S)-3-methyl-1-sulfonyl)piperazine. This work is a demonstration of an aryl-Narasaka acylation, where the C(Ar)-Si bond cleavage is promoted by the torsional strain of α, α’-disubstituted silafluorene.Atropisomers are important organic frameworks in bioactive natural products, drugs as well as chiral catalysts. Meanwhile, silanols display unique properties compared to their alcohol analogs, however, the catalytic synthesis of atropisomers bearing silanol groups is challenging. Here, we show a rhodium-catalyzed torsional strain-promoted asymmetric ring-opening reaction for the synthesis of α-silyl biaryl atropisomers. The reaction features a dynamic kinetic resolution of C(Ar)-Si bond cleavage, whose stereochemistry was controlled by a phosphoramidite ligand derived from ( Methods to access atropisomers bearing silanol groups are of value in organic synthesis. Here, the authors report a rhodium-catalyzed torsional strain-promoted asymmetric ring-opening reaction for the synthesis of α-silyl biaryl atropisomers. Among them axially chiral biaryls have caught extensive attentions due to their wide existence in bioactive natural products, drugs5; furthermore it is one of prior skeletons for chiral ligands or catalysts in asymmetric catalysis.Atropisomers are an important class of compounds, which showcased chirality due to the restricted rotation around the single C–C, C–N, or C–O bonds6. The aryl–aryl cross-coupling or oxidative coupling is the most straightforward method, which delivered extremely useful axially chiral ligands and catalysts, such as BINOLs18. In addition, other useful methods, including (dynamic) kinetic resolution26, point to axial chirality transfer29, de novo aryl ring synthesis34 have been developed and significantly broadened the diversity of biaryl atropisomers. Recently, organo-catalyzed transformation showed its strong power in organic synthesis and it have been successfully applied to axially chiral molecules construction40.Despite the importance of biaryl atropisomers, the catalytically asymmetric synthesis was challenging due to the steric hindrance around the axis, which usually required three or four ortho substituents for achieving stable axial chirality42. Recently, Zhang and co-workers realized Ir-catalyzed asymmetric hydrogenation of Bringmann’s lactones43. Under the catalysis of nickel, copper, and palladium, the groups of Hayashi, Gu and others realized the ring-opening of dibenzothiophenes44, diaryliodoniums48, and 9H-fluoren-9-ols , provided a potential solution for C–Si bond cleavage of silafluorene -3-methyl-1-sulfonyl)piperazine. Computational studies show that the storioanl stain of 7,7-diphenyl-7H-dinaphthosilole is about 12.58 kcal mol−1, which efficiently accelerate the C–Si bond cleavage.Here, we report an aryl-Narasaka acylation reaction catalyzed by the chiral rhodium complex which is in situ formed of 2/L1) to 4a and benzoic anhydride gave conspicuously decreased ee value . Thus, further optimization based on the modification of phosphoramidites were carried out by using Si,Si-diphenylsilafluorene 4b and benzoic anhydride phenylsulfonamide derivative L7 afforded the desired product 6b in 92% ee . 2-Methoxyacetic anhydride steadily underwent this acylation reaction to give 6g in excellent yield and enantioselectivity. The reaction of cyclopropanecarboxylic anhydride afforded the product 6h in 81% yield with 85% ee. The acrylic anhydride derivatives also reacted with 4b uneventfully to furnish the products in excellent ee values (6i–6k). The aromatic anhydrides were also compatible substrates. The p-methylbenzoic anhydride gave a slightly decreased yield, while the electron-withdrawing substituents were advantageous for both yields and stereoselectivity (6l–6o). Subsequently, other substituted aromatic acid anhydrides, including 3,4,5-trimethoxybenzoic anhydride, were submitted to the standard conditions to produce the corresponding silanols 6p–6s with excellent ees. Lastly, the 2-naphthoic or heteroaromatic anhydride were checked, and both of the reactions proceeded smoothly with the ee values were marginally changed (6t and 6u).Under the optimal conditions, a number of acid anhydrides were tested with ate Fig. . The aliL1 was found more efficient for substrate 4a, which reacted with propionic anhydride and butyric anhydride to give 6v and 6w in 85% and 90% ee, respectively. The substituted benzene rings attached to the silicon atom have marginal effect on the selectivity, all the silanols were formed in excellent stereoselectivity (6x–6bb). The silafluorenes with biphenyl skeleton gave relatively lower stereoselectivity (6cc–6ee). The diastereochemistry of compound 6cc was determined to be R by single crystal X-ray diffraction analysis (CCDC 1968743). The starting material α,α′,β,β′-tetramethyl biphenylsilafluorene showed slight poor stability, as a result, the corresponding product 6ff was isolated in only 45% yield with 87% ee.Silafluorenes bearing different substituents were further investigated Fig. . Ligand 4j, the reaction gave 1:1 diastereomers, although both 6gg (CCDC 2013379) and 6gg′ were obtained in high enantioselectivity   was performed with acetic anhydride as limiting reagent. It afforded a 1.5:1 mixture of 6a and 6c, indicating 4a was slightly more active than 4b under our conditions  in excellent diastereoselectivity  and 4c (CCDC 1968985) were obtained and analyzed by X-ray diffraction. It clearly shows the biphenyl structure in 4c is almost planar, while the increased size of compound 4a makes the naphthyl rings no longer coplanar. It is a twisted molecule with a distortional angle being 31.8° of the binaphthyl skeleton ] of 4a, which is around 20.4 kcal/mol. Thus, the calculated half-life of 4a is around 94 s at room temperature Cl(L7)]2 by releasing two molecules of CO. Recrystallization of this complex in ethyl acetate/hexanes gave a reddish orange crystal that was suitable for X-ray crystallography analysis (CCDC 1969002) 02) Fig. . In this66, a brief catalytic cycle was tentitatively proposed  complex. The oxidative addition of Rh(I) with silafluorene 4b cleavaged C(Ar)–Si bond to form 10, which gave optically active biaryl intermediate 11 via reductive elimination forming a Si–O bond. Subsequently, the second oxidation between 11 with acid anhydride 5 delivered Rh(III) complex 12. Reductive elimination of 12 furnished acylation product 13, which would give the final product 6 after hydrolysis of tri(aryl)silyl benzoate moiety.Based on the above results and previous studies on Narasaka acylationsed Fig. . The cooIn conclusion, we reported a Rh-catalyzed asymmetric ring-opening acylation reaction for the synthesis of α-silyl biaryl atropisomers, a class of chiral bulky silanols. The torsional strain of five-membered silafluorenes enabled the success of selective cleavage of C(Ar)–Si bond, thus accomplishing an aryl-Narasaka acylation variant. Additional notable merit of this work is the developed sulfonylpiperazine derived phosphoramidite ligands, which showed high stereo-induction in C(Ar)–Si cleavage/ring-opening reaction.2Cl]2 , L1  and dioxane (2 mL) at room temperature and was stirred for 30 min. The solution was transferred via cannula carefully to another Schlenk tube charged with 4a , acetic anhydride , Na2CO3 , and dioxane (2 mL). The tube was capped with a screw cap and stirred at 70 °C for 24 h. After being cooled to room temperature, the mixture was filtered through Celite and the filtrate was concentrated in vacuum and purified by flash column chromatography (PE/EtOAc 90:10) on silica gel to afford 6a . 2Cl2). HPLC conditions: Chiralcel AD-H, isopropanol/hexane = 10:90, flow: 1.0 mL/min, λ = 254 nm. 1H NMR  δ 8.04 , 7.99 , 7.94 , 7.92 , 7.87 , 7.80 , 7.55–7.50 , 7.49–7.44 , 7.31–7.26 , 7.26–7.22 , 7.17 , 7.13 , 2.70 , 1.94 , 0.13 , −0.47 . 13C NMR  δ 205.2, 141.5, 138.3, 137.9, 137.4, 134.2, 133.7, 133.5, 132.7, 130.4, 128.7, 128.2, 128.0, 127.6, 127.5, 127.0, 126.6, 126.5, 126.2, 123.9, 30.5, 0.6. HRMS (ESI) calcd for C24H22O2SiNa [M + Na]+ 393.1287, found 393.1288.Under nitrogen atmosphere, to a Schleck tube was added [Rh(CO)Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


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.


