This effect relies both on Ca2+ release from the endoplasmic reticulum (ER) and extracellular Ca2+ entry. The latter depends on stromal interaction molecules (STIM)-Orai1 interaction or transient receptor potential channel (TRPC) involvement. After Ca2+ entry, the activation of outward K+-Ca2+-dependent currents, mainly the KCa3.1 currents, provides a membrane polarizing influence which offset the depolarizing action of Ca2+ elevation and indirectly maintains the driving force for optimal Ca2+ increase in the cytosol. In this review, we address the main endocrine and extraendocrine actions displayed by TLQP-21, highlighting recent findings on its mechanism of action and its potential in different pathological conditions. VGF gene encodes for a neuropeptide precursor of 68 kDa composed by 615 (human) and 617  residues, expressed prevalently in the central nervous system (CNS), but also in the peripheral nervous system (PNS) and in various endocrine cells. This precursor undergoes proteolytic cleavage, generating a family of peptides different in length and biological activity. Among them, TLQP-21, a peptide of 21 amino acids, has been widely investigated for its relevant endocrine and extraendocrine activities. The complement complement C3a receptor-1 (C3aR1) has been suggested as the TLQP-21 receptor and, in different cell lines, its activation by TLQP-21 induces an increase of intracellular Ca However, it can also enter directly into the cells from the extracellular space to deliver information, without the intermediation of other first or second messengers. The most distinctive feature of Ca2+ is its Janus-faced action: while it is crucial for the homeostasis and the correct functioning of the cells, an uncontrolled excess of its influx or release from intracellular stores can turn it into a toxic agent leading to cell apoptosis or necrosis [2+ concentrations across extracellular and intracellular compartments of the cells is essential for cellular homeostasis. At resting state, free intracellular Ca2+ levels are strictly kept low (10–100 nM), in comparison to the endoplasmic/sarcoplasmic reticulum (ER/SR) levels (60–500 µM) [2+ signals are generated within a wide spatial and temporal range through a huge variety of Ca2+ channels as well as pumps and exchangers located in the PM and/or ER/SR. The increase of the intracellular Ca2+ concentration can result from the activation of cation channels at the PM, like: (i) Transient receptor potential channels (TRPC), a large group of non-selective cation channels activated by a several stimuli and mostly permeable to Ca2+; (ii) Voltage-gated ion channels (VGCs), activated by changes in the electrical PM potential near the channel proteins or mechanosensitive channels; (iii) ER/SR-associated inositol-3-phosphate receptor (IP3R) and ryanodine-receptor (RyR) channels; and iv) Ca2+ release activated Ca2+ (CRAC) channels composed of two cellular proteins, Ca2+-sensing stromal interaction molecule 1 (STIM1) and pore-forming, Orai1. The depletion of intracellular Ca2+ stores is sensed by STIM which interact with CRAC-Orai1, triggering store-operated Ca2+ entry (SOCE) currents, exploited to regulate basal Ca2+, to refill intracellular Ca2+ stores, and to execute a wide range of specialized activities. On the other side, the systems for Ca2+ extrusion consist of PM exchangers, as well as the low-affinity high-capacity sodium-calcium exchanger (NCX), the high-affinity low-capacity Plasma Membrane Calcium ATPase (PMCA), and the sarco-endoplasmic reticulum calcium ATPase (SERCA) pump, designed for Ca2+ sequestration in the ER/SR [2+ surge, which in turn activates a variety of signaling pathways. Calcium  . Ca2+ sihe ER/SR . Binding2+. This review aims to summarize the recent advances in TLQP-21 investigations. We then provide an integrated view on the recent reports supporting the role of Ca2+ in mediating TLQP-21 mechanism of action.VGF is a large neuropeptide precursor that generates a family of peptides endowed with different biological activities . Among tVGF was discovered in 1985 in the framework of the studies designed to study neurotrophins and growth factors role in the development, maintenance, and normal functioning of the peripheral and central nervous systems (PNS and CNS) . VGF , and variably extended to the VGF precursor C-terminus. The number is the amount of amino acid residues composing the peptide [By convention, the acronym TLQP designates the 4 N-terminal amino acid sequence of the peptides, i.e., Thr-Leu-Gln-Pro, resulting from the cleavage on the VGF precursor, at the specific R-P-R (Arg-Pro-Arg) processing site found at rat/mouse VGF peptide , whilst  peptide ,20. The  peptide . Consist peptide ,23, high peptide .In recent years, TLQP-21 has been widely investigated for its biological effects and functions. Studies in this field were prompted by the observation that VGF knockout mice (KO) are lean, hypermetabolic, hyperactive, and possess an obesity resistant phenotype, characterized by markedly reduced leptin levels and fat stores . These fTLQP-21 appears to be a multifunctional peptide involved in various endocrine functions. Depending from the source of its release (neurons or endocrine cells), it is indeed difficult to establish a clear endocrine effect or a paracrine or autocrine action. Despite the half-life after a single i.v. injection is initially of 0.97 min, the complete terminal value is about 110 min due to tissue uptake and renal clearance . Parente2+ mobilization from thapsigargine (TP) sensitive stores  and prolactin (PRL), TLQP-21 induces differentiation toward a more lactogenic phenotype, increasing PRL and decreasing GH production . This isores see . There aVGF immunofluorescence was detected in gonadotropic pituitary cells where it was modulated during estrous cycle . Two difVGF and its derivatives are highly expressed in pancreatic islet β-cells where VGF plays a critical role on biogenesis of the secretory granules, increasing insulin cargo transport ,33. A paTLQP-21 is highly expressed in enterochromaffin and neuronal cells of the stomach . One of On pancreatic lobules TLQP-21 induced ex vivo an increase of pancreatic amylase release, but failed to stimulate directly the acinar cells, suggesting an indirect mechanism of action .3-adrenergic receptor (AR), peroxisome proliferator activated receptor (PPAR)-δ and uncoupling protein (UCP)-1 in the white adipose tissue (WAT) suggested an additional peripheral catabolic action [TLQP-21 showed a specific role in nutrition and metabolism . Chronicc action . Indeed,c action ,40. The c action . By intec action . In Sibec action . VGF ovec action . Moreovec action . This coc action ,44. It is challenging to exactly define TLQP-21 actions, since the source of VGF-derived peptides have not been completely ascertained. In this section we will address TLQP-21 targets that are not directly involved with endocrine functions.VGF expression has been implicated in different models of neuroprotection and antidepressant action . Only fe2+-dependent mechanisms [TLQP-21 increased cell survival and decreased apoptotic death in primary cell culture of cerebellar granule cells . The cerchanisms . Concerning VGF and its derived peptides, recent studies indicated a strong role in cerebellar development and function ,48. VGF Finally, recent evidence shows that TLQP-21 could be considered a biomarker for neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS) . Indeed,Among the extraendocrine actions, a link between VGF-derived peptides and mood disorders has been proposed. Recent studies, in fact, have highlighted a possible role for VGF-derived peptides in depression and neuropsychiatric disorders, for which social stress is a known key-player ,55,56,57These interesting results have brought to surface two important aspects. First, the results presented by Bartolomucci and coworkers appear somehow in contrast with other observations showing that the lack of VGF gene, and therefore assuming the absence of VGF-derived peptides, increased depression-related behavioral changes. In the same direction is the observation that the acute treatment with some of VGF peptides  is associated to anti-depressant like effects in rats and mice . HoweverThe importance of the route of administration in TLQP-21 actions is underlined also by a study showing that TLQP-21 could regulate pain ,63. By mAnother interesting study provided evidence showing that TLQP-21 participates in mediating spinal neuroplasticity after inflammation and nerve injury . Fairban2+ surge, in a dose dependent manner, in macrophages and primary microglia [2+ mobilization in other cell lines, including N38, N41, and N42 cells [2+ release is specific for TLQP-21 and was not replicated with other VGF-derivatives or with LRPS-21, a peptide that contains the same 21 amino acid residues of TLQP-21, but arranged in a scrambled order to avoid any homology with other known proteins [Considering the plethora of actions displayed by TLQP-21, the need of identifying a specific receptor became compelling to focus on novel targets for pharmacological intervention. The first efforts made in this direction have shown that TLQP-21 was able to induce a transient intracellular Caicroglia , GH3 piticroglia , primaryicroglia  Chinese icroglia ,66, N9 microglia , and RAWicroglia , indicat42 cells ,65,66;  as the target site of TLQP-21 . C3aR1 i2+ increase only after ATP priming: this process seemed not to be coupled to cAMP or inositol phosphate accumulation, but likely mediated by a Go protein, since it could be blocked by pertussis toxin (PTX) [q protein-mediated mechanism, by which the rapid Ca2+ mobilization induced by TLQP-21 is explained by the release of Ca2+ from intracellular stores, in particular the ER, whereas the mitochondria did not appear involved [2+-ATP-ase pump responsible for Ca2+ sequestering in the ER and depletes the store by irreversibly preventing its refilling, reduced the Ca2+ mobilization stimulated by TLQP-21 [2+ secretion from ER store and the subsequent Ca2+ entry from the outside of the cell. The Ca2+ influx could depend on STIM-CRAC-Orai1 interaction, as demonstrated by data showing that SKF-96365 and YM-58483, two inhibitors of STIM and CRAC-Orai1, respectively, both inhibit TLQP-21-stimulated Ca2+ release [2+-chelator, prevented TLQP-21-induced lipolysis mediated by HSL activation via Ca2+ entry from the extracellular space [2+ [2+ entry stimulated by TLQP-21, the activation of outward K+-Ca2+-dependent currents, mainly the KCa 3.1 current, provides a membrane iperpolarizing effect which offsets the depolarizing action of Ca2+ elevation and indirectly maintains the driving force for optimal Ca2+ increase in the cytosol [In CHO-K1 cells the binding of TLQP-21 on C3a1R stimulated an intracellular Cain (PTX) . Howeverinvolved . Actuall TLQP-21 . Accordi release . In agrear space . Howeverpace [2+ . Experim cytosol . Figure 2+ can be induced by stimulation with C3a, the natural ligand of C3aR1, and C3a(70–77) peptide, an active fragment of C3a, as well as with TLQP-21 and JMV5656, a novel short analogue of TLQP-21 (TLQP-219-21) [2+ release elicited by subsequent stimulations with JMV5656, a likely index of homologous receptor desensitization; 2) repeated C3a(70−77) stimulations, instead, consistently elicited full intracellular Ca2+ responses; 3) the use of siRNAs against C3aR decreased intracellular Ca2+ mobilization stimulated by C3a but not JMV5656 [Although C3aR1 has been proposed as the putative receptor for TLQP-21 , some da-219-21) . However JMV5656 . AlthougRAW264.7 express also significant amounts of mRNA for gC1qR , the recThe VGF-derived peptide TLQP-21 has been extensively investigated because of its various actions and properties in different experimental settings. Some of its properties have been characterized, but others should be object of further studies in future, with the aim to better understand its specific role. In this context, the knowledge of the potential activity of TLQP-21 in preventing or reducing motor neuron death should be a stimulus to engage further studies aimed to address the possible relevance of TLQP peptides for new therapeutic approaches in neurodegenerative diseases. On the other hand, the possible role of TLQP-21 and other VGF-derived peptides as potential biomarkers of the early phases of disease onset and/or progression in ALS is also worth of further investigations. Finally, several tiles of the complex mosaic of TLQP-21 mechanism of action have been sketched out, but many others have yet to be clarified. The identification of the real specific receptor(s) is a starting point to boost the pharmacological research for the development of novel agonist and antagonist of TLQP-21.


