A key factor determining the fate of individual cells within an epithelium is the unique microenvironment that surrounds each cell. It regulates location-dependent differentiation into specific cellular sub-types, but, on the other hand, a disturbed microenvironment can promote malignant transformation of epithelial cells leading to cancer formation. Here, we present a tool based on a microfluidic biochip that enables novel research approaches by providing a means to control the basolateral microenvironment of a confined number of neighbouring cells within an epithelial monolayer. Through isolated single pores in a thin membrane carrying the epithelial cell layer only cells above the pores are stimulated by solutes. The very thin design of the biochip (<75 μm) enabled us to apply a high-resolution inverted confocal fluorescence microscope to show by live cell imaging that such a manipulation of the microenvironment remained locally restricted to cells located above the pores. In addition, the biochip allows access for the force probe of an atomic force microscope (AFM) from the apical side to determine the topography and mechanical properties of individual cells, which we demonstrated by combined AFM and fluorescence microscopy imaging experiments. Taken together, the presented microfluidic biochip is a powerful tool that will enable studying the initial steps of malignant transformation of epithelial cells by directly manipulating their microenvironment and by real-time monitoring of affected cells with fluorescence microscopy and AFM. We developed a microfluidic biochip that enables one to locally change the basolateral microenvironment of epithelial cells within a polarised monolayer. Most luminal surfaces of the human body are lined by epithelia, which control the permeation and secretion of solutes in and out of organs.3,4 Through these junctions epithelial cells within a monolayer permanently receive signals ensuring their growth arrest and polarised state.3Key to maintaining a functional epithelium is the microenvironment that surrounds individual epithelial cells and determines their fate. The microenvironment is defined by soluble molecules, which are sensed by cellular receptors, and cell junctions. The latter enable epithelial cells to recognise and anchor to the extracellular matrix, such as focal adhesions, and to link to neighbouring cells, such as tight junctions and adherens junctions. Cell junctions are highly dynamic structures; their components are constantly turned over by an intracellular trafficking network that organises recycling, degradation and delivery of newly synthesised adhesion molecules.An additional important aspect is that most epithelia found in the human body do not consist of homogenous cell types, but show a patterned organisation of many cellular sub-types that fulfil specific functions. One example is the organisation of the alveolar epithelium in the lung, which consists of type I cells that are responsible for gas exchange and type II cells that secrete pulmonary surfactant. Microenvironments are pivotal for the establishment of appropriately patterned epithelia, because from them individual cells derive the signals that lead to their location-dependent differentiation into the correct sub-type.3,5,6 The outstanding importance of epithelia for cancer development is illustrated by the fact that approximately 90% of all human cancers derive from epithelial cells.5On the other hand, if for example injury or damaging environmental conditions disturb the microenvironment, or the related delicate balance between signalling and trafficking of adhesion molecules is changed by mutations, severe consequences can ensue. Then, a malignant transformation can occur through only partially known mechanisms that enables affected cells to escape their polarised and growth-arrested state in the epithelium and to start proliferating, potentially leading to cancer.7,8 as is our understanding of the very first steps of malignant epithelial cell transformation and cancer establishment.9–12 An approach to shed light on these questions would be to extrinsically adjust and control the microenvironment of selected cells within an epithelium. Whereas the apical side of epithelial cells is freely accessible, which allows utilising existing techniques to locally apply molecules through e.g. microfluidic probes13 or microfluidic pipettes,14 the situation on the basolateral side, at which the cells are attached to a substrate, is more challenging. Yet, the basolateral membrane contains microenvironment-sensing cell adhesion receptors, such as integrins or cadherins, which have a pivotal role in cancer development and progression, and also many hormones and nutrients from the blood stream are reaching epithelial cells at the basolateral side via the underlying basement membrane and connective tissue.However, our knowledge about the microenvironment of epithelial cells is still incomplete,in vitro15 and numerous biochips have been developed to reconstitute aspects of the physiological organisation of epithelia.16–19 A previously successful design is based on a porous membrane that carries the epithelial monolayer and separates two chambers or channels that allow independent access to the apical and basolateral side of the epithelium. To achieve our goal, we varied this design by using a membrane as support for the epithelial monolayer that contains only single isolated pores. Hence, only the cells that grow above a pore can be selectively stimulated with solutes from the basolateral side, thus allowing to effectively control the microenvironment of these cells. In addition, our chip is sufficiently thin to enable real-time high-resolution fluorescence imaging of individual cells with an inverted microscope. Since the mechanical properties of cells tightly correlate with their malignant potential,20–22 our chip also provides access for the force probe of an atomic force microscope (AFM) from the apical side, which can be used to visualise the topography and to mechanically characterise individual cells directly within the cell layer in parallel to fluorescence imaging.Here, we present a microfluidic biochip that enables delivering molecules in a locally confined manner to the basolateral side of cells within an epithelium and hence changing their basolateral microenvironment. Microfluidic biochips are well suited to replicate the natural architecture of tissue The biochip design is schematically presented in In order to ease the alignment between layer 1 and layer 2 during production , which typically have small working distances (<150 μm), can be used to perform high-resolution microscopy.via a pipette. In addition, the gap between the access ports on each side (layer 3) was designed sufficiently wide (>20 mm) so that the cell culture area in the centre can be directly reached by an AFM cantilever mounted on a holder.From the apical side the cells are freely accessible for exchanging the buffer manually In conclusion, the chip architecture fulfils all requirements to stimulate a confined number of cells within an epithelial monolayer from the basolateral side and to enable parallel real-time observation by fluorescence microscopy and AFM.The manufacturing process of the biochip is schematically outlined in Fig. S1.To measure the true thicknesses of layers 1 and 2, 0.1 μm fluorescent TetraSpeck beads (Thermo Fisher) were applied between each layer during production. Fig. S2AIn addition – since the architecture of the on-chip valves is non-standard and is based on the 12 μm thick membrane of layer 2 – the functionality of the valves was verified by filling the channels with differently coloured fluorescent solutions Fig. S3.2.All cell culture reagents were purchased from Thermo Fisher unless stated otherwise. Wild type (wt) Madin–Darby canine kidney strain II ) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% fetal calf serum (FCS) at 37 °C and 5% CO−1 G418 for 2 weeks and clones showing homogeneous overexpression of ML-GFP were selected. Clones were cultured for 4 days on transwell filters  and the trans-epithelial electrical resistance (TEER) as well as the distribution of the polarity markers β-catenin  and ZO-1 (tight junction marker) was compared to wt MDCK cells. A clone showing the best agreement of these parameters with wt MDCK cells was chosen and used for further experiments. These experiments also demonstrated that ML-GFP labelled apical and basolateral plasma membranes of MDCK cells.The plasmid pML-GFP encoding for the plasma membrane marker GFP tagged with a Lyn-derived myristoylation signal (ML-GFP) and G418 resistance was a gift from Christian Wunder . MDCK cells stably expressing ML-GFP were created by transfecting wt MDCK II cells with pML-GFP using Lipofectamine 2000. Next, cells were selected with 1 mg ml23 To this end, bovine fibronectin (Sigma Aldrich) was diluted in phosphate buffered saline (PBS) to a concentration of 10 μg ml−1 and a drop of the fibronectin solution of sufficient volume to cover the cell culture area (approximately 10 mm diameter) was dispensed at the chip surface and incubated for 1 h at room temperature. After washing the chip three times with PBS and filling the channels with cell culture medium, MDCK cells were seeded on the chip by dispensing 500 μl of cell culture medium containing 5 × 105 cells. The cells were allowed to attach for 1 h at 37 °C and 5% CO2 and then non-attached cells were washed away. A PDMS frame surrounding the cell culture area with 5 mm height was placed on the chip in order to form a well. 1 ml of cell culture medium was applied to the well and the cells were cultured for 3 days with daily exchange of the medium to form a polarised monolayer.For growing polarised MDCK monolayers on the biochip, the biochips were first washed with ethanol to remove soluble components from PDMS and then autoclaved for sterilisation. The cell culture area in the centre of the chip see  was coat24 (‘WAGO Controller’) using solenoid valves and a custom-written GUI in Matlab .Solutions were pumped through the channels with a microfluidic flow control system . The valves were operated by a home-build controller−1 glucose, 2 mM l-glutamine). Fluorescence imaging was performed with a Nikon A1R confocal microscope equipped with a 40× water immersion objective  and an incubator housing maintaining a constant temperature of 37 °C (Okolab). To prevent evaporation, the cell culture well on the biochip was sealed with a PDMS slab during measurement.For imaging live cells, the cell culture medium was replaced with live cell imaging medium  supplemented with 20 mM HEPES, 1% FCS, 4.5 g lBurkholderia ambifaria25 tagged with Cy5 (BambL-Cy5) was flushed into the channel at a constant pressure of 20 mbar.After flooding the flow channel with live cell imaging medium containing 1% bovine serum albumin (BSA) to reduce unspecific adsorption to PDMS, live cell imaging medium supplemented with 10 μM of the cell-binding lectin BambL from Cells were fixed by incubation with 4% formaldehyde for 15 min at room temperature. After permeabilising the cells for 30 min with PBS supplemented with 1% saponin and 3% BSA, the sample was incubated with primary antibodies for 1 h followed by washing and incubation with appropriate secondary antibodies for 30 min. Nuclei were counterstained with DAPI. The following primary and secondary antibodies were used: rabbit anti-β-catenin (Abcam), rat anti-ZO-1 (Millipore), Cy3-tagged donkey