We found that NPR1 does not regulate stomatal density, but the npr1-1 mutant failed to close stomata when under pathogen attack, resulting in more pathogens entering the leaves. Moreover, the ROS levels in the npr1-1 mutant were higher than in the wild type, and several proteins involved in carbon fixation, oxidative phosphorylation, glycolysis, and glutathione metabolism were differentially changed in abundance. Our findings suggest that mobile SAR signals alter stomatal immune response possibly by initiating ROS burst, and the npr1-1 mutant has an alternative priming effect through translational regulation.Stomatal immunity is the primary gate of the plant pathogen defense system. Non-expressor of Pathogenesis Related 1 (NPR1) is the salicylic acid (SA) receptor, which is critical for stomatal defense. SA induces stomatal closure, but the specific role of NPR1 in guard cells and its contribution to systemic acquired resistance (SAR) remain largely unknown. In this study, we compared the response to pathogen attack in wild-type Arabidopsis and the  SAR occurs when pathogens attack one part of the plant, and the plant responds by sending mobile signals from that primary site of infection throughout the vascular system to uninfected parts of the plant, allowing remote tissues to have a robust defense against pathogens ,2,3,4,5. disease . SA is a defense .2O2), and nitric oxide. During biotic stress, ROS are generated by either plasma-membrane localized NADPH/NADH oxidases , which generate superoxide that is converted to H2O2, or cell-wall-localized peroxidases that generate H2O2 into the apoplastic spaces [The molecular functions and structure of the NPR1 protein have been unveiled. The N-terminus of NPR1 consists of a Broad-Complex, Tramtrack and Bric a brac (BTB) domain. This BTB domain interacts with NIMIN3 as part of SAR response, and the BTB domain may be an adapter for substrate ubiquitination by Cullin 3 E3 ligase, such as NPR3 and NPR4 with similar BTB domain structures, although NPR1 does not seem to interact directly with Cullin 3 ,9. Insidc spaces ,12.Pseudomonas syringae pv. Tomato (Pst) has evolved to counter stomatal defense, and it synthesizes a chemical called coronatine, a biochemical mimic of a plant hormone, jasmonic acid (JA)-isoleucine (Ile), that signals the guard cells to open stomata. The pathogen-associated molecular patterns (PAMP)-triggered stomatal immunity starts with the recognition of flagellin peptide by a FLAGELLIN SENSITIVE2 (FLS2) receptor kinase [Pst infection [Pst DC3000, a SMALL PHYTOCYTOKINES REGULATING DEFENSE AND WATER LOSS (SCREWs) triggers the PLANT SCREW UNRESPONSIVE RECEPTOR (NUT)-dependent phosphorylation of ABA INSENSITIVE1 (ABI1) and ABI2, which leads to an increase in the activity of ABI phosphatases towards OPEN STOMATA 1 (OST1), thereby promoting stomatal opening [Stomata are a first line of defense against pathogen entry, and they play an active role in limiting bacterial invasion as part of the plant innate immune system. This innate immune system is regulated by many factors, e.g., ROS ,13,14. Ar kinase . Throughr kinase . Plant hr kinase . Like JAnfection ,17. A re opening . Coronat opening . As oppo opening .Pst [npr1 mutant. Guard cells primed for SAR could override the coronatine signal if they maintained increased levels of SA and ROS.We have previously found that SA is increased in the guard cells of primed Arabidopsis leaves ,22. We hPst . As a kePst ,9,10,24.npr1-1 mutants to the bacterial pathogen Pst DC3000 under primed (SAR) or mock-treated conditions. We found that the npr1-1 mutant had no significant difference in stomatal density during vegetative development compared to WT. Priming led to reduced stomatal apertures in WT Arabidopsis but not in the npr1-1 mutant. Bacterial entry experiments of primed plants demonstrated that npr1-1 systemic leaves were more susceptible to bacterial colonization compared to WT, suggesting stomatal aperture affects bacterial entry. Our results indicate that mobile SAR signals alter stomatal aperture, possibly by initiating ROS bursts in the apoplastic space surrounding guard cells. Proteomic analyses revealed molecular differences between the genotypic difference, priming effects, and Pst DC3000 response of WT and npr1-1 systemic leaves. Although the npr1-1 mutant does not have SAR activation with priming, we discovered that ribosomal proteins were overly abundant after Pst infection in npr1-primed leaves. The npr1-1 mutant plants are deficient in immune response, making npr1-1 more susceptible to pathogen attack. Elucidating guard-cell-specific responses to SAR is important for the development of transgenic crops that have enhanced pathogen defense without negative effects on growth and development.Here, we investigated the differences in stomatal movement between wild-type (WT) and Pst exposure , followed by a decline from weeks 6 to 8. Notably, the only discrepancy in total stomatal density between the npr1-1 and WT plants occurred at week 6  or 10 mM MgCl2 (mock) into one mature rosette leaf of 5-week-old plants with a needleless syringe. After 3 days, we exposed the opposite distal rosette leaf to the pathogen (0.2 OD600) and compared the response of the stomata in the primed npr1-1 mutant and WT plants to each other and to the mock-treated plants. As previously reported [Pst and then reopen after 3 h of Pst treatment. This basal stomatal response occurred in the mock-infiltrated WT leaves, where the average stomatal aperture was approximately 2 µm at 0 h, decreased to approximately 1.3 µm after 1 h, and then increased to approximately 2 µm at 3 h after exposure to Pst. Surprisingly, the primed WT plants did not exhibit this typical stomatal immune response. The distal leaves from the primed WT plants had stomata with an average aperture of approximately 1.3 µm from 0 to 3 h after exposure to Pst. There was no significant difference in the stomatal aperture measurements from the primed leaves taken at 0, 1, and 3 h after exposure to the pathogen , DAB, and NBT staining. As shown in T leaves .npr1-1 mock group before subsequent Pst exposure, and WT and npr1-1 showed very different proteomic changes after the subsequent Pst exposure  cutoff of 1% . Principexposure . Thus, ip-value ≤ 0.05) between the npr1-1 mock group and the WT mock . Specifically, 301 proteins were significantly increased in the npr1-1 mock group, while 151 proteins were significantly decreased. The fold changes in the differential proteins ranged from 1.1-fold to about 40-fold. By mapping these differentially abundant proteins (DAPs) to KEGG Arabidopsis pathways, we found these proteins are related to metabolic pathways (150/452), biosynthesis of secondary metabolites (84/452), carbon metabolism (39/452), and biosynthesis of amino acids (26/452). Interestingly, of the 20 most DAPs, 18 of them were decreased in the npr1-1 mutant .There were 337 DAPs in the oup only . Of the npr1-1 mock, and npr1-1 primed groups after Pst DC3000 infection. GSTU5, which was co-regulated with the glutathione reductase activities [npr1-1 primed group and decreased most in the WT mock group. The abundance of the isocitrate/isopropylmalate dehydrogenase (AT5G14590) decreased after Pst DC3000 infection in WT, and increased in npr1-1 mutant, regardless of whether the systemic leaves were primed or not. Furthermore, it was observed that this protein exhibited a significant decrease in the npr1-1 mutant compared to the WT  vs. a mock solution (control). Surprisingly, we found that the WT distal leaves from primed plants had a much smaller stomata aperture when compared to the control plants. Interestingly, the stomata from primed leaves were able to maintain the same narrow aperture after 3 h of exposure to Pst . Interestingly, at 3 h after exposure to Pst, the npr1-1 stomata of both the mock and primed plants had larger apertures than either WT mock or primed plants. The larger stomatal apertures of the npr1-1 mutant corresponded to increased bacterial entry. On the other hand, the narrow stomatal apertures of the primed WT plants corresponded to lower numbers of bacteria that were able to enter the leaves  AT4G12830, is the most decreased protein in WT Arabidopsis after priming and is also the most decreased protein in biotics. . ABHs subiotics. . Althougnpr1-1 primed systemic leaves after Pst DC3000 infection. Importantly, prior to pathogen exposure, these ribosomal proteins did not show significant differences in abundance between the npr1-1 mock and npr1-1 primed groups, nor did they exhibit any significant differences in the WT primed groups after pathogen exposure. This suggests that these ribosomal proteins play important roles in pathogen defense due to the failure of being primed in the npr1-1 plants. We further propose that translational regulation may serve as a bypass mechanism for plant response to priming in the absence of an effective SAR signaling pathway (in npr1-1), as indicated by significantly higher levels of MDA in npr1-1 primed leaves compared to npr1-1 mock leaves. Based on the proteomic results, the translational regulation seems to be selective in the protein profiles but ineffective in the output of stomatal immune response  environment. Controlled chamber temperatures during the light and dark periods were 22 °C and 20 °C, respectively. Incandescent bulbs capable of emitting 160 µmol m−2 s−2 were used in the growth chambers, with a relative humidity of approximately 55%. A dome was placed over the flat until seeds began germination. After two weeks of growth, seedlings were transferred from the original pot into flats with moistened soil contained in 32 pots per flat . Each Arabidopsis seedling was allocated to one pot. The transferred plants were grown in the same growth chamber. Plants were watered weekly, with a supplement of nitrogen/phosphorus/potassium fertilizer. The plants were kept in the chambers until needed, and a mature rosette (stage 3.9) could be observed at 5 weeks [The  5 weeks .Pst DC3000, the model pathogen used for Arabidopsis SAR induction [2HPO4 (s), 0.75 g MgSO4 (s), 10 mL glycerol, 15 g agar, and deionized H2O for a total volume of 1 L). The King’s B media was autoclaved, and antibiotics rifampicin (25 mg/L) and kanamycin (50 mg/L) were added to the King’s B media once it cooled down. Pst was streaked on this medium and incubated for 3 days at 28 °C. For liquid King’s B media, the composition was the same as for the plates except without agar. One fresh colony was used to inoculate the liquid culture, which was shaken overnight at 28 °C.nduction  was strenpr1-1 plants were used for stomatal movement analysis. Primary inoculation occurred via needleless syringe injection, where the plants were either primed with Pst DC3000 (OD600 = 0.02) or mock-treated with 10 mM MgCl2. At three days post-inoculation, the leaf opposite to the injected leaf was detached for secondary treatment. In the secondary treatment, the plants were either floated in 10 mM MgCl2 or in Pst DC3000 (OD600 = 0.02) solution in small Petri-dishes. Three leaves were used for each time point and secondary treatment group, and only one leaf was collected from each plant. Stomatal apertures were measured at three time points: 0 h, 1 h, and 3 h. After each time point, the leaves were collected and peeled twice using the tape-peel method [https://imagej.net/, 28 April 2023).Five-week-old WT and l method . The abanpr1-1 were grown for 5 weeks and prime-treated via injection with either Pst DC3000 (OD600 = 0.02) or mock-treated with 10 mM MgCl2. Three days after the infection, the leaf opposite to the one infected was detached and floated in Pst (OD600 = 0.02) solution for both mock and primed plants. After three hours of floating in solution, the leaf was placed in Falcon tube with 0.02% Silwet [2O. The leaf disk was then ground using a plastic grinding tip, and 10 µL was collected to make a 1:1000 serial dilution. A total of 100 µL was collected from the dilution and plated on agar media containing rifampicin (25 mg/L) and kanamycin (50 mg/L). After 3 days of incubation at 25 °C, the colonies on the plate were counted.To measure how many bacteria entered the apoplast after 3 h, nine independent biological replicates of WT plants Col-0 and nine replicates of % Silwet , vortexePst growth assay was used to determine how much bacteria grew in the apoplast bypassing the stomatal barrier. Nine independent replicates of WT and nine independent npr1-1 replicates were grown for 5 weeks and either mock- or prime-treated as described above. After three days of treatment, an opposite leaf was infiltrated with Pst DC3000 (OD600= 0.02), and the plant was left in the growth chamber for three days. The opposite leaf was then detached and washed in 0.02% Silwet, and a disk was taken from each leaf to make a 1:1000 serial dilution and plate it on media. Colonies were counted to determine how many bacteria were able to grow in the apoplast.npr1-1 and three WT plants were collected, and two leaves were removed from each independent plant. With a total of six leaves for each genotype, three were used for he abaxial peel, while the other three were used for the adaxial peel. For the abaxial peel, tape was placed on both surfaces of the leaf and then removed from the adaxial side. Another layer of tape was placed and removed again. The tape containing the abaxial side of the leaf was then put on a microscope slide and imaged using a Leica DM 6000 B microscope . For the adaxial peel, adhesive was pipetted thinly onto a microscope slide, and the leaf was placed with the adaxial side touching the adhesive. The leaf was left for 10 min, and then, using a scalpel, the surface of the leaf was peeled in order to leave the adaxial surface on the microscope slide. Stomata were imaged in the same way as above. Two images of each of the leaf quadrants were taken per leaf, and the number of stomata were counted manually.Stomata density was measured every week from the moment the plant was 2 weeks old until 8 weeks old. Every week, three 2O2 was detected in situ in rosette leaves of WT and npr1-1 mock and primed plants by staining with 3,3ʹ-diaminobenzidine (DAB) , and superoxide (O2-) was detected by nitrotetrazolium blue chloride (NBT)  staining using a previously described method [2O2 in the presence of peroxidases, generating a dark brown precipitate that can be used as a stain to detect H2O2 presence and distribution in plant cells. NBT reacts with endogenous O2- and forms a formazan compound that is reflected by the presence of a dark blue stain. DAB and NBT staining solutions were prepared fresh in amber-colored bottles as follows: For NBT staining solution, a 50 mM sodium phosphate buffer (pH 7.5) was prepared by mixing 16 mL of 1M sodium phosphate monobasic with 84 mL of 1M sodium phosphate dibasic. Then, 0.1g of NBT was dissolved in 50 mL of the 50 mM sodium phosphate buffer in an amber-colored bottle to make a 0.2% NBT staining solution. DAB staining solution was prepared by dissolving 50 mg of DAB in 50 mL of sterile distilled water, and pH was adjusted to 3.8 using 0.1 M HCl. Three fully expanded distal leaves were collected from three individual 5-week-old Arabidopsis plants for each treatment group , placed in 15 mL tubes, and immersed in either DAB or NBT staining solution. Tubes were wrapped in aluminum foil and kept overnight at room temperature. The next day, the staining solution was poured off, and chlorophyll was removed from the leaves by boiling in absolute ethanol for 10 min with intermittent shaking. Leaves were transferred to paper towels saturated with 60% glycerol and photographed. The experiment was repeated twice with similar results.Hd method . Accordig at 4 °C for 10 min. The absorbance of the supernatant was detected at wavelengths of 532 and 450 nm. The MDA content (µmol) was calculated as 6.45 * OD532—0.56 * OD450.MDA content was measured using fully expanded leaves harvested from control and primed plants. They were ground to a fine powder in liquid nitrogen, followed by adding 3 mL of 10% trichloroacetic acid (TCA) to 0.2 g leaf tissue powder and incubated at 4 °C overnight. After centrifugation at 10,000 g at 4 °C for 20 min, the supernatant was transferred to a new tube, and 2 mL 0.6% thiobarbituric acid was added. The mixture was vortexed thoroughly, heated in boiling water for 15 min, cooled to 4 °C immediately and centrifuged at 10,000× 2 (mock) or the Pst DC3000 at OD600 of 0.02 (primed) to reveal molecular mechanisms underlying the priming and response to pathogens. After mock and priming, the distal leaves were exposed to either control or Pst DC3000 at OD600 of 0.2. Proteins were extracted using a TCA method [Three biological replicates of each sample type were prepared for the proteomic experiments. For mock and primed samples, distal leaves were collected and immediately frozen in liquid nitrogen. These leaves were 72 h after one local leaf of the plant was injected with either 10 mM MgClA method . Briefly® 100 particles with 3 μm size and 100 Å pores) and then eluted using a C18 analytical column . The flow rate was set at 300 nL/minute with solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid and 99.9% acetonitrile) as the mobile phases. Separation was conducted using the following gradient: 2% of B over 0–0.5 min; 2–35% of B over 0.5–45 min, 35–98% of B over 45–46 min, and isocratic at 98% of B over 46–59 min, and then from 98–2% of B from 59 to 60 min. The equilibration time at 2% B was 15 min. A full MS1 scan (m/z 350–2000) was performed on the Orbitrap with a resolution of 120,000 at m/z 400. The automatic gain control (AGC) target is 2e5, with 50 ms as the maximum injection time. Monoisotopic precursor selection (MIPS) was enforced to filter for peptides. Peptides bearing +2 to +6 charges were selected with an intensity threshold of 1e4. Dynamic exclusion of 15 s was used to prevent resampling the high abundance peptides. The top-speed method was used for data-dependent acquisition within a cycle of 3 s. The MS/MS was carried out in the ion trap, with a quadrupole isolation window of 1.3 Da. Fragmentation of the selected peptides using the collision-induced dissociation (CID) was carried out at 35% of normalized collision energy with 10 ms activation time. The MS2 spectra were detected in the linear ion trap with the AGC target as 1e4 and the maximum injection time as 35 ms.The bottom-up proteomics data acquisition was performed on the EASY-nLC 1200 connected to the Orbitrap Fusion equipped with a nano-electrospray source . The peptide samples were loaded to a C18 trapping column  absolute threshold of 0.4, and a fragment ion cutoff percentage of 0.1. Precursor mass tolerance was set to 10 ppm and fragment mass tolerance was set to 0.8 Da. Selected dynamic modifications included N-ethylmaleimide (+124.048 Da (C)), oxidation (+15.995 Da (M)), and phosphorylation ). The percolator node for protein identification had the parameters of a strict target FDR of 0.01 and a relaxed target FDR of 0.05. Consensus workflow node selection was MSF Files, PSM Grouper, Peptide Validator, Peptide and Protein Filter, Protein Scorer, Protein Grouping, Feature Mapper, and Precursor Ions Quantifier. Peptides were filtered to include only those with high confidence of identification (1% FDR), and quantification of unique peptides was performed using total area sum intensities. Quantified intensities were exported into Microsoft Excel, and ratios were calculated from the median-normalized peak intensity values.Proteome Discoverer™ 2.5  was used for protein identification by searching the raw LC-MS/MS files against the Uniprot npr1-1 Arabidopsis subjected to mock and priming pre-treatments and subsequent Pst exposure. While NPR1 does not affect stomatal density, the npr1-1 mutant does not display Pst-triggered stomatal closure in systemic leaves, and the reopening mechanism, presumably driven by the SCREW-NUT functions, remains active. Further examination of proteomic differences among genotypes and treatments, the effects of priming, and subsequent Pst response, led to the conclusion that in WT Arabidopsis, stomatal closure in systemic leaves occurs via an NPR1-dependent ROS burst. In the npr1-1, stomata in systemic leaves remain open, promoting an alternative response characterized by translation regulation. This research underscores the critical role of NPR1 in systemic acquired stomatal immunity, thus enhancing our understanding of plant defense mechanisms at the first line of defense—stomatal guard cells.In this study, we explored the role of NPR1 in systemic acquired stomatal immunity using WT and


Hyperglycemia leads to serious damage to the body, especially the blood vessels and nerves. This study aimed to determine the prevalence and factors associated with hyperglycemia in a rural population of Tanvè and Dékanmey in Benin in 2019.This was a cross-sectional, descriptive and analytical study, nested in the Tanvè Health Study (TAHES) cohort. It covered all residents of the villages of Tanvè and Dékanmey, aged 25 years and above, and having given their written consent. Data were collected in the households during the fourth annual monitoring visit in 2019 using the WHO STEPS Wise approach. Hyperglycemia was defined as a fasting capillary blood glucose value ≥ 110 mg/dL. Data were analyzed with R Studio software version 3.5.1.A total of 1331 subjects were included in the study with a 60% female predominance and a sex ratio  of 0.7. The median age was 40 years  with a range of 25 and 98 years. The prevalence of hyperglycemia was 4.6%. In multivariate analysis, advanced age , male sex , monthly income> 105,000 FCFA , abdominal obesity  were statistically associated with hyperglycemia.The prevalence of hyperglycemia is not negligible in rural areas in Benin. Our study found that older age, male gender, high income, abdominal obesity, and obesity are determining factors in its occurrence. Hyperglycemia is a precursor stage of diabetes, which corresponds to an increase in fasting glucose from 110 mg/dL [The International Diabetes Federation (IDF) estimated in 2013 the number of adults suffering from diabetes to around 382 million in the world, meaning a global proportion of 8.3%, with around 80% of these people living in Low and Middle Income Countries (LMICs). If nothing is done, by 2035, the number of persons living with diabetes will rise to around 592 million; meaning one in ten adults. Estimates based on these same figures, predict about three new cases every ten seconds, which corresponds to nearly 10 million per year [The World Health Organization (WHO) warned in 2012 that hyperglycemia caused around 2.2 million deaths in the world while diabetes was the direct cause of 1.6 million deaths a few years later in 2015 [In general, hyperglycemia and diabetes result in disabilities due to their manifestations and the expensive cost of treatment. The IDF estimated in 2017 that two-thirds of people ($327 million) living with diabetes are of working age; adding that in the same year, diabetes was the cause of $727 billion in health spending . It causIn the past, high prevalence of hyperglycemia was considered to be the prerogative of high-income urban populations; but today, studies show significant prevalence of hyperglycemia in rural areas and according to IFD in 2013, more people with diabetes live in rural areas, meaning 136 million compared with 246 million in urban areas [The situation is no less worrying in rural areas in Benin as evidenced by the Tanvè Health Study (TAHES) cohort erected as a rural population in South Benin since 2015 where prevalence of hyperglycemia has been estimated to respectively 3.5% and 4.5% in 2015 and 2017 . In lighOur study was a cross-sectional, descriptive and analytical, conducted during the fourth annual follow-up visit of the TAHES cohort . The study population included persons aged 25 years or older, residing in the villages of Tanvè and Dékanmey, and having given their free written consent after clarification to participate in the TAHES cohort.2, overweight by a BMI comprised