The capsaicin receptor TRPV1 is an outstanding representative of ligand-gated ion channels in ligand selectivity and sensitivity. However, molecular interactions that stabilize the ligand-binding pocket in its permissive conformation, and how many permissive conformations the ligand-binding pocket may adopt, remain unclear. To answer these questions, we designed a pair of novel capsaicin analogs to increase or decrease the ligand size by about 1.5 Å without altering ligand chemistry. Together with capsaicin, these ligands form a set of molecular rulers for investigating ligand-induced conformational changes. Computational modeling and functional tests revealed that structurally these ligands alternate between drastically different binding poses but stabilize the ligand-binding pocket in nearly identical permissive conformations; functionally, they all yielded a stable open state despite varying potencies. Our study suggests the existence of an optimal ligand-binding pocket conformation for capsaicin-mediated TRPV1 activation gating, and reveals multiple ligand-channel interactions that stabilize this permissive conformation. Unlike agonist) , TRPV1’sagonist) . TRPV1 fagonist) . Moleculagonist)  and a coagonist) . These rActivation of TRPV1 can be induced by capsaicin and related pungent plant compounds in two different ligand poses inside the ligand-binding pocket . The hig+1 contains one extra -CH2 group between the hydrogen-bond-forming groups, whereas Cap-1 contains one less -CH2 group between the hydrogen-bond-forming groups. Changes in the neck length of these compounds (±1 C-C bond length) are compatible to the 1.3 Å movement of the S4-S5 linker from the closed state to the open state. Importantly, these structural changes preserved all the key functional groups involved in direct ligand-channel interactions. Together with capsaicin, these compounds served as a set of molecular rulers for the conformational changes in the ligand-binding pocket. Binding of these capsaicin analogs to TRPV1 as well as their abilities to induce channel activation were investigated by a combination of computational modeling, pharmacological analyses, and mutational tests.In order to understand why different agonists take distinct binding poses, and how these poses allow ligands to stabilize the open conformation of the ligand-binding pocket, we designed two capsaicin analogs with an altered neck length : Cap+1 c+1 and Cap-1 strongly activated TRPV1 by binding to the same binding site as capsaicin . As previously reported  of 0.94  did not affect the maximal Po (as one might expect if the extended ligand is a better fit to the closed-state ligand-binding pocket), but instead reduced potency.We recorded current responses of mouse TRPV1 expressed in HEK cells to capsaicin and its two analogs at both macroscopic and single-channel levels . Both Careported , capsaic of 0.94 , compara of 0.94 . Cap-1 about 10% . After cesponses . High Poicacious . Cap+1 aapsaicin ; howeverPo level . Therefo-1 and Cap+1 inside the ligand-binding pocket. Using the RosettaLigand application within the Rosetta molecular modeling software suite  of the top 30 models are also shown   . The sim5 and S6 . The max-1 and Cap+1. The pore conformations were nearly identical and resembled that seen in the capsaicin-bound cryo-EM structure  cDNA was fused to its C-terminus to facilitate identification of transfected cells, as previously described . This coHEK293T cells were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 20 mM L-glutamine and 10% fetal bovine serum. No mycoplasma contamination was detected using Lonza MycoAlert Mycoplasma Detection Kit and the cell line was authenticated by ATCC using STR profiling. Cells were transiently transfected with cDNA using Lipofectamine 2000 (Life Technologies), and experiments were performed 18–24 hr after transfection.E)-N-(4-Hydroxy-3-methoxyphenethyl)-8-methylnon-6-enamide (Cap+1) and (E)-N-(4-hydroxy-3-methoxyphenyl)-8-methylnon-6-enamide (Cap-1) were synthesized by reacting (E)-8-methylnon-6-enoyl chloride with 4-(2-aminoethyl)-2-methoxyphenol or 4-amino-2-methoxyphenol, respectively (Supplementary Notes). The identity and purity of both Cap+1 and Cap-1 were confirmed by 1H, 13C NMR and high-resolution mass spectroscopy . Chemical structures were drawn using ChemDraw 19.0 (Perkin Elmer). All the other chemicals were purchased from Sigma-Aldrich.(rev) measurements, 140 mM NaCl was replaced with 70 mM MgCl2 or 70 mM CaCl2 in the intracellular solution, with EGTA excluded. Currents from inside-out patches were recorded with a 500 ms voltage ramp protocol consisting of a 0 mV holding potential, a pre-step to −100 mV for 100 ms, a voltage ramp from −100 mV to +100 mV for 500 ms, and a post-step at +100 mV for 100 ms before returning to the holding potential. Permeability ratios were determined using the Erev measurements with the Goldman-Hodgkin-Katz equation.Patch-clamp recordings were performed using a HEKA EPC10 amplifier with PatchMaster software (HEKA). Electrical signals were filtered at 2.9 kHz and sampled at 10 kHz. For macroscopic recordings, patch pipettes were fashioned from borosilicate glass and fire polished within the range of 3–5 MΩ. Both bath and pipette solutions contained 140 mM NaCl, 0.2 mM EGTA, and 5 mM HEPES (pH 7.2) unless otherwise stated. All recordings were performed at room temperature (~22°C) in either whole-cell or inside-out configurations. Currents were recorded with a protocol consisting of a holding potential at 0 mV, a step to +80 mV for 200 ms followed by a step to −80 mV for 200 ms before returning to the holding potential. Solutions were delivered in separate tubes onto the cell or excised-patch membrane using a rapid solution changer  to prevent solution mixing. Single-channel recordings under the inside-out configuration were performed 6–8 hr after transfection to maximize the chance of obtaining patches containing a single channel. Patch pipettes were fire-polished to resistance of 8–12 MΩ. All single-channel recordings were obtained with the membrane potential clamped to +80 mV. Conductance (g) was calculated at +80 mV. Single channel recordings were analyzed with Igor Pro 8 (WaveMetrics). For reversal potential . Current amplitude was fitted to a Hill equation to generate concentration-response curves which were used to obtain estimates of the EC-1 macroscopic open probability was determined by first correcting for the current difference due to single-channel amplitude.Single-channel recordings were analyzed using all-point histograms generated at +80 mV where a double-Gaussian function was used to fit the histograms. Event detection was performed using the threshold-crossing method to produce idealized event lists. From the peak values, single-channel current amplitude was determined for each compound. Single channel open probability was determined using QuB software. A dead time of 0.3 ms was imposed and traces were idealized using the 50% amplitude threshold-crossing method. Patches with more than one channel were discarded. Open probability of macroscopic recordings of each analog was determined by normalizing to that of saturating concentrations of capsaicin (10 μM). CAPt test. *, p<0.05.All statistical values are given as mean ± SEM. Statistical significance was determined using the Student’s Docking of capsaicin analogs was performed using RosettaLigand and RosettaScripts, an application within the Rosetta modeling software (version 3.8). The TRPV1 capsaicin-bound cryo-EM structure (PDB: 3J5R) was relaxed in a membrane environment using RosettaMembrane. Because the current version of Rosetta cannot handle explicit lipids, annular lipids or PIP2 were not modeled. Water molecules were also not modeled; their involvements in capsaicin binding were previously studied by RMSD measurements were calculated using RosettaScripts. Briefly, the top 1% lowest energy models were analyzed using the SimpleMetricsFilter within RosettaScripts. All measurements were calculated using the capsaicin-bound model as the reference. RMSD was calculated per residue and plotted using UCSF Chimera. Pore radius was calculated using the HOLE program version 2.0 . The topAvogadro was used to generate and optimize the geometry of the compounds and create input files. Using these input files, GAMESS software  was used In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:TRPV1 channels are sensitive to a wide variety of endogenous and exogenous chemical and physical stimuli. This paper employs subtle analogs of the toxin agonist capsaicin, the active component in chilli peppers, to show that despite vastly different ligand binding poses, the capsaicin analogs elicit very similar conformations of the TRPV1 ligand-binding pocket. The work thereby provides important insight on how ligands modulate TRPV1 activity.