anti-rabbit (Jackson ImmunoResearch), Alexa647-tagged goat anti-rat (Thermo Fisher).versus their position within the AFM-‘field of view’. All experiments were performed on formaldehyde-fixed cells in PBS. The AFM was operated in contact mode using soft cantilevers .The measurements were performed on an AFM (JPK NanoWizard 3 BioScience AFM) that was mounted on an inverted fluorescence microscope . The objective was a 40× water immersion objective (NA 1.25). The optical images were correlated with the AFM images by applying the DirectOverlay routine from JPK. In brief, images of the cantilever were recorded at predefined positions, which allowed calibration of the optical cantilever images 23 Therefore, the cell culture area of the chip was coated with the extracellular matrix protein fibronectin to facilitate cell attachment.23 For comparison, we cultured ML-GFP-expressing MDCK cells for 3 days on fibronectin-coated glass cover slips . Therefore we checked that pores were devoid of signal from ML-GFP before each experiment and used only samples that fulfilled this condition. Here, the fact that each chip contains four copies of basolateral channels with pores significantly increased the chance to find an appropriately overgrown pore on one chip. Importantly, if multiple cells are located over the area of the pore, intact ZO-1-labelled tight junctions still formed at this area . BambL binds with high affinity to fucosylated cell surface receptors and is rapidly taken up together with the bound receptors , 10 min,x–z sections). This indicates that indeed no paracellular leakage of BambL-Cy5 occurred during the experiment, as was expected from the fact that cells typically formed intact tight junctions above the pore . When A30In proof-of-concept experiments we showed that combined and correlated AFM and fluorescence microscopy is feasible on the biochip . In the Taken together, the combined AFM and fluorescence microscopy experiments demonstrate that the chip is thin enough to enable high-resolution optical microscopy, but still provides sufficient mechanical stability for AFM-based characterisation.All cells sense and respond to their microenvironment. Polarised epithelial cells are surrounded by a specific microenvironment that is determined by neighbouring cells, the extracellular matrix, and soluble molecules, which ensures that individual cells maintain their epithelial phenotype. On the other hand, an altered microenvironment also critically influences processes like malignant transformation and escape of individual cells from the epithelium during epithelial-mesenchymal transformation. However, these initial steps of tumour formation are currently only partially understood.Here, we present a microfluidic biochip that enables for the first time to actively control the basolateral microenvironment of a confined number of cells within an epithelium. The functional unit of the biochip is a single pore on the basolateral side of the epithelium through which solutes, such as chemokines, cytokines or growth factors, can be precisely applied. Since cells always maintain a small distance of a few ten nm between the substrate and the cell membrane, also pores that are located exactly under a single cell allow a diffusive spreading of soluble stimulants, although slowed down by the extracellular matrix. Therefore, the spreading of stimulants is limited by the density of the extracellular matrix and cellular receptors that immobilise the stimulants. Hence, the concentration and duration of stimulation for a given combination of stimulant and cells should be optimised to prevent excessive diffusive spreading. In addition, as we show exemplarily in In proof-of-principle experiments we demonstrated the feasibility of changing the microenvironment of a few adjacent epithelial cells by localised application of the cell-binding lecting BambL. The lectin was subsequently also taken up by epithelial cells contacting the pore, whereas at neighbouring cells that had no pore contact, no BambL was detected. In addition, no paracellular leakage of BambL occurred. Hence, all conditions are fulfilled to adjust and maintain the basolateral microenvironment of few neighbouring cells within an epithelial monolayer.In future, the described device will enable to study tumour initiation from epithelial cells in a novel way, since it makes it for the first time possible to manipulate the basolateral microenvironment of few epithelial cells within an otherwise undisturbed epithelium. The chip was designed in a way that it allows real-time monitoring of cells with fluorescence microscopy using high-resolution objectives with high NA and low working distances and, in parallel, with AFM. It will allow, for example, the utilisation of fluorescent reporter molecules in live or fixed cells to track changes in the microenvironment or the expression of oncogenes. In addition, long-term cell culture is possible on our chip, thus enabling to directly follow the fate of cells undergoing malignant transformation.via tight junctions.In addition, the localised application of solutes to the basolateral side of epithelia opens up further experimental possibilities, such as to locally probe and image paracellular transport There are no conflicts to declare.RA-008-C7RA11943G-s001


Amino acids are essential activators of mTORC1 via a complex containing RAG GTPases, RAGULATOR and the vacuolar ATPase. Sensing of amino acids causes translocation of mTORC1 to lysosomes, an obligate step for activation. To examine the spatial and temporal dynamics of this translocation, we used live imaging of the mTORC1 component RAPTOR and a cell permeant fluorescent analogue of di-leucine methyl ester. Translocation to lysosomes is a transient event, occurring within 2 min of aa addition and peaking within 5 min. It is temporally coupled with fluorescent leucine appearance in lysosomes and is sustained in comparison to aa stimulation. Sestrin2 and the vacuolar ATPase are negative and positive regulators of mTORC1 activity in our experimental system. Of note, phosphorylation of canonical mTORC1 targets is delayed compared to lysosomal translocation suggesting a dynamic and transient passage of mTORC1 from the lysosomal surface before targetting its substrates elsewhere.DOI:http://dx.doi.org/10.7554/eLife.19960.001 Cells in all organisms must constantly measure the amount of nutrients available to them in order to be healthy and grow properly. For example, cells use a complex sensing system to measure how many amino acids – the building blocks of proteins – are available to them. One enzyme called mTOR alerts the cell to amino acid levels. When amino acids are available, mTOR springs into action and turns on the production of proteins in the cell. However, when amino acids are scarce, mTOR turns off, which slows down protein production and causes the cell to begin scavenging amino acids by digesting parts of itself.Studies of mTOR have shown that the protein cannot turn on until it visits the surface of small sacks in the cell called lysosomes. These are the major sites within cell where proteins and other molecules are broken down. Scientists know how mTOR gets to the lysosomes, but not how quickly the process occurs.Now, Manifava, Smith et al. have used microscopes to record live video of the mTOR enzyme as it interacts with amino acids revealing the whole process takes place in just a few minutes. In the experiments, a fluorescent tag was added to part of mTOR to make the protein visible under a microscope. The video showed that, in human cells supplied with amino acids, mTOR reaches the lysosomes within 2 minutes of the amino acids becoming available. Then, within 3-4 minutes the mTOR turns on and leaves the lysosome. Even though the mTOR has left the lysosome, it somehow remembers that amino acids are available and stays active.The experiments show that mTOR’s brief interaction with the lysosome switches it on and keeps it on even after mTOR leaves. Future studies will be needed to determine exactly how mTOR remembers its interaction with the lysosome and stays active afterwards.DOI:http://dx.doi.org/10.7554/eLife.19960.002 Mammalian cells maintain elaborate ways to respond to amino acid availability and a prominent sensor is the protein kinase mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) . Under pThe mechanism by which amino acids are sensed by mTORC1 is beginning to be elucidated  are at the plasma membrane or inside the cell is not completely answered . The majThe exact spatial and temporal dynamics of the activation of mTORC1 upon amino acid stimulation are not understood owing to the difficulty of generating tagged versions of the kinase amenable to live imaging that still maintains physiological localization characteristics. In addition, making stable cell lines with other mTORC1 components such as RAPTOR also results in dynamics that differ significantly from the endogenous complex (unpublished results). We have tried to overcome this problem in two different cell lines and using two different strategies. In HEK-293 cells, we have down-regulated endogenous RAPTOR while simultaneously expressing exogenous tagged constructs; in the near haploid HAP-1 cells we have tagged the endogenous RAPTOR gene with GFP. We describe here the dynamics of activation of the mTORC1 complex in these cells, with special emphasis in the HAP-1 cells. To examine whether mTORC1 responds directly to lysosomal amino acids, we have also generated a cell permeant fluorescent analogue of leucine in order to image RAPTOR translocation in response to this compound.We compared the timing of the translocation of the endogenous mTOR to a punctate compartment in response to amino acids with the phosphorylation of S6K, one of mTORC1’s main substrates . As prevAn important protein in mTOR signalling is RHEB, a small GTPase absolutely required for mTOR activity . CurrentTo examine the dynamics of mTOR in response to amino acids we tried to express subunits of mTORC1 tagged with GFP in order to follow localization in live cells. Tagging either mTOR or RAPTOR with GFP at either N’ or C’ terminus and generating either transient or stable cell lines of varying expression levels did not provide us with model systems that recapitulated the dynamics of endogenous mTOR (data not shown). One approach that was partially successful was to transfect cells with siRNAs targeting the 3’ and 5’ untranslated regions of endogenous RAPTOR while simultaneously expressing transiently in the same cells RAPTOR-GFP . In thesAn alternative approach was to introduce to the endogenous RAPTOR a GFP tag. For this we used HAP-1 cells which are derived from the near haploid cell line KBM-7 via an unsuccessful attempt at reprogramming . Using aThe HAP-1 cells are not ideal for immunofluorescence analysis because they have a large nucleus and a small cytosol. Nevertheless when we examined the RAPTOR dynamics in these cells in response to amino acid starvation and re-stimulation we saw that, whereas the protein was primarily cytosolic in starved cells, it translocated to punctate structures upon amino acid stimulation . ImportaWe then set out to examine the RAPTOR-GFP dynamics in these cells by live imaging. After a round of amino acid starvation and re-stimulation, the protein very rapidly translocated to puncta that were also positive for the co-expressed mRFP-LAMP-2 lysosomal protein . This trAn important recent observation was that amino acid-dependent mTOR stimulation