set to Monday to Friday  and Saturday and Sunday (07:30–10:00 h). One of these outlets (milking machine) uses an electric element for domestic hot water and is used typically twice a day for 10 minutes, Monday to Friday.Process hot water is heated by an 18 kW electric immersion for use in the pasteurising process typically used for 3 times a week for 2 hours. Changes in pasteurization are process related energy efficiency measures, which can be applicable to specific dairy processes.via a plate heat exchanger.There is no air conditioning for comfort cooling on site. There are direct expansion (DX) units on site, but these units are used for the ice bank, fridges, and freezers, and four cold rooms. The ice bank is cooled by a DX unit; cold water then chills milk from the milking machinevia ON/OFF light switches.Lighting is a mix of surface mounted fluorescent tube fittings and surface mounted LED fittings. The yearly lighting energy is assumed to be 700 kWh. The lights are locally controlledThere is a large amount of equipment on site, primarily used for cheese manufacturing. The list of equipment is shown below inNo-cost EEMs. Four no-cost EEMs were identified and out of four suggested EEMs changing energy supplier was not agreed because they switched the energy supplier just before the SPEEDIER expert performed energy assessment at business-2. The list of selected EEMs is show below inFrom year 1, over EUR 2,500 can be saved and used to implement low cost measures.Night-time coolingIreland Climate overview, 2021).Assuming that the ice box is used only when it is needed. The ice box should be placed on a timer, so that it only cools at night. This will reduce costs by taking advantage of the cheaper night rate and saving energy by taking advantage of the cooler temperatures during the night .PHE ratio between milk and water is below 1:3, the cooling potential of the PHE is not being met. To reduce the cooling requirement, ensure that 3 times the amount of water enters the PHE. This can reduce cooling times, and reduce energy used by both pumps .Walk in fridges and Freezers need regular cleaning (2–3 a month) making sure that the evaporators are not being blocked. It also ensures that only the product is cooled. Fridges should have enough room for the air to circulate within, while freezers should be filled to about 70% to reduce the amount of air to cool  and can be used to springboard the implementation of high-cost measures.PHE insulationAlfa Laval, Gasketed Plate Heat Exchanger, 2021).Insulation of PHE and piping is essential to reducing heat loss (in heating systems) and heat gain (in cooling systems) inefficiencies throughout the system  should also be placed on each to identify when the temperature has gone over the optimal cooling temperature and how often .There is already an LPG boiler and an LPG 1000 L bulk tank on site. LPG has a lower g CODomestic hot water optimisationSEAI, 2019). When using hot water this can significantly reduce the amount of hot water required for hand washing, washing down areas which in turn will reduce the need for the boiler to heat water.Conventional wand wash basin aerators use more water than required, specialised aerators can save you up to 75% water compared to using the standard aerator . A pictorial representation of M&T system is shown inMonitoring and targeting has been found to have multiple benefits for industry, such as:• Energy cost savings, typically 5–15%, due to the ability to identify and rectify excessiveconsumption as it occurs;• Improved data for justifying capital investment;• Improved budgeting due to better prediction of future energy consumption;• Waste avoidance, not just energy, also water and materials;• Ability to benchmark against competitors and sister companies;• Measurement and verification of savings from capital investment activities; and• Compliance with ISO 50001 requirements.The yearly implementation plan for the next 3 years suggested by the SPEEDIER expert for business-2 is depicted inThe SPEEDIER expert suggested to implement no-cost measures  in year 1, low-mid cost measures  in year 2 and high-cost measures  in year 3.Business-2 agreed to invest their own money as capital cost for some of the low–mid-cost and high-cost measures in year-1 and suggested to implement measures related to plate heat exchanger (PHE) altogether in year-2 . After understanding the benefits and importance of monitoring & targeting system, business-2 also agreed to invest their capital cost and install monitoring and targeting system in year 1. Changing energy supplier was not agreed because they switched the energy supplier just before the SPEEDIER expert performed energy assessment.So, in year-1, business-2 agreed to implement below EEMs: -1. Boiler replacement (low-cost)2. Night time cooling (no-cost)3. Walk in fridge evaporator cleaning (no-cost)4. Walk in fridge sealing replacement (low-cost)5. Monitoring & targeting system (high-cost)In year 2, business-2 agreed to implement some low cost and some no cost measures. So, in year 2 business-2 will implement below EEMs: -1. Thermostat replacement2. Domestic hot water optimization3. Plate heat exchanger optimization4. Plate heat exchanger insulationBusiness-2 didn’t agree to install VSD because they are planning to replace the pump unit in the coming years, hence investing for VSD installation would be redundant by the time of pump replacement. The third case study refers to a small business in central Italy with revenues between EUR 2 million and EUR 5 million. The business owner engaged with the SPEEDIER expert to undergo through an energy audit. They have implemented a temporary monitoring