Prevention of pain in rabbits is a priority for both welfare and validity of scientific data. We aimed to determine if the rabbit grimace scale (RbtGS) could be used as a viable, rapid assessment tool in two breeds of rabbit, Dutch belted (DB) and New Zealand white (NZW), following orchidectomy, as an adjunct to behavioral analysis. All animals received analgesia. Rabbits were filmed and their behavior was recorded at multiple time points pre- and post-orchidectomy. Observers then scored specific pain associated behaviors for analysis. Time matched footage was also scored using the rabbit grimace scale (RbtGS). Following surgery, rabbits showed significant increases in the duration spent displaying key pain associated behaviors at 1 and 5 h post-surgery. DB rabbits that received low dose meloxicam (0.2 mg/kg) showed significantly more pain behaviors at 1 and 5 h post-surgery compared to those administered a combination of higher dose meloxicam (0.6 mg/kg) and a lidocaine/bupivacaine local infusion. DB rabbits showed an increase in RbtGS score at both 1 and 5 h post-surgery. In the NZW rabbits, an increase in RbtGS score was only observed at 1 h post-surgery. Using behavioral analysis as the gold standard for comparison, the RbtGS was an effective means of determining when rabbits are painful following orchidectomy. Higher dose meloxicam (0.6 mg/kg) combined with local anesthetic was a more effective method of reducing pain, compared to lower dose meloxicam (0.2 mg/kg) alone. Rabbits are a popular pet and a common laboratory and farm species globally. In the UK, over 900,000 rabbits are kept as pets  and overLeach et al.  carried Various grimace scales exist for evaluating pain in a variety of species –19, inclOne of the benefits of grimace scale scoring is that it is significantly less time consuming than standard behavioral analysis, therefore has the potential to allow more rapid means of assessing pain and therefore screening of analgesics for their effectiveness. Since humans have a natural tendency to look at the faces of animals, rather than their body , assessiThe primary aim of the current study was to determine if the Rabbit Grimace Scale (RbtGS) and Spontaneous Pain Behavior Scale (SPBS) provided an effective and rapid means of assessing pain following orchidectomy in rabbits. We also aimed to evaluate two methods of scoring the RbtGS; still images compared to scoring from video recordings to compare their effectiveness for scoring pain. Finally, we included two different multimodal regimes in two rabbit breeds. In Batch 1, use of meloxicam at the currently recommended dose rate (0.6 mg/kg) together with local infiltration of the surgical site with lidocaine and bupivacaine was evaluated in Dutch belted rabbits in comparison to meloxicam at a previously used dose (0.2 mg/kg)  as the sAll procedures were conducted in accordance with the Animals (Scientific Procedures) Act 1986 (PPL 60/4431), European Directive 2010/63 and with the approval of the Newcastle University Animal Welfare Ethical Review Body. This manuscript was prepared in accordance with the ARRIVE guidelines. This study employed a “rescue analgesia” policy whereby if any animal displayed >4 pain behaviors within 5 min (assessed by a veterinarian who was not otherwise involved in the study), 0.05 mg buprenorphine s.c. was administered immediately. No animals required this intervention. All animals recovered uneventfully from surgery.ad-libitum. Additionally, hay and cabbage or carrots were provided daily. The room was maintained at 21 ± 2°C, humidity 50% and a 12/12 h light/dark cycle (lights on at 07:00). A 14-day acclimation period was given before the start of the study for each Batch. During this time, the rabbits were habituated to the general daily activity of the animal care staff, handling, weighing, the presence of the observers, and the video monitoring equipment.This study used two separate batches of rabbits (Batch 1 and Batch 2). Batch 1 contained 16 male Dutch belted rabbits  aged 6 months at the start of the study. Batch 2 contained 16 male New Zealand white rabbits  aged 5 months at the beginning of the study. The animals were free from any common pathogens in accordance with FELASA health monitoring recommendations. These two breeds were selected for study as they are a typical pet breed (Dutch-Belted) and typical laboratory breed . On arrival, rabbits were individually housed in floor pens (1 × 2 m) with sawdust bedding . Each pen contained a cardboard box acting as both a shelter and a platform, a cardboard tube (large enough for the rabbit to enter), a cat litter tray and chew blocks for enrichment . Although kept in individual pens, rabbits were able to see, hear, smell and touch conspecifics through adjoining mesh pen walls. Food  and tap water were provided The rabbits were housed individually to prevent fighting between intact males and to prevent any changes in behavior or facial expressions because of transient separation from their pen mates during observations (see below). The rabbits were then housed in groups of 2–4 (with neighboring rabbits), at 2–3 weeks post castration, in preparation for rehoming as domestic pets. Groups of rabbits were housed in larger pens (2 × 2 m for pairs or 3 × 2 m for >3 rabbits) in the same manner as described above. Before rehoming, all rabbits underwent a veterinary examination, were formally released from the controls of the Animals (Scientific Procedures) Act 1986 and vaccinated against myxomatosis and viral haemorrhagic disease.The week before surgery, all rabbits were placed individually in the filming arena  which contained only sawdust bedding. The arena consisted of 3 clear Perspex sides with a mesh divider splitting into two filming pens (1 × 2 m). Two HD video cameras  were placed at a fixed distance from each of the two Perspex walls of each filming pen and remotely operated from outside the room at all times . The rabn = 8 per treatment group) in each Batch using a random number generator (www.random.org).The two treatments of meloxicam only or multimodal analgesia see  were ranAnesthesia and analgesia (AA) only control data were collected from rabbits in Batch 2. Video recordings were time of day matched with Baseline AM and Baseline PM recordings. Therefore, these rabbits acted as within-subjects' controls for the effect of treatment only. On the day of AA control data collection, rabbits received their allocated dose of meloxicam or multimodal analgesia . Thirty On the morning of surgery, rabbits were transported to theater using a pet carrier. All rabbits received their allocated dose of meloxicam or multimodal analgesia  30 min bFollowing surgery, the filming process was repeated, as described above at various time points, with time of day matched with Baseline AM, Baseline PM, AA-AM, and AA-PM. For Batch 1, filming was carried out at 1, 5, 24, and 48 h post-surgery. For Batch 2, filming was carried out at 1, 5, 24, and 29 h post-surgery. Following data collection for Batch 1 at 48 h, we felt that it was important to study an intermediate time point in the second Batch. By 48 h behavior had largely returned to baseline levels so we took the opportunity to study a time matched (with respect to baseline) point to determine how long the acute painful effects of surgery lasted. Dark phase activity was again monitored for those rabbits in Batch 2 on the day of surgery and surgery day +1.Individual body weight data were collected on Baseline, AA, Surgery day and Surgery day+1 for all rabbits. For Batch 2 only, this data was also collected on surgery day +2, together with food and water consumption measured at each time point, by weighing the water bottles and any food that remained in the pen.Fifteen minutes of manual behavioral analysis was carried out, for each rabbit, at each time point by treatment and time-point blinded observers (one for Batch 1 and two for Batch 2) using Cowlog 2.0 software , and an www.random.org). The chosen images were then randomly re-ordered and inserted into a custom-designed excel spreadsheet for scoring. Participants (10 for Batch 1 and 7 for Batch 2) who were blind to all aspects of the experimental design and purpose scored the images using the RbtGS. Each participant was provided with a RbtGS manual for reference when scoring in the images. Prior to scoring the images from this study, participants scored a small number of rabbit images, and these scores were compared to those from an experienced observer (ML). Participant scores were consistent with those of the experienced observer. The images were scored for all 5 of the FAUs comprising the RbtGS on a 3-point scale; 0 = not present, 1 = moderately present, 2 = obviously present. On occasions where the observer was unable to score a particular FAU, they were asked to mark this as “not visible,” following Keating et al. . In Batch 1, the video files were watched and paused every 20 s and scored using the RbtGS as described above made at the instance in time. To reduce the time taken for analysis, in Batch 2, four 30 s video sequences from across the 15 min video were extracted and scored. These video sequences were selected at random from each video file. Scoring was done based on the cumulative impression given by the 20- or 30-s clip observed.Video scoring was used to simulate remote live scoring in a clinical context. The same videos that were used for the production of the still images were scored by treatment and time-point blinded observers in a randomized order, assigned using a random number generator  via R studio . Generalized linear mixed models via the lme4 package . Tukey pairwise comparisons revealed a greater duration spent displaying active pain behaviors 1 h post-surgery compared to all other morning observations, irrespective of the treatment group. Similarly, for the duration of active pain behaviors during the afternoon observations (Baseline PM and 5 h post-surgery), the only factor found to have a significant effect was time , with a greater duration spent displaying these behaviors at 5 h post-surgery. Again, this was irrespective of the treatment group, with no significant difference found between the meloxicam alone and multimodal treatment groups (For the duration of time spent displaying active pain behaviors (composite pain duration) during the morning observations , the only factor found to have a significant effect was time , with rabbits administered meloxicam alone exhibiting significantly more pain behaviors compared to rabbits administered with multimodal analgesia. There was also a significant main effect of time  where rabbits exhibited significantly more pain behaviors at 1 h post-surgery compared to all other morning time points. Similarly, for the afternoon observations, a main effect of treatment  and time  were found, with significantly more pain behaviors exhibited by meloxicam only treated rabbits in comparison to multimodal and significantly more pain behaviors displayed at 5 h post-surgery than the baseline afternoon time point (For the frequency of active pain behaviors displayed (composite pain frequency) during the morning observations, there was a significant main effect of treatment . There was also an effect of time, irrespective of treatment group , with rabbits displaying more active pain behaviors at 1 h post-surgery compared to Baseline AM, AA-AM, and 24 h post-surgery. For the duration of active pain behaviors during the afternoon