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Vincenzo Carnevale (Reviewer #2); Vera Y Moiseenkova-Bell (Reviewer #3).Thank you for submitting your article "Novel Capsaicin Analogs as Molecular Rulers to Define the Permissive Conformation of TRPV1 Ligand-Binding Domain" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:The manuscript by Vu et al. reports on an interesting investigation of the structural underpinnings of TRPV1 activation by capsaicin analogs. In particular, thanks to a combination of chemical synthesis, kinetic modeling based on electrophysiology and molecular modeling, the authors conclude that two possible binding modes stabilize the gating-permissive conformation of the ligand-binding domain. This topic is relevant given that the microscopic mechanism of activation of TRPV1 is still not fully understood and the fact that novel TRPV1 channel modulators are sought-after for their potential use as pain killers. The experiment and computational approaches are sound and are generally carried out with the appropriate scientific rigor and the manuscript is well written. However, additional data is needed to confirm the validity of the structural model used. Further, the authors need to carefully revise the text with regard to the general applicability of their findings and expand on the discussion of a number of points, see below.Essential revisions:1) The structural model employed for the calculations (PDB: 3J5R) is a key concern. This very early structure of TRPV1 is not of high resolution/quality. There are several more recent studies and the functional state corresponding to this particular structure is still somewhat controversial. Therefore, using this model as a bona-fide open state raises important concerns. There is an abundance of recent structural information for members of the TRPV subfamily, including TRPV1 (some of these channels have been solved in both the open and closed conformations and thus provide suitable templates). It is therefore important to validate the robustness of the present findings by using a different model of the open state.2) Capsaicin is a powerful tool to study the activation mechanism of TRPV1. However, this molecule represents toxin activation of the channel, and the comparison with classical neurotransmitters is therefore not relevant (Introduction). The assumption that all TRPV1 agonists acting through the capsaicin binding site result in the same permissive open confirmation should be tested. Ideally, this should be conducted with endogenous activators that do not have a similar chemical structure as capsaicin, such as anandamide or 12-HEPTE. If such data cannot be generated in a reasonable time frame, the authors need to carefully emphasize throughout the manuscript that their present findings are only relevant to capsaicin and capsaicin analogs (including the title).3) Previous work on TRPV1 based on all atom molecular dynamics have shown that the interaction between capsaicin and the vanilloid binding site are partially mediated by tightly bound water molecules . Have the authors considered this possibility for the binding of capsaicin and the two analogs? It is our understanding that the Rosetta modeling strategy employed here may not allow for explicit description of such water molecules. If this is the case, the authors should acknowledge this limitation and, consequently, provide sufficient context with regards to the computational results in the Discussion.4) Another important concern relates to the fact that annular lipids seem to play a functional role in TRP channels in general and in the TRPV family in particular. Several structural determinations of members of this evolutionary family have found that lipids bind to crevices adjacent or overlapping with the vanilloid binding site in a state-dependent fashion . Assuming that a similar mechanism is at work in TRPV1, are these lipid molecules found in the open state of the channel compatible with both the binding sites? Again, for the purpose of providing a fair a complete context to the results, including limitations, pitfalls and possible alternative explanations, it is important that the authors extend the structural analysis to take into consideration this aspect as well.5) In part related to the previous point is the structural role of PIP2, which is a well-established positive regulator of TRPV1. Current experimental information indicates that this lipid molecule binds to the S4-S5 linker in a conformation that seemingly interferes with one of the two binding modes proposed in this manuscript . Is that the case? If so, what are the functional consequences of this?6) The term ligand-binding domain is somewhat misleading, given that the agonists do not bind to a discrete protein domain, but rather to part of the transmembrane core. There is no gain in introducing a new nomenclature and the authors should therefore use standard terminology .eLife. Your revised article has been evaluated by Richard Aldrich (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled “Novel Capsaicin Analogs as Molecular Rulers to Define the Permissive Conformation of mouse TRPV1 Ligand-Binding Pocket" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:In light of the observed differences between 3J5R and 5IRX, as well as the missing water, annular lipids and PIP2 , the authors are asked to mention these aspects more explicitly in the main text. Either as part of the results or as a brief separate section in the discussion. This will not take away from the impact of their study but will help the reader to put the work into context. Essential revisions:1) The structural model employed for the calculations (PDB: 3J5R) is a key concern. This very early structure of TRPV1 is not of high resolution/quality. There are several more recent studies and the functional state corresponding to this particular structure is still somewhat controversial. Therefore, using this model as a bona-fide open state raises important concerns. There is an abundance of recent structural information for members of the TRPV subfamily, including TRPV1 (some of these channels have been solved in both the open and closed conformations and thus provide suitable templates). It is therefore important to validate the robustness of the present findings by using a different model of the open state.The reviewer’s point is well-taken. We have in recent years studied binding of capsaicin , ginger compounds , and piperine  to TRPV1. These studies, initiated shortly after the report of 3J5R, were all based on this structural model. To be consistent, especially for direct comparison between binding of capsaicin and that of its analogs, we decided to use 3J5R in the present study as well. For modeling, we first relaxed 3J5R structure in a membrane environment using the RosettaMembrane application, as described in our report on capsaicin binding . The 3J5R structure represents a ligand-bound, closed channel (perhaps in a pre-open state?). The newer TRPV1 structure in complex with both RTX and DkTx, 5IRX, represents a ligandbound, open channel. When compared directly, their ligand-binding pocket conformations are similar whereas the pore region conformations, especially those of S5 and pore helix, are rather different. The comparison, as well as testing results, see We further ran a quick check on binding of capsaicin to 5IRX, using the same routines in Rosetta as we have applied to 3J5R in the present study. Initial results from 5IRX closely resembled what we have observed from 3J5R, with an unambiguous head-down tail-up pose secured by clearly identifiable neck-to-T551 interactions. Encouraged by the reviewer’s suggestion, we are initiating a thorough investigation of binding by capsaicin analogs to 5IRX and will verify modeling results with functional tests. While this effort will take much longer to complete, we will find ways to share the results once the study is completed.2) Capsaicin is a powerful tool to study the activation mechanism of TRPV1. However, this molecule represents toxin activation of the channel, and the comparison with classical neurotransmitters is therefore not relevant (Introduction). The assumption that all TRPV1 agonists acting through the capsaicin binding site result in the same permissive open confirmation should be tested. Ideally, this should be conducted with endogenous activators that do not have a similar chemical structure as capsaicin, such as anandamide or 12-HEPTE. If such data cannot be generated in a reasonable time frame, the authors need to carefully emphasize throughout the manuscript that their present findings are only relevant to capsaicin and capsaicin analogs (including the title).50/efficacy for TRPV1. Since capsazepine and iodo-resiniferatoxin  exhibited competitive inhibitory effects on channel activation by these compounds , they are assumed to bind to the same ligand-binding pocket. These functional findings are consistent with those for piperine and capsazepine that have drastically different molecular structures from capsaicin but bind to the same binding pocket. Given that the structures of endovanilloids deviate further from capsaicin than the analogs used in the present study, it is anticipated that they might bind differently, though modeling of these compounds  would require additional efforts beyond the scope of this project. We have modified the manuscript to clarify that the present manuscript focuses on the main conclusion of different ligand binding poses stabilizing the same open confirmation, and that endovanilloids are yet to be tested.Indeed, a number of “endovanilloids” with an overall similar