system  to be usThe auditing process followed with the SPEEDIER expert has comprised a five-step process. Initially, the business owner and the SPEEDIER expert have defined the scope of the auditing (step 1). After that an energy assessment including the billing analysis and the creation of a register of opportunities was performed (step 2). The selection of wanted energy efficiency measures (EEMs) was followed by their implementation (step 3). With the EEMs installed, energy consumption was monitored to evaluate the savings with respect to the baseline energy consumption (step 4). After evaluating the benefits, the business owner with the help of the SPEEDIER expert decided whether to repeat the whole process from the first step (step 5). The SPEEDIER auditing and consultancy service was delivered with the agreement that if the business owner decided not to execute the energy efficiency plan, he should be obliged to pay a consultancy fee. On the other hand, if the energy efficiency savings are obtained, they can be used to repay the consultant. This represents a strong incentive for the customer to commit with the energy efficiency project execution. Eleven EEMs were considered . They maChallenge, 2002). Moreover, an improved system management by means of installation of a new control unit will reduce the fluctuations in the air production, will establish a stable pressure, will improve the efficiency in the production of compressed air, will increase the overall stability of the system resulting in a better-balanced system  . AnotherIn the current state-of-the-art energy management, energy auditing and implementation of energy efficiency projects are based on the PDCA (Plan-Do-Check-Act) cycle  which enabled to achieve energy efficiency improvements and cleaner production in SMEs . In the This paper has presented a novel energy efficiency auditing process for SMEs including a ring-fencing mechanism for multi-stage planning of EEMs, which enables to finance the installation of new measures using the energy savings delivered by the EEMs previously implemented, which are accumulated until the end of the energy efficiency project. The ring-fencing model has been validated on three case studies which were part of the pilot demonstrations developed in the H2020 project SPEEDIER. The case studies show the effectiveness of the ring-fencing model in driving the selection of the most appropriate EEMs throughout multi-annual retrofitting projects. The auditing process delivered has been successful in convincing the decision-makers that a planning over multiple periods of EEMs’ installation is advantageous to overcome the barriers related to the limited access to capital, with respect to the case where the selection of the technologies and the investment on them is performed at once for the considered planning period. Furthermore, the auditing process substantiates the finding that decision making for energy efficiency in SMEs shows a complex decision structure and that decision-makers may not be able to optimise their choices because of lack of time, attention, and the ability to adequately process information. The ring-fencing method investigated in this paper is a tool to help overcoming bounded rationality issues  and improving opportunities for achieving the selection of the right EEMs among all those available, such that financial benefits are maximized .D9.4.The case study data is available as public deliverable of the project, which can be accessed via SPEEDIER websiteEthical approval and consent were not required. The manuscript investigates the impact of implementing a ring fencing mechanism approach to overcome financial barriers that companies face when implementing energy saving measures. The study's main contribution is to understand the implications of such methods in real case studies.  In current research practice, several studies already exist that investigate the implications of financing in energy saving projects. Overall, the study is well written, clear and tackles an important problem of financing mid to long-term projects. However, the only concern is the lack of clarity on how the energy savings have been quantified.  The paper could be improved if the previous concern is answered; thus, minor technical rework is necessary before it can be considered for indexing. Although most of the literature is appropriate, the introduction section could show cases when typical financing methods are implemented. This will provide more information about the limitations of such mechanisms, especially when applied to SMEs. Despite the work carried out, originality needs to be further explained or enhanced. The results section shows interesting results considering the chosen methodology. The section provides a concise discussion section reflecting a good understanding of the modelling outputs. The authors provide a critical reflection of the three case studies by providing insight for policy and decision-makers under the current economic environment.Where applicable, are recommendations and next steps explained clearly for others to follow? (Please consider whether others in the research community would be able to implement guidelines or recommendations and/or constructively engage in the debate)YesDoes the article adequately reference differing views and opinions?PartlyAre all factual statements correct, and are statements and arguments made adequately supported by citations?YesIs the rationale for the Open Letter provided in sufficient detail? YesIs the Open Letter written in accessible language? YesReviewer Expertise:Energy modelling, energy efficiency building sectorI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors propose a methodology to improve the implementation of energy efficiency measures in SMEs that perform energy audits. However, in order to be finally indexed, the paper should attend some questions related below. Abstract: The barrier of lack of capital for the implementation of energy efficiency measures can extendable to private developers, resident associations, small public administrations, etc. Accordingly, if only SMEs are referred, the explanation to this constraint should be justified or explained. In addition, the proposed ring-fencing approach should be better summarized. It can be noted that some measures may be very beneficial economically but not environmentally (good but not so good). And vice versa. Therefore, economic and environmental aspects should initially be treated separately. Introduction: References to justify the low participation of SMEs in energy audits should be updated, as the authors should ensure that this trend remains low 12 years later. The gap (to be solved) should be better highlighted. For instance, cost-optimal energy retrofit proposal provided by auditors could increase the rate measures proposed vs measures implemented. The quality of the Figure 1 should be improved because the factors are not ordered in the same way in all countries and some percentages are not clear. Many references date back 10, 11 or 12 years. In these years property owners and occupiers may have changed their position due to the energy context . And in the last 2 years, even more so. Energy efficiency projects reduce the amount of consumed energy but also make the consumption not eliminated come (partly) from renewable sources. ISO 50001 should be taken into account since it was conceived to provide a framework for more efficient use of energy. Ring Fencing Mechanism: The paper does not analyze the compatibility of these sequential proposals. This way of ordering interventions discriminates (or may discriminate) against those options that require larger investments to begin with. Are all larger investments ruled out? Larger by how much? Some measures are independent, but others are linked to previous decisions, and are made with future decisions in mind. While the idea sounds good, these drawbacks should be addressed. Methodology: This section should be joined to the previous one. The use of case studies as a methodology is widely used in the literature, but in this paper, the authors have not sufficiently justified and discussed for it. How they are chosen, how many are chosen, and what their characteristics should be . Case studies: Decision making about measures selection should be provided. This enhances the testing of the usefulness of the methodology. There are no alternatives to be followed. The methodology does not help to analyze which measures are independent and which ones are dependent. In the second case, alternatives should be raised. Time-dependent graphs should be presented that help to see how savings from the first measures can be used as available capital to undertake the next measures, predicting (through simulations) how much additional capital (if required) is needed to undertake each of the measures chosen from the proposed measures. In this regard, information provided in Figure 16 (for instance) could be a good initial point. Many data from this section could be laid out as supplementary material because its length is not proportional with the rest of the paper. Discussion: Cost-optimal and financial-optimal approaches are not considered to be compared with the chosen one (the proposed by authors). I mean, Is the best choice to begin always by the cheapest? Different sequences may be proposed, each of one can provide different savings distributed along years, helping or hindering future investments. The final savings through the remaining lifespan of the building should also be taken into account. Conclusion: Which are the improvements into the 3 case studies? The energy efficiency has improved, but no specific nor measurable findings are included at the end. Limitations are not considered. Future research is not suggested.Where applicable, are recommendations and next steps explained clearly for others to follow? (Please consider whether others in the research community would be able to implement guidelines or recommendations and/or constructively engage in the debate)PartlyDoes the article adequately reference differing views and opinions?PartlyAre all factual statements correct, and are statements and arguments made adequately supported by citations?PartlyIs the rationale for the Open Letter provided in sufficient detail? PartlyIs the Open Letter written in accessible language? YesReviewer Expertise:Energy Efficiency in Building, Building Technology, Sustainable Architecture, Green Building, Bioclimatic ArchitectureI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The article covers an important topic of how to encourage SMEs to invest in energy savings measures. It presents research to show the barriers that exist to this, and proposes a methodology that can provide a staged approach to energy management investment. The financial savings that accrue from low and no-cost measures can be used to pay for future higher cost measures. What is missing is an explanation of how such savings can be ring-fenced in practice. This may be a purely accounting exercise, or might involved money transfers to identified holding accounts. SMEs will have different methods for dealing with capital investments, and it is important that the accrued savings are clearly marked for future energy efficiency investments if this is to be successful. It would