Drosophila flight muscles to explore why many integrin associated proteins (IAPs) are needed and how their function is coordinated. These muscles revealed new functions for IAPs not required for viability: Focal Adhesion Kinase (FAK), RSU1, tensin and vinculin. Genetic interactions demonstrated a balance between positive and negative activities, with vinculin and tensin positively regulating adhesion, while FAK inhibits elevation of integrin activity by tensin, and RSU1 keeps PINCH activity in check. The molecular composition of myofibril termini resolves into 4 distinct layers, one of which is built by a mechanotransduction cascade: vinculin facilitates mechanical opening of filamin, which works with the Arp2/3 activator WASH to build an actin-rich layer positioned between integrins and the first sarcomere. Thus, integration of IAP activity is needed to build the complex architecture of the myotendinous junction, linking the membrane anchor to the sarcomere.We use the myotendinous junction of  Our body consists of many different types of cells that build our tissues and organs. To do so, cells need to be able to stick together. One family of proteins called integrins helps to keep cells connected. They sit across cell membranes and anchor cells to the networks of protein fibres outside cells that link and strengthen our organs, and also connect muscles to tendons.In fruit flies, the indirect flight muscle attach to the thorax of the insect, and create wing movements by ‘deforming’ the thorax. These flight muscles resemble the muscles of animals with a backbone, and consist of many different fibres. At the end of these fibres is a plaque of a protein important for muscle contraction, known as actin. Integrins attach to these actin plaques, allowing the ends of the muscle to anchor to the tendon.Integrins form complexes with so-called 'integrin-associated proteins' inside the cell, which regulate integrin. Integrins and integrin-associated proteins are essential for proper muscle development, but until now it was not fully understood how they interact with each other. Here, Green et al. explored the role of some of these proteins in the indirect flight muscles of fruit flies.This revealed that the connection between muscle and tendon is a balancing act. Some integrin associated proteins boost the attachment, whilst others block it. One protein, tensin, increased integrin attachment, whilst another, FAK, blocked tensin, decreasing attachment. Similarly, a protein called PINCH expands the attachment, whilst a protein called RSU1 reduced the activity of PINCH to the correct level. Moreover, the end point of the muscle fibres was discovered to be composed of four distinct layers, including a newly identified layer of actin, which is built by three other integrin-associated proteins.The flight muscles of fruit flies are similar in structure to the skeletal muscles that move our own limbs. An important next step is to discover whether these integrin-associated proteins work similarly in our muscles. A better understanding of how they work together could help with research into diseases of the muscles. Cell adhesion to the extracellular matrix (ECM) is essential for the development and homeostasis of multiple cell types and tissues . AdhesioDrosophila embryonic muscles talin is orientated similarly to fibroblasts, whereas in the developing wing both ends are close to the membrane  are the major muscles in the adult thorax that power flight . IFMs arelopment  and thenelopment . At the elopment . Furtherelopment , whereaselopment .We use the IFMs to discover functions for four IAPs  and vinculin) that surprisingly are not needed for viability of the fly, despite all colocalizing at integrin adhesion sites throughout development, and being conserved in all metazoa  diagram. We receHere we show that indeed IFMs reveals a new phenotype for each IAP examined, and genetic interactions reveal how they work together by balancing positive and negative control. Tensin is able to bind integrins , and conTo understand how IAPs contribute to integrin-mediated adhesion we sought to identify defects that occur in their absence. An unexpected finding is that some highly conserved IAPs can be genetically removed without impairing viability or fertility. Thus, we have been searching for biological processes that fail in the absence of this group of IAPs. We recently discovered that whereas the genetic removal of vinculin does not impair viability or fertility , it doesWe analysed the development of the myofibrils in each muscle by staining for filamentous actin at different stages of development and imaging by confocal microscopy . No grosWe then used transmission electron microscopy (TEM) to examine the MTZ defects in more detail . During To discover when the defects first appeared, we next examined the MTZs at different stages of development. During pupal stages, the MTZs in all mutants appeared normal . HoweverAs loss of tensin and RSU1 caused strong phenotypes, we hypothesised that their effects may be due to mislocalisation of integrins or other IAPs. We examined the distribution of integrins and other IAPs, using endogenous genes tagged with fluorescent proteins, all of which are fully functional (see Key Resources Table), but did not find any dramatic loss of those examined: βPS integrin, talin, PINCH, paxillin, GIT or vinculin . HoweverTo test for compensatory activities between these proteins we examined the consequences of removing two at a time. Flies lacking RSU1 and tensin died at pupal stages, however in these pupae the MTZs appeared normal , suggestmys[b28], a mutation in the hybrid domain (V423E) that strongly elevates affinity for ligand  MTZ actin expands, pushing the first sarcomere away; (2) MTZ actin mixes with sarcomeric actin; or (3) overall actin organization near muscle ends deteriorates. To resolve between these possibilities, we examined actin-binding proteins and components of muscle sarcomeres, which revealed three major insights.muscle actin regulatory layer or MARL; and (4) the first Z-line with high levels of ZASP and α-actinin, analagous to the stress fiber attached to the focal adhesion , whereas the ISL (e.g. paxillin) was not substantially altered . In addiThe MARL is distinguished by two actin-binding proteins associated with branched actin networks, Arp2/3 and filamin. Actin within the FTL is likely to link the MARL to the membrane, whereas the first Z-line assembles at the outer edge of the MARL, where it anchors the parallel actin filaments of the sarcomere. This highlights the ability of actin-binding proteins to associate with discrete actin structures in adjacent parts of the cell. Vertebrate muscles contain filamin-C at the myotendinous junction , suggestThe concentration of Arp2/3, its nucleation promoting factor WASH, and filamin in the MARL suggested that the actin in this structure may form a cross-linked network, and we sought to determine whether these proteins contribute to MARL actin assembly. Flies lacking filamin are viable, albeit female sterile due to defects in the actin-based ring canals during oogenesis . Loss ofMuscle-specific knockdown of Arp2/3 components is lethal and results in severe muscle defects . HoweverWe were intrigued by the observation that two proteins that aid MARL formation, vinculin and filamin, are mechanosensitive . We therWe further manipulated MARL proteins to determine how they worked together. None of the double mutant combinations of vinculin, filamin and WASH showed enhancement or suppression of the expected additive phenotype . As thesThe genetic interactions suggested that vinculin tail and filamin may interact directly or indirectly. To test this, we targeted a portion of filamin to the surface of the mitochondria with an ActA peptide  to test We can put these results together into a speculative model  whereby Drosophila have proved to be an excellent system to identify functions for integrin-associated proteins (IAPs) that are not essential for viability. The mechanical linkage between the last Z-line of each myofibril and the plasma membrane is a well ordered and multi-layered structure, ideal for elucidating the mechanisms by which actin can be organized into different structures at subcellular resolution. In the layer closest to the membrane, the integrin signaling layer, we find important counterbalancing between IAPs, with FAK inhibiting the activation of integrin by tensin, and RSU1 inhibiting excess PINCH activity, and binds the 5th LIM domain of PINCH . Loss ofDrosophila involve integrins on both sides of the interaction . UAS::vinculinHead-RFP  for the generation of βPS-GFP  using Not1 and Xba1. GFP was excised by digestion with Hind3 and Sac1 and replaced with mcherry. A guide RNA targeting the C-terminus of βPS was cloned into the pCFD3 plasmid as previously described  containing the genomic region of Drosophila zyxin was used as a template. A 9865 bp fragment containing the zyxin genomic region was excised from the BAC using Spe1 and Not1 and cloned into pBluescript II KS (addgene). A 1601bp region 5’ to the Spe1 site was PCR amplified from Drosophila genomic DNA and cloned into TOPO TA (Thermo Fisher Scientific). The forward primer added an Mlu1 site and had the sequence 5’-ACGCGTGGGAATAGCAAACGCCACA-3’, while the reverse primer was 5’-TGTGTACTTGCGCATTCACA-3’. pBluescript II KS containing the GFP sequence was used a template for GFP. A 738 bp product containing GFP was PCR amplified and cloned into TOPO TA. An Aat2 site was added with the forward primer 5’-GACGTCAGAACATTCGAGCTCATCGATGAGTAAAGGA-3’ and an Fsp1 site was added with the reverse primer 5’ TGCGCAATAAATAAAATGAGCACTCAATTTATTTGTATAGTTCATCCATGC-3’. GFP was cloned into the 3’ of the zyxin genomic sequence at Fsp1 and Aat2 sites. The resulting 10,603 bp zyxin-GFP sequence was excised using Spe1 and Not1, and the 1601 bp 5’ region was excised from TOPO TA using Mlu1 and Spe1, and both were cloned into Mlu1 and Not1 sites of the vector pWhiteAttPRabbit . The vector contains attB sites and the white gene. The constructs were inserted into position 51 on chromosome II.BACR13D24 . RFPmito was amplified from MrRFPmito plasmid  using prIFMs were dissected from pupae and adult flies as previously described . BrieflyConfocal images were taken with an Olympus FV1000 confocal microscope using a 60×/1.35 NA objective and 3x zoom. A single stack of 5 z-sections spaced by 0.5 µm was imaged per individual. SIM images were taken with a DeltaVision OMX BLAZE V3 (Applied Precision) using an Olympus 10 × 0.4 NA air objective or 60 × 1.4 NA oil objective, 488 nm and 593 nm laser illumination and standard excitation and emission filter sets. Raw data was reconstructed using softWoRx 6.0 (Applied Precision) software. Images were processed using ImageJ (NIH) and Adobe Photoshop.Half thoraces were dissected from 1 day old flies in PBS and fixed in glutaraldehyde/2% formaldehyde in 0.05 M sodium cacodylate buffer pH 7.4 for 4 hr at 4°C. Thoraces were osmicated in 1% OsO4/1.5% potassium ferricyanide/0.05 M NaCAC pH 7.4 overnight at 4°C and then treated with 0.1% thiocarbohydrazide/DIW for 20 min. Thoraces were osmicated again in 2% OsO4 for 1 hr and finally embedded in Quetol resin. 