observations , there was a significant interaction between time and treatment  and a main effect of time . Post-hoc analysis showed that rabbits administered meloxicam alone displayed active pain behaviors for significantly longer than those in the multimodal treatment group at 5 h post-surgery and spent significantly longer displaying these behaviors compared to Baseline PM (For the duration of time spent displaying active pain behaviors (composite pain duration) across the morning observations , those rabbits administered meloxicam alone displayed significantly more active pain behaviors than those in the multimodal treatment group at 1 h post-surgery . Although Tukey post-hoc analysis showed no significant differences between any individual pairwise comparisons, there was a trend toward rabbits displaying more pain behaviors at 1 h post-surgery compared to baseline AM observations. For the afternoon time observations, there was a significant interaction of time and treatment interaction . Tukey post-hoc analysis showed that rabbits administered meloxicam exhibited more pain behaviors at 5 h post-surgery compared to rabbits administered multimodal analgesia (For the frequency of pain behaviors observed (composite pain frequency) during the morning observations, the only factor found to have a main effect was time , and a time treatment interaction . Tukey post-hoc tests revealed that irrespective of treatment group, the RbtGS scores were greater at 1 h post-surgery than at any of the other morning time points. Rabbits administered meloxicam alone had a greater RbtGS score at 1 h post-surgery than any other morning time point. For the afternoon observations (Baseline PM and 5 h post-surgery), the total mean RbtGS scores were not significantly different between the two analgesic treatment groups. However, there was a significant effect of time . Rabbits had a significantly higher RbtGS score at 5 h post-surgery than at Baseline PM , there was no effect of treatment, but there was a significant effect of time  and a significant time treatment interaction . Irrespective of treatment, rabbits had the greatest RbtGS score at 1 h post-surgery compared to AA-AM. However, RbtGS scores were greater at both these time points compared to Baseline AM and 24 h post-surgery. Rabbits that had received meloxicam alone had significantly greater RbtGS scores one post-surgery compared to all other morning time points. In contrast, rabbits administered multimodal analgesia had a significantly lower RbtGS score at 1 h post-surgery compared to Baseline AM , there was no effect of treatment, but there was a significant effect of time  and time . Tukey post-hoc analysis revealed that irrespective of treatment group, rabbits had higher RbtGS scores at AA-PM and 5 h post-surgery compared to Baseline PM and 29 h post-surgery. Rabbits administered meloxicam alone had greater grimace scale scores at surgery +5 h compared to Baseline PM and surgery +29 h. Those rabbits administered multimodal analgesia did not have greater grimace scale scores following surgery , there was a significant main effect of both treatment , with no significant difference in the RbtGS scores between the treatment groups. Tukey post-hoc analysis showed that the rabbits were assigned higher grimace scale scores at surgery +1 h compared to all other morning time points. When analysing the afternoon time point data, a similar pattern was seen, where the only factor to show a significant effect was time , with a higher grimace score at surgery + 5 h compared to Baseline PM  and a significant time treatment interaction . Tukey post-hoc analysis showed that rabbits administered meloxicam alone had a significantly higher RbtGS score at 1 h post-surgery compared to Baseline AM and 24 h post-surgery. There were no significant differences in the rabbits administered multimodal analgesia. For the afternoon time observations, time was the only factor to have a significant effect , with rabbits assigned higher RbtGS scores at 5 h post-surgery compared to all other PM time points , with rabbits found to be significantly heavier 48 h post-surgery, compared to both baseline and 24 h post-surgery.No significant difference was found between the bodyweight of the rabbits in the two treatment groups at any time point. A significant difference in body weight was observed over time , with rabbits receiving meloxicam alone showing a greater reduction in food consumption relative to baseline levels than those rabbits administered multimodal analgesia. A significant effect of time was also found . No significant difference was observed between any time points or treatment groups in the volume of water consumed.There was a significant effect of treatment . The dose of meloxicam (0.6 mg/kg) used in the multi modal approach was higher than that of the meloxicam alone group, as we considered that improved analgesia could be achieved by doing so. We aimed to compare the use of the RbtGS with a range of analgesic regimens, thought likely to produce varying degrees of post-operative analgesia. Batch 1 evaluated the combination of meloxicam with a lidocaine/bupivacaine infiltration of the surgical site in Dutch belted rabbits. Batch 2 studied the combination of meloxicam and buprenorphine with a lidocaine/bupivacaine infiltration of the surgical site in New Zealand White rabbits. In Batch 1, Dutch belted rabbits that had received a lower dose of meloxicam alone showed significantly more pain behaviors at 1 h post-surgery and 5 h post-surgery compared to those that had been administered a combination of a higher dose of meloxicam and a lidocaine/bupivacaine local infusion. When the RbtGS was scored using still images, a treatment difference was only observed at 1 h post-surgery, suggesting that in its current format, the RbtGS may only be of benefit for comparing analgesics in the most acute phase of post-operative pain for this breed. However, no difference was seen between the two treatment groups at any time point when scoring the RbtGS from a video , despite differences being observed in the behavior of the rabbits. In Batch 2, the New Zealand white rabbits that had received the lower dose of meloxicam alone displayed pain behaviors for a significantly longer period than those that had received a combination of higher dose meloxicam and buprenorphine with a lidocaine/bupivacaine local infiltration at 1 and 5 h post-surgery. When assessed using the rabbit grimace scale, scores from still images and video were higher in the lower dose meloxicam only group than the multimodal group at 1 and 5 h post-surgery. This finding replicates the pattern of results obtained from the behavioral analysis, indicating that in this particular breed of rabbits, the RbtGS was effective at determining time points when pain is likely being experienced both in research and clinical settings. The multimodal analgesic regimen administered differed between the two breeds of rabbits. In Batch 1, meloxicam was administered alongside a lidocaine/bupivacaine local infiltration. Following this batch, we subjectively felt that the multi modal approach could be further refined, so in Batch 2, the same approach was taken but with the addition of 0.03 mg/kg buprenorphine. While breed differences cannot be ruled out because of the study design, those rabbits that received buprenorphine did not show an decrease in RbtGS score using either method of assessment.Across the time points studied, changes in behavior were only observed in the first 5 h following surgery. The next time point studied for full behavioral analysis was 24 h post-surgery when no significant differences were observed in pain behaviors compared to baseline. During this gap in behavioral analysis, dark phase activity was monitored in the New Zealand white rabbits. The methodology used was to count the number of transitions between zones in the pen, and no significant differences were found either between groups or between baseline and post-surgery time points. Further research is required to determine whether dark phase activity measured in this way was not sensitive enough to detect behavioral changes linked to pain following surgery or whether it was used beyond the period in which rabbits showed acute changes related to pain. Future research should focus on the time frame between 5 and 24 h post-surgery to determine the overall length of time pain is likely to be experienced by rabbits following orchidectomy.Using behavioral analysis as the “gold standard” for comparison, the RbtGS was an effective means of determining when rabbits are experiencing acute pain following orchidectomy. RbtRGS differentiated the effects of low dose meloxicam (0.2 mg/kg) from that achieved with a higher dose of meloxicam (0.6 mg/kg) combined with either buprenorphine (0.03 mg/kg) and local anesthetic infiltration  or local anesthetic infiltration (in Dutch Belted rabbits). The combinations using a higher dose of meloxicam were more effective in reducing pain compared to the lower dose of meloxicam alone.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All procedures were conducted in accordance with the Animals (Scientific Procedures) Act 1986, European Directive 2010/63 and with the approval of the Newcastle University Animal Welfare Ethical Review Body.AM, ML, PF, and JC: conception, design of the work, and interpretation of data. AM, ML, PF, JC, CQ, VN, CK, and AG-S: acquisition and analysis. All authors contributed to the article and approved the submitted version.This project was funded by NC3Rs, grant number: G1100563/1 Newcastle University provided funds for open access publication of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Arabidopsis TPC1 in the early stages of activation  Ca2+ binding site involving D454, which interacts with the voltage-sensing domain (VSD2). In the original protein crystal structures, the luminal Ca2+ binding site stabilized VSD2 in the resting state.To understand the changes in conformation that occur in ion channels upon activation it is necessary to have structures representing both active and inactive conformations. Two new reports in PNAS now show structures of tivation . Obtainiormation , 4, whil2+-permeable  ion channel was one of the first plant ion channels to be identified and studied using the patch-clamp technique  and is fermeable  and is aactivity  provided domains . TPC1 coevidence  and by tevidence , 4.Activation of TPC1 involves the physical movement of VSD2. VSDs are composed of four helical membrane spans, one of which has positively charged amino acid residues that move in response to membrane potential changes. In TPC1, VSD1 does not contribute to voltage regulation of the channel , 4. VSD22+ binding site. Ye et al. (WT) obtained in the presence of 1 or 50 mM Ca2+ which represents the Ca2+-inhibited inactive form of the channel with a triple mutant (D240A/D454A/E528A), designated AtTPC1ΔCai, at the same calcium concentrations. At 1 mM Ca2+ the AtTPC1WT structure showed a closed pore, with VSD2 in the resting conformation, and unoccupied EF hand domains. Surprisingly, the AtTPC1WT structure at 50 mM Ca2+ was the same, indicating that the resting VDS2 prevented occupation of the EF hands  mutant containing a D454N point mutation in AtTPC1  generate the membrane potential (cytoplasmic side negative) by pumping H+ into the vacuole lumen (− and NO3− (+/K+ antiporters in the NHX and CHX families (2+ gradient across the vacuole membrane, the cytoplasmic Ca2+ concentration in resting cells is around 0.1 to 0.2 µM and during signal transduction it can increase to around 2 µM. The Ca2+ concentration in the vacuolar lumen is in the range of 2 mM, generated by H+/Ca2+ antiporters in the CAX family and Ca2+-ATPases. Elevated cytoplasmic Ca2+ in the range of 1 µM was shown to activate K+-selective VK/TPK channels in the vacuole membrane (+ flux into the cytoplasm and depolarize the vacuolar membrane potential (2+ and membrane depolarization, the two conditions required for SV/TPC1 activation.To understand TPC1 activation in plant cells, we need to know what conditions could lead to depolarization of the vacuole membrane, also known as the tonoplast. The membrane potential of the vacuole membrane is controlled differently than at the plasma membrane. Proton pumps (vacuolar Hle lumen A. Howeveand NO3− –14. Thisfamilies , 15. Themembrane B which aotential . Thus, V2+:K+ of 3:1 and show tail currents that carry Ca2+ flux into the cytoplasm, with an ~7-fold higher single channel conductance for K+ compared to Ca2+ (2+ affinity. Calcium permeability of SV channels has also been observed by combining patch clamp and fura-2 fluorescence (2+ flux into the vacuole. When SV channels conduct K+ in one direction, a fractional Ca2+ current (+ flux using this approach (2+-induced Ca2+ release from the vacuole (SV/TPC1 channels have a permeability ratio for Ca to Ca2+ , consistrescence  but only current  through approach . A funct vacuole  is a hyp


