Odontites vulgaris Moench has the effect of clearing away heat, detoxification, dispelling wind, and clearing dampness. In this study, the potential anti-inflammatory compounds of O. vulgaris were investigated using ultra-high-performance liquid chromatography–mass spectrometry (UPLC–MS/MS) combined with the network pharmacology approach and further confirmed on an LPS-activated RAW 264.7 macrophage model. Monomer compounds were prepared from the active fraction using modern advanced separation and purification methods. UPLC–Q-Exactive HRMS was used to identify the chemical compounds in the active fractions of O. vulgaris. D-mannitol, geniposidic acid, salidroside, shanzhiside methyl ester, eleutheroside B, geniposide, 7,8-dihydroxycoumarin, gardoside methyl ester, arenarioside, vanillic acid, p-hydroxy-cinnamic acid, melampyroside, syringaresinol, tricin, and diosmetin were isolated from O. vulgaris for the first time. A compound database of O. vulgaris was established based on the existing literature to predict the mechanism of O. vulgaris in the treatment of rheumatoid arthritis. The results suggest that the PI3K-Akt pathway mediates O. vulgaris and deserves more attention in the treatment of RA. Finally, the anti-rheumatoid arthritis effects of the four target compounds were validated with the decreased levels of NO, TNF-α, IL-6 and IL-1β in RAW 264.7 macrophage cells treated with LPS. The present study explored the potential targets and signaling pathways of O. vulgaris in the treatment of RA, which may help to illustrate the mechanisms involved in the action of O. vulgaris and may provide a better understanding of the relationship between O. vulgaris and RA. This study provides novel insights into the development of new drugs and utilization of Mongolian traditional Chinese medicine resources. Thus fae system . The inte system . These Me system .Odontites vulgaris Moench  was kept in the herbarium of Baotou Medical College.β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were obtained from Beijing Solarbio Science & Technology Co. Ltd (China). Ultrapure water was obtained from Gen Pure . Acetonitrile (HPLC grade) was purchased from Thermo Fisher Scientific. All reagents were of analytical grade. Reference compounds for the 15 analytes were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . Purified water was obtained using a Milli-Q system .RAW 264.7 cells were purchased from the Cell Bank at the China Academy of Science . Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline, penicillin, and streptomycin were purchased from Gibco . Dimethyl sulfoxide (DMSO) and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Sangon Biotech . Lipopolysaccharides (LPS) and indomethacin were obtained from Sigma-Aldrich (United States). The nitric oxide (NO) kit was purchased from the Nanjing Jiancheng Bioengineering Institute . Enzyme-linked immunosorbent assay (ELISA) kits for interleukin-1O.vulgaris (6.04 kg) powder was successively extracted with 70% ethanol; approximately 1.2 kg of the extract was obtained. The extract was dissolved in water and extracted with petroleum ether, ethyl acetate, and n-butanol to obtain 17.56, 73, and 259 g of dry extracts, respectively. The aqueous phase contained 755 g of residue.Whole dried 2 and 37°C.RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS and a mixture of penicillin and streptomycin. The cells were incubated at 5% CO5 cells/ml were cultured at 37°C in a 5% CO2 incubator for 24 h. After adherence of the cells to the plates, a series of different concentrations  of the O.vulgaris extracts  was added. The control group (without the extract) was set at the same time. Five parallel wells were used in each group. After 48 h of culture, 10 μl of the freshly prepared 5 mg/ml MTT solution was added to each well, and the culture was incubated at 37°C for 4 h in the dark. The supernatant was discarded, and 150 µl of dimethyl sulfoxide (DMSO) was added to each well. After oscillation for 10 min, the absorbance (OD 570 nm) of each well was measured using a microplate analyzer .Logarithmic phase RAW 264.7 cells seeded in 96-well plates at a density of 1 × 105 cells/ml were placed in a 37°C 5% CO2 incubator for 24 h. After adherence to the plate, the cells were randomly divided into the control group, model group (LPS), and positive drug group (indomethacin group). Each group was placed in parallel holes. The cells were cultured with LPS (1 μg/ml) for 4 h, with the positive drug group being administered indomethacin solutions of different concentrations to co-interact with LPS cells for 20 h at the following concentrations: 6.25, 12.5, 25, 50, and 100 µM. The cell supernatant of each group was collected after culture, and NO release in the cell supernatant was detected according to the manufacturer’s instructions. The concentration of NO was determined using sodium nitrite as the control substance, and the 50% inhibition rate  was calculated according to the concentration of NO. Absorbance at 550 nm was determined using a microplate analyzer.Logarithmic phase RAW 264.7 cells seeded in 24-well plates at a density of 5 × 105 cells/ml in 24-well plates and placed at 37°C in a 5% CO2 incubator for 24 h until the logarithmic phase was reached. The group demarcation based on treatment in this study was as follows: control group with normal cell culture for 24 h without any treatment; inflammation model group with cells stimulated using 1 μg/ml LPS for 24 h; negative control group with cells treated using different extracts  of O.vulgaris at 400 μg/ml for 20 h; and the experimental group with cells stimulated with 1 μg/ml LPS for 4 h and different concentrations  of O.vulgaris extracts added to the cells for 20 h. The cell supernatant of each group was collected after culture, and NO release in the cell supernatant was detected according to the Griess test described in the NO kit. Absorbance was measured at 550 nm . After separation and purification, 7.6 mg of compound 1 and 2.4 mg of compound 2 were obtained. Fr. A2 was analyzed using atmospheric pressure silica gel column chromatography and confirmed using Sephadex gel column (Sephadex LH-20) chromatography. We obtained 5.3 mg of compound 3. Fr. A3 was further separated on an ODS column  into three compounds (B1–B3). Fr. B1 was repeatedly eluted with Sephadex LH-20  to yield 25.4 mg of compound 4 and 6.4 mg of compound 5. Fr. B2 was separated on a silica gel column  and eluted with methanol on a Sephadex LH-20 column to obtain 14 mg each of compounds 6 and 7. Compounds 8 (16.6 mg) and 9 (27.3 mg) were obtained from Fr. B3 using preparative HPLC. Compounds 10 (34.9 mg) and 11 (19.8 mg) were obtained from Fr. C4 by repeated elution with Sephadex LH-20 .The n-butanol extract (80 g) was separated on a D101 macroporous resin column , and eight fractions were obtained. Thin-layer chromatography was used to analyze the compounds. Three main fractions (Frs. A1–A3) were ultimately obtained by combining the fractions with similar polarity and compound composition. Separation was carried out on an octadecylsilyl (ODS) column  for chromatographic analysis, and nine fractions (D1–D9) were obtained. Compounds 12 (9 mg) and 13 (10 mg) were obtained using preparative liquid chromatography. Compounds 14 (34.0 mg) and 15 (22 mg) were obtained from Fr. D3–D5 using a silica gel column and a Sephadex LH-20 column. Compounds 16 (20.4 mg) and 17 (18.7 mg) were isolated from Fr. D8–D9 using RP-18, methanol-water gradient elution, Sephadex LH-20, and preparative HPLC.Ethyl acetate extract (60 g) was prepared using a small pore resin coagulant gel column  in methanol and the sample was filtered through a 0.22 µm microporous membrane to obtain the test solution, and 10 µl of the filtered solution was injected into the instrument.A C18 column  was used in the experiment. The mobile phase consisted of 0.1% formic acid aqueous solution (A), acetonitrile (B), gradient elution, 0–35 min, 95%–5% (A), 5–95% (B). The injection volume was 1 μl, flow rate was 0.3 ml/min, and column temperature was 25°C.2, full MS resolution 70,000; and dd-MS2 resolution 17,500, scanning range m/z 100–1,500. For MS/MS, the collision energies were 20, 40, and 60 eV. All MS spectra were collected using the Xcalibur 3.0 software of the UPLC–Q-Exactive quadrupole electrostatic field orbit trap high-resolution mass spectrometer.Mass spectrometry conditions were as follows: ion source HESI source, negative ion detection mode; sheath gas pressure, 206.84 kPa; auxiliary gas volume flow, 15.22 L/min; spray voltage, 3.5 kV; ion transmission tube temperature, 320°C; auxiliary gas temperature, 350°C; scanning mode full MS/dd-MSAfter the sample solution was analyzed and detected according to the above mass spectrum conditions, the Qualbrowser feature of the Xcalibur 3.0 software was used to complete data acquisition and processing, including the extraction of the ion chromatogram and fragmentation behavior information. The mass error of each main fragment ion was within 5 ppm. Full-scan mode high-resolution mass spectrometry data were analyzed using the Mass Frontier 7.0 software, and the possible structure formulae were deduced. The actual molecular weights of the detected compounds were then matched with the theoretical values. The Discoverer 2.1  database was used to search for the compounds, match the first and second mass spectra of the compounds, analyze the pyrolysis law, and identify the compounds.The compound database was established through literature retrieval and compound identification in Chinese and English databases . Compound information was collated in the PubMed database . The PubO.vulgaris with the targets of RA. Additionally, a Venn diagram was drawn to obtain the potential therapeutic targets of the compounds of O.vulgaris for RA.Using “rheumatoid arthritis” as the keyword, RA-related targets were identified in the OMIM, GeneCards, and DisgeNet databases. The TBtools software was used to match the targets related to the chemical compounds of O.vulgaris, a PPI network was constructed using the String Version 11.0 platform. We set a score greater than 0.7 to indicate high confidence (Homo sapiens (human), and hid the nodes that were disconnected from the network, leaving the rest of the parameters as default  was saved in SDF format. The Chem 3D software was used to convert the SDF file into a * Mol2 file, and the 3D structure of the target protein (PDB format) was downloaded from the PDB database (https://www.rcsb.org/)  and the ten key targets , the structure of the small molecule compounds downloaded from PubChem (sb.org/) . The PyMsb.org/) . The rec5 cells/ml were placed in a 37°C 5% CO2 incubator for 24 h. After the cells adhered to the plate, different concentrations  of the target compounds of O.vulgaris  were added to the culture. The normal control group (without the extract) was established at the same time. Five parallel wells were used for each group. After 48 h of culture, 10 μl of the freshly prepared 5 mg/ml MTT solution was added to each well and the culture was incubated at 37°C for 4 h in the dark. The supernatant was discarded, and 150 µl of DMSO was added to each well. After oscillation for 10 min, the absorbance (OD 570 nm) of each well was measured using a microplate analyzer.Logarithmic phase RAW 264.7 cells distributed as single-cells seeded in 96-well plates at a density of 1 × 10Chemical Materials and Extraction and Processing. The group demarcation based on treatment in this study was as follows: control group normal cell culture for 24 h without any treatment; inflammation model group with cells stimulated using 1 μg/ml