Therefore, glendonites in the sedimentary record are commonly used as paleotemperature indicators. However, several laboratory experiments have shown that the mineral can nucleate at temperatures>7 °C. Here we investigate the nucleation range for ikaite as a function of temperature and pH. We found that ikaite precipitated at temperatures of at least 35 °C at pH 9.3 −10.3 from a mixture of natural seawater and sodium carbonate rich solution. At pH 9.3, we observed pseudomorphic replacement of ikaite by porous calcite during the duration of the experiment (c. 5 hours). These results imply that ikaite can form at relatively high temperatures but will then be rapidly replaced by a calcite pseudomorph. This finding challenges the use of glendonites as paleotemperature indicators.Glendonites have been found worldwide in marine sediments from the Neoproterozoic Era to the Quaternary Period. The precursor of glendonite, ikaite (CaCO This explains why metastable hydrous and anhydrous carbonate minerals tend to form instead of calcite. A common example is the precipitation of aragonite instead of calcite in the oceans. The Mg/Ca ratio of the seawater controls this precipitation: when this ratio exceeds 2, aragonite is favoured because Mg acts as an inhibitor of calcite growth4.Calcium carbonate, one of the most common naturally occurring minerals, has an important role in the global carbon cycle. Calcite is the stable form of calcium carbonate at the Earth’s surface. However, growth of calcite is often inhibited by a range of external factors3·H2O) and ikaite (CaCO3·6H2O). Monohydrocalcite (MHC) has been found as calcareous incrustations and the main form of calcium carbonate in Lake Issyk-Kul, Republic of Kyrgyzstan5 and as beach rocks around two salt lakes6. Experimental results and investigation of natural samples indicate that the formation of MHC requires high Mg/Ca ratios and a pH >8 in the solution from which it forms8.Two hydrous forms of calcium carbonate occur instead of calcite under certain conditions: monohydrocalcite  of different ages (Neoproterozoic to Quaternary). Examples include Permo-Carboniferous marine sediments in Sydney Basin, Australia23, marine shales in the Marine Dwyka beds, South Africa24, Jurassic and Lower Cretaceous marine sediments in Northern Siberia25, deep and shallow marine sediments in Spitsbergen26, and organic rich shallow marine sediments in Northern Germany27. Glendonites can be large and have been reported up to >1 m in length28.In 1982, Suess 29. Glendonites have also been suggested as an indicator of extreme low temperature metamorphism in Neoproterozoic sediments30. However, in addition to cold temperatures, ikaite precipitation requires high alkalinity31, high pH32 and the inhibition of calcite growth which has been argued to occur due to the presence of phosphate31 or magnesium33. Moreover, in laboratory experiments ikaite has been reported to precipitate at a wider range of temperature than seen in natural environments. Clarkson et al. (1992) precipitated ikaite from supersaturated solutions at 15 °C with triphosphate as an inhibitor of calcite nucleation34. Stockmann et al. (2018) also precipitated ikaite at 15 °C but without phosphate35. In another set of experiments, magnesium from natural seawater inhibited nucleation of calcite in favour of ikaite33. The results from these experiments may suggest that ikaite nucleation can also occur at temperature >7 °C in the natural environment. A recent study by Popov et al. (2019) of fossil records in glendonite bearing strata suggested that ikaite nucleation probably occurred at water temperature >40 °C36. These findings call into question the use of ikaite as a paleotemperature indicator. In this study, we address this issue with a series of experiments on ikaite nucleation as a function of temperature and pH.The narrow temperature range of ikaite formation observed in nature has motivated using the mineral and its pseudomorphs as an indicator of paleotemperature conditionset al. (2018) and Tollefsen et al. (2018) with the following modifications35:In total 39 experiments were performed at different temperatures and pH to investigate the precipitation of ikaite. The experimental setup was similar to the methods described by Stockmann 2CO3 and 0.1 M NaHCO3 at different proportion, 1:5, 1:3, 1:2, 1:1 and 3:1, which represent solutions with pH 9.49, 9.70, 9.86, 10.14 and 10.59 at 5 °C respectively.The samples were precipitated from a 50–50 mixture of two solutions: Solution 1) was surface seawater collected nearby the island of Andøya, Norway  used the same approach and obtained comparable results for synthetic seawater and natural seawater from the same location. Solution 2 was prepared using Na2CO3 and NaHCO3 powders from Merck dissolved in 1 litre of ultrapure deionized water (MilliQ resistivity >18.2 MΩ cm).The seawater was filtered through Munktell Qualitative Filter Paper grade 3 before use and stored in a cooling room at 5 °C. This removes any particulate matter >10 µm in size. We consider it unlikely that finer particulate matter, if present, affected the outcome of our experiments. This is because Tollefsen The two solutions and their mixtures were kept in water baths during the experiments to maintain the desired temperature. We ran experiments at temperatures from 5 to 35 °C. The mixing rate was 0.67 ± 0.21 ml/min and when ~300 ml of mixture was obtained, the experiment was stopped. The resulting mixture was put in a cooling room (5 °C) overnight. The next day, the mixture was filtered through Munktell Qualitative Filter Paper 00 K and kept overnight to dry in the cooling room. Thereafter, the precipitate was collected, weighed and stored in a freezer (−18 °C).Precipitated minerals were identified by X-ray powder diffraction (XRD) using an X Pert Pro instrument from PANalytical and the program Data Colllector at the Swedish Museum of Natural History. The sample holder was kept in a freezer (−18 °C) for 10 min before analysis to preserve the ikaite crystals. The measurement program used was Absolute Scan 5–70°2θ with a runtime of 11 min. The program Topas version 6 (Bruker AXS product) was used to identify and quantify the different phases in the samples by Rietveld refinement. In 7 samples, amorphous calcium carbonate (ACC) was present, which was quantified by Rietveld refinement of the XRD data after addition of a quartz standard to the sample in weighed proportions. Distinguishing calcite from Mg-calcite was based on unit cell data obtained from the Rietveld refinement  was used to visualise 19 of 39 samples. The instrument used was a Carl Zeiss Merlin field-emission cryogenic scanning electron microscope, fitted with a Quorum Technologies PP3000T cryo-preparation system at Umeå University. The frozen precipitate was glued onto a support and placed into liquid nitrogen slush before transferring to the instrument chamber. The images were taken at temperature c. −140 °C using in-chamber secondary electron detector (ETD) at an accelerating voltage of 2 kV.37 version 3.4.0 for geochemical modelling of all the experiments to calculate values for pH and ionic speciation. Saturation indices (SI) for the carbonate minerals were taken from the PhreeqC llnl database, except for ikaite (CaCO3·6H2O), vaterite (CaCO3) and amorphous calcium carbonate (CaCO3·H2O). These data were added separately, using solubility constants from Bischoff et al. (1993) for ikaite31, Plummer and Busenberg (1982) for vaterite38 and Brecevic and Nielsen (1989) for ACC39. The SI was defined as follows: SI = log (IAP/Ksp), with IAP = ion activity product and Ksp = solubility constant.We used the program PhreeqC InteractiveWe dissolved and diluted 27 of 39 samples for cation analysis with an Inductively coupled plasma - atomic emission spectroscopy  with autosampler ASX520 from CETAC and using a spray nebulizer. The seawater sample from Andøya was also analysed with the same instrument. The concentrations of anions in the seawater were measured with an IC20 Ion Chromatograph from Dionex.2CO3 and 0.1 M of NaHCO3 in different proportions . The calculated pH of the parent solutions ranged from 9.09 to 10.31 and calculated alkalinities ranged from 59 to 89 meq/kg (PhreeqC).From the experiments, we obtained 39 samples precipitated from by mixing natural seawater Table  with a s3), Mg-calcite CO3), aragonite (CaCO3), monohydrocalcite (CaCO3·H2O), ikaite (CaCO3·6H2O), nesquehonite (MgCO3·3H2O) and one amorphous phase: Mg rich amorphous calcium carbonate (Ca(1−x)MgxCO3·H2O). We estimated the amount of Mg in the amorphous calcium carbonate (ACC) to be 35 ± 10 mol. % from ICP-AES measurements and measured proportions of ACC, ikaite (which can only incorporate traces of Mg) and nesquehonite in the sample. The refinement of Mg occupancy for the Mg-calcite was calculated to be ~10 mol. %. This is within the range observed in marine sediments and Mg-calcite cements40.Based on XRD analysis, we identified seven minerals , series 2 , series 3 , series 4  and series 5 . For simplicity, pH values reported in this study are those calculated at 5 °C using PhreeqC. In total, we ran 32 experiments and 7 duplicates  Table . Ikaite Ikaite precipitated in all experiments in series 3, 4 and 5. In series 3 pure ikaite precipitated at all temperatures up to 20 °C in all but one experiment in which calcite also precipitated, and together with calcite at temperatures of 25 °C and higher  together with Mg-calcite and/or MHC as indicated by the XRD analyses Table . PseudomIn three of the samples from series 2, we did not observe pseudomorphs after ikaite. In the first sample (exp. 9 run at 25 °C), we observed large spherical MHC and spherical Mg-calcite. In the second sample (exp. 10 run at 30 °C), the main phase was ikaite which occurred as euhedral crystals. In the third sample (exp. 7b run at 15 °C), ikaite and Mg-calcite were the main phases observed. Some of the ikaite crystals in this sample had cavities Fig. . This teIn sample 18 from series 3 (run at 35 °C), we observed ikaite but also partly replaced ikaite crystals Fig. . In one The PhreeqC software calculations for each of the experiments , ikaite precipitated at temperatures between 5 °C and 35 °C, and at pH 10.31 (series 5), ikaite precipitated together with ACC at temperatures between 10 °C and 35 °C. The amount of ACC increased with temperature and at 35 °C, the sample contained 94% ACC (Table At pH 9.32 (series 2), mixtures of ikaite, MHC, calcite, and Mg-calcite precipitated and pseudomorphs after ikaite were observed on Cryo-SEM images of seven samples . These samples were also the only samples that contained high amounts of calcite (13–90%), which suggests pseudomorphic replacement of ikaite by calcite.Because ikaite precipitated at 35 °C in series 2–5, we infer that the upper temperature limit for ikaite nucleation exceeds 35 °C. However, because ikaite content diminished with increasing temperature in series 4 and 5, we infer that this limit is close to 35 °C for these series. On the other hand, because ikaite was the main phase at 35 °C in series 2 and 3 and because Cryo-SEM images of these samples indicate that minor calcite  occurred as pseudomorphs after ikaite Figs.  and 4, wWe constructed a stability diagram for nucleation of some anhydrous and hydrous carbonate phases as a function of pH and temperature Fig. . We defi12. However, in our experiments, ikaite nucleated at much higher temperatures (up to 35 °C) in series 2–5. The upper temperature limit for ikaite nucleation was approached in series 4 (pH 9.82). In the experiment run at 35 °C, the main precipitate was ACC, which occurred as spheroids closely surrounding euhedral ikaite crystals  showed that CaCO30 is easily hydrated and initially forms ACC and, at low temperatures, ikaite41. The importance of the CaCO30 ion pair for ikaite nucleation is confirmed by our experiments: The main precipitates in series 1 (pH 9.09), which had the lowest CaCO30 concentration in the parent solution  in the parent solution  and Stockmann et al. (2018) have also previously suggested35.Based on previous experimental work which showed that ikaite nucleation is favoured by the presence of calcite growth inhibitors such as phosphate and MgIn some of our experiments, ikaite which had precipitated at temperatures up to 35 °C, was thereafter pseudomorphically replaced by calcite within the experimental runtime of 5 hours.45. These studies demonstrate that the ikaite unit cell expands anisotropically with increasing temperature. The CaCO30 ion pair in ikaite is surrounded by water molecules held together with hydrogen bonding. When ikaite is exposed to higher temperatures, the unit cell expands along the a-axis followed by the b and c-axis. This volume increase occurs just before the breakdown of the hydrogen bonds and the release of the water molecules from the ikaite structure45. In our experiments, this was observed in series 4 (pH 9.82). Here, unit cell data show a sudden increase along the a-axis and in volume at 35 °C .Based on the perfect shapes of pseudomorphs, porous nature of the replacement calcite and a sharp reaction front, we propose that ikaite replacement occurs by a coupled dissolution - reprecipitation mechanism at the ikaite-calcite interface. Putnis and Putnis (2007) describe this mechanism as a coupled dissolution and nucleation process within the fluid boundary layer at the parent solid surfaceet al. (2018) proposed 2 models for the ikaite to glendonite transformation, based on observations and data from their study and previous studies on glendonite49  made similar observation when they exposed ikaite crystals to air at 10 °C and at 20 °C. Ikaite crystals exposed at 10 °C were pseudomorphically replaced, whereas crystals exposed at 20 °C recrystallized to calcite and vaterite50. It could be that an abrupt change in temperature or other conditions would provoke partial dissolution of ikaite and crystallization of calcite or other phases, whereas a slight change in conditions favours pseudomorphic transformation.We observed two distinct textures of the ikaite crystals in the transition zone. The first type of texture Figs.  and 6B it al. 201 proposedIn our study we focused on ikaite nucleation, however we made some interesting observations on Mg-rich ACC formation that are worth discussing. With only one exception, we did not detect ACC below pH 10.31 in the parent solution Table , even th51. For experiments at pH 9.5 to 10.3 the Mg content in ACC in their study was 31–65% which is in agreement with our result of 35 ± 10%. It has also been found that the incorporation of Mg into ACC stabilises this otherwise unstable phase53. This would explain why we did not observe any transformation of ACC in our samples. In the experiments at pH 10.31 > 5 °C ikaite and ACC co-precipitate and both phases have similar predicted SI . However, ACC is favoured by the increase in temperature, whereas the amount of ikaite diminished (Table In series 5 (pH 10.31) ikaite precipitated together with Mg rich ACC at temperatures ≥10 °C and the amount of ACC increased with temperature. Blue and Dove 2015) demonstrated from experimental studies on ACC that the incorporation of Mg into ACC was controlled by Mg/Ca ratio and pH015 demon30 ion pair in the parent solution is an important control of ikaite precipitation.Pseudomorphs (glendonites) after ikaite are widely used as paleotemperature indicators because ikaite has only been observed in nature at temperatures below 7 °C. The results from our study show that ikaite can nucleate at temperatures up to 35 °C for pH ranging from 9.3 to 10.3. Based on geochemical modelling, we infer that the concentration of the CaCOEven though ikaite crystals precipitated at these higher temperatures do not survive, their pseudomorphs do, and can be misinterpreted as indicator of low paleo temperatures. Pseudomorphic replacement of ikaite by calcite can occur rapidly (<5 hours) in response to minor changes of pH and/or temperature. We therefore challenge the use of glendonite as a paleotemperature indicator.Finally, the broad temperature range (≤35 °C) for ikaite nucleation would suggest that ikaite could be a common transition mineral that competes with ACC formation in marine environments. ACC can incorporate Mg (<65%) whereas ikaite only contain traces of Mg. It is therefore possible that preserved low-Mg calcite commonly derives from ikaite in marine environments, whereas Mg-rich ACC is the major precursor of Mg-rich calcite.Supplementary Information.


A 66‐year‐old man, implanted Abbott dual‐chamber pacemaker, was admitted to our hospital due to recurrent palpitation. ECG was recorded on admission, which created a diagnostic confusion: What accounts for the appearance of the VP in the setting of a stable intrinsic atrioventricular (AV) conduction? In this case, we will focus on the logical reasoning in the analysis of Pacing ECG. A 66‐year‐old man, implanted Abbott dual‐chamber pacemaker, was admitted to our hospital due to recurrent palpitation. Programmed parameters were as follows: DDD pacing mode, Base rate 60 bpm, Pacing/Sensed AV interval 250 ms/200 ms, Post‐ventricular atrial refractory period (PVARP) 275 ms, Ventricular intrinsic preference (VIP) On, and VIP extension 100 ms.Figure 1Intermittent atrial undersensing appears to explain the confusion, but clues at R4 may not support it. There are spike‐like signal  and marked as AS.Step 2—P6 and R6: As the R6‐P7 interval (about 320ms) is longer than 275ms and less than 475ms (PVC response prolongs the PVARP by 200ms), the R6 cannot be marked as PVC, or P7 would be marked as atrial refractory event (AR). In Abbott, a VS in AR‐VS sequence will be marked as PVC when AR‐VS interval is longer than 280ms. Therefore, AR(P6)‐VS(R6) interval (grid bar) should be less than 280ms  to guarantee P6 marked as AR . The only sequence that fits the condition is AR(P5)‐VS(R5), and the P5‐R5 interval should be longer than 280ms. To guarantee AR(P5), the key point is VP‐VPb in R4. VPb recycles PVARP (white bar) which results in P5 falling in the PVARP and marked as AR, and AR(P5)‐VS(R5) interval (grid bar) can be longer than 280ms due to the measuring error. These coincidences  lead to the “Unreasonable” Ventricular Pacings.Inappropriate continuous ventricular pacing can cause adverse outcomes due to (a) atrioventricular asynchrony and (b) increased proportion of right ventricular pacing, which in turn cause ventricular filling and decreasing ejection volume and exacerbating heart failure (Patel & Mariani, Extending AV interval or VIP extension can avoid such inappropriate ventricular pacings. After extending VIP extension from 100 to 150 ms, the above situation did not occur. This case should be distinguished from intermittent atrial undersensing, but there is no clear evidence to support it in Figures 2The authors declared that they have no conflicts of interest to this work.All authors reviewed and approved the manuscript. Directed this study: Yubin Zhang, Tong Liu. Wrote the manuscript: Yubin Zhang. Gave suggestions on this study: Tong Liu, Panagiotis Korantzopoulos.The study was approved by the institutional review board of The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. The patient provided written informed consent.