be helpful if this article could outline how this ring-fencing occurs. The other missing piece is how the measurement and verification process should be implemented. This can be relatively expensive and is not a trivial exercise. The article should indicate which party is responsible for conducting this, and what level of costs are likely to be involved. Without these additions, any practitioner would have to decide on this approach for themselves. International Standards such as ISO 50015 would be important to mention in respect of the latter point. In a number of places, the language is difficult to follow. Overall the message is clear, but some arguments are a little difficult to follow. A thorough edit is advised.Where applicable, are recommendations and next steps explained clearly for others to follow? (Please consider whether others in the research community would be able to implement guidelines or recommendations and/or constructively engage in the debate)PartlyDoes the article adequately reference differing views and opinions?YesAre all factual statements correct, and are statements and arguments made adequately supported by citations?YesIs the rationale for the Open Letter provided in sufficient detail? YesIs the Open Letter written in accessible language? PartlyReviewer Expertise:Energy and carbon management and the application of renewable energy in buildings and the built environment. Energy monitoring and targeting, and measurement and verification. Optimisation of mechanical and electrical building services systems.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Recent advancements in additive manufacturing have made 3D design a desirable skill in combating the historically slow development of biomedical products. Due to the broad applicability of additive manufacturing to biomedical engineering, 3D design and 3D printing are attractive educational tools for biomedical engineering students. However, due to the multidisciplinary nature of biomedical engineering, finding a suitable spot in the curriculum to teach students basic and application-based skills for 3D manufacturing is difficult. Furthermore, prior training in fundamental 3D design skills may be needed to support the use of application-based supplementary content.We designed a SolidWorks Simulations toolkit to complement a sophomore (2nd-year)-level Biomechanics course and distributed this assignment to students with and without prior training in 3D design delivered in an introductory biomedical engineering course. Using short videos, example-based problem solving, and step-by-step tutorials, students completed this as an extra-credit assignment and completed a survey gauging student opinion on SolidWorks and 3D design, confidence in each target skill, and the effectiveness of assignment delivery. The compilation of survey responses suggests that the assignment effectively increased positive responses in student opinion on interest in and likeliness to use SolidWorks in both groups. However, confidence in the target assignment skills was higher in the trained group and fewer problems occurred in operating SolidWorks for trained students. Further, analyzing the distribution of student grades with respect to survey responses suggests that responses had no relationship with initial class grade.These data collectively indicate that prior training provided to the students had a positive impact on the effectiveness of the assignment although increases in student opinion on the utility of 3D design were observed in both trained and untrained students. Our work has generated and identified a useful educational supplement to enrich existing biomedical engineering course materials with practical skills.The online version contains supplementary material available at 10.1186/s13036-023-00339-7. Additive manufacturing, or 3D printing, enables the cost-effective, precise, and rapid synthesis of complex 3D structures and is increasingly supplanting many conventional 3D manufacturing methods. The biomedical industry has also experienced the impact of this revolution in device and material development. In recent years, biofabrication with 3D printing has been mobilized to replicate complex physiological environments, generate implantable devices or biomaterials, and manufacture drug delivery systems. The construction of microfluidic organ-on-a-chip devices has successfully demonstrated that bioprinted constructs can produce biomimetic in vitro 3D microenvironments with translational drug discovery applications –3. FurthWith the advent of 3D printing for biomedical applications such as in biomaterial development and in complex physiological models through organ-on-a-chip, the necessity of providing biomedical engineering (BMEG) students with training in 3D design techniques is becoming an increasingly relevant concern. BMEG, as the fastest growing engineering subdiscipline, presents a difficulty in developing pedagogical methods consolidating both the biomedical sciences and engineering principles in addition to a sufficient education in supplementary necessities such as ethics . IntegraCriticisms of the restrictiveness of a curriculum contingent on 3D design elements have been raised, warning educators against restricting imaginative and innovative thinking . DesigniIn this study, we investigated the impact of preexisting introductory 3D design training on students' pre-conceived notions of CAD and the ability of students to generate 3D simulations in SolidWorks. This study was performed on a cohort of students that experienced a traditional introductory BMEG course in their freshman (1st) year and a second cohort that was given a