Asclepias spp.) are important for sustaining biodiversity in marginal ecosystems, CO2 flux may afflict Asclepias species and cause detriment to native communities. Negative CO2-induced effects may be mitigated through mycorrhizal associations. In this study, we sought to determine how mycorrhizae interacts with CO2 to influence Asclepias biomass and root morphology. A broad range of Asclepias species (n = 6) were chosen for this study, including four tap-root species  and two fibrous root species (A. incarnata and A. verticillata). Collectively, the six Asclepias species were manipulated under a 2 × 2 full-factorial design that featured two mycorrhizal levels (−/+ mycorrhizae) and two CO2 levels ). After a duration of 10 months, Asclepias responses were assessed as whole dry weight  and relative transportive root. Relative transportive root is the percent difference in the diameter of highest order root (transportive root) versus that of first-order absorptive roots. Results revealed an asymmetrical response, as mycorrhizae increased Asclepias biomass by ~12-fold, while enriched CO2 decreased biomass by about 25%. CO2 did not impact relative transportive roots, but mycorrhizae increased root organ’s response by more than 20%. Interactions with CO2 and mycorrhizae were observed for both biomass and root morphology . A gene associated with CO2 fixation (rbcL) revealed that the two fibrous root species formed a phylogenetic clade that was distant from the four tap-root species. The effect of mycorrhizae was most profound in tap-root systems, as mycorrhizae modified the highest order root into tuber-like structures. A strong positive correlation was observed with biomass and relative transportive root. This study elucidates the interplay with roots, mycorrhizae, and CO2, while providing a potential pathway for mycorrhizae to ameliorate CO2 induced effects.While milkweeds ( Rcterized ,10,11,12cterized ,14, but cterized , and ecocterized ,17,18. Cdiameter ,20, but ot order , which cot order . Unravel2 may interact with mycorrhizae. Upon C fixation in the shoot, carbon is translocated to roots and metabolized by mycorrhizae [glomeromycotans, CO2 enrichment may not necessarily lead to an increase in arbuscule development [2 flux, fibrous and tap-root systems may vary in modes of nutrient foraging that are independent of mycorrhizal networks. Fibrous root systems are premier foragers at soil depths proximal to organic layers [3−, SO42−), minerals [Root morphology and COorrhizae , but amoelopment . Arbuscuelopment ,25, whilelopment . In respc layers . Meanwhiminerals ,28, and minerals . In compminerals ,31 and sminerals .2. An increase in atmospheric CO2 has been reported to improve lateral and tap-root sizes [2.Root function is a product of root branching order , but stiot sizes . Tap-rooot sizes . Lower oot sizes , which m2 and mycorrhizae on roots, and even fewer studies have addressed these factors in the context of tap- versus fibrous root systems. Here, we address the following questions, (1) are the combinatorial effects of CO2 and mycorrhizae additive or interactive? (2) Does the outcome of CO2 and mycorrhizae differ in tap- versus fibrous root systems? Here, it is hypothesized that (a) the effect of CO2 and mycorrhizae will not be additive, as roots are likely to trade-away excess carbon to mycorrhizae. It is also hypothesized that, (b) the root response to mycorrhizae and CO2 will differ across the tap- versus fibrous root trait continuum, particularly due to difference in root architecture.To date, very few studies have addressed the combinatorial effect of CO2 and mycorrhizae can influence root morphology and plant performance . Thus, 6 species of milkweed from genus Asclepias were chosen for this experiment (Asclepias species) are important to marginal ecosystems , biodiversity , and conservation interests.The aim of this study was to determine ways in which COperiment . Milkweeperiment . In addiAsclepias seeds were sourced from the midwestern region of the U.S.A. The 6 Asclepias spp. were A. sullivantii, A. verticillata , A. incarnata, A. syriaca, A. tuberosa, and A. viridis . The relatedness of each of these species was characterized using a chloroplast DNA sequence that codes for ribulose bisphosphate carboxylase (rbcL), a conserved gene involved in CO2 fixation. Since Asclepias spp. response to (CO2) manipulation is the nature of this study, assessing Asclepias relatedness with respect to rbcL gene was appropriate.Asclepias spp., mycorrhizal isolates were also sourced from the midwestern region of the U.S.A. Fungal consortia featured 4 isolates of mycorrhizae derived from Kankakee sands prairie reserve in Indiana, USA. Isolates included several species  that have comparable levels of colonization [Similar to nization . In addinization ,44, and nization .Mycorrhizae are lab isolates that were first isolated from prairie soil to start lab cultures . Identit3 (+ mycorrhizae). Meanwhile, sterile inocula void of live mycorrhizae was added to control pots (− mycorrhizae), also at a volume of 50 cm3. This assured that the soil structure and nutrient ratio of all pots was maintained. Finally, Asclepias seedlings were transplanted to cone-tainer pots , which were positive or negative for mycorrhizae.At the time of experimental use, mycorrhizal fungal inocula  was used to inoculate in between the top and bottom layers of heat sterile background soil (Kansas clay loam–sand (1:1)). The background soil was mixed with sand, primarily because sand enhances drainage in pots, reduces nutrient levels, and enhances plant’s mycorrhizal functional response. The volume of the added inocula  was 50 cm2 manipulation), Asclepias spp. were germinated on heat sterile potting soil  After five weeks, 5-week-old seedlings were transferred to pots that were positive or negative for mycorrhizae. The dimensions of the pots were 6.8 cm in diameter by a depth of 35.56 cm. In a random block design, pots were then placed into 4 Conviron CMP 6050 growth chambers . Two of the chambers were set to ambient (CO2) at 400 ppm, while the other two chambers were set to 3.75× ambient levels. The 3.75× ambient level (hereafter referred to as enriched CO2) was chosen because geological time records suggest (CO2) > 1500 ppm reduces plant stomatal index [2 concentrations were set to 400 ppm (ambient) and 1500 ppm (enriched). Plants were fertilized with nitrogen (0.2 g/L) periodically (average of once a month) at an application volume of 50 mL. Similar to Malik et al. (2016) [4NO3 compound, which was weighted to control for N mass. Using this ratio, we determined how much NH4NO3 is needed in 1 L of water to create 0.2 g/L of N.Prior to transplanting milkweed to experimental conditions  , this  × 2 CO2 conditions (ambient and 3.75× ambient) × 8 replicates per experimental treatment. In totality, the experiment was 192 observations . The experiment was performed under growth chamber conditions in a random block design. For 10 months, 6 Asclepias spp. were grown on −/+ mycorrhizal soil regimes, while under ambient and enriched (3.75× ambient) CO2 conditions. Plants were first germinated on heat sterile potting soil. After 5 weeks, seedlings were transplanted to −/+ mycorrhizal microcosms. The Asclepias–mycorrhizal microcosms were exposed to either ambient or enriched (CO2) for a duration of 10 months (December 2019–October 2020).Experimental design featured 6 2 on Asclepias spp. were observed. Roots were washed and scanned for root morphological responses. Briefly, roots were blot dried and positioned next to a scale, and an EPSON scan was used to make morphological observations. Roots were then stored in 75% ethanol and boiled in KOH, prior to trypan blue staining [After a duration of 10 months, the effects of mycorrhizae and COstaining . Stainin2 and mycorrhizae. Root morphological response was quantified with ImageJ1.46r [For image analysis, roots were washed and scanned to assess morphological response to COeJ1.46r , as pixeT) and absorptive (A) root diameters are divided by transport root (T) diameter. This value is then multiplied by 100.Relative transport root, or percent difference between transport and absorptive root, was appropriate because root diameter has been shown to vary across plant groups , includiR version 4.0.2. Shapiro–Wilk and Levene’s Test, as well as diagnostic plots, were used to assess homogeneity in variance and normality for dependent variables . CO2 level, mycorrhizae, species and growth chamber effects were set as predictors, while both relative transportive root and biomass were set as response variables. MANOVA analysis was employed, as relative transport root and biomass were response variables from the same set of plants. Briefly, for MANOVA analysis, relative transport root and biomass were made into a vector, and simultaneous inferences were made by the predictor variables. The analysis was followed by a set of ANOVAs that independently predict biomass and root morphology. Interactions among the predictors were also included in the model, this was critical for determining whether or not growth chamber(s) were a confounding factor. Post-hoc analysis included Step AICc, Tukey HSD, and a priori contrast using the ‘multcomp’ package. The ‘multcomp’ package allowed simultaneous test for general linear hypotheses [This study was analyzed in potheses . Finallypotheses . CorrelarbcL locus was aligned using Muscle, which assesses distance using kmer distance for unaligned pairs and kimura distance for aligned pairs . Evolutionary analysis was inferred using Maximum Likelihood Method and the JTT matrix-based model [Asclepias species was constructed according to the highest log likelihood. This enabled a tree that was drawn to scale with branch lengths measured as the number of substitutions per site. While this analysis involved 6 AA sequences, there were a total of 475 positions in the final dataset. This allowed evolutionary analysis to be conducted with MEGA X [Amino acid (AA) sequence for ed model . The phyh MEGA X .2  and mycorrhizae . Additionally, the combination of these traits led to significant interactions with CO2 and mycorrhizae , CO2 and Asclepias species , and mycorrhizae and Asclepias species . Both fibrous root species and tap-root species were enhanced by mycorrhizae . Similarly, root systems differed in relative transport roots by an estimate of 25.7% .The viability and effectiveness of the inoculum was confirmed after 10 months, as root–mycorrhizal structures  were observed to be present in experimental roots , while the presence of these structures was undetected in the control. MANOVA analysis revealed that the combination of root diameter and biomass significantly responded to COorrhizae . In tap-orrhizae . A prior2 by ~25% . In contrast, mycorrhizae significantly improved biomass by ~12-fold . Increase in biomass was broadly observed across the six Asclepias species  was decreased under enriched atmospheric CO species e–j. Inte2 did not play a significant role .With regard to the relative transport root, COp < 0.001, 2 regime  produced a 95% CI ellipse with a high degree of overlap and similar shape. However, the CO2 ellipses were not congruent as the ambient CO2 ellipse was more positive , a tree was constructed to the highest log likelihood (−1398.00) to examine relatedness of the six Asclepias species. Interestingly, the root system helped explain relatedness. Specifically, the two fibrous root species, A. incarnata and A. verticillata were sister taxa, and an outgroup, that was distant to the four other tap-root species  did not interact with CO2 chambers (p > 0.05). This provides confidence that results were not confounded by the experimental apparatus. In addition, Step AICc revealed that response variables including biomass and relative transport root were best explained when growth chamber interactions were removed from the model.Predictors , biodiversity, and prairie ecosystems; enriched CO2 decreased plant biomass by 25% , but this would not be sustainable without root absorption, subsurface foraging, nutrient transport, and organic content storage ,67,68. W2 , enricheosystems ,71. As is by 25% a. These  biomass i. This msclepias . Hence, 2 effect on biomass and root morphology may be context dependent. At a CO2 level of 650 ppm, plant growth and percent mycorrhizal colonization was reportedly increased [2 stimulates plant growth, percent mycorrhizal colonization is also stimulated. While percent mycorrhizal colonization was not quantified in this study, it may be the case that since enriched CO2 depressed plant growth  may be due to divergent life histories and localized adaptation. In particular, A. verticillata thrives in xeric soils, while A. incarnata thrives in hydric soils (A.incarnata and A. verticillata (two fibrous root species), were more closely related than the other four tap-root species.Irrespective of the main effect of COort root o. This sic soils . DespiteAsclepias spp. relatedness, as this observation does not appear to be confounded by rhizome trait(s). Hence, when rhizome traits were mapped onto a pre-existing phylogeny [Asclepias spp. relatedness did not assort according to rhizome morphology. However, as it relates to the present study, Asclepias spp. assort according to the root system  of the same community . This maructures , perhapsAs it relates to the ecosystem function, mycorrhizae may use different mechanisms to promote carbon storage depending on the plant root system. Our findings suggest that in tap-root systems, mycorrhizae may directly promote root carbon storage by helping to modify tap-roots into tuber-like structures . However2 had the opposite effect. Interactions with CO2 and mycorrhizae were observed for both biomass and relative transport root. The findings of this study shed insight on how CO2 and mycorrhizae may interact and influence plant eco-physiology. In particular excess CO2  can depress plant productivity, while mycorrhizae can act as a countering force to improve productivity.Mycorrhizae enhanced relative transportive root and biomass, while enriched CO