70 nm sections were imaged on a Tecnai G2 80-200kv transmission electron microscope at 6500x magnification.Phenotypes were analysed manually using ImageJ. The line tool was used to measure MTZ length, myofibril width, sarcomere length and the distance from the myofibril end to the first M-line. 10–30 myofibrils per individual were measured from 10 individuals per genotype. Average MTZ length of distance to first M-line was calculated per individual. Statistical significance was determined by Mann Whitney U test (p<0.05) using Prism software (GraphPad). Fluorescence intensity plots for SIM images were using the plot profile function in ImageJ (NIH). Profiles were processed and distance between peaks was calculated in Microsoft Excel.Fluorescence intensity of GFP tagged proteins at the MTZ were quantified using a Matlab script adapted from Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Nick Brown (nb117@cam.ac.uk). In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Drosophila" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Frank Schnorrer as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor.Thank you for submitting your article "Novel functions for integrin-associated proteins revealed by myofibril attachment in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Green et al., provide a comprehensive study of the distribution, and functional contribution of Integrin Associated Proteins  at the myotendinous junction (MTJ) of the indirect flight muscles (IFMs). Previous experiments revealed surprisingly mild phenotypes in flies upon deletion of those proteins. The authors show that individual deletions of FAK, RSU1, tensin and vinculin lead to distinct morphological defects in IFMs, and genetic interaction studies suggest various degrees of interdependency, with positive and negative roles for integrin activity.The experiments focus on a particular substructure of IFMs, called the modified terminal Z-disc (MTZ), which connects the myofibril to the neighboring tendon cell. The MTZ is affected to different extents by the loss of FAK, RSU1, tensin and vinculin. Unexpectedly, simultaneous deletion of FAK and RSU1 leads to the rescue of MTZ morphology, which is attributed to enhanced integrin activation (as a result of FAK depletion) since an active integrin mutant rescues the RSU1 phenotype as well. Activating integrins also partially rescued the phenotype of tensin-deficient flies, whereas FAK-overexpression leads to a tensin-mutant like MTZ morphology. The authors conclude from these experiments that FAK regulates integrin activity through tensin. The RSU1 phenotype can be  rescued by reducing levels of its binding partner PINCH, whereas PINCH overexpression causes a RSU1-like MTZ phenotype; it is concluded that RSU1 inhibits PINCH activity. Next, the role of vinculin is analyzed and the authors find that depletion of this protein has two distinguishable consequences. First, loss of vinculin affects talin-dependent adhesion in the MTZ; second, it impairs actin organization in a newly defined structure called MARL (muscle actin regulatory layer). Additional genetic studies imply that vinculin regulates MARL organization through filamin, and MARL assembly requires the presence of the Arp2/3 regulator WASH.Overall, the manuscript is interesting, the data are of high quality, and the amount of data is impressive. It adds important new structural information regarding the muscle-tendon junction structure (dividing it into 4 zones using super resolution and EM microscopy) and partially explains why so many IAPs are required to stably anchor myofibrils to tendons during the high force producing muscle contractions.Some important points should be addressed during the revisions to clarify the introduction and to ideally further support the conclusions or tone-down/modify some of the molecular conclusions drawn in the paper. It will also be interesting to show the consequences of IAPs deletions for flight muscle function.Essential revisions:1) Specificity of the phenotypes. The authors convincingly show a defect of the terminal Z-disc structure in the IAP mutants. However, many IAPs are likely also expressed in tendon cells. Is the observed defect indeed solely due to a role of the IAPs in muscle? This is already convincingly shown for tensin using the muscle specific RNAi, but should be shown for the other IAPs as well. This will rule out an indirect effect of the function of the IAPs in tendons on the muscle attachment morphology. The authors have also tools ready to perform a muscle-specific rescue for the IAP mutants.2) Functionality of GFP fusions. The authors gained important conclusions from their GFP-tagged IAP proteins. These are expressed from endogenous enhancers, thus over-expression artefacts are likely not an issue. However, the functionality of these GFP fusions should be tested wherever possible. Mutant rescue experiments with the IAP-GFP fusions used for the localisation studies appear straight forward. If not functional, it should be mentioned.3) Molecular mechanisms. The paper several times states potentially interesting mechanisms of the IAPs that are not much supported by molecular data. Can the authors provide more convincing data to support how FAK inhibits integrin activity via tensin? How is integrin activity defined? Ligand binding affinity, or integrin dynamics, or mechanical interconnection with IAPs, etc.?B72 mutant was used as previously described, but this line is not mentioned in the cited study .The authors state in the methods section that the βPS*: mysAlternatively, is there a way to support the hypothesis that RSU1 controls PINCH activity by molecular data? Pinch expression levels? IPP levels? IPP independent?Or, can the intriguing hypothesis that vinculin promotes MARL formation through inducing a conformational change in filamin be supported by molecular data? Can the authors show a direct association between vinculin and filamin ?4) Introduction/context. Some points in the intro should be made clearer. The development of the adult muscles in covered only very briefly in Dobi et al., 2015. A more appropriate recent review would be Gunage et al., 2017.It is important for the remaining parts of the paper to clearly state that IFMs are "fibrillar" muscles, with individual myofibrils attaching to tendons, in contrast to the normal "tubular" cross-striated adult muscles. See Josephson et al., or Schonbauer et al.,. This might explain the exclusive phenotype in the IFMs for the IAP mutants.Indirect flight muscles are built by multiple myofibers (not myofibrils as currently stated in the intro) and thus more similar to vertebrate muscles. Each myofiber contains many hundred myofibrils.The sarcomere not only consists of "interdigitated" actin and myosin filaments, which can be present in many cell types, not only in muscles. Please describe the sarcomere structure more accurately. "Interdigitations" are also used throughout the text to describe defects in sarcomere length 5) Force sensing structure. The description of the MARL domain is very interesting and novel. The authors suggest that this domain is induced by mechanotransduction. Can the authors reinforce this hypothesis by manipulating the force transmitted by the MTJ? One option might be to reduce myosin force by expressing a motor deficient version of myosin heavy chain under act88F control .Alternatively, they could try to reduce integrin function selectively in the tendon cells, which might make attachments less rigid.6) Flight muscle function. Please show the results from the flight tests at day 1 and day 14 of the IAP mutants instead of quoting "unpublished results". It is surprising that these mutants can fly normally despite the defective attachments, in particular at day 14 of age.Drosophila" for further consideration at eLife. Your revised article has been favorably evaluated by Didier Stainier (Senior Editor), and Frank Schnorrer (Reviewing Editor) after consultation with two additional reviewers.Thank you for resubmitting your work entitled "Novel functions for integrin-associated proteins revealed by analysis of myofibril attachment in The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:1) The authors state that vinculin mutants can fly . However, the day 14 vinculin mutant shows severe myofibril defects  and only about 20% of the flies appear to fly . This should be a very robust difference that should be mentioned .2) Interdigitations (old point 4). Sarcomeres are now explained correctly and not described as "interdigitated" actin and myosin filaments. However, "interdigitations" are used to describe the folded electron dense structure/membrane at the myofibril tips (zone 1 of the MTZ). This is used first in subsection “Indirect flight muscles (IFMs) reveal phenotypes for viable IAP mutants” without a clear description of what these interdigitations mean. Please explain it better, including its appearance in the model . What do "longer interdigidations" in the model mean? Longer than what?3) 'Web-like' structure of actin in SIM imaging (old point S4). If this is an artifact of the technique, it should be mentioned, at least in the figure legend.4) In many of the sarcomere stains with phalloidin  for example the recused FAK or tensin mutant  or many of the GFP-fusion genotype images in Figure 2 and Figure 2—figure supplement 2 the characteristic high concentration of phalloidin at the Z-disc is not visible. This likely due to an artifact when cutting the myofibrils during histology (these are under high tension). As sarcomere length was not determined in these samples the issue appears not too large. However, we recommend fixing