Rabbits are well-domesticated animals. As a crucial economic animal, rabbit has been successfully bred into wool-use, meat-use and fur-use breeds. Hair length is one of the most economically important traits affecting profitability in wool rabbits. In this study, to identify selection signatures with the long-hair trait, whole-genomic resequencing of long-haired rabbits (Angora rabbits) and short-haired rabbits  was performed.Dusp1, Ihh, Fam134a, Map3k1, Spata16, and Fgf5) were enriched in the MAPK signalling and Hedgehog signalling pathways, both of which are closely associated with hair growth regulation. Among these genes, Fgf5 encodes the FGF5 protein, which is a well-established regulator of hair growth. There was a nonsynonymous nucleotide substitution (T19234C) in the Fgf5 gene. At this locus, the C allele was present in all of the tested Angora rabbits, while the T allele was dominant in New Zealand and Rex rabbits. We further confirmed that the C allele was conserved in Angora rabbits by screening an additional 135 rabbits. Moreover, the results of functional predictions and co-immunoprecipitation revealed that the T19234C mutation impaired the binding capacity of FGF5 to its receptor FGFR1.By genome-wide selective sweeping analysis based on population comparison, we identified a total of 5.85 Mb regions (containing 174 candidate genes) with strong selection signals. Six of these genes (We discovered that the homozygous missense mutation T19234C within Fgf5 might contribute to the long-hair trait of Angora rabbits by reducing its receptor binding capacity. This finding will provide new insights into the genetic basis underlying the genetic improvement of Angora rabbits and benefit the improvement of rabbit breeding in the future.The online version contains supplementary material available at 10.1186/s12864-023-09405-2. Rabbit domestication was initiated in monasteries in southern France as recently as ~ 1500 years ago . As one Angora rabbit is a famous breed of wool rabbit and is largely bred for its high yield and quality of wool. Approximately 10,000 tons of Angora wool are produced every year, which makes it the third most-produced animal fibre in the world, after sheep wool and mohair , 4. The Phenotype is closely correlated with genotype. The variation in genotype is able to result in notable changes in phenotype, such as in behaviour, morphology, physiology, and reproduction. To further identify the genetic changes underlying the long-hair phenotypic developments of Angara rabbits, we used the whole genome sequencing approach to explore the genetic differences between long-haired rabbits (Angora rabbit) and short-haired rabbits  and identified genetic components related to the long-hair phenotypic trait. The results will guide us to improve rabbit breeding in the future.Three populations of domestic rabbits  were involved in this study Fig. a. Each pSequencing of 18 individuals generated 320.09 Gb of paired-end DNA sequence, of which 314.62 Gb (~ 98.29%) of effective reads were mapped to the rabbit reference genome assembly (Orycun 2.0) (Table We performed single-nucleotide polymorphism (SNP) calling and identified 24.83 million (M) SNPs from these 18 individuals. Of these, 161,407 coding SNPs leading to 57,721 nonsynonymous nucleotide substitutions  were detected  revealed genetic separation among the three rabbit breeds Fig. a. MoreovDusp1, Ihh, Fam134a, Map3k1, Spata16 and Fgf5) were included in the two pathways  and increased genetic distance to the short-haired rabbits  (Fst_A-RN). We calculated the Hp_A value and Fst_A-RN value, which were Z-transformed to ZHp_A and ZFst_A-RN, respectively. Then, we determined the selective sweep regions by using an empirical procedure and selected windows simultaneously with significantly low ZHp values  and significantly high ZFst values  of the empirical distribution as regions with strong selective sweep signals along the genome Fig. a, which Fgf5 gene resides on Chromosome 15 of rabbit and contains three exons. In this study, the Fgf5 gene was included in the selective sweep regions. By further analyzing SNPs in the Fgf5 gene, we found that there were a total of 15 SNPs; 13 SNP loci were located in introns, and 2 of them were present in extron 3  distributed in four major rabbit breeding provinces. The results showed that in Angora rabbits, the frequency of the C allele at this locus was 100%, but it was rare in New Zealand and Rex rabbits. Instead, the T allele was dominant in New Zealand and Rex rabbits, with frequencies of 93% and 100%, respectively , catagen (regression phase) and telogen (resting phase) . FGF5 isFgf5 was mutated from T to C, which resulted in leucine at residue 191 being replaced by serine. The FGFR1 and FGF5 or mu-FGF5 plasmids were co-transfected into HEK293T cells. Then, co-IP was conducted to detect the binding ability of FGF5 and mu-FGF5 on FGFR1. The results showed that the binding of mu-FGF5 and FGFR1 was much weaker than that of FGF5 and FGFR1  was constructed. In mu-FGF5, nucleotide 572 within the coding sequence of Dusp1, Ihh, Fam134a, Map3k1, Spata16 and Fgf5, underwent strong selection sweeps. Moreover, we discovered that the T19234C mutation in Fgf5 was conserved in Angora rabbits, which significantly impaired the binding capacity of FGF5 to its receptor FGFR1. This finding is beneficial for understanding the mechanism of hair development, thus promoting rabbit breeding.Hair fibre length is a critical economic trait for wool rabbits. Angora rabbit is a famous breed of wool rabbit for its high yield of long hair. In this study, based on next-generation sequencing data, we compared the genomes of Angora rabbit to the genomes of two breeds of short-haired rabbit: New Zealand and Rex rabbit. The SNPs and selective sweeping signals were exploited. Several genes associated with hair growth regulation, such as Angora rabbit, the famous breed of long-haired rabbit, is widely bred all over the world. Its active phase of hair growth is double that of normal rabbits. The annual world production of angora wool is estimated at 2500 to 3000 tonnes a year, with approximately 90% of the supply produced in China . In ChinShh mutant skin gives rise to large abnormal follicles, and no hair is formed [Dusp1, Ihh, Fam134a, Map3k1, Spata16, and Fgf5. These results indicate that these genes may be involved in the development of the long-hair trait in Angora rabbits by regulating MAPK signalling and Hedgehog signalling.The hair growth cycle consists of three phases: anagen, catagen, and telogen . In the s formed . Exogenos formed . Converss formed . In the Fgf5 has been reported to be closely related to hair growth in many species [Fgf5 underlie hair length regulation, such as deletion, duplication and missense mutations [Fgf5 resulted in Angora mice showing excessively longer fur formation due to a prolonged anagen phase [Fgf5 converts a conserved tyrosine at residue 174 to histidine (p. Y174H), which is predicted to impact the function of FGF5, consequently causing abnormal hair growth [Fgf5 have also been identified to be associated with hair length variation in cats [Fgf5 is responsible for the male-dominant long hair phenotype in Syrian hamster. The deletion leads to a truncated abnormal FGF5, which enhances prolonged anagen and/or retards catagen induction [Fgf5 [Fgf5, which led to stop codons at positions 159 and 82, respectively. The two truncated FGF5 proteins were predicted to lack the critical β strands that are necessary for the interaction between FGF5 and its receptor [Fgf5 were associated with the long-hair phenotype [Fgf5 exon 3 of Angora rabbits, which led to a missense mutation at residue 191 (p. L191S). The structural prediction of FGF5 showed that L191 was adjacent to the conserved ligand binding sites V94, N192, K193, K198, H209, V210 and S211. Moreover, L191 interacted with one of the ligand binding residues, S211. Therefore, we speculated that substitution of the hydrophobic leucine with a hydrophilic serine at residue 191 might impact the receptor binding ability of FGF5. Moreover, this speculation was further confirmed by a co-IP assay using FGF5 and mu-FGF5 plasmids. This demonstrates that the T19234C mutation is critical for Angora rabbits to develop their long hair trait and that the mutation weakens the interaction between FGF5 and FGFR1, a mandatory step to inhibit hair growth.In previous studies, RNA sequencing has been used to investigate the genetic principles of hair traits in rabbits. Messenger RNAs, long noncoding RNAs and microRNAs (miRNAs) have been discovered to control hair growth cycling by regulating protein-coding gene expression at the posttranscriptional and transcriptional levels –27. In t species , 28–34.  species . Increasutations . A serier growth . Five mi in cats , 36. A 1nduction . In addion [Fgf5 , 32, 37.receptor . In dogshenotype . In thisFgf5, other candidate genes identified in this study  also may contribute to the long hair trait in Angora rabbits. It has been reported that DUSP1 and MAP3K1 proteins are involved in high-quality brush hair in the Yangtze River Delta White Goat [Dusp1, Map3k1, Spata16, and Fgf5 are included in MAPK signalling pathway. Ihh and Fam134a participate in the Hedgehog signalling pathway. Both MAPK and Hedgehog signalling pathway are known to be involved in the regulation of hair growth cycle by promoting the proliferation and differentiation of hair-follicle stem cells, as well as increasing the production of growth factors [Dusp1, Ihh, Fam134a, Map3k1, and Spata16, respectively  were collected from Shaanxi, Shandong, Jiangsu and Anhui provinces of China. Following sampling, the rabbits were returned to their owners. There is no direct and collateral blood relationship within the last three generations among the sampled individuals. All research involving animals was approved by The Laboratory Animal Administration Committee of Xi’an Jiaotong University (Permit No. 2015–1317) and carried out according to the Guidelines for Animal Experimentation of Xi’an Jiaotong University and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .For whole genome sequencing, a total of 18 EDTA-blood samples of female rabbits, including 6 Angora rabbits, 6 New Zealand rabbits and 6 Rex rabbits, were collected from farms located in Shaanxi province of China. The samples were sent to Novogene Bioinformatics Institute  for genome sequencing. To validate the specific SNP locus by Sanger sequencing, EDTA blood samples of an additional 135 rabbits . Ended repair, added A-tail, ligation of paired-end adaptors, purification and PCR amplification with 350-bp inserts were sequentially processed to construct a paired-end sequencing library according to the manufacturer’s specifications (Illumina). Sequencing was performed on the Illumina HiSeq2500 platform, and 125 bp paired-end reads were generated with an insert size of approximately 350 bp. Raw FASTQ sequences have been deposited in the NCBI Short Read Archive under BioProject accession number PRJNA862254.ftp://ftp.ensembl.org/pub/release-78/fasta/oryctolagus_cuniculus/dna/) using BWA (Burrows‒Wheeler Aligner) software v0.7.8 [First, to ensure that reads were reliable and without artificial bias  in the following analyses, raw data (raw reads) in fastq format were first processed through a series of quality control (QC) procedure in-house C scripts. QC standards are as follows: (a) Removing reads with ≥ 10% unidentified nucleotides (N); (b) Removing reads with > 50% bases having phred quality < 5; (c) Removing reads with > 10 nt aligned to the adapter, allowing ≤ 10% mismatches; (d) Removing putative PCR duplicates generated by PCR amplification in the library construction process . Second, high-quality paired-end reads were mapped to the rabbit reference genome sequence  using a Bayesian approach as implemented in the package SAMtools . The gen2) between pairwise SNPs was computed using Haploview v4.269 software [2 value was calculated for pairwise markers in a 500-kb window and averaged across the whole genome. We found differences in the rate of decay and level of LD value, which reflected variations in population demographic history and effective population size (Ne) among breeds/populations. We also conducted principal component analysis (PCA) to evaluate genetic structure using GCTA software v1.24.2 [To estimate and compare the pattern of LD for different breeds, the squared correlation coefficient  was applied to quantify the polymorphism levels  and genetic differentiation (Fst) between Angora rabbit and the short-haired rabbit breeds . At each detected SNP position, we