Papillary urothelial carcinoma (PUC) is the most common malignant tumor of the urinary bladder. Urothelial tumors are notorious for frequent recurrences and follow a chronic relapsing course in most of the patients. In Pakistan, the incidence of PUC is showing a rising trend. Various immunohistochemical (IHC) markers including androgens have been studied as prognostic and predictive markers in PUC with conflicting results. Androgen is a steroid-based sex hormone and plays an important role in different body organs such as urinary bladder, prostate, muscles, and brain. We aimed to investigate the role of the IHC expression of androgen receptor (AR) as a predictor of recurrence in papillary urothelial carcinoma patients. Eighty-four patients were included in the study. Tissues from the biopsy specimens of these cases were stained with antibodies against AR; 17% of the cases demonstrated a positive AR IHC expression. The expression was slightly more common in low-grade carcinoma. The AR expression was not significantly associated with clinicopathological features. Recurrence was observed in 49% of the cases, and it was significantly more common in AR-negative cases . Eighteen out of 19 patients who died of disease were AR- negative, but no statistical significance was observed. We conclude that the IHC expression of AR can be used as a predictive marker for PUC as it correlates with the recurrence rate. Papillary urothelial carcinoma (PUC) is the most common malignant tumor of the urinary bladder, comprising 90% of all primary bladder carcinomas. It is more common in males than in females ,2. In thAccording to Globacon 2018, bladder cancer ranks eighth among all malignancies in Pakistan, with 4,610 new cases and 2,614 deaths reported in 2018 . UrothelStudy population and settingsThis retrospective study was conducted on patients diagnosed with low- or high-grade PUC between July 2009 and June 2019. In all cases, follow-up data was determined through data available for at least one year for patient survival or reported deaths from carcinoma, depending on whichever came first. The study was conducted at the Department of Histopathology, Liaquat National Hospital and Medical College, Karachi, Pakistan. Institutional board approval was taken from the Research and Ethics Committee of the hospital.MethodologyThe surgical pathology slides at the Department of Pathology reported as PUC during the study period and paraffin blocks were reviewed. The hematoxylin and eosin section for each case was reviewed by two pathologists for confirmation of the original diagnosis based on the 2004 WHO/International Society of Urological Pathology Classification. In selected cases, immunohistochemistry (IHC) was performed using a monoclonal mouse anti-AR antibody on paraffin-embedded tissue sections of transurethrally resected tissue samples. The Dako REAL EnVision Detection System, Peroxidase/DAB, Rb/Mo  was used for IHC staining. Briefly, 5-µm serial sections were cut for formalin-fixed, paraffin-embedded (FFPE) tissue onto Superfrost slides . The sections were deparaffinized in xylene and rehydrated in graded series of ethanol . Heat-induced antigen retrieval was performed in a 10-mM citrate buffer (PH6.0) for one hour in a boiling water bath. Endogenous peroxidase activity was blocked by immersing slides in 0.3% w/v H2O2 room temperature for 10 minutes. Next, the antihuman AR antibody  was applied for four hours at room temperature. After three washes for five minutes each in phosphate-buffered saline (PH7.4), horseradish peroxidase (HRP)-labeled secondary antibody was applied for one hour at room temperature. After washing, the substrate was added and DAB was used for visualization. Hematoxylin was used for counterstaining. The primary antibody was replaced by phosphate-buffered solution and prostatic tissue served as negative and positive controls respectively.ScoringThe AR expression was assessed by counting 500 tumor cells in the most immune-reactive area of the slides. The AR expression was considered positive when >10% of the tumor cells showed nuclear staining .Statistical analysisThe Statistical Package for Social Sciences version 20  was used to analyze data. Frequencies and percentages were computed for categorical variables. Mean and standard deviations were computed for quantitative variables. A chi-square test was applied to investigate the association of categorical variables. The odds ratio (OR) was calculated by univariate binary logistic regression. A p-value of ≤0.05 was considered statistically significant. Overall survival was taken as the time from surgical excision till death or the last follow-up, and disease-free survival was defined as the time between surgical excision and local recurrence or distant metastasis, death, or the last follow-up.A total of 84 PUC patients were included in our study, of which 66 were males (78.6%) and 18 females (21.4%). The mean age was 64.30 (±15.34) years (range: 8-92 years). Both low- and high-tumor grade PUC groups comprised 42 patients each. The mean follow-up duration was 13.32 (±10.55) months (range: 1-40 months). Recurrence was defined as the appearance of new growth in the bladder and/or ureter at least six months after the complete removal of initial growth. The recurrent growth was histologically confirmed to be PUC. Recurrence was noted in 41 (48.8%) patients. In our study, positive staining of AR was observed in 14 cases (16.7%). Detailed clinicopathological features of patients are presented below  patients, while among AR-positive patients, recurrence was observed in only 3 21.4%) patients. We found a significant association between AR-receptor negativity and recurrence (p: 0.025). AR was positive in 8 (19%) low-grade and 6 (14.3%) high-grade carcinomas; there was no significant association between AR expression and tumor grade (p: 0.558) , while older patients were more likely to develop recurrence in comparison with younger patients (OR: 2.78) Table .AR is a ligand inducible transcription factor that mediates the biological effects of androgen hormone . AndrogeIn Pakistan, bladder cancer incidence is rising . In our Androgen hormone-binding activates AR leading to the progression of bladder cancers . AndrogeAndrogen hormone and its receptor have been found to be strongly associated with bladder cancer progression and tumorigenesis ,19,20. IA multi-institutional based study on a large number of patients compared tumor grade with androgen status. It found no association between tumor grade and AR status . This waIn our opinion, such varied results in studies can be attributed to multiple factors, such as sampling and laboratory techniques. In sampling techniques, factors that can influence could include sample size and genetic make-up of the population being studied. The laboratory factors that can contribute include the fixation time of tissues in formalin, the use of antibodies from different companies, methods of dilution, different cut-off parameters used for positive and negative results, and IHC vs molecular-based techniques.Urinary bladder carcinomas belong to a group of tumors that have a high recurrence rate; therefore, regular surveillance with urine cytology and cystoscopy is an essential part of treatment protocol . There iOur study strengths include a large sample size and patient follow-up. Limitations were the usage of IHC methodology and transurethral resection samples.We are able to conclude that positive expression of AR correlates with decreased recurrence rate in PUC, irrespective of histologic grade. AR can be utilized as a new predictive marker in urothelial carcinomas, especially in developing countries.


AR) mutations and one of the causes of 46, XY disorder of sexual development (DSD). This study aimed to describe the clinical features and molecular defects of 36 Chinese patients with AR variants and investigate the functional alterations of novel variants in vitro.Androgen insensitive syndrome (AIS) is a rare genetic disease resulting from androgen receptor  of patients harbored additional variants other than AR. Mutations in the Arg841 residue were found in 7 unrelated patients. Postpubertal serum gonadotropin levels were significantly elevated in patients with complete AIS (CAIS) compared with those in patients with partial AIS (PAIS) (P < 0.05). All the novel variants initially predicted to be uncertain significance by in-silico analyses were reclassified as likely pathogenic for defective AR transcriptional activity in vitro, except p.L295P, which was found in an atypical patient with oligogenic mutations and reclassified as likely benign. c.368_369 ins T was observed to interfere with nuclear translocation.Eight novel and fifteen reported AR variants and re-examining the diagnosis of AIS in specific patients with oligogenic mutations, instead of in-silico analysis.Compared with PAIS patients, postpubertal CAIS patients had higher gonadotropin levels. Arg841 was disclosed as the location of recurrent mutations in Chinese AIS patients. Functional assays are important for reclassifying the novel The online version contains supplementary material available at 10.1186/s13023-021-01765-w. Androgen insensitivity syndrome  is the common cause of 46, XY disorders/differences of sex development (DSDs) –3, and iAR gene that lead to complete or partial resistance to the physiological effects of androgen in 46, XY individuals [The process of male sexual differentiation and maturation is strictly related to androgen and normal androgen receptor (AR) function . AR is aividuals .AR gene (NR3C4) is located at chromosome Xq11-12 and consists of eight exons [AR gene related to AIS [The ht exons . The entht exons , 10. To APOD (apolipoprotein D) gene in fibroblasts was used to evaluate the function of the androgen receptor [AR variants that were identified using targeted NGS and investigated the functional alterations of eight novel AR variants in vitro.DHT-dependent transcriptional induction of the androgen-regulated receptor , but the150 Chinese 46, XY DSD patients, who visited our hospital from 2009 to 2019 and could not be diagnosed according to the typical clinical phenotypes and routine candidate gene strategies, were recruited for this study. The phenotypes of these patients ranged from different degrees of undermasculinization to a nearly complete female appearance. The Ethics Committee  of the Ninth People’s Hospital of Shanghai approved all protocols. Patients provided written informed consent.A total of 80 related genes were selected in the NGS panel , the variants were classified into five categories: pathogenic, likely pathogenic, variants of uncertain clinical significance (VUS), likely benign and benign . Novel Aanalyses . Becausel assays .AR variants were enrolled for further study. The clinical data were collected at the subjects’ first visit to our hospital without any medication, however, patients 5 and 9 had already undergone ‘inguinal hernia surgery’. External masculinization score (EMS) was calculated to obtain a quantitative measure of the degree of undermasculinization [Thirty-six subjects with identified nization . Serum Tnization . All the4-Luc, which contains four synthetic tandem repeats of an androgen-responsive element (ARE), was a kind gift from Dr. Xin Yuan from Beth Israel Deaconess Medical Center, Harvard Medical School. Wild-type (WT) human AR cDNA was amplified from pCMV-hAR by PCR using specific primers with restriction enzyme sites (Xho I-BamH I) and subsequently cloned into the expression vector pEGFP-N2 to generate pEGFP-N2-AR-WT . p(ARE)Eight mutant AR expression vectors carrying the novel variants, including c.368_369 ins T, p.L295P, p.A588P, p.G590R, p.R761W, p.L812Q, p.F879C and p.I907V, were created by site-directed mutagenesis  using pCMV-hAR-WT and pEGFP-N2-AR-WT as the templates respectively. The primers used for mutagenesis are listed in Additional file 4-Luc (200 ng/well) and pRL-SV40 (10 ng/well) containing Renilla luciferase as an internal indicator. After 24 h, the LNCaP cells were incubated in RPMI 1640 containing 10% dextran-coated charcoal-stripped fetal bovine serum and 10–7 M DHT.Dimerized ARs translocate into the nucleus and bind to the ARE sequences found in various AR target genes to initiate a complex transcriptional program in target cells, such as LNCaP, the human prostate cancer cell line . Transcr–7 M, 10–6 M or 10–5 M DHT for 24 h.To eliminate the possible influence of the LNCaP cell line or DHT concentration on the functional activity of mutant ARs with p.L295P, transcriptional assays were repeated under the same conditions but in the embryonic kidney (HEK) 293T cells incubated with Dulbecco's modified Eagle's medium containing 10% FBS containing 10LNCaP and 293T cells were washed twice with PBS and lysed via incubation with Passive Lysis Buffer  for 20 min. The lysate (20 μl) in each well was sampled to measure the firefly and Renilla luciferase activities using the Dual-Luciferase Reporter assay system . The results were normalized to the internal Renilla control and presented as the relative luciferase activity. All the experiments were carried out in triplicate wells and repeated three times.