Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation . Using 32P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification in vivo. Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI . As patients suffering from AAN and BEN are always exposed to the plant extract , our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently , 2804–2818, 2020), we showed that the  Aristolochia and Asarum genera of the family Aristolochiaceae, namely Aristolochia clematitis in particular in Europe [d)-1,3-dioxolo-5-carboxylic acid, AAI) and aristolochic acid II -1,3-dioxolo-5-carboxylic acid, AAII) being the major components (Aristolochic acid (AA) is the natural plant extract of both the n Europe . The plamponents . AAI andanthro-3,-d-1,3-diN-hydroxyaristolactam I (N-OH-Alac I) and N-hydroxyaristolactam II (N-OH-Alac II), respectively, is required to exert their genotoxic properties. N-OH-Alac I and II generate cyclic acylnitrenium ions capable of binding to DNA, preferentially forming pre-mutagenic purine DNA adducts [N6-yl)aristolactam I (dA-AAI) is the most abundant DNA adduct detected [TP53 [AAI is considered to be responsible for AA-mediated nephropathy by directly causing interstitial renal injury ; however adducts ,24,31,32detected ,35,36,37ed [TP53 ,40,41,42in vivo, significantly higher adduct levels were detected in several organs of rats and mice when treated with AAI than with AAII [in vitro [in vivo and in vitro might be caused by differences in the enzymatic conversion of both compounds, which lead to their activation  and detoxification.Comparing DNA adduct formation with AAI and AAII ith AAII ,25,43,44in vitro ,28,30,45Aristolochia species growing both in Europe (Mediterranean) and Asia [Aristolochia plants contain more AAI, while in others AAII prevails, with amounts of AAI and AAII ranging between 40 and 60% [in vitro and in vivo; thus, investigating whether and how the ratios of AAI and AAII in plants influence the in vivo metabolism of AAI and AAII and subsequently the formation of AAI- and AAII-derived DNA adducts can help answer this question. Although the plant extract AA and its main components AAI and AAII are classified as group 1 human carcinogens by IARC, there are still important questions which need to be addressed to fully understand the underlying mechanisms involved in the development of AAN AND BEN. One key question is how AAI and AAII contribute to the toxicity (nephrotoxicity) or genotoxicity (carcinogenicity) of the plant extract, since only a small proportion of AA-exposed individuals develop AAN/BEN and associated UUC. In this context, it is noteworthy that the contents of AAs and their derivatives vary widely in and Asia ,47. Thisin vivo metabolism of AAI and AAII in rats is influenced by the presence of both AAs. Exposure to the AAI/AAII mixture affected the generation of their urinary metabolites formed by oxidation, reduction and conjugation reactions [32P-postlabelling. In addition, since AA metabolism dictates the levels of AA-DNA adducts formed, we also examined the activity of enzymes involved in AAI/AAII bioactivation and detoxification and how enzyme activity levels are influenced when rats are exposed to AAI, AAII or the AAI/AAII mixture. Specifically, we examined the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1), a key enzyme involved in the reductive activation of AA. Further, we evaluated cytochrome P450 (CYP) enzymes such as CYP1A1 and 1A2, which contribute not only to the reductive activation of AA, but also to its oxidative detoxification. The consequences of AA-mediated alternations in the activity levels of these enzymes on oxidation or reduction of AAI and AAII and subsequent AAI- and AAII-derived DNA adduct formation in vitro were also investigated.In this experimental model, rats were previously exposed to AAI, AAII and an AAI/AAII mixture . As parteactions . For ins32P-postlabelling in liver and kidney samples of male Wistar rats treated i.p. with a single dose of AAI (20 mg/kg bw), AAII (20 mg/kg bw) or AAI/AAII mixture (containing each 20 mg/kg bw AAI and AAII). The adduct patterns obtained by TLC 32P-postlabelling were distinct for each treatment group but qualitatively similar in both rat organs. As shown in N6-yl)-aristolactam I (dA-AAI) and 7-(deoxy- guanosin-N2-yl)-aristolactam I (dG-AAI). Moreover, in kidney but not liver DNA samples, another adduct spot (assigned as spot X) was detectable, the chemical structure of which has not yet been elucidated. To allow comparison, adduct spot X was excluded from the quantification of total AA-DNA adduct levels. After treatment with AAII, two major adduct spots were observed, which were previously identified [N6-yl)-aristolactam II (dA-AAII) and 7-(deoxyguanosin-N2-yl)- aristolactam II (dG-AAII)  B. After dG-AAII) C; thus, 8 nucleotides) (8 nucleotides for AAI versus ~130 adducts per 108 nucleotides for AAII)  D, adductor AAII) E. In botor AAII) D,E; howein vivo  AAI and AAII, their expression might dictate the formation of AA-DNA adducts; however, as CYP1A1/2 enzymes also oxidise AAI  but not AAII , their e compare . The act compare . Marker activity of the CYP1A (EROD) was detectable in both organs A,B. In tTreatment of rats with AAI, AAII and AAI/AAII mixture led to increased enzyme activity of cytosolic NQO1 in both organs K,L. The To study AAI detoxification, incubation with AAI in the presence of hepatic or renal microsomes was carried out under aerobic conditions. All microsomes oxidised AAI to AAIa, as demonstrated by one metabolite being detectable by HPLC (peak r.t. 24.329 min) C. The am32P-postlabelling assay was used again to determine AA-DNA adduct formation. The adduct pattern found on the TLC sheets when using incubations of hepatic and renal microsomes and cytosols with AAI and AAII was qualitatively identical to those observed in vivo in liver and kidney tissues of rats treated with either AAI or AAII  animals were used for comparison. NADPH-dependent DNA adduct formation after AAI and AAII incubation with cytosols and microsomes was used as a measure of AAI and AAII bioactivation by cytosolic NQO1 and microsomal CYPs, respectively. In these experiments, the  compare A,B. After dA-AAI ,29. For 8 nucleotides, with levels being up to ~5-fold higher for DNA adducts generated by incubation with AAI than AAII. Pre-treatment of rats with AAI, AAII or AAI/AAII mixture increased the levels of AAI- and AAII-derived DNA adducts when AAI and AAII were incubated with DNA and cytosolic subcellular fractions ex vivo. Increased AA-DNA adduct levels correlated with NQO1 enzyme activity in cytosolic fractions  in with AAI A,B and Awith AAI A,B. For with AAI A,C,E. FoAristolochiaceae family has been classified as carcinogenic to humans (group I) by the IARC [Aristolochiaceae species are still used in traditional herbal medicine, particularly in Asia, where potentially millions of people are exposed to the nephrotoxic and carcinogenic effects of AA [vice versa; however, no comprehensive study has yet been carried out to our knowledge. The extract of AA prepared from plants of the the IARC . It is rthe IARC ,15,16,17the IARC ,4,5,6,7,ts of AA ,57,58,59ts of AA ,38,40,42ts of AA ,30,33,34in vivo. Although previous studies showed AA-DNA adduct formation 24 h after administration, in the present study rats were sacrificed and analysed after 48 h in order to simultaneously measure and identify AA metabolites in urine [The aim of the present study was to investigate AAI and AAII metabolism, which are pathways of oxidative detoxification and reductive bioactivation, resulting in AA-DNA adduct formation in rats treated with AAI, AAII or AAI/AAII mixture. One key question of this study was how enzymes participating in AAI biotransformation are affected by co-exposure to AAII 32P-postlabelling method, which was successfully employed previously and has been proven to be a powerful tool for understanding pathways of AA biotransformation. Our present study not only demonstrates the predictable formation of AA-DNA adducts in all rat organs tested, as described previously [in vitro [gpt delta transgenic mice also showed that treatment with AAII induced higher levels of AA-DNA adducts in the kidneys than treatment with AAI [The ability of both AAs to form covalent DNA adducts was studied using the ultra-sensitive eviously ,60,61,62eviously ,23,25,44in vitro ,30,45,49with AAI . More imwith AAI . in vivo was overall in accordance with activity levels of AA metabolising enzymes determined in hepatic and renal microsomal and cytosolic fractions. AA can increase both the protein expression and enzyme activity level of cytosolic NQO1, the major enzyme reductively activating AA to reactive N-hydroxyaristolactam-nitrenium ions, resulting in AA-DNA adducts [In the present study, AA-DNA adduct formation  adducts ,64,65,66 adducts ,70,71,72 adducts ,73,74,75 adducts ,77; thusin vivo and in vitro was described in previous studies [in vivo, the increases in the levels of AA-DNA adducts formed in the kidneys and livers of the rats treated with AAI/AAII mixture compared to those treated with AAI alone does not fully correspond to the NQO1 enzyme activity detected in the renal and hepatic cytosols of these rats; thus, other mechanisms may contribute to the elevated levels of AA-DNA adducts in the liver and kidneys. This could include the ability of AAII to affect the oxidative detoxification of AAI. The oxidative metabolism of AAII has not been studied in detail, although this compound is present in high concentrations in some Aristolochia species and may even exceed the concentration of AAI [in vitro, unlike AAI [N-hydroxyaristolactam II and 7-hydroxyaristolactam II [in vitro, with an inhibition constant (iK) of 11.3 μM [in vivo. According to previous results obtained during the first phase of our study, the levels of AAIa and aristolactam Ia, the main detoxification metabolites of AAI, found in the urine of rats treated with AAI/AAII mixture were ~2-3-fold decreased on the first day after administration compared to those treated with AAI alone [The crucial role of NQO1 in AAI bioactivation  studies ,64,65,66n of AAI ,47. Somen of AAI  and indin of AAI ; howeverlike AAI , and usiactam II . These f 11.3 μM ; hence,  11.3 μM ,62. We sAI alone .The activation of AAI resulting in AA-DNA-adduct formation might also be modulated by the limited ability to detoxify AAI to AAIa ,62. OxidO-demethylate AAI to AAIa was demonstrated in vitro using hepatic and renal microsomes. Our results were in accordance with the elevated enzymatic activity levels of CYP1A1/2 and 2C6 determined in hepatic and renal microsomes isolated from rats treated with AAI or AAII. In hepatic microsomes, higher levels of AAIa were formed, confirming that the liver is the main organ responsible for AAI detoxification, as described previously [in vitro indicated that no hydroxylated (detoxification) product is formed . These findings are in agreement with previous studies [On the other hand, both AAI and AAII induced enzyme activity levels of other detoxification enzymes in hepatic microsomes of treated rats, such as CYP2C6 and CYP1A. Although the contents of these enzymes are lower in the rat liver (10% CYP2C6 and 2% CYP1A), their contribution to AAI oxidation in rat liver is not negligible (both at ~17%) . Here, teviously ,84,86,89 studies  and our  studies . This inCyp1a knockout mice), recombinant enzymes, specific inhibitors and inducers, in silico approaches and site-directed mutagenesis [ex vivo. Elevated levels of AA-DNA adducts were generated ex vivo in hepatic or renal microsomes when DNA was incubated with AAI or AAII. Increased AA-DNA adduct formation corresponded to the determined CYP enzyme activity, particularly in hepatic microsomes; however, while CYP1A1/2 mediated AA-DNA adduct formation in microsomes, in cytosols, AA-DNA adduct formation was mediated by NQO1. NQO1 enzyme activity was found to be elevated both in hepatic and renal cytosols isolated from rats pre-treated with AAI, AAII or AAI/AAII mixture.While the treatment with AAI or AAII alone resulted in similar increases in AAIa formation (~1.5-fold) in hepatic microsomes compared to control (untreated) rats, treatment with AAI/AAII mixture led to a greater increase . This indicates that despite the inhibition of CYP2C11 enzyme activity leading