structure to capsaicin have been reported to have high EC3) Previous work on TRPV1 based on all atom molecular dynamics have shown that the interaction between capsaicin and the vanilloid binding site are partially mediated by tightly bound water molecules . Have the authors considered this possibility for the binding of capsaicin and the two analogs? It is our understanding that the Rosetta modeling strategy employed here may not allow for explicit description of such water molecules. If this is the case, the authors should acknowledge this limitation and, consequently, provide sufficient context with regards to the computational results in the discussion.As the reviewer correctly pointed out, water molecules must be important for ligand-channel interactions in TRPV1, which was previously studied by Elokely at al., 2016. At the time when we carried out this project, Rosetta did not allow for explicit description of water molecules. While this new function is being added to Rosetta, its performance has not been thoroughly tested in the field or Yarov-Yarovoy/Zheng labs. We have revised our manuscript to state this important limitation of our modeling.4) Another important concern relates to the fact that annular lipids seem to play a functional role in TRP channels in general and in the TRPV family in particular. Several structural determinations of members of this evolutionary family have found that lipids bind to crevices adjacent or overlapping with the vanilloid binding site in a state-dependent fashion . Assuming that a similar mechanism is at work in TRPV1, are these lipid molecules found in the open state of the channel compatible with both the binding sites? Again, for the purpose of providing a fair a complete context to the results, including limitations, pitfalls and possible alternative explanations, it is important that the authors extend the structural analysis to take into consideration this aspect as well.We agree that annular lipids are important for TRPV1 functions and wish to better understand their role in ligand activation. However, currently Rosetta cannot handle explicit lipids. We wish to test potential involvements of annular lipids in the future when such functionality is added to Rosetta, and effective functional tests are developed for the same purpose. This limitation is clearly stated in the revised manuscript.5) In part related to the previous point is the structural role of PIP2, which is a well-established positive regulator of TRPV1. Current experimental information indicates that this lipid molecule binds to the S4-S5 linker in a conformation that seemingly interferes with one of the two binding modes proposed in this manuscript . Is that the case? If so, what are the functional consequences of this?We agree that lipids including PIP2 are important regulators of TRPV1 and may play a role in ligand activation. As mentioned above, modeling of explicit lipids is not currently possible in Rosetta. In the revised manuscript we made clear that PIP2 was not modelled.6) The term ligand-binding domain is somewhat misleading, given that the agonists do not bind to a discrete protein domain, but rather to part of the transmembrane core. There is no gain in introducing a new nomenclature and the authors should therefore use standard terminology .In our original manuscript, we used “ligand-binding domain” and “ligand-binding pocket” interchangeably, in the same sense as LBD has been used for glutamate receptors . Nonetheless, the reviewer’s point is well-taken. We have now gone through the manuscript to make sure the commonly used term for TRPV1 “ligand-binding pocket” is consistently applied.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:In light of the observed differences between 3J5R and 5IRX, as well as the missing water, annular lipids and PIP2 , the authors are asked to mention these aspects more explicitly in the main text. Either as part of the results or as a brief separate section in the discussion. This will not take away from the impact of their study but will help the reader to put the work into context.As suggested, we have added a paragraph in the Results section on limitations of our structural modelling approach.


Self-separation or peeling of an idiopathic epiretinal membrane (ERM) in an eye with partial posterior vitreous detachment (PVD) is a rare event. A 56-year-old woman presented to our clinic with complaints of floaters in her right eye. Best-corrected visual acuity (BCVA) was 9/10 in this eye. Fundus examination and Spectral domain optical coherence tomography (SD-OCT) revealed an idiopathic ERM and grade 3 PVD in this eye. Four months later, she had complaints of metamorphopsia in her right eye. BCVA was 7/10, while SDOCT images of the right macula were similar to previous images. One week after the last visit, she presented again due to the sudden disappearance of her metamorphopsia complaints. BCVA had improved to 10/10. Fundus examination demonstrated that the ERM had spontaneously separated from the retinal surface as a flap floating in the vitreous and the foveal contour had returned to normal. The etiologic mechanism may be explained as the contracting forces within an immature ERM being stronger than its adhesion to the retina. Spontaneous self-separation of an idiopathic ERM from the retinal surface is a rare event. It was reported in the literature that spontaneous separation may occur in eyes with secondary ERM, especially in young patients.In this article, we present a case of idiopathic ERM that spontaneously separated from the retinal surface in the form of a flap in an eye with partial PVD, which was immediately followed by resolution of the patients’ visual complaints and anatomic findings.A 56-year-old woman presented with complaints of floaters in her right eye. She had no history of trauma or eye problems and her best corrected visual acuity (BCVA) was 9/10 and 10/10 in the right and left eye, respectively. Intraocular pressure was 16 mmHg in both eyes and anterior segment and fundus examination findings were normal. Spectral domain optical coherence tomography (SD-OCT)  revealed idiopathic ERM in the macula of the right eye and stage 3 PVD, in which the vitreous is attached only to the optic nerve head. Loss of foveal contour was noted and central macular thickness (CMT) was measured as 360 µm . SD-OCT 4 Spontaneous full-thickness, free, or partial flap separations are known to occur in cases of secondary ERM, especially in adolescents and cases of ERM that develop after inflammatory retinal diseases.7 However, the self-separation of an idiopathic ERM from the retinal surface is rare.5 In this article, we present an eye with partial PVD which developed an idiopathic ERM that later spontaneously separated from the retinal surface as a flap. In their series of 1248 idiopathic ERM cases, Yang et al.3 determined with SD-OCT that the prevalence of spontaneous separation in eyes with and without PVD was 1.5% and 13.4%, respectively, during the 33-month follow-up period. Meyer et al.6 reported spontaneous separation rates of 0.47 and 2.3% in eyes with and without PVD, respectively, during 2-13 months of follow-up in their series of 210 patients under the age of 30 who had idiopathic ERM. Our patient had stage 3 PVD at initial examination.Spontaneous ERM separation is an uncommon clinical phenomenon, reported to occur in 1-3% of all ERM cases.6 In a study reporting decreased CMT and increased BCVA following spontaneous idiopathic ERM separation, it was determined that new defects may form at the retinal inner segment/outer segment (IS/OS) line after spontaneous separation and that ERM recurrence is more common in such cases, emphasizing that the IS/OS line is more susceptible to vertical forces.6 The absence of any defects in the retinal layers in our patient could be attributed to the retinal traction being weak and of short duration.An interesting and previously unreported finding in the current case is that the base of the self-separated ERM flap was positioned inferiorly and toward the peripheral retina, as with peripheral retinal tears. In earlier cases of ERM with partial spontaneous separation described in the literature, whether idiopathic or secondary, the base of the flap is usually positioned toward the optic disc or the temporal or superior directions.6Two mechanisms have been proposed in the literature to explain the pathogenesis of spontaneous ERM separation. The first and most common mechanism is a process that occurs due to the induction of PVD.The ERM may spontaneously detach when the contractive forces within an immature ERM are stronger than its adhesion to the retina. The second mechanism is a process resulting from the tangential traction created by the cells in the inner retinal layers that occur in eyes in which PVD has already developed.We believe that the spontaneous idiopathic ERM detachment in our patient is consistent with the first mechanism. In our opinion, the likely mechanism is that the anteroposterior tractional forces in the concentrated vitreous overcome the tangential tractional forces, and due to this force, the base of the already thin and immature ERM detaches from the retina in the form of a flap in the periphery.