Diabetes is a global health challenge. Currently, an effective treatment for diabetes is to reduce the postprandial hyperglycaemia by inhibiting the carbohydrate hydrolysing enzymes in the digestive system. In this study, we investigated the in vitro α-glucosidase and α-amylase inhibitory effects of free and bound phenolic extracts, from the bran and kernel fractions of five sorghum grain genotypes. The results showed that the inhibitory effect of sorghum phenolic extracts depended on the phenolic concentration and composition. Sorghum with higher phenolic contents generally had higher inhibitory activity. Among the tested extracts, the brown sorghum (IS131C)-bran-free extract  = 18 ± 11 mg sorghum/mL) showed the strongest inhibition against α-glucosidase which was comparable to that of acarbose (IC50 = 1.39 ± 0.23 mg acarbose/mL). The red sorghum (Mr-Buster)-kernel-bound extract  was the most potent in inhibiting α-amylase but was much weaker compared to acarbose (IC50 = 0.50 ± 0.03 mg acarbose/mL). The key features of the disease are impaired body glucose metabolism and chronic hyperglycaemia, which can lead to damage to a range of body parts, including eyes, kidneys, nerves and blood vessels . One of e system . Synthete system . Howevere system . Researce system . In parte system ,7,8.Sorghum bicolor) grain, one of the top five most produced cereal crops worldwide [Sorghum  to attenuate hyperglycaemia. Kim, Hyun and Kim [α-glucosidase (from Bacillus stearothermophilus) and α-amylase , and found that some sorghum cultivars exhibited strong α-glucosidase inhibitory activity, while foxtail and proso millets had no detectable effect on these enzymes. However, most of the research has focused only on sorghum bran or sorghum tannin, or α-amylase inhibition. Knowledge on the effect of different sorghum genotypes and their polyphenols in inhibiting the digestive enzymes is still limited.Studies have shown that the administration of sorghum phenolic extract to streptozotocin-induced diabetic rats had a significant hypoglycaemic effect ,14. The , et al.  found th, et al.  showed t and Kim  comparedα-glucosidase and α-amylase digestive enzymes. We also determined the total phenolic , flavonoid  and tannin (by way of the vanillin-HCl assay method) contents to assist in understanding the phenolic compounds/fractions responsible for the enzyme inhibition.In our previous work, we investigated the phenolic profile of free and bound phenolic extracts of bran and kernel fractions from five Australian-grown sorghum genotypes , with a total of 20 extracts . In thisp-nitrophenyl-α-D-glucopyranoside (PNPG), bovine serum albumin protein standard, acarbose, p-nitrophenyl α-D-maltohexaoside (PNPG6), porcine pancreas α-amylase were obtained from Sigma-Aldrich . All other chemicals were obtained from Chem-Supply . All chemicals used in this study were of analytical or HPLC grade.Folin–Ciocalteu phenol reagent, gallic acid, (+)-catechin hydrate, vanillin, rat intestinal acetone powders, White colour Liberty (W), red colour Mr-Buster (RM), red colour Nuseed Cracka (RC) sorghum variety grains were kindly supplied by Nuseed Company  in 2019. Brown colour IS131C (BR) and black colour Shawaya Short Black 1 (BL) sorghum grains were grown and harvested from the experiment field of the Bentley campus of Curtin University, from January to April 2019 . All sorghum grains were abrasively decorticated by a TM05C SATAKE Testing Mill  to separate the bran and kernels. The grains were decorticated for 60 s to collect the bran, and the remaining grains were further decorticated for 45 s to remove uncleared bran to obtain the kernels. Both bran and kernels were ground, sieved 100% through a 500 µm sieve and then stored at −20 °C in the dark until analysis.v/v) under N2 gas, and the mixture was incubated at 25 °C with shaking in the dark for 2 h. The mixture was then centrifuged at 3500× g and 4 °C for 10 min to collect the supernatant, and the residue was re-extracted with 20 mL 80% methanol (v/v) two more times. Supernatants were pooled and evaporated at 40 °C under vacuum to dryness, and the resulting solid was re-dissolved in 10 mL of methanol and stored under N2 at −20 °C in the dark before analysis. The remaining residue was then used for extraction of bound phenolic compounds. The residue was mixed with 15 mL of 2 M HCl under N2 and incubated at 100 °C in a water bath for 1 h for hydrolysation. After cooling, 20 mL ethyl acetate was added and mixed thoroughly, and the ethyl acetate fraction was collected after partitioning. The hydrolysate was re-extracted with 25 mL ethyl acetate five more times, and all ethyl acetate fractions were combined and evaporated at 40 °C under vacuum to dryness. The resulting solid was re-dissolved in 10 mL of methanol and stored under N2 at −20 °C in the dark before analysis. The extraction was done in triplicate for all samples.Free and bound phenolic compounds were extracted according to the method described previously . For thev/v) for 5 min. Then, 600 µL of 7.5% Na2CO3 (w/v) solution was added, and the mixture was incubated at 25 °C in the dark for 2 h. The absorbance of the mixture was measured at 765 nm by a Multiskan GO UV-Vis spectrophotometer . Gallic acid was used as the standard (0 to 0.400 mg/mL), and the results were expressed as mg gallic acid equivalents (GAE)/g sorghum sample dry basis (db).The TPC was determined by the Folin–Ciocalteau method, as described previously . In brie2 (w/v) solution and were left to stand for 5 min. Then 45 µL of 10% AlCl3 (w/v) solution was added to the mixture and was left to stand for 6 min. Next, 600 µL of 0.5 M NaOH solution was added to develop the colour, and the mixture was then incubated at 25 °C in the dark with shaking for 10 min until it turned pink. The absorbance was measured at 510 nm by the UV-Vis spectrophotometer. (+)-Catechin hydrate was used as the standard (0 to 0.800 mg/mL), and the results were expressed as mg catechin equivalents (CAE)/g db.The TFC was measured according to the method of Zhishen, et al. . The extw/v) solution with acidified methanol solution . The phenolic extracts (800 µL) were mixed with 700 µL of vanillin-HCl reagent and then incubated at 25 °C for 20 min, and the absorbance was measured at 500 nm by the UV-Vis spectrophotometer. (+)-Catechin hydrate was used as the standard (0–0.800 mg/mL), and the results were expressed as mg catechin equivalents (CAE)/g db.The TTC was determined by the vanillin-HCl assay method ,22. Vaniα-glucosidase enzyme solution. In brief, rat intestinal acetone powder (1.0 g) was suspended in 25 mL potassium phosphate buffer-I . The mixture was maintained in cold conditions in an ice water bath and sonicated at 50 Hz for 5 min by a Q55 sonicator . Then, the sonicated mixture was centrifuged at 18,000× g and 4 °C for 15 min, and the supernatant containing the α-glucosidase was collected and stored at −20 °C until analysis. The protein content of the enzyme prepared was determined to be 4.99 ± 0.27 mg/mL using the Bradford assay method with bovine serum albumin as the protein standard.Rat intestinal acetone powder was used to prepare a mammalian α-glucosidase inhibitory activity of sorghum phenolic extracts was evaluated by the method of Ng, et al. [α-glucosidase solution were mixed in individual wells in a 96-well plate. Then, 20 µL of 25 mM p-nitrophenyl-α-D-glucopyranoside (PNPG) solution was added to start the enzymatic reaction. All reagents were kept cold in an ice water bath during the preparation. A program was set up to incubate the 96-well plate (covered with lid) at 37 °C for 60 min using the Multiskan GO UV-Vis spectrophotometer. After incubation, the lid was removed, and the absorbance was measured at 410 nm. The inhibition of α-glucosidase was determined using the formula below, where Asample = sample + enzyme + PNPG, Asample background = sample + enzyme + PNPG solvent, Acontrol = sample solvent + enzyme + PNPG and Acontrol background = sample solvent + enzyme + PNPG solvent. All the measurements including control and background samples were conducted in triplicate.The , et al. , and modα-amylase inhibitory activity of sorghum sample extracts was evaluated using the chromogenic substrate p-nitrophenyl α-D-maltohexaoside (PNPG6) and adapted to a 96-well microplate. Porcine pancreas α-amylase was diluted to 250 units/mL in sodium phosphate buffer-III  to prepare the α-amylase working solution. PNPG6 solution (5.0 mg/mL) was prepared with sodium phosphate buffer-IV . The reaction mixture containing 20 µL of sorghum phenolic extract, 100 µL sodium phosphate buffer-IV and 50 µL of the α-amylase working solution was added to wells in a 96-well plate. Then, 50 µL of the PNPG6 solution was added to initiate the reaction. All reagents were kept cold in an ice water bath