The purpose of this work is to describe the use of Fibrin-Plasma Rich in Growth Factors (PRGF) membranes for the treatment of a rabbit alkali-burn lesion. For this purpose, an alkali-burn lesion was induced in 15 rabbits. A week later, clinical events were evaluated and rabbits were divided into five treatment groups: rabbits treated with medical treatment, with a fibrin-PRGF membrane cultured with autologous or heterologous rabbit Limbal Epithelial Progenitor Cells (LEPCs), with a fibrin-PRGF membrane in a Simple Limbal Epithelial Transplantation and with a fibrin-PRGF membrane without cultured LEPCs. After 40 days of follow-up, corneas were subjected to histochemical examination and immunostaining against corneal or conjunctival markers. Seven days after alkali-burn lesion, it was observed that rabbits showed opaque cornea, new blood vessels across the limbus penetrating the cornea and epithelial defects. At the end of the follow-up period, an improvement of the clinical parameters analyzed was observed in transplanted rabbits. However, only rabbits transplanted with cultured LEPCs were positive for corneal markers. Otherwise, rabbits in the other three groups showed positive staining against conjunctival markers. In conclusion, fibrin-PRGF membrane improved the chemically induced lesions. Nonetheless, only fibrin-PRGF membranes cultured with rabbit LEPCs were able to restore the corneal surface. Ocular chemical burns are one of the most devastating situations for the ophthalmologist, which require an immediate and intensive evaluation and treatment ,2. ChemiAcidic or alkaline agents usually cause ocular chemical burns. Alkaline substances are more damaging to the eye than acidic substances because they have the inherent ability to penetrate the ocular structures due to their hydrophilic and lipophilic properties ,9. The dSeveral medical treatments and different surgical procedures  have been used for the treatment of ocular alkali-burns ,14,15. TThe limbus is the transitional zone between the sclera and the cornea where epithelial corneal stem cells are located ,20,21,22LSCD has a multifactorial etiology with several primary or secondary causes, with severe chemical burns being a common cause of this disease ,20,25. THuman amniotic and fibrin membranes are commonly used in the latter two procedures as a substrate providing support to the transplanted cells or limbal explants, respectively ,42. AltePlasma Rich in Growth Factors (PRGF) is an autologous platelet-rich plasma that contains a greater number of growth factors than the human serum and other platelet-rich plasmas, which has great versatility, allowing the preparation of different therapeutic formulations including eye drops and fibrin clots . In ophtThis work aims to prove the efficacy of fibrin-PRGF fibrin membranes to treat an alkali-burn lesion in an animal model with different therapeutic approaches. For this purpose, we studied the transplant of in vitro expanded autologous and heterologous LEPCs on fibrin-PRGF membranes, the Simple Limbal Epithelial Transplantation (SLET) technique with a fibrin-PRGF membrane and the role of the fibrin-PRGF membrane without cultured cells as adjuvant therapy.After two days of culture, LEPCs began to migrate from the limbal explants to the culture plate. Cells were flat and round, forming a monolayer immediately adjacent to the explant. After five days, cultures were semiconfluent, and cells were trypsinized and subcultured onto rabbit fibrin-PRGF membranes (groups 2 and 3). These membranes were able to support the cellular growth of rabbit LEPCs while displaying their typical polyhedral morphology. Moreover, immunofluorescence analysis showed that LEPCs expressed a positive stain against the p63 marker .Seven days after the alkali-burn lesion, all rabbits showed opaque cornea, new blood vessels across the limbus penetrating the cornea, and epithelial defects shown by fluorescein stain .Results of clinical evaluation  showed tAll rabbits were evaluated once a week and at the end of the follow-up period .Results of the clinical evaluation  showed tIn contrast, rabbits in the other treatment groups improved the initial clinical diagnosis. Comparing the final and initial clinical evaluation, this improvement was different between groups. Rabbits transplanted with autologous or heterologous rabbit LEPCs (groups 2 and 3) and rabbits transplanted with fibrin-PRGF membrane SLET (group 4) showed a totally clear cornea (score 0), and only in two cases (in rabbit number two of groups 2 and 4) a mild haze (score 2) was observed. In the case of the rabbits transplanted with fibrin-PRGF membrane without rabbit cultured LEPCs (group 5), all the rabbits showed a haze of minimal density (score 1). Similarly, all transplanted rabbits showed no epithelial defects (score 0) or epithelial defects that affect less than 1/4 of the corneal surface (score 1).The major differences between groups were observed in the neovascularization and in the number of fibrovascular pannus. In the case of the neovascularization area, the rabbits transplanted with autologous (group 2) or heterologous (group 3) rabbit LEPCs showed an improvement, reducing the affected corneal area by half (from score 3 or 4 to score 1 or 2). In the case of rabbits transplanted with fibrin-PRGF membrane SLET (group 4) and rabbits transplanted with fibrin-PRGF membrane without rabbit cultured LEPCs (group 5), this improvement was not so evident, with one rabbit maintaining the same vascularization (score 3) and two rabbits decreased the vascularized corneal area (score 4 to score 2 or 3). Finally, the improvement in the number of fibrovascular pannus was again most evident in rabbits transplanted with cultured LEPCs (groups 2 and 3), showing a decrease from score 3 or 4 to score 0 or 1. The other groups, rabbits transplanted with fibrin-PRGF membrane SLET (group 4) and rabbits transplanted with fibrin-PRGF membrane without rabbit cultured LEPCs (group 5), also reduced the number of fibrovascular pannus, but to a lesser extent.One corneal edema was observed in rabbit number two transplanted with fibrin-PRGF membrane cultured with autologous LEPC (group 2).p < 0.05) were found in corneal neovascularization and fibrovascular pannus, between rabbits transplanted with fibrin-PRGF membrane cultured with autologous (group 2) or heterologous (group 3) rabbit LEPCs, and rabbits treated with only medical treatment (group 1).On the other hand, the percentage of change before and after treatment for each clinical variable measured was compared between the groups . StatistResults of the histological evaluation  showed tIn contrast, rabbits transplanted with autologous (group 2) or heterologous (group 3) rabbit LEPCs had a normal cornea with a multilayered peripheral and central epithelium with 3–5 layers of epithelial cells, with a slight inflammatory cell infiltration on the matrix and the limbal area showed a normal morphology without the presence of goblet cells. A small peripheral ulcer was observed in rabbit number three of both groups (data are not shown).However, in the groups of rabbits transplanted with fibrin-PRGF membrane SLET (group 4) and rabbits transplanted with only fibrin-PRGF membrane (group 5), the limbal area included goblet cells penetrating in the peripheral (both groups) and central cornea (rabbit number one of group 4). A small peripheral ulcer was observed in rabbit number three treated with fibrin-PRGF membrane without rabbit cultured LEPC (data are not shown). In the rest of the rabbits, the appearance of the central cornea was normal, in both groups.Immunofluorescence analysis  corroborMoreover, in the groups of rabbits transplanted with fibrin-PRGF membrane cultured with autologous (group 2) or heterologous (group 3) rabbit LEPCs and in the group of rabbits transplanted with fibrin-PRGF membrane without rabbit cultured LEPCs (group 5), CK3 was strongly positive in the intermediate and superficial layers of the central corneal epithelium . In the Ocular burns account for approximately 15% of eye events  and requThe majority of ocular burns do not require surgical interventions, and long-term morbidity rates have been reported as low as 4.5% with medical management alone ,58,59. HSurgical treatment of the ocular burns ranges from the application of several membranes to promote epithelization and to reduce inflammation, scarring and neovascularization, in patients with mild to moderate ocular injuries ,62,63, oIn this work, the efficacy of a fibrin-PRGF membrane for the surgical treatment of an alkali-burn lesion was studied in an animal model. Fibrin-PRGF membrane was used as a culture substrate because of its ability to reduce inflammation, vascularization and to stimulate the adhesion and proliferation of LEPCs ,50,73.The first step in the treatment of the ocular chemical injury was to irrigate the ocular surface to remove the NaOH. After that, medical treatment with antibiotics, analgesics and anti-inflammatories was administrated to prevent the infections and to reduce the inflammation during the acute phase (7 days). Several classification systems have been described to predict the prognosis of the lesion by grading the severity of the injury ,75,76. SClinical evaluation of the rabbits showed that all of them had moderately or severely dense corneal opacity, total corneal neovascularization, fibrovascular pannus and epithelial defects seven days after the chemical lesion. A clinical diagnostic with an unfavorable prognosis, since it depends on the extent of corneal, limbal and conjunctival involvement ,5,7,12. In this study, the fibrin-PRGF membrane was used with different therapeutic approaches: as a graft that provides a basement membrane for epithelization (group 5), as a membrane that supports the expansion of the autologous (group 2) or heterologous (group 3) rabbit LEPCs to maximize the probability of generating a normal corneal epithelial surface, and