The fabrication of nanofibers with a mechanical force has attracted increasing attention owing to its facile and easy fabrication. Herein, we demonstrate a novel and facile fabrication technique with the mechanical force, needle spinning, which utilizes a needle tip to draw a polymer solution to form fibrous structures. We studied the effect of the processing parameters to the nanofiber structure, namely, the pulling away speed, pulling away distances, needle size, and polymer concentration, which were systemically controlled. As the needle spinning provides an effective route to adjust those parameters, highly uniform nanofibers can be achieved. There are clear tendencies in the diameter; it was increased as the polymer concentration and needle size were increased, and was decreased as the pulling away distance and pulling away speed were increased. Needle spinning with a precise control of the processing parameter enables us to readily fabricate well-defined nanofibers, with controlled dimensions in diameter and length; plus, single nanofibers also can be easily formed. Those features cannot be realized in common spinning process such as electrospinning. Therefore, this technique will lead to further development of the use of mechanical force for nanofiber fabrication and will expand the range of nanofibers applications. Nanofibers have emerged as one of the promising one-dimensional nanomaterials, owing to their unique physicochemical properties, for example, their exceptionally large surface area to volume ratio and their long length with a small diameter in the nanometer scale ,2. Also,To address the challenges in electrospinning and the other techniques, there have been tremendous efforts to develop novel strategies to effectively fabricate nanofibers without the use of electrical force ,15. MechTo achieve a controlled nanofiber size with a high uniformity, while keeping the benefits of hand spinning, we developed a method using needles instead of plates as a tool to draw the polymer solution with mechanical force, namely needle spinning. The schematic illustration of needle spinning and a photograph of a needle spinning apparatus are presented in SPA, mm/sec), pulling away distance , needle size , and polymer concentration . The size and length of all of the nanofibers were highly uniform, the standard deviation of the nanofiber diameter was typically under 10% of the average diameter, and the length of all of the nanofibers was determined by the pulling away distance. The length of the nanofiber was extended up to 0.5 m with our simple setup, strongly suggesting that an exceptionally high aspect ratio can be achieved. We further found that the diameter of the nanofiber can be systematically controlled by varying the process parameters mentioned above. The following clear tendencies were observed: the nanofiber diameter was increased as the polymer concentration (and viscosity) and needle size were increased, and the diameter was decreased when the pulling away distance and pulling away speed were increased. These studies provide an effective route to obtain well-defined nanofibers with desired dimensional parameters, and also elucidate the fundamental principles for a mechanically driven nanofiber formation process. Our method should be useful for fabricating well-defined single nanofiber or nanofibers, with a high uniformity compared to other conventional methods.Herein, we demonstrate the fabrication of poly (ethylene oxide) nanofibers by needle spinning. The fabricated nanofibers exhibited a smooth surface and well-controlled diameter from ~1.4 to ~0.4 μm, by controlling the processing parameters, namely: the pulling away speed (A certain amount of poly (ethylene oxide)  was dissolved in deionized water. The concentration of the PEO aqueous solution was varied from 2 wt. % to 3 wt. %. Sodium dodecyl sulfate  was added to the prepared solution in order to lower the surface tension. The mixture was further homogenized by sonication for 3 h.SPA, mm/s), pulling away distance , and the diameter of needle . The default values of the parameters SPA, dPA, dN, and the concentration of the polymer solution  were set to 1000 mm/sec, 500 mm, 0.12 mm, and 2 wt. %, respectively. In order to examine the effect of each parameter on the nanofiber formation, we conducted the needle spinning with a variation of SPA from 50 to 1000 mm/sec, and dPA from 100 to 500 mm, dN from 0.12 to 1.22 mm, and a polymer concentration from 2 wt. % to 3 wt. %. The resulting individual nanofibers were collected, and were then subjected to morphological studies using scanning electron microscopy . The size of the nanofiber was characterized by measuring the diameter of the nanofibers from the SEM micrographs, using image analysis software . The nanofibers were taken from the same operation on each of the five needles, and were repeated 10 times. The average diameter and standard variation values were calculated from 50 fibers, and the representative scanning electron microscopy (SEM) images have been presented.The experimental setup for needle spinning is shown in The most important improvement of the needle spinning, when compared to other nanofiber fabrication techniques, is that it is capable of fabricating highly uniform nanofibers. With our homemade needle spinning apparatus, we aimed to precisely control all of the processing parameters, including the meniscus shape on the needle tip, which is a significant factor in the formation of a nanofiber. The morphology of five different individual nanofibers simultaneously formed by five needles is shown in in D) extracted from representative SEM images. As can be seen from the SEM images, all of the needle-spun nanofibers exhibited a bead-free morphology with a smooth surface. The plot was fitted with an exponential function of the concentration, D (nm) = να·c . It is known that D of the electrospun nanofiber scales to the solution viscosity with the relationship of D ∝ ηβ [The needle spinning process can precisely control the varying processing parameters, that is, the concentration of the polymer solution, size of needle, pulling away distance, and pulling away speed. Firstly, we varied the concentration of the PEO aqueous solution from 2 wt. % to 3 wt. %. The concentration is known to be a significant factor in other spinning process . In elecf D ∝ ηβ . As the f D ∝ ηβ , it impldN), was found to also significantly affect the diameter of the nanofiber. In the needle spinning, the size of the needle determines the shape of the drawn fiber filament on the tip, when the polymer solution is drawn by the tip. In the spinning process, firstly, the needle tip is slightly immersed in the polymer solution. Then, the needle is pulled out of the solution. Through the drawing of the needle, the polymer fiber filament is formed between the needle tip and the surface of the solution. The diameter of the filament formed at initial moment of needle spinning is likely equal to the size of the needle tip. Subsequently, pulling out the needle from the solution gradually reduces the fiber diameter. Hence, the size of the needle plays a critical role in the control of the nanofiber diameter. In order to examine this aspect, we varied the diameter of the needle tip to carry the needle spinning out. D (μm) = 0.40 + 0.69 dN (mm). These results strongly suggest that the size of the nanofiber can be precisely controlled by simply changing the needle size.Another processing parameter, the diameter of the needle  were found to be 1.94 ± 0.061 μm (100 mm) and 0.43 ± 0.024 μm (500 mm), respectively. A simple linear fitting of the relation between D and dPA results in an empirical equation of D (μm) = 2.398 − 0.004·dPN (mm). It should be noted that the pulling away distance largely affects the size of resulting fiber compared to the other parameters, that is, the concentration and needle size. The increase of the distance by five times, from 100 to 500 mm, led to a decrease of about four times in the diameter, from 1.94 to 0.43 μm. In cases of a variation of the concentration and needle size, the highest diameter can be found to be in the range of two to three times that of the lowest diameter value.As mentioned above, the fiber filament is initially formed between the needle tip and the surface of the polymer solution. This initially formed filament is elongated by drawing the needle along the z-axis. In this process, the diameter of the fiber becomes reduced to smaller size as it is pulled further out. To examine the effect of the pulling away distance variation, we fabricated the filaments with the variation of dPA, indicating that a careful control of the processing conditions should allow for achieving the pre-selected diameter in the nanometer scale and length in the meter scale. The nanofibers with the diameter from the micro- to nano-meter range and the maximum fiber length of ~50 cm, can be achieved with current needle spinning setup. As the precise control of the fiber length was challenging in electrospinning, it is apparently a significant improvement.We note that the length of the nanofibers should be equal to SPA of 500 and 1000 mm/sec is about 100 nm. That is, the pulling away speed does not significantly affect the fiber size, compared to the other parameters. Similar to other processing parameters, the fiber diameter also linearly scales to the pulling away speed, and the empirical equation generated by a linear fitting analysis was D (μm) = 0.54 − 9.08 × 10−5SPA (mm/sec). As shown in the equation, the small slope indicates that the drawing speed is not critical for determining the diameter.The simple but innovative method, needle spinning, offers an effective and facile route for fabricating highly uniform nanofibers with a smooth surface morphology and the desired dimensions in diameter and length. The method uses needles as a drawing tool to make the shape of the meniscus at the tip extremely regular, which cannot be realized in hand spinning. The effect of the processing parameters of needle spinning, that is, the pulling away speed, pulling away distance, needle size, and polymer concentration, was carefully investigated. They significantly affect the fiber structure; the nanofiber becomes thicker as the polymer concentration and needle size increase, and becomes thin as the pulling away speed and pulling away distance increase. Those relationships were depicted by four equations, which can be an important and useful tool for designing nanofibers and for achieving the desired property in nanofibers. Those clear tendencies enable us to achieve well-defined nanofibers in diameter and length, with exceptional uniformity. Moreover, this technique is highly advantageous for making a single nanofiber or nanofibers with a higher uniformity than other conventional methods. Plus, it keeps the benefits of hand spinning, for example, wide options for the polymer and the solvent selections, the ability to align the anisotropic particles in the nanofiber, a low cost, and excessive use of energy, by avoiding a high electrical voltage in the production of the nanofibers. We envision that these advantages of needle spinning will enable us to move forward to more advanced micro- and nano-structure fabrication technology.