–7 M DHT except for one dish transfected with WT AR. The medium was removed after 24 h. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min. Nuclear counterstaining was performed with Hoechst  for 5 min, and images were obtained by confocal laser scanning microscopy .The pEGFP-N2 vector, pEGFP-N2-AR and 8 novel mutants were transfected into 293T cells in a confocal dish with Lipofectamine 2000 (5 μl). The cells were incubated with 10P value < 0.05 indicated a significant difference.Data are presented as medians with interquartile range. The Mann–Whitney U test was performed for comparisons of hormone levels between CAIS and PAIS patients and for AR functional assays using GraphPad Prism 8.0 software . A two-sided AR gene were identified in 36 patients from 31 unrelated families (Table AR mutation. Seven unrelated patients (19.4%) harbored missense mutations at codon 841, with different amino acid changes (p.R841C or p.R841H). Patient 20 had 2 different missense AR mutations (p.P392R and p.R841C). Patient 36 was observed to have large deletion from exon 2 to exon 8 of AR by capture-based NGS. Moreover, 30.6% (11/36) of patients carried more than 2 variants other than AR, especially patient 3, who harbored variants in four other related genes  and PAIS (n = 24) group. All the CAIS patients were reared as females. Sixteen patients in the PAIS group were raised as male, six were reared as females, and two patients underwent gender reassignment after puberty. Gynecomastia was found in 95% (21/22) of postpubertal patients with AIS. Patients 5 and 9 underwent ‘inguinal hernia surgery’(orchiectomy) before their first visit to our hospital. Thirteen patients had a family history of similar clinical presentation.P < 0.05) and Serum LH  were significantly higher and EMS was significantly lower (P < 0.05) in postpubertal patients with CAIS than those in patients with PAIS  or ASI  between the CAIS and PAIS groups , except p.L295P   Fig. a.Fig. 3TGFP-tagged WT ARs showed nuclear localization with a uniform distribution. A similar nuclear localization pattern was observed for the seven novel missense mutants . However, the AR mutant c.368_369 ins T showed a distinct cytoplasmic localization, which suggests impaired nuclear translocation Fig. .Fig. 4NuAR variants were initially predicted to be VUS. However, according to the guideline of ACMG, based on the results of in vitro functional assays (PS3) and absence from controls in 1000 Genomes Project (PM2), all of the novel variants were reclassified as likely pathogenic except P.L295P. This variant met ‘Well-established in vitro functional studies show no damaging effect on protein function’ and ‘Variant found in a case with an alternate molecular basis for disease’ (BS3 and BP5) and was reclassified as likely benign, despite it was also predicted to be damaging by SIFT, Polyphen-2 and MutationTaster analysis (Table All the novel AR gene [P < 0.05) , suggesting the Arg 841 residue is a mutation ‘hotspot’ in Chinese AIS patients.A severe AIS phenotype may be related to LBD mutations, especially mutations between amino acid residues 688–712, 739–784 or 827–870, which comprise the greater part of the ligand-binding pocket , 19. HerSRD5A2 mutations causing 5ɑ-reductase type 2 deficiency and variants in the CBX2 gene reported in prenatal 46, XY phenotypic females [AR mutation was detected  paralleled by reduced AR expression, mutations outside the AR coding sequence, and variants in AR cofactors crucial for proper AR activity were also presumed potential causes [AR mutations themselves, causes AIS. However, most of the reported AR mutations detected in DSD patients were predicted to be pathogenic or likely pathogenic via in silico tools instead of functional assays in vitro. In general, most algorithms are 65–80% accurate for missense variant prediction when examining known disease variants [SCN5A variants initially predicted as VUS by in-silico analyses were reclassified as benign/likely benign (n = 14), likely pathogenic (n = 35) and still VUS (n = 12) via high-throughput automated patch clamping [AR variants, which were initially predicted to be VUS, significantly impaired the transcriptional activity of AR and were reclassified as pathogenic/likely pathogenic, except p.L295P  [AR mutations  [AR mutations (p.Q799E and p.C807F), in vitro assays showed that these two mutations impaired AR function and exerted a synergistic negative effect on the AR transcriptional ability in combination [AR mutations (p.P392R and p.R841C), which was consistent with the results of the transcriptional assays of these two mutations in vitro.So far with AIS . The redl causes , 13, 26.variants . Accordiclamping . In our  system) , similar system)  and was p.E213E) . Howeverp.E213E) . Anotherp.E213E) . In a CAbination . Unlike NR5A1 and p.A1411T of MYH, by targeted NGS  promoter. The transfected LNCap cells were incubated with 0.1% dimethyl sulfoxide  or variable concentrations of DHT. The relative luminescence activity was expressed as a ratio of firefly to Renilla luciferase. The error bars represent the standard error. *, vs 0.1% DMSO P < 0.05; #, vs 10−7 M DHT P > 0.05


The mutation was mapped to the phytochromobilin synthase gene AUREA (AU), in which a single amino acid substitution from asparagine to tyrosine occurred. In addition to the classic function of AU in phytochrome and chlorophyll biogenesis in leaves, we uncovered its novel role in mediating AR formation on stems. We further observed temporal coincidence of the two phenotypes in the rf mutant: chlorosis and spontaneous AR formation and revealed that AU functions by maintaining heme homeostasis. Interestingly, our grafting results suggest that heme might play roles in AR initiation via long-distance transport from leaves to stems. Our results present genetic evidence for the involvement of the AU–heme oxygenase-1–heme pathway in AR initiation in tomato. As fruit production and yield in the rf mutant are minimally impacted, the mutation identified in this study may provide a target for biotechnological renovation of tomato germplasm in future breeding.Flooding tolerance is an important trait for tomato breeding. In this study, we obtained a recessive mutant exhibiting highly enhanced submergence resistance. Phenotypical analyses showed that this  Compared to the huge efforts in rice, tomato, a commercially important vegetable, has received little attention with regard to waterlogging resistance3. The widespread popularity of tomato around the world has led to its cultivation in a variety of environments. As with many terrestrial plants, most tomato varieties have no flooding resistance and are very sensitive to submergence4. Thus, identifying genes that enhance flooding tolerance without affecting productivity is an important strategy to improve tomato adaptability.Frequent flood occurrences due to global climate change have posed a challenge to crop yield and agricultural productivitySub1A, which mediates the response of plant hormones to lower carbohydrate consumption when plants undergo submergence damage5. A more common way in most plants, however, is to enhance the formation of adventitious roots (ARs), which facilitate the absorption of dissolved oxygen and the uptake of minerals in the waterlogging condition6.Plants have developed different strategies to address submergence. Some crops, including rice, form aerenchyma in root cortex tissues to ensure gas exchange, reducing hypoxic injury. A number of beneficial genes have previously been identified, including 8. In tomato, waterlogging has been shown to trigger the formation of ARs9. The mechanism underlying waterlogging-induced ARs often involves multiple endogenous phytohormones10. A high level of auxin promotes AR initiation, while an increased ethylene level enhances the sensitivity of plants to the auxin response12. Other hormones including cytokinin, GA, JA, and ABA all participate in AR formation and often interact with each other during AR development16. In addition to these phytohormones, a gaseous signaling molecule, carbon monoxide (CO), mainly derived from the decomposition of endogenous heme by heme oxygenase-1 (HO-1) during phytochrome synthesis, has been shown to affect AR formation in incised plant tissues18. Despite these early physiological works and exogenous treatments, no genetic evidence has been found, and it remains unclear whether endogenous heme or CO functions in vivo to affect AR formation.ARs are generally observed in herbs and woody plants, but the natural occurrence of ARs in dicot vegetables is rareAUREA (AU) that not only led to leaf chlorosis but also caused the spontaneous formation of ARs on stems. Under flooding stress, au mutants extensively formed ARs along the stem, while wild-type (WT) plants produced ARs only at the root–shoot junction. After submergence for 7 days, the resistant to flooding (rf) mutants showed higher sensitivity to flooding regarding AR initiation compared to WT. Interestingly, mutation of the AU gene appears to have minimal impact on tomato growth and fruit yield. Further analyses demonstrated that the flooding resistance of rf mutants mainly results from the accumulation of heme and enhanced HO-1 activity, both of which function in the phytochrome synthesis pathway. In addition, we show that the exchange of substances, including heme, between leaves and stems accounts for AR formation on stems. Our study provides genetic evidence that the AU–HO-1–heme pathway induces ARs in tomato, and our findings offer a target for trait improvement by biotechnology in future tomato breeding.In this study, we identified a mutation in the tomato gene 19 and isolated a mutant that exhibited high flooding tolerance. We named the mutant rf. At 7 DPF, the rf mutant remained alive with only slight wilting  were visible