between 25kg/m2 and <30kg/m2 and obesity as BMI ≥ 30kg/m2. Abdominal obesity was defined by a waist circumference of at least 80 cm in women and 94 cm in men according to IFD criteria.Fasting hyperglycemia was considered as the dependent variable and was defined by fasting capillary glycemia ≥110 mg/dL. The glycemia was defined as a diabetic-type in case of fasting capillary glycemia ≥126 mg/dL The independent variables were socio-demographic, economic, cultural, behavioural and biological variables. The definition of risk factors used in our study was those described in the WHO STEPS Survey Manual. Only current and former smokers were considered to be smokers. Consumption of less than five servings (400 grams) of fruits and vegetables (FV) per day was defined as insufficient consumption of fruits and vegetables. The practice of physical activity was categorized in 2 ways: sufficient and insufficient. Subjects were reported to have insufficient physical activity when they had moderate physical activity of at least 30 minutes or intense activity of at least 15 minutes during their activities or recreation for less than 5 days per week. This practice was considered as sufficient when it was done for at least 5 days a week. Alcohol consumption has been defined as a consumption on at least one occasion in the past 12 months. Without any notion of follow-up, systolic blood pressure ≥ 140 mm Hg and/or diastolic blood pressure ≥90 mm Hg was defined as High Blood Pressure (HBP). Hypertension has been defined as either having HBP or being on antihypertensive treatment. The body mass index (BMI) was calculated by dividing the weight in kilograms (Kg) by the square of the height in meters (m). Under-weight was defined as a BMI <18.5kg/mThe data were collected from 26th January to 17th February 2019 in the habitations using the WHO STEPS Wise approach. An individual door-to-door interview was done with each respondent (STEP1), with anthropometric measurements  and blood pressure that were after the interview, taken according to WHO standards with validated materials (STEP2). Capillary glucose was measured by finger on appointment the next morning after at least 8 hours of fasting using a glucose meter. The data were recorded using a standardized sheet adapted from the WHO STEPS instrument and integrated into the smartphone via the Kobo-collect application. The study was approved by the National Ethics Committee for Health Research and pregnant women were excluded from the study.The collected data were cleared and analyzed with R Studio 3.5.1 software. The proportions were compared with the Chi-two test; the means with the Student test and the medians with the non-parametric Kruskal-Wallis test. A step-by-step descending binary logistic regression of Wald was performed using a significance threshold p < 20% for initial model and p < 5% for final model. Prior to model execution, the interactions were verified and the suitability of the model was made by the Akaike Information Criterion (AIC).A total of 1331 subjects were included in the study. The median age was 40 years  with extreme values of 25 and 98 years. There was a female predominance (60%) with a sex ratio of 0.7. In the sample, 63.4% had no educational level, 40.1% had a monthly income of 40,000 FCFA. Poor fruit and vegetable consumption was found in 96.4%, alcohol consumption in 63.9%, tobacco consumption in 5.0% and insufficient physical activity in 62.5%. Concerning the biological risk factors, the average waist circumference was specifically 83.4 ±10.8 cm (extremes: 53 cm and 136 cm) in male subjects and 86.8 ±12.6 cm (extremes: 52 cm and 162 cm) in female subjects. The average BMI was 23.3 ±5.2 Kg/m2.Abdominal obesity was found in 49.6% of the population. Almost one in ten persons (9.8%) was found to be obese and 19.5% of them were overweight. High blood pressure was found in 29.5% and hypertension was found in 32.1%. In addition, around 2 in 10 persons (21.1%) reported taking a glycemic control less than one year ago while 78.9% of them reported doing so within a year or more. A total of 61 subjects had fasting hyperglycemia, meaning a prevalence of 4.6% (IC95%: 3.6–5.8). The average fasting blood glucose value was 93.5 ±33.8 mg/dL with extreme values of 51.0 mg/dL and 333.0 mg/dL. This value was 85.6 ±36.3 mg/dL in male subjects and 82.2 ±31.9 mg/dL in female subjects (p = 0.073). On the other hand, 2.6% had diabetic-type hyperglycemia and 3.8% had a known history of diabetes. The Behavioral and Biological Risk Factors are described in Tables We found in our study a prevalence of fasting hyperglycemia of 4.6% (95%CI: 3.6–5.8). This prevalence corroborates those of Amidou et al and Houehanou et al who respectively found within the same cohort in 2017 and 2015, a prevalence of 4.5% and 3.5% , 11. MorThe prevalence of diabetic-type hyperglycemia in our study was 2.6% and that of diabetes was 3.8%. This result is similar to that obtained by Fen L et al in China in a study of 2,967 residents of the Han ethnic group aged 20 to 80 years, selected by multi-stage cluster sampling in Guizhou and which reported a prevalence of 3.77% among rural residents . BahendeOur study also revealed that 281 (21.1%) persons reported having taken a glycemic control less than a year ago whereas 1050 78.9%) of them reported having taken it more than a year ago. These results showed that this rural population is becoming more aware of its health. In fact, the 21.1% who did this check-up, were supposed to have done it in the absence of an annual check-up visit as part of the TAHES Cohort since the last visit before that of our study had been done in 2017. Similar to our findings, BeLue et al in Senegal reported in their study a proportion of 24.8% in type 2 diabetic patients that were regular in taking their glycemic control at the M’Bour Hospital Complex . On the % of themIn our study, factors associated with fasting hyperglycemia in multivariate analysis were: advanced age, male sex, abdominal obesity, weight status and high monthly income.The prevalence of hyperglycemia increased significantly with age (p = 0.004). In fact, the risk of being hyperglycemic increased by 1.03 with each increase of one unit of age (95%CI: 1.02–1.73). Bahendeka et al had reached out to the same conclusion in their study where they reported that the prevalence of hyperglycemia increased significantly with age (p = 0.003) . FurtherOur study showed that abdominal obesity was associated with hyperglycemia ); that is consistent with the finding of Aynalem et al in Ethiopia in 2016 who also found an association between hyperglycemia and abdominal obesity. In their study, people with abdominal obesity were more exposed to hyperglycemia compared to people with a normal waist circumference (AOR = 4.107 (1.108–15.231)) . SimilarObesity is established as an associated factor of hyperglycemia. In our study we found a significant association between hyperglycemia and obesity in univariate analysis (COR = 1.97(1.05–3.69); p = 0.030)). After adjustment, this significant association remained, though unstable (AOR = 1.68 (0.75–3.59); p = 0.004). Okumiya et al reported in their study that obese people were 1.77 times more exposed to hyperglycemia compared to people with normal BMI  . MillogoPeople with relatively high incomes tend in a given society to adopt eating habits or lifestyle habits in general, similar to a life of ease. The latter can sometimes lead to the occurrence of hyperglycemia which can progress to diabetes. Our study found that the prevalence of hyperglycemia was significantly higher among participants with a monthly income above 105,000 FCFA, compared to those with a monthly income below the Beninese’s minimum wage . Furthermore, hyperglycemia was 2.63 times more frequent in people monthly earning at least the minimum wage, compared to those earning below this amount . Millogo et al had made the same observation by pointing out in their study that subjects of high socio-economic level were more affected by hyperglycemia (13.6%) than others  . HarshfiThe results of this research must challenge us and draw our attention to a phenomenon that begins to emerge gradually even in the rural regions.We conducted a comprehensive recruitment of subjects who met the inclusion criteria with the benefit of a large enough sample size. The data were collected by investigators accustomed to the STEPS surveys with the STEPS wise tool of the WHO transcribed for needs in the main language of the medium "fon". Measurements of blood glucose, blood pressure and anthropometric parameters were made by trained investigators using WHO recommended methods and with valid and standardized equipments. All this has contributed to the minimisation of measurement errors and potential information biases.However, blood sugar and blood pressure measurements being taken during a single visit, are likely to be influenced by external factors such as infection, ongoing treatment, stress or lack of rest; which may overestimate our results.The prevalence of hyperglycemia was not negligible in the rural populations of Tanvè and Dékanmey in Benin. Advanced age, males, abdominal obesity, obesity and high income were significantly associated with its occurrence. Effective action against this health problem requires concerted political and health action at a much more multi-sectoral level considering the diverse origins of its risk factors.


The biological evaluation results showed that all compounds c1~c29 exhibited definite tyrosinase inhibitory activity; especially, compound c27 was the strongest tyrosinase inhibitor (IC50: 3.07 ± 0.26 μM), being ~4.6-fold stronger than the positive control, kojic acid (IC50: 14.15 ± 0.46 μM). Inhibition kinetic studies validated compound c27 as a reversible mixed-type inhibitor against tyrosinase. Three-dimensional fluorescence and circular dichroism (CD) spectra results indicated that compound c27 could change the conformation and secondary structure of tyrosinase. Fluorescence-quenching results showed that compound c27 quenched tyrosinase fluorescence in the static manner with one binding site. Molecular docking results also revealed the binding interactions between compound c27 and tyrosinase. Therefore, cinnamic acid–eugenol esters, especially c27, could be used as lead compounds to find potential tyrosinase inhibitors.Tyrosinase plays crucial roles in mediating the production of melanin pigment; thus, its inhibitors could be useful in preventing melanin-related diseases. To find potential tyrosinase inhibitors, a series of cinnamic acid–eugenol esters ( Tyroshenolase  + calcd for C19H18O3+: 295.1337; found: 295.1326; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(o-tolyl)acrylate (c2). White soild; yield 55% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 265.1–266.3 °C; 1H NMR  δ 8.16 , 7.65 , 7.31 , 7.25 , 7.23 , 7.04 , 6.83 , 6.81 , 6.61 , 5.99 , 5.16–5.09 , 3.84 , 3.41 , 2.48 ; 13C NMR  δ 165.3 (C=O), 151.1 (Ar-C), 144.2 (CH), 139.1 (Ar-C), 138.1 (CH), 138.0 (Ar-C), 137.2 (Ar-C), 133.3 (Ar-C), 131.0 (Ar-C), 130. 