these artifacts wherever possible. The use of relaxing solution during the preparation should also help. A clean, parallel cut is important to preserve myofibrils. Cut myofibrils should wherever possible not be imaged.5) Arrows in Figure 1A are yellow not white (legend). Same for Figure 7A. Figure 6: Obscurin/Unc-89 not Unc-86 (legend).6) Weitkunat et al., 2014 do not show any spontaneous muscle contractions during pupal stages of the developing flight muscles (currently stated in the Introduction). Spletter et al., 2018 found these contractions, so this could be cited.7) Subsection “Genetic interactions reveal inhibitory action and compensation between IAPs”: "RSU1 and integrin keep PINCH levels at the correct level; in its absence the […]"; in which absence? PINCH? Essential revisions:1) Specificity of the phenotypes. The authors convincingly show a defect of the terminal Z-disc structure in the IAP mutants. However, many IAPs are likely also expressed in tendon cells. Is the observed defect indeed solely due to a role of the IAPs in muscle? This is already convincingly shown for tensin using the muscle specific RNAi, but should be shown for the other IAPs as well. This will rule out an indirect effect of the function of the IAPs in tendons on the muscle attachment morphology. The authors have also tools ready to perform a muscle-specific rescue for the IAP mutants.We have performed muscle specific RNAi of all the IAPs and all of these phenocopy the mutant phenotype. This data can be found in Figure 1—figure supplement 1 and Figure 7—figure supplement 1. We do not show RNAi of vinculin as we had already shown muscle specific rescue in Figure 4—figure supplement 4.2) Functionality of GFP fusions. The authors gained important conclusions from their GFP-tagged IAP proteins. These are expressed from endogenous enhancers, thus over-expression artefacts are likely not an issue. However, the functionality of these GFP fusions should be tested wherever possible. Mutant rescue experiments with the IAP-GFP fusions used for the localisation studies appear straight forward. If not functional, it should be mentioned.The GFP-tagged proteins are expressed from 'genomic rescue constructs' or insertions into the endogenous locus (by gene trapping or homologous recombination), and all have previously been shown to rescue mutant alleles fully, or if at the endogenous locus, not cause a phenotype when homozygous viable. We have added a line stating this in the Results section.3) Molecular mechanisms. The paper several times states potentially interesting mechanisms of the IAPs that are not much supported by molecular data. Can the authors provide more convincing data to support how FAK inhibits integrin activity via tensin?We have not been able to advance our understanding of the details of this mechanism within the revision time.How is integrin activity defined? Ligand binding affinity, or integrin dynamics, or mechanical interconnection with IAPs, etc.?B72 mutant was used as previously described, but this line is not mentioned in the cited study .The authors state in the methods section that the βPS*: mysb27 allele is discussed at length in Kendall et al., 2011 . In this paper the authors show that this allele has "extremely elevated affinity" for ligand, testing by putting this mutant form of integrin into cells in culture and measuring binding to the ligand mimetic antibody TWOW. The similarity in loss of FAK and the mysb27 allele rescue of RSU1 and vinculin is what supports the idea that FAK is negatively regulating integrin activity. We lack antibodies that report on Drosophila integrin activity within tissues (TWOW does not work for this), so unfortunately, we do not have a way of directly assessing the amount of active integrin in Drosophila tissues.It is b27, not b72. The mysAlternatively, is there a way to support the hypothesis that RSU1 controls PINCH activity by molecular data? Pinch expression levels? IPP levels? IPP independent?We have added additional data to address this mechanism, but have not got as far as fully solving it. We have quantified IAP levels in Figure 2 and included this in the revised Figure. This showed that in the RSU1 mutant Paxillin is elevated 2.5 fold (as mentioned in the text previously), ILK levels are reduced 2 fold, but PINCH levels did not change. We followed up to test whether elevating Paxillin would mimic elevation of PINCH, but it did not and instead produced a new phenotype that we felt was outside the scope of the paper. We then tested two truncation mutants of PINCH, for their ability to mimic the RSU1 mutant phenotype when overexpressed, as full-length PINCH does. Deletion of LIM4,5 reduced its function, but we still got a phenotype. This rules out a model where RSU1 acts by competing with another protein that binds to LIM5. We also found that overexpression of PINCH ∆LIM1 did not cause a phenotype, suggesting that PINCH still needs to interact with ILK to produce its gain of function phenotype. We have included this data in the Results section, and in a revised Figure 3—figure supplement 1 and modified the model in Figure 3.Or, can the intriguing hypothesis that vinculin promotes MARL formation through inducing a conformational change in filamin be supported by molecular data? Can the authors show a direct association between vinculin and filamin ?Yes, we have been able to show an association between the vinculin tail and the filamin C-terminus that is independent of whether filamin is open or closed . For this we have used an assay where we target the filamin C-terminal 9 Filamin repeats (the isoform filamin90) to the mitochondria and show that it recruits vinculin tail, as well as actin and WASH. We prefer this to the IP as it shows they can interact within the intact cell, but like IP it does not distinguish direct from indirect. We tested whether purified filamin MSR region binds to the vinculin tail in vitro and it does not but have were not able to make and test other parts of filamin90. FRET would be a good approach, but not possible within the timeframe of revisions as we would need to map the interactions precisely enough to bring the FRET pairs in proximity and generate appropriate transgenic animals. The ability of filamin90 to recruit WASH made us reconsider whether they are in the same or different pathways and we realized that the majority of the data is consistent with WASH being downstream of mechanically opened filamin.We have also been able to strengthen our analysis with TEM pictures for vinculin, filamin and WASH, which give a clearer view of the impact of the loss of these proteins on the structure of the MARL.We thought it would be helpful to assemble all the data into a model that while speculative is consistent with all our data, which is described in subsection “Filamin and WASH contribute to MARL formation” and shown in Figure 8.4) Introduction/context. Some points in the intro should be made clearer. The development of the adult muscles in covered only very briefly in Dobi et al., 2015. A more appropriate recent review would be Gunage et al., 2017.It is important for the remaining parts of the paper to clearly state that IFMs are "fibrillar" muscles, with individual myofibrils attaching to tendons, in contrast to the normal "tubular" cross-striated adult muscles. See Josephson et al., or Schonbauer et al.,. This might explain the exclusive phenotype in the IFMs for the IAP mutants.Indirect flight muscles are built by multiple myofibers (not myofibrils as currently stated in the intro) and thus more similar to vertebrate muscles. Each myofiber contains many hundred myofibrils.The sarcomere not only consists of "interdigitated" actin and myosin filaments, which can be present in many cell types, not only in muscles. Please describe the sarcomere structure more accurately. "Interdigitations" are also used throughout the text to describe defects in sarcomere length  Force sensing structure. The description of the MARL domain is very interesting and novel. The authors suggest that this domain is induced by mechanotransduction. Can the authors reinforce this hypothesis by manipulating the force transmitted by the MTJ? One option might be to reduce myosin force by expressing a motor deficient version of myosin heavy chain under Act88F control .We did this experiment but found that the sarcomeres are so disrupted that it was difficult to make any firm conclusions see . The MTZAlternatively, they could try to reduce integrin function selectively in the tendon cells, which might make attachments less rigid.We appreciate the suggestion but think it would require extensive work to demonstrate that it was an alteration of forces that was having any effect observed.6) Flight muscle function. Please show the results from the flight tests at day 1 and day 14 of the IAP mutants instead of quoting "unpublished results". It is surprising that these mutants can fly normally despite the defective attachments, in particular at day 14 of age.Surprising but true, now quantified in Figure 1—figure supplement 1, Figure 2 and Figure 7—figure supplement 1. Only loss of filamin caused a substantial reduction in the number of flies that will fly in the test, and this is likely to be due to the defects in sarcomere structure as recently reported by Frieder Schöck and colleagues.1) The authors state that vinculin mutants can fly . However, the day 14 vinculin mutant shows severe myofibril defects  and only about 20% of the flies appear to fly . This should be a very robust difference that should be mentioned .We have revised subsection “Vinculin has two distinct functions in the MTZ” as follows:“One day old flies lacking vinculin were able to fly normally but after 14 days, only 20% were able to fly . However, muscle specific knockdown of vinculin did not reduce flying ability, yet caused comparable IFM actin defects, indicating that loss of flight in flies lacking vinculin is not due to the IFM defects.”Unfortunately, we were not able to perform sufficient numbers of independent repeats to merit a p-value.2). Interdigitations (old point 4). Sarcomeres are now explained correctly and not described as "interdigitated" actin and myosin filaments. However, "interdigitations" are used to describe the folded electron dense structure/membrane at the myofibril tips (zone 1 of the MTZ). This is used first in subsection “Indirect flight muscles (IFMs) reveal phenotypes for viable IAP mutants” without