San-ao decoction (SAD) has been widely used in Chinese medicine against respiratory diseases, such as asthma and rhinallergosis. The bioactive compounds for such pharmacological action remain unknown.β2-AR) onto magnetic fine particles, the capture of target compounds by the immobilized receptor, the identification of the receptor bound compounds by reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry.We developed a methodology to isolate the bioactive compounds of SAD. The assay involved the immobilization of beta 2-adrenoceptor  contains supplementary material, which is available to authorized users. Drug discovery involves identifying compounds that prevent or control diseases and play an important role in maintaining human health –3. Despiβ2-AR) chromatography was established for the identification of bioactive compounds of the receptor [β2-AR may misfold during the high pressure environment. Here, we established a bio-affinity technique by the utilization of magnetic material to remove the isolation and identification bottlenecks in identifying procedures [Natural products are the main source of virtually all medicinal preparations, and between 1981 and 2010, 34% of approved small molecule drugs were from natural products . The impreceptor . This stocedures –21. Magnocedures –24. Subsocedures .Herba Ephedrae, Semen Armeniacae Amarum and Radix Glycyrrhizae. Pharmacologically, respiratory ailments are mainly mediated by the β2-AR, which belongs to the G-protein coupled receptor (GPCRs) superfamily [β2-AR. In this work, we immobilized β2-AR onto MFPs to construct a new biologically relevant isolation material. The immobilized β2-AR MFPs were used to rapidly screen bioactive compounds of the receptor from SAD. The compounds of interest were subsequently isolated and identified by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Molecular docking confirmed the binding between the bioactive compounds and β2-AR. The receptor-functionalized MFPs provide a convenient and effective strategy for isolating ligands from traditional Chinese medicines.San-ao decoction (SAD) is a basic prescription for treating respiratory diseases in Chinese practice. The formula is indexed in the Pharmacopoeia of the People’s Republic of China 2015, and consists of three herbs: erfamily , 27. We The Minimum Standards of Reporting Checklist contains details of the experimental design, and statistics, and resources used in this study  (99%) and ferrous chloride tetrahydrate (FeCl2·4H2O) (99%) were purchased from Tianjin Tianli Chemical Reagents Ltd . Analytical-grade sodium hydroxide, trisodium citrate, ethanol and aqueous ammonia (25 wt%) were acquired from Shanghai Chem. Reagent Co. . Reference standards of salbutamol (batch no. 100328–200703), terbutaline (batch no. 100273–201202) and shaftoside (batch no. 111912–201703) were purchased from the National Institutes for Food and Drug Control . Vicenin (batch no. B21399), isoshaftoside (batch no. B21563), liquiritin apioside (batch no. B25831) and isoliquiritin apioside (batch no. B20987) were purchased from Shanghai yuanye Bio-Technology Co., Ltd . Herba Ephedra, Semen Armeniacae Amarum and Radix Glycyrrhizae were obtained at a local medicinal market and identified by Professor Xiao Chaoni in the College of Life Sciences of Northwest University.Ferric chloride hexahydrate  equipped with an isocratic pump, a column oven and an ultraviolet–visible detector. Separation and identification of the bioactive compounds were carried out on an Agilent 1100 series of high-performance liquid chromatography  coupled with an SL trap mass spectrometer . The size distribution of MFPs was determined by dynamic light scattering (DLS) using a Malvern ZS ZEN 3600 system .An AKTA10 low-pressure chromatographic system from GE Healthcare Life Sciences  was utilized for Herba Ephedrae, Semen Armeniacae Amarum and Radix Glycyrrhizae) were weighed according to the Pharmacopoeia formula dosages. The mixture was decocted three times with 8 volumes of water (v/w) and 1.0 h for each time. The three filtrates were combined and concentrated to a certain volume via evaporation. Appropriate volumes of ethanol were added to the concentrated solution to give a final ethanol concentration of 80%. The mixture was stored for 12.0 h under an ambient atmosphere and was collected by filtration. The filtrate was concentrated to 1.0 g/mL under vacuum at 40 °C and stored at 4 °C for further experiments.SAD was extracted by heat reflux . Briefly3+ + Fe2+ + OH− → Fe3O4 [3·6H2O  and FeCl2·4H2O  in 150 mL of distilled water. Oxygen was removed by a nitrogen stream for 1.0 h. An aliquot of 20 mL aqueous NaOH solution  was added to the mixture slowly and uniformly using a drop funnel. The solution was mechanically stirred for 1.0 h until the color turned black. The black solution was heated to 90 °C and then maintained at 90 °C for 1.0 h to ripen the particles. Decreasing the temperature to 25 °C, we collected the magnetic material using a magnet. The collected material was dispersed in 200 mL of 0.3 M sodium citrate solution and heated for 1.0 h at 80 °C. The solvent was removed after collecting the magnetic Fe3O4 FPs using a magnet. The Fe3O4 FPs were thoroughly rinsed with acetone and kept at 30 °C until all the solvent was removed. The dried Fe3O4 FPs were modified with aminopropyltriethoxysilane (APTES) to synthesize Fe3O4@NH2 FPs [Magnetic fine particles were synthesized by chemical coprecipitation based on the reaction of Fe → Fe3O4 . We dissβ2-AR was prepared and purified according to the methods in our previous work [E. coli BL21 (DE3)-pET32a-β2-AR) were incubated in 50 mL of Luria–Bertani medium containing 100 μg/mL penicillin. When the OD value reached 0.4–0.6, isopropyl β-d-thiogalactopyranoside was added to the medium at a final concentration of 2.0 mM to induce the expression of the receptor. Ni-chelated Sepharose high-performance affinity column and Quaternary Sepharose Fast Flow column were utilized to purify β2-AR by sequential. The fraction of interest was collected by eluting the Ni-chelated Sepharose high-performance affinity column using 50% phosphate buffer . The purification of the collected fraction was performed on the Quaternary Sepharose Fast Flow column. The elution containing β2-AR was collected from the column by 20 mM phosphate buffer  with gradient elution ranging from 18 to 50%. The purity of β2-AR was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The fraction with greater than 90% purity was used for further investigation  and gently shaken for 30 min. The unbound salbutamol was discarded after collecting the fine particles using a magnet. The result Fe3O4@β2-AR/salbutamol FPs were rinsed twice with 1.0 mL ammonium acetate buffer  and incubated with 100 μL competitive agent  for 30 min to release the bound salbutamol [3O4@NH2 FPs, The drug concentration in the collected solution was determined by HPLC using an Agilent C8 column  with a mobile phase of 10% (v/v) methanol/water containing 0.1% (v/v) formic acid, a flow rate of 0.6 mL/min and a detection wavelength of 276 nm.Salbutamol and terbutaline are both specific agonists of lbutamol . The sup3O4@β2-AR FPs was suspended in 200 μL of 0.2 g/mL SAD extract. This suspension was gently shaken for 30 min at room temperature. Following the removal of solvent, we washed the fine particles twice using 1.0 mL of 10 mM ammonium acetate. Bioactive compounds bound to the immobilized receptor were collected by treating the cleaned fine particles with 100 μL terbutaline (0.01 mg/mL) as a competitor. Terbutaline, a strong β2-AR binder, has capacity to target all the three types of binding sites of β2-AR [An aliquot of 50 mg Feof β2-AR . As a re18 column  was used with 40% (v/v) methanol/0.1% (v/v) formic acid as mobile phase. The flow rate was 0.6 mL/min and the column temperature was 30 °C. The nebulizing gas pressure was set at 50 psi. The flow rate and temperature of the dry gas were 10.0 L/min and 350 °C, respectively. Mass spectra were acquired in negative mode with scan range of 50–1000 amu.The released bioactive compounds were further separated and identified by HPLC–MS/MS with electrospray ionization (HPLC–ESI–MS/MS). An Agilent TC-Cβ2-AR was constructed by homologous modeling according to a previous work that utilized the methylated β2-AR–Fab complex (PDB ID: 3KJ6) as the template. The structures of vicenin, shaftoside, isoshaftoside, liquiritin apioside and isoliquiritin apioside were created by ChemDraw Ultra 8.0 software , and was converted to pdbqt format using Chem3D Ultra 8.0 software  followed by MM2 energy minimization. Diverse receptor–ligand complexations were generated by AutoDock 4.2 which was downloaded from the Scripps Research Institute website . A grid box size of 62 × 56 × 50 points in the x, y and z directions was built to locate the receptor ligand binding domain. The grid center was set at x = 0.644, y = − 2.121 and z = − 7.094, which was large enough to accommodate the three known types of binding sites in β2-AR.The crystal structure of 3O4@NH2 FPs and Fe3O4@β2-AR FPs were analyzed by DLS with intensity-weighted size distributions  after complete rinse of the particles. Figure 3O4, sodium citrate solution-modified Fe3O4, APTES-modified Fe3O4 and Fe3O4@β2-AR FPs. The FT-IR adsorption band at 575 cm−1 corresponded to Fe–O bond vibrations in iron oxide. The strong absorption band at 3417 cm−1 was produced by O–H bond stretching on the Fe3O4 surface. The absorption peak at 1396 cm−1 was the typical absorption peak of carboxylate, providing an evidence for the presence of –COOH on the Fe3O4 surface. Taking together, the citrate modification of Fe3O4 was occurred through chemical reaction rather than physical interactions. The spectrum of Fe3O4-APTES FPs displayed two broad bands at 3441 cm−1 and 1630 cm−1 due to N–H stretching and NH2 bending modes of free NH2 group. The strong absorbance observed at 3441 cm−1 was attributed to hydrogen bond formation. These results demonstrated that the immobilization of β2-AR was accomplished by covalent reaction rather than physical absorption.To determine whether the immobilization of 3O4@β2-AR FPs was evaluated using salbutamol as a probe. A denaturing buffer solution  was used to release the bound drug from the particles. The amounts of salbutamol adsorbed on the control and Fe3O4@β2-AR NPs surfaces were determined to be 3 ± 1 and 52 ± 2 ng, respectively. The adsorption of metoprolol and prazosin  on Fe3O4@β2-AR FPs was examined under the same conditions. The amounts of the two antagonists on the Fe3O4@β2-AR were 2 ± 1 and 3 ± 1 ng. On the basis of the pharmacological properties of the drugs, we concluded that the Fe3O4@β2-AR FPs have the bioactivity of recognizing specific ligands of the immobilized receptor. The stability of the immobilized β2-AR was investigated by measuring the amount of adsorbed salbutamol over 2 weeks. The relative standard deviation (RSD) of the content of the absorbed salbutamol was 2.1%  to remove non-specific binders on the immobilized receptor. In the final step, we incubated the suspension of the clean particles with 2.0 μg/mL terbutaline for 30 min. This aimed to release the bound ligand from the receptor by competitive displacement. Figure 3O4@β2-AR FPs. Compared with the original solution, a clear loss of salbutamol was observed in the supernatant after the incubation with Fe3O4@β2-AR FPs. This loss raised from the specific binding of potential ligand onto the immobilized receptor. Figure 3O4@β2-AR FPs. The control particles gave no peaks on the chromatograms, while the Fe3O4@β2-AR FPs resulted in a weak peak after the same rinsing treatment to control particles. The presence of this peak is expected because there will be a continuous ligand-dissociation/ligand-association from/with the ligand–protein complex at equilibrium [3O4@β2-AR FPs with terbutaline (competitive agent) solution. Two intensive peaks were observed at 12.2 min and 13.1 min in the chromatogram. Using the reference