The Na+/Ca2+ exchanger (NCX) connects the cellular Ca2+ and Na+ homeostasis. The NCX is an electrogenic transporter, which shuffles 1 Ca2+ against 3 Na+ ions over the plasma membrane in a forward or reverse mode. Here, we studied how the impact of NCX activity on PSC migration is modulated by cues from the TME. NCX expression was revealed with qPCR and Western blot. [Ca2+]i, [Na+]i, and the cell membrane potential were determined with the fluorescent indicators Fura-2, Asante NaTRIUM Green-2, and DiBAC4(3), respectively. PSC migration was quantified with live-cell imaging. To mimic the TME, PSCs were exposed to hypoxia, pressure, acidic pH (pH 6.6), and PDGF. NCX-dependent signaling was determined with Western blot analyses. PSCs express NCX1.3 and NCX1.9. [Ca2+]i, [Na+]i, and the cell membrane potential are 94.4 nmol/l, 7.4 mmol/l, and − 39.8 mV, respectively. Thus, NCX1 usually operates in the forward (Ca2+ export) mode. NCX1 plays a differential role in translating cues from the TME into an altered migratory behavior. When NCX1 is operating in the forward mode, its inhibition accelerates PSC migration. Thus, NCX1-mediated extrusion of Ca2+ contributes to a slow mode of migration of PSCs.Pancreatic stellate cells (PSCs) that can co-metastasize with cancer cells shape the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) by producing an excessive amount of extracellular matrix. This leads to a TME characterized by increased tissue pressure, hypoxia, and acidity. Moreover, cells within the tumor secrete growth factors. The stimuli of the TME trigger CaThe online version contains supplementary material available at 10.1007/s00424-023-02847-3. It plays a role among others in the formation of new lamellipodia and the formation of new focal contacts i and [Na+]i) and the membrane potential. Glass bottom dishes were coated with a 1:10 diluted collagen matrix. This matrix (1 ×) consists of 10.4 g/l RPMI, 10 mM HEPES, 40 µg/ml laminin, 40 µg/ml fibronectin, 800 µg/ml collagen I, 12 µg/ml collagen III, and 5.4 µg/ml collagen IV. The pH was set to 7.4 with NaOH [Ionic imaging experiments were performed for measuring the intracellular Caith NaOH . The mat[Ca2+]i measurements were performed with the ratiometric Ca2+-indicator Fura-2-AM . The cells were stained at 37 °C for 30 min. After washing twice, the cells were superfused with Ringer’s solution  at 37 °C. We employed a polychromator with a beam splitter 400DCLP and a D510/40m emission filter. The excitation wavelengths were 340 nm and 380 nm. The emission was measured at 510 nm. Images were taken in 10-s intervals. The fluorescence intensity was measured with VisiView software following background subtraction. For calibration, the cells were consecutively superfused with modified Ringer’s solutions with 0 Ca2+ (containing 5 mM EGTA) or 3 mM Ca2+. Both calibration solutions contained 2 µM ionomycin (Thermo Scientific). The [Ca2+]i was calculated as described by Grynkiewicz [Kd is the dissociation constant of 224 nmol/l and Rmin and Rmax are the fluorescence ratios at zero and saturated Ca2+ after excitation with 340 and 380 nm. β is a factor of F380 with zero Ca2+ and F380 at saturated Ca2+ solutions.nkiewicz :\documen[Na+]i measurements were done with the Na+ indicator Asante NaTRIUM Green-2 AM . The cells were stained with 5 µM ANG-2 at 37 °C for 1 h. After washing twice, the cells were superfused with Ringer’s solution at 37 °C. We used a beam splitter T525lpxr and an emission filter ET560/50 m. The excitation wavelength was 520 nm. Images were taken in 10-s intervals. For calibration modified Ringer’s solutions with 0, 10 and 20 mM Na+ were added to the cells. The Ringer’s solution with 0 Na+ contained N-methyl-D-glucamine (NMDG)-chloride 140 mM, HCl 122.5 mM, CaCl2 1.2 mM, MgCl2 0.8 mM, KCl 10 mM, HEPES 10 mM, and glucose 5.5 mM, pHe 7.4. The 10 mM and 20 mM Na+ solution were produced by mixing the 0 Na+ solution with the respective amount of Ringer´s solution (composition see above). The calibration solutions contained 50 µM amphotericin B (Sigma). The mean fluorescence intensity of the projected cell area was determined after background subtraction. Following linear regression of the three calibration steps, the [Na+]i concentration was calculated. Cells were only analyzed when the correlation factor of the linear regression was > 0.9.Membrane potential was measured with the fluorescent dye DiBAC4(3)  trimethine oxonol; AAT Bioquest). Murine PSCs were stained with 2 µM DiBAC4(3) at 37 °C for 20 min. Two micromoles of DiBAC4(3) were also added to all solutions used during the course of the experiment. Cells were excited with a wavelength of 490 nm. We used an emission filter D535/25 m. For calibration, three modified Ringer’s solutions with 2 mM NaCl, 35 mM, and 140 mM NaCl were added to the cells; when necessary, NaCl was isosmotically replaced by NMDG. The 2 mM and 35 mM NaCl solution were produced by diluting the 0 Na+ solution (composition see above) with the respective amount of Ringer’s solution. Each calibration solution contained 1 µM gramicidin (Sigma-Aldrich). The Na+ concentrations of the calibration solutions were measured with a radiometer . Mean fluorescence intensity of the projected cell area was determined after subtracting the background intensity. Following linear regression of the fluorescence intensities of the calibration steps, we used the Goldman-Hodgkin-Katz equation to calculate the membrane potential of PSCs. We assumed an intracellular K+ concentration of 155 mM and an intracellular Na+ concentration of 7 mM as determined during this study. Cells were only analyzed when the correlation factor of the linear regression of the calibration was > 0.9.NCX driving force: The driving forces of the NCX are calculated using the following formula i measurements. The excitation wavelength was 357 nm, and the emission was detected at 510 nm i amounted to 94.4 ± 7.5 nM, and the [Na+]i was 7.4 ± 0.4 mM . However, inhibition of the NCX caused a significant decrease of the velocity of murine PSCs. We observed a similar tendency in human siNCX1-PSCs  [+2+]i, [Na+]i, and the cell membrane potential of PSCs following activation of Piezo1 channels with 20 µM Yoda1 in Ringer’s solution. The [Ca+2+]i and [Na+]i rose to 232.6 ± 21.9 nM and 11.5 ± 0.5 mM  . Opening (TREK1) . We show2 reaches the cells, and they have to adjust their metabolism i was removed from the cell. If this was no longer possible upon NCX1 inhibition, an increase of the [Ca2+]i would occur. A moderate increase of the [Ca2+]i led to faster migration [2+]i and a concomitant activation of KCa3.1 channels i upon NCX1 inhibition potentially constituted too much stress for PSCs so that their migration decreased. We had seen a comparable cytoskeleton-dependent behavior earlier. Piezo1 activation elicited increased force generation by PSCs at pH 7.4, while force generation decreased when Piezo1 was activated in an acidic environment i and of the [Na+]i as we showed in our experiments. Accordingly, driving forces for NCX1 forward mode could be expected to decrease following the activation of mechanosensitive ion channels such as Piezo1. Thus, NCX1 activity was possibly too low to achieve a strong effect on migration.When pressure was applied to the cells, different responses were observed in murine and human cells. Pressure likely activated mechanosensitive ion channels such as Piezo1 and thereby led to an increase of the i from the cell. When NCX1 is inhibited or inactive, the [Ca2+]i rises so that faster and further migration occurs.In summary, this work provided evidence for the functional expression of NCX1 in murine and human stellate cells. It was also shown that NCX1 modulated the migration of PSCs in a way that depended on the prevailing environmental conditions Fig. . Thus, NSupplementary file1 (PPTX 991 KB)Below is the link to the electronic supplementary material.