A synchronized dialogue between maternal and embryonic tissues is required for successful implantation. Low uterine receptivity is responsible for two-thirds of implantation failures and leptin is effective in the physiology of reproduction by binding to specific receptors. In this study, we investigate leptin receptor expression in cases of embryo transfer to endometrial coculture.  Biopsy materials were taken from 20 females with indication for coculture application and were cultured in an appropriate medium after the epithelial cells were isolated. The grown cells were cultured in chamber slides as the first group. For the second group, day 3 embryo was added to chamber slides and the development was observed. The embryo was transferred 1 or 2 days later and other cells (after the transfer process) were used to form the second group. After fixation, immunohistochemical staining was performed with anti-leptin primary antibody.  Regarding the coculture without embryo transfer, moderate leptin receptor immunoreactivity was seen in the perinuclear region and the cell membrane. Also, regarding the coculture with embryo transfer, moderate leptin receptor immunoreactivity was seen in the cytoplasm and strong leptin receptor immunoreactivity was seen in the cell membrane. Embryo transfer to endometrium coculture triggers leptin receptor expression.  Infertility is an important public health problem. Around 40%–50% of cases are due to female infertility, whereas 30% of cases are linked to male infertility. There is no identifiable causative factor defined in 15%–25% of the couples [1]. Reproductive biotechnologies such as artificial insemination (AI), embryo transfer (ET), and embryo production via in vitro fertilization (IVF) have improved significantly in recent years [2]. However, the rate of live births per embryo transfer through assisted reproductive technologies remains as low as 30%, so there is still a substantial need to improve current human IVF-ET procedures by determining optimal embryonic culture conditions [3]. For patients with repeated IVF failures, the methods to improve IVF outcomes include focusing on improvement in embryo quality and implantation rates, because repeated IVF failure occurs due to unsuccessful implantation, according to the generally accepted view [4]. A receptive endometrium, a normal and functional embryo at the blastocyst developmental stage, and a synchronized dialogue between maternal and embryonic tissues are required for successful implantation [5]. Among these requirements is the window of implantation, which is the interaction between an embryo and a receptive endometrium during a well-defined period in the secretory phase of the menstrual cycle [6]. This embryo-maternal crosstalk is mediated by a variety of biochemical factors and cytokines produced by the endometrium and the blastocyst . Progesterone (P) receptors, estrogen receptors, leukemia inhibitory factor, transforming growth factor beta, and tumor necrosis factor alpha are among these factors. Integrins, mucins, and cytokines such as interleukin 15 and leptin also play essential roles as adhesion molecules [8–10].Several techniques have been used to improve culturing conditions to closely mimic these important interactions in the in vivo environment in IVF. Embryo coculture systems using helper cells are one of such strategies. They have been applied for patients with a history of poor embryo quality or with repeated implantation failures. The target of embryo coculture systems is to improve embryonic development during the critical period of maternal-to-zygotic transition and to increase implantation potential . The release of embryotrophic factors and the detoxification of the culture medium by removal of heavy metal cations, free radicals, or metabolic inhibitors are some of the mechanisms targeted by coculture .the leptin receptor expression in cases of embryo transfer to endometrium coculture.Based on this information, in this study our aim is to investigateIn accordance with routine IVF procedures, endometrial materials of approximately 0.5 cm3 were obtained via vacuum aspiration from 20 patients with indication for coculture application. These patients did not have PCOS syndrome. The materials were placed into Falcon tubes that included sterile u291b modified Eagle’s medium (DMEM). They were kept in an oven for a period of about 1 h, during which time the suspension was shaken for 5–10 min and was then centrifuged at 1600 rpm for 10 min. Afterwards, the supernatant was removed, and the medium was put on the pellet, which was then followed by resuspending. The tissue suspension was planted into a 100-mL dish. The next day, the holding cells were obtained and the top medium as well as the tissue debris were removed, after which fresh medium was added. The daily development of the cells was observed. The bottom of the dish was covered by cells within 3–4 weeks, and the cells were removed from the dish by trypsin. This cell suspension was centrifuged, and the supernatant was removed. Fresh medium was added to the pellet and thus a new cell suspension was obtained. The new suspension was planted into 5 different central well dishes. It was expected that the cells would proliferate and fill the wells in 3 to 7 days. If the IVF cycle started in a short time, the suspension was used immediately. A day 3 embryo was put into the first central well dish. The dishes were changed every day, and day 5 embryo transfer was performed. After the culturing process, the grown cells were cultured in chamber slides as in the first group. For the second group, a day 3 embryo was added to the chamber slides and the development was observed, which was followed by transfer 1 or 2 days later. The other cells (after the transfer process) were used to form the second group. The first group of cells was kept in the same environment under equal conditions for the same period of time. After creating the groups, the slides were fixed in 4% paraformaldehyde for about 30 min. Then the slides were washed twice in phosphate-buffered saline (PBS) solution for 15 min. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide  (diluted with PBS) for 15 min and the slides were washed three times in PBS solution. The epitopes were stabilized by applying serum blocking solution for 15 min. The slides were incubated with the anti-leptin receptor primary antibody for 60 min at room temperature. The slides were washed three times in PBS solution, after which the biotinylated secondary antibody was applied for 10 min. The slides were then washed three times in PBS solution. Thereafter, streptavidin peroxidase was applied to the slides for 10 min. After washing with PBS, 3,3’-diaminobenzidine (DAB) was used as a chromogen. Then all the slides were counterstained with Harris’s hematoxylin. The slides were examined with a photo-light microscope  and a Leica DFC280 Plus camera. The number of immunopositive cells was measured manually using the Qwin software program in consecutive areas. The following semiquantitative scoring system was used to assess the immunolabeling intensity: (0) no staining, (1) weak, (2) weak to moderate, (3) moderate, (4) moderate to strong, and (5) strong labeling. Two independent observers who were blind to the treatment protocol performed the evaluations of immunolabeling scores independently. The H-score was calculated using the following equation: H-score = ∑Pi (i + 1), where i is the intensity of Leptin-receptor labeling with a value of 0, 1, 2, 3, 4, or 5, and Pi is the percentage of labeled cells for each intensity, varying from 0% to 100%. The results are expressed as mean ± SD.Statistical analysis was performed using unpaired Student t-tests for the H-score, using SigmaStat v3.5 software. The results are expressed as mean values ± SD, and P < 0.05 was considered statistically significant.Cells were obtained with centrally located nuclei and normal cytoplasmic appearance in the coculture without embryo transfer. In a group of cells, a double nuclear formation was observed due to amitotic division. The distribution of the leptin receptor was evaluated in this group. Moderate immunoreactivity was seen in the perinuclear region and the cell membrane .Regarding the coculture with embryo transfer, the cell morphology was not significantly different from the previous group. However, when the distribution of leptin receptor was evaluated in this group, moderate leptin receptor immunoreactivity was seen in the cytoplasm. Strong leptin receptor immunoreactivity was also seen in the cell membrane .These findings were also supported by statistical analysis . The data were expressed as mean ± SD for each group. Statistical significance was determined with unpaired Student t-tests, *P < 0.05, versus Group 1.Ob) gene, is primarily produced by adipose tissue. The leptin system (leptin and its receptors) plays a role in food intake, basal metabolism and reproductive functions/fertility [14]. Indeed, leptin is an autocrine and paracrine regulator of the human implantation process [15]. For example, according to several studies, leptin causes synergistic stimulation of angiogenesis and vascular permeability via FGF-2 and VEGF also increases the expression of MMP-2 and 9. Angiogenesis and metalloproteinases are essential for the success of implantation, so leptin and leptin receptors have been detected in implantation sites and this detection is very important for preimplantation and implantation physiology [16].Leptin, a 16-kDa product of the Obese (Several cytokines like leptin and adiponectin are localized at the maternal/fetal interface, and they are significant modulators of human embryo implantation and placentation [17].According to some studies, leptin-deficient mice cannot reproduce , and leptin promotes mouse preimplantation embryo development [20]. Moreover, in a study by Yang et al., leptin was found to enhance the adhesion and outgrowth of mouse blastocysts on fibronectin [21]. Some of these findings were also supported by studies in which human blastocysts were cocultured with endometrial epithelial cells due to modulation of leptin secretion [15].At present, little is known about leptin and adiponectin system regulation in the human endometrium. We found strong immunoreactivity, especially for the cell membrane in embryo-transferred coculture, so our results showed that embryo transfer to endometrium coculture triggered leptin receptor expression.According to a study, the deficiency of leptin synthesis is related to obesity and sterility in ob/ob knock-out mice [22]. Moreover, another study suggested that higher leptin concentrations have been secreted by competent blastocysts than in the case of arrested embryos. Gonzales et al. also found that leptin and leptin receptor mRNA and protein were revealed in secretory endometrium and in endometrial epithelial cells that were cocultured with human embryos [15]. Taken together, the results of this study suggest that day 3 embryo transfer to endometrium coculture triggers leptin receptor expression as one modulator of human embryo implantation.