5 (Ar-C), 126.7 (Ar-C), 126.5 (Ar-C), 122.8 (Ar-C), 120.8 (Ar-C), 118.1 (CH), 116.3 (CH), 112.9 (Ar-C), 56.00 (OCH3), 40.2 (CH2), 20.0 (CH3); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H20O3+: 309.1487; found: 309.1483; IR (cm−1): 2937 (OCH3), 2967 (CH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(m-tolyl)acrylate (c3). Yellow soild; yield 67% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 326.6–327.8 °C; 1H NMR  δ 7.88 , 7.44 , 7.42 , 7.34 , 7.28 , 7.07 , 6.86 , 6.84 , 6.70 , 6.02 , 5.22–5.11 , 3.87 , 3.44 , 2.43 ; 13C NMR  δ 165.4 (C=O), 151.1 (Ar-C), 146.8 (CH), 139.0 (Ar-C), 138.8 (CH), 138.1 (Ar-C), 137.2 (Ar-C), 134.4 (Ar-C), 131.6 (Ar-C), 129.1 (Ar-C), 129.0 (Ar-C), 125.6 (Ar-C), 122.8 (Ar-C), 120.8 (Ar-C), 117.0 (CH), 116.3 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.3 (CH2), 21.5 (CH3); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H20O3+: 309.1487; found: 309.1484; IR (cm−1): 2937 (OCH3), 2961 (CH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(p-tolyl)acrylate (c4). Yellow soild; yield 57% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 266.8–267.7 °C; 1H NMR  δ 7.85 , 7.49 , 7.48 , 7.23 , 7.21 , 7.02 , 6.82 , 6.80 , 6.63 , 5.98 , 5.15–5.08 , 3.83 , 3.40 , 2.39 ; 13C NMR  δ 165.5 (C=O), 151.2 (Ar-C), 146.6 (Ar-C), 141.2 (Ar-C), 139.0 (CH), 138.1 (Ar-C), 137.2 (Ar-C), 131.7 (Ar-C), 129.8 (Ar-C), 128.4 (Ar-C), 122.8 (Ar-C), 120.8 (Ar-C), 116.3 (CH), 116.1 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.3 (CH2), 21.7 (CH3); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H20O3+: 309.1487; found: 309.1483; IR (cm−1): 2937 (OCH3), 2968 (CH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(2-chlorophenyl)acrylate (c5). White soild; yield 65% (the crude was purified using a mixture of PE/EtOAc 60:1); mp: 247.4–247.7 °C; 1H NMR  δ 8.29 , 7.71 , 7.44 , 7.34 , 7.31 , 7.04 , 6.83 , 6.81 , 6.67 , 5.98 , 5.16–5.09 , 3.84 , 3.41 ; 13C NMR  δ 164.8 (C=O), 151.1 (Ar-C), 142.2 (CH), 139.2 (Ar-C), 138.0 (CH), 137.2 (Ar-C), 135.3 (Ar-C), 132.6 (Ar-C), 131.45 (Ar-C), 130.4 (Ar-C), 128.0 (Ar-C), 127.3 (Ar-C), 122.7 (Ar-C), 120.9 (Ar-C), 119.8 (CH), 116.3 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.2 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17ClO3+: 329.0947; found: 329.0937; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 726 (Cl).4-allyl-2-methoxyphenyl (E)-3-(3-chlorophenyl)acrylate (c6). Yellow soild; yield 73% (the crude was purified using a mixture of PE/EtOAc 60:1); mp: 278.3–278.9 °C; 1H NMR  δ 7.80 , 7.57 , 7.45 , 7.39 , 7.36 , 7.03 , 6.82 , 6.81 , 6.67 , 5.98 , 5.15–5.09 , 3.83 , 3.40 ; 13C NMR  δ 164.9 (C=O), 151.0 (Ar-C), 144.9 (Ar-C), 139.2 (CH), 138.0 (Ar-C), 137.2 (Ar-C), 136.2 (CH), 135.0 (Ar-C), 130.5 (Ar-C), 130.3 (Ar-C), 128.2 (Ar-C), 126.5 (Ar-C), 122.7 (Ar-C), 120.9 (Ar-C), 118.7 (CH), 116.3 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.2 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17ClO3+: 329.0947; found: 329.0937; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 741 (Cl).4-allyl-2-methoxyphenyl (E)-3-(4-chlorophenyl)acrylate (c7). White soild; yield 62% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 282.3–283.3 °C; 1H NMR  δ 7.82 , 7.52 , 7.50 , 7.40 , 7.38 , 7.02 , 6.82 , 6.80 , 6.64 , 5.98 , 5.17–5.09 , 3.83 , 3.40 ; 13C NMR  δ 165.0 (C=O), 151.0 (Ar-C), 145.1 (CH), 139.2 (Ar-C), 137.2 (CH), 136.7 (Ar-C), 136.6 (Ar-C), 132.9 (Ar-C), 129.6 (Ar-2C), 129.4 (Ar-2C), 122.7 (Ar-C), 120.8 (Ar-C), 117.8 (CH), 116.3 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.2 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17ClO3+: 329.0947; found: 329.0936; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 755 (Cl).4-allyl-2-methoxyphenyl (E)-3-(2-fluorophenyl)acrylate (c8). Yellow soild; yield 69% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 270.6–271.2 °C; 1H NMR  δ 8.00 , 7.61 , 7.39 , 7.20 , 7.13 , 7.04 , 6.83 , 6.81 , 6.77 , 6.04–5.94 , 5.16–5.08 , 3.84 , 3.41 ; 13C NMR  δ 165.1 (C=O), 162.6 (Ar-C), 160.6 (Ar-C), 151.1 (CH), 139.1 (Ar-C), 138.1 (CH), 137.2 (Ar-C), 132.2 (Ar-C), 132.1 (Ar-C), 129.4 (Ar-C), 124.7 (Ar-C), 124.7 (Ar-C), 122.7 (Ar-C), 120.8 (CH), 119.8 (CH), 116.3 (Ar-C), 112.9 (Ar-C), 56.0 (OCH3), 40.2 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17FO3+: 313.1243; found: 313.1231; IR (cm−1): 2937 (-CH3O), 1704 (C=O), 1165 (Ar-C=C), 1317 (F).4-allyl-2-methoxyphenyl (E)-3-(3-fluorophenyl)acrylate (c9). Yellow soild; yield 45% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 279.6–280.4 °C; 1H NMR  δ 7.82 , 7.39 , 7.35 , 7.28 , 7.12 , 7.03 , 6.83 , 6.81 , 6.66 , 5.98 , 5.16–5.09 , 3.83 , 3.40 ; 13C NMR  δ 164.9 (C=O), 164.1 (Ar-C), 162.2 (Ar-C), 151.1 (Ar-C), 145.1 (Ar-C), 139.3 (CH), 138.0 (Ar-C), 137.2 (CH), 130.7 (Ar-C), 124.4 (Ar-C), 122.7 (Ar-C), 120.9 (Ar-C), 118.7 (Ar-C), 117.6 (CH), 116.3 (CH), 114.7 (Ar-C), 112.9 (Ar-C), 56.0 (OCH3), 40.2 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17FO3+: 313.1243; found: 313.1231; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 1325 (F).4-allyl-2-methoxyphenyl (E)-3-(4-fluorophenyl)acrylate (c10). Yellow soild; yield 50% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 270.4–270.6 °C; 1H NMR  δ 7.83 , 7.58 , 7.57 , 7.12 , 7.10 , 7.03 , 6.82 , 6.81 , 6.60 , 5.98 , 5.16–5.09 , 3.83 , 3.40 ; 13C NMR  δ 165.1 (C=O), 163.2 (Ar-C), 151.1 (Ar-C), 145.2 (CH), 139.2 (Ar-C), 138.0 (Ar-C), 137.2 (CH), 130.7 (Ar-C), 130.4 (Ar-C), 130.3 (Ar-C), 122.7 (Ar-C), 120.8 (Ar-C), 117.0 (CH), 116.4 (CH), 116.3 (Ar-C), 116.2 (Ar-C), 112.9 (Ar-C), 56.0 (OCH3), 40.3 (CH2). HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17FO3+: 313.1243; found: 313.1231; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 1323 (F).4-allyl-2-methoxyphenyl (E)-3-(2-bromophenyl)acrylate (c11). Yellow soild; yield 54% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 296.3–296.5 °C; 1H NMR  δ 8.25 , 7.70 , 7.64 , 7.36 , 7.26 , 7.05 , 6.83 , 6.81 , 6.63 , 6.04 , 5.16–5.09 , 3.84 , 3.41 ; 13C NMR  δ 164.7 (C=O), 151.1 (Ar-C), 144.8 (CH), 139.2 (Ar-C), 138.0 (CH), 137.2 (Ar-C), 134.5 (Ar-C), 133.7 (Ar-C), 131.6 (Ar-C), 128.0 (Ar-C), 127.9 (Ar-C), 125.7 (Ar-C), 122.7 (Ar-C), 120.8 (Ar-C), 120.0 (CH), 116.1 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.2 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17BrO3+: 373.0442; found: 373.0430 IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 568 (Br).4-allyl-2-methoxyphenyl (E)-3-(3-bromophenyl)acrylate (c12). White soild; yield 61% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 272.6–272.8 °C; 1H NMR  δ 7.78 , 7.73 , 7.54 , 7.50 , 7.29 , 7.02 , 6.82 , 6.80 , 6.66 , 5.98 , 5.16–5.09 , 3.83 , 3.40 ; 13C NMR  δ 164.8 (C=O), 151.0 (Ar-C), 144.8 (CH), 139.2 (Ar-C), 138.0 (Ar-C), 137.2 (CH2), 136.5 (Ar-C), 133.5 (Ar-C), 131.1 (Ar-C), 130.6 (Ar-C), 127.0 (Ar-C), 123.2 (Ar-C), 122.7 (Ar-C), 120.9 (CH), 118.8 (CH), 116.3 (Ar-C), 112.9 (Ar-C), 56.0 (OCH3), 40.3 (CH); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17BrO3+: 373.0442; found: 313.0433; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 559 (Br).4-allyl-2-methoxyphenyl (E)-3-(4-bromophenyl)acrylate (c13). White soild; yield 58% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 239.9–340.9 °C; 1H NMR  δ 7.80 , 7.56 , 7.54 , 7.45 , 7.43 , 7.02 , 6.82 , 6.80 , 6.66 , 5.98 , 5.15–5.09 , 3.83 , 3.40 ; 13C NMR  δ 165.3 (C=O), 151.3 (Ar-C), 145.4 (CH), 139.5 (Ar-C), 138.2 (Ar-C), 137.4 (CH), 133.6 (Ar-C), 132.6 (Ar-2C), 130.0 (Ar-2C), 125.3 (Ar-C), 123.0 (Ar-C), 121.1 (CH), 118.2 (CH), 116.6 (Ar-C), 113.1 (Ar-C), 56.3 (OCH3), 40.5 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17BrO3+: 373.0442; found: 373.0429; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 571 (Br).4-allyl-2-methoxyphenyl (E)-3-(3-hydroxyphenyl)acrylate (c14). White soild; yield 51% (the crude was purified using a mixture of PE/EtOAc 40:1); mp: 322.2–322.4 °C; 1H NMR  δ 7.80 , 7.25 , 7.12 , 7.03 , 7.00 , 6.86 , 6.82 , 6.81–6.78 , 6.61 , 6.01–5.92 , 5.14–5.08 , 3.82 , 3.38 ; 13C NMR  δ 165.7 (C=O), 156.3 (Ar-C), 151.0 (Ar-C), 146.7 (Ar-C), 139.3 (CH), 138.0 (Ar-C), 137.2 (CH), 135.8 (Ar-C), 130.2 (Ar-C), 122.7 (Ar-C), 121.0 (Ar-C), 120.9 (Ar-C), 118.0 (Ar-C), 117.3 (CH), 116.3 (CH), 114.8 (Ar-C), 112.9 (Ar-C), 56.0 (OCH3), 40.2 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H18O4+: 311.1286; found: 311.1275; IR (cm−1): 3376 (OH), 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(4-hydroxyphenyl)acrylate (c15). White soild; yield 47% (the crude was purified using a mixture of PE/EtOAc 40:1); mp: 296.3–296.5 °C; 1H NMR  δ 7.81 , 7.46 , 7.45 , 7.03 , 6.84 , 6.82–6.81 , 6.79 , 6.51 , 5.97 , 5.83 , 5.15–5.07 , 3.82 , 3.39 ; 13C NMR  δ 166.0 (C=O), 158.3 (Ar-C), 151.1 (Ar-C), 146.6 (CH), 139.2 (Ar-C), 138.0 (CH), 137.2 (Ar-C), 130.4 (Ar-2C), 127.0 (Ar-C), 122.8 (Ar-C), 120.9 (Ar-C), 116.3 (CH), 116.1 (Ar-2C), 114.3 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H18O4+: 311.1286; found: 311.1273; IR (cm−1): 3371 (OH), 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(2-nitrophenyl)acrylate (c16). Yellow soild; yield 42% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 295.2–295.4 °C; 1H NMR  δ 8.31 , 8.07 , 7.73 , 7.69 , 7.58 , 7.04 , 6.84–6.82 , 6.81 , 6.59 , 5.98 , 5.14–5.09 , 3.84 , 3.40 ; 13C NMR  δ 164.1 (C=O), 151.0 (Ar-C), 148.5 (Ar-C), 141.7 (CH), 139.3 (Ar-C), 138.0 (Ar-C), 137.2 (CH), 133.8 (Ar-C), 130.7 (Ar-C), 130.6 (Ar-C), 129.4 (Ar-C), 125.1 (Ar-C), 122.7 (Ar-C), 122.4 (Ar-C), 120.8 (CH), 116.3 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17NO5+: 340.1181; found: 340.1176; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1424 (NO2), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(3-nitrophenyl)acrylate (c17). White soild; yield 51% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 255.5–256.1 °C; 1H NMR  δ 8.44 , 8.26 , 7.90 , 7.88 , 7.61 , 7.03 , 6.83 , 6.81 , 6.79 , 5.98 , 5.16–5.08 , 3.83 , 3.40 ; 13C NMR  δ 164.4 (C=O), 151.7 (Ar-C), 148.8 (Ar-C), 143.5 (CH), 139.4 (Ar-C), 137.9 (Ar-C), 137.1 (CH), 136.1 (Ar-C), 133.9 (Ar-C), 130.2 (Ar-C), 124.9 (Ar-C), 122.8 (Ar-C), 122.6 (Ar-C), 120.8 (Ar-C), 120.5 (CH), 116.4 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17NO5+: 340.1181; found: 340.1178; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1421 (NO2), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(4-nitrophenyl)acrylate (c18). Yellow soild; yield 58% (the crude was purified using a mixture of PE/EtOAc 80:1); mp: 291.2–292.3 °C; 1H NMR  δ 8.28 , 8.27 , 7.89 , 7.74 , 7.72 , 7.03 , 6.83 , 6.81 , 6.79 , 5.97 , 5.16–5.09 , 3.83 , 3.40 ; 13C NMR  δ 164.3 (C=O), 151.0 (Ar-C), 148.8 (CH), 143.5 (Ar-C), 140.5 (CH), 139.5 (Ar-C), 137.8 (Ar-C), 137.0 (Ar-C), 129.0 (Ar-2C), 124.4 (Ar-2C), 122.6 (Ar-C), 121.6 (Ar-C), 120.9 (CH), 116.4 (CH), 112.8 (Ar-C), 56.0 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H17NO5+: 340.1181; found: 340.1178; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1424 (NO2), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(2-methoxyphenyl)acrylate (c19). White soild; yield 49% (the crude was purified using a mixture of PE/EtOAc 80:1); mp: 250.3–250.8 °C; 1H NMR  δ 8.17 , 7.57 , 7.38 , 7.03 , 6.99 , 6.94 , 6.82 , 6.81 , 6.78 , 5.99 , 5.16–5.08 , 3.90 , 3.83 , 3.40 ; 13C NMR  δ 165.8 (C=O), 158.7 (Ar-C), 151.2 (Ar-C), 142.1 (CH), 139.0 (Ar-C), 138.2 (CH), 137.3 (Ar-C), 131.9 (Ar-C), 129.5 (Ar-C), 123.4 (Ar-C), 122.8 (Ar-C), 120.9 (Ar-C), 120.8 (Ar-C), 117.7 (CH), 116.3 (CH), 112.9 (Ar-C), 111.3 (Ar-C), 56.0 (OCH3), 55.6 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H20O4+: 325.1437; found: 325.1432; IR (cm−1): 2937, 2873 (OCH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(3-methoxyphenyl)acrylate (c20). White soild; yield 60% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 246.3–246.4 °C; 1H NMR  δ 7.84 , 7.33 , 7.18 , 7.11 , 7.03 , 6.97 , 6.83 , 6.81 , 6.67 , 5.98 , 5.16–5.09 , 3.85 , 3.83 , 3.40 ; 13C NMR  δ 165.2 (C=O), 160.0 (Ar-C), 151.1 (Ar-C), 146.5 (CH), 139.1 (Ar-C), 138.1 (CH), 137.2 (Ar-C), 135.8 (Ar-C), 130.1 (Ar-C), 122.8 (Ar-C), 121.2 (Ar-C), 120.9 (Ar-C), 117.5 (Ar-C), 116.7 (CH), 116.3 (CH), 113.1 (Ar-C), 112.9 (Ar-C), 56.0 (OCH3), 55.4 (OCH3), 40.2 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H20O4+: 325.1437; found: 325.1432; IR (cm−1): 2937, 2867 (OCH3), 1704 (CO), 1165 (PH-C=C).4-allyl-2-methoxyphenyl (E)-3-(4-methoxyphenyl)acrylate (c21). White soild; yield 62% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 328.6–329.0 °C; 1H NMR  δ 7.83 , 7.55 , 7.53 , 7.03 , 6.94 , 6.93 , 6.82 , 6.80 , 6.54 , 5.98 , 5.17–5.08 , 3.85 , 3.83 , 3.40 ; 13C NMR  δ 165.6 (C=O), 161.7 (Ar-C), 151.2 (Ar-C), 146.3 (CH), 139.0 (Ar-C), 138.2 (CH), 137.2 (Ar-C), 130.1 (Ar-2C), 127.2 (Ar-C), 122.9 (Ar-C), 120.9 (Ar-C), 116.3 (CH), 114.6 (CH), 114.5 (Ar-2C), 112.8 (Ar-C), 56.0 (OCH3), 55.5 (OCH3), 40.2 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H20O4+: 325.1437; found: 325.1432; IR (cm−1): 2937, 2871 (OCH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(2-(trifluoromethyl)phenyl)acrylate (c22). White soild; yield 47% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 339.4–340.1 °C; 1H NMR  δ 8.25 , 7.73 , 7.61 , 7.54–7.49 , 7.05 , 6.83 , 6.81 , 6.65 , 5.98 , 5.16–5.09 , 3.84 , 3.41 ; 13C NMR  δ 164.4 (C=O), 151.1 (Ar-C), 141.9 (Ar-C), 139.2 (CH), 138.0 (Ar-C), 137.2 (CH), 133.3 (Ar-C), 132.3 (Ar-C), 130.0 (Ar-C), 129.3 (Ar-C), 128.2 (Ar-C), 126.4 (Ar-C), 125.1 (Ar-C), 122.7 (Ar-C), 121.6 (CF3), 120.9 (CH), 116.3 (CH), 113.9 (Ar-C), 56.0 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H17F3O3+: 363.1205; found: 363.1198; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1332 (CF3), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(3-(trifluoromethyl)phenyl)acrylate (c23). Yellow soild; yield 69% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 339.1–339.3 °C; 1H NMR  δ 7.88 , 7.83 , 7.76 , 7.67 , 7.55 , 7.03 , 6.83 , 6.81 , 6.74 , 5.98 , 5.16–5.09 , 3.83 , 3.40 ; 13C NMR  δ 164.7 (C=O), 151.0 (Ar-C), 144.7 (CH), 139.3 (Ar-C), 138.0 (CH), 137.2 (Ar-C), 135.2 (Ar-C), 131.8 (Ar-C), 131.4 (Ar-C), 129.7 (Ar-C), 127.1 (CF3), 125.0 (Ar-C), 122.7 (Ar-C), 120.9 (Ar-C), 119.3 (Ar-C), 116.4 (CH), 112.9 (CH), 56.0 (Ar-C), 55.9 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H17F3O3+: 363.1205; found: 363.1199; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1330 (CF3), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(4-(trifluoromethyl)phenyl)acrylate (c24). White soild; yield 42% (the crude was purified using a mixture of PE/EtOAc 60:1); mp: 343.2–343.3 °C; 1H NMR  δ 7.88 , 7.68 , 7.68 , 7.68 , 7.68 , 7.03 , 6.83 , 6.81 , 6.75 , 5.98 , 5.16–5.06 , 3.83 , 3.41 ; 13C NMR  δ 164.7 (C=O), 151.0 (Ar-C), 144.6 (CH), 139.4 (Ar-C), 137.0 (Ar-C), 137.8 (CH), 137.1 (Ar-C), 132.0 (Ar-C), 128.5 (Ar-2C), 126.1 (Ar-2C), 126.0 (CF3), 122.7 (Ar-C), 120.9 (Ar-C), 119.9 (CH), 116.4 (CH), 112.9 (Ar-C), 56.0 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H17F3O3+: 363.1205; found: 363.1200; IR (cm-1): 2937 (OCH3), 1704 (C=O), 1331 (CF3), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(4-(dimethylamino)phenyl)acrylate (c25). Yellow soild; yield 29% (the crude was purified using a mixture of PE/EtOAc 40:1); mp: 328.6–329.1 °C; 1H NMR  δ 7.80 , 7.49 , 7.47 , 7.02 , 6.81 , 6.79 , 6.70 , 6.68 , 6.44 , 5.98 , 5.17–5.07 , 3.83 , 3.39 , 3.04 ; 13C NMR  δ 166.2 (C=O), 152.0 (CH), 151.3 (Ar-C), 147.2 (Ar-C), 138.8 (CH), 138.4 (Ar-C), 137.3 (Ar-C), 130.2 (Ar-C), 123.0 (Ar-C), 120.8 (Ar-C), 116.2 (CH), 112.8 (CH), 112.0 (Ar-C), 111.3 (Ar-C), 56.0 (OCH3), 40.3 ((CH3)2), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C21H23NO3+: 338.1754; found: 338.1746; IR (cm−1): 2937 (OCH3), 2925 (CH2), 2968 (CH3), 1704 (C=O), 1609 (N), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(4-hydroxy-3-methoxyphenyl)acrylate (c26). White soild; yield 56% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 274.3–274.8 °C; 1H NMR  δ 7.80 , 7.13 , 7.10 , 7.02 , 6.94 , 6.82 , 6.80 , 6.52 , 5.99 , 5.95–5.87 , 5.16–5.08 , 3.93 , 3.83 , 3.40 ; 13C NMR  δ 165.6 (C=O), 151.2 (Ar-C), 148.4 (Ar-C), 146.9 (Ar-C), 146.7 (CH), 139.0 (Ar-C), 138.2 (CH), 137.2 (Ar-C), 127.0 (Ar-C), 123.6 (Ar-C), 122.8 (Ar-C), 120.8 (Ar-C), 116.3 (Ar-C), 114.9 (CH), 114.5 (CH), 112.9 (Ar-C), 109.5 (Ar-C), 56.1 (OCH3), 56.00 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H20O5+: 341.1386; found: 341.1380; IR (cm−1): 3239 (OH), 2937, 2854 (OCH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-acrylate (c27). Yellow soild; yield 37% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 265.5–266.5 °C; 1H NMR  δ 7.72 , 7.04 , 7.00 , 6.98 , 6.82 , 6.79 , 6.77 , 6.42 , 5.98–5.91 , 5.14–5.07 , 3.79 , 3.37 ; 13C NMR  δ 166.1 (C=O), 150.8 (Ar-C), 146.9 (CH), 146.8 (Ar-C), 143.9 (Ar-C), 139.0 (Ar-C), 137.8 (CH), 136.9 (Ar-C), 127.0 (Ar-C), 122.7 (Ar-C), 122.5 (Ar-C), 120.7 (Ar-C), 116.1 (Ar-C), 115.3 (CH), 114.4 (CH), 114.0 (Ar-C), 112.7 (Ar-C), 55.8 (OCH3), 40.0 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H18O5+: 327.1236; found: 327.1225; IR (cm−1): 3217, 3186 (OH), 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C).4-allyl-2-methoxyphenyl (E)-3-(4-chloro-3-fluorophenyl)acrylate (c28). Yellow soild; yield 40% (the crude was purified using a mixture of PE/EtOAc 80:1); mp: 264.2–264.9 °C; 1H NMR  δ 7.76 , 7.63 , 7.45 , 7.19 , 7.02 , 6.82 , 6.80 , 6.60 , 5.98 , 5.16–5.09 , 3.83 , 3.40 ; 13C NMR  δ 164.7 (C=O), 160.4 (Ar-C), 158.3 (Ar-C), 151.0 (CH), 143.8 (Ar-C), 139.2 (CH), 137.9 (Ar-C), 137.2 (Ar-C), 131.8 (Ar-C), 130.4 (Ar-C), 128.3 (Ar-C), 122.7 (Ar-C), 120.9 (Ar-C), 118.4 (CH), 117.4 (CH), 116.3 (Ar-C), 112.8 (Ar-C), 56.0 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C19H16ClFO3+: 347.0853; found: 347.0841; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 1046 (F), 728 (Cl).4-allyl-2-methoxyphenyl (E)-3-acrylate (c29). White soild; yield 55% (the crude was purified using a mixture of PE/EtOAc 100:1); mp: 267.2–268.1 °C; 1H NMR  δ 7.77 , 7.09 , 7.06 , 7.02 , 6.84 , 6.82 , 6.80 , 6.49 , 6.02 , 6.01–5.90 , 5.15–5.06 , 3.83 , 3.40 ; 13C NMR  δ 165.5 (C=O), 151.2 (Ar-C), 150.0 (Ar-C), 148.5 (Ar-C), 146.3 (Ar-C), 139.1 (CH), 138.1 (Ar-C), 137.2 (CH), 128.9 (Ar-C), 125.0 (Ar-C), 122.8 (Ar-C), 120.8 (Ar-C), 116.3 (CH), 115.1 (CH), 112.9 (Ar-C), 108.7 (Ar-C), 106.8 (Ar-C), 101.8 (CH2), 56.0 (OCH3), 40.3 (CH2); HRMS (ESI-MS) m/z: [M + H]+ calcd for C20H18O5+: 339.1229 found: 339.1224; IR (cm−1): 2937 (OCH3), 1704 (C=O), 1165 (Ar-C=C), 953, 946 (OCH2).c1~c29 were tested for their tyrosinase inhibitory activities according to previous reports [Compounds  reports ,54. Eachc27  was recorded at 475 nm, respectively.The kinetics studies were carried out using the same experimental method as described above. For enzyme kinetics, the absorbance of the mixture containing different concentrations of tyrosinase  and compound c27  was recorded at 475 nm, respectively. For substrate kinetics, the absorbance of the mixture containing different concentrations of L-DOPA  and compound c27 mixture was measured according to previous reports [c27 (2 mM) was added and incubated for 5 min. Then, the 3D fluorescence spectra analyses were conducted in room temperature. The excitation and emission wavelengths were 200–600 nm and the slit width was 2.5 nm.The 3D fluorescence spectra of tyrosinase-compound  reports . To 3 mLc27 mixture were measured according to previous reports [c27  was added and incubated for 5 min. Then, the CD spectra were measured at 195~250 nm at room temperature.The CD spectra of tyrosinase-compound  reports . To 190 c27 solution was titrated into 2 mL of tyrosinase and incubated for 5 min. Then, the fluorescence spectra were determined at excitation wavelength of 280 nm and temperatures of 303, 307, and 310 K, respectively. The quenching mechanism and parameters were investigated by the Stern–Volmer equation.An amount of 1 μL of compound c27 was conducted using SYBYL software according to previous reports [c27 was treated with hydrogenation and energy minimization with the Gasteiger–Hückel program. The crystal structure of tyrosinase (PDBID: 2Y9X) was obtained from the RCSB Bank. Tyrosinase protein was analyzed with the internal program and we generated the active pocket using ligand mode. Then, the docking program was conducted in the default format to simulate the docking between tyrosinase and compound c27. In the docking process, 20 poses were generated for compound c27. We selected the pose with the highest score to further analyze the detailed interaction.Molecular docking of tyrosinase–compound  reports ,58,59,60c1~c29 and assayed their tyrosinase inhibitory activities. The results showed that all cinnamic acid–eugenol esters c1~c29 exhibited definite tyrosinase inhibitory activities. Compound c27 was the strongest tyrosinase inhibitor (IC50 = 3.07 ± 0.26 μM), and thus validated as a reversible mixed-type inhibitor against tyrosinase. Three-dimensional fluorescence and CD spectra results indicated that compound c27 could change the conformation and secondary structure of tyrosinase. Fluorescence quenching results showed that compound c27 quenched tyrosinase fluorescence in a static manner with one binding site. Molecular docking results also revealed the binding interactions between compound c27 and tyrosinase. Therefore, cinnamic acid–eugenol esters, especially c27, could be used as lead compounds to find potential tyrosinase inhibitors.In this study, we synthesized cinnamic acid–eugenol esters


Does a preoperative walkthrough of a patient’s day of surgery using augmented reality (AR) effect the patient’s anxiety levels?In this randomized clinical trial that included 95 patients, patients who received the preoperative AR experienced a significant decrease in preoperative State-Trait Anxiety Inventory (STAI) score compared with the control group that received standard education materials. There was no significant difference in STAI scores after operations.These findings suggest that a preoperative AR walkthrough may be a useful tool for reducing preoperative anxiety, but its impact on postoperative anxiety is less clear. This randomized clinical trial assesses whether the use of an augmented reality walkthrough of the day of surgery reduces patient perioperative anxiety compared with a standard educational control. Both augmented reality (AR) and virtual reality (VR) have had increasing applications in medicine, including medical training, psychology, physical medicine, rehabilitation, and surgical specialties, such as neurosurgery and orthopedic surgery. There are little data on AR’s effect on patients’ anxiety and experiences.To determine whether the use of an AR walkthrough effects patient perioperative anxiety.This randomized clinical trial was conducted at an outpatient surgery center in 2021 to 2022. All patients undergoing elective orthopedic surgery with the senior author were randomized to the treatment or control group. Analyses were conducted per protocol. Data analysis was performed in November 2022.AR experience explaining to patients what to expect on their day of surgery and walking them through the surgery space. The control group received the standard educational packet.The main outcome was change in State-Trait Anxiety Inventory (STAI) from the screening survey to the preoperative survey.P = .01). All patients postoperatively experienced a mean decrease in anxiety score compared with both the screening survey  and preoperative survey . Of 42 patients in the AR group who completed the postoperative follow-up survey, 30 (71.4%) agreed or strongly agreed that they enjoyed the experience, 29 (69.0%) agreed or strongly agreed that they would recommend the experience, and 28 (66.7%) agreed or strongly agreed that they would use the experience again. No differences were observed in postoperative pain levels or narcotic use.A total of 140 patients were eligible, and 45 patients either declined or were excluded. Therefore, 95 patients  were recruited for the study and included in the final analysis; 46 patients received the AR intervention, and 49 patients received standard instructions. The AR group experienced a decrease in anxiety from the screening to preoperative survey , while the standard care group experienced an increase (mean score change, 2.6 ; In this randomized clinical trial, the use of AR decreased preoperative anxiety compared with traditional perioperative education and handouts, but there was no significant effect on postoperative anxiety, pain levels, or narcotic use. These findings suggest that AR may serve as an effective means of decreasing preoperative patient anxiety.NCT04727697ClinicalTrials.gov Identifier:  These technologies have gained much interest with applications in gaming, home layout, and navigation.14 Within medicine, the use of AR and VR is still in its infancy, but they have begun to be used in a multitude of settings, including both medical training and patient care.15 Limited reviews within the literature have suggested that AR and VR are successful tools for patient education and satisfaction.17Advances in the preparation and delivery of patient care and education have begun to incorporate