a clear description of what these interdigitations mean. Please explain it better, including its appearance in the model . What do "longer interdigidations" in the model mean? Longer than what?We have added a sentence defining the interdigitations in the Introduction. We have also added ‘interdigitations’ to the model in Figure 5 and Figure 9. Finally, we have added “longer than wildtype” to the legend for Figure 9.3) 'Web-like' structure of actin in SIM imaging (old point S4). If this is an artifact of the technique, it should be mentioned, at least in the figure legend.We have added a sentence to the Figure 5 legend.4) In many of the sarcomere stains with phalloidin  for example the recused FAK or tensin mutant  or many of the GFP-fusion genotype images in Figure 2 and Figure 2—figure supplement 2 the characteristic high concentration of phalloidin at the Z-disc is not visible. This likely due to an artifact when cutting the myofibrils during histology (these are under high tension). As sarcomere length was not determined in these samples the issue appears not too large. However, we recommend fixing these artifacts wherever possible. The use of relaxing solution during the preparation should also help. A clean, parallel cut is important to preserve myofibrils. Cut myofibrils should wherever possible not be imaged.As pointed out, the morphology of the sarcomeres is not the focus of the paper, and we have not noted any effects on the attachment structure, nor IAP distribution, of the contractile state of the sarcomeres. For example, we did not notice any changes when dissecting in relaxing solution. Nonetheless, to provide images that are as similar as possible we have replaced those images with a dark Z-line with available alternative images and did new stainings to replace the rescued FAK mutant .5) Arrows in Figure 1A are yellow not white (legend). Same for Figure 7A. Figure 6: Obscurin/Unc-89 not Unc-86 (legend).Thanks. These have now been fixed in the figure legends.6) Weitkunat et al., 2014 do not show any spontaneous muscle contractions during pupal stages of the developing flight muscles (currently stated in the Introduction). Spletter et al., 2018 found these contractions, so this could be cited.Sorry, our mistake, we were meaning to say the increased tension as measured in Weitkunat et al., 2014.7) Subsection “Genetic interactions reveal inhibitory action and compensation between IAPs”: "RSU1 and integrin keep PINCH levels at the correct level; in its absence the […]"; in which absence? PINCH?We have removed “its” and replaced with “in the absence of RSU1”.


There is a growing body of evidence to support the use of telehealth in monitoring HbA1c levels in people living with type 2 diabetes. However, the overall magnitude of effect is yet unclear due to variable results reported in existing systematic reviews. The objective of this study is to conduct a systematic review and meta-analysis of systematic reviews of randomised controlled trials to create an evidence-base for the effectiveness of telehealth interventions on glycemic control in adults with type 2 diabetes.Electronic databases including The Cochrane Library, MEDLINE, EMBASE, HMIC, and PsychINFO were searched to identify relevant systematic reviews published between 1990 and April 2016, supplemented by references search from the relevant reviews. Two independent reviewers selected and reviewed the eligible studies. Of the 3279 references retrieved, 4 systematic reviews reporting in total 29 unique studies relevant to our review were included. Both conventional pairwise meta-analyses and network meta-analyses were performed.Evidence from pooling four systematic reviews found that telehealth interventions produced a small but significant improvement in HbA1c levels compared with usual care . The greatest effect was seen in telephone-delivered interventions, followed by Internet blood glucose monitoring system interventions and lastly interventions involving automatic transmission of SMBG using a mobile phone or a telehealth unit.Current evidence suggests that telehealth is effective in controlling HbA1c levels in people living with type 2 diabetes. However there is need for better quality primary studies as well as systematic reviews of RCTs in order to confidently conclude on the impact of telehealth on glycemic control in type 2 diabetes.The online version of this article (10.1186/s12913-018-3274-8) contains supplementary material, which is available to authorized users. Diabetes is a serious, chronic condition that is recognised as an important cause of premature death and disability worldwide. In particular, the prevalence of type 2 diabetes is emerging as one of the greatest global public health challenges in twenty-first century . In the The aim of diabetes management is to keep blood sugar levels as close to normal as possible to improve symptoms and minimise the risk of long-term complications . This reThere is a growing body of evidence that supports the uses of advanced and innovative technologies, such as telehealth, to monitor and manage people with diabetes at a distance and as frequently as it is needed –7. TelehOverall, existing evidence suggests that telehealth has the potential in improving HbA1c for patients living with diabetes but the overall magnitude of effect is unclear due to variable results reported in existing systematic reviews. Given that the literature already contains multiple systematic reviews on telehealth and type 2 diabetes , 9, 10, For the purpose of this study, we defined telehealth as remote patient monitoring (RPM), which involves the transmission  of self-monitored blood glucose (SMBG) readings to a healthcare professional or a specialist team at an offsite monitoring center for evaluation and feedback.Studies that met the following criteria were included in this review: i) systematic reviews and/or meta-analyses of RCTs with our definition of telehealth as an intervention; ii) adults ≥18 years of age with a diagnosis of type 2 diabetes; iii) comparison of standard outpatient care  or other RPM telehealth interventions; and iv) reported HbA1c outcome. Systematic reviews and/or meta-analyses of RCTs on RPM telehealth interventions were excluded if they: i) were non-English publications; ii) included a mixed study population (type 1 and type 2 diabetes) and results were not reported separately for type 2 diabetes; or iii) do not provide feedback to patients following the transmission of SMBG data. In systematic reviews where RPM telehealth was one part of a wider intervention, these were only included where the effects of the RPM telehealth component were individually reported. In addition, if the same authors had produced several publications of the same review, the most updated and/or the full report of the review were included, and other versions excluded.The literature search was conducted from April 1 to 8, 2016 and the electronic bibliographic databases including The Cochrane Library, MEDLINE, EMBASE, HMIC and PsychINFO were searched. All searches were restricted by date range to 1990 – April 2016. Limiting the search period from 1990 is likely to identify all apart from a very small minority of systematic reviews that were carried out earlier , 12. A bBased on the eligibility criteria, two reviewers (AL and YP) independently screened the list of titles/abstracts identified through searches for systematic reviews. Selected systematic reviews at this stage were further included for a full-text review by the same two reviewers. Any disagreements between the reviewers about the inclusion and exclusion were resolved by discussion until a consensus was reached. The same two reviewers using Eppi-Reviewer software 4 then extracted data from the resulting final list of selected systematic reviews independently. The two sets of extracted data were then compared for quality and validity purposes. Consensus was achieved without negotiation.The assessment of the methodological quality and strength of each systematic review was based on the AMSTAR tool, which is a validated measurement tool available for evaluating multiple systematic reviews . The AMSEach of the 11 items is given a score of 1 if the specific criterion is met by a “yes” answer, or a score of 0 if the criterion is not met, unclear or not applicable. The overall AMSTAR score is calculated by adding all the individual item scores together. As defined by AMSTAR, quality is categorised into three levels: high quality if the total score is between 8 and 11, medium quality if the total score is between 4 and 7, and low quality if the total score is between 0 and 3.The same two reviewers independently assessed each potentially relevant review for inclusion. Any disagreements between the reviewers were resolved by discussion and when required, a final opinion from a third reviewer was sought.To examine the overall magnitude of effect in using telehealth for controlling HbA1c levels in Type 2 diabetes, where possible, we conducted conventional pairwise meta-analyses as well as network meta-analyses (NMAs) of the included reviews. While the pairwise meta-analyses allowed us to investigate the difference of effect between telehealth interventions vs. usual care, the NMAs enabled us to explore if there is any specific telehealth application that is superior. NMAs involve the statistical combination of both direct and indirect evidence about pairs of interventions that originate from two or more separate studies to provide estimates of relative effectiveness for all comparators.Care was taken to not include data from individual studies more than once by unpicking each of the included reviews and the subsequent combination of data of the individual primary studies included in the reviews. For HbA1c, where change from baseline data were reported in the trials and were accompanied by a measure of variation (for example standard deviation), these were extracted and used in the meta-analyses. Where measures of spread for change from baseline values were not reported, these trials were excluded from the meta-analyses.Furthermore, due to the various telehealth applications (technologies) used as well as feedback methods provided in the interventions, we performed subgroup meta-analyses to assess whether their impact on glycemic control differed.I2 value for the whole network, which is a weighted average of the I2 value for all comparisons where there are