is sensitive to inhibitors to the vacuolar ATPase . Indeed,Translocation of RAPTOR-GFP to punctate structures such as shown in The observed kinetic difference between RAPTOR-GFP translocation and phosphorylation of its substrates suggests either that mTOR activity persists long after the complex has come off the lysosomal surface or that de-phosphorylation of S6K continuously antagonises the phosphorylation step in a way that does not allow the phosphorylated protein to accumulate at the early time points. To address the second possibility directly we examined the rate of dephosphorylation of S6K after a round of stimulation for different time points. First cells were starved and then re-stimulated with amino acids and growth factors for either 20 min or 50 min, at which point a mTOR inhibitor was added to block any further phosphorylation. The cells were then allowed to incubate for different times before phosphorylation of S6K was measured by immunoblots. In those experiments we saw that phosphorylation of S6K was rapidly lost within 5 min of incubation with the mTOR inhibitor and the dynamics of this were identical for the early and late points after stimulation . To inveWe also addressed directly the temporal aspect of RAPTOR-GFP translocation in response to amino acid stimulation. Among amino acids, leucine has a prominent role in mTORC1 activation, and it has also been reported that a leucine methyl ester that enters cells and accumulates in lysosomes can activate mTORC1 on its own . PrompteTo our knowledge, the leucine analogue is the first fluorescent reagent capable of activating mTORC1 and at the same time visible by microscopy. In order to understand its mechanism of action further we set up parallel experiments in HEK-293 cells  and in the edited HAP-1 cells expressing RAPTOR-GFP . Given the recent work that Sestrin2 is a cytosolic sensor of leucine for mTORC1 activation  we deterN-ethyl-N-isopropyl) amiloride (EIPA), especially at later times after stimulation  and were tested for mycoplasma contamination every 2 years in our lab using Mycoguard . HAP-1 cells and the RAPTOR-GFP edited cells were obtained from Haplogen (now part of Horizon Genomics https://www.horizondiscovery.com/) and were tested for mycoplasma contamination every 2 years. The following antibodies were used. Mouse anti-β-COP (a kind gift from the late Thomas Kreis), mouse anti-LAMP2 , rabbit anti-phosphoS6K1 , rabbit anti-phosphoS6 , rabbit anti-mTOR , rabbit anti-RAGC , rabbit anti-RAPTOR , mouse ant-RHEB , rabbit anti-phospho4EBP1 , rabbit anti-Giantin , rabbit anti-phosphoULK1 , rabbit Sestrin2 . The following Plasmids were used. pRK5 HA GST Rheb1 from D Sabatini (Addgene 14951), RAGB from D Sabatini (Addgene 19301), myc-RAPTOR from D Sabatini (Addgene 1859). RAPTOR-EGFP was generated from myc-RAPTOR by excising the RAPTOR open reading frame, tailing it with restriction sites for Xho1 and EcoR1 and cloning it into the pEGFP-C1 vector. To target endogenous RAPTOR we used the following oligos:HEK-293 cells were obtained as part of an adenovirus expression system from Microbix  containing either 1% BSA or 1% dialysed FBS prior to incubation with this medium for 60 min. At the end of 60 min, the medium for HEK-293 cells was replaced with new starvation medium containing 2X solution of MEM amino acid solution , and incubated in this solution for the indicated times. For HAP-1 cells, the starvation medium was replaced with new starvation medium containing 1 mM glutamine, MEM amino acids as above, 2X solution of NE amino acids , insulin (1:500 dilution of liquid supplement at 9–11 mg/ml sold by Sigma-Aldrich) and EGF (20 ng/ml) as indicated in the Figure legends. In some experiments using the fluorescent analogue, we also added 0.4 mM arginine to the above solution.For starvation (both HEK-293 and HAP-1), cells were washed twice with pre-warmed starvation medium  Being able to follow Raptor translocation with endogenous levels of protein could be valuable, but it appears that the Raptor-GFP fusion protein is a hypomorph, and does not fully recapitulate the function of native Raptor. For example, amino acids alone failed to activate mTORC1 in the Raptor-GFP HAP-1 cells unlike the parental HAP-1 cells, and maximal activation of mTORC1 in Raptor-GFP HAP-1 cells required both amino acids and growth factor stimulation, and even then it was not as strong as in parental HAP-1 cells. In this regard, both growth factors and amino acids are generally required for the full activation of mTORC1 in in mammalian cells, and therefore it is not clear why mTORC1 was fully activated in HAP-1 cells just by replenishing amino acids.2) The lag in S6K phosphorylation behind Raptor recruitment to lysosomes looks real, but the authors need to demonstrate that the anti-pT389 S6K blotting signal is linear before drawing a firm conclusion .3) In contrast to the authors' conclusion, the immunofluorescence images in 4) Several figures: When assessing S6K, 4E-BP1 and S6 phosphorylation by immunoblotting, the authors should also blot for total levels of these proteins.5) 6) To determine the functionality of RAPTOR-GFP, the authors exclusively rely on S6K phosphorylation measurements by immunoblot. It would be informative to measure other readouts such as 4E-BP1.7) 8) The authors did not provide mechanistic explanation for the observations presented in Alternatively, if SESN2 depletion has an effect, how can the authors argue that accumulation in the lysosome lumen is physiologically relevant? The glutaminolysis model, which provides a sensing mechanism for leucine and glutamine in the mitochondria, should also be mentioned .[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]Thank you for choosing to send your work entitled "Dynamics of mTORC1 activation in response to amino acids" for consideration at eLife. Your resubmission has been evaluated by Tony Hunter and the original Reviewing Editor who handled your submission. The editors are generally satisfied with the revisions made, but would like to ask you to address the following points.On reading the resubmitted paper in depth, the conclusion drawn from the new data in 1) The new data in 2) It would strengthen these data if GFP-Raptor translocation to lysosomes FA Leu and amino acid-induced were monitored in the Sesn2 knock HAP1-RAPTOR-GFP cells.In sum, please provide a response to these issues and explain the apparent paradox that cells remain responsive to Leu after removal of a Leu sensor from the system. [Editors’ note: the author responses to the first round of peer review follow.]The reviewers found your work of interest but were very critical about the lack of controls and incorrect interpretation of the data. If you believe that you could improve the work, we would be happy to consider the work as a new submission. However, given the extensive requirements specified by the reviewers, we would understand if you choose to submit this work elsewhere.We thank the reviewers for the thorough evaluation of the manuscript and for suggesting important experiments to follow up. Below are our responses to specific comments.One comment we want to make that may not have been clear in the original manuscript. The dynamic range of the fluorescence signal of the RAPTOR-GFP edited cells is much lower than a typical cell overexpressing a GFP-tagged protein, owing to the fact that expression in HAP-1 cells rely on the endogenous promoter. This limits to some extent the types of quantitations that we can do since small differences are unlikely to be reproducibly measured. The measurements that we reported are done with a lot of confidence but we cannot push the system further. To make this point clear I am attaching photos from the RAPTOR-GFP cells and HEK-293 cells expressing GFP-ATG13 together with the dynamic range of their intensity. Please note that the range for RAPTOR-GFP is 102-250 whereas for GFP-ATG13 is 5 times larger at 379-1088.1) Being able to follow Raptor translocation with endogenous levels of protein could be valuable, but it appears that the Raptor-GFP fusion protein is a hypomorph, and does not fully recapitulate the function of native Raptor. For example, amino acids alone failed to activate mTORC1 in the Raptor-GFP HAP-1 cells unlike the parental HAP-1 cells, and maximal activation of mTORC1 in Raptor-GFP HAP-1 cells required both amino acids and growth factor stimulation, and even then it was not as strong as in parental HAP-1 cells. In this regard, both growth factors and amino acids are generally required for the full activation of mTORC1 in in mammalian cells, and therefore it is not clear why mTORC1 was fully activated in HAP-1 cells just by replenishing amino acids.There are two issues here.A) The edited cells being hypomorphs. This is correct and we have carefully documented it. However, we still were able to set up these cells to depend critically on amino acids for activation, and to re-capitulate the translocation of the mTORC1 component RAPTOR. Wherever possible, we have also supplemented the data with experiments from HEK-293 and parental HAP-1 cells. The original Sancak et al. paper describing for the first time mTOR translocation to lysosomes was published in 2008, but no live imaging for this phenomenon has been published since then to our knowledge. This is probably indicative of the problems in setting up this experiment. We think that the fact that the edited cells are hypomorphic must be evaluated against the fact that they are the first reagent to allow live imaging of this phenomenon.B) Interplay between amino acids and grown factors in mTORC1 activation. Here we have included a new experiment  to show 2) The lag in S6K phosphorylation behind Raptor recruitment to lysosomes looks real, but the authors need to demonstrate that the anti-pT389 S6K blotting signal is linear before drawing a firm conclusion .We have addressed this point by including a dose response blot for phospho-S6K in The last comment is of course the ideal experiment. It would require transfecting to the edited cells a FRET reporter for S6K activation and then looking at activity and translocation on a cell-by-cell basis. Several years ago a paper reported such a construct  MBC 22: 4647-56) and we obtained it at the time from the lab that published it with a view to use is as described above. We tried at the time to see if the reporter would show FRET after a round of amino acid starvation and re-stimulation, or after overnight serum starvation and re-stimulation with growth factors but it did not. We think that this reporter was oversold and that such a FRET reporter for S6K does not exist at present.3) In contrast to the authors' conclusion, the immunofluorescence images in all mTOR and all LAMP-1, but different between them to optimize the accuracy of the measurement. What we are claiming here is that whereas the intensity of LAMP-1 is constant, the intensity of mTOR peaks and then comes down. It is true that whatever pool of mTOR is still left in the intense staining region co-localizes with LAMP-1: what we are saying is that this amount/pool of mTOR is not constant but it peaks and comes down. For this submission we have repeated this experiment with somebody else’s hands and we get the same result.We clarify what the experiment in In order to provide an alternative quantitative description of the translocation of RAPTOR to lysosomes we derived a Pearson’s coefficient from live cells expressing RAPTOR-GFP and LAMP2; this is shown in new In