counted the numbers of alleles corresponding to the most and least frequently observed alleles  in each pool. Hp for each window was calculated by the equation Hp = ∑nMAJ*∑nMIN /(∑nMAJ + ∑nMIN)2 . Individ2  and Z-tr2 , 50. P vFgf5, the genomic DNA of an additional 135 rabbits was extracted using a TIANamp Genomic DNA kit (TIANGEN Biotech), followed by amplification of the Fgf5 partial fragment (containing the specific site) using 2 × Accurate Taq Master Mix (Accurate Biotechnology) and the following Fgf5 primers: Fgf5-F1 (5’- CCTATGCCTCAGCAATACATAGAACT -3’) and Fgf5-R1 (5’- ATCCAAAGCGAAACTTGAGTCTG -3’). Amplified PCR products were purified and then sequenced by Sangon Biotech . The BioEdit program (version 7.0.9.0) [To validate the nonsynonymous nucleotide substitution in exon 3 of 7.0.9.0)  was usedhttp://provean.jcvi.org/seq_submit.php) [http://genetics.bwh.harvard.edu/pph2/) [The effect of the missense mutation (p. L191S) on FGF5 protein function was predicted using two functional mutation prediction programs: PROVEAN (mit.php)  and Polyu/pph2/) , with thIn addition, the three-dimensional structure of the FGF5 protein was predicted by I-TASSER v4.2 . HomologFgf5 (c.572) corresponds to the 19,234 site within the whole gene sequence of Fgf5. Therefore, in the mu-FGF5 plasmid, c.572 was substituted with cytosine, which led to the leucine at amino acid 191 being mutated to a serine. Both FGF5 and mu-FGF5 are equipped with a C-terminal DYKDDDDK tag (Flag tag), which can be recognized and bound by an anti-Flag antibody. HEK293T cells were co-transfected with FGFR1 and FGF5 or mu-FGF5 plasmids. Forty-eight hours later, the cells were lysed with lysis buffer  supplemented with protease inhibitor . The lysate was clarified by centrifugation at 14,000 X g for 5 min at 4 °C. The supernatant was transferred to a new tube and incubated with Anti-DYKDDDDK (Flag) IP Resin  overnight at 4 °C. The final pellet from IP was subjected to elusion with 2 × Laemmli sample buffer  for 5 min at 95 °C. The IP samples were subjected to immunoblotting with antibodies against FGFR1  and Flag .The plasmids of FGF5 (XM_008267686) and FGFR1 (XM_008273980) were purchased from GenScript . The mutant plasmid of FGF5 (mu-FGF5) was also constructed by GenScript. Nucleotide 572 within the coding sequence of Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by immunoblotting as described previously . The sepAdditional file 1. The entire original pictures of blots in Figure Additional file 2. Table S1. KEGG enrichment analysis of the 174 identified candidate genes.Additional file 3:Table S2. Exonic SNPs of Dusp1, Ihh, Fam134a, Map3k1 and Spata16.


Neurodegenerative diseases affect millions of people worldwide. Neurodegenerative diseases result from progressive damage to nerve cells in the brain or peripheral nervous system connections that are essential for cognition, coordination, strength, sensation, and mobility. Dysfunction of these brain and nerve functions is associated with Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis, and motor neuron disease. In addition to these, 50% of people living with HIV develop a spectrum of cognitive, motor, and/or mood problems collectively referred to as HIV-Associated Neurocognitive Disorders (HAND) despite the widespread use of a combination of antiretroviral therapies. Neuroinflammation and neurotransmitter systems have a pathological correlation and play a critical role in developing neurodegenerative diseases. Each of these diseases has a unique pattern of dysregulation of the neurotransmitter system, which has been attributed to different forms of cell-specific neuronal loss. In this review, we will focus on a discussion of the regulation of dopaminergic and cholinergic systems, which are more commonly disturbed in neurodegenerative disorders. Additionally, we will provide evidence for the hypothesis that disturbances in neurotransmission contribute to the neuronal loss observed in neurodegenerative disorders. Further, we will highlight the critical role of dopamine as a mediator of neuronal injury and loss in the context of NeuroHIV. This review will highlight the need to further investigate neurotransmission systems for their role in the etiology of neurodegenerative disorders. The prevalence of neurodegenerative disorders (NDs) is increasing in the United States. It is projected that 13.8 million people will be affected by Alzheimer’s disease (AD) by 2050 . The curWhile the etiology of different NDs varies, a unifying feature of NDs is the activation of downstream pathways that activate cellular death, including but not limited to apoptosis and pyroptosis. Activation of these pathways is brought about by neurotoxic molecules which promote inflammation and/or mitochondrial dysfunction. Importantly, dysregulation of NTs induces such neurotoxic molecules. For example, methamphetamine (meth) dysregulates DA homeostasis via interaction with the DA transporter (DAT), leading to autooxidation of DA and the production of neurotoxic reactive oxygen species (ROS). The meth-induced production of ROS from dysregulation of DA metabolism has been directly linked as the cause of meth-induced neurotoxicity .2+ levels. Increased Ca2+ can then perturb mitochondrial function and activate mitochondrial-induced apoptosis ..2+ level2+, and disruption of mitochondrial function (with or without Ca2+ disturbances) can mediate mitochondrial-induced apoptotic pathways. Therefore, understanding how NTs disrupt intracellular Ca2+ to then (indirectly) initiate mitochondrial-mediated apoptosis is of great interest.Various neurotransmission systems are disrupted in NDs. Independent from this, mitochondrial function is highly dependent on homeostasis Ca2+ regulate one another. As described previously, local influxes of Ca2+ via Cav channels are required for the exocytotic release of NTs into the synaptic cleft for neurotransmission. Conversely, NTs can regulate intracellular Ca2+ levels through activation of GPCRs, which are expressed pre- or post-synaptically, as well as near the soma. GPCRs regulate NT release through modulation of Ca2+, primarily through inhibition of voltage-gated calcium channels by Gβγ subunits [2+ levels [3 (Gαq path) to release intracellular stores of Ca2+ [B receptors [2+ levels through overactivation of GPCRs. Considering the role of GPCRs in regulating internal Ca2+ levels and the importance of Ca2+ in promoting mitochondrial-induced apoptosis, overactivation of GPCRs by disturbances in NT levels is a major concern for the pathophysiology of NDs [Extracellular levels of NTs and intracellular levels of Casubunits . However+ levels  by gener of Ca2+ . There aeceptors ,107,108.y of NDs .2+ is regulated by several factors, including GPCRs such as mGluRs and mAChRs [2+ steady-state levels [2+ release to mitochondrial oxidative phosphorylation. However, while this feature helps SNr neurons to meet energy demands, it puts these neurons at higher risk for mitochondrial stress and ROS [2+ levels. IP3 kinase B (ITPKB), which inactivates IP3, a secondary messenger that increases the release of Ca2+ from ER stores, was found to negatively regulate αSyn aggregation. Considering how NTs and Ca2+ levels regulate one another, disruption of NTs to increase cytosolic Ca2+ may contribute to αSyn aggregation. Understanding how disturbances in NTs may translate to changes in GPCR activation is needed to understand how NTs may contribute to the neurobiology of AD and PD.In AD, increased intracellular Cad mAChRs ,111. Indd mAChRs . In the e levels . In the  and ROS . IndepenHIV-1-associated neurocognitive disorders collectively referred to as “HAND” include asymptomatic neurocognitive impairment (ANI), HIV-associated mild neurocognitive disorder (MND), and HIV-associated dementia (HAD) . The mosDopaminergic neurotransmission has been implicated as a mediator of HAND pathology. Brain volume measured in patients with HAD post-mortem showed a reduced volume of basal ganglia, which correlated with the severity of HAD . Further2+ homeostasis by initiating IP3-mediated Ca2+ release from ER stores [2+-mediated mitochondrial generation of ROS and caspase activation in hippocampal neurons [2+ homeostasis, which induce mitochondrial-mediated neurotoxicity, in addition to NLRP3 inflammasome-mediated pyroptosis, may also be a major contributor to the neurobiology of HAND [It has long been evidenced that Tat and gp120 are neurotoxic proteins, inducing neuronal apoptosis ,141,142 R stores ,146. Fur neurons . Therefo of HAND .2+ disturbances are well evidenced at this point, mechanisms by which these two key neurotoxic HIV-1 proteins initiate these effects are still coming to light. Tat-induced increases in intracellular Ca2+ levels have been attributed to the overactivation of L-type Ca2+ channels [2+ influx [2+ release from ER stores through a ryanodine receptor (RyR) dependent mechanism [2+ through multiple Ca2+ regulatory mechanisms, it is not known whether Tat directly interacts with these channels to alter their function. Tat-induced disturbances of Ca2+ have been considered as the underlying mechanism for Tat-induced alterations in NT systems [2+ dysregulation contributed to the Tat-induced disruption of NT systems, considering the critical role of Ca2+ in exocytotic NT release. However, direct interaction between Tat and molecular players has, to our knowledge, only been confirmed with monoamine transporters DAT and NET [2+ homeostasis are downstream effects of Tat-induced disturbances in NT systems, we argue, is a possibility. NTs can dysregulate Ca2+ homeostasis by signaling through GPCRs and downstream signaling cascades triggered by activation of specific GPCR subtypes. Tat-induced effects on ACh release in cortical synaptosomes were shown to involve the mGluR1. These receptors, upon activation, provided regulatory feedback to increase Ca2+ release from the ER, increase Ca2+ influx from NMDARs, and finally increase noradrenaline release [3-mediated release of Ca2+ to depolarize the neuronal membrane [3-mediated Ca2+ release in macrophages via D5 receptors coupled to Gq proteins [2+ stores but also due to DA-induced neuroinflammation and downstream activation of pyroptosis in response to cytokine release from macrophages. Thus, preventing the effects of proteins such as Tat from disturbing NT systems is of great interest in mitigating the neuronal loss and damage underlying the neurobiology of HAND  activate NLRP3 inflammasomes. Additionally, inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α), an ER stress sensor, regulates the release of inflammatory cytokines which can activate the NLRP3 inflammasome while also further producing ROS, which induces mitochondrial damage [In this review, we have primarily covered two major pathways involved in the pathophysiology of neurodegeneration in NDs: NLRP3 inflammasome-mediated pyroptosis and Cal damage . Crosstal damage  and renal damage . In astrl damage . Both pyDifferent types of cell death, including (but not limited to) pyroptosis and apoptosis, are a featured hallmark in NDs. The machinery necessary for these forms of cell death is ubiquitously present in all cell types, which begs the question—why do some cells die and not others? More specifically, how is cell-specific loss/injury brought about in various NDs? We propose that the unique pattern of neurodegeneration evidenced in various NDs such as AD, PD, and HAD may be partially explained by their disruption of specific NT systems. In the context of NeuroHIV, extensive studies demonstrate a clear link between perturbation of monoaminergic transmission by exposure of the CNS to HIV-1 viral proteins and risk for development of HAND. Importantly, abused substances directly target NT transmission and exacerbate NT-mediated development of neurodegenerative diseases. Further work is needed to determine whether the viral protein-induced dysregulation of NT systems is sufficient to induce neuronal damage through the activation of downstream pathways, such as mitochondrial-mediated apoptosis or the release of inflammatory cytokines from macrophages to induce pyroptosis. Currently, there are no promising therapeutic strategies for neurodegenerative disease. Considering the progressive nature of neurodegenerative diseases, it is probably unsurprising that aging is a risk factor for NDs, and as populations live longer, NDs are becoming more prevalent . It shou