Visual Patient is an avatar-based alternative to standard patient monitor displays that significantly improves the perception of vital signs. Implementation of this technology in larger organizations would require it to be teachable by brief class instruction to large groups of professionals. Therefore, our study aimed to investigate the efficacy of such a large-scale introduction to Visual Patient.In this study, we aimed to compare 2 different educational methods, one-on-one instruction and class instruction, for training anesthesia providers in avatar-based patient monitoring.We presented 42 anesthesia providers with 30 minutes of class instruction on Visual Patient (class instruction group). We further selected a historical sample of 16 participants from a previous study who each received individual instruction . After the instruction, the participants were shown monitors with either conventional displays or Visual Patient displays and were asked to interpret vital signs. In the class instruction group, the participants were shown scenarios for either 3 or 10 seconds, and the numbers of correct perceptions with each technology were compared. Then, the teaching efficacy of the class instruction was compared with that of the individual instruction in the historical sample by 2-way mixed analysis of variance and mixed regression.P<.001; effect size –0.55). No significant difference was found for the 10-second scenarios. There was a statistically significant interaction between the teaching intervention and display technology in the number of perceived vital signs . The mixed logistic regression model for correct vital sign perception yielded an odds ratio (OR) of 1.88  for individual instruction compared to class instruction as well as an OR of 3.03  for the Visual Patient compared to conventional monitoring.In the class instruction group, when participants were presented with the 3-second scenario, there was a statistically significant median increase in the number of perceived vital signs when the participants were shown the Visual Patient compared to when they were shown the conventional display (3 vital signs, Although individual instruction on Visual Patient is slightly more effective, class instruction is a viable teaching method; thus, large-scale introduction of health care providers to this novel technology is feasible. Monitoring and continuous evaluation of vital signs by anesthesia providers is central to perioperative patient safety . With 31We designed a simulation study where participants who had no previous experience with Visual Patient underwent either individual or classroom-based instruction and were then asked to interpret conventional displays and avatar-based Visual Patient displays. We hypothesized that the 2 instruction methods would be comparable in efficacy as an introduction to avatar-based monitoring with Visual Patient.On January 19, 2019, 42 nurse anesthetists were recruited to the classroom instruction group. Since the study did not include any real patient data or any human material, the research project did not fall into the scope of the Human Research Act and did not require ethics committee approval. However, we obtained written consent from all participants to use the collected data for scientific purposes.We delivered a 30-minute plenary presentation to all participants in the classroom instruction group. The presentation included an introduction to the concept and technology of Visual Patient as well as an educational video on how the system is used .Data are provided as medians and interquartile ranges (IQR) regardless of normality or estimated marginal means for linear models. Normality was assessed with the Shapiro-Wilks test and visual inspection of quantile-quantile plots of dependent variables. Binary variables are presented as frequencies with percentages. The Wilcoxon signed-rank test was conducted to determine the effects of the Visual Patient display on the ability to correctly perceive vital signs after seeing the display for either 3 or 10 seconds. The different scores were approximately symmetrically distributed, as assessed by box plots. For both scenarios, post hoc descriptive graphs were created detailing whether each vital sign was perceived correctly, incorrectly, or not at all.P>.05) and covariances (P>.001), as assessed by the Levene test of homogeneity of variances and the Box M test, respectively.To compare the effects of classroom instruction and individual instruction, 2-way mixed analysis of variance (ANOVA) was calculated with the factors of display technology (within-subject) and instruction method (between-subject). There was a single outlier, as assessed by inspection of a box plot for values greater than 1.5 box lengths from the edge of the box. As the studentized residual for this outlier was only 3.06, it was retained in the analysis. Homogeneity was observed for variances , the duration of the task (3 seconds vs 10 seconds), and the previous experience of the participants.P value <.025 was considered to indicate statistical significance.Analyses were conducted in SPSS 25 (IBM Corporation) and R version 3.6.1 . Figures were created using GraphPad Prism 8.1.1 . As group differences were calculated separately for both scenarios, a Bonferroni adjusted The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.The 42 nurse anesthetists participating in the study reported a median professional experience of 12 years (IQR 3-31). Of the participants, 28/42 (67%) were female. As they were presented in randomized order with 2 sets, each consisting of a Visual Patient scenario and a matched conventional display scenario, 84 direct within-subject comparisons were performed. After the study, most participants in the class instruction group rated the introductory presentation as very good  or as good , whereas 9 participants did not take part in the follow-up survey.The selected sample of 16 participants from a previous study, who received individual instruction on the Visual Patient, consisted of 8 (50%) physician anesthetists and 8 (50%) nurse anesthetists, where 11 (69%) were female. P<.001) with a large effect size of –0.55  with the Visual Patient and a median of 3 vital signs (IQR 2-4) with the conventional monitoring display. The Wilcoxon signed-rank test determined a significant median increase in the perception of vital signs (3) when participants were shown the Visual Patient compared to when they were shown the conventional display  as determined by Wilcoxon signed-rank test  with the Visual Patient and a median of 6 vital signs (IQR 5-7) with the conventional monitoring display. Thus, there was no statistically significant median increase in the perception of vital signs . Post-hoc univariate analysis yielded a statistically significant difference between the 2 teaching interventions for the Visual Patient  but not for conventional monitoring .The 2-way mixed ANOVA indicated a statistically significant interaction between the teaching intervention and display technology for perceived vital signs (FP<.001).In the classroom instruction group, the estimated marginal means of the perceived vital signs increased from 3.3 (95% CI 2.9-3.8) with conventional monitoring to 6.2 (95% CI 5.6-6.8) with the Visual Patient. In the individual instruction group, the estimated marginal means of the perceived vital signs increased from 4.1 (95% CI 3.4-4.9) with conventional monitoring to 8.5 (95% CI 7.5-9.5) with the Visual Patient. As shown in P<.001) for correct vital sign perception after individual instruction. Moreover, the model displayed very strong evidence for the superiority of Visual Patient, with OR 3.03  for correct vital sign perception with the Visual Patient . No significant difference was found for the 10-second scenarios. These results are similar to those of a previous study by our research group on Visual Patient, where more vital signs were perceived with the Visual Patient after both 3 and 10 seconds [In daily clinical practice, the superiority of the Visual Patient when seeing a monitor for 3 seconds may already be very relevant. It has been shown that anesthesia providers tend to look at patient monitors in short glances . These gParticipants were introduced to the Visual Patient according to our prespecified necessary criteria for general implementation. The teaching was conducted with 30 minutes of plenary classroom instruction , which wPost hoc analysis of perception of specific vital signs showed that with both technologies, the pulse rate and oxygen saturation were nearly always perceived correctly; also, the blood pressure was perceived correctly in approximately 69% of cases. In conventional monitoring displays, these figures are often the largest displayed and are thus easily perceived. The vital signs with more pronounced differences  may be displayed in smaller sizes or in less prominent places. One advantage of the Visual Patient is that all vital signs are displayed in close proximity to each other and sometimes repeatedly . This is supported by an eye-tracking study on Visual Patient, which showed that participants were able to visually fixate on more vital signs with Visual Patient than with conventional monitoring . The cloAlthough the introduction to Visual Patient seemed to be sufficient and the monitoring capability improved, further progress may be possible with more detailed teaching or continued clinical use. The vital sign–based analysis showed that while correct perception of vital signs increased with the Visual Patient, incorrect perception increased as well. This may be due either to the design of these parameters or to inexperience with the Visual Patient. More detailed user perception studies are required to evaluate this result; however, it is more likely to be due to inexperience. The Visual Patient parameters and their display were calibrated using a Delphi process and generally show high interrater reliability, with a previously reported Fleiss kappa >.94 ; howeverThis study had some limitations. The study was simulation based; thus, translational evidence for clinical practice may be limited. Further studies in a high-fidelity simulation environment or in clinical practice are required. However, it is plausible that the effects would persist if used in a clinical setting, as the basic physiological specifications of information intake do not change. The results are in line with those of similar avatar-based monitoring systems, such as the Visual Clot, an animated blood clot that represents coagulation disorders . This stAlthough individual instruction on the Visual Patient is slightly more effective, class instruction is a viable teaching method; this increases the feasibility of large-scale introduction of health care providers to this novel technology. This study further contributes to the growing evidence of the superiority of avatar-based monitoring to conventional monitoring in certain situations.


