Myocardial ischemia reperfusion injury (MIRI), the tissue damage which is caused by the returning of blood supply to tissue after a period of ischemia, greatly reduces the therapeutic effect of treatment of myocardial infarction. But the underlying functional mechanisms of MIRI are still unclear.We constructed mouse models of MIRI, extracted injured and healthy myocardial tissues, and performed transcriptome sequencing experiments (RNA-seq) to systematically investigate the dysregulated transcriptome of MIRI, especially the alternative splicing (AS) regulation and RNA binding proteins (RBPs). Selected RBPs and MIRI-associated AS events were then validated by RT-qPCR experiments.Eif5, Pdia6, Tagln2, Vasp, Zfp36l2, Grsf1, Idh2, Ndrg2, and Uqcrc1. These nine DE RBPs were correlated with RASGs enriched in translation process, cell growth and division, and endocytosis pathways, highly consistent with the functions of all RASGs. Finally, we validated the AS ratio changes of five regulated alternative splicing events (RASEs) derived from important regulatory genes, including Mtmr3, Cdc42, Cd47, Fbln2, Vegfa, and Fhl2.The differentially expressed gene (DEG) analyses indicated that transcriptome profiles were changed by MIRI and that DEGs’ enriched functions were consistent with MIRI’s dysregulated pathways. Furthermore, the AS profile was synergistically regulated and showed clear differences between the mouse model and the healthy samples. The exon skipping events significantly increased in MIRI model samples, while the opposite cassette exon events significantly decreased. According to the functional analysis, regulated alternative splicing genes (RASGs) were enriched in protein transport, cell division /cell cycle, RNA splicing, and endocytosis pathways, which were associated with the development of MIRI. Meanwhile, 493 differentially expressed RBPs (DE RBPs) were detected, most of which were correlated with the changed ratios of AS events. In addition, nine DE RBP genes were validated, including Our study emphasized the critical roles of the dysregulated AS profiles in MIRI development, investigated the potential functions of MIRI-associated RASGs, and identified regulatory RBPs involved in AS regulation. We propose that the identified RASEs and RBPs could serve as important regulators and potential therapeutic targets in MIRI treatment in the future.The online version contains supplementary material available at 10.1186/s12920-023-01706-5. Acute myocardial infarction (AMI) is an acute ischemic heart disease caused by sudden occlusion of the coronary artery, resulting in myocardial ischemia, injury, and necrosis . AMI is RNA binding proteins (RBPs) are a large group of proteins that can bind double- or single-stranded RNA and form ribonucleoprotein (RNP) complexes within cells. RBPs primarily function by binding with RNA and are often associated with RNA transcription initiation. At different stages of its life span, RNA binds to various types of RBPs to form RNP complexes, which regulate their maturation, transport, localization, and translation processes. Thus, diverse RBPs enable pivotal regulatory functions in almost all physiological and pathological processes . SpecifiMany RBPs can bind pre-mRNA to regulate alternative splicing (AS) and produce proteins with various functions. AS refers to a process in which the 5′ and 3′ splice sites on the pre-mRNA of a transcribed gene are spliced together in different ways to generate multiple mRNA isoforms, resulting in protein products with different amino acid sequences that serve diverse physiological functions . RBP-medTherefore, we proposed the scientific hypothesis that differentially expressed RBPs are involved in the post-transcriptional regulation processes after MIRI, which in turn causes variable splicing of pre-mRNAs. We hypothesised that the RBP-AS regulatory axis plays a molecular regulatory role in the MIRI process. In this study, we performed a systematic analysis of RNA-seq in mice with MIRI to obtain differentially expressed RBPs and variable splicing pre-mRNAs; we also combined with co-expression analysis to explore the genome-wide landscape of the RBP-AS regulatory network. The present study reveals a global picture of gene expression and splicing profiles as well as functional pathways in mouse models of MIRI. At present, there are a small number of reports on the role of RASEs and RBPs in the development of MIRI. In this view, we will discuss RBPs and its regulation of AS, and provide new mechanism insights for the development in MIRI.2O, the respiratory rate 88 times/min, and the ventilation volume 1 L/min. The skin of the surgical area was disinfected and cut off on the left side of the sternum between the third and fourth ribs. The subcutaneous tissue and muscles were passively separated layer by layer. The thoracic cavity was opened. The intercostals were opened by the chest opener to fully expose the heart. The pericardial sac was cut open by carefully identifying it under a stereomicroscope. A pink blood vessel appeared at the lower edge or left side of the auricle, which is the anterior descending branch (LAD) of the left coronary artery. Use a needle to pass a 6 − 0 suture through the surface layer of myocardium at 1 mm from the lower edge of the auricle and ligate the LAD under pressure. The traditional method group: before ligation, put a 0.2 ~ 0.3 cm 10 # polyethylene tubing under the line, and then ligate the LAD. After confirming that myocardial ischemia has been induced by ligation, close the chest cavity layer by layer by suturing the skin and muscle, and place two coils on both sides of the incision, leaving them outside the body. After 40 min of ischemia, the polyethylene tube was drawn out for perfusion for 24 h. The control group was treated by sham operation by receiving the same surgical interventions. For each group, five biological replicates were prepared. We used 0.12ml/10 g of 1% pentobarbital sodium for anesthesia, and after death from anesthesia, we bled the abdominal aorta to extract the heart tissue.The 6-week male C57BL/6 mice were originated from Hunan Slak Jingda. The mice were raised in the absence of specific pathogen free (SPF). The temperature of the feeding environment is 22 ~ 26 °C, the relative humidity is 50% ~ 60%, and the artificial light is dimmed for 12 h. For experiments, the mice were anesthetized by intraperitoneal injection of 0.12ml/10 g 1% sodium amytal. The hair on neck and chest was removed and the surgery site was disinfected. After oral tracheal intubation, the mice were connected to the ventilators to maintain respiration. The peak airway pressure was 14.40 cm HThe slices of heart tissue were placed in TTC incubation solution  preheated at 37 ℃ in advance, and incubate them in a 37 ℃ water bath in dark for 30 min. We then gently shaked the tissue at intervals of 10 min to evenly contact the staining solution, and observed the staining results.We used liquid nitrogen grounding method for myocardial tissue homogenization, and 10 to 50 mg of each sample was used to extract RNA. Total RNA was extracted using TRIZOL (Ambion). DNAs were removed by treating total RNA with RQ1 DNase (Promega). To detect the quality and count the number of the purified RNAs, the absorbance was measured at 260 nm/280nm (A260/A280) using smartspec plus (BioRad). Then, the integrity of total RNA was validated by 1.5% agarose gel electrophoresis.Next, 1 µg total RNA was used to construct RNA-seq libraries for each sample. mRNAs were captured using oligo-d(T) capture Beads . The purified RNA was used to prepare directional RNA-seq libraries by Stranded RNA-seq Kit for Illumina platforms . Polyadenylated mRNAs were subjected to purification and fragmentation. The cDNA synthesis was done by reverse transcription kit at 42˚C for 5 min, 37 ˚C for 15 min, 85 ˚C for 5 s that was performed on the thermocycler. After end repair and A tailing, the DNAs were subjected to ligation with Diluted Roche Adaptor (KK8726). After purifying the ligated products and fractioning the size to 300-500bps, the ligated products were subjected to amplification, purification, and quantification and were stored at -80℃ for sequencing. The second cDNA strand, which is marked with dUTP, is not amplified for sequencing specific strand.For high-throughput sequencing, the libraries were constructed according to the manufacturer’s protocols and were sequenced with the Illumina NovaSeq 6000 platform for 150 nt paired-end sequencing.Raw reads with no less than 2-N bases were first dropped. Then raw reads were trimmed using FASTX-Toolkit to remove low-quality bases and adaptors (Version 0.0.13). The short reads with no more than 16nt were also discarded. Subsequently, use HISAT2  to alignp-value < 0.05 and fold change > 2 or < 0.5 as the cut-off criteria.DEGs were screened by using the R Bioconductor package DESeq2  and idenABLas pipeline was used to define and calculate the alternative splicing events (ASEs) and RASEs between the samples as described previously , 16. In t-test was performed to assess the significance of the ratio alteration of AS events. Those events which were significant at a p-value cutoff of 0.05 and a false discovery rate (FDR) of 5% were considered RASEs.To evaluate the ASEs regulated by RBPs, Student’s p-value < 0.01 and Pearson’s correlation coefficient > 0.95 were set as the criteria for DE RBP-RASE pairs.To construct the correlation network between RBPs and RASEs, we performed Pearson’s correlation analysis between the RASE ratios and the expression levels of differentially expressed RBPs (DE RBPs). The −ΔΔCT method [For the validation of DEGs and RASEs, we performed reverse transcription and quantitative polymerase chain reaction (RT-qPCR) experiment. Specific primers were designed for each DEGs and RASEs and were provided in supplemental Table T method . Four bihttps://cn.string-db.org/; Version: 12.0).For protein-protein interaction analysis, the symbols of selected genes were served as input to calculate and construct protein-protein interaction network through STRING database .To sort out the DEGs’ functional groups, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms. were identified by utilizing KOBAS 2.0 server . To defiMyocardial ischemia-reperfusion, clinical treatment for myocardial infarction, could induce myocardial injury and cardiomyocyte death, reducing its curative effects . The undp-value < 0.05. PCA by calculating RASE ratios showed that the first component clearly separated the model and healthy samples, and that the first component could explain 68.3% of total variation  , but theion Fig. A. Then tion Fig. B. Anotheion Fig. B, suggesion Fig. C. We anaion Fig. D. KEGG pion Fig. E. These les Fig. F. In sumEif5, Pdia6, Tagln2, Vasp, Zfp36l2, Grsf1, Idh2, Ndrg2, and Uqcrc1. With four models and four healthy samples as input, the results showed significant expression differences between models and healthy samples. The results of RNA-seq and RT-qPCR demonstrated consistency in the changed patterns . Finally, in this network, 848 RASEs correlated with 483 RBPs were obtained . Analyses of the functions of the RASGs from the 848 RASEs indicated that they were associated with cell growth/division and important metabolism pathways . Bubble plot also demonstrated that these RASGs were enriched in endocytosis, protein transport, and mitochondrial respiratory chain complex I assembly, the top three GO BP pathways . Thermogenesis, Alzheimer disease, and bacterial invasion of epithelial cells were the top three KEGG pathways . Dilated cardiomyopathy (DCM) pathway, including Lama2, Tnnt2, Actg1, Atp2a2, Gnas, Tpm3, Itga6, Tpm1, and Pln, was enriched in the KEGG pathway . To further ensure the credibility of the result, we focused on the nine validated RBPs in Fig. Cd47, Fbln2, and Vegfa were repressed in MIRI model; 5pMXE event of Fhl2 was repressed in MIRI model  [Mtmr3 regulates the formation and size of autophagosome, and