to a considerable decrease in AAI detoxification in the rat liver, treatment with AAI/AAII mixture positively affects CYP enzymes detoxifying AAI, particularly CYP1A1 and 1A2. Nevertheless, both CYP1A1 and 1A2 play a dual role in AA metabolism, since they are capable of both detoxifying and activating AAI. Previous studies have highlighted this phenomenon using genetically modified experimental animals  and AAII (CAS number 475-80-9) were purified from the commercially available plant extract AA  by reverse-phase chromatography as reported previously . The purn = 3/group) were treated i.p. with a single dose of either 20 mg/kg body weight (bw) AAI, 20 mg/kg bw AAII or 20 mg/kg bw of AAI plus 20 mg/kg bw of AAII (AAI/AAII mixture). Animals in the control groups received the vehicle (corn oil) only. After administration, rats were housed in metabolic cages, which allowed the separated collection of urine and faeces. The results of these investigations were published previously [32P-postlabelling were isolated by phenol–chloroform extraction [n = 3 rats/group) were used in further experiments. All microsomal and cytosolic samples were free of residual AAI, AAII or AAI/AAII metabolites, as determined by HPLC [The study was conducted as described previously  in accoreviously . On the traction . DNA quatraction ,101. Pro by HPLC .32P-postlabelling analysis with separation by thin-layer chromatography (TLC) on polyethylenimine–cellulose (PEI) plates was carried out as described previously [The nuclease P1 enrichment version of eviously ,102. TLCeviously .c and NQO1 activity, expressed as nmol cytochrome c reduced [p-nitrophenol from a 5′-phosphoadenosine 3′-phosphosulfate (PAPS)-regenerating system [O-deethylation (EROD) for CYP1A1/2 and methoxyresorufin O-demethylation (MROD) for CYP1A2 [c as a substrate [In hepatic and renal cytosols, NQO1 activity was measured using menadione  as a substrate; the assay was improved by the addition of cytochrome  reduced ,99. SULTg system ,103,104.r CYP1A2 . CYP1A1 r CYP1A2 . CYP enzubstrate ,107. Theubstrate ,109.i) without microsomes, (ii) without NADPH or (iii) without AAI. AAI and its metabolite AAIa were separated by reverse-phase HPLC, identified by mass spectrometry and quantified as described previously [18, 250 × 4.0 mm, 5 mm (Macherey–Nagel) column, using a linear gradient of acetonitrile (20 to 60% acetonitrile in 55 min) in 100 mM triethylamonium acetate with a flow rate of 0.5 mL/minutes. A Dionex HPLC pump P580 with a UV/VIS UVD 170S/340S spectrophotometer detector set to 254 nm was used. Peaks were integrated with CHROMELEON™ 6.01 integrator. A peak eluting at a retention time (r.t.) of 24.329 min was identified as AAIa using mass spectroscopy analysis [Incubation mixtures contained 100 mM potassium phosphate buffer (pH 7.4), 1 mM NADPH, 0.5 mg rat hepatic or renal microsomal protein (microsomes of untreated (control) rats or rats treated with AAI, AAII or AAI/AAII mixture) and 10 µM AAI in a final volume of 500 μL, which were incubated at 37 °C for 10 min. AAI oxidation to AAIa was determined to be linear up to 25 min. Control incubations were carried out either  without cytosol or microsomes, (ii) without NADPH, (iii) without DNA or (iv) without AAI or AAII. After extraction with ethyl acetate, DNA was isolated from the residual water phase by the phenol–chloroform extraction method, as described above. DNA adduct analyses using 32P-postlabelling were performed as described above.The de-aerated and nitrogen-purged cytosolic or microsomal incubation mixtures contained 50 mM Tris-HCl buffer (pH 7.4), 0.2% Tween 20, 1 mM NADPH, 0.5 mg rat hepatic or renal protein  and 0.5 mg calf thymus DNA (2 mM dNp), as well as 0.5 mM AAI, AAII or both in a final volume of 750 μL. Incubations with cytosols or microsomes were performed at 37 °C for 60 min. AAI- and AAII-derived DNA adduct formation was found to be linear up to 2 h ,110. Cont-test. All p-values were two-tailed and considered significant at the 0.05 level.Statistical data analysis was performed used Student’s


Surface reconstruction engineering is an effective strategy to promote the catalytic activities of electrocatalysts, especially for water oxidation. Taking advantage of the physicochemical properties of precatalysts by manipulating their structural self-reconstruction levels provide a promising methodology for achieving suitable catalysts. In this review, we focus on recent advances in research related to the rational control of the process and level of surface transformation ultimately to design advanced oxygen evolution electrocatalysts. We start by discussing the original contributions to surface changes during electrochemical reactions and related factors that can influence the electrocatalytic properties of materials. We then present an overview of current developments and a summary of recently proposed strategies to boost electrochemical performance outcomes by the controlling structural self-reconstruction process. By conveying these insights, processes, general trends, and challenges, this review will further our understanding of surface reconstruction processes and facilitate the development of high-performance electrocatalysts beyond water oxidation. Energy conversion and storage systems are the most promising solutions to address the energy crisis and environmental pollution ,2,3,4. AParticularly, first-row transition metal-based materials, such as oxyhydroxides, oxides , and metal phosphides/chalcogenides reportedly show outstanding OER performance, given their flexible electronic structure and enriched active sites ,39,40,410.5Sr0.5Co0.8Fe0.2O3–δ (BSCF) as an example, this is a benchmark OER material for perovskite oxides in alkaline media 2 is consacancies b,c [102]mi level a. In addmi level d, the comi level e. Moreovmi level f. The W03+/Co4+ couples and Fe4+ ions) can relieve the electro-derived oxidation process and exhibit good structural/operational stability levels, as demonstrated with recently reported high-performance oxides, such as SrCoO3, Na0.67CoO2, Ba4Sr4(Co0.8Fe0.2)4O15, and CaCu3Fe4O12 [The OER reaction is an oxidation process, during which low-valence metal sites tend to be oxidized to higher valence states along with metal dissolution into the electrolyte ,92,93,98u3Fe4O12 ,108,109.u3Fe4O12 ,111,112.2 and BaCl2) were introduced into the perovskite structure with high-valence-state transition-metal ions simultaneously to achieve an optimized cation/anion leaching effect and to keep the surface crystalline matrix unchanged during the harsh reaction conditions [−2, which is approximately 137 mV lower than that of the pure-phase material 3Cl that exhibited a gradual phase transition due to the etching of lattice Cl− ions, alone with continuously increased OER activities [−2 in an alkaline solution, which is comparable to that of noble metal oxide IrO2. The irreversible etching of the lattice Cl− brought about numerous structural defects and high-valence Co species, which are demonstrated by aberration-corrected high-angle annular dark-field scanning transmission electron microscopy and operando synchrotron radiation-based X-ray spectroscopic characterizations, respectively (xFy nanosheets are easily changed into Ni(Fe)OxHy compared with the initial NiFe precatalysts, which was demonstrated by operando Raman, ex-situ XPS spectra, and HRTEM analyses  after surface reconstruction can serve as main active sites, offering high activity capabilities [Inactive and low-cost materials as potential catalysts should be activated as high-performance OER electrocatalysts. In situ-generated active species  spectroscopy  cycle. For perovskite oxides, if lattice oxygen participates in the reaction, a descriptor of the O 2p level relative to the Fermi level can be involved during the reaction process. However, with regard to CoFe0.25Al1.75O4, a ≈5 nm reconstructed surface was found after the first cycle, remaining quite stable during subsequent cycling .Wu et al. reported Fe-doped CoAll oxides . After Ftroscopy a,b. Modu cycling c and rev cycling d. Furthe lattice e. Simila2 oxide and requires a higher potential level to trigger surface reconstruction to Li1±xCo2O4 and longer cycles for stabilization . Finally, the generated nanoporous and loose layer played a crucial role in triggering the full diffusion of electrolytes into the inner structures, by which the goal of a complete change was achieved (4·xH2O), and complete reconstruction. The potential-dependent in situ Raman spectra further demonstrated the reaction mechanism. Bond breakage, a co-leaching effect, and active species generation could be confirmed by the disappearance of peaks (at ≈ 355 cm−1 corresponding to MoO4 vibration and at ≈820 and 950 cm−1 corresponding to Mo-O-Ni stretching in NiMoO4·xH2O) and the generation of new peaks with relatively high intensity levels  (4@NiOOH with a core-shell structure) and a commercial noble-metal-based Ir/C catalyst. More importantly, a negligible change of the potential during the 1350 h stability test according to chronopotentiometric measurements was observed for the completely reconstructed catalyst, suggesting vast superiority of the in situ-formed microstructure by the complete reconstruction and also excellent potential as a cost-effective alternative to noble-metal oxides  c. The suolution) d and an olution) e. The col oxides f.Rational control of the surface reconstruction process is an effective strategy to develop high-performance OER electrocatalysts . This caThe most widely reported active layers after surface reconstruction are oxyhydroxides . Thus, t3O4 catalyst [3O4 catalyst, when an oxygen vacancy was introduced into Co3O4, the oxygen vacancy could initialize the surface reconstruction process at a relatively low potential, even before the occurrence of the OER process, compared with that of the pure spinel Co3O4 catalyst [Though in situ-formed species during structural self-reconstruction work as active sites for the OER, the reconstruction mechanism is closely related to the physicochemical properties of the precatalysts used. For example, unlike the conventionally reported irreversible surface change, Bergmann found the structurally reversible change of the crystalline Cocatalyst . In contcatalyst . Therefo−2 is typically used) [3+ ions could further be oxidized to Co4+ ions, as confirmed by a positive energy shift and a shorter Co-O bond compared with that of the ex situ Co XANES. This work provides a novel strategy to design highly active and stability electrodes.The dissolution of metals  into electrolytes to induce surface reconstruction is widely reported in relation to the design of high-performance electrocatalysts ,127,128.ly used) ,131. Forly used) ,132,133.ly used) . Specifi3 was reported to be quite stable under cycling conditions [0.25Al1.75O4 catalyst, there was a quick self-termination process during the OER, [2CoMnO6 and SrIrO3 thin films) [Depending on the types of materials used  and the different physicochemical properties of the precatalysts, the actual surface reconstruction mechanism can differ ,63,79,97nditions ,69,134. the OER,  while thn films) ,103. Furn films) . Therefon films) ,136,137.Generally, it is difficult to capture intermediates with a short lifetime, or to observe dynamic changes by ex situ characterization approaches. To track the surface change during an electrochemical reaction, in situ/operando techniques, specifically in situ/operando XRD/XPS/TEM/XAS/Raman methods, are the most powerful tools ,138,139.Considerable efforts have been devoted toward designing high-performance electrocatalysts in fundamental research. Typically, activity evaluations on the laboratory scale are conducted at room temperature . However


