Arabidopsis thaliana TAPETUM DETERMINANT1 (AtTPD1) and its rice (Oryza sativa) homolog, OsTPD1-like (OsTDL1A), are required for cell specialization and greatly affect pollen formation and development. Little is known about the role of the TPD1 homolog in banana pollen development.Pollen formation and development is important for crop fertility and is a key factor for hybrid development. Previous reports have indicated that Musa itinerans) and examine their role in pollen development by overexpressing the closest homolog, MaTPD1A. MaTPD1A exhibits high expression in stamen and localizes in the plasma membrane. MaTPD1A-overexpressing plants produce no pollen grains and smaller and seedless fruit compared to wild-type plants. Transcriptome analysis showed that in plant hormone, starch and sucrose metabolism, and linolenic acid metabolism-related pathways were affected by overexpression of MaTPD1A, and the expression of several key regulators, such as PTC1 and MYB80, which are known to affect anther development, is affected in MaTPD1A-overexpressing lines.Here, we report the identification and characterization of TPD1 homologs in diploid banana (Our results indicate that MaTPD1A plays an important role in pollen formation and fruit development in diploid banana, possibly by affecting the expression of some key regulators of pollen development. Pollen development, release, and pollination are vital for successful plant sexual reproduction and genetic diversity in the world. The development of hybrid lines with genetically modified (GM) pollen might group multiple important agronomic traits together and result in great hybrid vigor. In the anther of flowering plants, pollen formation results from the differentiation and interaction of reproductive cells (microsporocytes) with somatic anther wall cells (tapetum) . In ArabOryza sativa) [Zea mays) [TPD1-overexpressing plants showed that increased expression of TPD1 activates cell division and results in increased cell number in the carpel through interacting with the EMS1/EXS receptor protein kinase [In Arabidopsis, rice and Maize, a pair of protein-protein interactors that complex TAPETUM DETERMINANT1 (AtTPD1): EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS (AtEMS1/EXS) in Arabidopsis –7, OsTDL sativa) –10, and ea mays) , 12 has ea mays) , 13, andn kinase .Musa acuminata (not parthenocarpic) and edible diploid bananas (parthenocarpic) [Banana is an important fruit crop in tropical and subtropical areas. The wild banana trees are diploid, fruits develop if pollinated, and are fully seeded . Parthenocarpic) . Edible ocarpic) . The ovaocarpic) . In wildocarpic) . Wild baocarpic) . Banana ocarpic) , 22.MaTPD1A in the wild cultivar M. itinerans. Characterization of MaTPD1A-overexpressing (MaTPD1A-OE) plants indicated that TPD1A impairs fruit and pollen formation, and transcriptome analysis disclosed that many previously known anther development-related genes exhibit differential expression in the MaTPD1A-OE plant compared to WT. These data suggest that MaTPD1A modulates fruit and pollen development by affecting the expression of genes involved in these processes.Pollen formation and vitality is an important factor for crossbreeding, but little is known about the molecular mechanism of pollen development and formation in banana. Here, we identified four homologs of TPD1 based on the released banana A genome. To investigate the role of MaTPD1A, the closest homolog of AtTPD1, we overexpressed TPD1 homolog genes were identified and named MaTPD1A , which has been reported to have an important role in anther development [Using the Arabidopsis TPD1 (AAR25553) protein sequence as a query, a TBLASTN search was conducted against the banana A genome database. Four elopment   from two banana varieties and weak expression in non-embryogenic cell suspensions of three banana varieties . These results indicate that MaTPD1A might play an important role in plant regeneration from the embryogenic callus.The qRT-PCR analysis showed that ues Fig. a. To furues Fig. b. Additiies Fig. c. MoreovAgrobacterium tumefaciens-mediated transformation. As a control, GFP fluorescence was observed in the entire cell. When the MaTPD1A-GFP vector and plasma membrane marker vector were cotransformed into tobacco leaves, the MaTPD1A-GFP fusion protein colocalized with the plasma membrane marker, which suggests that MaTPD1A localizes exclusively to the plasma membrane . The localization was determined by transient expression of these constructs in tobacco epidermis cells by MaTPD1A in banana, the cDNA of MaTPD1A was overexpressed in wild-type plants under the control of the constitutive cauliflower mosaic virus 35S promoter (35S). Constructs for overexpression were used to transform diploid banana embryogenic cell suspensions via Agrobacterium-mediated transformation. TPD1A homolog-overexpressing plantlets were transferred to soil and grown until anthesis. There are no apparent differences between wild-type and MaTPD1A-OE plants during the vegetative growth stage. In the reproductive growth stage, MaTPD1A-overexpressing plants can produce normal buds . The fragments per kilobase of transcript per million fragments mapped (FPKM) values were presented from three biological replicates for each sample  assay. The gene expression results of RT-qPCR exhibited similar trends with those of the transcriptome data with some variation in the magnitude . Regulation of metabolic process, cellular process, single-organism process, biological regulation, catalytic, binding and transporter activity related GO terms were enriched in differentially expressed genes in TPD1A-OE plants  in diploid banana. In some parthenocarpic bananas, The developmental program controls a series of key genes to support the formation of embryos without fertilization and male sterility, which was reminiscent of male sterility induced by MaTPD1A overexpression. A similar program could be activated in MaTPD1A-OE plants. Therefore, we believed that in these two genotypes plants there are some overlapping genes that for controlling similar phenotype. Further research to compare the expression profile of MaTPD1A-OE plants with that of parthenocarpic bananas would be beneficial for understanding the underlying molecular mechanism. In addition, the expression profile of female and neutral flowers is also helpful for us to disclose the role of MaTPD1A on pollen and fruit development.In Arabidopsis, the ut seeds , and ovesiliques . MaTPD1Ads Figs. , 5. One ts Table . In our sed Fig. , Table 2n grains , and a sn grains , similare supply . Linoleie supply . Hormonee supply . It has rawberry , and malrawberry . Our qRTMaTPD1A in diploid banana affects the expression level of numerous genes that related to pollen development, and cause male sterile phenotype. The underlying mechanism is still obscure. Mutation of MaTPD1A with CRISPR/Cas9 mediated gene editing or RNAi of MaTPD1A will provide more information for its role of MaTPD1A in pollen formation and development. This study provides a basis for further studies to confirm its roles in anther and pollen development and for manipulation of banana fertility in the future.Taken together, our results indicate that overexpression of MaTPD1A on pollen and fruit development in diploid banana. MaTPD1A is the most closest homolog of TPD1 in banana and localized to the plasma membrane. Overexpression of MaTPD1A in diploid banana results in the missing of pollen grains in male flowers, and producing small and seedless fruit. Further transcriptome analysis indicated numerous genes previously reported to be involved in pollen development were up- or down-regulated in MaTPD1A-overexpressing plants. Therefore, our results revealed that MaTPD1A modulates pollen formation through regulating the expression of some relative regulators, which provides a basis for further studies to confirm its roles in anther and pollen development and for manipulation of banana fertility in the future.In this research, we examined the effect of overexpressing M. itinerans, known also as Yunnan banana, is wide distribution across subtropical China, and can be found in moist ravines to mountainous areas in China (https://banana-genome-hub.southgreen.fr/organism/Musa/Itinerans). The M. itinerans that we used was collected from Jinji Mountain, Pingxiang City, Guangxi Province in China, propagated and maintained in national banana germplasm repository, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences . The sample collection got permission from the local administration department. After identification by Dr. Ou Sheng who is working on banana germplasm collection and identification for many years, the multiple bud clumps of M. itinerans have also been deposited in public in vitro repository for clonal banana germplasm in IFTR, GAAS, and the deposited number was NYBGZXJ01404. Its genome sequence has been determined and released in 2016 [M. itinerans maintained in the national banana germplasm repository. For banana plantlets cultivation potted banana plantlets were grown up to the five true leaves stage in a greenhouse before they were transplanted into the experimental field for further cultivation under the following condition: natural photoperiod (11/13 h) with a temperature of 18–30 °C under 60–80% relative humidity. The field trial in plastic house obtained permission from the biosecurity regulators in Guangdong Academy of Agricultural Sciences  to conduct this trial. in 2016 . The embTPD1A homolog open reading frame (ORF) was amplified with the following primer set: forward (5′-ATGGCCTGCTTCCACGGTTTG-3′) and reverse (5′-TCAGCAAGTGACGGAGGAGAC-3′). The reaction condition was as follows: 94 °C for 3 min; 32 cycles of 94 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s, and the PCR products were separated and extracted in 1.0% (w/v) agarose gels. This ORF was introduced into the pMD18-T vector  for sequencing. The correct MaTPD1A ORF fragment was collected and subcloned into the pBI121 vector after double digestion with Sac I  and Xba I . The pBI121:MaTPD1A plasmid was digested with EcoR I  and Hind III and cloned into the pCAMBIA1301 vector (CAMBIA). The obtained pCMABIA1301:MaTPD1A construct was transformed into A. tumefaciens strain EHA105 for transformation with a previously described method [M. itinerans were cocultured with A. tumefaciens for 24 h at 26 °C with gentle shaking in the dark. The ECSs were collected by removing the supernatant, washed twice with sterile water, and cultured in proliferation medium supplemented with 25 mg/L hygromycin B and 200 mg/L cefotaxime at 26 °C with shaking at 110 rpm in the dark. Next, the ECSs were cultivated in regenerated medium and sub-cultured every 3 weeks for 3 times total until resistant mature somatic embryos appeared. The regenerated seedlings were confirmed with PCR amplification of the hygromycin resistance gene, and positive banana plants were multiplied using meristems of in vitro plantlets. The rooted plantlets were transplanted into the greenhouse and cultivated for further study.The full-length d method . BrieflyMaTPD1A was cloned in frame with GFP in the pCMABIA1300-GFP vector. The pCMABIA1300-GFP vector was modified from binary pCMABIA1300 vector, and the CaMV 35S promoter, GFP, RBS terminator were introduced into pCAMBIA1300 with EcoR Iand Sac I, Pst I, Pst Iand Hind III, respectively. The obtained construct was transformed into A. tumefaciens strain EHA105 for further use. The transient expression of MaTPD1A:GFP was performed as previously described [The full-length ORF of escribed . Images UBQ2 (HQ853254.1) expression level was selected as an internal control [Total RNA was extracted from different samples using a plant RNA isolation kit  according to the manufacturer’s instructions. The different tissue samples including leaf, root, corm, pseudostem, fruit and male flower were collected from the banana plant at 30 days after flowering. For banana inflorescence the female flowers appear first, the distal portion of the inflorescence elongates and produces clusters of male flowers. When the female flowers develop into fruit, we collected male flowers and immature fruit samples at the same time. The cDNA template was synthesized from 1 μg total RNA using PrimeScript RT reagent kit with gDNA Eraser . For qRT-PCR assays, 5 μL of TB Green Premix Ex Taq II (TAKARA), 4.0 μL of diluted cDNA, 0.8 μL of gene-specific primers, and 0.2 μL of ROX Reference Dye were mixed together and the reaction was run in a StepOne Plus (ABI) with the following program: 30 s at 95 °C followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The  control . The rel control  relativeTPD1A-OE1# line plants were collected at about 90 days after flower and ground into fine powder using liquid nitrogen. There were three replicates for each sample. Total RNA was extracted using the methods described above. RNA quality and concentration were evaluated by a NanoDrop 2000 UV-Vis Spectrophotometer , and the integrity of the RNA was determined with an Agilent 2100 Bioanalyzer . The paired-end library was prepared using a TruSeq RNA Sample Preparation kit , and the resulting sequencing libraries were used for RNA-seq analysis by Biomarker Technologies Corporation  with the HiSeq × 10 platform.The male flower bud of wild-type and Additional file 1: Table S1. Primers used in this study.Additional file 2: Table S2. Full genes that examined in transcriptome analysis.Additional file 3: Table S3. Differentially expressed genes in MaTPD1A-OE plants.Additional file 4: Table S4. top 20 enriched pathways.Additional file 5: Table S5. Differentially expressed genes involved in plant hormone signal transduction.Additional file 6: Table S6. Differentially expressed genes involved starch and sucrose metabolism.Additional file 7: Table S7. Differentially expressed genes involved linolenic acid metabolism.Additional file 8: Table S8. Differentially expressed transcription factors in MaTPD1A-OE plants.