finally affects the autophagic activity [Cdc42, a cell division cycle regulatory gene, may play a role in myocardial I/R by affecting the cardiacmyocyte apoptosis rate through JNK, Bcl-2 and Bax signal pathway [In addition to the dysregulated RBPs, global AS profile was also significantly regulated in MIRI model. AS regulation has been recently reported in CVDs; circRNAs and products of irregular AS events were emphasized in the origination and development of MIRI . In the ry (IRI) , suggestry (IRI) . Among tactivity . The enr pathway . IrregulCd47, Fbln2, Vegfa, and Fhl2. CD47, a transmembrane protein, has been used to explore MIRI treatment by delivering anti-apoptotic miRNAs via forming complexes with extracellular vesicles (EVs) [−/− mouse, altered immune infiltration was found to respond to myocardial ischemia, contributing to smaller infarcts and better hemodynamic performance after MIRI [Finally, we constructed the regulatory network between DE RBPs and RASEs. We found over 30% RASEs were correlated with DE RBPs, suggesting the regulatory roles of RBPs in AS profiles. Then nine validated DE RBPs were selected together with RASEs correlated with them, and several important RASGs were found to emerge, including es (EVs) . Meanwhies (EVs) . Goltz eter MIRI . Vasculater MIRI . The splIn summary, our study highlights the important roles of AS and RBPs in the pathogenesis of MIRI using mouse models. We propose that DE RBPs and their regulated AS genes participate in RNA splicing, metabolic processes, and immune/inflammatory response pathways to modulate MIRI progression. Several important RBPs, AS events and genes could serve as novel therapeutic targets in treating MIRI by designing accurate molecular medicine in the future. Our study extends the understanding of the molecular pathogenesis of MIRI and emphasizes the important roles of post-transcriptional regulation.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Hippocampal area CA2 plays a critical role in social recognition memory and has unique cellular and molecular properties that distinguish it from areas CA1 and CA3. In addition to having a particularly high density of interneurons, the inhibitory transmission in this region displays two distinct forms of long-term synaptic plasticity. Early studies on human hippocampal tissue have reported unique alteration in area CA2 with several pathologies and psychiatric disorders. In this review, we present recent studies revealing changes in inhibitory transmission and plasticity of area CA2 in mouse models of multiple sclerosis, autism spectrum disorder, Alzheimer’s disease, schizophrenia and the 22q11.2 deletion syndrome and propose how these changes could underly deficits in social cognition observed during these pathologies. Several decades of human and animal lesion experiments have established that the hippocampus plays a central role in learning and memory processes . LorenteInterneurons play a critical role in tuning the balance between excitation and inhibition in all brain areas. If it can be said that one area relies more heavily on inhibitory transmission than any other, then area CA2 may qualify as a winner of the contest. In comparison to areas CA1 and CA3, there are very few studies examining the cellular composition of hippocampal area CA2. The pyramidal neurons in this region have larger soma than CA1 and dendritic arborizations that more closely resemble CA3 pyramidal neurons, except that CA2 pyramidal neurons lack the distinctive thorny excrescences that form the post-synaptic contact with mossy fibers from dentate granule cells . There iStratum oriens (so) of CA2, and that PV+, Reelin+ and Calbindin+ cells were the highest density in Stratum pyramidale (sp), as compared to areas CA3 and CA1. The high density of PV+ cells bodies in sp and so was also observed in mice using RGS14 staining as a boundary to define CA2 , and calbindin-positive cells in primate hippocampal CA2 was first reported in 1991 . A thorofine CA2 . The morfine CA2 . Based ofine CA2 . Of thesfine CA2 . The bisfine CA2 . There ifine CA2 . Similarfine CA2 . These cOne of the remarkable aspects of the local CA2 network is the large magnitude of feed-forward inhibition that is recruited by CA3 input stimulation, especially compared to the amount of inhibitory transmission recruited in area CA1 . We postAnother puzzling property of CA2 pyramidal neurons that is independent of the local inhibitory network is the resistance of these cells to activity-induced long-term potentiation (LTP) at the excitatory CA3-CA2 synapse. This property was first uncovered and demonstrated in-depth by So what gives? How can an entire hippocampal region be devoid of synaptic plasticity? While the glutamatergic synapses between CA3 and CA2 are resistant to activity-dependent plasticity, it is now clear that the inhibitory synapses in area CA2 express activity-induced synaptic plasticity that can powerfully shift the balance of excitation and inhibition and allow CA2 pyramidal neuron action potential firing. Specifically, there are two separate mechanisms allowing activity dependent long-term depression of GABAergic transmission (iLTD) in area CA2. One is mediated by the activation of delta-opioid receptors (DORs), and the other by cannabinoid type I receptors (CB1Rs). Importantly, there is evidence that both of these plasticities play a role in social recognition memory. How is this possible? Hippocampal CA2 pyramidal neurons project strongly to ventral hippocampal CA1 , a regioThe DOR-mediated iLTD that occurs in hippocampal area CA2 is specific to a subset of PV+ inhibitory neurons . This waEvidence has been presented that enkephalin, the endogenous ligand for DORs, is potentially released by local vasoactive intestinal peptide-expressing (VIP+) interneurons . DOR-iLTThere is evidence that the DOR-mediated iLTD in area CA2 contributes to social memory formation but may not be absolutely required. Selective removal of DORs in area CA2 does impair social recognition memory as tested by a 5-trial dishabituation task . In thisThe PV+ interneurons in hippocampal area CA2 receive very strong excitatory input from the hypothalamic supramammillary nucleus (SuM) . This hyIt has recently been reported that endocannabinoid-mediated synaptic plasticity is playing an important role in hippocampal area CA2 function. This plasticity results from the release of endocannabinoids (eCBs) by CA2 pyramidal cells and activation of cannabinoid type 1 receptors (CB1Rs) located on GABA-ergic terminals . As estaWe hypothesize that the eCB-iLTD is critical and necessary for area CA2 to allow for social memory formation, and likely playing a more prominent role than the DOR-iLTD. The DOR-mediated iLTD was found to be occluded after a single social interaction . HoweverGiven the critical role of inhibitory transmission and plasticity in controlling pyramidal cell firing in area CA2, it is not surprising that inhibitory transmission has been found to be altered in several neurodevelopmental and neurodegenerative diseases . While mMultiple sclerosis (MS) is an autoimmune disease often associated with severe cognitive symptoms including deficits in social cognition and facial emotion recognition. Interestingly, these symptoms can be observed early in the disease progression, even before appearance of other cognitive symptoms . Some ofAutism spectrum disorders (ASD) include neurodevelopmental diseases characterized by deficits in verbal and non-verbal communication, stereotyped behaviors and impaired social cognition. Many genes involved in synaptic function have been linked to the development of ASD, including mutations or deletions in genes encoding for Neuroligins (NGLs). NGLs are expressed by the postsynaptic elements and interact with their presynaptic partners Neurexins. Mice lacking NGL3 have been used as a model for a non-syndromic form of autism and NGL3 KO mice display strong deficits in social behaviors.in vivo and ex vivo, a recent study performed with these mice has focused on the potential alterations in hippocampal area CA2  was increased in CA2 pyramidal cells . ConversFinally, this study also shows that NLGN3 KO have a deficit in social memory formation, i.e., they spend the same amount of time exploring a novel and a familiar mouse. However, while these mice might indeed have an impairment in the capacity to remember a conspecific, an effect that would be consistent with a disfunction in the CA2 network, caution should be taken when interpreting this result. Indeed, the same study clearly showed that NLGN3 KO mice also have a deficit in sociability, an effect that could potentially result from a deficit in olfactory functions as described previously in a NLGN3-deficient mice . Thus, iAlzheimer’s disease (AD) is a neurodegenerative disease leading to dementia in the elderly population. Symptoms include impairments in acquisition and retrieval of memories that are linked to alterations in hippocampal function . In partA recent study focused on area CA2 has shown that PV+ and PNN+ cell density is strongly reduced in area CA2. This was accompanied by a large decrease in inhibitory transmission between CA3 and CA2, leading to a large increase in EPSP amplitude and the emergence of action potential firing in CA2 PNs in response to CA3 input stimulation. Impaired inhibitory transmission from PV+ interneurons likely contribute to the reduced inhibitory transmission because DOR-iLTD as well as the DOR-mediated disinhibitory increase in PSP amplitude was strongly reduced in this mice. In agreement with a role of this plasticity in social memory formation, social recognition memory was completely abolished in Tg2576 mice.trans-synaptic signaling through interaction between the Neuregulin 1 (expressed by PNs) and its receptor ErbB4 expressed by PV+ INs  have confirmed key data observed in human tissues from epileptic patients and also provided a detailed description of the anatomical and functional changes observed in area CA2 during the course of the disease. Specifically, aberrant mossy fiber sprouting in area CA2 was observed in the kainate mouse model of TLE . This exte gyrus . Subsequte gyrus . Anotherte gyrus . Furtherte gyrus .Epilepsy has long been thought to result from an imbalance between excitation and inhibition. Although anatomical and functional changes have been observed in all hippocampal areas, CA2 is unique in that it is much more resistant to cell death in comparison to area CA1 or CA3. Furthermore, while acutely reducing inhibitory transmission in rodent hippocampal slices was by itself sufficient to trigger the generation of epileptic-like activities that originated in area CA2 , it is lEarly studies on post-mortem hippocampal tissues from schizophrenic patients have reveal that CA2 is uniquely altered. In particular, a large decrease in the density of interneurons was reported in area CA2 during schizophrenia . A meta-Stratum oriens of CA2 and CA3  inputs that project to the basal dendrites of CA2 and CA3 pyramidal cells. This manipulation during adolescence results in a lasting decrease in the density of interneurons expressing PV, calretinin and calbindin located in  and CA3 . The denradiatum . Furtherradiatum .Changes in interneuron properties have also been observed in mouse models of the 22q11.2 deletion syndrome . This spIn vivo recordings have shown that CA2 PNs can encode social information . In Df(16)A mice, this coding was impaired while spatial coding, initially weak in WT mice, was enhanced  and lost-of-function  have been reported and they both ultimately result in social cognition deficits. This suggests that a fine, bidirectional tuning of CA2 activity is required for the normal function of this region. Unquestionably, inhibition plays a major role in controlling CA2 pyramidal cell activity in normal condition, and alterations in inhibitory transmission and plasticity likely contributes to the deficits in hippocampal function observed during these neurodegenerative and neurodevelopmental disorders. Alterations have often been reported in PV+ and CCK+ interneurons, but other classes of interneurons are also affected. Thus, understanding the precise contribution of diverse populations of interneurons during each specific pathology is one of the challenges of future studies.Both authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Haemaphysalis longicornis (17.9%) and Ornithodoros moubata (12.8%) were the most common tick models in vaccination trials. Experiments with rabbits have revealed that some proteins  can induce immune responses against various tick species. In addition, in some cases it was possible to determine that the vaccine efficacy in rabbits was similar to that of experiments performed on natural hosts . In conclusion, results showed that prior to performing anti-tick vaccination trials using natural hosts, rabbits can be used as suitable experimental models for these studies.Studies evaluating candidate tick-derived proteins as anti-tick vaccines in natural hosts have been limited due to high costs. To overcome this problem, animal models are used in immunization tests. The aim of this article was to review the use of rabbits as an experimental model for the evaluation of tick-derived proteins as vaccines. A total of 57 tick proteins were tested for their immunogenic potential using rabbits as models for vaccination. The most commonly used rabbit breeds were New Zealand (73.8%), Japanese white (19%), Californians (4.8%) and Flemish lop-eared (2.4%) rabbits. Anti-tick vaccines efficacy resulted in up to 99.9%.  