augmented reality (AR) and virtual reality (VR). AR has been defined as the superimposition of interactable virtual objects onto a physical environment in space.18 physical medicine, and rehabilitation19 and more broadly as a tool for pain and anxiety reduction when used as a distraction to decrease the perception of reality during hospitalization, venipuncture, wound care, chemotherapy, physical therapy, and routine dental and endoscopic procedures.26 This has even been demonstrated to be an effective adjunct in surgical procedures using wide-awake local anesthesia, regional anesthesia, and spinal anesthesia.31AR and VR have shown therapeutic benefits in psychology,While AR and VR have been used to improve patient education about the specifics of their procedure, to our knowledge, no studies have been performed that examine AR’s ability to affect patient anxiety and experience in the outpatient surgical setting by providing the patient a step-by-step walkthrough of their day of surgery. We hypothesize that by applying the AR to perioperative patient education and experience, we can decrease perioperative patient anxiety and improve patient experience and satisfaction.CONSORT) reporting guideline. The trial protocol and statistical analysis plan are presented in This randomized clinical trial was approved by the institutional review board at the University of Miami. All participants provided verbal informed consent or assent. This study followed the Consolidated Standards of Reporting Trials . Recruitment began in November 2021, and the follow-up for the final patient was completed in October 2022.To investigate the effect of a preoperative AR training experience on patient anxiety, we randomized 140 patients to receive either the AR experience or the standard set of preoperative instructions as a control. The interactive AR experience was a custom application developed in-house in collaboration with our institution’s Center for Computational Science by Xennial Digital Studios. The AR intervention visually walks patients through their trip to the operating room with narration from their surgeon while using an AR headset. The development was funded by an institutional grant of $10 000 that covered the costs of the application design and development. The AR experience takes approximately 3 minutes. Inclusion criteria consisted of all patients aged 12 years or older who were scheduled to have an ambulatory procedure with our senior author. Patients were excluded if they did not speak English (as the AR application was recorded only in English) or if they had circumstances that would prevent them from meeting or performing study requirements, such as canceling surgery or not completing the required surveys. After patients were indicated for surgery and given a surgical date, verbal informed consent was obtained. Patients were then randomized into 1 of 2 groups (AR or control) using a randomization table by whoever was performing the enrollment  . Patient32 It was administered at 4 different time points to both the control and AR groups  to quantify levels of patient anxiety. The STAI was administered at all points and for all groups via an online survey system. The screening STAI was administered immediately after enrollment to the study for both groups and served as a baseline anxiety index for each patient. After completing the preintervention STAI, patients in the AR group used the AR headset to receive preoperative educational training. Patients assigned to the control group did not receive any type of additional verbal, written, video, AR, or other source of education. On completion of the AR preoperative education intervention for the AR group and after a brief waiting period for the control group (3-5 minutes), the postintervention STAI was administered to measure any immediate effect of the preoperative education between the groups. This concluded their preoperative visit. The night before their scheduled procedures, patients in both groups were contacted and asked to fill out the presurgery STAI to assess anxiety at that time. Following their surgical procedure, patients in both groups were contacted at the time of their first postoperative visit to complete the postoperative STAI survey. Our primary end point was the difference in STAI from screening survey to preoperative survey, with secondary end points screening to postintervention survey, screening to postoperative survey, and preoperative to postoperative survey. We additionally collected patient-reported pain scores based on the visual analog scale  and the number of pain pills used on postoperative day 1 and at the time of their postoperative visit 7 to 10 days after surgery, in addition to overall feedback and satisfaction with the experience.34To quantify anxiety levels, we used the State-Trait Anxiety Inventory , which is a validated and widely used survey to determine patients’ anxiety.10An a priori power analysis was conducted to determine the number of patients needed to detect a difference in STAI scores between the groups. To detect a clinically meaningful difference of a change of 5 points in the STAI with a power of 0.8, a significance level of 0.05, and an SD of 8.9, 51 patients were needed in each group. The SD used was based on a separate study involving patients undergoing procedures under general anesthesia.To determine whether we were appropriately powered to detect a clinically meaningful difference of 2 points in VAS, we conducted a post-hoc power analysis: with 80% power to detect α = .05, based on our VAS SD of 3.12, we required 40 patients in each group; as there were only 24 patients who completed the pain portion of the postoperative survey, we did not reach appropriate confidence for definitive conclusion for this comparison.U tests were used to compare continuous data, and Fisher Exact tests were used to compare categorical data, unless otherwise indicated. Significance was assessed at P < .05, and all P values were 2-tailed. All data analysis was performed using Python version 3.8.10 (Python Software Foundation) and R version 4.2.1  software, with the libraries RPy2 3.5.4, NumPy 1.23.3, and SciPy 1.9.1 for Python and Stats 4.2.1 for R. Data analysis was performed in November 2022.Continuous variables are presented as mean and SD. Categorical variables are presented as count and percentage. Mann-Whitney Of 140 eligible patients, 45 patients either declined or were excluded; therefore, 95 patients  were recruited for the study and included in the final analysis, with 46 patients randomized to receive the AR intervention and 49 patients randomized to the standard instructions control group. There were no significant differences between AR and control groups with respect to age , sex , ASA score , smoking history , psychiatric comorbidity (1 patient [2.2%] vs 6 patients [12.2%]), surgical location , days between the preoperative appointment and date of surgery , or screening STAI score  . A totalP = .01). There also was a significant mean difference between the groups regarding screening to preoperative surveys, with the AR group again demonstrating a decrease in STAI  while the standard group showed an increase . These data suggest that the AR intervention decreased preoperative anxiety not only immediately—as evidenced by the initial decrease in STAI—but also with a lasting effect until the time of surgery.To determine whether patients who received the AR experience had a change in their preoperative anxiety, we compared the differences in postintervention and preoperative STAI scores vs screening scores . There wWe additionally compared the changes from the screening and preoperative surveys to postoperative surveys . There wTo determine whether the AR intervention influenced postoperative pain or narcotic use, patients were asked their pain levels as well as narcotic use both on postoperative day 1 and at the time of the postoperative appointment (7 to 10 days after surgery). There were no significant differences between groups with respect to postoperative pain or narcotic pill use on postoperative day 1 or at the time of the postoperative appointment (eTable in P = .71). Of patients who received the AR intervention, there were no adverse effects, such as visually induced motion sickness, experienced.Among patients who underwent the AR intervention, 42 patients were additionally asked about their overall experience with the AR application. Most patients either agreed or strongly agreed that they enjoyed the AR experience (30 patients [71.4%]), would recommend the experience (29 patients [69.0%]), and would do it again (28 patients [66.7%]) . HoweverOur randomized clinical trial focused on the effect of AR on the patient’s entire experience at our institution’s outpatient surgical center, from initial outpatient preoperative visit to completion of the scheduled surgery, and its effects on perceived anxiety and satisfaction. Our custom AR experience allowed a patient to visually walk through their day at the surgery center with narration from their surgeon, and it was designed to emulate what the patient would encounter on the day of surgery. This experience included a visual representation of the building, layout of the surgical center, progression from check-in to preoperative care, to the operating room, and finally postoperative care. Furthermore, a strength of our AR technology was that patients had the opportunity to revisit modules of the AR experience if they wanted to view them again. Users could also interact with the AR application by zooming in and out and rotating the model for a more personalized experience. The ultimate goal of our AR experience was to educate and prepare the patient for what to expect, potentially mitigating the cognitive and physiological burden of anxiety they perceived surrounding their surgery.We found that our AR experience significantly decreased patients’ preoperative anxiety even immediately from the time of screening to after intervention completion, with lasting effects until the time of surgery. This is contrasted with the control group, who had little significant change in their screening to postintervention surveys but did experience an increase in anxiety from screening to preoperative surveys. These changes subsided after surgery, with all patients showing a decrease in anxiety from their screening survey, with no significant difference between the groups. The focus of our AR application was on the day-of-surgery events and the immediate preoperative experience, which might explain the lack of observed postoperative differences. However, this study was underpowered, so this lack of statistical power increases the risk of type II error, potentially masking a significant effect.35 While our study showed no significant difference in pain or narcotic use, the required number of participants was not met, so a type II error may be present.We also examined the AR experience’s effect on postoperative pain and narcotic use. There were no significant differences between groups on VAS pain scores or narcotic use. These results are limited by the fact that they were patient-reported at the time of their postoperative survey and could thus be influenced by recall bias. Our study could have been strengthened by contacting patients on postoperative day 1 to determine VAS and narcotic use at that time point. We conducted a post-hoc power analysis to assess whether this study was appropriately powered to detect a clinically meaningful difference in VAS, and we did not reach appropriate confidence for definitive conclusion for this comparison. Previous studies have suggested that pain scores might be improved with the assistance of VR.39While most patients who completed the AR experience did enjoy it, some did not. Indeed, approximately 10% to 15% of patients disagreed or strongly disagreed that they enjoyed the experience, would recommend it, and would do it again. Specific factors cited for their criticism were disorientation associated with using the AR headset, as