Transient receptor potential canonical 6 (TRPC6) channels are non-selective cation channels that are thought to underlie mechano-modulation of calcium signaling in cardiomyocytes. TRPC6 channels are involved in development of cardiac hypertrophy and related calcineurin-nuclear factor of activated T cells (NFAT) signaling. However, the exact location and roles of TRPC6 channels remain ill-defined in cardiomyocytes. We used an expression system based on neonatal rat ventricular myocytes (NRVMs) to investigate the location of TRPC6 channels and their role in calcium signaling. NRVMs isolated from 1- to 2-day-old animals were cultured and infected with an adenoviral vector to express enhanced-green fluorescent protein (eGFP) or TRPC6-eGFP. After 3 days, NRVMs were fixed, immunolabeled, and imaged with confocal and super-resolution microscopy to determine TRPC6 localization. Cytosolic calcium transients at 0.5 and 1 Hz pacing rates were recorded in NRVMs using indo-1, a ratio-metric calcium dye. Confocal and super-resolution microscopy suggested that TRPC6-eGFP localized to the sarcolemma. NRVMs infected with TRPC6-eGFP exhibited higher diastolic and systolic cytosolic calcium concentration as well as increased sarcoplasmic reticulum (SR) calcium load compared to eGFP infected cells. We applied a computer model comprising sarcolemmal TRPC6 current to explain our experimental findings. Altogether, our studies indicate that TRPC6 channels play a role in sarcolemmal and intracellular calcium signaling in cardiomyocytes. Our findings support the hypothesis that upregulation or activation of TRPC6 channels, e.g., in disease, leads to sustained elevation of the cytosolic calcium concentration, which is thought to activate calcineurin-NFAT signaling and cardiac hypertrophic remodeling. Also, our findings support the hypothesis that mechanosensitivity of TRPC6 channels modulates cytosolic calcium transients and SR calcium load. The chathan Na+ . TRPC6 cthan Na+ , a produthan Na+ . Other s(2+ pool . TRPC6 c(2+ pool . SACs aned heart . Stretch[Ca2+]i) . Also, T stretch . Further stretch . Despite stimuli .2+]i and force, as well as arrhythmogenicity  infected with adenoviral constructs to modulate TRPC6 expression. We applied confocal microscopy and super-resolution imaging to identify TRPC6 localization. We measured [Ca2+]i transients and Ca2+ load of sarcoplasmic reticulum (SR) using Ca2+-sensitive dyes and spinning disk confocal microscopy. We applied computational modeling to qualitatively reproduce experimental findings and predict effects of upregulation or increased activity of TRPC6 on Ca2+ signaling.In this study, we aimed at understanding effects of upregulation of TRPC6 on CaAll studies were conducted at the University of Utah in accordance with National Institute of Health Guidelines for the Care and Use of Animals and reviewed by the Institutional Animal Care and Use Committee.2 in a 48 well tissue culture plate containing No. 0 coverslips treated with fibronectin. NRVMs were infected 24 h after plating with an adenoviral vector containing human TRPC6 attached to enhanced green fluorescent protein (eGFP) fused to the C-terminal and 6X HIS tag fused to the N-terminal (TRPC6-eGFP) at 10 multiplication of infection (MOI). We applied also an eGFP vector (Cat No. 1060) at 20 MOI as control. Furthermore, we infected NRVMs with shRNA TRPC6 with eGFP tag  at 100 MOI to silence native TRPC6 expression. The virus was removed 24 h after infection. Infected and washed cells were maintained and cultured for an additional 24–48 h in a humidity and CO2-controlled incubator at 37°C. All viral vectors were produced by Vector Biolabs  with a backbone of type 5 (dE1/E3). All eGFP tags were under a CMV promoter.Sprague Dawley rat dams were obtained from Charles River . Ventricular myocytes were enzymatically isolated from 1-day old rats . NRVMs were separated from fibroblasts by cell suspension to take advantage of fibroblast rapid adhesion and recovering myocyte suspension. Myocytes were counted and plated at 75,000 cells per 0.95 cmCultured cells were lysed in RIPA buffer. Protein concentration in each sample was determined using Pierce BCA protein assay kit . RIPA buffer, 2-Mercaptoethanol (BME), and 6x gel loading dye were added to the protein lysate to create gel ready samples at 1 μg/μl. Samples were heated at 70°C for 10 min to reduce protein. Protein was loaded on an 8% Bis-Tris Plus Gel  and electrophoresed in MOPS running buffer  at 200 V for 35 min. Protein was transferred onto 0.45 μm nitrocellulose membrane in Tris-Glycine-Methanol buffer at 250 mA for 1 h. The transferred blot was blocked in 5% (w/v) non-fat milk in Tris-Buffered Saline with Tween 20 (TBS-T) for 60–90 min at room temperature. Blots were incubated with primary anti-TRPC6 (LS-C19628) or anti-eGFP (AB6556) antibodies in blocking solution @1:1000 dilution and left overnight at 4°C. After washing in TBS-T, goat anti-rabbit secondary antibody was applied at 1:50 k in TBS-T, while Precision Protein StrepTactin-HRP  was applied at 1:100 k to stain for the molecular weight ladder. Blots were incubated in WesternBright ECL HRP substrate kit  for 2 min and imaged on Bio-Rad Image (Bio-Rad).Coverslip plated NRVMs were fixed with 1% paraformaldehyde (PFA) for 15 min at room temperature and then washed in phosphate-buffered saline (PBS) and stored at 4°C for immunolabeling. NRVMs were permeabilized with 0.3% Triton X-100  for 18 min and bathed in image-iT Fx Signal Enhancer  for 30 min. Cells were blocked in 10% normal donkey serum  for 60 min at room temperature. Primary antibody for TRPC6  was incubated overnight in 2% normal donkey serum incubation solution at 4°C. The next day primary antibodies were triple-washed in PBS for 15 min. Secondary antibodies were applied for 60 min at room temperature. For confocal microscopy, the secondary antibody used was a donkey anti-rabbit conjugated to Alexa Fluor 647 . Non-specific secondary labeling was controlled by similarly labeling NRVMs while omitting primary antibody. Cells were then incubated with DAPI  for 15 min to stain the nuclei and rinsed in PBS to be held at 4°C for imaging. For super-resolution microscopy, an anti-GFP nanobody conjugated to Alexa Fluor 647 was applied .Fixed and labeled NRVM coverslips were placed in PBS and imaged using a Leica SP8 confocal microscope . Two dimensional images were acquired using a GaAsP-HyD detector and a 40x oil immersion lens  with a 0.1 × 0.1 μm pixel size. DAPI was excited with a 405 nm laser and emission collected at 410–550 nm. Fluorescence of eGFP was excited with a 488 nm laser and emission collected at 491–610 nm. Alexa Fluor 647 conjugated antibody was excited with a 633 nm laser and emission collected at 638–775 nm. All samples were imaged with identical imaging parameters. We used sequential framing to avoid simultaneous excitation of fluorophores and minimize cross-talk. Images were processed for noise reduction and background correction, then visualized with the same intensity ranges for comparison.Three-dimensional single-molecule localization microscopy was performed using a Vutara 352 . Fixed and labeled TRPC6-eGFP NRVM coverslips were immersed in a blinking solution containing 20 mM MEA, 1% (v/v) 2-Mercaptoethanol, and an oxygen scavenging system  in a buffer of 50 mM Tris, 10 mM NaCl, and 10% (w/v) glucose. We applied a 640 nm excitation laser and 405 nm activation laser. A 60x water immersion lens  was used to collect 10,000 images with 20 ms exposure. We acquired image stacks with a size of approximately 18 μm × 18 μm × 2 μm. Localizations with less than median photon count and greater than median radial precision were filtered out.2+]i in infected NRVMs 48–72 h post-infection was measured using the ratiometric Ca2+ fluorescent dye indo-1 . We applied an X-Light V2 spinning disk equipped with a Photometrics Prime 95B camera and an OptoSplit III LS . The spinning disk setup was built on a Zeiss confocal microscope  equipped with a 40x oil lens and controlled with MetaMorph software . We used a 365 ± 5 nm UV laser  for excitation of indo-1. Excitation and emission signals were split by a 387 nm long pass dichroic mirror . Dual-wavelength emissions for the bound and unbound dye were split using a 440 nm dichroic mirror before band pass filtering .i transients under each pacing rate. SR load was determined by measuring the amplitude of the caffeine-induced peak : 126 NaCl, 4.4 KCL, 1 MgCl2, 11 D-Glucose, 24 HEPES, 12.9 NaOH, 10 EGTA, 0.7 probenecid, and 0.01 ionomycin  for 15 min at 37°C and then rinsed in the same solution without ionomycin. Cells were imaged as described in Section “Measurement of [Ca2+]i in Neonatal Rat Ventricular Myocytes,” while in 0 mM Ca2+ solution containing 5 μM ionomycin and 40 mM 2,3-butanedione 2-monoxime (BDM) for 2–3 min before rapidly switching to a Tyrode solution containing 2 mM Ca2+ and 40 mM BDM. F at 0 and 2 mM Ca2+ determined Fmin and Fmax, respectively, in the calibration equation  was modeled as an additional sarcolemmal leak Ca2+ current:We used a mathematical model of NRVM electrophysiology for qualitative comparison to our experimental results . CurrentGTRPC6 is the Ca2+ conductance for TRPC6 channels in the sarcolemma, Vm is the membrane voltage and ECa is the Ca2+ Nernst voltage. ECa was determined by:where R, temperature T, Faraday constant F, extracellular Ca2+ concentration [Ca2+]o and subsarcolemmal Ca2+ concentration [Ca2+]subSL.with the gas constant GTRPC6 to simulate increased TRPC6 expression. For pacing, i.e., triggering of action potentials, we applied intracellular current of −80 pA/pF for a duration of 0.5 ms at 0.5 Hz or 1 Hz for 1 min. [Ca2+]i transients from the final beat were analyzed. DecayT was calculated by fitting an exponential function to the decay phase of the transient. Application of caffeine was simulated 5 s following cessation of 1 Hz pacing. The effect of caffeine was modeled as previously described  through sarcolemmal Ca2+ channels and exchangers were converted to Ca2+ fluxes (Jchannel):We also used the model to explore effects beyond those measured in our experiments. CaAcap is the capacitive membrane area (1.38544 × 10−5 cm2), Cm is the specific membrane capacitance (1 μF/cm2), and F is Faradays constant of 96.5 C/mmol. The units of the resultant fluxes are μM/s, where the concentration is defined per cytosol volume. Fluxes through RyR and SERCA channels were converted to account for this definition. We integrated each flux over 2 and 1 s for 0.5 and 1 Hz pacing, respectively, to determine its effect on [Ca2+]i.where t-test and considered significant for p less than 0.05.Data are presented as mean ± standard error. Data analysis was performed in MATLAB version R2019a or higher . Differences in experimental data were assessed using the nimages = 10, ncells = 25). Fluorescence in eGFP  and shRNA-TRPC6-eGFP  infected cells was present in the entire myocyte (nlitters = 3), similar to the anti-eGFP western blot , eGFP signal exhibited a diffuse, in part sarcomeric pattern, and also localized to the nuclear membrane and nucleus , the TRPC6-eGFP signal was associated with the sarcolemma and nuclear membrane as well as near the nucleus , eGFP signal was similar to signal in eGFP infected cells , 0.08 ± 0.008 (ncells = 8), and 0.087 ± 0.009 (ncells = 5), respectively , TRPC6-eGFP , and shRNA-TRPC6-eGFP . We present examples of F405/F485 transients from NRVMs expressing eGFP and TRPC6-eGFP under 0.5 Hz (F405/F485 increased from 0.097 ± 0.004 in eGFP to 0.113 ± 0.005 in TRPC6-eGFP (p = 0.03) at 0.5 Hz, and from 0.105 ± 0.004 to 0.12 ± 0.005 at 1 Hz (p = 0.02) (F405/F485 of TRPC6-eGFP cells increased to 0.144 ± 0.005 compared to eGFP 0.124 ± 0.005 (p = 0.004) at both 0.5 Hz and 1 Hz  or 1 Hz . Also, SR load was not different before calibration (We measured r 0.5 Hz  and 1 Hzr 0.5 Hz . Diastol = 0.02) . Systoliand 1 Hz . The ampibration .F405/F485. NRVMs expressing TRPC6-eGFP cells exhibited a positive shift in cytosolic [Ca2+]i transients compared to eGFP cells at both 0.5 and 1 Hz pacing rates  and 1 Hz   and 1 Hz  (2+]i transients was not significantly