LPS for 24 h; negative control group with cells treated using different target compounds  of O.vulgaris at 100 μM for 20 h; experimental group with cells stimulated using 1 μg/ml LPS for 4 h and different concentrations  of different target compounds  of O.vulgaris for 20 h. Three parallel wells were set in each group. The cell supernatant of each group was collected after culture, and NO release in the cell supernatant was detected according to the Griess test described in the NO kit. Then, the absorbance was measured at 550 nm  of O.vulgaris at 100 μM for 20 h; experimental group with cells stimulated using 1 μg/ml LPS for 4 h and different concentrations  with different target compounds  of O.vulgaris for 20 h. Three parallel wells were set in each group. The supernatant of each group was collected, centrifuged for 5 min, and the supernatant was carefully removed for storage. The expression levels of TNF-α, IL-6, and IL-1β were detected using an ELISA kit.Logarithmic phase RAW 264.7 cells made into single-cell suspension cells under the scratch seeded in a 6-well plate at a density of 5 × 10p < 0.05.All data were analyzed using SPSS 25.0, and the results were expressed as mean ± standard deviation (SD) of the three independent experiments. Differences were considered statistically significant if p < 0.0001), and the inhibitory effect was proportional to the concentration of indomethacin . The IC50 value of indomethacin was 58.82 μM in linear regression analysis. Therefore, 60 μM indomethacin was selected as the positive drug concentration for subsequent experiments  (p > 0.05). When the concentration of the different polar fractions of O.vulgaris was greater than 200 μg/ml, the viability of the ethyl acetate- and n-butanol extract-treated cells decreased significantly (p < 0.01). The experimental results showed that the different polar fractions of O.vulgaris had no effect on cell proliferation and cytotoxicity at 25, 50, 100, and 200 μg/ml concentrations; thus, they were selected for subsequent experiments.The effects of different polar fractions  on the proliferation activity of mouse macrophage RAW 264.7 cells were detected. There were no significant differences in cell proliferation between the control group and the RAW 264.7 cells subjected to the different polar fractions of O.vulgaris on LPS-induced inflammation in RAW 264.7 cells showed that the release of NO in the LPS model group was significantly higher than that in the control group (p < 0.0001). Compared with the LPS model group, the ethyl acetate (p < 0.05) in a concentration-dependent manner. This indicated that the ethyl acetate and n-butanol fractions could improve the inflammatory response in LPS-induced mouse macrophage RAW 264.7 cells in the experimental concentration range. IC50 values were calculated according to the concentrations of the ethyl acetate and n-butanol fractions of O.vulgaris. The n-butanol fraction (135.9 μg/ml) showed a higher anti-inflammatory effect than the ethyl acetate fraction (166.8 μg/ml). The alcohol, water, and petroleum ether fractions (p < 0.05). There was no statistically significant inhibitory effect at other concentrations (p > 0.05).Analysis of the protective effects of the different crude extracts of  acetate  and n-bu acetate  at conceractions  had an iO.vulgaris were identified by  techniques: D-mannitol (compound 1) (compound 2) (compound 3) (compound 4) (compound 5) (compound 6) (compound 7) (compound 8) (compound 9) (compound 10) (compound 11) (compound 12) (compound 13) (compound 14) (compound 15) (compound 16) (compound 17) (O.vulgaris for the first time.The structures of the following 17 compounds isolated from pound 1) , genipospound 2) , salidropound 3) , shanzhipound 4) , eleuthepound 5) , genipospound 6) , 7,8-dihpound 7) , gardosipound 8) , arenaripound 9) , verbascound 10) , isoacteound 11) , vanilliound 12) , p-hydroound 13) , melampyound 14) , syringaound 15) , tricin ound 16) , and dioound 17) . The strO-glucuronide, apigenin, apigenin-7-O-glucoside, tricin, chrysoeriol, quercetin, diosmetin, hydroxygenkwanin), iridoids , phenolic acids , and others . The total ion chromatograms of the ethyl acetate and n-butanol extracts of O. vulgaris, both in the positive ion mode, are presented in Using UPLC–MS, 35 compounds were identified from the n-butanol and ethyl acetate fractions, of which 16 were identified by the reference substance. The chemical compounds of the n-butanol and ethyl acetate fractions were preliminarily determined and were mainly divided into four categories: phenylethanoid glycosides , flavonoids  was determined to be C22H26O10. Accordingly, a second-order spectrum was found, and fragment ion peaks at m/z 213.1622, 173.0118, and 121.0283 were recorded. Caffeic acid (peak 8) was detected in the ethyl acetate fraction of the herb, and its fragment ion peaks were at m/z 135.0441 and 107.0350. Peak 20 of n-butanol showed a molecular ion peak at m/z 623.1974, which was identified as that of verbascoside. There were fragment ions peaks at m/z 313.8787, 161.0232, and 133.0282 in the secondary spectrum, which was consistent with the fragmentation mode of the reference substance and that in previous reports. The molecular ion peak at m/z 623.1971 of peak 21, which was speculated to be that of the isomer of verbascoside, also showed a similar fragmentation mode. On comparison with the fragmentation mode of the reference substance, peak 21 was identified as that of isoacteoside. Peak 27 at m/z 301.0355 in the ethyl acetate fraction was identified as that of quercetin. The fragment ions with peaks at m/z 151.0029, 121.0282, 107.0122, and 65.0017 were found in the secondary spectrum, which was consistent with the fragmentation mode of the reference substance.The characteristics of the representative compounds in the ethyl acetate and n-butanol fractions of the The known and predicted targets of the 36 compounds, after eliminating duplicates, were examined . In thisO. vulgaris to affect RA target genes was comprehensively obtained, and a total of 198 target genes were reported . The top 10 significantly enriched terms in the BP , MF (Molecular Function), and CC (Cellular compound) categories were selected. Among them, BP , MF (167 records), and CC (92 records) accounted for 89.36, 6.86, and 3.78%, respectively. In the BP category, the target proteins were associated with cellular response to chemical stress (GO:0062197), response to oxidative stress (GO:0006979), and rhythmic processes (GO:0048511). In the MF category, the target proteins were associated with protein tyrosine kinase activity (GO:0004713), insulin-like growth factor I binding (GO:0031994), and phosphatase binding (GO:0019902). In the CC category, the target proteins were associated with membrane rafts (GO:0045121), membrane microdomains (GO:0098857), and vesicle lumen (GO:0031983).To verify the biological characteristics of 198 targets of O. vulgaris on RA treatment, KEGG pathway enrichment analysis was performed on 198 genes. O. vulgaris on RA treatment were the PI3K-Akt, HIF-1, VEGF, estrogen, MAPK, Fc epsilon RI, Rap1, FoxO, and Prolactir signaling pathways, suggesting that these pathways mediated the effect of O. vulgaris on RA.To determine the potential pathway underlying the effects of O. vulgaris, the Cytoscape 3.7.1 software was used to construct the network of the “compound-target” and “target and pathway” of O. vulgaris in the treatment of RA for visual analysis. In this network, the orange nodes represented compounds, blue and pink nodes represented targets, green nodes represented pathways, and red and purple nodes represented O. vulgaris and RA, respectively. The potential target of a compound was linked by an edge , TNF and prostaglandin (PG), IL, NO, and other inflammatory mediators. It has potential medicinal value for the treatment of RA  in the PPI network and four compounds  for analysis of molecular docking. The docking result is depicted as a heat map display in O. vulgaris at 6.25, 12.5, 25, 50, and 100 μM (p > 0.05). When the concentrations of melampyroside and tricin were more than 100 μM, cell viability decreased significantly (p < 0.0001). The different target compounds of O. vulgaris had no effect on cell proliferation and cytotoxicity at 25, 50, and 100 μM concentrations; therefore, the 25, 50, and 100 μM concentrations were selected for subsequent experiments  on the proliferation activity of mouse macrophage RAW 264.7 cells were examined. The results showed that there was no significant difference between the proliferation of control cells and that of RAW 264.7 cells upon treatment with different target compounds of eriments .p < 0.0001). The effect of different target compounds on NO release was not significant in the negative control group, and there was no significant difference compared with that in the normal control group (p > 0.05). Compared with that in the LPS model group, treatment with 6.25, 12.5, 25, 50, and 100 μM of verbascoside (p < 0.05) in a concentration-dependent manner. Melampyroside (p < 0.05). The IC50 values were calculated according to the concentrations of the different target compounds of O. vulgaris. The IC50 values were as follows: melampyroside, 129.7 μM; verbascoside, 129.9 μM; isoacteoside, 114.5 μM; and tricin, 101.6. Thus, the anti-inflammatory effect of the four target compounds was in following order: tricin > isoacteoside > verbascoside > melampyroside.NO release in the LPS model group was significantly higher than that in the normal control group , indicating that LPS caused an inflammatory response in RAW 264.7 cells. Compared with that in the model group, TNF-α content decreased significantly after treatment with different concentrations  of melampyroside, verbascoside, isoacteoside, and different concentrations (50 and 100 μM) of tricin. A concentration dependence was observed in the effective concentration analysis (p < 0.05). Compared with that in the model group, IL-1β content decreased significantly after treatment with different concentrations  of verbascoside, and different concentrations (50 and 100 μM) of tricin, isocalycoside, and melampyroside. A certain concentration dependence was observed (p < 0.05). Compared with that in the model group, IL-6 content decreased significantly after treatment with different concentrations  of melampyroside, verbascoside, and tricin, and different concentrations (50 and 100 μM) of isoacteoside. A concentration dependence was observed (p < 0.05). The results showed that melampyroside, verbascoside, isoacteoside, and tricin could downregulate the levels of inflammatory factors and exert anti-inflammatory effects. There was no significant difference seen between the verbascoside treated groups and the positive control group (indomethacin) in IL-1β content at 100 μM (p > 0.05). There was no significant difference in IL-6 content between the verbascoside, and tricin groups and the positive control group (indomethacin) at 100 μM (p > 0.05).Compared with those in the control group, the levels of TNF-O. vulgaris, although O. vulgaris has enormous therapeutic potential, its full chemical characterization remains incomplete. Therefore, the purpose of this study was to evaluate the biological activity of O. vulgaris and analyze its chemical compounds to lay a foundation for the identification of new drugs.Based on the symptoms of RA and the pharmacodynamic basis of O. vulgaris with compounds 1–9 and 12–17 being isolated from O. vulgaris for the first time. The UPLC–Q-Exactive assay was used to identify 35 compounds from the ethyl acetate and n-butanol extracts of O. vulgaris.In this study, anti-inflammatory activity was determined in terms of the NO content of RAW 264.7 mouse macrophages induced by LPS. The results showed that the ethyl acetate and n-butanol extracts exhibited better activity. We isolated 17 compounds from the n-butanol and ethyl acetate extracts of O. vulgaris on RA. In this study, the PI3K-Akt pathway was found to be particularly important for the treatment of RA by O. vulgaris. The PI3K-Akt signaling pathway is closely related to the occurrence and development of RA and plays an important role in the regulation of differentiation and formation of osteoblasts  were identified. The docking scores of all compound-target pairs were less than −5 kcal/mol, indicating that each of the four selected compounds  had good binding affinity to all 10 targets. The binding energies of isoacteoside, tricin to SRC were −9.2, −9.9, and −8.6 kcal/mol, respectively. The binding energies of verbascoside and melampyroside were −9.6 and −8.5 kcal/mol, respectively. The molecular docking simulation strengthened all the above investigations to some extent.