standards, we identified the two peaks as salbutamol and terbutaline. This result indicated that terbutaline has the capacity to competitively displace the bound salbutamol on the particles. The method is feasible to be utilized for screening potential ligand and simultaneously probing the exact binding sites of the ligand on the receptor.Competitive experiments were performed to prevent false positives which were generated by non-specific binding. Salbutamol was used as a ligand while terbutaline served as a competitive agent. The competitive displacement procedure consisted of three steps: loading, washing, and displacing. In the loading step, we added 200 μL salbutamol (10 μg/mL) to the suspension of Feilibrium . As antiβ2-AR were illustrated in Fig. 3O4@β2-AR FPs with SAD extract. No other peaks in SAD extract were detected in the same sample. These results demonstrated the immobilized receptor has the specificity to capture its ligands from complex matrices. The five peaks were identified by MS/MS analysis and the comparison with mass spectrometric behaviors of reference standards 256 were found to be the main binding sites of vicenin, shaftoside, isoshaftoside, liquiritin apioside and isoliquiritin apioside to β2-AR. The driving force of this interaction was the formation of hydrogen bonds between the receptor and the drug.The interaction between the five compounds and β2-AR was first immobilized onto Fe3O4@NH2 FPs to achieve an affinity selection supporter. Owning to the large surface area and high biocompatibility of the particles, this immobilization improved the stability of β2-AR. The Fe3O4@β2-AR FPs were used to select bioactive compounds binding to the receptor in a complex prescription. The bound compounds were released from the receptor by competitive displacement using a specific agonist. Finally, HPLC–MS/MS was used to identify the compounds that were released from the Fe3O4@β2-AR FPs. Such procedure is attractive since the recognition and isolation of the target compounds are achieved at the same time.This was aimed to develop a rapid methodology that enables us to screen the ligands of GPCRs. Development of such methodologies is challenging due to the instability of the receptors when they were removed from their native environment. As illustrated in the scheme of Fig. β2-AR demonstrated that both the types of particles displayed a relatively uniform size distribution. Compared with Fe3O4@NH2 FPs, the Fe3O4@β2-AR FPs produced a size growth of 41.6 nm. We attributed this growth to the immobilization of β2-AR based on the size of the receptor [3O4 FPs was determined to be 384 ± 6.94 μmol/g by acid–base titration. After amino group modification, the hydroxyl content decreased to 43.18 ± 3.21 μmol/g due to reversible Schiff base formation, confirming the covalent modification of Fe3O4 FPs by APTES. This change became more significant when APTES-modified FPs were treated with β2-AR, providing a proof of chemical immobilization of the receptor on the particle surface. The molecular weight of the purified protein was determined to be 66.5 kDa by SDS-PAGE. According to this result, we identified the purified protein to be β2-AR. The quantity of immobilized β2-AR was determined by the bicinchoninic acid assay. A calibration curve with a regression equation of y = (0.825 ± 0.025)x − (0.0127 ± 0.002) and a correlation coefficient of 0.996 was plotted using bovine serum albumin as the reference standard. Using this curve, the amount of β2-AR immobilized on the particle surface was determined to be 8.439 ± 0.784 nmol/g. Compared with the immobilization using silica gel as a supporter [Morphological characterization of the immobilized receptor . The hydupporter , we decl3O4@β2-AR FPs two times with ammonium acetate was optimal during the washing step. This buffer was compatible with the receptor, and was effective to remove nonspecifically bound moieties and remain high speed without false positive and false negative results.Affinity magnetic materials are primarily limited by false positive and false negative results. False positives are caused by nonspecific ligand adsorption, while false negatives are caused by excessive washing steps or manipulation. Considering the mentioned competitive displacement results, we concluded that flushing ligand- Feβ2-AR is the main drug target [β2-AR signal pathway. Molecular docking results aligned with a previous report states that three sites contribute to the binding of an agonist to β2-AR. The sites included an aspartate residue on the third domain, two serine residues on the fifth domain and two phenylalanine residues [3O4@β2-AR FPs in screening bioactive compounds of the receptor from complex matrices including traditional Chinese medicine.As a common prescription, SAD is mainly utilized for relieving cough and asthma symptoms in practice . Pharmacg target , 40. Theresidues . The agr3O4@β2-AR FPs to fish out bioactive compounds of the receptor from SAD. Bioactive compounds in SAD that targeted β2-AR were identified as vicenin, shaftoside, isoshaftoside, liquiritin apioside and isoliquiritin apioside by LC–MS/MS. The compound–receptor interactions occurred at Ser169, Ser170, and Phe256. These results indicated that the affinity magnetic particles have the ability to recognize and separate the target compounds plus the probe of their exact binding site on the receptor. This is probably constitute an effective and rapid method for separating and identifying ligands of GPCRs from complex system such as traditional Chinese medicine.In this work, we synthesized FeAdditional file 1. Minimum standards of reporting checklist.


P < 0.05) increased the levels of ROS, OHdG, Ar accumulation, and lipid peroxidation, and decreased levels of enzymatic and nonenzymatic antioxidants. Notably, a significant (P < 0.05) increase was observed in markers of apoptosis, DNA damage, TUNEL-positive cells, and dark staining of ICAM-1 in renal tissue with decreased PI3K/Akt/Nrf2 gene expression. The biochemical findings were supported by histopathological and electron microscopy evaluation, which showed severe renal damage in rats treated with Ar. Pretreatment with SFN significantly (P < 0.05) attenuated renal ROS, OHdG, lipid peroxidation, and DNA damage, and increased phase II antioxidants via PI3K/Akt-mediated Nrf2 activation in renal tissue. These results show that dietary supplementation with SFN protects against Ar-induced nephrotoxicity via the PI3K/Akt-mediated Nrf2 signaling pathway in the rat kidney.Oxidative stress plays a significant role in the pathophysiology of numerous kidney diseases, generally mediated by reactive oxygen species (ROS). Arsenic (Ar) is known to exert its toxicity through the generation of ROS and inflammation. The current study investigates the protective effects of sulforaphane (SFN) against arsenic-induced renal damage via PI3K/Akt-mediated Nrf2 pathway signaling. Thirty-two male albino Wistar rats were randomly divided into four groups of eight animals each, designated as control, arsenic (Ar), sulforaphane plus Ar (SFN+Ar), and sulforaphane alone (SFN), with oral administration of Ar (5 mg/kg BW) and SFN (80 mg/kg BW) daily for 28 days. Ar administration significantly ( Arsenic (Ar) is a major toxicant that is ubiquitous in the environment, and a high level of exposure carries an increased risk of cancers of the skin, liver, lung, and kidney, and cardiovascular and reproductive abnormalities , 1,1,3,3-tetramethoxypropane, and bovine serum albumin were obtained from Sigma-Aldrich . Sulforaphane (SFN) was purchased from Sigma Chemicals . The solvents, chemicals, and other biochemicals were of certified analytical grade and procured from SD Fine chemicals  or HiMedia Laboratories (Mumbai). Reagent and kits for this study were obtained from Span Diagnostics, Mumbai, India. Antibodies including caspase-3, caspase-9, cytochrome C, Nrf2, PI3, Akt, and β-actin were purchased from Cell Signaling Technology  and Santa Cruz Biotechnology .Sodium arsenite  and humidity with an alternating light and dark cycle. The animals were fed with commercially available pelleted rat chow  and water ad libitum. After a week of acclimatization, rats were divided into control and test groups. Eight rats were used in each treatment group. The study was approved by the Institutional Animal Ethics Committee and carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Bharathiar University (ref. no BU/IAEC/2017/03), Tamilnadu, India.2 (sodium arsenite) was administered intragastrically at a dose of 5 mg/kg body weight each day for 4 weeks, which is one-eighth of the oral LD50 values in rats Group 2: administration of arsenic alone (5 mg/kg BW)Group 3: treatment with SFN + Ar An initial investigation was performed with three different doses of SFN  to determine the effective dose for Ar-treated rats. We found that SFN 80 mg/kg BW (body weight) notably reduced the levels of AST, ALT, LDH, and GGT in the serum of arsenic-treated rats in comparison to the other two doses (20 and 40 mg/kg BW). Therefore, we selected 80 mg/kg BW for this study. After an acclimatization period, the animals were divided into four groups of eight rats each Fig. , as follg for 10 min. The major plain supernatant was stored and utilized for various biochemical and molecular assays.The total study duration was 4 weeks. Water and food intake was documented regularly. At the end of the period (28 days), the rats were starved overnight and sacrificed under light ether anesthesia by cervical decapitation, and the abdomen was opened. The renal tissue was excised, weighed, and washed in ice-cold saline. The tissue was then pulverized and homogenized in Tris–HCl buffer and centrifuged at 8000×The amount of ROS in blood and renal tissue was quantified by 2′,7′-dichlorofluorescin diacetate (DCF-DA), as outlined previously . Creatinine clearance as an indicator of the glomerular filtration rate in serum was estimated from serum creatinine in a 24-h urine sample. Bilirubin in serum was calculated using a commercial kit  according to a previously described method Jung .The concentration of 8-OHdG in the urine and kidney was determined using a highly sensitive ELISA kit .The arsenic concentration in the kidney was calculated using a hydride vapor generation system (PerkinElmer MHS-10) fitted with an atomic absorption spectrophotometer , as described previously  and lipid hydroperoxides (LOOH), as reported previously (Niehaus and Samuelsson Reduction of glutathione (GSH) was performed using a previously described technique  level was estimated using the method described by Kakkar et al.  in whichRNA was isolated from rat kidney (100 mg) using TriZol Reagent  following using the PrimeScript™ RT reagent kit with gDNA Eraser , qPCR of kidney was carry out using SYBR Green reagent  on PCR detection system . Rat mRNA from kidney was isolated using TRI Reagent (Sigma-Aldrich) and assayed with TaqMan control reagents . The following primer pairs were used for this analysis: SOD1: forward: 5′ TAACTGAAGGCCAGCATGGG 3′; reverse: 5′ CATGGACCACCATTGTACGG3′; CAT: forward: 5′ CACTCAGGTGCGGACATTCT 3′; reverse: 5′ TCCGGAGTGGGAGAATCCAT3′; Bax: forward: 5′ GGATGGCTGGGGAGACACCTGAG3′; reverse: 5′ CGGCCCCAGTTGAAGTTGCCATCAG 3′; Bcl-2: forward:5′ TGAGAGCAACCGAACGCCCG3′; reverse:5 ′GCACCCAGAGTGATGCAGGCC 3′; β actin: forward: 5′ AGCCTTCC TTCTTGGGTATGGAATC 3′; reverse: 5′ GGAGCAATGATCTT GATCTTCATGG-3′. These primers were designed using Primer3 and synthesized by Integrated DNA Technologies, Inc. . All real-time PCR assays were performed in triplicate. Relative quantitative analysis was carried out by comparing threshold cycle numbers for target genes and a reference β-actin mRNA.w/v) skim milk powder in 1× TBS containing 0.1% Tween-20 for 1 h, then incubated with rabbit polyclonal antibodies for (proapoptotic markers) anti-caspase-3, anti-caspase-9, cytochrome-C, anti-PI3K, anti-Akt, anti-Nrf2, and β-actin overnight at 4 °C . Membranes were washed and incubated for 1 h at room