Astragali Radix (AR) is one of the well-known traditional Chinese medicines with a long history of medical use and a wide range of clinical applications. AR contains a variety of chemical constituents which can be classified into the following categories: polysaccharides, saponins, flavonoids, amino acids, and trace elements. There are several techniques to extract these constituents, of which microwave-assisted, enzymatic, aqueous, ultrasonic and reflux extraction are the most used. Several methods such as spectroscopy, capillary electrophoresis and various chromatographic methods have been developed to identify and analyze AR. Meanwhile, this paper also summarizes the biological activities of AR, such as anti-inflammatory, antioxidant, antitumor and antiviral activities. It is expected to provide theoretical support for the better development and utilization of AR. Astragali Radix (AR), known by the Chinese name of Huang-qi (Ougi in Japanese name), is a traditional herbal medicine that has been used for over 2000 years in China [Astragalus membranaceus (Fisch.) Bge. or Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao (Fabaceae) [Astragalus membranaceus (Fisch.) Bge. and mainly grows in Shanxi, Inner Mongolia, Shaanxi, Gansu and other provinces in China as well as Korea and Mongolia [in China . Accordiabaceae) , which iMongolia . TherefoMongolia . In modeMongolia ,6,7, andOver the years, extensive research has been conducted on the chemical components of AR. It is known that AR root contains saponins, polysaccharides, flavonoids, amino acids, and trace elements . Among tAccording to available studies, more than 100 compounds have been isolated and identified from AR. Based on structure, it can be mainly divided into four groups, including polysaccharides, saponins, flavonoids and others. Enhancing the classification of different compounds in AR might facilitate the understanding of the pharmacological effects of AR.A. membranaceus. AG-1 is a water-soluble α (1→4) (1→6) glucan, and the composition ratio of α (1→4) (1→6) glycosidic glycosyl groups is 5:2. AG-2 is a water-insoluble α (1→4) glucan. AH-1 is a water-soluble acidic polysaccharide. After hydrolysis, hexuronic acid, glucose (Glc), rhamnose (Rha) and arabinose (Ara) uniting at 1:0.04:0.02:0.01 were detected. AH-2 was water soluble, with Glc and Ara uniting at 1:0.15, detected after hydrolysis. The polysaccharides APS-I and APS-II were isolated from the water extract of A. membranaceus. APS-I consisted of Ara and Glc, in a molar ratio of 1:3.45 and APS-II consisted of Rha, Ara and Glc uniting at 1:6.25:17.86 [A. membranaceus [The polysaccharides from AR are well-known for their hepatoprotective effects . As earl25:17.86 . Fang etranaceus . They weMw), identification methods, classifications, sources, and reference.The analysis of the polysaccharide from AR revealed three sugars: Glc, Rha, and Ara. Among them, Glc was the major component, followed by Ara and Rha . Table 11), Astragaloside II (4), Astragaloside IV (6), Astragaloside IV isomer/III (7) and Astragaloside V/VI/VII  in the roots of A. membranaceus using high-performance liquid chromatography–tandem atmospheric pressure chemical ionization mass spectrometry (HPLC–APCI–MS) [11) and Soyasaponin I (12) were detected and analyzed in AR using high-performance liquid chromatography-electrospray ionization-quadruple-time of flight-mass spectrometry (HPLC–ESI–QTOF/MS) [15) was found by Hirotani et al. [13) and Agroastragaloside IV (14) were determined by Zhou’s group [2), Isoastragaloside II (5) and Acetylastragaloside I (3) with NMR [1, 4, 6, and 13, Yu et al. also obtained alexandroside (16) from AR stem and leaves [A lot of saponins have previously been isolated and authenticated from AR. In 2007, Xu et al. isolated and identified Astragaloside I (APCI–MS) . In addiQTOF/MS) . Moreovei et al. ; Agroast’s group ; Kitagawwith NMR . These cMw, parent ions, fragment ions, sources, and others.1), ononin (2) and calycosin-7-O-β-d-glycoside (6) were separated from AR and analyzed using UPLC–DAD–ELSD [O-β-d-glucoside-6”-O-acetate (4), calycosin-7-O-β-d-glucoside-6”-O-acetate (8), pratensein (9), pratensein-7-O-β-d-glycoside (10), biochanin-A (11) and (3R)-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan (12) were isolated rapidly and identified with the help of HPLC–APCI–MS/MS [O-β-d-glucoside-6”-O-malonate (5), calycosin-7-O-β-d-glucoside-6”-O-malonate (7), -3-hydroxy-9,10-dimethoxypterocarpan (13), -3-hydroxy-9,10-dimethoxypterocarpan-3-O-β-d-glycoside (14) and astraisoflavanglucoside-6”-O-malonate (15) using LC–ESI–MS [3), wogonin (16), 3-hydroxyflavanone (17), medicarpin (18), isorhamnetin (19), mangiferin (20) and naringin (21) with the help of liquid chromatography–tandem mass spectrometry (LC–MS) from AR [Until now, many flavonoids have been isolated and identified from AR. Formononetin , active small molecules—riboflavin, caffeic acid, chlorogenic acid, niacin, linoleic acid, coumarin, folic acid, linolenic acid, lupeol, daucosterol, vitamin P, starch E, sterols, betaine, and choline—and other components. The chemical structures and molecular formulas of parts of these compounds are shown in γ-aminobutyric acid, asparagine, cystine, aspartic acid, threonine, glutamic acid, valine, alanine, methionine, isoleucine, serine, leucine, and so on. Among them, lysine, threonine, valine, leucine, methionine, isoleucine, and phenylalanine are essential amino acids for the human body [So far, it has been demonstrated that there are more than twenty kinds of biogenic amino acids in AR, such as glycine, man body .Since the components in AR can be divided into macromolecule compounds and small molecules, we discuss the different extraction methods used in the exploration of AR components mainly from two aspects: macromolecules (polysaccharides) and small molecules (saponins and flavonoids).Astragalus polysaccharides are a kind of bioactive ingredient extracted from Astragalus membranaceus, mainly composed of fructose, rhamnose, arabinose, hexuronic acid, glucose, galacturonic acid and glucuronic acid [nic acid . The difnic acid .Astragalus polysaccharides are soluble in water and insoluble in alcohol [ alcohol . At presApplying statistics-based experimental designs to optimize the extraction process of AP. The optimal conditions were: extraction time of 3 h, liquid-to-solid ratio of 4, and particle size of 33.8 mesh. Under these conditions, the maximal extraction rate of AP was 16.32% . The watThe microwave has a good auxiliary effect on the extraction of AP. The thermal effect of microwaves can break the cell wall . CellulaThe microwave-assisted extraction method for AP was studied, with water as the extraction solvent. The optimal extraction conditions were: the ratio of water/material— 12:1; pH—9.0, regulated by saturated limewater; microwave power—300 W; 2 extractions, each 10 min. The final yield was 14.6% and the purity was 88.1% .Astragalus membranaceus. The extraction time, heating temperature, pH value of extraction solution and solid–liquid ratio were optimized. The optimal operating process parameters were determined as follows: microwave heating time—20 min; heating temperature—120 °C; solid–liquid ratio—8:1; and pH value—10. The crude polysaccharide yield was 32% and the purity was 44.4% [Microwave extraction technology was also used to extract AP from the dried roots of Ultrasonic extraction utilizes ultrasonic waves to convert electromagnetic energy into thermal energy, makes openings and cracks appear on cells, and then breaks the plant cell wall and cell membrane structure, finally resulting in the release and dissolution of intracellular substances . CompareUsing the ultrasonic extraction method to extract polysaccharides, the optimum extraction conditions of AP in the single-factor test were: λmax = 490 nm, particle fineness—0.25 mm~0.5 mm; ultrasonic time—25 min; ratio of solid to liquid—1:25(vol); and extraction temperature—30 °C. The content of AP extracted could reach 4.92%.Astragalus polysaccharides is cellulase [Cellulose, the main component of AR cell walls, is the main barrier used to prevent the release of macromolecules such as intracellular polysaccharides. The traditional enzyme used to extract ellulase , which cellulase .Astragalus polysaccharide was optimized. A response surface methodology and orthogonal tests were applied to optimize the conditions: the combined enzyme concentrations of cellulase, pectinase, and papain were 1.5%, 1%, and 0.5%, respectively; the extraction time was 94.5 min; the extraction temperature was 49.9 °C; the pH value was 5.1 [The combined enzyme extraction of  was 5.1 .A response surface method was used to optimize the glucose oxidase-assisted extraction process to obtain the maximum yield of crude APS. The optimized extraction conditions were as follows: enzyme dosage, 3.0%; enzyme treatment time, 3.44 d; enzyme treatment temperature, 56.9 °C; extraction solvent pH of 7.8. Under these conditions, the experimental yield was 29.96 ± 0.14% . The conAstragalus resources and the identification of AR medicine quality. Presently, the main extraction methods of saponins and flavonoids from AR include reflux extraction, microwave-assisted extraction, and enzymolysis-assisted extraction [Saponins and flavonoids are the major active constituents in AR. Saponins mainly consist of astragaloside IV, astragaloside I and soyasaponin I and others; flavonoids mainly include calycosin, formononetin and ononin and others. The study of the extraction methods of saponins and flavonoids in AR has a positive effect on the screening of high-quality traction .Ethanol reflux extraction is applied as the main method for extracting saponins and flavonoids currently. This method is simple, stable, and feasible, and more importantly, it can extract active constituents such as saponins as much as is possible in AR.Astragalus mongholicus by the alcohol reflux extraction method. According to the results of single-factor experiments, the optimum conditions were confirmed as: temperature—75 °C; time—2.5 h; ethanol concentration—90% (v/v); and ratio of solvent to raw material—20 mL/g. The best yield was 0.934 mg/g [Astragalus. The contents of total extracts and flavonoids were regarded as indicators. The optimal process for the ethanol reflux extraction of total flavonoids from Astragalus was: extracted with 10 times 70% ethanol two times for 60 min each time [Yang et al. (2011) optimized the ethanol reflux extraction procedure parameters  using an orthogonal design method, with the astragaloside IV extraction rate as the detection index. The optimal conditions of alcohol extraction were: 60% ethanol; ratio of solid/liquid—1:6 (g/mL); and 3 extraction cycles, each 1 h . High-pe934 mg/g . Orthogoach time .A. membranaceus according to the extraction rate of flavonoids were obtained as followed: concentration of ethanol—95%; extraction time—20 min; ratio of material to liquid—1:15 g/mL; and temperature—90 °C. Under these conditions, the extraction rate of flavonoids was the highest (0.489%) [An MAE method was used to extract astragalosides I-IV from AR. MAE gave the highest extraction efficiency within the shortest time. The optimal conditions of MAE were: employing 80% ethanol as solvent; ratio of ethanol to the material—1:25 (g/mL); temperature—70 °C; irradiation power—700 W; and 3 extractions, each 5 min . The