different between eGFP and TRPC6-eGFP at 0.5 or 1 Hz (data not shown). DecayT was larger for eGFP than TRPC6-eGFP cells at 0.5 Hz , but not at 1 Hz    . Systoli = 0.01) . The ampp = 0.5) . Further= 0.048) .405/F485 in shRNA-TRPC6 was 0.09 ± 0.004 (p = 0.3) and 0.104 ± 0.005 (p = 0.89) for 0.5 and 1 Hz, respectively (F405/F485 was 0.125 ± 0.006 (p = 0.82) and 0.127 ± 0.006 (p = 0.66) at 0.5 and 1 Hz, respectively (2+]i for shRNA-TRPC6-eGFP was 76.5 ± 31.8 (p = 0.37) and 176.6 ± 48.1 (p = 0.92) for 0.5 and 1 Hz pacing, respectively (2+]i was 447.9 ± 85.2 (p = 0.9) and 502.3 ± 107.8 (p = 0.55) for 0.5 and 1 Hz pacing, respectively  or 1 Hz  vs. eGFP.NRVMs infected with shRNA-TRPC6-eGFP showed similar features as eGFP infected cells  and diffectively . Systoliectively . Calibraectively . Systoliectively . TDecay TRPC6 from 0 to 3 or 6 μS/μF. Increased GTRPC6 raised [Ca2+]i during both diastole and systole  was 30.2, 28.6, and 27.4 μM at 0.5 Hz and 24.4, 23.5, and 22.6 μM at 1 Hz  was developed from a 14 amino acid synthetic peptide from amino acids 50–100 of the human TRPC6 sequence. The antibody was validated by the manufacturer for western blot and immunofluorescence in human samples as well as immunochemistry in mouse samples. Comparison of the TRPC6 protein sequence from human and rat  shows 93.88% homology. Weak homology was in particular within the first 60 and last 100 amino acids. Comparison of the protein sequence from mouse and rat  exhibited 98.17% homology, with amino acids 50–100 being conserved in both species.Successful detection of our TRPC1-eGFP construct in western blots supports sensitivity of the antibody for human TRPC6 . However2+]i from indo-1 signals required correction for camera background, autofluorescence, and eGFP bleed-through. Contribution of eGFP to F485,cell, but not F405,cell signal, varied across the NRVMs depending on MOI. The difference in F485,cell across our experimental groups strongly affected calculation of F. TRPC6-eGFP and eGFP infection at 10 and 20 MOI yielded similar levels of bleed-through as we found in uninfected NRVMs (data not shown). In contrast, shRNA-TRPC6-eGFP infection at 100 MOI almost doubled the bleed-through, suggesting that a high eGFP expression causes a fluorescence offset beyond the offset primarily explained by autofluorescence. We suggest that our approach for correction is useful in other studies with eGFP markers at high MOIs, where changes in the F485 signal must be accounted for.Quantification of [Ca2+ allowed us to calculate [Ca2+]i accounting for the non-linear ratiometric fluorescence properties of indo-1. After corrections, eGFP, TRPC6-eGFP, and shRNA-TRPC6-eGFP infected cells yielded similar Fmin, Fmax, and Sf2/Sb2 (2+]i.Calibration using ionomycin and varying extracellular Ca Sf2/Sb2 . This in2+]i of 119 ± 34 nM at 0.5 Hz and 183 ± 43 nM at 1 Hz are close to previously measured diastolic [Ca2+]i of 140 ± 11 nM for spontaneous beating 2- to 7-day-old NRVMs (2+]i ranged from 323 to 480 nM, comparable to the measurement of 430 ± 100 nM in our 4- to 5-day-old NRVMs. Other studies on paced rat cardiomyocytes reported systolic [Ca2+]i in the range from 319 ± 37 to 454 ± 70 nM in 18-day fetal and rise to 743 ± 64 nM in adult rat (DecayT at 0.5 Hz pacing ranged from 0.47 ± 0.1 s−1 in the 18-day fetus to 0.2 ± 0.02 s−1 in the adult rat, similar to our measured DecayT of 0.42 ± 0.02 s−1 at 0.5 Hz for eGFP NRVMs. Application of caffeine in our eGFP myocytes caused a 279 ± 44 nM rise in cytosolic Ca2+, which is within the range of example responses for 2- and 7-day-old NRVMs (In our eGFP NRVMs, diastolic [Cald NRVMs . In thesdult rat . Previould NRVMs .2+]i of TRPC6-eGFP compared to eGFP cells (2+]i increased, but the amplitude did not, indicating only a offset in the overall transient. We qualitatively compared experimental findings with results from mathematical modeling assuming that TRPC6 causes sarcolemmal Ca2+ leak. In the model, diastolic and systolic [Ca2+]i increased with increasing GTRPC6 (2+]i also increased, but to a small degree, which could explain why our measurements were not significantly different in the experimental groups.Raw and calibrated measurements indicated an increase in i of NRVMs, which is in agreement with previous findings in adult rat (2+]i vs. 0.5 Hz pacing for GTRPC6 of 0, 3, and 6 μS/μF, respectively (2+]i was also higher with 1 Hz pacing (2+]i transient remained mostly unchanged for both pacing rates for all GTRPC6 settings. Additionally, DecayT reduced with increased TRPC6 in both, experiments (DecayT was reduced for increased pacing rate.We observed a pacing rate-dependent increase in diastolic i is often used as a measure of SR Ca2+ load. SR load was increased in TRPC6-eGFP vs. eGFP NRVMs (GTRPC6 (Caffeine reduces the CaFP NRVMs . Simulat (GTRPC6 .2+ leak. The simulations also helped in mechanistic explanation of the experimental findings and predicted effects of TRPC6 activity not measured in our experiments. With the model, we evaluated Ca2+ flux through ion channels, pumps and exchangers. Ca2+ flux through TRPC6 channels was small (2+ fluxes. As a result of increased GTRPC6, LCC fluxes decreased (2+ through NCX was increased, indicating increased activity of the exchanger (2+ flux through RyR and SERCA also increased with increased GTRPC6 (DecayT in both the model and experiments (Vm. With increased TRPCI6 leading to elevated [Ca2+]i, NCX extrudes more Ca2+ (2+ ion for three Na+ ions and resulting in a net increase of positive charge inside the cell (Vm was marginally increased by increased GTRPC6 (Vm is closer to Na+ channel activation threshold, so less current is required to trigger an action potential.Overall, the simulation results aligned with experimental findings, supporting the description of TRPC6 channels as sarcolemmal Caas small ,F vs. LCas small ,D,G,H buecreased ,G. The ixchanger ,H. Integd GTRPC6 , which ed GTRPC6 . Increaseriments ,6E. Sincore Ca2+ , exchangthe cell . Restingd GTRPC6 ,E. Thus 2+]i transients in cardiomyocytes. It was suggested that increased diastolic [Ca2+]i increases diastolic tension and lowers the extent of cell relaxation (2+]i and force in cardiomyocytes, we anticipate that the TRPC6-associated increase of [Ca2+]i increases contraction. Similar effects can be expected in stretched cells, since TRPC6 is thought to be activated through membrane stretch (2+]i (2+ in response to stretch, thereby increasing contraction.Our study shows direct effects of increased TRPC6 expression on [Calaxation . Due to  stretch . TRPC6 kch (2+]i . Our stu2+]i load (2+]i increase that is sufficient to activate the calcineurin-NFAT pathway. A positive feedback in which Ca2+ influx through sarcolemmal TRPC6 channels activates calcineurin-NFAT leading to upregulation of TRPC6 and increased Ca2+ influx was suggested (2+]i, and we explain the effects by increased Ca2+ influx and SR load. Reduction of TRPC6 expression and/or activity is thus promising in disrupting the positive feedback loop as possible treatment for hypertrophic remodeling. Modulation of TRPC6 activity through pharmacological blockers is an active area of research (Previous studies linked TRPC6 to hypertrophy and increased calcineurin-NFAT signaling . NFAT wa+]i load . It is tuggested . Our sturesearch . High-afresearch .We note several limitations related to our studies. We did not find differences of our TRPC6 functional measures between NRVMs infected with shRNA-TRPC6-eGFP and eGFP . While o2+ release from the SR of rat cardiomyocytes is highly temperature dependent (2+]i. For this reason, we only used the model for qualitative comparison of trends rather than actual measurements.Limitations of the applied mathematical model are discussed in . Most imependent . The modThe datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by IACUC, University of Utah.AA and FS designed the study. AA, FS, and MS drafted the manuscript. AS and MS acquired and analyzed image data. MS implemented the modeling and analyzed simulation data. CH isolated and infected cells. AA and CH performed western blotting. All authors critically revised the manuscript and approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


After years of development, the complexity of the biological sequence alignment algorithm is gradually increasing, and the lack of high abstract level domain research leads to the complexity of its algorithm development and improvement. By applying the idea of software components to the design and development of algorithms, the development efficiency and reliability of biological sequence alignment algorithms can be effectively improved. The component assembly platform applies related assembly technology, which simplifies the operation difficulty of component assembly and facilitates the maintenance and optimization of the algorithm. At the same time, a friendly visual interface is used to intuitively complete the assembly of algorithm components, and an executable sequence alignment algorithm program is obtained, which can directly carry out alignment computing. Bioinformatics is an interdisciplinary subject involving life sciences, mathematics, and computer science. Its main research work lies in the acquisition, processing, and storage of biological information, and further includes distribution, analysis, and interpretation. Its research methods are to use various technologies and tools of computer, biology and mathematics to mine and understand the biological significance contained in the massive data  has put forward the definition of software crisis. And then the research of software engineering (Wang et al., The idea of software reuse is to reuse the existing software in accordance with the specifications in the development process. When developing other systems in the same field, it is not necessary to develop from scratch, but on the basis of reusable resources to carry out efficient reuse development. In this process, abstraction is the basic element Zhu, , and effComponent assembly technology Zhang,  is the cThe component assembly forms mainly include black box assembly, white box assembly and gray box assembly. The main difference is whether the components need to be modified before assembly. Black box assembly is the most suitable assembly method for component encapsulation, but it also reduces the adaptability of components. White box assembly emphasizes the adaptability of components. The assembly is flexible and can achieve greater composability. However, due to too many implementation details exposed, the ease of use of components will be reduced, and improper modifications will occur, so that the final assembly cannot achieve the expected. Gray box assembly is the most widely used assembly method currently. It combines black box assembly and white box assembly and can be adapted to a variety of application scenarios.The difference between sequence alignment algorithm component and software component is that the former often has higher coupling degree, and the algorithm component often needs to be modified to adapt to the relevant application scenarios. However, the idea of sequence alignment algorithm is complex. If the assembler does not have a good understanding of the algorithm, new errors will be made when modifying it. Therefore, before assembling algorithm components, it is necessary to conduct a detailed domain analysis, formally describe the algorithm component, and form a structure framework to guide the algorithm component assembly. Finally, the gray box assembly of sequence alignment algorithm components is completed under the guidance of domain model, formal specification and algorithm framework.The goal of CBSD is that the software system can be automatically generated from a series of software components according to the system requirements supported by the generator. The purpose of developing the component assembly platform for sequence alignment algorithm is also to reduce the manual assembly workload as much as possible and improve the automation level of the whole component system.The platform mainly includes component transformation, component