Extracellular zinc ([Zn2+]o) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms that permit Zn2+ influx are unknown. Fluctuations in intracellular zinc ([Zn2+]i) were measured in fluozin-3-loaded platelets using fluorometry and flow cytometry. Platelet activation was assessed using light transmission aggregometry. The detection of phosphoproteins was performed by Western blotting. [Zn2+]o influx and subsequent platelet activation were abrogated by blocking the sodium/calcium exchanged, TRP channels, and ZIP7. Cation store depletion regulated Zn2+ influx. [Zn2+]o stimulation resulted in the phosphorylation of PKC substates, MLC, and β3 integrin. Platelet activation via GPVI or Zn2+ resulted in ZIP7 phosphorylation in a casein kinase 2-dependent manner and initiated elevations of [Zn2+]i that were sensitive to the inhibition of Orai1, ZIP7, or IP3R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn2+]o via influx into the cytosol through TRP channels and the NCX exchanger. Platelet activation results in the externalization of ZIP7, which further regulates Zn2+ influx. Increases in [Zn2+]i contribute to the activation of cation-dependent enzymes. Sensitivity of Zn2+ influx to thapsigargin indicates a store-operated pathway that we term store-operated Zn2+ entry (SOZE). These mechanisms may affect platelet behavior during thrombosis and hemostasis.Zinc (Zn Similarly, Zn2+ regulates Ca2+ release from stores and may therefore be a central factor in regulating platelet activation i buffering systems are important mechanisms for regulating Zn2+ bioavailability [2+]i are regulated by changes in the platelet redox state, suggestive of a role for redox-sensitive proteins, such as metallothioneins i) are maintained by store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) pathways, where the dense tubular system (DTS) is the primary Ca2+ store [2+ concentration ([Ca2+]rest) is maintained at approximately 100 nM by the action of channels and exchangers expressed along the plasma membrane and the surface of the DTS [2+]i; although, it is not clear whether the equivalent IP3R in platelets is modulated in a similar manner i via either pathway results in ZIPA, which is sensitive to a range of inhibitors and metal ion chelators o influx. OAG and the PKC inhibitor, GO6983, have previously been used to potentiate TRPC6 activity in HEK293 and smooth muscle cells o uptake, and we hypothesized that these mechanisms might also be responsible for Zn2+ influx. Washed platelet suspensions were loaded with FZ-3 or Fluo-4, and thrombin- or thapsigargin (TG)-induced fluctuations of [Ca2+]i or [Zn2+]i were investigated using flow cytometry . This effect was partially restored with the addition of 2 mM [Ca2+]o , confirming the nature of the [Ca2+]i response. Pre-treatment with the intracellular Zn2+ chelator, TPEN (50 µM), had no significant effect on thrombin-induced [Ca2+]i signals, but abolished the reductive effect of [Zn2+]o on [Ca2+]i signals  stimulation elevated [Ca2+]i to 2.0 ± 0.1 a.u . In the presence of [Zn2+]o, Ca2+ influx was reduced by 72% . In the presence of both [Ca2+]o and [Zn2+]o, peak TG-induced [Ca2+]i signals were reduced to 3.0 + 0.3 a.u. (p < 0.01) suggesting a competition between Zn2+ and Ca2+ for cation entry mechanisms. As expected, BAPTA pre-treatment abolished Ca2+ responses to TG treatment, irrespective of extracellular cation status (p < 0.05). The peak signal evoked by 1 μM TG upon TPEN (25 μM) pre-treatment was 1.8 ± 0.1 a.u, compared to the vehicle control . These findings implicate Zn2+ in the regulation of cation influx.To investigate whether o influx. To test this, platelets were loaded with FZ-3 and stimulated with thrombin or TG, and Zn2+ influx was quantified using flow cytometry. In the absence of [Zn2+]o, thrombin stimulation did not affect FZ-3 fluorescence. However, with 30 μM [Zn2+]o, thrombin stimulation led to an increase in FZ-3 fluorescence  demonstrating an activation-dependent [Zn2+]o influx. FZ-3 fluorescence was unaffected by [Ca2+]o. Further experiments employing the cation chelators BAPTA and TPEN abrogated increases in FZ-3 fluorescence, confirming that Zn2+ was responsible for the FZ-3 signal. The pre-treatment of platelets with KB-R or 2-APB had no significant effect on the [Zn2+]i signal evoked in the presence of [Zn2+]o or [Ca2+]o, or a combination of both cations . TPEN or BAPTA abolished TG-evoked increases in FZ-3 fluorescence  compared to the vehicle control . This was also the case for [Zn2+]o/Ca2+]o, where the peak signal was 1.9 ± 0.1 a.u (p < 0.001).TG was used to deplete internal stores, and Zn cations g,h, and rescence g,h. TG-m control e,f, ns. 2+]i as a result of Zn2+ influx from the extracellular medium in a similar manner to SOCE. We described this mechanism as store-operated Zn2+ entry (SOZE).These data indicate a mechanism where the depletion of cation stores in platelets following SERCA inhibition results in increased i by facilitating Zn2+ entry in platelets.Znesponses ,17. In ntivation ,51. Exogd by CK2 . We inve2+ (100 µM) led to an increased expression of ZIP7 on the platelet surface demonstrated by flow cytometry (p < 0.001). The stimulation of platelets with CRP-XL, but not the TP agonist U46619, resulted in increases in ZIP7 phosphorylation consistent with the role of GPVI in Zn2+ signaling (p < 0.001) [2+-mediated ZIP7 phosphorylation was abrogated by pre-treatment with the CK2 inhibitor CX-4945 (2+ release from stores. The role of ZIP7 in Zn2+ influx was further investigated using the ZIP7 inhibitor NVS-ZP7-4. The stimulation of FZ-3-loaded platelets with 100 µM Zn2+ following NVS-ZP7-4 inhibition (5 μM) resulted in reduced FZ-3 fluorescence in a concentration-dependent manner (p < 0.001). These findings demonstrate the role of ZIP7 in transiently elevating [Zn2+]i in response to extracellular signals, and the role of ZIP7 phosphorylation involving CK2.The activation of platelets with CRP-XL (1 µg/mL) or Zn< 0.001) . CRP-XL- CX-4945 c, consis2+]o influx or [Zn2+]i release from stores to [Zn2+]i signals remains unclear. Therefore, we utilized flow cytometry to investigate the influence of Ca2+- or Zn2+-release mechanisms on activation-dependent [Zn2+]i signals in the presence and absence of [Zn2+]o, to isolate the contribution of [Zn2+]i to intracellular stores.The relative contributions of [Zn2+]o stimulation compared to CRP-XL  o resulted in FZ-3 signals that were insensitive to the inhibition of IP3R, ZIP7, or Orai1  or ZIP7.As expected, a higher FZ-3 signal was observed following o uptake and the elevation of [Zn2+]i are mediated by the involvement of Zn2+-permeable ion channels, exchangers, and Zn2+ transporters, including NCX and TRP channels o-induced Zn2+ influx , whilst PKC has a Ca2+-binding domain i . Conversely, thrombin or thapsigargin stimulation results in increases in [Ca2+]i in the absence of any extracellular cation, consistent with Ca2+ from intracellular stores. Therefore, SOZE does not result in the liberation of Zn2+ from intracellular stores.Depletion of Ca [Zn2+]i . As this2+ signal and providing evidence that SOCE is not dependent on Zn2+. As neither SOCE nor SOZE were affected by KB-R, NCX is not involved in these processes. This is consistent with the previous data which indicate that NCX is not involved in the regulation of [Ca2+]i in neuronal cells o, consistent with competition for cation entry mechanisms. Conversely, the rate and extent of Zn2+ influx were unaffected by [Ca2+]o, suggesting that Zn2+ entry mechanisms, whilst being cation non-specific, favored Zn2+. Competition between Ca2+ and Zn2+ during intestinal absorption has been previously described where the two ions share a common transport pathway o/[Zn2+]o . Both pathways result in increases in [Zn2+]i with Zn2+ stimulation generating a stronger signal, consistent with a greater contribution from Zn2+ influx. Other published works have implicated IP3R in the persistent, rapid [Zn2+]I increases in cortical neurons i increases in the response to [Zn2+]o, but not CRP-XL. This profile is consistent with the roles of IP3R, ZIP7, and Orai1 in Zn2+ influx, but not for agonist-dependent Zn2+ release in the absence of [Zn2+]o. Therefore, Zn2+ is not stored and released in a similar manner to Ca2+, and ZIP7 was only effective in Zn2+ influx following activation-mediated externalization. This is consistent with the observation that [Zn2+]i release, but not Ca2+ release, is sensitive to the platelet redox state [2+ store in platelets. The reason why IP3R inhibition reduces [Zn2+]o influx is unclear; however, it is suggestive of a role for Ca2+ in regulating Zn2+ entry. This is consistent with the observation that both TG- and thrombin-mediated increases in [Ca2+]i were reduced in the presence of [Zn2+]o . ZIP7 is externalized following platelet activation. Both influx and Zn2+ release from stores both contribute to increases in [Zn2+]i. It is likely that other cell systems employ similar mechanisms to detect and respond to changes in [Zn2+]o.In conclusion, this study provides evidence that platelets are able to detect and respond to increases in ethyl-1H-imidazole hydrochloride), KB-R7943 mesylate phenyl]ethyl]isothiourea mesylate), SN-6 (2-[[4-[(4-Nitrophenyl)methoxy]phenyl]methyl]-4-thiazolidinecarboxylic acid ethyl ester), SAR7334 -2-[(3R)-3-Amino-1-piperidinyl]-2,3-dihydro-1H-inden-1-yl] oxy]-3-chlorobenzonitrile dihydrochloride), 9-Phenanthrol, AM0902 (1-[[3-[2-(4-Chlorophenyl)ethyl]-1,2,4-oxadiazol-5-yl]methyl]-1,7-dihydro-7-methyl-6H-purin-6-one), Teriflunomide (2-Cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]-2-butenamide), and integrilin were from Tocris . Flufenamic acid (FFA), 1-Oleoyl-2-acetyl-sn-glycerol (OAG), thrombin, calcium chloride, and zinc sulphate were supplied by Sigma , and thapsigargin (TG) was from Calbiochem . AC1903 and NVS-ZP7-4 were from MedChem express ; AMG9810 ((2E)-N--3-[4-phenyl]-2-propenamide) was from Abcam . AnCoA4 was from Merck . CX-4945 was from Stratetech, Ely, UK). Unless stated, all other reagents were from Sigma Aldrich.2+ or Zn2+ mobilizations, aliquots of platelet-rich plasma (PRP) were retained. Platelets were resuspended using a calcium-free Tyrodes buffer (CFT) containing, in mM, 140 NaCl, 5 KCl, 10 HEPES, 5 Glucose, 0.42 NaH2PO4, and 12 NaHCO3, titrated to pH 7.4 with NaOH.Preparation of washed human platelets: ethical approval for this study was obtained from the Faculty Research Ethics Committee at Anglia Ruskin University. Human blood was collected from healthy volunteers who had not taken medication for two weeks, following informed consent in accordance with the Declaration of Helsinki. Blood was collected in 11 mM of sodium citrate and washed platelets were prepared as described previously . For stu4 following pre-incubation (up to 15 min) with specified channel blockers or vehicle control (0.1% DMSO).Light transmission aggregometry: platelet aggregation was monitored as described previously using an AggRam light transmission aggregometer  . Washed g, 15 min), resuspended in CFT, and rested at 37 °C for 30 min prior to use. FZ-3 fluorescence data were acquired under 494 nm excitation and 516 nm emission using a Fluoroskan Ascent Fluorometer .Fluorometry: platelets in PRP were loaded with Fluozin-3  or Fluo-4  for 30 min, at 37 °C. Platelets were collected by centrifugation . Platelets were stimulated by thrombin (1 U/mL) in the presence of 2 mM [Ca2+]o, 30 μM [Zn2+]o, or a combination of both. Where stated, platelets were incubated with 1 µM TG, a SERCA pump inhibitor, for 5 min in the absence of [Ca2+]o to assess Ca2+ release from the DTS. For experiments assessing the role of NCX or TRP channels, platelets were incubated with inhibitors for 30 min at 37 °C, before stimulation with 100 µM Zn2+, and the FZ-3 signal was measured for 5 min. For experiments assessing [Zn2+] release from internal stores, platelets were pre-treated with inhibitors against Orai1, IP3R, or ZIP7 for 30 min at 37 °C, prior to stimulation with 100 µM Zn2+ or 1 μg/mL CRP-XL .Flow cytometry: PRP was loaded with Fluo-4 or FZ-3 (1 µM) and elevations of [CaIIbβ3 inhibitor integrilin (5 μM) for 5 min prior to stimulation with 30 μM or 1 mM Zn2+ for the given time periods, where platelets were then lysed with RIPA buffer . Lysates were separated on 8% SDS-PAGE or 4–12% Gradient NuPAGE Bis-Tris (BioRad) gels and transferred to PVDF membranes. Zn2+-induced protein phosphorylation was assessed using antibodies against phospho-β3 and Y759 , PKC substrate (AbCam), or p-MLC S19 , or anti-Phospho-ZIP7 antibodies , followed by anti-mouse, anti-rabbit, or anti-goat HRP-conjugated secondary antibodies  were included as loading controls. Blots were representative of 3–4 platelet preparations.Western blotting: Western blotting was performed as described previously . Brieflyt-test, where p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), p < 0.05 (*), and p > 0.05 (ns) were indicated.Data analysis and statistics: maximum aggregation and mean fluorescence values were calculated using Microsoft Excel 365 . Representative traces of FZ-3 or Fluo-4 fluorescence responses were generated using Flow Jo . Western blots were analyzed using ImageJ . Data were analyzed in GraphPad Prism by a two-way ANOVA of Student’s