ORAI1 calcium channel in neurons of murine brain led to spontaneous occurrence of seizure-like events in aged animals of transgenic line FVB/NJ-Tg(ORAI1)Ibd . We aimed to identify the mechanism that is responsible for this phenomenon. Using a modified Ca2+-addback assay in the CA1 region of acute hippocampal slices and FURA-2 acetomethyl ester (AM) Ca2+ indicator, we found that overexpression of ORAI1 in neurons led to altered Ca2+ response. Next, by RNA sequencing (RNAseq) we identified a set of genes, whose expression was changed in our transgenic animals. These data were validated using customized real-time PCR assays and digital droplet PCR (ddPCR) ddPCR. Using real-time PCR, up-regulation of hairy and enhancer of split-5 (Hes-5) gene and down-regulation of aristaless related homeobox (Arx), doublecortin-like kinase 1 (Dclk1), and cyclin-dependent kinase-like 5 ) genes were found. Digital droplet PCR (ddPCR) analysis revealed down-regulation of Arx. In humans, ARX, DCLK1, and CDLK5 were shown to be mutated in some rare epilepsy-associated disorders. We conclude that the occurrence of seizure-like events in aged mice overexpressing ORAI1 might be due to the down-regulation of Arx, and possibly of Cdkl5 and Dclk1 genes. Previously, we showed that the overexpression of  They include maintaining Ca2+ oscillations [2+ stores, and a few other [2+ concentration inside the endoplasmic reticulum (ER) [2+ channels at the plasma membrane (PM) [2+-selective Ca2+-release-activated Ca2+ channels (CRACs) – ORAIs [Orai1 reduces, whereas deletion of Orai2 increases SOCE, which indicates that ORAI2 is a less conductive subunit of CRAC channels and enables shaping of the magnitude of SOCE and modulation of the immune response [Orai1 and Orai3 in DRG neurons reduced SOCE compared with Orai1 or Orai3 knockdown alone [, whereas ORAI3 is abundant in many solid organs and its enhanced gene expression is reported in cancerous cells [ORAI1 suffer from severe combined immunodeficiency-like disease that is accompanied with chronic, often lethal infections and a variety of non-immunological symptoms [Store operated calcium entry (SOCE) is a well-described process in non-excitable cells. It was initially known under the name “capacitative Ca+ entry”  and has + entry” ,4,5. In  current ,7,8. Dur current ). Reductllations , mitochollations , refilliew other . The esslum (ER) ,14 and Oane (PM) ,16. Upon – ORAIs . CRAC ch – ORAIs ,19. Hete – ORAIs  and in m – ORAIs , whereas – ORAIs , dorsal  – ORAIs  and in o – ORAIs . In muriresponse . Ecksteiresponse . In contwn alone . These mwn alone . All of wn alone  and ORAIwn alone , whereasus cells . Physiolus cells ,29. Patisymptoms ,31. ORAI1 in the murine brain leads to spontaneous occurrence of seizure-like events in aged animals [ORAI1 in neurons (FVB/NJ-Tg(ORAI1)Ibd; Nencki Institute of Experimental Biology) by the analysis of Ca2+ response in the hippocampal CA1 pyramidal cells and RNA sequencing (RNAseq) gene expression profiling. Differential expression of selected genes was next confirmed by real-time PCR and validated using the digital droplet PCR (ddPCR) approach. The observed changes in Ca2+ signals after glutamate treatment or after ER Ca2+ store depletion suggest a new potential site of action for ORAI1. Our observations were supported by the identification of a set of genes, whose expression was changed in FVB/NJ-Tg(ORAI1)Ibd mice. For some of these genes, such as cyclin-dependent kinase-like 5 Cdkl5, aristaless related homeobox (Arx), and doublecortin-like kinase 1 (Dclk1), mutations in their human homologoues that lead to rare epilepsy-associated disorders were reported. Together, our results provided novel insight into the Ca2+ dependent actions evoked by CRAC channels that might underlie the occurrence of seizure-like episodes in FVB/NJ-Tg(ORAI1)Ibd mice.Our recently published results indicate that overexpression of  animals . In the ORAI1 mice were analyzed by fluorescent imaging. Calcium measurements were performed using Fura-2 acetomethyl ester (AM) indicator that was loaded into the cells by gradual pipetting over the slice with the use of an automated perfusion system. To assess Ca2+ homeostasis in the ORAI1 overexpressing neurons, we used a Ca2+ addback assay that was modified to enable the measurement of Ca2+ release from the ER [p-values [2+ was slightly higher in neurons of transgenic ORAI1 mice. Stimulation by glutamate led to comparable peaks in all genetic variants tested (ORAI1 overexpressing neurons (2+ levels persisted during the subsequent application of ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA)  or Ca2+ uptake into the ER. Moreover, a tendency towards the increased influx of Ca2+ ions upon Ca2+ addback was observed both in ORAI1 and STIM2 overexpressing neurons. Acute brain slices of hippocampi of wild-type and transgenic m the ER . Accordip-values . Also, ts tested B,F. Howe neurons D. The red (EGTA) A,C. Thes)  and of FVB/NJ-Tg(ORAI1)Ibd  females and performed RNAseq experiments. Based on the collected RNAseq data, we performed functional  analysis of the genes that were differentially expressed between the hippocampi of wild-type and of FVB/NJ-Tg(Orai1)Ibd females. As shown by the Gene Ontology Biological Process groups (GO BP)  A, left,  (GO BP) A, right. (GO BP) . Among tnic mice C. The linic mice B, was fuWe next performed real-time PCR analysis to verify the RNAseq data using customized arrays designed for the 23 genes that are listed in Uba-2 , Gapdh , and Actin  were selected, which meet the criteria of the reference gene selection tool . RNA was isolated from hippocampi of wild-type  and of FVB/NJ-Tg(ORAI1)Ibd females . The majority of genes showed a similar level of expression  , Dclk1 (Serine/threonine-protein kinase) , and Cdkl5 (Cyclin-dependent kinase-like 5) . Human mutations in these genes are associated with epilepsy. Also Gabrb2 (Gamma-aminobutyric acid receptor subunit beta-2) and Scn9a  genes, which are implicated in epilepsy, showed altered expression in the transgenic animals . Because the observed changes in 2−∆∆Ct values were relatively modest, to validate the changes in expression of the selected genes we used a novel technique, digital droplet PCR . This powerful method is based on partitioning of one PCR reaction mixture into up to 20,000 independent reactions, which resulted in high precision, sensitivity, and accuracy of the data [Uba-2 gene as a reference, we observed a reduction in the copy number of Arx  in transgenic females. The differences in the copy number of Cdkl5 and Dclk1 were insignificant . As the reference genes, pression  that wasthe data . To fulfIn the next set of the real-time PCR analysis based on RNAseq data, we designed an additional 15 pairs of primers .Uba-2 as a reference gene, we identified an up-regulation of Hes-5 (Transcription factor HES-5) expression  (Strap (Serine-threonine kinase receptor-associated protein). The protein is a part of the survival of motor neurons (SMN) complex that acts as a catalyst in the assembly of small nuclear ribonucleoproteins (snRNPs), the building blocks of the spliceosome .Using the matrixes that were derived from previous experiments (6 wild-type vs. 6 transgenic mice) and  0.0039) . Among t2+ channels, is far from being understood. It becomes clear that not only ORAI and transient receptor potential cation channels (TRPCs) [2+ microdomains that appear upon opening of store operated calcium channels activate downstream effectors like calcineurin or adenylyl cyclase in non-excitable cells. The wide scope of processes mediated by Ca2+ nanodomains that are generated by SOCE is well described in a recent review by Barak and Parekh [The role of SOCE in neurons and its components, such as STIM proteins and Ca (TRPCs) , but als (TRPCs)  and α-am (TRPCs) ,39 are s (TRPCs) . On the 2+ that is delivered by nSOCE is responsible for Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathway activation, which contributes to spine maintenance of mushroom spines [2+ disruption leads to synaptic loss and cognitive decline in aging and Alzheimer’s disease [2+ stores, whereas ryanodine-sensitive stores are VGCC dependent. The authors postulate that their observations may help to explain the distinct roles of inositol triphosphate receptor (IP3Rs) and RyRs for various forms of synaptic plasticity. In hippocampal neurons nSOCE is believed to be mediated mainly by STIM2 together with Orai2 and Trpc6. It was suggested that Cam spines . Ca2+ di disease ,42,43. T disease . They deORAI1 transgene overexpression in mice used in our studies, we modified the level of ORAI1 in neurons, especially in the hippocampus, in which the predominant isoform is ORAI2. The observed trend of a higher amplitude of nSOCE in neurons of Tg(ORAI1)Ibd transgenic mice is in line with the observation by Vaeth et al. (2017), who showed that ORAI2 was a less conductive subunit of CRAC channel in immune cells [2+, in which we observed faster decay of the signal in ORAI1 overexpressing neurons . They showed the importance of ORAI2-mediated nSOCE in refilling of IP3 sensitive Ca2+ stores [2+ release from the ER, subsequent ORAI-dependent refilling of ER Ca2+ stores might be altered in neurons of our transgenic line [As a consequence of h et al. 17, who s neurons . Because+ stores . Due to nic line . These o2+ microdomains that are generated by nSOCE serve as a regulatory component of the translation process [RNAseq analysis of the material from Tg(ORAI1)Ibd transgenic line identified up-regulation of the genes that belong to the GO biological processes that are involved in the ribonucleoprotein complex biogenesis, ribosome biogenesis, and assembly, which are all components of translational machinery. It was demonstrated in acinar polarized cells that overexpression of STIM1 increased the density of ribosome-free terminals in rough ER (RER), and facilitated the formation of ER-plasma membrane (ER-PM) junctions and SOCE propagation. If our future validation of these RNAseq data by real-time and ddPCR is positive, it may suggest that Ca process ,46. Arx, Cdkl5, and Dclk1 genes that we observed in the present work is in line with the report that loss of function mutations of human aristaless related homeobox (ARX), CDKL5, and DCLK1 are found in epileptic patients [2+ nanodomains that appear locally by the activity of CRAC channels affect the expression of these genes via downstream pathways.As previously reported by our group, overexpression of ORAI1 in neurons induced seizure-like symptoms in aged female mice . Applicapatients  Ibd line. In conclusion, our data indicate that overexpression of ORAI1 changed the subunit composition of CRAC channels, which caused differences in the observed CaThe mice were housed under standard conditions on a 12 h/12 h light/dark cycle with food and water available ad libitum. All of the animal experiments were approved by the Local Commission for the Ethics of Animal Experimentation No. 1 in Warsaw  and performed in accordance with the European Communities Council Directive . All analyses described in the text