Nrp1 expression is absent in the SS cortex while abundant in the motor area, creating a sharp border. During the following 3 weeks, Nrp1 is transiently upregulated in subpopulations of SS L2/3 neurons, earlier and more abundantly in S2 than in S1. In vivo knock-down and overexpression experiments demonstrate that transient expression of Nrp1 does not affect the initial development of callosal projections in S1 but is required for subsequent S2 innervation. Moreover, knocking-down Nrp1 reduces the number of S2L2/3 callosal neurons due to excessive postnatal refinement. Thus, an exquisite temporal and spatial regulation of Nrp1 expression determines SS interhemispheric maps.Axons of the corpus callosum (CC) mediate the interhemispheric communication required for complex perception in mammals. In the somatosensory (SS) cortex, the CC exchanges inputs processed by the primary (S1) and secondary (S2) areas, which receive tactile and pain stimuli. During early postnatal life, a multistep process involving axonal navigation, growth, and refinement, leads to precise CC connectivity. This process is often affected in neurodevelopmental disorders such as autism and epilepsy. We herein show that in mice, expression of the axonal signaling receptor Neuropilin 1 (Nrp1) in SS layer (L) 2/3 is temporary and follows patterns that determine CC connectivity. At postnatal day 4,  The cerebral cortex executes higher order functions by integrating information processed in different brain regions . During In sensory perception, the CC allows complex responses by informing sensory areas of the sensory inputs received in the other hemisphere. In the somatosensory (SS) cortices, primary (S1) and secondary (S2) areas process first-order (tactile) and higher order (nociceptive) inputs, respectively . HomotopNrp1 is transiently expressed in SSL2/3 subpopulations during the postnatal weeks that define the patterns of contralateral columnar connectivity, being earlier and more frequently detected in S2 than in S1. Gain and loss-of-function experiments show that changing Nrp1 levels do not affect the initial formation of the S1/S2 column. However, both experimental conditions blocked two important steps of CC development occurring after the first postnatal week: exuberant arborization in S1/S2, and the formation of the S2 column. These experiments demonstrate that transient Nrp1 expression determines the patterns of inter-areal callosal connectivity in SS.Neuropilin-1 (Nrp1) is a receptor that mediates early steps of CC development through its binding to various ligands in association with signaling coreceptors . In the Nrp1 mRNA expression is excluded from L2/3 and 4. Rather than a gradient, the absence of Nrp1 in SS L2/3 and L4 neurons creates a sharp border between motor and SS cortices. The exclusion area coincides with the prospective SS barrel cortex    . First, misphere . To analmisphere . S1 barr barrels . First, escribed . The maiescribed . Knockinescribed . To quanmethods) . First, nditions , and ten2 column  and the 2 column . We obseondition . These eondition . Differenditions . Thus, i+CTB+ L2/3 CPNs at P30 (+CTB+ neurons in S1 vs. the number in S2 (homotopic projections vs. heterotopic projections). This analysis showed that in controls most axons that form the S1/S2 column are homotopic projections from S1 since GFP+ S1L2/3 neurons were labeled 1.5 times more frequently than those in S2  vs. those labeled in S1 (heterotopic) (+ S2 column (nearly 2.5 ratio)  in the cortical plate of the non-electroporated hemisphere. This procedure labels the subset of neurons projecting to the site of injection and identifies the location of their soma in the S1 or S2 areas of the opposite hemisphere . We injes at P30 . As a res at P30 , while os at P30   . In the 5 ratio) . Thus, i5 ratio) . These dnditions  and indinditions . TogetheNrp1 and CAG-Nrp1 brains. To this end, we analyzed and compared axonal distributions of P10, P16, and P30 animals electroporated at E15.5 , coinciding with changes in Nrp1 expression. At P16, the proportion of S1L2/3 CPNs is very similar to that of the adult, while S2L2/3 populations still undergo some CPN refinement between P16-P30 , all SSL2/3 neurons contain low levels of Nrp1 mRNA. Because they express Sema3A, they repel the axons of high Nrp1 expressing neurons located in the motor cortex, which serves to establish the initial dorsoventral routes of navigation  C57BL/6JRccHsd  mice were used in all experiments. The morning of the day of the appearance of a vaginal plug was defined as embryonic day 0.5 (E 0.5). Animals were housed and maintained following the guidelines from the European Union Council Directive (86/609/European Economic Community). All procedures for handling and sacrificing complied with all relevant ethical regulations for animal testing and research. All experiments were performed under the European Commission guidelines (2010/63/EU) and were approved by the CSIC and the Community of Madrid Ethics Committees on Animal Experimentation in compliance with national and European legislation .P4 and P7 brains were fixed in 4% paraformaldehyde (PFA)  diluted in phosphate buffer (PBS 1 X) for 2 and 4 hr at room temperature (RT), respectively. P16 and P56 brains were collected from animals perfused intracardially with sterile PBS 1 X followed by 4% PFA and post-fixed with 4% PFA overnight (O/N) at 4 °C. After fixation, brains were PBS 1 X washed and cryoprotected in 15% sucrose /PBS 1 X. Lastly, they were embedded in 7.5% gelatin /15% sucrose /PBS1X and frozen at –80 °C. Coronal cryostat sections were cut at 16 μm thickness.Nrp1 probe was kindly provided by V. Gil-Fernández (In situ hybridization (ISH) was carried out as previously described by ernández .For double ISH and immunofluorescence (IF) staining, ISH was carried out prior to IF as previously described . The folNrp1 in pLKO.1 vector . In utero electroporation was performed as previously described (Nrp1) was injected into the embryo’s left lateral ventricle using pulled glass micropipettes. Five voltage pulses  were applied using external paddles oriented to target the somatosensory cortex or anterior cingulate cortex. After birth, P2 GFP+ pups were selected and allowed to develop normally until P10, P14, and P28. After sectioning, analyses were performed only in animals in which the electroporated area included both S1 and S2.Plasmids used were pCAG-GFP , pCAG-Nrp1 (gift from Prof. Mu-ming Poo), and shescribed . BrieflyThe tissue was freshly collected from electroporated mouse brains and homogenized with 3 mm stainless steel Lysing beads  in PBS·DEPC (diethyl pyrocarbonate. #D5758. Sigma-Aldrich. Merck. Darmstadt. Germany) for 1 min at 30 Hz with a TissueLyser . Total RNA was isolated using NZY Total RNA Isolation kit  following the manufacturer’s instructions. cDNA was obtained from 1 μg of total RNA with First-Strand cDNA Synthesis kit  in a 15 μl reaction volume.Nrp1-Forward 5’-ACACAGAAATTAAAATTGATGAAACAG-3’, Nrp1-Reverse 5’-GGATGGGATCCAGGGTCT-3’, GFP-Forward 5’-CAACCACTACCTGAGCACCC-3’, GFP-Reverse 5’-GTCCATGCCGAGAGTGATCC-3’, Gus-Forward 5’-AGCCGCTACGGGAGTCG-3’ and Gus-Reverse 5’-GCTGCTTCTTGGGTGATGTCA-3’.Quantitative real-time qRT-PCR was performed using GoTaq qPCR Master Mix  following the protocol of the manufacturer in a CFX-384 Touch Real-Time PCR Detection System . The following gene-specific primer pairs were used: Nrp1 and GFP expression levels were quantified in triplicates and normalized to Gus expression levels. Resultant data were analyzed using the comparative Ct method.–1. Animals were anesthetized during the surgical procedure with isoflurane/oxygen and placed on a stereotaxic apparatus . CTB particles ) were injected with a Drummond Nanoject II Auto-Nanoliter Injector using 30 mm pulled glass micropipettes . Mice were intrapericardially perfused with formalin two days after the surgery and brains were extracted and fixed O/N in formalin at 4 °C. After fixation, brains were cryoprotected with 30% sucrose (#S0389. Merck. Darmstadt. Germany) and frozen in Tissue-Tek O.C.T. Compound .Retrograde labeling from the CC and the cortical plate was performed by injecting subunit B of cholera toxin (CTB) conjugated to Alexa Fluor 555 . Injections were performed in the CC, close to the midline, as previously reported , or in tFifty µm free-floating brain cryosections were used for immunofluorescence. Rabbit polyclonal anti-GFP  was used as primary antibody and goat anti-rabbit-Alexa 488  as the secondary antibody. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) .In situ hybridization chromogenic and immunofluorescence images were obtained with a DMCTR5000 microscope equipped with a DFC500 color camera (Leica. Wetzlar. Germany). Confocal microscopy was performed using a TCS-SP5 (Leica. Wetzlar. Germany) Laser Scanning System on Leica DMI8 microscopes. Up to 50 μm optical z-sections were obtained by taking 3.5 μm serial sections with LAS AF v1.8 software (Leica. Wetzlar. Germany). Tilescan mosaic images were reconstructed with Leica LAS AF software. All images were acquired using a 512 × 512 scan format with a 20 x objective. All coronal sections correspond to rostro-caudal coordinates between Bregma −0.82,–1.46 mm . For thehttps://github.com/FMartin30/Macros, copy archived at swh:1:rev:290118c15f4bd80e241fd3090035432afc5e0edb; Quantification of the total number of electroporated neurons was done in a semi-automated manner using an ImageJ custom macro written in Java  and non-electroporated  hemispheres. The values in selected areas were measured manually delimitating ROIs, adjusting the threshold above the noise (making a binary image), and measuring the integrated density (using Fiji-ImageJ + over GFP+ cells in the primary (S1) and secondary (S2) somatosensory areas was performed on single plane confocal images from z-stacks  hemisphere. For quantification of GFP- populations, the proportions of CTB+ cells were calculated over randomly selected DAPI+ cells, excluding GFP+ cells. Functional areas of the adult mouse brain were identified using the atlas of Paxinos . Results were compared using two-way ANOVA and one-way ANOVA with Tukey Posthoc comparison. Statistical tests were performed using Prism eight software . The source data underlying main  Your study highlights a novel role of Neuropilin 1 in regulating callosal connectivity at the level of the areal map with important insights on how areas mature and develop. The revisions of your manuscript have now clarified some of the methodological issue and we believe that it will be an important contribution to the field. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and John Huguenard as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Role of Nrp1 in controlling cortical interhemispheric circuits" for consideration by The Reviewing Editor has drafted this to help you prepare a revised submission. Your study highlights a novel role of Neuropilin 1 in regulating callosal connectivity at the level of the areal map with important insights on how areas mature and develop. However, several reviewers have stressed the need for additional controls and experiments, in particular to address the following points:Essential revisions:– All reviewers underlined the importance to characterize in more depth the effects of Nrp1 ShRNA electroporation, in terms of impact on progenitors/ migration and on the level/timing of Nrp1 expression. It would thus be essential to address this point in the revised manuscript.– Several reviewers stressed the need to clarify and change some of the quantifications and normalisations that should be taken into account .eLife targets a broad audience, it would be important to modify the manuscript to make it more accessible for non-specialists and clarify some statements, as mentioned by two of the reviewers.