I recently reviewed an original paper with a title including the words “physiological stimulation.” In this particular case, it turned out that the stimulation was far from physiological. The concentration of the hormone used for activation was way above the maximal level ever observed in vivo, and this made me think about the use, and misuse, of the word “physiological.” It is a word that we (physiologists) employ frequently and perhaps too frequently. Papers in physiological, and other, journals often refer to “physiological conditions,” which sometimes is taken to indicate experiments in vivo, but also frequently just means that experiments on single cells or tissue fragments were carried out with stimulation protocols and under circumstances that are not unlike those that could happen in vivo.We have lived through a long and productive period of single-cell biology. Very important discoveries of real significance have been made, but it is now becoming increasingly clear that there are many critically important interactions between different adjacent cell types in most tissues. To characterize these processes, it is necessary to observe simultaneously more than one cell type in individual organs or tissues. Furthermore, the behavior of a particular cell type in isolation may not be the same as when it is embedded in its normal environment. Of particular concern is the tacit assumption in many studies that processes in cell lines reflect those in normal cells in situ. It may therefore be useful to reflect on the usefulness of working under real physiological conditions, notwithstanding the obvious difficulties of doing so. In what follows, I’ll try to illuminate these issues by examples from immunology, epithelial physiology, and neuroscience.2+ signaling studies in immune cells have been immensely successful in unravelling key Ca2+ transport events and, in particular, the properties of the Ca2+ release activated Ca2+ (CRAC) channel of the Orai type and its molecular control mechanism.1 Unlike the situation in epithelial cells, where Ca2+ signaling studies have largely been conducted on normal cells,2 very many studies on immune cells have been conducted on cell lines and have focused on defining molecular pathways rather than describing physiological signaling patterns. The studies of Markus Hoth and collaborators have, however, revealed important aspects of physiological Ca2+ signaling.4 One critical element of the physiology of T cell activation is the role of the immunological synapse.4 This is a specialized junction between a T cell and an antigen-presenting cell that establishes efficient communication between the cells, thereby promoting activation of the immune response. The generation of the immunological synapse focuses the Ca2+ signaling domain at the inner mouth of the CRAC/Orai1 channel by translocating the mitochondria close to interaction points between the CRAC channels and the endoplasmic reticulum Ca2+ sensor STIM1. The critical role of the immunological synapse dictates that the T cell becomes functionally polarized and, like epithelial cells,2 can generate both local and global Ca2+ signals.4 The immunological synapse allows the creation of large Ca2+ signals locally, immediately below the plasma membrane, in such a way that the hotspot of Ca2+ entry occurs at some distance from the site of the Ca2+ pumps in the plasma membrane, preventing immediate expulsion of Ca2+ entering through the CRAC channels.4Ca2+ signaling has recently been studied in normal macrophages embedded in the exocrine pancreatic tissue.5 These endogenous immune cells display oscillatory Ca2+ signals in response to activation of various purinergic receptors, but do not normally respond to stimulation with IgG. However, after induction of alcohol-related pancreatitis, when there is major recruitment of macrophages into the pancreas, these cells generate repetitive Ca2+ spikes when challenged in this way.5 In pancreatitis, many products, including ADP and ATP from dying acinar cells, appear in the immediate environment of the endogenous macrophages, activating these cells.5Ca2+ signaling studies in normal cells.2 Such studies gave rise to the concept of Ca2+ signal generation by primary release of Ca2+ from intracellular stores5 and later to the identification of IP3 as an intracellular messenger.2 Furthermore, the important concept of physiological local Ca2+ signaling and pathological global Ca2+ signaling arose from single-cell studies and was later verified by work on lobule preparations in which the normal microscopic structure of the gland was preserved.2 Recently, Takahiro Takano and David Yule have carried these studies further by examining Ca2+ signal generation in pancreatic acinar cells in the intact pancreas in the living mouse.6 This in vivo study showed that physiological activation, either by electrical stimulation of the vagal nerve, infusion of cholecystokinin (CCK) or intake of a meal, elicited Ca2+ signals in the acinar cells. At low levels of stimulation, these signals were predominantly local, confined to the apical granule-containing region, whereas at higher intensities of stimulation, the signals became globalized.6 Thus, this new in vivo study confirmed, in a real physiological setting, earlier work on isolated cells and pancreatic lobules.2 An important new and unexpected finding from the in vivo study was the appearance of low level Ca2+ signaling events, even in the complete absence of external stimulation, in a sub-population of acinar cells. This was due to the endogenous basal CCK level in the blood, since it was abolished by a CCK antagonist, but not by atropine.6In epithelia, and particularly exocrine gland epithelia, there has been a long tradition for Ca7 Technically, this proved the usefulness of fiber optics to image cortical sensory activity with high resolution and, importantly, the results of these studies demonstrated differential processing of sensory input depending on behavior.8The integrative neuroscientists have frequently been more advanced technically than other physiologists and have perhaps also been particularly concerned about studying physiologically relevant situations. Thus, already more than 15 yr ago, Carl Petersen and his group showed that the cortical representation of the physiologically important whisker touch could be studied by voltage-sensitive dye imaging in freely moving mice.2+ signaling in astrocytes and neurons.9 Whereas the neuronal Ca2+ concentration increased almost immediately after the onset of locomotion and faithfully followed such episodes, the astrocytic Ca2+ signals were delayed by several seconds, developing on a different timescale.9 These findings provide important fresh evidence for the special and distinct roles played by the astrocytes in sustaining neurons and supporting their function.10In a very recent study of neurons and astrocytes in awake and behaving mice, Alex Verkhratsky and collaborators have shown that locomotion induces fundamentally different patterns of CaThese are just a few examples of findings that could only have been made by studies paying close attention to the establishment of real physiological conditions. When I started out in research, more than 50 yr ago, we generally did work in vivo under proper physiological conditions, but we did not have tools to get mechanistic information about cellular control mechanisms. This became possible in single-cell studies carried out in the 1980s, 1990s, and 2000s. Now, we have tools that allow us to observe molecular events simultaneously in multiple cell types in intact tissues and increasingly in living behaving animals. In the future, we may perhaps be able to do some of these studies safely in humans and might then also carry out investigations under critical pathological conditions. This would open up amazing new vistas of great consequence for diagnosis and treatment.


Under pathophysiological conditions, disturbances in these pools have been linked to altered intracellular communication and cellular dysfunction. To facilitate studies of subcellular Ca2+ signaling in beta cells and, particularly, signaling between the endoplasmic reticulum (ER) and mitochondria, we designed a novel dual Ca2+ sensor which we termed DS-1. DS-1 encodes two stoichiometrically fluorescent proteins within a single plasmid, G-CEPIA-er, targeted to the ER and R-CEPIA3-mt, targeted to mitochondria. Our goal was to simultaneously measure the ER and mitochondrial Ca2+ in cells in real time. The dsK of G-CEPIA-er and R-CEPIA3-mt for Ca2+ are 672 and 3.7 μM, respectively. Confocal imaging of insulin-secreting INS-1 832/13 expressing DS-1 confirmed that the green and red fluorophores correctly colocalized with organelle-specific fluorescent markers as predicted. Further, we tested whether DS-1 exhibited the functional properties expected by challenging an INS-1 cell to glucose concentrations or drugs having well-documented effects on the ER and mitochondrial Ca2+ handling. The data obtained were consistent with those seen using other single organelle targeted probes. These results taken together suggest that DS-1 is a promising new approach for investigating Ca2+ signaling within multiple organelles of the cell.The free calcium (Ca The approach is promising, in our view, because the overall strategy we employed to make DS-1 can be used to make other dual or even triple Ca2+ probes or probes for other metabolites of interest such as ATP [To the best of our knowledge, this is the first report showing that the ER and mitochondrial free Cah as ATP , which aAn obvious limitation of CEPIA-based constructs is, that being intensitometric probes, they only report relative signal levels rather than ratios. Ratiometric probes can be more sensitive to changes in levels of the analyte and may be less susceptible to changes in the optical path length or variations in probe concentration ,84. In a2+ levels of distinct intracellular compartments in pancreatic beta cells. Future work will incorporate additional compartments and different fluorophores for real-time imaging of metabolism in beta cells and other cell types.In summary, we have developed a novel means to simultaneously monitor the Ca