were performed on 7 ±1-months-old female mice. XhoI and subcloned into the XhoI site of the modified mouse Thy-1.2 expression cassette [PvuI and NotI and used for typical pronuclear microinjection [The details on the generation of the transgenic lines were described elsewhere . In briecassette ,67. The njection . Offspri2PO4, 1.25, NaHCO3, 30, MgSO4, 10, CaCl2, 0.5, Na-ascorbate, 5, Na-pyruvate, 3 N-acetyl-L-cystein , 3. Sections that were 350 μm thick were obtained with the use of a vibratome . During the slicing procedure, the tissue was submerged in ice-cold NMDG-aCSF solution that was bubbled with carbogen (95%/5% O2/CO2). Next, the sections were incubated in carbogenated NMDG aCSF for 10 min (temperature: ~33 °C). A 2M NaCl solution was gradually added to the solution according to [2PO4, 1.25, NaHCO3, 30, MgSO4, 2, Na-ascorbate, 5, Na-pyruvate, 3, and 12 mM NAC (temperature: ~25 °C). The recordings were started after 1 h of incubation in HEPES-aCSF. The pH of all of the solutions was between 7.3 and 7.4. The reagents were purchased from Sigma unless indicated otherwise. Hippocampal sections were prepared according to the protective recovery method . Mice ofrding to . The sli2PO4, 1.25, NaHCO3, 25, MgSO4, 1, and CaCl2, 2.5; the pH was adjusted to 7.3–7.4) to a final concentration of 50 µM. The solution was supplemented with Pluronic F-127  at a final concentration of 2.5%. The dye solution was applied locally above the hippocampal slice with the use of Perfusion Pencil , for 20 min. During the loading procedure, the slices were perfused with aCSF solution at a rate of 4–5 mL/min, at 33 °C, to facilitate incorporation of the dye molecules by the cells. After the loading, the slices were allowed to rest for 20 min, under a constant perfusion of aCSF at a temperature of 25 °C. The consistency of the loading procedure was checked by acquiring fluorescent Fura-2 signal at its isosbestic point (λ = 360 nm). The intensity of the signal excited at this wavelength should not depend on Ca2+ concentration and thus only reflect the quantity of the incorporated and de-esterified dye alone. The signal was comparable across preparations. Moreover, we did not detect any correlation between the loading efficiency and the magnitude of the recorded Ca2+ responses.A 1 mM stock of Fura-2 AM (Thermo-Fisher) that was dissolved in DMSO was diluted in artificial cerebrospinal fluid (aCSF) . For the first 10 min of the recordings , 2.5 mM CaCl2 was included in the solution that was perfused with the use of the pump. Next, the solution was manually changed to Ca2+-free aCSF that contained 0.5 mM (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) EGTA. The pH of aCSF was adjusted to 7.4 at room temperature with carbogen and HCl. The extracellular solution was carbogenated throughout the recordings and heated with an in-line heater  to maintain the temperature in the recording chamber close to 25 °C. The rate of solution flow was 4–5 mL/min.The measurements were performed using MetaFluor software, a Zeiss Examiner Z.1 microscope (40×/1.0 objective), and the Retiga ELECTRO™ CCD Camera . A peristaltic pump was used to perfuse the hippocampal slices with aCSF that contained 126 mM NaCl, 2.6 mM KCl, 20 mM glucose, 1.25 NaH2+ homeostasis in the CA1 pyramidal neurons of the hippocampus, a Ca2+ addback assay that was modified to allow for the quantification of the Ca2+ efflux from the ER [2+ was perfused for 5 min, which was followed by the local application of 100 µM Na-glutamate for 10s. Next, the signal relaxation was monitored for another 5 min in 2.5 mM Ca2+ aCSF. To deplete the extracellular milieu from the Ca2+ ions, the solution was changed to aCSF that contained 0.5 mM EGTA for 15 min, which was followed by the local application of 20 μM cyclopiazonic acid (CPA) for 5 min. Next, Ca2+ was re-added to the solution at a concentration of 5 mM Ca2+ for 5 min, in the presence of 20 μM CPA. To limit activity-driven Ca2+ entry, 1 μM Tetrodotoxin (TTX) and 10 μM nifedipine were included in all solutions that were used from the 10th min of the experiment onwards. To assess Cam the ER  was used2, 2, MgCl2, 1, pH = 7.3–7.4) was perfused for 5 min, which was followed by application of Ca2+-free Ca2+ depleting solution (Ringer’s solution supplemented with 0.5 mM EGTA and 20 μM CPA) for 15 min. Next, Ca2+-free Ringer’s solution supplemented with 2 mM Mn2+ (as MnCl2) was locally applied above the cells of interest, in the absence of EGTA and presence of 20 μM CPA. To limit activity-driven Ca2+ entry, 1 μM TTX and 10 μM nifedipine were included in all solutions. To quantify the rate of Mn2+ influx, linear part of the curves following Mn2+ addition (which corresponded to the first 25 s of treatment) were approximated with a linear function and the decay slope was calculated .For Fura-2 quenching experiments, hippocampal slices were loaded with the dye as described above. For baseline recording, Ringer’s solution . Student’s p-value below 0.05), separately for genes up- and down-regulated in transgenic animals. The functional analysis of data was performed using clusterProfiler [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138370).Total RNA was extracted from three individual hippocampi per variant using RNeasy Lipid Mini Kit  and treated on-column with DNase using the RNase-Free DNase Set (Qiagen: 79254). Preparation of cDNA libraries and sequencing by Illumina HighSeq 1500  were carried out in cooperation with Core Facility of IIMCB and the Nencki Institute. Sequencing reads (fastq) were filtered by Trimmomatic program, discarding all reads contaminated by sequencing adapter sequence as well as discarding reads whose mean quality in a bin of 20 base pair (bp) was below Q30. Minimal length of read used for mapping was set as 65 bp. Quality filtered reads were aligned to mouse genome (mm10) by STAR program. Statistical analyses, including comparisons to other samples, were done in R environment using NOIseq library . FunctioProfiler  to find Profiler  and are Uba-2), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and Actin (Actb) were used as reference genes. The reactions were performed using the CFX Connect™ Real-Time PCR Detection System (Bio-Rad). Bio-Rad CFX Maestro 1.1 software was used to analyze the data . The reference genes’ stability was acceptable . For calculation of the expression levels, equal efficiencies were assumed for all reactions and the 2−∆∆Ct method was used. For each gene, the average normalized expression was calculated from the following formula:(−CtGOI).Hippocampi were dissected from three adult female mice ) per variant for real-time polymerase chain reaction arrays. The material was homogenized in Qiazol  and subjected to total RNA isolation according to manufacturer’s protocol of RNeasy Lipid Tissue Mini Kit . The quality of RNA was checked by absorbance measurements at 260, 280, and 230 nm. For the analysis, only samples whose A260/280 nm and A230/280 nm values exceeded 1.8 were used. There were 1000 ng of RNA templates used to synthesize first-strand cDNA, with the use of iScript reverse transcription supermix . The 96-well custom plates that contained primers for target and reference genes  and SsoAdvanced Universal SYBR Green Supermix  were used to perform teal-time polymerase chain reaction . The cDNA (50 ng) was used for each reaction. Small Ubiquitin-like Modifier (SUMO)-activating enzyme subunit 2 , followed by Tukey’s Honestly Significant Difference (HSD) post-hoc test.SUMO-activating enzyme subunit 2 (Uba-2) was chosen as reference gene. The specificity of the reactions was determined based on dissociation curve analysis. Relative gene expression was calculated using the 2−ΔΔCt method and Bio-Rad CFX Maestro 1.1 software . Six adult female mice , per variant were taken to perform this experiment.The real-time PCR reactions were performed in duplicate for each sample using SsoAdvance Universal SYBR Green Supermix (Bio-Rad) with the following sets of specific gene primers  and diluted according to the kit instructions. For ddPCR technology  System—Bio-Rad, USA) 21 μL reaction mixtures containing 25 ng of cDNA (six transgenics mice vs. 3 wild-type mice variant), primers, and QX200™ddPCR™ EvaGreen Supermix (Bio-Rad: 186–4034) were used. Droplet generation and transfer of emulsified samples to PCR plates (Bio-Rad: 12001925) were performed according to the manufacturer’s instructions . The ddPCR plate was sealed with a foil heat seal  and the PX1™ PCR Plate Sealer . The following cycling protocol was established according to the manufacturer’s protocol: 95 °C enzyme activation step for 5 min followed by 40 cycles of a two-step cycling protocol (95 °C for 30 s and 58 °C for 1 min). The ramp rate between these steps was slowed to 2 °C/second. The post-cycling protocol was in accordance with the kit instructions . The absolute quantity of DNA per sample (copies/μL) was processed using QuantaSoft version 1.7.4.0917 . The data were next exported to Microsoft Excel for further analysis. Primers for


LASIK Xtra is a recently described technique which combines LASIK and accelerated corneal cross-linking(CXL) in the same setting. Its long-term outcome in Asians with high myopia is not well described.To compare the efficacy, predictability and safety of LASIK Xtra with LASIK in patients with high myopia.This is a retrospective study comparing 50 consecutive eyes undergoing LASIK Xtra for the correction of high myopia and/or myopic astigmatism  with a matched control group of 50 eyes undergoing LASIK alone for correction of high myopia . Mean follow-up was 5.7 months  for LASIK Xtra and 3.6 months  for LASIK only. Outcome measures included Uncorrected Distance Visual Acuity (UDVA), Corrected Distance Visual Acuity (CDVA), refraction and intraoperative and postoperative complications.p = 0.115). Efficacy indices were 0.99±0.17 for LASIK Xtra and 0.94±0.17 for LASIK only (p = 0.164). The proportion of eyes within ±0.50 D of attempted correction was 84% in the LASIK only group and 72% in the LASIK Xtra group at post-operative 3 months (p = 0.148). Safety indices were 1.11±0.19 and 1.11±0.18 in the LASIK Xtra and LASIK only groups, respectively (p = 0.735).At post-operative 3 months, all eyes achieved UDVA of 20/40 or better, and 80.0% of LASIK Xtra eyes achieved UDVA of 20/20 or better, compared to 66.0% of LASIK only eyes (LASIK Xtra achieved comparable safety, predictability and efficacy as LASIK in patients with high myopia. Good refractive stability was attained at 6-12 months. Further long term studies are required to determine whether simultaneous CXL is able to reduce postoperative LASIK keratectasia in high-risk individuals. It results in faster visual recovery and causes less discomfort compared to surface ablation techniques such as photorefractive keratectomy [Laser atectomy .LASIK keratectasia is a rare but serious complication of the LASIK procedure. Although the mechanisms of postoperative LASIK keratectasia remain unclear, LASIK may reduce corneal biomechanical stability by severing structural lamellae in the anterior cornea during