In the liver, clearance (Kβ-ox) and flux (Rβ-ox) of FFA into β-oxidation were estimated using -(R)-bromopalmitate/[U-14C]palmitate tracers. As expected, dapagliflozin induced glycosuria and a robust antidiabetic effect; treatment reduced fasting plasma glucose and insulin, lowered glycated hemoglobin, and increased pancreatic insulin content compared with vehicle controls. Dapagliflozin also increased plasma FFA, Ra, Rox, and Rst with enhanced channeling toward oxidation versus storage. In the liver, there was also enhanced channeling of FFA to β-oxidation, with increased Kβ-ox, Rβ-ox and tissue acetyl-CoA, compared with controls. Finally, dapagliflozin increased hepatic HMG-CoA and plasma β-hydroxybutyrate, consistent with a specific enhancement of ketogenesis. Since ketogenesis has not been directly measured, we cannot exclude an additional contribution of impaired ketone body clearance to the ketosis. In conclusion, this study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward β-oxidation.Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin  on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10- SGLT2 inhibitors, including dapagliflozin, are established treatments for patients with type 2 diabetes leading to improved glucose control as well as decreased risk of cardiovascular events and development of kidney disease , 5, 6, 7A consistent effect of SGLT2 inhibitors in patients is an increase in plasma ketone body levels , 10, 11,et al.  increased fasting ketone bodies and plasma FFA levels . Howeveret al. , generalAn alternative explanation for the enhanced ketogenesis could be a liver-specific effect of SGLT2 inhibitors. Thus, the major site of ketone body production in the body is the liver , and the3H(N)]-Palmitic Acid or -(R)-bromopalmitate/[U-14C]palmitate. This study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward β-oxidation.The aim of this study was to elucidate the mechanism behind the increased ketone body levels seen following SGLT2 inhibition by measuring whole-body and tissue-specific FFA metabolism and liver co-enzyme A intermediates. Obese Zucker rats were treated with the SGLT2 inhibitor, dapagliflozin , or vehicle before performing dedicated tracer studies using either  analyses. Randomization into study groups was performed based on body weight and HbA1c levels. Animals were orally gavaged (2.5 ml/kg/day) at 3 PM for 4 weeks with either vehicle alone  or dapagliflozin (1 mg/kg/day), dissolved in the vehicle. Body weight was monitored twice weekly. In all three studies the rats were given a final dose of either vehicle or dapagliflozin at 7 AM in the morning of the final experiments. Based on this protocol, three different studies were performed to examine plasma and liver biomarker responses, whole-body FFA oxidation and storage, and tissue-specific FFA utilization and storage.2-cooled tongs for determination of glycogen and CoA intermediates. A piece of liver tissue was then collected for triglyceride (TG) analysis, and a piece of pancreas was collected for measurement of insulin and protein content. Tissues were immediately placed in N2-cooled tubes and placed in liquid N2 before storage at –80°C while awaiting analyses. Spot urine was collected at the pretreatment and 4 week time points for determination of urine glucose levels.Before treatment and following 4 weeks treatment, in the 16-h fasted state, tail vein blood samples were collected at 2 PM for determination of plasma biomarkers. Immediately after collection of the final tail vein sample, tissues were collected under isoflurane anesthesia. Liver samples were rapidly collected with liquid NFollowing 4 weeks of treatment, animals were fasted overnight (from 10 PM) and the following morning (at 9 AM) they were anesthetized with Na-thiobutabarbitol dissolved in sterile water . A tracheotomy was performed and a tube inserted to aid spontaneous breathing. One catheter was placed in the carotid artery for blood sampling and for registration of blood pressure, and two catheters were placed in the jugular vein (for tracer infusion and for topping up anesthesia as needed). Arterial catheter patency was maintained by continuous infusion (<10 μl/min) of a sterile saline solution containing sodium citrate (20.6 mM). A catheter was also placed in the bladder. Following surgical preparations, animals were allowed a 2 h stabilization period before experimental protocols commenced around 12 PM. Throughout the experiment, body temperature was monitored and maintained at 37.5°C.3H(N)]-palmitic acid  was infused at a constant rate over 120 min. Repeated blood samples were collected during and after stopping the infusion for determination of unlabeled and labeled plasma FFA levels as well as 3H-labeled water following a lipid/water extraction procedure. Calculations of the rate of whole-body FFA oxidation (Rox), FFA storage (Rst), plasma FFA clearance into oxidation (Kox) and storage (Kst), and FFA appearance rate (Ra) and clearance (Kf) are shown in the Whole-body FFA oxidation was determined as previously described . Briefly3H-bromopalmitate (3H-R-BrP) and 14C-palmitate . 3H-R-BrP was prepared by custom synthesis at Pharmaron-UK, and the purity was confirmed essentially according to methods described in (3H-R-BrP) and stored FFA . Calculations of the tissue-specific clearance and flux parameters  are shown in the Rβ-ox) and clearance into (Kβ-ox) hepatic β-oxidation as described in the Rates of FFA utilization, storage, and oxidation were assessed based on a previously described method , using tribed in . BrieflyPlasma glucose and blood HbA1c levels were determined from blood samples by Accu-Chek® Mobile  or the Multi-test HbA1c system . Plasma FFA , β-HBA , TG , total cholesterol , and glucose  levels were measured by enzymatic colorimetric assays on the ABX Pentra 400 (HORIBA ABX). Plasma glucagon levels were measured with an ELISA  and plasma insulin levels were determined using a Multi-Spot® Assay System .g. Supernatants were analyzed using the reagent Triglycerides CP . Calibrator  and control .Tissue biomarker analysis: Liver triglycerides; A piece of liver tissue (40–60 mg) was put into a micro tube  with six grinding balls zirconium oxide 3 mm  and homogenized in 500 μl 2-propanol in a PreCellys 24 (Bertin Technologies), for 40 s at 5,000 rpm. Another 500 μl of 2-propanol was added and samples were allowed to extract for 1 h at 4°C. Samples were then centrifuged for 5 min at 4°C at 1,300 Liver glycogen was measured in 70–90 mg pieces of tissue homogenized as described above but in 0.1 M acetate buffer and allowed to extract overnight at 4°C. Samples were then centrifuged and the supernatant analyzed using a 4:1 ratio of 0.5 mg/ml amyloglucosidase  in 0.1 M acetate buffer and Glucose HK CP ABX Pentra .Pancreatic insulin content was assessed following homogenization of 40–60 mg tissue pieces in acidified ethanol (1 ml 1.5% pure HCl in 70% EtOH) and extracted overnight at −20°C. Samples were then centrifuged, and the supernatant was used for determination of insulin and total protein concentrations using a Multi-Spot® Assay System  and Pierce Coomassie Plus Assay Kit .2-freeze clamped tissue. Tissue pieces (25–50 mg) were placed in preweighed, N2-cooled tubes containing beads for homogenization. The frozen tube was weighed and stored at −80°C until analysis. Malonyl-, acetyl-, succinyl-, and HMG-CoA levels were analyzed by a cold, semiautomated homogenization and extraction method followed by separation by ion-pair HPLC and detection by negative electrospray tandem mass spectrometry . Results are reported as mean ± SD, and P < 0.05 was considered statistically significant.Student’s In the obese Zucker rats, dapagliflozin treatment  for 4 weeks increased glucosuria compared with the vehicle group A, consisRa, Kf) compared with controls (Rox) was significantly increased after dapagliflozin treatment compared with vehicle control , which includes oxidation and storage, as well as a measure reflecting only FFA storage (Rfs), in an extensive range of tissues (Rβ-ox) in a specific tissue would result in a relative decrease in the ratio Rfs/Rf∗ in that tissue. This only occurred in one tissue, the liver (Rβ-ox), from the Rf∗ and Rfs values (as described in Rβ-ox involved a large enhancement in hepatic oxidative clearance (Kβ-ox) of >2-fold in the dapagliflozin- compared with vehicle-treated group  and elevated systemic FFA availability. Enhanced whole-body FFA oxidation (Rox) could be completely accounted for by a liver-specific induction of β-oxidation (Rβ-ox). Elevated β-oxidation increased hepatic acetyl-CoA levels and drove ketogenesis, evidenced through elevations in liver tissue HMG-CoA and plasma β-HBA. Recent publications following dapagliflozin treatment of patients with type 2 diabetes support the translational value of this study as increased whole-body fatty acid oxidation, and specifically increased hepatic FFA uptake, was observed in these clinical trials (Consistent with effects seen in multiple clinical trials in patients with type 2 diabetes , in the l trials , 26.Fig.The enhanced FFA mobilization could theoretically be due to increased lipolysis resulting from a reduction in plasma insulin or a decrease in intra-adipocyte re-esterification, due to falling glucose and insulin levels. Of these alternatives the available evidence suggests that reduced re-esterification is the dominant driver since in the clinic another SGLT2 inhibitor has been shown to minimally influence lipolysis .The lackOur data reveal that dapagliflozin robustly enhances β-oxidation in the liver by a local, liver-specific effect. The rate-limiting step for this process is thought to be long-chain fatty acid flux into the mitochondria via CPT1 , 32. TheDapagliflozin promoted the mobilization of FFA, which drove an increase in nonoxidative disposal of FFA G. PreforDapagliflozin successfully prevented the development of diabetes in the initially prediabetic obese Zucker rats used in this study. In addition to preventing the deterioration in glucose control, as seen in the vehicle-treated animals, dapagliflozin treatment also increased pancreatic insulin content E, in linAlthough many of the results tie together and confirm results in earlier clinical studies using SGLT2i, there are a number of limitations of the current work. First, all of our studies are restricted to the fasting situation. This was motivated by our primary interest in exploring the mechanisms of ketogenesis, which are suppressed by the physiological actions of hyperinsulinemia and hyperglycemia. There are also two major species of ketone bodies in the circulation, β-HBA and acetoacetate. In this study we have only measured β-HBA, as results from a pilot study suggested that plasma acetoacetate was not stable under the conditions of plasma handling and storage applied. Furthermore, we have not directly assessed ketone body production and clearance. Therefore, we cannot exclude the possibility that a reduction in the clearance rate of plasma ketone bodies contributes to the dapagliflozin-induced elevation of plasma ketone bodies observed. The interpretation of liver CoA intermediate levels is complicated by the unknown partitioning of the metabolites between subcellular compartments: e.g., mitochondrial HMG-CoA is destined for ketogenesis while the cytosolic pool can be directed to de novo cholesterol synthesis. A lack of change in plasma cholesterol F, howeveIn conclusion, this study provides new insight into the mechanisms by which an SGLT2 inhibitor, dapagliflozin, enhances ketogenesis. Our tracer kinetic studies in the fasting state provide evidence that the ketogenic effect is driven by the combination of accelerated mobilization of FFA from adipose tissue stores and a robust liver-specific enhancement in β-oxidation. These responses qualitatively resemble the physiologic response to fasting. Thus, faced with a situation of negative carbohydrate balance, due to SGLT2 inhibition and glycosuria, the decreasing glucose and insulin levels are probably primary triggers via established physiologic mechanisms for the observed adipose and hepatic lipid metabolic responses.All data are available in the main text or the supplementary materials.This article contains All authors are or were employed by AstraZeneca and have shares in the company.