during the preparation. A program was set to incubate the 96-well plate covered with lid at 37 °C for 40 min by the UV-Vis spectrophotometer. After incubation, the 96-well plate was cooled in an ice water bath, and 100 µL dimethyl sulfoxide (DMSO) was added to each well to dissolve precipitate, and then the absorbance was measured at 410 nm. The inhibition of α-amylase was determined using the formula below, where Asample = sample + enzyme + PNPG6, Asample background = sample + enzyme + PNPG6 solvent, Acontrol = sample solvent + enzyme + PNPG6 and Acontrol background = sample solvent + enzyme + PNPG6 solvent. All the measurements including control and background samples were conducted in triplicate.The Since the crude sorghum phenolic extracts  have different phenolic concentrations and the inhibition was expected to be derived from the phenolic contents, the inhibitory activity of each sorghum phenolic extract sample was divided by its total phenolic content (TPC) to evaluate the phenolic inhibition efficiency ,25. Resuα-glucosidase and α-amylase activities was determined from the inhibitions obtained from a range of concentrations of sorghum phenolic extracts. The IC50 value was derived from the least-squares regression line of the plot of inhibition % versus log10 sorghum phenolic concentration [The half-maximal inhibitory concentration (IC50) against ntration . ResultsAll measurements were carried out in triplicate and results were expressed as means ± SD. The significant difference between means was determined by one-way ANOVA with Tukey grouping at 95% confidence level using Minitab . The partial least squares (PLS) correlation test  was condUnderstanding the sorghum phenolic profile is the essential step in identifying the key phenolic compounds that contribute to the enzyme inhibition and other bioactive functions. The total phenolic content (TPC), total flavonoid content (TFC) and total tannin content (TTC) of the free and bound phenolic extracts from the bran and kernel fractions of five sorghum genotypes, white Liberty (W), red Mr-Buster (RM), red Nuseed Cracka (RC), brown IS131C (BR) and black Shawaya Short Black 1 (BL), are shown in p < 0.05). Similarly, the TFC of BL (14.05 ± 0.67 mg CE/g db) and BR (15.92 ± 0.67 mg CE/g db) sorghums were also much higher than that of RC (3.95 ± 0.75 mg CE/g db) and RM (4.78 ± 0.92 mg CE/g db) sorghums, followed by W (1.97 ± 0.07 mg CE/g db) sorghum (p < 0.05). Regarding the TTC, BL (138.64 ± 7.06 mg CE/g db) and BR (129.93 ± 9.24 mg CE/g db) sorghums had the highest, followed by RC (69.27 ± 5.92 mg CE/g db) sorghum, then RM (47.90 ± 0.29 mg CE/g db) sorghum, and finally W (5.74 ± 0.50 mg CE/g db) sorghum had a very low level of TTC (p < 0.05). In addition, the phenolic contents were concentrated in the bran. As shown in p < 0.05) than the bound phenolic contents, especially in the BL and BR sorghums and dominated the phenolic contents in these sorghums; except W sorghum. In the kernels, no significant difference was observed between the free and bound phenolic contents (p > 0.05). The results indicated that the phenolic compounds in sorghum bran are easily extractable, and BR and BL bran have huge industrial potential due to their high phenolic contents.The phenolic contents were significantly different between sorghum varieties. The TPC of black BL (31.33 ± 0.48 mg GAE/g db) and brown BR (33.74 ± 0.90 mg GAE/g db) sorghums were much higher than that of red RC (10.98 ± 0.86 mg GAE/g db) and RM (11.62 ± 0.34 mg GAE/g db) sorghums, followed by white W (7.16 ± 0.11 mg GAE/g db) sorghum  generally have higher levels of phenolics ,29. Moreα-glucosidase in varying degrees. Black BL and brown BR sorghum extracts generally had stronger α-glucosidase inhibition than red RC and RM sorghum extracts, followed by white W sorghum extracts. In addition, the bran extracts generally had stronger inhibition than the kernel extracts. In the bran, the free form phenolic extracts showed much stronger α-glucosidase inhibition than the bound phenolic extracts (p < 0.05), especially in the BL  and BR  sorghums, which had the highest inhibitory activity among all extract samples. The result was in line with the phenolic contents    as discuα-amylase inhibition was quite different. As shown in α-amylase inhibition than red RC sorghum extracts, followed by white W sorghum extracts, with the exception of red RM sorghum which also showed strong inhibitory activity. In addition, the bran extracts generally had stronger α-amylase inhibition than the kernel extracts, except for red RM sorghum. RM sorghum, the RM-kernel-bound extract, had the highest α-amylase inhibition (71% inhibition) among all sorghum extracts. However, no obvious trend was observed between the free and bound extracts. The result is roughly consistent with phenolic contents   was very low and was not significantly different (p > 0.05) from that in other kernel extracts (The result of and TTC) , as sorgextracts , which cα-glucosidase and α-amylase, with the RB-kernel-bound extract having the highest inhibition efficiency (p < 0.05).The inhibitory activities of sorghum phenolic extracts were divided by their TPC to evaluate the phenolic inhibition efficiency ,25, i.e.α-glucosidase and α-amylase [α-amylase inhibition is closely related to the tannin content, and sorghum with higher degrees of polymerisation tannins have shown greater inhibitions [α-glucosidase and α-amylase inhibition potency. The correlation between the inhibitory activity and specific sorghum phenolic compounds will be discussed in Research has shown that sorghum rich in tannins has strong inhibitory activity against -amylase . The α-aibitions ,17. Our ibitions ,31. Therα-glucosidase and α-amylase were determined and compared with that of acarbose, shown in α-glucosidase, and comparable to acarbose (IC50 = 1.39 ± 0.23 mg/mL). While the RM-kernel-bound extract (IC50 = 160 ± 12 mg sorghum/mL) was the most effective in inhibiting α-amylase but was much weaker, about 320 times weaker, than acarbose (IC50 = 0.50 ± 0.03 mg/mL).The half-maximal inhibitory concentration (IC50) of sorghum extracts  against α-amylase inhibition were generally higher than that of α-glucosidase inhibition, suggesting the phenolics in our tested sorghums were more effective against α-glucosidase than α-amylase.Another IC50 (mg GAE/mL) value, in terms of the amount of  sorghum phenolic contents required per mL reaction, was also determined to compare the phenolic inhibition efficiency . The resα-glucosidase and α-amylase by sorghum phenolic extract, in comparison with acarbose, have shown inconsistent results. Kim, Hyun and Kim [α-glucosidase (from Bacillus stearothermophilus) inhibition by phenolic extracts from the whole grain of some Korean sorghum cultivars (IC50 = 1.1–102.7 µg sorghum/mL) and found that some sorghum cultivars exhibited slightly stronger inhibition than acarbose (IC50 = 2.1 µg/mL). Links, Taylor, Kruger and Taylor [α-glucosidase (from yeast) and α-amylase (from porcine) inhibition by phenolic extracts from tannin-rich sorghum bran (PAN 3860 cultivar) and showed that the phenolic extract (IC50 = 0.4 µg sorghum/mL) had far stronger α-glucosidase inhibition (about 2000 times stronger) than acarbose (IC50 = 8464.0 µg/mL), but the extract (IC50 = 554.5 µg sorghum/mL) had weaker α-amylase inhibition (about 180 times weaker) than acarbose (IC50 = 3.1 µg/mL). The inconsistency between these studies (and our study), in comparison with acarbose, may be due to the difference in sorghum phenolic concentration and composition. It should be noted that the data between these studies (and our study) were not directly comparable, as the enzymes, substrates and assay methods used were different. Besides, the data obtained using yeast or bacteria α-glucosidase is less relevant than using mammalian α-glucosidases used in the present study, as mammalian α-glucosidases are different and have a narrower inhibitory spectrum with lower inhibition by phenolics [Previous studies evaluating the inhibition of  and Kim  investigd Taylor  investighenolics ,33.The sorghum phenolic extracts in this work showed significant inhibitory activity against these digestive enzymes, although their inhibition strength was relatively weak compared to acarbose. Sorghum phenolic compounds are well-known for their high antioxidant and anti-inflammatory activities , which cα-glucosidase inhibition was significantly positively correlated with TPC (0.961), TFC (0.954), TTC (0.955), 3-deoxyanthocyanidin (0.895), flavan-3-ol (0.581), flavanone (0.892), flavonol (0.722) and proanthocyanidin dimer (0.842) ; and positively correlated with bran (0.617) (p < 0.01); but had no correlation with sorghum genotypes (p > 0.05). The results suggest that the α-glucosidase inhibition is closely related not