in the SLET technique, a simple surgical technique that avoids the consideration of advanced therapy (group 4). These treatments were possible since the fibrin-PRGF membrane has demonstrated to be easily manageable in different types of surgery ,49 and tRabbit LEPCs grown on fibrin-PRGF membranes were positive for the p63 marker, which has been used to determine the stem cell property of LEPCs  being beDuring post-surgery healing, corneal transparency gradually improved. Within 40 days after transplantation, ocular surfaces were utterly reconstructed with clarity and smoothness, under slit-lamp microscopy, compared to those of normal cornea in the group of rabbits treated with fibrin-PRGF membrane cultured with autologous (group 2) or heterologous (group 3) rabbit LEPCs and in the group of rabbits treated with fibrin-PRGF membrane without cultured LEPCs (group 5). In the groups of rabbits treated only with medical treatment (control group) and rabbits transplanted with fibrin-PRGF membrane SLET (group 4), epithelial defects were detected by slit-lamp examination. One rabbit in the group of fibrin-PRGF membrane cultured with autologous LEPCs showed a corneal edema, which could be explained by the absence of corneal endothelium in a cornea area (data are not shown).Following clinical observations, the cornea was covered by stratified epithelium displaying corneal phenotype (positive CK3), and the limbal epithelium resembled a normal appearance with limbal stem cells identified by p63 positive labeling in the groups of rabbits treated with fibrin-PRGF membrane cultured with autologous (group 2) or heterologous (group 3) rabbit LEPCs. In contrast, in the other three groups , goblet cells were observed in the limbal epithelium, indicating conjunctivalization into the limbal area. These results seem to indicate that the fibrin-PRGF membrane has a beneficial effect after alkali-burn injury, but it is not able to restore the correct function of a normal cornea by itself ,81.In accordance with other studies ,83, our Another therapeutic approach, such as the SLET technique, is an even more promising treatment option since it is an easy procedure that does not require in vitro cell culture techniques. Previous studies have shown satisfactory results using this technique ,40,84,85® by the European Medicines Agency (EMA), the first Advanced Therapy Medicinal Product (ATMP) containing limbal stem cells in a fibrin scaffold for the treatment of a moderate or severe chemical-induced LSCD [® therapy includes the use of feeder mouse cells and bovine serum, so it is contraindicated for patients with a hyper sensibility to xenogeneic proteins [In the last years, several in vivo studies and clinical trials have demonstrated that fibrin membranes are a suitable scaffold for the culture and transplant of LEPCs, leading in 2015 to the approval of Holoclarced LSCD . On the ced LSCD ,88. Howeproteins , and in proteins .In this study, the use of feeder mouse cells was avoided, and limbal explants were used for the expansion of rabbit LEPCs in a simple technique of cellular culture. Although a complete medium with SBF, cholera toxin, among other supplements were used for the expansion of rabbit LEPCs, which was the major limitation of this study, this fact could be modified in the translation of our results to the clinical practice, since human LEPCs have been satisfactorily cultured on a fibrin-PRGF membrane using PRGF as the unique supplement of the culture medium . Thus, aFifteen healthy male New Zealand white rabbits (2 months of age and body weight of 2.0–2.5 kg) obtained from the Animal Housing Facility of the University of Oviedo  were used in this work. All animals were treated in accordance with the ARVO Statement to use animals in ophthalmic and vision research and EU Directive 2010/63/EU for animal experiment. The protocols were approved (PROAE 33/2016) by the Committee on the Ethics of Animal Experiments of the University of Oviedo and the Animal Production and Health Service of Asturias. Rabbits were kept under a 12/12 day/night light cycle with food and water ad libitum and were monitored daily.An alkali-burn lesion was induced in all rabbits, and a week later, all rabbits were clinically examined and treated. Animals were randomly divided into five groups of three rabbits each: rabbits treated only with medical treatment (group 1: control group), rabbits treated with a fibrin-PRGF membrane cultured with autologous (group 2) or heterologous (group 3) rabbit LEPCs, rabbits treated with fibrin-PRGF membrane SLET (group 4) and rabbits treated with a fibrin-PRGF membrane without rabbit cultured LEPCs (group 5). Once a week, rabbits were evaluated , and after 40 days of clinical follow-up, all rabbits were euthanized, and excised corneas were subjected to histochemical examination.Small limbal biopsies (1 × 2 mm) were excised from the limbus of the rabbits’ right eye before the performance of alkali-burn lesion (groups 2 and 3). Limbal biopsies were stored in Leibovitz’s  supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin  until culture.v/v% fetal bovine serum (FBS) and 100 U/mL penicillin, 0.1 mg/mL streptomycin (Sigma-Aldrich). After five days of culture, rabbit LEPCs were trypsinized, counted in a hemocytometer and subcultured on a fibrin-PRGF membrane for 48 h (groups 2 and 3).For culture procedure, limbal biopsies were cut into small pieces and cultured for five days on a 12-well culture plate in a 2:1 mixture of Dulbecco’s modified Eagle’s medium  and Ham’s F12 (Thermo Fisher Scientific), supplemented with 5 µg/mL insulin, 8.33 ng/mL cholera toxin, 24 µg/mL adenine, 1.3 ng/mL triiodothyronine, 0.4 µg/mL hydrocortisone, 10 2 in a 35 mm diameter dish at 37 °C for 30 min. Once a gel was formed, fibrin-PRGF membranes were obtained by flattening for 30 s using a 500 µm fibrin membrane shaper .Rabbit fibrin-PRGF membranes were obtained from rabbit blood samples and were processed according to the methodology described by Anitua et al. . Briefly2, and the medium was changed every two days.All cultures were maintained in a humidified incubator at 37 °C with 5% COCellular growth was assessed using a Leica DMIL LED phase-contrast microscope , and photos were taken with an attached EC3 camera (Leica). Moreover, fibrin-PRGF membranes cultured with rabbit LEPCs were fixed in ice-cold methanol (Sigma-Aldrich) for 10 min for histological analysis.®  and Metacam® , and then were intubated and ventilated with isoflurane 2% . After topical administration of double anesthetic Colicursi® , the alkali-burn lesion was performed by applying a gauze soaked in 1 M NaOH  on the right eye  for 10 s. After that, eyes were rinsed with balanced salt solution (Alcon); the central epithelium was removed with eye spears and Tobrex®  and Cetraflux®  eye drops were administered before performing a tarsorrhaphy with 4–0 silk suture . Once a day, rabbits received subdermal injections of Metacam®, Bupaq® and Alsir®  until treatment.New Zealand white rabbits were treated with BupaqSeven days after the alkali-burn lesion, the ocular surface was photographed and evaluated by slit-lamp , and the extent of epithelial damage was assessed by fluorescein stain. Opacification, neovascularization, number of fibrovascular pannus and epithelial defects observed in the photographs were evaluated according to the criteria shown in ®  over a fibrin-PRGF membrane. Then, a second fibrin-PRGF membrane was sutured over the explants with 8–0 absorbable suture .Once the rabbits were evaluated, they were anesthetized according to the procedure described above, and the different treatments were carried out. For this purpose, intracorneal vessels were removed, and a fibrin-PRGF membrane was sutured with autologous (group 2), heterologous (group 3) or without rabbit cultured LEPCs (group 5) with cells facing the cornea. In the case of rabbits transplanted with fibrin-PRGF membrane SLET (group 4), a 2 mm limbal biopsy of the contralateral eye was taken, cut into 10 pieces, and glued with Tisseel®  and Dexafree®  eye drops and one subconjunctival injection of Celestone®  before performing a tarsorrhaphy with 4–0 silk suture. Once a day, rabbits received subdermal injections of Metacam®, Bupaq® and Alsir® 5% for seven days. Tarsorrhaphy was opened after seven days, and rabbits were treated with Vigamox®, and Dexafree® eye drops twice a day during the follow-up period. The rabbits in group 1 (control group) received only medical treatment.All rabbits received VigamoxRabbits’ eyes were monitored with slit-lamp examination, fluorescein staining and photography once a week during the 40 days of the follow-up period. Epithelial integrity, corneal neovascularization, fibrovascular pannus and corneal opacity were evaluated and scored by the same examiner in a masked fashion following the previously described criteria .After the follow-up period, rabbits were euthanized by an intravenous overdose of pentobarbital sodium  and corneas were excised, rinsed in PBS solution, and fixed in ice-cold methanol for 24 h for histological analysis. Fixed fibrin-PRGF membranes cultured with rabbit LEPCs and rabbit corneas were embedded in paraffin (Thermo Fisher Scientific), and 3 µm sections were cut with a microtome (Leica), placed on commercially treated slides (Thermo Fisher Scientific) and stained with hematoxylin-eosin (Sigma-Aldrich). Immunostaining of the corneal sections using antibody against cytokeratin 3 (CK 3)  was used to identify differentiated corneal epithelial cells and antibodies against MUC5ac  and cytokeratin 7 (CK 7)  were used to identify goblet cells and conjunctival epithelial cells, respectively. Moreover, the antibody against p63  was used to identify progenitor limbal stem cells in fibrin-PRGF membranes cultured with rabbit LEPCs and rabbit corneas. Briefly, samples were deparaffinized, hydrated and rinsed with PBS solution twice for 10 min and permeabilized in a PBS solution containing 0.3% Triton X-100  for another 10 min. Next, the samples were incubated with primary antibodies containing 10% normal goat serum  at 4 °C overnight. Subsequently, the samples were incubated with the corresponding secondary antibody  for 2 h at room temperature. Between incubations, samples were washed three times with PBS for 10 min. Immunolabeled cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI) to allow nuclei visualization. All the samples were examined in a Leica DM6000B fluorescence microscope (Leica), and photographs were taken in the central cornea, peripheral cornea and limbus, identified as the first area where stromal vascular vessels were observed.p < 0.05 was considered statistically significant.Statistical analysis was performed using IBM SPSS Statistics v.22 software . The mean, standard deviation and percentages were used for descriptive analysis. The Wilcoxon test was used to compare the clinical evaluations between the beginning and end of the follow-up period and the Kruskal–Wallis test to compare the results between the groups. A level of Fibrin-PRGF membranes improved chemically induced lesions in a rabbit alkali-burn model but only fibrin-PRGF membranes with autologous or heterologous cultured rabbit LEPCs were able to restore the corneal surface. SLET technique with a fibrin-PRGF membrane did not show satisfactory results in this study. Additional data should be collected with a larger sample size, at a longer follow-up and with a different experimental design.