The activity of calpain is tightly regulated because its aberrant activation will nonspecifically cleave various proteins in cells. Abnormally elevation of Ca2+ promotes the abnormal activation of calpain during myocardial ischemia-reperfusion, resulting in myocardial injury and cardiac dysfunction. In this paper, we mainly reviewed the effects of calpain in various programmed cell death  in myocardial ischemia-reperfusion. In addition, we also discussed the abnormal activation of calpain during myocardial ischemia-reperfusion, the effect of calpain on myocardial repair, and the possible future research directions of calpain.Calpain is a conserved cysteine protease readily expressed in several mammalian tissues, which is usually activated by Ca Mammali2.2.1.2+ acts as the second messenger and participates in multiple biological processes in cells. Ca2+ plays a decisive role in the systolic function of the heart. Generally, the intracellular Ca2+ content is much lower than that in extracellular  (2+ channels (LTCC) on the cell membrane are open, issues in the flow of extracellular Ca2+ into the cells. When intracellular Ca2+ concentration is higher than 10 μmol/L, ryanodine receptors (RyR) in the SR are activated, Ca2+ in SR flow to the cytoplasm, which is called Ca2+-induced Ca2+ release  or transferer to extracellular through Na+/Ca2+ exchanger (NCX) in the plasma membrane. However, during ischemia, ATP deficiency inhibited Na+/K+-ATPase and reduced Na+ excretion, anaerobic glycolysis led to cytoplasmic acidification, and the increased H+ was discharged through Na+/H+ exchanger (NHX) to promote extracellular Na+ influx. Together with Na+/HCO3− cotransporter and persistent Na+ channels, all mechanisms increase intracellular Na+ content  . When thechanism . In gene content . Abnormantration . Moreoveincrease . In addidial I/R . The lea2.2.2+ can directly activate. Due to the proteolysis ability of calpain being finite, calpain mainly plays a role in regulating rather than removing substrates in cells  and calpain. This interaction promotes the directional movement of calpain to the Golgi body or endoplasmic reticulum, which is responsible for calcium storage. Moreover, Ttm50 increases the sensitivity of calpain to Ca2+ and enhances the activation of calpain ,–17,2+ caectively . Usuallyalpain-2 . Calpainalpain-2 . Calpastalpain-2 . When thmembrane . Calpaine stress , 24. Theive form , stayed ive form . Therefoform  on the electron transport chain  has not been fully understood. Previous studies have indicated that LMP mainly occurs after mitochondrial outer membrane permeabilization, which constitutes an intrinsic apoptosis-associated phenomenon. However, under certain experimental conditions, lysosomal membrane permeabilization can precede mitochondrial permeability, thereby mediating lysosomal-dependent cell death. The mechanism may be related to the recruitment of BAX to the lysosome membrane for pore formation . The stain vitro . The conin vitro . Interesnd vitro , 102. HSnd vitro . Althougnd vitro .3.5.+) and ATP to form poly (ADP-ribose)-sylation to change chromatin structure. However, DNA base modification affected by oxidative stress, hypoxia, and inflammatory stimuli can lead to excessive activation of PARP-1  and is mainly activated by oxidative stress-induced DNA damage and chromatin decomposition . PARP-1 f PARP-1 . Over-acnecrosis . Some scnecrosis . Calpainnecrosis . The actnecrosis . The relnecrosis . In addinecrosis . Calpainnecrosis . Therefonecrosis   could cause a kind of “programmed” necrosis, namely necroptosis . After t3.7.2 terminal, which interacts to maintain the inactivation of GSDMD . GSDMD has an inhibitory -COOH terminal and death-promoting -NHof GSDMD . Cells fof GSDMD . Inflammof GSDMD . The occof GSDMD , 116. Inof GSDMD   At present. There is still a lack of specific inhibitors of calpain. Most studies have adopted a wide range of non-selective calpain inhibition methods, ignoring the different subtypes of calpain that may have different effects. Javier Inserte et al. also put forward similar views ; (ii) CaStudies have reported that the calpain cleavage site is structurally dependent, not sequence-dependent. Calpain cleavage is uncertain. Why calpain eventually mediates apoptosis rather than autophagy or pyroptosis in some cases? Is there a pathway affecting the cleavage trend of calpain? However, there is no report on the related mechanism. If it can be found that something affects the tendency of calpain cleavage, this may have a significant impact on the future treatment of calpain. In addition, since we cannot ignore the extensive cleavage caused by calpain, can we analyze the action environment of calpain? With the development of proteomics, spatial transcriptomics, and protein structure prediction, we may know all proteins that can be cut by calpain at a particular time. Suppose apoptosis-related proteins account for the vast majority of the proteins that can be cut by calpain in cells at a specific time. Will we inhibit apoptosis by inhibiting calpain at this time? Therefore, we guess that the action time window of calpain may also significantly impact the experimental results. There is still a large blank in the study of calpain. With the progress of science and technology and the further understanding of various death mechanisms, the regulatory role of calpain will be further analyzed. The mode of action, targeted therapy, and treatment time of calpain may be essential research directions in the future.


However, the composition and function of MAMs remain to be fully defined. Here, we focused on the transient receptor potential vanilloid 1 (TRPV1), a Ca2+-permeable ion channel and a polymodal nociceptor. We found TRPV1 channels in the reticular membrane, including some at MAMs, in a rat cardiomyoblast cell line (SV40-transformed H9c2) by Western blotting, immunostaining, cell fractionation, and proximity ligation assay. We used chemical and genetic probes to perform Ca2+ imaging in four cellular compartments: the endoplasmic reticulum (ER), cytoplasm, mitochondrial matrix, and mitochondrial surface. Our results showed that the ER Ca2+ released through TRPV1 channels is detected at the mitochondrial outer membrane and transferred to the mitochondria. Finally, we observed that prolonged TRPV1 modulation for 30 min alters the intracellular Ca2+ equilibrium and influences the MAM structure or the hypoxia/reoxygenation-induced cell death. Thus, our study provides the first evidence that TRPV1 channels contribute to MAM Ca2+ exchanges.The Ca TRPV1 inhibition did not lead to any change in [Ca2+]c, while activation timidly increased [Ca2+]c  hot spots) as shown in 2 and a marked increase at the mitochondrial surface. In contrast, only a small increase was observed in the cytosol at the edge of Fura2 sensitivity. From a spatial point of view, a naïve explanation could be that the small amount of Ca2+ released from the ER is diluted in the whole cytosolic volume. Using the parsimony principle, we alternatively propose that only a fraction of reticular TRPV1 channels out of MAMs are functional, conversely to those in MAMs.Since TRPV1 appeared to be partially located in MAMs, we wondered whether TRPV1 might be involved in the local Caytoplasm ,64,65. Tactivity . Taking membrane , we were2+ movement in the outer mitochondrial membrane environment. Finally, TRPV1 inhibition by iRTX was not accompanied by any Ca2+ from the ER directly to mitochondria.To sum up, our data show that TRPV1 activation can mobilize Ca2+ homeostasis, we wanted to verify its impact over time. Therefore, we analyzed the Ca2+ concentrations in the different cellular compartments after 30 min of TRPV1 modulation. As expected, the decrease in ER Ca2+ observed with an acute TRPV1 activation resulted in a diminished resting [Ca2+]r and a reduced reticular Ca2+ mobilization under SERCA inhibition with thapsigargin .To better evaluate the effects of the pharmacological TRPV1 treatment on Cargin TG; A–C afterol cells D–F. Inte2+ transfer between the ER and mitochondria, we expected that a 30 min prolonged activation of TRPV1 could have led to a rise in the mitochondrial Ca2+ levels. Contrary to our expectations, we observed a decreased resting [Ca2+]m , even in a 12 min recording [2+ in the ER. NCX only plays a role of 7%, while PMCA pumps and mitochondria only account for a maximum of 1% of cytosolic Ca2+ efflux [2+ equilibrium, they were insufficient to compensate for the prolonged TRPV1 activation.During this 30 min interval, could some compensatory mechanisms contribute to the new Caecording ,67. In m+ efflux . As with2+ stock. Moreover, the TRPV1-mobilized Ca2+ pool is not directly transferred to mitochondria via the MAM structure but instead remains in the cytosol.In summary, we demonstrated that a 30 min prolonged activation of TRPV1 empties the reticular Ca2+ transfer in MAMs, it may result in a disruption in MAM organization. Aware that each available approach to characterize ER–mitochondria interaction has strengths and weaknesses [2+ transfer [Since the prolonged TRPV1 activation decreased the Caaknesses , we usedtransfer    in MAMs . Since t in MAMs , it is e in MAMs . IP3R recontacts . HoweverOur combined data show that TRPV1 modulation triggers the remodeling of MAMs, which occurs in several pathologies, such as episodes of hypoxia–reoxygenation (H/R) ,46,77. Lastly, we examined whether TRPV1 modulation could protect the cells against H/R-induced death efficiently. Using a similar cellular model and a similar H/R sequence to that used in the present study, Sun et al. demonstrated that the activation of TRPV1 at the onset of H/R is deleterious to cell survival and that its inhibition protects cells from death . Here, w2+ release occurs through Ca2+-release channels [2+-permeable channels like TRPC6 [2+ overload. Upon reaching the maximal capacity of mitochondrial Ca2+ retention, the mitochondrial permeability transition pore (mPTP) opens and drives the cells to death [2+ release via reticular Ca2+ leak channels is a milestone in this cascade responsible for Ca2+ dysregulation and cell death. In a previous study, in adult mouse cardiomyocytes, we showed that the pharmacological activation of translocon before hypoxia emptied the ER Ca2+ stores that restricted the mitochondrial Ca2+ overload, protected the cells from in vitro H/R, and reduced infarct size in mice submitted to in vivo I/R cardiac sequence [2+ depletion under TRPV1 activation is protective when it occurs before H/R. Thus, these two studies highlight that the ER Ca2+ discharge upstream of an H/R episode is an effective protective mechanism against cell death. In H/R conditions, the results showed a protective effect of RTX by reducing cell mortality by ~18% compared to control H/R  pumps lose their activity during hypoxia due to progressive ATP depletion [2+ released by TRPV1. As a result, the cytosolic calcium rises and accumulates in the mitochondria [Conversely to preconditioning, per-conditioning with TRPV1 activation failed to protect cells from death, which implies that emptying ER Caepletion ,83 and cchondria ,83.2+ leakage and increasing the formation of MAMs, as per-conditioning via TRPV1 inhibition, are critical steps to stabilize Ca2+ coupling between the endoplasmic reticulum and mitochondria, reducing cytosolic overload and avoiding H/R cell death. Inversely, iRTX applied during the hypoxic period reduced cell death by ~19% (21% vs. 40%). It indicates that blocking reticular CaAt the onset of reoxygenation, neither activation nor inhibition of TRPV1 prevented H/R-induced cell death. Our strategy would only apply to episodes of programmed I/R, such as organ transplantation or cardiothoracic, vascular, and general surgery.In conclusion, our study revealed several important findings (see summarized results in 2+ homeostasis has focused on a limited number of cellular compartments. We afford here the first complete approach with Ca2+ measurements performed in the cytosol, reticulum, mitochondria, and mitochondrial surface hot spots. We show that TRPV1 is a novel player in the reticulum–mitochondria Ca2+ crosstalk, i.e., acute pharmacological activation of TRPV1 mobilizes Ca2+ from the ER to mitochondria. In addition, we provide the first evidence that prolonged pharmacological modulation of TRPV1 structurally and functionally may alter the reticulum–mitochondria Ca2+ coupling. Since MAM dysfunction plays an important role not only in Ca2+ signaling [All previous work, including ours, on the role of TRPV1 in Caignaling  but alsoignaling  or oxidaignaling , lipid mignaling , and celignaling , TRPV1 cignaling  to neuroignaling  and cancignaling .2+ discharge via Ca2+ leak channels, such as TRPV1, prior to an H/R insult is a valid strategy to improve cell survival. Finally, we confirm that reticular Ca2+ dyes in a homogeneous population [The cell model of our study, the SV40-transformed H9c2 cell line, has been obtained from rat neonatal ventricular cardiomyocytes ,96. Evenpulation  or reducFinally, based on single PI labeling, our findings provide a limited snapshot of necrosis-like SV40-transformed H9C2 death. Future studies could clarify which of the twelve major cell death routines  are invo