Therefore, we concluded that the developed dopamine sensing platform CAuNE can be used for many applications including early diagnosis of neurological diseases; function tests of dopaminergic neurons derived from various stem cell sources; and toxicity assessments of drugs, chemicals, and nanomaterials on human neuronal cells.Dopamine is a key molecule in neurotransmission and has been known to be responsible for several neurological diseases. Hence, its sensitive and selective detection is important for the early diagnosis of diseases related to abnormal levels of dopamine. In this study, we reported a new cylindrical gold nanoelectrode (CAuNE) platform fabricated via sequential laser interference lithography and electrochemical deposition. Among the fabricated electrodes, CAuNEs with a diameter of 700 nm, 150 s deposited, was found to be the best for electrochemical dopamine detection. According to cyclic voltammetry results, the linear range of the CAuNE-700 nm was 1–100 µM of dopamine with a limit of detection (LOD) of 5.83 µM. Moreover, owing to the homogeneous periodic features of CAuNEs, human neural cells were successfully cultured and maintained for more than 5 days  Since dopamine is critical for signal transmissions to the brain, an inadequate level of dopamine can lead to many neurological diseases/disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD) and Parkinson’s disease (PD)14.Dopamine is one of the most researched neurotransmitters because of its important functions in the human body 19. Electrochemical detection is a simple and convenient technique and has therefore attracted increasing interest for measurement of various neurotransmitters including dopamine, gamma-aminobutyric acid, and serotonin24. Unlike other biological molecules such as glucose, dopamine is highly redox active and can effectively be measured without the use of other redox couples and/or enzymes required to facilitate the reduction/oxidation of the target molecules30. However, one critical concern for the detection of dopamine in blood is its low concentration level5, which is further amplified by the signal interference from other biological molecules such as glucose, ascorbic acid (AA), and uric acid (UA). To this end, numerous studies have reported a variety of platforms by mostly integrating conductive micro-/nanomaterials or polymers on the surface of electrodes30. Owing to the high conductivity and excellent electrocatalytic properties, metals such as gold, silver, and platinum have been modified on the electrode surface, mostly in the form of nanoparticles/nanostructures, to take advantage of their high surface-to-volume ratios. The methods employed to modify these metallic nanostructures on the conducting substrates can be categorized into two groups: (i) synthesis of metal nanoparticles and their following attachments onto the electrode surface via chemical linkers and (ii) direct formation of metal nanostructures on the substrates via electrochemical/chemical deposition. Nanostructured electrodes integrated into sensing platforms have been found to be highly effective to enhance both sensitivity and selectivity not only toward neurotransmitter detection including dopamine but also toward other biological materials . However, comparing these nanostructured electrodes with conventional products, critical concerns have been raised, especially the instability of the electrical signals and the increase in the signal variations due to the difficulty in controlling the number, size, and shape of nanostructures modified on the electrode surfaces.To address this issue, there is an urgent need to develop platforms for the sensitive and selective detection of dopamine concentrations. To date, conventional colorimetric/column-based methods  have been reported to be effective for the detection of dopamine. However, these techniques are generally laborious and time consuming, which hinders the early diagnosis of several representative dopamine-related neurological diseases, and require several expensive analytical reagents31, here, we developed a simple cylindrical gold nanoelectrode with varying its size and height to find the best conditions for measuring both chemical dopamine and the dopamine release from human dopaminergic neurons (SH-SY5Y). Sequential laser interference lithography (LIL) and electrochemical deposition (ECD) were utilized to prepare homogeneous cylindrical gold nanoelectrode (CAuNE) platforms, which are critical to achieve stable dopamine-specific electrochemical signals and to reduce the signal variations  while the electrical current during deposition process was monitored , wavelength of the UV laser (360 nm), and the incidence angle (°), respectively. However, unlike the PR nanoholes, variations in the diameters of CAuNEs were within approximately 30%, which might be due to the difference in the diffusion of the gold chloride mixture solution used for ECD. It seemed that the parameters related to the concentrations of each component in the gold mixture solution  and the ECD conditions  should be carefully tuned to overcome obstacles in nanohole-assisted CAuNE fabrication.To overcome the challenges of current nanosized platforms, such as high cost, long fabrication time, and huge signal variationspa − Epc| values, were 0.044 V, 0.041 V, and 0.036 V, while the Ipa/Ipc values were 1.48, 1.44, and 1.58 for CAuNEs with diameters of 500 nm, 700 nm and 900 nm, respectively. Consequently, the calculated values indicated that the reduction and oxidation of dopamine on the electrode, that is, the forward and backward reaction of dopamine to dopamine-o-quinone, was quasi-reversible. Remarkably, based on the reduction peaks (Ipc) marked as red circles in Fig. pc value obtained for CAuNE-700 nm was 2.33 and 1.24 times higher than that for CAuNE-500 nm and CAuNE-900 nm, respectively. The CAuNE-700 nm platform showed that the conditions to achieve the highest dopamine redox signal were clearly different from those hypothesized by us, that is, the CAuNE with the smallest diameter (500 nm) would be the best for the detection of the target analyte. This could be partially explained by the small diameter of the PR nanoholes (500 nm), which make the gold solution difficult to diffuse at the bottom of the ITO electrode surface, thus ultimately resulting in a decrease and/or failure in the gold deposition in each nanohole array. Taken together, we confirmed that the CAuNE-700 nm platform was the best for electrochemical detection of dopamine and was suitable for further study, including the sensitivity and selectivity tests.CV was performed to investigate the effect of the diameter of CAuNEs on the ECD of dopamine, and the redox peaks achieved for each CAuNE were compared. Considering that the density of the nanoelectrode was critical in enhancing its sensitivity, mostly owing to the surface-to-volume ratio, we hypothesized that the CAuNE with a diameter of 500 nm would be the best for dopamine detection. To confirm this hypothesis, the fabricated CAuNEs with diameters of 500 nm, 700 nm, and 900 nm were analyzed by CV in the presence of 10 µM dopamine . As shown in Fig. 2 based on the cyclic voltammogram obtained using K3Fe(CN)6 as a redox couple  and (2) high selectivity toward the target molecule in the presence of other interfering molecules. First, the reduction peaks for varying concentrations of dopamine were obtained. As shown in Fig. pc = 0.09 V, Epa = 0.14 V vs. Ag/AgCl) were observed in the absence of dopamine (PBS only), and the background signal was found to be stable and constant for more than three cycles. Based on the reduction peaks (Ipc), the CAuNE-700 nm platform showed good performance in terms of dopamine detection, with a linear dopamine concentration range of 1–100 µM (R2 = 0.9944). The limit of detection (LOD) of the CAuNE-700 nm platform was found to be 5.83 µM based on the calculated parameters, the slope of the calibration curve, and the standard deviation of the response.To confirm the potential of the newly developed platform for practical sensing applications, the following critical factors must be considered: (1) highly linear correlation between the signal intensity and the concentration of the target analyte , while there was no remarkable difference in cell growth at 2 DIV. Remarkably, as a conducting cell culturing platform, a clear peak was observed in the voltammogram (Ip = 1.773 µA) with the addition of 100 μM dopamine in the presence of SH-SY5Y cells (3 DIV). Furthermore, to confirm the ability of the platform to measure the dopamine released from cells, the SH-SY5Y cells on the electrode surface were cultured with the medium containing 100 μM L-DOPA, a precursor of dopamine, for 2 hours and were triggered to release the dopamine with the addition of potassium chloride (120 mM KCl). As hypothesized, spike-like electrical signals were observed when the KCl solution was added to the cells , proving the excellence of the developed platform for sensitive and selective dopamine detection. Moreover, owing to the periodic homogeneous feature of the CAuNE platform, which was known to be preferable for cell adhesion, human neuroblastoma cells (SH-SY5Y) were successfully cultured on the artificial electrode surface and showed enhanced cell growth at 5 DIV when compared with that on a conventional TCP. Further, both chemical dopamine (100 μM) and the dopamine release of cells with addition of KCl as a triggering molecule were detectable in presence of the SH-SY5Y cells at 3 DIV, proving the potential of the developed CAuNE platform for in situ label-free monitoring of dopamine released from neuronal cells.Here, we reported a new dopamine detection platform composed of ITO and periodic gold nanocylinder arrays. By simply changing the incidence angles during interference lithography, homogeneous PR nanohole arrays of three different diameters  were successfully formed on the surface of ITO. Gold nanocylinder arrays were then fabricated via ECD, and their dopamine detection ability was examined. According to the CV results, nanocylinder arrays with a diameter of 700 nm and a deposition time of 150 s (186 nm in height) showed the best performance for dopamine detection. The linear range of the CAuNE-700 nm platform was found to be 1–100 µM dopamine with an LOD of 5.83 µM55. The platform is also useful for other applications, including to test the function of dopaminergic neurons derived from various stem cell sources  and for toxicity assessments of various chemicals and nanomaterials on human neuronal cells.In conclusion, our newly developed CAuNE platform is an excellent candidate for simple, convenient, and sensitive detection of dopamine and can be highly useful for early diagnosis of various neurological diseases ITO electrodes were purchased from U.I.D . All chemical materials used for LIL including hexamethyldisilazane (HMDS) and dimethyl sulfate (DMSO) were obtained from Sigma-Aldrich, and the PR (AZ nLof-2020), thinner (AZ 1500 thinner), and developer (AZ 300MIF developer) were obtained from AZ Electronic Materials, USA.Commercial products for gold deposition including gold (III) chloride trihydrate and ammonium sulfate were purchased from Sigma-Aldrich, and tripotassium citrate monohydrate was purchased from Wako Pure Chemical, Ltd, Japan.All materials used for electrochemical dopamine detection including dopamine hydrochloride and phosphate buffered saline (PBS) were obtained from Sigma-Aldrich. SH-SY5Y cells derived from human neuroblastoma were obtained from the Korean Cell Line Bank (KCLB). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM)/nutrient mixture F-12 (F-12) obtained from Thermo Fisher Scientific in a ratio of 1:1.A cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories for cell viability testing.2, 0.5 mm thickness) was washed in 1% Triton X-100, deionized water, and 80% ethanol for 15 min and sonicated and dried using N2.The homogenous polymer patterns were obtained by laser interference photolithography using a Lloyd’s mirror interferometer. The interferometer generated light interference between light coming from a source (MXL-FN-360) and the reflected light from the mirror. First, ITO-coated glass (10 Ω/cmThe washed ITO glass was coated with HMDS using spin coating and this HMDS-coated ITO glass was coated with the diluted PR and thinner (ratio of 6:4). This obtained substrate was then baked on a hot plate at 130 °C for 60 s (soft bake). The soft-baked substrate was exposed to UV  using the Lloyd’s mirror interferometer. The substrate was exposed twice to UV to obtain PR nanohole patterns. The exposed substrate was then baked again under the same conditions as the soft bake (post-exposure bake). Subsequently, the substrate was treated in the developer to remove the unexposed PR and was washed with DI water. Further, the washed substrate was baked at 135 °C for 60 s (hard bake).The PR patterns and CAuNE substrates were imaged by field emission scanning electron microscopy  at an acceleration voltage of 10 kV.4, 0.5 mM ammonium sulfate, and 1 mM tripotassium citrate monohydrate was used for the electrochemical gold deposition. The Ag/AgCl (1 M KCl) and a platinum wire was used as the reference and counter electrodes, respectively. Prior to the electrochemical deposition, the reduction and oxidation potentials of HAuCl4 on ITO electrode were first characterized by CV, as shown in the Supplementary Fig. 4 as reported by previous study written as below56.Before gold deposition, a plastic chamber was placed on the PR-nanopatterned ITO glass using polydimethylsiloxane mixed with a thermal curing agent (ratio of 10:1) as a glue. A solution containing 2 mM HAuClip = peak current, n = number of electrons involved, A = electrode area (m2), D = diffusion coefficient (m2/s), v = scan rate (V/s) and C = concentrations of analytes (mol/L).The deposition was performed using a DY2013 potentiostat  with −1.2 V potential for 25, 50, 75, 100, 125, 150, and 175 s. Then, the photoresist used as a template for gold deposition was chemically stripped for 60 s using DMSO as a solvent, followed by washing with DI water three times to remove residual DMSO. For calculation of the active surface area, the Randles-Sevcik equation was used described as,The CAuNEs were characterized by scanning electron microscope (SEM) SIGMA  and atomic force microscope (AFM) NX-10 .CV detection and amperometric detection were performed using a DY2013 potentiostat with 10 µM of dopamine hydrochloride solution diluted in PBS. The fabricated substrate consisting of the ITO electrode served as the working electrode. The reference electrode used was an Ag/AgCl (1 M KCl) electrode and a platinum wire was used as the counter electrode. For the CV analysis, dopamine hydrochloride solution in PBS was placed in the fabricated substrate chamber. The scan rate was 0.05 V/s, and the scanning range was −0.2 to 0.4 V.−6 A. For electrochemical detection dopamine in the presence of SH-SY5Y, the medium for the cells grown for 3 days was removed and replaced with 100 µM dopamine hydrochloride solution diluted with PBS. For the detection of dopamine release from cells, L-Dopa (100 µM) dissolved in DMEM/F12 was treated for 2 hours prior to the amperometric detection and 120 mM KCl was added to trigger dopamine release from cells. Subsequently, electrochemical detection was analyzed under the same conditions as the preceding conditions. All of the experiments were performed at a temperature of 25 °C.For the dopamine selectivity test, amperometric detection was performed at a sampling time of 0.05 s and an initial voltage of 0.3 V, and the sensitivity was found to be 10pc) detected by CV was quantitatively analyzed using the statistical program Origin 8 or Microsoft Excel 2013. Graphical data were presented as mean ± SE (N = 3). Statistical significance between the groups was analyzed by the unpaired Student’s t-test. Cell spreading was analyzed using Image J software.The intensity of the cathodic peaks (IThe cells (SH-SY5Y) were cultured in DMEM/F12 on the fabricated substrate coated with 0.01% poly L-lysine solution (Sigma-Aldrich) for 3 days. After 3 days of incubation, the cells were washed with PBS (pH = 7.4) and fixed with 4% formaldehyde solution for 10 min. Then the fixed cells were treated with 1% Triton X-100 diluted with PBS for 10 min and washed twice with PBS. Subsequently, the cells were stained with Alexa Fluor® 568 Phalloidin for 30 min at room temperature. The cells were washed twice with PBS and stained with Hoechst 33342  for 10 min. Finally, fluorescence imaging of the actin- and nucleus-stained cells was performed using an Eclipse 80i (Nikon Japan) microscope.Supporting information


The free form of 2+BDP-Fe in solution displayed weak fluorescence emission, while it showed strong fluorescence emission after interaction with dopamine due to the release of Fe2+ from 2+BDP-Fe, confirming the binding of Fe2+ to dopamine. The increase in fluorescence intensity was concentration-dependent, and a good linear relationship was observed between the fluorescence intensity and dopamine concentration. The detection limit of dopamine by 2+BDP-Fe was 1.1 nM, indicating a 20-fold higher sensitivity than that of previously reported compounds. The reaction of 2+BDP-Fe with dopamine was not affected by the presence of foreign substances, allowing the highly selective detection of dopamine in the human serum sample. The results of this study indicate that the novel compound 2+BDP-Fe is a reliable fluorescent molecular probe for the detection of dopamine and can be widely employed in diverse scientific areas.A new fluorescent probe ( Dopamine is an organic compound of the catecholamine and phenylethylamine families. Dopamine plays an important role as a neurotransmitter, and it is responsible for a wide range of cognitive functions, including motivation, behavior, learning, and memory. Its concentration affects emotions and reflects the state of one’s health ,2,3. It Various studies have been performed on the optical determination of dopamine from the viewpoint of the native fluorescence properties of dopamine, the efficiency of fluorescence quenching based on the reaction between dopamine and HRP (horseradish peroxidase), the utilization of quantum dots, and the derivatization of dopamine by ethylenediamine following oxidation using mercury(II) nitrate ,12,13,14However, these methods are neither highly selective nor highly sensitive; instead, they are time-consuming and require further separation processes such as ion exchange chromatography, thin-layer chromatography, or high-performance liquid chromatography.The generation of a strong fluorescence signal after reaction with the target molecule;The elimination of background noise for the highly sensitive detection of dopamine;The reduction of interference from foreign substances for highly selective dopamine assay. The prerequisites considered in the present study to design a fluorescent reagent for dopamine detection were as follows:2+ from the reagent and subsequent formation of a complex between Fe2+ and dopamine. However, the analysis using this fluorescent reagent had poor sensitivity and low photostability due to the chemical properties of the cyanopyranyl moiety, which could not be applicable in long-term analysis.We have previously developed fluorometric reagents for the detection of various biological substances containing dopamine ,19,20,212+ complex both as a fluorescent quencher and a ligand exchange moiety for the detection of dopamine. The BDP group has interesting spectral properties such as high quantum yields, large extinction coefficients, and high photostability—characteristics that satisfy the above concept of molecular design [2+ complex, BDP-Fe2+) are shown in 2+ acts as a reliable fluorescent probe, and it is useful for the high-throughput analysis of dopamine.In this study, a new fluorescent molecular probe has been developed with the boron–dipyrromethenyl (BDP) group as the fluorescent emitter ,23 and ar design ,25. The 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV-500 spectrometer.All the chemicals required for the synthesis and fluorometric measurements were of analytical grade and provided from Tokyo Chemical Industry , Wako Pure Chemical Industries, Ltd.  and Sigma Aldrich . Absorption spectra were recorded at 25 °C on a V-670 UV/Visible Spectrophotometer . Fluorescence spectra were recorded at 25 °C on a JASCO FP-6500 fluorophotometer and an FMP-825 microplate reader. 2+ was dissolved in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution (pH 7.0) to obtain a final concentration of 1.0 µM. After the addition of 1.0 mL of dopamine or foreign substance solution to 1.0 mL of BDP-Fe2+ solution, the reaction mixture was incubated for 5 min, and fluorescence spectra were recorded.The compound BDP-FeThe synthesis of the fluorescent reagent is illustrated in 2 atmosphere. After the addition of trifluoroacetic acid , the reaction mixture was stirred for 24 h at room temperature. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone , dissolved in a mixture of anhydrous THF (25 mL) and anhydrous dichloromethane (25 mL), was added dropwise over 15 min to the mixture, which was then stirred for 4 h at room temperature. Saturated aqueous NaHCO3 solution was added and the mixture was extracted with dichloromethane. The organic layer was washed with water and brine, dried over Na2SO4, and concentrated in vacuo. The crude product was purified by column chromatography  to obtain a brown solid with 27.5% yield.Anhydrous dichloromethane (40 mL) and 2,4-dimethyl pyrrole  were added to a solution of diethyl-2,2’-(5-formyl-2-hydroxybenzylazanediyl)-diacetate  and stirred for 30 min in an N1H NMR  1.24 , 1.27, 2.34, 3.53 , 4.04 , 4.21 , 5.88, 6.99 , 7.57 , 7.75 , 10.54 .3•Et2O (9.2 mL). The mixture was stirred for 3 h at room temperature. Saturated aqueous NaHCO3 solution was added and the mixture extracted with CH2Cl2. The organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The crude product was purified by column chromatography  to obtain a brown solid in 95.8% yield.Diethyl 2,2’- methyl)-2-hydroxybenzyl) azanediyl) (Z)-diacetate  was dissolved in anhydrous toluene (65 mL), triethylamine (9.2 mL), and BF1H NMR  1.25 , 1.36, 2.55, 3.54 , 4.03 , 4.22 , 5.95 , 6.98 , 7.58 , 7.76 , 10.55 .2SO4. The solvent was evaporated in vacuo. The brown solid obtained in 96.2% yield was used without further purification.A mixture of diethyl 2,2’--2-hydroxybenzyl) azanediyl) diacetate , and sodium hydroxide  in EtOH–water (40 mL–10 mL) was stirred for 24 h at room temperature. After the removal of EtOH, the solution was acidified to pH 6.0 using 1 N HCl, extracted with ethyl acetate, and dried over Na1H NMR  