temperature with HRP-linked secondary antibodies. After washing, membranes were identified by enhanced chemiluminescence (ECL) kit and exposed to X-ray films . The band intensities were quantified by densitometry analysis using an image analysis system . The marks were stabilized to the β-actin appearance in each group (mean ± SD) as a percentage of control. No differences were observed in β-actin manifestation between the tentative animal groups.The kidney tissue (100 mg) was prepared by homogenization in RIPA lysis buffer  containing complete EDTA-free protease inhibitor cocktail . The protein was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with blocking buffer  was performed by terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick-end labeling (TUNEL) assay according to the instructions of the manufacturer  and the number of apoptotic cells, as defined by chromatin condensation of nuclear fragmentation (apoptotic bodies), was counted. After TUNEL labelling, nuclei were labeled with Hoechst 33258  and the TUNEL-positive kidney cells were observed using an Olympus IX81 fluorescence microscope with a 20× objective at 2048 × 2048 resolution. The morphometric examination was performed by two independent, blinded investigators. The average number of apoptotic cells in each group was calculated by taking the average of TUNEL-positive apoptotic cells in 10 fields from each kidney sample with 200× magnification.Kidney tissues (100 mg) were fixed in 4% buffered formalin. Paraffin-embedded sections were cut, deparaffinized, and hydrated in descending gradations of ethanol, followed by antigen retrieval with citrate buffer. Next, sections were incubated in 0.3% H2O2 in PBS to block endogenous peroxidase activity. The sections were then incubated with anti-intercellular adhesion molecule 1 (ICAM-1)  overnight at 4 °C in a moist chamber. Biotinylated secondary antibodies and ABC reagent were added according to manufacturer instructions . Color development was induced by incubation with a DAB kit (Vector laboratories) for 3–5 min, and specific staining was visualized by light microscopy, as described by Pan et al. . After IFor the histopathology investigation, the kidney tissues were dissected and fixed in Bouin’s solution for 14–18 h, processed in a series of graded ethanol to xylene, and embedded in paraffin wax. The paraffin sections were cut at 5–6 μm thickness using a rotary microtome and stained with hematoxylin and eosin for light microscopy examination. The slices were viewed and photographed under an Olympus BX51 light microscope  with an attached digital camera .For electron microscopy, kidney tissues were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4 °C, washed in the same buffer for 1 h at 4 °C, and post-fixed with 1% osmium tetroxide in sodium phosphate buffer for 1 h at 4 °C. The tissues were then dehydrated in a graded series of ethanol starting at 50% each step for 10 min and after two changes in propylene oxide. The tissue specimens were embedded in araldite. Ultrathin sections were prepared with magnesium uranyl acetate and lead citrate for the electron microscopic (Jeol JEM-1010) evaluation.p < 0.05 were regarded as statistically significant.Data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) using a commercially available statistics software package . Results are expressed as mean ± SD. Values of P < 0.05) with increased organ weight ratio when compared to the control group. However, pretreatment with SFN significantly (P < 0.05) reduced food and water intake, body weight, and organ weight ratio as compared with the Ar-treated group. Administration of SFN alone had no effect on those parameters in the control group.The specific indicators of oxidative stress include body weight, organ weight, and food and water intake. Table P < 0.05) increased ROS in blood  decrease in levels of ROS and Ar accumulation as compared to the control and Ar groups. Administration of SFN alone produced no change in ROS levels compared to normal control rats.The generation of ROS is the primary pathogenic mechanism of Ar-induced oxidative damage to cellular macromolecules. Arsenic treatment of rats resulted in significantly  improved kidney serum parameters  as compared to control and SFN groups. Prior administration of SFN led to a significant (P < 0.05) decrease in the above estimated parameters as compared to the Ar group. No statistically significant difference was observed between the group receiving SFN alone and the control group.The levels of urea, uric acid, creatinine, and bilirubin were determined under Ar and SFN treatment in kidney serum samples depicted in Table P < 0.05) increase in renal 8-OHdG in urine and kidney tissue after Ar exposure when compared to controls. Interestingly, SFN pre-administration significantly (P < 0.05) reduced 8-OHdG in urine and kidney tissue as compared to that of control and SFN groups.To investigate how SFN prevents Ar-induced renal damage, 8-OHdG, as a hallmark DNA marker, was measured in control and experimental animals Fig. . There wP < 0.05) increased compared with the control group, while SFN pretreatment significantly (P < 0.05) decreased these lipid peroxidation markers in comparison with the Ar group. Rats treated with SFN alone exhibited no significant differences in lipid oxidation markers compared with controls.Lipid peroxidation is the oxidative degradation of lipids in which free radicals steal electrons from the normal cells. In the Ar-treated group, lipid peroxidation Fig.  markers P < 0.05) lower in the arsenic-treated group than the control group. In the group with SFN pre-administration, however, levels of nonenzymatic antioxidants were significantly (P < 0.05) higher than in the Ar-treated group. The SFN and control groups showed similar activity.Table P < 0.05) decreased with Ar administration compared to controls. SFN pre-administration in Ar-treated rats significantly (P < 0.05) increased the levels of enzymatic antioxidants compared with controls and rats treated with Ar alone.The effect of Ar and SFN on the enzymatic antioxidant levels in the kidney was determined, as shown in Table P < 0.05) decrease in mRNA including SOD  increase in SOD  up-regulated in Ar-affected rats when compared with controls. Co-administration of SFN and Ar resulted in significantly (P < 0.05) down-regulated expression of those apoptotic markers when compared to rats treated with Ar alone.Figure P < 0.05) decreased with respect to the decrease in Nrf2 gene expression when compared with controls. Densitometric analysis  up-regulated Nrf2 gene expression in Ar-treated rats after SFN exposure. Rats treated with SFN alone also showed significantly increased levels of PI3K when compared to the Ar-treated group.The effect of SFN on PI3K/Akt-mediated Nrf2 activation in the kidney of control and Ar-treated rats is presented in Fig. sis Fig. b of westP < 0.05) increased in the kidneys of Ar-treated rats  prevented Ar-induced enhanced cell death  increased the expression of kidney inflammatory intercellular adhesion molecule (ICAM)  attenuated by SFN pretreatment  Fig. b and c cAM) Fig. a. This ient Fig. d. SFN alent Fig. e.Fig. 9EFigure Figure Arsenic (Ar) is known to induce oxidative nephropathy via production of reactive oxygen species (ROS), reactive nitrogen species (RNS), and dimethyl arsenic peroxyl radicals Flora . Ar is aAr is known to inhibit growth by interference with a number of metabolic processes (Tu and Ma 2) in the kidney. The O2 is unstable and readily accepts a second electron to form H2O2, and at the same time disturbing a large amount of an important cellular waste. Pre-administration of SFN drastically diminished ROS on average in Ar-exposed rats. This is likely explained by the successful free radical scavenging and potent hydrogen-donating properties of SFN, which minimize the inflammatory process.It has long been believed that ROS are involved in several pathogenic infection-linked inflammatory process  is one of the predominant forms of free radical-induced oxidative lesions, and has therefore been widely used as a biomarker for oxidative stress and carcinogenesis and as an indicator of oxidative DNA damage promoted by different xenobiotics in the kidney  that possess reactive hydrogen atoms  is the process in which free radicals steal electrons from the lipids in cell membranes, resulting in cell damage. It typically affects polyunsaturated fatty acids, since they contain multiple double bonds, between which are methylene bridges  of the glomerulus that mediate the efflux Ar to be excreted by urine  are a family of phase II detoxifying enzymes that shield cellular macromolecules from unstable electrophiles . In particular, GSTs influence the conjugation of glutathione (GSH) to a wide array of internal and external electrophilic complexes. Glutathione reductase (GR) can act as a scavenger for singlet oxygen in various electrophiles, which accelerates the reduction of GSSG to GSH. In the current study, Ar administration significantly reduced enzymatic antioxidants such as SOD, CAT, GPx, GST, and GR in comparison to controls. This result was well in line with an earlier report by Jain et al.  leading to tubular cell death  play essential role in apoptosis and inflammation. Cytochrome C aids in the activation of Apaf-1 and caspase-9 in the apoptosome. Once activated, caspase-9 cleaves and activates caspase-3 and caspase-7, ensuing in extensive proteolysis and cell death /Akt pathway is involved in cell survival, proliferation, and metabolism, and is commonly associated with harmful modification processes in a variety of cancers, including kidney  assay as a technique for identifying apoptotic DNA fragmentation, quantifying apoptotic cells, or detecting excessive DNA breakage in individual cells Dong . It has Inflammation is part of a complex biological response of body tissues. Ar exposure has been found to be an important cause of renal failure associated with oxidative stress, proximal tubular dysfunction, and necrosis of proximal tubular epithelial cells in the kidneys of rats (Li et al. In the present study, histopathological examination of Ar-treated rats revealed alterations in renal tissue including tubular necrosis, inflammatory cell infiltration, tubular degeneration, hemorrhage, and vacuolization. Similarly, electron microscopy study confirmed Ar-induced degeneration of podocytes including dilated cisternae of the endoplasmic reticulum, mitochondrial swelling, and degeneration of the nucleolus. This may be due to the accumulation of free radicals and increased lipid peroxidation by activated free Ar ions in the Ar-intoxicated animals. The increased generation of lipid peroxides associated with ROS leads to loss of membrane integrity and other pathological changes including nucleolus disintegration in kidney tissue as confirmed by electron microscopy study. Administration of SFN reduced Ar-induced histopathological and TEM alterations quite substantially in both organs and tissues via its antioxidant and membrane-stabilizing effects in rat kidneys.In summary, SFN showed a variety of beneficial properties, including free radical scavenging, anti-inflammatory, membrane-stabilizing, and antiapoptotic activity as possible key factors in the nephroprotective efficacy against Ar-induced oxidative renal injury. Ultimately, SFN was effective in preventing Ar-induced nephrotoxicity by activating phase II antioxidant enzymes via modulation of Nrf2 gene expression through the PI3K/Akt signaling pathway, and thus may prove beneficial in patients at high risk of arsenic toxicity Fig. . Further


Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leishmania donovani parasites in vitro. However, compounds with nitrogen modification at the 10-position on the phenoxazine ring were not evaluated.Basic blue 3 is a promising anti-malarial lead compound based on the π-delocalized lipophilic cation hypothesis. Its derivatives with nitrogen atoms bonded to carbon atoms at the 3- and 7-positions on the phenoxazine ring were previously shown to exert potent antiprotozoal activity against Six acylphenoxazine derivatives (ITT-001 to 006) with nitrogen modification at the 10-position on the phenoxazine ring, which were synthesized from basic blue 3, were characterized and evaluated for anti-malarial activity in vitro with an automated haematology analyzer (XN-30) and light microscopy. Intensity of self-fluorescence was measured using a fluorometer. Localization of basic blue 3 was observed by fluorescence microscopy. Cytotoxicity was evaluated using human cell lines, HEK293T and HepG2 cells. Finally, anti-malarial activity was evaluated in a rodent malaria model.Plasmodium berghei NK65 strain.All the six derivatives showed anti-malarial efficacy even against chloroquine-, pyrimethamine-, and artemisinin-resistant field isolates similar to the sensitive strains and isolates in vitro. The efficacy of basic blue 3 was the strongest, followed by that of ITT-001 to 004 and 006, while that of ITT-005 was the weakest. Basic blue 3 showed strong self-fluorescence, whereas ITT derivatives had five- to tenfold lower intensity than that of basic blue 3, which was shown by fluorescence microscopy to be selectively accumulated in the plasmodial cytoplasm. In contrast, ITT-003, 004, and 006 exhibited the lowest cytotoxicity in HEK293T and HepG2 cells in vitro and the highest selectivity between anti-malarial activity and cytotoxicity. The in vivo anti-malarial assay indicated that oral administration of ITT-004 was the most effective against the rodent malaria parasite, The six ITT derivatives were effective against chloroquine- and pyrimethamine-resistant strains and artemisinin-resistant field isolates as well as the sensitive ones. Among them, ITT-004, which had high anti-malarial activity and low cytotoxicity in vitro and in vivo, is a promising anti-malarial lead compound.The online version of this article (10.1186/s12936-019-2873-0) contains supplementary material, which is available to authorized users. Plasmodium falciparum parasite is an obstacle in the fight against malaria phenoxazine (SSJ-183) was developed and its high anti-malarial efficacy was demonstrated in vitro and in vivo + Calcd for C22H29N3NaO2 390.2158; Found 390.2163  in CHCl−1; 1H-NMR  δ 7.35 , 6.42 , 6.35 , 3.31 , 1.20 , 1.14 ; 13C-NMR  δ 178.8, 153.5 (2), 146.7 (2), 125.8 (2), 121.0 (2), 105.8 (2), 99.6 (2), 44.4 (4), 41.3, 29.2 (3), 12.4 (4); HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C25H35N3NaO2 432.2627; Found 432.2627 , followed by acylation of the resulting phenoxazine with pivaloyl chloride  was carried out according to the same procedure as described for ITT-001 to give acylphenoxazine ITT-002  as a green solid. [eluent: hexane-AcOEt ]; Mp 96.1–97.3 °C; IR (KBr) ν max 2966, 1652, 1507 cm−1; 1H-NMR  δ 7.42 , 6.40 , 6.37 , 3.32 , 2.21-2.09 , 1.15 , 1.18-1.13 , 0.84-0.77 ; 13C-NMR  δ 172.1, 151.8 (2) 146.4 (2), 125.1 (2), 118.2 (2), 106.0 (2), 99.5 (2), 44.4 (4), 12.4, 12.3 (4), 9.1 (2); HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C24H31N3NaO2 416.2314; Found 416.2342 , followed by acylation of the resulting phenoxazine with cyclopropanoyl chloride  was carried out according to the same procedure as described for ITT-001 to give acylphenoxazine ITT-003  as a gray solid. [eluent: hexane-AcOEt ]; Mp 37.7–39.5 °C; IR (KBr) ν max 2969, 1659, 1507 cm−1; 1H-NMR  δ 7.35 , 7.26-7.17 , 7.05 , 6.33 , 6.11 , 3.21 , 1.05 ; 13C-NMR  δ 167.4, 151.3 (2), 146.4 (2), 136.1, 129.7, 128.8 (2), 127.8 (2), 125.0 (2), 118.7 (2) 106.1 (2), 99.5 (2), 44.5 (4), 12.4 (4); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C27H32N3O2 430.2495; Found 430.2524 , followed by acylation of the resulting phenoxazine with benzoyl chloride  was carried out according to the same procedure as described for ITT-001 to give acylphenoxazine ITT-004  as a turquoise solid. [eluent: hexane-AcOEt ]; Mp 126.3–127.2 °C; IR (KBr) ν max 2966, 1644, 1507, 1340, 1288, 1241, 1203, 1115 cm3 (16.0 mL) and H2O (16.0 mL) were added 1.0 M NaOH (12.5 mL) and Na2S2O4  at ambient temperature. After stirring for 1.5 h at the same temperature, the reaction mixture was added a solution of chloroacetyl chloride  in CHCl3 (24.0 mL). After stirring for 4 h at the same temperature, the reaction mixture was quenched with H2O, and extracted with AcOEt. The organic layers were washed with brine, and dried over Na2SO4. The volatile solvent was removed in vacuo, and the residue was used for the next step without further purification. To a solution of the obtained chloroacetylphenoxazine in THF (2 mL) was added Et2NH  and Bu4NI  at ambient temperature. After stirring for 16 h at 50 °C, the reaction was quenched with 2 M HCl. The aqueous layers were washed with AcOEt, and added 2 M NaOH to extract with AcOEt. The organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel using CHCl3-MeOH  as an eluent to give ITT-005 as a turquoise solid. To the solid was added MeOH–HCl, then concentrated in vacuo to give ITT-005·HCl  as a yellowish green solid.; Mp 117.5–119.7 °C; IR ν max 3435, 2676, 2597, 1685, 1503 cm−1; 1H-NMR  δ 7.35 , 6.54-6.45 , 3.41 , 3.11 , 1.23 , 1.18 ; 13C-NMR  δ 167.6, 154.5 (2), 149.9 (2), 126.9 (2), 108.5 (2), 1006 (2), 55.9, 51.5 (2), 46.5 (4), 13.8 (4), 11.2 (2).; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C26H39N4O2 439.3073; Found 439.3102  in CHCl3 6.0 mL an−1; 1H-NMR  δ 7.42 , 7.11 , 6.94 , 6.41 , 6.21 , 3.31 , 1.38 ; 13C-NMR  δ 166.3, 151.3 (2), 146.5 (2), 132.7, 131.2 (2), 125.0 (2), 118.6 (2), 115.6, 114.9 (2), 106.2 (2), 99.6 (2), 44.5 (4) 12.4 (4); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C27H31FN3O2 448.2400; Found 448.2422 , followed by acylation of the resulting phenoxazine with 4-fluorobenzoyl chloride  was carried out according to the same procedure as described for ITT-001 to give acylphenoxazine ITT-006  as a green solid. [eluent: hexane-AcOEt ]; Mp 148.9–150.8 °C; IR (KBr) ν max 2968, 1664, 1508 cmBasic blue 3 and ITT-001 to 006 were synthesized as described above. ART and chloroquine (CQ) were purchased from TCI . CQ was dissolved in saline, while all other chemicals were dissolved in dimethyl sulfoxide (DMSO)  to obtain 10 mM stock solution. All solutions were stored at − 30 °C and thawed immediately prior to experimentation. The stock solutions were subjected to a maximum of five freeze–thaw cycles before being discarded.Plasmodium falciparum laboratory strains, 3D7 [CQ- and pyrimethamine (PYR)-sensitive] and W2 (CQ- and PYR-resistant) were obtained from Prof. Masatsugu Kimura  and the Malaria Research and Reference Reagent Resource (MR4) as part of the Biodefense and Emerging Infections (BEI) Resources, respectively. The field isolates, MRA-1239 and MRA-1240 , were obtained from MR4, BEI Resources. For the assessment of anti-malarial activity of the compounds in vitro, the parasites were cultured in Roswell Park Memorial institute (RPMI) 1640 medium supplemented with 0.5 g/L l-glutamine, 5.96 g/L HEPES, 2 g/L sodium bicarbonate (NaHCO3), 50 mg/L hypoxanthine, 10 mg/L gentamicin, 10% heat-inactivated human serum, and red blood cells (RBCs) at a 3% haematocrit in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 37 °C as previously described  and specific reagents were used    was calculated using the Microsoft Excel program . The data were graphically displayed and ICCA, USA) .Fluorescence spectrum of basic blue 3 and ITT-001 to 006 (10 µM) was obtained in saline containing 1% DMSO at room temperature. The compounds were excited at 592 nm  and their emission spectra, 630 nm to 730 nm, were measured with a RF-5300PC Spectro fluorophotometer (Shimadzu).g for 5 min at room temperature and treated with 15 nM basic blue 3 in HBSS containing calcium and magnesium for 15 min under standard culture conditions. Simultaneously, DNA was stained with 50 µg/mL Hoechst 33342 (Dojindo) for 5 min. The parasites were washed with HBSS containing calcium and magnesium twice. Images were captured using a BZ-X710 fluorescence microscope . A BZ-X TexasRed filter  was used for basic blue 3.Non-synchronized parasites were washed with Hanks’ balanced salt solution (HBSS) containing calcium and magnesium  twice by centrifugation at 840l-glutamine and sodium pyruvate; Nacalai Tesque, Kyoto, Japan) supplemented with 10% (v/v) fetal bovine serum  in a humidified incubator with 5% CO2 at 37 °C. For the cytotoxic assay, the cells (5 × 103/well) were seeded in a 96-well plate. Basic blue 3, ITT-001 to 006, and CQ at gradually decreasing concentrations  were added to the cell culture after 24 h and the cells were subsequently cultured for 48 h. Cell viability was measured using a Cell Counting Kit-8 (Dojindo) according to the manufacturer’s instructions. Briefly, 10 µL CCK-8 reagent was added to each well containing culture medium and incubated for 2 h under standard culture conditions. The absorbance of the sample was measured at 450 nm using a PowerWave HT microplate spectrophotometer . The cell viability was expressed as a percentage of the absorbance of the untreated control cells after subtracting the appropriate background intensity. The CC50 (mean ± SEM) was calculated as described in IC50. The SI, which is the ratio between the cytotoxicity and anti-malarial activity, was calculated according to the following equation.HepG2 (JCRB1054) and HEK293T cell lines were obtained from the Japanese Collection of Research Bioresources  and Prof. Yoshiharu Matsuura  , respectP. berghei NK65 as described by Fidock et al. [7 iRBCs prepared from the pre-infected mice was injected intravenously into the mice. After 4 h, basic blue 3 and ITT-001 to 006 dissolved or suspended in 5% (w/v) methyl cellulose 400 solution  were orally administered (100 mg/kg body weight of basic blue 3 and ITT-001 to 006 and 50 mg/kg body weight of ITT-004) to the mice . The control group (five mice/group) was treated with approximately 100 µL of the vehicle. This treatment was repeated for 3 consecutive days. On day 4, peripheral blood samples were obtained from the tail veins of the mice and thin blood smears stained with Diff-Quik stain kit (Sysmex) were observed under a Leica DM6000B microscope . Parasitaemia was determined by counting over 10 fields of view. The suppression rate (%) was calculated as the difference in parasitaemia between the test and control groups. The mean survival day (MSD) was calculated from the number of the surviving mice on day 30. The surviving mice were cured on day 30. Kaplan–Meier curves were generated to graphically display the efficacy using GraphPad Prism version 5.0.The in vivo anti-malarial efficacy of ITT-001 to 006 was evaluated using the rodent malaria parasite k et al. , with sot test. The significance limit was set at P < 0.05 and calculations were performed using the GraphPad Prism version 5.0.The statistical significance was calculated using a two-tailed unpaired Student’s To characterize the effects of the modification of the nitrogen atom at the 10-position on the phenoxazine ring of basic blue 3 [Fig. 50 = approximately 10 nM) was lower than that of basic blue 3 (IC50 = approximately 4 nM); the IC50 of ITT-005 (about 40 nM) was approximately tenfold higher than that of basic blue 3. The efficacy of these compounds was comparable against 3D7 and W2 strains, although CQ significantly affected the 3D7 strain more than the W2 strain (Table For both 3D7 (CQ- and PYR-sensitive) and W2 (CQ- and PYR-resistant) strains, the in vitro anti-malarial assay revealed that the efficacy of ITT-001 to 004 and 006  = 0.021 and 1.0 µM for HEK293T and HepG2 cells, respectively), whereas ITT-003, 004, and 006 had the lowest cytotoxicity . The selective index (SI) indicated that ITT-003, 004, and 006 showed the highest effectiveness on the parasites and were the safest for both cultured cells in vitro  cytotoxic concentration  and prevent ATP synthesis . Both fito phase –33. In aovaquone . These fP. berghei parasite infection, with 4 out of 5 animals cured on day 30  in Fig.