Aortic dissection (AD) is a life-threatening tear of the vascular tissue with creation of a false lumen. To explore the mechanism underlying this tissue tear, this study investigated the delamination strength of AD model rats and the histological composition of the aorta at various stages of AD development. SD rats were administrated beta-amino propionitrile for 0 (Control), 3 (Pre-dissection), and 6 (Dissection) weeks. The thoracic aorta was harvested at 10–11 weeks of age. The Dissection group exclusively showed AD at the ascending aorta. The delamination strength, a force that separates the aorta in the radial direction, of the descending aorta decreased significantly in the order of the Control, Pre-dissection, and Dissection groups. A quantitative histological analysis of the aortic tissue demonstrated that, compared with the Control group, the area fraction of collagen was significantly higher in the Pre-dissection and Dissection groups and that of elastin was significantly lower in the Dissection group. The area fraction of the elastin fibers between the elastic laminas (interlaminar fibers) was significantly decreased in the order of the Control, Pre-dissection, and Dissection groups. Histological changes of the aortic tissue, perhaps a reduction in interlaminar fibers mainly aligned in the radial direction, decreased delamination strength, thereby causing AD. Aortic dissection (AD) is a life-threatening disease in which the aortic wall separates in the radial direction, thus allowing blood to flow through the created lumen  and often causing aortic rupture; the mortality rates before patients arrive at hospitals and at 24 h after rupture are reported to be 17.5% and 21.4%, respectively  and typeThe delamination strength, which is the force that separates the aorta in the radial direction, can be a useful indicator in the assessment of AD risk because the aortic wall of AD separates in the radial direction. To date, the delamination strength of the porcine thoracic aorta ,9,10, heAnimal models of AD should prove useful to understand the mechanism underlying the occurrence of this condition. Previously, AD rat models were generated via the administration of β-aminopropionitrile (BAPN) ,19,20, aIn this study, we aimed to elucidate the mechanism of AD generation. For this purpose, we measured the composition and delamination strength of the aorta as AD gradually formed, to investigate the relationship between these two parameters.All animal experiments were approved by the institutional review board for animal care of the Nagoya Institute of Technology  and were performed according to the guidelines specified by the Guide for Animal Experimentation, Nagoya Institute of Technology. In reference to a study that observed the occurrence of AD after treatment with 0.4% BAPN for 6 weeks , we usedz) direction to obtain a plate-like specimen of 3 × 6–8 mm in the z and circumferential (θ) directions. Prior to the delamination test, half of the specimen was delaminated with forceps, to separate the specimen into the intimal and adventitial sides at the media. A piece of sandpaper  was glued onto both sides of the delaminated part using a cyanoacrylate adhesive . The sandpaper was used to chuck the specimen into the delamination tester, as described below. The specimen was imaged using a digital camera . The specimen was kept in Krebs–Henseleit (KH) buffer until further experimentation.The rats were sacrificed at 10–11 weeks, and descending thoracic aortas were excised as described previously ,25,26. LT, was measured using a load cell  and recorded. The specimen was fumed with KH buffer to prevent it from drying during the test. The test was performed in air at room temperature (25 °C) within 24 h of sacrifice.A T-type delamination test was conducted using a tabletop tensile tester , as described previously . BrieflySpecimens that were prepared as stated in The stained samples were observed using a microscope  equipped with a digital microscope camera  and a 40× (LUCPlanFLN40X) objective lens.All analyses described below were implemented using the ImageJ image processing software  . The funSd, was obtained by fitting the following Sigmoid function:Tp, which was defined by:w is the sample width. Sd, a, b, and c in Equation (1) are constants to be fitted. The sample width, w, was determined as an average of the sample widths measured at five locations.The delamination strength of the sample, λela) and collagen (λcol), respectively.Tissue compositions were estimated from histological images as reported previously . First, 2 or more and a circularity of 0–0.3 using the “analyze particle” function . The delamination strength was tested via two-way repeated-measures analysis of variance (ANOVA) for the three groups of AD stage and the two directions. Data obtained between three groups were tested using the Tukey–Kramer test as the multiple comparison. The correlation coefficients, N = 13) in the Control, 247.0 ± 10.3 g (N = 6) in the Pre-dissection, and 186.3 ± 21.0 g (N = 8) in the Dissection group. Significant differences in body weight were observed among all three groups. The daily water-uptake volume was 147.1 ± 27.0 mL/kg/day (N = 6) in the Control, 98.9 ± 17.8 mL/kg/day (N = 6) in the Pre-dissection, and 113.4 ± 24.1 mL/kg/day (N = 8) in the Dissection group. The daily water-uptake volume of the Control group was significantly higher than that of the Pre-dissection and Dissection groups. These data were consistent with previous reports showing that the weight [Tp, increased up to about 10 s, remained almost constant for about 50–100 s, and dropped suddenly by failure. Each specimen required a different amount of time for specimen failure. In each group, the z-direction delamination test (θ-direction delamination test (The time variation of the peel tension per unit length is shown in ion test d–f of a ion test a–c. In tion test .n = 20) in the θ-direction and 21.1 ± 5.1 N/m (n = 21) in the z-direction. In the Pre-dissection group, the delamination strength was 15.8 ± 2.0 N/m (n = 11) in the θ-direction and 17.4 ± 2.8 N/m (n = 10) in the z-direction. In the Dissection group, the delamination strength was 12.5 ± 2.4 N/m (n = 14) in the θ-direction and 10.2 ± 3.3 N/m (n = 14) in the z-direction. A two-way repeated-measures ANOVA revealed significant differences only at the AD stages. Post-hoc testing identified significant differences among all three groups. These statistical examinations demonstrated that the delamination strength decreased in the order of the Control, Pre-dissection, and Dissection groups. Although significant differences were not observed between the z- and θ-directions, the average delamination strength in the z-direction appeared to be larger than that detected in the θ-direction in the Control and Pre-dissection groups, and smaller in the Dissection group.n = 9) in the Control, 57.1 ± 4.2% (n = 9) in the Pre-dissection, and 47.2 ± 2.8% (n = 9) in the Dissection group. The area fraction of elastin in the Dissection group was significantly lower than area fractions in the Control and Pre-dissection groups. This result indicates that the elastin density decreased at the Dissection stage.n = 9) in the Control, 23.3 ± 6.0% (n = 9) in the Pre-dissection, and 25.5 ± 5.6% (n = 9) in the Dissection groups. The area fraction of collagen in the Control group was significantly lower than that in the Pre-dissection and Dissection groups. This result indicates that the collagen density increased at the Pre-dissection stage.n = 9) in the Control, 8.6 ± 1.6% (n = 9) in the Pre-dissection, and 5.2 ± 1.2% (n = 9) in the Dissection groups. Significant differences were detected among all three groups. This result indicates that interlaminar fibers decreased gradually as the AD developed.z-direction delamination test (p = 0.10), the delamination strength correlated significantly with the area fraction of interlaminar fibers in the θ-direction delamination test. This result indicates that the reduction of interlamellar fibers decreases the delamination strength.We performed a delamination test on AD model rats and histologically investigated the aortic tissue at various stages of AD development. The main findings were as follows: as AD developed, (1) the delamination strength decreased, (2) the area fraction of interlaminar fibers decreased, and (3) the area fraction of collagen increased first, followed by a decrease in that of elastin. These results are novel, to the best of our knowledge.A reduction in the delamination strength is the likely cause of AD. The delamination strength decreased in the order of the Control, Pre-dissection, and Dissection groups, as shown in The area fraction of interlaminar fibers decreased in the order of the Control, Pre-dissection, and Dissection groups. This observation was congruent with the report of Nakashima et al. , who shor direction [r direction. In that case, the reduction of interlaminar fibers decreases the delamination strength in the r direction. Based on these findings, we hypothesized that the decrease in the interlaminar fibers caused a reduction in the delamination strength, and accordingly, the aortic wall could not bear the radial force possibly brought by high blood pressure, leading to the tear of the aorta.Both the interlaminar fibers and delamination strength decreased with the development of AD. The interlaminar fibers are mainly aligned in the irection , and a pr direction of the aorta, thus decreasing the delamination strength. Further reduction of the delamination strength would have been promoted in the Dissection group  was higher than that recorded by the θ-directional test (θ-delamination strength) in the Control and Pre-dissection groups  z-direction delamination strength. Yu et al. [z-direction delamination test (θ-direction delamination test, for unknown reasons. Aortic dissection progresses preferentially from a weaker part of the tissue. Because a sample with multiple dissection planes has more chances of having a dissection plane with fewer interlamellar fibers, delamination occurs more easily in the sample with multiple dissection planes; thus, this sample has weaker delamination strength. This effect would have been pronounced in the Dissection group, in which the interlamellar fibers were markedly reduced by BAPN administration. This combination of two factors resulted in the lowering of z-direction delamination strength compared with θ-direction delamination strength in the Dissection group.The average delamination strength determined by the n groups , similarus study , we perfu et al.  carried ion test c, whereaz-direction delamination test was shorter in some specimens compared with that in the θ-direction delamination test. One explanation for this observation would be the lateral deviation of the dissection plane caused by the administration of BAPN. Sommer et al. [The time to complete the delamination test differed between specimens, which was simply explained by the different lengths of the specimens in the delamination direction. Interestingly, the completion time in the r et al.  reportedθ-direction and 71.4 ± 32.1 N/m in the z-direction [There are some limitations to this study. To begin, data obtained from the rat AD model might not be true of the human AD. However, a decrease in interlamellar fibers was observed in both the rat AD model and human AD, indicating that the rat AD model used in this study has similar properties to human AD. Moreover, there have been no studies investigating the delamination strength of rat aorta, to our best knowledge; thus, we cannot compare these results to previous data. The delamination strengths of porcine specimens were 53.0 ± 17.6 N/m in the irection  was alsoThe delamination strength of thoracic aortas was significantly lower after the administration of BAPN compared with control cases. The area fraction of elastin fibers between the ELs decreased with the development of AD. BAPN administration first increased the area fraction of collagen, followed by a decrease in area fraction of elastin. These histological changes in the aorta are the likely cause of the decrease in its delamination strength, thereby triggering AD.