creation of the LASIK flap and removing additional structural lamellae during laser ablation, leading to this complication . Risk faTreatment options for postoperative LASIK keratectasia include rigid gas permeable contact lenses, intrastromal corneal ring segments, and penetrating or lamellar keratoplasty . CornealLASIK Xtra is a recently described technique which combines LASIK and accelerated CXL in the same setting. There are limited studies in the literature which evaluate the outcomes of LASIK Xtra and even fewer that evaluate long-term outcomes in Asians with high myopia. A few studies have reported that LASIK Xtra may confer additional benefits of early refractive and keratometric stability after LASIK, improving the predictability of refractive outcomes in patients with high myopia -14. In t2In this retrospective study, 50 consecutive eyes of 26 patients undergoing LASIK Xtra for the correction of high myopia and/or myopic astigmatism, defined as myopia greater than -6.00 diopters (D) of spherical equivalent, were recruited at the Laser Vision Centre, Singapore National Eye Centre (SNEC), from April 2013 to December 2015. These were compared with a matched group of 50 eyes of 36 patients which had undergone LASIK alone for the correction of high myopia (greater than -6.00 D) during the same period. Patients returned for 1 day, 1 week, 1 month, and 3 month follow-up after the initial procedure for both the LASIK only and LASIK Xtra groups. A longer follow-up was obtained for the LASIK Xtra group. A comprehensive 100% clinical audit of all LASIK and LASIK Xtra cases in SNEC was performed independently by our Clinical Audit Department. The study was approved by the Sing Health Centralised Institutional Review Board.Inclusion criteria for surgery were no soft contact lens wear for 1 week before surgery and rigid contact lens wear for 3 weeks prior; stable refractive error for 12 months before surgery; normal peripheral retina or after prophylactic treatment with photocoagulation; no previous ocular surgery, no corneal diseases, no glaucoma; and no history of ocular trauma. Exclusion criteria for surgery were keratoconus or forme fruste keratoconus as evidenced by corneal topography, active ocular or systemic disease likely to affect corneal wound healing, pregnancy, or nursing females. Patients requiring monovision correction were also excluded.All cases were performed by two surgeons (LL and MR). The cases had a 100-110μm LASIK flap created with the IntraLase IFS femtosecond laser  and LASIK ablation using either the WaveLight EX500  or WaveLight Allegretto Wave Eye-Q 400Hz , wavefront optimised profile. The nomogram supplied by the WaveLight EX500 was used for all cases of LASIK and LASIK Xtra, with a refractive target of plano to +0.5 D for all cases.Pre-operatively, we evaluated Uncorrected Distance Visual Acuity (UDVA), Corrected Distance Visual Acuity (CDVA), subjective refraction, corneal topography on Orbscan II corneal topography system , and Endothelial Cell Counts (ECC)  (only for LASIK Xtra eyes). Analyses were performed for all cases at post-operative 3 months, and for LASIK Xtra cases at post-operative 6 to 12 months.The efficacy of the surgeries was evaluated by computing the proportion of cases which achieved UDVA of 20/40 or better, and 20/20 or better, and the efficacy index (ratio between postoperative UDVA and preoperative CDVA). The predictability of the surgeries was evaluated by computing the proportion of cases within 1.0 D and within 0.5 D of attempted refractive correction. The safety of the surgeries was evaluated by recording any intra-operative or post-operative complications and by computing the proportion of cases with improved or unchanged post-operative CDVA compared to pre-operative CDVA, and the safety index (ratio between post-operative CDVA and pre-operative CDVA).2.12 was applied for 46 seconds , provided by the KXL® CXL system . These parameters were recommended by Avedro and have also been used in another study [For LASIK Xtra, after completion of the LASIK excimer ablation procedure, VibeX Xtra , consisting of 0.22% saline-diluted riboflavin solution, was placed on the bare stromal bed, and left to soak in for 45 seconds. Special care was taken not to allow the riboflavin solution to come into contact with the LASIK flap. After the 45-second riboflavin soak, the stromal bed was copiously irrigated to remove residual VibeX Xtra, and the LASIK flap was repositioned. Following flap repositioning, UVA fluence of 30 mW/cmer study .®, Alcon Laboratories, Inc, Fort Worth, TX, USA), dexamethasone phosphate 0.1% , and hypromellose 0.3% and dextran 0.1% . Patients were followed up on the first post-operative day and the contact lens was removed during that visit. Further follow-up examinations were performed at the end of week 1 and months 1 and 3 after surgery for both groups. A longer follow-up of 6-12 months was obtained for the LASIK Xtra group.A bandage contact lens was then placed on the ocular surface and the patient was treated with topical moxifloxacin  Version 20.0 . Fisher’s Exact Test was used to analyse the efficacy and predictability of the surgeries. Independent Sample T-Test and Non-parametric Mann-Whitney Test were used to analyse the spherical equivalent, efficacy index and safety index.33.1p=0.236), ethnicity (p=0.274), gender (p=0.960) and laterality (p=0.688).The mean age was 31±7 years  and 31±7 years  for the LASIK Xtra group and LASIK only group, respectively. Fifteen (57.7%) patients in the LASIK Xtra group and 21 (58.3%) patients in the LASIK only group were female. The majority of patients in both groups were Chinese (68% in the LASIK Xtra group and 86% in the LASIK only group) and female (58% in both groups). There was no statistically significant difference between the groups in terms of age (Myopia ablation treatment was performed on 50 eyes (100%) and 25 eyes (50%) with the Wavelight EX500  for LASIK Xtra and LASIK only groups, respectively. Twenty-five eyes (50%) in the LASIK only group and none in the LASIK Xtra group were treated with the WaveLight Allegretto Wave Eye-Q 400Hz .The mean follow-up was 5.7 months  for the LASIK Xtra group and 3.6 months  for the LASIK only group. All eyes had a mean follow-up of 3 months, while 27 eyes in the LASIK Xtra group had mean follow-up of 6-12 months. Comparison of visual outcome between the two groups was performed at 1 and 3 months’ follow-up.3.21). The attempted correction mean spherical equivalent was -9.40±1.47 D . The mean spherical equivalent were +0.36±0.42 D  and +0.33±0.46 D  at post-operative 3 months and post-operative 6 to 12 months, respectively. Stability of spherical equivalent refractive outcome at post-operative 6 to 12 months was achieved in the LASIK Xtra group with no regression (p=0.243) .For the LASIK Xtra group, the pre-operative mean spherical equivalent was -9.45±1.83 D  (Table 2).For the LASIK only group, pre-operative mean spherical equivalent was -9.42±0.97 D . The attempted correction mean spherical equivalent was -9.56±0.86 D . The mean spherical equivalent at post-operative 3 months was +0.26±0.34 D   and attempted correction mean spherical equivalent (p=0.497). At post-operative 3 months, the mean spherical equivalent were +0.36±0.42 D and +0.26±0.34 D in the LASIK Xtra group and LASIK only group, respectively (p=0.243).There was no statistically significant difference between the LASIK Xtra and LASIK only groups in terms of pre-operative mean spherical equivalent (3.3p=1.000). At post-operative 3 months, all eyes achieved UDVA of 20/40 or better, and 80.0% of LASIK Xtra eyes achieved UDVA of 20/20 or better, compared to 66.0% of LASIK only eyes (p=0.115) . The efficacy indices were 0.99±0.17 for LASIK Xtra and 0.94±0.17 for LASIK only (p=0.164).The proportion of eyes with pre-operative CDVA of 20/20 or better was 98.0% and 96.0% in the LASIK Xtra and LASIK only groups respectively, and this was not statistically significantly different  show the spherical equivalent refractive accuracy at post-operative 3 months for the LASIK only group and at post-operative 3 and 6 to 12 months for the LASIK Xtra group. A greater percentage of eyes were within ±0.50 D of the attempted correction in the LASIK only group (84.0%) than in the LASIK Xtra group (72.0%) at post-operative 3 months (p=0.148). Similarly, 100% of eyes in the LASIK only group were within ±1.00 D of the attempted correction, compared to 96.0% of eyes in the LASIK Xtra group (p=0.495).Figs. . At post-operative 3 months, 20 (40.0%) LASIK Xtra eyes and 18 (36.0%) LASIK only eyes achieved a 1 Snellen line gain in CDVA, while 1 (2.0%) LASIK only eye achieved 2 Snellen lines gain in CDVA. No change in CDVA was found in 25 (50.0%) LASIK Xtra eyes and 30 (60.0%) LASIK only eyes. A decrease in 1 Snellen line of CDVA was found in 5 (10.0%) LASIK Xtra eyes, and a decrease in 2 Snellen lines of CDVA in 1 (2.0%) LASIK only eye .The safety index was the same in the LASIK Xtra group (1.11±0.19) and the LASIK only group (1.11±0.18) at post-operative 3 months (The safety index was slightly higher at 1.13±0.16 in the LASIK Xtra group at post-operative 6 to 12 months. A gain in 1 Snellen line of CDVA was achieved in 8 (38.1%) LASIK Xtra eyes. There was no Snellen line lost for the LASIK Xtra group at post-operative 6 to 12 months. Post-operative recovery was generally uneventful with no major adverse events observed in either group. Mild post-operative haze was noted in 5 (10.0%) LASIK Xtra eyes, which resolved by the last follow-up visit in 3 of these eyes. 3 of the 5 eyes in the LASIK Xtra group which lost 1 line of CDVA returned for the post-operative 6 months follow-up. Of these, 2 had 1 line of CDVA improvement and 1 remained unchanged compared to the pre-operative CDVA.p=0.755) (Table 1).Grade 1 diffuse lamellar keratitis was found in 4 (8.0%) LASIK Xtra eyes and all resolved by the second post-operative day. In the LASIK Xtra group, ECC remained stable post-operatively (2893 ± 304), with no statistically significant difference compared to the pre-operative ECC (3010 ± 299) . [et al, compared 70 LASIK Xtra eyes and 64 LASIK only eyes in their series of Asian eyes and found that 98% of LASIK Xtra eyes achieved UDVA of 20/25 or better as compared to 61% of LASIK only eyes at post-operative month 3 (p<0.001). [et al had a significant difference between the LASIK Xtra and LASIK only groups in terms of age (p=0.014). [et al ’s study may confound the refractive and keratometric outcomes.In a prospective study comparing 73 LASIK Xtra eyes and 82 LASIK only eyes, Kanellopoulos =0.042).  Tan et aet al found that the predictability of the refractive outcomes in the LASIK Xtra group was statistically significantly greater at 3 months, with a greater coefficient of determination and significant reduction in standard deviation of postoperative spherical equivalent in the LASIK Xtra group (p=0.005). [Tan =0.005).  