These cells, including the Deiters’ cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca2+ waves via diffusion of Ca2+ mobilising second messenger IP3 between neighbouring cells. We have compared the ATP-evoked Ca2+ transients and the effect of two different gap junction (GJ) blockers  on the Ca2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB/c mice (P14–19). Octanol had no effect on Ca2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca2+ pump expressions and in IP3–Ca2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP3-mediated Ca2+ signalling inhibition by CBX cannot be excluded. ATP, as a paracrine signalling molecule, induces intracellular Ca The cochlea is found in the inner ear. It consists of a bony structure with a characteristic spiral shape which can be divided into apical, middle and basal regions in mice ,2. The s+ uptake [In the organ of Corti, hair cells are the primary auditory receptor cells . They ar+ uptake ,10, and + uptake ,8. The f+ uptake . However+ uptake ,12,13.2+ ions are implicated in all steps of the transduction process, mechanoelectrical transduction function, neurotransmitter release, cochlear amplification, ion homeostasis in the endolymph and developmental processes [2+ increase in the cytoplasm when stimulated by extracellular ATP [2+-mobilizing second messengers IP3, which may play a role in the K+ recycle function [3-mediated Ca2+ release by inhibiting IP3R activity and voltage-gated Ca2+ channels [In the mammalian cochlea, Carocesses ,15,16,17ular ATP . These cular ATP . It allofunction ,20,21,22function ,23,24,25channels . 2+ imaging experiment, we investigated the ATP-induced Ca2+ transients of DCs in hemicochlea preparation of hearing mice. In the presence of gap junction blockers (octanol and CBX), we stimulated the cells and measured changes in cellular reactivity to explore their coupling strength and the effect of gap junction blockers on the transients. We also developed a mathematical model that simulates the calcium patterns in DCs of the apical and middle regions. Our model is an extended model considering cytosolic Ca2+ concentration, the fraction of active inositol trisphosphate receptors (IP3R) and IP3 dynamics as variables. Since no model has been developed for DCs, we based our model on Taheri et al. [2+ dynamics in astrocytes. We also explore the gap junction coupling effect employing Wu et al. [3R activity in addition to acting as a gap junction blocker [Cellular-level investigation in the cochlea is mostly carried out on young mice P1–2) before the temporal bone calcifies. However, mice are born deaf, and their hearing organ does not mature until the end of the second post-natal week  before t,27,28. Ii et al. ,30 and Di et al.  models tu et al.  model. S blocker , we inveDCs from hearing mice (P14-P17 for the CBX experiment and P15–19 for the octanol experiment) were investigated in hemicochlea preparation A after sp = 0.233, p = 0.299, p = 0.261, Octanol did not modify the ATP-induced responses compared to control responses . Neither2+ transients of DCs from hearing, P14–17 mice . It describes IP3 dynamics by including in the model the IP3 production by agonist-dependent (PLCβ) and independent (PLCδ) enzymes, the IP3 degradation by IP3-3-kinases and phosphatases, and the IP3 diffusion through gap junctions. Ca2+ elevation results from Ca2+ influx from the endoplamic reticulum (ER) through IP3 receptors activated by the IP3 elevation and Ca2+ influx through P2X receptors activated by ATP. Plasma membrane Ca2+-ATPase (PMCA) and sarco-endoplasmic Ca2+-ATPase (SERCA) are decreasing free Ca2+ concentration . We obtained 501 and 6243 parameter sets for CBX-treated apical All transients were characterised by seven points: the time and value coordinates of the 5%, 10%, 50%, 90% and 100% of the intensity on the rising part of the transient and 90% and 50% of the intensity on the decreasing part of the transient. A parameter combination was accepted when the generated curve fitted to experimental data for at least three time points and three value points tested by t-tests. More parameter combinations could reproduce similar curves. We analysed all accepted combinations to outline possible differences in intracellular mechanisms.2+ concentration, IP3 receptor gating variable, and the intracellular IP3 concentration  have a uniform distribution in the accepted combinations and in the tested parameter space, which means that they would not limit the model success , and the PMCA parameters (KPMCA and vPMCA) showed that in controls of CBX-treated apical cells, lower influx rates are more prevalent than in other control groups, and KPMCA remained lower in the same group (2.1–2.4 µM) in 75% of accepted parameters. In both control groups, cells from the middle regions tend to obtain higher values (2.4–2.9 µM in 58% for controls of octanol- and in 88% for controls of CBX-treated cells). VPMCA showed great variability, and no clear tendency has been seen in either group (Parameters reflecting Caer group .2+-induced Ca2+-release (rC) and the SERCA parameters (vER and KER) . For the same reason, KER took the lowest values in this group (38% were between 0.05 and 0.08). In other groups, 0.1 and higher values were predominant.The parameters connected to the ER membrane activity are the rate of the Caand KER) . The rC 3 dynamics and the enzymes which are connected to the production and degradation of IP3 molecules were also investigated and analysed  was tested from 0.1 to 0.5. In most groups, nearly 50%–50% were the lower and higher parameter distributions, except in the CBX-treated control group, where 99% of the accepted values are higher than 0.3. PLCδ is activated by the higher cytoplasmic Ca2+ concentration, and their activation was presented by three parameters: KPLCδ, κδ, vδ. In the apical region, more than 90% of KPLCδ values are higher (from 0.1 to 0.15), and this is more prominent in the middle-located controls of octanol-treated cells where most (90%) values are over 0.13. The parameter Kδ in the middle region took lower values than detected in the apical region: for the control of octanol cells, the apical region parameters are less than or equal to 1.3 in 34%, while in 94% for the middle region. In the control of CBX-treated cells, 17% of the accepted parameters were within the same range of values for apical cells while 35% for middle cells. In contrast, the vδ parameter in the apical region had higher values than in the middle region. In the apically located cells, values were greater than or equal to 0.03 in 58% and 84%, while for cells of the middle region, they were 60% and 58% in the octanol and CBX controls, respectively.The detailed IPanalysed . During 3 molecules. For the 3K, three parameters were introduced to the system: KD, K3 and v3K. For KD, we mostly found lower values in the accepted parameter combinations. In all cases, more than 60% of the parameters were less than or equal to 0.05, and were even more predominant in the controls of octanol-treated cells (93% in both regions).Inositol-triphosphate-kinases (3K) and 5-phosphatases are involved in the degradation of IP3 parameter is the IP3 affinity of IP3-3K. For controls of octanol-treated cells, K3 had higher values . The values for the controls of CBX-treated cells were similar in the apical region (60%), but in the middle region, mostly lower values were accepted .The K3K parameter, the maximal degradation rate of the kinases, was higher in the apical region compared to the middle region .The v5P parameter. The degradation rate in the apical region was slower: 63% and 93% of all the accepted values were equal or lower than 0.05 compared to 45% and 12% in the middle region .The 5-phosphatases, the other degrading enzyme family, were represented by the r2+ transport through the plasma membrane were visibly different: the rate through P2X receptors decreased, and was even more prominent for the CBX treatment while the VPMCA parameter of the apical region increased  were differently changed: parameters for the plasma membrane were increased while parameters for the ER membrane were decreased, but only in CBX-treated cells  of the model after removing the equations representing the fluxes through gap junction channels and IP3 receptors  is 0.7 µM (for octanol-treated cells), and 0.3 and 1 µM (for CBX-treated cells). The IP3 affinity, K3, was 0.7 µM, and 0.0.9 and 1.2 µM for the octanol- and CBX-treated cells, respectively, while the maximal rate was 1 µM·s−1 for the octanol-treated cells, and 3 and 4 µM·s−1 for the CBX-treated cells. Again, these groups showed great variability in the middle region, but for the CBX-treated cells, gap junction equation removed groups were limited: 1 µM for the KD, 0.9 µM and 3 µM·s−1 for the K3 and v3K, respectively.For the IPr trends . In the 3 degradation, the 5-phosphatases, had similar limitations  both on supporting cells [2+ signalling cascade showed tonotopic differences.A higher amplitude in the middle region could imply a stronger and faster response to the stimulus, perhaps due to a difference in the number and type of purinergic receptors, PLC or IPng cells  and hairng cells ,40,41. T2+-removing mechanisms. This could be related to an increased number of SERCA or PMCA pumps. An interesting observation has been made on the ability of OHCs to handle Ca2+ load along the tonotopic axis of the cochlea. Two main differences between hair cells of low- and high-frequency regions  have been demonstrated: larger amplitude of the MET current and fewer PMCA pumps in high-frequency OHCs. Both are likely to explain the Ca2+ overload in OHCs of the basal turn [On the other hand, a shorter duration of the response would imply stronger and faster Casal turn . One wouApart from the number of SERCA and PMCA pumps, their isotypes could also be different. The whole inner ear and cell-specific sequencing studies in young (P0) mice have shown that two types of isoforms (ATP2A1 and ATP2A) are present in the organ of Corti, and both are also present in DCs ,43,44. H3 production and degeneration have also been found in the hearing organ via mRNA sequencing methods in young mice. We modelled the PLCδ and β, which are activated differently. Their isotypes are also found in DCs: PLCδ1, 3 and 4 and PLCβ 1, 2, 3, 4 were detected [Enzymes which play a role in IPdetected ,43,44. F3 receptor also has three different isoforms, and all three are present in the young mouse DCs—but the IP3 receptor-2 has the highest expression rate [2+ signalling mechanisms.In addition to enzymes, pumps and cell surface receptors, the IPion rate . Our mod2+ signals via the diffusion of Ca2+-mobilizing second messengers [2+ waves in supporting cells of P25-P35 mice were inhibited by 1 mM octanol, and thus it is unlikely that connexin expression is already decreasing in P14-P19 mice [3 release in the cochlear sensory epithelium of adult guinea pigs compared to 100 μM CBX [Cochlear supporting cells are coupled with gap junction channels that allow the propagation of intercellular Cassengers . Recent ssengers ,46. Intessengers . Althoug0 μM CBX . As ment0 μM CBX . Octanol0 μM CBX . Altoget2+ imaging experiment showed that CBX had a stronger effect on the middle region, suggesting that more connexins are present in the middle part of the cochlea than in the apical one. In the absence of CBX, the amplitude of the calcium responses was higher in the middle region, with a P2/P1 ratio being smaller. The P2/P1 ratio emphasises how CBX treatment influenced Ca2+ responses in each cochlear region  to P19. The apical regions P15–17 were used for CBX experiments and P15–19 for the octanol experiment. In the middle region, P14–17 mice were sacrificed for the CBX experiments and P15–19 for the octanol experiments. The experiment protocol was based on previous studies [2+ indicator Oregon Green 488 BAPTA-1 hexapotassium salt (OGB-1)  dissolved in distilled water . The pipettes were mounted onto an electrode holder, which was attached to a micromanipulator . As the pipettes approached the cells of interest, a single square wave current impulse  was applied, allowing the cells to be loaded with OGB-1. The current was generated by a pCLAMP10 software-guided stimulator system . studies ,8. The mThe calcium imaging procedure was performed at room temperature. Excitation light of 494 ± 5 nm  was used to illuminate the cells, and the light emitted was recorded after passing through a band-pass filter (523 ± 25 nm). An Olympus BX50WI fluorescence microscope  equipped with a Photometrics Quantix cooled CCD camera  was used and was controlled with the Imaging Workbench 6.0 software . The images were taken at a frame rate of 1 Hz during the drug-evoked responses, and the rest of the time, the frame rate was 0.1 Hz to reduce phototoxicity and photobleaching. In the perfusion chamber, the volume of the buffer was