α is considered the leading trigger for inflammation and joint destruction. It can stimulate the synovium, fibroblasts, osteoclasts, and other cells to produce destructive substances called matrix metalloproteinases, which can damage cartilage and gradually destroy bone tissue in patients with RA. Therefore, TNF-α level can be used as an indicator of inflammatory activity in RA (α and is considered to be an amplifying factor of some biological effects of TNF-α (O. vulgaris significantly reduced the secretion of TNF-α, IL-1β, IL-6, and NO, suggesting that O. vulgaris is a potential therapeutic agent for RA. The anti-inflammatory activity of O. vulgaris can be attributed to its bioactive compounds. Phenylethanolglycosides, flavonoids, and iridoids have a wide range of pharmacological activities, including anti-inflammatory activityies (O. vulgaris is relatively large. Therefore, pharmacological studies on O. vulgaris cannot be ignored. Tricin is a flavonoid, and its anti-inflammatory properties have been studied (in vitro experiments.As a major pro-inflammatory factor in the pathogenesis of RA, TNF-ty in RA . IL-6 isof TNF-α . At non-ivityies . Verbascivityies . As an i studied ; it is nO. vulgaris in the treatment of RA and detected its target compounds from the levels of inflammatory factors in vitro. The results of network analysis and prediction showed that the PI3K-Akt pathway may play an important role in O. vulgaris-mediated treatment of RA. Therefore, the development of anti-inflammatory drugs using O. vulgaris has considerable advantages. However, bioinformatics methods still have some limitations, for example, it is found that it is too one-sided to explore the efficacy of medicinal plants only from the perspective of network pharmacology. It is necessary to consider the relative content of compounds in medicinal plants, so as to give better play to the pharmacological role of medicinal plants. In addition, the binding energy of compounds in molecular docking experiments is not necessarily at the same level as their therapeutic potential in vivo, and further systematic molecular biology experiments are needed to verify the accurate mechanisms.Based on studies of previous chemical substances, we used bioinformatics to predict the targets and potential action pathways of O. vulgaris, and combined with computer pharmacology, it is speculated that it may have the activity of treating RA. In the development of Chinese and Mongolian medicine, O. vulgaris could be a potential source of novel drugs for the treatment of RA. Our study also provides a novel paradigm to identify the potential mechanisms of pharmacological effects derived from a natural compound.In summary, the results of this study support the potential anti-inflammatory activity of


The strain also displayed ureolytic activity and cultivation with urea as ammonia source resulted in a growth rate of 0.104 day−1 and a doubling time of 6.7 days. A high ammonia affinity (Km(app) = 147 ± 14 nM) and high tolerance to toxic nitric oxide could represent an adaptation to acidic environments. Electron microscopic analysis showed coccoid cell morphology with a large amount of intracytoplasmic membrane stacks, typical of gammaproteobacterial ammonia oxidizers. Furthermore, genome and transcriptome analysis showed the presence and expression of diagnostic genes for nitrifiers , but no nirK was identified. Phylogenetic analysis revealed that this strain belonged to a novel bacterial genus, for which we propose the name “Candidatus Nitrosacidococcus tergens” sp. RJ19.Ammonia oxidation was considered impossible under highly acidic conditions, as the protonation of ammonia leads to decreased substrate availability and formation of toxic nitrogenous compounds. Recently, some studies described archaeal and bacterial ammonia oxidizers growing at pH as low as 4, while environmental studies observed nitrification at even lower pH values. In this work, we report on the discovery, cultivation, and physiological, genomic, and transcriptomic characterization of a novel gammaproteobacterial ammonia-oxidizing bacterium enriched via continuous bioreactor cultivation from an acidic air biofilter that was able to grow and oxidize ammonia at pH 2.5. This microorganism has a chemolithoautotrophic lifestyle, using ammonia as energy source. The observed growth rate on ammonia was 0.196 day Ammonia-oxidizing microorganisms use ammonia monooxygenase (AMO) to convert NH3 to hydroxylamine (NH2OH) 4+), lesse (NO3−) , 6 using to NO3− , 8. Unde− and NO , 10, theCandidatus (Ca.) Nitrosotalea” species and the acid-tolerant bacterium “Candidatus (Ca.) Nitrosoglobus (Ng.) terrae” sp. TAO100 that grow at pH 4 and 5, respectively, NH3 oxidation usually takes place at pH > 5.5 /(Km + [S]) in which V was measured as oxygen consumption rate and [S] represents the ammonium concentration.Oxygen consumption data were analyzed in OriginPro 2018 (64-bit) b9.5.0.193 and fitted to a Michaelis–Menten type curve using nonlinear least-squares analysis, according to the equation Proteins were extracted by incubating pelleted biomass in 0.5 ml 0.5 M NaOH for 30 min at 90 °C, followed by neutralization by adding about one volume of 0.5 M HCl. For samples from the kinetics determination experiment, cell lysis was achieved by using the B-PER Bacterial Protein Extraction Reagent (Thermo Fisher Scientific) according to manufacturer’s instructions. Protein concentrations were measured with the BCA Protein Assay (Thermo Fisher Scientific) following the manufacturer’s protocol.Biomass was fixed with 3% (v/v) PFA and hybridized with a newly designed Cy3-labeled oligonucleotide probe specific for the novel ammonia-oxidizing bacterium  using 10% formamide, as described elsewhere [g, 4 min). The pellet was resuspended in 15 μL of supernatant, and subsequently samples of 0.6 μL were high-pressure frozen in a HPM-100 (Leica Microsystems) using gold-plated platelets . Samples were freeze-substituted in an AFS2 (Leica Microsystems) using anhydrous acetone  containing 0.2% uranyl acetate (Merck). The substitution started at −90 °C for 48 h, followed by a +2 °C/h temperature increase to −70 °C, where the sample remained for 12 h. Afterwards, the temperature was raised by 2 °C/h to −50 °C, where the sample remained for 12 h. The sample was washed twice with anhydrous acetone at −50 °C and stepwise infiltrated with Lowicryl HM20  in anhydrous acetone at −50 °C. After four changes of 100% HM20, the samples were polymerized by UV irradiation at −50 °C for 96 h. Next, the temperature was increased by 2 °C/h to 0 °C, where the sample was kept for 24 h. Ultrathin sections (ca. 50 nm) were cut using an ultramicrotome  and transferred to 100 mesh copper grids  containing a carbon-coated formvar film. Micrographs were recorded in a JEOL Jem-1400 Flash transmission electron microscope  operating at 120 kV.4 ml of biomass was harvested from the membrane bioreactor and concentrated by centrifugation . The exhaust air passed through a dust filter unit before entering the scrubber. The scrubber showed a stable performance at 18–19 °C and no acid was needed to maintain pH 2–3, indicating considerable microbial ammonia-oxidizing activity that is confirmed by scrubber performance  Ala and Ile tRNAs  were encoded in between the 16S and 23S rRNA genes.The circular genome consisted of 1,811,369 bp and contained 1776 coding sequences (CDSs). Average CDS length was estimated at 929 bp, with a protein coding density of 90.3%. The G + C content was 37% and no plasmids were identified. https://www.imngs.org/) . Downstream in the amoCAB operon additional genes are found, which show homology to amoD and amoE. The first oxidation step is followed by the conversion of hydroxylamine to NO by the hydroxylamine-ubiquinone redox module (HURM) [cM552 . Interestingly, the “Ca. Na. tergens” sp. RJ19 genome lacks the soluble cytochrome c554 (CycA), which is encoded in the same operon (haoAB-cycAB) in all other genome-sequenced AOB including comammox Nitrospira [Ca. Ng. terrae” [Ca. Ng. terrae” and “Ca. Na. tergens” it looks like in both cases the CycA encoding gene is lost. While in “Ca. Ng. terrae” some remains seem to be present (BAW79846 and BAW79847), we were not able to find remains in the “Ca. N. tergens” genome (between NSCAC_1125 and NSCAC_1125). The two genes are separated by only 247 bases. CycA was speculated to shuttle electrons between HAO and cytochrome cM552 [Ca. Na. tergens” sp. RJ19 and “Ca. Ng. terrae.” For the further oxidation of NO to NO2− the enzyme responsible has not been identified yet, but both, a NO-producing nitrite reductase (NirK) working in reverse and nitrosocyanin have been speculated to catalyze this reaction [Ca. Na. tergens” sp. RJ19 (NSCAC_0629), its genome does not contain a gene for NirK, as was also reported for “Ca. Ng. terrae” [Analysis of the genome  , consisttrospira , except reaction . HoweverCa. Na. tergens” sp. RJ19 genome did not possess any genes encoding for a NO-forming nitrite reductase, we detected the presence of a gene encoding a NO reductase (NSCAC_1563) for the conversion of NO to N2O. This NO reductase is a monomeric type, the only adjacent gene encodes for a formate/nitrite transporter. Furthermore, cytochrome P460 (NSCAC_1129) was present and might cause additional N2O production from hydroxylamine detoxification under oxygen-limited conditions [Although as mentioned above, the “nditions .Nitrosococcus oceani ATCC 19707 (Noc_2700 and Noc_2701) [Nitrosacidococcus strain, as, besides ammonium, also CO2 is produced as carbon source for assimilation. CO2 can be accumulated intracellularly via two types of carbonic anhydrases  and assimilated by a type I ribulose-1,5-bisphosphate carboxylase/oxygenase . The carbonic anhydrases present in the genome of “Ca. Na. tergens” do not have signal peptides. This implies a cytoplasmic localization of these enzymes and a possible involvement in proton scavenging and pH homeostasis [+/H+ exchanger (NSCAC_1736). Different types of prokaryotic Na+/H+ transporters were found to be differentially regulated in relation to pH [Canonical AmtB-type transporters for ammonia uptake were not detected in the genome. However, a CDS (NSCAC_1037) with 54% amino acid identity to a duplicated putative ammonia permease of oc_2701)  was ideneostasis , 51. In eostasis . In straon to pH , 54.2 concentrations relative to O2, is presumably converted to pyruvate by a modified glycolate salvage pathway. First, glycolate is formed by phosphoglycolate phosphatase  and further converted to glyoxylate by the secondary