even on the fifth distal internode , the tomato plants became wilted, and at 7 DPF, most plants died Fig. , c. Usining Fig. , with a ing Fig. . In conting Fig. . Interessis Fig. , right asis Fig. , left. Usis Fig. . In contons Fig. . We furt WT Fig. , but rf age Fig. . AR formode Fig. , right. ode Fig. .Fig. 1rfrf also contributes to flooding resistance, we artificially created etiolated seedlings by growing WT tomato in darkness and then treated them with flooding. As shown in Supplementary Fig. rf mutants.To further determine whether chlorosis in 2 mapping population by crossing the rf mutant with WT. F1 plants from the crossing exhibited the normal phenotype, and the segregation ratio of AR to non-AR plants in the F2 population conformed to Mendel’s separation law (3:1) based on the χ2 test  , we identified a linkage peak on the short arm of chromosome 01 , Solyc01 g008930 encodes a phytochromobilin (PΦB) synthase gene AU, which is homologous to AtHY2. The SNP is located in the Fe-bilin-red domain of the AU protein  Fig. . Thus, t 01 Fig. . We then 01 Fig. . The fun 01 Fig. , and our 01 Fig. . The mut 01 Fig. . Accordiein Fig. . PreviouAU participates in AR formation, we cloned the full-length cDNA of AU and expressed it under the 35S promoter in the rf mutant (OE-AU/rf). We obtained seven independent stably transgenic tomato lines with OE-AU expression in the rf mutant background (OE-AU/rf). We verified that all these lines significantly overexpressed the gene (increased by 3–700-fold). Different from the case in WT   and reached an approximately normal status after 7 DPR  , while the scion of rf had increased AR numbers (approximately eight ARs in) on the hypocotyl of rf stocks  Fig. , graftincks Fig. . Althougted Fig. . However rf Fig. . The HO-nts Fig. . Thus, w27. Auxin is one of the most important phytohormones that promotes ARs, and many previous studies on de novo root regeneration have indicated that different early signals need to be converted into the auxin response during AR formation29. In cucumber cuttings, exogenous NPA, an auxin transport inhibitor, not only repressed the auxin dependent AR numbers but also decreased the endogenous HO-1 activity as well as CO content. In addition, exogenous NO and CO solutions were both shown to increase the AR numbers30. Therefore, both HO-1 activity and CO content can have positive roles in the process of AR initiation.AR formation is a complex process and is controlled by multiple environmental and endogenous factors, such as light, phytohormones, temperature, sugar, and other small molecules31 and is derived from the decomposition of heme by HO-1. In this study, we demonstrated that the activity of HO-1 is positively associated with AR formation, consistent with a previous report that exogenous hematin induces HO-1 activity and increases AR numbers in cuttings32. Based on the change in HO activity in rf, the fact that CO is the major product of heme/HO, and the evidence that CO acts as the signaling molecule to induce AR formation, we speculate that CO is likely functional in the heme/HO pathway to induce AR initiation in rf mutants.In both animals and plants, HO is regarded as the primary source for endogenous CO production, which is an intermediate metabolite in the Fe branch of the tetrapyrrole metabolic pathway34. However, no genetic evidence has been presented in planta that the accumulation of endogenous heme and the activity of HO-1 affect AR induction. Here, we demonstrate that AU not only functions in leaf chlorosis but also in AR development in stems. Interestingly, the OE-AU/rf lines showed the same effect on stem diameter and leaf area as the rf mutants, both of which were significantly larger than that of WT  by increasing the level of phytochrome photoreceptors.Previously, it was also shown that HO-1 positively regulates lateral root (LR) initiationAU function and leaf chlorosis has been previously reported23, AU activity in AR initiation has not yet been discovered. The chlorophyll content of rf was lower than that of WT before the three-leaf stage, when ARs were not visible. However, after this stage, the chlorophyll content in rf started to increase, which was coincident with the occurrence of ARs. This phenomenon suggests a correlation between chlorosis and AR initiation. Nonetheless, chlorosis is not directly involved in AR formation and submergence resistance. Etiolated tomato seedlings formed no ARs on their hypocotyls, and these seedlings even exhibited weaker rf. We thus inferred that the extra ARs induced by the AU mutation might be the major factor that conferred submergence adaption.Although the connection between disrupted 37. Accordingly, any factor influencing the efficiency of tetrapyrrole metabolism would also affect phytochrome and chlorophyll synthesis. It has been known that the au mutation might induce a feedback response to repress the metabolic conversion from ALA to protoporphyrin IX in the tetrapyrrole pathway38. In addition, phytochrome is necessary to activate ALA synthesis23, and heme accumulation inhibits glutamyl-tRNA reductase activity40. Therefore, we conclude that the decreased chlorophyll content at the early stage was mostly derived from the repression of tetrapyrrole metabolism, which presumably resulted from AU mutation-induced heme accumulation and phytochrome deficiency. However, the high heme level enhanced HO-1 activity, which helped to release tetrapyrrole repression and promoted AR formation. This may be the reason that accelerated AR formation occurred along with the recovery of chlorosis in rf mutants. Together, we think that the ARs produced by rf mainly resulted from the defective Fe branch of tetrapyrrole metabolism by the null mutation of AU, which might affect chlorophyll synthesis of the Mg branch via a feedback effect on tetrapyrrole metabolism.Downstream of tetrapyrrole metabolism are two different pathways (Mg branch and Fe branch) that share a common substrate, protoporphyrin IXAU functions downstream of HO-1 in phytochrome biosynthesis41. HO-1 decomposes heme to biliverdin IX for AU catalyzed PΦB synthesis, and PΦB is the direct substrate for phytochrome synthesis in plants42. Thus, both AU and HO-1 are essential for controlling the types and quantities of phytochromes in plants. Phytochrome synthesis catalyzed by AU and HO-1 mainly occurs in the plastids of the green leaf, while AR formation is restricted to stems. This spatiotemporally distinct location prompted us to hypothesize that the upstream intermediates of AU, such as heme and CO, may flow from leaves to hypocotyls or stems, which is necessary for HO-1 activation and AR formation. This is supported by the result that HO-1 activity was enhanced in rf hypocotyls  were unable to support AR formation through the active absorption of Fe. In contrast, small molecules such as CO that are not absorbed from the environment can trigger AR formation. Thus, we inferred that CO rather than Fe is more likely to participate in AR initiation. In addition, Fe has less mobility in plants, and due to its readily diffusible property, CO is more likely to function in inducing AR in plants.Fe is one of the byproducts of HO-1-catalyzed heme decomposition. The application of exogenous Fe has been previously reported to stimulate AR initiation in leafy cuttings of Petunia hybridsAU possibly leads to heme accumulation and enhanced HO-1 activity in hypocotyls, stimulating AR initiation. Taken together, our results revealed a potential in planta pathway consisting of AU–(HO-1)–heme–CO in AR development.Here, we provide genetic evidence showing that mutation of 47. In this study, we conducted grafting between rf and WT to confirm substance exchange between leaves and hypocotyls in rf, which had a positive effect on AR induction. Although our results support that the heme from rf leaves is necessary for activating HO-1 and AR initiation in stems, it remains unclear why rf scions failed to stimulate HO-1 activity and AR primordia on WT stocks. It is likely that the heme from rf leaves alone is essential but insufficient to induce AR initiation in hypocotyls. The different HO-1 activity between rf/WT and rf/rf reinforces that genetic background also determines HO-1 activity in hypocotyls.Since the initial site of heme accumulation is presumably leaves, long-distance transport of heme appears to be necessary for AR formation on hypocotyls. Grafting is a technique that has been widely used to ameliorate the poor traits of cultivated varieties in agriculture and to improve communication between different genetic materials48. One significant role of AR is to enhance plant resistance under flooding stress. Although spontaneous ARs can occur in the hypocotyl of Arabidopsis and many factors have been shown to affect wound-induced ARs49, it has rarely been reported that leaf-derived chemicals act in a long-distance manner to influence AR formation on hypocotyls. It is noteworthy that the contribution of leaf-derived auxin and sugars to AR formation in the stems of cuttings has also been shown52. In this study, we highlight the importance of other metabolic substances by showing that accumulation of heme metabolic intermediates in leaves can activate HO-1 activity in hypocotyls. Indeed, grafting between rf and WT confirmed that the potential communication between leaves and hypocotyls plays an important role in AR formation. Thus, our results provide additional evidence suggesting that metabolites derived from leaves can affect the development of stems, such as the formation of ARs.As plants are sessile organisms, they have evolved complex mechanisms to facilitate adaptation to environments. Both the primary root and LR are mainly responsible for absorbing and anchoringAU mutation does not reduce the weight per fruit , they possibly had insufficient heme fluxed into stems for AR stimulation. Our findings provide potential genetic evidence supporting the possibility of the endogenous AU–HO-1–heme–CO pathway in AR induction in tomato  WT and mutant rf (TOMJPW1898-1) seeds were obtained from TOMATOMA (http://tomatoma.nbrp.jp/), and rf was isolated by screening an EMS library generated in the cultivar Micro-Tom (TOMJPF00001)19. All materials were grown in a greenhouse at 28 °C with 16 h in light and 18 °C with 8 h in darkness. All plants were planted in 9 × 9 × 9-cm pots with a standard substrate (PINDSTRUP). The seedlings were grown with Hoagland nutrient solution with light intensity at 5000 Lx.Tomato  with water deep enough to completely submerge the whole plants, with the water surface less than 1 cm above the plant top. Growth conditions were evaluated, and the total number of ARs on each plant was counted. At least seven plants for each variety were evaluated. For further flooding resistance verification, both WT and rf were grown in half-strength Hoagland nutrient solution and treated with 200 µM zinc protoporphyrin IX  for 5 days. The ZnPP IX powder was first dissolved in DMSO to prepare a 5 M stock solution. We then added a certain amount of the stock solution directly to the hydroponic nutrient solution for a final concentration 200 μM. The number of ARs was quantified, and the middle section of hypocotyls (~5 mm length) where the ARs occurred was collected for the measurement of HO-1 activity.Drug treatment was conducted at the five-leaf stage. WT and 54. The reaction was carried out at 37 °C for 30 min after NADPH was added to the enzyme extract. The final production of biliverdin IX was used to evaluate