multiple trials (both direct and indirect), and random-effects models were used if the I2 value was above 50% . A funnel plot generated in Review Manager 5 (RevMan 5) for HbA1c was used to visually assess publication bias.The conventional pairwise meta-analyses were conducted with reference to the Cochrane Handbook for Systematic Reviews, whereas NMAs were undertaken using the Netmeta package in R3.2.2. This uses a graph-theoretical method, which is mathematically equivalent to the frequentist network meta-analysis . HeterogItems on the PRISMA  checklist that is relevant for a systematic review of reviews was used to report the findings ; thus, we identified in total 29 unique studies relevant to our review. However, four of these studies did not provide extractable data for HbA1c and were therefore not included in our meta-analyses. The number of relevant trials included in each review ranged from five to 18 trials and the sample sizes of the various trials ranged widely from 30 to 1665 participants. The length of the interventions ranged from 3 to 60 months, with majority of the interventions lasting three months (13/29 studies) or between six and 12 months (13/29 studies). Only one study reported a five-year follow-up.In total, we found 78 studies coded in the four reviews, of which 51 were considered relevant to our review. Individual telehealth interventions that were deemed irrelevant and excluded included studies that did not involve or report results separately for participants with type 2 diabetes, studies that did not include telehealth interventions that met our review’s definition of telehealth or studies that did not involve the transmission of SMBG data followed by automatic and/or healthcare provider feedback. The 51 relevant studies contained 16 duplicates and four studies also had multiple publications . In addition to the wide range of technologies used, many of the studies also incorporated an educational component to their telehealth intervention to improve patients’ knowledge in diabetes self-management. Almost all studies indicated that the transmitted self-monitored blood glucose data were used to provide feedback, or modify treatment or behavior, although the details varied.For the purposes of subgroup meta-analyses, telehealth applications and feedback methods were classified into different categories. For telehealth applications, these were grouped into four categories according to the method of transmission used for transmitting self-monitored data to a receiving station remotely: (i) Internet/web (including any application or software on a computer or a mobile phone that uses data networks or the Internet); (ii) automatic transmission ; (iii) automatic mobile transmission ; and (iv) telephone .With regards to feedback methods, six categories were classified: (i) automated message ; (ii) human calls ; (iii) human calls only if necessary ; (iv) human message ; (v) human message + calls ; and (vi) videoconferencing . Automated and human messages could include messaging through Internet, SMS, a patient portal and/or a telehealth system.Among the four systematic reviews described in the current review, two were rated moderate quality reviews , 17. TheIn the two reviews , 17 thatAll four reviews primarily examined the effect of telehealth on HbA1c. Russel-Minda et al., 2008 reported that three out of their five studies on type 2 diabetes using cell phones with SMS and Internet  found a statistically significant improvement in HbA1c when compared to usual care. Tildesley et al., 2015 who identified nine randomised controlled Internet blood glucose monitoring systems (IBGMS) trials, reported that eight of them showed significantly improved HbA1c levels in the IBGMS group when compared with the usual care group. However, one of the studies only achieved significant HbA1c reduction at six months but not 12 months. Greenwood and colleagues (2014) identified and reviewed 16 teleheatlh remote patient monitoring interventions using one or a combination of technologies , that incorporated key elements of structured self-monitoring of blood glucose (SMBG) identified as essential for improving HbA1c. They reported that, compared to usual care, telehealth was shown to significantly improve HbA1c in seven out of the 15 reviewed studies. The authors also found that interventions that incorporated at least five out of the seven key elements of structured SMBG consistently achieved significant HbA1c improvements between study groups. In addition, studies that incorporated at least four of the seven key elements of structured SMBG and had a baseline HbA1c greater than 8% resulted in a decrease of at least 0.7% in HbA1c levels. Lastly, Huang et al., 2015 reported that, compared to usual care, 11 out of the 18 studies included in the review found a statistically significant improvement in HbA1c in the telehealth group. Furthermore, a meta-analysis of the 18 studies found that participants using telehealth had significantly improved HbA1c levels when compared to participants receiving usual care . The same review also conducted subgroup analyses that included feedback methods, duration of follow-up, study location, baseline HbA1c and sample size. They found that feedback by interactive telephone calls with a healthcare provider or researcher to be associated with the greatest improvement in HbA1c , followed by automated phone-based SMS and/or internet-based messaging . No improvement in HbA1c was reported with automated telephone calls . For the remaining subgroup analyses, a significant reduction in HbA1c was reported to be associated with Asian ethnic groups, small study sample sizes, and patients with a baseline HbA1c level of 8% or higher.In order to determine the overall effectiveness of telehealth on glycemic control in individuals with type 2 diabetes, we conducted additional meta-analyses that incorporated all the unique studies, with extractable data on HbA1c, identified in the four reviews.2 = 82%; Fig. A pairwise meta-analysis pooling evidence from 25 (out of 29) RCTs indicate that, compared to usual care, telehealth is associated with significant improvements in HbA1c in patients with type 2 diabetes  but with statistical heterogeneity to the variability in effect estimate . However, considerable between-study heterogeneity was present (In = 1), videoconferencing (n = 1) and human calls only if necessary (n = 3). This is most likely due to the very limited number of studies being available in these subgroups. Similar results were reported in the NMA , followed by human message  and then automated message   SMBG results to a receiving station or person to receive automated messages and/or healthcare provider feedback.Our systematic literature search identified, in total, 58 potential telehealth and diabetes systematic reviews but only four reviews met our inclusion criteria, of which two were of moderate quality and the other two of low quality according to the AMSTAR tool. All four reviews concluded that telehealth interventions have the potential in improving glycemic control in people with type 2 diabetes. However, when we pooled the HbA1c results from the 25 RCTs included in the four reviews together, only 14 (56%) studies reported a significant improvement in telehealth intervention versus usual care group.The greatest improvements in glycemic control with telehealth was reported in studies where participants had a mean baseline HbA1c level of 8% or greater, regardless if they were on insulin or not , 16, 18.It is important to note when interpreting the results from our review that this review is limited to capturing and reporting information presented in the included systematic reviews and meta-analyses. Two important limitations therefore exist in our review of systematic reviews. Firstly, we depended on the authors of the four included reviews in this review to have adequately included and critically appraised individual studies as well as correctly captured and interpreted the study results. We did not examine the full-texts of individual studies unless there were major data gaps we had to fill in the reviews or we felt that there may be discrepancies in the analyses of individual studies included in multiple reviews. Hence, potential omissions or errors that may be present in our coding and/or analyses and results, may be due to unreported errors in the original reviews and/or original primary studies included in those reviews.Secondly, the four reviews varied greatly in terms of the type of telehealth interventions, duration of follow-up, study sample size, baseline HbA1c levels, and/or insulin- and non-insulin-dependent population with type 2 diabetes. In addition, the reviews included minimal description on the additional telehealth components such as e-learning, virtual coach and/or networking support group and how these additional components may have impacted on the health outcomes. Details on feedback frequency and how it was used to help support and improve patient self-management skills were also limited. In addition, only one of the four reviews conducted meta-analyses. Hence, statistical pooling of results to assess the estimated mean effect of HbA1c with telehealth is limited. Although we attempted to pool and examine the findings on HbA1c from the four reviews, substantial heterogeneity among individual studies was evident from the overall meta-analyses, majority of the subgroup analyses (by transmission and feedback methods) as well as network meta-analyses due to the diversity of telehealth interventions and applications used in the trials. It is therefore difficult to confidently conclude which telehealth component or type 2 diabetes population is likely to benefit the most and from which telehealth intervention, especially in the long-term.We therefore agree with the recommendations made by the authors of the four reviews that more high quality, well-designed RCTs with large sample sizes and longer follow-up durations are needed to investigate the sustainability and to confirm the benefits of telehealth in type 2 diabetes management. In addition, to produce reliable pooled estimates of HbA1c, it would be useful for future studies to take into account the differences in baseline HbA1c level when recruiting study participants and drawing conclusions from findings. Greenwood et al., (2014) have also suggested that future telehealth research should explore the use and impact of