Cyathus and Hericium sp., the fusicoccane-type diterpenoids from Fusicoccum and Alternaria sp., the guanacastane-type diterpenoids from Coprinus and Cercospora sp., and the harziene-type diterpenoids from Trichoderma sp., often represent unique carbon skeletons as well as diverse biological functions. The abundances of novel skeletons, biological activities, and biosynthetic pathways present new opportunities for drug discovery, genome mining, and enzymology. In addition, diterpenoids peculiar to fungi also reveal the possibility of differing biological evolution, although they have similar biosynthetic pathways. In this review, we provide an overview about the structures, biological activities, evolution, organic synthesis, and biosynthesis of diterpenoids that have been specially produced by fungi from 2010 to 2020. We hope this review provides timely illumination and beneficial guidance for future research works of scholars who are interested in this area.Fungi have traditionally been a very rewarding source of biologically active natural products, while diterpenoids from fungi, such as the cyathane-type diterpenoids from  Fungi are widely distributed in terrestrial environments, freshwater, and marine habitats; more than one million distinctive fungal species exist, but only approximately 100,000 of these have been classified . There aDiterpenoids are a class of C20 compounds derived from isoprenoid precursor geranylgeranyl diphosphate (GGPP) under the catalysis of diterpene synthases (DTSs) ,9,10,11.Trichoderma, Penicillium, Cyathus, Hericium, and Crinipellis account for 60% of the total  diterpenes/diterpenoids isolated from fungi, (2) carbon skeleton obtained only from fungi or rarely from other sources, (3) studies on the biological activities of diterpenes/diterpenoids and their derivatives that had been carried out in vitro or in vivo, (4) studies on the biosynthesis of diterpenes/diterpenoids and their derivatives. The data exclusion criteria included: (1) carbon skeleton of diterpenes/diterpenoids obtained in abundance from other sources, such as plants, bacteria and so on, (2) duplication of data and titles and/or abstracts not meeting the inclusion criteria.Cyathus, Hericium, and Sarcodon (Cyathus africanus and Hericium erinaceus tetradecane (5/9/5 ring system) core skeleton, namely alterbrassicenes B–D 70–72, were obtained from the same fungus A. brassicicola ..190 has 190 with phomopsene synthase (PaPS) has been examined through systematically deuterium-labeled geranylgeranyl diphosphate (GGPP), starting from site-specific deuterium-labeled isopentenyl diphosphates (IPPs) using IPP isomerase and three prenyltransferases , Nocardia testacea (NtPS), and Nocardia rhamnosiphila (NrPS) -, [2-13C]-, and -acetates, respectively [226 was predicted, as shown in β-methyl and α-hydride occurred at the six-membered ring part, then, the ring inversion and cyclization progressed to form the 6/5/9 ring intermediate. A rearrangement proceeded to expand the ring from five to six membered, and the last step resulted in the formation of the 6/5/6/6 ring skeleton. The C-8 position of wickerol A 225 was oxidized by a cytochrome P450 to give wickerol B 226.The new skeleton of wickerols A ectively . The cyc227–232 are new diterpenes with a 6/5/6/6 tetracyclic system that have been isolated from the marine sponge-derived fungus Trichoderma harzianum [227 potently inhibited the growth of murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines with IC50 values of 7.9, 6.8, and 7.6 μM, respectively [Trichodermanins C–H arzianum ,209. Triectively .Diterpenoids show huge potential for drug discovery and development due to their extensive biological functions and structural diversity. Fungal diterpenoids are a diverse family of hybrid natural products with potent bioactivities and intriguing structural architectures. A large number of fungal diterpenoids have exhibited significant anti-inflammatory, cytotoxic, anti-MRSA, antimicrobial, antiviral, antihypertensive, and platelet aggregation-inhibitory activities. Consequently, these bioactive diterpenoids are always hot trending topics for the synthesis community ,174,186.


Chronic exposure to butyrate induced autophagy via higher expression of Beclin-1 and LC3B-II. The AMP-activated protein kinase (AMPK) was downregulated along with the activation of Akt and mammalian target of rapamycin (mTOR) and decrease in acetyl-CoA carboxylase (ACC) in BR colon cancer cells compared to those in their respective PT cells. Activation of AMPK by AICAR treatment in BR colon cancer cells suppressed cell proliferation by inhibiting Akt and mTOR and activating ACC. Taken together, chronic exposure to butyrate increased butyrate resistance in human colon cancer by inducing protective autophagy through the downregulation of AMPK/ACC and activation of Akt/mTOR signaling. Activation of AMPK restored sensitivity to butyrate by the inhibition of Akt/mTOR, suggesting that AMPK could be a therapeutic target for BR colon cancers.Butyrates inhibit cell growth in colon cancer cells by inhibiting histone deacetylases. However, chronic exposure to butyrates induces butyrate resistance in colon cancer cells. The mechanism underlying the acquisition of resistance is not yet fully understood. Here, butyrate-resistant (BR) colon cancer cells were developed in HCT116, HT29, and SW480 human colon cancer cells and were confirmed by the increase in the inhibitory concentrations of cell growth by 50% (IC AMP-activated protein kinase (AMPK) is a key regulator for balancing energy supply and maintaining homeostasis and ultimately protects cells from stressful conditions by rearranging multiple metabolic pathways . The actAutophagy is an essential homeostatic, programmed catabolic process that is activated by various stimuli and is important for the breakdown or recycling of proteins and cellular components ,8. DysreButyrate is a major short-chain fatty acid produced from dietary fiber through fermentation by intestinal microbes in normal colon cells and is further oxidized to acetyl-CoA in the mitochondria for use as an energy source for normal colon cells; however, in colon cancer cells, butyrate is a histone deacetylase inhibitor. It acts as a cell growth inhibitory agent and induces differentiation ,15,16. HIn this study, the effect of butyrate resistance on autophagy in colon cancer cells was evaluated. Changes in the AMPK signaling pathway as a mechanism of butyrate resistance in butyrate-resistant (BR) colon cancer cells were examined, and the effect on the fatty acid metabolism was also investigated during the acquisition of butyrate resistance.Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium DMEM, and Dulbecco’s phosphate-buffered saline (DPBS) were obtained from Invitrogen . Penicillin-streptomycin was purchased from GenDEPOT . Sodium butyrate, 3--2,5-diphenyl-tetrazolium-bromide (MTT), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) were supplied by Sigma-Aldrich . Antibodies against p21; cyclin A; cyclin D1; cyclin E; Bad; acetyl-CoA carboxylase (ACC); ATP citrate lyase (ACLY); fatty acid synthase (FASN); LC3B; Beclin-1; p62; phospho-AMPKα (Thr172); AMPKα; mammalian target of rapamycin (mTOR); phospho-mTOR (Ser2448); Akt; phospho-Akt (Ser473); phospho-ACC (Ser79); carnitine palmitoyltransferase 1A (CPT1); breast cancer resistance protein (BCRP); Bax; P-glycoprotein (P-gp); and poly ADP ribose polymerase (PARP) were purchased from Cell Signaling Biotechnology . Actin, short-chain acyl-CoA dehydrogenase (SCAD), and p53 antibodies were obtained from Santa Cruz Biotechnology . Long-chain acyl-CoA dehydrogenase (LCAD) antibody, propidium iodide, and RNase A were purchased from Abcam . All other chemicals and reagents were of analytical or HPLC grade and were used without further purification.2 incubator. To establish BR colon cancer cells , a previously published method was used [Parental HCT116, HT29, and SW480 human colon cancer cells were purchased from Koran Cell Line Bank  and were cultured in DMEM with 10% FBS, 100 U/mL penicillin, and 10 mg/mL streptomycin at 37 °C in a humidified 5% COwas used . The butTo determine the effect of butyrate-resistance and chemoresistance on the proliferation of colon cancer cells, HCT116/PT, HT29/PT, and SW480/PT colon cancer cells and their respective BR cells were seeded onto 96-well plates. Cells were treated with various concentrations of butyrate, doxorubicin, 5-fluorouracil (5-FU), and oxaliplatin. After 72 h incubation, the medium was removed, diluted MTT solution (100 μL) was added, and the plate was incubated at 37 °C for an additional 2 h. Subsequently, the purple formazan crystals were solubilized by adding dimethyl sulfoxide (DMSO), and the absorbance was read at 540 nm by using an ELISA reader .In order to investigate the effects of AMPK activation on cell proliferation in BR colon cancer cells, HCT116/BR, HT29/BR, and SW480/BR cells were treated with DPBS, 0.2 mM AICAR, 3.2 mM butyrate, and 0.2 mM AICAR for 1 h followed by 3.2 mM butyrate for 72 h. Other procedures were the same as described above.g at 4 °C, fixed with 70% ethanol at 4 °C, and 1 mL of propidium iodide solution  containing 100 U/mL of RNase A and 0.1% glucose was added for 30 min in the dark [In order to assess cell cycle progression, each PT colon cancer cell line and its respective BR colon cancer cells were plated onto 60 mm dishes. After overnight incubation, PT and BR colon cancer cells were treated with DPBS and 1.6 mM butyrate for 24 h. Cells were then trypsinized, centrifuged for 5 min at 500× the dark ,21. Flowthe dark ,21.PT, AT1.6, and BR colon cancer cells were fixed with 1.6 mM butyrate for 24 h and fixed with 4% paraformaldehyde (pH 7.4) at room temperature for 10 min and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature. Cells were washed with DPBS three times and incubated overnight at 4 °C with an LC3B antibody diluted at 1:50. After washing three times with DPBS, the cells were incubated with fluorescence-conjugated secondary antibody , diluted at 1:100 at room temperature for 1 h, and washed three times with DPBS. The cells were carefully mounted with a coverslip by using a mounting medium containing DAPI (Sigma-Aldrich). Fluorescence images were analyzed by using a confocal microscope .2O. Complementary DNA (cDNA) was prepared from RNA samples by using the High Capacity cDNA Reverse Transcription kit  according to the manufacturer’s protocols.Total RNA was isolated from PT, AT1.6, and BR colon cancer cells using Trizol (Invitrogen), followed by column purification using the RNeasy Mini kit  according to the manufacturer’s instructions. RNA was eluted from the spin column using RNase-free dHCPT1A, LCAD, SCAD, ACC, ACLY, FASN, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are listed in GAPDH was used to standardize the qRT-PCR results for CPT1A, LCAD, SCAD, ACC, ACLY, and FASN. The relative mRNA levels were estimated by using the 2−ΔΔCt method described previously [Quantitative real-time reverse transcription PCR (qRT-PCR) was performed using Power SYBR Green Master Mix (Applied Biosystems), and qRT-PCR was performed using the StepOne Real-Time PCR System (Applied Biosystems). Each sample final volume was 20 μL, containing approximately 100 ng of cDNA. The sequences of the oligonucleotide primers for eviously ,23.In order to evaluate protein expressions, PT, BR, AT1.6, and/or AT6.4 colon cancer cells were washed three times with cold DPBS, and the cells were harvested with lysis buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich). Proteins were quantified by using a BCA assay kit  according to the manufacturer’s protocol. To investigate the effects of AMPK activation in BR colon cancer cells, BR cells were treated with DPBS or 1 mM AICAR for 24 h. Other procedures were similar to those described above. Proteins (20–40 μg) were quantified using a BCA assay kit  according to the manufacturer’s protocol. Proteins (20–40 μg) were resolved on 7.5–15% SDS-PAGE and transferred to nitrocellulose membranes . The blots were incubated with primary antibody diluted at 1:1000 or actin diluted at 1:10,000. Actin was used as the loading control.post-test for comparison among three means or more after analysis of variance (ANOVA) or Student’s t-test between two unpaired data using GraphPad Prism 5.0 software . Statistical significance was set at p < 0.05.Data are presented as means ± standard deviation (SD). The significance of differences was estimated using Tukey’s 50) values of butyrate in HCT116/BR, HT29/BR, and SW480/BR cells were significantly increased by 15.2, 14.0, and 6.08-fold, respectively, compared to those of their respective PT cells  transporters are proteins that efflux the absorbed drugs from the small intestinal epithelial cells and are considered as one of the possible mechanisms of butyrate resistance. Therefore, we determined P-gp, BCRP, and MRP1 protein