NLRP3, caspase-1, 3CR1, CCL17, RORc and Tbx21interleukin (IL)-1β, CX at the CNS. Otherwise, mRNA expression for ZO-1 increased at the lumbar spinal cord. These effects were accompanied by the stabilization of blood-spinal cord barrier permeability. The results of this study indicate that early intervention with 1,25-dihydroxyvitamin D3 can control the neuroinflammatory process that is the hallmark of EAE and MS immunopathogenesis and should thus be explored as an adjunct therapy for MS patients.Multiple sclerosis (MS) is a progressive disease of the central nervous system (CNS) that involves damage to the myelin sheath surrounding axons. MS therapy is based on immunomodulatory drugs that reduce disease recurrence and severity. Vitamin D is a hormone whose immunomodulatory ability has been widely demonstrated, including in experimental autoimmune encephalomyelitis (EAE), which is an animal model of CNS inflammation. In this study, we evaluated the potential of very early intervention with the active form of vitamin D  to control neuroinflammation during EAE development. EAE was induced in C57BL/6J mice and 1,25-dihydroxyvitamin D3 administration began 1 day after disease induction. This procedure decreased prevalence, clinical score, inflammation, and demyelination. It also reduced MHCII expression in macrophages and microglia as well as the level of oxidative stress and messenger RNA (mRNA) expression for  Multiple sclerosis (MS) has been classically categorized as an autoimmune disease in which self-reactive T CD4+ (Th1/Th17), T CD8+, and B cells attack the central nervous system (CNS) and cause neurological dysfunction . AlthougDespite this presumed sequential pattern, it is well established that MS is clinically heterogeneous and shaped by a complex interaction between genetic background and environmental factors . AccordiMany disease-modifying therapies for MS are currently approved by the Food and Drug Administration. Most of them are immunomodulatory or immunosuppressive drugs that significantly reduce the frequency of relapses in RRMS. Unfortunately, their effectiveness in avoiding the conversion to the progressive disease remains very limited. Moreover, they are ineffective when the disease has already advanced to the progressive phase . There iin vitro studies using rat neuron cultures demonstrated that active vitamin D  increases glutathione levels in these cells. The reduced form of glutathione, which is supplied to nerve cells by astrocytes, is a key antioxidant that protects cells against reactive oxygen species (ROS) and apoptosis  has a protective effect when given alone or in combination with the specific autoantigen (myelin oligodendrocyte glycoprotein-MOG) . Nonetheth day after disease induction. The other assays  were performed at the EAE acute phase, which was 18 days after disease induction. All animals were perfused before collecting the material to be analyzed. All real time polymerase chain reaction (PCR) assays and histopathological analyses were conducted with the lumbar portion of the spinal cord. Oxidative stress was evaluated in homogenates from brain plus cervical and thoracic portions of the spinal cord, and flow cytometry was performed with cells eluted from the whole CNS.C57BL/6J female mice were allocated into three experimental groups: control ; EAE , and EAE/Vit D . Body weight and clinical scores were evaluated daily, while the tests concerning blood-brain and blood-spinal cord barriers were performed on the 10ad libitum basis. Animals were housed in ventilated cages  in pathogen-free conditions and handled according to the ethical procedures established by the National Council for Control of Animal Experimentation . The entire procedure was analyzed and approved by the Ethics Committee on Animal Experimentation of the Institute of Biosciences, UNESP, Botucatu (CEUA-Process 926).Female C57BL/6J mice were bought from the animal facility of the Medical School—University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil. Water and food were sterilized and offered on an 35–55  emulsified with complete Freund's adjuvant (CFA) containing 4 mg/ml of Mycobacterium tuberculosis . Mice also received two intraperitoneal (IP) doses of Bordetella pertussis toxin  at 200 ng each: one at the immunization day and the other 48 h after disease induction. Body weight and disease scores were recorded daily. Disease clinical scores were defined according to the following manifestations: no symptoms (0); limp tail (1); hind leg weakness (2); partially paralyzed hind legs (3); complete hind leg paralysis (4); and complete paralysis (5). Percentage of body weight loss was determined between day 0 when the animals were weighed for the first time and submitted to EAE induction and at the end-point of the experiment, i.e. day 18, when they were euthanized and the different parameters were evaluated. A vitamin stock was made by dilution in absolute ethanol. Mice were treated with eight doses  of 1α,25-dihydroxyvitamin D3 . This dose corresponds to approximately 5 µg/kg and was delivered by the IP route every other day, as previously described . Then, 5 μm-thick sections were obtained using a Leica RM2245 microtome and stained with hematoxylin and eosin (H&E) or luxol fast blue (LFB) to analyze the inflammation and demyelination, respectively. The intensity of the inflammatory infiltration was defined according to the criteria used by other authors , which ivia the IP route with 100 μl of a 10% sodium fluorescein  solution. They were then anesthetized 20 min later and blood was collected for further plasma separation. The animals were then perfused with 10 ml of 0.9% saline solution and the brain and entire spinal cord were collected and individually processed. After being weighed, these organs