1.37 , 2.56 , 3.55 , 4.04 , 5.96 , 6.99 , 7.57 , 7.75 , 10.54 .2 aqueous solution (1.3 mL) was added, and the mixture was stirred for 1 h at room temperature. After solvent removal, the residue was dried, and the product was recovered as a brown solid in 98% yield.To a solution of 2,2’--2-hydroxybenzyl)azanediyl)diacetic acid  in methanol (20 mL), 100 mM FeClThe excitation and emission spectra of BDP were recorded in a buffer solution (pH 7.0) at 25 °C to study the photophysical properties in vitro. Typical excitation and emission spectra of BDP are shown in 2+ with BDP, fluorescence spectra were investigated, and the signal was monitored by mixing BDP (1.0 µM) with Fe2+ (5.0 µM) in HEPES  to form BDP-Fe2+. The fluorescence intensities of BDP decreased following the addition of Fe2+ due to the considerable quenching effect, as shown in 2+, Co2+, Cu2+, Mn2+, Zn2+, Li+, Na+, K+, Cs+, Mg2+, Ca2+, Ba2+, and Pb2+) were added to the solution of BDP, and the fluorescence intensities were monitored. The observed order of the fluorescence intensity was as follows: Fe2+ (6.5) > Ni2+ (25.0) > Co2+ (156.0) > Cu2+(201.5) > Mn2+ (250.2) > Zn2+ (292.3). Fluorescence quenching did not take place for alkali metal ions, alkaline earth metal ions, and Pb2+, as shown in 2+ was a particularly strong quencher of BDP fluorescence. To take advantage of this phenomena, the BDP-Fe2+ complex was used in this study, because we used the ligand exchange mechanism, and a large fluorescence enhancement from quenching state was requested, enabling the highly sensitive detection of dopamine by the dissociation of Fe2+ from BDP-Fe2+.To study the reaction of Fe2+ to investigate the ligand exchange reaction. The concentration of BDP-Fe2+ was 1.0 µM and that of the dopamine solutions varied between 0 and 5.0 µM in HEPES buffer solutions . The data are shown in 2+ increased from 6.4 (free form) at 525 nm to 328.9 nm following the reaction with 5.0 µM of dopamine, corresponding to a 51.4-fold increase in fluorescence intensity. The detection limit was determined to be 1.1 nM of dopamine, which was calculated from the baseline noise (S/N = 3). The detection limit of the previously reported compound is 10.0 nM [2+. These observations indicate that the BDP-Fe2+ containing a dipyrromethenyl group shows brighter fluorescence and large fluorescence enhancement after reaction with dopamine, thereby improving sensitivity. Thus, the chemical structures of the fluorophore in BDP-Fe2+ play an important role in the detection of dopamine.Fluorimetric titrations were performed with dopamine by using BDP-Fe 10.0 nM , indicat2+ with dopamine. The fluorescence intensity saturated after 3.0~5.0 min, and 5.0 min was considered optimal for the reaction between BDP-Fe2+ and dopamine.2+ and dopamine, fluorescence signals were monitored at different pH values. 2+ with 5.0 µM of dopamine. At high pH values (10 to 11) and low pH (3 to 4), the reaction of BDP-Fe2+ with dopamine was inhibited by the solution pH, whereas the fluorescence intensity was nearly unchanged when the pH was in the range 5–9. As a result, the reaction of BDP-Fe2+ with dopamine was neither inhibited nor masked at a physiological level of pH values, and this assay should be appropriate to monitor the quantitative analysis of dopamine under such conditions.In order to understand how pH affects the reaction between BDP-Fe2+ in BDP-Fe2+ for the detection of dopamine, the fluorescent properties of the free form of BDP (not Fe2+ complex), were monitored before and after the addition of dopamine (0 to 5.0 µM). Fluorescence intensities of BDP at 525 nm hardly changed after the addition of dopamine, as shown in 2+ ) did not show any response after the addition of dopamine because no reaction occurred between these metals and the dopamine [2+ indicated significant fluorescence enhancement after the reaction with dopamine. From these results, Fe2+ in BDP-Fe2+ plays an important role in the detection of dopamine, and schematic representations of the reaction between BDP-Fe2+ dopamine are illustrated in 2+ emitted remarkably weak fluorescence, whereas the fluorescence enhancement by the interaction of BDP-Fe2+ with dopamine can be considered to be the release of Fe2+ from BDP-Fe2+, and the dopamine coordinated with Fe2+ in a 3:1 stoichiometry to form a stable complex by the ligand exchange reaction [To investigate the role of Fedopamine . On the reaction ,14.2+ at 525 nm before and after the addition of dopamine, and other foreign substances that may be present in biological samples. All the tests were carried out with a mixture of BDP-Fe2+ and an excess amount of foreign substances. The fluorescence enhancement of BDP-Fe2+, when dopamine was added, was considerably larger (I/I0 = 49.8) than those after the addition of adrenaline (2.5), noradrenaline (2.2), serotonin (1.3), and other compounds (~1.0). The obtained results indicated that the response of BDP-Fe2+ to dopamine was unaffected by the excess amounts of other biological substances due to the release of Fe2+ from BDP-Fe2+ and formation of a stable Fe2+-dopamine complex.2+ was compared with those of previously reported compounds (References 1 and 2) and is summarized in 2+ exhibited large fluorescence enhancement upon dopamine addition, whereas the fluorescence intensities of [2+. Thus, BDP-Fe2+ could be used to detect dopamine with greater sensitivity than in [2+ is a sensitive functional group and contributes to a larger fluorescence enhancement via ligand exchange between dopamine and BDP-Fe2+, and the chemical structure of the fluorescent dye in the fluorescent probe plays an important role in determining the fluorescent properties.The analytical performance of BDP-Fe2+ is important for the continuous monitoring of dopamine. The BDP-Fe2+ solution was irradiated by excitation light (490 nm), and the fluorescence intensities were observed for 24 h. Compared with BDP-Fe2+, the photostability of a previously reported compound containing the cyanopyranyl group [2+ was ~98% of the original value, whereas previously reported compounds showed ~64%. This result indicates that BDP-Fe2+ is more stable than previously reported compounds, and that this compound produces a reliable quantitative response to dopamine even after long periods of optical measurement.The lifetime of BDP-Feyl group  was obse2+ and the results were compared with dopamine assays in HEPES buffer solution. 2+ in human serum or in the buffer solution increased with increasing dopamine concentration, and a good linear relationship was observed. Moreover, fluorescence intensities at each dopamine concentration for assay in human serum were almost the same as those in HEPES buffer solution. Thus, BDP-Fe2+ could be used to detect dopamine with high selectivity in the presence of other substances as bovine serum albumin (BSA), inorganic salts, etc., and this assay is applicable to the detection of dopamine in the presence of impurities.As an application of this assay, the detection of dopamine in human serum was conducted using BDP-Fe2+ satisfies the requirements for highly sensitive, highly selective, and high-throughput assays of dopamine and can be widely used as a convenient method for dopamine detection in both research laboratories and medical applications.This assay was compared with another detection method—i.e., enzyme linked immunosorbent assay (ELISA)—and the results are summarized in In the present study, a new fluorescent sensing molecule that selectively couples with dopamine and exhibits a large increase in fluorescence intensity as a result has been reported. No interference in the detection of dopamine by the presence of foreign substances has been found. In addition, the detection of dopamine in human serum was successfully performed. This assay for dopamine was not only easy to apply, but it has been proved to be rapid, highly sensitive and selective. Based on the extremely positive results of this study, further efforts are ongoing to investigate other possible indicators for the detection of various living substances.


Insights into the molecular underpinnings of primary central nervous system tumors have radically changed the approach to tumor diagnosis and classification. Diagnostic emphasis has shifted from the morphology of a tumor under the microscope to an integrated approach based on morphologic and molecular features, including gene mutations, chromosomal copy number alterations, and gene rearrangements. In 2016, the World Health Organization provided guidelines for making an integrated diagnosis that incorporates both morphologic and molecular features in a subset of brain tumors. The integrated diagnosis now applies to infiltrating gliomas, a category that includes diffusely infiltrating astrocytoma grades II, III, and IV, and oligodendroglioma, grades II and III, thereby encompassing the most common primary intra-axial central nervous system tumors. Other neoplasms such as medulloblastoma, embryonal tumor with multilayered rosettes, certain supratentorial ependymomas, and atypical teratoid/rhabdoid tumor are also eligible for integrated diagnosis, which can sometimes be aided by characteristic immunohistochemical markers. Since 2016, advances in molecular neuro-oncology have resulted in periodic updates and clarifications to the integrated diagnostic approach. These advances reflect expanding knowledge on the molecular pathology of brain tumors, but raise a challenge in rapidly incorporating new molecular findings into diagnostic practice. This review provides a background on the molecular characteristics of primary brain tumors, emphasizing the molecular basis for classification of infiltrating gliomas, the most common entities that are eligible for an integrated diagnosis. We then discuss entities within the diffuse gliomas that do not receive an integrated diagnosis by WHO 2016 criteria, but have distinctive molecular features that are important to recognize because their clinical behavior can influence clinical management and prognosis. Particular attention is given to the histone H3 G34R/G34V mutant astrocytomas, an entity to consider when faced with an infiltrating glioma in the cerebral hemisphere of children and young adults, and to the group of histologically lower grade diffuse astrocytic gliomas with molecular features of glioblastoma, an important category of tumors to recognize due to their aggressive clinical behavior. Primary brain tumors encompass many distinct tumor types arising in the brain parenchyma or meninges, with varying prevalence based on patient age and tumor location. The most common malignant primary brain tumors are gliomas, a category of tumors arising from glial or glial precursor cells that includes astrocytomas (which may be diffuse or circumscribed), oligodendrogliomas, ependymomas, and other rare histologic groups. Together, gliomas account for about 75% of malignant primary brain tumors, and the vast majority are glioblastoma . This reThe article will address the histologic categories of diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, and the key histologic and molecular features seen with these morphologies. The diffuse gliomas require molecular information for classification, and these tumors are regularly encountered in routine practice. Understanding the relationship between diffuse glioma histologic and molecular features is critical for