The evaporation rate constant linearly increased as the ambient temperature enhanced (573~873 K). For n-decane/ethanol bi-component droplets, at low mass fractions (≤0.2), the isothermal evaporation processes were steady due to the good miscibility between n-decane and ethanol, like mono-component n-decane, whereas at high mass fractions (≥0.4), the evaporation process experienced ultrashort heating and fluctuating evaporation stages. During the fluctuating evaporation, the bubbles formed inside the bi-component droplets and expanded, resulting in the occurrence of the microspray (secondary atomization) and the microexplosion. The evaporation rate constant of bi-component droplets increased as the ambient temperature enhanced, and showed a “V-shaped” trend with the increase of the mass fraction, and the evaporation rate constant was the smallest at 0.4. The evaporation rate constants based on the numerical simulation by using the multiphase flow model and Lee model showed reasonable agreement with the experimental ones, suggesting a potential of application in practical engineering.The evaporation characteristics of n-decane-based bi-component or multi-component droplets have been veiled for application in advanced combustion. This paper proposes to experimentally investigate the evaporation of n-decane/ethanol bi-component droplets settled in the convective hot air, and numerically simulate the key parameters affecting the evaporation charactersitics. It was found that the evaporation behavior was interactively affected by the mass fraction of ethanol and the ambient temperature. For mono-component n-decane droplets, the evaporation process included the transient heating  and steady evaporation  stages. In the isothermal stage, the evaporation rate followed  N-decane has been considered as a promising substitute for aviation kerosene applied in aircraft engines because of good thermal stability and low saturated vapor pressure, etc. . Dagaut d2-law, which says that the square diameter of a droplet decreases linearly with the evaporation time [The evaporation characteristics of mono-component fuel droplets have been intensively studied by many researchers. During isothermal evaporation, the evaporation rate of mono-component fuel droplets is governed by the so-called ion time ,8,9. Forion time ,11,12. Iion time  conducteion time  establision time  investigIn addition, internal bubbling in the multi-component droplets is possibly initiated in the droplet lifetime, and the internal vapor pressure is built up. This is beneficial for the microexplosive event and can enhance secondary atomization effects ,16,17,18Although many studies have been carried out to build a simplified model and predict the droplet regression of bi-component droplets, the comparison of experimental and numerical results has been scarcely made, and, thus, the numerical simulation has not been substantially applied in practical engineering. Therefore, this work will focus on the experiments and simulation of the evaporation characteristics of n-decane/ethanol bi-component droplets in laminar high-temperature air, analyze and discuss the influences of the ethanol mass fraction and the ambient temperature on the droplet evaporation characteristics, obtain the droplet evaporation regression rates in the isothermal stage, and make a comparison to the evaporation rate constants between experimental and numerical results.The n-decane and ethanol were purchased from Macklin Biochemical Co., Ltd., Shanghai, China. The n-decane/ethanol bi-component fuels were prepared by using a two-step method: (1) A certain amount of n-decane and ethanol were successively measured by a measuring cylinder. (2) The ethanol was added into the n-decane and stirred by a glass rod, and then the mixture was vibrated by ultrasonic wave for a few minutes to make them miscible or dispersive.Y is the mass fraction of ethanol; eρ, nρ are the densities of the ethanol and n-decane (g/mL); and eV, nV represent the volume of the ethanol and n-decane (mL), respectively.The ethanol mass fractions were calculated according to the following formula:The experimental setup was demonstrated in our previous work ,17, incld, dL, and dS are the diameter, perimeter, and area of a droplet, respectively. β is the pixel scale factor, which can be calibrated by the microscope graticules.To obtain the isothermal evaporation rate constant, the diameters of droplets need to be measured by the digital imaging treatment reported in our previous work . The diad0 is the initial droplet diameter, t and eK represent the evaporation time and the evaporation rate constant, respectively. Thus, the evaporation rate constant can be obtained by the linearly fitting of Equation (3).To eliminate the effect of the initial diameter of droplets on the evaporation in discussion, during the isothermal evaporation, the evaporation rate constant of a single droplet could be normalized as:l and v represent the liquid-phase and gas-phase. ρ represents the density, α represents the volume fraction, k represents the average thermal conductivity, μ represents the average viscosity coefficient, T represents the temperature, t represents time. E represents the total energy,e represents the enthalpy. lS represents the mass source term. S represents the energy source term, L is the latent heat of vaporization.For mono- and multi-component droplet heating and evaporation, Sazhin  summarizsatT is the saturation temperature, coeff represents the mass-transfer intensity factor with unit s−1. For an empirical coefficient, coeff is given with different values for solving different problems. In this work, the controllable empirical coefficient coeff of 0.6 s−1 was adopted.In the existing models, the vapor-liquid phase change model proposed by Lee has been most widely used. For the Lee model, the mass transfer rate from the liquid phase to the gas phase is expressed as ,24:(7)SlFor the sake of the simplification of the droplet evaporation, it is assumed that the droplet is a symmetrical sphere along the axis and the gravity is not considered. The physical properties for the simulation are listed into To evaluate the effect of grid numbers for meshing on the calculation, grid independence verification was conducted. The grid numbers of 125,000; 150,000; 270,000; 370,000; and 510,000 were used to calculate the evaporation rate constants of mono-component n-decane at the same operation conditions (in the hot air of 873 K). The results  verifiedd2-law  can be expressed as Re and Pr represent Reynolds number and Prandtl number, respectively. This shows that the comprehensive physical and heat transfer parameters of droplets, such as the density, dynamic viscosity, thermal conductivity, specific heat capacity, boiling point, latent heat of vaporization, etc., affect the evaporation rate constant [As a droplet is suspended into the convective hot air, the evaporation rate constant of the droplet can be expressed as:Ke=4kgNuρgT) based on the experiments is followed by:In Equation (9), the surrounding temperature plays a significant role in the evaporation rate constant. In the ambient hot air of 573 K, 673 K, 773 K, and 873 K, the isothermal evaporation rates of mono-component n-decane droplets are plotted in For simulation, the relationship can be expressed as:−7 m2/s, respectively. In comparison to n-decane, the evaporation rates of the ethanol droplets were obviously much smaller at each corresponding ambient temperature, since the latent heat of vaporization of ethanol (8.54 × 105 J/mol) is three times higher than that of n-decane (2.77 × 105 J/mol). The evaporation rate of ethanol also has a linear function with the ambient temperature, i.e., For the evaporation of mono-component ethanol droplets in experiments, the evaporation rate constants in the ambient hot air of 573 K, 673 K, 773 K, and 873 K were 1.19, 1.55, 2.27, and 2.81 × 10d2-law. The ethanol mass fraction affects the heat and mass transport properties of the bi-component droplets. The more ethanol dissolves into n-decane, the lower the droplet evaporation rate constant is. Case 2: the ethanol mass fraction is higher than the saturated concentration, the mixture can be regarded as an incompatible two-phase fluid, and the excessive ethanol settles the bottom layer of bi-component droplet, owing to the larger density of ethanol. As the mixture evaporates, one part of ethanol evaporates prior to the n-decane due to the lower boiling point of ethanol, and the other part of ethanol possibly dissolves into the n-decane and then evaporates, resulting in a complicated heterogeneous heat and mass transfer inside the bi-component droplets [d2-law inadequately responds the evaporation behavior, but it possibly approximately depicts evaporation behavior in the part of isothermal evaporation stage.To explore different ethanol mass fractions on the evaporation characteristics of bi-component droplets, the solubility of ethanol in n-decane was considered as a main factor affecting it. The solubility determines whether the bi-component droplet with different ethanol mass fractions can be regarded as a new mono-component-alike fluid. The two cases were classified as Case 1 and Case 2. Case 1: the ethanol mass fraction is less than the saturated concentration, and the mixture of two components is considered as a new mono-component-alike fluid. During the isothermal evaporation, the internal heat and mass transfer of bi-component droplets are homogeneous, and the evaporation follows the droplets . The infTB decrease. According to the linearity of d2-law, the ethanol completely dissolves in n-decane, and the mixture can be considered as a new mono-component-alike fluid. It can be testified that the evolving curves and evaporation rate constants based on numerical simulation . The experimental results demonstrated a fluctuating evaporation, whereas the numerical results show that the evaporation of bi-component droplets is steady in the isothermal stage . The difDiekmann et al.  showed tIn special case the opposite ranking occurs: the droplets with an ethanol mass fraction of 0.2 at the ambient temperature of 673 K evaporate faster than the droplets with 0.1. It is possible that there are two reasons: (1) the mass transfer inside the droplets is complicated, although the evaporation of n-decane/ethanol bi-component droplets with the low mass fractions of ethanol (≤0.2) are steady. Occasionally, we observed the occurrence of droplet diameter burst due to the formation and growth of the bubbles inside the droplets. (2) The evaporation behavior was interactively affected by the mass fraction of ethanol and the ambient temperature. In general, the solubility of ethanol increases with the increase of temperature and decreases with the increase of pressure. vD becomes greater, and the heat exchange between the droplet surface and the air enhances, and, thus, the evaporation slope becomes larger. Besides the mass fraction of ethanol having a significant effect on the evaporation of bi-component droplets, the ambient temperature also largely affects the evaporation behavior. Obviously, at certain a mass fraction of ethanol, as the ambient temperature enhanced, the evaporation rate