only to the total phenolic content but also to certain (active) phenolic subclasses/compounds; the active compounds are widely present in all sorghum genotypes, and sorghum bran has a high proportion of these compounds. Moreover, a total of 27 individual phenolic compounds  with α-glucosidase inhibition, and most of them are common plant phenolic compounds and have been widely studied and documented expect for 3-deoxyanthocyanidins [α-glucosidase inhibition (p < 0.01) (α-amylase inhibition only had a weak correlation with W (−0.453) and RM (0.447) sorghum (p < 0.05 for both), and had no significant correlation with the total/subclass phenolic contents or other variables (p > 0.05), indicating that the phenolic compounds in the tested sorghum samples generally had weak α-amylase inhibition, with RM sorghum only had a relatively stronger inhibition.The PLS correlation coefficients between enzyme inhibition, phenolic content and other variables are presented in yanidins ,36,37,38 < 0.01) . It is wα-glucosidase inhibition), the main phenolic compounds were flavonoids, including taxifolin 3-glucopyranoside (1993 µg/g), taxifolin (1479 µg/g), procyanidin B1 (1283 µg/g), catechin (1210 µg/g), apigeninidin (372 µg/g), naringenin 7-O-glucoside (269 µg/g) and eriodictyol 7-O-glucoside (78 µg/g). These compounds were all significantly positively correlated with α-glucosidase inhibition  and could be the main contributors to the α-glucosidase inhibition  and flavonoids  were the major phenolic compounds [p-coumaric acid, ferulic acid have also been shown to modulate glucose and insulin receptor function and lipid metabolism to improve glucose and lipid profiles of certain diseases including diabetes [In the kernel samples (high inhibition efficiency), phenolic acids , however, only five phenolic compounds -β-D-glucose, trans-ferulic acid, 7-methoxycoumarin-4-acetic acid and α,β-dihydroresveratrol) were found, and these phenolic compounds were also found in other sorghum samples with higher concentrations [α-amylase inhibition. We propose that kafirin, an alcohol-soluble protein and the major sorghum storage protein, may also be extracted and involved in the enzyme inhibition. According to Links, Taylor, Kruger and Taylor [α-glucosidase and α-amylase, it can interact with sorghum phenolic compounds such as tannins, and undergoes conformation change, which may bind and inhibit the enzymes. Further research is required to uncover the cause and mechanism.Regarding the RM-kernel-bound sample . Sorghum samples with a higher phenolic content generally had higher inhibitory activity: black and brown > red > white coloured sorghum. Sorghum bran, where the phenolic content is concentrated , was generally more effective in inhibiting α-glucosidase and α-amylase than the kernel; except red RM sorghum whose kernel phenolic content was more effective in inhibiting α-amylase.In conclusion, our investigation demonstrated that sorghum phenolic extracts exhibited significant in vitro inhibitory activity against rat intestinal The results from this study also suggest that the inhibitory activity may also be related to the phenolic composition, and some specific phenolic compounds may have high inhibition potency. In addition, other non-phenolic compounds such as kafirin may also be involved in the enzyme inhibition, which requires further investigation.α-glucosidase, while the RM-kernel-bound extract was the most effective inhibitor against α-amylase. Despite all sorghum extracts showing weaker inhibition relative to acarbose, sorghum has distinct advantages as it has high levels of resistant starch and other health-promoting benefits, as well as its role as a staple food especially in the regions of Africa and South Asia. Further research is under the way to isolate, purify and analyse the individual sorghum phenolic compounds responsible for the digestive enzyme inhibition observed in this study.Among the sorghum phenolic extracts, the BR-bran-free extract was the most effective inhibitor against


These data show that both plasma and DBS are suitable to determine blood C26:0-LPC levels and that there is a strong correlation between C26:0-LPC levels in both matrices. Following this, we investigated how VLCFA and C26:0-LPC analysis compare in diagnostic performance for 67 controls, 26 ALD males, 19 ALD females, and 35 ZSD patients. For C26:0-LPC, all ALD and ZSD samples had C26:0-LPC levels above the upper limit of the reference range. For C26:0, one out of 67 controls had C26:0 levels above the upper reference range. For 1 out of 26 (1/26) ALD males, 1/19 ALD females and 3/35 ZSD patients, the C26:0 concentration was within the reference range. The C26:0/C22:0 ratio was within the reference range for 0/26 ALD males, 1/19 ALD females and 2/35 ZSD patients. Overall, these data demonstrate that C26:0-LPC analysis has a superior diagnostic performance compared to VLCFA analysis (C26:0 and C26:0/C22:0 ratio) in all patient groups. Based on our results we recommend implementation of C26:0-LPC analysis in DBS and/or plasma in the diagnostic work-up for peroxisomal disorders.Peroxisomes are subcellular organelles that are involved in various important physiological processes such as the oxidation of fatty acids and the biosynthesis of bile acids and plasmalogens. The gold standard in the diagnostic work-up for patients with peroxisomal disorders is the analysis of very long-chain fatty acid (VLCFA) levels in plasma. Alternatively, C26:0-lysophosphatidylcholine (C26:0-LPC) can be measured in dried blood spots (DBS) using liquid chromatography tandem mass spectrometry (LC-MS/MS); a fast and easy method but not yet widely used. Currently, little is known about the correlation of C26:0-LPC in DBS and C26:0-LPC in plasma, and how C26:0-LPC analysis compares to VLCFA analysis in diagnostic performance. We investigated the correlation between C26:0-LPC levels measured in DBS and plasma prepared from the same blood sample. For this analysis we included 43 controls and 38 adrenoleukodystrophy (ALD)  and 33 Zellweger spectrum disorder (ZSD) patients. In combined control and patient samples there was a strong positive correlation between DBS C26:0-LPC and plasma C26:0-LPC, with a Spearman’s rank correlation coefficient of  Peroxisomes are organelles that are involved in various important physiological processes such as the oxidation of fatty acids and the biosynthesis of bile acids and plasmalogens . PeroxisPEX genes. ZSD are characterized by a large variety in clinical presentation , which includes the method developed by In the current diagnostic landscape C26:0-LPC in DBS is used for ALD newborn screening. However, the gold standard in the diagnostic work-up for patients with peroxisomal disorders is the analysis of plasma VLCFA levels. The objectives of our study were: (1) To investigate the correlation of C26:0-LPC in DBS and C26:0-LPC in plasma, and (2) To compare the diagnostic performance of C26:0-LPC analysis and VLCFA analysis. We discuss the important factors in the consideration of these methods and provide an overview of possible discrepancies.ABCD1 mutations, 32 ZSD patients had confirmed PEX1 mutations, 2 ZSD patients had confirmed PEX6 mutations and 1 ZSD patient had a confirmed PEX26 mutation. Control samples consisted of samples that were screened for a peroxisomal disorder and resulted in a negative outcome. All samples were collected according to the institutional guidelines for blood sampling.All routine measurements of C26:0-LPC and VLCFA performed at the Laboratory Genetic Metabolic Diseases in the Amsterdam UMC between June 2018 and June 2019 were collected and used for this study. This set was expanded with measurements in additional ALD and ZSD samples. This resulted in samples from 67 controls, 26 ALD males, 19 ALD females and 35 ZSD patients. All ALD patients had confirmed 4-C26:0-lysoPC in 0.5 mL of methanol by ultrasonication for 5 min in a sonicator bath (Branson 3510) at room temperature. For plasma, 10 μL was extracted with 10 μL of an internal standard solution containing 1 μmol/L D4-C26:0-lysoPC in 0.5 mL of acetonitrile by ultrasonication for 5 min in a sonicator bath (Branson 3510) at room temperature. After centrifugation  the resulting methanol (DBS) or acetonitrile (plasma) layer was transferred to a new glass tube and evaporated under a constant stream of nitrogen at 40°C. The samples were then reconstituted in 50 μL methanol, transferred to a sample vial, and capped.Analysis of C26:0-LPC was performed as described earlier by T = 0 min: 36% A, 64% B, flow 0.4 mL/min toward T = 6 min: 0% A, 100% B, flow 0.4 mL/min; T = 6–11 min: 0% A, 100% B, flow 0.4 mL/min, and T = 11–11.1 back to 36% A, 64% B, flow 0.4 mL/min. Detection was done using a Quattro Premier XE  using electrospray ionization