levels by immunoblotting . There w1 and S phases were not significantly different compared to those in their respective PT colon cancer cells. However, the proportion of cells in the G2/M phase was significantly increased by 53.6%, 22.1%, and 19.4% in HCT116/AT1.6, HT29/AT1.6, and SW480/AT1.6 cells, respectively, compared to that in their respective PT colon cancer cells, but it was restored to the basal levels of PT colon cancer cells in BR colon cancer cells. No subG1 peaks were observed in PT, AT1.6, and BR colon cancer cells, indicating that no apoptosis took place in PT, AT1.6, and BR colon cancer cells.Butyrate induces cell cycle arrest and apoptosis in cancer cells ; therefo1 peak was observed.Immunoblot analysis supported these results C. The exIn order to determine the effects of butyrate resistance on the induction of autophagy, the expression of LC3B, an important constituent of the autophagosomal membrane , was anaIn order to explore whether the autophagy induced by chronic exposure with respect to butyrate was related to AMPK/ACC and Akt/mTOR signaling pathways, immunoblot analysis was performed in PT, AT1.6, AT6.4, and BR colon cancer cells of HCT116, HT29, and SW480 . The proIn order to examine the effect of increasing AMPK activity in BR colon cancer cells, BR cells were treated with 0.2 mM AICAR, a well-known AMPK activator [Protein expression of the AMPK signaling pathway demonstrated that phospho-AMPKα (Thr172) and phospho-ACC (Ser79) levels significantly increased, whereas phospho-Akt (Ser473) and phospho-mTOR (Ser2448) levels decreased in BR colon cancer cells of HCT116, HT29, and SW480 treated with AICAR B. The exIn order to investigate whether the decrease in ACC in BR colon cancer cells is related to fatty acid metabolism, the enzymes involved in fatty acid catabolism and synthesis were determined by qRT-PCR and immunoblot analyses. The gene and protein expression of enzymes involved in fatty acid catabolism, such as CPT1A, LCAD, and SCAD, did not appear to be consistent in both PT and BR colon cancer cells, especially SCAD. The gene expression of SCAD in BR colon cancer cells was significantly lower but protein expressions of SCAD were significantly higher than compared to their respective PT cells. On the contrary, gene and protein expression of enzymes involved in the fatty acid synthesis, such as ACLY, ACC, and FASN, significantly decreased , which i50 values, resulting in poor responses to anticancer drugs, such as cell motility, apoptosis, and invasion [Dietary fiber consumed as food is fermented by intestinal microflora and broken down into short-chain fatty acids, including butyrates that inhibit tumor cell growth and enhance differentiation while inducing glutathione S-transferase (GST), increasing anticancer drug resistance . We previnvasion .2 arrest shown in AT1.6 and/or AT6.4 disappeared gradually by chronic exposure of colon cancer cells to butyrate, resulting in the restoration of G2 arrest in BR cells to that observed in untreated PT cells, confirming the development of resistance to butyrate. This may occur because the drug is not taken up by cells due to an increase in efflux pumps, such as P-gp, MRP1, or BCRP. However, the expression of P-gp and BCRP in BR colon cancer cells was comparable to that in PT cells, indicating that P-gp and BCRP were not involved in butyrate resistance. The relationship between butyrate resistance and P-gp and/or BCRP levels seems to be controversial. It has been reported that butyrate increases the expression of P-gp through upregulation of signal transducer and activator of transcription 3 (STAT3) and mRNA stabilization of MDR1 [The significant effects on cell proliferation and G of MDR1 . In cont of MDR1 .As shown in 2 cell cycle arrest, autophagy was observed via the upregulation of LC3B-II in both AT1.6 and BR colon cancer cells. It was observed that the expression of Beclin-1 and LC3B-II proteins was higher in BR colon cancer cells than that in AT1.6 cells. In AT1.6 and AT6.4 colon cancer cells, treatment with butyrate inhibited cancer cell proliferation by an increase in phospho-AMPKα expression followed by a decrease in phospho-mTOR, resulting in the tumor-suppressive autophagy in AT1.6 colon cancer cells. On the other hand, it is thought that autophagy in BR colon cancer cells protects and supports BR colon cancer cells via inducing excessive activation of Akt and inhibition of AMPK phosphorylation and resulting in the induction of mTOR phosphorylation. This seems to use energy efficiently by removing unnecessary proteins from cancer cells for the survival of cancer cells that, thus, become resistant to butyrate or anticancer drugs [Contrary to the results of cell proliferation and Ger drugs ,32.Similar results have been reported for several anti-cancer therapies. Under normal conditions, phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling inhibits autophagy through the activation of EGFR, but autophagy is induced for cellular survival under stress conditions. Anti-EGFR therapies, such as gefitinib, erlotinib, and cetuximab, promote autophagy in non-small cell lung cancer as a survival mechanism and result in resistance to anti-EGFR therapy ,33. ProtThe activation of AMPK by treating AICAR in BR colon cancer cells restored the inhibitory effect on cell proliferation through the inhibition of Akt/mTOR signaling and reduced the induction of autophagy, indicating that the activation of AMPK reduces resistance to butyrate and restores the sensitivity of butyrate relative to inhibitory effects on cell proliferation; thus, AMPK may play an important role in the regulation of resistance to butyrate by Akt/mTOR activation under stressful conditions. Similarly, stress and EGF induced Akt activation and promoted breast cancer progression, which developed drug resistance through AMPK-mediated Skp2 phosphorylation in this process .ACC, a downstream target of AMPK, is an enzyme involved in the process of fatty acid synthesis from acetyl-CoA to malonyl-CoA . During However, as butyrate resistance is induced by chronic exposure to butyrate produced by digestion of dietary fiber, it can be viewed as a situation rich in butyrate as an energy source; thus, there is no need to catabolize fatty acid. Therefore, it is presumed that the expression of SCAD involved in β-oxidation of butyrate is considerably high with respect to obtaining energy from butyrate, and the expression of enzymes involved in fatty acid synthesis may be lower than those in PT cells since there is no need to induce lipogenesis due to its abundance in the BR colon cancer cells.Taken together, a summary of this study is presented in


The knowledge we currently possess on the molecular function of steroid receptors in breast cancer is incredibly vast. New research in the field is constantly emerging, including studies focusing on potential therapeutic application of steroid receptors other than estrogen receptor which already serves as a crucial therapy target. Therefore we believe that it is necessary to regularly summarize the data on this topic. The aim of this review is to provide breast cancer researchers with a clear explanation of the complex nature of steroid receptor function, including the most up-to-date information, in order to support the effective development of future hormone therapies.Breast cancer remains one of the most important health problems worldwide. The family of steroid receptors (SRs), which comprise estrogen (ER), progesterone (PR), androgen (AR), glucocorticoid (GR) and mineralocorticoid (MR) receptors, along with a receptor for a secosteroid—vitamin D, play a crucial role in the pathogenesis of the disease. They function predominantly as nuclear receptors to regulate gene expression, however, their full spectrum of action reaches far beyond this basic mechanism. SRs are involved in a vast variety of interactions with other proteins, including extensive crosstalk with each other. How they affect the biology of a breast cell depends on such factors as post-translational modifications, expression of coregulators, or which SR isoform is predominantly synthesized in a given cellular context. Although ER has been successfully utilized as a breast cancer therapy target for years, research on therapeutic application of other SRs is still ongoing. Designing effective hormone therapies requires thorough understanding of the molecular function of the SRs. Over the past decades, huge amount of data was obtained in multiple studies exploring this field, therefore in this review we attempt to summarize the current knowledge in a comprehensive way. In 2020, breast cancer became the most frequently diagnosed cancer worldwide, regardless of the sex. In women, it accounted for almost a quarter (24.5%) of all cancer cases and remained the most common cancer-related death cause . Steroidhttps://www.ebi.ac.uk/gxa/home ..128].AR can be acetylated in the hinge region , in the two latter by p300 and p/CAF, or by Tip60 and deacetylated by HDAC1 and SIRT1. This modification leads to increased transcriptional activity of the receptor and was shown to occur in several prostate cancer cell lines; comparably, deacetylation leads to decreased AR activity . The menAR coregulators encompass numerous proteins, with several of particular interest in cancer  162,163163.It is estimated that androgen receptors are present in about 80% of invasive breast cancer cells, with the highest occurrence (95%) in ER-positive, and the lowest (10–35%) in triple-negative breast cancers (TNBC) ,165. In AR expression reduces with tumor grade progression in both ductal carcinoma in situ (DCIS) and metastatic carcinoma, yet is expressed at a higher rate than ER at any tumor grade .Possible causes for ER and AR crosstalk include AR coexpression with ER, mutual stimulation by respective steroids, inhibitory binding of testosterone by ERα and correlated upregulation of ERβ . AR uponThe investigation of AR influence on TNBC yielded mixed results, ranging from increased mortality and metastases through no relation to better prognosis and lesser node involvement .Luminal AR TNBC subtype is known to present ERα-similar effects under AR stimulation. Both AR and ERα can form ternary complexes with SRC and MNAR (PELP1), activating various pathways downstream, including MAPK. Direct corepression is also possible, either with AR NTD binding ERα LBD in presence of estradiol or by AR futile interaction with EREs (mediated by AR LBD)  One possAR expression is positively correlated with HER2 expression; it was shown that AR presence has beneficial effects only in tumors expressing HER, with no influence on outcome of HER2- luminal B cancers . HyperacAndrogen receptor therapy considered in breast cancer includes two main strategies: agonist administration in ERα+ cancers and antagonist administration in AR+ TNBC (the ‘molecular apocrine disease’), in this case the evaluated drugs include bicalutamide, enzalutamide, abiraterone acetate and orteronel . ArchaicThe glucocorticoid receptor’s gene is located in the long arm of chromosome 5 (5q31Y32) and alternatively called NR3C1. When inactive, GR receptor is bound to several chaperone proteins, including hsp90 (by LBD) , hsp70, Due to its immense activity, the coactivators and corepressors of GR are numerous, several are described in At least seven sites of GR phosphorylation are known , but it Several isoforms of GR have been described to this day: GRα (the ‘standard’ form), GRβ—Which is present in the nucleus , has noAbout 299 genes were shown to be regulated by the ‘defective’ GRβ (in HeLa cells), its activation upregulated S100P, ECM1, CGA, FOLR3, ADSSL1, MAFG, ENPEP, SLPI, CACHD1, TRIM29, LOC389203, FGF12 and downregulated FBLN2, UBE2U, SRPK2, CDH2, SELS, VIM, PTPRJ, C8orf4, LIMA1, TCTN1, ABCB1, TGFB2, LUM, TPM1, LOC729113, CA5A, LAMA4, STX1B, UGCG and TNC. For GRα, these were SOX2, CANT1, NEK2, ACOX1, TWSG1, ABCG8, LATS2, ESR2, TRNT1, PLXDC1, CEACAM5, PRSS23, DUSP10, PDZD8, DNAL1, TLK1, PELI1, ERP29, FLJ22662, CPA3, MBL2 (upregulated) and FLJ36644, ZNF280B, CLASP2, TDG, IQCK, MPHOSPH9, NLGN1, NRIP1, FLJ10769, MLLT4 (downregulated) .In normal breast, GR activity is necessary for milk production—Including ultrastructural changes in secretory cells, expression of milk proteins and inhibition of gland involution .GRβ, the LBD-free form of GR, deserves special attention. It needs to be addressed that most studies treat GR as whole, disregarding the difference between receptors and thus disallowing conclusions on the exact action and relations of the isoforms.GRβ was shown to stimulate the proliferation of LNCaP-ARA70β prostate cancer cells and to have a cross-talk relation with AR. It can be inhibited by mifepristone (as can be GRα) (2), in turn, ARA70β, an AR coactivator, has been found in MCF-7 breast cancer and PC3 prostate cancer lines and was shown to intensively stimulate the expression of NR3C1, which leads to preferential overexpression of GRβ . GRβ actGR expression is positively correlated with ERα expression . In infiApproximately 62% of primary invasive breast cancers express GRα . GR mutaIn general, GR level in breast tumors decreases with progression. Worse long-term survival and more recurrence is expected in ER-negative tumors with greater expression of GR ,183, howGR target molecules induce F-actin and paxillin  expression and cell spreading in ABC28 cells and were evaluated to suppress their growth, on pair with aldosterone. Dexamethasone and aldosterone stopped the transition to S phase in mitotic ABC28 cells .Glucocorticoids are antiproliferative towards breast cancer cells and reduce the inflammatory response to the tumor , therefore are used in treatment of primary breast cancer. In comparison, GR activation blocks apoptosis in normal breast epithelium by upregulating MKP-1. It was discovered that with tumor progression GR is translocated to the cytoplasm and its amount in the nucleus is significantly lower than in normal breast cells, surprisingly, this correlates positively with lower level of COX-2 . The exact effect of COX-2 on breast tumorigenesis is unclear, some propose COX-2 elevation as an event in the beginning of breast tumorigenesis, as their studies show its correlation with angiogenesis, lymphoinvasion and cancer progression, but other studies deny that. Cytoplasmic GR expression correlates with lower patient age of onset and with less disease-free time . GRα hinGRα expression seems to increase chemotherapy resistance and hinder apoptosis in TNBC and normal breast epithelium ; in case of tumors with high expression, this may happen even under physiological levels of cortisol. Introduction of mifepristone, a GR antagonist, with paclitaxel reverses resistance in MDA-MB-231, BT-20 cell lines and in murine MDA-MB-231 xenograft model—GR inhibition alone grants no such effect. A comparable effect was shown in MYC–overexpressing ER-negative MCF10A cell line . In ER+ MR, along with GR, are an evolutionary result of a gene duplication of a primitive corticoid receptor—with MR retaining the ability to bind aldosterone, 11-deoxycorticosterone and cortisol and GR being selective for cortisol only. Both associate with GREs when activated .MR possesses the longest NTD of all steroid receptors ,185.NTD of MR contains five SUMOylation sites  . MR is SThe NTD of MR possesses neither the binding sequences present in the AR (FxxLF and WxxLF) nor those typical for all steroid receptors (LxxLL) for binding cofactors, thus the N/C-interaction of MR and cofactors is largely AF-2—independent . MR actiThe chaperone proteins of MR include heat shock protein 90 (hsp90), which interacts directly with the receptor and other, acting indirectly, namely heat shock protein 70 (hsp70), p23, p48, FKBP-59 immunophilins, CYP40 cyclophillin and other immunophilins ,187. TheTarget genes of MR include α/β/γ ENaC subunits, α1/β1-basolateral Na+,K+-ATPase pump subunits, CHIF, K-ras2, SGK1, ELL, GILZ , Grem2, Usp2-45, KS-WNK1 (worth of note because of further PI3K stimulation), NDRG2 (related to N-myc), ET-1, PAI-1, osteopontin, ACE, MDM2, EGF-R (indirectly: fibronectin), collagen I, III, IV (via ERK1/2), tenascin-X (TNX), ADAMTS1, hyaluronic acid synthase-2, RGS2, adrenomedullin, orosomucoid  and CNKSAldosterone exerts some rapid effects on tissues via AT-1 receptor. Active MR can induce SRC, EGFR and MAPK/ERK pathways, in case of the two latter by binding GPER and subsequent activation of EGFR and MEK/ERK. GPER is known to stimulate such pathways as MAPK/ERK, EGFR, PI3K/AKT, in addition to calcium mobilization and cAMP synthesis—resulting in cancer proliferation. By cooperation with HIF-1 GPER promotes VEGF expression and tumor angiogenesis. Various actions mediated by the GPER were assumed to be caused by its function as an aldosterone receptor, recently they are seen rather as the effect of binding with MR. Both MR and GPER are necessary for activation and increased expression of Na+/H+ exchanger-1 (NHE-1), a membrane protein involved in proliferation, migration and metastasis of breast cancer that acts in response to hypoxic, acidic tumor microenvironment. Furthermore, aldosterone caused K-RAS upregulation and Raf and Akt activation in renal carcinoma cells and activated another membrane protein, Na+/HCO3-cotransporter (NBC) . MR actsThe MR possesses several isoforms: the ‘classic’ MR and MR+4 variant with four additional aminoacids between the zinc fingers (caused by a cryptic splice site at the exon 3/ intron C splice junction), the hMRα and hMRβ , which both yield the same final protein and the only distinction between them can be shown in the mRNA phase as the different stability/efficiency in translation), the Δ5 or the Δ5,6 hMR isoforms (skipping exon 5 or exons 5 and 6) with greatly altered activity (eg. constant activity despite lack of steroids nearby). Two variants called MRA and MRB are caused by different start (Kozak) sequences chosen by ribosomes and are less prone to transactivation than the normal MR (with MRA more potent than MRB) . SeveralMR binds mainly aldosterone, although it may bind other mineralocorticoids and even glucocorticoids, based on the presence of 11β-hydroxysteroid dehydrogenase 2 in target cells  ,194. MR Aldosterone hinders transcription of G6PD, mitochondrial uncoupling proteins UCP1 and UCP3, UPAR and HAS2 . MR suppThe mineralocorticoid receptor (MR) has been shown to be present in both normal and cancer breast cells, with about 79–82.2%  specimenApart from the treatment of cardiovascular diseases, MR can be exploited in cancer therapy. Prolonged (≥10 years) use of ARB/ACEi (angiotensin receptor blockers/angiotensin-converting-enzyme inhibitors) in Western populations significantly reduces breast cancer risk, it is disputed to be caused by suppressing renin-angiotensin II pathway and causing consequent downregulation of TF, VEGF, NF-κB and CREB. The mentioned drugs block adhesion and invasion of cancer cells by reducing production of integrin α3 and β1 subtypes and blocking VEGF-A (the latter proven only in mice) . MR inhiThe information on different isoforms of VDR is scarce. Ten proven isoforms and two putative are known. It is supposed that their expression is tissue-related and that they have different target gene profile, although it has not been elucidated properly as of now ,201.VDR is more stable upon binding an appropriate ligand . VDR canVDR displays some peculiar differences caused by polymorphism of its gene—With the most studied mutations known as Cdx2, Fok1, Bsm1, Apa1, Bgl1, Taq1, and Poly (A). According to , codominVitamin D receptor is expressed by normal lobule, ductal epithelial cells , stromalAlthough it was shown that breast cancer cells have elevated expression of VDR, they produce exotic receptor variants  and less full-length variants ; truncated, downstream aberrant variants (yet unnamed) contribute to much of the VDR-related mRNA production. The V1 and V2 suppression may be caused by promoter hypermethylation ,201.VDR is rarely mutated in cancers, albeit can be disabled by aberrant histone deacetylation and methylation of its gene. VDR promoter region (−760–−480), 1a and 1d exons of VDR contain several parts (including three CpG islands) prone to methylation and overlapping with SP1, AP-2, NF-κB, and transcription start sites. In spite of the fact that methylation occurs in any normal breast samples, breast cancer cell lines  and breast cancer samples exhibited elevated VDR promoter region methylation and significant hypermethylation of CpGs (40–65% dinucleotides), as compared to immortalized normal breast line (HBL100) or normal breast samples. In fact, normal breast samples employ meagre levels of methylation (av. 15%) as compared to breast cancer samples (av. 65%). Demethylation of these sites by 5′deoxy-azacytidine led to decreased cell viability in response to calcitriol in all breast cell lines and to increased expression of VDR, β-actin, p21, C/EBP, CYP27B1 and CYP24A1 . CYP27B1The relation of ER and VDR transcription is unclear: some studies show their coexpression in breast cancer samples and note their related stimulation effects , howeverVDR was expressed in considerable amounts in 82.9–91.9% ,203 of iThe risk of death is equally elevated in patients with not only reduced, but also elevated level of seral vitamin D . The debVitamin D induced apoptosis via expression of autophagy marker LC3 (LC3A/B—despite its suppression by inactive VDR), Beclin1, via calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and subsequent AMPK activation . Its apoBT-20, MDA-MB-468, SUM-159PT, MFM-223, CAL-148, ZR-75-B, and BT-474 cell lines are all susceptible to VDR-induced apoptosis. BT-549, a notable exception, possesses no VDR .Inecalcitol, a vitamin D analog with less calcaemic potency, was shown to be eighteen times more effective as vitamin D in apoptosis induction in breast cancer cells, prompting growth halt even in VDR-deficient breast cancer cell lines resistant to vitamin D. VDR activation promotes apoptosis more easily in ER+ lines. This effect is independent of the cells’ AR or HER2 status. Both compounds reduced cell migration , in MDA MB 453—only inecalcitol). Apart from calcitriol-related genes, inecalcitol stimulated CYTH4, LOC553137, MYBPH, GPR78, FREM1, B4GALNT2 and showed preferential upregulation of CLMN, CYP24A1, TMRPSS2, ARHGEF6, FOS, IGFBP3, PISD, G6PD, ITPR1, KLK6, SHE, ACOX3, FSTL4, MERTK and PADI3 . ForskolAs TNBCs do express VDR or AR (about 2/3 of tumors) and some ER+ tumors gain resistance to targeted therapy, they can be treated with VDR agonists , especially coupled with AR agonists and conventional chemotherapy. This type of treatment leads to G1 phase arrest, increase of apoptosis, reduction of tumorsphere formation efficiency and cancer stem cell features , with epithelial transformation . In this case, vitamin D analogues are preferred to avoid side effects of normal vitamin D in high concentrations .Most patients in the advanced stage of the neoplastic disease spread and consequently develop numerous symptoms, which is the basis for considering systemic treatment . The most common indications for hormone therapy include cancers whose growth is dependent on estrogens (breast cancer) and progestins  in women and testosterone in men (prostate cancer). Reduction of the effect of hormonal factors on tumor cells can be achieved by inhibiting the synthesis of sex hormones, administering analogues of the luteinizing hormone releasing factor in the pituitary gland , blocking peripheral synthesis (aromatase enzyme inhibitors) or by limiting the direct peripheral influence of sex hormones on receptors located on tumor cells . Contrary to chemotherapy, which causes numerous side effects, hormone treatment is much better tolerated and is often continued for the rest of the patients’ lives. However, it is important to be aware of the limitations of hormone therapy, which include the subsequent therapeutic response, side effects and drug interactions .The huge therapeutic potential of the SRs together with possible risks associated with endocrine therapies yield a need for constant improvement. Areas to explore include e.g., targeting PR depending on the ratio of isoforms, application of AR antagonists in AR+ TNBC, use of MR antagonists to prevent cardiotoxicity associated with chemotherapy or targeting VDR with vitamin D analogues such as inecalcitol. Given the immense complexity of SRs function in breast cancer, which provides multiple potential therapy targets, it is easy to expect that in the near future many more novel concepts on therapeutic applications of steroid receptors will arise.