reaction of glycerate dehydrogenase . Finally, glyoxylate is detoxified by a transamination of L-alanine to pyruvate and glycine, catalyzed by alanine–glyoxylate aminotransferase . The enzymes necessary for glycolysis and gluconeogenesis were also predicted, as were the oxidative and non-oxidative branches of the pentose phosphate pathway  and succinate dehydrogenase (EC 1.3.5.1) complexes, as well as malate dehydrogenase (EC 1.1.1.37) and pyruvate carboxylase (EC 6.4.1.1), could not be identified. Instead, oxaloacetate is most likely replenished by phosphoenolpyruvate carboxylase  and subsequently used for L-aspartate biosynthesis . From L-aspartate, fumarate can be formed via either adenylo- or L-arginino-succinate intermediates . Together with the fumarate formed here, L-aspartate can also be used to form succinate, as the “Ca. Na. tergens” sp. RJ19 genome encodes a L-aspartate oxidase , which can use fumarate as an electron acceptor during L-aspartate oxidation [All genes for the Calvin–Benson–Bassham  for the conversion of homoserine to phospho-homoserine was identified, and for methionine the enzymes forming the intermediates cystathionine and homocysteine from either cysteine or aspartate were absent.In addition, the genome of “Ca. Na. tergens” sp. RJ19, we started to enrich and cultivate this novel nitrifier under aerobic conditions in a continuous flow membrane bioreactor supplied with 76.2 mmoles NH4Cl day−1 at a constant pH of 3.5. After 150 days, a stable enrichment was obtained and FISH analysis with the newly developed Nitrosacidococcus-specific probe Nater1117 (see below), showed that “Ca. Na. tergens” sp. RJ19 was the dominant microorganism in the culture. The high level of enrichment was also supported by transcriptomic data (see below). These data also proved that we enriched the dominant bacterium from the air scrubber. At high stirring speed, biomass appeared as a combination of yellow small flocs and planktonic cells; if stirring was decreased or stopped, biofilm formation and big cell aggregates were observed.To facilitate the physiological characterization of “3 solution. Since ammonia oxidation acidifies the medium, the consumption of KHCO3 can directly be correlated to growth, and KHCO3 consumption indeed increased exponentially over time ) was calculated by measuring the ammonia-dependent oxygen consumption of “Ca. Na. tergens” sp. RJ19. Increasing concentrations of NH4Cl were injected into a cell suspension-containing respiration chamber and oxygen consumption was recorded via an oxygen microsensor. Using Michaelis–Menten type curve fitting  at pH 4.7 of 5.2 ± 0.5 mM NH4+ + NH3 was obtained, which corresponds to 147 ± 14 nM NH3 according to the Henderson–Hasselbalch equation. The maximum O2 consumption rate (V) was calculated at 0.28 ± 0.01 µM O2 min−1 µg protein−1.The apparent NHCa. Na. tergens” sp. RJ19 revealed the presence of an operon for urea uptake and degradation, we tested whether urea could serve as ammonia and thus energy source at pH 3.5. A batch reactor was set up with medium containing urea as substrate and monitored over a period of 42 days with daily measurements of urea, NO2−, NO3−, and NO/NO2 concentrations, and protein content  with urea was calculated from NO3− formation, which followed an exponential curve  and indicated a μ = 0.104 day−1 and a doubling time of 6.7 days. The ratio between urea consumption and product formation was calculated by determining the amount of nitrogen metabolized by the culture over 8 h  showed higher expression levels in the culture fed with ammonium as substrate  was threefold higher expressed during growth on urea , NO reductase (norB) and, interestingly, also RuBisCO (cbbLS) had lower expression levels compared to the ammonium-fed culture  in both cultures , 96% of the reads for the ammonium and 92% for the urea reactor were recruited by the genome of this microorganism. These result, combined with the FISH pictures  for NH3 of 147 ± 14 nM. Lower values were only reported for specialized marine AOA and for the comammox species Nitrospira inopinata, which thrive at extremely low substrate concentrations [KS(app) of Nitrosacidococcus, we could not yet isolate this strain in pure culture using dilution to extinction or floating filter techniques [In this study, we characterized a species of a novel gammaproteobacterial ammonia-oxidizing bacterium, for which we proposed the name of “naterra”  and “Ca. terrae”  exhibiteo pH 2.2 . The abio pH 2.2 , 20. Howtrations –63. Despchniques , 65. We “Ca. Na. tergens” sp. RJ19, like many other ammonia oxidizers [3 and our calculations indeed showed that 89% of the urea fed into the system was not consumed. Moreover, when lower concentrations of urea were supplied, activity was lost from the reactor (data not shown). This could suggest that this organism had a low affinity for urea, or that transport efficiency into the cytoplasm was ineffective for some reason. Alternatively, the export of ammonia formed during urea degradation into the periplasm, or the pseudo-periplasmic space within the intracytoplasmic membrane stacks, might be inefficient due to the lack of canonical ammonia transporters. As the active site of the AMO complex is assumed to be located in the periplasm [3/NH4+ permease (NSCAC_1037) was upregulated during growth on urea . This was still significantly slower compared to the rates of the other acidophilic ammonia oxidizers, “Ca. N. devanaterra” (0.23–0.37 days−1 at pH 4.5) [Ca. Ng. terrae” sp. TAO100 (0.89 day−1 at pH 6) [Ca. Na. tergens” sp. RJ19 at optimal pH.xidizers , 22, enceriplasm , 66, thirea Fig. . The gro pH 4.5)  and “Ca.at pH 6) . However2− as well as chemically formed NO3−. NO is a toxic compound that can be reduced to N2O via NO reductase (EC 1.7.2.5) [Ca. Na. tergens” sp. RJ19  and expressed at levels of 955 and 525 RPKM in the ammonium and urea cultures, respectively. Nevertheless, this bacterium did not seem to be inhibited by high concentrations of NO, and the presence of N2O was not detected in the off-gas of the culture. Further studies will be necessary to understand if the expression of norB is actually linked to enzyme activity.The products of ammonia and urea oxidation included NO and NO1.7.2.5) , which wCa. Na. tergens” sp. RJ19 appeared to be incomplete. In the Calvin–Benson–Bassham cycle, the gene for sedoheptulose 1,7-bis-phosphatase (EC 3.1.3.37) could not be identified; however, fructose-1,6-bis-phosphatase  might be bifunctional and also function in sedoheptulose 1,7-bis-phosphate hydrolysis, as shown for the enzymes in Ralstonia metallidurans and Xanthobacter flavus [Several metabolic pathways identified in the genome of “r flavus , 69. Addr flavus . In addiCa. Na. tergens” sp. RJ19 did not contain any amtB-type ammonia transporters; this was also the case for the acid-tolerant “Ca. Ng. terrae” sp. TAO100, for which it was proposed that NH3 transport relied on passive diffusion over the membrane [Ca. Na. tergens” sp. RJ19, the gene NSCAC_1037 showed 54% identity to a putative NH3/NH4+ permease of Nitrosococcus oceani ATCC 19707 (Noc_2700 and Noc_2701) [p = 0.00) when the culture grew on urea, which might indicate that NSCAC_1037 is involved in NH3/NH4+ transport, although its role would be in ammonia export during urea degradation.Interestingly, the genome of “membrane , 14. In oc_2701) . This geNitrosococcus species [Ca. Na. tergens” sp. RJ19 does not seem to be distributed in marine environments. Metagenomic sequence data (IMNGS platform) showed that closely related sequences (based on 16S rRNA gene similarity) were found in a refuse dump of leaf-cutter ants [Contrary to  species , “Ca. Nater ants , in a soter ants , which a−1 with ammonium and 0.1 day−1 with urea, and its morphology was similar to the closest relatives within the genera Ca. Ng. and Nitrosococcus [Ca. Nitrosacidococcus tergens” sp. RJ19. Taken together, our findings expand the knowledge about acidophilic microorganisms and confirm prior observation of oxidation of ammonia at pH as low as 2.5.In conclusion, we were able to obtain a highly enriched culture of a novel ammonia-oxidizing bacterium that showed activity and growth even at pH 2.5. This autotrophic microorganism had a high ammonia affinity and was able to use urea as alternative ammonia and thus energy source. The growth rate at pH 3.5 was calculated at 0.2 daysococcus , 50. Phynitrosus, full of natron, here intended to mean nitrous; L. neut. n. acidum, an acid; from L. masc. adj. acidus, sour; M.L. masc. n. coccus, sphere; from Gr. masc. n. kokkos, grain, seed; N.L. masc. n. Nitrosacidococcus, an acid-loving nitrous sphere; ter’gens. L. v. tergere, to clean; L. part. adj. tergens, cleaning, named thus for its ability to clean the air of ammonia in an air scrubber. Cells are Gram-negative, round cocci with a diameter of 0.5 µm and contain an extensive system of intracytoplasmic membrane stacks. Obligate chemolithotroph that oxidizes ammonia to nitrite. Urea can be utilized as alternative energy source. At pH 3.5, growth rate is 0.1 day−1 on ammonia and 0.2 day−1 on urea. The optimum pH for growth is 6, but growth is observed between pH 2.5 and 7. Cells exhibit a Km(app) for NH3 of 147 ± 14 nM. The type strain RJ19T was enriched from an air-cleaning bioscrubber of a pig farm in Erp, The Netherlands. The DNA G + C content of the type strain calculated on the basis of the genome sequence is 37 % and the genome size is 1.8 Mbp.Ni.tro.sa.ci.do.coc’cus. L. masch. adj. Supplementary Information


Enrofloxacin has low oral bioavailability in cats. Additionally, its unpleasant taste is linked to profuse salivation and vomiting, and the cat’s refusal to accept the following dose. In this trial, the pharmacokinetics (PK) of a new pharmaceutical preparation of enrofloxacin-alginate dried beads (DABE) is presented. It eliminates the unpleasant responses of cats to standard oral enrofloxacin. Its PK was carried out under a naive pooled sampling model. PK of DABEs (10 mg/kg/day) concealed in the cat’s food or morsels, complies well with the PK/pharmacodynamics ratios required for most pathogens, i.e., Cmax/CMI > 10. No rejection of DABEs was observed. The retinopathy associated with enrofloxacin appears to be dose-dependent. However, the reported Cmax values observed after the SC administration of 5 mg/kg/day, are notoriously similar to the obtained Cmax value obtained for DABEs (2.3 µg/mL). Hence the higher dose utilized PO is likely to result in similar toxicity as the dose utilized after parenteral administration, with the added benefit of an effortless drug administration and lack of tissue reactions in the injection site.0–24 value of 12.4 µg·h/mL and an AUC0–∞ value of 19.2 µg·h/mL, which are comparatively greater than values previously referred for kittens. In contrast, λ and elimination t½ were almost identical (0.12 1/h and 6.1 h). Pharmacokinetics/pharmacodynamics ratios taking the breakpoint of Staphylococcus epidermidis as a surrogate (0.5 µg/mL), can be regarded as borderline or low, but perhaps adequate in cats, as higher concentrations may be linked to toxicity .Available pharmaceutical preparations of enrofloxacin injected SC or IM to cats are likely to cause adverse tissue reactions in the injection sites (pH of the drug preparations is ≥10.4). Tablets often induce abundant ptyalism and vomiting, casting doubt on the efficacy of the drug administration maneuver. In addition, the reported oral bioavailability is very low. In this trial, the oral pharmacokinetics of dried alginate beads of enrofloxacin (DABE) administered by concealing them in the cat’s moist food or morsels, is described. A