in positive mode. The source temperature was 130°C, and capillary voltage was 3.5 kV. Multiple reaction monitoring (MRM) was done on masses 636.50 > 104.10 and 640.50 > 104.10 with a dwell time of 0.03 s. Argon was used as a collision gas.Samples were injected using an ACQUITY UPLC system  on a 50 × 2.1 mm, 2.6 μm particle diameter Kinetex C8 column . The column was held at a constant temperature of 50°C. The composition of mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in methanol. The gradient used was as follows: 4-C22:0, 50 μmol/L D4-C24:0, 1 μmol/L D4-C26:0. Acidic hydrolysis was performed by adding 2 mL of a 0.5 mol/L HCl in acetonitrile and incubating at 110°C for 45 min. After cooling to room temperature, fatty acids were extracted into 4 mL of hexane. The resulting hexane layer was transferred to a new glass tube and washed with 3.5 mL 1M KOH. Next, the hexane layer was removed and 600 μL 25% HCl was added. Fatty acids were extracted with 4 mL of hexane. The hexane layer was then transferred to a new glass tube and dried under nitrogen at 50°C. Fatty acids were derivatized with 50 μL pyridine and 50 μL MTBSTFA at 80°C for 30 min. The solvent was then evaporated under nitrogen at 50°C and the derivatized fatty acids were reconstituted in 200 μL hexane and transferred to autosampler vials.Very long-chain fatty acids analysis was performed essentially as described earlier by Vreken et al. In a glass tube, 100 μL plasma was added to 100 μL internal standard solution dissolved in toluene that consisted of 50 μmol/L D∗0.25mm∗0.20 μm column. Temperature gradient was as follows: 60°C was held for 1.5 min after which the temperature increased to 240°C at a rate of 10°C/min. The temperature was then increased to 270, at 4°C/min. Finally the temperature increased to 300°C at 20°C/min. An injection volume of 1 μL was used in splitless mode using a splitless time of 1.5 min. The carrier gas was Helium (50 kPa). Single ion monitoring mode was used for detection of VLCFAs.Analysis was performed on an Agilent 7890B GC using a 5977A MSD mass spectrometer for compound detection. Separation was achieved on a Cp-sil 19, 25mr (114) = 0.962, p < 0.001. For controls alone, it was r (43) = 0.907, p < 0.001. For patients alone, it was r (71) = 0.861, p < 0.001. These data show that both plasma and DBS are suitable to determine blood C26:0-LPC levels and that there is a strong correlation between C26:0-LPC levels in both matrices. The strong correlation between plasma and DBS allowed for combining plasma and DBS C26:0-LPC data for comparison to VLCFAs as a diagnostic marker for peroxisomal disorders.To investigate the correlation between C26:0-LPC in DBS and C26:0-LPC in plasma we measured C26:0-LPC in DBS and plasma prepared from the same blood sample. For this analysis we included 43 control, 21 ALD males, 17 ALD females and 33 ZSD samples . The medn = 67), ALD males (n = 26), ALD females (n = 19) and ZSD patients (n = 35)  and C26:0-LPC analysis in plasma and DBS, we measured these metabolites in samples from controls ((n = 35) . For C26r (147) = 0.892, p < 0.001; for C26:0-LPC and C26:0 levels it was r (147) = 0.892, p < 0.001; and for C26:0-LPC and the C26:0/C22:0 ratio it was r (147) = 0.816, p < 0.001.Since the C26:0-LPC and VLCFA analyses were performed on samples generated from the same blood sample, we investigated the correlation between the 3 diagnostic markers with a Spearman’s rank-order correlation . When, cr (114) = 0.962, p < 0.001). C26:0-LPC in DBS is an effective biomarker for ALD and ZSD patients  and ZSD patients. Almost all patient samples showed increased C26:0/C22:0 ratios. None of the ALD males, 1/19 ALD females and 2/35 ZSD patients had a C26:0/C22:0 ratio within the reference range. The finding that 6 samples from the control group fell outside the reference range of C26:0/C22:0 indicates that false positives should be considered. For these samples the false positive results were due to low C22:0 and normal C26:0 concentrations. For this reason, plasma VLCFA analysis always includes both the analysis of C26:0 and the C26:0/C22:0 ratio as a means of improving the accuracy of the readout. The combination of these 2 markers increases the sensitivity of the analysis. Furthermore, false-positive results in VLCFA levels have been reported in individuals without a peroxisomal defect. For example, diabetic ketoacidosis, a ketogenic diet or hemolysis of the blood sample can result in false-positive VLCFA levels . ImportaIn conclusion, this study demonstrates that C26:0-LPC analysis performed in DBS and in plasma are strongly correlated. Furthermore, our results demonstrate the analysis of C26:0-LPC has superior diagnostic performance for ALD and ZSD patients compared to C26:0 and C26:/C22:0. Based on our results we recommend implementation of C26:0-LPC analysis in DBS and/or plasma in the diagnostic work-up for peroxisomal disorders.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YJ, SF, WK, ME, SG, FV, and SK conceived the project. ID and HL performed the VLCFA and C26:0-LPC analyses. YJ and SK performed the data analyses. YJ, SF, RB, ME, SG, FV, and SK wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


Plasmodium to known anti-malarial drugs, local African populations often resort to plant-based treatments and/or a combination of this and standard anti-malarial regimens. Emphasis has been laid in this review to present the anti-malarial virtue of the most recently published phytochemicals or natural products, which have been tested by in vitro and in vivo assays.African Traditional Medicine (ATM) is used for the healthcare of about 80% of the rural populations of the continent of Africa. The practices of ATM make use of plant-products, which are known to contain plant-based secondary metabolites or natural products (NPs), likely to play key roles in drug discovery, particularly as lead compounds. For various reasons, including resistance of strains of The data was based on the current version of the African Compound Libraries, which are constantly being updated based on inputs from journal articles and student theses (M.Sc/Ph.D) from African University libraries. Emphasis was laid on data published after 2012. In order to carry out the original data collection, currently being included in the African Compounds Database, individual journal websites were queried using the country names in Africa as search terms. Over 40,000 articles “hits” were originally retrieved, then reduced to about 9000 articles. The retained articles/theses was further queried with the search terms “malaria”, “malarial”, “plasmodium”, “plasmodial” and a combination of them, resulting in over 500 articles. Those including compounds with anti-malarial activities for which the measured activities fell within the established cut off values numbered 55, which were all cited in the review as relevant references.Pure compounds derived from African medicinal plants with demonstrated anti-malarial/antiplasmodial properties with activities ranging from “very active” to “weakly active” have been discussed. The majority of the 187 natural products were terpenoids (30%), followed by flavonoids (22%), alkaloids (19%) and quinones (15%), with each of the other compound classes being less than 5% of the entire compound collection. It was also observed that most of the plant species from which the compounds were identified were of the families Rubiaceae, Meliaceae and Asphodelaceae. The review is intended to continue laying the groundwork for an African-based anti-malarial drug discovery project. Plasmodium protozoans , although most severe infections are caused by P. falciparum . Th. ThPolya 29.1 μM .Ancistrocladus (Acistrocladaceae), and the closely related genera Triphyophyllum, Dioncophyllum and Habropetalum (Dioncophyllaceae). Cyclombandakamines A1 (18) and A2 (19)  by Lombe et al. . Th. ThMille71) and isoerythrin A-4′-(3-methylbut-2-enyl) ether (72) maximaisoflavone B (73) and 7,2′-dimethoxy-4′,5′-methylenedioxyisoflavone (74) from the root bark of Millettia dura (Leguminosae) harvested in Tanzania . Th. ThPolya120) was isolated from the stem bark of Neoboutonia macrocalyx (Euphorbaceae) and potently inhibited the CQ-resistant FcB1/Colombia strain of P. falciparum, with an IC50 value 19.02 µM . Th. ThCanthOtostegia integrifolia (Lamiaceae) are used in Ethiopian folk medicine for the treatment of several diseases including malaria [124) , was isolated from the roots of Caesalpinia bonducella  from Tanzania by Nondo et al. [50 value of 2.20 and 4.16 µM against the 3D7 and Dd2 strains, respectively [The leaves of  malaria . The kno24) Fig.  was isolo et al. . This coectively .126 to 129), and 2β-acetoxy-1α,6β,9β-tribenzoyloxy-4β-hydroxy-dihydro-β-agarofuran (130) , harvested in Cameroon by Mba’ning et