Here, dynamic confocal imaging on intact skeletal muscle cells isolated enzymatically from the adult honey bee legs allowed the first spatio-temporal characterization of subcellular calcium release events (CREs) in an insect species. The frequency of CREs, measured in x–y time lapse series, was higher than frequencies usually described in vertebrates. Honey bee CREs had a larger spatial spread at half maximum than their vertebrate counterparts and a slightly ellipsoidal shape, two characteristics that may be related to ultrastructural features specific to invertebrate cells. In line-scan experiments, the histogram of CREs’ duration followed a bimodal distribution, supporting the existence of both sparks and embers. Unlike in vertebrates, embers and sparks had similar amplitudes, a difference that could be related to genomic differences and/or excitation–contraction coupling specificities in honey bee skeletal muscle fibres. The first characterization of CREs from an arthropod which shows strong genomic, ultrastructural and physiological differences with vertebrates may help in improving the research field of sparkology and more generally the knowledge in invertebrates cell Ca Moreover, the high levels of mortality in honey bee’s populations observed worldwide in the last few years prompted researchers to further study this model with the hope of understanding origins of this decline. Honeybee cells provide valuable models to study calcium signalling and intracellular calcium homeostasis in insects11. Calcium is an essential intracellular second messenger which plays pivotal roles in neuromuscular cellular processes such as signal transduction and modulation in motor or sensory neurons, contraction in cardiac, smooth, and skeletal muscle cells. More generally, Ca2+ plays a major role in development/maturation, cell shape modulation and migration, cell division, cell-membrane repair, hormonal secretion, and gene expression in virtually all cell types. In the honey bee, macroscopic intracellular Ca2+ variations have been recorded with Ca2+-imaging techniques in neurons from central brain structures such as mushroom bodies4 or antennal lobes8. Recently, Ca2+ was also monitored in peripheral sensory neurons from the antennae, i.e. the ‘nose’ of honeybees10. Global Ca2+ fluctuations were also studied in photoreceptor cells from the honey bee compound eye11. Finally, cytoplasmic Ca2+ transients (i.e. transient Ca2+ increases that can be elicited by a stimulation) and excitation–contraction coupling have been described in bee skeletal muscle cells as well, using fluorescent dyes and electrophysiological techniques6. Recently, studying intracellular Ca2+ signalling in bees and pollinators gained renewed interest because new classes of insecticides targeting several molecular players involved in Ca2+ homeostasis have been launched on the pesticides market and are now widely used in agriculture. Notably, most recent diamide type insecticides such as flubendiamide and chlorantraniliprole induce detrimental effects in honey bees by anarchically mobilizing Ca2+ from intracellular stores through Ca2+ release channels in neurons from the antenna and in skeletal muscle cells from the leg10.Bees are very efficient pollinator insects, and are thus crucial both for plant biodiversity maintenance and agricultural food production. Amongst bees, owing to their easy domestication, honey bees have long been used as a research model in various fields, ranging from cell physiology to inter-individual communication and sociability2+ signalling is undoubtedly the ryanodine receptor. The molecular identity of this receptor was unravelled thanks to the botanical alkaloid ryanodine, present in root extracts of the plant species Ryana, which was used in the 50’s as a natural insecticide (now withdrawn owing its’s toxicity towards environment). Ryanodine receptors are huge macromolecular complexes functioning as ion channels to provide a route for Ca2+ to be massively transferred on demand from intracellular storage organelles (sarco and endoplasmic reticulums) to the cytoplasmic compartment13. Increasing the intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) from submicromolar to tenths of micromolar ranges thus allows Ca2+ to play its multiple regulatory roles15. At rest, [Ca2+]i is tightly regulated and maintained at around 100 nM to avoid the toxicity of sustained Ca2+ increases and this low resting [Ca2+]i provides both a wide dynamic range and a high signal-to-noise ratio. Optical and electrophysiological techniques evolved and allowed characterizing cytoplasmic Ca2+ fluctuations with continuously increased acuity along with recent years. Nowadays, laser-scanning confocal microscopes allow detecting optically subcellular cytoplasmic Ca2+ fluctuations in the µm spatial and in the ms temporal ranges with the help of fast and high-affinity Ca2+ dyes (Ca2+ indicators). These localized fluctuations are believed to originate from the release of Ca2+ by one or a cluster of several ryanodine receptors and in striated muscles, they are considered as the unitary bricks of voltage-induced global Ca2+ release16. Localized elevation in [Ca2+]i can also fuse to initiate propagating macroscopic Ca2+ waves and have specific cytosolic localizations such as the perinuclear space where they can modulate nuclear Ca2+ independently of the bulk cytosolic Ca2+17. So far, local fluctuations in [Ca2+]i have been studied only in muscle cells from vertebrate species. First discovered in cardiac myocytes18, and originally termed Ca2+ sparks, similar signalling events were identified later in smooth muscle cells, skeletal muscle fibres, neurons and oocytes. The role and regulation of mechanisms of these spatially localized fluctuations are however elusive and remain to be fully identified with a rather unique research field dedicated to their study: ‘sparkology’17. This research field is essential as these signals are frequently altered in genetic diseases linked to mutations in the ryanodine receptor genes (ryanodinopathies). Even under physiological conditions, it soon turned out that localized Ca2+ fluctuations had many temporal patterns, not only an archetypal steep increase (~ 5 ms) and a fast mono-exponential decrease, and new terms emerged to better describe this signals diversity e.g. embers, macrosparks, compound sparks, bursts, sparklets…19. A more general term is now used to depict these [Ca2+]i fluctuations : elementary Ca2+ release events (CREs).A major molecular player in Ca20.While CREs have been identified in many tissues from several vertebrate species , to our knowledge, they have never been identified in an invertebrate tissue. Here, using honey bee as a model, we describe for the first time elementary CREs in an insect species and compare their spatiotemporal characteristics in the light of interspecific physiological and ultrastructural cellular differences. Part of this work was presented to the Biophysical SocietyApis mellifera), as described earlier5, with slight modifications. Newborn bees were collected outdoor, directly on a frame of a hive and used during the following 4 days. Bees were chilled (~ 4 °C) until lethargic immobility and then killed by decapitation. Tibias from the third pair of legs were dissected in a Tyrode’s solution without calcium , cut longitudinally and pinned open. Tibias were bathed 20 min in a dissociation solution at 37 °C . After rinsed in Tyrode’s solution without calcium and normal Tyrode (2 mM CaCl2), muscle masses were triturated with a pipette and dispersed in the central well of a clean glass bottom dish . Cell were loaded for 30 min with the cell permeant dye Fluo-8-AM (5 µM).Muscle fibres were prepared from young adult domestic honey bees (XYT) images, captured at 1 Hz. The pixel size was 0.328 µm and the pixel dwell time was 1 ms. CRE detection and analysis were performed using methods and algorithms previously described22. In brief, the region of the fibre and the background was defined on the first image of each series. The averaged background fluorescence was subtracted from each pixel of all images in one series. Ca2+ release events were detected by the stationary wavelet method. The filtering was made by soft thresholding wavelet detection. Finally the amplitude and full width at half maximum (FWHM) of the sparks were calculated. Two FWHM values were determined: perpendicular to (X) and parallel (Y) with the Z-lines. To detect the Z-lines the frequency spectrum in each line of all images was calculated using fast Fourier transform. To remove the Z-lines from the images, inverse FFT of the frequency components corresponding to sarcomeres was used. Bleaching correction was performed on all images by calculating the change in fluorescence intensity of each pixel in time. Finally images were normalized using stationary wavelet transform. The event frequency was determined by counting the number of events on all images from a series. Events that appeared on consecutive images at the same location were counted as a single event. To get the frequency the number of events was normalized to fibre area and duration of recording. To assess the amount of Ca2+ released during a CRE, the Signal Mass (SM) was calculated as described earlier23. To estimate FWHM in the z direction  in two-dimensional measurements, the average of the two FWHMs in the focal plane (x and y directions) was used. Figure Fluo-8-AM loaded cells were kept in normal Tyrode’s solution and observed with a laser scanning confocal microscope Zeiss LSM 5 LIVE  equipped with a Plan-Neofluar 40 × oil immersion objective NA = 1.3). Fluo-8 was excited with the 488 nm wavelength of an argon laser and the emitted fluorescent light was measured through a band-pass filter (505–570 nm) and digitized at 12 bit. First the fibres were examined by eye and the fibres showing spontaneous activity were recorded with series of 512 × 512 (.3. Fluo-XT) were taken at 1 or 2 ms/line and the pixel size was 0.328 µm. In some cases 0.2 ms/line was used to study the temporal properties of CREs in high time resolution. The length of the line was variable and depended on the actual fibre width. The images  were normalized to baseline fluorescence (F0[x]). Line-scan images containing spontaneous calcium release events were analysed using an automatic event detection program21, which calculated the amplitude (ΔF/F0), rise time, full width at half maximum (FWHM), and full duration at half maximum (FDHM) of the identified events. An event was identified as an ember if it had a clear plateau and was longer than 40 ms in time. Otherwise, it was considered as a spark. Events with an amplitude smaller than 0.1 ΔF/F0 units were excluded from the analysis. All measurements were carried out at room temperature (22–23 °C).Line-scan images (2+ transients were elicited on CRE free (silent) intact fibres with field stimulation and recorded in line-scan mode. Two platinum electrodes were placed adjacent to the fibres and 50 ms long depolarizations with amplitude ranging between 40 and 80 V were used to evoke Ca2+ transients . Whole-cell Ca2+ transients were recorded in patch-clamp experiment as described earlier5.CaFibres were stained with Hoechst 33258 (5 μg/ml) in Tyrode during 30 min. After being loaded, fibres were rinsed twice in Tyrode’s solution and nuclei were visualized under a Zeiss LSM710 confocal inverted microscope with a 63 × 1.42 NA objective .Single muscle fibres were superfused with normal Tyrode’s at room temperature (24 °C). Membrane potentials were recorded using 3 M KCl filled sharp glass microelectrodes with tip resistances ranging between 30 and 40 MΩ only in cells with intact surface membrane and clear cross striations. The electrodes were connected to the input of a Multiclamp-700A amplifier  under current-clamp conditions. Membrane potentials were digitized at 100 kHz using Digidata 1322 A/D card (Axon Instruments) under software control  and stored for later analysis.Pooled data were expressed as mean ± standard error (SE) of the mean. The differences were assessed using one-way analysis of variance (ANOVA) and all pairwise multiple comparison procedures (Student–Newman–Keuls method). A p-value of less than 0.05 was considered statistically significant. Graphpad Prism 6.0 software  was used for statistical analysis and non-linear regression.5 instead of right underneath the sarcolemma as in other adult organisms like amphibians and mammals , but some higher amplitude events (up to 0.6 ΔF/F0) were observed as well . The CREs were wide in space since both FWHMs (X and Y) were greater than 3 µm in average . The probability histograms of FWHMs . The maximum of ember amplitude was 0.5 ΔF/F0 . Secondly, insect myofibrils are radially oriented around a central row of nuclei, (an arrangement which is not seen in adult vertebrate muscle fibres), giving a so-called ‘tubular’ morphology to the fibres. Accordingly, insect tubular muscle fibres have relatively straight t-tubules arranged in radial symmetry from the surface membrane to central nuclei in order to convey action potentials throughout the fibre