constants improved a. The imIt is worth noting that the evaporation rate constant of the n-decane/ethanol bi-component droplet is nonlinear to the ambient temperature , unlike d2-law. By contrast, the evaporation of bi-component droplets with high mass fractions of ethanol  fluctuates violently and demonstrates microspray or microexplosion, since the excessive ethanol makes the mixture form an incompatible two-phase fluid. One part of dispersive ethanol possibly evaporates, and the other part of dispersive ethanol possibly dissolves into the n-decane and then evaporates, resulting in a complicated heterogeneous mass and heat transfer inside the bi-component droplets.The evaporation characteristics of mono-component n-decane and n-decane/ethanol bi-component droplets were experimentally conducted and numerically simulated. In general, the evaporation process experiences non-isothermal and isothermal stages. In the isothermal stage, the evaporation of mono-component n-decane droplets and n-decane/ethanol bi-component droplets with low mass fractions of ethanol (≤0.2) are steady. The unsaturated mixture of mutually soluble n-decane and ethanol can be considered as a new mono-component-alike fluid, and its internal mass transfer during evaporation is homogeneous, and, thus, the evaporation of the new fluid follows classical The numerical simulation based on the multiphase flow model and Lee model well demonstrates the evaporation process, and the calculated steady evaporation rate constants agree well with the experimental ones, especially for mono-component n-decane droplets and n-decane/ethanol bi-component droplets with low ethanol mass fractions. The relative errors between the simulation and experiments for the fluctuating evaporation of bi-component droplets with high ethanol mass fractions are larger, since the simulation neglects the internal complicated heterogeneous heat and mass transfer between the n-decane and ethanol components, owing to the bubble formation. It is vital to consider the internal heat and mass transfer inside non-uniform immiscible two-phase or multi-phase droplets in future work.According to the experiments and simulation, the ethanol mass fraction and the ambient temperature have significant effects on the evaporation rate constants of bi-component droplets, and interact with the evaporation. The evaporation rate constant increases with increasing ambient temperature, and presents a “V-shaped” trend as the mass fraction of ethanol enhances, and the evaporation rate constant is the smallest at 0.4.


P = 0.000, 0.004, 0.000, 0.000, and 0.003). The patients in the autologous fibroblast group rated more noticeable NLF improvements than those in the HA filler group . No serious adverse reactions were recorded. Autologous fibroblast injections are safe and effective for treating NLFs. These injections also promise sustained growth of living cells, possibly leading to a greater persistence than shown by other fillers.Autologous cultured fibroblast injections for soft tissue augmentation are a potential alternative to other filler materials. No studies have compared autologous fibroblast injections and hyaluronic acid (HA) fillers for treating nasolabial folds (NLFs). To compare the efficacies and safeties of autologous cultured fibroblast injections and HA fillers for treating NLFs. This prospective, evaluator-blinded, pilot study enrolled 60 Thai female adult patients diagnosed with moderate to severe NLFs. They were randomized to receive either 3 treatments of autologous fibroblasts at 2-week intervals or 1 treatment with HA fillers. The primary outcome was the clinical improvement of the NLFs graded by 2 blinded dermatologists immediately after injection and at 1-, 3-, 6-, and 12-month follow-ups. Objective measurement of the NLF volume was evaluated. Patient self-assessment scores, pain scores, and adverse reactions were recorded. Of the 60 patients, 55 (91.7%) completed the study protocol. The NLF volumes improved significantly in the autologous fibroblast group at all follow-ups relative to baseline ( Histologically, aged skin has a thinner epidermis, atrophic dermis, and reduced amounts of subdermal adipose tissue, fibroblasts, and collagen2. The most common dermatological presentations of aging are xerosis, skin laxity, wrinkles, and benign lesions1.Skin aging is caused by genetic and environmental factors such as sun exposure, air pollution, smoking, alcohol abuse, and poor nutrition3. Several types of fillers have been approved by the US Food and Drug Administration (US FDA), and they are categorized as temporary, semipermanent, and permanent4. Bovine collagen was the first US FDA-approved injectable filler. It has a short-lasting effect and is associated with hypersensitivity reactions5. Hyaluronic acid (HA) fillers have become popular. They offer a low immunogenic profile, safety, a range of applications, and reversibility with hyaluronidase6. However, severe complications have been reported. They include hypersensitivity reactions, foreign body granuloma, vascular occlusion, skin necrosis, and blindness9.Dermal fillers are commonly used for skin rejuvenation since they fill up wrinkles and replace soft-tissue volume lost due to aging10. However, in a retrospective study, 7 patients developed retinal artery occlusion after AFT, and they had a worse final best-corrected visual acuity than other injectables11.Autologous fat transfer (AFT) is another treatment option. It does not produce hypersensitivity reactions and granuloma formation because of the biocompatibility of the adipose tissues used12. Autologous cultured fibroblast injections were safe and effective in improving wrinkles, acne scars, and other dermal defects for up to 12 months after administration with no known side effects13. Histologically, fibroblast injections stimulate collagen formation with a concomitant increase in the thickness and density of dermal collagen13. Other mechanisms include an induced proliferation of native fibroblasts, secretion of cofactors that can augment the dermis, and the growth of the transplanted fibroblasts14.In 2011, the US FDA approved Laviv  as an autologous fibroblast tissue filler to improve moderate to severe nasolabial folds (NLFs) in adultsThe objective of this study was to compare the efficacies and safeties of autologous cultured fibroblast injections and HA fillers for treating NLFs.were pregnant or lactatinghad a history of connective tissue disorder, skin cancers, or related diseaseshad previous autologous fibroblast treatmenthad treatment with fillers, lasers, or energy-based devices during the previous 12 monthshad treatment using transdermal drug delivery, chemical peeling, or topical retinoid during the previous monthwere allergic to collagen, meat, dairy products, gentamycin, amphotericin B, or related productsThis prospective, single-center, evaluator-blinded pilot study enrolled 60 Thai female adult patients. The women had expressed dissatisfaction with their NLFs, scoring − 1 or − 2 on the Subject Wrinkle Assessment Scale (Table The subjects were randomly divided following simple randomization procedures into 2 groups (“HA filler” and “autologous fibroblast”). The HA filler group was given a single injection of HA filler  on both NLFs (0.5 ml on each side) by using 22-guage cannula. The autologous fibroblast group was intradermally injected with autologous cultured fibroblasts on both NLFs (0.5 ml on each side) by using 30-guage needle. However, the fibroblast injections were administered in 3 sessions at 2-week intervals.2 (PCO2 = 40 Torr). Media was changed every 3 days and the collected fibroblasts were then expanded to 20 × 106 cells per 1 mL of normal saline solution. As observed by light microscopy, the dermal fibroblasts were identified based on their spindle-shaped morphological features. Prior to injection, all specimens underwent standard laboratory testing: sterility using membrane filtration, endotoxin test using Limulus amoebocyte lysate, mycoplasma detection using quantitative polymerase chain reaction, and Gram staining.Preoperatively, each patient from the autologous fibroblast group had tissue collected from the postauricular area. An injection of 2% lidocaine was given, and a 3-mm punch biopsy was performed. The donor site was closed with a single nylon (5.0) suture. The collected tissue was sterilized with povidone-iodine, and the fibroblasts were extracted with debris removal. Separation of epidermis from dermis was done using dispase in fibroblast basal medium at 2–4 °C for 1–4 h. Then, the dermis was cut into small pieces and digested in trypsin at 37 °C for 15 min to isolate dermal fibroblasts. Subsequently, the cells were cultured in fibroblast basal medium with 2% fibroblast growth medium, 1% penicillin (100 units/ml) and streptomycin (100 µg/ml) at 37 °C in a humidified air of 5% COThe primary outcome of the study was the clinical improvement of the NLFs. The improvements were graded using a 5-point scale: 2 = “much improved,” 1 = “improved,” 0 = “no change,” − 1 = “worsened,” and − 2 = “much worsened.” Two blinded dermatologists subjectively evaluated photographs at the 1-, 3-, 6-, and 12-month follow-ups. All clinical photographs were taken with identical camera settings, lighting, and positioning using a Canon PowerShot G9 standoff camera .In addition, the NLF facial volumes were objectively evaluated using three-dimensional photographs captured by a Vectra H1 Imaging System (Canfield Scientific Inc.) immediately after injection and at the 1-, 3-, 6-, and 12-month follow-ups. Patients also performed self-assessments using the same 5-point scale at each follow-up visit. In addition, the patient pain experienced while the injections were being administered was rated using a 10-point visual analog scale (VAS). Any adverse reactions were recorded.P) value < 0.05 was considered statistically significant.Statistical analyses were performed using PASW Statistics for Windows, version 18.0 . Descriptive analysis was used for demographic data. Nasolabial folds volume changes were calculated using repeated measures ANOVA and paired t-test. Subjective improvement evaluation was analyzed by using Wilcoxon signed-ranks test. A probability . The study was performed per the Helsinki Declaration of 1964 and subsequent amendments. Written informed consent was obtained from all patients before their enrollment.The study was approved by the Ethics Committee of the Siriraj Institutional Review Board . Written informed consent was obtained for the publication and use of images prior to patients’ enrollment in the study. This study was performed in accordance with the Helsinki Declaration of 1964 and its subsequent amendments and was registered at ClinicalTrials.gov .Of the 60 female patients recruited, 55 (91.7%) completed the study protocol and were included in the final analysis. Five patients were withdrawn because they could not attend all follow-up visits. There were 30 patients in the HA group  and 25 in the autologous fibroblast group .P = 0.000 and 0.000, respectively). In contrast, the autologous fibroblast group showed significant volume improvements at all follow-ups compared with baseline . Furthermore, there was a significant