the activity of HO-1. Fresh hypocotyl tissue (1 g) was quickly ground into powder in liquid nitrogen, extracted immediately with 30 mL ice-cold isolation medium, and then filtered into a centrifuge tube through four layers of filter cloth. The samples were centrifuged at 1300 × g for 30 min. The supernatant was transferred to a new centrifuge tube followed by centrifugation at 60,000 × g for another 30 min, and the final supernatant was used for HO activity measurements.We chose five tomato plants with similar growth conditions and collected their hypocotyls for the measurement of HO-1 activity. In all these experiments, we chose the hypocotyl of the rootstock for the measurement. HO-1 activity was assayed and calculated according to a previous methodHypocotyl sections were fixed in FAA  overnight at 4 °C. After dehydration in different concentrations of alcohol , the sample was embedded in wax. A Leica EG1150 was used to cut 10-μm-thick sections, which were stained with 0.5% toluidine blue until the tissue outline was clearly visible. All stained sections were used in hypocotyl AR primordium detection under a stereomicroscope (Leica M205 FA).rf, and OE-AU/rf lines were collected with major veins removed. Chlorophyll was extracted with 10 mL 96% alcohol according to a previous method with some modification55. During the extraction, the samples were kept in darkness and mixed many times until all the tissues became white. Finally, each sample was diluted tenfold with 96% alcohol, and the optical density was determined at 665, 649, and 470 nm with an ultramicro spectrophotometer (IMPLEN GMBH). Ninety-six percent alcohol was used for the control. We repeated this experiment three times for quantitative analysis.We selected 12 tomato plants with very similar growth conditions and mixed their leaves for chlorophyll measurement. Leaves (0.1 g) from WT, 47. WT and rf at the five-leaf stage were cut into two parts at the top of the hypocotyl; the upper part was used as the scion, and the lower part was used as the rootstock. The whole graft was then wrapped firmly with a grafting clip to ensure good contact. After 15 days, the grafts survived, and the total number of ARs on different types of grafted hypocotyl was counted.Grafting was conducted as previously described with minor modificationrf. Total RNA was extracted using the Promega RNA extraction kit, and cDNA was synthesized using HiScript II 1st Strand cDNA synthesis Kit (Vazyme) according to the manufacturer’s instructions. qRT-PCR was performed using a Stratagene Mx3005P (Agilent Technologies) with ChamQ Universal SYBR qPCR Master Mix (Vazyme). Each analysis was based on three biological replicates, and error bars represent the standard deviation from the mean of three biological replicates. Gene-specific primers are listed in Supplementary Table ACTIN was used as an internal control.Cotyledons and leaves from tomato seedlings at different leaf stages  were used to analyze gene expression in WT and −∆∆CT method. Actin was used as the internal reference.Relative transcriptional values were determined by the 2rf. The fruit shape index was calculated by the maximum length divided by the maximum width. All corresponding fruits were used for evaluating weight per fruit. At least 50 random fruits for each line were included, and error bars represent the standard deviation of the biological replicates.At the red ripening stage, some yield traits, including plant height, stem diameter, weight per fruit, and fruit shape index, were analyzed in WT and Plant height was measured from the soil surface to the plant terminal bud; the internode diameter and total fruit numbers per plant were also determined for the corresponding plants. The internode diameter at the middle position of each internode was measured using a Vernier caliper (with 0.01 mm resolution). The total fruit per plant was counted including all mature and young fruits. Quantification of each line included at least seven plants, and error bars represent the standard deviation of the biological replicates.To quantify leaf area, we measured the terminal leaflet area of the third compound leaf using a scanner (Epson Perfection 4990 PHOTO). For each measured group, we used 12 tomato plants with similar growth conditions. Error bars represent the standard deviation of the biological replicates.https://solgenomics.net) under the following accession numbers: AU (Solyc01 g008930) and SlHEMA1 (Solyc04 g076870).The genes used in this article can be found in the Solanum Genomics Network (Legends of supplemental Figure1-7


Ovarian cancer is common gynaecological malignancy and a leading cause of death among women. Despite the advances in treatment strategies, majority of patients present with recurrence after first- or second-line treatment. Targeted therapy that has proven to be effective in other advanced or metastatic solid tumors have also demonstrated its efficacy in ovarian cancer. Recent studies have shown that the androgen receptor (AR) signalling is involved in pathogenicity and progression of cancer. Current observations suggest AR could be a potential target in managing the disease. In this case report we present a patient with high grade serous ovarian cancer (HGSOC) with multiple relapses with excellent disease control on AR inhibition with bicalutamide. Ovarian cancer is the third most common gynaecological malignancy and is the fifth leading cause of death in women. Due to lack of proper screening and early diagnostics, 70% of the patients are diagnosed with advanced stage ovarian cancer. The standard treatment for ovarian cancer includes surgery and combination treatment with carboplatin and paclitaxel. Many patients respond to the first line therapy; however 60–70% and 80–85% of women with residual disease < 1 cm and large-volume residual disease show recurrence [et al. reported that the serous ovarian cancer have more prevalence of AR expression than the non-serous ovarian cancer [The presence of hormone receptors such as androgen receptors (AR), progesterone receptors (PR) and estrogen receptors (ER) has been positively correlated with progression of many cancers including ovarian cancer –4. The En cancer . AR diren cancer , 10.®) [In this study, we report a case of recurrent high-grade serous ovarian cancer patient who had a durable response to AR targeted therapy designed based on Encyclopedic Tumor Analysis (ETA) (Exacta®)  which isA 57-year-old female with a prior history of hypertension, was diagnosed with Stage IIIC high-grade serous ovarian carcinoma (HGSOC) nearly 5 years ago. The patient received neo-adjuvant chemotherapy with paclitaxel and carboplatin every 3 weeks for 3 cycles. A repeat scan done post 3 cycles showed partial response to treatment and patient underwent a cytoreductive surgery at an outside center. The surgical pathology was consistent with Stage IIIC disease noted earlier with lack of good response to carboplatin and paclitaxel given neoadjuvantly. Hence treatment was switched to gemcitabine, cisplatin and bevacizumab during adjuvant therapy for 3 cycles. A repeat scan done post completion of treatment showed complete resolution of disease.BRCA negative status on germline testing at the time. The patient developed progressive disease after 9 months and underwent a second surgery with excision of the vaginal vault and the pelvic mass and omentectomy. Histopathology was suggestive of metastatic HGSOC. For the ideal therapeutic options, the sample was sent for multi-analyte Exacta® analysis. The overview of the treatment given is summarised in , TP53 (p. N263fs), PKHD1 (p. R3107Q) and IGF2R (p. A1425A) mutations. Copy number alteration analysis showed a copy loss in chromosomal regions including 4p, 4q, 5p, 5q, 6p, 7p, 10q, 11p, 12q, 13q, 17p, 17q, 18q, 19p and a copy gain in 7q and 16p. Longitudinal mutation profiling of cell-free DNA (cfDNA) was performed for the serial monitoring of circulating tumor burden in patient. Mutation load varied in the range 0% to 0.13% until 2019. However marginal increase in the mutational load of 0.26% and 0.56% was observed in the next follow-up samples (TP53 (p. A159V and p. W146fs) gene mutation was observed in cell free nucleic acid analysis and are reported to be associated with early relapse and adverse prognosis in HGSOC. She was negative for any germline mutations.The integrative, multi-analyte Exactae < 0.05 . The gen samples . TP53  and PR (80% of tumor cells); and moderate staining for AR (30% of tumor cells) . Chemose® based novel regimen with weekly injection of temsirolimus and oral therapy with tab bicalutamide 50 mg once daily. Temsirolimus was used due to the presence of PTEN loss (10q23.2) on genomic analysis and due to chemosensitivity analysis showing response in vivo. Bicalutamide was used due to the immunohistochemistry showing AR expression and also due to the fact that PTEN loss is known to confer resistance to aromatase inhibitor based therapy targeting the ER/PR. She was also started on supplementary treatment with quercetin and vitamin E capsules as per Exacta® based recommendation. The patient received 3 cycles of the combination therapy. However, temsirolimus was stopped since she started experiencing vasomotor side effects and patient was continued on tab bicalutamide 50 mg once daily, quercetin and vitamin E. She remains with no evidence of disease and no disease relapse over the last 30 months of Exacta® guided therapy.Patient was then proposed treatment with ExactaBRCA negative status on germline testing at the time. It is known that patients with ovarian cancer who fail two lines of therapy have very poor prognosis. An exploratory multi-analyte testing was carried out at this juncture to understand the biology of this tumor better and in an attempt to find newer therapeutic options.Ovarian cancer accounts for 295,414 new cases and 184,799 deaths around world-wide (GLOBOCAN 2018). More than 90% of the malignant cancers are of epithelial in origin and 70% of these malignant cancers belong to HGSOC . The firTP53 is the most frequently mutated tumour-suppressor gene in human cancer with the highest frequency of 80% in HGSOC [TP53 mutations detected in cfDNA at diagnosis and residual disease or disease progression. Various TP53 hotspot mutations can have different implications on outcome of disease and in response to chemotherapy [Mutational status of cfDNA reflects the genetic characterization of tumor lesion. in HGSOC . Studiesotherapy . Tumor motherapy , 17. Eleotherapy . The pre® guided treatment as a result of the AR directed therapy with bicalutamide. This response is not thought to be from temsirolimus since the patient was only able to take 3 cycles of the drug due to severe toxicity issues.In the current study, we identified high expression of AR protein in the patient. AR, a steroid hormone receptor belongs to the nuclear receptor superfamily and is activated by androgen hormones. Although no specifics cut-offs are practiced, in breast cancer some studies have used at least 1% nuclear staining of any intensity (1+ to 3+) as a positive AR IHC assay . In a Ph® based analysis. The higher expression of AR in the patient provides strong evidence of pathogenicity. Further, targeting AR with bicalutamide based therapy resulted in a durable response in this patient. Our result suggests targeting AR using bicalutamide as an efficient treatment strategy in AR positive HGSOC.In conclusion, we present a patient with advanced HGSOC with multiple relapses with moderate AR expression detected on multi-analyte Exacta