assembly and code running. By means of C++ program generation system of PAR, the component transformation module can transform Apla components into C++ components, see details in Xue et al. . The comComponent library module includes two parts, one is the source code of algorithm components that have been transformed and stored in the files, and the other is the Apla component assembly code that needs to be manually developed or modified in the database. In addition, the component library module also plays a management role, such as adding, deleting, modifying, and checking components, supporting further component expansion and modification in the future.Component selection module reads the components in the component library and displays them on the platform interface according to their required and optional features and the type characteristics of their affiliation. After selecting the components, the validity of component composition is checked. According to the multi-choice one or multi-choice relationship of feature dimension and the dependency relationship between components, the component composition is constrained accordingly to prevent illegal combination from the subsequent process.After completing the component selection, code assembly module obtains the required Apla assembly code from the database, and the user can make appropriate modifications to correctly call the selected component in the component library. After the assembly code is developed, the Apla code is converted into the corresponding C++ code through the transformation system of PAR platform. Finally, the assembly and compilation of executable codes is completed in the sever of platform to generate executable algorithmic programs.After the user inputs the alignment data, the code running module executes the corresponding executable alignment algorithm program. It enables the user to directly perform sequence alignment operations on the platform, and displays the final algorithm running results on the page.The platform is developed using B/S architecture following J2EE specifications, the Java language is used, and the Spring Boot (Wang et al., The overall architecture of the assembly platform is shown in According to the requirement analysis, the functional architecture of the platform is shown in Through system function requirement analysis, overall architecture design, and functional architecture design, the sequence alignment algorithm component assembly platform is outlined. Next is to give a detailed design of the platform system based on the operating sequence of each module. The platform mainly includes component conversion process, component selection process, component assembly process and algorithm execution process. This section mainly describes the process of component assembly, as shown in The pairwise sequence alignment algorithm and heuristic multiple sequence alignment algorithm based on dynamic programming have been implemented in the platform, and the most critical one is the progressive multiple sequence alignment algorithm based on phylogenetic tree. The most classic ClustalW (Thompson et al., Pairwise sequence alignment. The sequence group is aligned between two pairs, and the distance matrix is established by the pairwise sequence alignment score to indicate the distance between the sequences.Generate a phylogenetic tree. Using the information in the distance matrix, a phylogenetic tree is established through the corresponding clustering algorithm to guide the subsequent multiple sequence alignment operations.Progressive alignment. The previous operation has generated a guide tree, and the last step is to gradually complete the alignment of all sequences in the form of keeping gaps, starting from the close evolutionary relationship according to the alignment sequence reflected by the guide tree.The components involved in the algorithm are sequence validity check component, pairwise sequence alignment component, distance matrix component, phylogenetic tree component, progressive alignment component, and alignment result output component. Since the distance matrix component and the pairwise sequence alignment component are highly coupled, the pairwise sequence alignment component is designed as a generic operation parameter of the distance matrix, and the corresponding distance matrix can be generated by instantiating different pairwise sequence alignment algorithms. The phylogenetic tree component also includes a clustering algorithm selection sub-component, which is also designed as a generic operation parameter. The commonly used instantiation algorithms are the NJ algorithm (Saitou and Nei, We will carry out an example of the assembly for the progressive alignment algorithm based on phylogenetic tree to demonstrate how the modules of the platform work together and how they interact with each other.Transform Apla components except those for assembling. The transformation system of PAR platform is used to convert the developed Apla components into C++ components and store them to the platform's local files.Visually select several existing components satisfying the composable constraints according to the established domain feature model and component interaction model. The components are grouped by the required or optional attribute. In order to prevent the selection of illegal combinations from the subsequent assembly, the distinction between multi-choice-one or multi-choice-multi is carried out in the optional components group. The component selection interface is shown in Based on the interaction relationship between the components, read the corresponding Apla assembly code in the database and display it on the page following the selection of component combination. The user can check and modify the component assembly code, and then submit an Apla conversion request and store the converted C++ assembly code as the local file.makefile script file for compilation is generated automatically, and is executed to compile and link the C++ components. The parameter input interface of sequence the After all the Apla component conversion and assembly code conversion, the After the user inputs the sequence data, and the replacement matrix as well as the penalty model required by the multiple sequence alignment, the algorithm program generated will be executed, and the alignment output displayed in the user interface. As shown in With the development of CBSD, component-based development technology has been verified in many practical applications. It can exactly improve the development efficiency and maintenance of software. In this paper, the component development technology is applied to the development of biological sequence alignment algorithms. Under the guidance of domain modeling, generative programming and PAR, the formal transformation of sequence alignment algorithm components is carried out, and a B/S-based visual assembly platform for the gray box assembly of algorithm components is constructed. On top of our previous study results, the components required by the sequence alignment algorithm are classified and displayed, and the corresponding combination constraints are designed and implemented. After the legal component combination is selected, the assembly code can be modified and compiled to form an executable algorithm program. In addition, the algorithm can run directly on the platform to facilitate users to conveniently conduct sequence alignment studies.Next, we will release out codes in GitHub. Future work also includes the improvement of the biological sequence alignment algorithm component assembly platform from the following aspects.The algorithm components of this platform will be further expanded to enlarge the scope of algorithms generated from component assembly.The combination constraints in the platform have not been explicitly implemented. We will restrict the combination constraints of algorithm components to XML files, and shape the corresponding combination constraint documents to make it easier for users to assemble.With the richer component library, the algorithm component library needs to have an efficient component search function. Recent years, the recommendation algorithm based on artificial intelligence has developed rapidly. The feasibility of introducing this technology into the platform to improve the ease and automation level of algorithm assembly platform will be carefully studied.The original contributions presented in the study are included in the article/HS instructed the whole research work and revised the paper. GW, XZ, and JW did the codes work and the experiments. HS did the experiments. SX proofread the full text. All authors read and approved the final manuscript and were agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We use comparative genomics and sensitive sequence and structure analyses to investigate the evolution of this system. Our findings reconstruct the core form of the Ca2+-stores system in the last eukaryotic common ancestor as having at least 15 proteins that constituted a basic system for facilitating both Ca2+ flux across endomembranes and Ca2+-dependent signaling. We present evidence that the key EF-hand Ca2+-binding components had their origins in a likely bacterial symbiont other than the mitochondrial progenitor, whereas the protein phosphatase subunit of the ancestral calcineurin complex was likely inherited from the asgard archaeal progenitor of the stem eukaryote. This further points to the potential origin of the eukaryotes in a Ca2+-rich biomineralized environment such as stromatolites. We further show that throughout eukaryotic evolution there were several acquisitions from bacteria of key components of the Ca2+-stores system, even though no prokaryotic lineage possesses a comparable system. Further, using quantitative measures derived from comparative genomics we show that there were several rounds of lineage-specific gene expansions, innovations of novel gene families, and gene losses correlated with biological innovation such as the biomineralized molluscan shells, coccolithophores, and animal motility. The burst of innovation of new genes in animals included the wolframin protein associated with Wolfram syndrome in humans. We show for the first time that it contains previously unidentified Sel1, EF-hand, and OB-fold domains, which might have key roles in its biochemistry.The origin of eukaryotes was marked by the emergence of several novel subcellular systems. One such is the calcium (Ca The process is then reversed and Ca2+ is pumped back into the ER by the ATP-dependent SERCA  pumps, members of the P-type ATPase superfamily  chaperones such as calreticulin, calnexin, and calsequestrin in the ER lumen  divea2+ flux ; 3) chanflux  response . Togethedivision .2+-stores and -signaling system have revealed that some components were either present in the last eukaryotic common ancestor (LECA)  or derived early in the evolution of the eukaryotes (e.g. IP3R)  . Other pg. IP3R) , or wereg. IP3R) . A substg. IP3R) . Most st2+-stores system, analyzing their known and predicted interactions and inferring the evolutionary depth of various components. We show that an ancient core of at least 15 protein families was already in place at the stem of the eukaryotic lineage. Of these, a subset of proteins is of recognizable bacterial ancestry, although there is no evidence of a bacterial Ca2+-stores system resembling those in eukaryotes. We also show that gene loss and lineage-specific expansions (LSEs) of these components shaped the system in different eukaryotic lineages, and sometimes correspond to recognized adaptive features unique to particular organisms or lineages. Further, we conducted a systematic domain analysis of the proteins in the system, uncovering three novel unreported domains in the enigmatic wolframin protein. These provide further testable hypotheses on the functions of wolframin in the context of the Ca2+-stores system and in protecting cells against the response stresses that impinge on the ER.Given our long-term interest in the origin and evolution of the eukaryotic subcellular systems, we