– Since We hope that these comments help you in your revisions and look forward to receiving your manuscript.Reviewer #1 (Recommendations for the authors):I have the following questions and suggestions to the authors that would solidify and/or clarify the conclusions made from the study:1) There is previous evidence that Nrp1/Sema3A signalling may be involved in the migration of cortical neurons, with the loss of Nrp1 perhaps affecting this process to a greater extent than overexpression . This, combined with evidence that shRNAs can sometimes cause off-target migration defects  makes it possible that some of the effects observed in the manipulations of this paper may be due to migration defects. I suggest cell counts of the electroporated hemisphere with a comparison of the distributions by bins/cortical layer at P16 and P30 to better understand whether changes in migration might underlie some of the branching and axon guidance phenotypes.2) In the final Results section, the authors present an interpretation: "Refinement of L2/3 CPNs, in both S1 and S2 areas, was not altered upon overexpression of Nrp1. However, electroporation of shNrp1 modified refinement . In P16 control brains, 50% of GFP+ S1L2/3 neurons were CTB+ (CPNs) . This number increased to 65% in shNrp1-targeted S1L2/3 , indicating that low Nrp1 expression delays axonal refinement." An alternative interpretation of this is that the Nrp1 gradient determines to some extent the number and location of neurons initially projecting across the midline. This could be feasible, given that lowering the expression of Nrp1 in S1 neurons may make them more "lateral-S1-like" in identity, and in wildtype brains the lateral S1 neurons end up more heavily projecting through the corpus callosum. I understand it may be difficult to show this experimentally, as it would necessitate earlier ages of collection that have not been performed, but at least an allusion to such an alternative interpretation would be appreciated.3) In figure 4, it is unclear how the shaded areas of significant difference in panels E, I, J, K and L have been determined. The posthoc with Tukey's test p values reported in the legend and signified by the asterisks and hashes are presumably following the two-way ANOVA and comparing the main effect of experimental condition , so how were the shaded areas determined?4) Figure 4 in general shows fairly subtle changes in midline distribution of the electroporated axon tract, which could be easily found by very slight alterations in the positioning of the electroporated cell field, especially given the n=3 animal numbers of this experiment. I therefore suggest a quantification of electroporated cell field's mediolateral extent and position, as a comparable size and positioning between conditions is particularly relevant to this figure, but also for figures 1 and 3.5) At times the phrasing of results and conclusions could be clarified as it is not always apparent what is meant, for instance "They cannot be merely explained by selective repulsion from the cortical plate, although they do not discard its contribution" (line 268) and "This again that not support axonal repulsion, may suggest a disadvantage at a possible axonal competition" (line 305). I suggest clarifying the language throughout the manuscript, as well as potentially adding more information to the summary figure 6 such that it encapsulates the other manuscript findings . There is a lot of very complex data in here that is potentially valuable to the field, but at the moment it may be difficult for a non-expert reader to access it in totality and thereby appreciate the novel advances.Reviewer #2 (Recommendations for the authors):While this is a potentially interesting study, the results are still very preliminary and superficial. Moreover, serious technical considerations limit interpretation of the results at this stage, such that the value of the message conveyed is unclear.Technical concerns: There are multiple severe technical concerns to this study which in our view preclude publication if not directly and fully addressed.1. The quantifications were not blindly performed (see Methods). Given the high experimental variability of the techniques used in this study (in utero electroporations and stereotaxic injections), analyses should be performed in a strictly blind setting.2. Normalization of the GFP signal in the ROIs should be done using the number of electroporated cells rather than the GFP signal in the electroporated site, which is highly dependent on the acquisition settings.3. On all graphs, the dots correspond to the sections analyzed and not the brains, so that the interindividual variability cannot be assessed. For all graphs and statistical analyses, the average of all sections for each brain should be used and not the value of each section. This is a critical point as it misleads the reader into assuming high power of analysis. At the very least three animals per condition  should be used to provide convincing conclusions. In addition, given the variability of GFP signal upon in utero electroporation, taking only 2 sections per brain seems very low. At least 3 to 4 sections should be quantified and averaged to avoid any bias.4. For CTB injections, the injection sites for all brains should be shown (or a quantification of VPM / Po cell body location) in the supplementary figure.5. For the CC analysis at the midline, is the dorso-ventral distribution similar all along the rostro-caudal axis? How is this parameter taken into account in the analyses? Are all electroporated sites equivalent in term of rostro-caudal position? This should be quantified, and the data should be normalized for this parameter.Other concerns1. Timing of down / up regulation of Nrp1 expressionHere the shRNA against Nrp1 was electroporated at E15.5: is the down-regulation still efficient when callosal axons are extending and invading the contralateral cortex ? The levels of Nrp1 should be addressed in electroporated neurons, for example by RT-qPCR or western blot after FACS .The neuropilin-semaphorin family was shown to play a role at the progenitor levels (Castellani lab). Since the constructs used here induce constitutive gain or loss of function, how can the authors be sure that the previous steps of development are not affected in L2/3 neurons?2. Role of Nrp1 in branchingThis is not directly addressed in the study and should be removed from the text. To formally conclude on this aspect, the authors should use sparse electroporation and morphological reconstruction of contralateral axons.3. Biological mechanism underlying the findingsNeuropilins do not act alone and need co-receptors of the plexin family. While this is an important point to understand the mechanisms, this is only superficially mentioned in the discussion (l. 317).Sema3A is proposed as a likely candidate for Nrp1 late function in branching. This could be addressed using Sema3A gain or loss of function.What could control Nrp1 expression levels at these postnatal stages? This should be mentioned in the discussion.Reviewer #3 (Recommendations for the authors):The paper is a difficult read for non-specialists in the field. I suggest simplifying the language for the sake of broader audience. The final diagram might include postnatal refinement data. Essential revisions:– All reviewers underlined the importance to characterize in more depth the effects of Nrp1 ShRNA electroporation, in terms of impact on progenitors/ migration and on the level/timing of Nrp1 expression. It would thus be essential to address this point in the revised manuscript.We have characterized in-depth the expression of Nrp1 through postnatal development to understand the effects of our manipulations. We have confirmed that shNrp1 or CAG-Nrp1 do not produce migration defects. We have also confirmed that the effects of shNrp1 in P16 brains are detectable after electroporation at E15.5 and that our manipulations of Nrp1 do not alter migration or laminar position. Importantly, the characterization of postnatal expression revealed unreported temporal and spatial patterns. In light of this, we can now offer a clearer interpretation of our data. We consider these additions to be an important improvement of the manuscript and thank the reviewers for these important comments.– Several reviewers stressed the need to clarify and change some of the quantifications and normalisations that should be taken into account .p values that did not allow discarding that the data was equal or different from controls. We also responded to the concerns about inter-individual variability, rechecked that this is not the source of our phenotypes, and corrected that each value of the analysis represents the average data from individual mice and not the two measurements of each section.We have made newly blinded quantifications and changed the normalization method to the number of electroporated cells. None of these change the results, thus validating our previous methods. In fact, the quantifications relative to the number of cells suggested by reviewer #2 increase precision. This increases the statistical power of the measurements. All the data remains the same except that the statistical analysis with the new quantification produces lower p-values in the comparisons of the measurements of the effects of shNrp1 in the S2 column. The data normalized to the GFP fluorescence signal in the ipsilateral electroporated hemisphere from the previous version is now shown as a supplement. Previously, mean data showed the same tendency towards reductions, but the statistical analyses showed – Since eLife targets a broad audience, it would be important to modify the manuscript to make it more accessible for non-specialists and clarify some statements, as mentioned by two of the reviewers.We have rewritten major parts of the manuscript and changed Figure 6 summarizing the data. We hope we have improved this aspect.Reviewer #1 (Recommendations for the authors):I have the following questions and suggestions to the authors that would solidify and/or clarify the conclusions made from the study:1) There is previous evidence that Nrp1/Sema3A signalling may be involved in the migration of cortical neurons, with the loss of Nrp1 perhaps affecting this process to a greater extent than overexpression . This, combined with evidence that shRNAs can sometimes cause off-target migration defects  makes it possible that some of the effects observed in the manipulations of this paper may be due to migration defects. I suggest cell counts of the electroporated hemisphere with a comparison of the distributions by bins/cortical layer at P16 and P30 to better understand whether changes in migration might underlie some of the branching and axon guidance phenotypes.We thank the reviewer for this constructive criticism. We have performed these analyses and counted the GFP positive cells throughout the radial axis of the cortex and plotted cell distributions in bins as suggested. The data show that our knocking down constructs do not alter the normal migration of neurons. The data is shown in Figure 2 figure supplement 3 and Figure 4 figure supplement 1. Chen et al. (2008) reported experiments showing that electroporation of RNAi Nrp1 constructs in rats and mice block radial migration. They also showed that the conditional deletion of exon 2 of Nrp1 in mice by in utero electroporation produced the same phenotypes. However, using the same mouse Nrp1 conditional line, the studies by Zhou et al. 2013, report no defects in migration. Similarly, Hatanaka et al., 2009 found that overexpression of a dominant-negative form of Nrp1 disrupts axonal development but not the migration of cortical excitatory neurons. We do not observe defects in migration and cannot explain the discrepancies or our and others with the studies original findings in Chen et al., 2008.2) In the final Results section, the authors present an interpretation: "Refinement of L2/3 CPNs, in both S1 and S2 areas, was not altered upon overexpression of Nrp1. However, electroporation of shNrp1 modified refinement . In P16 control brains, 50% of GFP+ S1L2/3 neurons were CTB+ (CPNs) . This number increased to 65% in shNrp1-targeted S1L2/3 , indicating that low Nrp1 expression delays axonal refinement." An alternative interpretation of this is that the Nrp1 gradient determines to some extent the number and location of neurons initially projecting across the midline. This could be feasible, given that lowering the expression of Nrp1 in S1 neurons may make them more "lateral-S1-like" in identity, and in wildtype brains the lateral S1 neurons end up more heavily projecting through the corpus callosum. I understand it may be difficult to show this experimentally, as it would necessitate earlier ages of collection that have not been performed, but at least an allusion to such an alternative interpretation would be appreciated.Thank you for this suggestion and this comment. In reading this comment we realized that we were not sufficiently clear on explaining how our previous publication  changes the knowledge of how callosal fates are acquired. Our publication demonstrated that callosal fate is discarded rather than obtained only by some populations. Virtually, all L2/3 neurons are CPN at birth and we demonstrated that they all project across the midline. Hence, Nrp1 expression in WT cannot affect the number and location of neurons initially projecting across the midline. We have explained this more concisely and boldly in the introduction- pg 4, line 67-74 and result section-pg 10-11 ln 271-279. We apologize for the confusion.3) In figure 4, it is unclear how the shaded areas of significant difference in panels E, I, J, K and L have been determined. The posthoc with Tukey's test p values reported in the legend and signified by the asterisks and hashes are presumably following the two-way ANOVA and comparing the main effect of experimental condition , so how were the shaded areas determined?The shaded areas represented segments where Tukey´s test values showed significance for continuous adjacent bins. Nevertheless, because