were macerated with 400 μl of saline solution and then centrifuged . Supernatants and plasma samples were then distributed in a 96-well black plate and read in a Synergy 4 fluorimeter . NaFlu uptake by the brain and spinal cord were calculated according to the following equation: (tissue sample RFU/tissue sample weight)/(plasma RFU/amount of cardiac blood). RFU: relative units of fluorescence.This evaluation was performed 10 days after disease induction and was based on the NaFlu protocol described by 2 5%), the cells were washed with RPMI and resuspended in a 30% Percoll solution . This cell suspension was gently dispensed over 70% Percoll and centrifuged at 950 g for 20 min. The ring containing mononuclear cells was collected, washed, resuspended (1x106 cells/ml), and incubated with the following fluorochrome-labeled monoclonal antibodies  according to Cells from the CNS were obtained according to 5 cells/ml and then cultured in complete RPMI medium . Cultures were incubated during 48 h in the presence of MOG (20 μg/ml), as previously described by Inguinal lymph node cells were obtained and adjusted to 2.5x102+ oxidation reaction in the presence of xylenol orange, sulfuric oxide, and butylated hydroxytoluene diluted in methanol. To evaluate the degree of protein carbonylation, the samples were incubated with 2,4-dinitrophenylhydrazine and centrifuged in the presence of trichloroacetic acid. The protein pellet was washed with an ethanol-ethyl acetate mixture and then resuspended in guanidine hydrochloride. Catalase activity was evaluated by sample incubation with hydrogen peroxide diluted in phosphate buffer, and glutathione peroxidase activity was determined by a NADPH2 oxidation reaction in the presence of hydrogen peroxide and glutathione reductase. Superoxide dismutase activity was determined in a pH10 medium by measuring the inhibitory enzymatic activity on nitro-blue tetrazolium reduction triggered by hydroxylamine-derived free radicals. Quantification of the aforementioned oxidative stress parameters was based on absorbances detected by a microplate reader .Oxidative and anti-oxidative parameters were measured at the CNS using the biochemical methodology previously described by NLRP3, ASC, Caspase-1, IL-1β, 3CR1CX, CCL17, RORc, GATA3, Foxp3, Tbx21, and GAPDH gene amplifications were performed using the TaqMan Master Mix with primers and probes inventoried and tested by Life Technologies. ZO-1 and GAPDH gene amplifications were performed by using the Power SYBR Green PCR Master Mix (Applied Biosystems). Primer sequences included: ZO-1 forward—GAGAGACAAGATGTCCGCCA, reverse—CCATTGCTGTGCTCTTAGCG (NM_001163574.1), and GAPDH forward—GCATCTTCTTGTGCAGTGCC, reverse—TACGGCCAAATCCGTTCAC (NM_008084.3). The amplification of specific transcripts was confirmed by the melting curve profiles generated at the end of each run. Assays were performed in duplicate in “Sequence Detection Systems” 1.2.3—7300 Real-Time PCR Systems (Applied Biosystems). The results were expressed based on fold difference using the delta delta cycle threshold (2−ΔΔCt) method for quantification with the negative control group as a calibrator, according to Total RNA was extracted from the lumbar spinal cord  and complementary DNA (cDNA) was synthesized using the Kit High Capacity . Results were expressed as mean ± standard deviation or as median (25–75%). Comparisons between two samples were made by a t-test and comparisons between more than three samples were made by one-way analysis of variance (ANOVA) followed by Tukey’s test to compare parametric findings. A Kruskal-Wallis test followed by Dunn’s test was employed to analyze and compare non-parametric findings. Disease prevalence was evaluated by the chi-squared test. Statistical analyses were performed using SigmaPlot 12.0 for Windows , and p < 0.05 was considered significant. The difference between clinical scores was analyzed by the area under the curve method.via the IP route. This intervention, which began 24 h after disease induction, significantly reduced EAE prevalence  (mean ± SEM): control: 1.3 ± 0.3, EAE: 10.6 ± 1.8, and EAE/Vit D: 6.1 ± 0.9; f: 9.9, infiltrating macrophages and activated microglia (CD45HighCD11b+) (mean ± SEM): 1.34 ± 0.2, EAE: 25.2 ± 5.5, and EAE/Vit D: 16.1 ± 3.6; f: 9.9, as shown in LowCD11b+) (mean ± SEM): control: 24.5 ± 3.0, EAE: 17.2 ± 1.4, and EAE/Vit D: 21.5 ± 1.7; f: 3.5, which was predictably lower in EAE animals, was similar to the frequency found in normal mice : control: 41.4 ± 7.1, EAE: 176.2 ± 14.2, and EAE/Vit D: 100.0 ± 19.6; Tbx21 and RORc, which are expressed by Th1 and Th17 subsets, respectively, were significantly downmodulated in mice supplemented with 1,25-VitD3  in the cell population constituted by macrophages and activated microglia (mean ± SEM): control: 4,977 ± 1,118, EAE: 29,060 ± 3,091, and EAE/Vit D: 18,510 ± 1,959; 3CR1CX mRNA  and of CCL17 mRNA (a chemokine involved in leukocyte trafficking) were also significantly downmodulated in mice that received 1,25-VitD3. 