volume34. This spatial configuration may lead to less restricted diffusion along long portions of straight T-tubules. The average ratio of FWHMs (X/Y) honey bee CREs was greater than 1 (1.23 ± 0.01), which indicates that ellipsoidal sparks diffuse slightly more freely in the X direction  than in the Y direction . Elongated sparks were detected in frog skeletal muscle as well35 and CREs non-circularity has been previously attributed to a ‘channelled diffusion’ due to tubes of mitochondria17, but this hypothesis remains to be explored in bee fibres. Thirdly, an interesting feature is that bee T-tubules are running at the junction of A and I bands, like in mammalian skeletal muscle fibres, so that two T-tubules run per sarcomere5, increasing the chance to recruit release sites for large CREs. Fourthly, electron microscopy and rotary shadowing techniques showed that RyR disposition in invertebrate muscle differ from their disposition in vertebrates36, an arrangement that may favour wider CREs in honey bees. Finally, ultrastructural differences were also identified in the invertebrate couplons, i.e. the functional arrangement of RyRs and voltage-gated calcium channels from the plasma membrane , and previous studies underlined that this architecture was reminiscent of the cardiac striated muscle37. Accordingly, other ultrastructural similarities can be pointed out in between bee skeletal and vertebrate cardiac muscles, such as the presence of dyads in both tissues instead of the triads observed in vertebrate skeletal muscles5. Besides structural and ultrastructural differences, that clearly settle the conditions for broad CREs in insect muscle fibres, genomic differences could favour it as well. Homology data on RyR sequences suggest that invertebrates and vertebrates show important genomic differences. First, insects have only one ryr gene whereas amphibians have two  and mammalians have three39. Two isoforms are actually expressed in vertebrate skeletal muscles , since the ryr 2 gene is expressed in cardiac mammalian muscle only. After the ‘honey bee genome sequencing consortium’ made available the honey bee (Apis mellifera) genome sequence40, the predicted amino acid sequence for the Apis mellifera RyR protein (AmRyR) has been obtained from an annotated genomic sequence NC_037639.1 (Gene 408680 on chromosome LG2) using traditional gene prediction methods . Like in other species, predicted AmRyR protein isoforms (X1 to X9) contain ~ 5000 amino acids. According to BLAST tools (NCBI database), the homology degree of the honey bee AmRyR with vertebrate RyRs is around 45%. Such a difference may well settle the basis for strong differences into the protein functions and regulations leading to morphological differences in bee CREs as compared to other species. In zebrafish skeletal muscle cells, two RyR isoforms have been shown to be essential to achieve normal sparks production41. The second frog RyR isoform may also be related to high spark production in this species42. In foetal mammalian muscles, the two isoforms of RyR (1 and 3) are expressed and it was suggested that they both contribute to generate sparks43. Conversely, at the mature adult stage, most intact mammalian skeletal fibres (which no longer express RyR3) require specific conditions to fire sparks and at a relatively low frequency. Our data show that a single ryr gene allows the production of a high CREs frequency with wide spatial spread in intact insect fibres.A major difference lies in the spatial spreading of CREs from adult bees, which are clearly wider than CREs from adult intact vertebrate muscle fibres  on a calcium induced calcium release (CICR) mechanism44. CICR is thus a crucial mechanism in the skeletal muscle from bees and other insects (which are devoid of TTX-sensitive voltage-gated channels) where voltage-gated calcium currents activate quickly to achieve a rapid action potential overshoot46. The role of CICR in excitation–contraction coupling implies that honey bee RyRs must be highly sensitive to Ca2+, a feature that favours broad CREs. CICR has been shown in frog muscle fibres as well  but it plays a null or minor role in rat fibres47. Despite a demonstrated CICR in frog, CREs from frog fibres are narrower than CREs from mammalian fibres, both in intact cells  current has been identified in addition to the nifedipine-sensitive high-voltage activated (HVA) current49. Both HVA- and LVA- calcium currents exists in mammalian fibres as well, but the density of the later gradually decreases postnatally to eventually disappear after 2–3 weeks of age50. Whereas DHPRs responsible for the HVA- (L-type) current are involved in triggering and/or repressing sparks in immature and adult mammalian muscle fibres32, pharmacological proofs indicate that the LVA- (T-type) calcium channel doesn’t modulate sparks frequency32. A role for the LVA- current in the genesis or regulation of honey bee CREs thus remains to be studied.Functional differences could also lead to the specific shape properties of bees CREs. In the honeybee muscle, as in other invertebrates, excitation–contraction coupling relies on i) Cals Table  or in peXYT experiments appeared smaller than in almost all other intact preparations , a similar analysis strategy  led to the detection of CREs up to 1.3 ΔF/F0, with a half cumulative frequency value (CF50) of 0.5351. The apparently low amplitude of honey bee sparks as compared to canonical sparks may thus be more related to species specificities and/or experimental conditions than to the detection method we used. Another striking difference between bees and vertebrates is that in bees, embers had almost the same amplitude as sparks, whereas embers are usually smaller than sparks in mammals, as shown in non-intact preparations31. This finding implies the idea that in bee muscle during a CRE, the same number of RyRs open and only the duration of their opening vary stochastically. The amplitude of sparks from honey bee is ~ eightfold smaller than the amplitude of voltage-gated global Ca2+ transients . In intact mouse primary myotubes and in the C2C12 mouse cell line, sparks are 1.4 and ~ fivefold smaller than macroscopic transients, respectively53. To the best of our knowledge, no study relates both sparks amplitude and macroscopic transients in intact non voltage-clamped mouse fibres. A ratio of ~ 6 can however be calculated from macroscopic Ca2+ transients recorded under similar conditions as those depicted in Table 54.Amplitude of honey bee sparks measured in ns Table , 3. This55 and the value 40 ms was retained as the threshold for ember detection. Importantly, the amplitude could not be used as the second separator parameter since CREs in bee fibres are more homogeneous in amplitude than in mammalian muscles where the amplitude of embers is much smaller than that of the sparks.Honey bee sparks had similar kinetics properties as other species. Most features of honey bee embers were reminiscent to other species ones as well. Previous studies classified the embers using their durations Table . Althoug43. A comparison of sparks frequency collected in XT line-scan experiments in intact cells shows that in honey bee, frequency is four fold to 600 fold higher than in any other mature or immature cells from frog, mouse, rat or zebrafish (Table XT). In XYT images series from adult bees’ fibres, the frequency is one or two orders of magnitude higher as compared to intact cells from adult or immature mammalian cells . Interestingly, the XYT frequency observed in bees is almost identical to what was reported in intact adult frog muscle recently22. Again, these differences in CREs frequency among species may thus be a consequence of the relative incidence that CICR has in excitation–contraction coupling or to ultrastructural differences discussed before. It is important to point out that frequency similarities between bees and frogs are not only related to the analysis strategy, because a similar procedure used in mouse fibres led to a 100 fold lower frequency51. Finally, the proportion of embers as compared to the total number of CREs in XT experiments shows that honey bee embers are much more frequent than in adult or foetal mouse cells (33% in honey bee vs 4% in mouse) and either higher or identical in myotubes from rat and mouse respectively . CREs were seen in < 20% of adult honey bee intact fibres. By comparison, the incidence of spontaneous CREs was less than 5% of intact adult mouse fibres whereas it was more elevated in embryonic fibresrep-mode, has been identified when voltage-clamped vertebrate fibres (cut fibres mounted in a Vaseline gap chamber) were stimulated by a low-amplitude depolarization56. This activity mode can also happen in chronically depolarized frog fibre right after a brief repolarization protocol. Such an activity was seen in intact adult mouse fibres as well43 and in embryonic and postnatal mouse fibres, where 29% of all events location had reoccurring Ca2+ release events32. A pharmacological manipulation with caffeine is also increasing the appearance of the repetitive CREs in intact frog fibres22. However, whereas CREs in rep-mode were short in duration in frog fibres, bee hot spots led to CREs with various kinetics ranging from sparks to burst and embers.In honey bee fibres, CREs appear most of the time in a stochastic manner and rarely at a given triad. However, a repetitive mode of activation was observed in specific hot spots. This second mode, previously termed 2+ signals have not been described yet. We have overcome the difficulty of isolating single fibres from insects (firmly attached to the insect exoskeleton through tonofilaments), and this methodological advancement will allow an in-depth characterization of sparks in insects in general. Our methods could in particular be transposed to common insect models used either in fundamental research  or models of interest in agronomy . This study would be of particular interest to characterize the interspecific difference in susceptibility to new insecticides targeting RyRs, the phthalic acid diamides and anthranilic diamides. Diamides are highly efficient to control a variety of pests. They are widely used to kill a number of insect species, including lepidopterans (moths), hemipterans (aphids), dipterans (flies) and so on. Their major mode of action is to interfere with normal function of ryanodine receptors. They have been designed to ensure a favourable vertebrate selectivity ratio, that is to say: being more toxic to invertebrates than to vertebrates. However, insects exposed to these compounds, whether they are considered either as pests or beneficial insects, show strong symptoms of intoxication . For instance, we have shown that sublethal doses of chlorantraniliprole were highly deleterious to honey bees that encounter long lasting locomotor deficits lasting for several days, leading to high susceptibility to other threats9. Higher doses kill the bees quickly. Insect ryanodine receptors are quite divergent from those of vertebrates (~ 45% a.a. identity), but RyRs from insect species from all orders show a strong molecular proximity (~ 80%). The honeybee RyR (XP_006569098.1) shows 83, 80, 79 and 78% amino acid identity with RyRs from the beetle Tribolium castaneum (NP_001308588.1), the moth Plutella xylostella (XP_037970554.1), the aphid Myzus Persicae (XP_022160123.1) and the fly Drosophila melanogaster (NP_476991.1), respectively. Although fruit flies D. melanogaster are considered as models, the intracellular homeostasis in insect tubular muscle fibres has so far not been studied thoroughly owing to the difficulty of isolating muscle fibres from the cuticle, which is a prerequisite for calcium imaging. The honey bee fibres provide a suitable model in this context and the dissociation procedure we developed here to study sparks will be helpful in many instances.In addition to provide the first interspecific comparison of sparks between vertebrates and invertebrates, the present study sets the basis for a future comparison of sparks in other invertebrates, where these subcellular localized Ca2+-release events of the honey bee, a detailed comparison of their major specificities as compared with their vertebrate counterparts has been carried out. The successful isolation of insect muscle fibres and the thorough characterization of subcellular Ca2+ signalling in the honey bee muscle paves the way for further in vitro studies, which are clearly needed to elucidate the origins of the established differential toxicity of the RyR-targeting diamides insecticides between vertebrates and invertebrates. Furthermore, while a so-called ‘vertebrate selectivity toxicological ratio’ already exists for diamides, no protective selectivity ratio has been established yet for useful insects. Pest insects and non-pest insects share a high degree of homology in their RyR and a comparative study of Ca2+ sparks among insects may help to better characterize the toxicity of diamides, which constitute a threat to useful insects such as pollinators.In addition to the characterization of localized CaSupplementary file 1.Supplementary Video 1.