AR and CgA. AR aberrations were analyzed by droplet digital PCR in pre-treatment plasma samples collected from two biomarker protocols [197 patients from a retrospective study (REC 2192/2013) and 59 from a prospective trial (REC 6798/2015)]. We subdivided patients into three groups according to CgA by receiver-operating characteristic (ROC) curves. In the primary cohort, plasma AR gain and mutations (p.L702H/p.T878A) were detected in 78 (39.6%) and 16 (8.1%) patients, respectively. We observed a significantly worse PFS/OS in patients with higher-CgA than in patients with normal-CgA, especially those with no AR-aberrations. Multivariable analysis showed AR gain, higher-CgA and LDH levels as independent predictors of PFS [hazard ratio (HR) = 2.16, 95% confidence interval (95% CI) 1.50–3.12, p < 0.0001, HR = 1.73, 95% CI 1.06–2.84, p = 0.026, and HR = 2.13, 95% CI 1.45–3.13, p = 0.0001, respectively) and OS . These data were confirmed in the secondary cohort. Pre-treatment CgA detection could be useful to identify these mixed tumors and would seem to have a prognostic role, especially in AR-normal patients. This association needs further evaluation in larger prospective cohorts.Recently, mixed forms between adenocarcinoma and neuroendocrine prostate cancer (NEPC) have emerged that are characterized by persistent androgen receptor (AR)-signalling and elevated chromogranin A (CgA) levels. The main aim of this study was to analyze castration-resistant prostate cancer (CRPC) patients treated with abiraterone or enzalutamide, assessing progression-free/overall survival (PFS/OS) in association with circulating  After an initial response to medical or surgical castration, the disease will progress to castration-resistant prostate cancer (CRPC)2. Many drugs with different mechanisms of action are currently available for CRPC patients and have led to a significant benefit in survival8. However, the identification of predictive factors could help clinicians to better select the next-line systemic therapy on the basis of molecular risk stratification. The implementation of these biomarkers in routine clinical practice would substantially facilitate patient-tailored treatment.Prostate cancer is the most commonly diagnosed cancer in men worldwide and a major cause of cancer death11. Resistance in CRPC is typically driven by the reactivation of androgen receptor (AR) signalling, occuring through AR splice variants (such as AR-V7), and AR point mutations or amplification, which can be detected in blood20. In recent years, cell-free DNA has emerged as a promising and non-invasive biomarker for the characterization of the tumor genome21. Approximately 3% of tumor DNA is released into the blood daily, most likely deriving from apoptosis/necrosis of malignant cells22. High levels of cell-free DNA  have been detected in patients with tumors, including prostate cancer22. Recently, AR gene aberrations detected in cell-free DNA of CRPC patients using PCR-based methods or next generation sequencing have been associated with worse outcome and resistance to abiraterone and enzalutamide, suggesting a potential predictive/prognostic role for plasma AR status17.The molecular landscape of CRPC provides insights into the mechanisms of tumor heterogeneity and treatment resistanceAR-independent mechanism of resistance to cancer therapeutics, especially androgen deprivation agents. Neuroendocrine prostate cancer (NEPC) is uncommon and characterized by decreased prostate specific antigen (PSA) secretion, and usually by the expression of neuroendocrine markers, such as chromogranin A (CgA), synaptophysin, and neuron specific enolase (NSE)24. In addition, NEPC usually shows loss of AR expression and suppressed AR signalling, while AR aberrations (point mutation and gain) are present at low levels, probably because of clonal selection of non-amplified prostate adenocarcinoma subpopulations through selective pressure, especially during anti-AR therapies24. De novo small-cell prostate cancer, a highly aggressive histologic variant, is present in <1% of untreated prostate cancers, whereas the frequency of treatment-related NEPC has been reported as occurring in up to 20% of patients during the course of CRPC progression25. A recent classification of neuroendocrine prostatic tumors26 showed variants of neuroendocrine prostate cancer, including a mixed form between NEPC and conventional adenocarcinoma, usually characterized by AR independence, However, mixed tumors have been also observed with both AR positive and AR negative cells and, on occasion, with dual expression of both neuroendocrine markers and AR in the same tumor cells due to inter- and intra-patient clinical and pathologic heterogeneity. Consequently, a more accurate clinical and molecular classification is needed for these overlapping clinical entities, and further research is warranted to identify their prognostic impact27.In CRPC, neuroendocrine differentiation (NED) represents an alternative 28 who show a shorter survival than those with normal CgA values30.In addition, elevated levels of serum CgA, commonly observed in NEPC, may increase in CRPC patients with adenocarcinoma histologyAR status and CgA level before the administration of anti-AR therapies and in different settings (chemotherapy-naïve and chemotherapy-treated patients) in prostate adenocarcinoma. We also evaluated the impact on treatment outcome of CgA levels in association with cell-free AR status.Our main objective was to identify the correlation between The primary cohort included 197 patients from a retrospective biomarker study (REC 2192/2013) and the secondary cohort consisted of 59 from a prospective biomarker trial (REC 6798/2015). Patients with available pre-treatment serum CgA and plasma DNA for detection of AR gene aberration were considered evaluable. All patients in both cohorts underwent prostate biopsy and/or prostatectomy at diagnosis with a confirmed histology of prostate adenocarcinoma without NED. Median age was 73 years  and 75  in the primary and secondary cohorts, respectively. The prospective biomarker trial was more recent than retrospective study and, consequently, included many more chemotherapy-naïve cases treated with abiraterone or enzalutamide  than the retrospective study . This substantial difference may justify the different baseline characteristics between the two cohorts .30. In the primary cohort, serum CgA level was considered normal in 92 (46.7%) patients, elevated but ≤ 3-fold the upper normal value (UNV) in 66 (33.5%) and ≥3-fold the UNV in 39 (20.3%) patients. A similar distribution of CgA levels was observed in the secondary cohort. We assessed AR status in cell-free DNA showing AR copy number (CN) gain in 78 (39.6%) and AR mutations (p.L702H or p.T878A) in 16 (8.1%) patients of the primary cohort. In the secondary cohort, plasma AR gain and mutations were reported in 11 (18.6%) and 2 (3.4%) patients, respectively. All patient characteristics are summarized in Table The median serum CgA level was 122 ng/mL  . However, the receiver-operating characteristic (ROC) analysis, one of the most commonly used methods to analyze the effectiveness of a diagnostic, was used to evaluate the role of pre-treatment serum CgA for assessing the response to no response to treatment with abiraterone or enzalutamide. The cutoffs for CgA in response to AR-directed therapies were calculated through the area under the ROC curve (AUC), as previously reportedAR status and CgA measurements, with the exception of a trend (p = 0.057) of higher CgA level in AR-gained chemotherapy-naïve patients of the secondary cohort , and our results did not reveal a significant difference between PSA and CgA levels (Supplementary Table AR copy number (CN) in the primary and secondary cohort  and 17.6 months , respectively.AR mutations on treatment outcome because cell-free AR mutations were uncommon, especially in chemotherapy-naïve patients, as shown in previous studies13, and the clinical relevance of rarer mutations is uncertain. Our droplet digital PCR (ddPCR) assay detected point mutations present in at least 2% of plasma DNA.We did not evaluate the impact of AR-normal patients treated with abiraterone or enzalutamide, more evident in post-docetaxel setting for the primary cohort or in pre-docetaxel for the secondary cohort  and elevated CgA levels in ort Figs  and S5.FAR gain and higher CgA and lactate dehydrogenase (LDH) levels as an independent predictors of PFS  were detected by multiplex ddPCR on a QX200 ddPCR system (Bio-Rad)14.All patients underwent a history evaluation and physical examination, and blood tests including complete blood cell count, serum PSA, alkaline phosphatase (ALP), and LDH the week before each treatment cycle. PSA and blood tests were performed on a monthly basis. Serum CgA levels were determined at baseline and upon disease progression. Radiographic evaluation was performed by computed tomography and bone scan at the time of screening and every 12 weeks thereafter. Response was evaluated according to Prostate Cancer Working Group (PCWG2) guidelinesAR CN analyses, we used the AR gene and at least two different reference genes: RNaseP, NSUN3, ElF2C1, and AP3B1 and ZXDB at Xp11.21 as a control gene.For AR aberrations and CgA levels. The secondary endpoints were PFS/OS and PSA response rate (RR) (>50% PSA decline after 12 weeks of treatment) stratified by circulating AR status and CgA level.The primary endpoint of this study was the association between circulating 29. Consequently, all patients were subdivided into three groups: (1) normal serum CgA level < 120 ng/mL, (2) < 3-fold the UNV 120–360 ng/mL, and (3) > 3-fold the UNV > 360 ng/mL). In the exploratory sub-analysis, we also used 5-fold the UNV to evaluate a potentially more stringent cut-off.Data were summarized by frequency for categorical variables and by median and range for continuous variables. The Wilcoxon rank sum test was performed to compare continuous variables and Chi-Square or Fisher’s exact test were performed to compare categorical variables, as appropriate. The cut-offs for CgA in response to therapies were determined through ROC curve analysis, as previously reportedAR CN, CgA level, chemotherapy status, number of previous treatment lines, Gleason score, site of metastasis, and baseline serum LDH and PSA levels). We also conducted landmark analysis to reduce the potential for time-dependent confounding in treatment by assessing the impact of changes in serum CgA level from baseline to progression on survival outcome.PSA response was considered as a ≥50% decline after a minimum of 12 weeks treatment confirmed by a second PSA test after a minimum of four weeks. PFS was calculated from the start of each therapy until the first date of progression, death from any cause, or last tumor evaluation. OS was calculated from the start of each therapy until death or last follow-up. Survival curves were estimated by the Kaplan-Meier method and were compared using the log-rank test. Univariate and multivariate Cox regression models were used to investigate potential predictors of PFS and OS and to estimate HR and their 95% CI. For these analyses, we included different clinically relevant factors as covariates for both cohorts .Dataset 1