order to satisfy the request of examining this phenomenon with RAPTOR staining, we ordered and tried 5 different RAPTOR antibodies . None works for iffl as shown by our siRNA experiments and we were unable to find in the literature reference to an antibody for RAPTOR validated for iffl.4) Several figures: When assessing S6K, 4E-BP1 and S6 phosphorylation by immunoblotting, the authors should also blot for total levels of these proteins.We have added the requested blots for the non-phosphorylated versions of these proteins.5) Wherever missing we have added a loading control. Please note that we tend to rely on the non-specific upper band revealed by the phospho-S6K antibody (marked with an asterisk in some blots), which, in our experience, is as good indicator of loading as any other band. We also load equivalent amount of protein in our blots after protein quantitation with the BCA reagent.6) To address this question we quantitated the extent of RAPTOR/LAMP2 co-localization using the Pearson’s coefficient in live cells and in fixed cells. The plot for live cells is in To determine the functionality of RAPTOR-GFP, the authors exclusively rely on S6K phosphorylation measurements by immunoblot. It would be informative to measure other readouts such as 4E-BP1.For most blots, and certainly for all the critical ones, we have re-blotted for phospho-ULK1 (Ser 757), which is a very reliable reporter of mTORC1 activity directly linked to autophagy. These new data re-enforce our conclusions derived from phospho-S6K blots.7) We have repeated this transfection and the new data are shown in 8) The authors did not provide mechanistic explanation for the observations presented in Alternatively, if SESN2 depletion has an effect, how can the authors argue that accumulation in the lysosome lumen is physiologically relevant? The glutaminolysis model, which provides a sensing mechanism for leucine and glutamine in the mitochondria, should also be mentioned .We have spent the majority of time trying to address this question and the results are shown in two new Figures . First w[Editors' note: the author responses to the re-review follow.]On reading the resubmitted paper in depth, the conclusion drawn from the new data in We appreciate greatly the comments made by Tony Hunter and the original Reviewing Editor and the seriousness of their thinking. We have corrected the mistake of referring to GATOR2 “a key negative regulator of mTORC1” instead of a “key negative regulator of GATOR1” which is correct. The rest of the sentence and the concept described are correct.1) The new data in With respect to question and comment 1, we think that the most likely explanation may be that Sestrin2 is in excess over GATOR2 and down-regulating it has an enhancing effect upon stimulation. Similar observations have been reported in two recent papers of which the relevant sections are noted below and which we have now added in the reference and discussed briefly in the text in subsection “Activation of mTORC1 by the amino acid analogue depends on Sestrin2 and on intact lysosomes”. [Our aim with the Sestrin2 data is not to resolve the debate as to its exact role but to provide evidence that both the edited cells and the fluorescent analogue work within the canonical pathway.]http://dx.doi.org/10.7554/eLife.12204.001(a) “Sestrin2 silencing in RKO cells enhanced the mTORC1 signaling in both starved and serumstimulated cells , indicathttp://dx.doi.org/10.1016/j.cell.2014.08.038(b) “The sensitivity of mTORC1 signaling to amino acids was dependent on Sestrin gene dosage: the fewer alleles of Sestrins, the less dependence of MEFs on amino acids” Note in 2) It would strengthen these data if GFP-Raptor translocation to lysosomes FA Leu and amino acid-induced were monitored in the Sesn2 knock HAP1-RAPTOR-GFP cells.In sum, please provide a response to these issues and explain the apparent paradox that cells remain responsive to Leu after removal of a Leu sensor from the system.With respect to question and comment 2, we had tried to see if in the Sestrin2 down-regulated cells we can observe a change in the dynamics of the RAPTOR translocation by live imaging. This was essentially what we tried to do before by modulating the levels of growth factors in the continuous presence of amino acids  since we


Higher accuracies attainable with modified sample processing methods and the latest version of flow cells will further enable the use of nanopore sequencers for diagnostics and research in space.Rapid DNA sequencing and analysis has been a long-sought goal in remote research and point-of-care medicine. In microgravity, DNA sequencing can facilitate novel astrobiological research and close monitoring of crew health, but spaceflight places stringent restrictions on the mass and volume of instruments, crew operation time, and instrument functionality. The recent emergence of portable, nanopore-based tools with streamlined sample preparation protocols finally enables DNA sequencing on missions in microgravity. As a first step toward sequencing in space and aboard the International Space Station (ISS), we tested the Oxford Nanopore Technologies MinION during a parabolic flight to understand the effects of variable gravity on the instrument and data. In a successful proof-of-principle experiment, we found that the instrument generated DNA reads over the course of the flight, including the first ever sequenced in microgravity, and additional reads measured after the flight concluded its parabolas. Here we detail modifications to the sample-loading procedures to facilitate nanopore sequencing aboard the ISS and in other microgravity environments. We also evaluate existing analysis methods and outline two new approaches, the first based on a wave-fingerprint method and the second on entropy signal mapping. Computationally light analysis methods offer the potential for  Sequencing technologies could prove critical for rapid responses to medical infections in space, for instance in deciding whether to use antibiotics and, if so, which ones. Single-molecule methods can also identify modified nucleic acids,8 a potentially important aspect to monitoring crew health. Nanopore sequencers, as general current sensing devices, could also assist in the search for extra-terrestrial life by increasing the range of detectable polymers beyond the canonical nucleobases of DNA and RNA.10Remote molecular diagnostics on Earth and in space necessitate portable technologies. In microgravity, microbes show increased virulence,11 Libraries consist of double-stranded DNA molecules with a hairpin adapter joining the strands at one end and a motor protein attached to the other end. This structure permits sequencing of both strands of the library templates when the template strand, hairpin adapter, and complementary strand pass through the pore in succession. Consensus information from the “2D” reads produces more accurate base calls than template or complement strands alone.The MinION sequencer from Oxford Nanopore Technologies (ONT) is a small sequencing device  that draws power from and transmits data to a computer through a single USB 3.0 connection.12 but traditional alignment software failed to map most reads;14 for the newest version of the pore (‘R9’), Metrichor implements a recurrent neural network for improved base calling.The nucleotides in the pore at a given time disrupt current flow with a signal specific to their identity. “Events” are called at time points where raw electric current measurements change significantly, which should reflect the entry of a single new nucleotide into the pore. However, the current detection process remains noisy and dependent on reaction conditions like temperature. Past versions of the pipeline from ONT used a hidden Markov model (HMM) algorithm with a Viterbi decoder algorithm by Metrichor to call bases from event data,13). However, the long reads, on the order of several thousands of bases or more, are often sufficient to permit taxonomic classification at the species or genus levels.15 Furthermore, researchers using modified sample processing methods have shown accuracies of >95% with the R7 pores and MAP-006 sequencing kits.16 In addition, recent data have shown that sequencing yield and quality are likely to improve as the technology develops; the latest pores (R9) give accuracies of 95% for 2D reads and 85% for 1D reads.Significant challenges remain in the production and interpretation of nanopore sequencing data because of the high error rates ; Escherichia coli (K12 MG1655); and mouse . In a modification to the recommended protocol, we filled syringes (with plastic pipette tips) with 450 μl DNA libraries .Expanding on a proof-of-principle study regarding liquid handling in microgravity,Prior to the parabolic flight, we inserted one of the flow cells into the MinION . During We initiated sequencing after sample loading while there were still ten parabolas remaining in the flight  and continued sequencing through transport back to Johnson Space Center . After hn=16) was much lower than the maximum of 2,048 pores (512 channels with four pores each). We have observed wide variation in performance over many ground runs with older flow cell and kit versions, including the MAP-005 kit used in these experiments, and thus normal flow cell variation could account for low pore activity on a flow cell. In addition, flow cells are optimally used within 8 weeks of their receipt, whereas the flow cells used for the parabolic flight and ground control were ~12 weeks old. Nonetheless, enough of the pores were active to generate data (below). Finally, we did not perform a pre-flight scan of available pores for comparison: a key quality control step we are implementing for all flow cells we send to the ISS. Our QC runs on new R7 flow cells indicate those we sent to the ISS will likely have 1,000–1,500 available pores  of the original pores remained active  was higher than in any of the ground data reads, and the other two flight data reads demonstrated more moderate levels of 0.94 and 0.91 pA, respectively . For com18 The shortest of the three reads did not map to any species in the sample, whereas the longest aligned to multiple mammalian species including mouse and human, but with only 8% query coverage for the top mouse hit. The medium-length read mapped moderately well to E. coli, with 67% identity and 92% query coverage. BLAST results for the template strands were typically poor, with an average identity of 78%, but only 37% query coverage  using blastn settings on the base-called reads to evaluate species detection from the mixed sample.coverage . Almost  as none . The mea15 The WIMP pipeline attempts to classify only 2D reads that pass a certain quality threshold . We ran bacterial and viral classifications on the ground data to compare with our BLAST results  for the Kraken taxonomic classifier, which uses k-mer alignments to determine sample identities down to the strain level. results . WIMP “B19 Control experiments following an optimized protocol  show fewer unmapped reads using GraphMap  of Metrichor. As seen in Our first approach, titled Uncovering Nanopore's fingerprints of genomes (UNFOG), attempts to construct fingerprints of reference genomes and reads based on their most informative frequencies over time. This employs methods from Shazam, a mobile application that identifies songs based on short audio clips of user input.Enterobacter cloacae, 21We first ran a series of benchmarking tests using