SPAG family members and their clinical significance in acute myeloid leukemia (AML).Recently, an increasing number of studies have reported that sperm-associated antigen (SPAG) proteins play crucial roles in solid tumorigenesis, and may serve as potentially helpful biomarkers for cancer diagnosis and prognosis. However, very few studies systematically investigated the expression of SPAGs and their prognostic significance in AML were determined by a systematic analysis on data gathered from public databases, and the results were validated in clinical samples.The expression of SPAG1 expression negatively associated with survival in AML by Cox regression (P < 0.001) and Kaplan–Meier analysis (P < 0.001). The prognostic value of SPAG1 expression was further confirmed in other independent cohorts. Clinically, higher SPAG1 expression was significantly correlated with white blood cell counts (P = 0.014) and French–American–British (FAB) subtypes (P = 0.024). Moreover, higher SPAG1 expression was more common in + 8 patients (P = 0.034), rarely found with t (P = 0.014), and correlated with FLT3 (P < 0.001) and DNMT3A mutations (P = 0.001). Despite these associations, multivariate analysis confirmed the independent prognostic value of SPAG1 expression in AML (P < 0.001). Notably, AML patients with higher SPAG1 expression may benefit from hematopoietic stem cell transplantation (HSCT), whereas patients with lower SPAG1 expression appeared less likely to benefit. Finally, we further validated that SPAG1 expression was significantly increased in newly diagnosed AML patients compared with normal controls (P < 0.001) and with AML patients who achieved complete remission (P < 0.001). Additionally, SPAG1 expression could act as a potentially helpful biomarker for the diagnosis and prognosis of AML .Using public data, we identified only increased SPAG1 overexpression may serve as an independent prognostic biomarker and may guide the choice between HSCT and chemotherapy in patients with AML.Our findings demonstrated that The online version contains supplementary material available at 10.1186/s12920-022-01193-0. FLT3, CEBPA, NPM1, TP53, RUNX1 and ASXL1, have been well established to occur in AML, the current understanding of the molecular mechanisms involved in the development and progression of AML is still limited [Acute myeloid leukemia (AML) represents a cohort of clonal hematopoietic malignances that originate from myeloid precursors and is a highly heterogeneous disease in terms of molecular, cytogenetic and clinical features . Genetic limited . TherefoSPAG), of which 15 members  are CT antigens, has been identified [Evidence has proven that cancer–testis (CT) antigens may function in stemness due to their expression during germ cell and embryonic development, which promotes an important oncogenicity effect in cancer cells . To dateSPAG1 mRNA expression, among SPAG family members, is negatively associated with survival in AML. Moreover, the prognostic value of SPAG1 overexpression in AML was further confirmed by our data. High expression of SPAG1 mRNA was intrinsically connected to specific genetic (both cellular and molecular levels) abnormalities in AML. Despite these associations, SPAG1 overexpression could also function independently as a prognostic biomarker in AML, and it may serve as a reference for consolidation therapy selection between chemotherapy and hematopoietic stem cell transplantation (HSCT).To date, our research is the first to report that SPAG family members  from The Cancer Genome Atlas (TCGA) [SPAG1 in AML compared with controls was analyzed in GEPIA (http://gepia.cancer-pku.cn/).The identification cohort comprised 173 AML patients with RNA-Seq V2 data for SPAG1 expression in AML. Moreover, the association of the SPAG1 expression level with the prognosis of SPAG1 expression level on prognosis of 78 and 162 cytogenetically normal AML (CN-AML) patients was analyzed in the GSE12417 dataset with the public platform GenomicScape  [SPAG1 expression in two groups with median level of SPAG1 expression as the cutoff.Three independent cohorts from the Gene Expression Omnibus (GEO) database  were used to validate the prognostic value of The validation cohort included 131 AML patients, with 86 enrolled at diagnosis and 45 at complete remission (CR), treated at our hospital. Patients with antecedent hematological diseases or therapy-related AML were eliminated. The clinical characteristics of the cases are presented in Additional file Clinical bone marrow (BM) specimens were sampled from the validation cohort of AML cases and controls who were treated in our hospital. We separated BM mononuclear cells (BMMNCs) and then extracted total RNA by using Lymphocyte Separation Medium  and TRIzol reagent , respectively. cDNA was synthesized via RNA reverse transcription as described previously –13.SPAG1 and ABL1 (housekeeping gene) transcripts were unfolded by RT–qPCR via AceQ qPCR SYBR Green Master Mix . The primer sequences were 5′-TCTTCTGCGTCGTGCTAC-3′ (forward) and 5′-TTATCTCCACCGCCATCT-3′ (reverse) for SPAG1 as well as 5′-TCCTCCAGCTGTTATCTGGAAGA-3′ (forward) and 5′-TCCAACGAGCGGCTTCAC-3′ (reverse) for ABL1. The relative SPAG1 transcript level was calculated based on the 2−∆∆Ct method [Quantized data of t method –13.P < 0.05. All analyses were controlled for FDR by the Benjamini–Hochberg procedure. Gene Set Enrichment Analysis (GSEA) software was used for analysis, and the enrichment pathway was set to be significant based on the nominal (NOM) P < 0.05 and FDR Q < 0.05.All procedures referring to bioinformatics analysis were conducted as our previous reports , 15. To 2/Fisher’s exact test, respectively. Both the Kaplan–Meier method (log-rank test) and Cox regression were used to analyze the intrinsic connection between SPAG1 expression and survival time, including leukemia-free survival (LFS), event-free survival (EFS) and overall survival (OS). The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to determine the discriminating ability of SPAG1 expression for AML and controls. Two-sided P values < 0.05 in all statistical analyses were considered statistically significant.Comparisons of continuous and categorical variables were performed using the Mann–Whitney’s U/Kruskal–Wallis test followed by Dunn’s post–hoc test and Pearson’s χSPAG family members (SPAG1/2/3/4/5/6/7/9/10/13/16/17) in AML, we first determined the impact of each SPAG member on survival time (both OS and LFS) by Cox regression univariate analysis among AML in TCGA datasets. When analyzing the prognostic value of the SPAG family members in AML patients, each one was evaluated according to the difference between two groups of patients, divided by the median level of SPAG expression as the cutoff. As presented in Table SPAG1 expression had a significant connection with OS and LFS in both AML (both P < 0.001) and non-M3 AML (both P < 0.001) as well as CN-AML . Furthermore, Kaplan–Meier analysis also revealed that AML patients with higher SPAG1 expression showed significantly shorter OS and LFS than those with lower SPAG1 expression among AML (both P < 0.001), non-M3 AML (both P < 0.001), and CN-AML (both P = 0.004) patients  also confirmed the prognostic correlation of SPAG1 expression with OS in patients with CN-AML among two independent cohorts  counts, peripheral blood (PB)/BM blasts, FAB classifications, cytogenetics, and gene mutations are shown in Table SPAG1 expression had markedly higher WBC counts than did those with lower SPAG1 expression  and rarely t  or neither , non-M3 AML , or CN-AML patients    and Burkett lymphoma (BL) [Recent evidence has characterized e cancer , 18. SPAarcinoma , 20. SPAarcinoma –23. Moreferation . Interesg cancer . A direcarcinoma –32. Notaoma (BL) –37. ConsSPAG1 expression as uniquely associated with poor prognosis in AML among all SPAG family members through both analysis of public data and validation in a research cohort. It was demonstrated that SPAG1 expression could be a promising prognostic biomarker and could be used to optimize the choice of therapy between chemotherapy and HSCT in AML. Unlike SPAG6, SPAG1 expression has rarely been studied in hematological malignances. Conversely, several reports have examined at the relationships between SPAG1 and solid tumors. Shamsara et al. demonstrated that the amplification of SPAG1 was associated with decreased survival in SPAG1 is an early expressed gene in pancreatic tumorigenesis and can promote the activity of cancer cells [SPAG1 expression was a crucial variable related to many clinicopathological features and to RFS in breast cancer [SPAG1 acts as an inhibitor of breast cancer cell proliferation and colony formation during breast cancer pathogenesis and development [SPAG1 in AML, further mechanistic studies are essential for investigating the possible role of SPAG1 in leukemogenesis and AML development.This study was the first to reveal er cells . Lin et t cancer . Functioelopment . Since tSPAG1 expression and genetic (both cytogenetic and molecular) abnormalities in AML. We first found the associations of SPAG1 expression with FAB-M4/M5 disease, suggesting that SPAG1 expression may play a role in monocyte differentiation and monocyte leukemogenesis. In terms of cytogenetics, SPAG1 expression was positively correlated with +8 but negatively associated with t . Since the SPAG1 gene is located in 8q22.2, it is not surprising that aberrant SPAG1 expression was associated with these chromosome abnormalities. Notably, further studies are needed to determine whether the functions of these chromosomal abnormalities during leukemogenesis occur through aberrant SPAG1 expression. Regarding gene mutations, SPAG1 expression was associated with FLT3 and DNMT3A mutations in AML, but the exact relationship between SPAG1 expression and these gene mutations still unclear. Importantly, there is no evidence showing the association of FLT3 and DNMT3A mutations with the above chromosome abnormalities. Consequently, we need to obtain deeper insight into the underlying mechanism of SPAG1 expression in leukemogenesis caused by FLT3 and/or DNMT3A mutations.The current study also identified a significant association between SPAG1 hyperexpression may function independently as a prognostic biomarker and assist treatment selection between HSCT and chemotherapy in AML.In general, our discoveries suggested that Additional file 1: Clinic-pathologic characteristics of AML patients in our research cohort and Cox regression univariate and multivariate analysis of variables for leukemia free survival in AML patients in TCGA dataset.Additional file 2: Different expressed genes correlated with SPAG1 expression.