3CR1CX and CCL17 mRNA levels are presented in The local expression of f: 51.0 and protein carbonyl (mean ± SEM): control: 0.8 ± 0.05, EAE: 1.5 ± 0.2, and EAE/Vit D: 1.2 ± 0.1; f: 9.8, thus revealing the existence of an accentuated oxidative stress condition. Concomitant and clear reductions were observed in the level of enzymes involved in the control of this oxidative stress, such as glutathione peroxidase (mean ± SEM): control: 49.8 ± 2.3, EAE: 31.0 ± 1.2, and EAE/Vit D: 48.6 ± 1.9; f: 35.8, catalase (mean ± SEM): control: 24.5 ± 0.9, EAE: 13.1 ± 0.8, and EAE/Vit D: 21.2 ± 0.8; f: 49.2 and superoxide dismutase (mean ± SEM): control: 16.2 ± 0.8, EAE: 6.1 ± 0.3, and EAE/Vit D: 16.3 ± 0.6; f: 92.0, as shown in As displayed in NLRP3, caspase-1, and IL-1β, respectively. No change was detected in ASC expression : control: 0.08 ± 0.02, EAE: 0.19 ± 0.05, and EAE/Vit D: 0.06 ± 0.01; f: 3.1 of EAE mice, with spinal cord permeability being particularly disrupted : control: 0.05 ± 0.01, EAE: 0.09 ± 0.03, and EAE/Vit D: 0.06 ± 0.02; f: 5.6, IL-17 (mean ± SEM): control: 33.2 ± 1.4, EAE: 142.1 ± 34.3, and EAE/Vit D: 452.7 ± 109.4; f: 6.8, IL-6 (mean ± SEM): control: 31.7 ± 1.0, EAE: 67.0 ± 13.7, and EAE/Vit D: 90.0 ± 10.6; f: 5.5, and TNF-α (mean ± SEM): control: 26.4 ± 24.1, EAE: 152.3 ± 6.6, and EAE/Vit D: 247.1 ± 39.3; f: 14.1, respectively.Cytokine production by cells from inguinal lymph nodes clearly indicated an accumulation or increased activation of MOG-responsive cells in 1,25-VitD3-supplemented animals. As presented in The possibility of using active vitamin D3 as an alternative, adjunct, or even prophylactic therapy for MS has been investigated; however, it remains controversial. In this study, we used EAE, which is a widely adopted animal model, to evaluate the ability of this vitamin to control MS development, especially regarding the CNS. Our initial findings indicated that 1,25-VitD3 administration soon after disease induction was able to significantly arrest disease development by controlling the prevalence, disease severity, and the time required for the disease to manifest itself. All of these parameters are considered valid indicators of vitamin D or other drug efficacies in EAE . The absTh1 and Th17 lymphocytes, identified by Tbx21 and RORc mRNA expression, respectively, were significantly downmodulated by 1,25-VitD3. These findings are highly consistent with the pivotal role of these two T cell subsets in the immunopathogenesis of EAE and MS .LO, SZ-P, and AS contributed to the conception and design of this work. LO, SZ-P, TF-S, LM, LI, and AF performed experiments. LO, TF-S, LM, and LI contributed to data acquisition. LR, TF-S, LM, LI, and AS contributed to data interpretation. LR and AS wrote the paper and all authors reviewed the manuscript and approved the final version.This work was supported by São Paulo Research Foundation-FAPESP (grant number 2013/26257-8) and by the National Council for Scientific and Technological Development – CNPq (grant number 307269/2017-5).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The obtained nano-sized particles of WO3 and ZnO, PES, and nanocomposites (WO3-PES NC and ZnO-PES NC) were investigated. The average molecular weight of the cured PES was measured by employing the gel permeation chromatography (GPC) technique. Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) spectra assured the formation of the polymeric nanocomposites.WO3 and ZnO nanoparticles supposed a condensed porous spherical phase found implanted in the polymer structure, as detected by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) methods. These nano-scale systems achieved an electrical activity based on the conductive nanoparticles embedded matrix as a result of the ion–ion interactions. The microbial influence of the nanocomposites was examined against pathogenic bacteria; Pseudomonas aeruginosa,Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, and Fungi; Aspergillus niger, and Candidaalbicans. Results exhibited that these nanocomposites have antimicrobial effects from moderate to slightly high on bacteria and high on fungi which was confirmed by a clear zone of inhibition. This study contributes to the design of reasonable composites to be under evaluation for their catalytic effect.Nanoparticles of tungsten oxide (WO Recent  sensors ,22, dye- sensors , lithium sensors , photoch sensors , photoca sensors ,27, supe sensors , water s sensors ,30, wast sensors , dye rem sensors , smart w sensors , and ant sensors . As a re sensors . Newly,  sensors , photoca sensors ,38, phot sensors , microbi sensors , cancer  sensors , and wou sensors , food pa sensors  and wate sensors . Further sensors ,46,47,48Experimental fabrication of polyethylene sebacate resin has been reported . Sodium 3 and nZnO. The condensed porous crystalline phase was appeared with a particle size around 6–40 nm by using the Scherrer equation.Sodium tungstate and ferrous ammonium sulfate (mole ratio 2:1) were dissolved in DW individually and mixed under sturdy stirring. A dark-colored suspension appeared which was dissolved by the addition of oxalic acid . Eventua3 and ZnO nanoparticles dispersed PES resin was fabricated throughout sequential proceedings as described in 3 and ZnO as a solid powder was added quietly under vigorously stirring for 3 h (Wt.% 0.05 MOx/PES) [3-PES NC) and (ZnO-PES NC) (Nanocomposites of WOMOx/PES) . Ultimat-PES NC) b,c.1 radiation in the range of 2θ = 4–80° at a potential of 40 KV and a current value of 40 mA with a running step of 2° in 2θ/min. SEM analysis was performed by employing a Jeol 5410  instrument operated at a potential of 30 kV. TEM technique was performed by employing a Jeol instrument 2010 at a potential of 130 kV. The electrical activity was measured by using a Keithley test equipment-6517A Model-digit Electrometer . The microbial effect was examined by using the agar diffusion technique [Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 35556), Gram-negative bacteria (Escherichia coli ATCC 23282 and Pseudomonas aeruginosa ATCC 10145), Yeast , and Filamentous Fungus (Aspergillus niger ATCC 16404). The negative control was DMF showed no antimicrobial activity against the tested microorganisms, and the positive control was Erythromycin for bacteria and Metronidazole for yeast and fungus. All examinations were carried out in duplicates and the listed data are the average of the obtained results.Evaluation of weight throughout GPC analysis was performed by employing a Supremamax 3000 column at Polymer Standard Service with 2% acetic