SolidWorks introductory module in the Introduction to BMEG course. The Biomechanics sopohomore (2nd-year)-level course was the target to integrate 3D design simulation elements, which is primarily due to the problem sets for the course consisting of basic statics parameters. A simulation toolkit composed of 14 static simulations of homework problems and lecture examples was distributed with short videos guiding students through example simulation problems. Through the assignment, students would be able to familiarize themselves with SolidWorks design elements and simulations in addition to applying 3D visual learning to familiar biomechanics problems. This allows students to compare the simulation results to the analytical -by hand- solution of in-class problems due to the at times inaccurate assumptions of finite element analyses. Feedback from before and after the assignment was solicited through a pre- and post-assignment survey to gauge student confidence in operating the software, assess student interest and knowledge in 3D design, evaluate the effectiveness of the assignment delivery, and determine whether students experienced technical errors that occurred in accessing the assignment. This study aimed to accomplish an assessment of whether incorporating a SolidWorks Simulations component in the Biomechanics course could facilitate students’ ability to produce and analyze 3D models and whether previous basic 3D design training in the SolidWorks software would improve the effectiveness of 3D design integration at an ABET (Accreditation Board for Engineering and Technology)-accredited university.The target course for applying a simulation component to the original coursework was the sopohomore (2nd-year) second-semester Biomechanical Engineering course Fig. . The jusThe first cohort of students given the option to complete an extra credit assignment containing the Simulation Toolkit had worked through the original curriculum lacking SolidWorks training. However, in the second cohort of students that were enrolled in the Biomechanics course the subsequent year, an introductory basic SolidWorks component was briefly included in the Introduction to Biomedical Engineering course, which surveys the field to provide students with a broad perspective of the discipline.A second cohort of students was provided with training in SolidWorks basic modeling incorporated in their Introduction to Biomedical Engineering course, which was taken a semester prior to the Biomechanics course. This assignment was provided as an optional grade to replace their lowest homework grade at the end of the semester. The basic modeling tutorial required students to generate a functional Legos block with a fun fact about themselves written on the bottom of the model and a drawing illustrating multiple angles of the model Fig. .Fig. 2ScThe resulting Simulation Toolkit contains 14 similar statics simulations and 5 videos including a tutorial for accessing and navigating SolidWorks and three step-by-step example problems were included in a Box file and distributed to students. Box is a cloud-based file storage application that all students have access to and is an effective method of storing and sharing large files such as SolidWorks parts and simulation files. Notably, the second cohort of students that had taken the introductory SolidWorks module were also provided with written step-by-step instructions in addition to the demos based on the previous cohort reporting difficulties in following along with the video instructions, which does confound conclusions made in the post-assignment survey. Introductory demo videos instructed students on how to access SolidWorks on their computers. Students could access SolidWorks by (1) downloading the student license provided by the University on a windows-running computer or (2) through the Citrix Workspace cloud computing app. If their computer was insufficient for running the software or if they were encountering persisting errors, the students were invited to complete the extra credit assignment in an on-campus lab with computers capable of running SolidWorks. Students were also encouraged to explore and attempt running any remaining simulations provided in the toolkit.The instructions used to train students to run and analyze the example simulations was a linear process requiring critical thought at specific steps Fig. . The demWe derived the 3D SolidWorks models here from homework and lecture problems in the Biomechanics course, including moment, stress, axial loading, bending, shear, beam deflection, and static equilibrium problems. The SolidWorks Simulations software statics parameters generated by the students determine the displacement or stress values in each element of a discretely approximated object connected by a pre-defined coarseness of nodes through the finite element method (FEM). For students to define the exact bounds of an individual element, a mesh defining the polygonal representation of the object, including the number of nodes, and physical restrictions was applied by the students to the model before the simulation study was run. After running the simulation study the students independently designed and restricted, the students can determine the value of variables such as bending stress as requested by the problem statement by probing for values at specific locations. As most problems in the introductory Biomechanics course are 2D, additional approximations were made to convert models into a 3D problem in the models by the instructor, which further increases discrepancies between the 2D static solution and simulation result. These approximations were incorporated in