al. [P. falciparum with IC50 values varying from 1.71 to 2.63 µM [The novel sesquiterpenoids salaterpenes A–D (30) Fig. , were isg et al. . The inv 2.63 µM .Fig. 27S131) from the rhizomes of Scleria striatonux (Cyperaceae), harvested from Cameroon. According to the authors, this compound exhibited antimicrobial and antiplasmodial activities with IC50 values against the NF54 and K1 strains of 15.69 and 13.54 μM, respectively [Nyongbela et al.  isolatedectively .132) and 2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (133) were isolated from the leaves of the Ekebergia capensis (Meliaceae) harvested in Kenya [50 values of 27.1 and 56.1 µM, and against the D6 66.9 and 64.3 µM, respectively [The previously reported acyclic triterpenes; 2-hydroxymethyl-2,3,22,23-tetrahydroxy-6,10,15,19,23-pentamethyl-6,10,14,18-tetracosatetraene  harvested from Kenya, led Happi et al. to the isolation of the novel apotirucallane triterpenoids with antiplasmodial activities; prototiamins A–F (134–139) , prototiamin G (141) and seco-tiaminic acid A (142) [134–142) were also evaluated against the CQ-sensitive strain NF54. Compound 134 displayed strong selectivity for the NF54 strain against rat skeletal myoblast L6 cells (with a selectivity index of 104.7), while 136 and 138 had selective indices of 12 and 13, respectively. Compounds 135, 137, 139, and 140 were active against P. falciparum, with IC50 values ranging from 1.3 to 2.0 μM, and were less selective, while compound 142 inhibited the strain with an IC50 value of 19.3 μM.Phytochemical investigations of the root barks of 39) Fig. , as well A (142) . The obtNeoboutonia macrocalyx (Euphorbiaceae) is traditionally used to treat malaria in Southwestern Uganda around Kibale National Park, where the stem bark is widely used [143), 22α-acetoxyneomacrolactone (144), 6-hydroxyneomacolactone (145), 22α-acetoxy-6-hydroxyneomacrolactone (146), 6,7-epoxyneomacrolactone (147), 22α-acetoxy-6,7-epoxyneomacrolactone (148), 4-methylen-neomacrolactone (149), and neomacroin (150), Fig. 143–147, 149,150) exhibited antiplasmodial activities with IC50 of < 11 μM [The plant species ely used . The inv < 11 μM .Fig. 30C151)  collected from Egypt [P. falciparum with an IC50 value of 257.8 nM with no cytotoxicity up to the concentration of 9 μM. The compound also tested positive against the W2 strain with an IC50 value of 2000 nM [Ganoderic acid AW1 (51) Fig. , a new lom Egypt . This co 2000 nM .Fig. 31L152)  by Irungu et al. [P. falciparum. This compound exhibited weak antiplasmodial activity against the D6 and W2 strains with IC50 values of 84.7 and 150.2 µM, respectively [The known limonoid, proceranolide (52) Fig.  was founu et al. . The isoectively .Fig. 32L153), 12α-acetoxy-7-deacetylazadirone (154), mzikonone (155), 11-epi-toonacilin (156) and azadironolide (157), which were isolated from the stem bark of Turraea nilotica (Meliaceae), all showed potent antiplasmodial activity against the D6 and W2 strains with IC50 values ranged from 2.4 to 36.6 µM and from 1.1 to 40.5 µM, respectively [Additionally, three known limonoids; azadirone (ectively .158), 3-epi-oleanolic acid (159), oleanolic acid (160) and ekeberin A (161)  Fig.  were alsu et al. . The fou162) and oleanolic acid (160) from the twigs of Keetia leucantha (Rubiaceae). The authors re-tested the compounds, showing them to have in vitro activities on the 3D7 strain of P. falciparum with IC50 values of 32.4 and 59.4 µM, respectively [Mimusops caffra (Sapotaceae) growing in South Africa, ursolic acid acetate (163) and 3-oxo-ursolic acid (164), as well as the known compound 162 were isolated by Simelane et al. [50 values ranging from 3.81 to 15.54 μM [Bero et al. also isolated the known ursolic acid  and congoensin B (166), along with the known tirucallane-type triterpenoid gladoral A (167)  harvested from Cameroon by Happi et al. [50 values ranging from 2.4 to 6.1 µM [Two new tirucallane-type triterpenoids, namely congoensin A (67) Fig.  were isoi et al. . These co 6.1 µM .Fig. 34TTurraea nilotica (Meliaceae) growing in Kenya. Three known potent anti-malarial protolimonoids; niloticin (168), hispidol B (169) and piscidinol A (170) were isolated  and lupeol (172)  and Erythrina caffra (Papilionaceae), respectively [50 values of 112.94 and 97.73 μM, respectively [O-acetyl-26-deoxyneoboutomellerone (173) was isolated from the stem bark of Neoboutonia macrocalyx (Euphorbaceae) [P. falciparum, with IC50 value of 2.15 µM [The known hopane type triterpenoids; betulin (72) Fig.  were isoectively , 106. Thectively , 106. Thrbaceae) . The com 2.15 µM .Fig. 36O174) and the coumarin bergenin (175), Fig. Zanthoxylum heterophyllum (Rutaceae) and Diospyros conocarpa (Ebenaceae), respectively [50 = 39.04 µM [175 was recorded as 101.15 against the same plasmodial strain [176), plumbagin (177) and ismailin (178) from the stem bark of Diospyros canaliculata (Ebenaceae) harvested from Cameroon [P. falciparum with IC50 values ranging from 2.17 to 60.09 µM [These are summarized in Table ectively , 108. Wh39.04 µM , and perCameroon . The com60.09 µM .Table 8S179) was isolated from the stem bark of Erythrina caffra (Papilionaceae) collected in South Africa by Chukwujekwu et al. [50 value of 42.59 µM. The antiplasmodial activities of two lactones: morindolide (180) and lippialactone (181), obtained from roots of Vangueria infausta spp. infausta (Rubiaceae) and the leaves of Lippia javanica (Verbenaceae), respectively, were also evaluated [180 only inhibited the NF54 strain weakly, with an IC50 value of 109.99 µM, while compound 181 inhibited the D10 strain moderately with an IC50 value of 24.70 µM [The known ester erythinasinate  and 2-acetyl-1-hydroxy-8-methoxy-3-methylnaphthalene (183) isolated from the rhizomes of Kniphofia foliosa (Asphodelaceae) harvested in Ethiopia both inhibited the D6, W2, and 3D7 strains of P. falciparum with IC50 ranging from 6.32 to 67.32 µM [184) was isolated from the bark of Entandrophragma congoense (Meliaceae) collected in Cameroon by Happi et al. [50 value of 73.28 µM.Two naphthalene derivatives; dianellin , macluraxanthone (186) and allaxanthone B (187) were obtained from Allanblackia floribunda (Guttiferae) by Azebaze et al. [32 and FCM29 strains with IC50 values ranging from 0.91 to 70.33 µM for the first strain and from 0.68 to 67.22 µM against the second [Three xanthones; 1,7-dihydroxyxanthone  were described in the literature for the very first time. Besides, from Fig. A classification of the compounds by class into the plant families showed that most of the plant families represented their typical (chemotaxonomic) compound classes, often seen in the literature for species harvested from the African continent , 111–11450 values were converted to μM), and taking a cut off of 10 μM for the most promising secondary metabolites most likely to be lead compounds. The most active compounds within this range for at least one plasmodial strain, i.e. 25 out of 66 NPs were alkaloids ~ (38%), while 23 of them were terpenoids ~ (35%) and 11 were quinones ~ (17%). Taking a cut off IC50 value of at most 1 μM left us with 19 compounds, 14 of them being alkaloids. Besides, the majority of the 187 NPs were terpenoids (30%), followed by flavonoids (22%), alkaloids (19%) and quinones (15%), the rest of the compound classes only represent a negligible part of the current collection.Raw data retrieved from the literature showed activities reported in diverse units. A classification of the compounds by potencies (after all measured ICIn this review, an attempt has been made to document the anti-malarial/antiplasmodial activities of NPs derived from African medicinal plants in their various compound classes and source species, published between 2013 and 2019. A description of the in vitro and available in vivo activities for 187 compounds is shown, as well as their classification into the various known NP compound classes and plant families of origin. From the collected data, the most active compounds belong to the same compound classes as the malarial drugs of natural origin, e.g. the alkaloid class for quinine and the terpenoid class for artemisinin. A previous report from Titanji et al.  had showPlasmodium, out of which 39% were described as new NPs, with 29% having IC50 values ≤ 3.0 µM against at least one of the tested plasmodial strains [Although recently published reviews have described the activities of anti-malarial secondary metabolites of terrestrial and marine origins, input data from African sources has not been the focal point. Tajuddeen and van Heerden recently published a review of 1524 natural compounds from around the world, which have been assayed against at least one strain of  strains . However strains , 28, couAdditional file 1. List of journals consulted in building the initial data collection.