recognizing cases that require molecular studies. The morphologic basis for glioma classification was established in 1926 by Bailey and Cushing, who devised a set of histologic categories and introduced a naming convention based on tumor resemblance to normal cellular counterparts in the developing nervous system –7. This In recent decades, molecular studies of brain tumors resulted in an exponential rise in our knowledge of the molecular underpinnings of these neoplasms Fig. . From thThe aim of this review is to provide an overview of the molecular underpinning of the infiltrating gliomas, the most common brain tumors that should receive an integrated molecular diagnosis by WHO 2016 criteria. Various testing methods exist for obtaining molecular data, and some advantages, limitations, and potential pitfalls of the more common approaches will be discussed. Building on this foundation, we will discuss some molecular entities within infiltrating gliomas that should be considered when a pathologist encounters an unusual tumor that does not seem to fit into an existing molecular category. Molecular pathology of brain tumors as a general topic applies to many other glial and non-glial neoplasms which are not discussed in this review. Readers are referred to several recent focused reviews which have addressed other areas of the molecular pathology of other brain tumors, such as glioneuronal tumors, meningothelial tumors, other mesenchymal tumors, tumors of the sellar region, and lymphomas and histiocytic tumors .In 2014, the International Society of Neuropathologists (ISN) convened an expert group of over two dozen neuropathologists in Haarlem, the Netherlands, to address challenges in standardization of reporting integrated diagnoses. This group incorporated their expertise with input from over 150 neuro-oncology specialists to set boundaries and priorities for molecular testing in brain tumors. The group prioritized defining entities as narrowly as possible in order to create homogeneous tumor groups, and created a four-layered system to standardize reporting of integrated tumor classification . The ISNMolecular testing will not be possible in all cases, as tissue quality and quantity are frequent limiting factors in brain tumor sampling and testing may subject to technical failure. The WHO does not endorse specific testing modalities, and each institution can choose from several platforms or approaches . Still, Acta Neuropathologica with an accompanying editorial in Brain Pathology, and both will be a valuable resource for keeping up to date on molecular pathology of brain tumors.The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) was established to create a mechanism for updates in between WHO editions, which have historically been separated by intervals of at least 7 years , 27. TheCCND2) locus [BRAF V600E mutations; the differential diagnosis with anaplastic pleomorphic xanthoastrocytoma can be challenging and the relationship between these entities is controversial [Diffusely infiltrating gliomas are defined by a growth pattern of individual tumor cells growing through the brain parenchyma, in contrast with the sharp, pushing border seen in circumscribed gliomas or metastatic tumors . Individ2) locus . A few g2) locus . These toversial , 35. Othoversial .Fig. 2TyWhile some infiltrating gliomas readily declare themselves morphologically as astrocytoma or oligodendroglioma, many are ambiguous and appear either as a mixture of both cell types, or with a constellation of nuclear features that defies reproducible categorization. Before 2016, such “in-between” diffuse gliomas were categorized as “oligoastrocytoma” and assigned WHO grade II, or grade III (anaplastic) in the setting of significant mitotic activity, necrosis, and/or vascular proliferation. Oligoastrocytomas had an intermediate prognosis between histologically defined diffuse astrocytomas and oligodendrogliomas, but diagnostic reproducibility for this category or tumors was poor , 37. TheIDH1) in a subset of glioblastoma cases, mostly from younger patients with a history of prior lower-grade astrocytoma  [IDH1 at position arginine 132, and less commonly in the homologous arginine 172 of IDH2. Overall, IDH1 R132 and IDH2 R172 alterations occur in about 80–90% of adult WHO grade II or III infiltrating astrocytomas, oligodendrogliomas, and secondary glioblastoma [IDH1 converting arginine at position 132 to histidine (IDH1 R132H). This mutant protein is detectable by a sensitive and specific antibody [IDH1 and IDH2 can be performed to assess for less common IDH1 mutations, or mutations in IDH2. These non-canonical alterations are very rare in patients over 54, so sequencing is generally not necessary in older patients with glioblastoma histology and no history of a prior lower grade glioma [In 2008, next-generation sequencing identified recurrent mutations in isocitrate dehydrogenase 1 (encoded by lastoma) . Subsequblastoma . Normallblastoma , 44. Theantibody , 46. Immantibody . If the e glioma , 47, 48.EGFR amplification, focal deletions on 9p encompassing CDKN2A/B, and focal or whole chromosome gains/losses can be detected. For laboratories not currently offering microarray methods, it may be possible to include additional, more proximal FISH probes (i.e. closer to the centromere) to increase confidence in whole arm codeletion, although this approach would increase technical and scoring complexity and potentially increases cost to a similar level as microarray platforms.Oligodendrogliomas now have a strict molecular definition and must show an IDH alteration and evidence for deletion of both the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q) for an integrated diagnosis. 1p/19q-codeletion results from a reciprocal translocation between chromosomes 1 and 19 -- t -- with subsequent loss of one derivative chromosome, leaving an imbalance and loss of 1p and 19q , 50. ForTERT gene on chromosome 5, dyskeratin, and template telomerase RNA component, which together act to extend telomeres through addition of a repetitive DNA sequence to the chromosome ends [TERT promoter were originally discovered in melanoma, and subsequently identified in other tumors, including at a high frequency in primary glioblastoma and oligodendroglioma [TERT promoter mutations generate a cryptic binding site for an E26 transformation-specific (ETS) family transcription factor called GA-binding protein, alpha subunit (GABPA), leading to telomerase re-expression and subsequent telomere elongation [TERT promoter region is the only method to detect this alteration; in several studies of multiple cancer types, including gliomas, TERT immunohistochemistry does not correlate with promoter mutation status [TERT expression, and this may have prognosis in certain pediatric brain tumors, where TERT promoter mutations are rare [TERT amplification and TERT promoter rearrangements [Replicative senescence occurs as a consequence of cell division. Incomplete replication of the chromosome ends leads to progressive shortening of the telomeres, eventually triggering a DNA damage checkpoint signal resulting in cell cycle arrest . Telomerome ends , 54. TERome ends . Recurreroglioma –58. Mechongation . Sequencn status , 60–62. are rare , 63, 64.ngements , 66.TERT promoter mutations are rare in IDH-mutant astrocytomas, which instead maintain telomere length through a mechanism involving mutations in the ATRX gene, usually accompanied by mutations in TP53. ATRX encodes α-thalassemia/mental retardation syndrome X-linked, a protein that, along with death-domain associated protein or DAXX, is involved in maintaining chromatin structure at the telomeres [ATRX alterations strongly correlate with a phenotype called alternative lengthening of telomeres (ALT), characterized by increased telomere homologous recombination and subsequent telomere elongation [IDH-mutant astrocytomas and histone H3 mutant gliomas.In contrast to oligodendrogliomas and IDH-wildtype glioblastoma, elomeres . ATRX alongation . Within ATRX mutations are usually accompanied by loss of nuclear staining for ATRX protein, and TP53 mutations result in stabilization of the p53 protein leading to strong nuclear staining in a significant proportion of tumor nuclei  is a frequent event in IDH-wildtype glioblastomas, while EGFR point mutations are comparatively rare. The EGFR gene also commonly incurs an intragenic deletion of exons 2–7 producing a constitutively active variant protein called EGFR-vIII, occurring in a quarter to half of glioblastoma. Expression of EGFR-vIII is almost always associated with amplification of the wild-type EGFR allele, but the clinical significance of either alteration is still unclear [EGFR amplification occurs on a background of chromosome 7 polysomy and chromosome 10 monosomy [Most glioblastomas occur in adults, and 90% are IDH-wildtype tumors that arise de novo, i.e. with a rapid clinical presentation and absent a precursor lower-grade lesion (primary glioblastoma). The remainder about 10%) are mostly IDH-mutant tumors, typically arising from lower grade infiltrating astrocytomas, and thus clinically referred to as secondary glioblastoma. The molecular features of IDH-wildtype glioblastoma have been studied extensively, however the prognosis remains poor [0% are mo unclear , 74. Conmonosomy , 76.6-methylguanine-DNA methyltransferase (MGMT) protein, encoded by the MGMT gene on chromosome 10, plays a role in repairing the DNA damage from alkylating agents, including temozolomide. Methylation of the MGMT promoter region is associated with silencing of gene expression, and occurs in about 40–50% of glioblastoma cases [MGMT promoter methylation as an independent favorable prognostic factor, and a predictive factor for survival benefit in patients treated with temozolomide and radiation therapy [MGMT promoter methylation as a predictive biomarker in glioblastoma, but the exact role in guiding clinical management is still being refined, partly due to challenges in testing methodology and establishing meaningful cutoffs for the assays [The Oma cases . Mechani therapy , 78. Sube assays .EGFR amplification, but is being phased out in favor of chromosomal microarray, which can detect the amplification with better total copy number estimates and provides additional copy number information across the genome. The deleted region for EGFR-vIII is small, at only 13 kilobases, and therefore not amenable to FISH, and furthermore mosaicism complicates microarray detection and interpretation, particularly against the background of an amplified gene. Currently, of the genomic technologies readily available in the lab, next generation sequencing with high read depth is the most robust approach for detecting this deletion. Partial as well as whole chromosome gains and losses are readily observed using SNP microarray, which can circumvent some of the limitations of targeted FISH. For example, homozygous deletion of the DMBT1 gene on chromosome 10  has been reported in glioblastoma and medulloblastoma [DMBT1 loss may be predictive of a poor outcome, although IDH status was not assessed in that study so the status of DMBT1 as an independent predictor of outcome is unclear [DMBT1 is located at chromosome 10q26.31, approximately 35 megabases distal to PTEN. Traditional FISH using a PTEN probe will not detect DMBT1 deletion; SNP microarray will.FISH readily identifies