acid/0.2 M buffer sodium acetate as eluent (1 mL/min). FTIR technique was performed by employing an ATI Mattson model Genesis Series , infrared spectrophotometer adopting KBr technique. XRD analysis was performed by employing a Philips Powder Diffractometer and monochromatized Cu Kαechnique . The tesn” of 2103, which takes into account all the polymeric molecules that exist. The attained mode “the highest peak” molecular weight distribution “Mp” value was 2422. Mass of the total polymeric chains “Mw” value was 2621. As well, the catalytic action diminished the glycol overtaking and accordingly minimized the whole period of the reaction. The PES reveals the crucial mechanism and kinetics of the catalytic esterification process [GPC values of the prepared PES were illustrated in detail in  process .3-PES NC, and ZnO-PES NC are shown in −1. Two common symmetric stretching bands for CH– and CH= were revealed at 2852 and 2917 cm−1. Associated with ester chain formation, strong and sharp spectra were present at 1739 cm−1. Moderate spectra are shown at 1465 cm−1 due to the methylene group. Functional OH bending appeared as a medium absorption at 1389 cm−1. Two bands at 1292 and 1217 cm−1 were due to C–O of ester. C=C bending of CH= was exposed as two peaks at 894 and 754 cm−1. Owing to the low content of nano-sized oxides in PES, there are no considerable distinctions among the diagrams in which the spectrometric techniques are mainly employed to detect the hydrocarbon components [FTIR spectra of PES, WO3-PES NC, and ZnO-PES NC reveal the presence of multiple diffraction patterns observed. The reflections of the crystalline structure were recorded in the 2θ range of 6–45°. The characteristic crystalline peaks appeared at around 2θ = 6.06° (30.4%), 21.75° (100%), and 24.64° (57.12%). The exposed value at 2θ = 21.75° is correlated to the high content of the ester series. Medium appearance at 2θ = 6.06° was due to rough form. The PES backbone contains prolonged polymer chains referring to various arrangements of polyester bindings [3 and ZnO. Otherwise, the characteristic strong and sharp diffraction peaks reveal the good crystallinity of the composites of PES-WO3 NC and PES-ZnO NC. The characteristic peaks at 2θ = 6.0°, 13.4°, 20.4°,21.7°, 24.5°, 29.8°, 30.6°, 38.8°, and 41.5° are related to the (020), (120), (032), (131), and (220) patterns of PES (JPDS: 04-0783). The high intensity and narrow peaks are detected at 2θ = 6.0°, 7.2°, 21.7°,21.9°, 24.6°, 24.7°, 30.6°, 38.8°,41.7°, and 45.4° are correlated to the (020), (020), (200), (120), (111), (120), (131), (200), (202), and (222) crystalline planes of the spherical phase of WO3 (JPDS: 43-0679). The strongest diffraction peaks observed at 2θ values of 5.9°, 8.0°, 11.4°, 17.3°, 20.2°, 21.6°, 24.5° 30.3°, 35.8°, 38.7°, and 41.2° corresponding to the planes (020), (120), (101), (100), (131), (002), (101), and (110) of ZnO (JPDS: 36-1451). The size of crystals was measured using the Scherrer equation to the peaks at 2θ = 24.64° and 2θ = 21.75° around 36 and 16nm.XRD plots  for PES,bindings . ASTM 243-PES NC and ZnO-PES NC), SEM images , Gram-positive bacteria (Staphylococcus aureus ATCC 35556 and Bacillus subtilis ATCC 6633), and fungi  at a concentration of 5 mg/mL. The bacteria and yeast were grown on nutrient agar while the fungus was grown on Czapek’s Dox agar medium. Data in The microbial impacts of PES and its nanocomposites were evaluated against pathogenic Gram-negative bacteria, (3 and ZnO nanoparticles dispersed PES nanocomposites through the precipitation process and esterification reaction. Mass, structure, composition, morphology, conductivity, and microbial activity of the obtained systems were evaluated. Spectra of FTIR and XRD techniques confirmed the fabrication of polymer nanocomposites. It reveals the existence of several patterns for PES composites. The grain size of WO3 and ZnO were found in the nano-crystalline regime. SEM and TEM images show condensed spherical shaped nanomaterials embedded in the PES lattice. The nanocomposites based on nWO3 and nZnO exposed an electrical conductivity effect due to the interaction of ions in which the values are increased under heat. The antimicrobial results revealed a moderate effect on bacteria and a high effect on fungi for the nanocomposites based on inhibition zone. As a result, the current research contributes to the fabrication of reasonable nanomaterials to be under evaluation for their catalytic activity.The current study presents the preparation of WO


Chest computed tomography is a precise way to help clinicians diagnose COVID-19 patients. (Method) Two datasets are chosen for this study. The multiple-way data augmentation, including speckle noise, random translation, scaling, salt-and-pepper noise, vertical shear, Gamma correction, rotation, Gaussian noise, and horizontal shear, is harnessed to increase the size of the training set. Then, the SqueezeNet (SN) with complex bypass is used to generate SN features. Finally, the extreme learning machine (ELM) is used to serve as the classifier due to its simplicity of usage, quick learning speed, and great generalization performances. The number of hidden neurons in ELM is set to 2000. Ten runs of 10-fold cross-validation are implemented to generate impartial results. (Result) For the 296-image dataset, our SNELM model attains a sensitivity of 96.35 ± 1.50%, a specificity of 96.08 ± 1.05%, a precision of 96.10 ± 1.00%, and an accuracy of 96.22 ± 0.94%. For the 640-image dataset, the SNELM attains a sensitivity of 96.00 ± 1.25%, a specificity of 96.28 ± 1.16%, a precision of 96.28 ± 1.13%, and an accuracy of 96.14 ± 0.96%. (Conclusion) The proposed SNELM model is successful in diagnosing COVID-19. The performances of our model are higher than seven