about 1.9 mL. The cells were stimulated by 100 µM ATP , and added to the perfusion for 30 s. Before ATP stimulation, a baseline period of at least 3 min was registered.For the investigation of the gap junctional coupling mechanism, we perfused the cells with gap junction blockers: octanol  and carbenoxolone disodium salt . After the 1st ATP stimulus, the respective blocker was added to the perfusion for at least 15 min before the 2nd ATP administration. After the ATP response had been completed, the blocker-containing solution was replaced with a perilymph-like solution for at least 15 min before exciting the cells with a 3rd ATP dose D. To excWe have performed our experiments on DCs in the apical and middle turns in the hemicochlea preparation. These turns include the most relevant frequency region of the basal membrane , and0 = (Ft − F0)/F0, where F0 is the fluorescence intensity of the baseline, and Ft is the fluorescence intensity at time t [0 values) were recorded: 100% , 5%, 10%, 50% and 90% of the maximal amplitude on the increasing part of the transients, and the 90% and 50% of the maximal amplitude on the recovery part of the transients. The duration of the transients was calculated as the time between 50% of the maximal amplitude at the increasing and descending part of the response.Fluorescence intensities were background-corrected using a nearby area devoid of loaded cells. The relative fluorescence changes were calculated as follows: dF/Ft time t . A totalStatistical analysis of the experimental results was performed in R 4.1.0. version. The data distribution was tested by Shapiro–Wilk test. Most of the data were not normally distributed; because of this, these were logarithmically transformed, and a linear regression model was used.2+ dynamics evoked by neurotransmitters. A gap junctional flux of IP3 was added to our model employing Wu et al. [Previous studies have highlighted similarities between auditory supporting cells and glial cells . Therefou et al.  model.2+ concentration can change due to exchange across the plasma membrane. We assumed that the spatial arrangement of calcium concentration is uniform in the cytosol and ER. The model consists of a system of three differential equations: the changes in cytosolic Ca2+ concentration (1), the activated fraction of IP3R (2) and the IP3 intracellular concentration (3):The model is an open-cell model in which the intracellular CaiJ represents the fluxes through pumps and channels embedded in the plasma membrane and ER membrane, respectively (P2XJ represents the influx of Ca2+ to the cytosol through P2X channels:The ectively . JP2X rePMCAJ is the outflux of Ca2+ across the plasma membrane through PMCA pumps. SERCAJ is the flux of Ca2+ from the cytosol into the ER through SERCA pumps:AndIP3RJ is the flux of Ca2+ from the ER to the cytosol through the IP3R channel. This model simplifies the opening probability of IP3R by assuming that the gating kinetics is mainly driven by three binding sites on the receptor: an IP3 binding site (m), a Ca2+ binding site for activation (n), and a Ca2+ binding site for deactivation (h). The dynamics of h are governed by the equation given previously, where IP3RJ is given by:WithleakJ is a nonspecific calcium leakage from the ER:3 is produced from the hydrolysis of PIP2 catalysed by two PLC isoenzymes, PLCδ and PLCβ. The binding of ATP to G-protein coupled P2Y receptors induces the activation of PLCβ, releasing IP3 (ATPv). PLCδ is mainly activated by intracellular Ca2+ elevation (δv). IP3 concentration depends on its production and its degradation via two major pathways. The first degradation pathway is through the dephosphorylation of IP3 by the inositol polyphosphate 5-phosphatase (IP-5P) (vP5). The second one is through Ca2+-dependent phosphorylation of IP3 by the IP3 3-kinase (IP3-3K) (vK3). Both reactions can be considered as Michaelis–Menten type. However, IP-5P is not likely to be saturated by IP3, so the rate of IP3 degradation can be linearly approximated. The equations are:IPGJCJ) into our model, taking into consideration that IP3 can diffuse through gap junctions. To this end, we used Wu et al. [i cells is considered. To simplify this model, we assumed the IP3 concentration of the neighbouring cells constant over time, at a concentration of 5 μM, and IP3 diffusing only from the neighbouring cells to the target Deiters’ cell. Thus, the diffusion rate of IP3 only depends on its production and degradation over time in the experimental target cell. In Equation (13), we multiplied GJCJ by 4 because DCs usually have four neighbouring cells, as follows:In addition, we incorporated a gap junctional flux , and then exponentially decays before becoming depleted.We opted to treat ATP as inputs to our model (14). In our experiment, DCs were stimulated by injecting ATP for 30 s in the perfusion chamber. We assumed that ATP exponentially saturates to a certain level , the volume of the chamber (2 mL), and the concentration of the injected ATP dose (100 μM). The estimated ATP values in the perfusion chamber were compared to the ATP model (14), and an estimation of the model parameter values was obtained using a non-linear least squares approach (function nls in R). The initial set of ATP model parameters were: Maximal amplitude (A) = 58.85 μM; rised = 30 s. r was fitted first, and decayd second, both using a non-linear least squares approach. The following values were obtained and used for the model: riser = 0.0296 s−1, decayd = 316.7 s.We implemented our model in R 4.1.0 using forcing functions of the deSolve package with the aim of adjusting the parameter values to fit the model to our experimental data.Model parameters were tested in a parameter space by random sampling .t-tests. If at least 3 time points and 3 value points showed a non-significant difference from the experimental data, the parameter combination was saved.From each result of the parameter combinations, the same characteristic points were recorded as in the case of the experimental data: 100% , 5%, 10%, 50% and 90% of the maximal amplitude on the increasing part of the transients, and the 90% and 50% of the maximal amplitude on the decreasing part of the transients. Then, these points were compared to experimental data using 2+ signalling with the mammalian cochlear development that generally proceeds from the basal region to the apex [Our experimental results showed that the ATP-evoked calcium transients in DCs in the apical turn were smaller  at an earlier developmental stage (P14–17 vs. P15–19), which may represent the correlation of the development of purinergic Cathe apex ,53,54.2+ store. The shorter duration of these calcium signals may be the result of a faster recovery caused by a higher number of SERCA and/or PMCA pumps, eliminating intracellular Ca2+.The larger ATP response in DCs in the middle turn and in the more mature (P15–19) apical turn may imply a higher number or different subtype pattern of purinergic receptors, PLC, IP3R or a larger endoplasmic Ca2+ signalling in these cells. Despite this, the use of 1 mM octanol did not have any effect on the ATP-induced Ca2+ transients. Theoretically, this may be linked to a maturation-dependent decrease in the expression of cochlear gap junction proteins Cx26 and Cx30, although this has not been described in P15-P19 mice yet. However, octanol is thought to be selective for the Cx43, Cx46 and Cx50 isoforms which raises a question of its efficiency in blocking gap junction channels of cochlear supporting cells. On the contrary, CBX inhibited the ATP response in both cochlear turns, more so in the middle one, which may be explained by the higher number of CBX-sensitive Cx26/30 in that turn. The inhibitory effect of CBX on the IP3-mediated Ca2+ release, described recently [DCs are coupled with gap junction channels, which influence the Carecently , cannot 3 degrading enzymes and the similarity of the CICR rate to the one in the astrocyte model.Based on astrocyte models, we successfully simulated the calcium dynamics in DCs. This supports the hypothesis that DCs are glia-like cells. Our model, using the parameters of our experimental results, suggests the tonotopical distribution of IP


Dear Editor,CEBPA (CEBPAmut) is one of the most common molecular abnormalities in acute myeloid leukemia (AML), especially in east Asian population . Thus, we defined CEBPAbZIP-inf AML patients characterized by immunophenotypic CD7-positive and wild-type WT1 as low-risk group (LR group: CEBPAbZIP-inf/CD7 + /WT1wt); correspondingly, 19/89 patients of CEBPAbZIP-inf AML were re-stratified into the high-risk group  compared with HR patients (n = 13), although the difference was not significant due to the limited sample size  was then performed and indicated that the outcome was significantly improved after the integration of CD7 expression and CEBPAmut patients from our cohort. With unsupervised cluster analysis, HOXA/B family genes were identified to be highly associated with poor prognosis in CEBPAmut AML  AML Fig. . Besidesnts Fig. ; HR patip = 0.23 and 0.10, respectively); whereas allo-HSCT in CR1 could significantly improve the outcome of HR patients, with 2-year OS and EFS of 100% and 84.6% vs. 53.6% and 23.1% in chemotherapy-only, respectively   Fig. . The theWT1mut status is convenient for clinicians to acquire for the identification of CEBPAbZIP-inf AML patients who are in risk of disease relapse (Supplementary Fig. CEBPAmut AML.Conclusively, CD7 immunophenotype and Supplementary Information


The number of primary follicles in estrus was significantly higher than in the other stages. Some genes, such as CYP19A1 and IGF1R, related to the estrous cycle in female rabbits were screened via ovarian transcriptome analysis. Overall, extensive information relating the behaviors, hormones, and follicle development in female rabbits at different estrus stages was investigated for appropriate postpartum re-estrus management strategies.Estrus involves complex physiological signs and behavioral changes, but the exact ovulation time is difficult to determine, which is very important to improve the fertility of rabbits. This study aimed to analyze female rabbits’ changing behavior and physiology at different stages of the estrous cycle. The female rabbits showed less foraging, drinking, grooming, and biting while in estrus. Related physiological hormones . Differences in reproductive hormone levels  and follicle development were measured at each stage via ELISA and HE staining. The FSH and LH levels showed an increasing trend and then decreased, with the lowest being in late estrus (p < 0.05). The P4 level was the lowest in estrus (p < 0.05), and E2 showed a gradually increasing trend. There was no significant difference in the number of primordial follicles at each stage, but the number of primary follicles in estrus was significantly higher than at the other stages (p < 0.05). To further understand the molecular regulation mechanism of the estrous cycle in female rabbits, we analyzed the ovarian transcription patterns of female rabbits in diestrus (D group) and estrus (E group) employing RNA-seq. A total of 967 differentially expressed genes (DEGs) were screened from the ovaries of female rabbits between the diestrus and estrus groups. A KEGG analysis of DEGs enriched in the estrogen signaling pathway, aldosterone synthesis, and secretion pathway, such as CYP19A1 and IGF1R, was performed. The rabbits’ behavior, related physiological hormones, and molecular regulation also differed at different estrous cycle stages. The results provide recommendations for the adequate management practices of postpartum re-estrus and breeding female rabbits.Estrus involves a series of complex physiological signs and changes in behavior before ovulation, which play a crucial role in animal reproduction. However, there have been few studies that evaluate behaviors during the different stages of estrus cycle in female rabbits. Therefore, more detailed information is needed on distinguishing the various stages of the estrous cycle. This study explored the behavioral and physiological differences at