blastoma . In tumo unclear . DMBT1 iTP53 gene, is a frequent event [Copy number neutral loss of heterozygosity (LOH) is another common event in glioblastoma and can be detected using SNP chromosomal microarray . LOH cannt event . RegionaMGMT promoter methylation testing include simple or quantitative methylation-sensitive polymerase chain reaction, pyrosequencing, multiplex ligation-dependent probe amplification, and analysis by DNA methylation array. The relative costs and advantages/disadvantages of each platform were recently reviewed [Molecular methods for reviewed . Our insH3F3A and H3F3B, while the histone H3.1 variants are highly expressed during DNA replication and is encoded by several genes including HIST1H3B and HIST1H3C. Two categories of histone mutations have been identified in brain tumors. Sequencing studies of pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas identified recurrent mutations causing a lysine to methionine substitution at position 27 -- hereafter K27M -- in H3F3A and HIST1H3B/C, which are hereafter grouped as H3 to encompass H3.3 and H3.1 [H3F3A, and a K27M alteration in histone H2 variant encoded by HIST2H3C [Histones are a family of proteins that form a protein/DNA complex to maintain chromatin structure and regulate gene expression, which can be mediated by post-translational histone modifications including methylation and acetylation. Histone octamers comprise two each of H2A, H2B, H3, and H4 proteins, and these are encoded by different genes. The histone H3.3 variant is expressed throughout the cell cycle and is encoded by and H3.1 , 84. VerHIST2H3C . The H3 HIST2H3C . Since tHIST2H3C . The H3 HIST2H3C . The WHOH3F3A (H3.3-mutant) from HIST1H3B/C (H3.1-mutant). This distinction may impact prognosis and eligibility for clinical trials, and in such cases sequencing of H3F3A, HIST1H3B/C, and possibly HIST2H3C may be required [Three examples of histone H3 mutant glioma are presented in Fig. required , 88, 89.required , 91. TheBRAF V600E, a common driver alteration in a variety of circumscribed gliomas and glioneuronal tumors [midline, diffusely infiltrating gliomas with a K27M alteration to qualify for the integrated diagnosis [When the WHO 2016 was published, H3 K27M mutations were thought to be specific for diffuse midline gliomas. However, H3 K27M mutations have now been reported in midline circumscribed gliomas such as ganglioglioma, pilocytic astrocytoma, unspecified glioneuronal tumors, and ependymoma, and in some series the clinical behavior of these tumors is more aggressive than histologically comparable H3 K27-wildtype counterparts, although it is not as dismal as for diffuse gliomas –95. Intrl tumors , 94. In iagnosis . Due to H3F3A mutations also occur at guanine position 34, substituting arginine (G34R) or valine (G34V – hereafter G34R/V) in histone H3.3, and an example of this is provided in Fig. HIST1H3B/C. The H3.3 G34R/V mutant tumors typically occur in the cerebral hemispheres in adolescents and young adults, and in a series of 81 cases almost always show loss of ATRX by immunohistochemistry (95%), as well as TP53 mutation (88%) [IDH1 and IDH2 wildtype. About one third of the H3.3 G34R/V mutant tumors show primitive neuronal features, with monomorphic cells showing a high nuclear to cytoplasmic ratio and largely lacking vascular proliferation or necrosis, and regardless of their histologic pattern  GFAP and OLIG2 expression may be limited  . Since temistry 9%, as welf tumors . Since treported . The morreported . MutatioThe WHO 2016 classifies glioblastoma into IDH-mutant and IDH-wildtype categories. This distinction has clinical utility in that multiple studies showed significantly better outcomes in IDH-mutant glioblastoma , 98. In EGFR amplification, (2) TERT promoter mutation, and (3) whole-chromosome 7 gain combined with whole chromosome 10 loss (hereafter + 7/− 10). Patients with a proven IDH-wildtype, histologically grade II or III infiltrating astrocytoma and any of these alterations can be diagnosed with “Diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV”, reflecting that their tumors are likely to behave in a manner similar to IDH-wildtype glioblastoma. These alterations may be seen alone or in combination, and they have varying sensitivity and specificity as individual parameters or in combination. Stitchel et al recently described these alterations across a large cohort of brain tumors that were categorized by global DNA methylation profiling (discussed below) [www.molecularneuropathology.org [EGFR amplification is the most specific but least sensitive marker, with 99.8% specificity and 36.0% sensitivity. In this context, EGFR amplification refers to high level copy number gains; low level copy number gains and immunohistochemical expression of EGFR are not sufficiently specific for this purpose [EGFR amplification (59.4%). Although partial arm deletions and various combinations of chromosomes 7 gain and 10 loss may be seen in glioblastoma, partial arm alterations are not considered sufficient by cIMPACT-NOW criteria at this time [TERT promoter mutations have a clear association with aggressive behavior in the setting of glioblastoma, and this is the most sensitive (66.7%) but least specific (89.4%) parameter, largely since TERT promoter mutations can be seen in other gliomas or systemic cancers [TERT promoter mutations occur in a vast majority of molecularly defined (i.e. IDH-mutant and 1p/19q-codeleted) oligodendrogliomas, assessment of 1p/19q status and exclusion of an IDH mutation by sequencing is usually required before designating a molecular glioblastoma.The c-IMPACT-NOW consortium recently addressed this diagnostic dilemma by recommending a set of molecular features that indicate aggressive behavior in histologically lower grade, IDH-wildtype diffuse astrocytic gliomas . Per thed below) . DNA metlogy.org , 104. As purpose . The + 7his time , 102. Fi cancers , 57. CriMYB/MYBL fusion, FGFR1 or FGFR3 alterations, or BRAF alterations are associated with a more indolent clinical course, but do not yet have their own diagnostic categorization and would appear as IDH-wildtype [CDKN2A/B, and loss of ATRX. These tumors have a better outcome than IDH-wildtype glioblastoma and are molecularly distinct from conventional glioblastoma groups, as defined by DNA methylation profiling [EGFR amplification, TERT promoter mutation, and the + 7/− 10 chromosomal alteration. In the absence of an identifiable molecular driver, such cases may be diagnosed by histologic features and designated as “Not elsewhere classified”.Another complicating factor is the presence of other genetic alterations which may preclude a molecular glioblastoma diagnosis because of their own prognostic implications. The presence of H3 K27M in an infiltrative astrocytoma is diagnostic of diffuse midline glioma, H3 K27-mutant, a separate molecularly defined entity. Similarly, the IDH-wildtype gliomas with histone H3.3 G34R/V mutation should be considered, especially in younger patients . Emerginwildtype . Recentlrofiling . FinallyIn summary, for these three alterations, their presence alone is not sufficient for the molecular glioblastoma diagnosis and does so only with the histologic exclusion of other molecular-pathologic entities that may mimic diffuse astrocytomas, such as oligodendroglioma, other genetically-defined gliomas, or focal areas of infiltration in pleomorphic xanthoastrocytoma or pilocytic astrocytoma. The lack of absolute specificity for most of the alterations mentioned in this section, and the complexity of interpreting molecular findings in context of the histology, underscores the need for periodic updates to the molecular classification system. One possible approach to identifying these cases is presented in Fig. Brain tumor classification is increasingly rooted in the molecular drivers underlying different tumor entities. Currently, the absolute number of integrated diagnostic entities is low relative to the rapidly growing body of research on molecular analysis of brain tumors. However, incorporation of molecular features into the classification of diffuse gliomas means that due to their frequency, a majority of the primary intra-axial brain tumors now require some degree of molecular data for accurate and up-to-date classification. Additional molecularly defined entities and integrated diagnostic approaches are expected as the field evolves. As the number and scope of brain tumor-associated molecular alterations increases, academic medical centers are moving toward next-generation molecular diagnostics, allowing for detection of a broad spectrum of molecular alterations , 108–112


CSMD1) demonstrate significant genome-wide association with SCZ. However, few studies have been conducted on the effect of antipsychotics on the expression levels of CSMD1. This study explored whether a change occurs in the expression of the CSMD1 gene before and after antipsychotic treatment in SCZ patients.Schizophrenia (SCZ) is a heritable, refractory, and devastating psychiatric disorder. Previous studies have shown that the variants of CUB and sushi multiple domains 1 .The study population comprised Han Chinese patients from eastern China, including 32 SCZ patients and 48 healthy controls. The expression of CSMD1 gene in the peripheral blood mononuclear cells (PBMCs) of SCZ patients were lower than those in the healthy controls. The expression levels of the CSMD1 gene in the PBMCs of the SCZ patients after antipsychotic treatment were higher than those in the baseline SCZ patients .The expression levels of the CSMD1 are correlated with the development and treatment of SCZ, providing further evidence for the involvement of CSMD1 in SCZ.Our results showed that the expression levels of  Schizophrenia (SCZ) is a hereditary, disabling, and common psychiatric disorder with a worldwide prevalence of approximately 1%. The typical symptoms of SCZ usually appear in youth, with chronic deregulation of thoughts, perceptions, emotions, and behaviors. Genetic factors are the most crucial mechanism in the etiology of SCZ, accounting for approximately 80%; however, these genetic mechanisms remain unclear .CSMD1) gene is a complement control-related protein located on 8p23.2, the variants of which were reported to have a strong correlation with SCZ risk in a recent study [CSMD1 gene has been reported to be a major genome-wide risk locus for SCZ [CSMD1 may be related to neuropsychological deficits in csmd1 knockout mice [CSMD1 has been reported as being involved in regulating the ratio between dopamine and serotonin metabolites in cerebrospinal fluid [CSMD1 gene is closely related to the neurogenesis, cognition, immunity, neuropsychology, and monoamine metabolism of SCZ. However, recent studies on the relationship between CSMD1 and SCZ have been inconsistent; studies have also found no association between rs10503253 and SCZ in patients of Japanese [The CUB and sushi multiple domains 1  and 48 healthy controls (HCs) , all of whom were from a Han Chinese population in Shanxi Province, China. No obvious statistical differences were observed between the SCZ group and the HC group in terms of sex, age, or race (Table n = 5), olanzapine (n = 10), quetiapine (n = 6), ziprasidone (n = 2), aripiprazole (n = 6), amisulpride (n = 3)) and were followed-up for 12 weeks. Based on the evaluation of a Positive and Negative