Pediatric myopia affects more and more children in Asia, USA and other countries. There is no standard protocol for the therapy but pediatric ophthalmologists try to decrease myopia progression using different methods. Myopia in children is more and more frequent and the onset age has decreased over time, leading to a greater chance to progression into high diopters for spectacles or contact lenses and also other ocular complications. Regarding this issue, the aim of this paper was to underline the new therapeutic regimens for correcting and slowing pediatric myopia progression. In the past years, the prevalence of this condition has reached dramatic proportions in East Asia . The ocular morbidity is linked to axial growth, representing an important cause of permanent vision disfunction . Myopia usually manifests in childhood, typically between the ages of 7 to 10 years old, and after that, progresses over the next 10 to 15 years [6]. The prevalence of severe myopia is increasing in urban areas of Asia [7] and recent studies showed a similar situation in the USA [8] and many other populations [9].Pediatric myopia affects approximately 23% of the worldwide population with a prevalence between 0.9% to 3.1% [10]. Because of that, treatment to limit the progression would be beneficial [11].Because the prevalence increases and the age of onset decreases, the condition has more time to progress and the risk of high myopic errors and other ocular complications to occur is more probable . Single Vision Lenses (SVLs) represent the most often used therapeutic option for treating myopia. Although this method helps improving the visual acuity, it does not decrease the myopic evolution or ocular axial growth. Moreover, it has been showed that the use of SVLs might favor myopia progression and axial elongation [14]. Chung et al. study, which compared the effects of under and full correction spectacles, was interrupted after two years due to significant progression of the disease in the undercorrected group (0.23D) and a 30% rate of axial growth [15].From that point it was taught that undercorrection of myopic eyes with eye glasses should slow myopic progression. This concept of under correction was also shown in a couple of infant animal studies. Unfortunately, this finding did not work on children. Recent studies comparing the effects of under and full correction of myopia, explained that undercorrection might actually increase myopia progression .Another treatment strategy is represented by part-time spectacle wear. There is a three-year study that analyzed the effects of different patterns of lens wear on myopia progression showing no benefits after part-time lens wear in myopia evolution . The largest study with this type of lens was the COMET study , which aimed to evaluate whether PALs limit the evolution of myopia versus conventional SVLs. The study explained that patients with more important delays of accommodation and near esophoria could benefit from this treatment, compared to the others. Because of the limited period of time, the study did not find clinically significant results [19].Another studied therapeutic alternative against the progression of myopia is the use of bifocals lenses or PALs. By reducing the accommodation associated with near viewing tasks, they eliminate the delay in accommodation and the consecutive hyperopic defocus at the fovea . A similar study was conducted in Japan and included patients with ages between 6 and 12 years old, with myopia from −1.25 to −6.0 D. The participants wore one type of spectacle lens for the first half period of the study, and then another type of eyeglasses for the second half. Results showed improvement of 0.17 D at 18 months for patients who used PALs first. Early treatment with PALs could have been more efficient than SVLs in these patients. Similar to the These lenses represent a single vision lens with specific myopia management design that is effective in preventing myopic evolution. The periphery of the lens is responsible for myopia control, whereas the central part provides a sharp vision correcting myopia. With SVLs, the central zone works fine but the margin of the lens often projects the image behind the visual area. This can lead to a signal to the eye to elongate, favoring the myopic evolution. MyoVision Lenses active zone in the margin corrects this defocus and in consequence can help limit the progression.21].A 12-month wearer efficacy trial amongst 210 Chinese school children resulted in limiting the myopic evolution by an average of 30% in children between 6 and 12 years old, with at least one parents with myopia. A statistically significant reduced progression of 0.29 D was identified in eyes wearing a new rotationally asymmetric lens versus control lenses [22].More recently, another 2 years study was conducted in Japan on 207 patients with ages between 6 and 12 years old with spherical equivalent refractions ranging from -1.5 to -4.5 diopters and with at least one parent diagnosed with myopia. This study could not verify the therapeutic effect of these kind of lenses in limiting myopic evolution in children from Japan. Additional studies are needed to design lenses that can lower peripheral hyperopic defocus individually and to analyze the benefit of these lenses in preventing myopic evolution [23]. The lack of central vision is not associated with myopia, indicating that peripheral vision has a more significant role. According to literature, peripheral retina and peripheral vision are both associated with the development of myopia .In the past, a soft contact lens called ‘25].These lenses represent a disposable, soft lens designed for one-time use like classic contact lenses, and are FDA approved. The efficiency and protection of the lens were examined during a 3-year randomized controlled clinical study conducted on 135 children with ages between 8 and 12 years old. This type of lenses induces peripheral myopic defocus. The contact lenses have a central part of full myopic power and also a peripheral plus “rings” to defocus. According to this study, myopic evolution is greatly reduced by the MiSight soft contact lens [26]. Considering all of that, MiSight contact lens may be an alternative for slowing the progression of myopic errors.Ruiz-Pomeda et al. conducted a 2-year clinical trial analyzing myopia progression with the MiSight lens versus single-vision spectacles. The mean change in axial elongation at the 12-month follow-up was 0.12 mm for the MiSight lens and 0.24 mm for the other group, and at the 24-month follow-up, the variation in axial growth was −0.16 mm .Contact lenses represent an alternative for treating myopia in children, slowing down its evolution and limiting ocular axial growth. Rigid gas permeable contact lens (RGPCLs), Orthok contact lens and soft bifocal contact lens are available for this type of treatment. The 28].Another study analyzed 100 myopic patients with ages between 8 and 13 years old, who were ﬁtted with RGP contact lenses and 20 patients who used spectacles. The evolution of myopia was signiﬁcantly different with 0.48D progression for RGPCLs group in comparison with the −1.53 D progression for spectacles wearers group. The temporary limitation of evolution was also due to the altering of the corneal curvature and not the ocular axial elongation . Berkeley Orthokeratology study, conducted on 80 subjects who used orthokeratology versus a control group, identified a signiﬁcantly higher myopic reduction in orthokeratology group versus the other group, but unfortunately the study had minimal clinical signiﬁcance [30]. These are promising studies that demonstrate the demand for a well-designed study on what is now called corneal reshaping therapy with sufficient subject numbers, adequate control groups and long-term follow-ups to obtain clinical importance. Of course, the complication of the lenses, like microbial keratitis, compliance of the patients and family and participation to follow-ups must be taken into consideration to fully evaluate this treatment for slowing myopia progression.Fig. 1,2). The red ring indicates that the night time lens had the expected effectiveness. Orthokeratology improved the quality of visual acuity and life. The risk of amblyopia can be lowered by using orthokeratology in children with myopic anisometropia [31].The night contact lenses used in orthokeratology are manufactured after the interpretation of the corneal topography  [33].Topical 34]. The study compared an atropine group with a placebo-control group. The 1-year washout period showed important rebound phenomenon in the refraction in the axial elongation and it was efficient at the end of the two-year therapy phase. This study was followed by a 5-year clinical trial that analyzed the efﬁcacy of smaller doses of atropine on limiting the myopic evolution, while also minimizing the adverse effects. Patients received either 0.5% or 0.1% or 0.01% atropine for 2 years, followed by a 1-year washout period. The conclusion was that all 3 doses of atropine remained efficient in lowering myopic progression and the concentration of 0,01% had the least side effects.Chua et al. reported similar results in a two-year study of 400 participants, 6 to 12-year-old myopic children in Singapore .Although we have clarified many uncertainties regarding the reduction of myopic progression in children, many questions are still unanswered. Children and their parents should always discuss the risks and benefits of the treatment with their ophthalmologist. Far more studies need to be conducted to solve all the questions, so that we can improve eye care for children and eventually prevent or maintain a lower amount of myopia, which may decrease the risk of other ocular complications.DisclosuresNone.Conflicts of interestNone.