a subset of 12 reference bacterial genomes, including highly related species, to determine how well the algorithm is able to classify fragments from a reference genome  will enable more robust fluid transfers, and should help resolve these issues by facilitating one-handed operation of the pipette. The crew procedure for use aboard the ISS will emphasize a completely perpendicular loading and firm pressure to completely seal the pore with the pipette tip before loading to prevent pooling on the flow cell surface. Future iterations of nanopore technology may eliminate the need for careful loading procedures. Technologies under development at ONT include the VolTRAX, which aims to automate sample preparation before docking to a sequencer, and the SmidgION, a smaller device that will be compatible with mobile phones.We propose several potential modifications to the instrument to facilitate use in reduced gravity. We observed that the attached MinION cover was difficult to control in microgravity when attempting to load the sample . Though g) for the single read generated over the parabolas. The production of reads during and following repeated exposure to increased gravity and microgravity in the parabolic flight experiment suggests that the device will be capable of sequencing on the ISS. As the technology improves, we also expect that bubble formation will be less of an issue. An independent experiment by the Loose Laboratory on the ground reversed the direction of the 1 g acting on the flow cell by flipping the sequencer upside-down twenty minutes into a 40-minute amplicon run and did not observe any differences in quality or current shifts, confirming that gravity does not affect nanopore sequencing.25 Of greater concern than gravitational effects for space missions is device stability over extended missions with exposure to higher levels of radiation.9 Using flow cells beyond their optimal use period for this experiment illustrated the potential issues with bringing sequencers on long-term mission , but also show that even expired reagents can produce data in microgravity. Further development of solid-state nanopores may be necessary for many applications in space research, since protein pores are sensitive to degradation.Future sequencing experiments on the ISS have a clear potential for success. We have optimized the protocol, as described under Materials and Methods for the later experiments with R7 pores, including making the loading procedure for flight a two-step process to cope with limited crew time. Launch vibration testing showed pores will likely survive the trip to the ISS. We also observed leakage from a vent on return to the Johnson Space Center. In the microgravity conditions on the ISS, leaking due to rotation or tilting of the sequencer is extremely unlikely to occur, because surface tension and cohesion should be the dominant forces. On the basis of our observations, the primary issue with the parabolic flight run was a low number of available pores, likely related to the age of the flow cell. Although we speculate that the variable gravitational conditions of the flight could create more opportunities for air bubbles to migrate into the nanopores and obstruct them, we did not observe any changes in signal quality across gravitational conditions . To facilitate sample loading during the microgravity intervals of the flight, we deviated from the recommended three-step sample-loading procedure, which entails loading 500 μl of running buffer containing fuel mix, waiting 10 min, and loading an additional 500 μl of buffer and fuel mix with a 10 min wait, followed by loading 150 μl containing running buffer, fuel mix and the sequencing library mix. Instead, we pre-loaded syringes for both the flight and ground control, each containing 450 μl volume of running buffer and fuel mix with 6 μl of pre-sequencing mix from each organism. The library and pre-loaded syringes were prepared two days prior to flight, stored at −20 °C until the day of the flight, and stowed at ambient temperature aboard the plane prior to loading the flow cell.Each of the three types of genomic DNA samples was prepared for sequencing according to the procedures specified by ONT for the MAP-005 kit, beginning with 1 μg of each sample of organismal DNA  was sheared to 10 kb size using a Covaris G-tube . DNA wasNucleotide-Nucleotide BLAST 2.2.29+ was run using default settings for “somewhat similar sequences,” connecting to NCBI’s most recent nt database (as of 1 November 2015). If there were hits for multiple sample species only the highest scoring was considered. Blast hits were preferentially counted towards the known sample species. Query cover was calculated for the counted hit by taking dividing the length of the primary alignment by that of the query sequence. This differed at times from the query coverage calculated using the online version of BLAST, which is able to calculate over an aggregation of compatible aligning regions, and thus found a query coverage of 26% for the longest flight data read in its alignment to mouse. Identity was as provided by the BLAST alignment report.Base-calling and species classification were performed using the WIMP Bacteria k24 for SQK-MAP005 (version 1.27) and WIMP Viruses k24 for SQK-MAP005  pipelines. Species counts were combined for E. coli (NCBI reference sequence NC_000913.3), and Mus musculus (GRCm38.p4) genome using GraphMap version 0.3.0, with the command “graphmap align -r $ref -d $fi -o $name.sam”, which saves the top result for each read.We aligned to a combined Enterobacteria lambda phage (NCBI reference sequence NC_001416.1), After testing multiple parameter sets, we chose a window size of 128 and overlap of 64 to compute the FFT. We tested several sampling rates; shown are the results using a sampling rate of 10 for the test data and 50 for the real data. Spectrogram peaks over an amplitude of 5 were paired if within 100 of one another on the time axis of the spectrogram for the benchmarking test version, this was changed for the real data version to an amplitude of 20 and time limit of 300 in an attempt to increase specificity. Paired peaks were stored as hashes, along with their offset times for retrieval and comparison.Enterobacter cloacae genome joined to their reverse complements to create 2D reads. Errors were induced prior to conversion of the reads from k-mers to currents to mimic possible rates in output base-called sequences. The best version of UNFOG during testing involved storing all instances of a fingerprint across each genome and later removing from consideration any fingerprints that appeared over 50 times in any reference genome. However, this modification did not improve results for the real data (possibly because exact matches are unlikely with high error rates) and significantly increased classification time; therefore, a previous version that saved only the final instance of each fingerprint was used for the real data. In the future, a step discarding the most common fingerprints will likely be incorporated into building the database. For each read, the species with the highest number of matches at a consistent offset time from the beginning of the reference sequence was counted. With both template and complement strands, the same offset time had to be found for both strands for a positive hit. In the case of ambiguous matches, the count for each potential species was increased by 1/(number of matches).The benchmarking tests were run using a thousand random 2,500-base fragments of 22 with a sliding window of 20 and overlap of 19. We then smoothed the signal by taking the mean over a similarly sized sliding window. These signal were then matched to the raw, entropy-converted nanopore data. Code for UNFOG/UNSMOG  is open-source and freely available; posted at http://pbtech-vc.med.cornell.edu/git/mason-lab/unfog.Entropy was calculated for each base using a generalized correlation integral approach


Initially proposed to be a possible treatment for certain types of neuromuscular disorders, cardio-metabolic diseases and cancer, H2 improved clinical end-points and surrogate markers in several clinical trials, mainly acting as an anti-inflammatory agent and powerful antioxidant. In this paper, the medicinal properties of H2 in musculoskeletal medicine are discussed with the aim to provide an updated and practical overview for health professionals working in this field.Molecular hydrogen (H However, several trials in the past 10 years reported beneficial effects of H2 in the clinical environment, revealing its possible role as a novel therapeutic agent in medicine5. Usually administered orally or via inhalation, H2 improves both patient- and clinician-reported outcomes, and biomarkers of different pathologies and disorders, from metabolic diseases to chronic systemic inflammatory disorders to cancer [for detailed review see Ref.9 reporting that H2 was able to restore the health and functional abilities of patients after acute injuries or chronic illnesses affecting the muscles and bones. Since musculoskeletal conditions account for a large proportion of a general practitioner's workload10, one might consider H2 as a promising medication or adjuvant that could alleviate these prevalent conditions. In this opinion paper, the medicinal properties of H2 in musculoskeletal medicine are discussed to provide an updated and practical overview for health professionals working in this field.As the oldest and the most abundant molecule in the universe, molecular hydrogen  and of short duration (≤ 12 weeks). Although limited in size and scope, those studies can provide early support for specific therapeutic claims about H2 in musculoskeletal medicine. In a first trial, a combination of oral and topical H2 resulted in a faster return to normal joint flexibility in 36 young men who had suffered sports-related soft tissue injuries, when administered for 14 days as a complementary treatment to a traditional medical protocol for soft tissue injuries7. H2 intervention  was found to augment plasma viscosity decrease after an injury, while other biomarkers of inflammation  and clinical outcomes  were not affected by the intervention7. Another study in Japan reported that drinking 530 ml of a liquid containing 4 to 5 ppm of H2 every day for 4 weeks significantly reduced disease activity in 20 patients with rheumatoid arthritis, as evaluated by changes in the degree of tenderness and swelling in 28 joints and C-reactive protein levels8. H2 was administered as an adjuvant to regular disease-modifying anti-rheumatic drugs and biological drugs, with the efficacy of H2 found to be not inferior comparing to abatacept, methotrexate or a combination of two. In total, 47.4% of patients went into remission, with anti-citrullinated protein antibody (ACPA)-positive patients  responding best to the treatment. Finally, the consumption of water containing a high concentration of H2 (31% saturation) for up to 12 weeks improved surrogate markers of muscle pain and fatigability in 22 patients with inherited and acquired myopathies treated with low-dose prednisone9. Taken together, the above studies seem to pave the way for a future use of H2 therapy in musculoskeletal medicine.Being prompted by the prominent effects of H15, H2 still has a long journey ahead before it can be recognized as a common remedy in this medical discipline. At the moment, H2 therapy is not adequately described in terms of approval, labeling, side effects, and