Propolis is a honeybee product known for its antioxidant, anti-inflammatory, anticancer, and antimicrobial effects. It is rich in bioactive molecules whose content varies depending on the botanical and geographical origin of propolis. These bioactive molecules have been studied individually and as a part of propolis extracts, as they can be used as representative markers for propolis standardization. Here, we compare the pharmacological effects of representative polyphenols and whole propolis extracts. Based on the literature data, polyphenols and extracts act by suppressing similar targets, from pro-inflammatory TNF/NF-κB to the pro-proliferative MAPK/ERK pathway. In addition, they activate similar antioxidant mechanisms of action, like Nrf2-ARE intracellular antioxidant pathway, and they all have antimicrobial activity. These similarities do not imply that we should attribute the action of propolis solely to the most representative compounds. Moreover, its pharmacological effects will depend on the efficacy of these compounds’ extraction. Thus, we also give an overview of different propolis extraction technologies, from traditional to modern ones, which are environmentally friendlier. These technologies belong to an open research area that needs further effective solutions in terms of well-standardized liquid and solid extracts, which would be reliable in their pharmacological effects, environmentally friendly, and sustainable for production. Propolis is one of the most known honeybee products, used in folk medicine since ancient times for its numerous health effects. Nowadays, it is a starting raw material for manufacturing various extracts that can be used as active pharmaceutical ingredients (APIs). Raw propolis is a natural, glue-like mixture collected by honeybees, mostly from flower and leaf buds of different plant species. In general, propolis consists of plant resins and essential oils, beeswax, and pollen. Organic compounds that have been identified in propolis are: polyphenols, terpenes, esters, amino acids, vitamins, minerals, and sugars ,2. SinceHoneybees use propolis primarily as a thermo isolation material by sealing the cracks in wooden walls and other parts of the hive and strengthening the construction of wax combs. Additionally, it is a crucial part of collective social immunity at the colony level.The bioactive molecule profile of raw propolis varies according to the geographical and botanical origin, season, bees’ genetics, and environmental factors ,4,5. TheThe propolis extracts’ chemical profile will also depend on the extraction solvent type, solvent ratio, and extraction procedures. Overall, there are over 500 bioactive molecules identified in propolis, and most of them are secondary plant metabolites . Many ofThis review aims to present propolis both as the active pharmaceutical ingredient (API) and as a valuable source of other potential APIs. The latest research on the biological activity of propolis and its compounds outlines their commonalities and a multitude of effects. Since the activity of propolis will also depend on the extraction of its active compounds, we give an overview of extraction methods—from traditional to modern ones—with emphasis on improvements necessary for their application in pharmaceutical research and development.Populus spp., mostly P. nigra L. Poplar propolis is rich with flavones, flavanones, phenolic acids, and their esters . Cub  [4]. Cu)   16]..Populus Bankova (1998) reported that Canarian propolis has a higher amount of furofuran lignans and sugars and sugar alcohols due to mucilaginous plants characteristic for the geographical area .Melipona. Dos Santos et al. (2017) determined polyphenols, flavanones, terpenoids, but also di- and trigalloyl and phenylpropanoid heteroside derivatives in hydroethanolic extracts of geopropolis [6) and ellagic acid (7)   19]..Melipona8), galangin (9), pinocembrin (10) and pinobanksin-3O-acetate (11). The most representative phenolic acids were caffeic acid (12), p-coumaric acid (13), and ferulic acid (14), as well as their derivatives, 3,4-dimethoxycaffeic acid  and caffeic acid prenyl (16), benzyl (17), phenylethyl , and cinnamyl (19) esters , gDue to so many propolis types with different bioactive molecules, Bankova (2005) proposed the standardization according to the botanical origin and the corresponding chemical profile . For pop6) (20) (12), p-coumaric (13), and ferulic (14) acids  and abiotic stress conditions, like drought and cold. Based on their molecular structure, they can be divided into flavonoids and nonflavonoids. Flavone chrysin, flavonol galangin, and flavanone pinocembrin are flavonoids, while most abundant nonflavonoids are phenolic acids subdivided into derivatives of benzoic acid, such as gallic acid (6) , protoca6) (20) , and der4) acids  21]..6) (20) 2−), hydroxyl radical (•OH), and hydrogen peroxides (H2O2). Oxidative stress could be induced by various metals . Th. Th8) 3  (9) [Enterococcus spp., and clinical isolates of P. aeruginosa. Galangin (9) had an inhibitory effect on 16 strains of 4-quinolone resistant S. aureus [9) effectively inhibits murein hydrolase activity and the growth of vancomycin-intermediate S. aureus strain with the thickened cell wall, Mu50 [9) has better antimicrobial activity than quercetin due to well-spaced hydrophobic and hydrophilic regions in the molecule [Flavonol galangin (9)  also sup9) (9) . The re-. aureus . The cyt. aureus ,53. Latell, Mu50 . Echevermolecule .10)   has manyrf2 axis . Reducinactivity ,64,65,66activity , but it activity . Recentlactivity . It doesactivity . Pinocem. aureus .12), p-coumaric (13), and ferulic (10) have many effects in common with flavonoids and other propolis components (Nonflavonoid phenolic acids like caffeic (mponents .12) is a noncompetitive inhibitor of PKC activity in partially purified human monocytes [12) attenuates apoptosis in non-small-cell lung cancer (NSCLC) cells via NF-κB, by upregulation of survival proteins survivin and Bcl-2 [12)  and paclitaxel in inducing apoptosis in NSCLC H1299 cells [Caffeic acid (onocytes . Althougonocytes . Lin et nd Bcl-2 . On the 99 cells . Caffeic99 cells .One of the most studied active compounds of a poplar-type propolis is caffeic acid phenethyl ester (CAPE) (18). It is a potent antioxidant extracted from propolis, with excellent anti-inflammatory, wound-healing, antidiabetic, organ protective, anticancer, and antimicrobial properties ,75. CAPECAPE’s antioxidant activity is mediated through Keap1/Nrf2/ARE pathway in the rat colitis model . This meWound-healing properties of CAPE result from antioxidant and anti-inflammatory effects but not in an early phase . The stuCAPE showed anticancer potential in numerous studies: from hematological, lymphoid, breast cancer, gastrointestinal, prostate, ovarian, cervical, head, and neck, to lung adenocarcinomas . CAPE iNeuroprotective effects of CAPE are mediated through already mentioned anti-inflammatory (mostly NF-κB inhibition) , antioxiCaenorhabditis elegans infected with C. albicans [S. aureus, Enterococcus faecalis, and Listeria monocytogenes [Paenibacillus larvae [Streptococcus mutans, Streptococcus sobrinus, Actinomyces viscosus, and Lactobacillus acidophilus) [Vibrio cholerae [E. coli [S. mutans [Leishmania parasites [CAPE has antifungal, antibacterial, and antiviral activity. It prolongs the survival of mice  and Caenalbicans , and woralbicans , and ampalbicans . CAPE isytogenes , bee infs larvae , common ophilus) ,103, andcholerae  and E. c[E. coli . Besides. mutans ,103. In . mutans , hepatit. mutans , and typ. mutans . In morearasites .p-Coumaric acid (13) and ferulic acid (14) (p-Coumaric acid (13) decreases the production of iNOS, COX-2, IL-1β, a TNF-α [14) also inhibits the activity of NF-κB, expression of IL-6, and modulates the oxidative Nrf2 pathway [Similarities with caffeic acid and CAPE can be seen in the biological activity of cid (14) . p-Couma a TNF-α  NF-κB, a a TNF-α . Ferulic pathway .Most of the effects mentioned above were studied for each compound separately. Still, it is crucial to consider the mixture effect of propolis since there are always synergisms and antagonisms between compounds in the mixture . The mixFor instance, caffeic acid and ethanolic extract of Brazilian propolis suppressed LPS-induced signaling pathways, like p38 MAPK, JNK1/2, and NF-κB in macrophages . ERK1/2 Eucalyptus propolis (EEEP) improve antioxidant gene expression only via ERK/-Nrf2 signaling pathway, while EtOH extracts of Baccharis propolis (EEBGP) strengthen the antioxidant system by activating p38 MAPK and accelerating nucleus translocation of Nrf2 [It seems that no matter the origin and composition, propolis extracts will exert antioxidant action, but possibly through slightly different mechanisms of action. The EtOH extract of Brazilian red propolis (EERP) suppressed ROS generation and cytotoxicity by activating the Nrf2-ARE intracellular antioxidant pathway . Zhang e of Nrf2 .Streptococcus and S. aureus, Bacillus subtilis, E. faecalis, and against yeasts of Candida species [Propolis extracts are active against yeasts, fungi, viruses, bacteria, and even parasites, being the most effective against Gram-positive bacteria, such as  species ,120. The species .Most probably, the activity of polyphenols in propolis should be attributed to interactions between them and other components and follows the Goldilocks principle. The other, not yet identified compounds like peptides could also affect polyphenols’ activity and stability ,122. HowInterestingly, although propolis content varies depending on extraction, it seems like different extract’s biological activities remain comparable. Galeotti et al. 2018) demonstrated that propolis solubilized in various solvents and liquid and solid forms has a similar chemical composition when produced from the same raw material, with differences in total polyphenol content but comparable antioxidant activity  demonstr. Maceratv/v aqueous EtOH is used as the ES at room temperature (r.t.), yielding the propolis tincture. Such a simple extraction process is known as maceration. It is conducted by the addition of aqueous EtOH onto the crude propolis chunks at the weight (propolis) to volume (ES) ratio of 1:3–20, most commonly 1:5–10. This obtained mixture is usually left to stand in a closed vessel at r.t. for 7–30 days. In a typical example of such procedure, the crude propolis is subjected to a r.t. maceration process for 30 days [The crude propolis is traditionally extracted with extraction solvents (ES) consisting of various EtOH and water mixtures. Typically, 25–60%  30 days . Usually 30 days . After tS. aureus as a model pathogenic microorganism, antioxidant effect, and inhibition of hyaluronidase activity. The results showed that the most active extracts were those obtained with 60–80% aqueous EtOH, while those with 40–60% and 80–95% EtOH showed significantly lower activities in all three comparative pharmacological activities [Park and coworkers (1998) described the extraction of the crude propolis with various mixtures of water (5–100%) and EtOH (0–95%) at 70 °C for 30 min . After ctivities . Obvious(i)The presence of a relatively aggressive solvent (EtOH);(ii)Alcohol-based products are not suitable for children, pregnant and breastfeeding women, and certain patients;(iii)And relatively high content of beeswax, which causes its separation upon the phase of mixing with water phase, during the manufacturing of pharmaceutical