fla(0.489%) .The active substances of plant-based herbs are often encased in lignins, and enzymes can effectively degrade lignin and greatly increase the extraction rate of traditional Chinese medicine.Astragalus. The optimum extraction process was determined as follows: enzymolysis time—120 min; hydrolysis temperature—30 °C; enzyme dosage—8 mg; and pH—4.5 [An orthogonal design was used to study the optimum technology for the enzyme-assisted extraction of total flavonoids from d pH—4.5 .Astragalus, and the optimal conditions were: pH—4.38; enzymolysis time—132 min; enzymolysis temperature—51 °C; enzyme dosage—8.84 mg; predictive extraction rate—1.89%; and actual extraction rate—1.87 ± 0.03%. The technological conditions for the extraction of total flavonoids from A. membranaceus mongholicus by cellulose hydrolysis-assisted ethanol extraction were studied. The optimum process parameters were: amount of added cellulase—7.5 μL/g; enzymatic hydrolysis pH value—4.0; enzymolysis temperature—55 °C; enzymolysis time—1.5 h; ratio of liquid to solid—30 mL/g; ethanol concentration—90%; extraction temperature—75 °C; and extraction time—2.5 h; under these conditions, the extraction rate of total flavonoids was 0.57%. It increased by 46.15% compared to traditional alcohol extraction (0.39%) [A response surface methodology was used to optimize the process of cellulase-assisted extraction of total saponins from  (0.39%) .Based on the above review, we compared the advantages and disadvantages of each extraction method as shown in .v/v) to purify mullein with a purity of 95.8% and a recovery rate of 85.9% [O-glucoside, astraisoflavan-7-O-β-d-glucoside, and calycosin-7-glucoside [Pan et al. used a two-phase solvent system of n-hexane ethyl acetate–ethanol–water  [Astragalus samples from Shanxi, Gansu, Neimeng and Sichuan. The results showed that there was almost no difference in the contents of these seven metal elements in the Astragalus samples from these four regions. It is worth mentioning that surface-enhanced Raman scattering (SERS) technology was used to distinguish between the authentic Astragali Radix  and the most common adulterant samples  with a high sensitivity (100%) and specificity (97.6%) [The content of polysaccharides in 07 mg/g) . Inducti (97.6%) .With the selective distribution of different substances in different phases, chromatographic analysis elutes with the mixture in the stationary phase relative to the flow. Different substances in the mixture move at different speeds along the stationary phase and eventually reach separation.O-β-d-glucoside, ononin, 2′,4′-dimethoxy-3′-hydroxyisoflavan 6-O-β-d-glucoside, 10-hydroxy-3,9-dimethoxypterocarpan, afromosin, calycosin, formononetin, and rutin) in the A. membranaceus were separated and determined by using micellar electrokinetic chromatography (MEKC) with a DAD. Using fused silica capillaries, the micellar phase composed of 25% (v/v) acetonitrile, 100 mM sodium cholate, 20 mM Na2B4O7 and 20 mM H3BO3 buffer (pH 9.2) was used to obtain the optimal resolution at +25 kV and 25 °C [AR was established, using a silica gel G thin board. Chloroform–methyl alcohol–water (13:6:2) was used as the developer. The detected wavelength and reference wavelength were at 530 nm and 700 nm, respectively. The results indicated that the linear relationship of astragalin curves was good. The average recovery rate was 98.72% with RSD = 3.5% [Eight flavonoids  with a two-step two-phase solvent system of ethyl acetate-2-propanol-water . The contents of astragaloside I and astragaloside II were detected as 30.2 mg/g and 16.5 mg/g crude extract, respectively [Astragaloside I and astragaloside II were identified by thin-layer chromatography–tandem mass spectrometry (TLC–MS/MS) in the ethyl acetate extract of ectively .Astragalus [A sensitive and rapid ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) method was established for the simultaneous quantitation of two major categories of bioactive substances (triterpenes and flavonoids) in tragalus . Liu et tragalus .Liu et al. (2014) identified the structures and determined the contents of 16 compounds  from AR using ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS) and high-performance liquid chromatography–diode array detector–evaporative light scattering detector (HPLC–DAD–ELSD), respectively . An UPLCAstragalus, as a traditional Chinese medicinal material, has been used in clinical treatment. The active ingredients in Astragalus are mainly saponins, polysaccharides and flavonoids. In addition, there are some other extracts, such as chlorogenic acid, caffeic acid, β-sitosterol and so on. Studies have found that Astragalus has many biological activities, such as anti-inflammatory, antioxidant, antitumor, antiviral, cardiovascular disease prevention, and anti-diabetic activities and so on [nd so on ,52,53 -induced endothelial cell damage and inhibit atherosclerosis by reducing oxidative stress and inflammation. In addition, other ingredients in Astragalus are also believed to have anti-inflammatory effects. Ruby et al. [Astragalus and found that the activation of NF-κB in macrophages was dose dependent with Rx. The mRNA expression levels of the inflammatory cell markers CD68 and F4/80, as well as the expression levels of the cytokines MCP-1, TNF-α and IL-6, decreased significantly, which shows that Astragalus can reduce the symptoms of glucose intolerance, insulin resistance and hypertriglyceridemia caused by obesity by anti-inflammatory. Studies have found that these active ingredients include calyx-7-bD-glucoside (0.9%), onionin (1.2%), calyxin (4.53%) and formononetin (1.1%). Xuehong Nöst et al. [α, IL-1β and IFN-γ in U937 cells, proving the anti-inflammatory effect of Astragalus; active ingredients such as verbascoside, verbascoside, astragaloside IV, glycyrrhizin, 18 β-glycyrrhetinic acid, paeoniflorin and leucoflorin were identified. The anti-inflammatory mechanisms of AR are presented in LI et al.’s  study foy et al.  treated t et al.  combinedAntioxidants have many positive effects on human health, protecting the body and cells from ROS and free radical damage and preventing the oxidation of biological macromolecules such as DNA, membrane lipids and proteins . As a re2O2-induced retinal ganglion cells and found that the level of ROS in the cells decreased, and the mitochondrial membrane potential decreased, resulting in cells from oxidative stress damage, indicating that astragaloside IV has an antioxidant effect. Yang et al. [Astragalus, it regulates the expression of Nrf2 and Bach1 protein in the nucleus, promotes the expression of HO-1 protein, and provides a protective effect. At the same time, Isamu Murata et al. [The antioxidant activity of AR has been extensively studied. The antioxidant activity mechanisms of AR are presented in g et al.  found tha et al.  studies Astragalus through a fixed stress model in mice and found that the antianxiety effects of Astragalus extract (500 mg/kg), astragaloside IV (20 mg/kg) and buspirone (1 mg/kg) are comparable. Verbasin has been verified to have antioxidant activity. Verbasin can inhibit the production of ROS by enhancing the activity of antioxidant enzymes, namely glutathione peroxidase, catalase and superoxide dismutase (SOD) [Du et al.  establisse (SOD) .Astragalus has antitumor effects [With the increasing side effects of chemotherapy on the treatment of tumors, the use of Chinese herbal medicine to treat tumors has become a new direction. Many studies have shown that  effects . The antα, thereby directly preventing the growth of cancer cells, and has antitumor effects on breast cancer mediated by macrophage activation. Cho et al. [A. membranaceus, and found that the components designated as AI are the most effective for the mitogenic effect of mouse spleen cells. Among them, AI can induce the monocyte differentiation of human and murine cells in vitro. AR administered in vivo can even partially restore the suppressed mitotic response in tumor-bearing mice. In addition, there are many studies on the antitumor effects of Astragalus and other Chinese herbal medicines. Li et al. [Li et al.  found tho et al.  isolatedi et al.  believe Astragalus-Edocarium extracts have inhibitory effects on the growth of Lewis lung cancer (LLC) in transplanted mice. Compared with the control group, Astragalus-Curcuma extract can significantly reduce tumor weight and tumor microvessel density (MVD), and the 3:1 treatment group has a similar MVD reduction effect to the cisplatin group. The study also found that the expressions of p38 MAPK, P-p38 MAPK, ERK1/2, P-ERK1/2, JNK, and P-JNK in the Astragalus-Edocarium treatment group were reduced compared to the control group. The expression of VEGF protein in the 2:1 and 3:1 treatment groups was significantly lower than that of the control group.Xu et al.’s  study foAstragalus total saponins have been shown to have antiviral effects. Wang et al. [Astragalus and found that the clinical efficacy of the AC treatment group was significantly better than that of the control group. The negative seroconversion of hepatitis B virus (HBV) antigen e and HBV DNA was also significantly higher than that of the control group. At the same time, a duck hepatitis B model was established and verified, and the findings were consistent with the clinical results. This shows that AC can promote the recovery of viral hepatitis and inhibit HBV replication. Kajimura et al.’s [Astragalus has a protective effect on Japanese encephtis virus (JEV) infection in mice. After applying Astragalus extract (AE), the number of mouse peritoneal exudative cells (PEC)  increased significantly. The protective effect of oral AE is based on a non-specific mechanism. In the early stages of infection, before turning to antibody production, macrophages play an important role in resisting JEV infection, for example, by inducing the production of reactive oxygen species (AO). Wang et al. [Polygonum cuspidatum and Astragalus have a synergistic (1:1) effect, which inactivates the HSV-1F strain and can inhibit the proliferation of HSV-1F and block the infection of HSV-1F. The antiviral effect of Astragalus was verified.g et al.  used astg et al.  studied et al.’s  study fog et al.  found thAn increasing number of experiments have confirmed the effectiveness of AR in inhibiting cardiovascular diseases such as myocardial ischemia-reperfusion injury , myocard2+- ATPase (SERCA2a) in the SR is responsible for sarcoplasmic calcium reuptake. Many studies have shown that the expression of SERCA2a is significantly reduced after myocardial injury. AR has been reported to promote SERCA2a expression, enhance calcium reuptake in the SR and inhibit calcium overload, thereby inhibiting oxidative stress-induced apoptosis and cytoskeletal damage in adriamycin-injured rat heart cells [Astragalus membranaceus total saponin (ASP) increased abnormal aortic NO production, increased superoxide dismutase (SOD) activity, decreased the concentration of the metalloproteinases MMP-2 and MMP-9, and significantly reduced abnormal aortic NO production, thereby improving vascular endothelial dysfunction in rats with hyperhomocysteinemia-induced cardiovascular disease [The sarcoplasmic reticulum (SR) plays an essential role in intracellular calcium regulation, and the sarcoplasmic reticulum Cart cells . Coronarrt cells . Endothert cells . In a prrt cells . In addi