Tetrahymena thermophila, histones from macronucleus (MAC) and micronucleus (MIC) have been comprehensively investigated, but the function of histone chaperones remains unclear. Histone chaperone Nrp1 in Tetrahymena contains four conserved tetratricopepeptide repeat (TPR) domains and one C-terminal nuclear localization signal. TPR2 is typically interrupted by a large acidic motif. Immunofluorescence staining showed that Nrp1 is located in the MAC and MICs, but disappeared in the apoptotic parental MAC and the degraded MICs during the conjugation stage. Nrp1 was also colocalized with α-tubulin around the spindle structure. NRP1 knockdown inhibited cellular proliferation and led to the loss of chromosome, abnormal macronuclear amitosis, and disorganized micronuclear mitosis during the vegetative growth stage. During sexual developmental stage, the gametic nuclei failed to be selected and abnormally degraded in NRP1 knockdown mutants. Affinity purification combined with mass spectrometry analysis indicated that Nrp1 is co-purified with core histones, heat shock proteins, histone chaperones, and DNA damage repair proteins. The physical direct interaction of Nrp1 and Asf1 was also confirmed by pull-down analysis in vitro. The results show that histone chaperone Nrp1 is involved in micronuclear mitosis and macronuclear amitosis in the vegetative growth stage and maintains gametic nuclei formation during the sexual developmental stage. Nrp1 is required for chromatin stability and nuclear division in Tetrahymena thermophila.Histone chaperones facilitate DNA replication and repair by promoting chromatin assembly, disassembly and histone exchange. Following histones synthesis and nucleosome assembly, the histones undergo posttranslational modification by different enzymes and are deposited onto chromatins by various histone chaperones. In The online version contains supplementary material available at 10.1186/s13072-021-00409-4. Eukaryotic cells compact genomic DNA into chromatin to fit inside the nucleus. The nucleosome is the basic subunit of chromatin. It contains two copies of core histone and DNA wrapping around the histone octamer with an approximate length of 146 bp . H1 assoSaccharomyces cerevisiae, NASP homolog Hif1 (histone acetyltransferase 1-interacting factor 1) interacts with histone acetyltransferase Hat1 and Hat2 to form the nuclear HAT-B complex, which recognizes the post-translational modifications of the H3 tail and catalyzes the acetylation of the histone H4 tail. Hif1 interacts with the H2A-H2B dimer and H3-H4 tetramer via distinct mechanisms [Schizosaccharomyces pombe, NASP homolog Sim3 (start independent of mitosis 3) specifically deposits centromere histone H3 [Caenorhabditis elegans hermaphrodites [Xenopus laevis, N1/N2 allows the progressive release of histones after fertilization and thus ensures nucleosomal assembly during rapid cell divisions in early development [Arabidopsis NASP binds the histone variant CenH3 and affects its abundance at the centromeres [According to binding specificities and sequences and structural similarities, histone chaperones are classified into several families , 10. Onechanisms –14. In Sstone H3 , 16. NAShrodites . In Xenoelopment , 19. Redelopment , 20. Aratromeres . Mammalstromeres –25. The tromeres –28.Tetrahymena thermophila is a unicellular ciliated protist that contains structurally and functionally distinct germ-line micronucleus (MIC) and somatic macronucleus (MAC). The polyploid MAC is characterized by transcriptional activity, and the diploid MIC is transcriptionally silent in vegetative growing cells [Tetrahymena cells are starved. During the sexual developmental stage, MICs perform meiosis and produce four meiotic products. One of them undergoes a prezygotic mitosis to produce two pronuclei. The exchange and fusion of the pronuclei produce a zygotic nucleus. The zygotic nucleus performs two mitoses and produces four products. The two products at the anterior part form new MACs (NMs), the two products at the posterior part become the new MICs. NMs undergo genome rearrangement and replicate. Finally, the conjugating cells separate and become exconjugants, and one of the MICs is resorbed. The exconjugant resumes proliferation under nutrition conditions [ng cells . DNA repnditions , 31. Thenditions . The MACnditions , 34. Thenditions . The MICnditions , 37. HisT. thermophila. Nrp1 localized to the MAC and MIC, and disappeared in the apoptotic parental MAC and the degraded MICs. NRP1 knockdown inhibited cellular proliferation and led to abnormal micronuclear mitosis and macronuclear amitosis during the vegetative growth stage. During conjugation stage, NRP1 knockdown led to gametic nuclear abnormality and nuclear selection failure. The interaction proteins of Nrp1 were identified by affinity purification combined with the mass spectrometry (AP-MS) analysis of endogenously tagged Nrp1-HA proteins. The physical direct interaction of Nrp1 and Asf1 was also confirmed by pull-down analysis in vitro. These results indicate that Nrp1 is required for chromatin stability and nuclear division in Tetrahymena.In the present study, we identified a new NASP-related protein 1 (Nrp1) in Tetrahymena genome contains a single NASP homologous gene NRP1 (TTHERM_01014770). It is 1,700 bp, and the open reading frame is 1533 bp encoding 510 amino acids. Nrp1 contains four tandem TPR domains (TPR1-4) and one C-terminal domain containing the nuclear localization signal (NLS). TPR1, TPR3, and TPR4 consist of α-helices and exhibit similar structures. By contrast, the TPR2 domain is inserted by acidic amino acids patches   domain. The TPR motifs form a helix-turn-helix arrangement and provide a structural scaffold for mediating protein-protein interactions . The TetNRP1-3HA-Neo4 was constructed and introduced into different mating type cells, CU428 and B2086. The endogenous NRP1 gene in the MACs was targeted with the HA tag at the C-terminal  epitopes was inserted into the 3′ end of the Nrp1 open reading frame Fig. A. The pNinterval , 41. NrpNASP is initially described in rabbits as a highly autoimmunogenic testis and sperm-specific protein and localized in the nuclear area of primary spermatocytes. During the subsequent meiotic divisions, NASP is partitioned into the cytoplasm and then reassociates with the reforming nucleus , 42. DurNRP1 knockout plasmid and transformed it into Tetrahymena cells  method. Double-strand RNA was induced under the MTT1 promoter by Cd2+ induction, and target gene transcripts were disrupted by small RNA [Tetrahymena cells . For WT cells, each single cell survived and proliferated. By contrast, 19.5% nrp1i cells died, and 36.5% nrp1i cells failed to proliferate  than that in WT cells . We also identified ubiquitin degradation pathway-associated proteins UAB14, UBC4, and UCN2. NASP is regulated by the ubiquitin-proteasome pathway in HeLa cells [Arabidopsis NASP complex [Human NASP interacts with H3-H4 in various multichaperone complexes, including combinations of other H3-H4 chaperones, such as CAF-1, HIRA, and Asf1 . To idenependent . Thus, Nependent . Hsc70 aependent . The H3-ependent . Herein,nal tail , 51. DNArahymena . NASP israhymena . The phoLa cells . In addi complex . We alsoT. thermophila [NRP1 and ASF1 were synthesized and expressed in E. coli BL21. His-Nrp1 and GST-Asf1 were expressed and purified by affinity chromatography , and micronuclear H1(Mlh1) in the Nrp1 complex. Similarly, Nrp1 is also devoid in the Hho1 and Mlh1 complex [Tetrahymena.In assembly . Hif1 buand Nap1 . In mammand Nap1 , 24. In  complex . PerhapsT. thermophila cells B2086 (mating type II), CU428 (mating type VII), and CU427 (mating type VI) were obtained from the National Tetrahymena Stock Center (http://tetrahymena.vet.cornell.edu/). Tetrahymena cells were cultured in Super Proteose Peptone (SPP) medium at 30 °C [5 cells/mL) of starved cells from different mating types.Wild-type at 30 °C . The celat 30 °C . Mating NRP1(TTHERM_01014770) sequences were obtained from the Tetrahymena Genome Database (http://www.ciliate.org). Alignments of amino acid sequences were performed with DNAMAN. Protein clustering analyses were accomplished using NSAP sequences from 34 different eukaryotic lineages by employing the neighbor-joining (NJ) method with 1,000 bootstraps in MEGA 5.1. Structural and functional domains were identified on the basis of the information from the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi). Tertiary structure prediction was carried out using the SWISS-MODEL server (https://swissmodel.expasy.org/interactive/).NRP1-HA-Neo4 plasmid was created as previously described [NRP1 were amplified from genomic DNA by PCR using primers the NRP1-HA-F1/NRP1-HA-R1 and NRP1-HA-F2/NRP1-HA-R2, respectively . The transformed cells were selected under paromomycin and identified by PCR using primers J-NRP1-HA-F/J-NRP1-HA-R, as described previously [The pescribed . Brieflyeviously .NRP1 HA tagged overexpression plasmid under MTT1 promoter, the complete coding sequence of NRP1 was amplified using the OE-NRP1-F and OE-NRP1-R. The amplified sequences were digested with BamH I and Asc I and cloned into the pXS75 vector digested with the same