telehealth on behavior change in people with non-insulin-dependent type 2 diabetes. This group of users would primarily use telehealth for lifestyle and behavior change to manage their diabetes in contrast to insulin users who would primarily use telehealth for monitoring and adjusting insulin treatment. Comparing the usage and impact of telehealth in these two sub populations of diabetes may provide some explanations as to which and how different telehealth components work and/or are responsible for improved glycemic control for people with diabetes. For example, are improvements in HbA1c level with telehealth dependent on insulin dose adjustments, or SMBG frequency or from increased self-motivation and/or patient-physician communication? Future research in this area could provide important knowledge for clinical practice for diabetes management .To further strengthen the current evidence-base for telehealth and the management of type 2 diabetes, future reviews should also consider assessing the cost-effectiveness and outcome measures that may influence the uptake and outcomes of telehealth interventions, such as healthcare provider satisfaction and patient health-related outcomes  as well as the usability and feasibility of self-monitored devices for diabetes management. These are all important evidence for current clinical guidelines and health-related economic policies.This review found that telehealth interventions produced a small but significant improvement in HbA1c levels compared with usual care, suggesting that telehealth has the potential to deliver beneficial change. However, there is a need for higher quality primary studies as well as systematic reviews of RCTs in order for us to draw any definite conclusions. Furthermore, in order to provide a complete evidence base for policy makers on the overall effectiveness of telehealth interventions for type 2 diabetes management, future reviews should also focus on the impact of telehealth in other areas of diabetes management such as quality of life, quality of care and cost-effectiveness.Additional file 1:Base literature search strategy. (DOC 37 kb)Additional file 2:PRISMA checklist. (DOC 62 kb)Additional file 3:Excluded references. (DOCX 119 kb)Additional file 4:Evidence tables. (DOCX 98 kb)Additional file 5:NMAs on HbA1c by telehealth transmission methods. (DOCX 89 kb)Additional file 6:NMAs on HbA1c by telehealth feedback methods. (DOCX 119 kb)


Chronic obstructive pulmonary disease (COPD) is one of the major morbidities in public health, and the use of mHealth technology for rehabilitation of patients with COPD can help increase physical activity and ameliorate respiratory symptoms.This study aimed to develop a comprehensive rehabilitation management platform to improve physical activity and quality of life in patients with COPD.The study comprised the following 2 stages: (1) a pilot stage in which a prototype app was developed; and (2) a fully-fledged platform development stage in which 2 apps and 1 COPD patient monitoring website were developed. We conducted a randomized clinical trial to investigate the efficacy of the apps developed in the second stage of the study. In addition, two 12-week exercise regimens (fixed and fixed-interactive) were tested for the trial. The clinical parameters of the respiratory function and patient global assessment (PGA) of the app were obtained and analyzed. Notably, Android was the chosen operating system for apps.P=.01), and signs of improvement were witnessed in the fixed-interactive group. In addition, the PGA score was moderate or high in all aspects of the user experience of the apps in both intervention groups.We developed 2 COPD rehabilitation apps and 1 patient monitoring website. For the clinical trial, 85 patients were randomized into the following 3 groups: 57 were allocated to the 2 intervention groups and 28 to the control group. After 6 weeks, the COPD assessment test scores were significantly reduced in the fixed group  Chronic obstructive pulmonary disease (COPD) is recognized as a major public health problem and might become a considerable burden worldwide in the near future . The samAccording to a 2016 survey of 7 tertiary hospitals in Korea that provided PR programs, only 5 hospitals had established protocols for PR programs, while 2 hospitals had only conventional rehabilitation programs. Inpatients were admitted to a 1- to 2-week PR program with an average of 3-5 sessions a week, and each session ran 10-60 minutes. Only 1 hospital had a 12-week PR program with exercise programs and patient education sessions focusing on the muscular endurance, cardiorespiratory fitness, and breathing training. Though most of the hospitals’ survey acknowledged the need for an extended PR program, the hardships included a lack of funding or certified facilities, and low national health insurance coverage. An alternative model that assists in overcoming these barriers is home-based rehabilitation (HBR) . A well-A significant body of studies exists proving that HBR using mobile technology is as effective as center-based rehabilitation programs ,18. SpecThere are, however, only a few available HBR mobile apps that incorporate evidence-based health recommendations . To achiefil breath, which combines a mobile PR app platform, including a wearable device, a personalized app, and a website for monitoring patients by health care professionals. Furthermore, a randomized clinical trial was conducted to investigate the effectiveness of the platform. Notably, the study is the first multicenter-based clinical trial of a home-based mobile PR program for Korean patients with COPD.This study aimed to develop a home-based mHealth PR for patients with COPD to improve their daily physical capacity and QoL. To achieve this goal, we developed The study was divided into the following 2 stages : a pilotThe study participants of Stage 2 were recruited from outpatient clinics of 4 secondary or tertiary hospitals in Korea. Patients with COPD were selected according to the following inclusion criteria: (1) age>20 years; (2) a postbronchodilator forced expiratory volume in 1 second of <80% compared with the reference range; (3) ability to walk >150 m in a 6MWT; and (4) an Android smartphone owner. Of note, patients who were unable to follow the exercise regimen were excluded from the screening process. All study participants were to sign a written informed consent.The study participants were randomized into 3 groups as follows: fixed exercise, fixed-interactive exercise, and control group. The fixed-interactive exercise group initiated a fixed exercise regimen for the first 6 weeks, followed by an interactive exercise protocol for 6 weeks. A random allocation (1:1:1) within each center was moderated by an independent coordinator; patients were stratified by the baseline forced expiratory volume in 1 second and COPD assessment test (CAT) scores. The 6MWT, self-perceived dyspnea assessment in relation to a physical disability , and CAT were acquired at the baseline (V1), 6 weeks (V2), and 12 weeks (V3). Patient global assessment (PGA) by a 5-point Likert scale  was measured at V3. Participants in both intervention groups were provided with a wearable pulse oximeter . The trial commenced in May 2017 and ended in December 2017 and was approved by the Institutional Review Board of each participating hospital. In this study, the primary endpoint was the change of respiratory function parameters  at V3 compared with the baseline.P<.05 as a statistically significant difference. Based on previous studies showing 6MWT improvement in clinical trials, we hypothesized a mean difference of 6WMT to be >50 m in the intervention group versus 0 m in the control group after 12 weeks [All statistical analyses were performed using SPSS, version 18.0 . One-way analysis of variance was used to compare the baseline characteristics of the 3 groups. In addition, one-way analysis of variance with repeated measures was performed to analyze changes between visits. We considered 12 weeks ,28. AssuTo collect user requirements for the home-based COPD rehabilitation app and wearable devices, we recruited 11 patients  for the interview. 2) and heart rate were measured and displayed to users during exercise.In tandem with patient interviews, a comprehensive literature review and in-person consultations were obtained from health care professionals regarding the requirements for the app, wearable devices, and the patient monitoring website. In addition, patients were asked to perform a 30-minute walk every day, and blood oxygen saturation , and calorie consumption should be locally and securely stored in the smartphone.Biometric parameters of the patient heart rate, blood oxygen saturation  during exercise.Alarm function should be provided to alert the patient of critical health status . However, the feedback at the end of the pilot study expressed the desire that the app should offer longer exercise regimens with varying intensity; this was confirmed by the fact that 45% (5/11) of participants after 2 weeks and 55% (6/11) after 4 weeks stated that the exercise target was set too low (which might give the opportunity to set new targets as the PR continued). Therefore, at Stage 2, we developed 2 apps, one with a fixed exercise regimen, and the other with an interactive regimen for mobile phones running on the Android operating system (version 4.4.4 or above), as shown in 2) were sent to the monitoring website. Furthermore, a 6MWT was performed using the apps before initiating the exercise regimen.The apps were linked to a wearable pulse oximeter via Bluetooth (version 4.0), and activity-related data  and those experiencing breathing difficulties  who need closer monitoring during the use of the app.The website provides a summary of the patient PR compliance of individual patients after enrollment. A total of 153 patients were screened, and 85 participants were randomized into 3 groups . Of thesP=.01) at V2, and some improvement was also observed in the fixed-interactive group (P=.06). The CAT scores at V3, however, did not show further improvement in both intervention groups. No significant change was observed in CAT scores in the control group throughout the trial. In addition, 6MWT and mMRC did not show statistically significant changes at V2 or V3 in all groups. PGA was measured at V3 for both fixed and fixed-interactive regimens, which showed a moderate level of satisfaction  