pharmacovigilance information in musculoskeletal medicine. There are no dose escalation studies yet, and the optimal and safest dose range for H2 remains unknown; furthermore, no federal agency or industrial entity provides appropriate patient counseling information about H2. The US Food and Drug Administration (FDA) recently issued a notice (GRAS Notice No. 520)16 of a claim that the use of H2 solubilized in water (up to a concentration of 2.14%) is generally recognized as safe (GRAS) when it is added to beverages and beverage containers in order to prevent oxidation. Based on the information provided by the H2 gas-manufacturing company, as well as other information available to the FDA, the agency had no questions about the conclusion that hydrogen gas is GRAS under the intended conditions of use. However, the FDA has not made its own determination regarding the GRAS status of the subject use of H2 gas16. This seems to be the only formal information currently available concerning the use of H2 in food or medicine! Despite this lack of formal approval, there are many formulations and devices widely available in the market that claim to supply H2 for the use in musculoskeletal disorders, from gas-producing machines to dietary supplements and beverages, with H2 amount varying greatly across the different products. Consequently, consumers might be exposed to easy-to-acquire but questionable products containing H2.Compared with conventional treatment protocols in musculoskeletal medicine, based on drugs and methods that are well-described with respect to efficacy and safety2 should be considered as a dietary supplement or a medicine, since the FDA declares that a product intended for inhalation (such as H2) is not a dietary supplement17. Therefore, considering H2, or at least some H2 forms, for much stricter assessment and regulation by formally recognizing it as a drug in the future, might be more appropriate for this promising bioactive gas. So, it will take many more studies and tighter regulation before H2 therapy can be endorsed as a routine protocol (or adjuvant to standard treatment) in musculoskeletal medicine. In the meantime, H2 should be regarded as an experimental agent and not recommended to treat muscle or bone conditions in the general population.Among other important medical issues that need to be addressed, including long-term safety or pharmacokinetics, the main question remains whether H The title is appropriate with reference to the content of the article. The article is a review of the literature with reference to utilizing molecular hydrogen to enhance sports related injuries. After a detailed review of the literature, the conclusion is there is not enough information to make any solid recommendation concerning utilizing molecular hydrogen to treat sports related injuries, so the implication is probably molecular hydrogen doesn't improve recovery from sports related injuries enough to make any difference. This appears to be a good review of the related literature.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This opinion paper provides an undated and practical overview on the properties of molecular hydrogen in musculoskeletal medicine. The paper focuses on the preliminary studies of H2 on musculoskeletal medicine, and the concerns over the general use of products containing H2. I sympathize the author’s prudent attitudes, which toward the hydrogen should be regarded as an experimental agent and not recommended to general use provisionally. However, I think this paper should also mention the long-term diving practices which high pressure hydrogen inhalation involved to prove the possible safe use of H2 gas.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Instead, it shows a sub-internal limiting membrane (sub-ILM) cavity. Such cavities are known to occur following the resolution of sub-ILM bleed due to various cause including Valsalva retinopathy,2 Terson syndrome, and also in some retinitis3 cases.4 In fact, some of these cavities may simulate a neurosensory retinal detachment or central serous chorioretinopathy on cursory clinical examination.5 To confirm that the features of the current patient1 are indeed related to the optic disc pit, it is necessary for the authors to provide an OCT scan which shows a connection of the presented cavity with the optic disc pit. Also, clear OCT scans of the fovea, both at presentation and at final follow-up would help our understanding of the visual recovery of the patient. The interval between the presenting (28 June 2012) OCT and final OCT (30 Nov 2012) is 5 months and not 6 months as described in the manuscript. For an effective comparison, both the presenting and final OCT scans should have been taken using either horizontal or vertical orientation over the macula. Though the spontaneous resolution of optic disc pit maculopathy is possible, visual recovery in usually unlikely and in such cases an alternate diagnosis needs to be excluded.I read with interest the article reporting spontaneous resolution of optic disc pit maculopathy in a boy. 1 We shall try to summarize our answer for his specific questions.We would like to thank Dr. Koushik Tripathy for his interest and constructive comments regarding our case report entitled “Spontaneous Regression of Optic Disc Pit Maculopathy in a Six-Year-Old Child’’ published in Turk J Ophthalmol. 2017 Jan;47(1):56-58.1 does not clearly show typical intraretinal schisis, it shows a sub-internal limiting membrane (sub-ILM) cavity. In our description of the case and in the discussion 1, we reported that OCT imaging revealed a schisis cavity and cystoid changes due to fluid collection under the ILM, not at multiple retinal layers. The term schisis cavity and cystoid changes has been used for fluid accumulation under ILM.Dr. Tripathy has stated that the optical coherence tomography (OCT) image in Figure 1BThe author has declared that the interval between the presenting and final OCT 5 months not 6 months. Our patient’s follow up was 6 months but better OCT scan was taken at 5 months and 2 days. Seventeen months after follow-up , foveal OCT showed a total regression of optic pit-induced maculopathy, and visual acuity was 20/20 in the right eye.3 Terson syndrome, or retinitis4 as the author mentioned.Finally, the author mentioned that the increase in visual acuity is usually unlikely and that alternative diagnosis should be considered. The patient’s visual acuity at presentation was 20/32 and at final improved to 20/20. The increase in visual acuity was thought to be due to rapid absorption of the fluid and no structural change in the retina. We had already excluded such cavities which are known to occur following the resolution of sub-ILM hemorrhage due to various causes including Valsalva retinopathy,Again, we appreciate Dr. Tripathy’s interest and constructive comments concerning our study.


Regular physical activity can not only help with weight management, but also lower cardiovascular risks, cancer rates, and chronic disease burden. Yet, only approximately 20% of Americans currently meet the physical activity guidelines recommended by the US Department of Health and Human Services. With the rapid development of mobile technologies, mobile apps have the potential to improve participation rates in exercise programs, particularly if they are evidence-based and are of sufficient content quality.The goal of this study was to develop and test an instrument, which was designed to score the content quality of exercise program apps with respect to the exercise guidelines set forth by the American College of Sports Medicine (ACSM).We conducted two focus groups (N=14) to elicit input for developing a preliminary 27-item scoring instruments based on the ACSM exercise prescription guidelines. Three reviewers who were no sports medicine experts independently scored 28 exercise program apps using the instrument. Inter- and intra-rater reliability was assessed among the 3 reviewers. An expert reviewer, a Fellow of the ACSM, also scored the 28 apps to create criterion scores. Criterion validity was assessed by comparing nonexpert reviewers’ scores to the criterion scores.Overall, inter- and intra-rater reliability was high with most coefficients being greater than .7. Inter-rater reliability coefficients ranged from .59 to .99, and intra-rater reliability coefficients ranged from .47 to 1.00. All reliability coefficients were statistically significant. Criterion validity was found to be excellent, with the weighted kappa statistics ranging from .67 to .99, indicating a substantial agreement between the scores of expert and nonexpert reviewers. Finally, all apps scored poorly against the ACSM exercise prescription guidelines. None of the apps received a score greater than 35, out of a possible maximal score of 70.We have developed and presented valid and reliable scoring instruments for exercise program apps. Our instrument may be useful for consumers and health care providers who are looking for apps that provide safe, progressive general exercise programs for health and fitness. Regular physical activity provides many health benefits -3 and isIn the past decade, there has been a tremendous increase in the availability and use of mobile phones and mobile phone apps ,22. A 20The US Department of Health and Human Services recommends that adults should perform at least 150 min of moderate-intensity aerobic activity or 75 minutes of vigorous-intensity activity per week in addition to performing muscle-strengthening activities at least two times per week . FurtherPresently, the quality and accuracy of available mobile exercise program apps and the theoretical foundations that underpin these apps are not clear. Modave et al  used an Our study included two focus groups (N=14) from whom we elicited input and guidance on survey item refinement and questionnaire design. Specifically, we asked the focus group participants to identify unclear words or sentences, suggest alternative ways of phrasing a question, recommend response formats, and consider how the questions would have worked in eliciting responses. University of Florida (UF) College of Medicine employees and students were recruited to participate in these focus groups.Before the focus groups, the study investigators wrote survey questions according to the published ACSM exercise principles to create an initial version of the scoring instrument.  