disease .Type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) are widely prevalent metabolic diseases with different types of pathology. T1DM manifests itself as a result of the autoimmune destruction of pancreatic beta cells, leading to reduced insulin secretion . T2DM orAstragalus polysaccharide (AP) is extensively used as an immunosuppressant. There is substantial evidence that Astragalus polysaccharide downregulates blood glucose levels, upregulates serum insulin concentrations, increases β-cell volume and decreases the percentage of apoptotic β-cells in streptozotocin-induced T1DM mice, leading to a downregulation of the Th1/Th2 cytokine ratio and upregulation of PPARγ gene expression [Astragalus polysaccharides on T1DM by correcting the imbalance between Th1/Th2 cytokines in non-obese diabetic (NOD) mice [T1DM is an autoimmune disease which is characterized by the destruction of insulin-producing beta cells in the pancreas and a complete deficiency in insulin secretion . The T lpression ,88. In aOD) mice . In addiOD) mice .Astragalus polysaccharides in db/db diabetic mice [Astragalus in the clinical management of DN. The results of the analysis showed that Astragalus injection had stronger renoprotective effects (Blood urea nitrogen (BUN), Serum creatinine (SCr), and Creatinine clearance  and improved systemic status  in DN patients compared to controls [It has been well documented that improving insulin tolerance is the main mechanism of AR and that improving insulin tolerance is the active ingredient in AR for T2DM . Zhang etic mice . Li et acontrols .Astragalus formula  reduced blood glucose levels, increased the expression of GLP-1 in the serum of the rats, increased the ratio of the Bacteroidetes/Fimicutes, and increased the relative abundance of Bacteroides, Clostridium butyricum, and Rothia Geory and Brown [Astragalus turtle shell soup, ATSS  黄芪鳖甲汤” to treat T2DM patients and found that the relative abundance of Brucella.spp decreased and that of Bacteroides increased after treatment, indicating that ATSS plays a positive role in regulating the dynamic balance of intestinal flora [The normal functioning of the intestinal flora in the human body maintains a dynamic balance between the intestinal microorganisms and the organism, and is closely related to the normal functioning of the host physiology . If thisnd Brown . Yao et al flora .There are at least three levels of standards regarding the standardization of AR, namely efficacy standards, bioactivity standards, and production standards. AR is complex in terms of its origin, substance base, preparation process and quality control indicators, and it is for this reason that there is a lack of established standards for AR, but rather applicable standards must be developed in conjunction with the specific characteristics of specific species .Efficacy criteria are the first standard to be considered for AR, especially clinical efficacy. This is determined by its essential properties and cannot be considered in a single, incomplete way by considering its chemical composition, bioavailability, quality consistency and other modern drug indicators. Of course, efficacy criteria need to be measured through many clinical applications and medical observations, and for this reason, a reasonable evaluation system must be established. Unfortunately, there are few clinical studies on AR, data from clinical samples are lacking, and more studies are only to the extent of cellular or animal experiments.The second criterion is the activity criterion, which is considered based on the physical and chemical properties of the AR . It is well known that it is not valid to talk about activity in isolation from dosage. Therefore, the relationships between dose and activity and between structure and activity need to be rigorously studied in order to establish AR activity criteria . However, what is even more important for AR is batch consistency, as an extract of a natural product, so batch consistency is crucial and arguably a prerequisite for efficacy and safety. Unfortunately, different extraction and isolation methods can have a very significant impact on AR activity, and this poses a major challenge for the development of activity standards.The production standards mentioned here cover both the cultivation and production of AR. There are no specific standards for AR cultivation and there is a lack of in-depth research. They are generally based on basic standards such as GAP  and GMP (Good Manufacturing Practice). However, for the future industrialization of AR, more in-depth research is needed for the development of standard production. For example, how does the climate and soil of different production areas affect the accumulation of AR ingredients, and how do the many processes of extraction, alcohol precipitation, extraction and chromatography affect the migration and transformation of target ingredients in the AR production process? Studying the migration and transformation of the target components during the extraction, alcohol deposition, extraction, and chromatography processes is required.This paper reviews the chemical composition, extraction methods and chemical analysis and biological activity of AR, a highly valued herb that is both medicinal and edible, and therefore phytochemical and pharmacological studies of AR are becoming increasingly attractive. Although research on AR has been conducted for a long time and an increasing number of interesting compounds have been isolated and identified based on the development of new extraction methods and analytical methods, many problems with the application of AR in clinical treatment are still evident due to its complex chemical composition and poor quality control. Further research is needed to clarify the compounds of AR and to standardize their quality control with the help of extraction methods and analytical methods, which may lay the foundation for the further development of traditional Chinese medicine. Regarding the standardizations of AR, there are at least three levels of criteria, namely potency criteria, bioactivity criteria and production criteria, which still need further researched in depth.All authors contributed equally to this research. All authors have read and agreed to the published version of the manuscript.


An ophthalmic cyclosporine (CsA) formulation based on Lipid nanocapsules (LNC) was developed for dry eye management, aiming to provide targeting to ocular tissues with long-term drug levels and maximum tolerability. CsA-LNC were of small particle size (41.9 ± 4.0 nm), narrow size distribution (PdI ≤ 0.1), and high entrapment efficiency (above 98%). Chitosan (C) was added to impart positive charge. CsA-LNC were prepared as in-situ gels using poloxamer 407 (P). Ex vivo mucoadhesive strength was evaluated using bovine cornea, while in vivo corneal biodistribution (using fluorescent DiI), efficacy in dry eye using Schirmer tear test (STT), and ocular irritation using Draize test were studied in rabbits compared to marketed ophthalmic CsA nanoemulsion (CsA-NE) and CsA in castor oil. LNC incorporation in in-situ gels resulted in an increase in mucoadhesion, and stronger fluorescence in corneal layers seen by confocal microscopy, compared to the other tested formulations. Rate of recovery  assessed over 10 days, showed that CsA-LNC formulations produced complete recovery by day 7 comparable to CsA-NE. No Ocular irritation was observed by visual and histopathological examination. Based on data generated, CsA-LNC-CP in-situ gel proved to be a promising effective nonirritant CsA ophthalmic formulation for dry eye management. According to the Tear Film and Ocular surface Society Second Dry Eye Workshop (TFOS DEWS II) , prevalessolved) ,5,6,7. Amulation . Both oimulation ,8.This study aimed to develop an aqueous ophthalmic, patient friendly, CsA formulation. Among preformulation issues considered was CsA poor solubility and permeability. Although hydrophobic, CsA is a class IV drug in the BCS , showingA further advantage of LNCs is their ability to encapsulate hydrophilic drugs such as fondaparinux  and doxoLNCs were previously reported to enhance drug delivery through oral ,23, pareAnother preformulation issue tackled is the ease with which most aqueous ophthalmic preparations are washed out by tears. Several strategies are known to improve precorneal retention, among them in situ gels. They have the advantage of both liquids and gels; providing easy instillation as well as an improvement of precorneal retention once they attain gel state in physiological conditions ,28.Poloxamers are triblock copolymers widely used as non-ionic surfactants and solubilizers . They arChitosan is considered a cationic agent of choice for ocular formulations; it is a biocompatible and biodegradable linear polysaccharide with several free amino groups imparting a positive charge capable of interacting with negatively charged cornea surface mucin. There is some evidence that chitosan might enhance cornea uptake of drug molecules ,33. It iThe objective of the present study was to develop a thermoresponsive mucoadhesive ophthalmic gel containing CsA encapsulated in LNCs and to characterize the developed formulation in vitro to assess pharmaceutical attributes, ex vivo using excised bovine cornea to assess mucoadhesion, and in vivo in rabbits to assess biodistribution, irritation potential, and efficacy in a dry eye model.All stated concentrations refer to concentrations in the final formulations.® lipophile WL 1349, Gattefossé S.A., Saint-Priest, France), macrogol 15 hydroxystearate , deionized water (1:1:3 by weight), NaCl 0.44% w/v and phosphatidylcholine  were heated under magnetic stirring to 85 °C followed by cooling to 60 °C. Heating/cooling was repeated for 3 cycles at a rate of 4 °C change/min.LNCs were prepared using the phase inversion and temperature cycling method . PropyleIn the last cycle, quenching was induced by a 3.5-fold dilution using cold deionized water (2 °C) added at the phase inversion temperature (PIT) around 75 °C, detected visually when the mixture appeared translucent. The dispersion was left under slow magnetic stirring for 5 min, filtered through a 0.45 µm syringe millipore filter, and kept at 4 °C until further characterization.For CsA-loaded LNCs , 10–50 mg CsA/10 mL formulation were added with LNC ingredients from the beginning.w/w were prepared by dissolving in cold deionised water (4–8 °C) with continuous stirring for 3 h and refrigerated overnight [Concentrations of Poloxamer 407 (P)  ranging from 17.5 to 25% vernight ,36.w/v was dispersed in 1% v/v acetic acid solution and stirred overnight. Poloxamer (17.5 to 25% w/w) was dissolved in the chitosan solution, stirred for 3 h, and refrigerated overnight [Chitosan (C),  0.5% vernight .in situ gel and CPin-situ gel), resulting in a clear solution.For CsA-containing in situ gels, CsA was dissolved in the cold in situ gel (PCompatibility between poloxamer and LNC ingredients was first investigated by FTIR .w/w) was dispersed in cold LNC dispersion and refrigerated overnight. Quenching volume used in LNC preparation was decreased to accommodate volume increase due to added poloxamer.For gel preparation, poloxamer  was added to LNC dispersion. Chitosan powder (0.5% w/v) was dispersed in the acidified LNCs and stirred overnight. Poloxamer was added to the LNC-chitosan