AR pre-mRNA and the inclusion of a cryptic exon containing a premature stop codon in the mRNA. The resulting loss of the ligand-binding domain allows AR-V7 to act as a constitutively active transcription factor. Here, we designed two antisense oligonucleotides (AONs) directed against cryptic splicing signals within the AR pre-mRNA. These two AONs, AON-ISE and AON-ESE, demonstrated high efficiency in silencing AR-V7 splicing without affecting full-length AR expression. The subsequent downregulation of AR-V7-target gene UBE2C was accompanied by inhibition of androgen-independent cell proliferation and induction of apoptosis in castration-resistant prostate cancer (CRPC)-derived cell line models 22Rv1, DuCaP, and VCaP. Our results show that splicing-directed AONs can efficiently prevent expression of AR-V7, providing an attractive new therapeutic option for the treatment of CRPC.One of the mechanisms by which advanced prostate cancer develops resistance to androgen deprivation therapy is the elevated expression of C-terminally truncated androgen receptor (AR) variants. These variants, such as AR-V7, originate from aberrant splicing of the  AR-splice variant 7 (AR-V7) is the most commonly and abundantly expressed variant in human CRPC tissues [Despite the clinical remission achieved by androgen deprivation therapy, advanced prostate cancer eventually progresses into recurrent or castration-resistant prostate cancer (CRPC) . CRPC is tissues , 5. AR-V tissues –7. AR-V7 tissues  and tumo tissues –11.AR-V7 originates from alternative splicing of the AR pre-mRNA. A typical splicing process requires the coordinated action of splicing factors and cis-acting regulatory elements. Intron 3 of the AR contains two splicing signals known as intronic and exonic splicing enhancers . Recognition of these cis elements by the splicing machinery results in the inclusion of a cryptic exon 3 (CE3) into the mRNA. This cryptic exon includes a premature stop codon leading to the synthesis of AR-V7 [AR mRNA (AR-FL) and this potentially could be used as a mean to re-sensitize tumor cells to current androgen deprivation therapy.of AR-V7 . BlockinAR-V7 mRNA synthesis in CRPC-derived cell line models 22Rv1, DuCaP, and VCaP. We show that splicing-directed AONs specifically and efficiently knockdown expression of this variant. The AON-mediated suppression of AR-V7 has an inhibitory effect of androgen-independent cell proliferation. Our results provide the first proof of principle for the use of splice-switching AONs in CRPC and highlights their potential as therapeutic agents.Antisense oligonucleotides (AONs) are single stranded, short molecules that can block (aberrant) splicing events by base-pairing with cryptic splice sites in the pre-mRNA in the nucleus , 14. In cis-acting splicing enhancer elements within the so-called CE3 sequence [For the identification of sequence , 5 and isequence  and ESEFsequence , 17 to psequence . Two ESEsequence  and 2′-Osequence , 21 to mAR minigene was created with CE3 and its flanking regions inserted in between exon 3 and exon 4 and flanking regions of the human AR gene  and an AR-V7 (exon 3–CE3) transcript were expressed, suggesting that canonical and alternative splicing occurs in the minigene-encoded AR transcript  detected two protein bands of about 75 kDa. The upper band had the same size as the translated product in AR-V7-transfected HeLa cells (data not shown), suggesting it corresponds to endogenous AR-V7 protein levels (67 kDa). This band was weakened after treatment with AON-ISE, similarly to band detected with AR-V7-specific antibody. Interestingly, the lower molecular size band (~66 kDa) was also reduced in VCaP cells, presumably corresponding to AR-V3  AR antagonist, inhibited it  gene to the ETS family member ERG is the most prevalent fusion in prostate cancer [TMPRSS2 [TMPRSS2-ERG mRNA levels . Thus, AR-V7-mediated cell viability cannot be solely attributed to the regulation of TMPRSS2-ERG.Another gene reported to be regulated by both AR-FL and AR-V7 is TMPRSS2 , 12, 24.he cases . Knockdoin vitro , and knose model . AON-ISEUBE2C knockdown downregulates cell proliferation and activates the cellular apoptosis pathway [UBE2C transcriptional activation by direct occupancy of its promoter [AR-V7 and the subsequent downregulation of UBE2C were accompanied by an increase in Caspase-3/7 activity and an inhibition of androgen-independent cell proliferation. This suggests a role for UBE2C as a key regulator of AR-V7-promoted cell viability.Previous studies have shown that  pathway . AR-V7 hpromoter . InteresAR-V7, which induces cell death in three CRPC-derived cell line models. These results certainly are promising and warrant further in vivo investigation.Sustained cell proliferation in CRPC cells under castrate levels of androgens clearly demands for the development of new AR-targeted therapies. AONs have been shown to mediate significant biological effects in vitro and in vivo, reverting diverse disease phenotypes –37. AON DMD mRNA and ameliorated symptoms in Duchenne muscular dystrophy patients [Targeting of internal organs like the prostate or disseminated prostate cancer cells, typical for CRPC, would require systemic administration of AONs. Systemic administration of splice-switching AONs has demonstrated significant clinical benefits before. AON treatment corrected the open reading frame of the dystrophin patients , 45. Phapatients , 47. TheAR intronic region known as CE3 and its flanking sequences (516-bp upstream and 2418-bp downstream of CE3) was screened for the presence of intronic and exonic splice enhancer motifs. The publically available computer-based algorithms ACESCAN2 (http://genes.mit.edu/ acescan2/index.html) and ESEFinder (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi) were used to predict potential ISEs and ESEs, respectively. The cryptic splicing acceptor site was detected by screening the same sequence with the NetGene2 server (http://www.cbs.dtu.dk/services/NetGene2/).A 4-kb sequence containing an http://sfold.wadsworth.org/cgi-bin/index.pl). The chimeric GapmeR (RNA5-DNA10-RNA5) was chemically modified and synthesized as described above for the RNA AONs. The GapmeR sequence described by Wheeler et al. [Two RNA AONs, a 22 nucleotides long AON-ISE and a 19 nucleotides long AON-ESE, together with two control sense RNA oligonucleotides SON-ISE and SON-ESE were synthesized and modified with a phosphorothioate backbone and 2′-O-methyl groups at the sugar chain . Oligos were dissolved in nuclease-free water. A 20 nucleotides long GapmeR AONs, GapmeR-AR-V7, was designed using SFold software  . Pancreatic carcinoma MIA-PaCa-2 cells (ATCC# CRL-1420) were grown in Dulbecco’s modified Eagle's medium (DMEM) (Invitrogen) with 4.5 g/ml glucose and 1 mM pyruvate, supplemented with 2 mM l-glutamine and 10% FCS and 2.5% of horse serum (Invitrogen). For hormone stimulation experiments, 0.1 nM of synthetic androgen R1881 (PerkinElmer) was added to the medium in combination with 2 µM enzalutamide  or 0.2% dimethyl sulfoxide (as a vehicle control). Results were reproduced in at least three independent experiments. All cultures were maintained in a humidified atmosphere at 37 °C and 5% CO2. Cell lines were authenticated in 2016 using the PowerPlex 21 system (Promega) by Eurofins Genomics (Germany). Cells were frequently tested for Mycoplasma infection, using a Mycoplasma-specific PCR, and propagated for no >6 months or 30 passages after resuscitation from the authenticated stocks.The CRPC-derived 22Rv1 (ATCC# CRL-2505), DuCaP, and VCaP , the prostate cancer-derived LNCaP (ATCC# CRL-1740), PC-3 (ATCC# CRL-1435), and the bladder carcinoma-derived 5637 (ATCC# HTB-9) cell lines were maintained as monolayer cultures in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen), supplemented with 2 mM AR exon 4 amplicon included 469 bp from the upstream flanking region. For SOEing PCR, all three fragments contain 20-bp overlapping sequences incorporated as overhangs in the forward and reverse primers. The assembled minigene was directionally cloned into the pEGFP-N3 vector (Clontech) between the BglII and NotI sites (thereby removing the eGFP region). For cloning of the eukaryotic expression vector pCMV-AR-V7, CE3 was amplified from human genomic DNA. The fusion between an exon 3 and CE3 was achieved by SOEing PCR using Phusion High-Fidelity DNA Polymerase (New England Biolabs). The forward primers for the sewed amplicons were complemented with a 5′-GAGATG-3′ overhang and a HindIII site, and the reverse primers with a 5′-GTTGTT-3′ following an MfeI restriction site. The insert was directionally cloned into the pEGFP-N3-derived CMV-driven expression vector backbone vector. Correct cloning was verified by Sanger DNA sequence analysis of PCR products, purified using Wizard PCR preps DNA purification system (Promega). Primer sequences for cloning and sequencing analysis are listed in Supplementary Table The AR minigene was built according to sequence coordinates described by Liu et al. . BrieflyOne day before transfection, 140,000 cells (DuCaP/VCaP) or 70,000 cells (22Rv1/MIA-PaCa-2) were seeded per well of a 24-well plate, in a total volume of 500 µl medium. After trypsinization, cells were collected and seeded in charcoal-stripped serum-containing medium to wash away traces of androgens previously reported to be present in FCS [HP1BP3), the hypoxanthine phosphoribosyltransferase 1 (HPRT1) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used for normalization and relative gene expression levels were calculated according to the mathematical model for relative quantification in real-time PCR [TMPRSS2-ERG fusion transcript levels, a forward primer directed to exon 1 of the TMPRSS2 transcript together with a reverse primer directed to exon 4 of the ERG transcript were used [Total RNA was isolated using TRIzol reagent (Invitrogen) according to manufacturer’s protocol. Concentration and purity of the RNA was determined on a Nanodrop-1000 spectrophotometer (Thermo Scientific). Subsequently, 2 μg of total RNA was treated with DNaseI and used to synthesize complementary DNA using random hexamer primers and SuperScript II Reverse Transcriptase (Invitrogen). Real-time PCR (qPCR) analysis was performed using LightCycler 480 SYBR Green I Master Mix (Roche) and gene-specific primers , 60 mM Tris-Glycine pH 6.8) supplemented with 1:50 β-mercaptoethanol (Merck). Lysates were homogenized by sheering them through a 0.5 × 25 mm syringe needle. Protein concentration was measured using the Odyssey CLx Imaging System (LI-COR) and Image Studio software (LI-COR), after staining with Coomassie brilliant blue (Merck) with serial dilutions of bovine serum albumin as a standard. A total of 100 µg of protein was subjected to SDS-polyacrylamide gel electrophoresis using 7.5% polyacrylamide gels. Proteins were electrotransferred onto polyvinylidene fluoride membranes . Membranes were blocked for 1 h in phosphate-buffered saline with Tween-20 (PBS-T)/5% non-fat dry milk and incubated overnight with the primary antibody. The mouse monoclonal-antibody anti-AR-V7 , the rabbit polyclonal AR antibody N20 , the rabbit monoclonal-antibody anti-PARP , and the mouse monoclonal-antibody anti-β-actin  were used, diluted 1:500, 1:50,000, 1:1000, and 1:5000 in PBS-T/5% non-fat dry milk, respectively. The Horseradish peroxidase-conjugated donkey-anti-rabbit antibody  or sheep-anti-mouse antibody  diluted 1:50,000 in PBS-T were used as secondary antibodies. Protein bands were detected using ECL and Hyperfilm (Amersham Biosciences). Results were reproduced in two independent experiments.One day before transfection, 1,200,000 (DuCaP/VCaP) cells were seeded in 10-cm dishes. When cells reached 70% confluency, oligonucleotide transfection was performed. Four days after transfection, cells were harvested and washed. Cell pellets were lysed using Laemmli lysis buffer (1 mM CaCln = 20) was obtained by transurethral resection of prostate tumor tissue (TURP). TURP specimens were snap frozen in liquid nitrogen. Regions of CRPC tumors (n = 20) with high percentage of epithelial tumor cells (>50%) were selected for cryo-sectioning. The use of patient materials was approved by the local ethics committee of the Radboud university medical center (CMO Arnhem-Nijmegen).CRPC tissue , 20,000 cells (DuCaP/VCaP), or 500 cells (MIA-PaCa-2) were cultured in 96-well culture plates. Transfection with oligonucleotides was done 24 h after seeding. Four days after transfection, cell viability was measured using 3-−2,5-dephenyltetrazolium bromide  assays. Alternatively, cell viability was measured using CellTiter-Glo luminescence assays (Promega), following the manufacturer’s instructions. Absorbance (at 490 nm) and luminescence were