Syndrome Scale reduction rate of over 25%, the clinical symptoms of the patients in the case group were all improved. The exclusion criteria included severe organic brain injury, mental retardation, epilepsy, alcohol or substance abuse, and other mental disorders.The case group comprised SCZ patients recruited from the First Hospital of Shanxi Medical University who conformed to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V), as confirmed by two experienced psychiatrists. Patients with SCZ were not treated with antipsychotic medication before they entered the group. A total of 32 SCZ patients were treated with oral second generation antipsychotics  [CSMD1 were GTCTGGGCTCGTGGATATGT (forward) and CAGGTCTCGGAAGGACAGAG (reverse).A 3-mL sample of peripheral blood was taken from each of the 48 HCs and 32 SCZ patients before and after antipsychotic treatment. Leukocytes were isolated from the fresh blood sample through centrifugation. We extracted total RNA from PBMCs by using TRIzol reagent  with on-column DNase I treatment, as described by the manufacturer. The purity and integrity of total RNA were evaluated using ultraviolet spectrometric measurements and denaturing agarose gel electrophoresis, respectively. A High-Capacity RNA-to-cDNA Kit (Invitrogen) was used to synthesize cDNA, as described by the manufacturer. The expression levels of RT-qPCR) , which w2 test and an independent-samples t test, respectively. The relative expression level of CSMD1 of each patient after normalization to the glyceraldehyde 3-phosphate dehydrogenase gene was analyzed using the comparative Ct (2−ΔΔCt) method. The expression levels of CSMD1 were compared between the HC and SCZ groups by using the Mann–Whitney U test, and the expression of CSMD1 was also compared before and after treatment in the SCZ group by using the Mann–Whitney U test. Two-tailed P values of less than 0.05 were considered to be statistically significant.We used the Statistical Package for the Social Sciences 20.0 for statistical analysis. Sex and age were compared between the SCZ and control groups by using the χCSMD1 gene in the SCZ patients before treatment, SCZ patients after the 12-week treatment, and the HCs were compared. The results revealed that the expression levels of the CSMD1 gene in the PBMCs of the SCZ patients were lower than in the HCs . However, after 12 weeks antipsychotic treatment, the expression levels of the CSMD1 gene in the PBMCs of the SCZ patients were markedly increased   for normalization in the RT-qPCR experiments, so the results may be influenced by changes in GAPDH.Our research has some limitations. First, the patient population was relatively small, which may have affected the statistical effect in comparing the CSMD1 is associated with both the development and antipsychotic treatment of SCZ, suggesting that the CSMD1 gene may be a promising biological marker for SCZ.Our results indicated that the expression of


A phase-sensitive fluidic biosensor based on a spectral-domain low-coherence interferometer is presented in this paper. With a fiber optic probe employing the common-path interferometric configuration, subnanometric changes in thickness of the molecular layers can be detected through phase analysis of the acquired interference signal from the sensor surface. Advantages of this biosensor include its picometer-scale thickness sensitivity, 13.9-ms time response, and tolerance to the fluctuation in concentration of the target solution. The capabilities of this biosensor in monitoring specific molecular binding and recognizing specific molecular was successfully demonstrated by using the reactions between the molecules of protein A and IgG. The calculated minimum detectable concentration of IgG is 0.11 µg/mL. Techniques capable of molecular recognition and detecting molecular interactions play important roles in multiple fields such as medical diagnosis, drug screening, and environmental monitoring. These techniques can be divided into two categories, which include the label-free methods and the label-based methods. Compared with those label-based methods  ,10,11, gAs one type of the label-free immunoassays detection technique, optical-interferometry methods have advantages such as high sensitivity, real-time detection, and the ability in characterizing molecular affinity and kinetics. The Mach-Zehnder interferometer (MZI) , integraIn this study, we have developed an SD-LCI-based fluidic biosensor for label-free and real-time immunoassays. This biosensor consists of a fiber optic probe employing a common-path configuration (which acts as an in situ detector) and a fluidic chip with a built-in reaction cavity (which acts as a holder of the target solution). When the thickness of the molecular layer at the probe surface is changed by the binding of the target molecules, the optical path length of the sample arm would be altered. As a result, the phase of the sample-arm light and, subsequently, the phase of the interference signal will be changed. Therefore, this thickness change and, thus, molecular binding can be detected by using our biosensor and resolving the phase dynamics of the interference signal acquired from the probe surface. Moreover, the common-path design of the interferometer employed in our biosensor precluded the common-mode phase noise and, therefore, permitted the measurement to be of picometer-scale sensitivity. The efficacy of this developed fluidic biosensor has been experimentally demonstrated by the successful detection of a specific molecular binding reaction. The specifications such as the sensitivity and specificity of the developed biosensor were also measured in our study.The entire experimental procedure includes functionalization of the probe surface and label-free molecular sensing. The functionalization requires a protein-free blocking reagent , a protein A  solution , and a freshly prepared dopamine-Tris solution (2 mg/mL). For preparing the dopamine-Tris solution, we dissolved 12 mg Tris-HCl (0.1 mmol) into 10 mL deionized water and subsequently adjusted the pH to 8.5 by using a 10% HCl solution , which was followed by dissolving 0.02 g dopamine  in this solution. The dopamine-Tris solution must be prepared freshly because it is easily oxidized and blackened in air. For molecular sensing, immunoglobulin G  solution at a concentration of 50 μg/mL in PBS buffer was prepared and sequentially diluted to concentrations of 30, 20, and 10 μg/mL.As shown in The fluidic chip was molded with polydimethylsiloxane (PDMS) following a stainless-steel template. The molding template with positive structures is shown in The customized detection system used in this study is shown in S(k). In Equation (1), k is the wavenumber and defined as λ is the wavelength of the broadband source ranging from 1230 nm to 1360 nm. rR and sR are reflectivity values of the fiber-epoxy interface and the coverslip-solution reflector, respectively. z0 denotes the position of the sensor surface, z(t) denotes the thickness change induced by molecular absorption at the probe surface, and n denotes the refractive index of the molecular layer. After eliminating the DC part and resampling the spectrum, Fourier transform was applied to acquire the path-length-resolved interference signal, I(z), which can be calculated by using the equation below.S(k) and k0 is the source center wavenumber. The obtained interference spectrum by the spectrometer can be expressed by the equation below.The path-length-resolved intensity signal is used for locating a specific interference signal acquired from the molecules-coupled probe surface. The magnitude of such a signal is determined by and reveals the reflectivity values of the reference reflector and the sensor surface while the phase dynamics of such a signal can be calculated by using the equation below.This reflects the change in thickness of the molecular layer at the probe surface. Molecular binding occurring at that surface, therefore, can be detected by monitoring the induced phase change at the probe surface. In our data processing, each phase value was averaged from 500 measurements to minimize the phase noise. Then the molecular thickness can be calculated by using the formula below.The assumption that the refractive index of the molecular layer is a constant of 1.45 was applied . As the Fluids were pumped through the flow channel using a syringe pump . The flow rate of the solution injection was 200 μL/min. The temperature was controlled as 25.0 ± 0.3 °C during the entire experiment. All the involved reagents were stored in 25 °C water bath for 1 h, then immediately followed the detection. Functionalization of the sensor surface was performed before the detection of molecules binding. The functionalization includes three steps: first, depositing a thin dopamine adhesion layer onto the sensor surface in order to improve the biocompatibility of the probe , then, mrR, which satisfies n1 and n2 are the refractive indices of the fiber and epoxy, respectively). When the peak value of the cosine signal is maximized and the valley value is approximately equal to zero, a maximum amplitude and, thus, a maximum SNR and minimum phase noise can be obtained. By this way, the performance of the biosensor was optimized with a measured SNR of 58 dB.For evaluating the stability of the phase characterization, we first measured the phase dynamics only with the injection of PBS buffer (without interactive or binding molecules) within 30 min. As shown in To demonstrate the capability of the developed fluidic biosensor in real-time molecular detection, the biosensor was used for detecting IgG molecules . To further verify the last progressive increase in the thickness of the molecules layer was only generated by the specific binding of IgG molecules to the probe molecules protein A (without nonspecific absorption on the probe surface), we performed another nonspecific binding experiment, which differed from the specific binding experiment by removing the protein A. The protein-free blocking reagent were injected into the flow channel directly after the deposition of the dopamine layer. The detected thickness dynamics revealed a larger thickness increase induced by the injection of blocking reagent molecules a, which In addition to the determination of specific molecular recognition, the sensitivity of this developed biosensor was estimated. The thickness increase induced by the specific binding of IgG molecules to protein A molecules appeared to be linearly proportional to the concentration of IgG . The coeIn conclusion, the fluidic biosensor based on a phase-sensitive SD-LCI has been demonstrated to be capable of real-time and label-free monitoring of molecular activities such as binding reactions. Such monitoring is conducted through phase analysis of the specific interference signal from the probe surface, which enables the subsequent detection and measurement of the subnanometric changes in the thickness of the molecular layers. Future research would refer to the improvements enabling multi-point detection, which may require multiple reaction cavities and fiber optic probes with coverslips of different thicknesses and, thus, simultaneously provide both pre-reaction and post-reaction information and further miniaturization of the fiber optic probe, which may require replacement of the ceramic ferrule with a thinner glass capillary.