mixture.For LNC-CPw/v. Selected formulations with CsA concentration 0.05% w/v were also prepared for in vivo testing.In all in situ gel formulations prepared, final CsA concentration was 0.1% Gelling temperature was measured on 2 mL samples using the inverted tube method . The samTime in seconds for sol-gel transition in blank in situ gels using100 µL samples was determined using an aluminum pan placed on a hot plate maintained at 37 °C. Gelation time was recorded when the sol ceased to flow when pan was tilted 90° .Viscosity was measured using cone and plate viscometer  at 10 rpm using spindle 40 for 5 min., at 25 °C and at 37 °C. Profiles were plotted at different shear rates  at 37 °C using the Rheocalc T software. Both upwards and downwards curves relating shear stress to shear rate were generated to detect possible thixotropic behavior.v/v with filtered deionized water prior to measurements. Data were processed using the Zetasizer software (version 7.02). The same procedure was applied for sizing LNC gels. Three samples per formulation were measured in triplicate (n = 9).Mean particle size and polydispersity index (PdI) of LNCs were determined by photon correlation spectroscopy (PCS), and the zeta potential  was measured by Laser Doppler Velocimetry (LDV) using a Malvern Zetasizer  at a fixed angle 173° at 25 °C using a 4 mW He-Ne laser at 633 nm. Samples were diluted 1:20 in situgel at optimized poloxamer concentration were taken by transmission electron microscopy  preceded by negative staining for 30 s of 4-fold diluted samples with deionized water using 2% w/v aqueous uranyl acetate solution. Photographs were taken at 30–40 K X magnification power at 80 kV.Images of CsA-loaded LNC dispersion and LNC-Pg at 25 °C  [in situ gel and LNC-CPin situ gel), 1:4 dilution with deionized water was done prior to ultracentrifugation. Entrapped CsA was calculated from the difference between total CsA concentration (Ct) and free concentration (Cf), divided by Ct.Percent EE was determined by measuring free (un-entrapped) CsA separated from LNC dispersion using an ultrafiltration/centrifugation technique for 30 min at 3663× 100,000) . For in To determine Ct, LNC dispersion (100 μL) was diluted with methanol (up to 2 mL), sonicated for 20 s and CsA assayed by a validated HPLC method .Drug payload was calculated by dividing Ct by dry weights of the LNC ingredients. The effect of initial amount of CsA on EE and on LNC properties was also determined.Formulations were refrigerated for 6 months at 4 °C. Particle size, PdI, and ζ-potential were assessed monthly during the first three months and after 6 months by Malvern Zeta sizer. Changes in%EE were also monitored.Mucoadhesion force was measured using the modified physical balance method . Bovine Animal studies were conducted at the Animal Experimentation Facility, Faculty of Pharmacy Alexandria University, Egypt, in accordance with Principles of Laboratory Animal Care and institutional Guidelines for ethical conduct in use of animals in research. Efforts were made to minimize animal suffering. The study protocols were approved by the institutional Ethics Committee.Male New Zealand colored rabbits weighing 1.2–1.5 kg and free from any visible ocular damage were selected. Rabbits were provided with food and water in a temperature-controlled room (25 °C).DiI  was used for fluorescent labeling. Labeling method is provided in in situ gel, DiI-castor oil and DiI solution as control). Rabbits were sacrificed 3 h post administration and the eyes were enucleated and stored in 10% v/v formalin. Histological sections, 5 µm thick, were prepared for laser scanning confocal microscopy  was compared with marketed CsA-NE and with CsA solution in castor oil (0.05%). Positive (untreated) and negative  control groups were included.Schirmer Tear test (STT)  was usedUsing Schirmer tear test strips, tear production was monitored over a total of 10 days. Induction of dry eye (starting on day 0) was done using 50 μL of 1% atropine sulphate (AS) eye drops, three times daily instillation in both eyes for 3 days . This wain situ gel (0.1%) or CsA-NE or CsA eye drops in castor oil. Self-control method was adopted; right eye was treated, and left eye was given the same volume of normal saline solution. Rabbits were treated twice daily for 14 days. Visual inspection of the eye took place over 14 days for any signs of ocular reaction  and continued for three days after stopping treatment [Nine rabbits were divided into three equal groups given either 50 µL of CsA-LNCs-CPreatment . Draize Rabbits were euthanized with an intravenous air injection and eyeballs were enucleated for histopathological examination using a light microscope. Enucleated eyeballs were fixed in 10% formalin. Five-micrometer-thick histological sections were stained with hematoxylin and eosin.p ≤ 0.05.Data comparison was performed by one-way ANOVA followed by pairwise comparisons (Tukey test) using SPSS 20.0; . Significant differences were considered at LNCs were successfully prepared using the phase inversion method  with no w/v) resulted in minimum change in colloidal properties (size 41.9 ± 4.0 nm) and zeta potential −4.0 ± 1.50 mV. PdI value remained less than 0.1 for 10–50 mg initial CsA. CsA-loaded LNCs were spherical in shape and monodispersed , determined total CsA (free and entrapped) ranged from 9.6 ± 0.6 to 47.6 ± 4.2 mg. EE was constant and above 98% , and CsA payload ranged from 0.83% to 4.18%. Beyond 50 mg initial CsA, signs of drug precipitation were evident.High entrapment of CsA is mostly due to its incorporation in the oily core of LNCs due to its lipophilic nature (log P = 3). Similar results have been previously reported with lipophilic drugs in LNCs, such as paclitaxel , ropivacin situ gel and assessed effects of chitosan and LNCs on gelling temperature, gelling time, and viscosity. The optimum in situ gels identified (shaded in Gelling data  identifip < 0.005). Similarly, addition of Chitosan (0.5%), resulted in a significant decrease in gelling temperature (at P 20 to 25%) (w/w) formed gel at 34.8 ± 0.32 °C, consistent with reported data stating that 25% w/w Pluronics are required to form a stiff gel upon ocular administration [Increasing P concentration resulted in a decrease in gelling temperatures ( to 25%) . P   (When comparing each in situ gel with its respective dispersion there is a significantly higher PdI ( < 0.01) a, which for SLNs . Additio < 0.05) a. This cNo incompatibility was detected by FTIR between P and LNC ingredients . Poloxamdispersion showed marginal increase from 42.2 ± 0.2 to 44.9 ± 1.4; corresponding data for LNC- P in situ gel and LNC-CP in situ gel were 64.96 ± 3.1, and increased to 85.9 ± 2.6 and 79.1 ± 8.1 to 115.45 ± 15.0, respectively . Changes were evident in the first two months and tended to stabilize beyond. A possible explanation for apparent size enlargement in case of in situ gels is progressive hydration of poloxamer sheath surrounding LNCs, while LNC size remained relatively unaffected, as seen with LNC dispersion.Particle size, PdI, ζ—potential, and entrapment efficiency were monitored over six months . Particlp > 0.05). ζ-potential data for LNC-CPin situ gel indicated no significant change over six months . Similarly, % EE showed no significant change over six months (p > 0.05); all formulations remained above 98% EE.PdI followed the same pattern as the particle size; in situ gels remained monodispersed over the storage period with pdI < 0.25 . Chitosan (0.5% w/v) addition to LNC dispersion increased mucoadhesive force to 629.4 ± 146.9 dyne/cm2, (p > 0.05). Value recorded for LNC-P in situ gel was 2127.5 ± 377.3 dyne/cm2 , due to increased gel viscosity at the study temperature, and also to the ability of polymers with hydrophilic functional groups such as hydroxyl groups in P to form electrostatic and hydrophobic interactions as well as hydrogen bonding with cornea surface [LNC  surface ,56.in situ gel, 3412.3 ± 442 dyne/cm2, p <0.001, vs. LNC-P in situ gel,) due probably to ionic interaction between chitosan amino group and anionic substructures of cornea mucus layer [Addition of chitosan to the in situ gels further increased mucoadhesive force  incorporated in LNCs compared to other fluorescent substances ,59.in situ gel followed by DiI-LNCdispersion (p > 0.05). Values for DiI-Castor oil and control DiI solution were lower. The reported stability of DiI in LNCs [Cornea distribution of fluorescent DiI was observed by confocal laser microscopy . Based o in LNCs ,59 suggeDiI-LNC formulations showed strong fluorescence in cornea layers, with fluorescence in corneal epithelium higher than in stroma ; previous reports have shown that nanocarrier particle size is inversely proportional to membrane permeability with particles below 400 nm showing greater penetration through polarized epithelial cells ,61. The Corneal accumulation may also be due to infiltration mechanism, where the nanocarrier (polycaprolactone nanospheres) were claimed to be absorbed onto the corneal surface, followed by transport of encapsulated drug into the corneal epithelial cell membrane .Schirmer Tear Test (STT) without anesthesia (type I) was used to measure aqueous tear secretion in response to both conjunctival stimulation and basal non-reflex secretion. Dry eye induction model was selected; it is simple and able to rapidly induce dry eye (DE) symptoms compared to other invasive models. Burgalassi et al.  reportedp < 0.4). Tear production was then followed over a period of 10 days . Differences between the study groups were insignificant  was recorded for the other five animal groups (15 rabbits) receiving 1% AS eye drops three times daily, measured on day 3 indicating successful induction of DE, maintained over the study period by three times daily instillation of 1% AS  receiving normal saline drops maintained a consistent STT value over 10 days (11 mm ± 2.65). A significant reduction in tear production (p > 0.5) for both CsA-NE and CsA/castor oil treated groups . LNC-CPin situ gels treated groups showed a statistically significant (p < 0.005) increase in STT values which changed from 5.5 ± 2.5 and 3.8 ± 0.8 mm on day 3 to 10.5 ± 4.2 and 12.4 ± 4.3 mm on day 5 for 0.05% and 0.1% CsA, respectively (Starting from day 5, an increase in STT value was observed for the four treated groups. Compared to day 3 values, changes were statistically insignificant (ectively .in situ gel, 0.05% CsA-LNC-CPin situ gel, and CsA-NE, respectively. With CsA in castor oil, the rate of improvement was less than 5%.Concerning comparative rate of recovery (difference in percent tear production from day 3 to day 5, in situ gel could be partly attributed to gel mucoadhesive properties enhanced by interaction between positively charged chitosan and negatively charged cornea mucin layer, hence increased CsA-LNC retention on cornea surface.In vivo efficacy of 0.05% and 0.1% CsA-LNC-CPCsA in castor oil failed to demonstrate comparative therapeutic response in the dry eye rabbit model; it is not clear why baseline tear values were not reached within seven days of initiating treatment. Coating of conjunctiva with residual oil could have prevented proper functioning of the Schirmer tear strip although measurements were taken one hour after instillation of all treatment formulations to allow for partial formulation clearance from conjunctiva. Alternatively, poor