conducted a comprehensive analysis of the core and regulatory components of the CaIterative sequence profile searches were performed using the PSI-BLAST program (RRID: SCR_001010)  against Protein-protein interactions (PPIs) were extracted from published data sources, updating any outdated gene/protein names and making substantial efforts to disambiguate between paralogs . NetworkQuantitative analysis of phyletic patterns and paralog counts for different proteins/families were first obtained using a combination of sequence similarity searches as outlined above. We filtered the counts to exclude multiple identical sequences annotated with the same gene name, using the latest genome assemblies for each of these organisms as available in NCBI GenBank (RRID: SCR_002760) as anchors. Proteomes with identifiable quality issues were removed from downstream analyses (e.g. genomes containing sequences with ambiguous strain assignment), leaving counts for 216 organisms. These counts and their phyletic patterns defined two sets of vectors, namely the distribution by organism for a given protein and the complement of proteins for a given organism. These vectors for the protein families and organisms were used to compute the inter-protein or inter-organism Canberra distance , which iThese distances were used to cluster the protein families and organisms through agglomerative hierarchical clustering using Ward's method . The resThe protein complement vectors for organisms were also used to perform principal component analysis to detect spatial clustering upon reducing dimensionality. The variables were scaled to have unit variance for this analysis. Similarly, a linear discriminant analysis was performed on these vectors using representatives of the major eukaryotic evolutionary lineages  counts the number of paralogs of some protein domain family p in some organism o, P is the set of all proteins studied, and O is the set of all organisms studied, then the polydomain score for an organism o∈O is defined as:Organism polydomain scores were calculated as follows: if f(p) for all proteins p∈P and f(p) is defined as:where Computations and visualizations were performed using the R language.2+ stores have been extensively studied, leading to the identification of several proteins directly or indirectly involved in calcium transport or signaling and regulation of these processes. In order to apprehend the global structure of this system, we used published literature and the FunCoup database to derive a network of protein-protein interactions (PPIs) containing human genes that have roles in Ca2+ stores, centering the network on the three families of ER Ca2+ channels  (see Methods). The resulting network totaled 173 protein nodes and 761 interaction edges  EF-hand domain proteins such as calmodulin and sorcin that bind Ca2+ and consequently interact with and regulate the biochemistry of numerous other proteins; 3) proteins involved in folding and stability of other proteins, such as chaperones, redox proteins, and protein disulfide isomerases; 4) components of the protein phosphorylation response that is downstream of Ca2+ flux into the cytoplasm; and 5) proteins linking the network to other major functional systems, such as Bcl2, which is involved in the apoptosis response in animals, and beclin-1, which is involved in autophagy.Analysis of the proteins in this highly connected sub-network suggests that the 46 proteins can be broadly classified into five groups: 1) the channels and ATP-dependent pumps which constitute the core CaMethods). To understand better the emergence of this sub-network and the conservation of its nodes across eukaryotes, we systematically analyzed their phyletic patterns (Methods) revealed the presence of five distinct clusters (2+-stores system (e.g. clusters 4 and 5).This densely connected sub-network invites questions about its evolutionary origin, particularly given that the wet-lab results that inform the connections are predominantly drawn from mammalian studies  the SERCA Ca2+ pumps; 2) Ca2+-binding EF-hand proteins like calmodulin; 3) chaperones involved in protein folding that are mostly found in the ER and that sometimes act as either Ca2+ binding proteins  or as regulators of other components of the Ca2+-stores and -signaling system ; and 4) core enzymes of the Ca2+-dependent phosphorylation-based signaling system including the CaMKs and the calcineurin A phosphatase. This set of components is likely to have comprised the minimal ER Ca2+-stores and -signaling system in the LECA and suggests that there were already sub-systems in place to mediate: 1) the dynamic transport of Ca2+ ions across the emerging eukaryotic intracellular membrane system and, 2) the transmission of signals affecting a wide-range of subcellular processes based on the sensing of Ca2+ ions, and 3) a potential stress response system that channels Ca-dependent signals to regulation of protein folding  were both present in the LECA. They are most closely-related to bacterial P-type ATPases (+-Ca2+ antiporters), ion exchangers (Na+-H+ exchangers), permeases, and other distinct P-type ATPases in conserved gene-neighborhoods  calstabin (FKBP1A/B in humans), which is a member of the FKBP family of PPIase chaperones. Phyletic pattern analysis indicates that calstabin was present in the LECA . After computing the pairwise Canberra distance between these vectors, we performed clustering using the Ward algorithm (see Methods). The resulting clusters recapitulate aspects of eukaryote phylogeny, with animals, fungi, plants, kinetoplastids, and apicomplexans forming distinct monotypic clusters  on these vectors to obtain a global quantitative view of the diversification of the Ca2+-stores system. Plotting the first two principal components/discriminants reveals discernable spatial separation of the metazoans from their nearest sister group, the fungi  and autotrophy (photosynthetic lineages). Hence, investigations on individual lineages are likely to unearth novel, lineage-specific regulatory devices, which potentially reflect adaptations to their respective environments and provide hints about the biological contexts in which they were found.In order to obtain some insights regarding the major developments in the evolution of the eukaryotic CaMethods), which captures the overall amplification of the Ca2+-stores system of an organism in terms of the contribution of the different constituent protein families of the system . Similarly, fungi, which use chitin as a central cell wall component, show lower polydomain scores relative to sister eukaryotic lineages. Conversely, we noted elevated polydomain scores in molluscs with calcareous shells. Likewise, ciliates, which have a evolved a distinct extension to the ancestral eukaryotic Ca2+-stores system in the form of the alveoli , calcineurin A (in ciliates and Entamoeba), calnexins (in Trichomonas and diatoms), ORAI  might correspond to or be involved in the regulation of Caormation . Similaric roles , might em stores .Emiliania might correspond to the need to regulate Ca2+ transport for mineralizing the calcareous coccoliths , which is close in proximity to the envelope of the coccolith and the site of biomineralization  family displays an unusual phyletic pattern of particular note, being found only in metazoans and land plants    in land genitors .2+-stores system. A notable exception is seen in the CaMKs: metazoan CaMKs show a higher architectural complexity via their fusion to several distinct globular domains. These domains act as adaptors in interactions with Ca2+-binding regulatory domains, effectively broadening the total range of signaling pathways in which the CaMKs participate in metazoa  Proteins or component domains which are involved in a broader range of functions but have been recruited to roles in Ca2+-stores regulation specifically in the animal lineages, such as the SAM domain in the STIM1/2 proteins, the PDZ domain in the neurabin protein, and the TRPC channels. 3) Proteins containing domains which appear to be either novel metazoan innovations, such as the KRAP domain of the Tespa1 protein, or whose origins have yet to be traced, such as VGCC and wolframin. Further, the origin of certain metazoan-specific proteins such as phospholamban, which inhibits the SERCA ATPase, remain difficult to trace because of their small size and highly-biased composition as membrane proteins. Inspection of the neighbors of these proteins in the constructed network suggests that in almost all instances, these proteins were added to the Ca2+ signaling network via interactions with one or more of the ancient components of the system  pathway for re-filling the ER from extracellular Ca+ stores . Of the lineages . The Jir2+-stores system is wolframin, a transmembrane protein localized to the ER membrane (Methods) as containing Sel1-like repeats , which are α-helical superstructure forming repeats structurally comparable to the tetratricopeptide repeats (TPRs) (Methods) unified the two remaining globular regions respectively with various EF-hand domains  and OB fold-containing domains .As noted above, one of the uniquely metazoan proteins in the Camembrane   . ProfileThe wolframin SLR possess several conserved residues seen in the classical SLRs , and add2+-binding DxDxDG motif or any comparable residue conservation  formed by cyanobacteria  certain contours of the eukaryotic tree , or the evolution of calcium-rich biomineralized skeletons and shells .Even within animal Ca decades . Assignm decades  supports2+-stores regulatory role at the ER (However, outside of a possible bacterial origin for the SLR region (see above, t the ER . Their rDespite observations that genic disruptions contribute to similar though not identical phenotypes , WFS1 an2+-stores regulatory system points to a core of domains inherited from distinct prokaryotic sources conserved across most eukaryotes. Lineage-specific differentiation of the system across eukaryotes is driven by complexities stemming from both the loss and/or expansion of the core complement of domains by the addition of components via LSEs or recruitment of domains of diverse provenance. We analyze in depth one such striking example of the latter, namely wolframin, which has been previously implicated in human disease. The evolution of wolframin provides a model for how regulatory components of the Ca2+-stores system emerged: through the combining of existing mediators of Ca2+ signaling, like the EF-hand domains, with other domains originally not found in the system. Such transitions often happened at the base of lineages that subsequently underwent substantial diversification. We hope the findings presented here open novel avenues for the ongoing research on the regulation of calcium stores across eukaryotes, including providing new handles for understanding the functional mechanisms of wolframin and its dysregulation in Wolfram syndrome.Reconstruction of the evolution of the eukaryotic CaAll datasets analyzed for this study are included in the article/Conceptualization: DS, AB, LA. Formal analysis: DS, AB, LI. Analytical tools: DS, LA. Project administration: AB, LI, LA. Visualization: DS. Writing—original draft: DS, AB. Writing—review and editing: AB, LI, LA.DS, LI, AB, and LA are supported by the Intramural Research Program of the NIH, National Library of Medicine.