For example, the single training session principle for aerobic exercise “Warm-up: 5-10 minutes of light/moderate intensity cardiovascular exercise” was written as “Does the app advise you to warm up for 5-10 minutes with light or moderate cardiovascular exercise before starting any aerobic exercise?” The responses were initially written on a 3-point scale with 0 indicating “Principle missing,” 1 indicating “Principle present but unclear ,” and 2 indicating “Principle present and clear.” The scoring instrument was divided into 3 sections: (I) aerobic exercise, (II) strength and resistance, and (III) flexibility. Sections I and II were further divided into 3 subsections: (1) safety, (2) program principles, and (3) single training session principles. Section III was divided into 2 subsections: (1) safety and (2) program principles.Next, we provided the participants with the initial instrument and asked them to independently rate 5 fitness apps randomly selected from the apps evaluated in . We instThe 3 main components of the ACSM exercise principles  were weighted 3:3:1 based on the time allocated by the ACSM within a standard exercise program for health and fitness. For each of the 3 principles, the subsections  were allocated the same weight due to the lack of evidence that the subsections should be emphasized differently. Therefore, the overall quality score was scaled to have a highest possible score of 70, with 30 points in aerobic exercise, 30 points in strength and resistance, and 10 points in flexibility.For testing the psychometric properties of the scoring instrument, we chose the same fitness apps evaluated in . The appr among pairs of scores was computed as a measure of reliability.We assessed the inter-rater and intra-rater (test-retest) reliability of the instrument. To assess inter-rater reliability, we asked 3 reviewers to score all 28 apps using the instrument concurrently but independently. The reviewers included 1 staff member and 2 college students from the UF Orthopaedics and Sports Medicine Institute. To assess intra-rater reliability, 5 apps were randomly selected from the 28 apps . Then, the same 3 reviewers rated the 5 apps again using the instrument approximately 1 month later. The Spearman correlation coefficient We asked the director of UF Human Performance Laboratory and UF Health Sports Performance Center  to rate all 28 apps on a scale of 1-10 (criterion scores). In addition, the expert reviewer rated 5 apps  using our scoring instrument. Criterion validity was then assessed by calculating the Spearman correlation coefficient between the criterion scores and those obtained with the instrument, given by the nonexpert reviewers. For the 5 apps rated using the instrument, we computed the Spearman correlation coefficient between scores from the expert and nonexpert reviewers. In addition, we computed a weighted kappa statistic to assess the agreement between the expert and nonexpert reviewers for each app. We used the following guidelines for interpreting kappa statistics suggested by Landis and Koch: <0=poor agreement, 0-.2=slight agreement, .2-.4=fair agreement, .4-.6=moderate agreement, .6-.8=substantial agreement, and .8-1=almost perfect agreement .The focus groups identified multiple challenges when using the initial instrument for scoring. The biggest challenge was the need to clarify meanings and definitions of domain-specific words and phrases. For instance, one of the questions asked was “Does the app advise you to train each major muscle group 2-3 times per week?” Focus group participants did not know what the “major muscle groups” were or what “advise” represented and thus could not judge whether the app provided a comprehensive strength training program or not. In addition, some participants did not understand some of the technical terms such as metabolic equivalent (MET) and Proprioceptive Neuromuscular Facilitation (PNF) stretching. To improve comprehension, participants were asked to suggest alternative strategies for phrasing the questions. Eventually, we removed terms that were too technical and added footnotes to most questions explaining the words that were unclear and phrases compiled from the focus groups.Participants’ preferences of response options were tested. The majority of the participants felt that the initial 3-point response scale did not fully differentiate the apps on quality since there was only one middle category “Principle present but unclear .” As a result, final response options were revised to include a 5-point Likert scale with 1 being “No,” 3 being “Partially,” and 5 being “Yes.”r=.75) and strength and resistance (average r=.79) sections had lower reliability than the flexibility section (average r=.88).Levels of inter-rater reliability for the principle section, subsection, and overall scores for each pair of reviewers are summarized in r=.66). The reliability for Reviewer 2 was only 0.47 for JEFIT. On the other hand, the intra-rater reliability was high for FitStar. All raters were able to provide the same scores for FitStar 1 month later.Results from the intra-rater (test-retest) reliability analysis are summarized in P<.001) for Reviewer 1, .69 (P<.001) for Reviewer 2, and .78 (P<.001) for Reviewer 3. The Spearman correlation coefficient between the criterion scores and average nonexpert reviewer scores obtained with the instrument was .72 (P<.001). The high correlations provided support for the validity of our scoring instrument.In evaluating the validity of the instrument, the Spearman correlation coefficient between the criterion scores and those obtained with the instrument was .70  or almost perfect (κ is between .8 and 1) agreement between the scores given by the expert and nonexpert reviewers. The correlations between the scores from the nonexpert reviewers and those from the expert reviewer were high, ranging from .58 to .99, with most coefficients being greater than .7. The agreement was relatively poorer for FitStar  and Sworkit  when compared with the other apps.The overall and section quality scores for the 28 apps evaluated using the scoring instrument are summarized in All subsection scores for the 28 apps evaluated in this study are summarized in We developed and presented a novel scoring instrument for evaluating the quality of exercise program apps with respect to the ACSM exercise prescription guidelines. Inter- and intra-rater reliability and criterion validity were assessed using the Spearman correlation coefficients and weighted kappa statistics. Our results showed excellent reliability and validity of our instrument, indicating that it provides accurate and stable measurement of the quality of exercise apps. In addition, our findings confirmed the preliminary study of Modave et al that very few of the most popular exercise prescriptive apps are of sufficient quality to provide evidence-based exercise prescription. This is a significant problem, particularly for novice exercisers who do not have the necessary expertise to develop their own program or assess whether a program is well-designed. In addition, the subscores pertaining to the aerobic, strength and resistance, and flexibility components suggest that most apps are of substandard quality even if we assume that they were designed to address a single aspect of exercise prescription.For inter-rater reliability, the least reliable question asked whether an app provides any safety warning before starting aerobic exercise. One potential reason for the low reliability is that fitness apps have a variety of ways of delivering safety warnings. Current fitness apps are very sophisticated in terms of user interface design and content presentation. Safety warnings may be presented as part of the introductory text that users need to read or delivered as a push notification that users must read and close before starting a training program. Furthermore, safety warnings may be presented as part of the exercise instructional videos, as many apps are delivering training programs in videos. This diversity in safety warning delivery methods creates some difficulty for app reviewers to find and judge the appropriateness of safety warnings, especially in sophisticated fitness apps.Although the overall intra-rater reliability is high, we have observed a few relatively lower reliability coefficients. Given that the test and retest were conducted 1 month apart, it is possible that some of the app contents and functions were updated, introducing additional unexpected measurement errors from the app reviewers. In order to survive and stay competitive in the crowded health and fitness apps market, app companies are constantly redesigning their product. It is not surprisingly to see fitness apps redesigning user interface, adding or removing functions, or disappearing from the app stores.The overall quality scores and principle section quality scores are low for the apps evaluated in this study. It indicates that most popular fitness apps do not fully follow the evidence-based exercise principles set forth by the ACSM. On the other hand, one reason for the low overall quality scores is that not all apps have all 3 components of aerobic exercise, strength and resistance exercise, and flexibility. As the number of fitness apps continue to grow, they have become more specialized and focused in functions, with many apps providing exercise programs only for one specific type of exercise. For instance, with a score of 24.0 (out of 30.0 points) for the strength and resistance section, StrongLifts 5x5 is an app of good quality for strength training. However, it does not provide sufficient prescriptive principles for the aerobic exercise and flexibility, making the overall quality score relatively low for this app. Therefore, we recommend that the section quality scores always be evaluated with the overall quality score, which provides a more comprehensive evaluation of the app quality. In addition, app designers may need to consider all 3 main components of the ACSM exercise prescription principles when designing exercise apps, which can improve the degree to which apps adhere to the ACSM standards.One limitation of the study is that evaluating the quality of fitness apps using the instrument in its current form can be time consuming. Our reviewers have reported spending on average 30-40 min to score one app. It can be tedious when a large number of apps need to be evaluated. However, the reviewers have also noted that the scoring process becomes fairly automatic after having reviewed a few apps. Thus, the scoring process can be shortened with some practice. However, future research is needed to develop reliable and valid short forms of the instrument. Another limitation of the study is the small sample size for intra-rater reliability testing. More apps need to be reviewed for computing and analyzing section-specific intra-rater reliability. Nonetheless, reliability coefficients from this study are all statistically significant, providing initial evidence that the instrument is reliable. Finally, we acknowledge that this scoring instrument was designed to capture app quality for overall exercise programs, not for specialized programs that emphasize one type of activity such as weight lifting, yoga, or running. Additional scoring methods are needed to determine quality for these specialized exercise programs.We have developed a reliable and valid instrument for evaluating the quality of exercise program apps according to the ACSM exercise prescription guidelines. This instrument can be used to determine which apps are useful for novice exercisers and health care providers who are looking for apps that provide safe, progressive general exercise programs for health and fitness. Given that the instrument provides separate scores for the 3 main components of ACSM principles, it can also be used to determine which apps are suitable for each of the components of an exercise routine.In future research, we plan to examine the relationship between scores from our instrument and those from other instruments for evaluating app quality, including the instrument published by Stoyanov et al. This would provide a more comprehensive evaluation of exercise apps and thus help users to select apps based on not only evidence-based exercise principles but also functionality and aesthetics. In addition, we plan to evaluate the relationship between app quality scores and behavioral outcomes  among app users. It is important to quantify the differential impacts of app quality aspects on these outcomes to design better exercise apps.