Ruta graveolens (Rutaceae) is a plant used in folk medicine to treat several diseases, including parasitic infections. In this study, we reported the in vitro schistosomicidal activity of the R. graveolens extract (Rg) and its active fraction (Rg-FAE). Also, the characterization of Rg-FAE by UPLC-ESI-QTOF-MS analysis and its in vitro antileishmanial activity against Leishmania braziliensis were also performed. In vitro schistosomicidal assays were assessed against adult worms of S. mansoni, while cell viability against peritoneal macrophages was measured by MTT assay. Rg (100 μg/mL) exhibited noticeable schistosomicidal activity, causing 100% mortality and decreasing motor activity of all adult male and female schistosomes, but with low activity against L. braziliensis. After chromatographic fractionation of Rg, fraction Rg-FAE was obtained, showing high activity against adult schistosomes. UPLC-ESI-QTOF-MS analysis of Rg-FAE revealed the presence of eleven alkaloids and one furanocoumarin. No significant antileishmanial activity was found for Rg, while Rg-FAE exhibited activity against L. braziliensis promastigotes. We demonstrated, for the first time, that the R. graveolens extract (Rg) and its alkaloid-rich fraction (Rg-FAE) are active against adult worms of S. mansoni, with no significant cytotoxicity on macrophages. Our findings open the route to further antiparasitic studies with the active fraction of R. graveolens and its identified compounds, especially alkaloids.Schistosomiasis is a neglected tropical disease that affects million people worldwide, mostly in developing countries.  Schistosoma parasites, mainly S. mansoni, that is associated with long-term undernutrition, anaemia, organ scarring, and fibrosis, resulting in disabling patient symptoms +. Peak 3 also showed a loss of CO, giving the m/z of 237.0768 [M-CH3-CO]+. As previously reported [m/z 207.0654 [M-CH2O]+. Mass fragmentation data for peak 3 are in agreement with the proposed structure of graveoline  showed an [M+H]+ ion at m/z 260.0886 and fragment ions at m/z 245.0661 [M-CH3]+ and 230.0430 [M-CH3]+ in MS2 mode, suggesting consecutive losses of 15 u, which may be due to the loss of methyl groups from methoxyl groups. In addition, fragment ions were observed at m/z 216 [M-CH3-CO]+ and 199 [M-CH3-H2O-CO]+. Molecular ion and fragmentation patterns are similar to those reported from literature [5 is skimmianine (6 (tR = 6.70) was identified as arborinine  showed a molecular ion [M + H]+ at m/z 315.1586 and an ion fragment [M-(CH3)2COH]+ at m/z 255. Based on its fragmentation pattern along with previous literature data [7, Peak terature , 29, indmmianine . Similarborinine  based on reports . In addim/z 186 and m/z 173 [m/z 186 and 173 ion fragments were used as diagnostic ion fragments to identify the quinolone alkaloids 8, 9, 10, 11, and 12 (8 (m/z 286.2171), 9 (m/z 300.2355), 10 (m/z 314.2481), 11 (m/z 328.2617), and 12 (m/z 342.2783) were identified, respectively, as quinolone alkaloids 1-methyl-2-nonyl-4(1H)-quinolone, 1-methyl-2-decyl-4(1H)-quinolone, 1-methyl-2-undecyl-4(1H)-quinolone, 1-methyl-2-dodecyl-4(1H)-quinolone, and dihydroevocarpine, respectively. All of these quinolone alkaloids  were previously identified in R. graveolens [According to literature, the McLafferty rearrangement occurs in quinolone alkaloids, leading to the formation of stable conjugate systems with ion fragments at  m/z 173 . Therefo, and 12 , which daveolens .Rg was evaluated against L. braziliensis. However, no significant antileishmanial results were found for Rg (IC50 > 50 μg/mL) against L. braziliensis promastigotes (data not shown). In contrast, previous antileishmanial study with a crude extract of R. graveolens against L. amazonensis showed an inhibition of 74.4% in the number of promastigotes at 100 μg/mL [Rg-FAE exhibited pronounced activity against L. braziliensis promastigotes in the antileishmanial assay, inhibiting the parasites growth in all concentrations, displaying an IC50 value of 5.90 μg/mL, which was better than the reference drug miltefosine . However, Rg-FAE showed low activity against intracellular amastigotes of L. braziliensis, diminishing the number of intracellular amastigotes by 26.58% at the maximum concentration used (50 μg/mL), while miltefosine showed an IC50 value of 2.95 ± 0.44 μg/mL. This difference in sensibility between both stages of parasite could be due to biochemical targets, the rate of division, exposure, and inactivation into the parasitophorous vacuole or drug metabolism [Rg-FAE cannot be considered as promising as well as the schistosomicidal activity, our data contribute with the ethnopharmacological use of a traditional medicinal plant from the Brazilian flora, such as R. graveolens, for the treatment of Leishmaniasis.In addition, the effect of the crude extract 00 μg/mL . On the tabolism . AlthougRg and Rg-FAE were also evaluated on cytotoxicity assay against murine macrophages. No significant toxic effects were observed for Rg (CC50 > 75 μg/mL) or Rg-FAE  to mammalian cells (S. mansoni and promastigotes of L. braziliensis, giving support to its potential in identifying lead compounds for the development of novel antiparasitic drugs.Moreover, considering their safety, an cells  at conceR. graveolens is an important medicinal plant that has been used as anthelmintic and to treat several diseases, such as leishmaniasis [Rg-FAE, several alkaloids, along with the identified furanocoumarin, could be related to the antiparasitic activity of this fraction.maniasis , 10. Alkmaniasis . Among tRg-FAE and its chemical composition, it was shown that 2-substituted quinoline alkaloids are highly active in vitro and in vivo against Leishmania sp. [R. graveolens possesses a wide pharmacological potential and may have low toxicity [Rg-FAE in treating schistosomiasis and leishmaniasis.Regarding the antiparasitic activity of ania sp. . Also, sania sp.  and adulania sp. , schistotoxicity , additioR. graveolens extract and its alkaloid-rich fraction are active against adult worms of S. mansoni in vitro, with no cytotoxicity on mammalian cells. Eleven alkaloids, together with a furancoumarin, were identified by UPLC-ESI-QTOF-MS analysis as constituents of the active fraction Rg-FAE. Our findings open the route to further antiparasitic studies with the active fraction and its isolated compounds, especially alkaloids.The present study has demonstrated, for the first time, that the


The half maximal effective concentration (EC50) of eckol for the dopamine D3 and D4 receptors was 48.62 ± 3.21 and 42.55 ± 2.54 µM, respectively, while the EC50 values of dopamine as a reference agonist for these two receptors were 2.9 and 3.3 nM, respectively. In silico studies revealed that a low binding energy in addition to hydrophilic, hydrophobic, π–alkyl, and π–π T-shaped interactions are potential mechanisms by which eckol binds to the dopamine receptors to exert its agonist effects. Molecular dynamics (MD) simulation revealed that Phe346 of the dopamine receptors is important for binding of eckol, similar to eticlopride and dopamine. Our results collectively suggest that eckol is a potential D3/D4 agonist for the management of neurodegenerative diseases, such as Parkinson’s disease.The G protein-coupled receptor (GPCR) family of proteins comprises signaling proteins that mediate cellular responses to various hormones and neurotransmitters, and serves as a prime target for drug discovery. Towards our goal of discovering secondary metabolites from natural sources that can function as neuronal drugs, we evaluated the modulatory effect of eckol on various GPCRs via cell-based functional assays. In addition, we conducted in silico predictions to obtain molecular insights into the functional effects of eckol. Functional assays revealed that eckol had a concentration-dependent agonist effect on dopamine D G-protein-coupled receptors (GPCRs) belong to the largest superfamily of cell surface proteins encoded by the human genome, and are valuable molecular targets for drug discovery. Design and implementation of high-throughput GPCR functional assays to identify novel drug candidates is an important aspect of the early drug discovery process because these receptors are involved in human pathophysiology and are also pharmacologically traceable . Althougs/olf-coupled D1-like (D1 and D5) and Gαi/o-coupled D2-like  receptors with regard to the stimulatory or inhibitory properties of the secondary messenger, cyclic adenosine monophosphate (cAMP) -Substance P, [Arg8]-vasopressin (AVP), (+)butacamol, clozapine, dopamine, -AVP, serotonin, SCH 2330, L-733,060, (S)-WAY-100635 and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich . All the other chemicals and reagents used were purchased from E. Merck, Fluka , and Sigma-Aldrich , unless otherwise stated, and were of highest grade available.E. stolonifera as described in our previous paper  obtained in the presence of the eckol.3 receptor (hD3R)-eticlopride complex (PDB ID: 3PBL) and human dopamine D4 receptor (hD4R)-nemonapride complex (PDB ID: 5WIU) were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB); the resolution of these complexes is 2.89 and 1.96 Å, respectively [Docking of the target receptor and eckol was successfully simulated using AutoDock 4.2 . X-ray cectively . The 3D Pharmacokinetic parameters of eckol such as absorption, distribution, metabolism, and excretion (ADME) was determined using the web-based software PreADMET  .3 receptor-eckol complex generated by the docking study employing the NAMD 2.9 package [+ and Cl− ions were generated with a condition of 5 Å between ions employing the AUTOIONIZE module of visual molecular dynamics (VMD) to approximate 150 mM NaCl in the system [2) was set to decrease gradually during 25 ns. Subsequently, NPnAT ensemble simulations were performed for 80 ns without constraint on the protein . Constant pressure (1 atm) was maintained by using the Langevin piston Nose-Hoover method [−1 for Langevin dynamics and the direct space cut off was 12 Å. The simulation was performed with a 2-fs time interval.We performed molecular dynamics (MD) simulations for the dopamine D package  with the package  force fi package . Paramet package . The pro package . Positioe system . We perfr method . The parr method . The damp < 0.05.All redsults are expressed as the mean ± standard deviation (SD) of triplicate experiments. Statistically significant values were compared using one-way analysis of variance (ANOVA) and Duncan’s test . Different alphabet letters indicate a significant difference between groups at 3 and D4 receptors. Similarly, in silico modeling and MD simulation suggested the mechanisms by which eckol bound to these receptors and exerted its agonist effect. Overall results of this study suggest that eckol is a D3/D4 agonist that has potential in the management of neurodegenerative diseases, especially PD.Eckol is a phlorotannin that is abundant in brown algae and that has a wide variety of biological activities. In the present study, we evaluated the effect of eckol on GPCRs. Cell-based functional assays revealed that eckol is an agonist of the dopamine D


Together, these advances have blurred the structural boundaries between the myocardium and the circulation, and brought us to appreciate the key signaling pathways that are common to various domains of the heart as well as the fluidity of molecular signals that play opposing roles depending on the spatial and temporal context.Classically, basic cardiovascular research has been delimited to investigating one of the two architectural territories of the heart; the myocardium and the coronary vasculature. Technological advances continue to foster mechanistic insights on homeostatic physiology and pathophysiology of the heart. In parallel, translational efforts in gene therapyIn this thematic series, four review articles bring spotlights on key conceptual advances in the cardiovascular research. It opens with Kwong and colleagues’ article on mitochondrial metabolism, and strategies to target specifically the mitochondrial reactive oxygen species for better therapeutic outcomes. De Couto connects cardiac regeneration/repair with one of the most dynamic cell types within the heart, the macrophages, and presents approaches to modulating the immune cells’ properties with extracellular vesicles. Kassiri and colleagues highlight the significance of the extracellular milieu in the context of aortic aneurism, and the importance of choosing the correct model to study specific matrix. Finally, Eom and Yoon bring together the molecular concepts and present the-state-of-the art in understanding an elusive form of heart failure that appears to be normal in its ability to pump blood effectively. All four articles underscore topics that are highly relevant now. A quick search on PubMed illustrate nearly an exponential increase in the number of peer-reviewed articles published in recent years on each topic Fig. . What ti