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Matched with advances in fluorescence imaging techniques, the improvements in the functional range and stability of various calcium indicators have significantly enhanced more accurate study of intracellular Ca2+ dynamics and its effects on cell signaling, growth, differentiation, and regulation. Nonetheless, the current limitations broadly presented by organic calcium dyes, genetically encoded calcium indicators, and calcium-responsive nanoparticles suggest a potential path toward more rapid optimization by taking advantage of a synthetic biology approach. This engineering-oriented discipline applies principles of modularity and standardization to redesign and interrogate endogenous biological systems. This review will elucidate how novel synthetic biology technologies constructed for eukaryotic systems can offer a promising toolkit for interfacing with calcium signaling and overcoming barriers in order to accelerate the process of Ca2+ detection optimization.Since the 1970s, the emergence and expansion of novel methods for calcium ion (Ca Calcium signaling plays a key regulatory role across numerous physiological processes, including cell growth, gene regulation, neuronal synaptic activity, immune system function, hormone secretion signaling, fertilization, and the biomechanics of contraction  with synthetic dyes. Single-wavelength indicators exhibit significant changes in fluorescence intensity when bound to Ca2+ without shifting excitation or emission wavelengths [2+], at the expense of increasing the spectral range [Basic design of calcium probes involves covalent linkage between the binding unit with an active site for Ca of Ca2+ . Currentelengths . This enal range . Fluores2+]. In order to assess dye fluorescence, the addition of an ionophore, commonly ionomycin, to a dye-loaded cell allows external and internal [Ca2+] to reach equilibrium before measurement  by measuring the ratio of fluorescence emitted at two different excitation or emission wavelengths and are still in use today to track intracellular release of Ca2+ in neuronal and immune cell cultures , and viability of neurons even after incubation for >24 h within a carefully controlled recovery chamber  in the organelle, the Kd of the probe, and the concentration of the probe itself . For example, as reviewed by McMahon and Jackson, high concentrations of Fura-2 loaded into cells can bind 99.9% of Ca2+ and leave the small percentage remaining as free ion  is in the micro to millimolar range, and lower concentrations of probes are sufficient for organelle Ca2+ detection without interfering with endogenous buffering . Va. Va98]. d 300 nM ,109, whiat 20 °C . This ha1000 s−1 . Studies1000 s−1 . Althougosophila . Cardenaosophila . The sloebrafish ,113.2+ findings in cell processes between older and new variants. For example, Ye et al. found that Ca2+ waves in processes along with microdomain Ca2+ transients were more readily detectable using GCaMP6f instead of GCaMP3, with only minor differences in kinetics and a similar dynamic sensing range  has also prompted new research into interrogation of Ca2+ homeostasis in the mitochondria and its effect on cell migration and mitochondrial metabolism ER dynamics. Calcium-measuring organelle-Entrapped Protein IndicAtor 1 in the ER (CEPIA1er) utilizes the well-established ER targeting scheme comprised of an N-terminal signal peptide calreticulin and the C-terminal KDEL retention motif, and this indicator has since been widely cited in multiple studies, including validation in ventricular myocytes  and between different cells in neurons and astrocytes . This can interfere with cell autofluorescence or optogenetic control . Blue waytes see a 158]..2+ imagi2+ detector studies, Dana et al. developed mRuby-based jRCaMP1a and jRCaMP1b and mApple-based jRGECO1a  rises, the development of CaMPARI, or calcium-modulated photoactivatable ratiometric integrator, moves away from online monitoring to a high-temporal-resolution “active snapshot” of large tissue volume  in the cell body and neurites of neurons.Calcium-responsive nanoparticles can potentially enhance imaging with preexisting dyes refer to . An impoolecules . This co2+ in vivo pushed researchers to find a molecularly specific analogue to functional magnetic resonance imaging (fMRI) in order to map brain activity. While fluorescent Ca2+ sensors have been used to measure extracellular Ca2+ levels, these preexisting compounds have binding constants which are too high for the targeted range of 0.1–1.0 mM and are ultimately unsuited for deep-tissue study [2+] fluctuations  in cells and living animals with high sensitivity.Near-field fluorescence (NFF) can improve the fluorescence properties of fluorophores when utilizing metal nanoparticles . When th2+ inside cells upon introduction with a CPP  while limiting undesired off-target activity by eliminating endogenous NFAT site binding, thus ensuring orthogonality. Ultimately, these various synthetic transcription factors which are activated by physiological Ca2+ can serve as an interface for endogenous cell systems and synthetic systems.In recent years, a number of different research groups have constructed engineered calcium-responsive transcription factors that focus on detecting or triggering transient Cachannels . An artilevation . An incr signals . The sig2+ integrators in eukaryotes is the use of Tobacco Etch Virus (TEV) proteases, having previously been deployed in neurons [2+ Activated Neuron Recorder (SCANR), is a modular Ca2+ reporter system which remains catalytically incompetent until a Ca2+ spike triggers reconstitution and activate transgenes of interest [2+ spikes in HEK293 cells or in neuronal signaling in cultured rat hippocampal neurons. SCANR’s modularity offers important flexibility for future synthetic biology research on incorporating optogenetic modules for spatial and temporal control of the enzyme’s activity.A significant advance in the realm of stable Ca neurons . While c neurons ,194. Theinterest . This en2+-mediated induced proximity of active calmodulin and an M13 peptide leads to TEV protease cleavage and release of a membrane-constrained transcription factor, which drives the expression of opsins and other genetically encoded tools when it senses coinciding elevated cytosolic Ca2+ levels and externally applied blue light. With modular design and minute-scale temporal resolution, FLARE offers an important system in neuroscience to measure responses to inputs as varied as calcium channels and engineered ligand-receptor pairs, finding numerous approaches that extend even beyond Ca2+ to result in transcription factor release [Wang et al. wanted to selectively characterize and manipulate neurons by engineering a different transcription factor system, Fast Light- and Activity-Regulated Expression (FLARE) . Ca2+-me release . These a release ), which  release .2+ releaser under the control of Opto-CRAC, a light-inducible Ca2+ release activated channel (CRAC) [2+ influx as a result of Opto-CRAC activates calcineurin, which dephosphorylates NFAT and consequently results in its nuclear translocation. Specifically, the OptoCRAC channel remotely controlled the Ca2+ signal so that an NFAT fragment fused with dCas9 and a VP64 transcription factor would translocate into the nucleus to control gene expression. In Xie et al.’s experiment, a synthetic promoter PNFAT2 containing NFAT repeats from murine IL-4 promoter was most responsive to chemically induced membrane depolarization by KCl [V1.2 channels, these channels fine-tuned calcium signaling so that cells exhibited amplified excitation-transcription coupling and higher sensitivity [2+ dependencies of NFAT isoforms permit agonists to recruit different combinations of isoforms as stimulus strength increases [One powerful tool to alter genomic DNA or the epigenome is the CRISPR/Cas9 system . Programl (CRAC) . Ca2+ inn by KCl . By simusitivity ,201. In ncreases . This me2+-sensing receptor (CaSR), a GPCR that normally manages Ca2+ homeostasis in humans, was instead ectopically expressed in mice. The researchers constructed a CaSR-dependent calcium-sensitive promoter (PCa4) containing three SRE and six NFAT operator modules 5′ of a minimal version of the constitutive human CMV immediate early promoter. Under the control of this promoter, HEK293 cells produced melanin in response to sustained Ca2+ sensed in blood, which was also validated in wild-type mice implanted with engineered cells. Such cell-based biosensors can provide cheap, portable, and simple methods of detecting molecules of interest, but a review by Hicks et al. discusses the lack of reliability and long-term stability of biosensors so far as barriers to scaling up applications [Beyond in vitro systems, Tastanova et al. demonstrated a working hypercalcemia biosensor in both HEK293 cells and in vivo mice . The Ca2ications .2+-driven pathways, this raises the important possibility of expanding the array of options for understanding endogenous cell responses to Ca2+. A majority of the calcium indicators discussed so far rely on fluorescent imaging, which come with phototoxicity and photobleaching effects discussed in previous sections. While longer wavelength or bioluminescent probes can ameliorate some of these issues, a newer foray of synthetic biology into DNA-based cellular recorders may offer an opportunity to preclude a fluorescent imaging requirement altogether. Of the hundreds of readouts for calcium signaling broadly available, these synthetic biology-based recording devices specifically have the potential to capture stimulus data from whole animal cell populations by using a DNA-based output detectable with HTS techniques  to name a few Caques see a, obviatques see b–d. Whenll death . The DNA imaging ,207.2+ detection will be vital to biosensor optimization [2+ detection by introducing intracellular synthetic circuits not limited to natural sources of Ca2+-sensing machinery. Directed evolution has existed for decades in protein engineering, but Saito et al. recently used advances in machine learning to develop an artificial intelligence-guided mutagenesis platform that could alter the properties of GFP [Just as GECIs and synthetic dyes have undergone their own optimization, cell-based biosensor optimization via synthetic biology needs to similarly apply rational methods. More strategies that can expand the range of de novo allosteric transcription factors for Camization . In parts of GFP . With twInformation derived from mathematical models can point to necessary modifications that need to occur with various genetic parts. These performance models should account for concentration levels, biosensor kinetics based on enzymes involved in each part of the system, and alteration of copy number of promoters to improve sensitivity . When de2+ and the drawbacks or confounding factors that can influence data collection. We have an expansive body of research on Ca2+ detection methods, from synthetic calcium dyes to GECIs, which has enabled significant optimization of these tools. However, even within recent years, more research has revealed that introducing these calcium sensors into cells has significant effects that are not orthogonal to endogenous systems. These include potent suppression of Na+- and K+-dependent adenosine triphosphatase activity by chemical calcium indicators [2+ signals [Calcium signaling dynamics continue to have key implications across all reaches of biological applications, from neuronal communication to T-cell activation to muscle bioenergetics. It is therefore essential to evaluate the best possible detection methods for Cadicators , interrudicators , or othe signals ,89.2+-binding properties, capacity to control localization in desired subcellular compartments, fluorescence lifetime, and stability to photobleaching. Of course, these concerns naturally arise due to the fact that the current, popular methods of detecting Ca2+  all rely on a fluorescent readout. The resultant phototoxicity and inability to capture deep tissue all present limitations to biological applications, particularly in vivo [2+ sensors will need further modification and optimization in order to represent the same dynamic range and SNR exhibited by the most recent iterations of GCaMP6s or dyes. Future research on current Ca2+-binding proteins and dyes should focus on multicolor calcium sensors which achieve fast kinetic responses and Ca2+ affinity optimized for a range that encompasses the various concentrations of Ca2+ in cellular components and the cytosol [When considering the development of successful probes, researchers should consider Ca in vivo ,184,185. cytosol . Synthet cytosol , and num2+ detection and provides a potential toolkit to do so. Moving in the direction of synthetic biology, a cytosolic Ca2+ sensor would take advantage of endogenous Ca2+-binding properties while removing the limitations presented by the more popular fluorescent methods. Although more work has been done regarding the design principles of transcription-factor-based biosensors in prokaryotic systems, the same issues must be tackled in order to have the desired precision and range in eukaryotic systems. As synthetic biology tools become increasingly common at the intersection of biology, chemistry, and physics, novel classes of detection methods for Ca2+ are ripe with research possibilities. We should wholly embrace the diverse and versatile array of experimental methods utilizing synthetic biology that continue to be refined and expanded, and the concomitant ability to achieve more precise spatiotemporal measurement of Ca2+ dynamics will enhance the capacity to analyze the diverse effects of Ca2+ on biological systems in basic science and medical applications.The advantage of future applications of synthetic biology lies in a combinatorial approach, which synthesizes these desired features for Ca