enzymes. The constructs were introduced into CU428 and B2086 cells by biolistic transformation and mutants were screened under increasing paromomycin concentrations until cells failed to grow. The mutants were confirmed by PCR amplification using primers the OE-J-NRP1-F/OE-J-NRP1-R.To create the NRP1 were amplified from genomic DNA by PCR using the primers KO-NRP1-5′F/KO-NRP1-5′-R and KO-NRP1-3′-F/KO-NRP1-3′-R, respectively. The 5′ and 3′ flanking sequences were digested with Sac I/Not I and Xho I/Kpn I, respectively, and then ligated with the pNeo4 vector digested with the same enzymes. NRP1 knockout strains were selected based on paromomycin resistance and PCR amplification using the primers J-KO-NRP1-F and J-KO-NRP1-R.The 5′ flanking sequence and 3′ flanking sequence of NRP1 knockdown construct was created by cloning the 490 bp region of the NRP1 into the RNAi vector phpNeo. The forward and reverse fragments were amplified by primers RNAi-NRP11F/RNAi-NRP11R and primers RNAi-NRP12F/RNAi-NRP12R, respectively. The two fragments digested with the enzymes were cloned into the RNAi vector phpNeo digested with the same enzymes, respectively. The recombinant plasmid was digested using Not I and Xho I and introduced into the starved Tetrahymena cells. The transformed cells were selected under increasing paromomycin concentrations until cells failed to grow [NRP1 knockdown efficiency was detected by qRT-PCR.The  to grow , 44. NRP5 cells with a Trizol reagent . cDNA was synthesized with a random primer using the PrimerScriptTM RT reagent kit . qRT-PCR was performed with the RT-NRP1-F/RT-NRP1-R primer pairs using an SYBR Green II mix . The steps are as follows: heat for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and an extension for 30 s at 60 °C. The relative quantifications of the NRP1 mRNA expression levels were normalized using 17S rRNA as an internal control.Total RNA was extracted from approximately 8 × 102:100% ethanol) overnight at 4 °C. The fixed cells (50 μL) were immobilized on cover glasses coated with poly-L-lysine (Sigma) and dried for 45 min at room temperature (RT). Coverslips were washed with phosphate-buffered saline (PBS), and fixed with 0.1% Tween-20 (PBST) for 10 min at RT. The samples were blocked in the blocking solution  for 1 h and then incubated with mouse anti-HA antibody , anti-α-tubulin mouse monoclonal primary antibody , anti-γ-H2A.X mouse monoclonal antibody  at 4 °C overnight. After washing three times with PBST, the samples were incubated with TRITC conjugated anti-mouse IgG antibody  for 1 h at RT. Coverslips were washed three times with PBST, then stained with 1 μg/mL DAPI for 15 min at RT. Digital images were collected using a Delta Vision Elite deconvolution microscope system , confocal microscope  or fluorescent microscope .For HA-Nrp1 localization, 5 mL of cell culture was collected (3500 rpm/4.5 min) and fixed in Lavdowsky’s fixative  overnight at 4 °C. For the alpha-tubulin or γ-H2A.X immunofluorescence staining, cells were fixed in 10 mM Tris pH 7.4 with 20 μL of Schaudinn’s fixative  after 4 days of culture at 30 °C.Tetrahymena was grown in 100 mL of 1 × SPP to a final concentration of 3.5 × 105 cells/mL and was collected. Then, 24 h starved cells of different mating types were mixed in equal numbers to induce conjugation. After 2 h post-mixing, cells were collected, the pellets were re-suspended in 100 μl Tris-HCl  with 1 μL of 100 × Inhibitor Cocktail (Thermo) and 1 μL of 100 × 0.5 M EDTA, then frozen at − 80 °C. The pellets were thawed on ice and lysed by sonication. The whole cell lysate was clarified by centrifugation at 13,000 g for 20 min. The resulting soluble material was incubated with 20 μL of packed anti-HA agarose (Thermo) at 4 °C overnight. The anti-HA agarose was washed once with 3 mL of TBS plus 0.05% Tween-20. Then, 0.5 ml of TBS-T was added to each column and the columns were centrifuged for 10 s. The wash was repeated five times. The washes were saved for future analysis. Exactly 25 μL of 2 × non-reducing sample buffer was added to the anti-HA agarose. The spin column was heated at 100 °C for 5 min, and the sample was collected by centrifugation for 10 s. The sample was detected by SDS-PAGE and Western blot analysis. Another part of the sample was analyzed by MS. 5 μL product of protein digestion was used for LC-MS/MS analysis and separated using a nanoliter HPLC EASY-nLC1000 system  couple with a Q Exactive mass spectrometer . The mobile phase A was 0.1% formic acid solution with 2% acetonitrile, and the mobile phase B was 0.1% formic acid solution with 84% acetonitrile. The chromatographic column EASY column SC200 150 μm × 100 mm (RP-C18) was balanced with 100% A solution. The sample was loaded onto the EASY column SC001 traps 150 μm × 20 mm (RP-C18) through an automatic sampler and separated by a chromatographic column at the flow rate of 400 nL/min. The gradient elution procedure was as follows: the percentage change of the mobile phase B for 0–100, 100–108, and 108–120 min is 0–45%, 45–100%, and 100% of the linear change, respectively. After the capillary separation by HPLC, the product of protein digestion was determined using Q-Exactive mass spectrometer with the following parameters: duration: 120 min; detection method: positive ion detection; parent ion scanning scope: 300–1800 m/z; MS1 resolution at M/Z 200: 70,000; and MS2 resolution at M/Z 200: 17,500. The mass-to-charge ratios of polypeptides and polypeptide fragments were obtained by collecting 20 fragment patterns  after each full scan. The data of MS were analyzed by Mascot2.2 Software. MS assay was supported by Shanghai Applied Protein Technology . All the proteins detected in the WT data sets were removed. Proteins in tables were selected either by a high number of hits or by a previously described functional or biochemical association with Nrp1 in other eukaryotes.ASF1 and NRP1 coding sequence was synthesized for expression in Escherichia coli. The synthesized ASF1 and NRP1 were cloned into pGEX-4T-1 and pESUMO, respectively. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) and expressed under 0.1 mM IPTG induction at 37 °C or 16 °C. Recombinant proteins were purified by affinity chromatography [2EDTA, 200 mM NaCl) containing 2 µg of His-Nrp1 and 1 µg of GST-Asf1. After overnight incubation at 4 °C, the beads were washed four times with 5 mL of A300 . The binding protein was eluted and determined by 12.5% SDS-PAGE and Western blotting.The full-length tography . Pull-doAdditional file 1. Additional figures S1–S8 and tables S1, S2.


Hybrid burn centers are challenged to meet the criteria of the ABA verification process. When burn care occurs in different units across a hospital, it is difficult to ensure all areas are aware of the verification criteria and ensure timelines and expectations of optimal burn care are understood. Our center had difficulty ensuring rehabilitation referrals were placed on admission to allow the maximum amount of time for these services to establish a plan of care. Staffing issues were identified as contributing to the inability to consistently establish a rehabilitation program within 24 hours of admission.We had a QI goal of 100% compliance for establishing a comprehensive rehabilitation plan within 24 hours of admission. Several methods of education were provided to all providers and other burn team members to encourage immediate consultation to physical therapy and occupational therapy. Consultations to these services were frequently delayed. In 2017, 63% of the patients admitted had a rehabilitation plan of care established within 24 hours of admission. To address this, a burn order set was developed. This burn order set had the orders for PT and OT pre-populated to have the order placed on admission. The order set went live in November of 2017. In 2018, the percentage of patients receiving a comprehensive rehabilitation plan within 24 hours of admission had increased to 80%. Further education for use of the order set was provided, including additional presentations during our resident "boot camp" in 2018, our burn program manager reviewing all burn admissions and providing feedback to providers to use the burn order set, and established the role of a dedicated burn NP along with an everyday burn clinic in 2020. Staffing patterns were assessed in collaboration with the lead PT and OT. Rehabilitation established more coverage on the weekends and holidays to ensure staff was available 7 days per week to respond to the rehabilitation referrals for our patients.The compliance with the ABA verification criteria for establishment of a comprehensive rehabilitation program within 24 hours of admission had increased with consistent use of the burn order set and increased rehabilitation staffing. In the last 4 quarters of 2020-2021, our compliance has increased to 92%. In 2 of the last 4 quarters our compliance was 100%.The use of a comprehensive burn order set can help a hybrid burn center ensure they are set up to meet ABA verification criteria. These order sets when used consistently can not only ensure consults are ordered, but also help remind providers of the unique needs of the burn patients and improve efficiency.