well as the quality of the animations present. Patients with glasses sometimes had difficulty with the headset and fitting their glasses inside it so that they could see it project across the room. Although visually induced motion sickness is a well-described phenomenon with VR and AR, no patients in our study experienced any.We additionally examined whether patient experience with AR had any association with their anxiety levels. Interestingly, not enjoying the AR experience was not associated with the intervention’s efficacy of decreasing preoperative anxiety, as there was no significant difference in STAI score from screening to the preoperative survey between patients who underwent the AR experience and were neutral, enjoyed, or strongly enjoyed the experience compared with those who did not enjoy or strongly did not enjoy it. This suggests that the mental exercise of experiencing the operative day walkthrough in AR has an anxiolytic effect and is not dependent on ultimate enjoyment.35 performed a single-blind randomized clinical trial to investigate whether VR as a preparation tool for elective outpatient surgery in children was associated with lower levels of anxiety, pain, and emergence delirium from anesthesia compared with a control group receiving traditional care without VR.35 On the day of surgery, children receiving VR were exposed to a realistic, child-friendly immersive virtual version of the operating room so that they could get accustomed to the environment and anesthesia procedures. No differences between groups were found in self-reported anxiety, pain, emergence delirium, or parental anxiety, suggesting that VR in the pediatric population is not able to overcome the fear and anxiety experienced by a child before surgery. One possible explanation for this could be that a child’s brain has not fully developed the higher-level processing capabilities needed to fully integrate a VR experience into their reality or the necessary coping skills to navigate the experience of being a surgical patient. Of note, all patients in the study by Eijlers et al35 were aged 4 to 12 years, all of whom were younger than our youngest patient (age 14 years).To our knowledge, limited randomized clinical trials have investigated the utility of an AR educational experience to prepare a patient for their day of surgery. A 2019 study by Eijlers et al40 Thus, to our knowledge, our study is the first of its kind to investigate AR’s application to the patient’s perioperative experience and demonstrate that AR decreased perioperative anxiety.Other studies have looked at VR and AR and patient education focusing on digital imaging and 3-dimensional models to educate the patient on the specifics of their involved anatomy and provide a detailed visual representation of the steps of the surgery, yet these were primarily interested in patient understanding of the disease process and not on the effect on perioperative anxiety.Future directions include the inclusion of multiple centers with multiple surgeons and expansion to other languages. Our current experience focused exclusively on the day-of-surgery experience and did not provide any postoperative instructions, such as wound care or physical therapy, so adding these components might also impact patient experience and potentially even outcomes.Our study has some limitations. First, this study was performed at a single institution with a single surgeon, and it was custom built to depict the exact process our patients experience. Generalizability is thus limited, since patients at other centers or with other surgeons may not experience a benefit. Additionally, we did not control for the presence of previous surgical procedures between the groups: a patient who has undergone surgery previously might not experience as much anxiety as someone who has never undergone surgery and is not as engaged in the experience. Furthermore, since participation was voluntary, our results may have been influenced by self-selection bias. Once enrolled, some patients no longer wished to complete the surveys, and this accounted for a small but noticeable attrition rate between patients who completed the preoperative surveys and those who did not complete either the entire or portions of the postoperative survey. Additionally, the study was underpowered in both groups, which increases the risk of type II error and reduces our confidence in the findings that were not statistically significant.In this randomized clinical trial, the administration of a preoperative AR experience decreased preoperative patient anxiety, and with most patient enjoying the experience, but there was no significant effect on postoperative anxiety, pain levels, or narcotic use. These findings suggest that the use of AR may serve as a means of decreasing preoperative patient anxiety.


In King Oyster mushroom slices, 0.08% phytic acid combined with 0.1% glutathione inhibited the PPO activity by 97.6%, suppressed browning by 78.09% after 6 h of drying at 60 °C and inhibited browning by 69.93% and oxidation by 78.75% after 12 months of storage at ∼ 20 °C. Results indicated that using phytic acid combined with glutathione may inhibit browning and suppress the oxidation of King Oyster mushroom slices during drying and storage.Phytic acid and glutathione can inhibit polyphenoloxidase (PPO) activity and suppress browning. This study investigated the effects of phytic acid alone (Treatment-1) or combined with glutathione (Treatment-2) on inhibiting browning and oxidation resistance of King Oyster mushroom  Afterwards, the supernatant was collected to yield crude enzyme extract. One gram of acetone powder was added to 20 mL of 0.025 mol/L phosphate-buffered solution (pH 7.2), homogenised using a magnetic stirrer at 0 ℃ for 30 min and centrifuged at 12,000 × g for 30 min. Then, the supernatant was taken as a crude enzyme extract. NH4SO4 was added into the crude enzyme extract to 80% saturated solution and stirred for 30 min. Next, the mixture was placed overnight, centrifuged at 12,000 × g for 30 min, precipitated and dissolved in 0.025 mol/L phosphate buffer (pH 7.2) and dialysed with 10 mmol/L Tris-HCl solution (pH 8.3) for 18 h. During dialysis, Tris-HCl solution was replaced three times, and the final product was purified PPO.One hundred grams of King Oyster mushroom was added to 200 mL of glacial acetone (approximately − 20 ℃) and homogenised using a homogeniser for 5 min. The mixture was filtered using a medium-speed filter paper, and the residue was washed repeatedly with glacial acetone and filtered until it became a white powder, which was acetone powder. One gram of acetone powder was added to 20 mL of 0.025 mol/L phosphate-buffered solution (pH7.2), homogenised using a magnetic stirrer at 0 ℃ for 30 min and centrifuged at 12,000 × 2.32.75 mL of 0.025 mol/L phosphate-buffered solution (pH 7.2) was added into a 25 px colorimetric cup containing 0.1 mL of 0.15 mol/L catechol solution, mixed well and placed at room temperature for 3 min. Then, 0.1 mL of enzymatic solution was added. After 5 s, the change of the A420 value within 1 min was recorded. The amount of enzyme required to change the light absorption value by 0.001 per minute was one activity unit.2.4l-DOPA (45 °C) were added to initiate the reaction, which was performed at 45 °C. Absorbance was recorded at 475 nm for 3 min. The control experiments were performed using the same method; however, the modulating agent used was replaced with deionised. l-DOPA was replaced with distilled water to prepare the blank sample. An increase in the absorbance by 0.001 at 475 nm/min/mL was used to define a unit of PPO activity (1 U). The inhibitory activity was calculated in accordance with the following equation Different concentrations of phytic acid solution (0% to 0.12%) were used to assess PPO activity. One hundred microliters of crude PPO extract was mixed with 100 µL of phytic acid solution. After the mixture was incubated at ∼ 25 °C for 30 min, 400 µL of 0.05 mol/L of assay phosphate buffer (pH 6.0) and 600 µL of 15 mmol/L where A is the initial enzymatic activity in the control, and B is the residual enzymatic activity in the presence of an inhibitor.The maximum inhibition level of phytic acid (0.08%) combined with different concentrations of glutathione solution (0% to 0.12%) was evaluated for PPO inhibitory activity.2.5Fresh King Oyster mushrooms of the same size were washed and cut into slices with a similar thickness of 5 mm. Purified water (control), 0.08% phytic acid (Treatment-1) and 0.08% phytic acid + 0.1% glutathione (Treatment-2) were used as dipping solutions. A total of 400 King Oyster mushroom slices were randomly and evenly assigned to the control and treatment groups. The King Oyster mushroom slices were soaked in the two dipping solutions at 5 °C for 20 min. After draining the residual dipping solutions, the King Oyster mushroom slices were dried in an air-drying oven at 60 °C for 6 h, during which the colour of King Oyster mushrooms was measured every 1 h. The dried King Oyster mushroom slices were cooled and stored at ∼ 25 °C for 10 months, during which the colour and malondialdehyde (MDA) content of King Oyster mushrooms were measured every 2 months.2.6E) of the King Oyster Mushroom slices was calculated in accordance with Equation The colour of the King Oyster Mushroom slices was measured by using a colorimeter . The colour parameters were L* (brightness), a* (red/green) and b* (yellow/blue). The total colour difference . All data with the same drying or storage time were statistically analysed by using one-way analysis of variance and Tukey Kramer multi-range test.33.1The PPO activity can be inhibited by varying agents such as a reducing agent, chelating agent, complexing agent, acidulants, enzymatic inhibitor and enzymatic treatment agent . The inh3.2E values of the King Oyster mushroom slices during drying are presented in E values of the King Oyster mushroom slices in the control group increased sharply with drying time (P < 0.05). In addition, the ΔE values of the King Oyster mushroom slices in the treatment groups increased slowly, and they were lower than those of the control group after 6 h of drying, indicating that phytic acid alone or phytic acid combined with glutathione effectively suppressed the enzymatic browning of King Oyster mushroom slices during drying. The differences in the ΔE values between treatment-1 and treatment-2 groups increased during 6 h of dry; this could be due to the PPO activity and browning inhibition ability of glutathione . However, the ΔE values the King Oyster mushroom slices in the treatment groups increased slowly (from 5.12 to 18.51 for treatment-1 and from 2.28 to 8.29 for treatment-2), and they were lower than those of the control group after 12 months of storage (P < 0.05), indicating that phytic acid combined with glutathione effectively suppressed the enzymatic browning and non-enzymatic browning of King Oyster mushroom slices during drying. The differences in the ΔE values between treatment-1 and treatment-2 groups increased during 12 months of storage; this also could be due to the PPO activity and browning inhibition ability of glutathione . Nevertheless, the MDA contents in the King Oyster mushroom slices in the treatment groups increased slowly (from 1.07 µmol/L to 9.58 µmol/L for treatment-1 and from 0.61 µmol/L to 3.08 for treatment-2 µmol/L), and they were lower than that of the control group after 12 months of storage (P < 0.05), indicating that phytic acid alone or combined with glutathione effectively inhibited the lipid oxidation of King Oyster mushroom slices during drying. The differences in the MDS contents between treatment-1 and treatment-2 groups increased during 12 months of storage; this could be due to the antioxidant activity of glutathione (Dried King Oyster mushroom contains a small amount of easily oxidised lipids . The oxitathione  and show4Browning and oxidation decrease the commercial value of King Oyster mushrooms. In this study, 0.08% sodium phytic acid