Ticks are obligate blood-sucking ectoparasites that parasitize a large number of terrestrial and semi-terrestrial vertebrates, including humans ,2,3. AltTraditional methods to control these arthropods are mainly based on the use of synthetic acaricides ,9,10. HoMetarhizium spp., Beauveria spp.) [Heterorhabditidae and Steinernematidae) [Solenopsis germinata, S. saevissima, Camponotus rengira, and Ectatomma quadridens) [These issues raise the need to develop alternative control methods, including the selection of parasite-resistant breeds ,13; biolia spp.) ,15; entomatidae) ,17; regudridens) ,19; pestdridens) ,21; and dridens) ,23,24. Rhipicephalus microplus [® and GAVAC® [R. microplus as well as other tick species.Several proteins have been studied to date as candidates for the development of tick vaccines ,26. The icroplus ,32, and d GAVAC® . This lad GAVAC® , paved tThe evaluation of tick vaccines in natural hosts has limitations, mainly due to the high costs of maintaining and using farm or wild animals in experiments. For this reason, animal models such as hamsters, guinea pigs, and rabbits are commonly used ,35,36. TThe use of hamsters, guinea pigs, and rabbits in tick vaccination experiments generally has low maintenance costs, minimal space requirements, short reproductive cycles and larger numbers of pups produced per year compared to some natural hosts ,42,43. HOryctolagus cuniculus) [Another limitation in experimental animal models is the number of ticks that can be used when performing the infestation. Studies in rabbits have reported that these animals can support a higher burden of adult ticks ,47 companiculus) . The Ameniculus) , among wniculus) ,42,57.Laboratory rabbits have proven to be the most suitable and accessible hosts for all life-stages of various tick species during infestation and vaccination experiments ,58. ThisMoreover, it is evident that rabbit-based experiments are more cost-effective when comparing trials conducted using large animals such as bovines. Various factors contribute to the overall costs, including animal prices, the extended maintenance period, a higher demand for feed, as well as the size and complexity of the animal facilities. Bovines require a greater amount of physical space and specialized infrastructure, along with large feed quantities. As a result, more demanding waste management systems are necessary for bovine experiments. Nonetheless, the determination of the real cost difference is difficult due to various local factors. This is because facilities, locations, and services vary among different regions and installations. Therefore, although rabbits incur lower costs, the precise amount varies.Dermacentor variabilis larvae induced immunity that prevented subsequent larval infestations [Historically, the evaluation of tick-stimulated immune responses in rabbits began by studying the skin reactions caused by tick bites. A study by Trager observed that a single infestation of rabbits with stations . This wostations . The maiAmblyomma, Dermacentor, Hyalomma, Haemaphysalis, Ixodes, Ornithodoros, and Rhipicephalus  and hetorexone (R. sanguineus s.l. and R. appendiculatus) lifecycles, preferring domestic hosts such as bovines, canines, and some wild animals, respectively. They feed on humans as incidental hosts [cephalus . Rhipicen Africa ,83. Rhiphe globe ,84. Theyal hosts .R. appendiculatus, R. microplus, and R. sanguineus s.l. in experiments carried out in Brazil, Cuba, Kenya, Japan, and Mexico, which proved to be successful in evaluating anti-tick proteins. Regarding the evaluation of proteins for the development of vaccines against Rhipicephalus species using rabbit as an animal model, a total of 15 molecules, to date, have been evaluated in rabbits for vaccine development against Rhipicephalus species, the most frequent tick genus in this kind of study.New Zealand white, Californians, Japanese white, and Flemish lop-eared rabbit breeds were studied as model hosts for R. appendiculatus tick was used to immunize Japanese white rabbits, which resulted in a reduction in the weight of ticks, followed by a 50% reduction in egg mass [R. sanguineus s.l. infestations [The voraxin α homologue of the egg mass . On the stations .R. microplus cystatin 2c and reported a reduction in the number of fully engorged adult female ticks, causing damage to R. appendiculatus tissues such as the intestines, salivary glands and ovaries [R. sanguineus s.l.; a decrease in nymphs and larvae fed on vaccinated rabbits was observed [R. microplus salivary gland serpins in New Zealand rabbits, obtaining an 83% reduction in adult R. microplus engorgement compared to the control [Parizi et al. using the New Zealand white rabbit for immunization with the  ovaries . A studyobserved . In turn control . These rR. microplus ticks, and demonstrated in a rabbit model that it is both immunogenic and protective. A study used New Zealand rabbits to verify the vaccinal efficacy of the 50-kDa protein from Rhipicephalus haemaphysaloides, reporting a 74.7% protection in feeding ticks just 24 h after infestation [Additionally, for cattle ticks, Lagunes-Quintanilla et al. initially studied the recombinant peptide derived from the serpin RmS-17 protein as a vaccine in New Zealand rabbits in order to later vaccinate cattle. The results showed that the experimental vaccination reached 79% efficacy, limiting the number of infested adult ticks, the oviposition, and the fertility of the eggs . Rabbitsestation . The proIxodes ricinus and Ixodes scapularis are ixodid ticks that are characterized by a heteroxenous life cycle, and infest cattle, deer, dogs, and a wide variety of vertebrates, including humans [Ixodes, 55 are distributed in the neotropical regions of the planet [I. ricinus and I. scapularis can be found only in the northern hemisphere [I. ricinus and I. scapularis using the New Zealand rabbit breed were reported in the U.S., Spain, and the Netherlands  against different tick species. For example, high vaccine efficacy against both n/akirin .Amblyomma hebraeum. There is an amino acid sequence similarity between the voraxin α of A. hebraeum (85%) and that of D. variabilis (92%) and R. appendiculatus (85%) [Similarly, experiments on rabbits using voraxin α, a protein derived from the male tick and transferred to the female through copulation to stimulate female blood-feeding , have yius (85%) . The immus (85%) .I. ricinus (IrFER2) yielded an efficiency of 98%, while the efficiency of the same protein used in bovines infested with R. microplus and R. annulatus (RmFER2) was 64% and 72%, respectively [R. microplus of between 67% and 79% in rabbits [R. appendiculatus [The use of rabbits as animal models in the discovery of anti-tick molecules has been fundamental in enabling the testing of these molecules before inoculation of the natural hosts. It was verified that rabbits present an immune response similar to that of the natural hosts. For example, the use of the ferritin 2 protein to immunize rabbits infested with ectively . Additio rabbits ,87; in biculatus .R. microplus, obtaining a 62% reduction in rabbits [R. microplus obtained a recovery of 33% in rabbits [Studies carried out with the Bm86 antigen in rabbits and cattle have shown that rabbits and cattle have a very similar reduction efficacy against  rabbits , and a 6 rabbits . These r rabbits , compare rabbits , 0.4% in rabbits , and 1.8 rabbits .R. microplus infestations. The BM95-MSP1a vaccine reduced oviposition and fertility of R. microplus similarly to that of the commercial vaccine Bm86, having a significantly greater immune response in vaccinated rabbits compared to the controls [On the other hand, in a vaccination experiment on rabbits, Canales et al. reported for the first time that the recombinant bacterial membrane fraction containing the BM95-MSP1a chimera was effective in the control of controls . The resR. microplus nymphal-to-adult stages on rabbits takes an average of 30 days to complete [R. microplus, in rabbits could be a valuable alternative animal model for the evaluation of candidate vaccines and new molecules with acaricidal activity against this ectoparasite. A possible explanation of why the life cycle takes longer in the nymphal-to-adult life stages of R. microplus in rabbits could be due to the inflammatory cellular response caused by tick bites at the beginning of feeding, which prevents them from accessing the blood source, which can in turn lead to increased mortality and feeding times [The infestation time of the complete , while tcomplete . These dng times . Anotherng times ,130.R. sanguineus s.l. or I. ricinus developed local inflammatory immune responses involving leukocytes, basophils, eosinophils, lymphocytes, mast cells, and macrophages. In turn, bovines immunized with 64TRP and challenged by R. appendiculatus showed dermal migration of dendritic cells, actively degranulating mast cells, basophils, and eosinophils [R. appendiculatus, with infiltration of neutrophils, macrophages, eosinophils, and basophils in both rabbits and cattle [Rabbits immunized with 64TRP and infested with inophils . These ainophils . Furtherd cattle .H. longicornis fed on immunized rabbits suggests that these proteins may be involved in mediating key physiological functions in the tick [The immune responses generated by the different proteins studied in rabbits could vary depending on the challenges of ticks in immature or mature life stages. For example, the response generated by the p29 and HL34 proteins in the life stages of larvae, nymphs, and adults of the tick ,71. Alththe tick , while tR. microplus in immature and mature states have different sensitivities for acquiring resistance against anti-tick molecules. While there was severe intestinal damage in adult females and males feeding on cattle infested with R. microplus-derived antigens, there was no effect on tick larvae feeding on the same protected cattle [H. longicornis ticks fed on rp29-vaccinated rabbits could be consistent as well on natural hosts.This result can be supported by Kemp et al. who recorded that d cattle . TherefoR. sanguineus s.l. and I. ricinus; they target both “exposed” and “hidden” antigens, preventing attachment, and feeding by affecting the feeding site, as well as cross-reacting with ‘hidden’ midgut antigens, resulting in the death of engorged ticks [Additionally, studies