Postpartum weight retention (PPWR) causes intergenerational harm, negatively affecting a mother’s cardiovascular health and ability to have future healthy pregnancies. Low-income minority women are at highest risk for PPWR with little guidance concerning timeline or strategy to lose weight after delivery. An academic-community partnership conducted observational and focus group work to develop an intervention for PPWR among low-income mothers. This study’s objective is to determine the feasibility of implementing a PPWR intervention trial in partnership with a community-based organization (CBO) serving low-income families with social service support.We analyzed five implementation outcomes in this feasibility study: acceptability, adoption, appropriateness, penetration, and sustainability. Other secondary outcomes were the change in psychosocial and clinical outcomes from baseline to one year following the intervention delivery.An academic-community partnership developed and piloted a postpartum weight retention intervention among 17 participants that included 1) six weeks of interactive daily health texting, 2) exercise assistance with baby carrier, home exercise program, and pedometer provision, 3) two live healthy eating and baby feeding workshops, and 4) two 45-min home visits over one year to provide social support and acquire followup data. Implementation outcomes demonstrate an intervention supported by the organization and accepted by end-users, with increased capacity of the CBO to test and deliver an effective intervention. Weight loss was achieved by the majority of participants at one year (Md − 5 pounds (IQR = − 14.5 - 0.3).We made protocol enhancements to the developed intervention based on the analysis of this study, and now prepare for a funded randomized controlled trial (RCT) in a community-based setting. Our central hypothesis is that low-income women who participate in a multi-component, low cost-intervention delivered by a CBO will have less postpartum weight retention than those women who do not participate in the program.The trial was retrospectively registered, ID NCT02867631, 8/11/16. Postpartum weight retention (PPWR) is associated with future unhealthy pregnancies, long-term obesity, and chronic cardiovascular disease –4. DespiIn the literature, several postpartum weight retention interventions have been studied. Intervention delivery was undertaken by a range of health professionals in various settings, but no definitive pattern was seen in successful interventions, and few took place in community-based settings –17. PhelIn addition to focusing on socioeconomically disadvantaged mothers –24, raciAs part of a larger research project to reduce postpartum weight retention by determining the effectiveness of a community-based intervention among low income mothers, the University of Pennsylvania research team partnered with a community-based organization providing social services to low income women with a home visitation model implemented by community health workers (CHWs). This partnership performed observational  and focuThe curricula therefore included meal planning, avoidance of sugar-sweetened beverages, and actionable healthy texts that include 100-cal snack ideas. The healthy feeding curricula delivered task-oriented instruction focused on lactation support provided in class and by phone if requested, bottling education, and goals of infant solid food introduction. The addition of the infant feeding instruction was prompted by focus group work demonstrating many client’s requests to know more about ways to combat pediatric obesity and its intergenerational transmission , 34.The current study focuses on determining the feasibility of implementing a randomized clinical trial of these intervention components among clients of the partnering community-based organization, primarily focusing on five out of eight implementation outcomes defined by Proctor et al. including acceptability, adoption, appropriateness, penetration, and sustainability . Since wThe intervention components are informed by the Theory of Planned Behavior as described. The academic-community partnership developed the overall study design, and chose the outcomes based on a literature review of the behaviors we aimed to change: increase in meal planning, increase in eating fruits and vegetables, decrease in sugar-sweetened beverages, decrease in feelings of social isolation, increase in walking and incorporating the baby into exercise, and increase in breastfeeding time. The primary outcome is therefore postpartum weight retention . The secondary outcomes will be improved dietary composition , decrea2, delivery of single, live term (> 37 weeks gestation) infant, and cell phone with text messaging capabilities (92% clients in preliminary work). Women were excluded from the pilot study if their pre-pregnancy BMI was < 18.5 kg/m2 (underweight). Additionally, we terminated a mother from the study if BMI was not maintained above 18.5 kg/m2, or infant weight loss was noted at any of the weigh-ins, with referral to her healthcare provider. Our goal was to recruit 15 people which represented 10% of our initial proposed sample size needed for the RCT (n = 150), to detect a difference of two pounds of weight between groups with a standard deviation of four.The CHWs were engaged at staff meetings and through email, and encouraged to refer their pregnant and postpartum clients to the study team for a feasibility study of the intervention. The target population was a convenience sample of low-income postpartum clients of the CBO, 100% of which are WIC-eligible. Inclusion criteria included English or Spanish-speaking recently postpartum non-pregnant participant, with self-reported pre-pregnancy BMI ≥ 18.5 kg/mOver three months, 26 women were referred of which 17 were eligible and in the window of 6–12 weeks postpartum at the time of the first study session. This window was chosen to engage women in the intervention after attending their recommended six week postpartum follow up visit with their health care provider to ensure safety to exercise. Women were recruited from 3 of the CBO sites and attended an initial ninety-minute class at their neighborhood site. They returned for a follow up class six weeks later, with 45-min home visits at six and twelve months post-delivery. Cab rides to attend classes were provided, as well as childcare for older children, and an invitation for a partner or support person to accompany the participant. The most intensive part of the intervention was between the two classes during which time women were encouraged to use the baby carrier with associated 20-min exercise program and pedometer while receiving and answering forty two days of text messages. The timeline for receipt of these intervention components is noted in Table Acceptability of the intervention trial was defined in a few ways: 1) rate of initial enrollment of those referred; 2) rate of attrition over the one year follow-up period; 3) engagement with the environmental aids given ; 4) post intervention self-reported utilization of environmental aids; and 5) post intervention reported satisfaction; and 6) positive attitudes towards planned randomization.Adoption was defined qualitatively by the CBO leadership’s support of the study as demonstrated by the internal and external communication of the intervention and organizational development around the study.Appropriateness was defined qualitatively by the academic-community partnership as the ability to recruit and retain participants at a rate comparable to other community-based programs, with the use of one research assistant, an interventionist, and the current geographic resources.Good penetration was defined as the widespread referral of clients from the eight geographic sites of the CBO.Sustainability was defined as the ability to acquire grant funding for a larger RCT and the ability of the CBO to assume all administrative and logistical components of the intervention with minimal outside assistance.Nutrition knowledge and psychosocial factors were evaluated by a comprehensive tool previously validated by the academic-community partnership , and wasVital sign assessment including maternal and child height and weight was measured at baseline, six weeks, six months, and twelve months.A CHW and research assistant collected paper surveys at each class, and at six and twelve months postpartum. A script was written for both the classes and home visits to ensure fidelity of the delivered intervention. Anthropometric training was provided to the research assistant using the National Health and Nutrition Examination Survey videos and protocol to ensure the collection of research-standard measurements, including maternal weight on an electronic scale measured to the nearest 0.1 kg (Seca Robusta 213), and height measured by a portable stadiometer (Seca 213). The infant weight was acquired by measuring both mother and baby on the scale. Infant length was acquired using the Measure Mat II Infantometer . Participants reported their pre-pregnancy weight and gestational weight gain. All survey data were entered into REDCap (R6.16.4) for later analysis by the Stata statistical package .Univariate analysis was completed with chi-squared  and t-tests (continuous variables) to determine differences among those participants who were retained and those who dropped out. Mean and median postpartum weight change was calculated with interquartile ranges.Acceptability and penetration: During the intervention pilot, we focused on operations and protocol enhancement by looking at the implementation outcomes described. Initially, we enrolled 17 women, with 5/17 (30%) attrition by six weeks, 6/17 (35%) at 6 months, and 8/17 (47%) attrition at one year. For this initial pilot recruitment, we relied on referrals from community health workers, with disparate rates of referral across the neighborhood sites. For example all women came from 3 sites where the CHWs had a previous interest in health and nutrition and had an increased level of success with recruitment. 5–8 women were referred by these advocates monthly, and demographics mirrored those of the population the CBO serves and those studied in focus group work. At the time, 3 other sites were being renovated and the CHW’s were busy managing the transition beyond their normal caseload and didn’t refer any clients from these sites. Yet another site had an ongoing transition of leadership and did not refer any clients during the study period. In terms of engagement by the mothers, during the intensive portion of the intervention, 8/12 (66%) reported use of the baby carrier more than half the days of the week; 9/12 (75%) reported pedometer use more than half the days of the week. Of those followed through six months, 8/11 (73%) continued to use the baby carrier and pedometer provided at least once a week, and 7/11 (64%) viewed the packet of exercise, eating, and baby feeding tips at least once a week. Engagement with texting back footsteps and baby carrier time was variable, with 9/11 (82%) of women texting at least 3 times a week. 100% said they were satisfied and would tell a friend about the program. Those who dropped out of the study by one year were similar to those retained in regards to starting body mass index (BMI), self-efficacy, perinatal depression risk, racial/ethnic background, and days spent returning texts. Those who dropped out did have more children living at home as an important difference . However, self-efficacy returned to baseline by six and twelve months without significant change. Of those retained, more than half  lost weight over the course of the six week intensive intervention (Md − 1.0 pounds (IQR = − 4.1-1.8)), with 7/9 (78%) losing at one year post-delivery (Md − 5 pounds (IQR = − 14.5 - 0.3)). 25% of women were at risk for perinatal depression at the start of the program, and this point prevalence remained stable over time.Psychosocial and weight assessment: Mean self-efficacy for healthy behaviors improved over the course of the six week intensive intervention for the 12/17 who were retained at six weeks(from 6.9 (2.7) to 5.2(2.6), Based on the results of the pilot intervention, we made key protocol changes to effectively deliver the components during RCT testing. For example, during the pilot, we relied on CHW referrals to generate our study subjects. However, they had a large number of responsibilities they accomplish for the CBO home visitation program, and recruitment was subject to the environment at the neighborhood site. Recruitment for our study was limited by the amount of time the CHWs had, renovation or leadership changes at the neighborhood site, and the severity of an individual community health worker’s case load, as well as personal interest in health and nutrition. Since the level of penetration and attrition during the pilot was unacceptable to us and more than the 40% often encountered by community-based intervention studies, for our RCT intervention study, we will instead use a database of all postpartum clients managed by the CBO to recruit our participants, with CHWs providing input about safety information or mental health status regarding the participant. This change will allow us to contact every postpartum client as potential study subjects, and will allow the CHWs to focus on their primary responsibility of direct service provision. We can also ensure better penetration by contacting pregnant women from all geographic sites. We now know the importance of budgeting future funding to allow certain CHWs to be designated wellness champions with a reduced case load to allow for research study participation and program implementation, ensuring sustainability of the program once implemented. We will improve our emergency contact procedures for depressed participants as we found such a high prevalence during the pilot, and we will incentivize engagement with the intervention by providing an extra gift card to the woman who texts back the most in her cohort.We also decided to increase the dose of the intensive intervention by lengthening class time to 120 min and home visit education to 60 min as we saw a significant initial change in self efficacy which returned to baseline at six and twelve month follow-up. The weight outcomes at six and twelve months post-delivery helped us to determine effect size and plan for our sample size in the RCT. To detect a 4 pound difference in weight change at one year post delivery using the standard deviations acquired (8), we would need a control population of 85 and an intervention population of 85 . Attrition rates also aided in planning for our proposed recruitment sample. We used several previously described strategies for retention of our subjects in this community setting and anticipate the attrition seen in other community-based interventions (no more than 40%) . We emplThis pilot study was a key step in planning for an RCT to study efficacy and cost-effectiveness of the holistic intervention, thus deepening the knowledge of best practices that exists for the combat of postpartum weight retention and its intergenerational downstream effects. At every point, community insight and collaboration has been considered. We were encouraged by this feasibility study that serves as a proof of concept and readies us for further testing with improved implementation outcomes and sample size to detect differences among control and intervention groups. Additionally, in this small group of women, we were able to assist women in decreasing their weight in the postpartum period while increasing their self-efficacy, thus improving their personal and familial health.