In our set al found a reduced refractive shift in the LASIK Xtra eyes compared to LASIK only eyes, though not statistically significant (p=0.063). The mean postoperative Manifest Refraction Spherical Equivalent (MRSE) at postoperative year 1 was -0.19±0.17 D in LASIK Xtra eyes and -0.27±0.23 D in LASIK only eyes. [et al similarly reported a trend towards reduced refractive drift in LASIK Xtra eyes (-0.04 D) compared to the LASIK only eyes (-0.13 D) (p=0.051). [et al reports that corneal keratometry results were stable for LASIK Xtra eyes and slightly regressing in LASIK only eyes (p=0.039). [1). However, LASIK Xtra eyes were found to achieve good refractive stability and less regression with spherical equivalent of +0.35±0.45 D at post-operative 6 to 12 months . This finding may support the concept postulated by Tan et al of a CXL-induced early stabilization of the cornea following corneal wavefront-guided stromal ablation. [Other studies have found that LASIK Xtra may provide the additional advantages of refractive and keratometric stability after LASIK. , 13 Kane=0.051).  FurthermThe LASIK Xtra group in our study showed a good safety profile. One Snellen line decrease in CDVA was found in 5 (10.0%) LASIK Xtra eyes at post-operative 3 months, which may be due to postoperative corneal haze. The corneal haze in these eyes resolved in 3 out of 5 eyes by post-operative 6 to 12 months (2 eyes lost to follow-up).Limitations of the study include a relatively high rate of subjects lost to follow-up and short term follow-up period, which is typical of patients undergoing keratorefractive surgery, precluding long-term follow-up. Corneal biomechanical properties were not measured in this study. Corneal hysteresis and corneal resistance factor, measured by the Ocular Response Analyzer , before and after corneal CXL were found to have no statistically significant difference. , 22In conclusion, LASIK Xtra achieved comparable visual outcomes compared to LASIK in Asians with high myopia in terms of safety, predictability and efficacy. Good refractive stability was attained at 6-12 months for LASIK Xtra. Further long term studies are required to determine whether simultaneous CXL is able to reduce postoperative LASIK keratectasia in high-risk individuals.


The interest toward alginate and nanoemulsion-based hydrogels is driven by the wide potential of application. These systems have been noticed in several areas, ranging from pharmaceutical, medical, coating, and food industries. In this investigation, hydrogels prepared through in situ calcium ion release, starting from lemongrass essential oil nanodispersions stabilized in alginate aqueous suspensions in the presence of the nonionic surfactant Tween 80, were evaluated. The hydrogels prepared at different concentrations of oil, alginate, and calcium were characterized through rheological tests. Flow curves demonstrate that the hydrogels share shear thinning behavior. Oscillatory tests showed that the strength of the hydrogel network increases with the crosslinker increase, and decreases at low polymer concentrations. The hydrogels were thixotropic materials with a slow time of structural restoration after breakage. Finally, by analyzing the creep recovery data, the hydrogel responses were all fitted to the Burger model. Overall, it was demonstrated that the presence of essential oil in the proposed hydrogels does not affect the mechanical characteristics of the materials, which are mainly influenced by the concentration of polymer and calcium as a crosslinker. The emulsion/dispersion technology frequently uses an internal gelation process for designing alginate particles  = 6 mM), medium ([Ca2+] = 8 mM), high ([Ca2+] = 10 mM), and medium* ([Ca2+] = 4 mM).Homogeneous Ca-alginate nanodispersion gels were prepared at room temperature, through the in situ gelation of sodium alginate induced by the release of Caurve see . The mol−1) over 300 sec at 20 °C [Rheological measurements of the gelled nanodispersions were carried out through a rotational rheometer, Haake MARS III  10 days after their preparation. All rheology measurements were made using a 20 mm diameter cone (1° angle) and plate geometry. The temperature was controlled at 20 °C by a cooling and heating system  in combination with a Peltier heating system. The samples were carefully placed onto the surface of the lower plate, and the upper cone was lowered to a 0.052 mm gap distance. Before testing, samples were left equilibrating for five minutes, in order to allow mechanical and temperature equilibrium. Flow curves were made in control rate mode (CR), varying the shear rate  determined through amplitude sweep measurements), and in a frequency range from 0.1 to 10 rad/s.−1 for 30 s. Step 3 was made in a controlled stress oscillation at τ 0.005 Pa and a frequency of 1 Hz for 6 min.In the oscillatory three-step thixotropy test, step 1 was made in controlled stress oscillation at τ 0.005 Pa at a frequency of 1 Hz for 1 min. Step 2 was made in CR at a shear rate of 1000 sCreep recovery experiments were carried out by applying a constant stress  for 180 s, following the deformation (loading phase). The stress was then removed, and the recovery phase was registered for 360 s.2, essential oil, and alginate concentrations. The aspect of the hydrogel formed from the alginate-based nanodispersions is shown in As reported in 2+ concentration are displayed (samples 1–9). In particular, samples 1, 2, and 3 were gelled with 6 mM of Ca2+; samples 4, 5, and 6 with Ca2+ 8 mM; and samples 7, 8, and 9 with Ca2+ 10 mM. Moreover, samples 1, 4, and 7 did not contain essential oil; samples 2, 5, and 8 contained 0.1 wt % of essential oil; and samples 3, 6, and 9 had an oil content of 0.5%. At the bottom of 2+ 4 mM, and various oil content  are reported . Remarkably, considering the alginate and Ca2+ concentration, these last samples were prepared with the same alginate/Ca2+ ratio as samples 4, 5, and 6. As can be seen from 2+ and alginate concentrations. Indeed, high and medium calcium content with a higher alginate percentage gave flat stiff interfaces, while decreasing both the ingredients resulted in hydrogels with less stiff interfaces.At the top of Steady-state flow curves of the alginate-based hydrogels demonstrate that all the hydrogels showed shear rate . The linear viscoelastic (LVE) region for each hydrogel can be identified, in G’ results are independent of the applied deformation. Considering the hydrogels prepared at 1 wt % alginate, their mechanical strength was evaluated by comparing the values of G’ in the LVE region [2+ used as cross-linker. Hydrogels with low levels of Ca2+ had G’ values of about 20 Pa, the presence of medium levels of Ca2+ increased the value of the elastic modulus around 50 Pa, and high levels of Ca2+ provided a further stronger gel, with a G’ value around 100 Pa. In all these samples, the strength measured was strongly dependent on the continuous phase, since no difference was evidenced by the presence of the oil-dispersed phase. Very close G’ values were also recorded for hydrogels prepared with 0.5% alginate. Although the green symbols seem more differentiated in G’ values ranged between 2 and 5 Pa, resulting in those with weaker structures being among the produced hydrogels.The results of the dynamic oscillation tests gave important information concerning the effect of CaE region , and theG’) that was always higher than the loss modulus (G”), denoting that the elastic character is always dominant when a load is applied. A very low dependence of the elastic modulus on the frequency was found for all of the hydrogels, while the G” modulus showed a certain dependence in hydrogels having low Ca2+ content . The compliance parameter is given by the ratio of the deformation γ to the applied stress τ:J is G, which is considered as the rigidity of the material. As a consequence, if a material has a high compliance it has a low rigidity, and vice versa, has high rigidity when exhibiting low compliance. From 2+ concentration , made by a spring and a dashpot in parallel, having shear modulus G2 and viscosity η2, respectively; and a dashpot (viscous element) with viscosity η3.In order to analyze quantitatively the materials during the loading step (creep phase), according to the shape of the creep curves the data were fitted to the Burger model (four-parameter Voigt model) ,31. The J(t) with the following equation:G1 is 1/J1, which in the creep curve is the time-independent elastic jump; λ2 is the retardation time of the viscoelastic element; and η3 is the viscosity of the viscous element. In other words, the compliance, J(t), in the Burger equation is the sum of an instantaneous elastic response, a viscoelastic (delayed elastic) component, and the unrecovered viscous flow. The elastic response of the viscoelastic element is given by the G2 parameter from Equation (2) and the viscosity (η2) is calculated by the retardation time that is given by the ratio of the viscosity to the elastic storage modulus of the second element, λ2 = η2/G2. The parameters obtained by fitting the creep curves to Equation (2) are listed in The Burger model expresses the creep compliance G1 of the hydrogels from the fittings had a lower value with low alginate concentration, and the G1 values increased in hydrogels with 1 wt % alginate together with Ca2+ content, albeit the values were quite similar for medium and high Ca2+ concentrations. The elastic response of the viscoelastic component G2 follows the same trend as G1, and a similar trend was also registered for η2 and for the viscosity of the third element η3.The elastic component The data extrapolated, overall, indicates that the presence of the oil in the composition of the hydrogels at a mechanical level did not cause substantial changes, but the differences observed were principally due to the different calcium levels. In the last column of In this study, the rheological properties of hydrogel-based nanodispersions made of alginate and lemongrass essential oil were analyzed. Different alginate, essential oil, and calcium concentrations were considered. The flow curves of the hydrogels revealed a shear thinning behavior with the presence of a Newtonian plateau at low shear rates in hydrogel formed at low and medium calcium concentrations in the absence of oil, and which persisted in the presence of an oil phase only at low alginate concentrations. Strain sweep tests allowed for determining the LVE region of the hydrogels and the evaluation of the gel strength. Frequency sweep tests confirmed that the characteristics of the hydrogels were mainly influenced by the alginate and calcium concentrations. The thixotropic tests revealed that after the breakage of the hydrogel structure, the complete structure restoration is a slow process; finally, through the creep recovery experiments, the hydrogels were found to be fit with the Burger model.Among the hydrogel systems studied, the only one that appeared in some way to be influenced by the presence of oil was the one with low alginate concentration, which in creep–recovery experiments deformed differently with the oil concentration increase. In order to give more details on the structural differences, other studies should be carried out.