various estrous cycle stages in female New Zealand White rabbits. The continuous recording method was employed to record the daily behaviors of twenty postpartum female rabbits during the estrous cycle. Compared with the diestrus stage, the duration of foraging and drinking behavior in estrus decreased significantly, and the frequency of grooming and biting behaviors increased ( Estrus, as the initial stage of animal reproduction, holds significance in animal conception, as it largely determines the probability of female rabbit pregnancy. However, various endogenous hormones and exogenous factors influence the presence of uncontrollability in reproductive efficiency. The estrous cycle of female rabbits is divided into diestrus, early estrus, estrus, and late estrus. The conception rate during the estrous cycle is the highest . The estDuring estrus, the female rabbit’s appetite decreases, the mood becomes agitated, and it will sniff around and dig nest burrows among the range of activities . TypicalFollicle-stimulating hormone (FSH) can promote the rapid development of follicles after the action of dominant follicles [2) levels inhibits the secretion of FSH through negative feedback so that non-dominant follicles cannot enter the rapid growth stage. Progesterone (P4), a natural hormone secreted by the ovary’s corpus luteum, inhibits follicular development and steroidogenesis in ovaries [2, which is the key condition for follicle maturation [The estrous cycle of a female rabbit is closely related to the endocrine system. Reproductive hormones act as messengers to regulate the coordination of various systems and organs to maintain homeostasis . Folliclollicles . Subsequ ovaries . When thturation ,10,11. TBMPR1B in granulosa and theca cells is the most prominent in follicular development, which may be influenced by FSH and LH [CYP19A1 may regulate the estrous cycle by mediating reproductive hormone synthesis and follicular development through positive or negative regulation [CYP1A1 is expressed the most during ovulation in the ovary, indicating an increased hydroxylation of estrogen and the elimination of negative estrogen feedback control over LH secretion, which play direct or indirect roles in ovulation [The molecular regulation mechanism of the mammalian estrous cycle is very complex, involving several key genes and signaling pathways. The expression of H and LH . CYP19A1gulation ,14. CYP1vulation . The keyTo investigate the changes in the behavior and physiology of female rabbits at different stages of the estrous cycle, we compared and studied the daily behaviors, hormones, follicle development, and differentially expressed genes (DEGs) in female rabbits at various estrus stages. The parameters regulating the estrous cycle of female rabbits were investigated using behavioral, endocrine, and molecular methods to provide the foundation for further research into their reproductive potential.The present work acquired ethical approval from the Animal Protection and Use Committee of Yangzhou University (no. 202109221) and was conducted following rigorous adherence to animal management regulations. A total of 20 female New Zealand White rabbits, aged 8 months, were selected based on their similar body size and overall good health in the third parity.Twenty postpartum female rabbits were transferred to individual cages (70 cm × 90 cm × 50 cm) with the camera shooting and allowed to rest for 1 day to adapt to the new environment. On the 2nd day after the transfer, light stimulation was used to induce estrus from 7:00 a.m. to 10:00 p.m. every day. All female rabbits’ daily behaviors  were monitored and recorded at 8:00 a.m. and 8:00 p.m. for 15 days. Then, the blood samples were collected from the female rabbits at different estrus stages at 7:00 a.m. every day during the 2nd estrous cycle. During the 3rd estrous cycle, ovarian tissue was collected at different estrus stages.The estrus state of the female rabbits was determined according to the vaginal mucosa color daily. There are four estrus stages in the entire estrous cycle. As shown in A total of four surveillance cameras were strategically positioned at a distance of 2.6 m from the cage walls, ensuring an equal spacing of 3 m between each camera. The orientation of the cameras was modified to ensure a comprehensive coverage of the actions of the five female rabbits by each camera. The estrous cycle of the female rabbits was observed and documented bi-daily, with continuous monitoring conducted throughout the day, resulting in the storage of comprehensive records for all the female rabbits. The frequency and duration of each behavior were observed using a continuous recording method for 15 days. The software used to watch the video was PotPlayer (1.7.21796), which individuals watch at double speed and record individually. The number of behaviors occurring per unit of time was recorded as the frequency, while duration referred to the time from occurrence to the completion of a certain behavior. Before the formal recording of the observation, the female rabbits were pre-observed for 3 days. The specific behaviors and definitions of the female rabbits are listed in 4, and E2 in the plasma were determined using enzyme-linked immunosorbent assay (ELISA) kits for rabbits, FSH kit , LH kit , P4 kit , and E2 kit . Each well’s absorbances  at 450 nm were measured using a microplate reader  with the blank as a zero reference. The sample concentration was calculated according to the standard curve.Female rabbit blood was obtained from the auricular vein, centrifuged at 1500 r/min for 5 min, and the supernatant was absorbed. The contents of LH, FSH, POvaries were collected and fixed with 4% paraformaldehyde. The tissue size was trimmed and dehydrated with ethanol and xylene at different concentration gradients, tissue wax was immersed and embedded using an embedding machine, and blocks were sliced with a thickness of 5 μm using a microtome. The prepared sections were then dewaxed, rehydrated, stained with HE, sealed with neutral gum, observed, and photographed. The number of follicles  was observed and counted.2FoldChange| > 1 and p-value < 0.05 were set as conditions of screening DEGs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed for functional annotation and pathway analyses of the DEGs.The ovaries of female rabbits in the diestrus (D1–D3) and estrus (E1–E3) stages were collected for RNA-seq analysis. After total RNA isolation and library preparation, next-generation sequencing (NGS) based on the Illumina sequencing platform was used. The filtered reads were compared with rabbit genomes using HISAT2(2.1.0) software. Gene expression was calculated using fragments per kilobase of transcript per million mapped reads (FPKM). DESeq was used to analyze the differential expressions between groups. |log® qPCR Master Mix  on a QuantStudio® 5 Real-Time PCR System. The primer sequences are listed in −ΔΔCt method.Total RNA was extracted using TRIzol  following the manufacturer’s instructions. Real-time PCR was performed using ChamQ™ SYBRt-test. GraphPad Prism 8 was used for plotting. The results are represented as the mean ± standard deviation. * p < 0.05 indicated significant difference and ** p < 0.01 indicated extremely significant difference.The differences in behavior, reproductive hormone levels, and follicle count were analyzed utilizing a one-way ANOVA using SPSS (ver. 25.0) software. The RT-qPCR data were analyzed using a p < 0.01) and early estrus stages (p < 0.05). The duration of foraging and drinking behaviors were in contrast to that of grooming behavior, and the duration in diestrus was significantly higher than in estrus . There were no significant differences in the duration of relaxing, locomotion, standing, observing, and biting behaviors (p > 0.05).The estrus stage was determined according to the characteristics of the vaginal mucosa of the female rabbits. Daily behaviors of female rabbits at different estrous cycle stages were observed and recorded . As mentp < 0.05, p < 0.01). The frequency of drinking behavior was significantly higher in early estrus than in late estrus (p < 0.05). The frequency of locomotion behavior was the highest in estrus and significantly higher than in other stages (p < 0.05). The frequency of biting behavior increased first and then decreased with the estrous cycle, with the highest frequency observed in estrus.The frequencies of behavior differences at different stages of the estrous cycle are mentioned in p < 0.05). The differences in LH levels were similar to those of FSH. The LH level was the highest in estrus and significantly higher than in diestrus and late estrus (p < 0.05). The P4 levels in diestrus and late estrus were significantly higher than in estrus (p < 0.05). The E2 level was opposite to that of P4 and was significantly lower in early estrus than at the other stages (p < 0.05).As shown in p > 0.05). The number of PFs in estrus was significantly higher than at other estrus stages (p < 0.05). The number of SFs in estrus was significantly higher than in diestrus and early estrus (p < 0.05). The number of TFs was higher in estrus and late estrus than in diestrus and early estrus (p < 0.05) (As shown in  < 0.05) C.BMPR1B, CYP19A1, PMFBP1, CYP1B1, OVGP1, IGF1R, EGFR, ESR1, ADCY7, ITPR2, SPP1, FGF23, etc. Among these, ESR1, IGF1R, CYP19A1, CYP1B1, BMPR1B, and HOXA9 were randomly selected to verify the accuracy of the transcriptome results via RT-qPCR, and the results were consistent with the sequencing results  and estrus (E group) using RNA-seq. A total of 967 DEGs were screened out between the D and E groups, among which 623 were up-regulated and 344 were down-regulated A,B. Seve results C. GO enr results D. KEGG a results E.Animal behavior is the most direct manifestation of environmental change, and behavioral changes often reflect the internal physiological changes of the organism . Animals2 in the whole estrous cycle of female rabbits was the lowest in early and peaked in late estrus. This study showed the hormonal differences in female rabbits at different estrous cycle stages for estrus identification.The growth and development of the follicles and the formation of the corpus luteum after ovulation are closely related to reproductive hormones . The resBMPR1B, CYP19A1, LHCGR, etc. The overexpression of the BMPR1A and BMPR1B genes in ovarian granulosa cells inhibits the occurrence of mouse granulose tumors, while the knockout of BMPR1A revealed less spontaneous ovulation and reduced fertility in mice [BMPR1B regulates the differentiation of granulosa cells and follicle development by participating in the signal transduction of BMP2, BMP4, BMP6, BMP7, BMP15, and GDF5, thus affecting the reproductive cycle of mammals [CYP19A1 can convert androgens into estrogen, and estrogen can inhibit apoptosis in granulosa cells and promote its differentiation by increasing the expression of LHCGR, the LH receptor, thus regulating follicle development [CYP19A1 was significantly higher during estrus compared to diestrus, with an increased change in plasma LH levels and a decrease in foraging and drinking times. These genes are potentially important in regulating the estrous cycle in female rabbits.The estrous cycle of female animals is affected by external and internal physiological factors ,35. To e in mice . Studies mammals ,38. In pelopment ,40. In t4, and E2) and follicular development with different estrous cycle stages also differed. Extensive information on behaviors, hormones, and follicle development in female rabbits at different estrus stages was investigated for appropriate postpartum re-estrus management strategies. The results provide recommendations for the adequate management practices of postpartum re-estrus and breeding female rabbits.The female rabbits showed less foraging and drinking, and more grooming and biting in estrus. Related physiological hormones (FSH, LH, P