Toona ciliata Roem (T. ciliata). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions.Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta (H. robusta) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 (TcMYB3) gene.The experimental results showed that T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.This is the first report to verify the best reference genes for normalizing gene expression in  Toona ciliata Roem. belongs to the Meliaceae family, which is widely distributed in China, Australia, and India. Because of its straight trunks and russet wood, T. ciliata has the title of “Chinese Mahogany” [T. ciliata has great economic value, for example, its wood is often used to produce high-end furniture, instruments and crafts [T. ciliata include ketones, steroids, and coumarins, many of which have antifungal, anti-glycation, or anti-tumor activities [T. ciliata is low. In addition, in previous research, it has been found that T. ciliata is very susceptible to the moth pest Hypsipyla robusta Moore [H. robusta is distributed, such as Australia and Brazil, T. ciliata also faces serious damage from H. robusta [H. robusta effectively, and current pest control methods are time- and labor- consuming, thus not applicable to large-scale forest plantations [T. ciliata. So far, the knowledge base ICG (http://icg.big.ac.cn) has collected reference genes from more than 120 plant species including Arabidopsis [T. ciliata. Nor are there any literature references about the housekeeping genes in T. ciliata, which can be used for the standardization of gene expression.ahogany” . But itsd crafts . More imd crafts . Compountivities –7, and itivities . Howeverta Moore  that eat robusta –12. At pntations . It may ntations , 15. Genbidopsis , peanut bidopsis , cucumbebidopsis , and soybidopsis , except RT-qPCR has good repeatability, high sensitivity, accurate quantification, and fast reaction, making it a powerful tool to carry out the entire PCR process and the most commonly used method of determining gene expression levels . HoweverACT), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), and tubulin (TUB) [The commonly used housekeeping genes are those consistently express under all conditions, such as genes encoding actin (in (TUB) . Howeverin (TUB) . Softwarin (TUB) , NormFinin (TUB) , and Besin (TUB) , are widin (TUB) , 28. Thein (TUB) .T. ciliata transcriptome database generated by our group were investigated to determine the most suitable T. ciliata candidate gene(s) as the reference(s) for gene expression analysis using RT-qPCR technique under specific conditions including different tissues  and treatments , including actin 7 (ACT7), phosphoglycerate kinase (PGK), 60S ribosomal protein L13 (60S-13) and L18 (60S-18), histone deacetylase 1 (HIS1) and 6 (HIS6), protein phosphatase 2 C57 (PP2C57) and C59 (PP2C59), ubiquitin-conjugating enzyme E2 5B (UBC5B) and 17 (UBC17), S-adenosylmethionine decarboxylase proenzyme (SAMDC), elongation factor 1 (EF1) and 2 (EF2), peptidyl-prolyl cis-trans isomerase CYP95 (PPIA95) and CYP26–2 (PPIA26), 18S rRNA (18S), tubulin alpha-3 chain (TUB-α), tubulin beta-5 chain (TUB-β), membrane-anchored ubiquitin-fold protein 1 (MUB1), and TIP41-like protein (TIP41). In addition, the TcMYB3 gene was used to confirm the reliability and validity of the reference genes screened. MYB proteins, which constitute one of the largest family of transcription factors in plants, play important roles in plant growth and development, biotic and abiotic stress responses, and circadian rhythm regulation [Arabidopsis transcription factor MYB102 increases plant susceptibility to aphids [T. ciliata under different conditions, laying a foundation for studying molecular mechanisms in T. ciliata through gene expression analysis.In this study, 20 candidate genes from gulation , 31. Foro aphids , 33. OurR2 > 0.99). The amplification efficiency for the 20 candidate reference genes ranged from 90.41% for PPIA95 to 102.44% for PGK. Further details of primers are given in Table Reverse-transcribed cDNA from each sample was used as a template with primers for standard PCR amplification. Electrophoresis verified all PCR products were specific with single bands in the gel Fig. S. The melH. robusta treatment, 4 °C treatment, MeJA treatment, and PEG6000 treatment. The expression levels of candidate genes were very different across the samples. The maximum cycle threshold (CT) value was 31.66, and the minimum was 13.18  of the normalization factor after the introduction of a new reference gene, and determines the optimal number of reference genes based on this ratio. The default Vn/n + 1 value for the software is 0.15. If the ratio is less than 0.15, the number of internal gene combinations that can meet the requirements for relative quantification is n, otherwise another reference gene needs to be introduced. In our study, the values of pairwise variation V2/3 under conditions with H. robusta treatment, 4 °C treatment, MeJA treatment, PEG6000 treatment, and different tissues, were all less than 0.15, indicating that the optimal number of reference gene combinations is two  was 0.180, V3/4 was 0.15, and V4/5 was 0.126, indicating that the addition of the third and fourth reference genes has different impacts on the results. It always better to use fewer reference gene due to the time and cost economy consideration, hence the best reference gene combination was EF2, EF1, ACT7 and UBC5B for all the samples.In general, it is more reliable to use multiple reference genes are more reliable than a single reference gene for quantitative gene analysis. Given this, geNorm calculates the pairwise variation , HIS1 (0.105), and PP2C59 (0.161) , UBC5B (0.042), and TIP41 (0.109) in young stems , UBC5B (0.206), and PPIA95 (0.250)  and TUB-α (0.112) had lower stability values across different tissues since they showed the most stable expression, which is consistent with the results of geNorm analysis , TUB-α (0.082), and PGK (0.099) were the most stable candidate genes under 4 °C treatment  and UBC17 (0.100)  and HIS6 (0.082) , 18S (0.316), and PGK (0.335) were the three genes with the lowest stability values for all samples  Fig. a, while  ACT7 0.02, UBC5B  ACT7 0.02, UBC5B  ACT7 0.02, UBC5B  ACT7 0.02, UBC5B  ACT7 0.02, UBC5B  ACT7 0.02, UBC5B  ACT7 0.02, UBC5B UBC17 (1.16 ± 0.23) and 18S (1.62 ± 0.36) were the most stable genes in leaves under H. robusta treatment, and the expression of HIS1 (1.40 ± 0.31) and UBC17 (1.70 ± 0.33) were the most stable in young stems. Genes with the most stable expression across two tissues were UBC17 (1.73 ± 0.34) and 18S (2.12 ± 0.47), as was the case in leaves. HIS6 (5.41 ± 1.38) and MUB1 (5.48 ± 1.16) were the most stably expressed genes in the different tissues, 18S (0.73 ± 0.16) and 60S-18 (0.83 ± 0.18) in 4 °C treatment, 60S-18 (0.67 ± 0.14) and EF1 (0.83 ± 0.14) in MeJA treatment, PPIA26 (3.89 ± 0.87) and 60S-18 (4.48 ± 1.02) in PEG6000 treatment. For all samples, MUB1 (3.95 ± 0.87) showed the highest value for expression stability.BestKeeper takes as input the CT data for each gene-primer pair combination and calculates the coefficient of variation (CV) and standard deviation (SD), as shown in Table H. robusta treatment on leaves and under MeJA treatment was consistent with the results of geNorm analysis. HIS1 ranked first for young stem tissue under H. robusta treatment. The first-placed genes were 60S-18 and HIS6 under 4 °C treatment and PEG6000 treatment, respectively. TUB-α was the most stable gene in different tissues and all samples. The expression of PPIA26 was the most unstable under all experimental conditions except PEG6000 treatment.In this study, three algorithms were used to analyze the expression stability of 20 candidate reference genes. The gene ranking tables generated by them are different because of their different algorithms. RankAggreg is an algorithm designed to aggregate large ranking lists. It performs aggregation of ordered lists based on the rankings via the Cross-Entropy Monte Carlo algorithm or a Genetic Algorithm . To provTcMYB3 gene was quantified using either the two most stable genes, alone and in combination, or the two most unstable genes in the consensus ranking. Under H. robusta treatment, the relative expression of TcMYB3 in leaves and young stems reached a peak at 12 h when the most stable genes and their combinations were used for standardized. But the relative expression of TcMYB3 was abnormally increased when standardized with the most unstable genes  was down-regulated and the depression multiple was basically the same. But the expression level of TcMYB3 in the flower was the highest using the most unstable gene (PPIA26) as the reference gene. In addition, the expression level and trends were very similar when the most stable two reference genes and their combination were used for relative quantification under other stresses, including 4 °C treatment  stress; TUB4 and TUA4 were stable under two-spotted spider mite (TSSM) stress [GmELF1A and GmTUA5 were stable reference genes for normalization of expression data obtained from soybean roots infected with Meloidogyne incognita, and GmCYP2 and GmELF1A were the best reference genes in soybean leaves infested by Anticarsia gemmatalis [H. robusta stress, the reference genes that