naïve polled sampling approach was chosen with fourteen adult healthy cats. Neither their housing nor their feeding habits were altered. A single pharmacokinetic profile was obtained with 5 samples per designated bleeding time, sampling each cat 2–3 times only. None of the cats rejected their medicated food or morsels. Plasma profile of enrofloxacin exhibited an AUC Mycoplasma haemofelis (previously Haemobartonella felis) infections that respond to treatment with enrofloxacin at 10 mg/kg/day, for 21 days [Although enrofloxacin has been on the market for cats for more than three decades and is recommended for various bacterial infections in this species ,2,3,4, a 21 days .0–24/MIC ≥ 120, respectively), and achieving these ratios is associated with higher clinical efficacy [The literature often refers to retinopathy, as the most important adverse drug reaction to enrofloxacin in cats ,7. Yet, efficacy ,10. Howeefficacy ,11, and efficacy .Recently, it was shown that the pharmaceutical preparation of dried-alginate beads of enrofloxacin (DABE) concealed in a morsel resulted in both adequate pharmacokinetic (PK) parameters and PK/PD ratios in dogs. It appears that these beads are tasteless to dogs, and can be concealed in morsels such as a piece of sausage or pate, without affecting its pharmacokinetics . In the All study procedures and animal care activities were carried out following the Institutional Committee for Research, Care, and Use of Experimental Animals of the National Autonomous University of Mexico (UNAM), under Official Mexican Regulation NOM-062-ZOO-1999. Taking into account the difficulties of working under the SARS-COVID-19 pandemic conditions, and to have an approximation of the variations that may occur when dosing enrofloxacin-alginate beads concealed in the cat’s food when these animals are within their customary environments, this pharmacokinetics study was designed as a naïve pooled sample PK study . This waAlginate-dried beads of enrofloxacin were manufactured as previously described . Thus, 2g. The supernatant was discarded and the organic phase was evaporated in a nitrogen environment. Residues were reconstituted in acetonitrile:methanol:water (17:3:80), with phosphoric acid (4% v/v), and trimethylamine (4% v/v) as the mobile phase. This product was then analyzed by high-performance liquid chromatography (HPLC) using a Hewlett Packard 1046A fluorescence detector at λexc 280 nm and λem 460 nm. A Jasco XLC HPLC system  with a Symmetry-C18 column  was used. Injection volume was 50 μL and flow was 0.6 mL/min. Data were analyzed by using Empower 3 from Waters . The chromatographic method was validated, and the analytical procedure was demonstrated as specific. The method produced a linear result from 0.01 to 20.48 μg/mL . Recovery of enrofloxacin was calculated by applying linear regression analysis. Samples had an r2 = 0.978 . Precision was demonstrated by the inter-day coefficient of variance (3.1) and inter-assay error value (<3.9). The lower quantification limit for enrofloxacin in plasma was 0.01 μg/mL with a detection limit of 0.008 μg/mL, and linearity was established up to 10 μg/mL. For robustness and tolerance, an absolute difference of 1.8 and a coefficient of variance of 2.2% (<3.0%) were obtained.The method of enrofloxacin quantification in plasma samples was developed and validated in our laboratory and was based on the method described by Idowu and Peggins . An aliqλ ; AUC0–24 (area under the plasma concentrations versus time curve in 24 h); AUC0–∞ (area under the plasma concentrations versus time curve from 0 to ∞); AUMC0–∞ (area under the moment curve from 0 to ∞) and MRT (mean residence time).The concentrations versus time data were analyzed both by compartmental and noncompartmental analysis , assuming that the plasma concentrations of enrofloxacin vs time data obtained were from a single subject. Results presented include Cmax (maximum plasma concentration); Tmax (time to reach Cmax); T½max/MIC ≥ 10, and AUC0–24/MIC ≥ 125) at a level of ≈90% or higher, and using 1000 sham cats . The choice of the bacterial sensitivity to enrofloxacin was adopted arbitrarily from the available literature [Monte Carlo simulation was used to generate the probabilities of target attainment (PTA) of both accepted PK-PD ratios for enrofloxacin  [All participant cats received a 10 mg/kg dose of enrofloxacin as DABE, concealed in their food or a morsel, and none of them rejected their medicated food or exhibited behavior that could indicate that the food had changed in taste. 5 µg/mL) ,16, phar5 µg/mL) .0–24/MIC ≥ 125 ratios in each MIC value are presented in 0–24/MIC ≥ 125 ratio, it was found that PTA was 90–100% with MICs from 0.03 to 0.5 µg/mL. When MIC value is 1 µg/mL, only 65% PK/PD adequacy was predicted, and 10% for a MIC value of 2.0 µg/mL. For the Cmax/MIC > 10 ratio the PTA was 100% for MIC values of 0.03 to 0.125; 94% for 0.25 µg/mL, and 89% for 0.5 µg/mL. Hence, the PK/PD cut-off point was approximately 0.5 µg/mL, considering a dose of 10 mg/kg of enrofloxacin as DABEs. As far as Monte Carlo simulations are concerned, the percentage of sham patients who achieved Cmax/MIC ≥ 10, and AUCA review of the main databases to search for PK studies of enrofloxacin in cats revealed only one study in this species. It was carried out in kittens and authors found that after oral administration of the drug, bioavailability was so low that the serum concentrations obtained, were negligible ,18. The In this trial, useful serum concentrations were found after dosing the cats with 10 mg/kg of enrofloxacin-alginate dried beads (DABEs), which were easily mixed/administered in the cats’ food or morsel. Cmax values in this trial were 2.3 µg/mL vs. 0.8 µg/mL found in kittens . This stProteus mirabilis to 0.45 µg/mL for Pseudomonas aeruginosa, and for Gram-positive pathogens from 0.84 µg/mL (Staphylococcus spp.) to 0.19 µg/mL for Staphylococcus intermedius. These MIC values fall into the ranges utilized for the Monte Carlo simulations carried out in this study. Enrofloxacin is lipid-soluble and a zwitterion, and as such, it is reasonable to think that it has good distribution to many tissues, as it happens with other species [max values to mitigate the selection of new fluoroquinolone-resistant strains and consistently reach PK/PD indices associated with adequate clinical efficacy. This occurs only at the highest dose possible [For susceptible isolates, enrofloxacin consistently reached PK/PD indices associated with clinical efficacy, but in this case, a relatively high dose was utilized i.e., 10 mg/kg. Boothe et al. [ species ,21. Thispossible ,23. HencThe incidence of retinal degeneration and blindness in cats medicated with enrofloxacin occurs sporadically in cats with a proposed rate of 0.008%  and is mTo obtain more regular PK profiles of enrofloxacin in cats the drug should be administered orally on an empty stomach as food is known to affect the absorption of fluoroquinolones ,26. Howemax after dosing 10 mg/kg achieves a similar Cmax as the reported value of this parameter, after the SC injection of enrofloxacin (5 mg/kg). Finally, as with many other drugs, the potential benefits of administering enrofloxacin as DABEs should be pondered against the known side effect of this active principle, but it must be considered that the described key PK parameters of DABEs at 10 mg/kg/day are almost identical to the ones obtained after the SC or IM administration of standard solutions of enrofloxacin but without the inflammatory reactions in the injection sites.Standard enrofloxacin has low oral bioavailability in cats; additionally, it has a bitter-unpleasant taste that induces a notorious ptyalism and often vomiting. The new pharmaceutical preparation of enrofloxacin-alginate dried beads (DABE) showed that cats presented no reaction when administered concealed in their food or within a morsel. Its PK through naive pooled sampling and Monte Carlo simulations shows that DABEs administered at 10 mg/kg/day comply well with the main PK/PD ratios required for most pathogens, and C


Regarding vitamin C content, the highest amount was found in the cultivar “Sabrina” (25.08 mg 100 g−1). Ellagic acid was the main phenolic acid in all examined cultivars (except the “Plared” cultivar), and the highest amount was detected in the “Fortuna” cultivar (3.18 mg 100 g−1). We found that the cultivar FL-127 had the highest total phenolic content . Regarding antioxidant capacities, the highest value (88.92%) was found in the cultivar Victory among all the cultivars studied. The detailed analysis of volatile compounds was performed by gas chromatography/mass spectrometry (GC/MS) and 34 compounds were detected. Among them, esters, acids, and alcohols were found to be the major volatile compounds in strawberry fruits. In conclusion, strawberry fruits belong to ten cultivars showed abundant phenolic compounds and at the same time have high antioxidant activity.The aim of this study was to compare certain important fruit quality parameters such as sugars, organic acids, total phenolic content, antioxidant capacity, and volatile compounds of 10 commercial strawberry cultivars grown in the research and experimental area in Yaltir Agricultural Company located in Adana provinces of Turkey. As for the sugar content of strawberry fruits, fructose was identified as the dominant sugar and the highest value (4.43%) was found in the cultivar “Rubygem”. In terms of organic acid, among the examined strawberry cultivars, “Calinda” had the greatest level of citric acid (711.45 mg g Over the past two decades, interest in research to determine health compounds in fruits has increased . ColorfuIt is well known that phenolic composition strongly affects the quality of the fruits and contributes to both their sensory-organoleptic properties and their nutritional values. They affect the taste of products and create a sour taste. Anthocyanins are the most notable and quantitatively the most important type of polyphenol in strawberries. Anthocyanins is one of the phenolic compounds, are responsible for the bright red color of the fruit. The concentration and composition of anthocyanins are important for the sensory quality of fruits and products, in addition to their possible health benefits. Recent studies have focused on determining the bioactive compound of diverse strawberry varieties and reveal the best one and determining the factors that affect the composition of this unique fruit ,11,12,13Carbohydrates (sugars) and organic acids are the major factors that influence the organoleptic properties of fruits. The organic acid–sugar ratio is also an important criterion to characterize fruit flavor. Organic acids show antioxidant properties, which explains their extensive use for pharmacological purposes. The organic acid content of strawberry fruits changes according to the genotypic background of the plant and the variety has a decisive role on the taste of the fruit by affecting the acid–sugar balance ,15,16.Fruit volatile compounds and taste are the results of a special assortment and mixture of different metabolites. While sugars and acids contribute to sweetness and tartness, the aroma is derived from combinations of volatile molecules. The different proportions of the volatile components and the presence or absence of trace components often determine aroma properties. The flavor of cultivated strawberries is mainly determined by a complex mixture of esters, aldehydes, alcohols, and sulfur compounds ,18. BecaTurkey has become one of the world’s largest strawberry producers in recent years . StrawbeThe study aimed to determine the