the reviewer was right in pointing out that we should revise if the lateral to the medial position of the cells was a factor accounting for the observed differences, we have modified this figure and moved this figure to a supplementary figure- (see below).4) Figure 4 in general shows fairly subtle changes in midline distribution of the electroporated axon tract, which could be easily found by very slight alterations in the positioning of the electroporated cell field, especially given the n=3 animal numbers of this experiment. I therefore suggest a quantification of electroporated cell field's mediolateral extent and position, as a comparable size and positioning between conditions is particularly relevant to this figure, but also for figures 1 and 3.We are very thankful to the reviewer for this observation. We have performed quantifications of the distributions of the electroporated cells in S1 and S2. These confirmed that we analyze brains with similar distributions in S1 and S2 of electroporated brains . We statistical tests demonstrated that distributions in S1 and S2 are not a source of variability when concerning innervation of the cortical plate. Differences in innervation are accounted for only by the variable condition  . This is in agreement with all neurons behaving in an equal way when they express similar levels of Nrp1 when electroporating shNrp1 and CAG-Nrp1 constructs. However, minor changes in the exact number of cells in medial and lateral positions are unavoidable and the reviewer was right, these analyses showed that minor differences in the distributions of electroporated neurons explain the subtle changes in the distribution of axons at the midline we reported in previous figure 4. We, therefore, performed a new analysis with only the brains with the most similar distributions along the lateral to the medial axis . This analysis shows no differences between conditions. This is in agreement with that the location of the projecting neuron in the somatosensory has a greater impact on the dorso-ventral level path of callosal projection than its level of Nrp1 expression, possibly because it is determined by Sema3A expression -which repels the Nrp1 expressing axons of motor neurons. It also makes sense because now we show that in SS L2/3 neurons – Nrp1 expression is not activated until the post-crossing stages.The analysis at the midline is valid and it still serves to demonstrate that shNrp1 and CAG-Nrp1 projections cross the midline using the ventral path as control axons, as was also concluded in the previous version but does not support refinement. We sincerely thank the reviewer for correcting us on this.5) At times the phrasing of results and conclusions could be clarified as it is not always apparent what is meant, for instance "They cannot be merely explained by selective repulsion from the cortical plate, although they do not discard its contribution" (line 268) and "This again that not support axonal repulsion, may suggest a disadvantage at a possible axonal competition" (line 305). I suggest clarifying the language throughout the manuscript, as well as potentially adding more information to the summary figure 6 such that it encapsulates the other manuscript findings . There is a lot of very complex data in here that is potentially valuable to the field, but at the moment it may be difficult for a non-expert reader to access it in totality and thereby appreciate the novel advances.We appreciate this comment. We have edited the manuscript trying to avoid long complex wording. We have tried to summarize the information of the model in the discussion and also changed the figure of the model , also because certain interpretations have been revised.Reviewer #2 (Recommendations for the authors):While this is a potentially interesting study, the results are still very preliminary and superficial. Moreover, serious technical considerations limit interpretation of the results at this stage, such that the value of the message conveyed is unclear.We would like to thank the reviewer for his/her suggestions. We have performed control analysis and changed quantifications. The overall results do not change. The added description of Nrp1 expression across postnatal stages sharpen the interpretation of the results. We think this improves the manuscript.Technical concerns: There are multiple severe technical concerns to this study which in our view preclude publication if not directly and fully addressed.1. The quantifications were not blindly performed (see Methods). Given the high experimental variability of the techniques used in this study (in utero electroporations and stereotaxic injections), analyses should be performed in a strictly blind setting.We have repeated all quantifications in blind conditions and automated some measurements (performed by new authors) and the results do not change.2. Normalization of the GFP signal in the ROIs should be done using the number of electroporated cells rather than the GFP signal in the electroporated site, which is highly dependent on the acquisition settings.We previously described and validated our method of quantifications using GFP signal in the ipsilateral hemisphere and describe how to set conditions that ensure that the GFP signal is proportional to the number of cells . This is the method we had employed. In this revised version and following the reviewer's suggestion, we have now counted the number of GFP electroporated cells and performed alternative normalization against the number of cells instead. The results show little variation but the reviewers advice was valid, the measurements show less variability when normalizing against the number of electroporated cells. They are more precise. The new quantifications provide an increase in statistic power when measuring S2 differences. Previous analysis revealed a tendency to decreases in S2 innervation in the shNrp1 condition. However, p values did not allow to conclude differences with control conditions. Normalizing against the number of GFP cells shows that the reductions in S2 innervation we observe upon knocking-down Nrp1 are significant. This sharpens our interpretations. We have changed the figures and text showing all data according to this method of normalization. We sincerely thank the reviewer for this suggestion.We have developed a Macro for automatic counting that can be found as described in Material and Methods. We hope this is useful for other researchers.3. On all graphs, the dots correspond to the sections analyzed and not the brains, so that the interindividual variability cannot be assessed. For all graphs and statistical analyses, the average of all sections for each brain should be used and not the value of each section. This is a critical point as it misleads the reader into assuming high power of analysis. At the very least three animals per condition  should be used to provide convincing conclusions. In addition, given the variability of GFP signal upon in utero electroporation, taking only 2 sections per brain seems very low. At least 3 to 4 sections should be quantified and averaged to avoid any bias.The reviewer is right. We have corrected this mistake. In all figures, we now show the average obtained from the sections of each mouse as individual data points and then the mean and SEM value of all mice. Previously we showed the mean of all sections. Nevertheless, the data always included at least three mice per condition and stage. The number of animals necessary for conclusions is determined by statistical analysis. The number of animals per condition and the total number of mice in each analysis is now reflected in the figure legends.4. For CTB injections, the injection sites for all brains should be shown (or a quantification of VPM / Po cell body location) in the supplementary figure.We have included Figure 3 supplement 2 with all the injection sites.5. For the CC analysis at the midline, is the dorso-ventral distribution similar all along the rostro-caudal axis? How is this parameter taken into account in the analyses? Are all electroporated sites equivalent in term of rostro-caudal position? This should be quantified, and the data should be normalized for this parameter.In WT animals the dorso-ventral CC routes change some within the rostro-causal axis. However, opur quantifications are performed only at the level of the somatosensory area, where CC axons only follow ventral routes. Our electroporation protocol targets the SS cortex and our experiments and only include brains with equivalent size and location of electroporations at rostrocaudal and mediolateral positions. We have now included a new figure describing this Brains with obvious dissimilar electroporated areas are always discarded. Brains are selected visually after dissection under the dissecting scope and subsequently we confirm the correct location of the electroporated area by the analysis of the processed sections following histological landmarks . We have included quantifications of the average distributions of electroporated cells in the radial axis, as well as in the mediolateral axis and in each section, and confirmed that the brains we analyzed in all groups are similar and not the source of our phenotypes . There are minor changes in the mediolateral distribution of electroporated cells that were the source of minor changes in the dorsoventral path at the midline level. This is now corrected (see the response to reviewer#1).Other concerns1. Timing of down / up regulation of Nrp1 expressionHere the shRNA against Nrp1 was electroporated at E15.5: is the down-regulation still efficient when callosal axons are extending and invading the contralateral cortex ? The levels of Nrp1 should be addressed in electroporated neurons, for example by RT-qPCR or western blot after FACS .We electroporated the shRNA in plasmids. We and others have shown that expression of shRNAs pKLO lentiviral plasmids electroporated at E15.5 last to P21 postnatal stages , similar to the expression of CAG-GFP used as a reporter. See for example reduced Cux1 or Cux2 proteins in GFP-expressing layer 2/3 neurons of the P21 cortex electroporated at E15.5 with shRNA in supplementary materials in Cubelos et al., 2010. Unfortunately, detecting Nrp1 protein in the cell somas was not possible in our hands (manuscript pg 13 ln 323-325). We confirmed that the effects of shNrp1 in P16 brains are detectable after electroporation at E15.5 by assaying the effects of the constructs on the cingulate cortex, which express high levels of Nrp1. Whole tissue was dissected and Nrp1 levels assesed . Importantly, this comment has provided insightful revision. In addressing it, we have analyzed the patterns of Nrp1 throughout postnatal development and found patterns that help us to refine the interpretation of our data.The neuropilin-semaphorin family was shown to play a role at the progenitor levels (Castellani lab). Since the constructs used here induce constitutive gain or loss of function, how can the authors be sure that the previous steps of development are not affected in L2/3 neurons?+ cells in all conditions and at all stages and we found no differences.As mentioned above, we have quantified the number and laminar distribution of GFPWe now include analysis at earlier stages. This analysis at P10 demonstrates no differences between conditions. Importantly, it has revealed that the S2 column is formed after the S1 column and later than P10 in WTs and all our electroporating conditions. This finding, together with the description of nrp1 expression has allowed us to refine the interpretation of our data.2. Role of Nrp1 in branchingThis is not directly addressed in the study and should be removed from the text. To formally conclude on this aspect, the authors should use sparse electroporation and morphological reconstruction of contralateral axons.+ signal but not the number of CPNs. They block axonal increases that in controls are known to be caused by terminal branching. We have rewritten the text trying to make this clear and in the discussion specify that this is a model that requires further investigation.The role in branching is principally concluded from the comparative analysis of innervation patterns at different stages . Our manipulations reduce contralateral GFP3. Biological mechanism underlying the findingsNeuropilins do not act alone and need co-receptors of the plexin family. While this is an important point to understand the mechanisms, this is only superficially mentioned in the discussion (l. 317).We now discuss this more extensively (pg 15 ln 378-392).Reviewer #3 (Recommendations for the authors):The paper is a difficult read for non-specialists in the field. I suggest simplifying the language for the sake of broader audience. The final diagram might include postnatal refinement data.We have rewritten most of the manuscript trying to address a broader audience. We have changed Figure 6 showing the diagram of the model.