Regarding 2, 2, and RR during the rest period and in each motion, 2 and 2 in males were significantly greater than those in females. RR in the female participants had greater value than that in males. Among the maneuvers, 2, 2 or E during a hair-washing motion with both hands were greater than those during hair-washing motion with one hand. 2, 2, RR, or E during a hair-washing motion with both hands fast speed was greater than those during a hair-washing motion with slow speed. In conclusion, this study showed the effects owing to the differences in motion maneuvers and gender during UAE in healthy young people. These suggest a need to consider motion maneuver or gender when teaching motion methods of activities of daily living on the patients.It is known that patients with chronic obstructive pulmonary disease experience dyspnea during unsupported arm exercise (UAE). We examined the respiratory variables in during a hair-washing motion for healthy young people requiring the UAE to find the effects across gender, motion, and speed. In this study, 33 healthy young people were enrolled. Participants performed the following four types of hair-washing motions: both hands with fast speed, both hands with slow speed, one hand with fast speed, and one hand with slow speed. The respiratory variables such as oxygen uptake ( Patients with COPD frequently experience dyspnea during unsupported arm exercise (UAE). It is known that respiratory function is altered in the UAE during activities of daily living (ADL). Accordingly, patients with early-stage COPD already have significant ventilatory constraints and increased dyspnea during ADL3. Nevertheless, it is a motion that involves repetitive movements with UAE. Therefore, it is necessary to provide guidance on the motion that reduces dyspnea. Moreover, a few studies have examined the effects of differences in ventilatory parameters between one hand and both hands or fast and slow speed.Patients with COPD regarded personal body care, including hair-washing, as a routine task with a relatively fixed form and a symbolic means of self-preservation6. However, these differences between males and females during low-intensity exercise with UAE are unclear.The previous studies have suggested differences in the respiratory function between males and females in maximal exerciseIn this study, we attempt to elucidate the effects of differences in the UAE and gender on respiratory variables in healthy people. A hair-washing motion was chosen for this study as ADL include UAE. The aim of this study was to investigate effects on respiratory variables during hair-washing motion across gender and motion type in healthy young people.A total of 33 healthy young people  were enrolled in this study : both hands with fast speed (motion A), both hands with slow speed (motion B), one hand with fast speed (motion C), and one hand with slow speed (motion D). These motions were divided into 2 sets and continued for 3 min with resting periods before and after. There was a break time between the two sets , carbon dioxide output , minute ventilation , or respiratory rate  were measured by breath-by-breath methods using a portable metabolic measurement system , and heart rate  was continuously measured using a heart rate monitor . Oxygen (O2) and carbon dioxide (CO2) in the participant’s exhaled gas were detected by the analyzer’s O2 and CO2 meter. From these data, the device calculated 2, 2, E, RR, etc. The response time for the O2 and CO2 analyzer was less than 250 ms . The measurement range of the flow sensor was 0–840 L/min, and its measurement accuracy was within 3% of the indicated value. The precision of this analyzer has been reported in a previous study7. A complete calibration routine was performed before each experiment. The respiratory variables were measured as standard temperature and pressure, dry. We used the mean value of the data for the last 1 min of each motion and each rest period. The mean value of the respiratory variables during the last 1 min of the first rest period was used as a baseline to compare the increases in the parameters for each motion.Respiratory variables such as oxygen uptake . The data were analyzed using IBM SPSS Statistics software, version 24.0 . Student’s t test was used to analyze differences in respiratory variables between males and females. Three-way analysis of variance (ANOVA) with repeated measures was used to compare the effects across (1) gender and (2) motion (one hand or both hands), and speed (fast or slow) on respiratory variables. Bonferroni’s correction was used for the post-hoc analysis. The level of significance was set at p < 0.05 for all statistical comparisons.This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Yamagata Prefectural University of Health Sciences, Yamagata, Japan (#1801-23).2, 2, HR, RR, and E) were measured in the rest period and each motion.Participants performed 4 type hair-washing motion . Respiratory variables  during a hair-washing motion were analyzed by three-way ANOVA and post-hoc analysis.Effects of gender, motion and speed on respiratory variables . Post-hoc analysis showed 2 and 2 in males was significantly greater than those in the female during rest period and motion A–C, RR in females was significantly greater than those in males during motion A–C , and there was significantly main effect of speed on 2, 2, RR, E . 2, 2, HR, and E during a hair-washing motion with both hands were greater than that during hair-washing motion with one hand. 2, 2, RR, E during hair-washing motion with both hands fast speed were greater than those during a hair-washing motion with slow speed.When considering the differences in the maneuver of motion, there was a significantly main effect of motion on 2 and 2/weight   may be restricted and RR may increase to supplement oxygen demand during UAE. The mobilization of the sternocleidomastoid muscle during upper arm elevation suggests a decreased respiratory function12. These possibly cause dyspnea during upper arm elevation. The present study revealed that respiratory variables during each hair-washing motion was increased compared to the rest period. These finding indicated that UAE during ADL caused increasing ventilatory demands in healthy young people.Hair-washing motion requires UAE, that cause significant metabolic and ventilatory demands2 and 2 in males were significantly greater than those in females. It has been known that males have a greater oxygen uptake than females in the rest period, so observed differences in 2 between males and females might be affected by their muscle mass. In motion A–C, 2, and 2 in the males were significantly greater than those in females. On the other hand, 2 divided by body weight showed no significant effect on gender. This result shows that differences in body weight between males and females affect the differences in 2. Previous studies suggested that differences between males and females for peak 2 depend on the size of the contracting muscle mass14. In this study, differences between the males and females in 2 and 2 during each motion were observed. This result indicated that 2 during the hair-washing motion is affected by gender owing to the differences in contracting muscle mass, as well as during maximal exercise.In the rest period, E during rest period and each motion showed no main effect on gender. This result suggests that chest kinematics may be significantly influenced by gender as revealed by the differences in the ventilatory values during the performance of hair-washing motion. T and E in females are mechanically constrained during exercise, since the lung volume of females is smaller than that of males15. Bellemare et al. suggested that the rib cage muscle contributes more to the inspiratory pressure swings in females than males. Altogether, females respond to increased oxygen demand by increasing their RR with their rib cage muscle efforts.In the females, RR during the rest period and each motion was significantly greater than that in their males. On the other hand, 18. This study suggested that there are differences in respiratory function during UAE between males and females, even though motions did not require maximal effort for healthy young people. Further studies are need to reveal the effects of gender during ADL with UAE for elderly people and patients with respiratory diseases.Previous study showed differences on respiratory variables between males and females during maximal exercise2, 2, RR, HR, or E, during the performance of motion with both upper arms. Also, the speed of motion may have influenced these ventilatory parameters.The results of this study show significant increases in the ventilatory parameters, such as 2 and 2 during motion A. Noteworthy, the increase in 2 in males was greater than that in females when comparing the motion with one hand and both hands. Motion with both hands increased oxygen uptake. Therefore, males might be effect on motion with one hand or both hands compared with females. This result extends our knowledge of effects of differences motion and gender during the UAE.Motion A and B required elevation of both upper limbs, motion C and D required elevation of one limb. Motion A and C were performed at fast speed compared with motion B and D. Therefore, the results showed the highest values in 19. This suggests that the hair-washing motion with fast speed increases oxygen demand compared with hair-washing motion with slow speed.When the participants performed hair-washing motion with fast speed, the waveform amplitude of their inferior fibers of the trapezius increased21. LRC refers to the phase-locking of locomotor and respiratory frequencies. The mechanism underpinning LRC has not been elucidated yet, but LRC has been suggested to increase respiratory airflow22. In this study, we could not confirm that the hair-washing motion with fast speed caused LRC. Nevertheless, it is possible to suggest that LRC occurs with fast-speed motion owing to the action of relieving the respiratory load caused by the UAE.In the results of this study, RR increased on fast-speed motion compared with slow speed motion. We deduced that the increase in RR was caused by two factors. One of these is limited ventilation owing to upper arm elevation, and another is locomotor–respiratory coupling (LRC). It is known that LRC is initiated between exercise rhythm and RR during exerciseHair-washing motion with one hand and slow speed showed the lowest value in each motion for 3 min. This study suggests that we should choose motion maneuvers considering to factors such as gender and physical function.We showed the effects owing to the differences in motion maneuvers and gender during the UAE in healthy young people. The findings from this study made several contributions to reveal changes of respiratory demands during ADL. These suggest a need to consider motion maneuver or gender when teaching motion methods of ADL on the patients.