AR) signaling is a critical event in the progression of prostate cancer (PCa) and hormone therapy consisting of androgen deprivation (ADT) or AR inhibition is therefore used to treat advanced cases. It is known that N-cadherin becomes upregulated following ADT and can directly induce PCa transformation to the castration-resistant stage (CRPC). However, the relationship between AR and N-cadherin is unclear and may promote better understanding of CRPC pathogenesis and progression. Here, we demonstrate a new axis of N-cadherin/c-Jun/N-myc downstream regulated gene 1 (NDRG1) that N-cadherin promotes c-Jun expression and suppresses NDRG1 to promote invasion and migration of PCa cells through epithelial to mesenchymal transition (EMT). Targeting N-cadherin in combination with enzalutamide (ENZ) treatment synergistically suppressed PC3 cell proliferation in vivo and in vitro. Further studies showed that compared to lower Gleason score (GS) (GS < 7) cases, high GS (GS > 7) cases exhibited elevated N-cadherin expression and reduced NDRG1 expression, corroborating our in vitro observations. We further demonstrate that c-Jun, AR, and DNA methyltransferase-1 (DNMT1) form a complex in the 12-O-tetradecanoyl phorbol-13-acetate (TPA) response elements (TREs) region of the NDRG1 promoter, which suppresses NDRG1 transcription through DNA hypermethylation. In conclusion, we demonstrate an underlying mechanism for how N-cadherin collaborates with AR and NDRG1 to promote CRPC progression. Controlling N-cadherin/c-Jun/NDRG1 axis may help to overcome resistance to commonly used hormone therapy to improve long-term patient outcomes.The dysregulation of androgen receptor ( AR) which is critical to their survival. However, after a period of treatment, the cancer becomes resistant to hormone therapy, which is termed castration-resistant PCa (CRPC). CRPC is more aggressive than hormone-sensitive PCa and is associated with poor survival rates Prostate Cancer (PCa) is a common malignancy and leading cause of cancer-related mortality in men over the age of 50. In 2019, there were 174,650 new cases reported along with 31,620 deaths, indicating that PCa remains a serious health concern for older men and that more research is needed to ultimately improve long-term outcomes Elucidating the mechanisms underlying transformation to CRPC is extremely crucial for preventing treatment resistance and improving long-term patient outcomes. Dysregulation of the AR signaling axis is well known to be a critical event in both the development and pathogenesis of CRPC. AR has been reported to perform dual effects on PCa progression NDRG1) signaling may play an important role in PCa progression Our previous study suggested that reduced AR/N-myc downstream regulated gene 1 (CDH2), is a transmembrane protein that mediates cell-cell adhesion and cell migration N-cadherin, also called Cadherin-2 . Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS)  and 1% antibiotic-antimycotic (AA) . Cells were cultured in a humidified atmosphere at 37°C and 5% COTo stably overexpress/downregulate specific genes, lentiviruses harboring specific gene vectors or specific short hairpin RNA (shRNA) sequences were generated by GENECHEM . PCa cells were infected with the lentiviruses with 5 μg/ml polybrene for 24 hours (h), and the medium was changed. After 72 h, the successfully transduced cells were selected with 1-2 μg/ml puromycin.To transiently knockdown the expression of specific genes, small interfering RNAs (siRNAs)  specific to these genes were transfected into PCa cells using Lipofectamine 3000  according to the manufacturer's instructions. Information regarding these genes is provided in 3 cells were seeded in 96-well culture dishes and cultured for 48-96 h. At the detection time point, the supernatant was discarded, medium containing 10% Cell Counting Kit-8 (CCK-8) (MedChemExpress (MCE), Monmouth Junction, NJ, USA) solution was added, and the sample was incubated (37°C) for 1 h. The absorbance at 450 nm in each well was measured through Varioskan Flash .In total, 2 × 103 for LNCaP and 2 × 103 for PC3) were evenly seeded in 6-well plates and cultured for 15 days to form visible colonies. Then, the cells were fixed  and stained with 0.01% crystal violet solution . The cells were fully decolorized, and the number of colonies was counted.Cells (3 × 104) in RPMI medium (serum-free) were seeded into the upper chamber with an insert coated with Matrigel . The lower chamber was filled with 500 μl medium with 20% FBS as a chemoattractant. After 48 h of incubation, the cells on the upper surface of the Transwell membranes were removed, and the membranes were fixed , stained ), and counted.As previously described 5) at more than 90% confluence were seeded in 6-well plates. A scratch was generated in the middle of the wells using a 200-μl pipette tip, and the cells were cultured for 48 h. Cell migration was imaged by light microscopy at 0 h, 24 h, and 48 h. The cell-free areas were analyzed with ImageJ software , Bethesda, MD, USA).Cell migration was evaluated through a wound healing assay as previously described TRIzol™ reagent (Invitrogen) was used to isolate total RNA. Complementary DNA (cDNA) was synthesized through One-Step gDNA Removal and cDNA Synthesis SuperMix  with anchored oligo (dT) primers according to the manufacturer's instructions. qPCR was performed using Top Green qPCR SuperMix (TransGen Biotech) on an SDS 7500 FAST Real-Time PCR system . The endogenous reference gene used for normalization was GAPDH or 18S ribosomal RNA. The relevant primer sequences are shown in WB analysis was performed as previously described Co-IP assay was performed as previously described An EZ-ChIP kit  was used to perform the ChIP assay according to the manufacturer's instructions. Information regarding the antibodies used for immunoprecipitation is provided in Cells were seeded in 24-well plates and transfected with relative vectors using Lipofectamine 3000 (Invitrogen). The promoter activity was evaluated with a Dual-Luciferase Reporter Assay System  according to the manufacturer's instructions. Information regarding the relevant promoters and vectors is provided in The genomic DNA of PCa cells was extracted through an EasyPure® Genomic DNA Kit (TransGen Biotech) and quantified using NanoDrop One C (Thermo Fisher Scientific).Bisulfite conversion and pyrosequencing assays were performed by Shanghai Geneland Biotech Co., Ltd. . The bisulfite conversion of DNA was performed using EpiTect Bisulfite Kits  according to the manufacturer's instructions.For the pyrosequencing assay, the bisulfite-converted DNA was PCR amplified through Platinum KAPA Taq DNA Polymerase  according to the protocol. The primers were designed using PyroMark Assay Design Software 2.0.2 (Qiagen), and the sequences are listed in 7) were mixed with 200 μl of phosphate-buffered saline (PBS) containing 30% Matrigel (BD Biosciences). The cells were subcutaneously inoculated into the right axilla of mice, and the mice were intraperitoneally injected with 25 mg/kg/day enzalutamide (ENZ) or the same volume of dimethyl sulfoxide (DMSO). Tumor size was measured every 5 days with a caliper, and tumor volume was calculated using the following formula: volume (mm3) = (length × width2) × 0.5. Fifty days post-inoculation, the mice were euthanized, and pathological analyses of the dissected tumors were performed. All experiments were performed in accordance with the institutional ethical guidelines of Capital Medical University.Xenograft assays were performed as we previously reported For pathological analyses, tumors were paraffin-embedded and sectioned at 5 μm. The sections were stained with hematoxylin and eosin (H&E) or subjected to antigen retrieval. Then, the antigen-retrieved sections were incubated with the appropriate primary and secondary antibodies. Ki-67 was used to assess tumor cell proliferation, while cleaved caspase-3 (C-Casp-3) and terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL)  were used to assess apoptosis.Statistical analyses were also performed as we previously described Sixty patients who were pathologically diagnosed with PCa  and underwent prostatectomy between 2016 and 2019 were recruited in accordance with the Ethics Committee of Beijing Chaoyang Hospital, affiliated with Capital Medical University. The tissues were paraffin-embedded and stored in liquid nitrogen until use.2) test. The values shown in the graphs represent the means ± standard errors of the mean (SEMs), and P < 0.05 was considered statistically significant. Pearson correlation and linear regression analyses were performed to analyze the associations between two genes. Absolute value of Pearson correlation coefficient (R) > 0.3 and P < 0.05 were considered indicative of a significant association between two genes. In the survival analysis, the Kaplan-Meier method and Cox regression model were used. The optimal cutoff value of the two groups was determined using X-tile software (https://x-tile.software.informer.com/). Statistical analyses were performed using SPSS software version 22 , GraphPad Prism 7 software , and Microsoft Excel 2010 software.We performed all experiments in triplicate or more to ensure statistical significance. Comparisons of the means (continuous variables) of two groups were performed with a two-tailed Student's t-test or Mann-Whitney U test. Comparisons among ≥ 3 groups were performed with one-way or two-way analysis of variance (ANOVA), followed by post hoc Tukey's or Dunnett's multiple comparison tests. The data of categorical variables were analyzed with a chi-square (χCgA), synaptophysin (Syn), and neuron-specific enolase (NSE), indicating a potential relationship between EMT and the development of NEPC , androgen-dependent PCa (ADPC) (LNCaP), and CRPC (PC3) cell lines. The mRNA and protein levels of N-cadherin were relatively high in BPH-1 and PC3 cells, and the expression of NDRG1 and AR was significantly high in LNCaP cells  and divided into the following four groups: shNC-siNC, shCDH2-siNC, shNC-siNDRG1, and shCDH2-siNDRG1. The experiments confirmed the efficiency of the NDRG1 siRNA in PC3 cells and showed that it was slightly influenced by N-cadherin expression  < 0.001) and higher Gleason score (GS) (GS > 7) (P(χ2) = 0.074, P(M-W) = 0.028). Clinical T stage (P(χ2) = 0.282, P(M-W) = 0.064), lymph node metastasis (P(χ2) = 0.08), and distant metastasis (P(χ2) = 0.421) did not significantly differ between the groups. However, the percentages of T3 clinical stage (low:high = 20.0%:33.3%), lymph node metastasis (low:high = 16.7%:36.7%), and distant metastasis (low:high = 6.7%:16.7%) were higher in the group with high N-cadherin levels.In total, 60 PCa patients were recruited from Beijing Chaoyang Hospital. The clinical characteristics are shown in Table All PCa patients were divided into three groups based on the GSs as follows: GS < 7, GS = 7, and GS > 7. The expression of N-cadherin/AR/NDRG1-related markers was detected via IHC, qPCR, and WB analyses. The IHC staining percentage and staining index indicated that N-cadherin was upregulated while NDRG1 was downregulated in the GS > 7 group compared with those in the GS < 7 group P < 0.05)  were converted to log base 2 (or negative log base 2). The results showed that the mRNA expression of N-cadherin was high and that of NDRG1 was low in the CS > 7 group compared with the GS < 7 group Fig. Ca-b. We CDH2 = 0.1685, HR(MH)CDH2(low/high) = 0.6196 (0.3134-1.225)) NDRG1(low/high) = 2.397 (1.242-4.625))  was assessed through Kaplan-Meier survival and Cox regression analyses. The optimal cutoff values of the two groups were determined as follows: low 49 and high 91 in N-cadherin and low 66 and high 74 in NDRG1 (determined through X-tile software) . The resWe selected 16 patients with GS < 7 and 16 patients with GS > 7. The patients were randomly divided into four groups, each with 4 patients with GS < 7 and 4 patients with GS > 7. WB was performed to compare the protein levels of N-cadherin and NDRG1 between the patients with GS < 7 and GS > 7 in each group. The results showed that in each group, the expression of N-cadherin was higher and that of NDRG1 was lower in the patients with GS > 7 compared with those with GS < 7 Fig. E.To achieve high expression of both c-Jun and AR in PCa cells, c-Jun was overexpressed in LNCaP (LNCaP-oe-cJun) cells, and AR was overexpressed in PC3 (PC3-oe-AR) cells through lentiviral transduction. Gene regulation was assessed through WB analysis Fig. Aa-b. A CTwo adjacent TREs (TRE (1) and TRE (2)) and an ARE were found