measured using a Victor3 multilabel reader (PerkinElmer). Medium only was used as background control. To calculate the relative cell viability, cell viability values for each condition were normalized to the average of the cell viability values for control oligo-transfected cells. Each experiment was performed in triplicate and repeated at least three times.In parallel to the cell viability assays, cells were seeded into 96-well plates for assessment of Caspase-3/7 activity using the Apo-ONE Homogenous Caspase-3/7 Assay (Promega), following manufacturer’s instructions. After 4 h of incubation, luminescence was measured on a Victor3 multilabel reader (PerkinElmer). The luminescence signal from medium alone was used as background. Caspase-3/7 activity was normalized to values in control oligo-transfected cells. Each experiment was performed in triplicate and repeated at least three times.One day before transfection, 280,000 cells (DuCaP/VCaP) were seeded per well of a 12-well plate. The next day, cells were transfected with oligonucleotides as described above. Samples were harvested at different time points after transfection. Briefly, cells were harvested and washed with 0.9% NaCl. Cell pellets were resuspended in Hank's balanced salt solution (Invitrogen) and cells were fixed with ice-cold ethanol (58%). Fixated cells were centrifuged, resuspended in PBS, and treated with RNase A  for 40 min at 37 °C. Subsequently, cells were stained with propidium iodide  for 15 min in the dark. The samples were analyzed on a FC500 Flow Cytometer (Beckman-Coulter) and histograms were created and analyzed using Kaluza® Flow Analysis software (Beckman-Coulter). Results were reproduced in two independent experiments.t-tests were performed using GraphPad Prism . Pearson correlation coefficients were used to determine the relationships between relative gene expression profiles, considering a 95% confidence interval. A p-value of < 0.05 was considered statistically significant and p < 0.05 is represented by one star (*), p < 0.01 is represented by two stars (**), and p < 0.001 is represented by three stars (***).The data are presented as means ± SD from at least three independent experiments. Two-tailed paired and unpaired Supplementary TablesAR mRNA expression and AR copy number in CRPC tissue and in cell line modelsAR-FL and AR-V7 signalingGapmeR-mediated knockdown of AR-V7Effect of AON-ISE-mediated AR-V7 knockdown in 22Rv1 cellsAssessment of cell death in MIA PaCa-2Supplementary Figures and Tables Legends


Augmented reality (AR) is a technology that integrates digital information into the user’s real-world environment. It offers a new approach for treatments and education in medicine. AR aids in surgery planning and patient treatment and helps explain complex medical situations to patients and their relatives.This systematic and bibliographic review offers an overview of the development of apps in AR with a medical use case from March 2012 to June 2017. This work can aid as a guide to the literature and categorizes the publications in the field of AR research.From March 2012 to June 2017, a total of 1309 publications from PubMed and Scopus databases were manually analyzed and categorized based on a predefined taxonomy. Of the total, 340 duplicates were removed and 631 publications were excluded due to incorrect classification or unavailable technical data. The remaining 338 publications were original research studies on AR. An assessment of the maturity of the projects was conducted on these publications by using the technology readiness level. To provide a comprehensive process of inclusion and exclusion, the authors adopted the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement.The results showed an increasing trend in the number of publications on AR in medicine. There were no relevant clinical trials on the effect of AR in medicine. Domains that used display technologies seemed to be researched more than other medical fields. The technology readiness level showed that AR technology is following a rough bell curve from levels 4 to 7. Current AR technology is more often applied to treatment scenarios than training scenarios.This work discusses the applicability and future development of augmented- and mixed-reality technologies such as wearable computers and AR devices. It offers an overview of current technology and a base for researchers interested in developing AR apps in medicine. The field of AR is well researched, and there is a positive trend in its application, but its use is still in the early stages in the field of medicine and it is not widely adopted in clinical practice. Clinical studies proving the effectiveness of applied AR technologies are still lacking. Augmented reality (AR) is a technology that extends the user’s reality using digital information. It has become a publicly discussed topic in our society and a prime field for new kinds of apps in the medical sector. AR can be seen in many aspects of medicine; for example, Kamphuis et al reported that AR technologies have started maturing in the field of anatomical and physiological education . There iThe focus of this systematic and bibliographic review is to provide insight into the research conducted in the field of AR. The scope lies in its advances in the medical field, centering on the medical specialty, technical impact, maturity of projects, and publications on the topic of AR in medicine.Recently Chen et al  publisheWithin the review presented here, the publications have been manually categorized and structured according to medical branches, applied technologies in hardware and software, and strong indicators of the maturity of the developed technology.For the classification of AR apps, the taxonomy proposed in the Handbook of Augmented Reality by Hugues et al  was usedThe aim of this review paper was to build a foundation and guide to the literature and to be used as a motivation for scientists, researchers, and developers in the field of AR in medical settings. The main objectives were to assess the current state of research, identify possible future trends, and provide an overview. Another outcome of this research was an interactive table  of the rThis section offers a brief overview of AR and its definitions, differentiating it from VR. Additionally, a short overview of its technical development and its applications will be given.There are several definitions of AR, depending on how it enhances our environment with artificially added information and whether one can interact with this information .AR is a variation of VR . In contAnother term used in this context is mixed reality (MR), which could be explained by the “reality-virtuality continuum” explained by Milgram et al . MR showMilgram et al used the term MR to distinguish different MR displays and design a taxonomy for categorization of MR systems . In addiFrom a historical viewpoint, the first approach to AR was the Sensorama, a machine that was supposed to provide a cinematic experience with all senses . It was With the invention of handheld devices like smartphones and tablets, there were opportunities to advance to a bigger audience. In 2013, Google presented Google Glass, an HMD that provides hands-free interaction via a voice interface and enables the user to call, send texts, or search the internet. In 2015, Microsoft presented the HoloLens; this device allows one to see and interact with holographic 3D virtual objects via voice, gaze, and gestures.This section provides an overview of the methods used to acquire relevant publications in addition to the restrictions and inclusion and exclusion criteria used. Thereafter, an introduction to the principles used to analyze the publications and the relevant variables are presented.To gather the initial data, a query was designed to collect publications from the PubMed  and ScopIn three stages, the results of the query were filtered using a set of factors to determine the validity and relevance for this review. After each exclusion stage, the remaining papers were analyzed, and the corresponding variables were entered into the results file. We then processed each batch individually. To ensure reliability during the classification phase, a subset of 85 (25%) of 338 publications were cross-checked by the authors, and conflicts were resolved by consensus.In the next step, data were analyzed and prepared for visualization, where applicable. Analysis and visualization were conducted using R  .The initial queries returned 1309 results , from whThe second main reason for exclusion was a false positive result, a criterion that includes publications without any connection to AR because they either focus entirely on VR or separately contain the tags “augmented” and “reality.” This included the paper by Yoo et al about the effect of training alone or training with VR, both augmented by electromyography, for children with cerebral palsy .The exclusion criteria “Other” refers to papers where only the abstract could be found, papers where the authors did not reply, conference posters, collections of papers, and books. One additional criterion for exclusion was veterinary publications, for example, the paper by Sutton et al about the glass knife-fish and the way it uses “electrosensory feedback” to hold a position in a moving environment .A short overview of the variables used is presented in To classify the publications, several factors were chosen to create a comparable dataset within the research of AR applied to medicine. Besides the classification methods from the metadata, such as the year of publication and geographical location of the first author, several other classification factors were manually introduced into the dataset.For medical classification, the Medical Subject Headings (MeSH) terms were used. These terms, only applicable to the PubMed database, offer a terminology for categorization of biomedical apps and can be used, for example, to categorize publications in PubMed. For this review, the 2017 version of MeSH terms was used.For additional classification, data from ClinicalTrials.gov  were useThis variable was used to assess how collaboration using the AR device was realized. This could be done via a remote connection. For example, this category included studies in which two medical professionals present at different locations see the same AR environment and are allowed to interact with it.The technology readiness level assessment is a method to estimate the maturity of technology . UsuallyThe scope of the published apps was manually split into two subgroups—treatment and training—as shown in In this section, we present a short introduction to the AR display technology. Schmalstieg et al  divided The HMD is the most well-known display technology used for AR. These displays are further classified into “Optical-See-Through Head-Mounted Display” and “Video-See-Through Head-Mounted Display” . The OptThe commonly known smartphone and tablet are best examples of handheld AR devices and are mostly known for games like Pokémon Go. The back camera of such devices is used to provide the user with a video see-through image. In a medical context, this could be an app to visualize anatomical structures in a book.A display located in the world has many advantages for AR. These displays can be divided into desktop display, virtual mirror, projector-based display, and stationary display.A desktop display can be used as an AR display by adding a webcam, which then provides the necessary input of reality. The display then shows both reality and additional information within it. A virtual mirror uses the front camera of the device and shows a picture of what is in front of the camera. Another kind of an augmented mirror is the visual showcase, but it does not show the user; it is a stationary variant of the optical see-through, which allows the user to see through alongside additional information. The last stationary displays are projector-based displays. They can be dependent or