the model supplied to students to allow them to focus on generating the simulation rather than developing a suitable model. Supports that are assumed rigid in the static solutions are assumed to be composed of the same material and simply supported objects are assumed to be geometrically fixed. Students were asked to enforce these assumptions in generating the simulation parameters and restricting the model to provide them with perspective on the degree of assumptions needed to obtain their simulation data and corresponding potential sources of simulation errors.Students were provided with three demo problems asking the student to derive support reactions, confirm that a uniform distributed load does not exceed a given maximum bending stress, and determine displacement due to a point force, respectively. Students would determine support reaction FEM estimations by defining the face at which a result force should be calculated in SolidWorks Simulations. As an example of the types of problems designed for the simulation toolkit, the first problem Fig. a is regaThe second example Fig. a is fromThe surveys and experiments conducted in this study were performed under formal approval from the University of Arkansas Institutional Review Board IRB #2,012,306,631). A survey was distributed to students participating in the extra credit assignment through the Qualtrics software . The questions included in the survey were sorted into three broad categories  while trained students had no statistical significance except for SolidWorks Simulations.From the initial cohort of students without training, 26 students out of 51 total students participated in the extra-credit assignment while the second cohort with introductory SolidWorks knowledge only had 21 participants out of 55 students. For each student cohort, participating students had a 100% completion rate. Student responses gauging the previous knowledge and experience possessed in CAD-related software and simulations were assessed to determine a baseline for each cohort. In the untrained cohort, 88.5% of students had zero prior experience with any 3D design software while 11.5% had some experience in 3D design and CAD programs . The trained cohort of students experienced a similar trend and distribution of students expressing an interest in SolidWorks and CAD. Students in the trained group decreased in negative responses from 38.09% to 9.52% after completing the assignment .Likert-like data based on responses that allowed students to rate their likeliness, interest, confidence, or difficulty in completing an assignment are represented by assigning each response a number increasing in positivity from 1 to 5. Overall, 51% of untrained students in the Biomechanics course participated in the simulation assignment while 38% of trained students participated. A larger proportion of untrained students participated in the extra credit assignment, and further in-depth analysis of the degree of interest participating students expressed was further investigated using survey data. Student interest prior to the extra credit assignment in the untrained group majorly fell within the range of no interest to a slight interest in CAD-based software Fig. . Howeverent Fig. . Positivp < 0.001)  and after (p < 0.01) assignment delivery.The question gauging the likeliness of students to use SolidWorks in their future career followed a similar pattern in the untrained group with responses indicating extremely or somewhat unlikely decreased from 65.38% to 19.23% while responses indicating extremely or somewhat likely increased from 11.54% to 65.39% (p < 0.01) and analyzing simulations (p < 0.05) in the trained group. Untrained and trained students exhibited similar confidence in creating simulations, indicating a deficiency in the assignment design for developing this particular skill. Internally, the untrained students were statistically significantly inclined to the slight or no confidence in all skillsets (p < 0.05), while the trained students exhibited no statistical significance in a Kruskal–Wallis test. These data indicate that confidence levels were more evenly distributed, but overall more positive, in the trained cohort.To diagnose the confidence in 3D design and simulation skills students had obtained upon completion of the simulation toolkit assignment, we also asked students to rank their confidence in each skill using a Likert scale. Student confidence in operating SolidWorks, analyzing simulations, and creating simulations between the trained and untrained groups demonstrated a stronger consensus in response compared to other questions. The student confidence in using SolidWorks, analyzing simulations, and creating simulations for the untrained cohort before was heavily biased toward 65.38%, 69.23%, and 57.69%, respectively, of students responding with no or slight confidence . Internally, the trained students had no statistical significance between each group while the untrained students were significantly responded positively.Students assessed the usefulness of SolidWorks Simulations, and their ability to complete the assignment, and provided additional feedback after the assignment through the post-survey. Consistent with the increasing positive opinion of SolidWorks and CAD in previous questions, 84.6% of untrained students indicated that SolidWorks Simulations is a very or extremely useful tool Fig. . The trap > 0.05).Most students participating in the study also encountered errors but were ultimately able to compare the results of the simulation with their theoretical solutions for the problem, which reduced in the trained group with a consistent 90.5% of students