performed best in leaves and young stem tissues were different in our study. PP2C59 and UBC5B showed high stability of expression in leaves, while only PP2C59 ranked high for young stems. Once again, the appropriate reference genes for different species under different conditions and in different tissues vary. Hence, it is necessary to identify the best reference genes for specific conditions via RT-qPCR. Protein phosphatase can reverse the phosphorylation of protein kinases, thereby dynamically controlling protein phosphorylation and protein phosphatase 2Cs (PP2Cs) is the most abundant type of phosphatase in plants [Santalum album [PP2Cs can be considered as a candidate reference gene.Other researchers have studied the best reference genes for plants under pest stress, and estation . ABCT an) stress . Mirandammatalis . Under Hn plants . Althougum album . TherefoPPIA26, which was the best reference gene recommended by BestKeeper for use under PEG6000 stress. Another gene in this family, PPIA95, ranked first in geNorm analysis for both 4 °C cold stress and MeJA treatment. For leaves and young stems under H. robusta treatment and MeJA stress, PPIA95 ranked third in NormFinder analysis. In BestKeeper analysis, PPIA95 ranked third under 4 °C cold stress and PEG6000-induced drought stress, and among all samples it ranked second. Overall, the PPIA gene family is a promising reference gene set in T. ciliata. The PPIA gene family encodes proteins with functions in immune responses, as well as resistance to cancer, autoimmune diseases, protozoan, and viral infections [PPIA family are rarely used as internal reference genes, but they are abundantly expressed in the T. ciliata transcriptome data and the expression level in each sample is very similar, which is the main reason for choosing them. As reference genes, they are also stably expressed in animals. For example, in different heart and disease conditions, PPIA is recognized by ReFinder as the best reference gene in different skeletal muscles of mice, and it ranked first for human endometrial cancer [PPIB is believed as the optimal reference gene in analyzing the blood of Machado-Joseph disease (MJD) patients [Under most experimental conditions in this study , the reference gene with the worst performance was fections . In planl cancer , 53. PPIpatients .T. ciliata, showing that the optimal reference genes were TUB-α and PGK across all samples; PP2C59 and UBC5B in leaves and HIS1 and ACT7 in young stems under H. robusta treatment; TUB-α and PPIA95 in different tissues; 60S-18 and TUB-α under 4 °C treatment; UBC17 and PPIA95 under MeJA treatment; HIS1 and MUB1 under PEG6000 treatment, respectively. We believe this research is important for accurate quantification and expression analysis of genes under different conditions in T. ciliata. It will play a vital role in the molecular breeding work of T. ciliata, such as the research on the H. robusta-resistant and drought-resistant varieties, as well as the research on the metabolic pathways of precious compounds in plants in the future.This study is the first report about screening and verification of expression stability analysis of a series of reference genes under different conditions in T. ciliata, grown in a greenhouse in South China Agricultural University (SCAU). For samples from different tissues, mature leaves, young leaves, flowers, shoots, and young stems were collected at 9:00 am on August 25, 2019. Before treatment with the H. robusta, PEG6000, 4 °C, and MeJA, all the seedlings were pre-incubated in incubator for 7 days with 16 h of light at 28 °C and 8 h of dark at 22 °C to mimic the wild environment. For H. robusta treatment, seedlings were exposed to herbivores, and leaves and young stems were harvested after 0, 6, 12, 24, and 36 h. After seedlings were treated with 0, 5, 10, 20, and 30% (w/v) of PEG6000 for 7 days, leaves were collected. For 4 °C treatment, seedlings were placed at 4 °C, and samples (leaves) were taken at 0, 6, 12, 24, and 36 h. Seedlings sprayed with MeJA (100 μM) were sealed in plastic bags and leaves were collected at 0, 6, 12, 24, and 36 h. Three biological replicates were taken for each sampling point, and all samples were immediately frozen in liquid nitrogen and stored at − 80 °C.Five different experiments were conducted for data collection Table . ExperimTotal RNA was extracted from all samples using a HiPure HP Plant RNA Mini Kit (Magen) with DNase treatment to remove genomic DNA. The quality of the RNA was determined with NanoDrop ND1000 (Thermo Scientific). RNA samples with absorbance ratios of A260/A280 and A260/A230 both around 2.0 were selected for further analysis. To synthesize cDNA, 0.5 μg of total RNA was used according to the manufacturer’s instructions for the HiScript II Reverse Transcript kit (Vazyme). Five-fold diluted cDNA was used for subsequent RT-qPCR experiments.T. ciliata leaf transcriptome database by reviewing previous literature: PGK, 60S-18, 60S-13, HIS1, HIS6, PP2C57, PP2C59, UBC5B, UBC17, ACT7, SAMDC, EF1, EF2, 18S, TUB-α, TUB-β, MUB1, PPIA26, PPIA95, TIP41. Since there is no genomic sequence data available for T. ciliata, we designed primers based on the sequences in the T. ciliata transcriptome database. Firstly, the Coding Sequence (CDS) and genomic DNA sequences (gDNA) of the candidate reference genes were amplified separately (Table Shttps://www.ncbi.nlm.nih.gov/tools/primer-blast/). Apart from 60S-18, SAMDC, TUB-β and PPIA26, RT-qPCR primers were designed across introns. Details of these genes and primers are shown in Table Twenty candidate reference genes were selected from the 2O. The PCR reaction procedure was as follows: 95 °C for 3 min, 35 cycles of 95 °C for 15 s, 55 °C for 15 s, 72 °C for 15 s, followed by 5 min extension at 72 °C. The RT-qPCR reaction mixture consisted of 10 μL ChamQ Universal SYBR qPCR Master Mix (Vazyme), 2 μL of cDNA, 0.4 μL of each primer (10 μM), and 7.2 μL ddH2O to a final volume of 20 μL, and it was performed on LightCycler480  with optical 96-well plate. To test the specificity of the RT-qPCR primers, the products of PCR were analyzed by nucleic acid electrophoresis on a 2% (w/v) gel and the melting curve was included after amplification. All samples used for RT-qPCR analysis had three biological replicates, each containing three technical replicates. In order to calculate the gene-specific PCR efficiency (E) and correlation coefficient (R2) of each gene, a standard curve was generated from the mixed complementary DNA (cDNA) using a fivefold dilution series.The volume of each PCR amplification reaction mix was 20 μL, containing 10 μL of Phata Max Buffer, 2 μL of five-fold diluted cDNA, 2 μL of each primer (10 μM), 0.5 μL of dNTP, 0.5 μL of Phata Max Super-Fidelity DNA Polymerase, and 5 μL ddHCT values were obtained by RT-qPCR, and used to evaluate the expression levels of candidate genes in different experimental conditions and tissues. Three commonly employed algorithms, geNorm , NormFinn / Vn + 1. If the value of Vn / Vn + 1 is less than 0.15, the number of optimal internal reference genes is n. If the value of Vn / Vn + 1 is greater than 0.15, the number of optimal reference genes is n + 1. NormFinder selects the most suitable internal reference gene by calculating a stability value for gene expression of the candidates. The lower the stability value is, the more stable the gene is. Using BestKeeper, the SD and CV of expression of each candidate gene can be obtained. The CV ± SD values of different genes were then compared to determine the relative stability of expression of the candidates. Finally, in order to generate an overall ranking of candidate genes from the data generated by geNorm, NormFinder, and BestKeeper, we used the RankAggreg (version 0.6.5) software package in R as previously described [rho set at 0.1, the seed at 100, and the “convIn” argument at 50.The package geNorm (Version3.5) screens stable reference genes by calculating the M value of the stability of each candidate gene, and the criterion is that the smaller the M value is, the higher the stability of the candidate gene is. It also calculates pairwise variations of the normalized factor after introducing a new internal reference gene, and determines the number of optimal internal reference genes based on the ratio Vescribed , 55–57. escribed . The ranTcMYB3 under 4 °C, MeJA, PEG6000, in different tissues, and under H. robusta treatment (leaves and young stems). Finally, we used the 2-△△CT method to calculate the relative expression levels of the verified genes, where △CT = CT (target gene)-CT (reference gene), △△CT = △CT (treatment)-△ CT (control), 2-△△CT = relative expression. Three technical replicates were performed for each biological sample [In order to verify the accuracy of the rankings and the stability of expression of the selected reference genes, the two most stable reference genes, alone and in combination, and the two most unstable reference genes, as recommended by RankAggreg were used to verify the relative expression of l sample .Additional file 1 Figure S1: Amplification products of the twenty candidate reference genes and TcMYB3.Figure S2: Melting curves of candidate reference genes and TcMYB3.Additional file 2 Table S1. Coding sequences and genomic DNA sequences of candidate reference gene.