regarding the rehabilitation education content (Q2), helpfulness of content toward physical activity (Q3), and management of exercise and physical strength (Q4). The overall satisfaction of the apps (Q1) received an average of 2.8 points for both regimens.Regarding primary endpoints , CAT scoMobile PR for use by patients with COPD has great potential in improving health outcomes for patients, especially when it incorporates standardized guidelines for PR, and malleable exercise programs that can accommodate the diverse physical capacity of patients. Therefore, this study aimed to develop a mobile PR platform for COPD patients that offers an opportunity to observe the improvement or maintenance of daily physical capacity and QoL. The final technological aspects of this study consisted of (1) 2 mobile apps, one with a fixed exercise regimen and the other with flexible exercise regimen in accordance with the level of physical capacity; (2) a secure COPD patient management and monitoring server with a database used for better patient care. Moreover, our first randomized multicenter-based clinical trial in Korea demonstrated the benefits of applying mobile technology to the HBR of COPD patients.P=.002) in the sum of patients in both groups after 6 weeks. Meanwhile, the majority (80%) of study participants in all groups had mild or moderate disease severity , and their 6MWT ranged 350-400 m at the baseline; this might explain why the 6MWT did not show significant improvement at V2 or V3. In addition, mMRC, a change-resistant outcome measure, did not show meaningful improvement in our trial, yet correlations between the measured CAT and mMRC changes were present in this study. Useful as it may, mMRC is more of a subjective value ranging from 0 to 4. Considering our patient population, the baseline respiratory function, exercise regimen, and study period, CAT may be the better outcome measure than mMRC in this type of study or even future trials [Among respiratory function parameters, CAT scores showed significant improvement after 6 weeks in the fixed exercise group. As a matter of fact, both intervention groups underwent a fixed exercise regimen for the first 6 weeks. The statistical significance of improvement of CAT scores was high (e trials .Regarding the demographics of participants, we first speculated that the interactive regimen or app may present some difficulty in its use, owing to a relatively more complex interface. However, the difference in outcomes between the fixed and fixed-interactive regimens was interestingly largely insignificant. Despite the results, further refinement of the user interface would be necessary to assist users of our platform.There are several limitations in the trial. First, study subjects were aware to which group they were allocated to during the study. We attempted to minimize further bias by blinding the person who obtained the primary endpoints or analyzed the data. The dropout rate in the intervention groups was noted to be slightly higher than expected, indicating that ongoing patient education and feedback would be required to maximize the benefits of adherence and better secure the merits of mobile PR. Hence, more studies are needed to direct the suitable outcome measure to assess the effectiveness of the utilization of appropriate mobile PR platforms, especially in use for patients with COPD.efil breath is the first attempt in Korea at developing a comprehensive mHealth management platform for the rehabilitation of patients with COPD. Further research is required to study the long-term benefits of compatible mobile COPD rehabilitation services and to investigate the benefit of mobile-only COPD rehabilitation services for patients without access to local or regional clinical health care centers or health care providers.mHealth technology is on the verge of being sufficiently robust to be incorporated as an ancillary component of chronic disease management and rehabilitation. Its efficacy and relevance as an alternative or supplemental means for COPD rehabilitation can be strengthened by accumulating evidence in mobile rehabilitation programs and services; this will enable the prescription of flexible exercise regimens and commensurate with patients’ physical capacities. Moreover, a well-designed rehabilitation monitoring and management of patients with COPD will fill the gap left open by traditional center-based rehabilitation programs. Our


Introduction stating the aim of the study: Robot-assisted laparoscopic pyeloplasty (RALP) is gaining acceptance among pediatric urologists. Few studies have evaluated the retroperitoneal approach for RALP. We share our experience from the first 2 years of a multidisciplinary pediatric robotic program in our center.Patients  and Methods: We performed a retrospective analysis of prospectively collected data of children undergoing RALP for ureteropelvic junction obstruction (n = 50). Diagnosis was confirmed by ultrasound and Tc-99m mercaptoacetyltriglycine renal scan or MRI; the same criteria were used to evaluate outcome. Surgical approach was chosen according to a specific algorithm. Transperitoneal approach (n = 13) was reserved for horseshoe kidney, ectopic kidney, and redo surgery. We analyzed the 37 cases performed by a lateral retroperitoneal approach. Dismembered pyeloplasty was done for all cases and anastomosis was performed using a running monofilament 6/0 absorbable suture. All were drained by double J stent. Patient data, operating room parameters and postoperative course were recorded.Results: The median age was 7.9 years (5.1–13.8); the youngest was 2 years old. The median weight was 23 kg (17–41) with the smallest weighing 12.4 kg. Aberrant crossing vessels were present in 18 children. Median set-up time, from skin incision until the end of the 4-port insertion, was 33 min (29–48). Median surgeon's console time was 151 min (136–182). No conversion to an open procedure was necessary. The postoperative course was free of complications, except urinary tract infection in 6 children. All but 4 patients were discharged on day one. Median follow-up was 9 months (5–13). Redo pyeloplasty was not required. Practical training of other colleagues was possible after 10 cases performed by the same surgeon.Conclusion: These preliminary results suggest that retroperitoneal RALP in children is feasible, safe and effective. It is an excellent option with ideal anatomical exposure. Longer term results as well as continued practice will identify and overcome any challenges and enable surgical mastery of this procedure which is still evolving. The European Association of Urology Pediatric guidelines acknowledge for pyeloplasty procedure that “in good and experienced hands, the open, laparoscopic, or robotic approaches have the same good outcome” . This stIt remains controversial whether to use a transperitoneal or retroperitoneal approach for pyeloplasty in children.A large single-center series with long-term follow-up addressed the impact of 10-year retroperitoneal laparoscopic pyeloplasty experience in a pediatric teaching center, demonstrating that it is a safe, reliable, and efficient procedure with an excellent outcome . SurgeonWe present our findings in terms of safety and efficacy during the first 2 years of a multidisciplinary pediatric robotic program.Between December 2016 and November 2018, with the Da Vinci Xi Surgical System, we performed 50 robot-assisted laparoscopic pyeloplasties (RALP): 37 by a retroperitoneal approach and 13 by a transperitoneal approach for redo procedure, horseshoe kidney, ectopic kidney, based on a previously published algorithm that we have since modified  (5).All 50 cases were either done by or with the assistance of one surgeon (TB) who had no prior experience in robotic surgery but who had extensive experience in the retroperitoneal approach . Before starting robotics, the main author had performed almost one hundred laparoscopic retroperitoneal or transperitoneal pyeloplasties (Unpublished data). He also observed an experienced operator in robotics (one of the co-authors Christophe VAESSEN), who later on assisted him with his first procedure.ClinicalTrials.gov identifier NCT03274050.This retrospective analysis of prospectively collected data received approval from an independent ethics committee . The sponsor was Assistance Publique—Hôpitaux de Paris  and this project was funded by a grant from Necker Hospital. It is registered with the The diagnosis of UPJO was confirmed by renal ultrasound, Uro-magnetic resonance imaging (Uro-MRI) and technetium Tc 99m mercaptoacetyltriglycine-3 (MAG-3) renal scan (RS). Those with equal differential renal function (DRF) on RS were symptomatic  with pyelocaliceal dilatation on ultrasound.The child is placed in the lateral position close to the edge of the table, with minimum flexion, using lumbar padding to stretch the costo-iliac distance without flexing the operating table. Non-stretch adhesive banding secures this position and prevents displacement either forwards or backwards. The upper leg is stretched while the lower leg is flexed, with no contact between them to avoid compression.® iFS System assistant port are placed in an imaginary line drawn from the iliovertebral angle to the iliac fossa  by blunt dissection and gas insufflation dissection, with no need for finger or balloon dissection.The second port is inserted under direct vision at the angle of the iliac crest and the lateral border of the paraspinal muscles.To avoid transperitoneal insertion of the 3rd 8-mm robotic port in the iliac fossa, on the edge of the rectus abdominis muscle, the retroperitoneal working space is fully developed by identifying the deep surface of the anterior wall muscles and pushing the peritoneum medially with a laparoscopic bipolar forceps .