tolerance by the eye, rapid clearance from ocular surface ,66 in adThe Schirmer tear test was able to demonstrate effectiveness of the LNC formulations under the same conditions as the marketed CsA-NE but could not demonstrate greater effectiveness of the 0.1% compared to the 0.05% LNC formulation; both gave similar results. The 0.1% concentration is recommended for severe cases of DE, which may not apply to the dry eye rabbit model.in situ gel in comparison with marketed CsA-NE and CsA/castor oil eye drops.Modified Draize test  was condNo behavioral reaction such as eye rubbing was observed in all animals tested following twice daily instillation of any of the three formulations studied. Rabbits showed no symptoms of ocular irritation such as redness, tearing, inflammation, or swelling following twice daily instillation over a period of two weeks. The eye lids showed healthy rosy color with no swelling, and the cornea and conjunctiva showed no pathological alterations in comparison to the control eye receiving only NSS.To further validate observational results, animals were sacrificed, eyeballs were removed, and histopathological examination of the ocular tissues was done after H & E staining. No signs of changes at cell and tissue levels were detected . Photomiin situ gel could serve as an effective patient-friendly formulation for the management of dry eye condition. Results of the present study provide incentive for a future clinical study to determine efficacy and tolerability in humans. The inclusion of chitosan in the gel enhanced mucoadhesion and offers a positively charged promising LNC formulation, possibly useful in other applications.A mucoadhesive thermoresponsive ophthalmic in situ gel containing CsA loaded in LNCs was developed and characterized. In vitro, ex vivo, and in vivo data generated provided evidence that CsA- LNC-CP


P < 0.01), when held at profound-hypothermic-temperature for 24 h before culture. Further, cell viability and testosterone levels in cultured cells from profound-hypothermic group were comparable to corresponding ultraprofound-hypothermic group but with moderate reduction in postmeiotic cells (P < 0.01). In conclusion, holding ITT at ultraprofound-hypothermic-temperature is most suitable for organotypic culture, whereas short-term exposure at profound-hypothermic-temperature may compromise postmeiotic germ cell yield post in vitro culture. This data, albeit in mouse model, will have immense value in human prepubertal fertility restoration research.Cryopreservation of immature-testicular-tissue (ITT) prior to gonadotoxic treatment, while experimental, is the only recommended option for fertility preservation in prepubertal boys. The handling and manipulation of ITT before cryopreservation could influence the functionality of cells during fertility restoration, which this study explored by evaluating cellular niche and quality of mouse ITT subjected to various temperatures and time durations in vitro. ITT from 6-day-old mice were handled at ultraprofound-hypothermic, profound-hypothermic, and mild-warm-ischemic temperatures for varying time periods prior to 14-day organotypic culture. Viability, functionality, synaptonemal complex and chromatin remodeling markers were assessed. Results have shown that cell viability, testosterone level, and in vitro proliferation ability did not change when ITT were held at ultraprofound-hypothermic-temperature up to 24 h, whereas cell viability was significantly reduced (The online version contains supplementary material available at 10.1007/s43032-020-00396-z. The prevalence of childhood cancer has increased significantly over the last decades, with the likelihood to be diagnosed with cancer being higher in boys than in girls . This siCryopreservation of immature testicular tissue (ITT) prior to gonadotoxic treatment is an experimental procedure but the only recommended option for fertility preservation in prepubertal boys , 4. The Fertility preservation centers offering ITT banking are limited worldwide, which implies that the place where the testicular biopsy is performed might not be the same as the place of tissue banking. This could lead to longer transit time and risk of exposing ITT to suboptimal conditions such as warm temperature. Due to constant turnover of stem cells, prepubertal testis is more sensitive to cancer treatment than that of adults . HoweverIf the retrieved ITT can be cryopreserved with minimal manipulation, the chance to recover optimum number of cells for fertility restoration techniques might increase . Hence, All experiments and animal handling were conducted in accordance with the institutional guidelines for animal experimentation after obtaining prior approval from the Institutional Animal Ethics Committee . A total of eighty-two, male prepubertal Swiss albino 6-day postpartum (dpp) mice were used in the study. Animals were sacrificed by cervical dislocation, and the testes were collected in alpha minimum essential medium  containing 1% (v/v) penicillin-streptomycin  and 5-μg/mL Nystatin . Testes were made fat-free using fine needles, under the stereomicroscope, and later randomly distributed/categorized for either holding-phase or cultured directly (described below or as depicted in Supplementary fig. The holding phase temperature for testes ex vivo was categorized as ultraprofound-hypothermic (~ 4 °C), profound-hypothermic (22–24 °C), and mild-warm-ischemic (34 °C) based on earlier recommendations . For exp3. ITT were subjected to baseline validation of viability, functional characteristics, or for organotypic culture.The holding phase interval was scheduled as 6 h (corresponding to short range shipment) and 24 h (corresponding to long range shipment). This time points are selected based on our experience from human gonadal tissue cryopreservation in fertility preservation programs. Following the holding phase, the testes were released back into a falcon dish containing media preequilibrated at respective conditions. The testes were then processed by removing tunica albuginea and each testis was divided into eight equal pieces of 2–5 mmThe ITT manipulation was performed at specific temperature corresponding to the holding temperature. Briefly, ITT held at ultraprofound-hypothermic condition were handled on frozen-ice pack, whereas profound-hypothermic, and mild-warm-ischemic groups were set on a stereomicroscope heating plate  maintained at 22 °C and 34 °C, respectively. The total handling-phase time, including removal of tunica albuginea, cutting, tissue fixation for baseline validation, or transferring to agarose bed organotypic culture system was < 30 min.3 in size) were picked using sterile microforceps, and placed on a half-soaked agarose bed (at gas-liquid interphase) for organotypic testicular culture as explained earlier [2 for 14 or 35 days, and the culture medium was completely replaced by fresh medium on the seventh day of the culture.The equally cut testicular fragments  at × 4 magnification. Cultured ITT were assessed for structural, functional, and genetic parameters.Cultured ITT were fixed in Bouin’s solution for histological analysis. Excessive fixative was removed by washing in 50% and 70% alcohol for 15 min each. The fixed tissues were then manually processed for dehydration by acetone, clearing by xylene followed by infiltration, and impregnation by paraplast wax. The tissue was then embedded in paraplast wax and cut into 5-μm sections on slides. After deparaffinization and rehydration, the sections were stained with Hematoxylin and Eosin (H&E) and examined for the integrity of the tubules and cells.The PI staining of testicular cells was similar to the protocol described by Bose et al. . Brieflyg for 8 min. The resultant pellet was resuspended in 100 μL of 1X Annexin-binding buffer, 5 μL of FITC Annexin-V and 1 μL PI (100 μg/mL) and incubated for 15 min at room temperature in dark. Following this, 400 μL of 1X Annexin-binding buffer was added with a target of one million cells per tube and mixed gently. An unstained control was run with each batch to identify autofluorescence. A fresh sample control at room temperature without incubation or culture was run to determine the live cell population. Flow cytometric analysis was performed on BD FACS CANTO II . The cells stained with Annexin-V FITC and PI was excited using 488 nm laser. Debris and doublets were eliminated from the analysis using light scatter characteristics. Fluorescence signals of FITC were collected using 530/30 BP and PI using 585/42 BP. The early apoptotic cells bind only Annexin-V and late apoptotic cells bind both Annexin-V and PI. The double negative cells are considered as live cells. Biexponential scale was applied for the data analysis  for the reproducibility and statistical evaluation.Fluorescein isothiocyanate (FITC) Annexin-V/propidium iodide (PI) dead cell apoptosis kit  was used to detect cell viability according to the manufacturer’s instructions with minor modifications. Briefly, ITT were treated with trypsin-collagenase mixture (1:1) in α-MEM + Glutamax medium at 37 °C for 30 min and then mechanically dispersed using pipette. The suspension was passed through a nylon mesh into a sterile tube containing equal volume of α-MEM + Glutamax medium supplemented with 20% fetal calf serum. Single cell suspension was washed twice with medium followed by washing in cold phosphate buffered saline (PBS) at 110g for 10 min, and the testosterone levels were measured in the cell lysate. Precoated wells were loaded with 25 μL of cell lysate and 200 μL of enzyme conjugate, mixed thoroughly and incubated for 60 min at room temperature. Wells were washed with wash buffer and incubated in 200 μL of substrate solution for 15 min. The reaction was terminated using stop solution and optical density was recorded at 450 nm using Multiskan™ FC Microplate Photometer .Testosterone levels in ITT were measured using Demeditec testosterone ELISA kit , according to the manufacturer’s instructions with minor modifications. Briefly, ITT was homogenized in 1 mL of PBS followed by centrifugation at 2700Total RNA was extracted from ITT using TRIzol reagent . Total RNA (1 μg) was reverse transcribed using random primers by high capacity cDNA RT kit  according to manufacturer’s protocol.Sycp1 and Sycp3) and chromatin remodeling proteins (Tnp2 and Prm1) were used (Supplementary Table Actb and Gapdh housekeeping genes according to ΔCT methodology.Quantitative polymerase chain reaction (qPCR) was carried out using Premix Ex Taq kit , in StepOne™ Real-Time PCR System . TaqMan assay  for postmeiotic markers  or Kruskal–Wallis test if failed normality test (followed by Dunn’s test) by GraphPad InStat 3.0 statistical package . The graphs were plotted using Origin 8.0 .Five quantifiable populations in ITT, subjected to ultraprofound-hypothermic, profound-hypothermic and mild-warm ischemic handling conditions, based on the DNA content were determined by DNA flow cytometry . These iP < 0.001) in the ultraprofound-hypothermic group, while compensatory decrease in 2C population was observed (P < 0.001) and 4C population increased by 2-fold (P < 0.05) compared to 6 dpp ITT  (Table P < 0.01). Based on this observation, in vitro culture was restricted to only 14 days for subsequent experiments.Prior to the holding phase, the fresh ITTs were manipulated/handled in ultraprofound-hypothermic, profound-hypothermic-temperature, and