in the promoter of NDRG1 Fig. Ba. To evThen, we investigated whether the expression of c-Jun affected DNA methylation near TREs of NDRG1 promoter. DNA methylation rates were quantitatively assessed by pyrosequencing analysis across an extended region of 356 bp spanning six CpG sites (CpG (-1164), CpG (-1136), CpG (-1059), CpG (-922), CpG (-836), and CpG (-810)), which are close to the TRE sequences in the NDRG1 promoter  and TRE (2) were deleted Fig. Df and DhIn this study, we elucidated a novel mechanism of the effect of the c-Jun/NDRG1/N-cadherin axis on the progression of PCa. This study provides the first evidence of complex formation between c-Jun, AR, and DNMT1, which is promoted by N-cadherin. Furthermore, we showed that this complex epigenetically suppresses AR/NDRG1 signaling to promote the progression of CRPC.N-cadherin has been reported to promote PCa cell invasion and migration via several pathways, such as through ErbB signaling To solve this problem, we used the ADPC cell line LNCaP and the CRPC cell line PC3. PC3 was previously reported as an AR-negative cell line We found that N-cadherin significantly promoted the expression of other EMT-related markers  and neuroendocrine markers  in PCa cells, suggesting that N-cadherin may promote PCa cell proliferation, invasion, and migration by regulating EMT and NEPC transformation.According to accepted theories that N-cadherin functional activation and AR repression by ADT are major causes of CRPC progression However, in the course of further study, we found that N-cadherin, a downstream effector, could reversibly inhibit the expression of AR and NDRG1 in PCa cells, suggesting that N-cadherin is not only a downstream effector but that an interplay mechanism may exist between N-cadherin and NDRG1. We further demonstrated that N-cadherin promotes PCa invasion and migration through suppressing AR/NDRG1 signaling and that other mechanisms may participate in N-cadherin-induced cell proliferation, which is consistent with the notion that NDRG1 does not affect PCa cell proliferation The specific mechanism by which N-cadherin affects AR/NDRG1 signaling is unclear. We found that N-cadherin significantly promoted the expression of c-Jun, which is a transcription factor that binds to the TRE to regulate several target genes The regulatory effect of c-Jun on transcription repression is achieved through DNA methylation We initially hypothesized that N-cadherin induces c-Jun to bind to the promoter of AR and suppress AR expression through DNA methylation. However, only one TRE sequence (position -3193 to -3187) was found in the promoter region of AR (position -6000 to -1), and no CpG site was found at approximately 150 bp of TRE, suggesting that c-Jun is unlikely to induce DNA methylation at the AR promoter by binding to the TRE.We then considered another mechanism by which N-cadherin induces c-Jun to bind to the TREs of NDRG1 and competitively regulate its transcriptional activity with AR. In the NDRG1 promoter region (position -2000 to -1), we found two TREs (TRE (1) in position -1124 to -1118 and TRE (2) in position -789 to -783) located near the ARE (position -984 to -952). c-Jun has been reported to form a complex with AR The influence of the AR-ARE interaction on AP-1 is controversial because c-Jun has been reported to either competitively inhibit In vitro and in vivo assays both confirmed the synergistic antiproliferative effects of N-cadherin downregulation and ENZ treatment. The expression of apoptosis marker (TUNEL) was also significantly upregulated under both treatments, suggesting that N-cadherin knockdown may induced the transition of CRPC to ADPC in PC3 cells.In our previous study, the sensitivity of CRPC cells to ENZ treatment was restored by epigenetically stimulating AR/NDRG1 signaling In PCa patients, GS ≥ 7 has been defined as an important predictor of PCa aggressiveness Several limitations in this study warrant further research. First, N-cadherin was confirmed to suppress AR expression, but the specific mechanism underlying the relationship was undetermined, which may be the reason that functional amplification of c-Jun competitively repressed AR transcriptional activity and further led to degeneration of AR. Additionally, the specific mechanism by which N-cadherin promoted c-Jun is unclear in this study. Finally, the N-cadherin-induced promotion of PCa cell proliferation is independent of NDRG1 expression, and other molecular mechanisms may participate in this process.In this study, we elucidated a mechanism underlying CRPC progression through the N-cadherin/c-Jun/NDRG1 axis. N-cadherin is highly expressed in malignant PCa tissues and epigenetically suppresses AR/NDRG1 signaling through c-Jun. c-Jun forms a complex with AR and DNMT1 on the TREs of the NDRG1 promoter and not only promotes DNA methylation through DNMT1 but also competitively suppresses AR-induced transcriptional activity on the ARE in the NDRG1 promoter. Preventing this vicious cycle by repressing the expression of N-cadherin may shed light on ways to overcome CRPC progression and reverse CRPC to ADPC.Supplementary figure and tables.Click here for additional data file.


In Japan, the national medical insurance system and long-term care insurance (LTCI) system cover rehabilitation therapy for patients with acute, convalescent, and chronic stroke. Medical insurance covers early and multidisciplinary rehabilitation therapy during acute phase hospitalizations. Patients requiring assistance in their activities of daily living (ADL) after hospitalization are transferred to kaifukuki  rehabilitation wards (KRW), which the medical insurance system has also covered. In these wards, patients can receive intensive and multidisciplinary rehabilitation therapy to improve their ADL and transition to a smooth home discharge. After discharge from these hospitals, elderly patients with stroke can receive outpatient (day-care) rehabilitation and home-based rehabilitation using the LTCI system. The Japanese government has proposed building a community-based integrated care system by 2025 to provide comprehensive medical services, long-term care, preventive care, housing, and livelihood support for patients. This policy aims to promote smooth coordination between medical insurance services and LTCI providers. Accordingly, the medical insurance system allows hospitals to receive additional fees by providing patient information to rehabilitation service providers in the LTCI system. A comprehensive database on acute, convalescent, and chronic phase stroke patients and seamless cooperation between the medical care system and LTCI system is expected to be established in the future. There are only 2,613 board-certified physiatrists in Japan, and many medical schools lack a department for rehabilitation medicine; establishing such a department at each school is encouraged to teach students efficient medical care procedures, to conduct research, and to facilitate the training of personnel in comprehensive stroke rehabilitation. Historically, stroke has been the number one cause of death among Japanese people, but has shifted to the fourth most common cause in recent years due to decreasing mortality rates, with cancer as the first, heart disease second, and senility third. These numbers reflect fewer deaths due to improved emergency medical services and advances in treatment methods, such as the use of recombinant tissue-type plasminogen activators (rt-PA) and mechanical thrombectomy. However, the overall number of patients with stroke remains high. In Japan, ~220,000 people experience a new stroke and ~290,000 people have recurring strokes annually . EndovasThe clinical importance of rehabilitation therapy for patients with stroke is well-established. Rehabilitation must be provided in a timely and appropriate manner during acute, convalescent, and chronic phases. It is also important to transition seamlessly between the treatments for each phase to improve and maintain the function of patients with stroke. In Japan, the national medical insurance system and long-term care insurance (LTCI) system were established to provide rehabilitation services for patients with stroke . In factThe purpose of this mini-review is to outline the transitional and long-term care system in Japan and the current challenges for the rehabilitation of patients with stroke at each phase.The main purpose of acute rehabilitation treatment for stroke is to prevent complications associated with immobility, to prevent systemic complications, and to promote functional reorganization of the brain. For this reason, mobilization, basic movement training, gait training, and activities of daily living (ADL) training are implemented as early rehabilitation. In Japan, in recent years, the importance of early rehabilitation for the treatment of acute stroke has been increasingly recognized, and its practice has become widespread. Evidence for these treatments is being collected, which leads to improved knowledge about early rehabilitation therapy . In obseFor acute rehabilitation, multidisciplinary cooperation is important for the functional recovery of patients with stroke. For example, early assessment of swallowing function after the stroke onset helps prevent aspiration pneumonia and to promote early oral intake . Aoki etAfter acute hospitalization, patients who require ADL assistance are transferred to the kaifukuki  rehabilitation wards (KRW), which have been covered by the medical insurance system since 2000 , 14. PatThe KRW association annual survey reported almost 85,000 KRW beds in 1,500 hospitals in 2019 , with anA variety of cutting-edge rehabilitation therapies have been developed and practiced in the KRW. According to a cohort study analyzing 2,325 patients , intensiJapan has an unprecedented aging population that affects health and long-term care systems. The LTCI system was introduced in Japan in 2000 to address the demands of older people with disabilities based on a user-oriented social insurance system supporting independence . Older pThere are two main types of rehabilitation services in the LTCI system: home-based rehabilitation and outpatient (day-care) rehabilitation , 27. HomTo use LTCI services, a person must be certified as needing long-term care based on their physical and cognitive functions, as well as the status of their nursing care and medical treatment. For certified patients, the system requires that they use LTCI rehabilitation services instead of medical insurance, except when they are hospitalized or in the early stages of their illness. In addition, the medical insurance also covers the outpatient rehabilitation for younger patients with stroke and elderly patients who were not certified as needing long-term care in the chronic phase. Among rehabilitation services after returning home from the KRW, outpatient rehabilitation through medical insurance, outpatient (day-care) rehabilitation through LTCI, and home-based rehabilitation through LTCI each account for ~10% of discharged patients .In the LTCI system, using information and communication technology devices to collaborate with other professions is encouraged. If service providers provide information from medical insurance service centers to LTCI rehabilitation centers, they can charge an additional fee for linking the information using information and communication technology. Furthermore, holding conferences with other professionals within LTCI rehabilitation services is recommended, which also would allow the use of these technologies, such as video teleconferencing systems.Scientific evidence is necessary to promote high-quality long-term care services. Therefore, the Japanese Ministry of Health, Labor, and Welfare launched a database for LTCI services, the Long-Term Care Information System for Evidence (LIFE), in April 2021. This database stores information on diseases, physical and cognitive functions, rehabilitation goals and interventions, ADL, instrumental ADL, and nutritional status. The purpose of this database is to provide feedback for users and facilities, as well as promote high-quality evidence-based services. Furthermore, by using the LIFE database, service providers can charge another additional fee within the LTCI system. Data from the LIFE database could allow the establishment of evidence for higher quality rehabilitation services for elderly patients, who also suffer from stroke. Although it is possible to link the National Database of medical claims data with the LIFE database in the LTCI system , there a“The