state-of-the-art COVID-19 recognition models. The polymerase chain reaction (PCR) can effectively detect its existence; however, the cluster of false-positive . U4 = {u4(k)} with the size of each image as . The final grayscale image u4(k) is then stacked along channel direction to output the color image u(k): u(k) is now a1 × a2 × 3.The grayscale range of 33.1u(k), then the horizontally mirrored image (HMI) is defined as HMIu(k) as b1 different data augmentation (DA) methods u(k) and HMIu(k). Suppose each DA generates b2 new images. Finally, the whole generated images Λ(k) are defined as: Compared to normal individual DA methods, the MDA fuse the separate DA methods together and thus can yield better performances .3.2SqueezeNet (SN) is chosen since it can achieve a 50× reduction in model size compared to AlexNet and maintain the same accuracy . This lis1×1, e1×1, and e3×3, which stand for the number of 1 × 1 kernels in the SL, and the number of 1 × 1 and 3 × 3 kernels in the EL.The fire module (FM) is the core component in the N. It contains a squeeze layer (SL), which uses only 1 × 1 kernels, followed by an expand layer (EL), which contains several 1 × 1 and 3 × 3 kernels . The strCompared to ordinary convolutional neural network (CNN) architectures, the SN  has threThere are different variants of SN. Özyurt et al.  used van3.3The SN features after global avgpool See  are usedi-th input sample be xi = T ∈ Rn; i = 1, …, N. The output of an ELM with L hidden neurons is: h stands for the activation function, jα = T the input weight, jβ the bias, iO = T the output of the model for the i-th input sample. Afterwards, the model is trained to yield Let the Let us rephrase the above equation as αj, jβ and jλ. ELM can yield a solution quickly via the pseudo inverse: M† signifies the Moore-Penrose .Input SN features [αj and the bias jβ randomly.Step A Initialize values of input weight M using Step B Compute the output matrix λ using the pseudo inverse in Step C Compute the output weight 3.4T runs of I-fold cross-validation (CV) are carried out. Assume the test confusion matrix  over t-th run and i-th fold is: i = 1, …, I stands for the fold index, and t = 1, …, T the run index. The  signify true positive, false negative, false positive, and true negative, respectively. At i-th trial, the i-th fold is employed as test, and the left folds {1, …, i – 1, i + 1, …, I} altogether are employed as training, as shown in I-fold CV consists of I trials.t, i) is gauged based on the i-th fold, which is the test set. We afterward take their summation across altogether I trials, as shown in t-th run Θ(t) is attained as Θ: 1 sensitivity, κ2 specificity, κ3 precision, and κ4 accuracy as: At κ5 is F1 score: κ6 is Matthews correlation coefficient (MCC), which is a more reliable statistical rate that produces a high score only if the prediction obtained good results in all of the four entries in the TCM [κ7 is the Fowlkes–Mallows index (FMI).  the TCM . (18)κ6. This study finally chooses κ6 as the most important indicator due to its larger range (–1 ≤ κ6 ≤ +1) than that of κ4 (0 ≤ κ4 ≤ 1).There are two indicators I-fold CV. The experiment runs the I-fold CV T runs. After all runs, the mean and standard deviation (MSD) of all seven indicators T runs. μ signifies the mean value and σ the standard deviation. The values of MSD are recorded in the format of μ ± σ.The previous process is for one run of 44.1b1 = 9 different DA methods on both raw image and HMI. Every DA produces b2 = 30 images. The AFMDA is b3 = 542. Activation function in ELM is chosen the sigmoid function. The number of hidden neurons in ELM is set to L = 2000. We run ten runs of 10-fold CV to report the robust results.The hyperparameters are listed in 4.2The MDA result of b3 = 542 makes the training burden of our model 542 times as much as that of the model without MDA. Nevertheless, in the test stage, there is no need to apply MDA to the test images, so our model is the same quick as the model without MDA.Meanwhile, the AFMDA value 4.3t = 1, 2, ... , 10 means the run index. For the dataset D1, SNELM attains a sensitivity of 96.35 ± 1.50%, a specificity of 96.08 ± 1.05%, a precision of 96.10 ± 1.00%, an accuracy of 96.22 ± 0.94%, an F1 score of 96.22 ± 0.95%, an MCC of 92.45 ± 1.87%, and an FMI of 96.22 ± 0.95%. For the dataset D2, SNELM attains a sensitivity of 96.00 ± 1.25%, a specificity of 96.28 ± 1.16%, a precision of 96.28 ± 1.13%, an accuracy of 96.14 ± 0.96%, an F1 score of 96.13 ± 0.96%, an MCC of 92.29 ± 1.91%, and an FMI of 96.14 ± 0.96%.4.4After combining the ten runs altogether, we can draw the overall TCMs and the ROC curves of the two datasets. The top row of 4.5The SNELM model is compared with seven state-of-the-art COVID-19 recognition models over two datasets. The comparison models consist of FSV , 3SBBO , CNNSP , GCMSVM Error bar (EB) can assist in observing the differences in the model’s performances. 5This study proposes an innovative SNELM model for COVID-19 detection. The MDA is used to increase the size of the training set. The SN with complex bypass is employed to generate SN features. ELM is used as the classifier. This proposed SNELM model can produce higher results than seven state-of-the-art models.There are three deficiencies of the proposed SNELM model: (i) Strict clinical validation is not tested. (ii) The SNELM model is a black box. (iii) Other chest-related infectious diseases are not considered.In our future studies, our team first shall distribute the proposed SNELM model to the online cloud computing environment (such as Microsoft Azure or Amazon Web Services). Second, we intend to incorporate Gram-CAM into this model to make it explainable. Third, chest-related infectious diseases, such as tuberculosis or pneumonia, will be added to our task.