Accumulating evidence suggests androgen receptor splice variant 7 (AR-V7) may be associated with the prognosis of castration-resistant prostate cancer (CRPC) received novel hormonal therapy while its characteristic and prognosis value in hormonal sensitive prostate cancer is unclear.We aimed to evaluate the prognostic role of AR-V7 by progression free survival (PFS) and overall survival (OS) in hormonal sensitive prostate cancer (HSPC), and the AR-V7-positive-proportion difference in HSPC and CRPC. A search of PubMed, Embase, and the Web of Science was performed using the keywords prostate cancer, prostate tumor, prostate neoplasm, prostate carcinoma; AR-V7, AR3, androgen receptor splicing variant-7, or androgen receptor-3. Seventeen trials published due December 2019 were enrolled.AR-V7-positive proportion in CRPC was significantly larger than newly diagnosed prostate cancer (PCa) . Subgroup analyses indicated significantly higher AR-V7-positive proportion in CRPC derived from RNA in situ hybridization , exosome RNA  and tissue RNA . AR-V7-positive patients had a significantly shorter PFS than those who were AR-V7-negative treated with first-line hormonal therapy  and prostatectomy . OS  were better in AR-V7-negative than AR-V7-positive HSPC patients treated with first-line hormonal therapy. The limitations of our meta-analysis were differences in study sample size and design, AR-V7 detection assay, and disease characteristics.AR-V7-positive proportion was significantly higher in CRPC than that in newly diagnosed PCa. AR-V7 positive HSPC patients portend worse prognosis of first-line hormonal therapy and prostatectomy. Additional studies are warranted to confirm these findings. Prostate cancer (PCa) is the most common cancer in Western countries among male and the fifth leading cause of cancer death worldwide . Most meAR-V7 was firstly reported as an abnormally spliced mRNA isoform of the androgen receptor, which lacks the C-terminal ligand-binding domain but retains the transcriptional active N-terminal domain, and was constitutively active in driving the expression of androgen-responsive genes regardless of androgen level –7. AccumThough growing evidences suggest the prognostic value of AR-V7 in CRPC, it remains unclear whether AR-V7 may also serve as a prognostic biomarker in hormonal sensitive prostate cancer (HSPC). Several studies reported the predictive role of AR-V7 in the first-line hormonal therapy outcomes in HSPC , 15. DueThis meta-analysis reviewed 17 clinical trials to integrated different measurements and compared the expression of AR-V7 in newly diagnosed PCa and CRPC, evaluated the treatment effectiveness of first-line hormonal therapy, and estimated the prognostic value of AR-V7 in HSPC. In order to understand the differences between various AR-V7 detection methods, subgroup analyses were further performed. The prognostic value was assessed by the impact on progression free survival (PFS) and overall survival (OS) of patients with different AR-V7 status. These are preliminary clinical evidences of the prognostic role of AR-V7 in HSPC, and further studies in larger cohorts are warranted.This meta-analysis was conducted out in accord with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . Search The titles and/or abstracts of the retrieved studies were screened, and the full text of those that satisfied the selection criteria were reviewed. Eligible studies met the following criteria. 1. The study reported on prostate cancer and AR-V7. 2. The results included AR expression and the proportion that consisted of AR-V7 (the AR-V7-positive proportion) in both newly diagnosed PCa and CRPC patients. Other reported results included PFS or OS after radical prostatectomy or first-line androgen deprivation therapy (ADT). 3. The results were obtained in a clinical trial, including prospective or retrospective cohort studies or comparative series. Studies were excluded if they 1. reported the AR-V7-positive proportion in, or only enrolled, newly diagnosed PCa or CRPC patients; 2. did not report PFS or OS; or 3. were animal or in vitro studies. 4. Studies in languages other than English were excluded unless a translator was available. 5. Case reports; letters; comments; editorials; and review papers were excluded. When more than one report of the same trial was available, the most recent information, with longer follow-up and a larger patient population were included in the analysis.P value.1. The patient characteristics extracted from each included trial included age, tumor stage, Gleason score, baseline PSA and alkaline phosphatase, and median time from diagnosis to sampling. 2. The description of study design included the country in which it was conducted, the treatment received, the primary end point, and the hypothesis tested. 3. The numbers of patients enrolled, assigned to treatment with radical prostatectomy or first-line ADT, and followed up, and the median follow-up time were extracted. 4. The AR-V7-positive proportion in CRPC and newly diagnosed PCa patient specimens and the AR-V7 detection assay were recorded. 5. Survival data for patients included the number of patients with PFS median PFS, and hazard ratios (HRs) with 95% confidence intervals (CIs), and P-values 6. OS: number of deaths in each study, median OS, HR with 95% confidence interval (CI), After data were abstracted, analysis was performed using Review Manager Software . In all the included trials, efficacy data from all randomly assigned patients were analyzed on an intention-to-treat basis. The AR-V7-positive proportion in newly diagnosed PCa and in CRPC was evaluated. The primary end point of the meta-analysis was OS and the secondary end point was PFS.2 statistic. Odds ratio (OR) and HR estimates were weighted and pooled using the Mantel–Hansel random effect model. All statistical tests were two-sided, and statistical significance was defined as P < 0.05. No correction was made for multiple statistical testing.The main analysis compared PFS and OS for first-line ADT in HSPC by different AR-V7 status. For both OS and PFS, the summary measure was HR (95% CI). A random effect model was applied. Statistical heterogeneity among studies was evaluated using the Chi square test and the IThe study selection process is shown in Fig. Fifteen trials enrolling 1731 patients were included in the AR-V7-positive proportion meta-analysis, their characteristics are shown in Table 2 = 88%), ORs and 95% CIs were calculated by a random-effects model. Subgroup analysis of different AR-V7 detection assays was further performed, and there was no significant subgroup differences . The AR-V7-positive proportion was significantly higher in CRPC derived from RNA in situ hybridization (RISH) , exosome RNA  and tissue RNA  while other detection assays showed no statistical difference.Overall, 433 of 781 CRPC and 249 of 950 newly diagnosed prostate cancer patients were AR-V7 positive. As shown in Fig. 2 = 60%, P = 0.06), so the random effect model was applied to calculate the HR and 95% CI. The subgroup analysis found that the HR for the PFS of AR-V7-positive patients was 3.63  in patients treated with first-line ADT, 2.49  in patients received radical prostatectomy, and there was no significant subgroup differences .In the entire study population Fig. , the AR-2 = 6%, P = 0.30), a fixed effects model was used to calculate the HR and 95% CI.As shown in Fig. First-line hormonal therapy has been the standard-of-care for metastatic and locally advanced prostate cancer, but most patients eventually progress to castration-resistant prostate cancer within 2–3 years, which is a lethal stage of the disease. , 18. TheWe evaluated differences in the AR-V7-positive proportion in newly diagnosed prostate cancer and in CRPC. This systematic review and meta-analysis demonstrated that the AR-V7-positive proportion is frequently up-regulated in CRPC as compared to newly diagnosed PCa , and may emerge as an adaptive response to therapies targeting the AR-signaling axis, which is consistent with previous reports . A numbePFS and OS were evaluated to compare the prognosis of first-line hormonal therapy and prostatectomy in HSPC respectively with different AR-V7 status. Four studies were included, and treatment was analyzed in subgroups. AR-V7-positive patients had an increased risk of worse OS compared with AR-V7-negative patients of first-line hormonal therapy; same as PFS was associated with AR-V7 status in HSPC treatment. Since Antonarakis et al. –10 and SOur systematic review has limitations. The statistical power was limited by the small and distinct sample sizes of these studies, which ranged from 16 to 224 participants. Several factors might have contributed to seemingly contradictory results reported in these studies. Firstly, smaller studies certainly lead to less reliability because of the size effect and portend to publication bias. Funnel plots shown in Additional file We made several important efforts to handle with the limitations. First, a systematic, comprehensive, and reproducible search strategy was applied for the relevant studies in multiple online databases to minimize publication bias. Secondly, the eligibility criteria were clear and critical to limit bias from the varieties in AR-V7 detection methods, determination of cutoff points and definition of prognosis. Selection bias was acknowledged, but we believe that it was minimized by our specific restrictions of the qualification of studies in each step of the meta-analyses. Thirdly, further subgroup analyses of results concluded from different AR-V7 detection assays were performed. Fourthly, study design details, disease stage, AR-V7 detection method, types of therapy, baseline PSA, and follow-up period are tabulated and available for further analysis and reference Tables  and 2.The individual optimal therapy strategy of prostate cancer remains highly concern. Early combination of docetaxel to androgen deprivation therapy in hormone-sensitive metastatic prostate cancer indicated benefit in several randomized clinical trials –38. ClinIn conclusion, our meta-analysis clearly showed the AR-V7-positive proportion was significantly higher in CRPC than that in newly diagnosed prostate cancer. AR-V7 positive HSPC patients portend worse prognosis of first-line hormonal therapy and prostatectomy as shown by PFS and OS. AR-V7 might be a predictive biomarker in HSPC, indicates more aggressive and AR-V7 targeted therapy strategies to AR-V7-positive patient. Expanded, cross-institutional studies designed to further validate AR-V7 as a treatment selection marker is warranted and future studies aimed to improve AR-V7 measurements to assess the clinical utility of AR-V7 in HSPC are expected.Additional file 1: Figure S1. The funnel plot for meta-analysis of the AR-V7 positive proportion in newly diagnosis prostate cancer and CRPC. Figure S2. The funnel plot for meta-analysis of the progression free survival of first-line hormonal therapy in HSPC of different AR-V7 status. Figure S3. The funnel plot for meta-analysis of the overall survival of first-line hormonal therapy in HSPC of different AR-V7 status. Table S1. Target specimens and AR-V7 detection assay of studies in the positive proportion meta-analysis in newly diagnosed PCa and CRPC. Table S2. Definition of PSA response, PFS and OS in the studies included in the meta-analysis of prognosis for hormonal therapy and chemotherapy in different AR-V7 states.