® iFS System (ConMed Corporation) assistant port is then inserted in between the camera port and the iliac fossa port. This system is advantageous in providing a stable pneumoperitoneum, constant smoke evacuation and valve-free access.The 8-mm AirSeal2 flow rate is 5 liters/min.Insufflation pressure does not exceed 12 mm Hg and the COThe Hasson cone allows for stable and constant traction of the robotic camera port, which is a major advantage with a retroperitoneal limited working space and all the trocars are “burped” as much as possible upwards and outwards, to give an overall 1 to 2 cm space needed for safe maneuvering and to reduce the risk of breaching the peritoneum.After docking, when the instruments are inserted, the Gerota's fascia is widely opened in a caudo-cranial manner close to the quadratus lumborum muscle with the monopolar curved scissors. Insufflation and gravity push the kidney medially, which correspondingly appears on the upper section of the screen.Minimal dissection is done to free up the UPJ, and a 4/0 PDS transparietal stay stitch is placed at the junction to limit tissue handling, to give stability and to facilitate suturing. The stay stitch provides variable traction on the ureter and renal pelvis. The ureter is largely spatulated, the UPJ is opened and the pelvis, if required, is resected.® magnetic stent). The Black-Star®  is a 4.8 French ureteral stent (length 10–24 cm) with a small magnet fixed with a string at the distal Double-J ureteral stent loop. To remove it, a customized catheter-like retrieval device, lubricated with 2% lidocaine jelly, with a magnetic Tiemann tip is inserted. Both indwelling magnets connect and the catheter can be removed together with the Double-J in an outpatient setting.The ureteropelvic anastomosis (Anderson-Hynes pyeloplasty) is initiated with a 6/0 monofilament absorbable running suture with a 3/8-circle needle. After finishing the anterior line of anastomosis, we insert a 4.7F polyurethane double-J stent through the assistant port  at induction.The posterior anastomosis is then performed, and the redundant renal pelvis is partially trimmed. No drainage tube is left In the presence of aberrant polar vessels, the ureter is completely divided and the UPJ and pelvis are delivered anterior to the vessels with the help of the stay stitch. Then the anastomosis is performed as described.Finally, a plain abdominal film verifies the double-J stent lower end, permitting relocation if it is positioned in the urethra, before waking the patient.Set-up time is counted from skin incision until the end of the 4-port insertion.Console time is defined as the time taken to perform the procedure by the surgeon at the master console.Anastomosis time is the time needed to perform the anterior line of anastomosis, insert the double-J stent and perform the posterior line of anastomosis.Complications are regarded as any deviation from the expected postoperative course according to the five-grade Clavien classification .FU consists of a clinical visit associated with renal ultrasound at 1 month after stent removal and then at 6 months, 1, 2, and 5 years  and improvement of drainage on MAG-3 without further impairment of renal function in patients who had preoperative reduced DRF.http://cran.r-project.org).All statistical analyses were performed using R software  for continuous variables, and as numbers and percentages for categorical variables.t-test (or Wilcoxon test if appropriate). P-values below 0.05 were considered statistically significant.Operative time was divided into two categories (< 150 min or ≥150 min). Factors associated with console time were compared between these 2 groups with Chi2 test (or Fisher test if appropriate) and Student The cumulative sum (CUSUM) method was performed to assess the consecutive performances in terms of operative delay over time with reference to pre-defined target . The CUSUM series was defined as Sn = Σ(Ti–T0), where Ti was the time for an individual i and T0 was the target time.The median age was 7.9 years (5.1–13.8); the youngest was 2 years old. The median weight was 23 kg (17–41) with the smallest weighing 12.4 kg. Aberrant crossing vessels were present in 18 children.There was no significant blood loss.The peritoneum was opened once during the 3rd port placement, and once during the procedure. To overcome this difficulty, the assistant lifted up the kidney through the assistant port, with no need for conversion to open surgery or laparoscopyNeither parenchymal injury nor vascular injury in case of polar vessel occurred.In one patient, preoperative evaluation  missed a HSK which was discovered during RALP via the retroperitoneal approach and the procedure was continued using an anterior extraperitoneal approach to the left renal pelvis.® magnetic stent was inserted in 17 children.The Black-StarMedian set-up time, from skin incision until the end of the 4-port insertion, was 33 min (29–48). Initially taking more than an hour, over time the operator became more than twice as proficient .The CUSUM methodology demonstrated a biphasic learning curve . In the Median surgeon's console time was 151 min 136–182). For 5 patients, console time was < 120 min; while for 4 patients, console time was >210 min. Median complete anastomosis time was 79 min (68–90). Despite a trend of a flat negatively sloping curve , 6, the –182. ForThe median hospital stay was 1 day (1–1); all but 4 patients (90%) were discharged the day after the procedure.® magnetic stent was removed after 34 days (31–35) in the outpatient clinic.The one blind-ended JJ stent was removed after a median of 37 days (31–44) under general anesthesia as a day procedure and the Black-StarAll patients have been followed up regularly (median follow-up 9 months (5–13) and remain asymptomatic. All had a decrease of the AP diameter on ultrasound in the postoperative course . Based oSix children (4 girls and 2 boys) were treated for 8 febrile UTI in total (Clavien Grade 2) with oral antibiotics. These were not immediate postoperative UTIs, rather they occurred during the period in which the Double-J catheter was indwelling.p = 0.03) (Weight was the only significant factor predicting console time [19 kg (16–27) for time < 150 min vs. 31 kg (22–48) for time > = 150 min,  = 0.03) .The preliminary results of retroperitoneal RALP in children in the debutant 2 years of a multidisciplinary pediatric robotic program in our center demonstrates that this approach can achieve results comparable with those reported with open pyeloplasty, laparoscopy or RALP using the transperitoneal approach –13.There is still some controversy concerning which approach to choose: transperitoneal or retroperitoneal .While the transperitoneal approach offers the advantage of a larger working space, retroperitoneal approach accesses the urinary tract directly and detection of crossing vessels is easier. The potential risk of intra-abdominal organ injury during transperitoneal access, though a rare event, is also avoided. It is a matter of personal preference, based on the experience of the surgeon who should ideally be familiar with both approaches. Since the beginning of the multidisciplinary pediatric robotic program in our center, we have modified the previously published algorithm .Olsen and Jorgensen published the only series of retroperitoneal RALP cases in 13 pediatric patients . Median As already highlighted by Olsen et al. previous experience with retroperitoneal pyeloplasties using standard laparoscopic instruments facilitated our transition to this new technology . Both apFor laparoscopic pyeloplasty, we use 3 ports (5-3-3 or 10-5-5 mm)  port is optional given that suction/irrigation devices can be passed through the instrument ports of the system in the case of bleeding or need for double-J stent or suture material, it allows us also to operate in greater security and maintain time efficiency. It is very important to keep meticulous hemostasis maintaining a clear vision and thereby orientation in a space with the psoas muscle being a major landmark which is always located on the lower part of the screen.For robotic pyeloplasty, we have chosen our port placement based on surgical techniques in adult centers and not as described by Olsen et al in 2004 . We use The robotic trocar diameter is larger (8 vs. 3 or 5 mm) which limits its application to very small children. In our study, the youngest child was 2 years old and 12 kg, older than the infants operated in retroperitoneal laparoscopic pyeloplasty . In the However, we feel that instrument size is not the limiting factor as the procedure is performed in a restricted workspace with limited instrument movement, meaning a low risk of collision of the various parts of the robot. The Da Vinci Xi arms are less bulky and therefore much more adapted to pediatric surgery. Thakre et al. have evaluated the performance of robot assisted laparoscopic skills in different workspace sizes with the first generation of the da Vinci Surgical System. While small cubes measuring 40 and 45 mm in diameter have posed difficulties to the surgeon due to arm collisions rendering it impossible to perform drills, these same skills have been replicated with difficulty but no arm contact in cubes measuring 50 and 60 mm, and finally executed with ease in cubes larger than 70 mm, the latter being comparable to the retroperitoneal space in a small child .We suggest that the major limitation lies more in the length/depth needed to operate the robotic instrument in the restricted area that is the retroperitoneal space in small children.The robotic system mainly speeds up performance of the anastomosis. The LC in robotic surgery is increasingly being analyzed though, for the moment, operative time has been the single parameter used to assess proficiency in studies. Given the disparities within this measure, a case load ranging from 15 to 58 has been thus far proposed as the LC target , 18.We have demonstrated a significant learning curve for the set-up time and a moderate but insignificant learning curve for anastomosis and console times.In essence, OT is not a reliable measure of learning, and OT alone may not be a useful indicator of good practice . KassiteThe role of comfort should not be underestimated in lengthy complex laparoscopic procedures. Pediatric laparoscopic surgeons, who work in very bad ergonomic environments due to the small size of their patients, may have a further increase in static postural stress . In 2013Retroperitoneal RALP in children is feasible, safe and effective. Developing competence and ease with bulky instruments poses a significant learning curve on the surgeon. Longer term follow up as well as continued practice will allow surgical mastery and address any challenges associated with this procedure which is still in its early days.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.ClinicalTrials.gov identifier NCT03274050.This retrospective analysis of prospectively collected data received approval from an independent ethics committee . All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.