Panax notoginseng and to examine the regulatory mechanisms involved in an adaptation to the simulated sunflecks. When P. notoginseng was exposed to the simulated sunflecks, non-photochemical quenching (NPQ) increased rapidly to the maximum value. Moreover, in response to the simulated sunflecks, there was a rapid increase in light-dependent heat dissipation quantum efficiency of photosystem II (PSII) (ΦNPQ), while the maximum quantum yield of PSII under light (Fv′/Fm′) declined. The relatively high fluorescence and constitutive heat dissipation quantum efficiency of PSII  in the plants exposed to transient high light  was accompanied by the low effective photochemical quantum yield of PSII (ΦPSII) after the dark recovery for 15 min, whereas the plants exposed to transient low light (50 μmol m–2 s–1) has been shown to lead to significant elevation in ΦPSII after darkness recovery. Furthermore, PSII fluorescence and constitutive heat dissipation electron transfer rate  was increased with the intensity of the simulated sunflecks, the residual absorbed energy used for the non-net carboxylative processes (JNC) was decreased when the response of electron transfer rate of NPQ pathway of PSII (JNPQ) to transient low light is restricted. In addition, the acceptor-side limitation of PSI [Y(NA)] was increased, while the donor-side limitation of photosystems I (PSI) [Y(ND)] was decreased at transient high light conditions accompanied with active cyclic electron flow (CEF). Meanwhile, when the leaves were exposed to transient high light, the xanthophyll cycle (V cycle) was activated and subsequently, the JNPQ began to increase. The de-epoxidation state [(Z + A)/(V + A + Z)] was strongly correlated with NPQ in response to the sunflecks. In the present study, a rapid engagement of lutein epoxide (Lx) after the low intensity of sunfleck together with the lower NPQ contributed to an elevation in the maximum photochemical quantum efficiency of PSII under the light. The analysis based on the correlation between the CEF and electron flow devoted to Ribulose-1, 5-bisphosphate (RuBP) oxygenation (JO) indicated that at a high light intensity of sunflecks, the electron flow largely devoted to RuBP oxygenation would contribute to the operation of the CEF. Overall, photorespiration plays an important role in regulating the CEF of the shade-tolerant species, such as P. notoginseng in response to transient high light, whereas active Lx cycle together with the decelerated NPQ may be an effective mechanism of elevating the maximum photochemical quantum efficiency of PSII under light exposure to transient low light.Light is highly heterogeneous in natural conditions, and plants need to evolve a series of strategies to acclimate the dynamic light since it is immobile. The present study aimed to elucidate the response of light reaction of photosynthesis to dynamic sunflecks in a shade-tolerant species  Arabidopsis thaliana might optimize electron transport and PSI photoprotection mediated by phosphorylation of vesicle-like proteins in response to the dynamic sunflecks  and long  timescales , 2018a,bunflecks . It is, unflecks .Nicotiana tabacum) and A. thaliana are exposed to constant high light, violaxanthin (V) is de-epoxied to form antheraxanthin (A), which is further de-epoxied to form zeaxanthin (Z)  is one of the most effective ways for plants to dissipate excess light energy. The mechanism of NPQ associated with heat dissipation is not well understood, but PsbS proteins and the xanthophyll cycle play an important role in regulating the NPQ processes . When tothin (Z) . Z is usthin (Z) . Howeverficiency . NPQ is ficiency . It has unflecks , 2009. HNPQ) is high and the quantum yield of electron transport in PSII (ΦPSII) is low in rice (Oryza sativa) grown under constant high light , thereby protecting PSII and PSI from photooxidation  is thought to protect plants from the damages that occurs due to the over-reduction of the thylakoids under fluctuating light . The CEFxidation ,c. This gh light . The eviitiation ,b. The pitiation . Moreoveitiation . Thus, titiation . SurprisPanax notoginseng (Burkill) is a perennial herb belonging to the Araliaceae family, which is a typically shade-tolerant species  is preferred for the growth of P. notoginseng  the greater increase in CEF activity in response to the transient low light may accommodate the electron flows; (2) Lx cycle exist in the typically shade-tolerant species in response to the transient low light, and Lx may play the role of improving light-harvesting efficiency; (3) NPQ coupled to the de-epoxidation in the V cycle might attribute to the energy dissipation in response to the transient high light; (4) the photorespiration pathways may play a role in regulating the photosynthetic electron flow under transient high light condition. species . In agrioginseng . It has  of PSII . The phous study . HoweverP. notoginseng. Light intensity in the shade house was collected every 10 s from 6:00 to 19:00 on a clear, cloudless day using a Li-1500  light quantum meter. FL intensity was measured simultaneously as a control. The light transmittance of the shade house was about 10% FL, which is a suitable light intensity for P. notoginseng growth. The soil was local raw soil (red soil). According to the method of –1, total phosphorus (P), 7.273 g kg–1, hydrolysable N, available P, 48.39 mg kg–1, available K, 1910.00 mg kg–1, pH, 6.42, and organic matter, 57.35 g kg–1.The experiments were conducted at the experimental farm of Yunnan Agricultural University in Kunming, Yunnan Province, China . There is plenty of rain from May to October and very little rain from November to April. A shade house was constructed using a special shade net for P. notoginseng was disinfected with 0.1% potassium permanganate for 5 min. In January, the seedlings were planted in white pots contained with 10 kg disinfected raw soil, and there are three seedlings in each pot. Fertilization was conducted from April to July. Compound fertilizer, monopotassium phosphate, and potassium sulfate were applied with 0.4, 0.2, and 0.3 g/pot, respectively. During the growing period, insecticides  were sprayed to control the disease. In August (linear phase), P. notoginseng was used to determine the chlorophyll fluorescence and photosynthetic pigment content.Healthy 1-year-old P. notoginseng was placed in an opaque incubator  for dark adaptation (more than 1 h), and the minimum fluorescence of PSII (Fo) and maximum fluorescence (Fm) were measured at 0 μmol m–2 s–1 light intensity. According to the previous studies, the light saturation point of P. notoginseng is 100–150 μmol m–2 s–1 (P. notoginseng was suddenly exposed to the simulated sunflecks for 30 min with the light intensity of 50 μmol m–2 s–1 (transient low light), 100 μmol m–2 s–1 (light saturation point), 400 μmol m–2 s–1 (transient moderate light), 800 μmol m–2 s–1 (transient high light), 1,600 μmol m–2 s–1 (transient high light), respectively, followed by the dark adaptation for 15 min, and the whole process lasted for 45 min.According to the method of  m–2 s–1 . Thus, iFo′), maximum fluorescence (Fm′), and light-adapted steady-state fluorescence (Fs) after light adaptation were taken two times at 30 s intervals at the beginning, then at intervals of 1 min. Five replicates were randomly selected from each treatment for the analysis of chlorophyll fluorescence. The chlorophyll fluorescence parameters of PSII are calculated as follows (Fv′/Fm′) = (Fm′ − Fo′)/Fm; NPQ coefficient of PSII (NPQ) = (Fm − Fm′)/Fm′; photochemical quenching coefficient of PSII (qP) = (Fm′ − Fs)/(Fm′ − Fo′); effective quantum efficiency of PSII (ΦPSII) = Y(II) = (Fm′ − Fs)/Fm′; fluorescence and constitutive heat dissipation quantum efficiency of PSII  = Y(NO) = Fs/Fm; light-dependent heat dissipation quantum efficiency of PSII (ΦNPQ) = Y(NPQ) = Fs/Fm′ − Fs/Fm; electron transfer rate of photochemical quenching pathway of PSII (JPSII) = ΦPSII × PFD × 0.84 × 0.5; fluorescence and constitutive heat dissipation electron transfer rate of PSI  = Φf,d × PFD × 0.84 × 0.5; electron transfer rate of NPQ pathway of PSII (JNPQ) = ΦNPQ × PFD × 0.84 × 0.5; the electron transfer rate of PSII [ETR(II)] = Y(II) × PFD × 0.84 × 0.5.Photosystem II chlorophyll minimum fluorescence  is similar to that of the maximum chlorophyll fluorescence, except that 10 s of far-red light exposure was required before the determination of the highest P700 signal. PSI reaction center P700 maximum fluorescence (Pm′) was similar to Fm′, except that actinic light was used instead of far-red light. The calculation of PSI parameters was referred to the method of Pm′ − P)/Pm; donor terminal heat dissipation efficiency of PSI [Y(ND)] = 1 − P700red = P/Pm; receptor terminal heat dissipation efficiency of PSI [Y(NA)] = (Pm − Pm′)/Pm; the electron transfer rate of PSI [ETR(I)] = Y(I) × PFD × 0.84 × 0.5; cyclic electron transfer size of PSI (CEF) = JPSI – JPSII = ETR(I) – ETR(II).The light absorption of PSI was measured with reference to the measurement method of 2 fluorescence leaf chamber. The gas exchange parameters of healthy 2-year-old P. notoginseng were measured from 9 to 11 a.m. on a sunny day, with five replicates per treatment (n = 5). The CO2 content in the chamber was maintained at 400 μmol mol–1 during the measurement of photosynthetic light response curves. The leaf was induced under the light intensity of 500 μmol m–2 s–1 (red light: blue light = 9:1) for 10 min. The automatic measurement procedure of the instrument was started after the data of all parameters were relatively stable. The gradient of light intensity was listed in the following order: 500, 300, 200, 150, 100, 80, 60, 40, 20, 0 μmol m–2 s–1. Induction is stabilized for 120–180 s at each light intensity, during which the gas exchange parameters are collected and the data are saved after the measurement. The relationship between the net photosynthetic rate (Pn) and photosynthetic photon flux density (PFD) was fitted, Pn = Pmax − PmaxC0e–αPFD/Pmax , and C0 is the index where the net photosynthetic rate level off to 0 under low light. According to the parameters in the formula, the following parameters can be calculated, respectively: dark respiration rate (Rd) = Pmax − PmaxC0; light compensation point (LCP) = Pmax ln(C0)/α; light saturation point (LSP) = Pmax ln(100C0)/α.The steady-state gas exchange parameters were determined using the photosynthesis system  with the 2 cmPFD/Pmax . In the –2 s–1 and the leaf was induced under the CO2 intensity of 400 μmol mol–1 for 10 min. The gradient of reference cell CO2 concentration was set in the following order: 400, 300, 250, 200, 150, 100, 50, 400, 600, 800, 1,000, 1,200, 1,500 μmol mol–1. According to the method of 2 response curve where the internal leaf CO2 concentrations (Ci) were below 250 μmol mol–1 to obtain the carboxylation efficiency (CE), CO2 compensation point (Γ∗), and to calculate the rate of photorespiration (RL) = CE × I∗. The maximum carboxylation rate (Vcmax) and maximum electron transfer rate (Jmax) were calculated by using the method of The light intensity in the chamber was maintained at 500 μmol mP. notoginseng was moved into an opaque incubator  for dark adaptation, during which the plants were covered with a black bag. The CO2 concentration in the reference chamber was controlled to be 400 μmol mol–1. The leaves of P. notoginseng were put into the leaf chamber, and the light intensity in the leaf chamber was 0 μmol m–2 s–1. An automatic measurement was started after the data were stabilized. During measurement, the leaves were irradiated at 0 μmol m–2 s–1 light intensity for 2 min, and then induced with 400 μmol m–2 s–1 light intensity for 30 min. Data were collected every 30 s, and the gas exchange parameters were simultaneously collected.Before measuring the light induction curve, 2 response curve, and light induction curve, the following relevant parameters can be obtained: net photosynthetic electron transfer rate (JCO2) = Pnet × 4; the residual absorbed energy used for the non-net carboxylative processes (JNC) = JPSII − JCO2 (JC) and oxidation (JO) were calculated: JO = 2/3 × [JT – 4 × (Pn + Rd)], JC = 1/3 × [JT + 8 × (Pn + Rd)]; total photosynthetic electron flow through PSII (JT) = PFD × Y(II) × 0.84 × 0.5.According to the light response curve, the COI − JCO2 . AccordiPanax notoginseng was placed in a dark incubator  for 12 h at a temperature of 25°C. After 12 h, P. notoginseng was placed under the light intensity of 50, 100, 400, 800, and 1,600 μmol m–2 s–1, respectively. The process lasted 90 min, with the first 30 min being light treatment and the last 60 min being dark treatment. The leaves were collected at 0, 5, 15, 30, 35, 45, 60, and 90 min, respectively, and the cleaned and dried leaves were wrapped in tin foil and quickly stored in liquid nitrogen. Afterward, the tin foil-wrapped leaves were stored in liquid nitrogen in a sealed tin box at −80°C in the refrigerator. To measure the photosynthetic pigment content, 0.2 g leaves were weighed in the dark condition, repeatedly extracted with cold acetone, and fixed to 10 ml. According to the method of –1 Tris). 0.83 ml of 36–38% HCl solution was fixed into a 100 ml volumetric flask with pure water, (0.1 mol L–1 HCl). Further, 50 ml of 0.1 mol L–1 Tris solution and 40.3 ml of 0.1 mol L–1 HCl solution were mixed and the pH was adjusted to 7.5 with Tris mother liquor and HCl mother liquor to obtain 0.05 mol L–1 Tris–HCl buffer. Mobile phase A was prepared with acetonitrile and 0.05 mol L–1 Tris–Hc1 buffer at 70:3; mobile phase B was prepared with methanol and n-hexane at 5:1. The extracts were filtered through a 0.22 μm pore size membrane and sampled on a ZORBAX SB-C18  column with a column temperature of 25.0°C and a flow rate of 1 ml min–1 at a detection wavelength of 445 nm. The standards were purchased from Sigma and ChromaDex  with a purity of >98%.P < 0.05), and the graphical data were presented as mean ± SE. GraphPad Prism 8.3.0  software was used for graphing.The data were counted using Microsoft Excel 2007 software . The experimental site was divided into 15 plots, and 12 pots were placed per plot. We obtained five replicates  randomly selected from each plot for the analysis of photosynthetic parameters. Also, the results were displayed as mean values of five independent plants. One-way ANOVA was performed using SPSS 19.0 software , and Duncan’s test was applied for multiple comparisons of significant differences  recovered to the higher levels with respect to Fv′/Fm′, and the others recovered to lower level. These results indicated that the transient high light leaded to lower PSII activity.The leaf exhibited a significant difference in PSII activity in response to the simulated sunflecks . The Fv′–2 s–1) contributed most of the energy dissipation to the qP pathway (–2 s–1), the plants dissipated more energy through NPQ in the early stages   .–2 s–1 transient low light, the light energy received by PSII was mainly allocated to the photochemical reaction pathway ] is low, and the effective quantum efficiency of the PSI [Y(I)] decreases with the increase of transient light  increased rapidly; while during darkness recovery, Y(ND) was close to the initial level  rose rapidly during darkness recovery ], except for the transient light of 50 and 100 μmol mnt light . When P.al level . Meanwhi (–2 s–1 . In contrecovery .–2 s–1 (–2 s–1 (0.0027) > 1,600 μmol m–2 s–1 (0.0024) > 800 μmol m–2 s–1 (0.0023) > 400 μmol m–2 s–1 (0.0021) > 100 μmol m–2 s–1 (0.0014), suggesting that the high cyclic electron transfer activity plays an important role in photoprotection.Electron transfer rate II [ETR(II)] increased rapidly when plants were exposed to transient high light . The ETR–2 s–1 . The cha–2 s–1 . When pl–2 s–1 . The phoJPSII increased rapidly when P. notoginseng was suddenly exposed to transient light , the electron flow that was largely devoted to RuBP oxygenation would contribute to the operation of the CEF /(V + A + Z) was higher in the plants exposed to transient high light . The NPQ–2 s–1) and 3 min high light , Fv′/Fm′ and ΦPSII gradually increased with the extension of alternating time under the light intensity of 100 μmol m–2 s–1  did not change significantly in the alternation of fluctuating light, but Fv′/Fm′ in low light gradually increased with the alternation of fluctuating light ΦPSII, indicating that the fluctuating light has an important effect on the recovery of PSII activity in the plants. Moreover, during alternating fluctuating light, ΦPSII at the light intensities of 400, 800, and 1,600 μmol m–2 s–1 was at a low level. The increase in ΦPSII becomes greater when the plants were transferring from high light  to low light (50 μmol m–2 s–1), suggesting that in the process of alternating fluctuations, ΦPSII was inhibited under high light, and ΦPSII is recovered and electron-replenished under low light.In the simulated sunflecks environment with alternating fluctuations between 4 min low light (50 μmol m m–2 s–1 . Fv′/Fm′P. notoginseng in darkness was transferred to 4 min of low light (50 μmol m–2 s–1), the NPQ gradually decreased, followed by a rapid increase when P. notoginseng was exposed to high light , CEF was activated , but gradually decreased over time. CEF at light intensities of 100 and 400 μmol m–2 s–1 increased over time.When ctivated . The CEFLight is the ultimate resource for photosynthesis, and its intensity and spectral composition considerably vary in nature. These traits compel plants to maintain the balance between light absorption needed for photosynthesis and excess light dissipation for photo-protection . Excess NPQ  can be explained by a relatively high photo-damage as estimated from the low ΦPSII after dark recovery for 15 min , while Y(ND) was decreased in P. notoginseng (P. notoginseng exposed to the simulated sunflecks of 50 μmol m–2 s–1 (+) is also a mechanism to ensure the integrity of PSI. PSI photo-inhibition with large differences between the transient high light and transient low light can be explained by the photosynthetic alternative electron flows interacting with PSI. The greater increase in CEF activity in responses to the transient low light may accommodate the electron flows, consequently avoiding the response of photo-oxidative damage  was increased with the intensity of the simulated sunflecks as presented by the elevation of Φf,d at simulated sunflecks of 400, 800, and 1,600 μmol m–2 s–1 (JNC) predominated at the simulated sunflecks of 1,600 μmol m–2 s–1  enabling optimal V de-epoxidase activity (–2 s–1 suppressed the (Z + A)/(V + A + Z) (trans-thylakoid pH (–2 s–1)  , this coakoid pH , CEF was(–2 s–1) , the optincrease , 6.P. notoginseng to transient high light was accompanied by a slow operation of the Lx cycle, therefore, based on the correlation between NPQ and the degree of de-epoxidation in the V cycle, the de-epoxidation in the V cycle might primarily attribute to energy dissipation /(V + A + Z) to exhibit rapid and enhanced NPQ . Under tsipation . In partce yield . Reducedce yield . In the ce yield , 6C. TheA. thaliana under the simulated sunflecks has been preliminarily investigated by previous work on CEF and O2-dependent alternative electron sinks , the electron flow that was largely devoted to RuBP oxygenation would contribute to the operation of the CEF (The regulation of photosynthetic electron flow in on sinks . The adaunflecks . The pre pathway , 2015a. ntration . A. thaly of CO2 , the facy of CO2 . Diminispiration . Similar the CEF . Thus, fEnergy dissipation through NPQ predominates in response to the dynamic sunflecks, the V cycle plays an important role in regulating the NPQ processes, leading to the dissipation of excess light energy. Meanwhile, CEF was highly stimulated to protect PSI from photo-inhibition in response to the dynamic sunflecks. Additionally, photorespiration plays a role in regulating the CEF in response to the transient high light, whereas the Lx cycle together with the decelerated NPQ may be an effective mechanism of elevating the maximum photochemical quantum efficiency of PSII under the light in response to the transient low light. Overall, our results indicate that NPQ and CEF may protect the photosynthesis apparatus from dynamic sunfleck in a typically shade-tolerant species.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.J-WC directed the whole process of the experiment and made suggestions for the writing of the manuscript. J-YZ participated in the whole experiment, analyzed the relevant experimental data, and wrote the manuscript. Q-HZ and S-PS measured the light absorption in photosystem I and chlorophyll fluorescence. ZC and H-MW participated in the determination of photosynthetic pigment content and steady-state gas exchange measurements. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Helicobacter pylori. We also show how the database can be used to design a new antimicrobial peptide that shows better Candida albicans clearance and lower hemolysis than its template homologs. Finally, we demonstrate how TP-DB can suggest point mutations in helical peptide blockers to prevent a targeted tumorigenic protein-protein interaction. TP-DB is made available at http://dyn.life.nthu.edu.tw/design/.The systematic design of functional peptides has technological and therapeutic applications. However, there is a need for pattern-based search engines that help locate desired functional motifs in primary sequences regardless of their evolutionary conservation. Existing databases such as The Protein Secondary Structure database (PSS) no longer serves the community, while the Dictionary of Protein Secondary Structure (DSSP) annotates the secondary structures when tertiary structures of proteins are provided. Here, we extract 1.7 million helices from the PDB and compile them into a database  that allows queries of compounded patterns to facilitate the identification of sequence motifs of helical structures. We show how TP-DB helps us identify a known purification-tag-specific antibody that can be repurposed into a diagnostic kit for  Here, we present TP-DB; a pattern-based search engine based on 1.67 million helices from the Protein Database (PDB). We demonstrate the utility of TP-DB in identifying microbe-specific antigens, as well as the design of antimicrobial peptides and Protein-protein interaction blockers. Especially at the interface, either water-lipid interface at the cell membrane, or protein-DNA/protein–protein interface, these helices have disparate physicochemical properties at their two sides to properly play their functional roles. Take CM1513, an amphiphilic, helical antimicrobial peptide (AMPs) for example, for its proper bactericidal function, the positively charged lysines and hydrophobic residues need to stay at opposite sides of a helical wheel, the 2D projection of amino acids that one would see if viewed from a helix’s longitudinal axis. As a result, after Lys3 (K3), there would not be any positively charged residues till K6 and K7 appear at the same side , or after Leu4 (L4) and Phe5 (F5), there are no hydrophobic residues till Ile8 (I8) is present at the same side. In this example, what the peptide needs for proper function is a stretch of amino acid sequence consisting of a pattern of (K3)x1x2(K6) or (F5)x3x4(I8), where x1 and x2 (x3 and x4) can be any hydrophobic (positively charged) amino acid that still holds a helical structure. From the point of view of protein design, either to design a better AMP2 or to propose stronger interface blockers11 for therapeutic purposes, it is not an easy task to allow constituent residues to address physicochemical needs, meeting a specific sequence pattern, while maintaining the required structural integrity. Suitable tools need to be developed to facilitate the process.Secondary structures comprise a subset of amino acid sequences where each constituent residue forms hydrogen bonds (H-bonds) in specific orders with other constituent residues, at their peptidyl backbones, which grants a special mechanical property in our life-thriving nanomachines—proteins. Particularly, α-helices, where the 14, while the Dictionary of Protein Secondary Structure (DSSP)15 annotates the secondary structures when tertiary structures of proteins are provided16. However, both DSSP and protein data bank (PDB) do not provide a search engine for specific types of secondary structures  for >170,000 structurally solved sequences, nor do they provide any search function that recognizes specific sequence patterns (see the beginning of “Methods” for our definition of “patterns”). To better integrate functional structural fragments into therapeutics19, we extracted ~1.7 million  structurally resolved helices and their contacting/interacting helical partners from the PDB, from which helical propensity (HP) and coordination (contact) number are derived (Table 20 that takes the input of both residue pattern and the template sequence). We use this Therapeutic Peptide Design dataBase (TP-DB) to develop a new anti-fungal peptide/AMP, helix–helix interface blockers that could potentially disrupt the interaction between Shugoshin-1 (Sgo1) and protein phosphatase 2A (PP2A) and a diagnostic reporter to detect Helicobacter pylori infection.Currently, both (A) pattern-based (only) search engines locating desired functional motifs in primary sequences regardless of their evolutionary conservation and (B) dedicated secondary structural databases indexed for (A)’s purpose are lacking. Protein Secondary Structure database (PSS) proposed 30 years ago no longer serves the community10 helices) is to find stretches of protein sequences that match the queried pattern and adapt a helical conformation. Since protein sequences assume structures in the context of their 3-dimensional environment full of residue contacts, it is quite likely the helices are not as helical as when they are part of their parent proteins. We herein design two metrics to evaluate and prioritize the peptides, in the query results, which are highly likely to still stay helical when expressed or synthesized alone—HP and contact number. A higher HP and/or lower contact number would suggest a good helicity for peptides even in their isolated forms. For the former, we use a normalized HP (see “Methods”), HPNA, defined as HPa.a./NAa.a., where HPa.a. is the HP of a given amino acid type and NAa.a. is natural abundance of that amino acid  from the ExPASy tool “ProtScale”21. A log-odds ratio, log (HPNA) or log (HPa.a./NAa.a.) is found for each amino acid type, and the reported HP for each peptide in TP-DB is the sum of log (HPNA) for every constituent residue in this peptide . The distributions of the HP, log (HPNA), and the average contact number of all the TP-DB helices can be found in Supplementary Fig. TP currently comprises 1,676,117 helical peptides  extracted from 130,000+ experimentally solved protein structures stored in PDB. The apparent goal of establishing search engines for protein secondary structures . Considering both flexibility and search comprehension, we let the constituent pattern be [A1m A2n A3] where A1, A2, and A3 are the single-letter codes for amino acids, in between which are the spacings represented by the whole numbers m and n. We currently allow m and n to be from zero to four. A key “Y3G2K” or its search pattern [Y 3 G 2 K] would indicate a “Y” is (3 + 1) residues upstream to a “G” that is (2 + 1) upstream to a “K”. A key is paired with identifiers  referring to where this key can be found in all the stored peptides , which could be searched by a simple pattern [A1m A2n A3] or a pattern compounded by several of these simple patterns , Glycine (G), and Lysine (K) are called ‘anchoring residues’ of the pattern. This is equivalent to the regular expression “Y-x(3)-G-x(2)-K” in the PROSITE format and also used in the PHI-BLASTdes Fig. . Specifides Fig. . These krns Fig. .Fig. 1InTo query the database, users need to specify at least three anchor residues and the gaps that separate them, such that “ADE” or “K****VA”  can be queried by [A 0 D 0 E] and [K 4 V 0A], respectively Fig. . For mor23. To showcase the prowess and disparate utility of the TP-DB, we demonstrate below how a purification tag (herein FLAG-tag) and its antibody (herein M2 antibody) can be repurposed into a diagnostic kit for detecting human pathogens. The relevant experimental methods are listed in Supplementary Information. FLAG-tag is known to have a sequence DYKDDDDK24 with the pattern D-Y-K-x-x-[DE] being experimentally confirmed as the main affinity determinant motif25. “DYK” here is the strongest determinant while the last D (or E) is of secondary importance. We would like to search TP-DB for proteins that (i) are not included in the PHI-BLAST search results and (ii) belong to proteins in human pathogens but not in non-pathogenic bacteria.To demonstrate our search engine’s utility of finding patterns without considering residues’ evolutionary similarity, we would like to identify the sequences matching the queried pattern which can only be found by our TP-DB but not by other tools, including PHI-BLAST that considers both the pattern matching and evolutionary similarity between residues in the sequence alignment. The latter has been the main (if not only) bioinformatics tool to find biological sequences that match a specific pattern while inferring homologyE-value <100. On the other hand, among the 19 TP-DB-identified sequences, HP-NAP, containing the sequence “DYKYLE”, can be found in H. pylori strain 26695  but not in regular non-pathogenic bacteria. If the anti-FLAG M2 antibody can recognize this randomly selected pathogenic protein for its containing a FLAG-determinant motif (herein D-Y-K-x-x-[DE]), we can potentially use the same concept to repurpose the original use  of any known pair of antibody and antigen recognition motif  into its alternative use, herein the potential diagnostic kit for human pathogens. As expected, the anti-FLAG M2 antibody was indeed found to be capable of recognizing HP-NAP as analyzed by enzyme-linked immunosorbent assay  with the pattern D-Y-K-x(2)-[DE] (in PROSITE format) and FLAG sequence “DYKDDDDK”, we were not able to find any sequence with an MBP Fig. , indicatH. pylori, through recognizing native HP-NAP by ELISA or the HP-NAP monomer as a 17 kDa band by western blot.As a result, the antibody (herein M2 antibody) of a purification kit can be repurposed into a diagnostic kit for detecting human pathogens, herein 26. A discouraging forecast has suggested that by 2050, 10 million people could die from these superbugs each year, with half of them in Asia27. AMPs that target bacterial lipid membranes, evolving at a much slower pace than bacterial protein receptors, appear to be a promising alternative for antibiotics. There have been substantial efforts made to study AMPs’ insertion mechanism31 and analyze the determining characteristics for their efficacy33. To join these collective efforts and showcase the good use of TP-DB, we first examined in detail the insertion processes of two previously reported helical AMPs, W3_p1 (sequence: KK WRK WLK WLA KK)34 and W3_p2 (sequence: KK WLK WLK WLK KK)2 by MD simulations. The two amphiphilic AMPs, membrane-selective and mediated through no known protein receptors, have three equally spaced tryptophans at their hydrophobic face, while positively charged lysines/arginines are on the other side, which is a typical feature and requirement for helical AMPs. The goal of the simulations is to understand each residue’s physicochemical role in the bacteria-membrane insertion process while earlier reports37 have suggested the deeper a peptide penetrates the lipid membrane, the higher the bactericidal potency it can have.Pan-drug-resistant bacteria are no less a threat to mankind than viral infections that could cause a pandemic38 and also common to most of the membrane anchoring signals39. N-terminus seems to better interact with the phosphate groups of lipids  and transiently interact with the lipid tails better than the C-terminus.With microsecond simulations, we revealed types of contact modes and time-resolved atomic interaction between AMPs and lipid bilayers before AMPs’ final insertion into the bacterial membrane , we replace residues flanked by these anchoring residues using TP-DB to find functionally favored substituents that are ensured to have desired secondary structure. The pattern “W**W**W” or W 2 W 2 W] as the input query in TP-DB is used as the search query. We are interested in finding other helical peptides in TP-DB with the following three characteristics—(1) containing the W2W2W motif, (2) maintaining helicity in isolation (with relatively high HP), and (3) enriched with positively charged residues so that the corresponding part in W3_p1 or W3_p2 can be replaced by the TP-found helical stretches  containing 11 unique “W**W**W” motifs can be found through the query W 2 W 2 W] (Table  W 2 W (T90) at which the growth of 90% of the pathogenic fungus Candida albicans SC5314 and Escherichia coli ATCC 25922 can be inhibited. Experiments are performed in pentaplicate and MIC90 is determined as the majority results of the 5 replicates (see “Methods”). It is worth mentioning that we could reproduce the reported MIC90 values for three published AMPs when using the same culture protocol to culture E. coli ATCC 25922. They are CM15, Anoplin-R5KT8W, and FK13 with reported MIC values of 4.013, 15.140, and 27.541 μg/ml, respectively, while we obtained 6.3, 12.5, and 25 μg/ml for the same. Our results  and W3_p2 (7.5 μg/ml), while it is a level lower in its bactericidality with a MIC90 of 30 μg/ml than that of W3_p1 (15 μg/ml) and W3_p2 (15 μg/ml). The latter result is consistent with the MD-based natural insertion results  inserted into the bacterial membrane by the end of 1 μs simulation (Supplementary Movie 5) to lyse 5% of the RBCs, where W3_db5’s MHC5 can be much larger than 240 μg/ml, which implies even a high concentration of W3_db5 can still be potentially used in medication or personal hygiene products.Although our newly designed W3_db5 has similar anti-bacterial and anti-fungal activities as its mother templates’, it has surprisingly low cytotoxicity as compared with the template AMPs. As shown in Fig. 90 given the concentration range we have tested (the highest at 120 μg/ml) (see Table th unique sequence in Table 90 within the concentration range we have tested (the highest at 120 μg/ml) -W-x(2)-W” and S as the sequence of W3_p1 are used to search the PDB via the NCBI PHI-BLAST website. A single-chain protein (PDB: 4F54) from the DUF4136 family with a relatively high resolution (1.60 Å) contains a stretch of “WGN WGG W”, located at the end of a helix connecting toward a beta-sheet through a short loop. We synthesize a peptide “KK WGN WGG WRL KK”, coined as “W3_n1”, which replaces the “WKC WAR W” part in W3_db5 with the “WGN WGG W”. When examining its MIC, we fail to find its MICIt should be noted that the original protein  that contains this potent stretch “WKC WAR WRL” in W3_db5 is not a transmembrane or membrane-bound protein; the helical stretch is situated close but not fully exposed to the water-protein interface. Therefore, TP-DB brings an interesting opportunity for researchers to put together structurally resolved elements, matching the desired pattern, for the rational design of therapeutics, herein showcased by a new class of AMP that is 6 point mutations away from its original templates—W3_p1 and W3_p2.42. The resulting AMP, W3_db5, can still penetrate the peptidoglycans, reach the lipid-water interface, stay amphiphilic and helical, and eventually insert into the cell membrane. This is difficult to achieve by mutagenesis experiments or MD simulations without the guidance of TP-DB.From the design point of view, these 6 point mutations do not seem to change much the required insertion thermodynamics43. This low helicity is consistent with the fact that the “WGN WGG W” stretch in W3_n1 takes mostly coil and sheet structures in its original PDB file (PDB ID: 4F54). The online software DichroWeb44 provides a helicity percentage prediction for Tat, W3_n1, W3_n2, W3_db5, W3_p1, W3_p2, and CM15 based on their spectra data over the wavelength 190 to 260 nm wavelength −6.5, 2.0, 18.3, 60.2, 57.8, 51.2, and 54%, respectively. Interestingly, the helicity percentage has a correlation coefficient of 0.78 with the HP (log(HPNA)) of aforementioned peptides −0.406, 0.011, 1.418, 1.675, 2.012, 1.940, and 0.852, respectively.Taking our peptides, we would like to see whether the sequences extracted from TP-DB can be more helical in its isolation than similar ones (sequences with the same pattern) suggested by other methods . Our circular dichroism (CD) data for W3_db5, W3_n2, and W3_n1, flanked by the same residues in the template , showed that the three have different degrees of helicity . However, with the pattern-based search engine  made available in TP-DB, one can do point mutation(s), by choosing the residues suggested by TP-DB, in a helix to abolish or enhance (see Supplementary Information) its interaction with certain drugs, peptides or proteins while still maintaining the helicity of the helix, as well as to abolish the catalytic function of a residue on an α-helix while maintaining its binding affinity with certain substrate or ligand in the scenario where a structural biologist co-crystalizes an enzyme with its cognate substrate. In the Supplementary Information, we demonstrate an in silico study on how peptide blockers can be designed by TP-DB to block a helix–helix interface between Sgo1 and PP2A.Our statistics in Table 45 and Lifson–Roig46 coil–helix transition models suggested that the propagation of a helix can occur more easily than nucleation of a helix from the coil state. This suggests that, given our helical templates, point mutations (substituted residues) can be more likely in their helical forms than it following a coil structure. In our AMP and anti-tumor peptide design, a pattern with functional importance is first known and then pattern-matched helical stretches are found by TP-DB to suggest structurally “suitable” residue substitutions for the next-round selection to further address the functional need. The anchoring residues  have to be in the found helical stretches and consistently serve as the first neighbors of “x” before and after the stretch replacement . The H-bond formation between any residue and its third or fourth neighbors throughout the “WKC WAR WRL” is evidenced in earlier structural determination. On the other hand, whether “WCK WRA WRL”, having the same HP as W3_db5, would result in the same helicity is less assured than what was experimentally determined , is not necessarily helical in 0.1 M NaCl solution based on previous circular dichroism data47 despite the tryptophan having the second-highest HP  value, HP, for each of these 37 peptides. In Fig. To further quantitatively estimate how helical the TP-DB peptides in their isolation can be, including AMPs, already in isolation, and TP-DB-stored peptides, we conducted the following aiming to provide the helicity percentage estimate as a function of HP, peptide concentration, and average residue contact in peptides’ original proteins. Here, helicity percentage is simply defined as the percentage of the residues in a peptide being in the helix form. First, from a PDB search and literature survey, we compiled an “isolated peptide set” comprising 37 NMR-resolved helical peptides with a helicity percentage from 37% to 96%  has 65 and 35% helicity percentage when structurally resolved by NMR and CD, respectively49. The CM15 we used for control in the CD experiment was previously found to have a 65% helicity percentage (compared to our measurement of 54%) by CD50 but 80% helicity by NMR1. To reflect such a concentration dependency of helicity percentage, we surveyed the concentration of AMPs in their structural determination for our isolated peptide set (Supplementary Table NA) is considered, and an estimated error  has a higher helicity percentage than that of the same measured by CD (at a lower concentration). For instance, an intragenic AMP (PDB ID: 6VLA) has a helicity percentage of 94% according to its NMR-resolved structure but 59% by CDFor the above analyses, we did not take the average residue contact into account because all these AMPs were already in isolation when they were structurally solved and had an average contact ranging from 6.0 to 7.2 (95% of the data). We also noted that the isolated peptide set did not exhibit expected dependency between helicity % and concentration, evidenced by the −0.03 coefficient for concentration. However, as only 4 CD-measured AMP data were included in the correlation, the coefficient started to grow toward zero (−0.009). In fact, if another three non-AMP peptides  were included, the pre-concentration coefficient would become +0.043. Given the unbalanced data where our equations resulted from more NMR- than CD-solved structures, these equations could be more suitable to predict helicity% observed by NMR experiments.NA), we chose 8 TP-DB peptides having an average residue contact that is within −2 S.D. lower and +4 S.D. higher than the average value (0.89). Together with this, another 15 peptides were chosen to have a contact fixed at 8.0 to 8.25 and log(HPNA) values ranging from −2 S.D. lower and +2 S.D. higher than the average value (0.38). The 23 TP-DB peptides, each of which has a length of 13 or 14 residues, are detailed in Supplementary Table 51 and have the lowest log(HPNA) in Table 52 to report the averaged helicity percentage  for each peptide. Our linear regression showed that Helicity Percentage = 0.597 − 0.037 × Avg. Res. Contact + 0.076 × Helical Propensity . In a similar vein, the coefficient before average residue contact is −0.037, which also agrees with our earlier surmise that higher tertiary contact in peptides’ original protein environment may lead to a slightly lower helicity% in their isolation.In the above regression models, the coefficients before HP for the isolated peptide set and TP-DB peptide set are +0.067 and +0.076, respectively, agreeing with our expectation that observed helicity% grows with log values of all the 4 residues among all the 4-residue windows in a peptide, representing the strongest folding nucleus in that sequence, and examined the correlation between Max_sum4 and MD-revealed helicity percentage. We indeed found a correlation of 0.63 when examining the “wide type” + 5 mutants and 0.73 for the 5 mutants , in addition to the fact that the W2W2W motif can already be flanked by residues in their helical form and/or other positively charged residues in the N/C-termini.To conclude, the pattern-based (without relying on sequence homology) and BLAST-based (relying only on sequence homology) search engines for the protein sequences that have been observed to have a specific type of secondary structures, herein α-helices, were established and implemented online with efficiency to find pattern-matched sequences from 1.67 million peptides within usually half a minute. With these search engines, to introduce mutations for gain of function while securing a desired secondary structure was made possible for the first time. With such a facility, we introduced how one can utilize our database to design a new helical AMP that was later verified to be anti-fungal and anti-bacterial, while its high helicity% was verified by circular dichroism spectra. We also demonstrated that one can use this database to design helical-peptide blockers to prevent a tumorigenic protein–protein interaction. Improved therapeutics can therefore be designed not only with known functional sequence motifs but also with desired secondary structures, while the helicity% for our TP-DB in isolation, if needed, can be estimated from our parameters including HP and average residue contact. We also foresee its future extension to incorporate search engines for beta-sheet elements containing specific sequences and/or patterns to meet desired physicochemical traits with a design purpose.23 files of 130,000+ experimentally determined protein structures as of June 2017 and extracted the locations of secondary structures from the header part of the PDB files. There are 96,711 unique PDB IDs/files containing at least one alpha-helix documented at the header area to result in 1,676,087 structurally resolved helices having a length longer than 5 residues . In addition, for a given helix, its contacting neighbors in 3D space and the adjacent helical peptides that are within 4 Å (per heavy atoms) were also collected and analyzed.To obtain the amino acid sequences that fold into helices in nature, we processed the PDBa.a. is the HP of a given amino acid type and NAa.a. is natural abundance of that amino acid , and NAa.a. is the count of that amino acid in the 130,000+ proteins in PDB, divided by the total number of residues in these proteins. A log-odds ratio, log (HPNA) or log (HPa.a./NAa.a.) can be found for each amino acid type, and the reported HP for each peptide in TP-DB is the sum of log (HPNA) for every constituent residue in this peptide  their normalized HP (default) or by their contact scores. Using the data collected in the TP-DB, each of the 20 amino acids has a normalized HP, HPNA that is HPa.a./NAa.a., where HPid Table . Both HP29 were prepared by CHARMM-GUI server53. The AMPs and lipid bilayers were solvated in explicit TIP3 water molecules54. Neutralizing ions were added to maintain a concentration of 100 mM. The initial area per lipid of PC was equilibrated to stay close to its experimental values measured at 303 K, which is 68.3 ± 1.5 Å/[Acontrol+ − Acontrol−], where Acontrol+ and Acontrol− are the measurement at wavelength 450 nm for the positive and negative controls, that are 100 μl 10% RBCs suspended in 100 μl PBS buffer with and without 2% (v/v) Triton X-100 (a known detergent to lyse the RBCs), respectively. The final hemolytic percentage for AMPs at various concentrations was determined by the mean values of three repeated experiments, and MHC5 value, defined as the minimal concentration of AMPs leading to at least 5% hemolysis of RBCs, can be determined.The hemolytic toxicity was determined by the hemolysis against human RBCs as in previous publicationThe hemolysis protocol was pre-approved by the Research Ethics Committee of the National Taiwan University Hospital . The blood collections were performed by trained phlebotomists to confirm the safety of the donors. The relevant informed consents were provided to the donors to obtain their agreement on this experiment prior to the process, and the nutritional supplements as compensation for research participants were also provided after the operation.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Movie 1Reporting Summary


The total tocopherol content ranged from 118.92 μg g−1 to 344.02 μg g−1. Accessions from the USA had the highest average concentration of γ- and total tocopherols , whereas a higher level of α-tocopherol (12.82 μg g−1) was observed in the Russian accessions. The maturity group of the accession significantly (p < 0.001) influenced all tocopherol components, and higher levels of α-, γ-, and total tocopherols were observed in early maturing accessions, while late-maturing accessions exhibited higher levels of δ-tocopherol. The inclination of tocopherol concentrations with various MGs provided further evidence of the significance of MG in soybean breeding for seed tocopherol components. Furthermore, the correlation between the seed tocopherol components and geographical factors revealed that α-, γ-, and total tocopherols had significant positive correlations with latitude, while δ-tocopherol showed an opposite trend. The elite accessions with high and stable tocopherol concentrations determined could be used to develop functional foods, industrial materials, and breeding lines to improve tocopherol composition in soybean seeds.Tocopherols are natural antioxidants that increase the stability of fat-containing foods and are well known for their health benefits. To investigate the variation in seed tocopherol composition of soybeans from different origins, 493 soybean accessions from different countries  belonging to 7 maturity groups (MG 0–VI) were grown in 2 locations (Beijing and Hainan Provinces of China) for 2 years (2017 and 2018). The results showed that significant differences ( Glycine max (L.) Merr.] seeds are consumed worldwide, owing to their nutritional values and health benefits. Soybean seeds are the principal source of natural tocopherols. Tocopherols are natural antioxidants that improve the stability of fat-containing foods and have vital biological functions as well as protect food from oxidation processes induced by free radicals [Soybean [radicals . They airadicals ,3,4,5. Tradicals . The antradicals . Alpha- radicals . BecauseTocopherol content and composition in soybean seeds are affected by both genetic and environmental factors. High temperature and drought stress during seed maturation enhance the content of α-tocopherol in soybean seeds by two to three times, with a decrease in δ-tocopherol content ,10. On tIt is widely acknowledged that introducing new germplasms is essential for increasing genetic diversity and strengthening breeding stock resources. Traditional breeding techniques have been using a diverse set of germplasm to produce novel soybean lines with desirable tocopherol composition. The availability of germplasm resources plays a crucial role in the success of breeding techniques. This will allow breeders to develop new cultivars with improved tocopherol profiles and broaden soybean cultivar genetic diversity, which has been narrowed down due to the overuse of a few superior cultivars in breeding programs .Germplasm collections greatly vary in their origins; how the effect of country of origin in combination with other factors such as MG, influences tocopherol composition features of soybean seeds simultaneously have not been adequately explored. In an attempt to fill this apparent gap, using similar environmental conditions, this study utilized four different soybean collections that originate from the USA, China, Russia, and Japan with varying MGs to ensure maximum sample heterogeneity. Therefore, we hypothesized that the country of origin and MG influence had a significant effect on the tocopherol composition of soybeans when they are grown in similar environments, compared to the null hypothesis that both the country of origin and MG influence did not significantly affect composition of tocopherol in soybeans.Therefore, the current research was carried out in order to (i) investigate the variation in seed tocopherol composition and evaluate the content of tocopherols in soybean germplasms of different origins, (ii) evaluate the impact of MGs on seed tocopherol composition, (iii) visualize the geographical distribution of MG materials across different latitudes, and (iv) identify elite soybean accessions with a high and stable concentration of tocopherols across environments. Furthermore, the geographical distribution map facilitated the identification of hotspot regions representing the origin zone of the desired MGs and the selection of target MG accessions with the intended tocopherol composition. The findings of this study will open up new possibilities for the food industry and soybean breeders in terms of developing new soybean varieties with high and stable tocopherol content.p < 0.001) among the soybean accessions  from the USA, probably due to the presence of comparatively higher content of γ-tocopherol in the USA soybean accessions  [−1) from Russia  ,20 and tm Russia , represe−1 to 33.38 μg g−1 with an average of 10.84 μg g−1 (−1) from China, whereas ZDD12828 (3.15 μg g−1) from China had the lowest level, indicating a more than 10-fold difference between the highest and lowest-containing accessions. This finding is consistent with earlier research [For the individual tocopherols, the α-tocopherol levels ranged from 3.15 μg g4 μg g−1 . The higresearch ,16. Acceresearch . Moreoveresearch ,22. −1 to 220.91 μg g−1 with a mean of 143.15 μg g−1 (−1) from the USA, which may be attributed to the presence of γ-tocopherol as the most prevalent form of vitamin E in the US diet [−1) from Russia  from Russia, perhaps due to the presence of low δ-tocopherol in the accessions of high-latitude regions [−1) from China  among the germplasm origins for α-, γ-, δ-, and total tocopherol contents (p < 0.05) observed between Chinese and USA soybean accessions in terms of individual and total tocopherol contents , followed by the Japanese (228.50 μg g−1) and Chinese accessions (227.22 μg g−1), whereas the Russian accessions revealed the lowest total tocopherol content (203.58 μg g−1)  . The tot−1), followed by the Chinese (11.07 μg g−1) and Japanese accessions (10.91 μg g−1), while the lowest level of α-tocopherol (9.94 μg g−1) was found in the USA accessions, which differed significantly from the other three germplasm origins , followed by the Japanese (145.81 μg g−1) and Chinese accessions (139.64 μg g−1), while the lowest γ-tocopherol (136.75 μg g−1) was recorded from the Russian accessions , followed by the USA (75.35 μg g−1) and Japanese accessions (71.78 μg g−1), while the Russian accessions contained the lowest mean δ-tocopherol level (54.01 μg g−1) (−1) and the lowest mean content of δ-tocopherol (54.01 μg g−1). This demonstrates the possibility of decreasing δ-tocopherol levels with increasing α-tocopherol levels. Such an inverse connection between α- and δ-tocopherols was also evident in numerous previous studies [The Chinese accessions displayed the maximum mean content of δ-tocopherol (76.52 μg g μg g−1) . The Rus studies ,17,21,22The Russian and Chinese accessions exhibited no significant differences in α- and γ-tocopherol contents due to the closeness of the Heilongjiang province of China and the far-eastern region of Russia, which are the central regions for soybean production in China and Russia and share similar MGs . Overallp < 0.001) for individual and total tocopherol contents among different maturity groups  with maturity groups    . The meaectively . There w in MG 0 . Therefo 0.91**) , indicat 0.91**) . These fPCA is a clustering technique commonly used to discover variables between populations of multiple categories, identify key influencing parameters, and reveal interactions between parameters . The appThe PCA results revealed that the first two components (PC1 and PC2) accounted for 83.4% of the total variance in tocopherol compositions. The first axis (PC1) explains 50% of the total variance, and the principal contributor to the variance in PC1 was α-tocopherol (50.10%), followed by δ-tocopherol (27.08%) and γ-tocopherol 22.81%). The second axis (PC2) explains 33.4% of the total variance, and most of the variance was by γ-tocopherol (54.23%), followed by δ-tocopherol (45.76%), while α-tocopherol had the lowest contribution to PC2 variability (0.00002%). From the biplot, δ-tocopherol is inversely correlated with α-tocopherol; such a contrasting relationship has been observed previously [.81%. TheLength of growing period or maturity is a vital feature of crops since it influences the geographical adaptation of a variety . Soybeanr = 0.66***, 0.49***, and 0.13**, respectively)  , indicatctively) .r = −0.56***)  , reflect0.56***) . These f0.56***)  clearly Moreover, we also observed that α-, γ-, and total tocopherol level of soybean accessions planting in Hainan were significantly higher than in Beijing . This coSoybeans are cultivated across a broad latitude range (about 20 to 53° N) under a wide range of environmental circumstances in China . The sta−1) with lower CV (18.14%), followed by another Chinese accession ZDD23615, as its α-tocopherol content was 31.35 μg g−1 with a lower CV of 7.42%. Other accessions with higher and stable α-tocopherol content are presented in Accessions having a higher content of α-tocopherol and lower CV values A are mor−1) followed by another USA accession WDD01715 (199.44 μg g−1), but their CV values  were higher than the average CV values (11.96%) of γ-tocopherol, indicating that these genotypes were less stable (−1) with lower CV (5.08%), followed by WDD02019 from the USA, which had a higher γ-tocopherol content (185.16 μg g−1) with a lower CV of 10.32%. Other stable accessions with higher γ-tocopherol levels are shown in A collection of the USA, Chinese, Japanese, and Russian accessions with high γ-tocopherol content are shown in s stable B. The re−1 with a CV of 9.29%, followed by ZDD06595 from China, which had a higher δ-tocopherol content (129.54 μg g−1) with a lower CV of 9.30%. Other accessions with higher δ-tocopherol levels demonstrating greater stability are listed in Accessions with a higher level of δ-tocopherol and lower CV values C are als−1), but it seems unstable due to a higher CV value (14.25%) (−1) at different sites  D. Based nt sites . Other ant sites  showed tSoybean is a short-day plant that is extremely sensitive to photoperiods . This hiStandards of α-, γ-, and δ-tocopherols purchased from Sigma-Aldrich Chemical Company  were used for the quantification of tocopherol components. High-Performance Liquid Chromatography (HPLC) grade ethanol  and methanol  were used as an extraction solvent and mobile phase, respectively. All other chemicals used in the present study were of analytical grade.A total of 493 soybean accessions, including 326 Chinese, 123 USA, 25 Japanese, and 19 Russian accessions covering 7 MGs from MG 0–VI, were employed in this investigation. The accessions were chosen based on their availability at the soybean genetic resource research group of the Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS). The accessions were divided into distinct MGs as MG 0 (68 accessions), MG I (83 accessions), MG II (79 accessions), MG III (96 accessions), MG IV (82 accessions), MG V (54 accessions), and MG VI (31 accessions). MG III contained the maximum number of accessions that were collected from various locations. Accession number, accession name, origin, and MG of all accessions are listed in −1, 68.7 mg kg−1, and 12.31 g kg−1, respectively, at Changping and 98.59 mg kg−1, 39.68 mg kg−1, and 80.78 g kg−1, respectively, at Sanya. The experimental details, as well as plot management operations, have already been reported [−1, which contained 30 kg N ha−1, 60 kg P ha−1, and 50 kg K ha−1. Weeds were controlled by postemergence application of 2.55 L ha−1 of acetochlor, as well as hand weeding during the growing season. When plants reached physiological maturity, plots were harvested manually. After harvesting, all the seeds of each accession were pooled together, and about 100 seeds were randomly picked from each accession to determine tocopherol levels.Field experiments were carried out in two different locations: Changping, Beijing (40°13′ N and 116°12′ E), and Sanya, Hainan (18°24′ N and 109°5′ E) in 2017 and 2018. The accessions were planted on 12 June 2017 and 14 June 2018 in Changping and 14 November 2017 and 16 November 2018 in Sanya. The locations differ significantly in climate. The monthly temperature (°C), precipitation (mm), and sunshine (h) readings for the experimental sites in Hainan and Beijing are presented in reported . Briefly−1 of the soybean seed sample in fresh weight.Tocopherol components were detected from matured soybean seeds using a reverse-phase HPLC  system following the method described by Dwiyanti et al. (2007)  with somr), scatter plots, and boxplots were analyzed and visualized using R statistical software version 3.5.0 . Scatter plots were utilized to show the association between different MGs and tocopherols and the relationship between the mean of each tocopherol component and its CV. Differences were considered significant in all cases when the P-value was less than 0.05 (p < 0.05). The discriminative tocopherol components in soybean seeds were visualized and identified using PCA. The correlation analysis was carried out between the components of seed tocopherol and geographical variables . A geographical distribution map of world soybean MGs was developed with ArcGIS 10.0  using ordinary kriging interpolation. ArcGIS is extensively used in geographic information system (GIS) technologies since it can be effectively used to create maps, compile geographical data, and preserve spatial data.Descriptive statistics were primarily used to interpret the data for each tocopherol isomer across environments. Minimum, maximum, mean, standard deviation (Std.), and coefficient of variation (CV) of individual and total tocopherol contents were estimated using Excel software . Analysis of variance (ANOVA) for seed tocopherol composition was performed using the general linear model technique (PROC GLM) in SAS 9.2 . Three sThe variation observed in tocopherol composition among four worldwide germplasm accessions may provide elite genetic resources for soybean breeding. The accessions from the USA had the highest average concentration of γ- and total tocopherols, whereas a higher level of α-tocopherol was observed in the Russian accessions, which illustrates the influence of ecoregions on individual and total tocopherol contents of soybean accessions. This study may also provide relevant information to consumers and food sectors when evaluating soybean accessions from different MGs in terms of tocopherol content. To the best of our knowledge, the present study is the first to address the response of soybean tocopherols to MGs where it was indicated that higher levels of α-, γ-, and total tocopherols were observed in early maturing accessions, while higher levels of δ-tocopherol could be obtained from cultivating late-maturing accessions. MGs are useful in evaluating this chemical component, which mainly determines the shelf life and flavor of soybean oil. Furthermore, the geographical map can be used to identify hotspot regions where MGs possess greater levels of desired tocopherol components. Overall, the soybean accessions with high stability and desirable tocopherol content identified in this study could be useful for consumption and cultivar development.These findings would help make decisions early in the selection of elite soybean with desired levels of both individual and total tocopherols. This may encourage researchers to develop future investigations for further molecular and metabolic research works to validate the usefulness of this bioactive chemical component in improving human feeding.


The responses of tropical forests to heat and drought are critical uncertainties in predicting the future impacts of climate change. The 2015–2016 El Niño Southern Oscillation (ENSO) resulted in unprecedented heat and low precipitation across the tropics, including in the very poorly studied African tropical forest region. We assess African forest ENSO responses using on-the-ground measurements. Across 100 long-term plots, record high temperatures did not significantly reduce carbon gains from tree growth or significantly increase carbon losses from tree mortality. Overall, despite the climate anomaly, forests continued to gain live biomass over the ENSO period. Our analyses, while limited to African tropical forests, suggest that they may be more resistant to climate extremes than Amazonian and Asian forests. −1 y−1) despite extreme environmental conditions. Our analyses, while limited to African tropical forests, suggest they may be more resistant to climatic extremes than Amazonian and Asian forests.The responses of tropical forests to environmental change are critical uncertainties in predicting the future impacts of climate change. The positive phase of the 2015–2016 El Niño Southern Oscillation resulted in unprecedented heat and low precipitation in the tropics with substantial impacts on the global carbon cycle. The role of African tropical forests is uncertain as their responses to short-term drought and temperature anomalies have yet to be determined using on-the-ground measurements. African tropical forests may be particularly sensitive because they exist in relatively dry conditions compared with Amazonian or Asian forests, or they may be more resistant because of an abundance of drought-adapted species. Here, we report responses of structurally intact old-growth lowland tropical forests inventoried within the African Tropical Rainforest Observatory Network (AfriTRON). We use 100 long-term inventory plots from six countries each measured at least twice prior to and once following the 2015–2016 El Niño event. These plots experienced the highest temperatures and driest conditions on record. The record temperature did not significantly reduce carbon gains from tree growth or significantly increase carbon losses from tree mortality, but the record drought did significantly decrease net carbon uptake. Overall, the long-term biomass increase of these forests was reduced due to the El Niño event, but these plots remained a live biomass carbon sink (0.51 ± 0.40 Mg C ha S1). The 1970 to 2017 temperature record (T) includes the monthly adjusted CRU data from 1970 to 1978 and ERA-I monthly records from 1979 to 2017.We compare each of the 1982–1983, 1997–1998, and 2015–2016 El Niño events with the climate of the prior decade over African forests. This requires a continuous record from the early 1970s to 2017. We use mean monthly temperatures (0.25° resolution) from the ECMWF Re-Analysis-Interim (ERA-I) dataset for dates from 1979 to 2017 . For the2 using WorldClim Version 2 (Tdiff = Tμ − TWorldClim) of the mean climate Tdiff is then used to create a plot-level monthly temperature 1970 to 2017: Tplot = T – Tdiff. Temperature values were then additionally adjusted for any difference in altitude between the plot and the altitude of the 1-km grid cell used for WorldClim interpolation using a lapse rate, so that Tplotalt = Tplot + 0.005 × (AWorldClim – Aplot), where T is temperature (degrees Celsius) and A is altitude (meters above sea level).We also compare each El Niño with the prior decade for only the monitored plot locations by downscaling our climate data to 1 kmersion 2 . We downSI Appendix, Fig. S2). Hence, the precipitation record (P) includes data from TRMM from 1998 to 2017 and prior to this, adjusted GPCC data.Similarly, continuous precipitation records are required from 1970 to 2017. We use 0.25°-resolution data from the Tropical Rainfall Measurement Mission  from 1998 to 2017 . Prior t2 resolution using WorldClim . The GPersion 2 , the monThe drought intensity experienced by plots was estimated as the maximum cumulative water deficit (MCWD). The MCWD is calculated as in ref. WD = water deficit, n = month, ET = evapotranspiration, and P = precipitation.where −1 as has been used in previous studies . For the 2015–2016 El Niño census interval, we select the maximum annual MCWD value, as we are interested in the most extreme climate conditions within the El Niño sampling interval for each plot.https://www.forestplots.net/ . When only one accurate measurement was available, growth was estimated by applying the mean growth rate (for diameter classes from 100 to 199 mm and from 200 to 399 mm) or median growth rate for size classes with few stems .Field data were checked against rules to identify potential errors, identically for all 100 plots, consistent with previous large-scale analyses –8, 32. WWe estimate tree aboveground mass using the allometric equation AGB=0.067−3), D is stem diameter (cm) at 1.3 m or above buttresses, and H is height (m). We estimate the aboveground mass of palms using the equation in ref. https://datadryad.org/stash) (https://www.ville-ge.ch/musinfo/bd/cjb/africa/index.php?langue=an) to maximize matches (where ρ is stem wood density (g cmg/stash) , 52, and matches .SI Appendix, Table S2) were used to estimate tree height from tree diameter for all stems for input into the allometric equation.Tree heights were measured in 93 plots: typically, the 10 largest trees and 10 trees in each of the diameter classes 100 to 199, 200 to 299, 300 to 399, 400 to 499, and 500 to 599 mm were measured, with trees selected only when the top was visible . We fit We estimate the aboveground biomass in live stems (AGB) in megagrams dry mass per hectare, at each census of each plot; the additions of biomass to each plot over the census interval, as aboveground woody productivity (AGWP), were in megagrams dry mass per hectare per year, and the losses of AGB from the plot, termed AGB mortality, were also in megagrams dry mass per hectare per year. Plot-level carbon gains and losses are increasingly underestimated as census length increases; therefore, to avoid any census interval bias, we corrected for this using the Kohyama method . We thusWe calculate an analogous set of parameters to AGB carbon gains and carbon losses on a stems basis. We estimate stem density (the number of stems per hectare at a census), stem recruitment , and stem mortality . Again, to avoid census interval effects, we estimated stem recruitment and stem mortality following Kohyama et al. , accountWe use the BiomasaFP R package to calculate AGB, AGWP, AGB mortality, stem density, stem recruitment, and stem mortality, including the calculation of the census interval corrections. Pre-El Niño means of these variables are time weighted using the census interval lengths. We express AGB, AGWP, and AGB mortality in carbon terms  using the mean carbon fraction of aboveground biomass for tropical angiosperms, 45.6% . The dif1/6 + Area1/8 − 1; Δ carbon gains, Monitoring length1/4; Δ carbon losses, Monitoring length1/3; Δ net stems, Monitoring length1/2 + Area1/4 − 1; and no weighting for both recruitment and stem mortality. As most plots are relatively similar in size and census monitoring length, the weighting does not affect the results .We weight the plots when testing the impacts of climate  on biomass carbon  and stems  using linear regression  and 3 beWe test the impacts of climate  on biomass carbon  and stems  using multiple linear regression. We include pre-El Niño climate in models to test whether plots that were already hotter (pre-El Niño temperature) or plots that were already drier (pre-El Niño MCWD) were more or less resistant to environmental change . We include interactions in our models as we might expect greater impacts in locations that are hotter pre-El Niño and experience a greater temperature anomaly or locations that are drier pre-El Niño and experience a greater MCWD anomaly; plus, high temperatures may exacerbate water deficits. Variables were standardized to allow effect size comparisons. All possible combinations of effect terms were calculated and then restricted to a 95% confidence set (Akaike information criterion [AIC] weights of models sum to 0.95), thereby excluding highly unlikely models. We then model averaged the coefficients of terms (using the AIC weights of each model), meaning that terms with limited support exhibit shrinkage toward zero . This muFinally, we test whether the results are robust to the methodological choices necessary to analyze the data. We repeat the main analyses with alternatives; we do not weight the plot data based on plot size and pre-El Niño census monitoring length; we extend the pre-El Niño censuses back to 1984; we test our assumptions associated with the MCWD drought metric; we use an alternative census interval correction procedure; we use an alternative R package, BIOMASS , to estiOur 50-y climate record shows that the long-term climate trends across African tropical forests are rising temperatures, decreasing precipitation, and stronger seasonal moisture deficit . The thr−1, compared with the prior decade, and increased seasonal drought intensity, measured as a more negative MCWD, by ∼30 mm y−1 compared to the previous decade . These anomalies are smaller than those for the 12-mo El Niño year because the plot El Niño mean census length was 2.7 y, so the climate anomaly is diluted by the inclusion of months of more usual conditions. The 100 monitored plot locations are hotter, wetter, and less droughted than the region as a whole because the wider region also includes currently degraded fringes of the biome, but both the plots and region have similar trends of increasing temperature, decreasing rainfall, and stronger seasonal drought , low total annual precipitation , and a record low MCWD, with mean −261 ± 2 mm (April to May year) . Compari drought .P ≤ 0.05)  B. Thus, esponses . DroughtSI Appendix, Table S6. The responses of carbon gains, carbon losses, and the net carbon sink in live biomass remain very similar if we do not weight the plots by size and pre-El Niño monitoring length , include a longer pre-El Niño plot monitoring length back to 1984 , employ an alternative census interval correction procedure , use different drought metrics , use an alternative R package to analyze changes in carbon stocks , or remove potential allometric uncertainties by analyzing changes in basal area . In these cases, the slopes of the temperature relationships remain very similar and are not significant . However, for the temperature anomaly–carbon gains relationship, if we exclude four plots that cooled during their El Niño census interval , then the temperature–carbon gains linear model has a significant positive slope (P = 0.045) , although when our multimodel inference excluding the same four plots is repeated, the positive temperature–carbon gains effect is nonsignificant (P = 0.1) . In the case of MCWD, again slopes remain very similar and relationships are always at the margins of significance under the various methodological choices, with stronger drought decreasing carbon gains and increasing carbon losses . Calculating MCWD using variable evapotranspiration (which includes temperature) does not alter the slope of the relationships, but it improves significance of the relationship with carbon gains and losses , further suggesting that drought impacted African forests but only modestly.The lack of a negative temperature response and the modest negative drought response are both robust to the various methodological choices necessary to analyze the data, summarized in −1 y−1; paired t test, P = 0.07) (P = 0.03) . Growth responses by size class indicate that small trees grew less during the El Niño census interval. Small trees had significantly lower median growth rates in the El Niño compared with the pre-El Niño measurement period , as did the medium size-class trees . The median growth rate of large trees (400+-mm diameter) was also lower but did not significantly decline . Hence, the growth of smaller trees appeared to be more negatively impacted by the El Niño conditions than the growth of large trees.Plot-level carbon dynamics over the 2015–2016 El Niño were similar to those pre-El Niño. Carbon gains from tree growth and the recruitment of new trees were 4.4% lower in the interval spanning the 2015–2016 El Niño (mean 2014.3 to 2017.1) compared with the pre-El Niño censuses (mean 2008.4 to 2014.3), a marginally significant difference (2.59 ± 0.15 to 2.48 ± 0.14 Mg C ha = 0.07) A. Exclud−1 y−1; paired t test, P = 0.4) (P = 0.30) .There was a nonsignificant 10% increase in carbon losses from mortality in the El Niño compared with pre-El Niño censuses (1.79 ± 0.26 to 1.97 ± 0.39 Mg C haP = 0.4) A. Exclud−1 y−1  (P = 0.11) . Despite the extreme conditions, the 100 plots remained a significant net aboveground live biomass carbon sink over the census interval that included the 2015–2016 El Niño, at 0.51 ± 0.40 Mg C ha−1 y−1. The El Niño had modest impacts on the overall carbon dynamics of the 100 plots studied. While drought significantly impacted the aboveground live biomass carbon sink in plots  A. Excludin plots , not enoin plots .SI Appendix, Fig. S3). Indeed, recruitment is the process most responsive to the climate anomaly, responding positively to temperature and negatively to drought . The increase in stem recruitment with temperature could potentially have been driven by recruitment of low wood density stems, which might have benefited from more light reaching the understory, but there is no evidence of this as the median wood density of recruits did not change . Somewhat surprisingly, stem mortality showed no significant relationship with either temperature or drought anomalies .An analysis of stem dynamics, rather than biomass carbon dynamics, and the potential drivers of change shows, surprisingly, stem recruitment increasing with increasing temperature, and as expected decreasing with more negative MCWD     B. Stem m < 0.01) B. Togeth< 0.001) B.t test, P ≤ 0.05), but this was mostly due to small  and medium , rather than large trees . Furthermore, the median diameter of dying trees decreased significantly from 190-mm diameter pre-El Niño to 174 mm in the El Niño census, a decline of 16 ± 14 mm (P ≤ 0.05). When separated into size classes, the median diameter of dying trees in the smallest size class, 100- to 199-mm diameter, decreased by 4 ± 4 mm , while the median diameter of the other size classes did not change . The median size of surviving trees overall also does not change , and there is no change in the median wood density of dying  nor surviving trees . Overall, stem mortality increased significantly over the El Niño census interval, and it was predominantly smaller trees that died.The significant increase in stem mortality without simultaneous significant increases in carbon losses and the larger increase in stem mortality than carbon losses from mortality  implies −1 y−1. Maintenance of the live biomass carbon sink suggests that African forests were resistant to this short-term extreme climate anomaly, albeit that the carbon sink was maintained at a lower rate. Alternatively, it is theoretically possible that impacts were more substantial but we failed to detect them, perhaps due to limited sampling or because the main impacts of the El Niño are delayed beyond the time of the plot remeasurement, and so the impact is ultimately larger than we estimate.African tropical forests experienced record heat and drought in the 2015–2016 El Niño, yet the 100 inventory plots monitored through this extreme climate anomaly maintained net biomass carbon uptake over the 2.7-y period they were measured, at 0.51 ± 0.40 Mg C haSI Appendix, Figs. S6–S13 and Table S6). Critically, halting the live biomass carbon sink requires large increases in tree mortality, as seen during the Amazon 2005 and 2010 droughts and Bornean 1997–1998 El Niño event . If the impacts were concentrated in just the strongest dry season months, then these forests were a short-term source of 0.58 Mg C ha−1 y−1 for those 3 mo. Under this potential scenario, the immediate return of the forest to a strong sink after being a very temporary strong source could then be interpreted as African tropical forests being resilient rather than resistant to environmental change. However, the lack of strong correlations between the biomass carbon sink and temperature or drought anomalies  and soils. The net of these fluxes is net biome productivity. From an atmospheric perspective, the full impacts of the reduction in the live biomass carbon sink from slowing carbon gains are experienced immediately, but the contribution from rising carbon losses is delayed because dead trees do not decompose instantaneously, meaning that the contribution of biomass losses to the net biome productivity response to the El Niño is also likely to be modest. For intact tropical forest soil carbon, fluxes to the atmosphere are typically lower during drought conditions , 67, sugand 2016 . This in−1  for the years 2000 to 2010 . While mindful that the reduced sink captured by plot measurements does not include necromass and soil fluxes and is extrapolated to selectively logged forests  as well as intact forests, our results are in broad agreement with a recent satellite-derived estimation of the carbon sink in African humid forests . Hence, increased heterotrophic respiration could potentially contribute to the muted response of carbon gains to the climate anomaly that we see. It should be noted that the Liu et al.  or 0.2% of aboveground carbon stocks given the initial aboveground biomass carbon of 164 Mg C ha−1. Droughts in Amazonia had larger impacts. During the 2005 drought (−1 y−1 (0.41 Mg C ha−1 y−1 predrought sink to a loss of 0.32 Mg C ha−1 y−1 during the drought) or 0.5% of their aboveground biomass carbon assuming aboveground biomass carbon of 140 Mg C ha−1 (−1 y−1 (0.61 Mg C ha−1 y−1 predrought sink to a loss of 0.20 Mg C ha−1 y−1 during the drought) or 0.6% of aboveground biomass carbon assuming 140 Mg C ha−1 aboveground biomass (−1 y−1 (0.52 Mg C ha−1 y−1 pre-El Niño sink to a loss of 0.94 Mg C ha−1 y−1 during the El Niño drought) or 0.7% of aboveground biomass carbon assuming 197 Mg C ha−1 aboveground biomass , while peak temperature increases across African tropical forests were relatively short-lived. Nevertheless, these were record conditions regionally, and they failed to cause intact African tropical forests to lose, on average, any live biomass . One reason why African forests may be more drought-adapted than most Amazonian or Southeast Asian forests is due to biogeographic history having favored the persistence, expansion, and perhaps diversification of more drought-adapted species , which implies that rates of hydraulic failure in African tropical forests during the 2015–2016 El Niño only increased slightly, if at all. The nonsignificant 6% increase in stem mortality for trees with 400+-mm diameter is much smaller than the 70 to 100% increase seen as a result of previous droughts observed in Amazonia and Borneo when considering droughts of similar intensity  (P = 0.03) . The African forest decrease in biomass gains, 0.11 ± 0.12 Mg C ha−1 y−1, is a more muted response than the Amazonian forest response to the 2010 drought, where gains reduced by 0.50 Mg C ha−1 y−1 [0.31 to 0.78, 95% CIs; P < 0.001 (P ≤ 0.05), suggesting resistance. While an unexpected result, this could occur if higher temperatures increase mineralization rates and nutrient release  A and a 5 release , particu release , 67, 74. release , 76. Ano release .2 concentration data in Africa showing a carbon sink in 2015 and 2016 in the regions where most of the AfriTRON plots are located  Afri located ; or 3) c located . Our detSupplementary File


Chemoresistance, especially temozolomide (TMZ) resistance, is a major clinical challenge in the treatment of glioblastoma (GBM). Exploring the mechanisms of TMZ resistance could help us identify effective therapies. Adenosine deaminases acting on RNA (ADARs) are very important in RNA modification through regulating the A-to-I RNA editing. Recent studies have shown that ADARs regulate multiple neurotransmitter receptors, which have been linked with the occurrence and progress of GBM. Here, data from several bioinformatics databases demonstrated that adenosine deaminase RNA specific B1 (ADARB1), also named ADAR2, was upregulated in both GBM tissues and cells, and had the prognostic value in GBM patients. Moreover, ADARB1 was found to be involved in AKT-mediated TMZ resistance in GBM cells. The KEGG analysis of ADARB1-associated co-expressed genes showed that ADARB1 was potentially involved in the mitochondrial respiratory chain complex. TISIDB and GEPIA databases were further used to analyze the role of ADARB1 in tumor-immune system interactions in GBM. These findings deepened our understanding of the function of ADARB1 in tumorigenesis and therapeutic response in GBM. Glioblastoma (GBM) is the most malignant and aggressive brain tumor in adults, with a median survival of 12–15 months . The tumAdenosine deaminases acting on RNA (ADARs) are important in post-transcriptional RNA modification, especially A-to-I RNA editing . A-to-I Protein kinase B (AKT) plays an important role in tumorigenesis. A high level of phosphorylated AKT (p-AKT) has been reported to be associated with poor prognosis in GBM patients . InhibitGBM is also a profoundly heterogeneous tumor that facilitates immune evasion. Immunotherapeutic strategies for GBM have a long history, including immune stimulation, antibody-mediated immunotherapies, adoptive cellular immunotherapies, and vaccines . PreviouOur study aims to investigate the molecular mechanism underlying the aberrant expression of ADARB1 in GBM biology. In our study, ADARB1, also named ADAR2, was found to influence the TMZ chemotherapy response. In addition, higher expression of ADARB1 was identified in GBM tissues and cell lines, which was associated with poor prognosis.https://www.ncbi.nlm.nih.gov/gds) (The effects of ADARB1-related chemoresistance of GBM were investigated using multiple bioinformatics databases. The databases are listed in gov/gds) . Oncomingov/gds) . Thus, fgov/gds)  and GSE4gov/gds) , to evalgov/gds) . Chemothgov/gds) , GSE4412gov/gds) , and GSEgov/gds) , were usNext, 19,915 ADARB1-associated co-expressed genes in GBM pathology were downloaded from the cBioportal database . We thenhttps://www.xiantao.love/products). The TISIDB database is an integrated repository portal for tumor-immune system interactions  targeting ADARB1  was purchased from Genepharma , and a scrambled siRNA  was used as the negative control. Transfection was performed using Lipofectamine 3,000 reagent  according to the manufacturer’s protocol. After the indicated incubation times, the cells were harvested and subjected to protein extraction or other cellular experiments.Altogether 10 pairs of formalin-fixed, paraffin-embedded (FFPE) specimens of primary and recurrent GBM tumor tissues were collected from Department of Pathology, Xiangya Hospital . The ethics of our study has been approved by the Ethical Committee of Xiangya Hospital of Central South University. The ethical approval number is 202,110,206. Total RNA was extracted from FFPE tissue specimens using PureLink FFPE RNA Isolation Kit  according to the manufacturer’s protocol, followed by cDNA synthesis using a PrimeScript RT reagent kit . The qPCR was conducted with iTaq Universal SYBR green Supermix . The forward and reverse primer sequences are as follows: CXCL12: 5′- ATTCTCAACACTCCAAACTGTGC-3′ and 5′-ACTTTAGCTTCGGGTCAATGC-3’; B2M: 5′- GAGGCTATCCAGCGTACTCCA-3′ and 5′-CGGCAGGCATACTCATCTTTT-3’; TABPB: 5′- TGGACCGGAAATGGGACCT-3′ and 5′-CCCCAGAAGGGTAGAAGTGG-3’.-△△CT method. All reactions were run at least three times.Total RNA extracted from glioma cell lines was using TRIzol reagent  according to the manufacturer’s protocol. Afterward, total RNA was reverse transcribed to cDNA using the PrimeScriptTM RT reagent kit . The qPCR assay was performed using iTaq Universal SYBR Green Supermix , with b-actin as the internal control. The forward and reverse primer sequences are as follows: β-actin: 5′-CATGTACGTTGCTATCCAGGC-3′ and 5′-CTCCTTAATGTCACGCACGAT-3’; ADARB1: 5′-GTGAAGGAAAACCGCAATCTGG-3′ and 5′-CAGGAGTGTGTACTGCAAACC-3’. Relative expression levels were calculated by the 2Protein extraction was performed as previously described . Afterwa3/well. The value of cell survival was detected at 490 nm using a microplate reader  after a 1-h incubation with MTS solution . The experiments were repeated at least three times, and each experiment was performed at least twice.Cell viability and cell survival were analyzed by MTS assay. After 24 h of transfection, cells were re-seeded in 96-well plates at a density of 3 × 10p < 0.05, and ∗∗p < 0.01 were defined as statistically significant.Statistical analysis was performed with SPSS 12.0 software . All experiments were performed at least three times, and the mean ± SD was subjected to Student’s t-test. Kaplan-Meier analysis was performed to analyze survival rates for GBM. The differential mRNA expression between cancer and non-cancer tissues or between the control group and treatment group were analyzed using Student’s t-test. The associations between ADARB1 expression and clinicopathologic characteristics in GBM patients were assessed using the Kruskal-Wallis rank test or the Mann–Whitney U test. Correlations between genes were analyzed using Spearman’s correlation coefficient. ∗The flowchart of the whole analysis process was shown in p = 0.009) , which were previously constructed in our lab. The colony formation assay results showed that T98G-R and U118-R cells were more resistant to TMZ than their parental cell lines T98G and U118 cells, respectively . The IC5= 0.009) . These rPrevious studies have shown that the AKT signaling pathway is important in the treatment of GBM . TherefoTo further investigate the potential role of ADARB1 in GBM pathogenesis, we performed a functional enrichment analysis of ADARB1 co-expressed genes using the cBioportal database . Studyinhttps://www.xiantao.love/products). Using the TISIDB database, we investigated the correlations between ADARB1 expression and immune-associated biomarkers, such as lymphocytes, immunomodulators, chemokine, and receptors. p = 1.41e-04), NK cells , Treg cells , and NKT cells .Previous studies reported that several immune pathways were associated with the pro-tumorigenic phenotypes of immune cells and protection of tumor cells from the immune attack, ultimately favoring the development and metastasis of tumors . The disp = 1.46e-04), CXCL12 , IL6R , and LTA . p = 0.0493), LAG3 , CD160  and CTLA4 . p = 1.54e-03), TAP2 , TAPBP , and HLA-DMB .Most immunomodulatory therapies have focused on increasing T-cell responses by targeting inhibitory pathways with immune checkpoint inhibitors or by targeting activating pathways. Although these approaches have led to prominent successes, only a minority of patients with cancer benefit from these treatments, highlighting the need to identify new cells and molecules that could be exploited in the next generation of immunotherapy . Immunomp = 2.96e-03), CXCL12 , CXCL1  and CX3CL1 . p = 0,219), CXCR1 , CCR10 , and CCR6 .As molecular messengers, cytokines allow immune system cells to communicate with each other to produce coordination of target antigens, it have regulatory and effector functions in many diseases, thus cytokines and their receptors can be used in immunotherapy . SupplemImmune checkpoint inhibitors (ICIs) have developed in the field of tumor therapy. and ICIs are now first-line therapies for various solid and liquid tumors . FurtherWe also verified the expression of several immune molecules in TMZ response and resistant human GBM tissues. As shown in TMZ resistance in GBM is still a major clinical issue in its treatment . Our stuA-to-I RNA editing is the most abundant RNA editing mediated by ADAR enzymes in mammalian tissues . It has AKT is a serine/threonine kinase activated by a dual regulatory mechanism that requires translocation to the plasma membrane and phosphorylation . High leDue to the limited efficacy of existing therapies, immunotherapy is being widely investigated in patients with GBM. In recent studies, increasing evidence has indicated that tumor microenvironments and immune infiltration are important in tumor development and chemoresistance . ImmunotEmerging studies have demonstrated the important roles of mitochondrial dysfunction in TMZ resistance of GBM patients. TMZ resistance in GBM has been proved to be associated with increased mitochondrial coupling and decreased concentration of reactive oxygen species . RegulatIn conclusion, this study reports the association of the RNA editing gene ADARB1 with the TMZ chemoresistance of GBM for the first time. Aberrant expression of ADARB1 is a promising biomarker for the prognosis of GBM patients. Furthermore, ADARB1 is found to be involved in AKT-mediated TMZ resistance in glioma cells. Therefore, ADARB1 may play an important role in the therapeutic response and immune regulation of GBM.