Hyalomma anatolicum is the principal vector for Theileria annulata, T. equi, and T. Lestoquardi in animals and the Crimean–Congo hemorrhagic fever virus in humans. Due to the gradual loss of efficacy of the available acaricides against field tick populations, the development of phytoacaricides and vaccines has been considered the two most critical components of the integrated tick management strategies. In the present study, in order to induce both cellular and humoral immune responses in the host against H. anatolicum, two multi-epitopic peptides (MEPs), i.e., VT1 and VT2, were designed. The immune-stimulating potential of the constructs was determined by in silicoinvestigation on allergenicity ), physicochemical properties (instability index 27.18 and 35.46), as well as the interaction of constructs with TLRs by docking and molecular dynamics analysis. The immunization efficacy of the MEPs mixed with 8% MontanideTM gel 01 PR against H. anatolicum larvae was determined as 93.3% and 96.9% in VT1- and VT2-immunized rabbits, respectively. Against adults, the efficacy was 89.9% and 86.4% in VT1- and VT2-immunized rabbits, respectively. A significant (p < 0.001) reduction in the anti-inflammatory cytokine (IL-4) and significantly higher IgG response was observed in a VT1-immunized group of rabbits as compared with the response observed in the control group. However, in the case of the VT2-immunized rabbits, an elevated anti-VT2 IgG and pro-inflammatory cytokine (IL-2) (>30 fold) along with a decreased level of anti-inflammatory cytokine IL-4 (0.75 times) was noted. The efficacy of MEP and its potential immune stimulatory responses indicate that it might be useful for tick management. Rhipicephalus microplus and Hyalomma anatolicum are the two most economically significant species infesting Indian livestock [H. anatolicum is the principal vector of Theileria annulata, T. equi, and T. Lestoquardi [Ticks are obligatory blood-feeding ectoparasites of animals and humans. They are the most potent vector of animal diseases caused by protozoans, spirochetes, bacteria, rickettsia, and viruses  than anyivestock . Among ttoquardi ,6. It altoquardi ,8,9. Thetoquardi ,11. The toquardi ,13,14. Gtoquardi  opined tR. microplus in field conditions, the efficacy of the vaccines against different strains of the tick species was evaluated with variable results [After the successful introduction of two vaccines for the control of  results ,17,18,19 results ,21,22.Upon reviewing the literature, it appears that the selection of specific tick protein antigens (epitopes) is a usefuland advantageous strategy for vaccine production. The peptides targeting epitopes which augments precise immune responses to the essential immunogenic segment of a protein antigen can be designed . MalonisR. microplus were used to immunize cattle and an efficacy of >81% against challenge infestations was reported using SBm7462 peptide [R. microplus and R. sanguineus challenge infestations on the bovine and canine host, respectively [R. sanguineus and R. microplus, respectively.Previously, three synthetic peptides  derived from Bm86 of  peptide . A synthectively ,27. Furtectively  reportedH. anatolicum larvae and adults.In the present study, an attempt was madeto design multi-epitope peptide constructs of immunodominant peptides by immunoinformatic approach. The peptides were preferentially picked based on previous laboratory data ,30,31 anBos indicus × B. taurus) male calves were maintained in atick-proof cattle shed withinthe Division of Parasitology of the institute as per the standard guidelines.Twenty-eight young NewZealand white (NZW) rabbits  were raised in clean rabbit-rearing steel cages (25 × 20 inches) by providing ad libitum feed  and fresh water. In addition, cross-bred (Theileria annulata-free H. anatolicum (IVRI-2 strain) was maintained by the ear bag protocols of Ghosh and Azhahianambi [The acaricide-susceptible and hianambi . NZW rabBased on our previous findings, the proteins were selected and amino acid sequences of ferritin-2 (FER2) and tropomyosin (TPM) were retrieved from the NCBI database (ALJ92580.1 and AMB19058.1) . The tichttp://www.cbs.dtu.dk/services/SignalP/ (accessed on 15 January 2020)) [http://www.cbs.dtu.dk/services/DeepLoc/ (accessed on 15 January 2020)) to predict localization [To ascertain the occurrence and cleavage site locus of the peptides, the proteins were characterized using SignalP 6.0 server (y 2020)) . Subsequlization .BepiPred-2.0 and ABCpred were used to predict the B-cell epitopes of varying lengths ,36.http://crdd.osdd.net/raghava/ifnepitope/ (accessed on 17 January 2020)) [The cytotoxic T lymphocyte (CTL) and helper T-cell (HTL) epitopes were predicted using a specific server. Nine residual lengths of CTL epitopes were deliberately selected using NetMHCpan 4.1  . While 1y 2020)) .Two MEP (multi-epitopic peptide) sequences were constructed using conserved, exposed potent epitopes. The MEPs were constructed by combining different epitopes using GPGPG linkers. To make the constructs more functional, PRIDE , CPP (cell-penetrating peptide), and EAAAK linker at N and C terminals were also added .http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html (accessed on 20 February 2020)) and AllerTOP v2.0 tools were used to predict the antigenicity and allergenicity of the proteins [http://web.expasy.org/protparam/ (accessed on 20 February 2020)) [TheVaxiJenV2.0 (proteins . The phyy 2020)) .p-value, uGDT , and GDT , and root mean square deviation (RMSD) [The input sequence was processed by the RaptorX server, which enables anticipation of 2D and 3D protein structures coupled with solvent accessibility (ACC) and disordered regions (DISO). Moreover, the server provides insight into the quality of constituted 3D models by elaborating different confidence score indices, viz. n (RMSD) .http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=REFINE (accessed on 22 February 2020)) [https://www.ebi.ac.uk/thornton-srv/databases/sas/ (accessed on 22 February 2020)) [The 3D model of the multi-epitopic peptides were later refined by GalaxyRefine server (y 2020)) . After sy 2020)) , SWISS-My 2020)) .The CPORT server was used for the prediction of active sites in toll-like receptors (TLRs) and vaccine constructs . The prehttp://imods.Chaconlab.org/ (accessed on 1 April 2020)). The server processed the assigned job by NMA  approach in dihedral coordinates  for setting biological macromolecules in naturally reproduced collective motion. Another (molecular dynamics simulation) MDS study was also carried out to obtain information on construct stability using the MDWEB server (https://mmb.irbbarcelona.org/MDWeb//index.php (accessed on 1 April 2020)).The molecular dynamics simulation study was conducted for the MEPs–TLR complex by recruiting the iMODSwebserver . The molecular weight of the VT1 and VT2 was estimated as 7997.09 Da and 6893.98 Da, respectively , and were soluble in water.v/v) and kept at 4 °C until use.The lyophilized MEPs (VT1 and VT2) were dissolved in autoclaved Milli-Q water (Milli-QW) to prepare a concentration of 8 mg/mL and kept at −20 °C until use. Peptides were mixed 24 h before immunization with 8% Montanide™ gel 01 PR  in a 40:60 ratio  for each MEP were used. For both the MEPs, two dosages were used, and in each dose, 2 rabbits were immunized. Rabbits of the control group were injected with adjuvant mixed with the same volume of autoclaved Milli-QW . A 500 µw/v BSA) and kept in an incubator set at 27 °C for 2 h. Subsequently, 100 µL of sera samples  in phosphate-buffered saline with 0.1% Tween 20 (PBS-T)) were added in each well and the plates were incubated at room temperature for 2 h with gentle continuous shaking. The HRP-conjugated anti-rabbit IgG and IgM  were added and incubated for 1 h at room temperature with gentle continuous shaking. A 100 µL of TMB  substrate solution was added into each well and incubated at 27 °C for 30 min in the humidified chamber in the dark. The reaction was stopped by adding 50 µL of stop solution/well (12.5% H2SO4) and the absorbance was measured at 450 nm wavelength using an ELISA reader . Each concentration of antigen, sera, and adjuvant was tested in triplicate well for each antigen. All other controls were maintained to compare the data.All the variables were optimized by employing the checkerboard method . IndirecY-axis) vs. log sera concentrations (X-axis) for each antigen concentration (µg/mL) was drawn and an optimum concentration at which the highest signal-to-noise ratio that presentsan acceptable background was selected. The immunization dose was selected on the basis of maximum IgG and IgM response obtained in the dose optimization experiment.A plot of OD (v/v). Rabbits were immunized (VT1 and VT2) with the selected doses given at14 day intervals.Based on the dose standardization experiment, doses for VT1 and VT2 were selected. For the challenge study, eighteen NZW rabbits were divided into three groups of six animals in each. Rabbits of the control group were injected with 8% Montanide™ gel 01 PR with Milli-QW in a 40:60 ratio (H. anatolicum larvae (generated from 25 mg of eggs/rabbit). Subsequently, on the 112th day, the immunized and control rabbits were again challenged by 10 pairs of adults to evaluate the immunization efficacy [All the rabbits of immunized and control groups were challenged on the 80th day of the first immunization with 14–18 day old efficacy ,47.The engorged nymphs dropped from each immunized and control rabbit were counted and kept at 28 °C and 80% humidity for moulting into adults . The numAfter three inoculations of two concentrations of VT1 and VT2, ELISA was conducted as mentioned above, employing optimized conditions. The blood was collected from day 0 to day 112 post-immunization (DPI), sera were separated and processed for monitoring IgG and IgM responses at different time points. The mean optical density was calculated at each point in individual immunized and control groups of rabbits and analyzed statistically.Relative quantification of the targeted genes in immunized and control rabbits were measured by Q-PCR method. The primers for the targeted genes were designed using Primer3Plus online tool  for optimum annealing/extension temperatures, the presence of any nonspecific reaction such asself-primer dimer, cross-primer dimer, and hairpin. The reaction mixture was prepared for a 10 µL reaction using the following components: cDNA generated from 4 µg of total RNA, 2X SYBR green PCR master mix and 1pg both of the primers were incorporated. The reaction conditions were optimized with initial denaturation of 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 52–54 °C for 1min.