carbohydrates (sugars), organic acids, total phenol content, individual phenolic compounds, total antioxidant capacity, and volatile aroma substances on ten strawberry varieties grown in Adana province of Turkey. These parameters are important in determining the quality of strawberries.Rubygem, Fortuna, Festival, Calinda, FL 127, Plared, Sahara, Sabrina, Victory, and E-2 strawberry cultivars were used in the study. All cultivars are grown together at the experimental field of Yaltir Agricultural Company in Adana provinces of Turkey. About 30 fruits were homogeneously collected from each cultivar during the harvest period of May 2019. The samples were placed in cloth bags and then transferred to the laboratory for future analyses and stored at −80 °C in deep freeze. “Rubygem” is a short-day strawberry cultivar that was obtained from cross-breeding between cv. Earlibrite × cv. Carlsbad in Australia. It has an excellent sugar–acid balance gives it a sweet-tasting flavor. Attractive medium-sized generally conical to cordiform and short wedge-shaped fruits give good yields. The fruit flesh is firm, sweet, and flavorful. The fruit is glossy bright red on the outside and medium red on the inside. Resistance to Fusarium Wilt is displayed. “Fortuna”, originated from cross-breeding Winter Dawn × FL 99-35 by the University of Florida. Fortuna is moderately resistant to Colletotrichum crown rot and anthracnose fruit rots but is moderately susceptible to Botrytis fruit rot and is highly susceptible to Phytophthora root rot. It has large, firm, glossy fruit. The “Festival” is a cross between Rosa Linda × Oso Grande. The cultivar is distinguished by the numerous runners it produces in the fruiting field, the long pedicels attached to its fruit, and the production of fruit that are flavorful, firm-fleshed, deep red on the outside, bright red on the inside, conically shaped, and have large, showy calyces when grown in Dover, Florida or the other areas that have a subtropical climate similar to that of Dover.“Calinda” is obtained in a planned breeding program conducted in The Netherlands and Bonares, Andalusia, Spain. Calinda is an early-season short-day cultivar that produces exceptionally attractive, flavors berries. “FL127” is cross between FL 05-107 and FL 02-58 in Florida, USA. FL 127 is distinctive for its combination of high early and total yields, large fruit size and excellent fruit quality due to high sugars, fruity aroma, firm but the juicy texture and extended shelf life. The plant is upright and moderately vigorous. This cultivar is resistant to anthracnose fruit rot and moderately resistant to charcoal rot and Colletotrichum crown rot but is susceptible to Botrytis fruit rot, powdery mildew, and Phytophthora root rot.“E22” is distinct for its day-neutral flowering habit, low chill requirement and compact plant habit. Its fruits have excellent rain tolerance, very consistent shape and color, and very good flavor. It is resistant to Phytophthora crown rot but is susceptible to powdery mildew and anthracnose fruit and crown rots.“Plared”, the variety of strawberry was created in a breeding program by crossing two parents in 2009 in Cartaya (Huelva), Spain between 2 unpatented progenies, “09-024” and “03-98”. It has a pleasant scent with a nice balance between sugar and acidity and it has an intense bright color and large fruit size. It is a short-day variety.“Sahara” variety was created in a breeding program by crossing two parents in 2006 in (Huelva), Spain by 02.125 (unpatented) and 03.98 (unpatented). The Sahara fruits resemble the Sabrosa ones, but they are much earlier. It is a short-day variety.“Sabrina” is obtained in a breeding program by crossing two parents, namely 9719 (unpatented) and 94-020 (unpatented). It has very firm and resistant skin, it enables long periods of conservation and transport. This variety produces red colored, conical-shaped, firm, large fruit.“Victory” is an early variety with regular production throughout the whole season. It is a rustic variety that stands out mainly for the post-harvest life of its fruits, being able to reach the most distant destinations. The seedling cross was selected from a controlled breeding plot in Ventura County, California, the USA in the winter of 2004.The production of certified strawberry varieties is carried out in the tissue culture laboratory by the meristem culture method. In another word, the production system is based on virus-free meristem seedlings. Solarization or fumigation is done during field experiments against soil-borne diseases. The experiment plantations included 2 years old strawberry plants. Licensed pesticides are used for strawberries throughout the production season. During the summer period, the control of the spider and aphid was done. After taking flowers and stems, fungicide is applied. In winter, spraying is done twice against Botrytis, the first at 25% of flowering and the second at the beginning of fruiting. Great attention has been paid to the red spider and thrips. Fresh and mature strawberry fruits were harvested at the ripe stage. A total of 30 fruits (10 fruits per replicate) from each variety were selected, with uniform size, shape, color without any visible damage, a disease for further study. The experimental area’s soil is processed deeply (at least 50–60 cm) and the plow layer (hard layer) is broken. Disinfection is done. The soil is aerated, plowed with a cultivator and the clods are broken. Very well burned animal manure was given 3–4 tons per decare (before soil disinfection). Base fertilizer is given as per da: 4 kg Nitrogen, 7 kg Phosphorus and 20 kg of Potassium. Fertilization begins 3 weeks after planting. 0.20–0.25 kg/da Nitrogen is given per decare per day. This practice continues until November. No water or fertilizer is given between November and January. Starting from February, 0.20–0.25 kg/da of Nitrogen and 0.25–0.40 kg/da of Potassium are given daily depending on the soil temperature. As soon as chlorosis (yellowness) is seen from planting, 500 gr. Sequestrene Fe (Iron) or others are given by drip irrigation and this process is repeated as needed. w/v) Na2CO3 were supplemented, and it was incubated 2 h at room temperature. Then, the absorbance was measured at 760 nm against a blank. Quantifications were calculated through a calibration curve daily prepared with known concentrations of gallic acid (GA) standards, and results are expressed as mg GAE 100 g−1 fresh weight (FW) of strawberry.The total phenolic content was determined by the Folin-Ciocalteau method  with somThe radical scavenging activity of DPPH  was done as described by  with sli−1. Elution was isocratic with ultrapure water. Individual sugars were calculated based on their standards and expressed in % of fresh weight (FW).Glucose, fructose, xylose, sucrose, and total sugar content in the juice obtained from the harvested strawberries were determined according to the method developed by Crisosto et al. . Before −1.Organic acids in strawberry fruit juice were determined by the HPLC analysis by Bozan et al. . The malp-coumaric acid, ellagic acid, quercetin, and kaempferol were dissolved in methanol at a concentration of 1 mg mL−1, and five dilute solutions from these stock solutions were used to prepare calibration curves of each standard. Three replicates from each sample were used for HPLC analyses. All samples and standards were injected three times, and mean values were used.For the extraction and hydrolysis procedure of phenolics of homogenized samples, boiling refluxed was used for 1 h . After i2 was added in headspace vial for 30 min at 40 °C. The SPME fiber 85 μm CAR/PDMS  was used for analysis. The adsorbed flavor compounds of the strawberry fruits were analyzed using a Shımadzu GC-2010 Plus Gas chromatography-mass spectrometer (GC/MS). HP-Innowax Agilent column  was used and helium was the carrier gas. The GC oven temperature was kept at 40 °C and programmed to 260 °C at a rate of 5 °C/min, and then kept constant at 260 °C for 40 min. The injector temperature was at 250 °C. The MS was taken at 70 eV. The mass range was m/z 30–400. A library search was carried out using the commercial Wiley, NIST, and Flavor GC–MS Libraries. The mass spectra were also compared with those of reference compounds and confirmed with the aid of retention indices from published sources. Relative percentage amounts of the separated compounds were calculated from total ion chromatograms. Volatile compounds were extracted by solid-phase microextraction (SPME). One gram of homogenate strawberry was weighed and 1 mL of CaClp < 0.05. Principal component analysis (PCA) was applied to examine the relationship between phenolic compounds data and strawberry samples. Mathematica program for Windows version 4.0 was used for data processing .All the result data were processed with the SPSS package program version 16.0 . All data were presented as the mean ± standard error (SE) and analyzed by one-way analysis of variance (ANOVA). Differences were considered significant at There were statistically significant differences among the strawberry varieties in terms of studied all biochemical traits . p < 0.05. The total phenolic content of strawberry varieties varied from 99.02 to 158.37 mg GAE 100 g−1 FW (−1 FW), while Plared had the lowest (99.02 mg GAE 100 g−1 FW). The previous study [The results of total phenolic of 10 strawberry cultivars are given in 0 g−1 FW . FL-127 us study  showed tp < 0.05). The findings indicated that the Victory and Rubygem varieties had the highest antioxidant capacity with 88.92% and 88.58% among the studied varieties, respectively. Researchers have demonstrated that a high antioxidant activity correlated with high phenolic content [−1, the total flavonoid amount 121.7 and 36.5 mg kg−1, respectively. They reported 91.0% and 58.6% in terms of DPPH scavenging activity among varieties. Our results in accordance with [There were significant differences in terms of antioxidant capacities of examined varieties grown in the Adana region in the present study .Carbohydrate of strawberries are the main taste compounds and are considered as one of the major fruit quality parameters preferred by both producers and customers. In the present study, the values of carbohydrates  in fruits of ten strawberries were measured. Data related to sugars, total sugar, and °Brix are shown in −1 FW and 2.17 ± 0.13 to 4.43 ± 0.21 g 100 g−1 FW, respectively. While the content of sucrose and xylose is relatively much lower with the values ranging from 0.36 ± 0.11 to 1.94 ± 0.10 g 100 g−1 FW and 0.01 ± 0.00 to 0.16 ± 0.19 g 100 g−1 FW, respectively. Here, we found the highest values of both glucose and fructose in “Rubygem”, while the lowest content of glucose and fructose were both detected in “Plared” variety. The majority of the rest varieties like “Calinda”, “Sahara”, “Sabrina”, “Victory” and “E-22” showed similar glucose and fructose content varied from 2.60 to 3.10 g 100 g−1 FW, respectively. The results also indicate the content of glucose and fructose in strawberries are closely related to each other and varieties with high glucose contents also showed high fructose values. For sucrose, the values ranged from 0.36 ± 0.11 to 1.94 ± 0.10 g 100 g−1 FW, and the lowest and the highest sucrose content value was obtained in “Festival” and “Victory”, respectively. Xylose content is relatively lower compared to fructose, glucose, and sucrose. The highest xylose content was in E-22 with a value of 0.16 ± 0.19 g 100 g−1 FW and the lowest was in FL-127 with the value of 0.01 ± 0.00 g 100 g−1 FW. Actually, except “Calinda” and “E-22”, xylose content in the rest of the varieties was similar and quite low. Literature studies showed some differences and little consistency in sugar concentrations compared to our study [−1 FW) was found to be the predominant sugar in strawberry fruit of two cultivars “Korona” and “Tufts” followed by glucose (1.79–2.25 g 100 g−1 FW) and sucrose (0.01–0.25 g 100 g−1 FW), respectively [−1 FW) in two strawberry varieties  and followed by sucrose (0.9–1.1 g 100 g−1 FW) and fructose (0.9–1.2 g 100 g−1 FW) [−1 FW and 3.5–6.7 g 100 g−1 FW and sucrose 0.6–0.9 g 100 g−1 FW, respectively [−1) and “FL-237” (8.46 ± 1.02 g 100 g−1). Varieties “Festival”, “Calinda”, “Sabrina”, “Victory”, and “E-22” presented medium values from 7.25 ± 0.33 g 100 g−1 to 7.86 ± 0.35 g 100 g−1, while varieties like “Fortuna”, “Plared”, and “Sahara” attained relatively low total sugar content (from 5.13 ± 0.15 to 6.70 ± 0.51 g 100 g−1 FW). Mahmood et al. [In the fruits of ten strawberry varieties, glucose and fructose are the major sugars with values ranging from 1.69 ± 0.01 to 3.93 ± 0.13 g 100 gur study ,30. Prevectively  indicateectively . The comectively ,33. The d et al.  reportedd et al.  the strad et al.  reportedp < 0.05). According to the organic acid results, citric acid is the major and predominant organic acid in fruits of 10 strawberry varieties, which ranges from 522.40 ± 1.5 to 711.45 ± 2.11 mg 100 g−1 FW. The highest citric acid content was obtained in “Calinda” and the lowest in “FL 127”. L-ascorbic acid was detected at concentration 13.90 ± 0.641 to 25.08 ± 0.35 mg 100 g−1 FW, malic acid ranges from 159.80 ± 2.84 to 266.65 ± 0.30 mg 100 g−1 FW, and succinic acid was from 24.27 ± 1.81 to 59.53 ± 2.92 mg 100 g−1 FW, respectively. Fumaric acid was also detected and presented in very small amounts (from 1.48 ± 0.05 to 2.10 ± 0.06 mg 100 g−1 FW), therefore it might not significantly influence the fruit flavor of strawberry. Several previous studies showed similar results with our present data and revealed that citric acid is widely found as the principal organic acid in strawberries [−1 FW, however, this result is much lower than our study. L-Ascorbic acid has important antioxidant and metabolic functions [Organic acids are known to affect the formation of taste and many physiological processes in fruits that depends on the variety. The sugar–acid balance and contents are very important in determining the taste characteristics of the fruit. Oxalic acid, L-ascorbic, citric, malic, fumaric, and succinic acid contents in fruits of 10 strawberry cultivars were examined in this study and results are given in wberries ,35,36. Mwberries . Only Mawberries  reportedwberries . Succiniwberries  comparedunctions . The finunctions ,39,40,41Phenolic compounds are plant metabolites that spread throughout the plant kingdom. The recent focus on phenolic compounds stems from their potential protective role against oxidative damage diseases such as coronary heart disease, stroke, and cancer, through the ingestion of fruits . Phenoli−1 FW, gallic acid contents between 0.45 and 1.06 mg 100 g−1 FW, quercetin contents between 0.84 and 1.15 mg 100 g−1 FW, myricetin contents between 0.19 and 1.94 mg 100 g−1 FW, caffeic acid contents between 0.13 and 0.23 mg 100 g−1 FW, p-coumaric acid contents between 0.05 and 0.35 mg 100 g−1 FW, and kaempferol contents between 0.04 and 0.18 mg 100 g−1 FW, respectively  were selected giving 54% of total variability. As indicated in Differences among fruits for the quantity and the composition of phenolic compounds can be due to factors as genotype, environmental conditions, agro techniques, and storage conditions as observed by ,51,52,53In the present study, a total of 34 volatile compounds were observed in 10 strawberry varieties by using different HS/SPME/GC-MS techniques and the results were given in The varieties were also rich in butanoic acid and hexanoic acid, which are important volatile substances in strawberries. Hexanoic acid was the predominant acid in 10 varieties, followed by acetic acid and butanoic acid. Among the ten varieties, Calinda had the highest concentration of aldehydes including 2,4-dimethyl-benzaldehyde. Ester compounds, including diethyl ester, ethyl ester, and methyl ester are important aroma compounds. The esters detected in the ten strawberry varieties were diethyl ester phenylmethyl ester acetate, octyl ester ethyl ester, and methyl ester. In this study, especially esters such as diethyl ester and methyl ester were the most abundant and high percentage of esters. Esters provide fruity and floral characters in fruits and the amount of these compounds provides an important basis for classification ,55. SimiIn this study, bioactive and aroma properties of different commonly grown strawberry varieties in Adana province in Mediterranean were examined and nutritional values and importance of these varieties for human health were determined using advanced analysis techniques. The region of study is one of Turkey’s most important regions in terms of citrus production. Demand for fruit types containing more antioxidants is increasing due to the identification of biochemical compounds that have an important impact on human health in recent studies. This situation increases the importance of strawberries in terms of human health. In this study, 10 different varieties were evaluated under the same climatic and cultural conditions and provide a valuable reference for further studies how genotype affects bioactive and aroma composition in strawberries. Studies on the biochemical properties of strawberry cultivars are important to find the most suitable for commercial production and human health. According to the results, it was determined that Rubygem, Victoria, and FL-127 varieties stand out in terms of bioactive properties including phenolic profiles, the antioxidant capacity and have higher sugar composition and acidity (%) values. The results show high potential of strawberry fruits for health benefits. Finally, differences found among varieties highlight a great variability and should be considered for the choice of the variety and in breeding programs aimed at selecting varieties with improved antioxidant capacity and nutraceutical properties.


Recently, there has been an increasing interest in using 360° virtual-reality video for an ecologically valid assessment of executive functioning in the neurologic population. In this framework, we have developed the EXecutive-functions Innovative Tool (EXIT 360°), an original 360°-based instrument for a multicomponent, ecologically valid evaluation of executive functioning in Parkinson’s Disease (PD). This work aimed to test the usability and user experience of EXIT 360° in patients with PD (PwPD). Twenty-seven PwPD and twenty-seven healthy controls underwent an evaluation that involved: (1) usability assessment by the System Usability Scale and (2) evaluation of user experience using the ICT—Sense of Presence and User Experience Questionnaire. Results showed a satisfactory level of usability for patients (mean = 76.94 ± 9.18) and controls (mean = 80 ± 11.22), with good scores for usability and learnability. Regarding user experience, patients provided a positive overall impression of the tool, evaluating it as attractive, enjoyable, activating, and funny. Moreover, EXIT 360° showed good pragmatic  and hedonic quality . Finally, PwPD considered EXIT 360° as an original tool with high ecological validity (mean = 4.29 ± 0.61), spatial presence (mean = 3.11 ± 0.83) and engagement (mean = 3.43 ± 0.54) without relevant adverse effects. Technological expertise had no impact on performance. Overall, EXIT 360° appeared to be a usable, easy-to-learn, engaging, and innovative instrument for PD. Further studies will be conducted to deepen its efficacy in distinguishing between healthy subjects and patients with executive dysfunctions. Over the years, virtual reality-based (VR) tools have appeared to be a promising solution in neuropsychological assessment, providing an ecological evaluation to detect everyday cognitive impairments ,2,3. SpeED constitutes a typical non-motor symptom in PD, from the early stages of the disease ,9,10,11,A preliminary study conducted using a virtual supermarket showed the presence of a deficit in planning and the switching mechanism required to process, in parallel, a large volume of information ,28. VR aIn the following years, the virtual version of the Multiple Errand Test (VMET) allowed for the detection of several executive disorders typical in patients with PD ,29,30. IIn light of these interesting results, Borgnis and colleagues (2021) have developed a 360° instrument to allow for an ecologically valid and multicomponent evaluation of executive functioning: the EXecutive-functions Innovative Tool 360° (EXIT 360°) . In EXITSeveral studies have shown the need to consider the evaluation of “usability” and “user experience” as crucial elements in developing technological tools ,35,36,37Despite the evident importance of paying attention to usability and user experience, only one study has been conducted to evaluate the usability of VR-based instruments for the assessment of neurocognitive abilities in Parkinson’s disease. Pedroli and colleagues conducted a preliminary study involving 21 healthy control subjects and three patients with PD in an evaluation with VMET. Data showed that healthy participants gave good usability for the VMET, while the patients with PD showed that there needs more than an improvement to VMET to be considered usable. Moreover, results showed that a good training phase before the test is crucial to apply the virtual protocol to PD patients .This work aimed to test the usability and user experience of EXIT 360° in patients with PD. t test [Twenty-seven patients with PD (PwPD) (M:F = 11:16) and 27 healthy control subjects (HC) (M:F = 11:16) matched for age and education were consecutively recruited at IRCCS Fondazione Don Carlo Gnocchi ONLUS in Milan. All participants had to follow specific inclusion criteria: (a) age between 18 and 90 years; (b) education ≥5 (primary school); (c) absence of cognitive impairment as determined by the Montreal Cognitive Assessment test  t test ; (b) milThe study was approved by the “Fondazione Don Carlo Gnocchi-Milan” Ethics Committee on 7 April 2021, project identification code 09_07/04/2021. The neuropsychologist provided all participants with a complete explanation of the purpose and risk of the study before they signed the written informed consent based on the revised Declaration of Helsinki . All participants underwent a one-session evaluation at IRCCS Fondazione Don Carlo Gnocchi ONLUS in Milan that involved three main phases: (a) pre-task evaluation; (b) EXIT 360° session; and (c) post-task evaluation .In pre-task evaluation (a), all participants underwent an assessment of their global cognitive profile through the MoCA test, a sensitive screening tool able to exclude the presence of cognitive impairment ,42. MoreYou will now wear a headset. Inside this viewer, you will see some 360° rooms of a house. To visualize the whole environment, I ask you to turn on yourself; you are sitting on a swivel chair for this reason. Within these environments, you will be asked to perform some tasks.”After the preliminary screening, all participants underwent an evaluation session with EXIT 360°. The psychologist started the administration by inviting participants to sit on a swivel chair and wear the mobile-powered headset. Before wearing the headset, participants received a general instruction of the task: “You are about to enter a house. Your goal is to get out of this house in the shortest time possible. To exit, you will have to complete a path and a series of tasks that you will encounter along your way. Are you ready to start?”. All instructions were provided in a standardized way, as they have been previously recorded and inserted within the virtual environments. After that, participants started an initial phase to familiarize themselves with the device and virtual environment and to control any adverse effects . Participants were immersed in a neutral 360° living room, exploring the settings and finding specific objects. At the end of this preliminary phase, participants had to indicate the presence of any negative sides. If the subjects did not report side effects, they were immersed in another 360° living room. They started the real experimental session, hearing the following instruction: “During their evaluation, the subjects were engaged in many domestic 360° environments explorable through the head’s movement as in real-life situations . In thesBriefly, “Let’s Start” requires subjects to observe a map, choosing the path that allows them to reach the “finish” in the shortest possible time. In the second subtask, “Unlock the Door”, the participants have to open a door choosing between “key, telephone, and drill”. The task “Choose the Person” requires the participant to explore a living room and select a specific person according to a particular instruction. In task 4, “Turn on the Light”, the subjects are immersed in a dark room because “the power went out,” and they must choose the object that allows them to continue the journey. In the following task (“Where are the Objects?”), participants have to identify the piece of furniture on which four specific objects  are placed in a bedroom .In task 6, subjects must complete a rebus. Finally, they must memorize a sequence of numbers in the last task, “Create the Sequence”, reporting them in reverse. EXIT 360° was designed to allow participants to respond to each task by choosing between three or more alternatives simply by moving their head and positioning a small white dot that they saw in the headset on the answer for a few seconds . TherefoParticipants had to perform all seven subtasks, obtaining one point for a wrong answer or two for a correct one. Therefore, we evaluated the usability and user experience of the whole task. Overall, EXIT 360° allowed for the collection of Total Score (range 7–14) and Total Reaction Time . After the EXIT 360° session, participants underwent an evaluation that assessed usability and user experience quality. Regarding usability assessment, we used the System Usability Scale (SUS), a short questionnaire of 10 items on a 5-point scale from “completely disagree” to “strongly agree”, often used to evaluate the overall usability of technological instruments ,50,51. Ft test for independent sample (parametric or non-according to variables) and Chi-square were conducted to verify possible differences between pathological group and healthy controls in main demographic and clinical characteristics and technological expertise. Moreover, Pearson’s correlation was applied to compare the usability scores, user experience, and technological experience. At the same time, a t test for the independent sample was conducted to evaluate any significant differences between groups in the same variables. All statistical analyses were performed using Jamovi 1.6.7 software. A statistical threshold of p < 0.05 was considered statistically significant.Descriptive statistics included frequencies, percentages, and median and interquartile range (IQR) for categorical variables and mean and standard deviation (SD) for continuous measures. The normality of data distribution was assessed using the Kolmogorov–Smirnov test. A n = 27) was predominantly female (M:F = 11:16) with a mean age of 68.2  and age of education =13 . HC was predominantly female (M:F = 11:16) with a mean age of 66.4  and age of education = 13 . The comparison between the PwPD and HC showed the absence of significant differences in all main demographic and clinical characteristics. All participants included in the study showed no cognitive impairment .The ad hoc five-point scale for evaluating the perceived level of familiarity with technologies showed similar results between patients and healthy controls, with a mean score of 3.15 ± 0.89 and 3.14 ± 1.12 . Moreover, the mean score of the ad hoc five-point competence questionnaire was 2.68 ± 1.01 for PwPD, indicating a score near to “little”. HC obtained a mean score of 3.04 ± 0.98 . t test (52) = −1.377; p = 0.174) and familiarity (t test (52) = 0.045; p = 0.964) with technologies.Analyzing the possible differences between groups, data showed the absence of significant differences in levels of competence (t test (52) = −4.95; p < 0.001) and Total Reaction time (t test (52) = 7.12; p < 0.001). Specifically, HC obtained a higher total score (mean = 12.3 ± 1.07) and completed the test in less time (mean = 457.3 ± 73.60). Furthermore, a significant difference also appeared in the FAB score (p = 0.006), showing that HC achieved a higher performance (17.46 ± 1.003).Preliminary analyses have shown that EXIT 360° could be considered as an effective tool in discriminating between PwPD and HC. t test (52) = −1.09; p = 0.279). PwPD provided a mean score of 76.94 ± 9.18, while HC showed a mean score of 80 ± 11.22. Both scores indicate a satisfactory level of usability, according to the scale’s score acceptability ranges (cut off = 68) and adjective ratings (included between “good” and “excellent”).t test (52) = −1.96; p = 0.055) and learnability (t test (52) = 1.89; p = 0.064). Specifically, PwPD showed a mean score of 2.98 ± 0.47 for usability (vs 3.24 ± 0.50) and 3.37 ± 0.63 for learnability (vs. 3.06 ± 0.59). Only 11.1% of patients and 3.7% of controls showed low scores (<2.5) at usability, while only 3.7% for each group in learnability.Specifically, according to the cut-off score (cut-off = 68), more than 74% of PwPD and 92% of HC showed scores above the cut-off. In addition, according to the adjective rating, 29.6% of subjects evaluated EXIT 360° as “OK”, 59.3% as “good”, 7.4% as “excellent”, and 3.7% as “best imaginable” ,58. As rp = 0.978). In addition, the other two items allowed for evaluation of the level of challenge of EXIT 360°, also based on their own abilities, such as balance/appropriate  for both groups, without significant difference (U test (52) = 326; p = 0.191).The first item of the Flow Short Scale showed a high score in the perceived level of skill in performing EXIT 360° both for PwPD and HC , without significant difference between groups (U test (52) = 363; Specifically, t test (52) = −3.44; p < 0.05). As regards the domain “negative effects”, only a few participants (three PwPD and three HC) reported the presence of minor adverse effects (score < 3), such as vertigo or nausea. Finally, despite the two groups that obtained a significant difference in the domain “engagement”, most of the participants indicated a good level of engagement while performing EXIT 360° , with only six patients and one control that showed low scores.The UEQ questionnaire showed positive evaluation (>0.8) in all 26 items in both groups. Finally, the means of each UEQ scale of PwPD were compared to existing values from a benchmark dataset  . Resultsp = 0.576), but not for age . Moreover, Pearson’s correlation has underlined the absence of significant correlation between SUS total score and technological expertise measured by the ad hoc questionnaire of competence, both for patients  and controls . As regards the relationship between usability and user experience, patients showed no linear correlation between SUS total score and three ICT—SOPI domains, spatial presence , engagement , and ecological validity . HC obtained similar results, except for ecological validity that appeared significantly correlated with usability score . Moreover, data showed the presence of a significant and negative correlation between the SUS total score and the ICT—SOPI domain negative effect only in PwPD . Finally, the statistical analysis showed the absence of significant correlations between usability and the three dimensions of UEQ: attractiveness , pragmatic quality  and hedonic quality .Overall, Pearson’s correlation showed the absence of significant linear correlation between the SUS total score and education  and adjective ratings (included between “good” and “excellent”) . SpecifiIn addition to the good usability level, EXIT 360° showed promising results in terms of user experience in both groups. First, patients with PD showed high scores in the perceived level of skill in performing EXIT 360° and evaluated the EXIT 360° subtasks as balanced/appropriate concerning their abilities. Second, they supported a positive general impression of the EXIT 360°, considering it attractive , activating, funny and not boring. Moreover, EXIT 360° demonstrated a good pragmatic quality as it appeared: (1) efficient, fast, practical, and organized (efficiency); (2) understandable, easy to learn, and straightforward (perspicuity); and (3) predictable, supportive, and secure (dependability). The two groups obtained similar scores in these variables, except for dependability, in which patients obtained a lower score. However, this domain was influenced by the item “meets expectations” because patients referred to having negative expectations due to traditional long and complex evaluation, and they claimed to be pleasantly surprised. In addition, EXIT 360° showed excellent hedonic quality in terms of stimulation  and novelty . Interestingly, results showed that the scales stimulation and novelty evaluated better than existing values from benchmark data ,61.Overall, the present results appear promising in terms of usability and user experience of EXIT 360° in patients with PD, in line with results obtained in the previous study on healthy controls. Briefly, EXIT 360° allowed for an ecologically valid evaluation without relevant sicknesses  that lead to unpleasant experiences for the patients, impacting their performance and significantly decreasing the test results’ validity . MoreoveThe present work has some limitations. First, the head-mounted display used was entry level: the 360° mobile-powered devices currently available on the market have higher quality , which would allow for improving the quality of 360° images, providing a better and more realistic experience. Second, possible misdiagnosis with syndromes showing high resemblance with PD ) could have an impact on enrollment and results. Possible misdiagnosis is an important concern for experienced movement disorder specialists, particularly in the early stage of the disease in which several clinical manifestations could overlap ,63. To mThe present study results are promising and interesting in terms of the usability and user experience of EXIT 360° in patients with PD. Overall, patients provided a positive global impression of the instrument, evaluating it as usable, easy-to-learn, understandable, enjoyable, attractive, and friendly. Moreover, EXIT 360° is an efficient, fast, and organized tool, with excellent hedonic quality regarding stimulation (exciting and interesting) and novelty. Finally, EXIT 360° also appeared to be an engaging and challenging tool with good spatial presence, excellent ecological validity, and irrelevant adverse effects. Technological expertise did not influence the encouraging results. Further studies will have to be conducted to deepen the efficacy of EXIT 360° in discriminating between healthy control subjects and patients with executive dysfunctions.