and other products, where such an extract is employed as an API.Several disadvantages characterize such liquid propolis extracts:Due to these deficiencies of such traditional liquid propolis extracts, various improved solutions are developed and described in numerous scientific and patent literatures. Some selected, typical improved solutions are presented in this article.Except by a simple maceration process in a single extraction vessel, optionally equipped with a suitable stirrer for adequate agitation of the propolis mass during the maceration process, the extraction can be performed by percolation or with a Soxhlet apparatus . These t(i)Repeated extraction at cold processing conditions ;(ii)Elevated extraction temperatures ranging from 50–95 °C ,132,133;(iii)Soxhlet extraction technique ;(iv)Or ultrasonic-assisted extraction (UAE) .Certain progress has been introduced by the use of pure water as a sole extraction system ,132,133.n-PrOH), 2-propanol (2-PrOH), 1-butanol (n-BuOH), 2-butanol (s-BuOH), tert-butanol (t-BuOH), diethylether (Et2O), 1,2-propylene glycol , dimethylsulfoxide (DMSO), ethylene glycol (ETG), benzyl benzoate (BnBz), polyethylene glycol (PEG), acetone, and glacial acetic acid (HOAc) at a low (1:2) ES-to-propolis weight ratio (w/w) [Sosonowski described the preparation of the liquid propolis extracts obtained by maceration of the crude propolis with various alternative organic solvents (OS) such as: methanol (MeOH), 1-propanol (io (w/w) . Such liAmong more polar extraction solvents, especially convenient are 1,2-PG, PEG , or glycerol (GL), which are relatively non-toxic, safe, and widely used as pharmaceutical excipients or as diluents and humectants in numerous pharmaceutical products ,135,136.w/w) with water resulted in significantly increased total phenolic content (TPC) in comparison to pure aqueous propolis extracts [1,2-PG was found to be very effective propolis ES, giving high percentage of extracted polyphenols at DER ratio 1:10 to 1:20 at 50–60 °C for a short period of time (2 h), or during the maceration at r.t. for several days . PEG 400extracts . When coextracts . Still, extracts .w/w, at 90–160 °C, with subsequent filtration. Such glycerol extracts are water-soluble and suitable as API for the production of various pharmaceutical products [Additionally, Tsukada and co-workers (1991) disclosed the use of GL as the ES at very low weight ratio of propolis-to-ES, 1:2 products .The least polar ES that could be employed for the propolis extraction are various fixed and essential oils. The former are various triglycerides, plant oils such as sunflower, rapeseed, sesame, olive, and soybean oils. They are able to extract predominantly non-polar propolis compounds from the starting crude propolis. In this manner, Galeotti and coworkers 2018) described the extraction with plant oil and obtained the corresponding liquid extract of similar TPC like with GL as the ES [ describeSimilarly, Savickas and coworkers (2001) described a unique waterless ES based on a mixture of EtOH (96%) and sunflower oil . Such a w/v ratio of the crude propolis to the ES during 48 h at r.t. with constant stirring. After subsequent filtration, the resulting liquid extract was of a high total phenolic content  and high total flavonoid content (mg). The former parameter was about 50% lower, while the latter was around 28% lower than at 70% EtOH-based extract [Keskin (2020) described the use of orange peel essential oil (OPEO) predominantly based on d-limonene as a green and renewable ES for propolis . The ext extract .(i)The choline chloride (CC):1,2-PG in a molar ratio (n:n) 1:1 or 1:2;(ii)2O in 1:2:2 or 1:1:1, n:n:n;CC:lactic acid (LA):Hare very effective ESs for propolis extraction, comparable to aqueous ethanol (70%) as a golden standard ES [L-lysine  is relatively effective ES for propolis, yielding the corresponding liquid extract with about 50% of the component’s concentration obtained with aqueous EtOH (70%) [Further progress has been made in the field of the utilization of natural deep eutectic solvents (NADES), which exhibit good solvent properties, while safe, of low toxicity, and from renewable resources ,140. ThuOH (70%) ,140.2) as the extraction solvent [2 is transformed into a supercritical fluid, whose solvent properties are roughly similar to acetone. It is able to dissolve different non-polar to slightly polar propolis compounds. After this supercritical extraction (scE), the liquid scCO2 propolis extract is subjected to the filtration within the closed system to remove undissolved propolis residues [2 propolis extract is simply converted to a dry powdery propolis extract by evaporation (and regeneration) of CO2 by cooling the system and reducing the crystallization unit’s pressure [2 extraction is relatively mild extraction conditions, easy removal of the ES, no residual solvent impurity, and a highly positive environmental impact due to the complete absence of organic solvents [Modern green-chemistry technology for the manufacturing of solid propolis extracts includes supercritical carbon dioxide (scCO solvent . When exresidues . The respressure . The maisolvents .w) propolis components by forming host-guest molecular complexes. A typical example of such a complexing agent is hydroxypropyl-β-cyclodextrin (HP-β-CD). The latter facilitates the propolis extraction via decreasing the equilibrium concentration of low Mw propolis compounds within the supernatant liquid phase of the extraction suspension by their complexation into the hydrophobic HP-β-CD cavity. This causes the dissolution of a higher amount of propolis compounds of relatively lower polarity and thus increases the extraction process’s overall efficacy [Another unique approach to propolis extraction is applying reagents dissolved in the ES that are capable of complexing low molecular weight , and compared their efficiencies with a traditional maceration process and conventional heat reflux extraction (HRE) [Pellati and coworkers 2013) described ultrasonic-assisted (UAE) and microwave (MW)-assisted (MAE) extractions of propolis with a mixture of EtOH and water  under MAE conditions  resulted in approximate extraction efficacy and higher (73–75%) yield when compared with UAE that took 30 min yielding 41–53%, or with traditional maceration that required 72 h and gave 55–58% yield [Trusheva and coworkers (2007) described that the use of UAE and specially MAE do result in dramatically reduced extraction processing time with proportional extraction efficiency and somewhat higher chemical yields . For ins8% yield .In contrast to these results, Oroian and coworkers (2020) described that UAE was more efficient in terms of the chemical yield of the extraction and higher content of active propolis substances against the corresponding products obtained with traditional maceration and MAE. Perhaps 2 × 1 min of their MW (2450 MHz) at 140 W was too mild for more effective extraction than 2 × 15 min duration of UAE (20 kHz) process .An additional modern approach to the propolis extraction is based on a high-pressure extraction , in a batch-type high-pressure extraction vessel, which stimulates the transport of ES into the solid crude propolis particles ,146. ThiThe use of suitable emulsifiers introduced further significant improvements in environmentally friendly, water-based technologies. They facilitate the solubilization of relatively less polar propolis active substances into the water ES medium. In this manner, Paradkar and coworkers (2010) described the process for the propolis extraction by the use of an aqueous polysorbate (PS) solution at 40–90 °C during 2–24 h, yielding the corresponding liquid propolis extract . PSs arep-coumaric acid (9), 75–800 μg/mL trans-ferulic acid (10), 25–300 μg/mL caffeic acid (8), and 40–400 μg/mL 2-phenylethyl 3,4-dihydroxycinnamate (14) [In this field, Radic et al. (2019) described a unique non-aqueous ES system based on the solution of plant lecithin (LE), e.g., rapeseed lecithin, in liquid polyethylene glycol such as PEG 400, which effectively extracts the crude propolis at r.t. or at elevated temperatures (up to 150 °C) yielding the corresponding liquid extract (after appropriate filtration), which is suitable as the ACI or API for the production of various cosmetic, pharmaceutical, and veterinary medicinal products . This prate (14) .There is a vacuum resistive heating extraction (VRHE) process among special new technologies, which is based on a vacuum Ohmic heating process . LastriyDescribed selected typical technologies for propolis extraction are given in v/v), EtOH (96% v/v), 2-PrOH, acetone, or similar low toxic organic solvents. The latter are usually further processed through the spray-drying or fluid bed technology onto a suitable powdery carrier such as food- or pharma-grade maize-, wheat- or potato-based maltodextrin, with or without subsequent addition of a small (0.2–2% w/w) amount of magnesium stearate, colloidal silicon dioxide, or similar anti-caking agent. Alternatively, powdery propolis extracts can be prepared directly via scCO2-based extraction [The preparation of powdery propolis extracts usually starts from suitable primary liquid extracts, obtained with volatile ES such as water, aqueous EtOH , and additives that enhances extraction chemoselectivity such as emulsifiers (LE). The propolis extraction technologies still belong to an open research area that needs further effective solutions in terms of well-standardized liquid and solid extracts, which would be reliable in their pharmacological effects and environmentally friendly and sustainable strategies for their production.To better describe propolis extraction technologies’ characteristics, we overviewed more traditional ones to modern and environmentally friendly processes. In contrast to existing reviews, the special attention was paid to environmentally friendly extraction processes, non-toxic ES (H


Community-acquired pneumonia (CAP) causes substantial morbidity and mortality. There is a lack of data on the comprehensive burden of CAP across the life span in Canada. We estimated the incidence of all-cause CAP in all age groups in Ontario and British Columbia (BC), Canada. We identified hospitalized and outpatient CAP episodes from the Discharge Abstract Database (DAD) and physician billing claims databases  in both provinces. The National Ambulatory Care Reporting System was used to identify CAP episodes from emergency department visits in Ontario. CAP recorded with a primary or secondary diagnosis was identified using International Classification of Diseases 9  and 10  codes. We estimated the age and sex adjusted annual incidence of CAP overall, and by age groups  according to routine childhood pneumococcal conjugate vaccine (PCV) immunization periods from 2005–2018 in Ontario and from 2002–2018 in BC. Poisson regression models were fitted with population denominators from Statistics Canada to estimate the incidence rates. Ontario had 3,607,186 CAP episodes from 2005–2015 with a mean annual incidence of 2,801  per 100,000 population; incidence declined from 3,077/100,000 in 2005 to 2,604/100,000 in 2010 before increasing to 2,843/100,000 in 2018. BC had 1,146,172 CAP episodes from 2002–2008, with a mean annual incidence of 2,146 ; the incidence increased from 2,005 /100,000 in 2002 to 2,199/100,000 in 2018. A high incidence of CAP was observed in children aged 0–4 years and older adults, particularly in adults aged ≥85 years in both provinces across all PCV program periods .Figure 1: Age group-specific incidence of all-cause community-acquired pneumonia according to childhood pneumococcal conjugate vaccine (PCV) program periods in Ontario (PCV7 [1 Jan 2005–30 Sep 2009]), PCV10 [1 Oct 2009–31 Oct 2010] and PCV13 [1 Nov 2010–31 Dec 2018]) and British Columbia (PCV7 [1 Sep 2003–31 May 2010] and PCV13 [1 Jun 2010–31 Dec 2018]), CanadaCAP continues to be a public health burden in Canada despite publicly funded pneumococcal vaccination programs. Ontario seems to have higher CAP burden than British Columbia that warrants further investigation. The youngest cohort of children and older adults contribute significantly to the CAP burden.Manish Sadarangani, BM BCh, DPhil, GlaxoSmithKline (Grant/Research Support)Merck (Grant/Research Support)Pfizer (Grant/Research Support)Sanofi Pasteur (Grant/Research Support)Seqirus (Grant/Research Support)Symvivo (Grant/Research Support)VBI Vaccines (Research Grant or Support) Allison McGeer, MSc,MD,FRCPC,FSHEA, GlaxoSmithKline (Advisor or Review Panel member)Merck Pfizer  James D. Kellner, MD, FRCPC, FIDSA, Pfizer, Merck, GSK, Moderna (Grant/Research Support) Shaun Morris, MD, MPH, DTM&H, FRCPC, FAAP, GSK (Speaker’s Bureau)Pfizer (Advisor or Review Panel member)Pfizer (Grant/Research Support) Shaza A. Fadel, PhD MPH, Merck  Fawziah Marra, BSc(Pharm), PharmD, Pfizer Canada (Research Grant or Support)