Much of the literature surrounding bilingual spoken word recognition is based on bilinguals of non-tonal languages. In the Mandarin spoken word recognition literature, lexical tones are often considered as equally important as segments in lexical processing. It is unclear whether and how lexical tones contribute to bilingual language processing. One recent study demonstrates that tonal bilinguals require the availability of both tonal and segmental information to induce cross-language lexical competition during bilingual lexical access, even without phonological overlap between the target and non-target language. The current study investigates whether overt phonological overlap between the target and non-target language would equally require both tonal and segmental information available to induce cross-language lexical competition. We employed two auditory lexical decision experiments with both Mandarin-English bilinguals and English monolinguals to test whether inter-lingual homophones (IH) would induce lexical competition from the non-target language, L1 Mandarin. Our results show that cross-language lexical competition was only observed with the presence of lexical tones, in addition to segmental overlap. In thisd Marian  reportedlanguage status (i.e. whether the target language is L1 or L2). Adopting the same eye-tracking technique, they found that Dutch-English bilinguals fixated longer to competitor objects only when the target language was L2 English , but not when the target language was L1 Dutch . This asymmetry suggests that co-activation in bilingual word recognition is not unconditional, but rather depends on the characteristics of the auditory input. Second, using the same experimental paradigm, Ju and Luce 33] and iThus, Experiment 1 did not show cross-language lexical competition effects with inter-lingual homophones when lexical tones were absent. To further investigate this, Experiment 2 was designed to understand whether the same stimuli would produce cross-language lexical competition with the presence of lexical tones. In order to test this hypothesis, we superimposed Mandarin tones onto the English words and non-words in Experiment 2.Participants were recruited from the same population as those in Experiment 1, consisting of 22 Mandarin-English bilinguals and 21 English monolinguals. The self-reported English proficiency with respect to the four skills  and IELTS scores are reported in my or /maɪ/ corresponds to at least nine Chinese characters, the most frequent of which is 买 ‘to buy’ /maɪ3/ (Pinyin: mai3). Thus, the third tone was chosen for the superimposition of my. As the non-IH was selected to match the IH item-by-item in both frequency and phonological neighbourhood density, we superimposed the same tone used for its matched IH. The four tones in Mandarin were assigned randomly for nonwords.The materials and design of Experiment 2 were the same as Experiment 1, except that Mandarin tones were superimposed onto all stimuli. For the IHs, the tone chosen for superimposition was that of the most frequent Chinese character corresponding to the English IH in pronunciation, based on the SUBTLEX-CH database . For exaha/ [Pinyin: pa], , was played to the speaker as an example to follow. Thus, the bilingual speaker pronounced each given English word and non-word, and then pronounced each word/non-word with 4 Mandarin tones in the sequence from Tone 1 to Tone 4. Throughout the entire recording session, no Mandarin was used at all. Furthermore, recordings were independently judged by another native speaker of Mandarin so that tonal tokens evaluated as ‘awkward’ were re-recorded. These re-recorded items were all nonwords. Only toned syllables matched with experimental items/designs were used in testing. All the auditory stimuli were trimmed for programming purposes and normalized to ensure all the tokens have the same amplitude .To test whether native speakers of Mandarin are sensitive to tonal information when processing English, we superimposed Mandarin tones of the IHs onto English in Experiment 2. That is, each homophone was superimposed with a Mandarin tone that corresponds to the tone of its inter-lingual counterpart. Tokens were recorded by the same simultaneous bilingual speaker as in Experiment 1. To create the experimental tokens, we followed the same procedure of tone superimposition adopted in , and useThe procedure of Experiment 2 was the same as Experiment 1.z = 1.67, p = .094). However, the monolingual results showed significant difference between the IH and non-IH conditions . This means that monolinguals found the IH words more difficult to process than the non-IH words, while this was not the case for bilinguals. A combined analysis of both groups in error rate showed a marginal main effect of Group . However, there was neither main effect of Word Type  nor interaction between Word Type and Group . These results indicate that bilinguals encountered more difficulty during lexical processing in their L2 than monolinguals.The data trimming and analysis procedure were the same as in Experiment 1. As a result, 1 bilingual participant and 1 monolingual participant were excluded from the analyses because their error rates were larger than 30%. Similarly, along with the results from Experiment 1, mean response times and error rates for IHs and non-IHs are presented in t = 2.57, p = .01 < 0.05) and Group . There was no interaction between Word Type and Group . Similar to Experiment 1, these results showed that the bilingual participants responded to L2 English spoken targets much more slowly than their monolingual counterparts. However, different from Experiment 1, the IH items became more difficult for bilinguals with the presence of lexical tones, compared to the non-IH items (125ms difference). Restricting the analysis to just the bilingual group, the mixed-effects analysis of the RTs showed a main effect of Word Type . Unlike the results from Experiment 1, with the presence of lexical tones, the bilinguals treated IHs and non-IHs differently by responding to the IH words significantly more slowly than the non-IH words. This result suggests that the Mandarin lexicon was activated so as to create lexical competition in processing English words. These results are consistent with previous studies .–65.59–65strategy , which pThis analysis is crucial in understanding the IH inhibitory effects observed in bilinguals in Experiment 2, further confirming the inhibition was due to cross-language lexical competition induced by both lexical tones and segments. The logic is that if bilingual listeners were sensitive to coda or to segmental overlap between L1 Mandarin and L2 English, we would expect to observe inhibition on the IH items in Experiment 1. However, this was not what we observed here. The contrast demonstrated between Experiments 1 and 2 in bilinguals suggests that language-specific cues  are crucial in bilingual lexical access with bilinguals whose one language is tonal while the other is non-tonal. That is, both tonal and segmental information need to be available to induce language co-activation. These findings differ from those from previous studies of bilingual lexical access by showing that segmental overlap was insufficient to activate the non-target Mandarin lexical representations. This is in line with the results reported by Lee , who fouIn addition, at the theoretical level, our current findings shed light on the mechanism of pitch processing. A large but under-researched theoretical debate in the literature of Speech Perception is whether the processing of pitch contours during word recognition is language context sensitive ,68. HereOne limitation of the current study is the unnaturalness of Experiment 2, as English stimuli were superimposed with lexical tones. As demonstrated earlier, the error data indicate that Experiment 2 turned out to be more difficult than Experiment 1 overall and IH words became more difficult than non-IH words with the superimposed pitch contours; in other words, the Mandarin pitch contours carried by English monosyllables appeared to ‘interfere’ with lexical processing in general terms for both monolinguals and bilinguals. Our post-experiment debriefing found that some participants reported difficulty in recognizing some items in Experiment 2. One way to improve this study methodologically is to manipulate pitch contour in the way that is consistent with English input in natural speech. For instance, English words can be pronounced with a pitch contour that is similar/comparable to Tone 2 or Tone 4 in Mandarin, whereas; Tone 3 is rarely observed in English pitch contour. Thus, our stimuli superimposed with Tone 3 can bias bilinguals more towards the Mandarin lexicon and generate more lexical competition. On the other hand, they could be a lot more difficult for monolinguals to process due to their unnaturalness in English speech.The current study adds to the bilingual literature by providing empirical evidence of a linguistic dimension  as an important representational and processing mechanism in bilingual spoken word recognition. The contrast between Experiments 1 and 2 shows that lexical tones are a critical cue inducing cross-language lexical competition, in addition to segmental overlap across languages, as demonstrated in previous studies , 33]. I. I33]. IIn summary, our findings support the requirement of a precise input-representation match for language co-activation and hence cross-linguistic interactions, echoing Ju and Luce  in the aS1 Data(ZIP)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.