For patients whose prostate cancer spreads beyond the confines of the prostate, treatment options continue to increase. However, we are missing the information that is needed to choose for each patient the best treatment at each step of his cancer progression so we can ensure that maximal remissions and prolonged survival are achieved. In this review, we examine whether a better understanding of how the activity of the target for the default first treatment, the androgen receptor, is regulated in prostate cancer tissues can improve prostate cancer treatment plans. We consider the evidence for variability of androgen receptor activity among patients and examine the molecular basis for this variable action. We summarize clinical evidence supporting that information on a prostate cancer’s genomic composition may inform on its level of androgen receptor action, which may facilitate choice for the most effective first-line therapy and ultimately improve prostate cancer treatment plans overall.Inhibiting the activity of the ligand-activated transcription factor androgen receptor (AR) is the default first-line treatment for metastatic prostate cancer (CaP). Androgen deprivation therapy (ADT) induces remissions, however, their duration varies widely among patients. The reason for this heterogeneity is not known. A better understanding of its molecular basis may improve treatment plans and patient survival. AR’s transcriptional activity is regulated in a context-dependent manner and relies on an interplay between its associated transcriptional regulators, DNA recognition motifs, and ligands. Alterations in one or more of these factors induce shifts in the AR cistrome and transcriptional output. Significant variability in AR activity is seen in both castration-sensitive (CS) and castration-resistant CaP (CRPC). Several AR transcriptional regulators undergo somatic alterations that impact their function in clinical CaPs. Some alterations occur in a significant fraction of cases, resulting in CaP subtypes, while others affect only a few percent of CaPs. Evidence is emerging that these alterations may impact the response to CaP treatments such as ADT, radiation therapy, and chemotherapy. Here, we review the contribution of recurring somatic alterations on AR cistrome and transcriptional output and the efficacy of CaP treatments and explore strategies to use these insights to improve treatment plans and outcomes for CaP patients. Prostate cancer (CaP) remains the most frequently diagnosed non-skin cancer and the second leading cause of cancer-related mortality in American men . When loThe concept and scope of ADT, which prevents activation of AR by androgenic ligands, has been reviewed extensively before ,10. OverA better understanding of the molecular basis for these diverse responses may improve and better inform treatment choices to maximize remissions and prolong survival. Such insights may be derived from taking a closer look at the regulation of AR activity. AR action, which is targeted specifically by ADT, is impacted also by radiation therapy and chemotherapy in CaP cells, and is subject to context- and gene-specific regulation ,21,22,23AR is a member of the nuclear receptor family of transcription factors. Its organization in three major domains is similar to that of other NRs and consists of an N-terminal domain  and a DNA binding domain  that is connected via a small hinge region to a ligand-binding domain  . AndrogeThat AR NTDs can adopt different conformations depending on their interacting protein partners, as suggested by the cryoEM study above, is consistent with previous reports of α-helical sheets forming in the otherwise unstructured AR NTD upon interactions with components of the  transcriptional machinery or with ligands ,35. SuchSupporting the possibility of target gene set-specific AR conformations, multiple literature reports indicate the differential output of AR at distinct targets genes, depending on its interaction with specific ligands, interactors, and DNA. It is well-recognized that the magnitude (fold change) and direction (induced or repressed) of androgen regulation of the hundreds of AR-binding genes that have been discovered to date vary widely. Moreover, the spectrum of AR target genes that are expressed differs between androgen-responsive tissues in general and with the stage of the cell cycle, duration of androgen stimulation, and/or coregulator dependency in CaP cells. For instance, integrated ChIP-Seq and gene expression analysis, after androgen stimulations that varied in duration from 30 min to 24 h, uncovered signatures of AR target genes whose expression was up-or downregulated early  or late  . These rTaken together, these observations support that the specific AR DNA-binding motif region, protein interactome, and (in)activating ligand can influence AR target gene transcription . The prebona fide AR target gene signatures [The use of genome-wide transcriptomics and ChIP-chip, ChIP-Seq, ChIP-Exo or CUT and RUN approaches, by themselves or in an integrated manner, has uncovered a compendia of direct AR target genes, i.e., genes that are androgen-responsive and contain an AR binding site, in CaP cells ,50,51,52gnatures ,56. The gnatures . Unsupergnatures ,54,57. Tgnatures .These different lines of investigation independently confirm that variability in AR transcriptional cistromes and transcriptomes exists among clinical CaPs, which raises the question of the molecular basis for such differences. As mentioned above, AR’s ligands, protein interactome, as well as DNA binding sites may all modulate its transactivation function . AR agonSince AR-binding ligands or proteins and DNA regions to which AR is recruited contribute to AR transactivation, and at least the concentrations, levels, or sequence integrity of the former two determinants are influenced by AR-targeting therapies, it is tempting to speculate that their status may help classify CaP cases based on AR activity. In this respect, it is important to keep in mind that steroid panels are not routinely determined on clinical CaP tissues, that most somatic alterations events occur at low incidence , that coThe most frequent somatic alterations in CaP are rearrangements that involve several members of the ETS family of transcription factors such as ERG, ETV1, and ETV4, which have important roles in embryonic development and cell proliferation. The most common of these rearrangements is a fusion between the AR-dependent promoter of the TMPRSS2 gene to the (majority of the) coding region of the gene encoding ERG. This gene fusion, which is present already in ~50% of CS-CaP, results in AR-dependent overexpression of ERG. The TMPRSS2-ERG fusion has been implicated in CaP invasion and migration, and (lack of) treatment response. In view of these findings, inhibitory peptides and peptidomimetics that bind ERG and lead to its proteolytic degradation of the ERG protein have been developed, which attenuate CaP cell invasion and proliferation, and CaP growth .Not surprisingly, information on such alterations has been considered for CaP subtyping. A predominant classification is based on The Cancer Genome Atlas (TCGA) characterization of 333 primary CaPs. Integrated WES, WGS, transcriptomics, and methylation assays on these CaPs led to seven major subtypes encompassing three-quarters of all clinical cases studied. The subtypes were based on the presence of gene fusions involving the ETS transcription family members ERG1, ETV1, ETV4, and Fli, as well as somatic alterations in the E3 ligase SPOP, the pioneering transcription factor FoXA1, and the isocitrate dehydrogenase IDH1 . Six of Indeed, since the first AR ChIP-chip data from promoter arrays on CaP cells in culture, binding sites for ETS factors have been recognized to be enriched in close proximity to AREs . MultiplFurther data analyses or deeper sequencing have revealed other clinically relevant sub-classifications, some of which have a bearing on AR transcription output. For instance, a subclass of ERG gene fusion-positive cases frequently showed deletions or mutations in the gene encoding PTEN . ReciproIt should be noted that alternative CaP classification methods have been proposed in which subclasses also vary in AR activity. As an example, the PAM50 classifier, which is now widely used to subtype breast cancer into molecular classes with varying prognoses, has been applied to localized CS-CaP . Three CThe fact that genomic alterations can impact CaP aggressiveness, progression, and treatment response, has long been recognized. For instance, localized CS-CaP with a higher number of copy number alterations have a shorter time to biochemical relapse, the most unfavorable prognosis, and more frequently metastasize after surgery ,87. BecaIn this regard, it is important to note that several of the genomic markers or distinct gene expression patterns used to subtype CaP have been associated with CaP progression and response to CaP treatments that directly target AR or are impacted by AR action. Indeed, although ADT drugs were designed to specifically prevent AR action, other treatment options that are currently considered for first-line treatment of metastatic CaP and were not developed as AR inhibition, also either target aspects of AR function or are impacted by AR inhibition. The best-recognized example is synergistic effects by combining ADT and radiation therapy to control disease progression in localized CS-CaP. The molecular basis for this clinical benefit has been AR’s control over a subset of AR target genes that are induced by DNA damage and mediate DNA repair. ADT thus prevents the resolution of double-strand breaks and resistance to DNA damage, enhancing the impact of radiation therapy ,88. The Whether this implies that genomic marks that are increasingly determined in a patient’s CaP and are linked to AR action can also predict treatment response to AR-targeting of impacting therapies is not yet entirely clear, although evidence is emerging that this may be the case. For instance, SPOP mutations were associated with higher activity of an AR target gene signature . SPOP alERG gene fusions represent the most frequent somatic alteration and are present already in half of localized CS-CaP ,26, yet A subclass of ERG+ cases is enriched for deletion of wild-type PTEN function, and PTEN deletions in ERG+ cases reflect a late event subtype with more aggressive features such as increased locoregional stage compared to ERG fusion-positive and PTEN deletion-negative cases ,93. LossWhile FoXA1 mutant CS-CaPs show higher AR activity , the linThe studies above focus on first-fine treatments for CS-CaP. Metastatic CRPCs that have already acquired resistance to at least one line of treatment were also characterized genomically. Evaluating the genomic and transcriptomics data from CRPC tissues and circulating tumor DNA from CRPC patients for their