CBS) gene, and is frequently associated with dyslipdemia. The aim of this study was to further elucidate the effect of mutated CBS gene on circulating lipids using a rabbit model harboring a homozygous G307S point mutation in CBS.Congenital hyper-homocysteinemia (HHcy) is caused by a defective cystathionine β-synthase  and lipid profile were analyzed.CRISPR/Cas9 system was used to edit the CBS-knockout (CBS-KO) founder lines with biallelic modifications were obtained. Mutation in CBS caused significant growth retardation and high mortality rates within 6 weeks after birth. In addition, the 6-week old CBS-KO rabbits showed higher plasma levels of Hcy, triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) compared to the age-matched wild-type (WT) controls. Histological analysis of the mutants showed accumulation of micro-vesicular cytoplasmic lipid droplets in the hepatocytes. However, gastric infusion of vitamin B and betaine complex significantly decreased the plasma levels of TG, TC and LDL-C in the CBS-KO rabbits, and alleviated hepatic steatosis compared to the untreated animals.Six CBSG307S rabbit model was generated that exhibited severe dyslipidemia when fed on a normal diet, indicating that G307S mutation in the CBS gene is a causative factor for dyslipidemia.A  Homocysteine (Hcy) is a non-essential sulphur-containing amino acid derived from methionine metabolism. The plasma Hcy range in healthy individuals is 5–15 μmol/L, and an increase to 16–30 μmol/L, 31–100 μmol/L and > 100 μmol/L result in moderate, intermediate and severe hyper-homocysteinemia (HHcy) respectively –5, atherCBS) or 5,10-methylenetetrahydrofolate reductase (MTHFR) [CBS gene. In fact, around 22 mutant alleles of CBS have been reported so far in individuals with CBS deficiency [The causative factors of HHcy include genetics, nutrition, medication, disease status, smoking and age. In addition, severe HHcy is often triggered by congenital deficiency of cystathionine β-synthase ( (MTHFR) , and theficiency –22, and ficiency . The mosficiency .CBS metabolizes Hcy to cystathionine in the presence of vitamin B6 [Hcy is re-methylated to methionine by 5-methyltetrahydrofolate-homocysteine methyltransferase (MHMT) and the cofactor folate-cobalamin, and the methyl group is donated by betaine via betaine-homocysteine methyl transferase (BHMT). In addition, tamin B6 –30. Intetamin B6 . While ptamin B6 . Althougtamin B6 . In additamin B6 , either Oryctolagus cuniculus) model offers several advantages over rodents, such as greater phylogenetic similarity to primates, adequate amount of blood for plasma biochemical analysis, suitable heart size for studying atherosclerosis in both aorta and coronary arteries [HHcy is routinely accompanied by life-threatening vascular complications, and individuals with high circulating levels of Hcy are at a higher risk of CVD and increased 5-year mortality . Howeverarteries , 40, andarteries .CBS mutations in humans are of the missense type caused by base pair substitutions, HHcy modeling require gene knock-in as opposed to knock-out. To this end, we generated CBS gene mutant rabbits including point mutation G307S using the CRISPR/Cas9 system in order to gain new insights into the HHcy pathogenesis. In addition, the CBS-KO rabbits were fed with vitamin B and betaine complex supplements to devise possible therapeutic approaches.Since most CBS were designed using http://crispr.mit.edu based on its sequence . Three CBS-targeting sgRNAs were screened   for rabbit New Zealand White rabbits (6–8 months old) were housed at the Animal Genetic Engineering Laboratory of Yangzhou University under a 12 h diurnal/nocturnal cycle, and fed twice a day with free access to water. All protocols were approved by the Care and Use of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China) and the Animal Care and Use Committee of Yangzhou University, Yangzhou, China (license number: SYXK(Su)2017–0044).Female rabbits were superovulated by intramuscular injection of follicle-stimulating hormone  twice daily. The dosage was 15 IU for the first two injections, 10 IU for the next two and 5 IU for the last two injections. After the final injection, both the superovulated and recipient females were injected with 100 IU human chorionic gonadotropin (HCG), and the former were mated with male rabbits. Approximately 18–20 h post-coitus, the female rabbits were anesthetized with pentobarbital , and the pronuclear zygotes were extracted and transferred into M2 medium  supplemented with 10% fetal bovine serum . Following cytoplasmic injection of 40 ng/μL Cas9 mRNA, 10 ng/μL of an sgRNA and 25 ng/μL ssODN, the embryos were transferred to complete (with 10% FBS) M16 medium  and incubated at 38 °C under 5% COCBS sequences were amplified using specific primers  was used to predict potential sites homologous to the 23-bp sgRNA + PAM sequence across the rabbit genome. These sites were then amplified in the genomic DNA of founder CBS-KO rabbits and sequenced. The off-target sites and primer pairs are listed in Supplementary Tables 1 and 2 , total cholesterol (TC), and high-density (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured using specific kits . In addition, Hcy levels were measured using an ELISA kit .The expression levels of the apolipoproteins (Apo) B, E and A-I in the plasma were analyzed by Western blotting as per standard protocols. The protein bands were probed with the goat anti-ApoE and anti-ApoB , and sheep anti-ApoA-I  antibodies. The secondary antibodies were HRP-conjugated donkey anti-goat IgG  and donkey anti-sheep IgG .CBS-KO and WT littermates were maintained under similar conditions, and fed according to their growth stage. The body weight of the rabbits was monitored from birth till 6 weeks of age. After euthanizing the animals with sodium pentobarbital overdose, the liver lobes were harvested and fixed in 4% paraformaldehyde. For histological analysis, the fixed tissues were embedded in paraffin and cut into sections that were stained using hematoxylin and eosin (HE) using standard protocols. The stained sections were viewed under a Leica light microscope.The P < 0.05 was considered statistically significant.Data were expressed as mean ± SD and compared by Student’s t test. GraphPad Prism was used for all statistical analyses, and CBS , 36.88% (45.00 ± 7.16 mg/dL vs 71.31 ± 6.70 mg/dL), 27.31% (89.33 ± 4.94 mg/dL vs 122.89 ± 11.83 mg/dL) and 39.33% (17.14 ± 0.56 mg/dL vs 28.25 ± 3.54 mg/dL) respectively in the WT rabbits compared to non-supplemented littermates. In contrast, no significant changes were seen in plasma HDL-C levels  lacked the glycine 307 residue in CBS protein, and displayed high serum levels of Hcy and lipids. Supplementing the normal chow with vitamin B and betaine alleviated the pathological symptoms of HHcy. Although there was some research reported on the G307S point mutation, it was mostly based on the heterologous gene expression system such as E. coli, S. cerevisiae, and Chinese hamster ovary cells. But it was unfortunate that the heterologous CBSG307S protein lacked enzyme activity [CBS-deficient animal model of G307S point mutation or deletion of glycine at position 307. The CBS-KO rabbit showed HHcy, dyslipidemia, a short life span, hepatic steatosis and other lesions. This indicates that HHcy and hyperlipidemia are likely the result of 307G deletion as well as G → S mutation. Compared with CBS-deficient mice in the previous research [CBS-KO rabbit model is suitable for studying the pathogenesis and therapeutic strategies of HHcy. Studies show that HHcy is an independent risk factor for CVDs [CBS-KO founder rabbits, and decreased significantly following betaine and vitamin B6 supplementation.Homocysteinemia is observed in 5–30% of the human population, and leads to vascular complications in one-fourth of the patients –45. We oactivity –48. At presearch , 50, thefor CVDs , 52. In for CVDs , 54. ConCBS-KO rabbits. Interestingly, the normal rabbits also showed Hcy levels higher than 15 μmol/L, which may explain the rapid induction of hyperlipidemia via high-fat diet consumption. Thus, the species and nutrient intake should be taken into account when assessing the effect of Hcy metabolism on dyslipidemia.Dyslipidemia is a major factor underlying CVDs, and normalizing blood lipid levels can lower the morbidity and mortality in patients with heart disease . The quaCBS-KO rabbits was filled with microvesicular cytoplasmic lipid droplets due to the influence of Hcy on the expression level of S-methyltransferase (BHMT). CBS-KO rabbits showed signs of growth retardation and short survival. Betaine and vitamin B supplementation reduced the fatty liver symptoms and prolonged survival of the mutants. Maclean [CBS alleviated liver steatosis and prolonged survival in a mouse model. We showed for the first time that the addition of betaine and B vitamins partly compensated for the lack of CBS. This is significant since the previously reported hypolipidemic effects of betaine were seen animal models with high-fat diet-induced hyperlipidemia.The fatty liver parenchyma of  Maclean  et al. rCBS.HHcy is an important factor in hyperlipidemia and cardiovascular disease. In this study, animal models of HHcy showed hyperlipidemia and cardiovascular disease symptoms. Although the lipoprotein metabolism and cardiovascular pathophysiology of rabbits are similar to that of humans , the inhCBS-KO rabbits with betaine and vitamin B supplementation had lower ApoB100, ApoB48 and ApoE levels in their plasma. ApoB is the major component of very low density lipoprotein (vLDL) and LDL particles. Namekata et al. [−/− mouse serum, and Salahi et al. [Compared to the untreated animals, the a et al.  detectedi et al.  found thi et al. . In addii et al. .In conclusion, CBS-KO rabbits have impaired growth and metabolism, although it remains to elucidated whether these changes are the direct effect of the mutation, or an indirect effect of hyperlipidemia or high homocysteine.CBS-KO rabbits with G307S mutation for the first time using the CRISPR/Cas9 system, and the model exhibits hyperhomocysteinemia and dyslipidemia on a normal chow diet. It is also the first report on the effect of betaine on the serum lipids of CBS-KO rabbits.We have generated CBS-KO rabbits were higher compared to that of the normal controls even after betaine and vitamin B complex supplementation.However, the short lifespan of these rabbits obviated the control data of C9♀, C14♂, C15♀ and C16♂ without betaine and vitamin B complex supplementation. Nevertheless, Hcy, TG, TC, LDL-C and other indices of most CBS mutated rabbit model is a promising tool for studying human dyslipidemia, despite their high mortality. The lesions of G307S homozygous CBS-KO rabbit should be useful to clearly understand the pathophysiology of the CBS gene deficiency and the G307S mutation rabbit provides a suitable animal model for drug development.In conclusion, the Additional file 1: Table S1. The sequences of potential off-target sites.Additional file 2: Table S2. The sequences of potential off-target loci PCR primers.