Multiple mechanisms contribute to the survival and growth of metastatic castration‐resistant prostate cancer (mCRPC) cells without androgen, including androgen receptor splice variants (AR‐V) and de novo intratumoral androgen synthesis. AKR1C3 is a critical androgenic enzyme that plays different roles in mCRPC, such as an EMT driver or AR coactivator. However, the relationship and regulatory mechanisms between AKR1C3 and AR‐V remain largely unknown. In this study, we observed a positive correlation between AKR1C3 and AR‐V7 staining in tissues from prostate rebiopsy at mCRPC. Mechanistically, AKR1C3 interacts with AR‐V7 protein in CRPC cells, which can reciprocally inhibit AR‐V7 and AKR1C3 protein degradation. Biologically, this complex is essential for in vitro and in vivo tumour growth of CRPC cells after androgen deprivation as it represses B4GALT1, a unique tumour suppressor gene in PCa. Together, this study reveals AKR1C3/AR‐V7 complex as a potential therapeutic target in mCRPC. AR‐V7 is generated from aberrant alternative splicing of the third intron of AR pre‐mRNA. AR‐V7 is always expressed with full‐length AR (AR‐FL) in most cases, and its positive rate and intensity can be enhanced by hormonal therapies.Aldo‐keto reductase 1C3 (AKR1C3), a pivotal androgenic enzyme, is highly expressed in metastatic and recurrent prostate xenografts and in sets of CRPC bone metastatic tumours acquired from patients in several studies.In this study, we observed a positive correlation between AKR1C3 and AR‐V7 staining in tissues from prostate rebiopsy at mCRPC. Mechanistically, AKR1C3 bound to the AR‐V7 protein in CRPC cells, which reciprocally enhanced protein stability of AR‐V7 and AKR1C3 by inhibiting ubiquitin activity. Biologically, this complex was essential for in vitro and in vivo tumour growth of CRPC cells under ADT by uniquely repressing B4GALT1 expression. Consistently, B4GALT1 expression was significantly down‐regulated in PCa clinical specimens, especially in high‐grade, metastatic and recurrent PCa, which could be used to predict a poor prognosis of patients. Together, this study showed a new mechanism by which the AKR1C3/AR‐V7 complex regulates CRPC tumour growth by repressing a novel tumour‐suppressor gene.22.1The human PCa cell lines  and the embryonic kidney cell line (HEK293) were kindly gifted by Dr Leland WK Chung . Highly tumorigenic 22RV1 T sublines were generated from the isolated xenograft tumours of 22RV1 cells in athymic BALB/c nu/nu mice. 22RV1, 22RV1 T, VCaP and HEK293 cells were cultured in Dulbecco's modified Eagle's medium  with 10% fetal bovine serum (FBS). LNCaP and C4‐2 cells were cultured in T‐medium (Invitrogen) supplemented with 5% FBS. All these cells were maintained at 37℃ with 5% CO2.2.2http://rnai.genmed.sinica.edu.tw/index). Clone ID was TRCN0000278350 and TRCN0000026564. The vector was pLKO TRC005. Oligo sequence for AKR1C3 sh350 was CCGGCCGGAGTAAATTGCTAGATTTCTCGAGAAATCTAGCAATTTACTCCGGTTTTTG. Oligo sequence for AKR1C3 sh564 was CCGGCCGGAGTAAATTGCTAGATTTCTCGAGAAATCTAGCAATTTACTCCGGTTTTT. GFP‐AR‐V7 was kindly provided by Professor Jun Luo (Johns Hopkins University School of Medicine). The cDNA encoding the full length AR‐V7 (NM_001348061.1) was inserted into the pEGFP‐C3 vector to express the GFP‐AR‐V7 fusion protein. pcDNA3.1 AKR1C3 plasmid was kindly provided by Professor Allen Gao (UC Davis). To overexpress AKR1C3, LNCaP and 22RV1 cells were transfected with the empty vector pcDNA3.1 and pcDNA3.1 encoding AKR1C3, and both cell lines were maintained in medium with 300 mg/mL G418. Two B4GALT1 siRNAs  and a control were purchased from Thermo Fisher Scientific . Transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.The source and stable transfection of AKR1C3 shRNA and GFP‐AR‐V7 plasmids were described in our previous studies.2.3MG132 and cycloheximide (CHX) were purchased from Selleck Chemicals . All antibodies used in this study are listed below: Mouse monoclonal Anti‐β‐actin , anti‐AKR1C3 , anti‐AR‐V7 , anti‐ubiquitin , anti‐Bcl‐2 ; rabbit polyclonal anti‐AR (N‐20)  and anti‐B4GALT1 .2.4in vitro.The indicated cells were resuspended in 50 μl of DMEM with 10% FBS, plated in 96‐well plates at a concentration of 1500 cells/well and cultured for 24 hours and then recorded as 0 hours. Then, the medium was changed with phenol‐red free DMEM with 5% charcoal‐stripped FBS to mimic ADT conditions 2.5One thousand of the indicated cells were plated in 6‐well plates in duplicate and cultured in DMEM with 10% FBS in a humidified incubator containing 5% CO2 at 37°C. Cells were fed fresh phenol‐red free DMEM with 5% charcoal‐stripped FBS every 4 days for 2 weeks. Colonies were washed with phosphate‐buffered saline (PBS), fixed with 4% paraformaldehyde for 10 minutes and stained with 0.5% crystal violet for 5 minutes. Colonies were photographed, and visible colonies were counted.2.6RNA was isolated using RNeasy Kit . cDNAs were reversely transcribed by the RevertAid kit , and a 25 µl system realtime PCRs were carried out in an iCycler thermal cycler  using SYBR Green Supermix  with the gene‐specific primers: AKR1C3, F: 5’‐GAGAAGTAAAGCTTTGGAGGTCACA‐3’, R:5’ ‐CAACCTGCTCCTCATTATTGTATAAATGA‐3’; AR‐FL, F: 5’‐CTTACACGTGGACGACCAGA‐3’, R:5’‐GCTGTACATCCGGGACTTGT‐3; AR‐V7, F: 5’‐ CCATCTTGTCGTCTTCGGAAATGTTATGAAGC‐3’, R: 5’‐ TTTGAATGAGGCAAGTCAGCCTTTCT‐3’; SLC30A7, F: 5’‐ TCTCTTTCGCTTTTGTGGAACT −3’, R:5’‐ CCAGAACTTCCGCTCTAACATAC −3’; B4GALT1, F: 5’‐ CCAGGCGGGAGACACTATATT −3’, R:5’‐ CACCTGTACGCATTATGGTCAT −3’; HIF1A, F: 5’‐ GAACGTCGAAAAGAAAAGTCTCG −3’, R:5’‐ CCTTATCAAGATGCGAACTCACA −3’; SNX14, F: 5’‐ CCGCCTCCCTGCTTCTTAAC −3’, R:5’‐ CTGAAGTCCTAACTGCTTGGG −3’; 18S, F: 5’‐GGAATTGACGGAAGGGCACCACC‐3’, R:5’‐GTGCAGCCCCGGACATCTAAGG‐3’ .2.7Cells were treated by RIPA buffer  with 1% cocktail, proteinase inhibitors and 1 mmol/l PMSF . Then, 20 μg of protein was separated by 8%‐12% SDS‐PAGE and transferred to nitrocellulose membranes. Then, the membranes were blocked in Tris‐buffered saline with 0.1% Tween 20 and 5% skim milk for 1 hour. After blocking, the primary antibodies were used to incubate the membranes overnight at 4℃. After washing 3 times with TBST, membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. Then, membranes were visualized by an ECL chemiluminescent detection system . A monoclonal β‐actin antibody was used to normalize the loading difference.2.8For immunoprecipitation, VCaP, 22RV1 T and HEK293 cells transfected with GFP‐AR‐V7 were washed twice with cold PBS and lysed in 1.5 ml of cold lysis buffer for 20 min on ice. The immunocomplex was first precipitated with Dynabeads Protein G  and then subjected to Western blot analysis as described previously.2.922RV1 cells were transfected with GFP‐AR‐V7 and its control. Cells were washed with PBS, fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X‐100 and blocked in 5% bovine serum albumin (BSA). Cells were then incubated with primary AKR1C3 antibody at 4°C overnight and Alexa Fluor 633‐conjugated secondary antibodies, followed by nuclear staining with 5 μg/ml 4′,6‐diamidino‐2‐phenylindole . Signals were examined using a fluorescence microscope with glycerinum, and then a microscope was used to observe slides and take photos.2.9.16 22RV1 sublines (shCon and sh564) in 100 μl medium containing Matrigel  into both flanks. The mice were killed 4 weeks after cell injection, and xenografts were stripped for measurement, Western blot analysis and immunohistochemistry (IHC) staining.Male athymic BALB/c nu/nu mice were used according to protocols approved by the Ethical Committee of Xi'an Jiaotong University. All mice were castrated at the age of 4 weeks. One week later, mice were subcutaneously injected with 5 × 102.9.2All the prostate rebiopsies of mCRPC were obtained from the Department of Urology, West China Hospital of Sichuan University, China.2.10http://gepia.cancer-pku.cn/) was used to analyse the RNA sequencing expression data of tumours and normal samples, the correlation of gene expression and patient survival analysis.P value <0.05 was considered statistically significant.GEPIA . To further explore the potential downstream genes, we validated the expression of four AR‐V7 unique target genes  that were previously reported.P < 0.05). Clinical data from the TCGA suggested that the expression of B4GALT1 was significantly lower in PCa tissues than in normal tissues . Consistently, PCa patients with higher Gleason scores, more metastatic lymph nodes and biochemical recurrence exhibited a lower expression of B4GALT1 , and these patients had worse disease‐free survival . Collectively, these data suggested that the AR‐V7 target gene B4GALT1, as a novel tumour suppressor in PCa, was negatively regulated by AR‐V7/AKR1C3 complex.The functions of the AR‐V7/AKR1C3 complex on tumour growth were further studied by using MTT and colony formation assays in vitro. Indeed, 22RV1 T cells with high tumorigenic ability exhibited increased cell proliferation and colony formation abilities compared with parental 22RV1 cells, while knock‐down of AKR1C3 partially reversed this phenomenon  and de novo intratumoral androgen biosynthesis.AKR1C3 is an important enzyme in the steroidogenesis pathway that can produce androgen intratumorally to maintain CRPC tumour growth after ADT.In addition, AKR1C3 has been shown to play different roles in mCRPC, such as acting as an EMT driver or AR coactivator.AR‐V7, as a truncated AR without a LBD, was believed to persist constitute AR target gene activation. However, Cato et al recently utilized cistrome and transcriptome analyses of CRPC data to show that AR‐V7 functions as a transcriptional repressor and heterodimerizes with full‐length AR at a subset of growth suppressive genes to support CRPC growth. Four genes, including SLC30A7, B4GALT1, HIF1A and SNX14, were specifically targeted by AR‐V7 and showed a negative effect on CRPC cell proliferation.B4GALT1 is a member of the b‐1, 4‐galactosyltransferase gene (B4GALT) family and plays important roles in many biological events, including morphogenesis, mammalian fertilization, brain development and cellular adhesion. Recently, it was identified as a potential biomarker in malignancies with controversial effects. On the one hand, B4GALT1 serves as an oncogene in clear cell renal cell carcinoma, muscle‐invasive bladder cancer, lung adenocarcinoma, breast cancer and leukaemia.Taken together, this study identified AKR1C3 as a new critical regulator that increases the expression and transcriptional activity of AR‐V7 by forming a complex to inhibit its protein degradation. Additionally, this protein‐protein interaction can in turn to enhance AKR1C3 protein stability. This complex is essential for CRPC tumour growth as it represses the expression of the downstream B4GALT1 gene ; Data curation ; Formal analysis ; Methodology ; Resources ; Software ; Visualization ; Writing‐original draft ; Writing‐review & editing . Shiqi Wu: Conceptualization (supporting); Data curation ; Formal analysis (supporting); Methodology ; Software ; Validation ; Writing‐original draft . Yong Fang: Data curation ; Formal analysis ; Methodology (supporting); Validation (supporting); Visualization . Guangxi Sun: Data curation (supporting); Formal analysis ; Software ; Validation (supporting). Dalin He: Conceptualization (supporting); Funding acquisition (supporting); Resources ; Supervision . Jer‐Tsong Hsieh: Conceptualization ; Resources ; Supervision . Xinyang Wang: Methodology (supporting); Resources . Hao Zeng: Software . Kaijie Wu: Conceptualization ; Funding acquisition (lead); Project administration ; Supervision (lead); Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing .Fig S1Click here for additional data file.Supplementary MaterialClick here for additional data file.