on rabbits have allowed us to broaden our knowledge about the “exposed” and “hidden” antigens of anti-tick proteins. For example, it was reported that HLS1 acts on the expression of hidden antigens, inhibiting the secretion of rHLS1 in rabbits during feeding . Also, 6ed ticks .R. appendiculatus ticks, resulting in reduced feeding weights [A. hebraeum, which was studied in infestations on rabbits immunized with voraxin α, where a reduction in feeding of up to 72% was obtained compared to that of the engorged ticks on the control rabbits [Only a few studies focused on identifying molecules that affect male feeding or reproduction. One of the proteins that was identified and tested in rabbits is voraxin . The pre weights . This ma rabbits . These rR. microplus, demonstrating that R. microplus saliva is rich in hemolipoproteins, lipocalins, peptidase inhibitors, antimicrobial peptides, glycine, and maintenance proteins [Results obtained from the study of the tick saliva proteome have shown a variety of proteins that protect ticks against host immune responses and antihemostatic mechanisms ,135,136.proteins . These pproteins ,138, whiproteins  by stimuproteins .I. scapularis and A. americanum stimulated in different hosts, including rabbits, dogs and humans, identifying a total of 276 proteins in I. scapularis and 340 proteins in A. americanum. Among these proteins, 55 (I. scapularis) and 67 (A. americanum) belonged to the same functional classes [A. americanum and I. scapularis use a core set of functionally similar proteins that regulate key host defense pathways to successfully feed. I. scapularis saliva had a high abundance of proteins related to heme/iron metabolism, followed by extracellular matrix/cell adhesion, oxidative metabolism/detoxification, cytoskeletal metabolism, proteasome machinery, nuclear regulation, conserved protein with unknown function, modification proteins, protein synthesis machinery proteins, and transport/storage. In turn, A. americanum saliva had a high abundance of extracellular matrix/cell adhesion proteins and proteinase inhibitors, followed by immune-related heme/iron metabolism, energy protein metabolism, cytoskeletal, protein synthesis machinery and proteasome machinery [Another study by Tirloni et al. examined the saliva proteome of non-fed adult ticks of  classes . These dachinery .A. americanum saliva proteins contain a diversity of protease inhibitors (PI), expressing a total of 155 PI proteins belonging to eight families. Approximately 74% of these PI (115/155) were secreted into saliva within the first 120 h of feeding, indicating that the functions of the PIs are associated with the regulation of the early stages of feeding in A. americanum, which could also include the transmission of TBD agents by A. americanum [The above results indicate that these two tick species could inject the same protein at different levels into different hosts, and that the protein composition of the saliva of different tick species feeding on the same host is likely to be different. Furthermore, the results suggest that ticks of the same species differentially express tick salivary proteins when stimulated to start feeding on different hosts, expressing unique protein profiles in their saliva. There is evidence that ticks differentially express specific sets of genes when stimulated to start feeding ,143. Forericanum .H. longicornis, obtaining 30 proteins in the saliva of nymphs, 74 proteins in the saliva of fully engorged adult females, and 31 proteins that were detected at both stages [On the other hand, Tirloni et al. identified differences in the expression of proteins in the development stages of nymphs and adult females of h stages . Proteinh stages ,145. TakThe current review summarized the evaluation of 57 antigens as anti-tick vaccines in different rabbit breeds. These breeds include New Zealand, Japanese white, Flemish lop-eared, and California rabbits. The most widely used is the New Zealand breed, in countries located in Africa, Asia, America, Oceania, and Europe. Rabbits are not the natural host of most tick species; however, this has not been a limitation in obtaining vaccination results very similar to those of natural hosts for different tick species. Likewise, the use of rabbits has provided valuable insights on the immunological responses generated by novel antigens prior to vaccination trials in natural hosts. Rabbits stand out among other animal models used in vaccination experiments because they are suitable and commercially accessible alternative hosts for challenging the larval, nymphal, and adult life-stages of various tick species.


Invasive blood pressure measurement is considered the gold standard, however it is also technically challenging, requires specialised equipment and carries certain risks. For these reasons, non-invasive blood pressure measurement devices are commonly used and are expected to provide accurate and reliable results. This requirement is particularly true for rabbits, in whom peri-anaesthetic hypotension is commonly observed and in whom perioperative mortality remains disproportionally high. Several authors have compared different non-invasive devices with invasive measurements in rabbits and have reported contrasting results. However, to date no comparison between invasive measurements and the PetMAP™ in rabbits. We hypothesised that PetMAP™ would show acceptable agreement with the invasive measurements according to the American College of Veterinary Internal Medicine guidelines.The aim of the study was the comparison of invasive blood pressure measurement with PetMAP™ cuff was applied distal to the elbow according to the manufacturer’s guidelines. For each measurement with PetMAP™, three invasive blood pressure values were recorded. The mean of the three invasive values was compared with one value measured with PetMAP™.Sixteen client-owned rabbits presenting for various surgical interventions were included in the study. Invasive measurements were performed by cannulation of an auricular artery. The PetMAP™ device showed significant overestimation of systolic, diastolic and mean arterial pressure, which were measured in the auricular artery. In addition, the bias was not constant, implying that the device poorly predicted changes in blood pressure.Data collected from 16 rabbits were used for statistical analysis. In the clinical setting, the PetMAP™ device did not meet any of the American College of Veterinary Internal Medicine recommendations.The PetMAP WhilTo ensure accuracy of measurements, the American College of Veterinary Internal Medicine (ACVIM) has issued a consensus statement recommending validation of NIBP measurement devices .™ is an oscillometric ABP measuring device that was designed specifically for veterinary medicine. It features species and cuff position optimizations  aged 41 +/− 19 months were included in the study. The median weight was 1.9 kg (minimum 1.3 kg and maximum 6.3 kg). Four rabbits were presented for ovarihysterectomies and 12 for dental procedures . Gradati2.1.®, Streuli Pharma AG, Switzerland), butorphanol , medetomidine , midazolam  and ketamine . They were administered in different dosages, combinations and routes as represented in A preanaesthetic examination and basic blood tests were performed in all rabbits. Based on the findings, an appropriate anaesthetic protocol was selected by an experienced anaesthesiologist. Drugs used for premedication included methadone  was applied to the skin above the vein prior to insertion of the cannula.Once adequate sedation was achieved, both ears were clipped, cleaned and disinfected, and an intravenous (IV) catheter  size 24G or 26G, respectively was placed in the left or right medial or lateral auricular vein. In three rabbits EMLA®, Jurox Animal Health, Australia) titrated to effect IV. Orotracheal intubation was performed in 13 rabbits, a laryngeal mask was placed in one rabbit, and a tight-fitting face mask was used in two rabbits for administration of inhalant anaesthetics in oxygen. The respective airway device was connected to a non- rebreathing system and anaesthesia was maintained with sevoflurane  or isoflurane  delivered in oxygen. All rabbits were allowed to breathe spontaneously during anaesthesia. If applicable, loco-regional anaesthesia was performed. In case of nociception (defined as an increase in heart rate and IBP > 20% from baseline) a fentanyl  bolus 2–6 μg kg−1 IV was administered as a rescue analgesia.Prior to induction of anaesthesia all rabbits were preoxygenated with 100% oxygen at 2 L/min administered via a tight-fitting face mask. Induction was performed with alfaxalone . Lactated Ringer’s Solution (B. Braun) was infused at 10 mL kg hour−1 IV. All parameters were constantly assessed by an experienced anaesthetist and the inspired fraction of volatile anaesthetic was adjusted to maintain adequate anaesthesia.The electrocardiogram, end-tidal carbon dioxide tension, body temperature, and arterial oxygen saturation (SpO2) were monitored during anaesthesia using a multiparametric monitor  40 μg kg−1 IV, at the discretion of the responsible anaesthetist. Episodes of hypotension were treated with boluses of Ringer’s Solution 5 mL kg−1 or Voluven  2 mL kg−1 IV.In case of perianaesthetic complications, appropriate treatment was administered as needed. Episodes of bradycardia were treated with either glycopyrrolate  20 μg kg®, Zoetis, United States). Naloxone  40 μg kg−1 IV was used in one rabbit and flumazenil  0.05 mg kg−1 IV in eight rabbits. Meloxicam  was given 1 mg kg−1 orally in two rabbits and 0.2–0.5 mg kg−1 subcutaneously in eight rabbits. One rabbit . The transducer was positioned at the level of the heart, connected to a multiparametric monitor and zeroed to atmospheric pressure before IBP measurements were taken for each new subject. To minimise errors, the IBP monitoring system was carefully checked and periodically flushed to avoid clots and to remove air bubbles that could alter the damping coefficient of the system.A 24G size catheter was placed in the left or right auricular artery . Ear was extended backwards and care was taken not to bend the artery (a roll of bandage was placed under the ear and the ear was further taped if needed to keep it in position). The arterial catheter was then connected to a disposable pressure transducer  with an arterial pressure monitoring line. A dynamic pressure response test was performed to detect over- or under-damping of the system prior to the start of the procedure. A fast flush test was subjectively assessed. Quantification of the damping coefficient was not possible in this study. The system was connected to a pressurised bag filled with 0.9% NaCl  and containing 5 IU mLBefore the start of each anaesthesia, the accuracy of the pressure transducer was checked with a manometer (mmHg) . The manometer was previously tested at different pressures in the hypotensive, normotensive and hypertensive range with a mercury column.2.2.2.™ device. Care was taken to ensure that the cuff was at the level of the heart. If this was not possible, the distance between the cuff and the heart was measured, converted to mmHg (1 cm H2O = 0.73554 mmHg), and the value corrected accordingly.The size of the cuff was selected based on the manufacturer’s instructions. The cuff was then snuggly applied distally to the left or right elbow  and connected to the PetMAP™ , three IBP values were recorded. The mean of the three IBP values was compared with one value measured with PetMAP™. Throughout the procedure, simultaneous measurements of SAP, DAP and MAP were taken every 5 min with the IBP and PetMAP™ monitors. A successful measurement was defined as SAP, DAP and MAP values being all displayed on the monitor and the heart rate on the PetMAP™ monitor matched the heart rate on the IBP waveform.A single operator was responsible for all measurements. The procedure was the same for each measurement. During the ABP measurement with the PetMAP2.3.Statistical analysis was performed on 16 rabbits, comparing IBP and NIBP measurements for SAP, DAP and MAP. To limit the width of the 95% confidence interval for the within-subject population standard deviation to 10%, at least 97 measurements were required for IBP and NIBP. To compare invasive and non-invasive measurements Bland Altman analysis and linear regression were performed with Excel 2016 . Descriptive statistics were performed with SigmaPlot 11.0 .3.A total of 18 rabbits were included in the study, two were excluded due to failure to place an arterial line or due to life threatening complications, respectively . Data coFour rabbits developed bradycardia, which was treated with glycopyrrolate (two rabbits) or atropine (two rabbits). Hypotension (defined as a MAP <60 mmHg) developed in five rabbits and was treated with boluses of Lactated Ringer’s Solution (three rabbits) or Voluven (two rabbits). Additional analgesia (fentanyl bolus) was required in seven rabbits.Bland–Altman analysis included 158, 154, and 155 comparative IBP-NIBP measurements for MAP, DAP, and SAP, respectively. The number of measurements per rabbit ranged from 4 to 23 with a median of 10 [8–12 (25–75%)]. The number of successful measurements for each pressure and measurement technique is shown in ™ device overestimated the IBP measurements of SAP, DAP and MAP which can be seen from the bias . The best agreement in between the two devices was found for DAP, however PetMAP™ still overestimated invasive DAP by up to 54 mmHg (average bias −19 mmHg). As linear regression shows, an overestimation increased with higher pressures for SAP, DAP and MAP.In the present study the ABP measurements taken by PetMAPIn rabbits, different oscillometric ABP measurement devices have already been compared to IBP measurements – with contrasting results . A studyThe ACVIM has published recommendations for the validation of noninvasive blood pressure monitoring devices . The rec™. In anesthetised cats, regardless of cuff position, PetMAP™ met the AAMI criteria for agreement with IBP . The choice of the anterior limb as the site of cuff placement was based on previously published data showing that the position of the NIBP cuff on an anterior limb resulted in better agreement between noninvasive and invasive blood pressure measurements , 13.Still, the shape of the rabbit’s limbs may have affected the measurements. Sphygmomanometric techniques for measuring ABP work by occluding a bigger superficial artery with inflation of a suitable cuff. Therefore, an unsuitable limb shape could hinder the compression of the artery when using a standard cuff . This ma™ tended to greatly overestimate the IBP measurements  cream was used in some of the rabbits. In humans it was demonstrated that Emla cream produces a biphasic vascular response (Invasive blood pressure measurements are considered a gold standard but can also be a source of error , 21. In response . It is tThis study has several limitations.™ would be even greater. And even while the agreement for SAP and MAP between the both methods would be better, PetMAP™ would still not meet the recommendations for validation of NIBP measurement devices.For once, IBP was measured in a peripheral artery as compared to a central one. This decision was based on a risk–benefit evaluation for the clinical patient. While the more invasive catherterisation of a central artery was not considered appropriate, this choice may have affected the IBP measurements. Several authors , 11, 20 Another possible limitation was the selection of clinical patients. Rabbits in this study varied in size, age and ASA status and anaesthetic protocols as well as positioning of the patients differed. In rabbits, the effect of different anaesthetic protocols on the agreement between high definition oscillometric device and invasive blood pressure measurements was examined by others  and the Finally, the damping coeficient was not determined in the present study. Overdamping or underdamping in a pressure system will result in falsely low or falsely high IBP readings . Because5.™ device showed significant overestimation of SAP, DAP, and MAP, which were measured in the auricular artery. In addition, the bias was not constant, implying that the device poorly predicted changes in blood pressure. The PetMAP™ device did not meet any of the ACVIM recommendations.In the clinical setting, the PetMAPThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Institutional Ethics and Animal Welfare Committee of the University of Veterinary Medicine Vienna. Written informed consent was obtained from the owners for the participation of their animals in this study.JS, AR, and N-GK prepared study design and were involved in clinical management of the cases. AR and N-GK supervised the experiment. JŽ performed statistical analysis. All authors reviewed and edited the manuscript, have read, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The component-based software system has a core that is based on architecture design. Predicting the reliability growth trends of a software system in the early stages of the development process is conducive to reducing waste and loss caused by blind development. Restricted by the lack of information and data in the design and integration phase, it is difficult to implement reliability prediction research at this stage. In this article, we focus on a software system in which the reliability of each component follows the G-O model. First, two system-level parameters, which are the total number of system faults and the detection rate of the system faults, are defined. Then, by studying the relationship between the total number of faults and the detection rate of faults between the components and the system, the defined system parameters are calculated from the known component parameters. On this basis, and by incorporating the system parameters, we construct a reliability growth model for the software system, called the component-based generalized G-O model (CB-GGOM). Besides, two approximate models of CB-GGOM are proposed to facilitate reliability evaluation of the software system in the early and stable stages of the integration test. An engineering explanation of the proposed models is also provided, and their effectiveness is verified through simulation and with an authentic example. Since the proposed models are formulated without any integration test data, they are beneficial for developers to optimize test strategies of the software system and implement defect prevention in advance. With the development of software integration techniques, component-based software engineering (CBSE), which is a technique for software reuse, has become widely used in the development of software systems. The goal of CBSE is to construct a new software system by integrating several existing components that are connected loosely and interact with each other in an interoperable way . The conAt present, the scale of component-based software systems is becoming larger, and the applications of this type of system in aviation, military, medicine, and other key fields are constantly growing . HoweverTo evaluate and predict the reliability level of software, traditional analysis methods usually regard software as a black box and establish software reliability growth models (SRGMs) by collecting the fault data of software in the testing or operation stage. These black box methods mainly applied to early small-scale software and were the prominent approach for software reliability evaluation until the early 21st century . HoweverThe lifecycle of component-based software systems generally includes the stages of analysis/design, implementation/acquisition, integration, test, maintenance/distribution, and administration/support. Also, software architecture design forms the core of the software system. For such a system, the cost of making modifications in the later development stages is massive, while some designs cannot be modified at all. In some specific software system development projects, such as the development of extremely large systems, the core architecture of the system needs to be designed at the outset. Based on passing the test of the core architecture, peripheral components are gradually added to it. To reduce waste and loss caused by blind development, evaluating the reliability of software system in the upstream stage of development has become the focus of many scholars in the field of component-based software system reliability evaluation . HoweverThis article aims to infer the reliability growth trend of a software system during integration testing by using available system architecture and the historical reliability growth trends of components. For this purpose, we focus on a software system in which the reliability of components follows the Goel & Okumoto model (G-O model) . First, The remainder of this article is organized as follows: Section 2 introduces research work in related fields and the research motivation of this article. In Section 3, we analyze the available information before system integration and propose some basic assumptions. In Section 4, the estimation models of two system parameters and the reliability growth models of the system are proposed. Also, the engineering interpretation is provided. Section 5 verifies the effectiveness of the proposed models through simulation and authentic example. Section 6 discusses the applications of the proposed models. Section 7 discusses the threats to the validity of the proposed models and future work.At present, there are several reliability evaluation models for component-based software systems that can be divided into static and dynamic models.Static reliability evaluation models generally focus on the modeling of system architecture under the assumption that component reliabilities are known, thereby obtaining the point estimation of software system reliability. These models can be further categorized into path-based methods and state-based methods. Path-based methods employ all executed paths to model the system architecture and apply the reliabilities of all components in the path to represent path reliability. Then, they utilize the path reliabilities and path execution probabilities to calculate overall system reliability . State-bTo achieve time-dependent system reliability evaluation, some dynamic evaluation methods, such as the dynamic probability transition graph  and stocTo shorten the reliability evaluation time, Component-based software reliability growth models (CB-SRGMs) are models that focus on the growth process of software system reliability. They generally use SRGMs to model the growth processes of component reliabilities, thereby establishing a relationship between components and the software system. Subsequently, they evaluate and predict system reliability. The function of the cumulative number of system faults is the kernel of CB-SRGMs. Additive models are representative CB-SRGMs. By assuming that the fault process of each component followed the non-homogeneous Poisson process (NHPP) and the system had a serial architecture, In general, although there are many reliability evaluation methods for component-based software systems, the guiding ideology of most studies is to simplify the models as much as possible to reduce time and cost while ensuring evaluation accuracy. However, most existing methods are employed in the integration test stage or operation stage, when test data are abundant. Restricted by the lack of information and data in the design and integration stage, few system reliability prediction models are applied at these stages, and the evaluation and prediction performances are not satisfactory.A new system assembled from components is substantially different from single software developed from scratch since each component has historical test information. This information can be used to establish SRGMs that reflect historical fault trends of components. Integration tests have not been carried out, so any data generated during system operation cannot be obtained. However, the system architecture is designed in advance and the integration test is usually performed by simulating the operating behavior of users. This means that random interaction in integration testing should follow a certain statistical regularity. Therefore, we can utilize the known system architecture and historical fault trends of the components to infer the fault trends of the software system during the integration testing process. The NHPP is the most popular theory for describing the fault trend of a single software, while the G-O model has important theoretical value. Because of the above considerations, we intend to conduct research under the assumption that component reliability follows the G-O model.1) The initial time of the software system integration is 0.i.e.:2) The fault process of the component follows the NHPP. The component fault trend in the unit test stage can be modeled using SRGM, which is known and is assumed as the G-O model , i.e.:Components can be diversely defined depending on the discipline . In the Ti is the last unit testing fault time of component i, mi(t) represents the expected number of faults detected from component i when the unit testing time reaches t, and once a fault is detected during unit testing, it immediately causes component failure. The constant ai denotes the total number of faults in component i, and the constant bi represents the fault detection rate of component i in the unit testing stage. Here, ai and bi are estimated through unit testing fault data.3) The component interfaces are considered to be completely reliable. A larger-scale component interface can be abstracted as a separate component, and its reliability follows the G-O model with known parameters, while the remaining smaller-scale component interface is assumed to be simple enough.4) The fault processes between components are independent, and the interaction processes between components are also independent. The operation profile model is known.I = {i|i = 1,2,…,n} as a discrete set of states, where element i represents the ith component, including the component interface. Also,  is the continuous operation time interval between any two system failures, while the random variable vi is the residence time of the system on the active component i. S = {s1,s2,…,sm,…} is the set of transition moments during system operation, which satisfies 0 < s1 < s2 < …< sm <…< s. Also, X(sm) = i is the component activated after the mth operation transition. P{X(sm+1) = j|X(sm) = i} = pij is a one-step transition probability from component i to component j at the m+1th operation transition, and P is the transition probability matrix. Based on the above assumptions, the state transition process at each operation transition time has no aftereffect. Thus, it can be described by a time-discrete Markov chain, which is time-homogeneous. The value of pij is determined by the architecture design of the software system and the operating behavior of users. If component i and component j are designed not to interact directly, pij is assigned as 0. Otherwise, the value of pij is given by the operation profile model. Thus, the control transition of a simple software application is shown in Next, we define When the interaction processes of components are assumed to be completely independent and the fault process of each component follows the NHPP, then the entire fault process of the system is an independent incremental process that can also be described using the NHPP . This fet and the number of remaining latent faults in the software has not yet been detected. When the reliability of each component follows the G-O model, the total number of faults in the component is assumed to be constant and no new faults are introduced during the debugging process. Therefore, the total number of faults of the entire software system should also be constant, which can be written as the notation As. The relationship between As and a1, a2, …, an is written as a function of f.The total number of software faults refers to the sum of the cumulative number of faults that have been removed when software testing reaches time As does not change as integration testing proceeds, it is considered equal to the sum of the total number of faults in each component at the start of integration. Each component is tested before integration with many internal faults already having been removed. Therefore, if i when unit testing reaches time t, i at the initial time of integration. This satisfies:Because the value of Ti is the final unit testing fault time of component i. Thus, As can be expressed as:mi(t) from f, which is the estimator of As:By substituting the biTi) measures the sufficiency of component i being tested in the unit test phase.t is closely related to the FDR of the component currently being executed and also changes with the transition of the executed component. Thus, we consider the FDR of the system as a function of time and express it as Bs(t). Moreover, the relationship between Bs(t) and b1, b2, …, bn is a function g.The fault detection rate (FDR) describes the efficiency of software fault detection in the current testing environment. Because the testing process of the software system is considered a control transition process, the FDR of the system at time t  and ms(t) respectively denote the fault intensity function and the cumulative fault function of the software system at time t. Besides, according to NHPP theory, Λ(t) = m′s(t). Since As has already been expressed by the component parameters, if a relationship between the cumulative fault function of the system and its components can be subsequently established, Bs(t) can also be expressed by b1, b2,…, bn and the model of function g is automatically obtained.ms(t) should equal the sum of the cumulative fault functions of the components when integration testing reaches time t. However, the components are no longer continuously activated but are instead intermittently exchanged during integration testing. Meanwhile, the function mi(t), which is created in a unit testing environment, cannot be directly used in the integration testing phase. To measure the execution state of the component in , we first regard n components as n states, then use the transition probability matrix P to obtain a steady-state distribution of the n states, which we express as τi as the expectation of the random residence time vi, that is Evi = τi. Thus, τi represents the average task time of component i in one execution. Its value is determined by the functional attributes of the component and can be obtained from the statistics of unit testing data. Consequently, the factor:Obviously, i during the stable state of the system  can be modeled as follows:e system . Owing tms(t) can be estimated as:By substituting the G-O model into Bs(t) is:Finally, by integrating ωi(t) as:If we write the factor the estimator of ωi(t) is the proportion of the remaining faults in component i to the remaining faults in the software system at time t. Therefore, if we name ωi(t) the proportional factor of the remaining faults in component i, then bi is estimated according to the component fault data generated in the unit test stage. It is a known parameter that reflects the historical fault detection level of component i; the fault detection level of component i in the integration test stage is unknown, for ease of expression, we use the notation biIT(t) to represent it. Therefore, according to ωi(t)bi is the prediction for biIT(t); because integration testing is a process of control transition, each component is not allocated the entire integration testing time but is assigned a proportion of the total test time as πi. Thus, the weighted sum of the probability of detecting faults in each component within  and its actual contribution proportion to the system is an estimation of the system fault detection probability within . We can imagine that without πi, the test of the software system is a parallel test rather than a control transition process. Also, if there is no ωi(t) but only bi to predict biIT(t), the test of the software system then becomes a process of n newly developed components being independently tested. In such a testing environment, the number of initial faults in each component is the largest, each component is on an independent testing platform with its own exclusive testing resources, and the test for each component is continuous, just the proportion of tested time is different. However, every component entering the integration testing stage has been tested, so actually there are few residual faults; moreover, these faults are no longer concentrated in a single component but are unevenly distributed within each component. With the transition of system operation, n components share a set of test resources. A reduction in the number of latent faults and changes in the distribution of latent faults are the main differences between the two test stages of the components. The meanings of “remaining faults” and “proportion” of ωi(t) reflect the above component changes from unit testing to integration testing. Therefore, using ωi(t)bi to predict the fault detection level of component i in the integration test stage is a correction of prediction as bi.By observing To further investigate the rationality of ωi(0) can be calculated as:ωi(t) continuously fluctuates. When the testing enters a stable state, the limit of At this time, the number of remaining faults in the software system is the largest, so the fault detection ability of the system is strong. As testing proceeds, the weight Bl), where the superscript l indicates that the minimum πibi is generated by the lth component. Thus, We define ωi(t), the probability of detecting faults in a component without fault tends to zero, and the fault detection rate of the system is equal to min(πibi).Now we can explain the FDR of the system in a steady state. When the integration test enters the steady state, the internal faults of almost every component are eliminated and only the component with the slowest fault detection has undetected faults. At this stage, the system still activates the components according to the operation profile, but it is impossible to detect faults in the components that have no faults. After the task is completed, the system quits the active component. Therefore, the FDR of the system is reflected entirely by the calling frequency and the FDR of the last faulty component. The task of establishing a software reliability growth model generally includes modeling the cumulative fault function and the reliability function. When the fault process of the software obeys the NHPP, the latter can be fully represented by the former. In this section, according to the concept of engineering approximation, we model the cumulative fault function and reliability function for the component-based software system by incorporating the system-level parameters proposed in the previous section.The G-O model uses a negative exponential structure to express the cumulative fault function, so it is also known as an exponential SRGM. Since many SRGMs are derived from the G-O model and essentially belong to the exponential category, the G-O model is considered a model with good flexibility and adaptability. When the reliability of each component follows the G-O model, the debugging process of each component is assumed to be an instantaneous process without the introduction of any new faults. Based on previous assumptions, the debugging process of the system is the same as that of the components. Thus, we can use a negative exponential structure to express the cumulative fault function of the software system. It can be modeled as:t, then the continuous duration of operation after startup is s. According to the definition of software reliability, the probability is that the software will run continuously for a given period without fault. The reliability of the software system within  can be expressed as:Therefore, if the last startup time of the software system is Bs(t) is a function rather than a constant. Therefore, As and Bs(t) are estimated by the component parameters.ξ in the interval  and use the corresponding constant Bs(ξ) to replace the function t, t + s) can be obtained:Since ξ is either 0 or ∞, and consider two special cases of In this section, we verify the effectiveness of the proposed models through simulation and by applying the models to an example. The significance of proposed models for the early evaluation of software system reliability is also demonstrated.Rate-based discrete-event simulation (RBDES) is an important reliability simulation technique that is applied to individual software. It simulates the random fault behavior of software based on rate-driven event process theory . By combWe utilized the software application reported in t. As a demonstration, we present the values of t is taken as 1 × 104, 1 × 105, and 1 × 106 respectively, as First, based on the above-known information before integration, the values of 6 time units, then simulated the fault detection process of the system. By repeating the simulation 30 times and eliminating random errors, we obtained 25 groups of simulated system fault data , where ti represented the simulated time of the ith failure of the software system, and yi represented the cumulative number of system faults at ti, which is a positive integer. Then, we calculated the values of Next, we verified the prediction effects of the proposed models through simulation. We set the discrete time step of the simulation as one unit of time and the total simulation time as 4 × 10Finally, to illustrate the significance of the CB-GGOM, we used traditional SRGMs to fit the simulated system fault data. The selected SRGMs were the G-O model and the Yamada & Ohba model (Y-O model) , which ims(t) of the CB-GGOM in the form of the G-O model. Next, since the fitting error of the G-O model based on LSE was only 0.9928, we compared its parameter values  with those of the CB-GGOM. We found that the G-O model’s parameter values had little deviation from those of CB-GGOM. However, the parameters of the G-O model were estimated based on 25 pieces of simulated system fault data, which means that the G-O model can only be used in the late stages of the integration test with 4 × 106 time units. This duration is almost ten times the length of the component unit test . In comparison, the parameters of the CB-GGOM are calculated without any integration test data so that the CB-GGOM can predict the growth trend of system faults in the design and integration stage. Therefore, the simulation experiment verifies the accuracy of CB-GGOM, and demonstrates its value in the early evaluation of software system reliability.In We further examined the performance of the proposed models using a subsystem of a management information system for a patent affairs process as an example . This suBy applying the data in Besides, we determined the proportion vector for the time spent on each component:b1IT(0), b2IT(0), …, b5IT(0) are 0.303 × 10−6, 1.249 × 10−6, 1.142 × 10−6, 0.602 × 10−6, and 0.775 × 10−6 based on Following this, we calculated that the values of Next, we used the data in It can be seen from i in the integration test stage ) predicted by CB-GGOM-E has one more proportional factor ωi(t) than that predicted by the CB-NHPP model. According to our explanation given in Section 4, the test described by the CB-NHPP model is the process in which n newly developed components are independently tested. Thus, the value of n components. With this in mind, we note that the value of b1, b2, …, bn. In comparison, the values of b1, b2, …, bn. It is widely known that the time spent on integration testing is actually much slower and longer than that spent on unit testing. Thus, from the quantitative analysis alone, the system fault detection level predicted by the CB-GGOM is closer to the objective reality.The main reason for these differences is that the fault detection rate of component ith component in the integration test with a constant bi, no matter how long the integration test lasts. Thus, if we assume that the integration test has entered a stable state, according to the prediction of the CB-NHPP model, the tester willdetect faults from a component that has no faults with the possibility bi. Obviously, this situation is contrary to objective fact. Therefore, the system fault detection level predicted by CB-NHPP should be faster and faster than the actual system fault detection level as the test proceeds, resulting in increasingly premature predictions for the cumulative number of system faults. In comparison, CB-GGOM uses ωi(t)bi to predict the fault detection rate of component i in the integration test stage. Under the constant correction of ωi(t), the probability of a component without faults being detected tends to zero. Therefore, CB-GGOM can reasonably predict the growth process of system faults in the middle and late stages of the test. The findings presented in Next, we analyzed the rationality of CB-GGOM theoretically. The CB-NHPP model always predicts the fault detection rate of the R(s|t) using Finally, we calculated As The two main innovations of this article are the system parameter estimation models and the software system reliability growth models that incorporate these system parameters.First, the proposed system parameter estimation models can be calculated according to the known information in the integration phase. Their main function in this article is to serve as the input of the software system reliability growth models. Due to the incorporation of system parameters, the proposed growth models can evaluate and predict the reliability growth trends of the software system during the integration testing process without integration test data. Therefore, the reliability growth models are useful for software system developers to grasp the maturities of the current design scheme and test scheme, thereby assessing software development costs and planning the project schedule.Another advantage of the proposed system parameter estimation models is that they are expressed by the sum of the product of the weight factors. To focus on the topic of software system reliability evaluation, we only discuss the interpretability of these factors of the proposed models and do not dig into their other values in this article. Actually, accurate identification of the weakest components of the software system in the design phase is also a beneficial work, because this identification can helps the software system developers to formulate targeted prevention strategies and eliminate potential hazards in the design stage and guarantee the quality of the software system economically and efficiently . HoweverbiIT(0), we can predict that the probability of faults detected in the component at the beginning of the integration test is c2 > c3 > c5 > c4 > c1 in descending order, and the probabilities of c2 and c3 are almost four times that of c1. Since the overall probability of faults being detected in the software system at time t is dependent on the probability of faults being detected in each component, the component with the highest biIT(0) value is the one with the highest probability of faults being detected first at the initial stage of the integration test. From this, we know that c2 and c3 are the two most fully tested components before delivery, but they may be the two components that are most likely to fail first in the early phase of the integration test due to the longer test time allocated in integration testing. Therefore, c2 and c3 should be the focus of software system developers. Based on this inference we can provide further suggestions for the optimization of software system testing strategies. We know that within the specified time interval, smaller fault detection probability reflects a more reliable software system. For the Bs(t) proposed in this article, the value of bi is determined by the unit testing environment before component delivery, and the value of πi is determined by the user’s operating habits and the functional attributes of the components, that is, these two factors are not easy to be modified by the software system developers. Therefore, an effective method is to strengthen testing for weakest component first and then implement integration testing according to the original scheme. This testing strategy actually reduces the value of Bs(0) by only reducing the value of ωi(0) of the weakest component. Theoretically, it can maximize the level of software system reliability without modifying the structural design and the original integration test scheme, so it is an operable and low-cost testing method.We can take the instance in Part B of Section 5 as a concise demonstration. First, by applying In this article, we propose a reliability growth model named CB-GGOM, as well as two approximate models, CB-GGOM-E and CB-GGOM-S, for a software system whose component reliability follows the G-O model. The G-O model assumes that once a fault is detected, it immediately causes software failure. Thus, the models proposed in this article also regard that once a fault in a component is detected, the component and the entire software system both immediately fail. This assumption is rigorous and does not correspond exactly with reality, so the evaluation results of the proposed models are conservative. Fortunately, some SRGMs of single software that consider failure delay and degraded operation have been studied, and many of them have been enhanced according to the G-O model. Therefore, we can improve the CB-GGOM based on these research results, to obtain evaluation results that are closer to reality.To facilitate the establishment of the mathematical model, we propose some assumptions in the third section. The analyses and citations in this section are to illustrate that the proposed assumptions are derived from the engineering approximation, so the deviation between the assumption and the actual situation is not too large to be accepted. Besides, there are some situations that are not covered by the proposed models, such as the constraints of the test cost, the introduction of new faults during the debugging process, and the adjustment of test strategies caused by the participation of multiple debugging personnel. The solutions of these situations usually involve other theories, relevant references and integration test data, so they need to be explored in the subsequent research, and reasonable engineering approximation is still an effective means for subsequent model construction.To verify the effectiveness of the proposed models, we use mean square error to measure the deviation between the cumulative fault function and the test data in Section 5. Since the cumulative fault function is also an increasing function, it is beneficial to know its supersaturation to the horizontal asymptote in the Hausdorff metric. This metric allows researchers to choose an appropriate model for approximating specific data from different branches of scientific knowledge . HoweverIn Section 6, we provide a tentative discussion and a brief demonstration of how the proposed system parameter estimation models could be applied to infer the weakest components of the software system and optimize testing strategies. Certainly, the validity and rationality of these inferences and suggestions should be further verified in diverse situations. Assorted simulation techniques should also be adopted to substantiate the value and contribution of the proposed models. We think that this application of system parameter estimation models should be explored as a special topic in the future.10.7717/peerj-cs.1247/supp-1Supplemental Information 1Click here for additional data file.