Background: Non-alcoholic fatty liver disease (NAFLD) is linked with metabolic syndrome. Previous studies showed that obesity may disrupt adrenal function and adversely affect its counter-regulations against shock. This study hence evaluated adrenal function abnormalities in NAFLD with shock. Methods: Sprague-Dawley rats were fed with regular chow-diet (control) or high fat diet . Blood tests were performed at the end of the 4th, 6th and 8th week, respectively. Experiments were performed at the end of the 8th week. Results: HFD rats developed NAFLD. HFD rats had 27% and 51% increase in plasma corticosterone at the 6th and 8th week in usual status. However, HFD rats had 5 times more reduction of mean arterial pressure in response to lipopolysaccharide-induced sepsis as compared to control rats. The corticosterone increment ratio was also lower in HFD rats, even after ACTH administration. 11β-HSD system tended to generate more corticosterone in HFD rats under hemodynamic stable status without shock and the trend was lost in HFD rats with septic shock. Conclusion: Rats with NAFLD had profound septic shock due to inadequate corticosterone response. This is, at least partly, due to 11β-HSDs dysregulation in sepsis. NAFLD  and NASH  are major global public health problems linked with diabetes, obesity, insulin resistance and metabolic syndrome. It has been reported that over 90% of obese patients develop NAFLD . NAFLD iIt is noteworthy that cortisol-adrenocorticotropic hormone (ACTH) feedback may be defective in obesity and male obesity patients suffer from stress-stimulated hypothalamus-pituitary-adrenal (HPA) axis dysfunction . FurtherThe body usually combats critical illness via HPA axis activation, which is highlighted by increased serum corticotropin and cortisol levels . This isIsozymes of 11β-hydroxysteroid dehydrogenase  play pivotal roles in adrenal function by interconverting cortisol and cortisone , in which cortisol and corticosterone are active corticosteroids. 11β-HSDs modulate the amount of active corticosteroids in a tissue-specific manner. 11β-HSD1 catalyzes both dehydrogenation  and reverse oxo-reduction . Reductase activity can be maintained or induced via NADPH catalyzed by glucose-6-phosphate dehydrogenase (G6PD) . On the Comparing to clinical studies, animal experiments are feasible ways to survey the mechanism. This study, therefore, aimed to use a high fat diet (HFD)-fed rat model of NAFLD with or without lipopolysaccharide (LPS)-induced septic shock to address the aforementioned aspects.Six-week-old male Sprague-Dawley rats without significant difference in body weight (BW) were fed with either regular chow-diet  or high fat with calories adjusted diet  ad libitum. The body weight was recorded weekly.The animal works were performed at Taipei Veterans General Hospital and had been approved by Taipei Veterans General Hospital Animal Committee . All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health .Rats were fed with regular chow-diet as control group or HFD. Body weight was recorded weekly and blood samplings were performed at the end of the 4th, 6th and 8th week, respectively. At the end of the 8th week, rats were subjected to the following experiments: (A) body weight (BW), systemic and portal hemodynamic parameters measurement; (B) Plasma glucose, triglyceride, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), insulin, corticotropin-releasing hormone (CRH), ACTH, aldosterone and corticosterone concentrations measurement; (C) Determination of protein expressions of G6PD, 11β-HSD1 and 11β-HSD2 in visceral adipose tissue and adrenal gland.At the end of the 8th week, after BW, systemic and portal hemodynamic parameters measurements and blood withdrawn for corticosterone levels determination. Control and HFD group were subdivided to receive isotonic saline (vehicle) or LPS , respectively. The hemodynamic parameters were monitored continuously for 120 min. After that, visceral adipose tissue and adrenal gland were dissected for protein expression analyses.One hundred and twenty minutes after LPS injection, ACTH  was administrated. Blood before and 30 min after ACTH stimulation was withdrawn for calculation of corticosterone increment ratio. The hemodynamic parameters were recorded during the 30 min. Corticosterone response shown as corticosterone increment ratio was defined as the difference of corticosterone concentrations before and after ACTH stimulation, divided by the corticosterone level before ACTH stimulation .The rats were anesthetized with ketamine hydrochloride . The right femoral artery was cannulated with a PE-50 catheter connecting to a Model P23XL transducer . Continuous recordings of mean arterial pressure (MAP) and heart rate (HR) were performed on a MP45 two channel data acquisition system . The data were recorded and analyzed by BIOPAC BSL 4 (BIOPAC). The abdomen was then opened with a mid-line incision and the superior mesenteric vein was cannulated with the soft needle of 20G intravenous indwelling cannula connected to a Model P23XL transducer. Continuous recording of portal pressure (PP) was performed on a MP45 two-channel data acquisition system. The data was recorded and analyzed by BIOPAC BSL 4.The superior mesenteric artery (SMA) was identified at its aortic origin and a 5-mm segment was gently dissected free from surrounding tissue. An ultrasonic flow transducer was placed around the SMA and the flow was detected through a small animal blood flow meter .Cardiac output (CO) was measured by thermodilution, as previously described . Briefly® 3 Cell, Bio-Rad Laboratories, Hercules, CA, USA). Prestained proteins markers  were used for molecular weight determinations. Proteins were then transferred to a polyvinylidenedifluoride membrane  by a semi-dry electroblotting system  for 1.5 h at 4 °C. To block non-specific binding, membranes were blocked for 30 min with 5% non-fat dry milk in TBS-T, pH 7.4 (25 mmol/L Tris base-137 mmol/L NaCl-2.7 mmol/L KCL-1% Tween 20). Blots were incubated with the primary antibody , diluted with 5% non-fat dry milk in TBS-T then washed. After that, the blots were incubated with the secondary antibody diluted with 5% non-fat dry milk in TBS-T  and washed. Subsequent detection of the target proteins  were performed by enhanced chemi-luminescence (Millipore). With a computer-assisted video densitometer and digitalized software , the blots were scanned, photographed then the signal intensity  of the appropriate band was analyzed.Liver, visceral adipose tissue, kidney and adrenal gland were immediately frozen in liquid nitrogen and stored at −80 °C until required. The protein extracts were made