Acute myeloid leukemia (AML) is one of the most lethal blood cancers, accounting for close to a quarter of a million annual deaths worldwide. Even though genetically heterogeneous, all AMLs are characterized by two interrelated features—blocked differentiation and high proliferative capacity. Despite significant progress in our understanding of the molecular and genetic basis of AML, the treatment of AMLs with chemotherapeutic regimens has remained largely unchanged in the past 30 years. In this review, we will consider the role of two cellular processes, metabolism and epigenetics, in the development and progression of AML and highlight the studies that suggest an interconnection of therapeutic importance between the two. Large-scale whole-exome sequencing of AML patients has revealed the presence of mutations, translocations or duplications in several epigenetic effectors such as DNMT3, MLL, ASXL1, and TET2, often times co-occuring with mutations in metabolic enzymes such as IDH1 and IDH2. These mutations often result in impaired enzymatic activity which leads to an altered epigenetic landscape through dysregulation of chromatin modifications such as DNA methylation, histone acetylation and methylation. We will discuss the role of enzymes that are responsible for establishing these modifications, namely histone acetyl transferases (HAT), histone methyl transferases (HMT), demethylases (KDMs), and deacetylases (HDAC), and also highlight the merits and demerits of using inhibitors that target these enzymes. Furthermore, we will tie in the metabolic regulation of co-factors such as acetyl-CoA, SAM, and α-ketoglutarate that are utilized by these enzymes and examine the role of metabolic inhibitors as a treatment option for AML. In doing so, we hope to stimulate interest in this topic and help generate a rationale for the consideration of the combinatorial use of metabolic and epigenetic inhibitors for the treatment of AML. Th. Th14). + or zinc cofactor , under the control of at least 17 acetyltransferases (HATs) that require an acetyl-CoA cofactor and of at least 18 deacetylases (HDACs) that require an NADcofactor . Severalcofactor . On the cofactor  and histcofactor .in vitro models. These studies highlight the importance of and distinction between the enzymatic activities and the scaffolding functions of HATs in promoting AML.Some of the most dramatic examples of impaired acetylation regulation involve chromosomal rearrangements that fuse HAT enzymes to other genes. Multiple rare AML subsets contain genetic fusions with HAT partners . For ins− progenitors  driven AML, administration of Panobinostat triggered proteasomal degradation of the pathogenic AML1/ETO fusion protein leading to terminal myeloid differentiation and excellent survival benefit in mice , wh, wh37), ll lines . Howeverll lines , 45. To ll lines . Dactolill lines . Along wll lines , 49.Even though single drug therapies have so far been the major focus in the attempt to restore the balance of histone acetylation in secondary and refractory AML, the most promising therapeutic leads have come from the simultaneous inhibition of HDACs and the mTOR/Akt axis. Entinostat, which primarily inhibits HDAC 1 and 3, partially inhibits Akt/mTOR signaling in HL60 AML cells. However, combination of Entinostat with the mTOR inhibitor Everolimus yields excellent synergism in inducing differentiation and apoptosis in myeloblasts . More rePost-translational histone methylation of lysine and arginine residues is well-documented and arguably more complex in regulation and function than histone acetylation . While aOf all the known HMTs, KMT2A or the MLL methyltransferase has the most well-documented role in the development of acute leukemia. MLL translocations are observed in ~10% of all AML cases  and are The H3K4 mono and di-methylation specific, FAD-dependent histone demethylase LSD1  has also been found to be crucial for both normal hematopoiesis and AML malignancy . While Lin vivo as a monotherapy compared to the current standard of care chemotherapy, prompting the combination of multiple epigenetic inhibitors as a treatment option for AML , is frequently mutated in myeloid malignancies. EZH2 is responsible for di- and tri-methylation of Lys27 in H3 to establish a repressive chromatin state at promoters of important hematopoietic transcription factors such as HOXA9 . Althoug for AML . However for AML .G, N′G-asymmetric dimethylarginine (ADMA), Class II catalyzes omega-NG, N′G-symmetric dimethylarginine (SDMA), and Class III stops at the production of omega-NG-monomethylarginine (MMA). The first identified arginine methyltransferase, PRMT1, has been shown to directly associate with a variety of oncogenic fusion proteins. Shia et al. demonstrated that PRMT1 interaction with RUNX1 fusion proteins mediates transcriptional activation through increasing promoter H4R3me2a of multiple genes regulating proliferation  have been identified in mammals so far. Class I generates omega-Nferation . Subsequferation . The H4Rloblasts . Intrigu in vivo . The safG12D AML cells  still needs to be investigated, because clues from these metabolic studies will provide new and exciting pathways that can be targeted in conjunction with well-studied epigenetic pathways as future therapeutics in leukemias.Lysines demethylases are equally adept at responding to the metabolic state of a cell and are known to rely on two types of cofactors; either flavin adenine dinucleotide (FAD) in case of LSD1/LSD2, or 2-oxoglutarate (α-ketoglutarate), oxygen and iron Fe(II) in case of the Jumanji domain containing demethylases . The rolnditions . A more nditions . On the nditions . This fiDS1/LSD2 . How theThus far, we have reviewed evidence demonstrating how metabolic states can directly influence chromatin-based epigenetic programs in AML. The shared biochemistry of mitochondrial and chromatin regulation is common to all eukaryotic cells. However, the rapid tissue turnover in the blood requires orchestration of rapidly changing cell identity programs, creating unique mitochondrial dynamics. From the quiescence of HSCs to the proliferative bursting of progenitors to cell cycle exit upon terminal differentiation, blood cells can phase through markedly different epigenetic and metabolic states in a short period of time. However, to become leukemic, these cells must not only elevate proliferative potential, but must also disable differentiation programs. We propose the possibility that altered metabolic dynamics may help simultaneously confer both of these features upon AML cells, as metabolic alterations not only enable the rapid proliferation of blasts, but also directly skew the epigenetic programs that regulate differentiation. This scenario would be distinct from solid tumors, in which the metabolic switch to aerobic glycolysis is primarily thought to enable rapid proliferation. Indeed, it is possible that metabolic alterations in fact represent a very key mechanism through which the AML differentiation block is constructed.in vivo may impair their differentiation programs in a manner that resembles the AML differentiation block. There is support for this prediction. For example, a mouse knockout study investigated the blood phenotypes resulting from loss of the mitochondrial phosphatase PTPMT1, which is highly expressed in HSCs (in vivo. Such studies demonstrate that metabolic alterations in healthy myeloid cells can induce an AML-like differentiation block. It will be of interest to see how exactly the corresponding alterations in chromatin regulators may contribute to the gene expression program driving the observed differentiation arrest.One prediction of this hypothesis is that experimental manipulation of cellular metabolic programs in normal blood cells  in HSCs . Hematop in HSCs  found thFinally, the connection between metabolism and chromatin regulation in AML may have therapeutic significance. Epigenetic regulators have emerged as attractive therapeutic targets for AML, and we have highlighted several inhibitors of chromatin factors that have shown potential in AML models. However, as with most cancer therapeutics, these drugs may work better in combination than as monotherapies. We propose that it would be of particular interest to explore AML drug combinations that pair an epigenetic inhibitor with a metabolic inhibitor. Our rationale is that metabolic inhibitors will have profound influences on chromatin regulators, perhaps opening up marked epigenetic vulnerabilities. For example, if a metabolic inhibitor decreases the pool of nuclear acetyl-CoA, cells may struggle to maintain proper histone acetylation levels—which in turn could make them exquisitely vulnerable to HAT inhibitors, as any further impairment of histone acetylation could be intolerable. The number of possible combinations between metabolic and chromatin regulators is large, but advances in screening methodology—both genetic and small molecule—is starting to make such endeavors feasible.AD and AB conceptualized the idea and wrote majority of the manuscript. BZ and FY helped write parts of the manuscript and prepared the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using High-Resolution FluoRespirometry, we also tested whether mitochondrial permeability transition pore (mPTP) inhibition or anoxia affects the mitochondrial Ca2+ flux. Ca2+ movement evoked by CaCl2 or anoxia was assessed with CaGreen-5N dye using Blue-Fluorescence-Sensor in isolated liver mitochondria, liver homogenates and duodenal biopsies. Exogenous CaCl2 (50 µM) resulted in an abrupt elevation in CaGreen-5N fluorescence followed by a decrease (Ca2+ uptake) with simultaneous elevation in O2 consumption in liver preparations. This was followed by a rapid increase in the fluorescence signal, reaching a higher intensity (Ca2+ efflux) than that of the initial CaCl2-induced elevation. Chelation of Ca2+ with EGTA completely abolished the fluorescence of the indicator. After pre-incubation with cyclosporin A, a marked delay in Ca2+ movement was observed, not only in isolated liver mitochondria, but also in tissue homogenates. In all samples, the transition to anoxia resulted in immediate increase in the level of extramitochondrial Ca2+. The results demonstrate that the CaGreen-5N method is suitable to monitor simultaneous O2 and Ca2+ fluxes, and the opening of mPTP in various biological samples. In this system the duration of stimulated Ca2+ fluxes may provide a novel parameter to evaluate the efficacy of mPTP blocker compounds.Our aim was to develop a method to detect extramitochondrial Ca Various channels, transmembrane proteins and receptors have been identified in the regulation of mitochondrial Ca2+ influx, efflux and storage, and the net result of these processes fundamentally influences the activity of intracellular regulatory systems. Low micromolar concentration range (~0.1–10 µM) of Ca2+ activates various enzymes  in the mitochondrial matrix2, stimulates the outer mitochondrial membrane-bound monoamine oxidase A (MAO-A), the enzyme responsible for the degradation of biogenic amines3, and influences the action of the mitochondrial antioxidant system through the regulation of manganese superoxide dismutase (MnSOD) activity4. In this range, Ca2+ fine-tunes oxidative phosphorylation and ATP synthesis, and the electron transport system (ETS) can be either stimulated or depressed7. Higher concentrations (~50 µM) may, however, lead to the opening of the mitochondrial permeability transition pore (mPTP), with non-selective Ca2+ efflux, collapse of mitochondrial membrane potential and apoptosis-mediated cell death9. The multiple roles of Ca2+ in cellular and mitochondrial function and the mechanism of mPTP activation in various hypoxia-associated pathologies are in the focus of intense research interest and laboratory investigation10. However, despite its importance, current technical and analytic options have prevented the collection of essential data on simultaneous Ca2+ and O2 fluxes in cells or mitochondria12. Therefore, our aim was to design a method, where the respiratory chain activity and mPTP opening can be detected together with extramitochondrial Ca2+ concentrations. More directly, our aim was to examine the Ca2+-activated mPTP opening and mPTP-mediated Ca2+ release, in association with O2 consumption changes in isolated mitochondria, tissue homogenates and tissue biopsy samples. Since detailed protocols and technical information on the subject were not available, the effect of mPTP inhibition was also tested together with anoxia-induced changes in Ca2+ flux when the function of ETS was inhibited.Mitochondria are main controller units of calcium , a single wavelength fluorescent dye, was employed to measure extramitochondrial Ca2+. It has been shown that this probe has a low affinity for Ca2+, making it suitable for evaluating relatively high Ca2+ concentrations  and insensitive to Mg2+  with negligible bleaching and without shifting its excitation or emission wavelengths14.For this in-depth study, High-Resolution FluoRespirometry was used for the combined detection of Caad libitum access to standard rodent chow and water. The experiments were performed in accordance with National Institutes of Health guidelines on the handling and care of experimental animals and EU Directive 2010/63 for the protection of animals used for scientific purposes. All animal experimental protocols were reviewed by the National Scientific Ethical Committee on Animal Experimentation  and was approved by the Animal Welfare Committee of the University of Szeged . Tissue samples of liver and duodenum were taken after ketamine and xylazine  anaesthesia.Male Sprague–Dawley rats (330–360 g) and SKH-1 hairless mice (20–30 g) were used. The animals were housed in plastic cages (21–23 °C) with a 12/12 h dark-light cycle and http://wiki.oroboros.at/images/f/fc/Gnaiger_2014_Mitochondr_Physiol_Network_MitoPathways.pdf).CaGreen-5N  was purchased from Thermo Fisher Scientific . All other reagents, including respiratory substrates and inhibitors, were purchased from Sigma Aldrich . Manual titration of these substances for 2 mL volume was carried out with Hamilton syringes. , Ca2+. This probe has a low affinity for Ca2+13, making it suitable for evaluating relative high Ca2+ concentrations . CaGreen-5N was dissolved through magnetic stirring in anhydrous dimethyl sulfoxide (DMSO), and 2 mM stock solution were prepared according to manufacture’s instructions. Stock solution was aliquoted (20 µL) in sterile Eppendorf vials, covered with strips of aluminium foil and stored at −20 °C until further use.CaGreen-5N, a single wavelength fluorescent dye, was used to measure extramitochondrial Ca3) affected fluorescence markedly; here, a nearly 50% decrease in signal intensity was observed  with a steep increase in the CaGreen-5N signal resulted in a fluorescent artefact as well  and inhibitors . After 40 min, the O2 signals were stable with the O2 slope (uncorrected) close to zero (at gain 1 for sensor and 800 mV polarization voltage). Noise of the O2 slope was within ± 2 pmol s−1 mL−1 at a data recording interval of 2 sec and 40 data points selected for calculation of the slope. Calibration and measurements were performed during continuous stirring (750 rpm) at 37 °C in a 2 mL respiration medium9 containing 210 mM mannitol, 70 mM sucrose, 0.2 mM KH2PO4 and 5 mM Tris-HCl adjusted to pH 7.4. The DatLab software  was used for online display, FluoRespirometry data acquisition and analysis. Blue Fluorescence-Sensor  was connected15 to the windows on the glass chambers and the instrument  to measure flourescence. Ca2+-related changes were expressed as the rate of changes in fluorescent signal and average resting fluorescence using the following formula: ∆Ca2+ = ∆F/F = (F − Frest)/Frest, where F is the indicator fluorescence at any given time during the experiments and Frest is the average fluorescence signal before treatment (e.g. exogenous Ca2+) or before the start of in vitro anoxia in the respiration chamber.All mitochondrial measurements were performed using High-Resolution FluoRespirometry . On the day of the experiment, a 40 min stabilization period was allowed for air calibration and temperature equilibration of the incubation medium, visualized as stabilization of the Peltier power  was used for mitochondria isolation (See http://wiki.oroboros.at/images/d/dc/MiPNet20.08_IsolationRatLiver-mt.pdf). The liver tissue was dissected and placed in ice-cold phosphate buffered saline , and wet weight was measured (1.5 g of liver tissue was used for isolation). Liver samples were cut into small pieces with a sharp scissors, suspended in 10 volumes of ice-cold isolation medium  and transferred to a pre-cooled Potter-Elvehjem. After homogenization (10 strokes), samples were centrifuged at 1000 g for 10 min at 4 °C  was activated with complex II substrate . To prevent accumulation of oxaloacetate (a known endogenous inhibor of succinate dehydrogenase), complex I was blocked with 0.5 µM rotenone (Rot) prior to succinate administration. Stimulation of oxidative phosphorylation (OXPHOS) with adenosine diphosphate (ADP) was omitted from the protocol due to its inhibitory effect on mPTP opening16. After stable respiration, Ca2+ movement was assessed with CaGreen-5N fluorescent dye . Mitochondrial Ca2+ influx and subsequent mPTP-mediated Ca2+ efflux were stimulated with the addition of 50 µM calcium chloride (CaCl2). When maximum fluorescence was reached, 1 mM EGTA was used for Ca2+ removal. ETS-independent respiration  was determined after complex III inhibition with antimycin A . Duration of Ca2+ fluxes (t1 influx and t2 efflux) was expressed in seconds (s), whereas volume-specific O2 flux  was expressed in pmol sec−1 mL−1.A modified method described by Sumbalova  °C Fig. . Then 1.2+ flux and O2 consumption in homogenate samples , cut into small (<20 mg) pieces, washed five times with PBS (pH 7.4) and then homogenized  in isolation media containing 225 mM mannitol, 5 mM sucrose and 0.2 mM EDTA (pH 7.4). All the mitochondrial samples were energized with complex II substrate (10 mM succinate) after complex I blockade with rotenone (0.5 µM). Stimulation with ADP was omitted from the protocol due to its inhibitory effect on mPTP opening16. After reaching a stable respiration, Ca2+ movement was assessed with 2 µM CaGreen-5N fluorophore. Ca2+-induced Ca2+ flux was stimulated with the addition of 50 µM CaCl2. At the end of mPTP-mediated Ca2+ release, 1 mM EGTA was used to chelate Ca2+. ROX was determined after complex III inhibition with antimycin A (2.5 µM). Duration of Ca2+ signals (t1 and t2) was expressed in seconds (s), whereas volume specific O2 flux  was expressed in pmol sec−1 mL−1 normalized to 8 mg wet weight per chamber.The experiments were expanded to determine the simultaneous Cales Fig. . A modif−1 O2 concentration in O2k chambers at 37 °C), samples were allowed to consume dissolved O2 available in respiration media. Thus, the chambers were kept closed throughout the experiments (to avoid any contact with air under anoxia), and oxygenation  or addition of O2 (e.g. O2 gas mixture injected through a needle) was avoided through the central capillary17. Black cover-slips were placed on top of stoppers to prevent light penetrating the capillary and avoid disruption of the fluorescence signal. The preparation protocol, instrument set-up and respiration media were identical to those for mitochondria exposed to CaCl2, as described above. Samples were treated with 0.5 µM rotenone, 10 mM succinate, 2.5 mM ADP and 2 µM CaGreen-5N before anoxia. After anoxia, responsiveness of fluorescent dye was monitored by exogenous 15 µM CaCl2. The Ca2+ signal was abolished with the addition of 1 mM EGTA.To elucidate the mechanism of transition from hypoxia to anoxia  and that they are responsive to ATP synthase inhibitor (2.5 µM oligomycin), as compared to other intestinal segments  or with succinate and 2.5 mM ADP  and used for analysis. Exogenous CaCl2-induced and anoxia-induced Ca2+ fluxes were determined from these samples in the presence of 2 µM CaGreen-5N using Fluorescence-Sensor Blue. Finally, 1 mM EGTA was injected into the chambers for the chelation of Ca2+. The instrumental set-up, including excitation, gain and polarization voltage, was identical with the set-up used for isolated mitochondria and homogenate.Mouse doudenal samples were used to detect Ca2+ flux. In the experiment setting, non-specific Ca2+ release through mPTP was inhibited by 1 µM cyclosporin A. The inhibitor was pipetted to energized mitochondria (rotenone + succinate), and then samples were incubated for 3 min at 37 °C. After chamber closure, Ca2+ movement was stimulated with 50 µM CaCl2, and Ca2+ signal was abolished with 1 mM EGTA, according to the previously described protocol.We used isolated mitochondria and homogenate for the pharmacological modulation of extramitochondrial Cat test and ANOVA for repeated measures using Fisher’s LSD post hoc test, as appropriate. Data analysis was performed with a statistical software package . A value of P < 0.05 was considered statistically significant.All data were expressed as mean ± SD; differences between means were compared using Student’s 2+ fluxes registered in isolated rat mitochondria are shown in Fig. 2+ flux was stimulated with 50 µM exogenous CaCl2  was similar, not exceeding 30 seconds. Simultaneously with these fluxes, a marked elevation in O2 consumption was found after addition of Ca2+. Non-electron transport system-derived respiration was negligible in our preparations, since residual O2 consumption was close to zero , particularly during stimulation with exogenous Ca2+.Figure 2+ that were evaluated at zero O2 flux and O2 consumption  and completion of fluxes (II) are indicators of mPTP activation.Representative registration of CaCl2 Fig. , resulti2+-evoked quantitative changes in Ca2+ flux, O2 consumption and O2 concentration in rat liver homogenate. In each sample, the duration of both Ca2+ influx and efflux was fast, with a maximum value of ~40 s  were recorded . However, the inhibitory effect of rotenone and stimulatory effect of succinate were manifested on these duodenal segments. The CaGreen-5N signal was stabilized before anoxia; however, anoxic transition immediately increased its fluorescence, and this increase remained continuous more than 30 min throughout the measurement  and EGTA (1 mM) under anoxia , was indicated by slow elevation of fluorescence after the maximum Ca2+ uptake. Secondly, Ca2+ influx was also increased, since a more pronounced decrease in CaGreen-5N signal was found after stimulation. Third, the Ca2+-induced respiratory stimulation occurred in a different manner. In contrast with the vehicle-treated mitochondria, where a single injection of CaCl2 resulted in a single peak in O2 consumption, CsA pretreatment resulted in two well-distinguishable O2 peaks or a delayed peak in maximum O2 consumption. Figure 2+ fluxes in rat isolated mitochondria and liver homogenate. The duration of Ca2+ uptake and that of subsequent Ca2+ release were very similar  without CsA, whereas in the presence of CsA, a delayed Ca2+ efflux and distinct t1/t2 rate were observed (t1: 53 ± 14 and t2: 506 ± 250 in isolated mitochondria and t1: 66 ± 17 and t2: 402 ± 259 in homogenate) both in rat isolated mitochondria and homogenate , and external Ca2+ was added to activate mPTPs and also to promote non-selective Ca2+ release through these channels. Both isolated mitochondria and tissue homogenate were responsive to a higher exogenous Ca2+ (50 µM), whereas the duodenal biopsies did not exhibit marked changes in CaGreen-5N fluorescence and increase of O2 consumption after the same insult. The reason behind the tissue-specific differences may stem from the more complex cellular milieu of intestinal punch biopsies. Duodenal segments contain various type of cells from all layers of bowel. It may well be that a higher external Ca2+ concentration should be used for the induction of Ca2+ fluxes. Secondly, we have no information on the magnitude of cellular Ca2+ influx  and circumstances that may potentially contribute to elimination of Ca2+  or modulate Ca2+ homeostasis .In the first set of experiments, liver mitochondrial preparations and intestinal biopsies were kept under normoxic conditions  was accompanied by a decline in respiration. Of note, EGTA abolished the signal within 3 sec independently of the type of sample. The duration and rate of Ca2+ influx and subsequent Ca2+ efflux were also very similar in isolated mitochondria and homogenate in the absence of the mPTP inhibitor. When the samples were treated with CsA, the duration of stimulated Ca2+ fluxes, in particular Ca2+ efflux (t2), was prolonged, the Ca2+ influx was elevated (indicated by an abrupt decline in fluorescence), and dyscoupled respiration was delayed, indicated either by two O2 peaks or a single, delayed peak in maximum O2 consumption. The prolonged duration of mitochondrial dyscoupling  may be associated with a delayed inner membrane depolarization and inhibiton of membrane potential disruption.Stimulated fluxes in liver-isolated mitochondria and homogenate, however, exhibited similar Ca2+ and O2 flux mediated by mPTP inhibition, both in isolated mitochondria and tissue homogenate. In fact, fluorescent techniques are available to determine mPTP opening, including spectrofluorimetric determination of Ca2+ retention capacity in isolated mitochondria (CRC12), fluorescence microscopy protocols in living cells using the calcein-cobalt technique or mitochondrial membrane potential changes (TMRM) and ionomycin-induced swelling and mitochondrial network fragmentation assay with mitochondrially-targeted GFP20. However, all these methods focus on immediate changes in a single mitochondrial parameter. In addition, the function of ETS is critically dependent on the O2 concentration dissolved in a medium and the O2 availability at the surface of the sample. The limitation of O2 is a significant shortcoming of all in in vitro studies with mitochondrial preparations or cells because the insufficient O2 decreases the maximum capacity of OXPHOS and decreases the uncoupler- or Ca2+-triggered respiratory stimulation as well.To our best knowledge, these are the first results to describe and characterize simultaneous changes in Ca9, but several challenging questions have remained unanswered. In this respect, our most important technical modifications were as follows: 37 °C was used instead of 25 °C, the succinate pathway control state was activated instead of the NADH electron transfer pathway state, the composition of the medium used for isolation was improved, and the fluorophore tracer concentration was increased (from 1 µM to 2 µM). In pilot studies, we ascertained that these changes will make the detection of exogenous Ca2+-triggered Ca2+ fluxes possible. Based on these findings, it appears that the composition of the respiration buffer is perhaps the most critical factor in Ca2+ flux detection. In the multicomponent respiration medium (MiR05), Ca2+ fluxes were undetectable, even in the absence of a chelator, but a mannitol- and sucrose-based respiration buffer (without chelator) made the Ca2+ fluxes readily distinguishable from the addition of CaCl2. It may well be that some components of MiR05, such as lactobionic acid, taurine or bovine serum albumin, bind Ca2+ directly, or they make mitochondria resistant to external Ca2+ stress through membrane stabilization (antioxidant or inhibition of fatty acid oxidation) rendering Ca2+ flux detection impossible.FluoRespirometric measurements with CaGreen-5N have previously been reported2+ influx or efflux after single Ca2+ injections instead of multiple additions of relatively low concentrations of Ca2+ (as in the case of the CRC method) may be an alternative option to reducing the interval between individual additions, thereby minimizing differences originating from multiple Ca2+ exposure. These differences include distinct alterations in the depolarization of the inner mitochondrial membrane that may occur before mPTP opening. Not only can duration of fluxes be detected after a single Ca2+ injection, but also changes in fluorescence; thus, two parameters, time  and relative fluorescent changes in intensity of Ca2+ indicator (∆F/F), can be evaluated at the same time and compared to the inhibitory effect of CsA. Further investigations are necessary to optimize the concentration of exogenous Ca2+ and specifically to determine whether more or less than 50 µM should be used to open the mPTP in isolated mitochondria in organs other than the liver. Based on our data, we suggest that simultaneous measurement of Ca2+-triggered Ca2+ and O2 flux may aid in testing and comparing (I) the effect of novel mPTP blockers , which lack immunosuppressive effects, and (II) biologically active gases that are known to inhibit these channels, such as nitric oxide22, hydrogen sulfide23, isoflurane24, sevoflurane25 and noble gases26. Since mPTP-related fluxes were similar in isolated mitochondria and homogenate (both in the presence and absence of CsA), a time-consuming mitochondrial isolation procedure with low-speed and multiple high-speed centrifugation can be replaced by the use of homogenate, at least in the liver. Tissue homogenate provides numerous advantages over isolated mitochondria; for example, (I) the preparation protocol is faster; (II) tissue heterogeneity is well-preserved; and (III) a small amount of tissue is required for functional mitochondrial studies.Measuring the duration of stimulated Ca2+ in the respiration buffer occur, and, secondly, what are the channels through which this Ca2+ can leave the organelle? Since our primary aim was to investigate the methodological aspects of Ca2+ fluxes, further experiments are needed to clarify the precise mechanism of influx and efflux in this model. A number of mitochondrial Ca2+ influx mechanisms have recently been identified in the literature, for instance, (I) rapid uptake mechanism-related Ca2+ uptake ; (II) mitochondrial Ca2+ uniporter (which operates at a higher micromolar concentration of Ca2+28); (III) mitochondrial N-methyl-D-aspartate receptors29 in rat heart; (IV) leucine zipper-EF-hand containing transmembrane protein 1 ; (V) uncoupling protein (UCP31) 2 and 3; (VI) mitochondrial ryanodine receptor (mRyR32); and (VII) electron transport system-mediated Ca2+ uptake .The question arises: how does uptake by mitochondria of external Ca2+ transport: (I) mPTPs8, (II) mitochondrial Na+/Ca2+ exchanger (mNCX34) and (III) Ca2+-proton (H+) exchanger (mHCX)35. Among Ca2+ entry mechanisms, the mitochondrial Ca2+ uniporter (MCU) seems to be a candidate since it operates at micromolar Ca2+ range (˃10 µM36), and the duration of Ca2+ uptake is slower than RaM-mediated influx (~ns). According to a potential model, it is suggested that Ca2+ transport through MCU is inhibited through MICU1 and MICU2 regulatory protein (“gatekeepers”) at a lower concentration of Ca2+, whereas it allows Ca2+ entry into the matrix at a higher concentration of Ca2+ as a result of conformational change in the protein via Ca2+ binding of EF hands of MICU1/MICU236. Moreover, activation of mPTPs has been shown to be inhibited in MCU knockout mitochondria or after channel blockade with Ru36040. We hypothesize that RAM mechanism-mediated Ca2+ entry plays a negligible role or is undetectable in our measurements because changes in CaGreen-5N fluorescence occur at a higher (0.5–50 µM) concentration of Ca2+, whereas RAM operates at nanomolar concentration range42.As concerns efflux, so far three mechanisms have been documented in Ca2+ can influence mitochondrial ROS formation43. Despite dissipation of mitochondrial membrane potential and mPTP activation, 50 µM CaCl2 did not affect the rate of extramitochondrial H2O2 generation in pilot studies using liver-isolated mitochondria  to stimulate the fatty acid oxidation pathway control state with octanoylcarnitine, palmitoylcarnitine (F-pathway) or the N-pathway with glutamate, malate and pyruvate to measure Ca2+-induced mitochondrial ROS generation.An increase in the level of intracellular Ca2+-triggered Ca2+ flux, anoxia-related endogenous Ca2+ release occurred in all the types of samples studied. A continuous increase in CaGreen-5N fluorescent intensity was started immediately after the commencement of anoxia, with no decrease in its intensity after 30 min. The mechanism behind anoxic Ca2+ release may involve the possibilities noted above, i.e. mNCX, mHCX or mPTPs, or their combined action. In addition, responsiveness of fluorophore to exogenous CaCl2 was detected, and then the signal was successfully abolished with EGTA several hours (~3–4 h) later under anoxia . The different pathways for ATP production would largely affect mitochondrial function, including membrane potential and Ca2+ transport as well.The study design has certain limitation because it is solely based on the High-Resolution FluoRespirometry technique. Besides, organelle-targeted biosensors can also be used to confirm the correlation between respiration activity and Ca2 and Ca2+ fluxes and the opening of mPTPs in various biological samples (in isolated mitochondria and tissue homogenate) after stimulation with external Ca2+. It can also be used to monitor anoxia-induced changes in Ca2+ release. Measuring the duration of stimulated Ca2+ fluxes may provide a novel parameter to evaluate the efficacy of mPTP blocker compounds.In summary, the new method is suitable to monitor simultaneous OSupplementary infornation