Together, our experimental and modeling approaches have further defined the relative contributions of X-ROS signaling and the length-dependent calcium buffering by troponin in shaping the myoplasmic calcium transient.The stretching of a cardiomyocyte leads to the increased production of reactive oxygen species that increases ryanodine receptor open probability through a process termed X-ROS signaling. The stretching of the myocyte also increases the calcium affinity of myofilament Troponin C, which increases its calcium buffering capacity. Here, an integrative experimental and modeling study is pursued to explain the interplay of length-dependent changes in calcium buffering by troponin and stretch-activated X-ROS calcium signaling. Using this combination, we show that the troponin C-dependent increase in myoplasmic calcium buffering during myocyte stretching largely offsets the X-ROS-dependent increase in calcium release from the sarcoplasmic reticulum. The combination of modeling and experiment are further informed by the elimination of length-dependent changes to troponin C calcium binding in the presence of blebbistatin. Here, the model suggests that it is the X-ROS signaling-dependent Ca Stretching increases the affinity of troponin for Ca2+, increasing Ca2+ buffering and force generation, i.e., buffering by troponin promotes the contraction. On the other hand, X-ROS signaling provides an increase in Ca2+ mobilization during stretching that contributes to increases in the measured [Ca2+]i level and hence may contribute to contraction. It does this by offsetting a possible reduction in measured [Ca2+]i due to the buffering of Ca2+ by troponin.The combination of experiments and modeling suggest that stretching offers two antagonistic effects on the level of measured [Ca2+ binding of sites on troponin and (2) stretch-dependent changes in Ca2+ affinity for both the low- and high-affinity Ca2+ binding sites on troponin.We previously developed a model for excitation–contraction coupling in the rat ventricular myocyte that included a novel 4-state formulation for the RyR2 that simulates the increase in open probability of an RyR2 channel due to its oxidation by stretch-induced ROS  5]. To . To 5]. 2+ or Ca2+ most of the time. Therefore, a more realistic model for binding of Ca2+ to troponin buffer, described by Jafri et al. i transient was evaluated with Fluo-4 by 15 min incubation with 2 mM Fluo-4-AM ester and 0.01% Pluronic F127. Cells were allowed an additional 10 min for de-esterification and then scanned using a 488 nm argon ion laser in confocal line-scan mode at 1.92 ms per line. Cells were paced to steady state using 0.5 Hz field stimulation through platinum electrodes. After 20 s of 1 Hz stimulation, all cells showed steady state transients and contractions. At this point, five calcium transients before, during and after the release of stretching were acquired. This protocol was repeated twice in each myocyte, and data were pooled for analysis.Experiments were performed in custom fabricated cell chambers  mounted on a Zeiss LSM 510 inverted confocal microscope with a 40 Oil 1.2 NA objective. The i)  B as we p[Ca2+]i) C occurre2+ affinity driven by a 10% increase in length [2+ spark frequency increases abruptly by ~1.7-fold which closely recapitulates our experimental data [2+ sensitivity which predisposes a sudden decrease in [Ca2+]i, the simultaneous increase in X-ROS elicits Ca2+ sparks, which offsets the effect  X-ROS signaling that increases SR Caon, 2+]i B, and (22+ concentration (ΔF/F0) was quantified before, during, and after, stretching to one-half of the cardiomyocyte. Transients within the 10 s duration of each period were averaged and normalized to the period before stretching , or colchicine  gave a better least squares error than a linear fit for both the short and long sarcomere lengths, the result being that length-dependent differences disappear as the blebbistatin concentration increases . When no X-ROS was produced (gp91ds), the peak normalized ΔF/F0 decreased by 4.6%. The agreement of the experiment and simulation indicate the relative contributions of X-ROS signaling and myofilament calcium buffering by troponin to stretching-induced changes in [Ca2+]i transient amplitudes.Using the adaptive fit described above, the simulation data were used to simulate the experiments displayed in troponin . With thin [2+]i A. Removaretching B over coretching A, similaretching C. The permalized D. These rmalized E. When s2+ bound to troponin ([Ca2+]trpn) was determined in each condition , displayed in 2+]trpn was 158 µM in the control condition, increased with myocyte stretch, and returned to its initial level when the cell was returned to resting length , a rapid activation of Ca2+ spark activity could occur. Given the importance of length-dependent myofilament Ca2+ binding and activation in the regulation of cardiac contractility, the burst of X-ROS signaling with stretch appears to be an important mechanism that offsets the consequences of increased Ca2+ affinity by troponin. In contrast, during the return of the myofilaments to resting length from stretching, the kinetics of the decrease in Ca2+ binding to troponin and the decrease in Ca2+ spark activity due to the decline of X-ROS signaling are critical to the time-course of [Ca2+]i. Despite the impact of the X-ROS burst rapidly waning, the acute reduction in the Ca2+ buffering by troponin may transiently increase myoplasmic [Ca2+]. If so, CICR would increase and there would be a brief delay in the return of Ca2+ spark activity to the levels before stretching  [2+, which might be arrhythmogenic [2+ binding affinity lessens this response through increased buffering during stretch but will not abolish it completely. In other diseases such as Duchenne muscular dystrophy, X-ROS signaling has been suggested to play a role in triggering spontaneous Ca2+ release, which can lead to arrhythmias [Studies have suggested that in cardiac myocytes, there is an abrupt increase in force after stretching (rapid response) and a slower response that evolves over several minutes (slow force response). A potential mechanism for the slow force response has been suggested, including the activation of stretch-activated non-specific cation channels and paracrine/autocrine signaling. The slow force response has been shown to be helpful in normal function and detrimental in diseases such as heart failure . In the roponin) . Our preroponin) . The X-Rhmogenic ,5. The ihythmias .Another mechanism in which stretching can modify heart function is mechanoelectrical feedback. Mechanoelectrical feedback is thought to play a role in health and disease. In pacemaker cells, it has been suggested that the acute stretch cause by blood returning to the heart can lead to more rapid depolarization, increasing heart rate. In atria, stretching has been associated with sustained atrial fibrillation. In ventricles, stretch has been associated with increased arrhythmias . X-ROS s2+ dynamics in these cells. Our work yielded new insight into the interaction between X-ROS signaling and Ca2+ buffering by troponin; these insights could not have been realized with either approach alone. In the future, expanded models exploring the spatial aspects of X-ROS signaling are planned to better understand potential targets of X-ROS signaling and how X-ROS signaling may play a role in Ca2+ entrained arrhythmias.This study extends our previous work on the physiologic roles of X-ROS signaling in cardiac myocytes  and demo


Meanwhile, LETM1 has been associated with the regulation of fundamental cellular processes, such as development, cellular respiration and metabolism, and apoptosis. This mini-review summarizes the diversity of cellular functions impacted by LETM1 and highlights the multiple roles of LETM1 in health and disease.Nearly 2 decades since its discovery as one of the genes responsible for the Wolf-Hirschhorn Syndrome (WHS), the primary function of the leucine-zipper EF-hand containing transmembrane 1 (LETM1) protein in the inner mitochondrial membrane (IMM) or the mechanism by which it regulates mitochondrial Ca Dependie brucei . A TXXR e brucei . Electroe brucei . The N-te brucei .+/H+ exchanger (KHE) as originally surmised or a Ca2+/H+ exchanger (CHE), as subsequent studies have shown m, loss of K+ acetate-induced swelling, and dissolution of cristae structure and mitochondrial morphology  came from studies in mitochondria expressing MDM38rphology . These e. brucei . The K+ chondria .T. brucei, LETM1 orthologs of higher organisms possess EF-hands, which may act as Ca2+ sensors and implicate LETM1 in Ca2+ regulation. Accordingly, LETM1 was identified as a putative CHE (LETM1-CHE) in a genome-wide RNAi screening in D. melanogaster cells, where mitochondrial Ca2+ influx/efflux was coupled to H+ extrusion/influx, respectively. LETM1-knockdown (LETM1-KD) decoupled the Ca2+/H+ antiport while LETM1 overexpression increased pH-induced Ca2+ transport 5-fold  following Ca2+ depletion in the sarco/endo-plasmic reticulum (SR/ER) and/or failure of Ca2+ sequestration through the sarco/endo-plasmic reticular Ca2+ ATPase , ultimately affecting NCLX-mediated Ca2+ efflux  and to nucleoprotein complexes called mitochondrial nucleoids, which are mitochondrial DNA (mDNA) complexed with regulatory proteins . Consequ+ transport using ionophores suggested that the absence of a LETM1-CHE/KHE activity may underlie the distorted morphology, lack of respiratory supercomplex formation, and decreased ATP production  is a progressive neurodegenerative disorder, associated with mitochondrial dysfunction . LETM1 ieostasis . The stuactivity . Future D. melanogaster exhibit motor defects, which correlates with reduced release of neurotransmitters , LETM1 expression is implicated in mitochondrial dysfunction. For example, a common mouse model used to study ALS, the end-stage superoxide dismutase SOD1(G93A) transgenic mice, exhibited a 2-fold increase in LETM1 expression in motor neurons of the hypoglossal nucleus (hMNs). This correlated with a decrease in mitochondrial Caeostasis .2+ dysregulation suggests that the loss of a LETM1-CHE activity and resulting Ca2+ dysregulation may underlie pathophysiology. Interestingly, however, LETM1-KD in the epilepsy model also decreased the expression of mitochondrial cytochrome b, a component of complex III , presumably encoded by LETM1, increased with matrix Ca2+ load and that CHE may be able to sense matrix Ca2+ buffering (2+ handling in the absence of NCLX. Future studies will need to address these knowledge gaps as to how the LETM1-CHE function in in vitro conditions is modulated in the presence or absence of one or more EF-hands in the LETM1 protein or factors such as the presence of other ionic exchangers.The diverse impact of LETM1 deficiency, as outlined above, suggests that LETM1 expression and function is critical for short-term dynamic processes such as cellular metabolism as well as long-term processes important for cell survival and development. Although the primary function of LETM1 as CHE or KHE is still being debated, it is evident that loss of LETM1 function adversely impacts mitochondrial Ca in vivo . Convers in vivo , do not uffering . The stuin vitro experiments that insertion of LETM1 complexes into membrane bilayers alone led to the formation of membrane invaginations (LETM1 interacts with proteins, such as CTMP, BCS1L, and MRPL36, and regulates processes like Akt signaling and protein import and assembly. Changes in LETM1 expression also affect these processes, implicating it in the pathophysiology of several disease phenotypes, including metabolic, neurodegenerative, and neoplastic diseases . The lininations . Four reinations , which iinations . These rinations , this suinations , this asAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.