by pulverization in grinder with liquid nitrogen, mixed with a ratio of 1 mL of lysis buffer ) and 0.05% protease inhibitor cocktail solution  for each 100 mg powdered tissue sample. Protein concentration was determined for each sample by the Bradford method. An aliquot of 20–40 µg protein from each sample that dissolved in sample buffer  was separated on denaturing SDS-10% polyacrylamide gels by electrophoresis  were stained with Hematoxylin and Eosin for the evaluation of tissue architecture and damage. Oil red O staining was applied to survey the extent of liver steatosis.All results are expressed as mean ± S.D. Statistical analyses were performed using an unpaired Student’s t-test or ANOVA with the Least Significant Difference test as appropriate. Results were considered statistically significant at a two-tailed P-value of less than 0.05. The SPSS 21 statistical package for Windows  was used.p < 0.001, n = 8, 8). Liver histology showed ballooning change of hepatocytes and increased extent of oil red O staining, compatible with liver steatosis : 33.6 ± 21.4 v.s. 62.4 ± 23.8, p = 0.028; HOMA-IR (insulin × glucose): 9.1 ± 8.3 v.s. 21.7 ± 11.7, p = 0.030). The cholesterol and triglyceride levels also significantly increased in HFD group (cholesterol (mg/dL): 63.8 ± 6.0 v.s. 96.8 ± 11.3, p < 0.001; triglyceride (mg/dL): 66.6 ± 11.4 v.s. 88.4 ± 12.2, p = 0.002). ALT and AST levels were not significantly different (AST (U/L): 103 ± 17 v.s. 111 ± 14, p = 0.355; ALT (U/L): 57 ± 7 v.s. 53 ± 24, p = 0.730). The results supported that rats fed with HFD for 8 weeks developed histology and biochemistry features of NAFLD.At the 8th week, BW increased significantly in HFD-fed rats : 26.1 ± 3.3 v.s. 24.1 ± 4.3, p = 0.359; SVR (mmHg/mL/min/100 g): 23.9 ± 4.0 v.s. 25.9 ± 5.9, p = 0.470; SMA flow (mL/min/100 g): 3.0 ± 0.4 v.s. 2.7 ± 0.4, p = 0.104; SMA resistance (mmHg/mL/min/100 g): 34.7 ± 6.2 v.s. 41.2 ± 6.3, p = 0.075) except that PP increased significantly in HFD group (PP (mmHg): 7.0 ± 1.30 v.s. 10.1 ± 1.03, p < 0.001, n = 7, 7).p = 0.204, n = 7, 7). On the other hand, the plasma ACTH and corticosterone levels increased in HFD group : 2.3 ± 0.5 v.s. 3.6 ± 0.4, p < 0.001; corticosterone (ng/mL): 150 ± 27 v.s. 226 ± 42, p = 0.002).The plasma CRH, ACTH and corticosterone levels were determined on the 4th, 6th and 8th week, respectively . CRH levp = 0.002, n = 8, 9). At the same point, PP was elevated in control group but reduced in HFD group ((mmHg): 0.55 ± 0.55 v.s. −0.22 ± 0.44, p = 0.006). HR was not significantly different. The SVR, SMA resistance and CI were also measured before and at 120 min after LPS injection. SVR was not significantly different between the two groups before LPS injection. At 120 min, SVR was maintained in control group but decreased significantly in HFD group ((mmHg/mL/min/100 g): 5.8 ± 1.0 v.s. 4.6 ± 0.8, p = 0.020). The SMA resistance increased significantly in control group but not in HFD group : 55 ± 15 v.s. 85 ± 16, p = 0.002). The CI was unaffected before and after LPS injection in both groups.LPS was injected to simulate bacterial infection-related stress. MAP, PP and HR were monitored for 120 min after LPS administration . MAP drop = 0.021). The results suggest a poorer corticosterone response to septic shock in the HFD group.The corticosterone increment ratio after LPS injection was significantly lower in HFD group . The ΔPP showed consistent results. There was no significant difference in HR throughout the whole experimental period.ACTH or vehicle  was injected, 120 min after LPS injection, to investigate the cardiovascular and corticosterone responses of control and HFD rats. The MAP, PP and HR were monitored for another 30 min after ACTH injection. p = 0.025). This finding reinforced the impaired corticosterone response to septic shock and ACTH stimulation in NAFLD rats.p = 0.008; 11β-HSD2: 1.00 ± 0.20 v.s. 0.70 ± 0.13, p = 0.007; G6PD: 1.00 ± 0.27 v.s. 3.56 ± 0.96, p < 0.001; adrenal gland, 11β-HSD1: 1.00 ± 0.11 v.s. 1.33 ± 0.15, p = 0.001; 11β-HSD2: 1.00 ± 0.20 v.s. 0.68 ± 0.14, p = 0.005; G6PD: 1.00 ± 0.07 v.s. 1.43 ± 0.18, p < 0.001). These results suggested that the up-regulations of 11β-HSD1, 11β-HSD2 and G6PD in NAFLD enhanced corticosterone production when hemodynamic condition was relatively stable. The balance between corticosterone (active form in rats) and its inactive form is regulated by 11β-HSD isoenzymes. While G6PD help 11β-HSD1 facilitate corticosterone production, 11β-HSD2 exerts the opposite function. p = 0.001; aorta iNOS: 1.00 ± 0.13 v.s. 1.16 ± 0.17, p = 0.078, n = 7, 8).COX1, COX2, iNOS, eNOS and phospho-eNOS protein expressions of aorta and SMA are presented in Animal models including genetic deficiencies, food, toxin or surgery have been developed to induce NAFLD or NASH. Genetic deficiency models are mostly applied to mice, such as ob/ob mice, db/db mice, SREBP-1c mice and so on. Regarding diet-modification methods, MCD diet (methionine and choline-deficiency), CD diet (choline-deficiency), intragastric overfeeding, high sucrose diet and high fat diet have been used. Each method has different limitations: MCD diet-fed rats do not develop obesity (they even get weight loss) and insulin resistance; CD diet induces liver steatosis and fibrosis without significant weight change; intragastric overfeeding needs specific training and equipment; high sucrose diet may induce mild liver inflammation without significant steatohepatitis and fibrosis; high fat diet may require longer feeding period to induce fatty liver diseases. Among them, high fat diet may be the most feasible one to simulate the lifestyle and intake pattern of NAFLD or NASH patients. A previous research from Japan revealed that 35% high fat diet (about 60% energy from fat) induced hepatic steatosis in four weeks .The current study demonstrates that the weight of HFD rats was significantly higher than the control rats since the third week of feeding. In addition, HFD rats had higher blood glucose and total cholesterol levels at the 4th, 6th and 8th week and higher plasma insulin concentration and HOMA-IR level, the index of insulin resistance at the 8th week, supporting that HFD successfully induced features of metabolic syndrome. There were no significant differences of plasma aldosterone levels at the 4th, 6th and 8th week and also in MAP, HR and CO. The findings suggest that HFD feeding for 8 weeks does not significantly influence systemic hemodynamics.The HFD rats had higher PP. Similarly, a previous study with rats fed with a cafeteria diet  for 1 month developed features of metabolic syndrome including overweight, arterial hypertension, hypertriglyceridemia, hyperglycemia, insulin