successfully completing the assignment with few errors Fig. . Howeverp < 0.01). This significant increase was also present between the trained and untrained groups for ease of navigating demo videos (p < 0.05). Between scores for written instructions and video demos, there was no significant difference between ease of use if the trained group is only considered (p > 0.05). However, ignoring the potential confounding variable of written instructions, the trained and untrained score for navigating video demos is significantly lower than for navigating written instructions (p < 0.05).Questions diagnosing the difficulty students had in completing and working through the assignment and the confidence of students in operating CAD-related software in the future were included to determine the effectiveness of the content delivery method. For the untrained group of students, while most responded that their ability to follow along with the demo videos was easy (50.0%), a large percentage of students also displayed difficulty in following along (34.61%), which may indicate that the videos need either a more in-depth tutorial or a slower delivery Fig. . Furtherp > 0.05) , indicating that standing in the course did not influence confidence responses for 3D design skills  Fig. . Similarlls Fig. .Fig. 13DBased on the student feedback solicited after the completion of the assignment, the errors encountered were primarily due to long runtime, the inability to access SolidWorks through Citrix, and other technical difficulties in the untrained cohort Fig. . The traStudent responses regarding previous experiences in 3D design or CAD-related software reflect the likeliness of students to independently study 3D design software. The student cohort without introductory training contained ~ 89% of students with no experience in any type of 3D design software Fig. . BecauseThe impact of initial training on the effectiveness and effect on student opinion of the assignment is exhibited in the results of the pre-and post-surveys. Previous training in basic SolidWorks part creation had no impact on students' self-assessed interest in SolidWorks pre- and post-assignment compared to the untrained group post-assignment Fig. . These dDespite the positive student responses in likeliness to use and interest in SolidWorks post-assignment, the reported confidence in operating SolidWorks and SolidWorks Simulations was observed to be low in both the trained and untrained groups Fig. . In the The demo videos provided through the assignment were distributed in a Box file that students could additionally use to download the SolidWorks part/assembly and Simulations files. Based on the difficulty students had in navigating the assignment format Fig. , a more Linear regressions between final grades in the course and the student confidence reported for each 3D design and simulation skill demonstrate that confidence is not associated with in-class performance. Therefore, the assignment effectiveness was not dependent on whether the student was performing well in the class and could target students at all stages Figs.  and 14. We designed a SolidWorks Simulations Toolkit that was developed for a sophomore (2nd-year) biomedical engineering course to enrich existing content with 3D design applications for students with and without explicit SolidWorks basic training. The Simulation Toolkit effectively demonstrated the benefits of SolidWorks Simulations with a sufficiently short tutorial that increased positive student opinion in both cohorts. The untrained group of students was more significantly impacted by the assignment with a more severe opinion shift post-assignment, but positive opinion was overall greater in the trained students. Despite an increased interest in and likeliness to use the software, student confidence in operating SolidWorks and SolidWorks Simulations remained low in both groups. Students with previous training felt more competent with SolidWorks but expressed a similar lack of confidence as the untrained group in SolidWorks Simulations. The assignment delivery method could be the cause of low confidence due to both groups reporting that the instructions and tutorials lacked sufficient detail that would facilitate their ability to extrapolate this skill elsewhere. However, notably, students in the trained cohort found navigating and completing the assignment easier compared to the untrained group, which was further reflected in an increase in positive comments. Without the initial training in part design and general SolidWorks tools, the assignment yields little tangible benefit for the untrained group aside from an increased interest in using SolidWorks in the future. Further, we were able to demonstrate that grade distribution had no correlation to confidence responses, which suggests that students at all skill levels were engaging with the assignment. These results are of interest for future investigations into students’ motivations for participating in the extra credit assignment.st and 2nd-year Biomedical Engineering courses improves student opinion of 3D design software and increasing student confidence in a marketable skill. Taken together, increased awareness of the utility of 3D design software and the ability to use the software can be improved through providing short basic tutorials followed by enriching existing courses with 3D design applications. However, the assignment delivery of the toolkit needs to be perfected such that the ease of assignment completion is increased compared to the current delivery method.This work confirms that implementing basic 3D design tutorials in 1Additional file 1: Survey questions.