mild-warm-ischemic temperature, were cultured up to 14 and/or 35 days. At the end of 14 day culture, the HC and 1C populations were approximately 11-fold higher (ITT subjected to varying holding time and temperature was assessed for the cell viability using Annexin-V/ propidium iodide (AV/PI) staining. The flow cytometric measurement of AV corresponding signal provides a very sensitive method for detecting cellular apoptosis, while PI is used to detect nonviable population characterized by the loss of the integrity of the plasma and nuclear membranes Figs.  and 2.P < 0.01), which was accounted for dead cells population (P < 0.001). The early apoptotic and the late apoptotic cell number did not vary significantly across the groups similar to the ultraprofound-hypothermic group , and at the end of 24 h, respectively, where only 33.4 ± 1.8% of cells were viable (P < 0.001). Conversely, the dead cell population increased by 10.4-fold at 6 h (P < 0.01), and 16.7-fold at 24 h (P < 0.001). On the other hand, the number of early and late apoptotic cells did not vary significantly across the groups     . Similarly, 1C population in 6 h holding time at profound-hypothermic-temperature was significantly lower compared to 0 h (P < 0.01). However, no significant changes were observed between other groups  in ultraprofound-hypothermic and profound-hypothermic-temperature were cultured up to 14 days. S-phase population was significantly higher at 6 and 24 h holding time in both ultraprofound-hypothermic and profound-hypothermic groups compared to 0 h . The number of transcripts in 0 and 24 h groups was comparable between ultraprofound-hypothermic and profound-hypothermic-temperature groups of cultured ITT. However, the number of transcripts in 6 h group was significantly higher than 0 h (P < 0.05)   Fig. . On the 05) Fig. . Though,Tnp), and then by protamine (Prm). Tnp2 expression was studied in cultured ITT held at ultraprofound-hypothermic and profound-hypothermic-temperatures for varying time periods. Though, 0 h group did not show any difference in the number of transcripts between ultraprofound-hypothermic and profound-hypothermic-temperatures of in vitro cultured ITT, a significant decline (P < 0.01) in the number of transcripts was observed in both cultured ITT at 6 h holding time  in which ITT is usually transported and maintained prior to the storage. Profound-hypothermic-temperature  mimics tissue handling outside the ultraprofound-hypothermic-temperature or when time taken for shipment is long. Cell viability did not change significantly until 6 h of holding at ultraprofound and/or profound-hypothermic-temperature whereas a marginal loss in viability was evident at the end of 24 h, in profound-hypothermic group. This raises concern about the quality of ITT when shipment to the banking facility is delayed. Earlier study in human ITT showed no change in the structural integrity up to 3 days of holding at 4 °C . HoweverSycp1 (Sycp1−/−) in spermatocytes, the homologous chromosomes do not synapse, causing the cells to undergo apoptosis at pachytene stage [Sycp1 and Sycp3 transcripts were higher at 6 and 24 h respectively, in ultraprofound-hypothermic and profound-hypothermic-temperatures. The elevated levels of Sycp transcripts could be due to the significantly higher S-phase population in the group, resulting in delayed progression to postmeiotic population compared to 0 h post organotypic culture. The prolonged/delayed S-phase could be the time taken for cell population to repair the DNA damage before duplication. The meiotic phase of spermatogenesis is followed by elongation phase of spermiogenesis, which involves elongating and condensing spermatids undergoing extensive chromatin remodeling, where the histones are first replaced by transition proteins (TP), and in turn replaced by protamines (PRM) [Tnp2 in 6 h profound-hypothermic group could account for a significantly lower 1C population in the group. However, the nonsignificantly different Prm1 transcripts among all the groups and intervals studied might be an indication of nuclear condensation in the spermatids.During the meiotic prophase I, synapsis of homologous chromosomes is achieved by the formation of a large zipper-shaped protein complex known as the synaptonemal complex . SYCP2 ane stage . In the es (PRM) , 39. TheOn the other hand, even up to 6 h exposure to warm-ischemic-temperature before the organotypic culture resulted in a significant impairment in the cell viability and testosterone levels. Warm ischemia is known to have adverse effects on mammalian spermatogenesis leading to increased apoptosis or DNA damage in germ cells , 41. HanStudies using several experimental models have shown that holding and manipulation at 4 °C is optimal for ITT , 43, 44 The main limitation of our study is that only fresh ITT were tested without cryopreservation hence, does not mimic the human clinical situations. However, it is convincing to note that fresh and cryopreserved prepubertal testicular fragments have comparable functionality in relation to spermatogonial survival and proliferation, intratubular cell apoptosis, Sertoli cell maturation and proliferation, and testosterone production by Leydig cells . SimilarIn conclusion, we report that holding ITT at ultraprofound-hypothermic-temperature is the most suitable condition for organotypic culture. Though, cell viability and endocrine function are not affected by short-term exposure of ITT at profound-hypothermic-temperature, impaired germ cell proliferation may compromise the number of postmeiotic germ cells derived from in vitro culture. We believe that the data derived from our experiments albeit in mouse model will be of immense value in future prepubertal fertility restoration research.Supplementary Figure 1(PNG 848 kb)High Resolution Image (TIF 169 kb)Supplementary Figure 2(PNG 4620 kb)High Resolution Image (TIF 1122 kb)Supplementary Table 1(DOCX 13 kb)


Arthrogryposis multiplex congenita (AMC) is a group of rare congenital disorders characterized by multiple joint contractures present at birth. Contractures can affect different body areas and impact activities of daily living, mobility and participation. Although early rehabilitation is crucial to promote autonomy and participation in children with AMC, empirical evidence to inform best practice is scarce and clinical expertise hard to develop due to the rarity of AMC. Preliminary research involving stakeholders in AMC  identified priorities in pediatric rehabilitation. Scoping reviews on these priorities showed a lack of high quality evidence related to rehabilitation in AMC. The objective of this project is to provide rehabilitation expert guidance on the assessment and treatment of children with AMC in the areas of muscle and joint function, pain, mobility and self-care, participation and psychosocial wellbeing.An integrated knowledge translation approach will be used throughout the project. Current rehabilitation practices in AMC will be identified using a clinician survey. Using the Grading of Recommendations, Assessment, Development and Evaluations framework (GRADE) approach, a panel of interdisciplinary expert clinicians, patient and family representatives, and researchers will develop expert guidance on the assessment and treatment for pediatric AMC rehabilitation based on findings from the scoping reviews and survey results. Consensus on the guidance statements will be sought using a modified Delphi process with a wider panel of international AMC experts, and statements appraised using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) tool. Theoretical facilitators and barriers toward implementing clinical guidance into practice will be identified among rehabilitation clinicians and managers to inform the design of dissemination and implementation strategies.This multi-phase project will provide healthcare users and providers with research-based, expert guidance for the rehabilitation of children with AMC and will contribute to family-centered practice. Arthrogryposis multiplex congenita (AMC) is a group of rare disorders where a child is born with stiff joints. Joint stiffness can be present in different parts of the body, making it difficult for the child to move, to walk and to participate in activities. Rehabilitation interventions starting early in life are very important to maximize autonomy. AMC being rare, it is difficult for clinicians to develop experience with this population and there is little research specific to rehabilitation interventions in AMC. In a previous research project, a group of youth with AMC and their families identified five areas of priority for rehabilitation: muscle and joint function, pain, mobility and self-care, participation and psychosocial wellbeing. The purpose of this project is to provide guidance for the evaluation and treatment of children with AMC, in these priority areas. The project involves multiple steps. First, we will perform an online survey with international rehabilitation practitioners to learn about current evaluation and treatment approaches used with children with AMC. We will then bring together expert clinicians, patient and family representatives, and researchers to develop the guidance statements. Then, we will perform an online survey with a larger group of international experts to validate and finalize the guidance statements. Finally, we will interview rehabilitation clinicians and managers to find out about what could help them or limit them in integrating this guidance into their practice. This project will provide healthcare users and providers with research and expert-based guidance for the rehabilitation of children with AMC. Rehabilitation in rare diseases is often under-represented in research due to lack of funding , 2, therA recent qualitative study identified rehabilitation needs for youth with AMC, their parents, and clinicians through semi-structured interviews . These nPreliminary work consisted of a series of scoping reviews on the five priority areas as a lack of empirical studies in rehabilitation precluded a systematic review , 19–21. Developing rehabilitation expert guidance using the current literature and expert opinion addresses the important knowledge gap in this field. Expert guidance can inform key end-users, such as clinicians, youth, and family representatives, on the assessment tools and treatment indicated for this population. Thus, the overall aim of this project is to develop rehabilitation expert guidance, on the assessment and treatment of children with AMC to improve muscle and joint function, pain, mobility and self-care, participation, and psychosocial wellbeing.The research team, composed of key stakeholders , is involved in all phases of this integrated knowledge translation (iKT) project. Members’ clinical expertise and lived experience ensures that the project is in line with the priorities and needs of end-users. The research team, including patient and clinician partners, has been meeting on a monthly basis to develop the research question and determine the methodology and targeted outcomes. The team will continue to meet monthly during the research process and members will be assigned some tasks to complete between meetings. Research partners will provide methodological and technical expertise and guidance in all steps of the study. Knowledge