difference between the 2 groups immediately after the HA filler injection and the first of the 3 fibroblast injections (P = 0.034).The objective evaluations of the volume differences in the NLFs using the Vectra H1 Imaging System are presented in Table P-value of 0.00.The subjective evaluations made by the 2 blinded dermatologists of the clinical improvements using the 5-point scale are illustrated in Fig. Patient self-assessment scores were recorded at the 1-, 3-, 6-, and 12-month follow-ups . By contrast, the autologous fibroblast group had significant volume improvements at all follow-ups compared with baseline . These findings prove that using autologous fibroblast injections can produce sustained clinical improvements in skin that has suffered collagen degradation19. A previous study biometrically assessed the skin changes caused by autologous fibroblast injections. The research revealed significant increases in epidermal and dermal thicknesses after 6 months relative to the pretreatment values20.In the present study, the HA group demonstrated a significant volume improvement immediately after the HA injection and at the 1-month follow-up compared with baseline  compared with baseline. However, no changes were detected at the 3-, 6-, and 12-month follow-ups. The autologous fibroblast group showed improvements only at the 3- and 6-month follow-ups.21. In our study, the HA fillers degraded over time, but the autologous fibroblast injections resulted in gradual improvements, as evidenced by our patient self-assessments. Fibroblast injections have previously been shown to require a more extended period before their effects are observed (at least 1–2 months following the completion of treatment) but without any risk of hypersensitivity reactions13. The findings of the current investigation are consistent with those of another study that used autologous fibroblasts in NLFs. That research observed sustained soft tissue augmentation at 3 months with continued clinical improvement at 6 months19. The more extended period before wrinkle improvement becomes apparent with autologous fibroblast injections than with HA fillers results from collagen deposition not using direct volume replacement. Consequently, fibroblast injections have a more gradual effect than HA fillers, which show immediate results14.Earlier investigations found that HA fillers in NLFs can show clinical results 1 month after injection, with satisfaction maintained at 6 months13.More autologous fibroblast patients than HA patients reported improvements in their NLFs at the 3-, 6-, and 12-month follow-ups. This finding is similar to the results of another study. It found that 81.6% of the patients treated with autologous fibroblasts demonstrated continued therapeutic benefits even at the 12-month follow-up22. In other research, autologous fibroblasts combined with plasma gel (Fibrogel) showed persistent improvements in the infraorbital area and lower face at the 3-, 6-, and 12-month follow-ups, with minimal adverse reactions23.Other novel autologous fibroblast combinations have been formulated to produce a faster onset of results with more prolonged clinical efficacy. For instance, Jiang et al. combined autologous fibroblasts and keratin as a soft tissue filler. The researchers found that 90% of their patients had significant NLF improvements at the 1-month follow-up. In addition, the improvements were maintained in 93.8% of cases at the 24-month follow-up20.Regarding adverse reactions in the present work, we had 1 patient in the autologous fibroblast group who experienced mild transient erythema. The condition resolved spontaneously within a day. Erythema is a commonly reported adverse reaction among patients injected with autologous fibroblastsCurrently, fibroblast injections are indicated for the treatment of NLFs. Autologous fibroblast treatment of NLFs is a promising option for patients. As autologous fibroblasts are not volume fillers, they are ideal for treating fine lines. On the other hand, possible disadvantages of autologous fibroblasts over HA fillers are their additional costs (which can be as high as 4 times when comparing to 1 cc of HA fillers) and the long lead time needed to harvest and culture the fibroblasts.Our study was limited by its small sample size, and the follow-up duration might not be long enough to determine the maximum efficacy of the treatment. We recommend that further studies be conducted with larger sample sizes and longer follow-ups to establish the longevity of autologous fibroblast injection therapy.Autologous fibroblast injections are safe and effective for treating NLFs. Unlike conventional dermal fillers, autologous cultured fibroblast cells are injected superficially and may require a more extended period to show improvement. They also promise sustained growth of living cells, possibly leading to a greater persistence than shown by other fillers.


Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca2+-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca2+ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca2+ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca2+]i and by modulating Ca2+-dependent flagellar waveform conversion.Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian  Sperm flagellar motility is crucial for fertilization success among most sexual organisms . SpermatCiona intestinalis i concentration and sperm motility during chemotaxis i and by modulating Ca2+-dependent flagellar waveform conversion.Here, we examined the role of sAC in the regulation of flagellar motility in C. intestinalis  was collected from Onagawa Bay near the Onagawa Field Research Center, Tohoku University, or obtained from the National BioResource Project for Ciona . Animals were kept in aquaria under constant light for accumulation of gametes without spontaneous spawning. Semen samples were collected by dissecting the sperm duct and kept on ice until use.The ascidian 2, 48.27 mM MgCl2 and 10 mM Hepes-NaOH (pH 8.0). Ca2+-free seawater (CaFSW) was as ASW without CaCl2 but with 478.2 mM NaCl and 2.5 or 10 mM EGTA. Inhibitors (KH-7) from Chemical Diversity Research Institute  were dissolved in dimethyl sulfoxide (DMSO) and added to an appropriate concentration in ASW. Other reagents were of analytical grades. SAAF was synthesized as described previously i increase along the asymmetric wave should be transient and end with a subsequent Ca2+ efflux. The regulation of the sAC and Ca2+ efflux in response to the SAAF-induced [Ca2+]i increase would be responsible for the intracellular signaling of chemotaxis (Ciona is caused by a transient calcium influx into flagella i (Asymmetric waves contribute to form swimming trajectories with a small circle, while symmetric waves contribute to straight swimming. During chemotaxis, sperm change their swimming direction by exhibiting these two waveforms in sequence, resulting in a chemotactic turn, as they swim away from the attractant source . We haveemotaxis . Sperm cflagella . The higthe SAAF , keeps tAF [2+]i , ‘Turn’.Ciona sperm i revealed that sAC plays a key role in sperm chemotaxis, possibly by regulating the clearance of [Ca2+]i and by modulating Ca2+-dependent flagellar waveform conversion. We found that both full-length and truncated isoforms of the sAC protein are expressed and localized in


Such amplification is integral to the action of the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, functional regulators of inter-organellar Ca2+ crosstalk between endosomes and the ER remain poorly defined. Here, we identify progesterone receptor membrane component 1 (PGRMC1), an ER transmembrane protein that undergoes a unique heme-dependent dimerization, as an interactor of the endosomal two pore channel, TPC1. NAADP-dependent Ca2+ signals were potentiated by PGRMC1 overexpression through enhanced functional coupling between endosomal and ER Ca2+ stores and inhibited upon PGRMC1 knockdown. Point mutants in PGMRC1 or pharmacological manipulations that reduced its interaction with TPC1 were without effect. PGRMC1 therefore serves as a TPC1 interactor that regulates ER-endosomal coupling with functional implications for cellular Ca2+ dynamics and potentially the distribution of heme.Membrane contact sites (MCSs) between endosomes and the endoplasmic reticulum (ER) are thought to act as specialized trigger zones for Ca Neostasis , 5. Dysfeostasis , 7, 8.2+ release from endosomes and lysosomes by targeting ion channels known as two-pore channels (TPCs) resident within endosomes and lysosomes. TPCs are evolutionarily ancient members of the voltage-gated ion channel superfamily . ER-resident Ca2+ channels (ryanodine receptors and IP3 receptors) are thought to be engaged by TPC-mediated Ca2+ signals at specific locales known as membrane contact sites (MCSs) where the membranes of both these cellular organelles occur in close apposition , a heme-binding transmembrane protein resident in the ER, as a TPC1 interactor that regulates the amplification of NAADP-evoked Ca2+ signals at MCSs between endosomal and ER Ca2+ stores.Given the functional impact of CaA), found in the endoplasmic reticulum that has been implicated in various cellular functions ) resolved the structural basis for this heme-dependent dimerization .PGRMC1 is a single-pass transmembrane protein  cells (A). Owing to the small amplitude of these endogenous NAADP-evoked Ca2+ signals, responses were also examined in cells overexpressing TPC1 (biased toward endosomes) or TPC2 (biased toward lysosomes), a known manipulation that enhances NAADP-evoked Ca2+ signals . Overexpression of RFP-PGRMC1 in this background further potentiated the NAADP-evoked Ca2+ mobilization (B). Potentiation of NAADP responses by PGRMC1 was not observed in cells overexpressing TPC2 (C). These data are consistent with the observed association of PGRMC1 with TPC1, but not TPC2 ), which contains the TPC1 backbone fused to the NH2-sequence of TPC2 that harbors lysosomal-targeting determinants, overexpression of PGRMC1 did not potentiate NAADP-evoked Ca2+ release (D). Quantitative analysis of these experiments is provided in Figure 2E. These results demonstrate that PGRMC1 acts to potentiate NAADP-evoked Ca2+ signals via TPC1.Our prior proteomic analysis of the TPC interactome identified PGRMC1 as a candidate TPC1 interacting protein ). To ass release , 25. In d) cells A. Owing  signals . In cellol cells B. Overexlization B. Potenting TPC2 C. These not TPC2 . An endo release D. QuantiB) that potentially serves as a protein interaction interface. Five PGRMC1 point mutants were generated (B): the PGRMC1 dimerization breaking mutant Y113F , and foA). Apo-PGRMC1 displayed decreased interactivity with TPC1 (B). Quantitative analysis of these experiments are shown in Figure 3C. Two of the proline point mutants , also displayed decreased interaction with TPC1. PGRMC1[Q159P] conferred the strongest inhibition (D and E). Interestingly both these mutants lie within the helical domain of PGRMC1 (B). In contrast, the proline point mutants designed outside the PGRMC1 helical domain failed to disrupt the interactivity between PGRMC1 and TPC1 (D and E). Quantitative analysis of these immunoprecipitation assays is provided in Figure 3F. These