−∆∆Ct) programs were used for relative quantitation with a reference (NTC) and endogenous control (GAPDH). MicroAmp fast optical plate (96-well) was used for running the reaction. Prepared cDNA, which was later taken as the template , was used in the reaction mixture along with SYBR® Green master mix2X (5 µL), primers (1 pg each forward and reverse), and NFW . For each sample and control, reaction was kept in triplicate to minimize technical error. The reaction condition was initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing at 53 °C for 1 min.For quantitative/real time measurement of the expression profile of Th1- type  and Th2 –type  cytokines, two step-qPCR was performed, keeping rabbit GAPDH as an endogenous control/reference. Quantitation-comparative , and 82  using ELISA kits  following the manufacturer’s protocol and the data were compared with the control group of rabbits.Relative quantification of gene expression was performedfollowing the method illustrated by Livak and Schmittgen . For thet-test was carried out for comparing the OD values of sera collected from individual groups of rabbits and post-challenge entomological data collected from the immunized and control group of rabbits.All the data were subjected to a statistical analysis using GraphPad Prism9. The Upon an evaluation of the nature of selected proteins using SignalP 6.0 server, it is revealed that the targeted proteins except TPM have signal peptides(sec/SP1) with a single identified cleavage site. Using DeepLoc 1.0 server, the targeted proteins TPM, FER2, and VgR were predicted as soluble and are localized at the Golgi apparatus extracellularly and in plasma membrane, respectively .After meticulously screening a large number of B- cellepitopes using BepiPred-2.0 web and ABCpred servers, 29 B-cell epitopes were selected based on properties such asallergenic, antigenic, and physiochemical properties . FinallyCTL epitopes were selected from three targeted proteins using NetMHCpan 4.1 (Allele of BoLA) and CTLpred servers. Seventy-one CTL epitopes were selected based on their binding affinity to different BoLA, allergenicity, and antigenicity . Out of Initially, a total of seventy helper T cell epitopes were selected using NetMHCIIpan 4.0 (HLA class II based) and IEDB MHC II servers . FinallyThe antigenicity of VT1 and VT2 constructs were 0.46 and 1.046, respectively, when compared with the parasite proteins . Both the constructs were non-allergenic on both AllerTOP v2.0 and AllergenFP servers. Predicted pIvalues of VT1 and VT2 were estimated as 4.97 and 4.61, respectively, representing acidic in nature. The predictive instability index and other physiochemical parameters of VT1 and VT2 indicate stability of the constructs .In the predicted secondary structure of the peptides, the percentage of alpha-helices  was higher than coils  followed by beta-strands  . Howeverp-value of 4.12 × 10−5. Similarly, the tertiary structure of the VT2 construct was generated by Raptor-X Property and SWISS-MODEL. The Swiss Model of VT2 was selected because of the better quality structure than Raptor-X. The models of VT1 and VT2 were refined and validated by ProSA. The validated VT1 and VT2 models were used for docking with different TLRs such asTLR-2, TLR-4, and TLR-9.Similarly, the tertiary structures were generated using Raptor-X Property and SWISS-MODEL for VT1 and VT2, respectively A,B. The Out of all of the refined models, model 1 (first model on the server among the five refined models) was selected for both the MEPs. Essential parameters such as MolProbablity, RMSDclash score, poor rotamers, and Ramachandran plot were better in this case .Moreover, Ramachandra plot analysis also signifies better structure analysis in VT2 than VT1 and it reveals a Ramachandran plot score of 98 and 88%, respectively . FurtherMolecular docking assesses the interactions between a ligand and a receptor molecule to determine the stability and binding affinity of their docked complex. The docking was significant in both the peptides with high negative energy values for top-ordered protein–protein docking complex A–F. The The VT1 and VT2 constructs with TLR-9 produced a reliably stable simulation, as shown by normal mode analysis (NMA). The simulation study was conducted to specify the motions of molecules and atoms in the construct. The deformability graph of the complex depicts peaks in the graphs, which characterizes the regions of the protein with deformability A. The B-The optimum antigen concentration in ELISA was determined as 1.0 µg/mL and 500 ng/mL for VT1 and VT2, respectively. The 1:100 dilution of sera was optimized for both VT1 and VT2 while secondary antibody concentrations were determined as 1:5000 and 1:10,000 for IgG and IgM, respectively .p < 0.05) than in the control group. Based on the antibody levels, the in vivo trial dose was selected as 50 µg and 100 µg/rabbit for VT1 and VT2, respectively.The IgG and IgM levels were higher in animals inoculated with 50 µg dose of VT1 than the 100 µg dose group . A 4.6-fNo observable clinical signs nor any inflammatory reactions were seen at the injection site in any of the rabbits after immunization with VT1 and VT2 antigens.Against larvaeOn average, larvae fed for 14 days in the controlanimals while a mean feeding duration of 16 days was recorded in the VT1-immunized rabbits. Yet, a substantial amount of 67.9% of fewer nymphs dropped from the VT1-immunized rabbits than from the control rabbits. . InsigniAgainst adultsThe unfed adults started feeding within 24 h of release on the rabbit. After 12–14 days of feeding, engorged females started to drop from rabbits of all the groups. Significant differences in the number of dropped females were recorded amongst the groups. Further, significant differences in the engorgement weight and egg masses were recorded when the corresponding data were compared between the ticks fed on VT1, VT2, and control rabbits. The RI values of the engorged ticks showed significant differences  among all the groups. The DT %, DR %, and E % were 43.6, 44.4, 89.9 and 43.6, 43.03, 86.4 for VT1- and VT2-immunized rabbits, respectively .On the day of immunization (day 0) all the rabbits showed a baseline IgG and IgM with an OD ranging from 0.09 to 0.12 and 0.16 to 0.19, respectively. After immunization, the rabbits immunized with VT1 showed significantly higher levels of anti-VT1 IgG  and antiThe mean IgG OD value of the sera from VT2-immunized rabbits rose to 2.8 and 8.3 times as compared to the control rabbits on the 14th and 28th DPI. A maximum VT2-specific IgG response was observed at 28 DPI. The response was reduced on the 51st day and maintained until112 DPI . The IgGThe mean IgM OD values of the sera collected on the 14th and 51st DPI were 0.27 ± 0.01, 0.49 ± 0.01 and 0.59 ± 0.021, 0.21 ± 0.01 in VT1 and VT2 groups, respectively, and the values were nearly 1.5–3.3 and 1.4–2.7 times higher in VT1- and VT2-immunized rabbits, respectively, in comparison to control. A fall in IgM response after 51 and 14 DPI was noted in VT1- and VT2-immunized animals, respectively .t-test; p < 0.05). Similarly, a reduction in IL-4 level (35.66 µg/mL) was also observed in VT2-immunized animals as compared with the responses observed in the control rabbits (47.81 µg/mL). In contrast, the mean IL-2 level in the VT1 group of rabbits was 40.59 pg/mL as compared with the pre-challenge level of 28.31 pg/mL. The mean IL-4 level was significantly (p < 0.01) reduced in VT1-immunized (32.90 µg/mL) rabbits as compared with the control rabbits (47.81 µg/mL) , and 82  were determined by quantification of cytokines  immunized compared with the response observed in control rabbits. No significant difference was noticed in the mean IFN-γ level between the immunized and control rabbits. However, IL-2 level was significantly reduced in VT2-immunized rabbits after challenge infestations as compared with the pre-challenge level (unpaired 1 µg/mL) A–F.p < 0.001) IL-2 gene expression was observed in both VT1- and VT2-peptide-immunized rabbits. There was also a significant 20-fold increase in the IFN-γ level in VT2-immunized rabbits than the control rabbits. A significant reduction of the IL-5 level was recorded in both the immunized groups of rabbits. The comparative cytokines transcript level is shown in Relative gene expression was also determined by Q-PCR in terms of fold change. Comparatively, a higher level of  followed by coils and then beta-strands, respectively. In nature, the unfolded protein portion and alpha-helical coils have been reported to be important forms of “structural antigens” . The VT2P. falciparum multi-epitope vaccine. The results obtained in the form of B-factor and deformability plots indicated that the molecular structures of both of the peptides were stable, and the overall stability of the VT2 constructs was higher compared to the VT1.Docking of vaccine constructs were performed with TLRs that show favorable docking. The docking results were further validated by simulation studies. MD simulation study was carried out to attain information on construct stability using the MDWEB server. The residue-based RMSD plot peaked at more flexible residues, such as L14, G33, and Y38 in VT1. Both structures fluctuated, indicating their considerable flexibility and validating it as suitable vaccine structures. Similarly, Atapour et al.  recordedAt the initial phase of any infection, the TLRs trigger innate immune responses after the recognition of pathogen-derived compounds/molecules. The TLRs are type-I integral membrane receptors, with an N-terminal domain for the recognition of ligands and the cytoplasmic signaling domain at the C-termini . All theI. ricinus-specific neuropeptides. Both the neuropeptides  and myoinhibitory peptide (MIP) produced a significant level of IgG response at 73 DPI.Classical whole antigen-based vaccines have several detrimental effects such as an autoimmune response or allergic reactions. Synthetic peptide-based vaccines can be an alternate choice to overcome it. However, these peptides have low immunogenicity, and thus additional immuno-stimulating agents such as adjuvant or carrier molecules  are to bThe VT2 was constructed with one B-cell epitope (INF-γ-inducing) of Fer2 and two immunodominant TPM epitopes (CTL and HTL/B-cell). Finally, to potentiate CMI and humoral immunity, CPP was fused at the C terminal end of the VT2. This CPP has been recommended as an efficient mediator for vaccine delivery as it may improve the uptake of antigens by APCs and is considered as a classical adjuvant .H. anatolicum challenge following immunization of cross-bred calves by rHaFER2 and rHaTPM, respectively.Although several candidate proteins have been identified as potent vaccine candidates in different tick species ,69,70, FR. Sanguineus challenge infestations. It is also easy to reconstitute the immunization dose using the adjuvant and is cost-effective as a veterinary vaccine with low adverse reactions, as described by Guzman et al. [The antigen dose and adjuvants are the two most important parameters to decide for a better immune response. In the present study, two dosages were tested with 8% Montanide™ gel 01 PR to stimulate both humoral (HI) and cellular immune (CMI) responses. This adjuvant has the potential to stimulate both the immune systems following immunization with peptide-based vaccine . It prodn et al. . SimilarIn the present work, immunization with VT1 confers strong immunity in rabbits, resulted in quick larval detachment, delayed tick feeding, low engorgement weights (0.55 times), and reduced egg masses (0.31 times) with an overall efficacy (E%) of 93.3% and 89.9% against larvae and adults, respectively. Rabbits inoculated with VT1-induced robust antibody (IgG) response and multiple T cell-related inflammatory cytokines and thus indicated the role of humoral and cell-mediated immunity in conferring protection against challenge infestations.p < 0.05) level of protection against H. Anatolicum larval (96.9%) challenge in terms of the reduction in percentages of healthy and fully replenished nymphs dropped, along with an increased feeding period of ticks fed on the immunized group of animals in comparison to ticks fed on a control group of rabbits. An overall efficacy (E%) of 86.4% was obtained against adult challenge in terms of rejection in the number of attached and dropped ticks (DT% = 43.7), tick weight (DR% = 43.0), and egg masses (DO% = −54.1).An increased anti-VT2 IgG level (IgG) and pro-inflammatory cytokine (IL-2) and decreased anti-inflammatory cytokine IL-4 are most likely responsible for conferring a significant (p < 0.001) reduction in the anti-inflammatory cytokine (IL-4) expression was observed in the VT1- and VT2-immunized group of rabbits as compared with the response observed in the control group of rabbits. This finding was contradictory to the results obtained by Sajid and co-workers [I. Scapularis engorgement and prevented transmission of Lyme disease. Another peptide-based vaccine against mosquito has already been under phase 1 trial on49 healthy human volunteers. The salivary protein A. gambiae (AGS-v) has been reported to producea significant IgG antibody response in addition to a significant increase in INF-γ levels expression by the PBMCs at 42 DPI [The importance of inducing both HI and CMI responses in conferring immunity against challenge infestations/infection is well documented . Likewis-workers , where a-workers ,57,58. A-workers , where pt 42 DPI .Although the minimum number of rabbits in place of cattle for immunization was used as a pilot experiment, the results obtained clearly demonstrated the suitability of MEP-based vaccine for a higher efficacy against challenge infestations.