Cerebrovascular and Cardiovascular Disease Control Act” was enacted in December 2019 to control national cerebrovascular diseases. “The Japanese National Plan for Promotion of Cardiovascular Disease Control” based on this law set out three goals: prevention of cerebrovascular diseases and dissemination of correct knowledge; improvement of the service delivery system for health, medical care, and welfare; and promotion of research on cerebrovascular diseases , 32. By Providing appropriate rehabilitation for patients with stroke requires active physiatrist participation. Physiatrists are usually involved in the clinical management of a multidisciplinary rehabilitation team that consists of nurses, physical therapists, occupational therapists, speech therapists, and medical social workers . The phyInnovative neurorehabilitation techniques, such as non-invasive brain stimulation, are effective in functional recovery, primarily in patients with chronic stroke. Our group developed a combined protocol using repetitive transcranial magnetic stimulation (rTMS) and IRT that can effectively improve the function of patients with chronic stroke . This prSK designed concept, drafted the manuscript with important intellectual input from TO and KM, and takes responsibility for whole work from inception to published article. MA provided technical and administrative support and critically assessed the manuscript. All authors have read and approved the final version of the submitted paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The colliding events are recorded by a newly designed detector, called the Beijing spectrometer (BESIII), based on state-of-the-art technologies. Physicists working at the BESIII experiment formed the BESIII Collaboration in 2005, which is a large international collaboration consisting of more than 500 physicists from 80 institutions in 17 countries. Over the course of more than ten years, about 400 physics papers have been published in internationally renowned journals covering a large variety of physics topics and notable achievements, including the following.The Beijing electron positron collider (BEPC) was constructed in the 1980s and its upgraded version (BEPCII) with a center-of-mass energy region of 2–5 GeV and a maximum luminosity of 1 × 10Zc(3900), Zc(4020) and Zcs(3985), the fine structure of Y(4260) and a new production mode for X(3872).Discovery of the ‘four-quark matter’ Vcd| and |Vcs| CKM matrix elements.Precision measurements of the standard model (SM) parameters, the τ-lepton mass and |J/ψ decays have provided a deeper understanding of the glueballs, the resonant structures at the proton-antiproton mass threshold, as well as possibilities of searching for other light exotic hadrons.A huge sample of e+e− annihilation cross sections and complex phases in D-meson decays provide important input for experiments at other energies, and measurements of the purely leptonic and semileptonic decay rates of D and Ds mesons provide sensitive tests of lattice QCD calculations.Precision measurements of low-energy J/ψ decays provides new and unique opportunities for high-sensitivity searches for non-SM sources of CP violation.The discovery of the polarization of hyperons produced in c baryons revealed unexpected threshold behaviors and motivated measurements for other stable baryons.Investigations of time-like electromagnetic form factors of nucleons, Λ, and ΛA collection of review articles in this issue of the journal will cover some of the most important results and future prospects for the next ten years, including charm physics, charmonium and charmonium-like exotic states, light hadron spectroscopy, QCD studies with light meson decays, properties of baryons and new physics searches. These represent the advancement of our understanding on tau-charm physics in the last decades. An interview with Luciano Maiani, who proposed the existence of the charm quark and was the Director General of CERN from 1999 to 2003, is also included in this Special Topic.J/ψ events, 3 billion ψ(2S) events, an integrated luminosity of 3 fb−1 at the ψ(3770) resonance and about 25 fb−1 at energies above ψ(3770), as was collected in the BESIII physics book in 2020 [BEPCII was originally intended to operate at a variety of energy points in accordance with the physics plan that was articulated in its 2009 yellow book . To dateBESIII as the world’s premier experiment for physics in the charm and tau energy region is an exemplary, large-scale and ever-expanding international collaboration that is paving the way for Chinese physicists to take a world-leading role in the pursuit of a basic understanding of nature.We thank the BESIII Collaboration, the BEPCII and the IHEP supporting staff, as well as funding agencies including MOST, NSFC, and CAS in China, and those of foreign countries in the BESIII Collaboration.


Fundus Fluorescein Angiography (FFA) is a specialist ophthalmic test to visualise the retinal vasculature. FFA is conventionally performed through intravenous injection of 10–20% Sodium Fluorescein dye, following which serial retinal imaging (~490 nm wavelength) is used to dynamically visualise retinal flow, circulation and vascular integrity. FFA is a clinically valuable test in many conditions, in particular those causing non-perfusion, leakage, oedema, or other vascular anomalies [As FFA typically requires intravenous injection of Fluorescein, this procedure is less tolerated by children and may have to be performed under anaesthesia. Furthermore, use of needles in children may have adverse psychological effects, which is a major consideration for children with chronic conditions. Intravenous FFA holds a moderate risk of adverse effects, rarely serious, while oral FFA has shown no serious adverse effects in large cohorts . HoweverWe outline our flow chart for the oral FFA technique in children in Fig. A 20% Fluorescein sodium solution dosage is calculated and the patient is provided a straw to consume the drink as fast as is comfortable for them, with special care taken to ensure the drink is not swirled within the mouth resulting in temporary tooth staining. Following ingestion, the patient is positioned in front of the image acquisition device, typically an ultra-wide field scanning laser ophthalmoscope , as this enables fast image acquisition for less cooperative children and visualisation up to 200 degrees of the retinal periphery .Our experience is that the time-to-onset using 20% Fluorescein solution typically takes 2–5 min for a choroidal flush to appear shortly followed by retinal circulation. Following this stage, the circulatory filling times are proportionately delayed, but main phases are typically complete within 20 min of consumption for diagnostic purposes (Fig. We describe our departments protocol for performing oral FFA in children, a specialised but valuable clinical technique in the evaluation of children with conditions affecting the eye. We hope in future to provide more robust reference timings of major circulatory phases in children undergoing this technique, alongside patient experience to disseminate our findings for other departments interested in adopting this technique.


In repeated social interactions, individuals often employ reciprocal strategies to maintain cooperation. To explore the emergence of reciprocity, many theoretical models assume synchronized decision making. In each round, individuals decide simultaneously whether to cooperate or not. Yet many manifestations of reciprocity in nature are asynchronous. Individuals provide help at one time and receive help at another. Here, we explore such alternating games in which players take turns. We mathematically characterize all Nash equilibria among memory-one strategies. Moreover, we use evolutionary simulations to explore various model extensions, exploring the effect of discounted games, irregular alternation patterns, and higher memory. In all cases, we observe that mutual cooperation still evolves for a wide range of parameter values. However, compared to simultaneous games, alternating games require different strategies to maintain cooperation in noisy environments. Moreover, none of the respective strategies are evolutionarily stable. In many instances of reciprocity, individuals cooperate in turns. Here, the authors analyze the equilibria and the dynamics of such alternating games, and in particular describe all strategies with one-round memory that maintain cooperation. Traditionally, researchers capture the logic of direct reciprocity with the repeated prisoner’s dilemma17. According to that model, two individuals—usually referred to as players—interact with each other over several rounds. In each round, both players can either cooperate or defect. Mutual cooperation yields a better payoff than mutual defection, but each individual has an incentive to defect. Theoretical and experimental work suggests that cooperation can evolve if there are sufficiently many interactions between the individuals18. This work has been used to explain a wide variety of behaviors, including why humans are more likely to cooperate in stable groups19, why certain animal species share food20, and why firms are able to achieve higher market prices when they engage in collusion21.Cooperation can be maintained by direct reciprocity, where individuals help others in repeated interactions22, vampire bats donate blood to their conspecifics20, sticklebacks engage in predator inspection23, or ibis take turns when leading their flock24. Such interactions are better captured by alternating games, in which players consecutively decide whether to cooperate28. When individuals decide asynchronously, they make their decisions based on different histories. The most recent events one player has in memory differ from the most recent events that the next player takes into account . We refer to this kind of repeated interaction as a simultaneous game Fig. a. For maunt Fig. b. Such a3. Memory-one strategies depend on each player’s most recent move. Our analysis involves two steps. First, we show that successful play in alternating games does not require a sophisticated cognitive apparatus. More specifically, when interacting with a given memory-one opponent, it suffices to respond with a reactive strategy that only depends on the co-player’s most recent move. This result is reminiscent of a previous finding of Press and Dyson for the simultaneous game39. They showed that against a memory-one strategy, there is nothing to gain from having a larger memory than the opponent. Our result for the alternating game goes one step further. Against a memory-one strategy, players can afford to have a strictly lower memory, without any loss to their or their co-player’s payoff. As we show, this result crucially depends on the alternating move structure; it is not true when players move simultaneously. In a second step, we show that in order to identify the best response to a given memory-one player, we only need to check the four most extreme reactive strategies: unconditional defection, unconditional cooperation, Tit-for-Tat, and Anti-Tit-for-Tat. Using this approach, we identify all Nash equilibria among the memory-one strategies.Here, we propose an analytical approach to describe when cooperation evolves in the alternating game. In line with the previous literature, we typically focus on individuals with so-called memory-one strategies41. In that case, there is a simple rule for how to sustain full cooperation: individuals should repeat their previous action if it yielded a sufficiently large payoff, and switch to the opposite action otherwise. In contrast, in the alternating game, all stable cooperative strategies require players to randomize. After mutual defection, they need to cooperate with some well-defined probability that depends on the game parameters and the error rate. Although the respective strategies in the alternating game are Nash equilibria, we show that none of them is evolutionarily stable. As a result, evolving cooperation rates in the alternating game often tend to be lower than in the simultaneous game, although this difference is smaller than perhaps expected from static stability considerations alone. We summarize our analytical findings in Fig. In the absence of errors, we find an unexpected equivalence. The very same memory-1 strategies that can be used to enforce cooperation in the simultaneous game also enforce cooperation in the alternating game. However, once we take into account errors, the predictions for the two models diverge. In