Recent evidence suggests that IP3R isoforms participate in synthesis and degradation pathways. This minireview will summarize the current findings in this area, emphasizing the critical role of Ca2+ communication on organelle function as well as catabolism and anabolism, particularly in cancer.In the last few years, metabolism has been shown to be controlled by cross-organelle communication. The relationship between the endoplasmic reticulum and mitochondria/lysosomes is the most studied; here, inositol 1,4,5-triphosphate (IP3) receptor (IP3R)-mediated calcium (Ca The RyRs (RyRs) , while Ichannels .2+ plays additional roles regulating different aspects of organelle function, including metabolism. The regulatory role of Ca2+ on metabolic networks is supported by physical interactions between organelles that allow ion and molecule fluxes  receptor is a family of three ligand-gated channels: type 1 IP3R (IP3R1), type 2 IP3R (IP3R2), and type 3 IP3R (IP3R3) , each ab channel . The sup channel ; neverth channel . The cen channel . The gat channel . This do channel .Inositol 1,4,5-triphosphate is produced by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in the cell membrane through phospholipase C (PLC) in response to extracellular stimuli such as hormones, growth factors, and neurotransmitters that bind to G-protein-coupled receptors , as well2+ release mediated by IP3R include free Ca2+ concentration in the cytosol (mentioned here as cytoplasmic Ca2+), as well as in the ER (mentioned here as reticular Ca2+), ATP, thiol modification, and phosphorylation by protein kinases (2+-dependent phospholipid-binding protein Annexin A1 (ANXA1)  , IRBIT ( (ANXA1) , and the (ANXA1) .2+  , which interacts with many regulator proteins that affect its permeability to Ca2+ , isocitrate dehydrogenase (IDH), oxoglutarate dehydrogenase (OGDH), and electron transport chain (ETC) complexes III and V  , which r (VDACs) . Additio (VDACs) , TOM70 ( (VDACs) , and the (VDACs) . Finally to Ca2+ . The Ca2II and V . If the t manner , 2017. St manner , and trit manner . Autophat manner . Additiot manner , and itst manner .2+ uptake. This is mediated by Ca2+-dependent protein phosphatase-4 (PP4) activity  , interacycolysis , it is n2+ fluxes between these two organelles have been described. In fact, the ER is the most important source of lysosomal Ca2+, which can be delivered by all three IP3R isoforms  and allows its translocation to the nucleus (+) and Ca2+ that form a complex with mTOR. Upon starvation, when mTOR is inhibited, TPCs are actively transferring Ca2+ to the lysosome, which is necessary to maintain the lysosomal pH and amino acid efflux  metabolism , which is a peptide (Cancer is a pathology like no other due to the presence of several layers of heterogeneity, which include different tissues and different oncogenic processes . For theew drugs . Regardiysiology . In this release , selecti release . In thisll death . Other Xll death . Similarll death . Anotherll death . Along wll death . On the  peptide , and GGT peptide .2+ channels for cancer cell survival. How different IP3R isoforms – homotetramers or heterotetramers – contribute to cancer development remains an area of research that requires more exploration. A better understanding of IP3R regulation and its effects on metabolism in normal and cancer cells will help to develop new therapeutic strategies to fight this devastating set of diseases in the future.In all cases mentioned above, IP3Rs have emerged as essential CaUA-C and CC designed and outlined the structure and contents of the review. UA-C, GB, ES-P, AP-H, AL, and CC contributed to the literature review, discussion, and writing of the manuscript. All authors contributed to manuscript revision and approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CaV1.2 and RyR2 (cardiac). The CaV channels transform action potentials into elevations of cytosolic Ca2+, by activating RyRs and thus promoting SR Ca2+ release. The high levels of Ca2+, in turn, stimulate not only the contractile machinery but also the generation of mitochondrial reactive oxygen species (ROS). This forward signaling is reciprocally regulated by the following feedback mechanisms: Ca2+-dependent inactivation (of Ca2+ channels), the recruitment of Na+/Ca2+ exchanger activity, and oxidative changes in ion channels and transporters. Here, we summarize both well-established concepts and recent advances that have contributed to a better understanding of the molecular mechanisms involved in this bidirectional signaling. The skeletal muscle and myocardial cells present highly specialized structures; for example, the close interaction between the sarcoplasmic reticulum (SR) and mitochondria—responsible for excitation-metabolism coupling—and the junction that connects the SR with T-tubules, critical for excitation-contraction (EC) coupling. The mechanisms that underlie EC coupling in these two cell types, however, are fundamentally distinct. They involve the differential expression of Ca Thus, 2+ channels (VGCCs), termed dihydropyridine receptors (DHPRs), which connect the firing of action potentials (APs) with the activation of RyRs, and thereby promote SR Ca2+ release and muscle contraction. More precisely, the SR Ca2+ release is finely tuned by Ca2+ release units: that is, interacting clusters of DHPRs and RyRs  tubules and contains ryanodine receptors (RyRs); network, which expands throughout the sarcomere and surrounds the myofibrils; corbular, an extension of the network SR but that is confined to regions away from the sarcolemma and also contains RyRs. The T-tubules contain a subgroup of voltage-gated Cacouplons ). These d triads ,4. Here, we discuss the critical role of DHPRs in mediating the forward and backward signaling between the sarcolemma and the SR, along with the contribution of other molecules involved. In the last section, the SR-mitochondria interaction is also briefly reviewed. The number of diseases related to alterations in the couplon is vast, and thus we only revise few and emblematic disorders. Due to space limitations, many original studies were not cited. Thus, we encourage readers to consult other recent and influential reviews. An abbreviations list was included to improve readability (see Abbreviations). 2+ channels (VGCCs) are oligomer complexes that consist of the following subunits: α1, β, α2, δ, and γ i). It begins to increase at nearly 1 µM, reaches the maximum at 100 µM, and then gradually decreases with concentrations in the millimolar range. This biphasic behavior is likely explained by the existence of two possible Ca2+-binding sites, of high and low affinity: the former could generate activation ) and the latter inhibition. Indeed, the inhibition of RyRs by high [Ca2+]i contributes to ending the positive feedback inherent to CICR , bu, buV1.1 nd Stac3 ,88. V1.1 i to baseline levels . 2+ is remarkably well-regulated. For example, it has been reported that high levels of SR Ca2+ content correlate with an elevated frequency of spontaneous Ca2+ release  but also by the entry of calcium through a single channel  or C-terminal (CaM34) lobes, Peterson and coworkers concluded that CDI is only supported by the CaM12 mutant. Thus, the C-terminal lobe of CaM was identified as the precise domain that binds Ca2+ for CDI i, can also be considered as a retrograde signal of the couplon  which participate in ion translocation i induces higher ROS production, which in turn exacerbates Ca2+ overload [4—an essential cofactor of the nitric oxide synthase (NOS)—to BH2, and the resulting increase in BH2 to BH4 ratio promotes “NOS uncoupling,” which implies that the enzyme synthesizes superoxide anions, instead of nitric oxide (NO). Thus, the NOS uncoupling entails not only greater ROS production, but also the depletion of NO. The latter effect represents another level of complexity, because NO signaling also regulates the function of EC coupling-related proteins, via either direct S-nitrosylation or through the modulation of cGMP- and cAMP-dependent protein kinases (PKG and PKA) [Under physiological conditions, the mitochondrion to SR coupling involves a delicate balance of several processes. This balance, however, can be disrupted when the rate of ROS production overtakes antioxidant defenses, activating positive feedback mechanisms that ultimately lead to pathological conditions . For exaoverload . Besidesoverload . Moreoveand PKA) ,156. 2+ are finely tuned by the activity of transporters and channels, whose expression is not only tissue-type specific but also properly targeted to distinct organelles of the junction. Many fundamental questions remain unsolved. For instance, what keeps the checkerboard pattern array of V and C RyRs in skeletal muscle? Is the CaV1.1-RyR1 interaction necessary or perhaps even the sole requirement for explaining this particular array? Concerning the luminal concentration of SR Ca2+: which are the primary mechanisms for explaining the role of luminal Ca2+ on SR Ca2+ release? Moreover, which of these mechanisms are more influential under physiological and pathological conditions? Equally appealing could be the identification of novel factors that may participate in the excitation–metabolism coupling, as well as their precise impact on the function of EC-coupling-related proteins. Undoubtedly, studies solving these, and other similar questions will significantly advance the field of sarcolemma–SR symbiosis and, thereby, will also pave the route towards discovering novel therapeutic strategies for couplonopathies. The T-tubule-SR junction is a remarkable signaling hub whose specialized functions depend on a complex array of organelles, enzymes, and ion transport systems. The DHPRs play a preponderant role, orchestrating downstream and upstream signaling pathways for correct muscle function. Thus, changes in the concentration of Ca