independent from the view.As another option, one can simply mount the projectors onto an HMD, creating a personal projection with retro-reflection screens. The location “World” can be found in any medical context; a known example here would be in the operating room, where images acquired prior to surgeries are either shown on a display or projected directly onto the patient.Tracking in an AR environment can be categorized by the different technologies used to track objects. In this paper, the applied categorization refers to whether the sensors are stationary or mobile and which type of optical tracking was used .Today, many systems use more than one tracking mechanism; the HTC Vive system uses stationary tracking in the form of stationary lighthouses in the room with beam detectors in the tracked devices and mobile tracking in the form of gyro sensors.Stationary tracking is a technique that uses either a mechanical device or an electromagnetic field, infrared light, or ultrasound to obtain position data. Commonly known stationary tracking systems are the SteamVR trackers, which use a time-based location estimation for the HTC Vive or other AR/VR solutions. Another option is Ultrasonic Tracking , which uWhile stationary tracking does not allow the user to move around much, mobile sensors allow tracking outdoors. The most popular mobile sensor is the Global Positioning System, which determines the position of an object by the time of flight of signals emitted by a satellite. Inside an already established wireless network, it is also possible to track an object simply by the base station used to connect to the wireless network.There are two ways to classify tracking in AR: “model-based versus model-free tracking” and “markers/fiducial versus natural features” . Model-bInterest points, also known as key points, are mostly used to track an object, but the key points should be easily detected and stable from all angles. An alternative for interest points is edge features, but here, it is necessary to distinguish the edge from the background.In this section, the parts of the “Functional Taxonomy” , introduSix subfunctionalities of augmented perception can be separated by considering the ability to assist in decision making for AR. The first is functionality 0, where real images and virtual entities are shown on the screen but have no relation. This conditioning indicates that there is one screen but two different boxes: One box shows real images and one shows a virtual entity.The functionality “Documented Reality and Documented Virtuality”  is a minIn “Reality with Augmented Perception or Understanding”  environmThe subfunctionality “Perceptual Association of the Real and Virtual”  is dividThe “Behavioural [sic] Association of the Real and Virtual”  is a ste“Substituting the Real by the Virtual or Virtualised [sic] reality”  is a subThe query results contained 104 review publications. Most of the reviews assessed the use of AR in a surgery setting. The review publications can be found in Khor et al  providedThese publications do not include neurosurgical reviews. However, Pelargos et al  refined In total, two publications assessed the use of AR technologies in Urology. One study  providedSeveral endoscopic devices are equipped with AR technologies. Mahmud et al  provide Smith et al described the use of AR in education in the fields of Obstetrics and Gynecology . There iAs shown in The publications from PubMed feature the title and abstract and are annotated with MeSH terms . These pThe factor clinical trials was included to analyze if the proposed app was tested in a clinical trial registered in ClinicalTrial.gov . As showResearch maturity, categorized by the technology readiness level index , shows aThe results of the Medical scope are presented in Of the 285 publications identified to be in the treatment category, 69.5% (n=198) projects dealt with scenarios in the operating room and 9.1% (n=26) projects were set in a rehabilitation setting. In addition, 21.4% (n=61) of the presented projects included direct involvement in the therapy of the patient.The results of the Display classification are shown in Within the stream “Body,” some of the applications used a “Tablet” to visualize 3D information from a medical imaging system. For example, a system used a tablet showing a previously virtually reconstructed tumor during a neurosurgical operation, to assist the surgeon while drilling . AnotherOf all analyzed (N=338) publications, most optical tracking mechanisms are accomplished via a marker (n=223), mostly through commonly known mechanisms such as color variation . This pattern was used by Edgcumbe et al , who appThe results for the stationary and mobile tracking mechanisms are presented in To avoid doubling numbers, for any device using more than one tracking mechanism, the stationary device was chosen. This occurred mostly in the Head Location or Video-See-Through scenarios, since there are devices like the HTC Vive or the Oculus Rift that depend on tracking with infrared, which are found in a stationary device or involve smaller movements that often rely on gyroscopes.In some cases, there was no tracking of movement, like the app for rehabilitation proposed in the study by Chinthammit et al , who oveIn the second most classified functionality—“Perceptual Understanding”—there is a connection between the real and virtual environment, similar to the app presented by Marker et al . They imThe functionalities “Behavioral Association” and “Substitution” are addressed in 35 of the analyzed papers. One example is the paper by Olivieri et al  about anEvaluating the recent publications in the analysis timeframe, we detected an increasing trend in the number of papers on AR. This is most likely because the technology is developing, and affordable systems are partially available in daily life. In most cases, the first author was located in the United States, followed by Germany, Japan, and France.Inspecting only the PubMed database and their MeSH annotations, the dominating terms were found to be Computer-Assisted Surgery, Three-Dimensional Imaging, Computed X-Ray Tomography, and Laparoscopy. All terms were connected to technology-intensive environments that make use of screens and advanced visualization technology. This finding was also observed in the analysis of the Medical Scope, as the results show that the main scope was Treatment, and within this area, primarily, the operating room.With only three clinical trials registered in ClinicalTrials.gov, the scope for clinical trials was unexpected. One possible explanation for the low number of registered trials could be that it is not mandatory to register a medical invention prior to a clinical trial. Furthermore, most techniques were at a technology readiness level of 6 to 7; thus, they were mostly prototypes but needed additional completion and proving, a step that would lead to a clinical trial. We often observed that trials were conducted, but with a low number of subjects (N<9), or that the trials conducted were animal trials, which are not registered.Only a very small subset of explored AR technologies featured a collaboration aspect. This could be due to the lack of widely available network coverage in sensitive environments or simply the size of AR technologies. Wearable technologies, in particular, are still too bulky to be used in a professional setting.The maturity analysis of the research projects showed that the majority of publications were distributed in a rough bell-curve shape around level 6 and the use of technology was demonstrated in a relevant environment. This indicates that either early research is not published or basic research is no longer necessary, because AR technology is underway to production readiness.From a medical perspective, the majority of papers were categorized under “Treatment.” This is due to the fact that the use of displays is already established in operating rooms, and this is a small step toward adding information to the images shown.This finding is additionally supported by the next categorization; the technological perspective showed that the majority of publications were written under the stream “World,” and in these cases, the majority were in the class “Display.”The training scope is dominated by surgery simulations. Since the technology is easy to apply in a training environment, this is an expected finding.The second most important role after the presentation of the augmented world is the tracking of real-life objects that are used in the augmented space. Most of the tracking mechanisms used “marker-based” optical tracking, a technique that, in most cases, is more robust and accurate than “markerless” optical tracking. Only 39 publications did not use any kind of optical tracking, as it was not necessary for their apps. The majority of projects used a mobile tracking system. This can be explained by the broader range of movements provided by mobile tracking systems, which is essential in most cases, especially in surroundings like a hospital room.Defining the different levels of augmentation based on the taxonomy by Hugues et al, majority of the apps were assigned to the class “Perceptual Association”; thus, most apps seek to make associations between the real and the virtual world, for example, the association between an organ seen in endoscopy and the visual image projected onto it. The second most observed definition was “Perceptual Understanding”; the virtual object draws a connection to what is seen and explains or shows certain objects in the real world.The strength of manually analyzing the publications is that data could be deducted, which is not explicitly written, and were therefore not detectable by automated text mining. For example, some articles stated which device was used but did not disclose the tracking technology or which display was used explicitly. Since specifications for most devices are known, the basic tracking and display technologies were added to the result, as derived from context, for example, the publication by Shi et al  who presAn additional strength of manual analysis is that whenever ambiguous terms were used, it could be compensated. If a system was introduced as AR but was, by the Milgram definition, a VR system, the paper was excluded. In contrast, publications were included even if they involved ambiguous use of the term “virtual,” as seen in the study by Lozano-Quilis , who proOne strength of this review, in contrast to that by Chen et al  who usedThe number of analyzed publications is, in contrast to the study by Chen et al , a datasThis work provides a detailed view into 5 years of AR research in medicine. AR in medicine is an emerging technology that can benefit medical practitioners, health care professionals, and patients. The assessment of the technology readiness level shows that the AR technology is beyond the testing phase, and practical applications are becoming more common.MeSH term analysis showed that the fields of Computer-Assisted Surgery, Three-Dimensional Imaging, and Computed X-Ray Tomography are the most explored. These fields already make use of advanced display technologies, and it is easy to integrate AR technologies into their workflow.There was also a clear trend in technologies assisting actual treatment of patients in comparison to technologies in a training environment. In the treatment/training aspect, a more balanced distribution of publications was expected.AR technology is an upcoming technology that will impact the treatment of patients in the future. With shrinking and more powerful hardware, the technology will be able to merge better into existing workflows and create opportunities for patients, doctors, and health care professionals.The aim of this review was to offer a foundation to researchers, which was met by offering a categorized list. A certain scope of research can be searched from our list by using the interactive table in In contrast to the study by Chen et al , the aut