the simultaneous game, Win-Stay Lose-Shift is evolutionarily stable when the benefit to cost ratio is sufficiently large and when errors are sufficiently rare29 and provide theoretical methods to further analyze repeated games in the future. Overall, we find that cooperation is still feasible in alternating games. However, the strategies that enforce cooperation can be neutrally invaded, and hence cooperation tends to be more short-lived than in the simultaneous game.Our work suggests that in most realistic scenarios, successful play in alternating games requires different kinds of behaviors than predicted by the earlier theory on simultaneous games. In this way, we corroborate earlier experimental work on human cooperationC) or defect (D). These repeated interactions can take place in two different ways. In the simultaneous game, there is a discrete number of rounds. In each round, both players make their decision at the same time, not knowing their co-player’s decision . As a result, we obtain the donation game3. Here, cooperation means to pay a cost c > 0 in order to provide a benefit b > c to the co-player. The donation game is a special case of a prisoner’s dilemma for whichFor the simultaneous game, the possible payoffs in each round can be represented by four parameters. Players receive the reward 42; they take into account the outcome of one previous round . The entry pij denotes the probability to cooperate in the next round. This probability depends on the player’s action i and the co-player’s action j in the previous round. The equivalent strategy class also exists in alternating games25. In alternating games, however, there is no longer a unique previous round to which both players refer. Instead, the last round that is taken into account depends on the perspective of each player. It consists of the respective last moves of the two players , Tit-for-Tat, TFT = , and Win-Stay Lose-Shift, WSLS = . In the alternating game, a strategy called Firm-but-Fair3, defined by FBF =  and also referred to as Forgiver27, has been successful in evolutionary competitions. Out of these examples, ALLD and TFT are reactive, whereas WSLS and FBF are not. We say a strategy is deterministic if each conditional cooperation probability is either zero or one. In particular, all of the above examples are deterministic. Otherwise, we call the strategy stochastic.Some well-known examples of memory-1 strategies for the simultaneous game include Always Defect, ε that the player defects by mistake. Conversely, a player who intends to defect may cooperate with the same probability. We refer to the case of ε = 0 as the game without errors, and to the case of ε > 0 as the game with errors. We note that even a strategy that is deterministic becomes fully stochastic in the game with errors because in that case, a player’s effective cooperation probability is always between ε and 1 − ε.Note that our analysis includes the possibility that players sometimes make errors. That is, when a player decides to cooperate, there is some probability 29. Second, when both players use memory-1 strategies, their average payoffs are simple to compute . To this end, suppose player 1 uses the strategy p and player 2 adopts strategy q. By representing the game as a Markov chain, we can compute the stationary distribution v = . The entries of this stationary distribution give the probabilities of observing each of the four possible combinations of the players’ actions over the course of the game. Based on this stationary distribution, we define player 1’s payoff as π = (vCC + vDC)b − (vCC + vCD)c, and similarly for player 2. While the baseline scenario focuses on memory-1 strategies, our results are more general. For example, when we describe which memory-1 strategies are Nash equilibria in the following, co-players are allowed to deviate to strategies with arbitrarily long (but finite) memory. Moreover, similar approaches can also be used to explore the evolutionary dynamics of memory-2 strategies, as we will discuss later.Considering memory-1 strategies is useful for two reasons. First, such strategies are straightforward to interpret, and the respective conditional probabilities can be easily inferred from experimentsq as a Nash equilibrium if π ≥ π for all alternative memory-1 strategies p , Always Cooperate (ALLC), Tit-for-Tat (TFT), or Anti-Tit-for-Tat (ATFT).The second step is to show that we do not need to consider all reactive strategies to find the best response against partner33 if (i) it is fully cooperative against itself, and (ii) if it is a Nash equilibrium (such strategies are referred to as ‘good’ by Akin36). We find that partners are exactly those memory-1 strategies q for which the following three conditions hold,q can either be invaded by ALLD or ATFT. Together, the three requirements in .Using the above recipe, we first explore which memory-1 strategies can sustain full cooperation in games without errors . If the co-player defected, partners need to cooperate with a well-defined probability, as defined by the last two conditions in  have been observed in early simulations of alternating games26. There, it was found that evolutionary trajectories often lead to strategies that are deterministic, except that they randomize after mutual defection. Our results provide an analytical justification: SFBF is the only such strategy that is a Nash equilibrium.As a result of these additional constraints, the three-dimensional polyhedron degenerates to a one-dimensional line segment Fig. b. On oneWSLS, can be evolutionarily stable in the presence of noise40. That is, in the simultaneous game, cooperation can be sustained with a simple deterministic strategy if b > 2c. In contrast, conditions . These results suggest that cooperation can still evolve in alternating games, but it may be less robust than in the simultaneous game.The above conditions in  provide nditions  imply thnditions  are NashN players. Each member of the population is equipped with a memory-1 strategy. They obtain payoffs by interacting with all other population members. To model the spread of successful strategies, we assume individuals with high payoffs are imitated more often50 . In addition, new strategies are introduced through random exploration . These random strategies are uniformly taken from the space of all memory-1 strategies. We capture the resulting dynamics with computer simulations. For details, see Methods.In order to test these equilibrium predictions, we next explore which behaviors emerge when the players’ strategies are subject to evolution. To this end, we consider a population of qCC ≈ qDC ≈ 1. Moreover, after mutual defection, they cooperate with some strictly positive probability. Both patterns are predicted by our equilibrium conditions  the game is infinitely repeated, (ii) players move in a strictly alternating fashion, (iii) games take place in a well-mixed population, and (iv) players use memory-1 strategies. In the following, we use simulations to explore the effect of each of these assumptions in turn. Here, we briefly summarize the respective results. For an exact description of the models, and for a more detailed discussion of the results, we refer to Supplementary Note Our baseline scenario represents an idealized model of alternating interactions. It assumes . We observe similar results as in the infinitely repeated game: The simultaneous game leads to more cooperation , and the second player’s strategy is q = . Depending on the most recent actions of the two players , we can compute how likely we are to observe each of the four outcomes in the following round. For the alternating game, we obtain the following transition matrix25,v = vMA. In most cases, the solution of this equation is unique. Uniqueness is guaranteed, for example, when the players’ strategies p and q are fully stochastic, or when the error rate is positive. In exceptional cases, however, the transition matrix can allow for two or more stationary distributions. In that case, the outcome of the game is still well-defined, after specifying how players act in the very first round.When two players with memory-1 strategies interact, their expected payoffs can be computed by representing the game as a Markov chainv = , we define the players’ payoffs asδ, or equivalently if future payoffs are discounted by δ . However, the respective matrix MA only allows for the first two invariant sets25. More generally, MS allows for equilibria where players cooperate in one round but defect in the next round. Such equilibria are impossible for MA p + ε(1 − p). To compute the payoffs of strategy p against strategy q in the presence of errors, we apply the formulas (pε and qε.We sometimes assume players commit errors. We incorporate errors by assuming that with probability formulas –10) to  to ε, a 50. This process considers a population of constant size N. Initially, all population members are unconditional defectors. Each player derives a payoff by interacting with all other population members; for each pairwise interaction, payoffs are given by Eq. , the player may engage in random strategy exploration. In this case, the player discards its strategy and samples a new strategy uniformly at random from the set of all memory-1 strategies. Second, with probability 1 − μ, the player considers imitating one of its peers. In this case, the player selects a random role model from the population. If the role model’s payoff is πR and the focal player’s payoff is πF, then imitation occurs with a probability given by the Fermi function63β ≥ 0 is called the strength of selection. It measures the extent to which players are guided by payoff differences between the players’ strategies. For β = 0, any payoff differences are irrelevant. The focal player adopts the role model’s strategy with a probability of 1/2. As β becomes larger, payoff differences become increasingly important. In the limiting case β → ∞, imitation only occurs if the role model’s payoff at least matches the focal player’s payoff.To model how strategies with a high payoff spread within a population, we consider a model in discrete time. In each time step, one player is chosen from the population at random. This player is then given an opportunity to revise its strategy. The player can do so in two ways. First, with probability 64. For those simulations, we require mutant strategies to either fix in the population or to go extinct before the next mutation occurs. Under this regime, the mutant’s fixation probability can be computed explicitly9. This in turn allows us to simulate the evolutionary dynamics more efficiently66.Overall, the two mechanisms of random strategy exploration and directed strategy imitation give rise to a stochastic process on the space of all population compositions. For positive mutation rates, this process is ergodic. In particular, the average cooperation rate (as a function of the number of time steps) converges, and it is independent of the considered initial population. Herein, we have explored this process with computer simulations. We have recorded which strategies the players adapt over time and how often they cooperate on average. For most of these simulations, we assume that mutations are sufficiently rare7-time steps each (measured in a number of introduced mutant strategies over the course of a simulation). For Fig. For the simulations in well-mixed populations, we used the following baseline parametersTo report which strategies the players use to sustain cooperation (or defection), we record all strategies that arise during a simulation that have a cooperation rate against themselves of at least 80% (in the case of self-cooperators), or a cooperation rate of less than 20% (in the case of self-defectors). In Fig. 52. Here, we consider a population of size N = 2500. Players are arranged on a 50 × 50 lattices with periodic boundary conditions. Players use memory-1 strategies . In every generation, every player interacts in a pairwise game with each of its eight immediate neighbors. After these interactions, all players are independently given an opportunity to update their strategies. With probability μ = 0.002, an updating player chooses a random strategy, uniformly taken from all memory-1 strategies . With probability 1 − μ, the updating player adopts the strategy of the neighbor with the highest payoff . This elementary process is then repeated for 20,000 generations. Figure Finally, for the simulations for spatial populations Fig. g–i, we cFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary