The structures of target compounds were characterized by 1H NMR, 13H NMR, 19F NMR and HRMS. The preliminary antifungal evaluation of all target compounds showed that some target compounds possessed moderate to good activities against G. zeae, P. sasakii, P. infestans and C. wilt. Among them, compounds 5L and 5o exhibited noticeable inhibition effects against G. zeae with the EC50 values (effective concentration for 50% activity) of 20.06 and 23.17 μg/ml, respectively, which were even nearly double effective than that of hymexazol (40.51 μg/ml). Meanwhile, compound 5q displayed a notable inhibitory effect toward P. sasakii, with the EC50 value of 26.66 μg/ml, which was better than that of hymexazol (32.77 μg/ml). In addition, compound 5r yielded the EC50 value of 15.37 μg/ml against P. infestans, which was less than those of hymexazol (18.35 μg/ml) and carbendazim (34.41 μg/ml). Eventually, compound 5p showed higher inhibitory effect against C. wilt, with EC50 value of 26.76 μg/ml, which was better than that of hymexazol (>50 μg/ml).A series of 1,4-benzoxazin-3-one derivatives containing an acylhydrazone moiety were designed, synthesized and evaluated for their  Phytopathogenic fungi can invade plants and cause plant diseases, which not only bring about dramatic financial lose, but also can cause food safety problem because mycotoxins produced by some plant pathogenic fungi can threaten the health of humans and animals . At presA1 is efficiently synthesized and strongly inhibited the growth of many different strains of phytopathogenic fungi   . Compounrevisiae . CompounCC 90030 . Compounactivity . Thus, tB1 has been proved that it possesses potential antifungal activity against multiple Candida spp.  core can be obtained by the condensation of a hydrazide with a ketone or an aldehyde, which is a particular type of Schiff base compound. As a result, acylhydrazone core has become one of the most extensively existed pharmacophores during the course of new drug research and creation due to their privileged structures and a large number of excellent bioactive properties, such as anticancer , antivirida spp. . Compounericin B . Compounumigatus . Compoun.5 μg/ml .in vitro antifungal activities against Gibberella zeae (G. zeae), Pellicularia sasakii (P. sasakii), Phytophthora infestans (P. infestans), Capsicum wilt (C. wilt) and Phytophthora capsica (P. capsica).Thus, to improve the antifungal activities of 1,4-benzoxazin-3-one derivatives, we committed to introducing the bioactive acylhydrazone moiety to 1,4-benzoxazin-3-one skeleton, then, designing and synthesizing a serial of novel 1,4-benzoxazin-3-one derivatives with an active acylhydrazone moiety  and appr5a–5s was presented in 5 was obtained by the cyclization, substitution, hydrazinolysis and condensation reactions with 36%–53% yields over four steps. The structures of the title compound 5 were confirmed by 1H NMR, 13C NMR, 19F NMR and HRMS, and all analytical data were consistent with the assigned structures. The 1H NMR, 13C NMR, 19F NMR and HRMS spectra of the target compound 5 , benzyl triethyl ammonium chloride  and dichloromethane (20 ml) was added to a 100 ml reaction flask equipped with a magnetic stir bar. After the mixture was stirred for 10 min, sodium hydrogen carbonate  was added step into mixture. The resultant mixture was allowed to stir at 0°C, and chloroacetyl chloride  was added via syringe over a period of 15 min. Then, the reactant mixture was refluxed at 40°C for 6–8 h. After the reactants were consumed , the resulting crude residue was poured into ice water (20 ml). The crude products were collected by filtration, washed with water, and recrystallized from methanol to obtain intermediate 2  and K2CO3  were added into a 100 ml reaction flask with 20 ml dimethylformamide (DMF). Then, ethyl bromoacetate  was dropwise added via syringe and the reaction mixture was allowed to stir at rt for 12 h. After the reactants were consumed , the resulting crude residue was extracted with ethyl acetate (3 × 20 ml). The organic layer was washed with water, dried over anhydrous Na2SO4, concentrated under reduced pressure to obtain intermediate 3  was dropwise added via syringe to this mixture. The reaction mixture was refluxed for 6–8 h, and then allowed to pour into ice water. The crude residue was filtered to yield the intermediate 4  in 5 ml ethanol was dropwise added via syringe into this solution. The reaction mixture was refluxed for 2–4 h, and allowed to cool to room temperature. The crude residue was filtered and recrystallized from toluene to get the title compound 5.Intermediate cis-trans isomers in acylhydrazones  spectroscopy is a practical strategy to identify the drazones . The anae method . The cheeratures . The metin vitro fungicidal activities against five types of plant pathogenic fungi including G. zeae, P. sasakii, P. infestans, C. wilt and P. capsica at the concentration of 50 μg/ml through the classical mycelium growth rate method according to the previously established procedure . Secondly, compounds 5L and 5q displayed stronger inhibitory activities against P. sasakii, with the inhibition rates of 64.38% and 73.32%, respectively, than that of hymexazol (60.70%). Meanwhile, compound 5r exhibited the excellent inhibitory activity against P. infestans, with the inhibition rate of 82.62%, which was better than hymexazol (72.86%) and carbendazim (58.57%). In addition, compound 5p had 71.33% inhibition rate against C. wilt, which was better than hymexazol (49.88%).All synthesized 1,4-benzoxazin-3-one derivatives in this work were screened for 50 values of some title compounds against G. zeae, P. sasakii, P. infestans, and C. wilt were tested and the result data were demonstrated in 5L and 5o exhibited commendable in vitro antifungal activities against G. zeae, with the EC50 values of 20.06, and 23.17 μg/ml, respectively, which are nearly double effective than that of hymexazol (40.51 μg/ml). Meanwhile, compound 5q displayed outstanding inhibitory activity against P. sasakii, with the EC50 value of 26.66 μg/ml, respectively, which is better than hymexazol (32.77 μg/ml). Furthermore, compound 5r also had excellent inhibitory activity against P. infestans, with EC50 value of 15.37 μg/ml, which is double more efficient compared to that of carbendazim (34.41 μg/ml). Then, the EC50 values of compounds 5b and 5p against to C. wilt were calculated, just the EC50 of 5p (26.76 μg/ml) was slightly close to that of carbendazim (26.08 μg/ml).To further validate the antifungal activities of the title compounds, the EC5a-5g , only compound 5e showed better activity against P. infestans, with 3-Br attached aryl of acylhydrazone, the introduction of other substituents, including 4-F-Ph, 2-F-Ph, 3-F-Ph, 4-Br-Ph and 2-furan could not obviously affect the antifungal activities. Then, some of target compounds showed better activity against fungal strains, when introduction of methyl attached on 1,4-benzoxazin-3-one skeleton (for compounds 5h-5n), for example, compound 5L (3-Br attached aryl of acylhydrazone) exhibited bioactive with the inhibitory rates 76.37% and 64.38% against G. zeae and P. sasakii, respectively. However, target compounds (5o – 5t), with 6-Cl attached on 1,4-benzoxazin-3-one skeleton, exhibited better antifungal activity than other substituents attached on 1,4-benzoxazin-3-one skeleton, such as compound 5o, 5p, 5q and 5s were exhibited excellent bioactive with the inhibitory rates 76.14%, 71.33%, 73.32% and 82.62% against G. zeae, C. wilt, P. sasakii and P. infestans, with EC50 value of 23.17, 26.76, 26.66 and 15.37 μg/ml, respectively. Especially, compound 5s had the best activity against P. infestans fungal strain. The replacement of the benzene ring (5o) to alkyl such as ethyl (5t) group could not improve the antifungal activity.For compounds in vitro antifungal activities against five species of fungi. The preliminary antifungal evaluation of all the title compounds indicated that some of them had considerable activities against tested fungi. Among them, compounds 5L and 5o possessed commendable outstanding antifungal activity toward G. zeae with EC50 values of 20.06 and 23.17 μg/ml, respectively. Meanwhile, compound 5q showed outstanding inhibitory activity against P. sasakii, with EC50 value of 26.66 μg/ml. Additionally, compounds 5e and 5r had preeminent inhibitory activity against P. infestans, with EC50 values of 26.77 and 15.37 μg/ml, respectively. Our present work indicated that 1,4-benzoxazin-3-one derivatives with an acylhydrazone moiety could result in potential fungicide agents.In conclusion, a total of 19 original 1,4-benzoxazin-3-one derived compounds containing an acylhydrazone moiety were synthesized and estimated for their


In order to evaluate the selectivity of these ligands towards G4 structures, their interaction towards DNA calf thymus, as a double-strand DNA sequence, were also studied. The data obtained by using different spectroscopic techniques, such as ultraviolet-visible, fluorescence, and circular dichroism, suggested good affinity of the free-base porphyrin and of its zinc(II) complex for the considered DNA structures, both showing a pattern of selectivity for the telomeric G4 structure. A pattern of aggregation in aqueous solution was detected for both Zn(II) and Ni(II) metallo dibenzoporphyrins and the ability of DNA sequences to induce ligand disaggregation was observed.Since the occurrence of tumours is closely associated with the telomerase function and oncogene expression, the structure of such enzymes and genes are being recognized as targets for new anticancer drugs. The efficacy of several ligands in telomerase inhibition and in the regulation of genes expression, by an effective stabilisation of G-quadruplexes (G4) DNA structures, is being considered as a promising strategy in cancer therapies. When evaluating the potential of a ligand for telomerase inhibition, the selectivity towards quadruplex versus duplex DNA is a fundamental attribute due to the large amount of double-stranded DNA in the cellular nucleus. This study reports the evaluated efficacy of three tetracationic  Chromosomic and genomic stability is ensured by the presence of special structures at each of the chromosome ends, the telomeres. The presence of these structures protects the chromosome ends from enzymatic degradation ,2,3,4, aTelomeres shortening occurs during each DNA replication and, in normal somatic cells, when a critical length is reached, telomeres can no longer guarantee chromosome integrity and cell senescence and death occur. A different situation happens in cancer cells due to the presence of telomerase, a reverse transcriptase enzyme, that is able to switch off this shortening process ,6. Since2AG3), and can form non-canonic DNA structures, such as the G-quadruplexes (G4). The high percentage of guanines in this sequence enables their self-assembly in a planar quadrangular arrangement via Hoogsteen hydrogen bonding, with the formation of G-quartets. The establishment of π−π interactions between G-quartets on top of each other will give rise to the formation of G4 structures 4+ molecular ions.The three tetracationic 2-β-TMePyBPH, Zn-β-TMePyBP, and Ni-β-TMePyBP were recorded in phosphate buffer saline solution (PBS)/DMSO (1%) medium at a concentration of 2.0 μM and 298 K and are shown in The absorption and emission spectra of the tetracationic derivatives 2-β-TMePyBPH derivative displays an absorption spectrum with a strong Soret band centered at 454 nm due to S0→S2 transitions and three Q bands ranging from 541 to 613 nm ascribed to S0→S1 transitions +.Ni-β-TPyBP  or 2H-β-TPyBP , methyl iodide (60 equiv.), and DMF (1.0 mL). The reaction was placed under magnetic stirring at 40 °C for 23 h. After cooling to room temperature, diethyl ether was added to the reaction. The precipitate obtained was filtered, washed with diethyl ether, and dissolved with a CH2Cl2/MeOH (95:5) mixture. The solvent was evaporated to dryness under reduced pressure. Ni-β-TMePyBP and 2-β-TMePyBPH were obtained pure in 94% and 97% yield, respectively, after recrystallization in a CH2Cl2/Hexane (95:5) mixture.Into a sealed tube was added Ni-β-TMePyBP1H NMR : 8.86 , 8.33–8.03 , 7.99–7.86 , 7.69 , 7.34 , 4.34  ppm. 13C NMR : δ 156.1, 145.8, 137.2, 136.4, 136.19, 133.6, 129.9, 129.3, 128.4, 128.2, 123.2, 48.1 ppm. UV-Vis (PBS): λmáx (ε) nm = 466 , 574 , 622  nm. MS-ESI(+): m/z 284.7 [M]4+.2-Hβ-TMePyBP1H NMR : δ 9.01 , 8.91 , 8.25 , 8.02–7.94 , 7.68 , 7.03 , 4.37 , −2.68  ppm. 13C NMR : δ 155.2, 148.4, 145.8, 142.5, 141.3, 138.9, 133.8, 133.0, 129.9, 129.2, 128.9, 128.5, 128.0, 119.6, 48.1 ppm. UV-Vis (PBS): λmáx (ε) nm = 454 , 541 (7937.4), 588 (8651.4), 613 (7595.5) nm. MS-ESI(+): m/z 270.7 [M]4+.2-β-TMePyBPH , 1.5 equivalents of zinc acetate  and methanol (2 mL) were added. The mixture was stirred at 60 °C until the total consumption of the starting porphyrin was verified by TLC control. The zinc(II) complex was precipitated with the addition of diethyl ether and centrifuged at 12,000 rotations for 3 min to separate the precipitate from the supernatant. The desired Zn(II) complex Zn-β-TMePyBP was attained in 98% yield after recrystallization in a CH2Cl2/Hexane (95:5) mixture.To a round bottom flask 1H NMR : δ 8.88 , 8.84 , 8.20 , 8.01–7.87 , 7.68 , 7.17 , 4.36 , ppm. 13C NMR : δ 155.4, 150.4, 145.7, 143.5, 142.9, 140.6, 133.4, 132.3, 132.0, 129.4, 128.6, 128.5, 128.4, 120.1, 48.1 ppm. UV/Vis (PBS): λmáx(ε) nm = 485 , 595 , 635  nm. MS-ESI(+): m/z 286.2 [M]4+.2PO4 1 M, and 200 μL of K2HPO4 1 M) and 100 mM of KCl was prepared with pH adjusted to 6.8. The resultant PBS solution was used as the solvent for oligonucleotide solutions. After solubilisation in PBS, each oligonucleotide was heated up to 85 °C for 10 min and left to cool overnight to assure the correct folding into G4 structures. The solutions were stored at −20 °C. In the case of calf thymus, the lyophilized was dissolved in water (1 mg/mL) with no sonication or stirring and keeping a gentle inversion overnight at −4 °C to completely solubilize the DNA. Calf thymus solution have been then stored at 4 °C.A PBS solution, containing 20 mM of phosphate buffer (10 mL of KH260), to avoid inaccuracies A260 ≤ 1.2. The concentration was then calculated using the formula: Concentration (µg/mL) = A260 × dilution factor × weight per OD of stock solution (in µg/OD). The obtained values were then converted to micromolar data for further analysis.For calf thymus, the sample concentrations were determined by measuring the absorbance of a diluted solution at 260 nm  to 1 mL solution of the dibenzoporphyrins with the initial concentration of 2 μM, in the range of 350–700 nm. Titrations were finished after observing three consecutive values of constant absorbance ,72. Blanfree − εbound)/εfree] × 100, where εbound was calculated using Beer’s Law (εbound = Abound/Cbound) and εfree is the molar extinction coefficient values experimentally obtained, in PBS.To ensure the reproducibility of results, all the experiments were performed in triplicate. The percentage of hyper/hypochromicity of the absorption band was determined using the equation % hypochromicity = [ were determined by fitting data obtained from the UV-Vis absorption titrations to the equation:obs corresponds to the absorbance observed after each DNA addition at the ligand maximum wavelength, Clig and CDNA correspond, respectively, to the total porphyrin and DNA concentrations after each DNA addition, and εlig and εDNA are the extinction coefficients. The extinction coefficients of the DNA structures are: εG4-Tel = 228.500 M−1 cm−1 and εCT = 13.200 M−1 cm−1 (base pair) at 260 nm [The binding stoichiometries were obtained by using the method of continuous variation, or Job plot ,84. Job t 260 nm . The stoFluorescence titrations were conducted using a Horiba Floromax-4 spectrofluorometer. The studied ligands were excited at their maximum absorption wavelength; the collection of the fluorescence emission data took place between 600–850 nm at 25 °C using a 1 cm quartz cuvette containing a 2 μM ligand solution. Our setup involved 5 nm excitation and emission slits.For G4-FID experiments, stock solutions of 35 μM of TO and 10 μM of each oligonucleotide were prepared. Mixing these solutions produced the desired TO-DNA adduct, and after 10 min of orbital shaking, the fluorescence using a Fluoromax-4 spectrofluorometer (Horiba) was measured. The excitation wavelength was set at 485 nm, and the emission range was at 510–750 nm, with 10 nm slits for both excitation and emission.A0. To investigate further, solutions with increasing concentrations of each ligand (ranging from 0 to 4 μM) were prepared, which were added to the TO-DNA solutions (17.5 μM TO–5 μM DNA). Again, we measured the fluorescence using the same experimental parameters. The percentage of displacement, DC50, was calculated using the following equation:A is the fluorescence intensity of each sample minus the background fluorescence from mili-Q water (FA = F − FH2O), and FA0 corresponds to the fluorescence intensity of the fluorescent probe bound to DNA without the added ligand minus the background fluorescence from mili-Q water (FA0 = F0 − FH2O).The initial fluorescence data were termed Fmax is the highest ellipticity, and CDmin is the lowest ellipticity.CD experiments were performed in a Jasco J-1500 spectrophotometer, equipped with temperature controller Jasco PTC-517. Prior to analysis, the equipment was purged with pure nitrogen gas. The solutions containing the DNA structures and the ligands (4 μM) were previously prepared by heating the solution for 10 min at 85 °C and then cooling it overnight. The CD melting spectra were then obtained by monitoring the ellipticity at 295 nm in the temperature range of 25–95 °C. The equation below was used to convert the data obtained into fraction folded (θ) plots : (4)ϴ=CDThe melting temperatures were then obtained by fitting sigmoidal curves with the Origin 8.0 software and the Boltzmann function. opp-dibenzoporphyrins 2-β-TMePyBPH, Zn-β-TMePyBP, and Ni-β-TMePyBP to selectively stabilise G4 DNA structures versus double-stranded DNA structures. The data obtained from the different analytical techniques were in general consistent and it suggests that the free-base 2-β-TMePyBPH and the zinc(II) complex Zn-β-TMePyBP have a high affinity and good selectivity for the telomeric G-quadruplex over double-stranded DNA structures. In the case of both complexes, a pattern of aggregation in aqueous solution was observed, which apparently can be reversed through a DNA-promoted ligand disaggregation process. In this study, ultraviolet-visible, fluorescence, and circular dichroism spectroscopic methods were used to evaluate the ability of the three cationic 6 M−1) and selectivity of the free-base cationic dibenzoporphyrin (KbG4 ~ 4 × Kbds) and of its zinc(II) complex (KbG4 ~ 1.9 × Kbds) towards telomeric G4 versus ds DNA. In the case of the nickel(II) complex, the similarity of KbG4 and Kbds values confirm the pattern of non-selectivity of this complex.The data from the UV-Vis supports, for all the ligands, the formation of ligand–DNA adducts and the obtained binding constants confirm the higher affinity  complexes during the spectrofluorimetric titrations, which is in line with the disaggregation process of ligands in the presence of DNA structures and also pointing to a higher affinity of The melting profiles obtained for the human telomeric G4 in the absence and in the presence of all the selected ligands also confirmed that cationic dibenzoporphyrins are able to stabilise the G4 telomeric.


Based on the structural and morphological analysis such as X-Ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), and RAMAN the composite of F0.1BiVO4/r-GO/Nafion was successfully prepared with no filth. It was used to selectively detect the environmental contaminant 4-chlorophenol (4-CP) on a modified glassy carbon electrode (GCE). The electron channeling ability of reduced graphene oxide (r-GO) with F0.1BiVO4 yielded a great electrochemical response (ER) in cyclic voltammetry compared to pure GCE and other modified electrodes. The differential pulse voltammetry response of 4-CP was highly sensitive with the detection of limit (LOD) of 0.56 nM and a wide linear response of 0.77–45.0 nM. Fluorine doping, in particular, was able to affect the crystal growth of BiVO4, which was the primary cause of the aforementioned improvement. On the other hand, r-GO acts as an electron bridge to improve charge transfer between electrolytes and F-BiVO4 due to its high electron transport rate. These results demonstrate the effectiveness of F0.1BiVO4/r-GO/Nafion/GCE for the electrochemical detection of 4-CP.This study describes the synthesis of fluorine-doped bismuth vanadate (F They are widely used as wood preservatives, industrial adhesives, disinfectants, pesticides, paints (in cans), and antiseptics in industrial applications as well as paper and plastics manufacturing and plant growth regulators, and intermediates in the production of pharmaceuticals4. Among chlorophenol derivatives, 4-chlorophenol (4-CP) is the most toxic and hazardous1. The high toxicity, mutagenic, and carcinogenic effects of 4-chlorophenol (4-CP) on the ecosystem and human health, as well as its entry into the food chain, have caused increased public concern in recent years2. Therefore, there is a need for a reliable, rapid and sensitive method to analyze the environment and potable water, and also to prevent harm to human health. Up to now, several quantitative strategies have been developed for the quantification of 4-CP, such as UV spectrophotometry, enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis, fluorescence spectroscopy, and high-performance liquid chromatography (HPLC)7. Nonetheless, the above-mentioned methods necessitate large and expensive equipment, skilled operators, and time-consuming processes that restrict the scope of applications. Among 4-CP detection procedures, methods based on the use of electrochemical sensors (ECSs) have enjoyed considerable attention because of their rapid response speed, high sensitivity, and low cost9. In recent years, developing ECSs for the detection of 4-CP through modified electrodes has enjoyed considerable attention. The main components in developing electrodes are noble metal nanoparticles (NPs) and enzymes11. Two drawbacks that lead to the low reproducibility of sensors are immobilization difficulty and instability, which are regarded as the main disadvantages of enzyme-modified sensors. Moreover, electrodes modified with noble metal NPs  are too expensive. One of the most important methods for increasing the sensitivity of sensors is the surface modification of a nano-sized core, which is a heterostructure with two or more materials at a nanometer scale in a hybrid structure. The unique physical and chemical properties of hybrid structures make them encouraging candidates for applications in various fields like biomedicine and electronics12.Globally, water pollution poses a threat to human health and is receiving increasing attention. Organic compounds like chlorophenols have been ranked among the most hazardous organic pollutants due to their widespread use in agriculture and industry16. For instance, Uddin et al.14 reported that ZnO/RuO2 nanoparticles exhibited excellent sensitivity toward 2-nitrophenol. In another study, Ge-dopped ZnO composite on flat GCE was fabricated by electrochemical method. It has provided a better response compared to simple flat GCE or other electrodes in detection of 4-aminophenol13. Several reports have also shown that bismuth vanadate (BiVO4) nanoparticle-based photoelectrochemical sensors exhibit a good response for the detection of nitrite anions (NO2−)17 and non-enzymatic H2O2 photoelectrochemical sensors19. In addition to having a narrow energy gap of 2.4 eV, it has other unique characteristics, including excellent physicochemical properties, easy fabrication, good conductivity, low cost, chemical stability in aqueous solutions and non-toxicity12. Despite having a high electrical conductivity, pure BiVO4 (P-BiVO4) tends to agglomerate and reduces the fast charge and discharge capabilities due to its small specific surface area20. To overcome these drawbacks, many researchers have applied graphene oxide as a substrate for BiVO4. The large surface area of electrode materials has been regarded as important for enhancing the sensing capability. The development of reduced graphene oxide (r-GO), has been recently carried out to avoid the irreversible agglomeration. Gr has unique properties22, which provide r-GO with advantages such as high electron transport rate, high mechanical strength, and large specific surface area24. These advantages make r-GO to be a promising sensing substrate, on which hydrophilic groups serve as an electron bridge for enhancing the charge transport between BiVO4 and electrolytes.In recent years, various metal oxides have received significant attention as electronic mediators with high potential for the fabrication of effective chemical sensors4 structure. For example, Rohloff and coworkers exhibited that the photo-electrochemical performance of Mo:BiVO4 photoanodes achieved 21%25. Another study3 has revealed that Au doping into BiVO4 structure significantly improved water splitting performance. According to this study, Au plays a crucial role in visible light absorption and internal defects formation. Fluorine (F) doping in particular is capable of affecting the crystal growth of BiVO4, as well as providing an internal electric field that is helpful for separating photogenerated electron–hole pairs. Jiang and coworkers26 reported that Fluorine-dopped BiVO4 exhibited higher surface area, better light-absorbing performance, and as result of that lower bandgap energies, which enhanced photodegrdation of phenol to 97%.Up to now, several studies have confirmed that metal and non-metal doping can enhance electron transfer efficiency in BiVO0.1BiVO4/r-GO/Nafion composite was prepared using an effective three-step method  of nanostructure F0.1BiVO4/r-GO onto GCE electrodes. One of the unique features of Nafion is its ability to facilitate proton transfer from its sulfonic groups to the perfluorinated hydrophobic backbone, which results in the formation of a highly conductive medium for protons to provide greater reticulation of the nanocomposites on the surface of the GCE. Finally, the modified electrode (F0.1BiVO4/r-GO/Nafion) was utilized as an ECS to detect 4-CP. The detection limit of F0.1BiVO4/r-GO/Nafion towards 4-CP was low, showing that it has the advantages of the combination of composite materials, such as fast electron transport rate, large specific surface area and high conductance.In this work, a novel F3⋅5H2O, 98.0%), was purchased from Fluka and ammonium vanadate , ethylene glycol (EG) , sodium fluoride (NaF), and the graphite powder (< 50.0 mg) were purchased from Merck and they were used in the hydrothermal synthesis of F0.1BiVO4/r-GO/Nafion composite. Also, 4-chlorophenol was purchased from Across Company. We prepared the Britton–Robinson buffer solution  through mixing the standard stock solution of Boric acid (H3BO3) . Diluted Phosphoric acid (H3PO4)  and Acetic acid (CH3COOH)  were used for the preparation of 3.0–10.0 pH during the study.In this work, bismuth (III) nitrate pentahydrate (Bi (NO)−1 using the KBr disk technique. A Renishaw instrument, model InVia reflex equipped with 532, and 785 nm lasers, was used to obtain the Raman spectra. The laser power was 10 mV. The spectral resolution was < 3 cm−1. For all spectra, acquisition time was 60 ms (4 × 15 ms). The Raman spectra of each sample were filed at diverse areas of the sample. The phase formation of samples was investigated by performing PXRD through a X'Pert diffractometer of PAN analytical with monochromated Co-Kα (λ = 1.78901 Å) radiation, in the 2θ range of 5° to 90°. The characterization of the electrode’s surface structure was investigated through a scanning electron  microscope together with an Energy-dispersive X-ray spectrometer . UV–Vis diffuse reflectance spectrometer  was employed for testing the UV–Vis absorption spectra of the composite. The electroanalytical experiments were performed through an EG&G PARSTAT 2273 electrochemical potentiated with a conventional three-electrode unit. The F0.1BiVO4/r-GO/Nafion-coated glassy carbon electrode (GCE) was the working electrode, and an Ag/AgCl electrode in aqueous 3.0 M KCl solution and Pt, respectively, were utilized as reference and counter electrode. The DPV scan was performed in a potential range of 0.0 to 1.8 V with a step potential of 8.5 mV, a pulse amplitude of 10 mV, a pulse width of 10 ms, a pulse period of 100 ms, and a scan rate of 0.01–0.07 V/s. The scan rate was calculated as the potential increment per unit time during the DPV scan.We recorded the FT-IR spectra on a thermoscientific NICOLET IR100 FT-IR Spectrometer which ranged from 400 to 4000 cm4 was synthesized using a hydrothermal reaction, according to the prior reports28. For the typical preparation, we dissolved 2.0 mmol of Bi (NO3)3·5H2O in 30.0 mL of ethylene glycol (EG) solution under continuous agitation at room temperature for 30 min. Next, we dissolved 0.234 g of NH4VO3 in 20.0 mL of EG for 15 min under steady stirring. We added NH4VO3 to the above mixture dropwise and a homogeneous solution was established following 1 h stirring. Then, we added NaF with the molar ratio of 0.5 wt% to the above solution and it was stirred for another hour. We kept the obtained solution in a Teflon-lined autoclave for 24 h at 180 °C. Following the reaction, we later collected the black precipitate through centrifugation using water and ethanol was used to wash it several times. We dried the obtained powders in a vacuum oven for 20 h at 60 °C and after that they were calcined at 450 °C for 5 h.Spherical-like BiVO29. The standard hydrothermal method was adopted to fabricate F0.1BiVO4/r-GO composites. In summary, we dispersed 0.05 g of F-BiVO4 powder in 50.0 mL of deionized water and ethanol through ultra-sonication for 15 min. Afterward, we introduced 50.0 mL of GO suspension (2.5 mg/mL) into the F0.1BiVO4 suspension dropwise, obtaining the weight ratio of GO to F0.1BiVO4 at 0.0025:0.05. After stirring at 60 °C for 5 h, a homogeneous suspension was created. The F0.1BiVO4/r-GO suspension was then rinsed with ethanol and deionized water several times and dried in a 60 °C oven. The synthesis method’s schematic view is presented in Fig. The synthesis of GO nanosheets was accomplished through the chemical oxidation of graphite flakes and then exfoliation, according to the improved Hummers’ method2O3 slurry and ultrasonically cleaned in distilled water and ethanol several times. Then, 2.5 µL of the prepared ink was loaded onto a polished GCE and dried. Different modified-electrodes were characterized by cyclic voltammetry (CV) in 5 mM [Fe (CN)6]3−/4− containing 0.1 M KCl.To prepare the working electrode, 2–10 mg of the prepared materials were dispersed in 1 mL of ethanol and isopropanol with a volume ratio of 1:1 and 15 µL of 0.1 wt% Nafion suspension under sonication for 30 min to form a homogeneous ink. Then, the bare GCE was polished with 0.3 μm Al4 and F-BiVO4 nanostructures. We can index the XRD patterns of P-BiVO4 to the characteristic peaks (CPs) of monoclinic BiVO4 , where the peaks located at 19.0°, 30.0°,30.5°, 35.2°, 46.7°, 53.3°, and 59.5° belong to the (011), (112), (004), (020), (204), (116) and (132) planes, respectively. The pattern of F-BiVO4 exhibited the main peaks of m-BiVO4. Meanwhile, the CPs of the BiVO4 at 30.6°, 32.8°, and 39.9° shift to lower angles after modification with fluorine  than o2 (1.28 A)32. Also, the XRD pattern of the F0.1BiVO4 shows an increase in the peak intensity at 2θ = 27.5°, 30° compared with that of BiVO4, suggesting that F-doping leads to the prompt of the crystallization of V2O5. Using the Debye–Scherrer equation, we obtained the average size of the crystal particles, which is equal to 45.5 nm. Moreover, the dipole moment (DM) of VO43− tetrahedron deviated from zero to non-zero DM after BiVO4 was doped with F, which can increase the separation of charge on photo-excitation and improve the electrochemical activity33. The 4-CP of GO (001) at around 11° almost disappears in the r-GO-BiVO4 nanocomposite, which might be because of the low content of GO used in the nanocomposites35.XRD in the 2θ range of 5° to 90° , usually appearing below 900 cm−1, were observed around 810 cm−1 and demonstrated that metal oxide interacted with Gr24.We recorded the FTIR measurements of P-BiVO4 in all of the samples that were synthesized. It was observed that BiVO4 had a monoclinic phase according to the attributed stretching vibrations and twisting vibrations of the VO43– tetrahedron42. The strongest Raman band around 831 cm was assigned to the symmetric V–O (Ag) stretching mode, but the weak Raman band at 706 cm was assigned to the antisymmetric V–O (Bg) mode stretch43. The Raman band near 372 represents the symmetric δs (VO43–) (Ag) bending mode and 331 cm−1 represents the antisymmetric δas (VO43–) (Bg) bending mode.An efficient method for inspecting the crystallization, electronic properties of the materials and investigating the local structures is Raman spectroscopy. Figure −1 was assigned as rotational and 126.58 cm−1 was assigned as translational. As shown in the inset of Fig. 4 indicates (defect formation) that the VO43− tetrahedron was weakly deformed, which was due to the fact that V ions were replaced with F ions in the F-doped sample, we could not observe extra peaks, but there was an enhancement in the intensity of the peak for F-doping, which was incorporated in the BiVO4 and also increased the crystallinity behavior observed in XRD data. On the other hand, the Raman profile of GO was dominated by 2 CPs of carbonaceous materials located at 1351 and 1590 cm−1, i.e., the D band peak because of the sp3 defects and the G band peak, which could be attributed to in-plane vibrations of sp2 C atoms45. The Raman spectrum of F-doped BiVO4/r-GO composite exhibited the same CPs as F doping in BiVO4 and G and D bands of GO. Additionally, peak G in F0.1BiVO4/r-GO was relatively blue-shifted by 20 cm−1 in contrast with GO. Therefore, this clearly demonstrated that GO was effectively reduced into r-GO. The intensity ratios of the D and G bands (ID/IG) in GO and F0.1BiVO4/r-GO were determined 0.93 and 0.95, respectively. The ID/IG ratios for F0.1BiVO4/r-GO was higher than those for GO, indicating that a significant number of structural defects were introduced to the graphene lattice in the reaction. Conclusively, the larger ID/IG ratios in the F0.1BiVO4/r-GO compared to GO indicate an increase in the amount of smaller sp2 domains and the revival of graphene network conjugation (re-aromatization). Moreover, the revived graphene network size was smaller than that of the GO starting material. This effect gave rise to an increased ID/IG ratio in the F0.1BiVO4/r-GO composite materials.The mode at 213.23 cm4, and F0.1BiVO4/r-GO samples. Figure 4 has spherical-like and homogenous particles with diameters of 40–90 nm. The magnified images in Fig. 4. After the composition of F0.1BiVO4 with GO  analysis. The results from mapping images in Fig. 4 structure.FE-SEM micrographs see Fig.  show ima0.1BiVO4/r-GO was dispersed in 1 mL of ethanol and isopropanol with a volume ratio of 1:1 and 15 µL of 0.1 wt% Nafion suspension under sonication for 30 min to form a homogeneous ink. Then, the bare GCE was polished with 0.3 μm Al2O3 slurry and ultrasonically cleaned in distilled water and ethanol several times. Then, 2.5 µL of the prepared ink was loaded onto a polished GCE and dried. For comparison, a similar procedure was used to prepare F0.1BiVO4/GCE, GO/GCE, and BiVO4/GCE. Different modified-electrodes were characterized by cyclic voltammetry (CV) in "5 mM" [Fe (CN)6]3−/4− containing 0.1 M KCl.To prepare the working electrode, 2–10 mg of the F4/GCE, (red) F0.1BiVO4/GCE, (Pink) GO/GCE, (Blue) F0.1BiVO4/r-GO/Nafion/GCE was thoroughly inspected through the measurement of the CV in solution of [Fe (CN)6]3−/4− with 0.1 M KCl with the frequencies at 50.0 mV/s scan rate. As shown, the values of anodic peak current of these electrodes were as follows: 2.46 μA (Bivo4/GCE), 7.9 μA (F0.1BiVO4/r-GO/Nafion/GCE), 8.8 μA (GO/GCE), and 16.5 μA (F0.1BiVO4/r-GO/Nafion/GCE). Using the CV results, it was concluded that the F0.1BiVO4/r-GO/Nafion on the electrodes surface exhibited the highest redox peak currents. This was attributed to the large surface area and the excellent electrical conductivity of the GO and F0.1BiVO4, which significantly increased the electron transfer rate  had a better electron transfer, followed by F0.1BiVO4 (0.095 V), BiVO4 (0.098 V), and GO (0.110 V). The indication is that F0.1BiVO4/r-GO/Nafion is surpassing GO in terms of conductivity47.The electrochemical behavior of (black) BiVO0.1BiVO4/r-GO/Nafion/GCE,and bare GCE electrodes in the presence of 0.1 nM of 4-CP (in 0.1 M KCl at pH∼7.0) were scrutinized through cyclic voltammetry at 0.1V/s scan rate. In the in the presence of GCE electrode, no redox peak was observed. But in the presence of F0.1BiVO4/r-GO/Nafion/GCE, a relatively large oxidation peak with the potential of 0.78 V and the peak current of about 58 μA was observed  on F0.1BiVO4/r-GO/Nafion/GCE sensor surface is controlled by adsorption.The results show that the electrochemical response process of 4-CP onto the F0.1BiVO4/r-GO/Nafion/GCE was evaluated in 0.1 M Britton–Robinson buffer (BRB) in the pH range of 3.0 to 10.0  peak potential became lower and lower by increasing pH. Also, we can express the dependence of pH on the shift in Epa as follows49.The impact of pH on the CV responses of F0.1BiVO4/r-GO/Nafion/GCE electrode is a one-electron and one-proton process and this is illustrated in Fig. 850.This slope value is approximately the same as the theoretical Nernstian number of − 59.0 mV/pH, which demonstrated that the number of involved protons and electrons in the electron transfer process of 4-CP was equal. Therefore, it can be inferred that the electrochemical oxidation of 4-CP at the F0.1BiVO4/r-GO/Nafion/GCE sensor for detecting a series of 4-CP solutions with varying concentrations was investigated by the DPV method. As exhibited in Fig. p) by increasing the concentration of 4-CP from 0.77 to 45.0 nM. As can be seen from Fig. Under the optimal conditions, the analytical ability of the F0.1BiVO4 and GO thin nanosheets, which were better than those of previously reported sensors.The detection limit for 4-CP was 0.56 nM (S/N = 3). Moreover, in Table 0.1BiVO4/r-GO/Nafion/GCE sensor was evaluated by studying the influence of some potential interferents including inorganic ions and organic phenolic compounds, which were coexisted in the electrochemical detection of 4-CP in the subsequent DPV experiments. As shown in Fig. 0.1BiVO4/r-GO/Nafion/GCE towards 4-CP was approximately 10 times larger than interferents, indicating the proposed sensor had good anti-interference ability, and therefore, can be used to selectively determine 4-CP.The selectivity of the F0.1BiVO4/r-GO/Nafion/GCE in BR buffer solution (Figs. 0.1BiVO4/r-GO/Nafion/GCE modified electrode effectively prevent the surface fouling effect caused by the oxidation products of the 4-CP. According to the result above, the developed F0.1BiVO4/r-GO/Nafion/GCE modified GCE is a suitable platform for the 4-CP analysis with good stability and repeatability.Additionally, The oxidation peak current of 1 nM 4-CP decreased with the successive potential scan on the Fon Figs.  and 3S.T0.1BiVO4/r-GO/Nafion/GCE modified electrode, real sample analysis was carried out using DPV technique. The standard addition method was followed to determine the 4-CP in tap water. The concentration of added PCMC and the recovery values are displayed in Table To study the practicability of F0.1BiVO4. In addition, an electrochemical method was applied to synthesize the F0.1BiVO4/r-GO/Nafion/GCE composite. Various techniques have been used to characterize the synthesized mixture for its morphological, structural and elemental properties. In addition, the 2D layered material synthesized with impregnated nanoparticles was exploited as an electrocatalyst for 4-CP detection. In addition, the quantitative determination of 4-CP through the proposed modified electrode provides a detection limit of 0.56 nM and a linear range of 0.77–45.0 nM, a wide range. Thus, the modified GCE demonstrated high sensitivity, better stability, and a lower detection limit for 4-CP detection. These properties demonstrated the potential practical applications of the F0.1BiVO4/r-GO/Nafion/GCE for 4-CP sensors. Furthermore, the new sensor is efficient, environmentally sustainable, cost-effective and simple to manufacture.In this study, the solvothermal method is applied to synthesize the catalyst FSupplementary Information.


Lopharalichus are described from parrots in Brazil.Understanding the current biodiversity of our planet is an ongoing challenge, as natural habitats are being destroyed at a faster rate than species are described. This is especially true for South America, which harbors over one-third of the parrot species in the world. A diverse yet poorly studied group of mites associated with birds are feather mites, which currently include about 2500 known species, and estimates range from 10,000 to 20,000 species. Herein, three new species of feather mites of the genus Lopharalichus Gaud & Atyeo, 1996 (Pterolichidae: Pterolichinae), formerly containing three described species, are associated with New World parrots (Psittaciformes: Psittacidae) of the subfamily Arinae. Three new species of this genus are described: Lopharalichus tuimsp. nov. from Forpus xanthopterygius , L. spinosussp. nov. from Ara ararauna , and L. chiririsp. nov. from Brotogeris chiriri . Type specimens of the previously described Lopharalichus species were examined, and a key to the known species is provided.Feather mites of the genus  Protolichus, Psittophagus, and Rhytidelasma groups [Protolichus generic group, incorporating nearly 100 described species in 24 genera, is the most diverse of these groups, with 11 genera found on parrots of the New World [Lopharalichus Gaud & Atyeo, 1996 belongs to this group and has included, to date, three species [Lopharalichus denticulatus  from Pyrrhura cruentata  from Brazil, L. cribiformis  from Forpus passerinus  from Guyana, and L. beckeri Mironov, Dabert & Ehrnsberger, 2005 from Conuropsis carolinensis , an extinct parrot of North America. Gaud & Atyeo [Thectocercus acuticaudatus  (formerly Aratinga acuticaudata) and Forpus modestus sclateri  (formerly Forpus sclateri). An undetermined Lopharalichus species was reported from Brotogeris chiriri  [Lopharalichus from Brazil, and these mites are described below.Three groups of feather mite genera from the subfamily Pterolichinae (Acariformes: Pterolichidae) are found on parrots (Psittaciformes): a groups ,2,3. The species ,6: LophaLopharalichus is the presence of prominent spiny crests on the femora and genua of legs I and II of both males and females, after which the genus was named . Other noticeable features are as follows: in both sexes, the lateral regions of hysterosoma have small cuticular spines, setae c2 are bifid, scapular setae si are subequal to or longer than setae se, setae se are very short (at maximum ½ the distance si:se), the prodorsal shield is entire , and setae h1 are absent. Additionally, in males, setae h2 and h3 are flatly expanded with a filamentous tip, setae e2 are bifid with a short basal spine (except in L. denticulatus), setae f2 are expanded (leaf-like), setae ps1 are broad, and in females, setae e2 and ps1 are short, expanded with minute spines.The most distinctive feature of the genus Lopharalichus are described from parrots of Brazil, and a key to the known species of this genus is presented.In this paper, three new species of Ara ararauna (Linnaeus) were also retrieved from dry museum skins deposited at the Museu de História Natural Capão da Imbuia (MHNCI), Curitiba, following the ruffling technique described in Gaud & Atyeo [The new mites studied herein were collected from either wild bird specimens found dead in the field or from taxidermied bird specimens (see below). In the former case, the birds were collected and frozen for a later study; in laboratory, they were washed in a plastic tray with water and detergent to remove the ectoparasites , and the & Atyeo . The mit & Atyeo , and hea & Atyeo  and Atye & Atyeo , respect & Atyeo . The nom & Atyeo .Lopharalichus cribriformis and L. denticulatus determined by W.T. Atyeo and are deposited at the Trouessart collection of the Muséum National d’Histoire Naturelle (MNHN), Paris, France. Photos of non-type specimens of L. beckeri deposited at the Zoology Institute, Russian Academy of Sciences (ZISP), St. Petersburg, were also examined.The species descriptions are given according to the formats proposed by Mironov et al.  and HernPterolichidae Trouessart & Mégnin, 1884Lopharalichus Gaud & Atyeo, 1996Lopholichus, Gaud & Atyeo 1996:121, sic)(Pterolichus (Pterolichus) denticulatus Mégnin & Trouessart, 1884, by original designation.Type species: Lopharalichus denticulatus Pterolichus (Pterolichus) denticulatus Mégnin & Trouessart, 1884 [rt, 1884 : 211.Pterolichus (Eupterolichus) denticulatus; Canestrini & Kramer, 1899 [er, 1899 : 37.Pterolichus denticulatus; Radford, 1953 [rd, 1953 : 201; GaPyrrhura cruentata  (Psittaciformes: Psittacidae) from BRAZIL, no further data, MNHN#969.236.3 (slide 35-I-6) ).Type material examined: Lectotype male ex P. cruentata, BRAZIL, Bahia state, Boa Nova, 5 June 1928, E. Kaempfer , MNHN#1060.31.2 (slide 65-D-6) (W.T. Atyeo det. 1993); one female ex P. cruentata, BRAZIL, Espírito Santo state, Lagoa Juparanã, 11 November 1929, E. Kaempfer , MNHN#1060.31.1 (slide 65-D-5) (W.T. Atyeo det. 1993).Additional material examined: One male ex Lopharalichus denticulatus stands out from other species in having, in males, setae ps1 roughly triangular, setae e2 simple and not bifurcate basally; in females, the prodorsal setal pair si is well spaced by about three-times the distance si:se A–FPterolichus (Pterolichus) denticulatus var. cribriformis Mégnin & Trouessart, 1884 [rt, 1884 : 213.Pterolichus (Eupterolichus) cribriformis; Canestrini & Kramer, 1899 [er, 1899 : 38.Pterolichus denticulatus; Radford, 1953 [rd, 1953 : 201.Forpus passerinus  (=Psittaculus passerinus), from GUYANA, MNHN#969.237.1 (slide 35-I-8).Type material examined: Syntypes 15 males and 19 females  ex Forpus passerinus cyanochlorus , BRAZIL, Amazonas state, Frechal, Rio Surumu, 6 September 1929, T.D. Carter col.  MNHN#1060.30 (slide 65-D-4) (W.T. Atyeo det. 1993).Additional material examined: one male and one female ex Lopharalichus cribriformis is very similar to L. beckeri Mironov et al. (2005), differing from that species in having, in males, the terminal cleft angular and the paragenital apodemes indistinctly developed, and in females, setae si distinctly longer and more robust than se (at least twice longer and twice thicker)  B, and ththicker) F. In malcircular C, and thtructure C, and sotructure G.L. cribriformis, setae e2 are twice as long as f2, and in females, setae f2 are “large and foliform, almost circular, and with a vein”. However, in the examined specimens of this species, setae e2 and f2 have about the same length in males, and setae f2 of females are roughly triangular, like in L. beckeri. The type series of L. cribriformis consists of a single slide containing 34 poorly clarified syntypes, still with the original label by E.L. Trouessart. The illustrations presented here are based on non-type material collected from the type host species and determined by W.T. Atyeo.Mironov et al.  stated tLopharalichus beckeri Mironov, Dabert & Ehrnsberger, 2005Lopharalichus beckeri Mironov, Dabert & Ehrnsberger, 2005 [Conuropsis carolinensis , USA, Florida, Tampa, no date, coll. W. Brewster.Material examined: Photos of 1 male and 1 female  ex Lopharalichus beckeri was described from Conuropsis carolinensis , an extinct parrot from North America. This species is very similar to L. cribriformis .Remarks: Lopharalichus tuim sp. nov.Zoobank registration: urn:lsid:zoobank.org:act:7BAEC958-A174-42FC-8C28-0198480DC854Forpus xanthopterygius  (Psittaciformes: Psittacidae), BRAZIL, São Paulo State, Pedreira, 22°44′ S, 46°54′ W, June 2012, D.V. Boas-Filho col. (#1074).Type material. Holotype male, paratypes 10 males, 31 females, and 1 nymph ex vi to the base of setae h3) 284 (284–297), greatest width at level of humeral shields 162 (163–176). Prodorsal shield shaped as an Erlenmeyer flask (elongated trapezoid), with rounded edges, posterior margin slightly sinuous, surface without ornamentation, 64 (64–72) in length from the level of setae vi to the posterior margin, 69 (69–78) in width at the widest part. Scapular setae si thin spiculiform, 9 (8–11) long, setae se piliform, reduced, distance between bases of scapular setae si:si 29 (25–27), se:se 57 (56–61). Hysterosoma 212 (211–226) in length from sejugal area to the bases of setae h3. Hysteronotal shield: anterior margin straight, length from anterior margin to bases of setae h3 207 (212–227), greatest width at the level of setae d2 138 (144–158), surface with numerous circular lacunae posterior to level of setae c1  blunt-angular, 28 (30–34) long, opisthosomal lobes with prominent tubercles at bases of setae h3, narrow interlobar membrane between bases of setae ps1. Setae c2 bifid, 12 (12–15) long; setae e2 lanceolate with short basal bifurcation, 45 (42–49) long; setae f2 lanceolate with outer edge minutely serrate, 54 (56–65); setae ps1 roughly parallelogram-shaped, 78 (77–84) long. Distances between hysteronotal setae: c2:d2 72 (84–90), d2:e2 84 (76–82), e2:h3 40 (45–50), d1:d2 10 (8–13), e1:e2 4 (5–13), ps1:ps1 45 (43–51), h3:h3 69 (66–76), h2:h2 82 (82–92), and ps2:ps2 106 (106–117).Male . Idiosoma length  long; coxal fields I–II without sclerotized areas. Genital apparatus situated between levels of trochanters III and IV, 24 (24–28) long, 11 (10–13) wide; paragenital apodemes as a pair of longitudinal sclerites lateral to the genital apparatus and bearing the genital acetabula. Distances between setae: g:4a 55 (51–58), g:g 8 (6–9). Cupules ih ventrally at the level of setae ps2. Adanal suckers 13 (13–15) in diameter, distance between centers of suckers 24 (22–26), corolla with 5–7 teeth on anterior half, posterior half without teeth , σIII 9 (7–9), φI 50 (50–55), φII 45 (43–47), φIII 33 (30–33), φIV 40 (35–43), ω1I 10 (10–12), ω3I 29 (29–32), and ω1II 19 (17–18).Femora I with 1–3 apicoventral spines or crests, femur II with 2–6 apicoventral spines. Acute apicoventral spines on genua, tibiae I, II. Length of tarsi excluding ambulacra: tarsus I 37 (35–38), tarsus II 44 (46–50), tarsus III 49 (49–54), tarsus IV 55 (55–57). Seta together D. Setae si spiculiform, 14–17 long, setae se piliform, reduced; distances between scapular setae si:si 21–28, se:se 59–63. Hysteronotal shield 239–247 in length, 162–171 in width at the widest part; surface with numerous circular lacunae posterior to level of setae c1. Setae c2 bifid, setae f2, ps1 flat, spiky leaf-like, setae c1, d1, d2, e1, e2 piliform. Terminal region of opisthosoma shaped as a semicircular concavity between a pair of tubercles bearing setae h2, h3, and with a small external copulatory tube in the center about 5–7 long located between setae ps1. Posterolateral margins of opisthosoma with small spines. Length of setae: c2 12–15, c3 16–31, e2 11–16, and f2 16–22. Distances between dorsal setae: c2:d2 95–101, d2:e2 89–93, d1:d2 11–20, e1:e2 40–46, ps1:ps1 17–21, h3:h3 37–40, h2:h2 52–56.Female (range for 6 paratypes). Idiosoma length 309–334, greatest width 173–185. Prodorsal shield-shaped as in the male, 73–79 long, 76–79 wide A; scapula:3a 36–42, 3a:g 20–30. Legs I, II as in the male, except for a shorter apicoventral spine on genu and tibia I. Length of tarsi excluding ambulacra: tarsus I 35–42, tarsus II 44–50, tarsus III 53–56, tarsus IV 59–65. Length of solenidia: σ2I 10–13, σIII 9–10, φI 61–62, φII 50–55, φIII 35–38, φIV 12–16, ω1I 11–13, ω3I 30–34, and ω1II 18–25.Epimerites I free. Bases of epimerites I, II inflated, dark-sclerotized B. EpigynL. tuim sp. nov., is very close to L. cribriformis  described from Forpus passerinus in having a blunt-angular terminal cleft in males. In males of L. denticulatus and L. beckeri, the lobar cleft is concave and semi-circular. The new species most clearly differs from L. cribriformis in the relative length and arrangement of prodorsal setae si: in females of L. tuim sp. nov., si reaches the base of se of the same side . In L. cribriformis females, setae si only reach about halfway to the bases of corresponding se (si is about the same length as se) (si and se are uniformly spaced (distance si:se = si:si).Differential diagnosis: The new species, ame side D, and in than se D. Also, nding se B, and inh as se) B, and inEtymology: The name of the new species is based on the Brazilian common name of the host (tuim) and is a noun in apposition.Lopharalichus spinosus sp. nov.Zoobank registration: urn:lsid:zoobank.org:act:12DC2DDA-DE5D-4356-AF19-53F77CB37A96Ara ararauna  (Psittaciformes: Psittacidae), BRAZIL, São Paulo State, Itatiba, 23°00′ S, 46°50′ W, 24 March 2007, U. Kawazoe col. (#152). Paratypes from the same host species: five males and four females, Pernambuco State, 29 September 1953 (MHNCI#1557), mites collected from the bird skin by FAH in November 2016.Type material: holotype male, seven male and six female paratypes ex vi to the base of setae h3 345 (338–350), greatest width at level of humeral shields 190 (190–193). Prodorsal shield shaped roughly as an isosceles trapezoid, with sinuous lateral margins and rounded edges, 78 (76–78) in length from the level of setae vi to the posterior margin, 96 (93–96) in width at the posterior margin. Scapular setae si as a short spike, about as long as the distance between their bases, 13 (11–13) long, distance between scapular setae si:si 25 (24–25), se:se 75 (73–77). Hysterosomal region 263 (256–265) in length from sejugal area to the bases of setae h3. Hysteronotal shield: anterior margin straight, length from anterior margin to bases of setae h3 260 (254–256), greatest width around the level of setae d2 165 (167–176), surface with numerous circular lacunae from the level of setae c1 to genua IV  long, opisthosomal lobes with prominent tubercles at bases of setae h3, narrow interlobar membrane between bases of setae ps1. Setae c2 bifid, 21 (17–21) long; setae e2 lanceolate with short basal bifurcation, greatest length 43 (40–43); setae f2 lanceolate with external margin minutely serrate, 66 (64–66); setae ps1 roughly parallelogram-shaped with sharp posterior edges, 89 (88–90) long. Distances between hysteronotal setae: c2:d2 111 (106–111), d2:e2 101 (95–98), e2:h3 52 (50–52), d1:d2 11 (13–14), e1:e2 11 (9–11), ps1:ps1 49 (45–52), h3:h3 78 (75–78), h2:h2 96 (92–96), and ps2:ps2 126 (116–126).Male . Idiosoma length from the level of setae genua IV A. A bow-c3, cp ventrally. Setae c3 thin piliform, 19 (17–19) long, coxal fields I, II without sclerotized areas. Genital apparatus situated between levels of trochanters III, IV, 30 (27–30) long, 12 (12–14) wide; paragenital apodemes as a pair of longitudinal sclerites roughly parallel to the arms of genital arch and bearing genital acetabula. Distances between setae: g:4a 66 (61–67), g:g 9 (7–9). Cupules ih ventrally at the level of setae ps2. Adanal suckers 15 (14–17) in diameter, distance between centers of suckers 27 (25–27), corolla with 5–7 teeth on anterior half, posterior half without teeth.Bases of epimerites I, II inflated, dark-sclerotized B. HumerakT present on tibia IV. Setae d, e minute spiculiform inserted together , σIII 10 (8–10), φI 49 (46–50), φII 43 (42–44), φIII 47 (39–42), φIV 45 (37–42), ω1I 11 (10–11), ω3I 30 (26–30), and ω1II 20 (17–20).Femora I, II with 3–5 apicoventral spines or crests. Acute apicoventral spines on genua, tibiae I, II. Length of tarsi excluding ambulacra: tarsus I 44 (40–42), tarsus II 55 (53–55), tarsus III 57 (58–60), tarsus IV 67 (62–65). Seta together D. Genualsi spiculiform, 17–20 long, setae se piliform; distances between scapular setae si:si 29–39, se:se 78–88. Hysteronotal shield 278–307 in length, 179–190 in width at the widest part at the level of setae d1; surface with numerous circular lacunae from the level between setae c1 to e2. Lateral hysterosomal setae c2 bifid, c1, d1, d2, e1, e2 thin piliform, setae f2, ps1 flat, spiky leaf-like. Terminal region of opisthosoma shaped as a semicircular concavity flanked by a pair of tubercles bearing setae h2 and h3. Posterolateral margins of opisthosoma with small spines. Terminal margin of opisthosoma between setae ps1 with small copulatory extension about 5–7 long. Length of setae: c2 14–20, e2 10–14, c3 20–29, and f2 23–32. Distances between dorsal setae: c2:d2 113–124, d2:e2 110–121, d1:d2 7–13, e1:e2 39–48, ps1:ps1 21–26, h3:h3 42–49, and h2:h2 59–65.Female (range for six paratypes). Idiosoma length 367–399, greatest width 197–212. Prodorsal shield shaped as in the male, 82–90 long, 94–106 wide A; scapula:3a 46–55, 3a:g 14–24. Legs I, II as in the male. Length of tarsi excluding ambulacra: tarsus I 39–47, tarsus II 56–61, tarsus III 61–66, tarsus IV 70–76. Solenidion σ1I present, minute, about 5 in length. Length of solenidia: σ2I 10–13, σIII 11–13, φI 56–64, φII 51–60, φIII 41–58, φIV 13–18, ω1I 10–14, ω3I 27–34, ω1II 19–21.Epimerites I free, bases of epimerites I, II inflated, dark-sclerotized B. EpigynLopharalichus spinosussp. nov. is close to L. beckeri and L. cribriformis in having, in males, well-formed cuticular spines in the lateral part of idiosomal anterior to setae e2. In both sexes of L. spinosus, however, those spines are much more numerous and occupy a larger area, from the level of setae cp to that of setae e2; in addition, in males of the new species, scapular setae si are spiculiform, noticeably more robust than se  referring to the numerous cuticular spines on the lateral margins of hysterosoma, more pronounced and numerous than in other known species.Lopharalichus chiriri sp. nov.Zoobank registration: urn:lsid:zoobank.org:act:6DD91CE5-499F-43AD-8210-7B84DF879959Brotogeris chiririType material: holotype male, 15 male and 8 female paratypes ex  (Psittaciformes: Psittacidae), BRAZIL, São Paulo State, Pedreira, 22°44′ S, 46°54′ W, October 2013, D.V. Boas Filho col. (#1113); paratypes 4 females and 1 nymph, same host species, Pará State, Santana do Araguaia, Fazenda Fartura, 09°40′ S/50°23′ W, 07 September 2011, D.V. Boas-Filho coll. (#1006).vi to the base of setae h3 285 (294–308), greatest width at level of humeral shields 160 (160–167). Prodorsal shield roughly as an isosceles trapezoid with rounded posterior corners, 76 (67–74) in length from the level of setae vi to the posterior margin, 74 (76–79) in width at the widest part. Scapular setae si piliform, 7 (6–7) long, distance between si:si 23 (23–26), se:se 59 (57–62), si:se 17 (17–19). Hysterosomal region 224 (213–219) in length from sejugal area to the bases of setae h3. Hysteronotal shield: anterior margin straight, length from anterior margin to bases of setae h3 207 (213–219), greatest width around the level of setae d2 150 (140–155), surface with sparse circular lacunae from the level of setae c1 to genua IV  long, opisthosomal lobes with prominent tubercles at bases of setae h3, and narrow interlobar membranes between bases of setae ps1. Setae c2 bifid, 12 (11–15) long; setae e2 lanceolate with short basal bifurcation, greatest length 35 (35–44); setae f2 lanceolate with outer margin minutely serrate, 54 (59–68); setae ps1 roughly parallelogram-shaped, 73 (73–82) long. Distances between hysteronotal setae: c2:d2 95 (94–100), d2:e2 81 (71–81), e2:h3 42 (41–50), d1:d2 16 (9–16), e1:e2 9 (8–14), ps1:ps1 42 (42–47), h3:h3 66 (67–72), h2:h2 82 (85–92), ps2:ps2 106 (107–115).Male . Idiosoma length from the level of setae genua IV A. A bow-c3, cp ventrally. Setae c3 thin piliform, 17 (12–15) long, coxal fields I–II without sclerotized areas. Genital apparatus situated between levels of trochanters III and IV, 13 (10–13) long, 11 (10–11) wide; paragenital apodemes as a pair of thin longitudinal sclerites roughly parallel to the arms of genital arch and bearing genital acetabula. Distances between setae: g:4a 51 (47–53), g:g 9 (7–13). Cupules ih ventrally at the level of setae ps2. Adanal suckers 13 (13–15) in diameter, distance between centers of suckers 24 (22–27), corolla with 5–7 teeth on anterior half, posterior half without teeth.Bases of epimerites I, II inflated, dark-sclerotized B. HumerakT present on tibia IV. Setae d, e minute spiculiform inserted close together. Solenidion σ2 of genu I apparently absent. Length of solenidia: σ2I 7 (7–9), σIII 9 (7–10), φI 50 (48–55), φII 43 (39–48), φIII 41 (34–45), φIV 40 (33–40), ω1I 12 (10–11), ω3I 26 (25–28), ω1II 19 (18–20).Femora I, II with 1–4 apical spines on. Acute apicoventral spines on genua, tibiae I, II (slightly more developed on legs II than in legs I). Length of tarsi excluding ambulacra: tarsus I 33 (31–36), tarsus II 43 (40–45), tarsus III 46 (42–49), tarsus IV 48 (48–50). Seta si short spiculiform, 9–11 long, setae se piliform; distances between scapular setae si:si 21–28, se:se 59–65, si:se 16:21. Hysteronotal shield 233–254 in length, 163–174 in width at the widest part around level of setae d1; surface with numerous circular lacunae from the level between setae c1 to supranal concavity. Lateral hysterosomal setae c2 bifid, c1, d1, d2, e1, e2 thin piliform, setae f2, ps1 flat, spiky leaf-like. Terminal region of opisthosoma shaped as a semicircular concavity flanked by a pair of tubercles bearing setae h2, h3, and a small external copulatory tube around 5–7 in length between bases of setae ps1. Lateral margins of opisthosoma with few small spines. Length of setae: c2 9–13, e2 8–12, c3 14–17, f2 22–25. Distances between dorsal setae: c2:d2 99–112, d2:e2 85–101, d1:d2 10–20, e1:e2 31–46, ps1:ps1 16–20, h3:h3 35–41, h2:h2 53–57.Female (range for six paratypes). Idiosoma length 296–338, greatest width 171–189. Prodorsal shield shaped as an Erlenmeyer flask (elongated trapezoid), 68–80 long, 74–83 wide A; scapula:3a 37–54, 3a:g 17–21. Length of tarsi excluding ambulacra: tarsus I 30–37, tarsus II 40–46, tarsus III 42–48, tarsus IV 51–58. Length of solenidia: σ2I 8–11, σIII 7–11, φI 54–64, φII 48–58, φIII 38–47, φIV 10–14, ω1I 10–13, ω3I 24–29, ω1II 18–24.Epimerites I free, bases of epimerites I, II inflated, dark-sclerotized B. EpigynLopharalichus chiriri sp. nov. is very similar to L. cribriformis due to the blunt-angular shape of terminal cleft in males but can be distinguished by the relatively longer distance between prodorsal setae si-si. In males of the new species, this distance is about 3.5-times the length of setae si, against 2.5-times that length in L. cribriformis. Also, the new species has smaller dorsal lacunae and relatively shorter solenidion on tibia IV in males, reaching only about half of the length of tarsus (it reaches at least ¾ of tarsus length in L. cribriformis). The new species is also distinguished from all previously known species in having, in both sexes, considerably longer solenidion on tibia III, roughly longer than the length of genu and tibia III combined. In females of L. chiriri, setae si are relatively shorter, their tips not touching each other ; setae e2 not bifid basally……. L. denticulatus 1. Both sexes: wide apicoventral spines on genua I, II around base of seta vi not dilated; cuticular spines present on posterolateral margins of opisthosoma; males with setae ps1 parallelogram-shaped ; setae e2 bifid basally ……. 21’. Both sexes: spines on genua I, II about as wide as those on tibiae I, II; setae si and se piliform, subequal in length , and d1 to f2  …. 43’: In both sexes, spines on the lateral margins of hysterosoma limited to the levels between setae φIII longer or equal to the length of genu + tibia III 5’ In both sexes, distance ce si:se B; in malLopharalichus, they mentioned that it occurred solely on parrots of the subfamily Aratinginae ). Herein, a new species is described from the genus Brotogeris, previosuly considered in yet another subfamily of Wolters, Brotogerinae. In the current classification of parrots [Lopharalichus are parrots of the family Psittacidae, subfamily Arinae, tribes Arini, Forpini, and Androglossini—it remains to be discovered whether Lopharalichus is also present on the tribe Amoropsittacini. Those three tribes account for nearly 140 parrot species (~93% of the arine species), and Lopharalichus spp. has been reported from only eight of those hosts so far, including two undescribed species illustrated by Gaud & Atyeo [By the time Gaud & Atyeo  establis Wolters ). Howeve Wolters  from par parrots , the hos & Atyeo .Lopharalichus, being found only in New World parrots, probably originating between those dates, and given its seemingly uneven distribution on three out of four arine tribes (see above), it probably independently colonized those hosts horizontally rather than vertically. Recent studies have demonstrated that horizontal transfer is an important means of colonizing new hosts e.g., [Lopharalichus being present on the arine ancestor and having independently become extinct from several hosts of the tribe Arini  and Androglossini  and did not retrieve Lopharalichus. As for the tribe Androglossini, only one Lopharalichus is known, L. chiriri sp. nov. from Brotogeris chiriri; the latter tribe contains 10 genera and at least 66 species [Lopharalichus species may be present in some of those hosts. In the past decade, only a few studies have examined feather mites associated with psittaciform birds in Brazil e.g., [According to Wright et al. , the Arits e.g., ,22. An aris, see ). In a sralichus ,26,27,28ralichus  examined species . Since mil e.g., ,32,33,34il e.g., .Protolichus group, the solenidion σ1 of genu I in Lopharalichus is highly reduced, vestigial, and depending on the position of the specimen on the slide, barely visible. Although the presence of this solenidion was confirmed for some Lopharalichus species , it was not possible to confirm its presence in the remaining species studied.As in other genera of the Lopharalichus species (except in L. denticulatus) is not unique to this genus. In other pterolichines belonging to the Protolichus generic group, like Aralichus Gaud, 1966 and Distigmesikya Atyeo, Gaud et Pérez, 1984, the immatures have numerous such spines—in Aralichus, they are mostly located caudally, and in Distigmesikya, they abundantly cover most of the dorsum) [Aralichus glaucogularis Atyeo et Pérez, 1990, and in females of Scolaralichus vazquezae Pérez et Atyeo, 1986, Aralichus menchacai Pérez et Atyeo, 1989, and Tanyaralichus elongatus Pérez et Atyeo, 1989. However, the immatures of the latter two species were not illustrated with cuticular spines [Despite the presence of cuticular spines in the adults, the two examined immature specimens belonging to the species described herein lack such spines. The retention of small cuticular spines on the posterolateral margins of opisthosoma in most adults of  dorsum) ,25. As tr spines ,24,28.Lopharalichus has effectively doubled its known species count, now encompassing six species: L. denticulatus  (type species), L. cribriformis , L. beckeri Mironov et al. 2005, L. tuimsp. nov., L. spinosussp. nov., and L. chiriri sp. nov. However, since most neotropical parrots remain uninvestigated for their feather mites, it is safe to assume that many other Lopharalichus species may exist and will eventually be discovered.With the description of three new species,


The oncoprotein KAI1 C-terminal interacting tetraspanin  promotes cell metastasis, invasion, and angiogenesis, resulting in shorter survival times in cancer patients. Here, we aimed to determine the effects of KITENIN on the energy metabolism of human colorectal cancer cells.The effects of KITENIN on energy metabolism were evaluated using in vitro assays. The GEPIA web tool was used to extrapolate the clinical relevance of KITENIN in cancer cell metabolism. The bioavailability and effect of the disintegrator of KITENIN complex compounds were evaluated by LC–MS, in vivo animal assay.KITENIN markedly upregulated the glycolytic proton efflux rate and aerobic glycolysis by increasing the expression of GLUT1, HK2, PKM2, and LDHA. β-catenin, CD44, CyclinD1 and HIF-1A, including c-Myc, were upregulated by KITENIN expression. In addition, KITENIN promoted nuclear PKM2 and PKM2-induced transactivation, which in turn, increased the expression of downstream mediators. This was found to be mediated through an effect of c-Myc on the transcription of hnRNP isoforms and a switch to the M2 isoform of pyruvate kinase, which increased aerobic glycolysis. The disintegration of KITENIN complex by silencing the KITENIN or MYO1D downregulated aerobic glycolysis. The disintegrator of KITENIN complex compound DKC1125 and its optimized form, DKC-C14S, exhibited the inhibition activity of KITENIN-mediated aerobic glycolysis in vitro and in vivo.The oncoprotein KITENIN induces PKM2-mediated aerobic glycolysis by upregulating the c-Myc/hnRNPs axis.The online version contains supplementary material available at 10.1186/s13578-023-01089-1. The molecular carcinogenesis of colorectal cancer (CRC) is complex and poorly understood . The patDrosophila VANGL1, KAI1 COOH-terminal interacting tetraspanin (KITENIN), has been shown to bind KAI1/CD82 via its COOH-terminal region [KAI1/CD82, a transmembrane glycoprotein in the tetraspanin family, was previously reported to be a prostate-specific suppressor of metastasis. Low KAI1/CD82 activity is associated with metastasis and a poor prognosis for cancers –6. A maml region , and to l region . Previoul region , 16–20.A hallmark of cancer is the reprogramming of energy metabolism , which iMYO1D increases colorectal and breast cancer cell motility and viability, and to upregulated KITENIN and ErbB4 expression . In a prThe expression and biological role of KITENIN in malignant tumors have not yet been characterized, nor has the relationship of KITENIN expression with the metabolism of CRC cells. Therefore, in the present study, we aimed to address these deficiencies, and show that the expression of KITENIN is closely related to aerobic glycolysis and its transcriptional regulators in CRC. These findings suggest that KITENIN represents a potential target for the development of novel cancer cell metabolism-based therapies.Human colorectal cancer cell CaCo2, HCT116 and murine CT26 cell were used. Cell line modified to Empty vector or KITENIN overexpression cells. All cells were cultured in DMEM  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution. 5% CO2 in a humidified atmosphere at 37 °C was used for incubation of the cells. A hypoxic state was created by incubation with 300 µM Cobalt (II) chloride (CoCl2).Cell invasion was assessed using Boyden chambers  coated with 1% gelatin. CRC cells in 100 µl DMEM containing 0.2% bovine serum albumin (BSA) were plated onto precoated inserts and incubated for 24 h. The lower chamber was filled with 600 µl DMEM containing 0.2% BSA and 10 µg/ml fibronectin  as a chemoattractant. After incubation, cells in the upper chamber were fixed and stained using a Diff-Quick kit . Observed using a Nikon Eclipse 400 fluorescence microscope . Invaded cells were counted in five random fields of each Boyden chamber and analyzed by iSolution FLAutosoftware.™ RNAiMAX. Cells were treated for 24 h or 48 h to allow for maximum knockdown, followed by qRT-PCR, Western blot analysis, invasion assays and Seahorse XF instrument analysis.siRNA interference experiments, we purchased from siRNA specific to human MYO1D (Thermo Scientific and Invitrogen), purchased from siRNA specific to human KITENIN (Thermo Scientific and Invitrogen), and siPKM2 specific to human (CCA UAA UCG UCC UCA CCA A) (Bioneer). The si-RNA duplexes were prepared and transfected according to a protocol provided by Lipofectamine™ LTX with Plus Reagent.PKM (Myc-DDK-tagged)-Human pyruvate kinase, muscle (PKM2), transcript variant 2 purchased from Origene. The plasmid was prepared and transfected according to a protocol provided by Lipofectamine3 cells/well, incubated overnight in culture medium (2% FBS), and then incubation for 48 h. On the day of analysis, the plated cells were washed twice and loaded (180 μL) with assay medium enriched with glucose, sodium pyruvate, and glutamine. While incubating the cells for 1 h at 37 °C in a non-CO2 incubator prior to analysis, 0.5 μM rotenone (Rot) + antimycin A (AA) and 50 mM 2-deoxy-d-glucose (2-DG) were loaded into the hydrated sensor cartridge for ECAR analysis (glycolytic rate assay) [To measure real-time changes in ECAR, an XF96 extracellular flux analyzer  was used. First, CRC cells were seeded at 5 × 10e assay) . ECAR wa3 cells/well, incubated overnight in culture medium (2% FBS), and then incubation for 48 h. 1 μM oligomycin, 1 μM carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 μM Rotenone (Rot) + antimycin A (AA) were loaded for OCR analysis (Cell Mito Stress Test) [To measure real-time changes in OCR, an XF96 extracellular flux analyzer  was used. First, CRC cells were seeded at 5 × 10ss Test) . OCR mea5 cells/well, cultured overnight, after that transfection si-MYO1D for 24, trypsinized, and washed with FACS wash solution. First trypsin solution was added and incubated for 10 min and then RNase inhibitor was added and incubated for 10 min at room temperature. Next, the samples were centrifuged, the supernatants were removed, and the pellets were resuspended in 100 µL of 4 mg/mL PI  and incubated for 2 h in the dark at 4 °C. Flow cytometry was performed with a CytoFLEX instrument .CaCo2/EV and CaCo2/KITENIN cells were seeded in six-well plates at a density of 2 × 10Total RNA was extracted from CRC cells using RNAiso Plus . cDNA was synthesized from one microgram of RNA by M-MLV . SYBR Green reagents  were used to determine relative expression of candidate genes. The list of primers used in this research is reported in Additional file http://gepia.cancer-pku.cn/) (http://gepia.cancer-pku.cn/) was extracted to estimate the differential expression level between VANGL1 and metabolic and transcription regulators in colon samples [GEPIA . Antibodies . Fraction purity was assessed by probing for tubulin or β-actin for the cytoplasm and Lamin B for the nucleus.Cytoplasmic and nucleus fractions were prepared by a subcellular protein fractionation protocol according to the Thermo Scientific instructions . For oral administration studies, the carotid artery was cannulated using a polyethylene tube for efficient blood sampling. For intravenous administration studies, the carotid artery and jugular veins were cannulated for blood sampling and drug administration, respectively. Each cannula was exteriorized to the dorsal side of the neck. Then, each animal was individually housed in a metabolism cage and allowed to move freely. A recovery time of 3–4 h from anesthesia was allowed before drug administration. For oral administration, DKC-C14S (4 mL/kg dissolved in 50% PEG400 water solution) solution was administered using a gastric gavage tube at doses of 10 mg/kg. For intravenous administration, the same solution was administered via jugular vein over 1 min at doses of 2 mg/kg. Animal blood samples were collected using automated blood sampling system  and the time point were as follows: 5, 15, 30, 60, 120, 240 and 360 min after administration. The blood samples were immediately centrifuged at 12,000 rpm for 5 min. Each plasma sample was transferred into a new microtube and stored at − 80 °C until used for LC–MS/MS analysis. Sample analysis was conducted by validated LC–MS/MS method. The liquid chromatographic system was Ultimate® 3000 HPLC unit  which connected to AB SCIEX API 3200 triple quadrupole mass spectrometer  with an electrospray ionization (ESI). All experimental procedures were approved by Dankook University’s Institutional Animal Care and Use Committee.Eight-week-old adult male ICR mice and SD rats were used in the present study. The animals were housed four per cage and kept in a vivarium maintained the temperature at 23 ± 2 °C and the humidity at 50 ± 10% with a 12 h: 12 h alternating light/dark cycle. The animals were fasted for at least 12 h prior to the start of the experiments and freely supplied water. On the morning of the test, the animals were anaesthetized with a mixture of alfaxan and rompun  were injected subcutaneously into the right