combined with 0.1% glutathione suppressed browning during drying and storage and inhibited the lipid oxidation of King Oyster mushrooms during storage. Thus, treatment with 0.08% sodium phytic acid combined with 0.1% glutathione may be a practical method to prepare and store dried King Oyster mushroom slices.Chao Gong: Investigation, Data curation, Formal analysis, Methodology, Software, Writing – original draft. Wenjuan Gao: Investigation, Methodology, Software, Validation, Visualization. Shengjun Wu: Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Project administration, Writing – original draft, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Persistent androgen receptor (AR) signalling is the main driver of prostate cancer (PCa). Truncated isoforms of the AR called androgen receptor variants (AR-Vs) lacking the ligand binding domain often emerge during treatment resistance against AR pathway inhibitors such as Enzalutamide. This review discusses how AR-Vs drive a more aggressive form of PCa through the regulation of some of their target genes involved in oncogenic pathways, enabling disease progression. There is a pressing need for the development of a new generation of AR inhibitors which can repress the activity of both the full-length AR and AR-Vs, for which the knowledge of differentially expressed target genes will allow evaluation of inhibition efficacy. This review provides a detailed account of the most common variant, AR-V7, the AR-V7 regulated genes which have been experimentally validated, endeavours to understand their relevance in aggressive AR-V driven PCa and discusses the utility of the downstream protein products as potential drug targets for PCa treatment. The proerfamily . The AR  TMPRSS2 Figure 1 TMPRSS2 . However TMPRSS2 .The androgen receptor itself is regulated by a number of transcription factors and related proteins. Transcription factors CREB, Myc, Sp1, Foxo3a, LEF1, NF-ĸB, Twist1, E2F, SREBP-1 have shown to positively regulate AR expression while c-Jun, p53 and Purα demonstrated negative regulation of AR expression. Other transcription cofactors, cytokines, signal transducers and RNA-binding proteins such as β-Catenin, Rb1, Smad2 and 3 and EBP1 also play a role in direct regulation of the AR. Further to this, there are known compounds and agents that alter AR expression by acting on transcription, translation and post-translational activity for example polyphenols acting on hormones and transcription factors . It is aGiven the mitogenic effects of androgens on prostate epithelial cells, the drugs used in the treatment of advanced PCa aim to suppress AR signalling. Androgen deprivation therapy (ADT) either surgically or chemically castrates patients to reduce the levels of free androgens in the blood , and thu1.2via heterodimerisation (There are ca. 20 different known AR-Vs which are truncated forms of the full-length AR (AR-FL) Figure 2risation , 18. In risation . Howeverrisation .The selection pressure of AR-targeting treatments induces genomic rearrangements and/or alternative splicing that form the AR-Vs –24. The There are a few studies that discuss how AR-Vs and specifically AR-V7, are regulated. Hu et al. concluded that AR-V protein expression is negatively regulated by AR-FL signalling ; Jones e1.3AR-V7 is produced in PCa cells as a result of aberrant splicing. It retains exons 1, 2, and 3 which code for the NTD, DBD, and part of the hinge region, as well as the cryptic exon 3 (CE3) that codes for an additional stretch of sixteen further amino acids Figure 2FOXA1)  , which mSince AR-Vs confer resistance to AR inhibition therapies, recent research has been focussing on how to inhibit their activity in CRPC. Targeting of AR-Vs has proven challenging, with new ways of inhibition being required for control in disease progression . Novel eSuccessful inhibition of AR-V7 can be experimentally verified by examining the gene expression signature of AR-V7 target genes and therefore, there is a need to identify AR-V7 exclusive target genes. This review will provide a comprehensive account of some of the key AR-V7 target genes and their relevance in ARI-resistant PCa.22.12.1.1EDN2) was first identified as an AR-V7 target gene in 2014 by Krause et al. The creation of AR-V7 expressing cell lines by transfection with lentivirus containing doxycycline (Dox)-inducible expression of AR-V7 enabled quantification of AR-V7-specific target gene expression by qPCR and therefore identified transcriptional up-regulation of EDN2 by AR-V7 (Endothelin-2 (by AR-V7 .EDN2 encodes the protein endothelin-2 (ET-2) which belongs to a group of secretory vasoconstrictive peptides that are part of the endothelin protein family and is co-regulated by the TF FOXA1 [FOXA1 has been described to co-regulate the expression of many genes with the AR  as another AR-V7 target gene  was first mentioned as an AR-V gene in metastatic PCa tissue  enzyme, UBE2C is essential for ubiquitination , 72. UBEgnalling , while agnalling , 74. Undgnalling , 75. Howgnalling .UBE2C is commonly upregulated in multiple cancers, inhibition of this gene has been explored, however inhibitors specifically for this protein are not widely used in the clinic. Bortezomib however is an FDA approved treatment for myeloma and has been shown to also prevent cell cycle progression in Colorectal Carcinoma   , 76.2.1.4NPR3) as an AR-V7- upregulated target gene  was compared to identify differentially expressed genes between the AR-Vs and AR-FL. These data identified Natriuretic Peptide Receptor 3 (get gene .NPR3 encodes for natriuretic peptide clearance receptor (NPCR). NPCR represents approximately 94% of natriuretic peptide receptors (NPRs) (via cyclic GMP activation (s (NPRs) , and rems (NPRs) . NPCR has (NPRs) , which hs (NPRs) , therefotivation , 84, whitivation . Indeed,tivation . Howevertivation . Thus, AInhibition of natriuretic peptide receptors has been investigated in literature, and a few antagonists of the receptors have been identified. Isatin has demonstrated inhibition of atrial natriuretic peptide receptor binding in mammalian cell models , while pNPR3 expression increases in PCa, and if NPR3 is specifically AR-V7 regulated. AR-V7 regulation of NPR3 expression, as described by Nagandla et al, would implicate NPR3 expression is upregulated in CRPC and is corroborated by Terada et al.  was identified as an AR-V7 target gene by Sugiura et al. following AR-V7-specific ChIP-sequencing investigating LNCaP-95 (PCa cell line expressing both AR-FL and AR-V7) vs LNCaP (PCa cell line expressing AR-FL only), where expression levels were more than two-fold higher in LNCaP-95   .SLC3A2 encodes for 4F2 heavy chain, aka CD98hc, a subunit of several heterometric amino acid transporters (SKP2)  . Other a cancers –99, incl cancers . Whilst on score , 100.BCH (2-amino-2-norbornane-carboxylic acid), a non-metabolisable amino acid, has shown to inhibit the disulphide bond-linked heterodimer that consists of CD98hc plus a catalytic light chain . This waSLC3A2 gene as specifically regulated by AR-V7 in LNCaP-95, an increase in SLC3A2 expression by androgen R1881 treatment has been reported which would suggest regulation by AR-FL as well   . Therefo2.1.6NUP210) was also identified as an AR-V7 gene by Sugiura et al., as it was expressed more than 2-fold higher in AR-V7-expressing LNCaP-95 than LNCaP. Specific knockdown of AR-V7 in LNCaP-95 caused a significant decrease in NUP210 expression. Furthermore, in the TCGA-PRAD dataset, NUP210 expression positively correlated with AR-V7 expression (NUP210 expression in the Grasso dataset (GSE35988) between benign prostate and primary PCa, where there is little AR-V7 expression  - which regulates nucleocytoplasmic transport, although it also has non-structural functions  with an aminocyclopropenone (ACP) has been explored, ACP-1n has demonstrated inhibition of NUP210 expression in colorectal cancer cells. This is due to NUP210 being a BRD4-driven nuclear complex component . Reducin2.1.7BIRC3), which encodes for Inhibitor of Apoptosis 2 (IAP-2) was first experimentally validated by Basil et al.   , which i2 (ELL2) , 118, an2 (ELL2) . TherefoThe endogenous antagonist of IAPs, second mitochondria-derived activator of caspase (SMAC) has been employed for drug design since the early 2000s and are now in their fourth generation. The fourth-generation optimised non-alanine agonist, ASTX600, showed growth repression in breast cancer mouse models and are now in phase I/II clinical trials , 120. SiBIRC3 is AR-V7 upregulated rather than AR-FL is compelling, BIRC3 can be regulated by other TFs in PCa. Wang et al. explored IAP-2 protein levels in several PCa cell lines including LNCaP, LNCaP-95 and VCaP  regulated gene  was experimentally shown to be an AR-V7 target gene in a variety of PCa cell lines including 22Rν1 and C4-2B-ENZ  2 and 1 respectively  and the transcription factor, E2F-1, along with other proteins. Nitrogen-containing bisphosphonates (N-BPs) however, seem to solely target and inhibit the expression of cyclin A2 at the transcriptional and consequently translational level in epithelial cell models . RXL pep2 is solely regulated by AR-V7. Cao et al. demonstrated that CCNA2 expression did not decrease with AR-FL knockdown or Enzalutamide  was first shown as an AR-V7 target gene by Jones et al., although had also been previously identified as a hub gene for AR-Vs  was first described as an AR-V target gene in a study by Liu et al. where metastatic PCa patient samples were collected and split into groups of low and high expression of AR-Vs and then gene profiles of each group were compared  and androgen insensitivity in castrate conditions by inducing increased testosterone excretion  gene was specifically regulated by AR-V7 in CRPC LNCaP-95 cells , activating the APC/CDC20 complex . The APCeuploidy , 174, thInhibition of CDC20 has been demonstrated in multiple cancer cell models including PCa by the compound Apcin –177. MecCDC20 to be generally upregulated by AR-Vs, none have specified which AR-Vs other than AR-V7 regulate this gene. Thus, further investigation is necessary in order to determine whether CDC20 is solely AR-V7 regulated  as an AR-V target gene  and synthetic cyclic peptides containing the cadherin CAR sequence HAV (e.g ADH-1) , 188. ADCDH2 is not expressed in HSPC , which encodes for zinc transporter member 7 (ZNT7)  .in vivo to be important for dietary zinc absorption and body fat accumulation (SLC30A7 in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse model of PCa accelerated prostate tumour formation   . B4GALT1B4GALT1) . Indeed,B4GALT1) , 199. B4eukaemia –203, buteukaemia , 60, 204eukaemia  and multeukaemia –209.There are no known B4GALT1 agonists, however one study found upregulation of B4GALT1 at the mRNA level by lectin stimulation  . This waB4GALT1 was downregulated by both Aldo-keto Reductase Family 1 member C3  and AR-V7, indicating a complex formed by these two proteins, transcriptionally repress B4GALT1  is the third gene that was found downregulated by AR-V7 by Cato et al.  is the last of the four AR-V7 downregulated genes identified by Cato et al. HIF1α is the alpha subunit of the HIF-1 TF and plays a large role in regulating the cellular homeostatic response in the event of hypoxia, through the transcriptional initiation of genes involved in angiogenesis, apoptosis, and increasing oxygen transportation ,. ,. AKT1 drts zinc , 258. HoThe fact that AR-Vs promote a more aggressive phenotype highlights the need for new therapeutics to inhibit them since the antiandrogen drugs currently in the clinic only target AR-FL. Many experimental drugs are currently being tested to determine their efficacy, such as those named in the review by Maylin et al.  and thusKM and IH wrote the majority of the manuscript with planning, direction and editing from ZM. ZM and CS wrote the discussion. ZM and IH prepared the figures. EA, CS, HP and MA further edited the manuscript and provided guidance throughout. All authors contributed to the article and approved the submitted version.