users will contribute in developing tools , identifying new partnerships and recruiting participants, interpreting findings, co-developing the expert guidance statements, and identifying strategies for knowledge dissemination . This prPhase 1: clinician survey to describe the current practice among rehabilitation clinicians working with children with AMC;Phase 2: develop expert guidance statements for the rehabilitation management of children with AMC in the five priority areas listed previously;Phase 3: validate and finalize the expert guidance statements using a modified Delphi-process with international clinical experts and family/patient representatives worldwide.Phase 4: appraise the final expert guidance and evaluate the rigor and robustness of the methodology by external reviewers using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) questionnaire ;Phase 5: identify the perceived facilitators and barriers to the uptake of the clinical guidance among rehabilitation professionals working with children with AMC, and their clinic managers to inform the development of knowledge translation strategies.Phase 1: Clinician survey To inform current clinical practice in pediatric rehabilitation in AMC, the experience of rehabilitation practitioners , social workers (SW), physiotherapy technologists) working with children with AMC worldwide will be sought through an electronic survey questionnaire. The survey will inquire about the different assessments and interventions that rehabilitation practitioners use with this population, in relation to the identified priority areas , for each age group . The questionnaire will be based on the findings from the scoping reviews and from the experience of clinicians on the team, and include the assessment tools and treatment strategies reported in the literature  . AlthougPhase 5: Identifying perceived facilitators and barriers to the uptake of clinical guidance & development of KT strategy Theoretical factors (facilitators and barriers) regarding the uptake of pediatric rehabilitation guidance will be identified among approximately 13 rehabilitation practitioners working with the pediatric AMC clientele and about five pediatric musculoskeletal clinic managers identified through convenience sampling. The Theoretical Domains Framework (TDF), a framework used to identify influences on behaviour change, will be used for this phase to assess what factors likely influence healthcare professionals’ behaviour regarding the uptake of clinical practice guidelines [1. Data will be collected through individual interviews to cater to different schedules and time zones as we are reaching out to professionals worldwide. Demographic data will be gathered . The interview guide will be adapted from existing TDF interview topic guides [idelines –36. The c guides . On averc guides . The quec guides . Data wic guides . Particic guides . Once coThe identified relevant TDF domains and the corresponding identified facilitators and barriers will be matched with behaviour change techniques using a matrix by Michie and colleagues . This maThis multi-phase project uses an iKT approach to guide the development of rehabilitation expert guidance for children with AMC, based on the needs and expertise of key stakeholders, including clinicians, youth and families with AMC, as well as decision makers, and contributes to family-centered practice . An iKT nd and 3rd International Symposia on arthrogryposis [This project will help synthesize the existing knowledge and current practice in pediatric rehabilitation in AMC, identify gaps, create consensus-based expert guidance needed to inform clinical decisions for AMC, and identify perceived facilitators and barriers to the uptake of clinical guidance in practice to guide KT strategies. The results will provide healthcare users and providers with research-based, expert guidance to address physical, activity, participation and psychosocial needs of children with AMC. The project addresses one of the research and clinical priorities articulated during the 2gryposis , 44, speAlthough input from international stakeholders will be sought for this project, the applicability of the clinical guidance may be limited by regional and cultural factors. Since AMC is an umbrella term including many different diagnoses with a variety of clinical presentations and severities , 8, cert


We report a new mechanism of androgen receptor (AR) mRNA regulation and cytoprotection in response to AR pathway inhibition (ARPI) stress in prostate cancer (PCA). AR mRNA translation is coordinately regulated by RNA binding proteins, YTHDF3 and G3BP1. Under ambient conditions m6A-modified AR mRNA is bound by YTHDF3 and translationally stimulated, while m6A-unmodified AR mRNA is bound by G3BP1 and translationally repressed. When AR-regulated PCA cell lines are subjected to ARPI stress, m6A-modified AR mRNA is recruited from actively translating polysomes (PSs) to RNA-protein stress granules (SGs), leading to reduced AR mRNA translation. After ARPI stress, m6A-modified AR mRNA liquid–liquid phase separated with YTHDF3, while m6A-unmodified AR mRNA phase separated with G3BP1. Accordingly, these AR mRNA messages form two distinct YTHDF3-enriched or G3BP1-enriched clusters in SGs. ARPI-induced SG formation is cell-protective, which when blocked by YTHDF3 or G3BP1 silencing increases PCA cell death in response to ARPI stress. Interestingly, AR mRNA silencing also delays ARPI stress-induced SG formation, highlighting its supportive role in triggering this stress response. Our results define a new mechanism for stress adaptive cell survival after ARPI stress involving SG-regulated translation of AR mRNA, mediated by m6A RNA modification and their respective regulatory proteins. FL and AR-Variant mRNA in preclinical models (Androgen receptor (AR) pathway inhibition (ARPI) induces profound and sustained responses in advanced prostate cancer (PCA). Unfortunately, recurrence is inevitable and associated with re-activation of the AR and progression to castration-resistant prostate cancer (CRPC) ,2, attril models  and humal models . While al models . Moreovel models . Cancer A highly conserved mechanism for regulation of translation during stress involves the sequestration and protection of mRNAs in stress granules (SGs). SGs are membrane-less ribonucleoprotein (RNP) complexes formed by liquid-liquid phase-separation (LLPS) of intrinsically disordered proteins and RNAs, comprised of mRNAs, ribosomal components (40S), RNA-binding proteins (RBPs) and signalling factors . SGs seq3) at the N6-position of adenosine (m6A) is an abundant and versatile RNA ETM (An important regulator of mRNA homeostasis involves RNA epitranscriptomic modifications (ETMs) . Methyla RNA ETM . Writers RNA ETM . Disturb RNA ETM ,40.In this study, we investigated AR mRNA regulation in the context of acute ARPI stress to define rapid adaptive changes that support survival under treatment stress. Depending on its m6A status, YTHDF3 and G3BP1 RBPs coordinately regulated the translation of AR mRNA. Under ambient conditions, YTHDF3 binds an m6A-modified, translationally active fraction of AR mRNA in polysomes (PSs), while G3BP1 binds an m6A-unmodified, translationally inactive fraction of AR mRNA. When AR-regulated PCA cells are exposed to ARPI stress, AR messages traffic away from PSs, and translation of AR mRNA is reduced. Translationally-repressed AR mRNAs assemble in the cytosol in SGs to associate in distinct clusters with YTHDF3 and G3BP1. Blocking this adaptive response by silencing of YTHDF3 or G3BP1 sensitizes PCA cells to ARPI stress. Interestingly, AR (or ESR1) silencing also blocked SG formation after ARPI (or ERPI) stress in prostate (and breast) cancer cells, highlighting a role for AR and ESR1 mRNA in triggering SGs when its receptor is antagonised. Our research defines a novel mechanism of AR mRNA translation mediated by m6A modification, its regulatory RBPs, and SG formation.LNCaP, V16-D and PC-3 cells were obtained from ATCC. The following primary antibodies were used: PARP (Cat. No. 9542), BAX (2D2) (Cat. No. 89477), β-Actin (Cat. No. 3700), BIOTIN (Cat. No. 5597), AR (Cat. No. 5153) and GAPDH (Cat. No. 2118) were from Cell Signaling; G3BP1 (Cat. No. 611127) was from BD Biosciences; YB-1 (Cat. No. ab76149), m6A (Cat No. ab151230) and G3BP1 (Cat. No. ab56574) were from Abcam; YTHDF3 (Cat. No. NBP2-94636) was from Novus; m6A (Cat. No. 202 003) was from Synaptic Systems; ERα (Cat. No. SAB4500810) and Vinculin (Cat. No. V9131) were from Sigma; Fluorescent secondary antibodies , TRIzol, RPMI, FBS, RNAiMAX transfection reagent, Dynabeads M-280 Streptavidin, DMEM, FBS, Click-iT Protein Reaction Buffer Kit, biotin-alkyne and L-azidohomoalanine (AHA) were from Life Technologies; FluorSave was from Merck; siAR siRNA (5′-UCAAGGAACUCGAUCGUAUUU-3′) targeting full-length AR was from Dharmacon; siESR1 (Cat. No. sc-29305), siMETTL3 (Cat. No. sc-92172), siWTAP (Cat. No. sc-63224), siKIAA1429 (Cat. No. sc-77700), siMETTL14 (Cat. No. sc-89054), and siControl (Cat. No. sc-37007) siRNAs were from Santa Cruz Biotechnology.2EDTA, 1 mM EGTA, 1% Triton X-100 and 1× protease inhibitor). Cell lysates were centrifuged at 5000 rpm for 10 min and the supernatant was saved. Protein concentration was determined using a Bradford assay (Bio-Rad Laboratories). Protein lysates were mixed with 2× loading dye, and equal amount of proteins were separated in 4–12% gradient SDS-PAGE and immunoblotted into nitrocellulose membrane using wet transfer as described previously  were treated with vehicle alone or exposed to ARPI stress. IF was performed as described previously . Cells w® 2.5 Chromogenic Assay (Single-plex and Duplex)’ from ACD. Briefly, LNCaP cells seeded in glass-bottom chamber slides were transfected with siControl or siAR siRNAs for 48 h. Cells were then subjected to +/–ARPI stress and fixed in 4% PAF. Cells were dehydrated in a Coplin jar using 50%, 70% and 100% ethanol, then rehydrated in 70%, 50% ethanol and 1× PBS (each for 10 min). Slides were treated with hydrogen peroxide, quick protease III to digest proteins to enhance probe accessibility to RNA, and then subjected to hybridisation using RNAscope® 2-Plex Detection Kit (Chromogenic). First, slides were hybridised with a mRNA specific probe for 2 h, then incubated with a series of probes (Amp 1–6) provided by ACD to enhance the hybridization signal, followed by treatment with Fast A substrate to reveal red spots corresponding to the regions of mRNA localisation. The ISH slides were then subjected to immunostaining with anti-G3BP1 or ant-YTHDF3 antibodies as described above and viewed using Zeiss LSM780, confocal microscope with 60× and 100× oil-immersion objective lenses. Images were captured using ZEN imaging software.For the localization of AR mRNA, manual in situ hybridization (ISH) was performed as described previously . Probes Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. Polysomal mRNA was extracted using a protocol described before . For thed-1-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 0.5 mM and protein expression was induced by further incubating the culture at 37°C for 5 h. The cells were harvested by centrifugation at 7000 rpm for 10 min, resuspended in 10 ml lysis buffer (10 mM imidazole in 1× PBS) and subjected to sonication. The suspension was then cleared by centrifugation at 10 000 