flank of each mouse. After 1 week later, mice were intraperitoneally injected with vehicle or DKC-C14S at a dose of 5 mpk once every 2 days. Using a fluorescence-labeled organism bioimaging instrument (FOBI) technology, near-infrared fluorescence bioimaging was promptly achieved . After sacrifice, mouse tumor tissues could be properly isolated, and each sample's tumor weight was calculated. The Chonnam National University Medical School Research Institutional Animal Care Committee provided oversight for all animal trials, and the committee also approved all experimental procedures (CNU IACUC-H-2020-12).Five- to six-week-old BALB/c mice were obtained from OrientBio  and housed in metal cages with free access to water and food. In brief, CT-26/KITENIN-iRFP-expressing cells . The statistically significant between two groups was compared using the student’s t test. Unless indicated otherwise, a p-value ≤ 0.05 was considered significant.vs. low expression of PKM2  level in patients  are involved in numerous biological processes, including cell survival, differentiation, and growth, and have been targeted for a number of cancer therapies –44. PrevWe next assessed the effects of MYO1D knockdown on the cell cycle in KITENIN-overexpressing cells, by comparison to cells transfected with empty vector, and found that MYO1D knockdown caused cell cycle arrest. Specifically, MYO1D knockdown in CaCo2/EV and CaCo2/KITENIN-overexpressing cells accumulated the G1 and S phases. Therefore, MYO1D knockdown caused the population to decline in the G2/M phase  under KITENIN overexpression condition Fig. A, B. NexTo determine whether si-PKM2 specifically reduces PKM2 expression, we performed Western blotting to measure the protein expression of PKM2, PKM1, and KITENIN in CaCo2/EV and CaCo2/KITENIN cells -RACK1-Dvl2 complex by binding to KSRP . To deteLater in this study, we created a skin xenograft model of DKC-C14S, the soluble form Fig. A of DKC1KITENIN is an oncoprotein that is associated with the metastasis of tumors and a poor prognosis in patients. Specifically, it promotes invasiveness, early hepatic metastasis, angiogenesis, and tumorigenicity , 58–60. Cellular metabolism has a key role in cancer and is an important area for research aimed at better understanding the molecular biology of this disease . The ideFirst, we analyzed the glycolysis and mitochondrial respiration of KITENIN-overexpressing cells and cells transfected with empty vector using a Seahorse XFe96 instrument and found that KITENIN-overexpressing cells predominantly use glycolysis for energy production. The level of mitochondrial respiration did not differ between KITENIN-overexpressing and empty vector-transfected cells. Next, we analyzed the mechanism of the effect of KITENIN on aerobic glycolysis. Glucose is catabolized to pyruvate and lactic acid during glycolysis, with very little ATP being produced . The actThe transport of glucose across cell membranes is mediated by facilitative and energy-dependent mechanisms involving the GLUT family or sodium/glucose-linked transporter family . GLUT1 iRTKs frequently regulate differentiation, cell growth, and survival, and are regarded as proto-oncoproteins because the overexpression or activation of many are associated with the development and growth of tumors –44. In oThe oncoprotein c-Myc promotes the transcription of hnRNPI, hnRNPA1, and hnRNPA2, which reduce PKM1 mRNA expression by binding to the RNA sequence encoded by exon 9, resulting in greater PKM2 (exon 10) isoform expression , 30. HerTranscriptional regulators affect the initiation and progression of cancer, including metastasis and cellular proliferation . NuclearChanges in aerobic glycolysis may be important for metastasis and other effects of the KITENIN oncoprotein. Previous studies have shown that miR-124 targets KITENIN and suppresses tumorigenesis , and supIn our previous study, we developed a KSRP-binding compound that modifies KITENIN expression in multiple types of cancer, and this was developed as a strategy to reduce distant metastasis and chemoresistance. We showed that KITENIN-phospho-Dvl2 and the AP-1/c-jun axis signaling pathway are inhibited by DKC1125 treatment . TherefoIn the present study, we have demonstrated a regulatory role for KITENIN in aerobic glycolysis. c-Myc regulates the expression of hnRNP isoforms, and their upregulation increases the expression of PKM2, which transactivates the genes expressing β-catenin, c-Myc, cyclin D1, CD44, and HIF-1α in KITENIN-overexpressing cells. MYO1D supports KITENIN-induced aerobic glycolysis by regulating the expression and activation of the ErbB4/KITENIN complex by enabling the cell surface receptor ErbB4 anchor them to plasma membrane. These changes in signaling that occur secondary to KITENIN expression enable the upregulation of aerobic glycolysis, resulting in poor survival. Furthermore, DKC compounds inhibit the KITENIN-induced upregulation of aerobic glycolysis and tumorigenesis. Thus, we have shed light on the mechanism of metabolic reprogramming in cancer, and this may lead to the identification of novel target molecules for its treatment.Additional file 1: Figure S1. The overexpression of KITENIN (VANGL1) is correlated with unfavorable prognosis in malignant tumors. (A) The gene expression profile across all tumor samples and paired normal tissues. (B) VANGL1 and transcriptional regulators regulating the metabolic pathway are highly regulated in colon cancer. VANGL1 and transcriptional regulators mRNA expression level determined by the GEPIA web tool. The boxplot analysis showed the expression level by log2 (TPM + 1) on a log-scale. ns, not significant; *, P < 0.05. Figure S2. KITENIN(VANGL1) and genes involved in the aerobic glucose pathway are correlated. The GEPIA web tool was searched for the correlation between the expression of (A) VANGL1-SLC2A1 (GLUT1), (C) VANGL1-HK2, (E) VANGL1-LDHA and (G) VANGL1-SLC16A1 in mRNA levels in COAD samples. Survival rates between high and low expression of (B) VANGL1-GLUT1, (D) VANGL1-HK2, (F) VANGL1-LDHA and (H) VANGL1-MCT1 levels from GEPIA database. Figure S3. Overexpression KITENIN induce the aerobic glucose on CT26 murine CRC cell line. (A-B) CT26/EV and CT26/KITENIN cells incubated 48h and then examinated for metabolism assays. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were detected using the Glycolytic Rate Assay Kit on a Seahorse XF96 extracellular flux analyzer. To measure glycolytic rates, the assay utilizes both ECAR and OCR measurements to determine the glycolytic proton efflux rate (glycoPER). GlycoPER was measured at two time points, followed by sequential injection of rotenone (1 μM)/antimycin A (1 μM), and 2-DG (50 mM). (C-D) To elucidate the mechanisms by KITENIN on glycolysis pathways. Relative protein expression of Warburg effect responsible key enzymes  in CT26/EV and CT26/KITENIN cells incubated for 48h. (E-F) Downstream effectors analyzed by Western blotting on KITENIN overexpression condition in CT26/EV and CT26/KITENIN cells. KITENIN induces the β-catenin, c-myc, and CyclinD1 protein level. (G-H) Hypoxia inducible factor-1α induce on KITENIN overexpression condition. α-tubulin or actin served as a loading control. Data represent means ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure S4. Relative protein expression of Warburg effect responsible key enzymes  under hypoxic condition on CaCo2/EV and CaCo2/KITENIN cells. Data represent means ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure S5. KITENIN knockdown reduced the Warburg effect and responsible key enzymes on HCT116 cell line. (A-B) HCT116 cells transfected si-KITENIN and then examined for metabolism assays. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were detected using the Glycolytic Rate Assay Kit on a Seahorse XF96 extracellular flux analyzer. To measure glycolytic rates, the assay utilizes both ECAR and OCR measurements to determine the glycolytic proton efflux rate (glycoPER). glycoPER was measured at two time points, followed by sequential injection of rotenone (1 μM)/antimycin A (1 μM), and 2-DG (50 mM). (C) KITENIN knockdown efficiency control on mRNA level of KITENIN and ErbB4. (D) Relative mRNA of Warburg effect responsible key enzymes  in HCT116 cells incubated for 48h. (E-F) Protein level of the GLUT1, HK2, PKM1, PKM2 on KITENIN knockdown condition. (G-H) β-catenin, CD44 and Cyclin D1 protein levels on KITENIN knockdown condition. Data represent means ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure S6. MYO1D knockdown reduced the KITENIN, Warburg effect key enzymes and c-Myc/hnRNPs protein expressions on CaCo2 cell line. CaCo2 cells transfected with si-MYO1D for 24h and then examined for Western blotting. (A-B) KITENIN, (C-D) GLUT1, HK2, PKM2 and LDHA, (E-F) c-Myc, hnRNPA1 and hnRNPA2 protein levels. Data represent means ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure S7. Flow cytometric analysis of the cell cycle distributions of Caco2/EV and CaCo2/KITENIN cells. (A–B) Cells were transfected with si-control or si-MYO1D and then incubated for 24 h, after which cells in the subG1, G1, S, and G2M phases were quantified. (C) Effects of si-MYO1D on the mRNA expression of MYO1D, assessed using two different primer sequences (MYO1D #1 and MYO1D #2), in CaCo2/EV and CaCo2/KITENIN cells. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure S8. Effect of si-PKM2 transfection on the level of PKM2, PKM1, and KITENIN proteins in CaCo2/EV and CaCo2/KITENIN cells.  PKM2, PKM1, and KITENIN levels. β-Actin or GAPDH served as the loading control. Figure S9. (A) KITENIN overexpression enhances c-Myc-PKM2 interaction. CaCo2/EV and CaCo2/KITENIN cells were immunoprecipitated with anti-c-Myc antibody and immunoblotted with the indicated antibodies. (B) Calculation of PKM2/c-Myc IPed was given relative to Caco/EV. * p < 0.05. Figure S10. Knockdown of KITENIN and MYO1D inhibit c-Myc and hnRNPI, hnRNPA1, hnRNPA2 mRNA levels on PKM2-myc transfected cells. HCT116 cells were transfected with si-KITENIN and si-MYO1D for 24h followed by transfection of the plasmid, a construct expressing myc-tagged PKM2 (PKM2-myc) for 24h and subjected to the qRT-PCR. Data represent means ± standard deviation, n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure S11. KITENIN overexpression is induced under hypoxic conditions treatment with 300 μM Cobalt (II) chloride (CoCl2) on CaCo2/EV and CaCo2/KITENIN cells. (A) Normoxic condition and hypoxic condition on the same blotting membrane, HIF-1A level. (B) Relative protein level of HIF-1α. (C-F) CaCo2 and HCT116 cells were transfected with si-KITENIN and si-MYO1D for 24h followed by transfection of the plasmid, a construct expressing myc-tagged PKM2 (PKM2-myc) for 24h and subjected to the β-catenin, CyclinD1 and HIF-1α mRNA levels. Data represent means ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure S12. KITENIN and its downstream signals protein ERK , GLUT1, HK2, PKM1, PKM2, c-Myc, hnRNPA1, HIF-1α, β-catenin, and Cyclin D1 were examined via immunoblot analyses tumors collected from both groups, vehicle and DKC-C14S (5 mpk). The quantitative amount of each protein was measured by densitometry and is represented as an arbitrary score . An asterisk indicates a significant difference between indicated groups. * p < 0.05; ** p < 0.01; *** p < 0.001, NS, no significant difference between groups. Table S1. Primer (Forward/Reverse) sequences. Table S2. Antibodies information.