Emaravirus of the family Fimoviridae. Moreover, when GEVA was graft-transmitted to SJ and “Beta” grapevines, all grafted plants showed the same symptoms, similar to those observed in the source of the inoculum. This is the first report to our knowledge of an emaravirus infecting grapevine and its possible association with chlorotic mottling symptoms.A novel negative-sense, single-stranded (ss) RNA virus was identified in a “Shennong Jinhuanghou” (SJ) grapevine showing severe chlorotic mottling symptoms by integrating high-throughput sequencing (HTS) and conventional Sanger sequencing of reverse transcription polymerase chain reaction (RT-PCR) products. The virus was provisionally named as “grapevine emaravirus A” (GEVA). GEVA had a genome comprising five genomic RNA segments, each containing a single open reading frame on the viral complementary strand and two untranslated regions with complementary 13- nt stretches at the 5′ and 3′ terminal ends. RNA1 , RNA2 , RNA3 , and RNA4  encoded putative proteins P1–P4 that, based on their conserved motifs, were identified as the RNA-dependent RNA polymerase, glycoprotein, nucleocapsid protein, and movement protein, respectively. However, the functional role of protein P5 encoded by RNA5  could not be determined. Phylogenetic trees constructed based on amino acids of P1 to P4, allocated GEVA in clade I, together with other species-related emaraviruses. These data support the proposal that GEVA is a representative member of a novel species in the genus  Emaravirus is the sole genus in the family Fimoviridae and encompasses plant viruses with multiple, negative-sense, single-stranded (ss) RNA genomes (Emaravirus: Actinidia chlorotic ringspot-associated virus (AcCRaV) (Emaravirus) , they have not been reported in grapevines. genomes . Viruses(AcCRaV) , blackbe(AcCRaV) , Europearavirus) , fig mosravirus) , High Plravirus) , pigeonpravirus) , PPSMV-2ravirus) , Pistaciravirus) , raspberravirus) , redbud ravirus) , rose roravirus) , Actinidravirus) , aspen pravirus) , lilac cravirus) , blue paravirus) , pear chravirus) , ti ringravirus) , Camelliravirus) , jujube ravirus) , and Perravirus) . Other travirus) , common ravirus) , and Chrravirus) . AlthougEmaraviruses consist of 4–8 RNA segments in their genomes. Each segment contains a single open reading frame (ORF) on the strand of complementary RNA. The RNA1, RNA2, RNA3, and RNA4 segments of emaraviruses encode an RNA-dependent RNA polymerase (RdRp), a glycoprotein (GP) precursor, a nucleocapsid protein (NP), and a non-structural movement protein (MP), respectively, as core elements . The proChina is one of the world’s leading grape-production areas . Grapevine virus diseases are major threats to grape production, with 21 species of grapevine viruses reported in China . ChlorotDuring a field investigation in 2016, the obvious symptoms of chlorotic mottling on the leaves of a “Shennong Jinhuanghou” (SJ) grapevine cultivar were observed. A preliminary investigation conducted using reverse-transcription polymerase chain reaction (RT-PCR) assay showed that the SJ grapevine did not harbor common viruses identified previously in China. To identify possible virus infections in the diseased-grapevine sample, small RNA sequencing (sRNA-seq) and RNA sequencing (RNA-seq) were used to identify viruses in diseased grapevine samples in 2018.Vitis vinifera L.), showing systemic symptoms of chlorotic mottling , as reported previously DNA library of sRNAs. sRNA-seq was carried out using an Illumina HiSeqeviously . “Clean”ously  according to the manufacturer’s instructions. Primers 5H/3C usually allow full-length amplification of emaravirus RNAs (expect for RNA1) by RT-PCR  7.03. Protein analyses for prediction of the transmembrane domains of proteins were conducted using TMHMM4  was used to search for potential ORFs in the genomic and anti-genomic RNAs of the virus. Nucleotide (nt) and amino acid (aa) sequences were compared with those of other emaraviruses using ClustalW2g TMHMM4 . PredictalP 3.06 .Graft transmissibility was assessed by grafting GEVA-infected grapevine buds onto SJ seedlings and “Beta” grapevine seedlings (2 years of age) in four replicates in July 2020. These seedlings had tested negative for GEVA and other major viruses reported in China: grapevine leafroll-associated virus-1 (GLRaV-1), GLRaV-2, GLRaV-3, GLRaV-4, GLRaV-7, GLRaV-13, grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine fleck virus (GFkV), grapevine fanleaf virus (GFLV), grapevine virus A (GVA), grapevine virus B (GVB), grapevine virus E (GVE), grapevine Pinot gris virus (GPGV), grapevine berry inner necrosis virus (GINV), grapevine fabavirus (GFabV), grapevine rupestris vein feathering virus (GRVFV), grapevine geminivirus A (GGVA), grapevine Syrah virus-1 (GSyV-1), and grapevine red globe virus (GRGV). Inoculated seedlings were maintained in a greenhouse under natural conditions for symptom development. Grafted grapevines were observed continuously for symptoms after inoculation. After 2 months, total RNAs were extracted from the leaves of the grafted SJ grapevine and Beta grapevine using the method described by To survey GEVA prevalence, 473 grapevine samples of 71 cultivars were collected from 21 provinces in China during 2018–2020. These samples were tested for GEVA by RT-PCR using the primers P3-1a/1b and P4-1a/1b. Amplification products from positive samples were cloned and sequenced as described above.Sequencing of a cDNA library of sRNAs from symptomatic leaves of the SJ grapevine  resultedRNA1, RNA2, RNA3, RNA4, and RNA5 had lengths of 7,090, 2,097, 1,615, 1,640, and 1,308 nt, respectively . The fir1132–1138) and C (HSDDS1257–1261) are part of the palm domain of the replicase, and are involved in the binding of divalent metal cations (1217–1225) is thought to be involved in RNA binding with the Gly residue, which allows for mobility in the peptide backbone; motif D (KKTY1308–1311) has a strictly conserved Lys residue, and is proximal to the Asp of motif A (1317–1322) is likely to be involved in “cap-snatching” in bunyaviruses as well as in possible endonuclease activity (95–97–PD134–135–EIK148–150), which is deemed to be involved in cap-snatching of viral mRNAs during genome expression  encoding a putative protein (P1) of 2,300 aa with a predicted molecular mass of 268 kDa. The sequence identity between GEVA P1 and RdRps of other emaraviruses ranged from 27.8 to 49.8% . P1 of G cations . Motif B motif A . Motif Eactivity . P1 alsopression . Such reIn silico analyses predicted four transmembrane helices  and four N-glycosylation sites  (28L/VNV and K192A/DDN) that would process the GP precursor into the C-terminal GP  and two N-terminal GPs, a larger Gn (19 kDa) and a smaller Gs (3 kDa)  encoding P2, a putative GP of 638 aa with a predicted molecular mass of 73 kDa. GEVA P2 shared identity of 19.0–43.1% at the aa level with GPs of the other emaraviruses. N439STI) . Two cle (3 kDa) , were al136–145, NRLA184–187, and GXEF205–208) similar to the NP motifs reported in other emaraviruses  encoding P3, a putative NP of 313 aa with a predicted molecular mass of 35 kDa. It shared aa-sequence identity of 18.5 to 43.5% with NPs of the other emaraviruses. Sequence alignment of P3 identified three conserved aa blocks  encoding P4, a putative protein of 363 aa with a predicted molecular mass of 41 kDa. It shared aa-sequence identity of 11.2–34.7% with the NPs of the other emaraviruses. We identified a signal peptide with a cleavage site (VKA23DD) using SignalP . SMART aRNA5 was of length 1,308 nt and had one ORF  encoding the P5 protein consisting of 255 aa with a molecular mass of 29 kDa. A BLASTp search against the GenBank database of NCBI indicated that GEVA P5 shared a limited aa-sequence identity of 25.8% with PiVB P6, but did not display significant sequence identity with proteins encoded by other emaraviruses or the proteins available in GenBank. Moreover, we did not identify a conserved domain in P5 through CDD analysis. Thus, we could not ascertain the function of this viral protein.Mapping results showed that 23,829 (0.17%), 61,088 (0.43%), 52,943 (0.37%), 86,093 (0.60%), and 79,211 (0.55%) of 14,348,642 clean reads were derived from RNA1, RNA2, RNA3, RNA4, and RNA5, respectively . The sRNPhlebovirus, Tenuivirus, Coguvirus, and Rubodvirus in the family Phenuiviridae, the genus Orthotospovirus in the family Tospoviridae, and the genus Orthobunyavirus in the family Bunyaviridae were used in the tree reconstruction. Regardless of the proteins under consideration, GEVA clustered together with emaraviruses with high bootstrap values, and was clearly separated from other viruses in several genera in the order Bunyavirales, thereby confirming a close phylogenetic relationship of GEVA with members of the genus Emaravirus  has become very popular in recent years for identification of potentially novel viruses associated with fruit disease . SeveralEmaravirus, as reported by Emaravirus. To ascertain if GEVA had more than five types of RNA, we used the primers 5H/3C to amplify the full-length of emaravirus RNA according to the method described by Five genomic components of GEVA from a SJ grapevine sample were determined by integrating data from sRNA-seq, RNA-seq, and conventional Sanger-seq. Each genomic RNA of GEVA possessed a single ORF on its complementary strand and 13-nt stretches with complementary sequences at its 5′ and 3′ termini, similar to those of other emaraviruses . The P1–Emaravirus. Analyses of the sRNAs derived from the GEVA genome revealed that most were 21 nt in length, suggesting that DICER-like (DCL) enzyme-4 had a major role in siRNA production , accession number were MW888853–MW888857.The data presented in the study are deposited in the NCBI repository (XF and YD conceived and designed the experiments. XF, ZZ, and HS collected the samples. XF, CL, FR, GH, BZ, and YD conducted the experiments and analyzed data. XF, CL, and YD discussed the results and drafted and revised the manuscript. All authors approved the final draft of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.