rpm for 30 min at 4°C and the resulting supernatant was passed through a 1 ml Ni-NTA column (Thermo Scientific). After washing the column with washing buffer (50 mM imidazole in PBS), recombinant proteins were eluted with elution buffer (250 mM imidazole in PBS) and dialyzed extensively against PBS to remove the imidazole.G3BP1_OHu02150C_pET-28a(+)-TEV, YTHDF3_OHu08859C_pET-28a(+)-TEV, YTHDF3-EGFP_YTHDF3_OHu08859C_pET-28a(+)-TEV, G3BP1-RFP_G3BP1_OHu02150C_pET-28a(+)-TEV were custom cloned from GenScript. The plasmids were transfected to BL21(DE3) pLysS competent cells (Promega). Positive colonies were picked up and grown at 37°C in LB media supplemented with 50 μg/ml of kanamycin until the early exponential phase (OD600 0.4–0.8). Isopropyl-β-2, 10 mM DTT, 5% glycerol and 2 μg tRNA) in a total volume of 20 μl for 30 min. Control reactions were set up using 200-fold molar excess of unlabeled RNA probe added together with labeled RNA. The reaction products were mixed with 5 μl of 5× loading buffer and subjected to electrophoresis in a 6% native polyacrylamide gel in 0.5× TBE buffer (100 V for 8 × 8 × 0.1 cm gel) until the bromophenol blue dye has migrated 3/4 down the length of the gel. The RNA–protein complexes were then transferred to a nylon membrane and cross-linked for 60 s at 120 mJ/cm2 using a commercial UV light cross-linker (Agilent Technologies) equipped with 254-nm UV light lamps. The membranes were then incubated with stabilized Streptavidin-Horseradish peroxidase conjugate and developed using chemiluminescent substrate.RNA EMSA to detect direct binding of G3BP1 or YTHDF3 to m6A-unmodified or m6A-modified probes was performed using a LightShift Chemiluminescent RNA EMSA kit (Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, 6.25 nM biotin tagged probes  were then precleared by incubating with 20 μl of RNasin (RNase inhibitor), 12 μg of yeast tRNA, and 100 μl Streptavidin magnetic beads in a total volume of 1 ml at 4°C for 2 h. In parallel, 100 μg of biotinylated RNA was incubated with 100 μl of Streptavidin magnetic beads at 4°C for 2 h on a rotator. The beads were washed and mixed with the precleared lysate and incubated at 4°C for 30 min in a rotator, then washed five times in the binding buffer, boiled in loading dye, and subjected to immunoblotting.The RNA affinity chromatography was performed as described previously . LNCaP c® N6-methyladenosine Enrichment Kit (NEB) from the supernatant and droplets fractions and the pulldown mRNAs from each fraction were subjected to qRT-PCR using AR specific primers. To check the effect of requirement of RNA in the phase-separation, recombinant G3BP1 or YTHDF3 were mixed with RNA in presence and absence of RNase A and subjected to phase separation and imaged as explained above.Phase-separation assays were conducted as described previously with modifications ,44. BrieLNCaP cells were transfected with siRNA for either G3BP1, YTHDF3, METTL3 or scrambled control at the time of seeding in 96-well plate using Lipofectamine RNAimax as per manufacturer's guidelines. Next day, the cells were transfected with 50 ng of reporter plasmid ARR3tk-luciferase using TransIT2020 transfection reagent . After 24 h of transfection, the cells were untreated or treated with ARPI stress. Luciferase activity was measured using the dual luciferase assay system  and normalized by Renilla activity or protein concentration .® S3 Live-Cell Analysis System for real time image acquisition. Cell proliferation data was quantified using the IncuCyte® basic analyzer.LNCaP cells were transfected with siControl, siG3BP1 or siYTHDF3 siRNAs using Lipofectamine RNAimax. The transfected cells were treated with 10 μM ENZA. After 24 h of ENZA treatment, cells were treated with 20 μM ARS. For rescue experiment, media in treated (ENZA+ARS) cells were replaced with fresh media without compounds. All cells were incubated in a IncuCyteP < 0.05, unless otherwise indicated. P- value significance is represented by the following: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Statistical analyses were performed in GraphPad Prism 8 or Microsoft Excel using one-way ANOVA and two-tailed unpaired Student's t-test unless otherwise indicated. The outcomes of all statistical tests including p-values and number of experiments are included in the figure legends. Significance was defined as any statistical outcome that resulted in a + PCA cell lines and clinical PCA tumors after ARPI stress.Since ARPI is stressful for AR-responsive PCA cells, we explored conditions whereby this treatment induced SG formation. When LNCaP cells were pre-treated with the AR-antagonist enzalutamide (ENZA), SGs efficiently formed when combined with a mild stress, including oxidative stress [arsenite (ARS) 20 μM for 1 h], endoplasmic reticulum (ER) stress [thapsigargin (TGN) 0.1 μM for 1 h], or osmotic stress [sorbitol (SBL) 50 μM for 1 h]. SGs were visualized by co-localization staining of SG marker proteins G3BP1 and YTHDF3 Figure . We defiAR protein levels have been reported to decrease after ARPI , which wm6A-modified mRNA has been reported to bind YTHDF3 and not G3BP1 . Since ATo determine the association of AR mRNA with YTHDF3 and G3BP1 in cells, both proteins were pulled down from untreated or ARPI-stressed LNCaP cells using ribo-immunoprecipitation (ribo-IP) and the associated AR mRNA was analyzed by qRT-PCR. We found that both G3BP1 and YTHDF3 binds AR mRNA in untreated cells. While AR mRNA-YTHDF3 interaction remained unchanged following stress, the AR mRNA-G3BP1 interaction increased after stress Figure  and D. TOur predicted potential m6A modification sites in AR mRNA Figure . As descTo investigate effects of m6A modification in recruiting AR mRNA to SGs, we developed an in vitro liquid-liquid phase-separation (LLPS) assay using recombinant YTHDF3 or G3BP1 that formed in vitro liquid droplets Figure  see Met,56–60. FWe next analyzed phase-separation of full-length AR mRNA in the YTHDF3 and G3BP1 droplets. In vitro mixing of YTHDF3 or G3BP1 recombinant protein with total RNA extracted from LNCaP cells induced LLPS to form liquid droplets Figure  and B asNext, to define differences in m6A-modified or -unmodified AR mRNAs phase-separated in YTHDF3 or G3BP1 droplets, m6A -modified messages from ‘droplet’ and ‘liquid’ fractions of YTHDF3 or G3BP1 were pulled down using anti-m6A antibodies, and subjected qRT-PCR to quantify AR mRNA levels (see scheme in Figure Since AR mRNA phase-separated with YTHDF3 or G3BP1, we set out to define functional roles for AR mRNA in SG formation in response to ARPI stress. We found that AR silencing initially delayed SG formation in ARPI stressed cells, Figure  and B, bSGs are complexes of several hundreds of RBPs comprised of a dense core linked with loosely packed regions termed as shells ,63. SG cYTHDF3 and G3BP1 immunostaining using STED microscopy in unstressed and ARPI-stressed PCA cells also identified distinct clustering of YTHDF3 and G3BP1 proteins in unstressed cells that converge to form large SGs after ARPI stress. Similar to Figure G3BP1 binding with m6A-unmodified fraction of AR mRNA might constitute an untranslatable AR mRNA pool, a postulate consistent with prior reports that G3BP1 associates with an untranslatable pool of cellular mRNAs , and datSince many residues are m6A-modified in AR mRNA Figure , we explSince METTL3 and YTHDF3 regulate AR mRNA translation, we checked whether downregulating the expression of these proteins could reduce AR downstream activity in LNCaP cells. AR transactivation assay  showed aSGs form acutely after ARPI stress, and disassemble when stress is removed Figure . SimilarThe AR is the key driver and most important therapeutic target for advanced PCA ,73. Trea+ PCA cell lines co-incident with reduced AR protein levels. Immuno-staining levels of SGs increased, while AR protein levels decreased, in post-ARPI treated PCA tissues, credentialing this stress response in clinical specimens. Total AR mRNA or protein degradation rates were not altered in ARPI stressed cells; rather the reduced AR protein expression was due to reduced AR mRNA translation as AR messages in the PSs were disassembled and recruited to SGs in ARPI-stressed cells. We defined m6A modification of AR mRNA as a key determinant for its partitioning between PSs and SGs, guided by its differential interaction with YTHDF3 and G3BP1. G3BP1 tends to not bind m6A-modified transcripts (We observed induction of SGs in ARPI-stressed ARnscripts ,78, whilnscripts . Additionscripts ,58. We inscripts ,68,79–81Following ARPI stress, levels of m6A-modified AR mRNA decrease, reflecting either lower m6A-writer or enhanced m6A-eraser activity . Under ASGs adopt heterogeneous structures formed by initial nucleation of the G3BP1-rich cores in juxtaposition with a more dynamic shell ,84. YTHDRNA and its modifications, including m6A, are critical elements for the size and composition of phase-separated RNA–protein condensates ,57,64. MSince SGs are dynamic complexes of hundreds of proteins and mRNAs, it is reasonable that other RBPs, in addition to YTHDF3 and G3BP1, are involved in regulating the translation of AR mRNA, and its association with SGs. In fact, a recent study reports that DDX3 regulates AR mRNA translation by binding AR mRNA and sequestering it to SGs, which reduces AR mRNA translation . These fARPI stress-induced SG formation and suppression of AR expression is dynamic and reversible, with SG disassembly coinciding with AR expression returning to baseline levels after treatment withdrawal. These oscillations of AR protein expression might be linked to the dynamic shuttling of AR mRNA between PSs and SGs. While small amounts of AR protein are translated after ARPI stress, possibly through selective mRNA translation of a part of m6A-modified AR mRNA in PSs or in SGs , this isUsing context-dependent and physiologically relevant anti-cancer stress in PCA or breast cancer cells, this study shows that AR or ER mRNA support the formation of SGs under specific stress of AR or ER antagonism. Other approved therapeutics, docetaxel or olaparib, did not induce SGs. While induction of SGs occurs at higher non-physiological concentrations of oxidative, proteotoxic, or metabolic stressors as a general stress response , our datIn conclusion, cytoprotective SGs are formed after acute ARPI stress in PCA cell lines and tissues. The RBPs YTHDF3 and G3BP1 coordinately regulated translation of AR mRNA, with YTHDF3 binding m6A-modified, translationally active AR mRNA in PSs, and G3BP1 binding m6A-unmodified, translationally repressed AR mRNA. Under acute ARPI stress, AR mRNA supported the phase separation and SG formation - m6A-unmodified AR mRNA bound to G3BP1 phase-separated to a core cluster in SGs, while YTHDF3- bound m6A-modified AR mRNA localized as a distinct shell cluster surrounding the SG core. ARPI-induced SG formation reduced global translation, while activating adaptive cytoprotective pathways; blocking this adaptive response by G3BP1 or YTHDF3 silencing sensitized PCA cells to ARPI stress. Our research defines a novel mechanism of AR mRNA translation involving AR mRNA m6A regulatory proteins and SG formation that may be exploitable in designing ARPI-based therapies.The data that support the findings of this study are available from the corresponding author, upon reasonable request.gkab1247_Supplemental_FilesClick here for additional data file.