impact of features on clinical outcome and specifically treatment response, it was shown that loss of wild-type p53 function was associated with a shorter time of treatment with second-generation antiandrogens. These findings established several of the genomic marks used to subtype CaP as drivers of resistance to first-line AR-directed therapy ,107.Moreover, CaP classes derived from methods other than the TCGA-based subtyping have also been suggested to display variable treatment responses. For instance, luminal- and basal-like PAM50 CS-CaPs demonstrate a difference in clinical behavior, in which luminal B CaPs respond better to postoperative ADT than do patients with non-luminal B tumors . These rTo ascertain if the information on such marks may facilitate better and informed choices between the multiple treatments or decisions on whether or not to combine such treatments for first-line CS-CaP treatment, we expanded our analyses of AR target gene expression patterns on 6532 CS-CaP clusters . To thisTaken together, the studies summarized above support marked differences in AR transcriptional output in clinical CaP. Evidence is emerging that somatic alterations that are linked to fractions of AR action and are routinely assessed in the course of a patient’s treatment course may be associated with preferential response to ADT, radiation therapy, and/or chemotherapy . AlthougEvaluating genomic subtypes may eventually outperform clinical parameters previously used or currently considered as predictive biomarkers. However, it is important to consider also that about a quarter of CS-CaP cases already do not fall in any of the seven TCGA subclasses identified , suggest


Macrophage-mediated inflammation plays an essential role in the development of atherosclerosis (AS). Long noncoding RNAs (lncRNAs), as crucial regulators, participate in this process. We identified that lnc-MRGPRF-6:1 was significantly upregulated in the plasma exosomes of coronary atherosclerotic disease (CAD) patients in a preliminary work. In the present study, we aim to assess the role of lnc-MRGPRF-6:1 in macrophage-mediated inflammatory process of AS.  The correlation between lnc-MRGPRF-6:1 and inflammatory factors was estimated firstly in plasma exosomes of CAD patients. Subsequently, we established lnc-MRGPRF-6:1 knockout macrophage model via the CRISPR/Cas9 system. We then investigated the regulatory effects of lnc-MRGPRF-6:1 on macrophage polarization and foam cell formation. Eventually, transcriptome analysis by RNA sequencing was carried out to explore the contribution of differential genes and signaling pathways in this process.  lnc-MRGPRF-6:1 was highly expressed in the plasma exosomes of CAD patients and was positively correlated with the expression of inflammatory cytokines in plasma. lnc-MRGPRF-6:1 inhibition significantly reduced the formation of foam cells. The expression of lnc-MRGPRF-6:1 was upregulated in M1 macrophage, and lnc-MRGPRF-6:1 knockout decreased the polarization of M1 macrophage. lnc-MRGPRF-6:1 regulates macrophage polarization via the TLR4-MyD88-MAPK signaling pathway.  lnc-MRGPRF-6:1 knockdown can inhibit M1 polarization of macrophage and inflammatory response through the TLR4-MyD88-MAPK signaling pathway. lnc-MRGPRF-6:1 is a vital regulator in macrophage-mediated inflammatory process of AS. Atheros2 (PGE2) .κB and MAPK signaling pathways in macrophages [α, TNF-β, and CXCL11 in CAD patients, which indicates that lnc-MRGPRF-6:1 may participate in the regulation of macrophage-mediated inflammation in AS.Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs with a length of more than 200 nucleotides, which have important functions in many biological processes . Previourophages . FOXP1-Irophages . TherefoDysregulation of phenotypic transitions in macrophages prolongs inflammation and impedes tissue repair. There is evidence that the polarization imbalance of M1/M2 macrophage may contribute to inflammatory diseases, including AS . The polCoincidentally, several researches have indicated that lncRNAs are involved in the dysregulation of phenotypic transitions in macrophages. Cao et al. demonstrated that lncRNA-MM2P could affect macrophage M2 polarization by regulating the dephosphorylation of signal transducer and activator of transcription 6 (STAT6) . Chi et Taken together, the regulation of macrophage polarization largely determines macrophage-mediated inflammation development and AS progression, and maybe some lncRNAs are the keys to the lock of macrophage polarization. Therefore, in the present study, we aim to explore the role of lnc-MRGPRF-6:1 in macrophage polarization and macrophage-mediated inflammation.2.THP-1 cells  were incubated in RPMI Medium 1640  with 10% fetal bovine serum . All cells were cultured in a moist environment at 37°C and 5% COThis study enrolled 20 CAD patients in the Cardiology Department of the Affiliated Jiangning Hospital of Nanjing Medical University. Inclusion criteria were patients undergoing coronary angiography for chest pain or suspected CAD. Coronary angiography of the patient showed a lesion stenosis ≥ 50% in any coronary artery. And patients with congenital heart disease, cardiomyopathy, liver and kidney insufficiency, blood system diseases, malignant tumors, and other concomitant diseases were excluded. 20 healthy controls were selected from physical examination people during the same period. Plasma samples were collected and stored at liquid nitrogen immediately after collecting and centrifugation.Plasma exosomes were isolated using an exosome extraction kit . The enrichment of exosomes was identified using transmission electron microscopy (TEM).Human monocyte was isolated from venous blood by Ficoll . CD14 microbeads  and magnetic beads conjugated with antibodies against human CD14 were used for the monocyte separation.5/well) were inoculated in 24-well plates with 1 mL per well and incubated with PMA  with a final mass concentration of 100 ng/mL for 48 h. Human monocyte was incubated with macrophage colony-stimulating factor (M-CSF)  with a final mass concentration of 50 ng/mL for 7 days. M1 polarization of THP-1-derived macrophage and human monocyte-derived macrophage was performed using 20 ng/mL recombinant human IFN-γ  and 100 ng/mL LPS . M2 polarization was performed using 20 ng/mL recombinant IL-4 . Nonpolarized PMA-activated cells were used as control. The polarization model of macrophage was established after 20 h of polarization. Based on the establishment of the macrophage polarization model, M1 macrophage was stimulated with 20 ng/ml IL-4 for 20 h and then induced to M2 polarization. M2 macrophage was induced to polarize toward M1 after 20 ng/mL IFN-γ and 100 ng/mL LPS stimulation for 20 h.THP-1 cells  for 24 h.http://crispor.tefor.net) [CRISPR/Cas9 system was used to generate lnc-MRGPRF-6:1 knockout stable macrophage. The sgRNA targeting lnc-MRGPRF-6:1 was designed using CRISPOR (for.net) . Two tar6/well) were inoculated in 6-well plates with 2 mL per well and incubated with 100 ng/mL LPS for 24 h.lnc-MRGPRF-6:1 knockout macrophage (KO) and control cells  , from the plasma using BIOG cfDNA Easy Kit  and from exosomes by RNA Purification Kit . Total RNA was reverse transcribed into cDNA by using PrimeScript™ RT Reagent Kit . RT-qPCR was conducted using SYBR® Premix Ex Taq™ II  on StepOnePlus Real-Time PCR System . U6 was used as an internal reference. The primers of all genes were synthesized by GenScript . Primer sequences are listed in α , TNF-β , and IL-10  in the cell culture medium was detected according to the protocols.The cell culture supernatant of each treatment group was collected, and the secretion of TNF-6 cells were dispersed with EDTA-free trypsin, washed with PBS, and resuspended in 500 μL binding buffer and were then stained with FITC-labeled annexin V and PI. Apoptotic cell rate (%) = annexin + /PI − cells (%) + annexin + /PI + cells (%). Flow cytometry was used to detect the fluorescence intensity of foam cell samples with ROS Assay Kit , and average fluorescence intensity (MFI) represented ROS content.Annexin-V-FITC apoptosis kit  was utilized for apoptosis determination. 10THP-1 cells were induced to differentiate into foam cells. Dye with filtered solution of Oil Red O  was used for lipid staining in foam cells. The cells were finally observed and photographed microscopically.A CCK-8 assay tested the proliferation of cells . After 48 h of incubation, the supernatant was refreshed with RPMI-1640 medium containing CCK-8 for another 2 h. An automated microplate reader was used to read the optical density (OD) at 450 nm.Protein samples were obtained by cleavage of cells using RIPA lysis buffer . After being measured with bicinchoninic acid kit , normalized volumes of protein samples were isolated on SDS-PAGE on a 10% gel and transferred onto polyvinylidene fluoride membranes. The transfer membrane was then washed in a wash buffer and sealed in 5% BSA TBST for 1 h, followed by overnight incubation with primary antibody at 4°C. Then, HRP-labeled goat anti-rabbit IgG was stained for 1 h. After the developer solution is added, the film is viewed under Bio-Rad chemiluminescence imaging system. The primary antibodies used in experiments were as follows: P38 , p-P38 , TLR4 , MyD88 , GAPDH , JNK , p-JNK , ERK , p-ERK , and Tubulin .http://www.bgionline.cn).Total RNA was extracted from the cells using Trizol . RNA-seq was performed by BGISEQ-500 platform of Beijing Genomics Institute (BGI). Data analyses were carried out with BGI Online platform  software and WPS Office 3  were used for statistical analysis. Quantitative data between the two groups were evaluated using Student's α, TNF-β, and CXCL11 in the plasma of CAD patients were significantly increased with U6 as an internal reference. Further research demonstrated that lnc-MRGPRF-6:1 was positively correlated with TNF-α , TNF-β , and CXCL11  in plasma  compared with macrophage infected by control lentivirus (control) . Subsequγ and LPS, both the levels of CXCL10, CXCL11, IL-1β, TNF-α, and TNF-β protein secretions were significantly increased in THP-1 macrophage, which suggested that THP-1 macrophage was polarized to M1 type (M1) successfully  culture medium could increase the levels of M1-related cytokines CXCL10, CXCL11, IL-1β, TNF-α, and TNF-β and decrease the levels of M2-related cytokines CCL17, ARG1, and IL-10  and control were significantly increased  after lnc-MRGPRF-6:1 knockout was significantly lower than that of the control group  and macrophage infected by control lentivirus (control). Transcriptome analysis showed that the expression levels of 32 genes were up-regulated and 191 down-regulated in THP-1 macrophage after lnc-MRGPRF-6:1 knockout with ∣LogP < 0.05 . Gene Onimmune system processes, innate immune response, and inflammatory response. In innate immunity, macrophages produce proinflammatory mediators by activating several receptors that recognize pathogens, including the toll-like receptor family (TLRs) [As shown in y (TLRs) . Coincidy (TLRs) . FascinaConsidering MAPK including JNK, ERK, and P38 signal transduction pathway, we still verified JNK and ERK pathways, although RNA-seq showed no significant changes in JNK and ERK expression in MAPK after lnc-MRGPRF-6:1 knockout.Subsequently, we verified the expression of the genes above-mentioned by RT-PCR and Western blotting. In Figures β) were significantly elevated in the KO group after LPS induction (Figures To further investigate that lnc-MRGPRF-6:1 regulates macrophage polarization through the TLR4-MyD88-MAPK signaling pathway, we used LPS, the activator of TLR4, to activate this signaling pathway. LPS induced intense activation of TLR4, MyD88, P38, phosphorylation of P38, JNK, and phosphorylation of JNK in the KO group Figures ; meanwhi Figures .AS, as a chronic inflammatory vascular disease, is the pathological basis of CAD, which is a serious threat to human health. It is demonstrated that lncRNAs play important regulatory roles in inflammatory diseases, including AS . In a prFoam cells derived from macrophages are associated with the initial stage of AS . TherefoPrevious studies demonstrate that macrophage-mediated inflammation plays an essential role in the development of AS. After overtaking ox-LDL, macrophage transforms into foam cell under the intima of the coronary artery and recruits many proinflammatory factors to initiate the disease . Afterwaα, TNF-β, and CXCL11 in CAD patients' plasma, which indicates that lnc-MRGPRF-6:1 may participate in the regulation of macrophage-mediated inflammation in AS.Interestingly, in our study, further research demonstrated lnc-MRGPRF-6:1 was positively correlated with TNF-Polarization is deeply involved in the regulation of macrophage-mediated inflammation , 26. MacInitially, we analyze the expression of lnc-MRGPRF-6:1 in both M1 and M2 phenotype macrophages. For further research, we analyze the expression of lnc-MRGPRF-6:1 in the repolarization models of M1 and M2 macrophages once again. lnc-MRGPRF-6:1 is significantly upregulated in M1 macrophage. It is worth noting that the unchanged thing is the expression of lnc-MRGPRF-6:1 which was upregulated in M1 macrophage. Moreover, we observe that lnc-MRGPRF-6:1 knockout inhibits the polarization of M1 macrophage. These facts suggest that lnc-MRGPRF-6:1 may modulate the macrophage-mediated inflammation through transforming its polarization.However, the problem is how lnc-MRGPRF-6:1 regulates macrophage polarization. We conducted transcriptome sequencing between lnc-MRGPRF-6:1 knockout macrophage and control macrophage. Subsequently, GO enrichment analysis indicates that functions and processes associated with immune were enriched after lnc-MRGPRF-6:1 knockout. Coincidentally, the toll-like receptor signaling pathway, which is closely related to immune, was enriched by KEGG pathway analysis.TLRs, regulating immune cell survival and proliferation, are involved in the first line of defense against invading pathogens in organisms and play important roles in inflammation . TLRs plβ, markers of M1, were also significantly increased.Fascinatingly, expressions of TLR4, MyD88, and P38 were significantly changed after lnc-MRGPRF-6:1 knockout according to RNA sequencing data. Further analyses demonstrate that the expression levels of TLR4, MyD88, p-P38, and p-JNK were significantly decreased after knockout. After LPS induced lnc-MRGPRF-6:1 knockout macrophage model, the TLR4-MyD88-MAPK pathway was activated; meanwhile, CXCL10, CXCL11, and IL-1Based on these findings, we speculate that lnc-MRGPRF-6:1 promotes macrophage-mediated inflammation through the TLR4-MyD88-MAPK signaling pathway which regulates macrophage M1 polarization.In summary, the present study clarifies that lnc-MRGPRF-6:1 promotes macrophage-mediated inflammation by regulating macrophage M1 polarization, which may play a crucial role in the development of AS. Keeping the balance of macrophage polarization for reducing foam cell formation and alleviating inflammation has become the ideas for AS treatment strategies, and lnc-MRGPRF-6:1 might become a potential therapeutic target for AS.


Albumin deficiency did not reduce whole body fat oxidation over a 24‐h period and did not alter exercise performance in an incremental treadmill test. The system‐level phenotypic changes in lipid and glucose metabolism were accompanied by reduced hepatic perilipin‐2 expression (p < 0.05), as well as increased expression of adiponectin (p < 0.05) and glucose transporter‐4 (p < 0.05) in adipose tissue. The results indicate an important role of albumin and plasma FFA concentration in lipid metabolism and glucoregulation.Circulating albumin is expected to play a significant role in the trafficking of plasma free fatty acids (FFA) between tissues, such as FFA transfer from adipose tissue to the liver. However, it was not yet known how disrupting FFA binding to albumin in circulation would alter lipid metabolism and any resulting impacts upon control of glycemia. To improve understanding of metabolic control, we aimed to determine whether lack of serum albumin would decrease plasma FFA, hepatic lipid storage, whole body substrate oxidation, and glucose metabolism. Male and female homozygous albumin knockout mice and C57BL/6J wild type controls, each on a standard diet containing a moderate fat content, were studied at 6–8 weeks of age. Indirect calorimetry, glucose tolerance testing, insulin tolerance testing, exercise performance, plasma proteome, and tissue analyses were performed. In both sexes of albumin knockout mice compared to the wild type mice, significant reductions ( Plasma free fatty acids (FFA) bind to albumin in the bloodstream to facilitate their transport from adipose tissue to other sites in the body. Albumin knockout mice exhibited reduced plasma FFA concentration, reduced hepatic lipids (triacylglycerol and diacylglycerol), and improved insulin sensitivity. The results indicate that disrupting the trafficking of FFA between tissues, through disruption of albumin expression, may lead to altered lipid metabolism and maintenance of lower blood glucose concentration. In healthy individuals, rapid mobilization of free fatty acids (FFA) into circulation, through TAG lipolysis and FFA export from adipose tissue, facilitates the use of FFA as an alternative fuel. Use of FFA is believed to spare blood glucose, and the capacity to achieve high plasma FFA concentrations likely evolved for defense of glycemia when stresses such as exercise or fasting are imposed  on the C57BL/6J background were purchased from the Jackson Laboratory ; the gene ablation was a whole body knockout model  after weaning at 3 weeks of age for their lifetimes and were acclimated to the facility for at least 1 week prior to any experiments. Food intake was monitored at approximately 8 weeks of age. As described below, metabolic assessments were performed in the final 2 weeks of life and mice were euthanized at 8 weeks of age.This protocol was approved by the Purdue University Animal Care and Use Committee. Male and female homozygous albumin knockout mice  was performed at approximately 7 weeks of age, following a 4–6 h fast. Glucose was administered orally (2 g/kg bodyweight) as a 20% wt/vol solution. Blood glucose was measured in a drop of blood from the tail using a point of care device (Prodigy Diabetes Care) at times 0, 10, 20, 30, 60, 90, and 120 min. Area under the curve (AUC) was calculated as a Riemann Sum , and incremental AUC was calculated by factoring out the baseline blood glucose level from the AUC calculation.2.3n = 6 mice per group) at approximately 8 weeks of age, following a 4–6 h fast. Insulin (0.75 U per kg bodyweight) was administered by intraperitoneal injection. Blood glucose was measured in a drop of blood from the tail using a point of care device (Prodigy Diabetes Care) at times 0, 10, 20, 30, 60, 90, and 120 min. AUC and incremental AUC were calculated as described above.Insulin tolerance testing (ITT) was performed on the same mice (2.42) and carbon dioxide production (VCO2) were measured over a 24‐h period, after an initial 24‐h acclimatization period in the metabolic cage. The respiratory quotient (RQ) was calculated as VCO2/VO2, and standard equations were used to calculate energy expenditure as well as percent of energy expenditure from fat and carbohydrate. For the purposes of the calculations, the contribution of protein to fuel oxidation was assumed to be negligible and thus assigned a zero value.At approximately 7 weeks of age, indirect calorimetry assessment was performed on each mouse using an Oxymax System (Columbus Instruments). Mice were allowed free access to food and water, while oxygen consumption  was set to a 25° incline, and the shock grid set at a low intensity . Exhaustion was defined as a mouse remaining on the shock grid for 5 s. The treadmill speed was started at 6 m/min for a duration of 5 min, and then increased by 3 m/min at each subsequent 2 min interval. When a mouse reached exhaustion, the time was noted and the mouse was removed from the test.2.62 inhalation followed by immediate collection of blood via cardiac puncture. Blood was collected in ethylenediaminetetraacetic acid (EDTA)‐coated tubes, centrifuged at 3260× g for 10 min at 4°C, followed by isolation, and storage of plasma at −80°C until analysis. Liver, skeletal muscle, and the gonadal fat pad  were quickly collected following blood draw, immediately frozen in liquid nitrogen then stored at −80°C until analysis.Food was withdrawn for 4–6 h before euthanasia. Mice were euthanized by CO2.77H2] pentadecanoyl‐ceramide internal standard for ceramide and addition of [7H2] oleoylcholesterol internal standard for CE analysis; analyses of ceramide and CE were by direct infusion into a 6410 model tandem mass spectrometer (Agilent Technologies) and MRM based upon the LipidMaps database.For FFA analysis, lipids were extracted from plasma with heptane, isopropanol, and 0.033N sulfuric acid. Heptadecanoic acid (17:0) was used as the internal standard, and heptane extracts were analyzed by liquid chromatography/mass spectrometry (LC‐MS) using a model 1260 LC and model 6160 single‐quadrupole MS (Agilent Technologies); reversed phase separation and selective ion monitoring were employed to quantitate the internal standard, myristic acid (14:0), myristoleic acid (14:1), palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), α‐linolenic acid (18:3), arachidonic acid (20:4), eicosapentaenoic acid (20:5), and docosahexaenoic acid (22:6) following our established method , 10 mg of liver, and 30 mg of skeletal muscle (triceps) were each processed with a bead homogenizer in Trizol Reagent. RNA was subsequently isolated with RNA isolation kits (Qiagen). cDNA was synthesized from DNase‐treated RNA using the AffinityScript qPCR cDNA Synthesis Kit (Agilent Technologies). SYBR green qPCR was performed using a QuantStudio 7 Real‐Time PCR System (Applied Biosystems) and PowerUp SYBR Green Master Mix (Applied Biosystems). Primers used for determination of relative mRNA levels was produced by Integrated DNA Technologies and are listed in Table 2.9g for 15 min at 4°C and the protein content of the supernatant was determined by the Bradford method , and β‐actin . Membranes were then washed with TBST, then incubated at room temperature for 1 h with IR Dye 680  or IR Dye 800  secondary antibodies dissolved in TBST containing 1% nonfat dried milk and 0.02% SDS (BioRad). Membranes were then washed with TBST and bands were visualized and quantified using a LI‐COR Odyssey Infrared Imaging System (LI‐COR Biosciences) with values normalized to β‐actin bands as the loading control. Insulin was measured in plasma by enzyme‐linked immunosorbent assay (Merdodia) in the Indiana University Translation Core.Approximately 40 mg of liver was homogenized using a bead homogenizer in 20 volumes Cell Lysis Buffer  containing Complete Mini Protease Inhibitor (Roche) and Halt Phosphatase Inhibitor Cocktail (Thermo Scientific). The homogenate was centrifuged at 10,000 2.10Protein concentration in plasma was determined by the Bradford method  and sex as the factors. Results were also analyzed by three‐way ANOVA, when appropriate, including time as a factor. For analysis of VO2, metabolic rate, Lox, and CHOox, the two‐way and three‐way analysis were carried out as an ANCOVA with body weight set as the covariate. ANOVA and ANCOVA were followed by Fisher's least significant difference post hoc test when appropriate. Statistical analyses were performed with JMP version 14 (SAS Institute Inc.) and p ≤ 0.05 was considered statistically significant.Data are presented as means ± SE. The results were analyzed by two‐way ANOVA, with genotype (WT vs. Alb3p < 0.05) with no significant main effect of sex or interaction. The main effect of genotype corresponded to lower plasma FFA in Alb−/− than WT. Each individual FFA that was measured  was significantly lower in Alb−/− than WT for both males and females  with no significant main effect of sex or interaction . Plasma TC  and HDL‐C  each exhibited a main effect of genotype (p < 0.05) with no significant main effect of sex or interaction. The ratio between HDL‐C and TC was not significantly different between genotypes. Food intake and exercise capacity are reported as animal characteristics in Table −/− and WT (no significant main effect of genotype), but it was significantly higher in males than females , and there was no significant sex‐by‐genotype interaction. The eWAT fat pad was weighed after necropsy and was not different between Alb−/− and WT (no significant main effect of genotype). The eWAT weight was significantly higher in males than females , and there was no significant sex‐by‐genotype interaction. Food intake was normalized to body weight and was not different between Alb−/− and WT (no significant main effect of genotype). Food intake was significantly higher in females than males , and there was no significant sex‐by‐genotype interaction. Exercise capacity  was not different between Alb−/− and WT (no significant main effect of genotype). Exercise capacity was significantly greater in females than males , and there was no significant sex‐by‐genotype interaction.For total plasma FFA Figure , the ANOn Figure . For pla2 , main effect of genotype (p < 0.05), main effect of time (p < 0.05), genotype‐by‐time interaction (p < 0.05), and no significant sex‐by‐genotype or sex‐by‐genotype‐by‐time interaction. Thus, the relative effect of albumin deficiency did not depend upon sex. Post hoc testing was performed to explore the genotype‐by‐time interaction, and this test indicated a significantly lower blood glucose concentration in Alb−/− than WT at all time points (p < 0.05). AUC and incremental AUC (I‐AUC) were then analyzed by two‐way ANOVA. For AUC, ANOVA of the OGTT data , main effect of genotype (p < 0.05), and no significant interaction. For I‐AUC . AUC and I‐AUC for blood glucose in the OGTT were significantly lower in Alb−/− than WT. In the analysis of the ITT data, three‐way ANOVA on blood glucose over time , main effect of genotype (p < 0.05), main effect of time (p < 0.05), and no significant interactions. The main effect of genotype corresponded to significantly lower blood glucose during the ITT in Alb−/− as compared with WT. As with OGTT, ANOVA results for ITT indicated that the relative effect of albumin deficiency did not depend upon sex. AUC , main effect of genotype (p < 0.05), and no significant interaction. AUC for blood glucose in the ITT was significantly lower in Alb−/− than WT, and this genotype difference did not depend upon sex. In the ANOVA for I‐AUC results from the ITT, there were no significant main effects or interaction; thus, the effect of albumin deficiency upon blood glucose following insulin injection was related to the lower fasting blood glucose in Alb−/−. The differences between groups in blood glucose during OGTT and ITT were not a result of fasting insulin levels, as we did not observe any significant main effects or interactions in the analysis of fasting plasma insulin .To test markers of insulin sensitivity, OGTT and ITT assessments were performed on live mice. In the analysis of blood glucose concentration for the OGTT Figure , three‐wa Figure  indicateC Figure , only a e Figure  indicateC Figure  and I‐AUC Figure  for ITT p < 0.05) and main effect of sex (p < 0.05) was observed with no significant interaction. The main effect of genotype corresponded to lower hepatic TAG in Alb−/− than WT, while the main effect of sex corresponded to overall higher levels in females than males. For hepatic DAG content  and main effect of sex (p < 0.05) was observed with no significant interaction. The main effect of genotype corresponded to lower hepatic DAG in Alb−/− than WT, while the main effect of sex corresponded to overall higher levels in females than males. For hepatic ceramide concentration, no significant main effects or interaction were observed in the analysis . For hepatic CE, a significant main effect of sex (p < 0.05) indicated significantly higher CE in females than males; however, no significant main effect of genotype nor any significant interactions were observed for hepatic CE . While the focus in lipid analysis was on the liver, to test if ectopic deposition in muscle exhibited similar responses, TAG was measured in skeletal muscle; for TAG in skeletal muscle, there was no significant main effects of sex or genotype, and no significant interaction .To determine if reduced plasma FFA concentration may lead to lower ectopic lipid deposition, specific lipids were measured in the liver. For hepatic TAG content Figure , a signit Figure , a signip < 0.05) were observed, with no significant main effects of sex or interactions. The main effects of genotype corresponded to lower Plin2 gene and protein expression in the liver of Alb−/− compared to WT. There were no differences between groups for other gene and protein expression levels that are reported in Figure To understand metabolic responses in the liver, we performed measurements of gene and protein expression on this tissue. Total ACC expression Figure , phosphop < 0.05) was observed with no significant main effects of sex or interaction. The main effects of genotype corresponded to higher expression of each gene in adipose tissue of Alb−/− compared to WT. Gene expression for LPL  but no significant main effect of sex or interaction; as potentially expected, total plasma protein was significantly lower in Alb−/− than WT . In addition to the expected albumin deficiency in Alb−/−, the data filtering approach of the plasma proteomics data led to discovery of 19 plasma proteins exhibiting significantly altered expression in Alb−/− compared to WT  is expected to remain intact, while FFA transport from cells to the bloodstream is slowed. This slowed export is expected because dissolution of FFA into the bloodstream is the final step of export from adipose tissue, but this dissolution relies upon albumin. While one might expect that reduced plasma FFA supply to tissues could lead to difficulty in maintaining the whole body metabolic rate and fat oxidation, remarkably we observed that the albumin knockout mice did not exhibit any depression in metabolic rate or fat oxidation. Thus, though the reduced plasma FFA concentration was sufficient to decrease hepatic lipids, it appears to have remained high enough to fuel resting substrate oxidation.Compensation for the slowed release of FFA from adipocytes could be through enhanced intracellular FFA re‐esterification within the adipocytes. Intracellular FFA re‐esterification via futile cycling in adipose tissue is an ongoing metabolic process in healthy individuals (Henderson et al., The global proteome analysis of plasma indicated some changes in the plasma protein distribution, and these alterations included higher plasma adiponectin abundance in albumin knockout mice. Adiponectin promotes insulin sensitivity (Berg et al., The present results indicate that albumin could theoretically be a therapeutic target to reduce plasma FFA concentration and improve insulin sensitivity. In the future if therapeutics for impeding FFA binding to albumin were developed, it would be reasonable to hypothesize that this intervention could lead to reduced plasma FFA concentration, reduced hepatic lipids, and improved insulin sensitivity. That is to say, one could consider the albumin knockout mouse model to be a laboratory tool for understanding whether reducing the binding of FFA to serum albumin could lead to favorable outcomes. Indeed the insulin sensitivity improvements that are expected from reducing plasma FFA concentration (Henderson, It has been speculated that ApoB and its related lipoproteins are elevated in CAA patients, based upon comparison to reference ranges (Del Ben et al., 5Plasma FFA elevation is a risk factor for various diseases, and generally reducing plasma FFA in both healthy and unhealthy individuals is expected to improve metabolic well‐being (Henderson, The authors declare no conflict of interest.Gregory C. Henderson conception and design of research; Afsoun Abdollahi, Brianna N. Dowden, Kimberly K. Buhman, Alyssa S. Zembroski, and Gregory C. Henderson performed experiments; Afsoun Abdollahi, Brianna N. Dowden, and Gregory C. Henderson analyzed data; Afsoun Abdollahi, Brianna N. Dowden, and Gregory C. Henderson prepared figures; Gregory C. Henderson and Afsoun Abdollahi wrote the manuscript; Afsoun Abdollahi, Brianna N. Dowden, Kimberly K. Buhman, Alyssa S. Zembroski, and Gregory C. Henderson edited and revised the manuscript; Afsoun Abdollahi, Brianna N. Dowden, Kimberly K. Buhman, Alyssa S. Zembroski, and Gregory C. Henderson approved the final version of the manuscript.