The molecular mechanisms of their fusion with the plasma membrane are only partially understood. In this review, we discuss the molecular players involved in the regulated exocytosis of CTL, highlighting the parallels and differences to neuronal synaptic transmission. Additionally, we examine the strengths and weaknesses of both systems to study exocytosis.Regulated exocytosis is a central mechanism of cellular communication. It is not only the basis for neurotransmission and hormone release, but also plays an important role in the immune system for the release of cytokines and cytotoxic molecules. In cytotoxic T lymphocytes (CTLs), the formation of the immunological synapse is required for the delivery of the cytotoxic substances such as granzymes and perforin, which are stored in lytic granules and released  These toxic substances are stored in lytic granules (LG), which undergo exocytosis upon T cell antigen receptor (TCR) signaling, releasing cytotoxic molecules at the contact zone with the target cells. This contact zone, where TCR-mediated signal transduction and secretory events take place, is named the immunological synapse (IS) . ExocytoInterestingly, very similar mechanisms govern neuronal synaptic transmission. The release of neurotransmitters by neurons is by far the most highly regulated form of exocytosis known. The speed, accuracy and temporal resolution found at synapses are unmatched. Due to their complexity and importance, the mechanisms involved have been the target of intense study. A variety of the molecules discovered at neuronal synapses have recently been found to have similar roles in LG exocytosis. Furthermore, a number of immunological deficits have been tied to mutations of proteins involved in neuronal synaptic transmission. The mechanisms underlying LG exocytosis in CTLs are not well characterized and their understanding has benefited greatly from the knowledge of synaptic transmission. The aim of this review is to describe the molecular process of neuronal synaptic vesicle release in neurons and compare it with that of LG exocytosis in CTLs. We highlight the similarities and differences between the two systems and identify gaps in our current understanding of these key cellular processes.2The core molecular machinery for membrane fusion is formed by the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. They were discovered by R. Schekman in yeast  and J. R2.1The molecular mechanism of synaptic vesicle exocytosis is highly complex and is described in detail in excellent reviews –11. Ther2+-calmodulin, this mesh dissolves releasing SVs that move toward the active zone at the pre-synapse along F-actin using myosin II or V as the motor protein (via Rab3 (2+ channels (VGCC) is promoted by the t-SNAREs and Munc18-1 . As the resolution of electron microscopy has improved, the analysis of electron micrographs has become more precise, and it is now accepted that docking equates to priming, which is why we refer to this process as docking/priming . For docmembrane –38. Bothmembrane –41.2+-dependent activator protein for secretion-1 (CAPS1) promotes priming via interaction with syntaxin-1 probably downstream of Munc13-1 or -2 . Finallntration –47. Few ntration  and addintration , 50.2.2via classical clathrin-mediated endocytosis, fast endocytosis or bulk endocytosis. The mode of endocytosis depends on the strength of the stimulus, i.e. the pre-synaptic Ca2+ concentration, and the amount and the speed of SV fusion  cells. The main difference between the two cell types lies in the recognition of the target cells. For this reason, we will discuss the mechanisms of LG exocytosis using data obtained on both cells  causing FHL type 2 (SH2D1A/SAP (coding for SLAM-associated protein), ITK (Tyrosine protein kinase) or CD27 (receptor for TNF) inducing FHL type 1   , Munc13-4  and Munc18-2   –93; 4. IP/BIRC4) , 95. In , FHL 4) , and thr, FHL 5) –100. All3.2via Fas-FasL that occurs via a different vesicular trafficking pathway  , 121. In2+ channels, that occurs simultaneously to an IP3/Ca2+ dependent activation and translocation of STIM proteins to the ER close to the IS. Activated STIM proteins interact with Orai, forming the store-operated Ca2+ release activated Ca2+ (CRAC) channel complex, which open leading to store operated Ca2+ entry (SOCE)  –130. Thechondria , 132 enschondria , 134. Thchondria , 136. Mevia two different protein complexes. The first is composed of Rab27a that associates with LG membranes in a GTP dependent fashion, the synaptotagmin like protein-2 (Slp-2) anchored in the plasma membrane and probably Munc13-4 . Mutations of these C2 domains that alter their Ca2+ sensitivity, abolished LG exocytosis in NK cells  in about 300 ms. Events, in which content diffusion was much slower, were ignored from the analysis as it was questioned whether they corresponded to proper LG exocytosis. However, in a groundbreaking work, Bálint et al. . This leads to distinct intracellular Ca2+ concentration ([Ca2+]i) increases. Stimulation of a neuronal synapse by one action potential induces a very short (> 10 ms), extremely localized (1-2 µm diameter) but steep (> 10 µM) [Ca2+]i increase, which is sufficient for the fusion of one to three SVs  increases in [Ca2+]i (see section 3.3). The rise of [Ca2+]i is relatively uniform along the IS and can reach values of about 2 µM , but even the LGs are diverse. As mentioned above, LGs can be divided into single-core granules, containing diffusible granzyme B, and multi-core granules, containing SMAPs  along the entire plasma membrane. Both organelles have well-defined shapes and sizes. The release machinery of LDCV is not fully understood, but appears to be very similar to that of SV exocytosis with some differences in docking/priming factors , 181. Thng SMAPs , 154. Thng SMAPs , 183. Fuer cells , 154.5Both the nervous and immune systems use synaptic contacts to transmit key intercellular information through regulated secretion of intracellular vesicles. They use a similar molecular machinery, with SNAREs at the core and Munc18 and Munc13 as the major facilitators. The protein paralogs in CTLs are different from those in neurons, but their individual domain structures are nearly identical, with the exception of Munc13. Thus, advancing knowledge of exocytosis in one system benefits the study of the other.A great advantage of studying secretion in immune synapses over neuronal synapse is that human CTL are readily available and can be easily handled. Accumulated research findings in CTL reveal subtle differences in the mouse and human T cell secretory machinery. One example for this diversity is the v-SNARE of LG that is required for exocytosis: VAMP7 is used in human CTL, while VAMP2/synaptobrevin2 is used by mouse CTL , 142. VAIn conclusion, in our review, we discussed how “synaptic inspection” in neuroscience and immunology can learn from each other and how important it is to define the precise identity and function of the multiple proteins involved in both SV and LG secretion.UB, H-FC, and CS conceived and wrote most of the paper. VP and EK wrote some parts of the paper. CS and VP generated the data shown


The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+ measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+ levels in the MAMs. However, sensitivity of the first generation Ca2+ biosensors on the surface of the outer-mitochondrial membrane (OMM) do not allow to measure μM or sub-μM changes in Ca2+ concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+ biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+ leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked.Genetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca These pd 565μM) , Yue et  the OMM . IP3R1 hKO cells , is expe2+ leak during hypoxia. When Hela cells were incubated under oxygen glucose deprivation (OGD), an ER Ca2+ leak occurred which could be measured indirectly through the sustained Ca2+ increase both in the cytosol, [Ca2+]cyto  the loss of IP3Rs activity and (ii) the modification in ER-mitochondrial contacts were involved in the observed decrease in the rate of Ca2+ accumulation on the surface of the OMMs during the hypoxia, we incubated WT Hela cells with U73122 to inhibit phospholipase C (PLC), which has a crucial role in the initiation of the activation of IP3Rs  .Blots were incubated with horseradish peroxidase (HRP)-coupled sheep anti-mouse IgG  and (HRP)-coupled goat anti-rabbit IgG , and developed with Clarity Western ECL Substrate . The band intensity was determined using Image Lab software (Bio-Rad).2+-free buffer containing 140mM NaCl, 5mM KCl, 1mM MgCl2, 10mM HEPES and 10 mM Glucose, adjusted to pH 7.4, supplemented with 1 mM EGTA.Living cells were imaged on an inverted epifluorescence microscope Leica DMi6000B using a 40x oil-immersion objective, with Lambda DG4 wavelength-switch xenon light source (Sutter Instruments), equipped with an ORCA-Flash4.0 digital CMOS camera C11440 (Hamamatsu). Pictures have been acquired with 200 ms acquisition time per frame, 10% fluorescence intensity manager (FIM) and an interval of 1 second for time-lapse. Cameleon fluorescent proteins were excited at a wavelength of 430nm and emissions were collected at 480nm and 530nm. Fluorescence ratio imaging was analysed using MetaFluor software (Molecular Devices). Experiments were carried out in controlled environment at 37°C and cells were placed in Ca2+-free buffer containing 140mM NaCl, 5mM KCl, 1mM MgCl2, 10mM HEPES and 10 mM Glucose, adjusted to pH 7.4, supplemented with 600μM EGTA. The Rmin was achieved by perfusing the cells with this medium containing 600μM EGTA and 5μM BAPTA-AM. The Rmax was achieved by perfusing the cells with a Ca2+ buffer containing 140mM NaCl, 5mM KCl, 1mM MgCl2, 10mM HEPES and 10 mM Glucose, adjusted to pH 7.4, supplemented with 10mM CaCl2. R% is calculated as R% = (R − Rmin)/(Rmax − Rmin) × 100.Dynamic range method: In H9C2 cells expressing N33D3cpv or N33D1cpv, Rmin and Rmax were obtained upon permeabilization of the cells with 5μM ionomycin and a Ca2+ titration assay: H9C2 cells expressing N33D3cpv were permeabilized with 100μM digitonin in a Ca2+-free medium containing 600μM EGTA for 60 sec and then washed 3 times with the same medium without digitonin. Cells were then perfused with Ca2+ buffer containing 140mM NaCl, 5mM KCl, 1mM MgCl2, 10mM HEPES and 10 mM Glucose, adjusted to pH 7.4, and known Ca2+ concentrations. At the end of each experiment, a saturating Ca2+ concentration (10mM) was applied. For [Ca2+] lower than 0,5μM, the buffer was supplied with BAPTA free acid and Ca2+. The free [Ca2+] was estimated using MaxChelator. The results obtained were plotted as log10[Ca2+] (x-axis) and R% (y-axis) and fitted using Prism 9.0 (GraphPad) with the following equation: y = (Rmax1 × xn1)/(kd1n1 + xn1)+(Rmax2 × xn2)/(kd2n2 + xn2).In situ Ca2+-containing buffer having 140mM NaCl, 5mM KCl, 1mM MgCl2, 10 mM HEPES and 2mM Na2S2O4, adjusted to pH 7.4, supplemented with 2mM CaCl2. Cells were placed in a specifically manufactured bio-incubator  connected with a 100% N2 bottle. Oxygen level and temperature were monitored at 0.5% and at 37°C respectively. An Okolab system with a specific hypoxic chamber was used to control the environmental constants .Cells were washed twice, placed in CaLiving cells were imaged on an inverted confocal microscope Nikon A1R+ system using 40x oil-immersion objective with Argon laser (488-514nm) for the Cameleon excitation, Diode (642nm) for the Mitotracker Deep Red and diode (560nm) for the ERtracker Red. The images were acquired on living cells plated on glass coverslips.t-test  or Mann–Whitney test  was used unless stated otherwise in the figure legends. p-values are indicated in figures. Data show mean with standard deviations (SD) calculated from at least three independent experiments. For single-cell imaging analysis, statistics were performed on n  =  number of cells to assess single-cell effect as well as heterogeneity between them. Both N and n values are indicated in the figure legends. A p value < 0.05 was considered significant.Data processing and statistical analyses were conducted with Prism 9.0 (GraphPad) software. Before proceeding to any analysis, the normality of the samples was evaluated (Kolmogorov-Smirnov test). Unpaired S1 Fig2+]OMM was estimated using N33D3cpv biosensor in WT and TKO IP3Rs HeLa cells treated with 100 μM Na, in absence of external Ca2+. Representative average FRET-ratio (F) normalized with the baseline FRET-ratio value (F0). (E) Steady-state [Ca2+]cyto in WT and TKO IP3Rs HeLa cells. (F) Steady-state [Ca2+]OMM in WT and TKO IP3Rs HeLa cells. The normality of the samples was evaluated (Kolmogorov-Smirnov test) and Mann–Whitney test  was used. (G) [Ca2+]OMM steady state  and peak measurements upon ATP, CPA and OGD treatment protocol with the N33D3cpv sensor.(A) Immunoblotting against IP3R1 isoform (IP3R-I Santa-Cruz sc-28614) and tubulin in WT and TKO IP3Rs HeLa cells. Data shown represent the mean with standard deviation (SD) of 3 independent experiments, (* p<0.05). (B) Immunoblotting against IP3R1 receptor (Anti-IP3 receptor antibody Abcam ab5804) in WT and TKO IP3Rs Hela cells. Proteins normalized by using 2,2,2-Trichloroethanol (TCE) to visualize total protein content. Data shown represent the mean with standard deviation (SD) of 3 independent experiments, (* p<0.05). (C) Immunoblotting against IP3R3 receptor  in WT and TKO IP3Rs Hela cells. Proteins normalized by using 2,2,2-Trichloroethanol (TCE) to visualize total protein content. (D) [Ca(TIF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.