data demonstrate that PGRMC1 associates with TPC1, and this interaction is selectively attenuated in cells when the heme binding and dimerization of PGRMC1 is prevented (PGRMC1[Y113F]), or when a helix-breaking mutant is introduced within the PGRMC1 helical domain (PGRMC1[Q159P]).The PGRMC1[Y113F] mutant, which prevents heme binding to PGRMC1 and thereby PGRMC1 dimerization, caused an increase in monomeric PGRMC1 A. Apo-PGith TPC1 B. Quantihibition , D and Ef PGRMC1 B. In conand TPC1 , D and E2+ imaging assays. Overexpression of PGRMC1[Y113F] or PGRMC1[Q159P] in U2OS cells failed to potentiate endogenous NAADP-evoked Ca2+ signals (G–I). Similarly, in U2OS cells overexpressing TPC1, the NAADP-evoked Ca2+ mobilization response was not potentiated by expression of PGRMC1[Y113F] or PGRMC1[Q159P] in contrast to the potentiation seen with wild type PGRMC1 (J–L). The PGRMC1 [S143P] mutation, which failed to disrupt TPC1 interactivity, retained potentiation of NAADP-evoked signaling, similar to wild-type PGRMC1 . A similar concentration inhibition was observed in cells exposed to AG-205 (D–F). Peak impairment of PGRMC1-TPC1 interactivity was observed following exposure to either drug (30 μM) for 1 h. These conditions were subsequently employed in functional assays of NAADP action. Under the same conditions, exposure to either drug inhibited the PGRMC1-dependent potentiation of endogenous NAADP-evoked Ca2+ signals (G–I). After drug treatment, no potentiation of the peak amplitude (J) or extent of NAADP-evoked Ca2+ release was seen (K). These results, consistent with the PGRMC1 mutational analysis described earlier  or bafilomycin A1 (B) respectively. These experiments revealed no difference in the magnitude of either Ca2+ store between control cells and cells expressing PGRMC1 (C and D). NAADP-evoked changes in cytosolic and ER Ca2+ were then examined using dual emission imaging to simultaneously resolve both the cytosolic Ca2+ signal and luminal ER Ca2+ dynamics. In U2OS cells expressing two genetically encoded Ca2+ indicators (E), the kinetics of the endogenous NAADP-evoked cytosolic Ca2+ signal (resolved using GCaMP6M) closely paralleled the kinetics of luminal ER Ca2+ depletion (resolved using RCEPIA1er). Following PGRMC1 expression, both signals increased in magnitude, with PGRMC1 expression associated with a larger, more protracted cytoplasmic Ca2+ signal in response to microinjection of NAADP (F). This potentiation was also observed at the level of the ER, with the larger cytoplasmic Ca2+ signal correlating with a larger depletion of ER Ca2+ (E). These effects were quantified in G and H. Therefore, the potentiating action of PGRMC1 on NAADP-evoked Ca2+ signals is manifested through enhanced mobilization of ER Ca2+.Does PGRMC1 enhance the size of the Casigargin A or bafimycin A1 B respectg PGRMC1 , C and Ddicators E, the kiof NAADP F. This p ER Ca2+ E. These 2+-activated Ca2+ release channels within the ER. Specifically, PGRMC1 may increase the number (or longevity) of membrane contact sites (MCSs) between TPC1-positive endosomes and the ER. One method for assessing the functional coupling between these organelles is through the measurement of EGF-evoked Ca2+ signals . Responses to EGF in control U2OS cells were robust and were dampened in those cells expressing PGRMC1 . Both the peak magnitude and size (AUC) of EGF-evoked Ca2+ signals were depressed in PGRMC1-expressing cells (C and D). However, no changes in Ca2+ signals evoked by acetylcholine were noted between the same cells (C and D). Therefore, by using EGF activity as a reporter for endosome-ER connectivity, these data demonstrate that the interaction between PGRMC1 and TPC1 enhanced functional Ca2+ crosstalk between TPC1-positive endosomes and the ER.A parsimonious explanation for this effect is that PGRMC1-TPC1 interaction between PGRMC1  and TPC1  enhances the functional coupling between NAADP-activated TPC1 channels in endosomes and Ca signals , 31. TheRMC1-RFP A. Respon2+ stores. Overexpression of PGRMC1 enhanced NAADP-evoked Ca2+ signals  partner . RTN3 red of 50 nM, . This residue, as a tyrosinate, acts as the 5-coordinating ligand for the heme iron atom  and theal roles , 47. It al roles , 49. PGRal roles , 48, 50. nM,  andal roles . Our datal roles B. This rron atom  such thaThe interaction between PGRMC1 and TPC1 establishes a regulable nexus supporting crosstalk between discrete organelles. Exploration of cues that impact PGRMC1 properties in the context of MCS function has merit for future study. These include physiological levels of carbon monoxide, which dissociate the PGRMC1 dimer, cancer-associated mutants in PGRMC1, as well as changes in PGRMC1 phosphorylation or protein-protein interactions.2+ sensor that activates store-operated Ca2+ entry following ER Ca2+ store depletion (2+ entry (2+ store depletion through association with the coiled-coil domain of STIM1 to interact with Orai1 channels at ER-plasma membrane junctions (2+ store depletion) transitions to a cell surface-ER junction (regulating Ca2+ store refilling) underscores the dynamic nature of MCS components and differential functional roles that are keyed to their specific spatial localization.One particular PGRMC1 interaction that is especially intriguing is with STIM1, the ER Caepletion . This fo2+ entry , 21. PGR2+ signals. This role establishes PGRMC1 function at an MCS interface, between endosomes and the ER, where PGRMC1 acts as a regulatory adaptor impacting Ca2+ dynamics and potentially heme transport.In conclusion, these data demonstrate a molecular interaction between PGRMC1 and TPC1 that regulates the amplitude and duration of NAADP-evoked CaAplysia ADP-ribosyl cyclase and purified by high-performance liquid chromatography, as previously described . NAADP was synthesized in-house by incubating nicotinamide adenine dinucleotide phosphate  with nicotinic acid (Sigma Aldrich) in the presence of escribed . The folescribed , and wer2 at 37 °C). U2OS TPC1 knockout cells were generated in collaboration with Synthego , using CRISPR-Cas9 with three independent guide RNAs targeting exon 4 . Deletion of exon 4 was validated by Sanger sequencing of isolated genomic DNA. For overexpression experiments, U2OS cells were transfected with constructs using Lipofectamine 3000 (Thermo Fisher Scientific). Briefly, 3 × 106 cells were seeded in a 75 cm2 flask in the absence of antibiotics. The following day, transfection complexes were produced by combining plasmid DNA (15 μg), P3000 reagent (30 μl) and Lipofectamine 3000 reagent (20 μl), consistent with the vendor protocol. The DNA-lipid complexes were then added to cells and incubated overnight. Cells to be used the following day for imaging experiments were trypsinized and reseeded into MatTek dishes . Cells being used for biochemical experiments were harvested 48 h post transfection. All cells are free of mycoplasma contamination and are routinely tested using standard PCR-based methods.Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC. U2OS cells were maintained in Dulbecco’s Modified Essential Medium  and cultured in a humidified incubator , 1% Triton X-100, complete EDTA-free protease inhibitor cocktail (Roche), and Halt phosphatase inhibitor cocktail (Pierce). Samples were lysed on a nutating mixer for 30 min at 4 °C prior to centrifugation at 16,000×RCF for 10 min at 4 °C. Soluble supernatants were collected, and protein concentrations were determined by Bradford assay (Thermo Fisher Scientific). For immunodetection of proteins, a sample (20 μg) of solubilized lysate was separated by SDS-PAGE and transferred to nitrocellulose membranes using standard methods. Nitrocellulose membranes were blocked in 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween 20 (TBST) for 1 h at room temperature. Membranes were then incubated with primary antibodies  overnight at 4 °C. The following day, membranes were washed three times in TBST, before incubating membranes with IRDye secondary antibodies (1:7000 dilution in 5% skim milk TBST) for 1 h at room temperature. Membranes were processed using a Li-COR Odyssey Imaging system. For co-immunoprecipitation experiments with TPCs, 1 mg of solubilized protein from U2OS cell lines overexpressing TPC1-GFP and PGRMC1-myc was incubated with either mouse IgG isotype control antibody (2 μg/ml) or anti-GFP antibody produced in rabbit  at 4 °C for 1 h before overnight incubation with protein G agarose beads (Roche). Beads were collected after brief centrifugation and washed three times with lysis buffer. Immunoprecipitated complexes were eluted by incubating beads with 2× Laemmli sample buffer at 95 °C for 10 min and analyzed by SDS-PAGE and immunoblotting as described above.To prepare cell lysates, U2OS cells were washed in PBS, harvested by mechanical scraping, and then centrifuged at 800×RCF. Cell pellets were suspended in a lysis buffer consisting of 110 mM KCl, 10 mM NaCl, 10 μM CaCl2+ at a flow rate of 1 ml/min. Isolated cells expressing GCaMP6M were identified by fluorescence. Cell morphology was assessed by acquiring z-stack images and reconstructing three-dimensional models of each cell to be injected. Regions that were distant from the nucleus and from the cell periphery were targeted for injection sites. Pipettes (Femtotips II) were back-filled with an intracellular solution composed of 110 mM KCl, 10 mM NaCl, and 20 mM HEPES (pH 7.2) supplemented with NAADP (100 nM). Cells were injected at a depth of approximately 70% of the cell thickness at the site of injection, with an injection pressure of 85 hPa, a compensation pressure of 40 hPa, a 0.5s injection duration, 45° injection angle, and a 600 μm/s injection speed. For single-channel injection experiments, fluorescence was monitored . Dual-color timelapse imaging was performed by alternating image acquisition of GCaMP6M  and R-CEPIA1er . All injections were performed using a Plan-Apochromat 60×/1.42 oil differential interference contrast objective.Single-cell microinjections were performed as previously described , using a2+ imaging experiments, cells were cultured in the absence of serum for 16 h prior to imaging, to prevent serum-mediated desensitization of EGF signaling. Cells were washed twice with HBSS with Ca2+, and incubated with fluo-4-AM (4 μM), pluronic F127 (0.4%), and probenecid (2.5 mM) for 25 min at room temperature. Cells were then washed twice with HBSS and left at room temperature for 30 min to allow de-esterification. Dishes were mounted on an Olympus IX81 microscope and cells were identified by fluorescence. Fluorescence was monitored  using a Plan-Apochromat 40×/1.4 oil differential interference contrast objective. For all Ca2+ imaging experiments, fluorescence changes were monitored using a Yokogawa spinning disk confocal (CSU-X-M1N), and an Andor iXon Ultra 888 EMCCD camera. Image acquisition and data collection was done using Metamorph version 7.10. Traces of fluorescence intensity over time were plotted and the area under the curve calculated using the curve integration function in Origin .For agonist-evoked CaAll data necessary for evaluating the conclusions of this study are present within the manuscript and the This article contains The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.