Differences between groups post-intervention were not statistically significant in the Short Physical Performance Battery. The tele-rehabilitation intervention proposed in this study is a valuable treatment option in the recovery process for older adults with hip fracture. ClinicalTrials.gov Identifier: NCT02968589.This study aimed to examine the effect of a multidisciplinary tele-rehabilitation program on functional recovery of older adults with hip fracture compared with home-based in-person rehabilitation. In this single-blinded, non-randomized clinical trial, we included older with hip fracture. The tele-rehabilitation group received a 12-week tele-rehabilitation program (supervised by their family caregivers). The control group received the usual postoperative rehabilitation provided by the Andalusian health system (Spain). The primary outcome was the patient-reported functional status assessed with the Functional Independence Measure. We also measured performance-based functional recovery using the Timed Up and Go Test and Short Physical Performance Battery. We performed both a per-protocol  and an intention-to-treat analysis . Participants who used the tele-rehabilitation program had higher Functional Independence Measure scores (high effect size: 0.98 Cohen’s d;  Osteoporosis is a common chronic condition associated with aging and is related to low-trauma or fragility fractures at the wrist, spine, or hip . Hip fraEarly rehabilitation in hospital and post-discharge can improve older adults’ functional recovery . Home reThere may be perceived barriers for older adults to access and use ICTs, as the average age for someone with hip fracture is mid-eighties. However, Crotty et al.  delivereThis was a single-blinded, non-randomized clinical trial conducted according to the established guidelines by the Helsinki Declaration and Law 14/2007 on Biomedical Research. The non-inferiority design was based on the evidence gap of the effectiveness of tele-rehabilitation programs for patients with a hip fracture on functional recovery  versus tAll consecutive patients admitted with hip fracture who met the following inclusion criteria were invited to enroll in the study: (1) had hip fracture surgery; (2) were 65 years or older; (3) had a high (self-reported) pre-fracture functional level the week before the fracture  index > 90 points); (4) could weight-bear at 48 h after surgery; (5) community-dwelling (in own home or with a relative) after hospitalization; and (6) had internet access and/or a family caregiver with access. Exclusion criteria were: (1) presence of severe cognitive impairment  usual care, an educational workshop, and OT and PT delivered home rehab (control group); or (2) usual care, an educational workshop, and the tele-rehabilitation intervention (@ctivehip intervention group). We chose this study design because of the pragmatic nature of our practice-based research, the novelty of the tele-rehabilitation intervention, and the importance of patients’ preferences in health care delivery under real-world settings .It was not possible to blind patients and their caregivers to group allocation. However, data collection was conducted by an OT, PT, and sport scientist who were blinded to group allocation. Data analysis was performed by a statistician, a PT, and an OT, also blinded to group allocation.After hip fracture, usual care consisted of a few sessions of rehabilitation (between 2–5 sessions) during a hospital stay. Caregivers were also invited to participate in one workshop on postoperative patient management and recommendations for home. The workshop was delivered by hospital staff (an OT and a PT) twice a week on the acute care unit at the Virgen de las Nieves University Hospital . In addition, an informational leaflet with recommendations and exercises for home was given to patients and caregivers during the hospital stay.Participants in the tele-rehabilitation group (patients and caregivers) received usual care during the hospital stay, the invitation to participate in the workshop described above, and a 12-week multidisciplinary tele-rehabilitation program. Details of the program are described elsewhere in the @ctivehip protocol , but a sThe design of the tele-rehabilitation program was based on: (1) a previous published home-based exercise program for patients with hip fracture ; (2) exeIn addition to usual care during the hospital stay and the caregiver workshop, patients in this group also received the usual postoperative home-based in-person rehabilitation delivered by the Andalusian health system .All patients and their caregivers enrolled in the study were assessed at three time points: (1) before hospital discharge; (2) one month after hospital discharge; and (3) three months after hospital discharge (end of the tele-rehabilitation program). We asked participants to prospectively self-report, via the online platform, any adverse or serious adverse events which were reviewed weekly by one OT.The main outcome measure was patient-reported functional status assessed with the FIM  at 3 monWe also assessed functional recovery using two performance-based tests: (1) the Timed Up and Go Test (TUG) measures the time that a person takes to perform the following tasks: get up from a chair, walk three meters, turn around, walk back three meters, and sit back down in the chair . ParticiSociodemographic data, such as age, gender, educational level, falls in the previous year, and place of residence, were collected during the interviews with patients and their caregivers. We collected clinical data from medical charts for hospital length of stay, health status (measured by the American Society of Anesthesiologists’ score) , 24 h deThe study was designed to have an 80% chance of detecting an 8.7% difference between groups for the primary outcome (FIM) according to data from a previously published home-based rehabilitation intervention on hip fracture patients . We set 2 test for categorial binomial variables.Before performing the analysis, continuous variables were checked for normal distribution via the visual inspection of histograms together with the Kolmogorov–Smirnov test. All the outcomes demonstrated a non-normal distribution and were transformed using the Blom formula . The chaThe main effects of the tele-rehabilitation program were tested with two models of analysis of covariance (ANCOVA). In Model 1, we used post-rehabilitation outcomes as dependent variables, group  as a fixed factor and baseline outcomes as a covariate. In Model 2, we additionally performed a sensitivity analysis to test the influence of potential confounders in the results, such as age, sex, educational level, health status, duration of the hospital stay, falls in the last year, and type of fracture. Baseline age, sex, and type of fracture were the only variables that demonstrated an additional predictive capacity to Model 1 and were included as covariables in Model 2 in addition to the baseline outcomes.The z-scores for each outcome at post-rehabilitation were also formed by dividing the difference of the post-rehabilitation raw score of each participant from the baseline mean by the baseline standard deviation /baseline SD). This way of reporting the effects has been used in recent leading RCTs  and has p < 0.05.All analyses were performed using the SPSS software , and the level of significance was set at We identified 417 potentially eligible older patients admitted to the hospital for hip fracture, of which 71 participants enrolled in the study and were allocated into the control (N = 36) or tele-rehabilitation (N = 35) groups. A total of 62 participants (28 in the tele-rehabilitation group and 34 in the control group) were included in the per-protocol analysis, while all the 71 participants were included in the intention-to-treat analysis. The reasons for exclusion and dropouts of participants during the data collection and per-protocol analysis are provided in the CONSORT 2010 flow diagram . There wp = 0.003), and in the TUG performance, the tele-rehabilitation group having a better performance (p = 0.027).The characteristics of the participants and dividing the sample into both tele-rehabilitation and control groups are shown in p < 0.001), and this result remained similar in Model 2 . Regarding the physical function evaluated through the TUG, the tele-rehabilitation group had a greater decrease in performance time in comparison with the control group , and this effect was slightly attenuated in Model 2 . Lastly, the tele-rehabilitation group had a better improvement in the SPPB score than the control group, although this difference was not statistically significant in neither Model 1  and Model 2 . All these results are graphically presented in p = 0.024), although the significance disappeared in Model 2 .The intention-to-treat represents the secondary analysis and is shown in This study compared the effects of a novel 12-week tele-rehabilitation program (delivered by caregivers) against a home-based in-person rehabilitation  on functional outcomes of older adults that suffered a hip fracture. Regardless of the intervention received, both groups improved function, but the tele-rehabilitation program was superior at three months for improving function, as measured by the FIM and TUG test.In this study, we noted that the tele-rehabilitation was superior to a more traditional home care rehabilitation. These results extend the limited evidence for tele-rehabilitation for this population ,13,15. HThere are some differences between our study and the few published tele-rehabilitation studies conducted with older adults with hip fracture ,14,15. PThe older adults included in this study shared some similarities to previous studies for patients with hip fracture, such as the absence of cognitive impairments or the higher proportion of women ,14,15, aWe observed variation for adherence to the tele-rehabilitation program, but in general, it decreased over time. In our study, only 15% of patients completed the full program (50–60 sessions), but 22 patients (63%) completed >20 sessions. Despite the low adherence to the full @ctivehip protocol, patients obtained very good functional recovery (96.8% of their previous pre-fracture FIM). Thus, it is possible that patients did not need as many sessions as were offered. We tried to tailor the intervention by prescribing the program after baseline assessment and re-evaluation at one month (either online or via telephone). But as we did not monitor patients daily  or more This study has two main strengths to highlight. First, to our knowledge, it is the first tele-rehabilitation program for patients with hip fracture offered during the acute phase and after hospital discharge. Second, the inclusion of caregivers (in both groups) to support patients’ active role during recovery. Nevertheless, the main limitation of the present study is the non-randomization in the allocation of the participants. All statistical analyses were adjusted for baseline values and sensitivity analysis were performed accounting for potential confounders  to address that limitation. Choice of group allocation could introduce other sources of bias, such as higher motivation or amount of support from caregivers. However, patients in the control group received in-person rehabilitation at home delivered by an OT or PT, while caregivers supervised the @ctivehip intervention. Thus, there were possible advantages to being in either group. Further, despite the possible bias introduced with choice-based allocation, this study provided the opportunity to appreciate patient preferences and program implementation in the real-world setting to support future clinical decision-making . FurtherThis study highlights that for older adults with hip fracture, a 12-week tele-rehabilitation program supervised by family caregivers had better results in functional independence and physical condition (self-report and performance-based) than traditional home-based rehabilitation. These results suggest the use of ICTs could be a management option in the recovery process for patients with hip fracture and their caregivers.