resistance and liver steatosis without inflammation or fibrosis. Furthermore, liver perfusion demonstrated that CafD rats had an increased portal perfusion pressure and decreased intrahepatic vasodilation . It is wThe effects of NAFLD on baseline HPA axis activity is controversial . In a syThere were higher protein expressions of 11β-HSD1 and G6PD and a lower protein expression of 11β-HSD2 in visceral adipose tissue and adrenal gland in HFD rats were noted. The major enzyme activity of 11β-HSD1 is exerted by oxo-reductase, which transfers 11-DHC to corticosterone by NADPH generated by G6PD or H6PDH ,24. Our It has been reported that the incidence of sepsis is significantly higher in severely burned pediatric patients that has fatty infiltration of the liver . HepaticThe limitations of this study are as the followings: The 11β-HSD1 and G6PD protein expressions were upregulated in the adipose tissue and adrenal gland while 11β-HSD2 expression was down-regulated in the HFD group. However, the specific cell type involved in the changes are not clear. In addition, the underlying mechanism ascribed for septic shock-induced 11β-HSD system dysregulation deserves further clarification. Taken together, more investigations are required.In conclusion, the current study reveals that HFD rats has a higher HPA axis activity, which is, at least partly, resulted from the up-regulation of 11β-HSD1 and G6PD and down-regulation of 11β-HSD2 in visceral adipose tissue and adrenal gland. HFD rats had profound shock in response to LPS challenge related to a poorer corticosterone reactivity and excess vascular iNOS production, which could be ascribed to the dysregulation of 11β-HSDs system in sepsis.


Naegleria fowleri. This amoebic species is able to live freely in warm aquatic habitats and to infect children and young adults when they perform risk activities in these water bodies such as swimming or splashing. Besides the need to increase awareness of PAM which will allow an early diagnosis, the development of fully effective therapeutic agents is needed. Current treatment options are amphotericin B and miltefosine which are not fully effective and also present toxicity issues. In this study, the in vitro activity of various sesquiterpenes isolated from the red alga Laurencia johnstonii were tested against the trophozoite stage of a strain of Naegleria fowleri. Moreover, the induced effects (apoptotic cell death) of the most active compound, laurinterol (1), was evaluated by measuring DNA condensation, damages at the mitochondrial level, cell membrane disruption and production of reactive oxygen species (ROS). The obtained results demonstrated that laurinterol was able to eliminate the amoebae at concentrations of 13.42 ± 2.57 µM and also to induced programmed cell death (PCD) in the treated amoebae. Moreover, since ATP levels were highly affected and laurinterol has been previously reported as an inhibitor of the Na+/K+-ATPase sodium–potassium ion pump, comparison with known inhibitors of ATPases were carried out. Our results points out that laurinterol was able to inhibit ENA ATPase pump at concentrations 100 times lower than furosemide.Primary amoebic encephalitis (PAM) is a lethal disease caused by the opportunistic pathogen,  Naegleria fowleri species, also known as brain eating amoebae is an opportunistic pathogenic amoebae which is able to colonize the central nervous system (CNS) causing an infection known as primary amoebic meningoencephalitis (PAM)3. The disease affects mainly healthy children and young adults who have reported previous nasal exposure to amoebae-contaminated water during the practice of risk activities such as diving, splashing, among others, in warm water bodies. Moreover, the incidence of PAM since the year 2000 has seen an increase worldwide as it has been recently reported4.5. Since then, PAM cases have been recorded worldwide and it is usually described as a rare disease with only 431 cases available in the literature4.Primary amoebic encephalitis was first reported in 1965 in AustraliaNaegleria fowleri infects humans by passing the nerves and entering the cribiform plate. After that, the amoebae have free entrance to the brain causing inflammation and necrosis8. Hence, the fulminant nature of this infection which normaly manifests in the patient with symptons such as intense headache, temperature, seizures and stiff neck. In the final stage of the disease, patients have been reported to suffer from hallucinations and paralysis previous reaching coma and death8. Furthermore, the average time of symptoms appearance after infection starts 1–9 days after exposure to contaminated water sources whereas patient death average is 1–18 after symptoms begin9.post-mortem because the clinical symptons are not specific. Therefore, late diagnostics correlates to delayed treatment which commonly ends with the lost of the patients11. Current therapeutic options involves a combination of drugs which includes amphotericin B, azithromycin, rifampin, azoles and lately miltefosine with or without the combination of hypothermia13. Even though, recent cases of PAM treatment have been reported to be successful16, there is still an urgent need to find novel anti-amoebic agents which are able to eliminate the pathogen also causing low toxicity.Regarding diagnosis of PAM, it is often undertaken 19. Moreover, and specially in the case of antiamoebic compounds, algae have recently revealed their potential as a source of compounds with therapeutic potential21.Natural products have been used for the treatment of different parasitic diseases, among them artemisinin, quinine and ivermectin are examples of important antiparasitic compounds from this originLaurencia is one of the richest sources of active compounds among red algae which have also previously revealed metabolites presenting antiprotozoal and antiparasitic properties24. However, no activity of these metabolites has been previously reported against Naegleria fowleri.The genus Laurencia johnstonii species in our group were re-isolated and evaluated against the trophozoite stage of a type strain of Naegleria fowleri. Furthermore, the most active molecule was further investigated in order to establish the mechanisms of programmed cell death induced in the treated amoebae.In the present study, previously identified sesquiterpenes from 1–3)  was the most active molecule with an IC50 of 13.42 ± 2.57 µM. Therefore this compound was selected for further studies focused on the study of the induced mechanisms of programmed cell death in the treated amoebae.A series of natural phenolic sesquiterpenes –3 Fig. –3 .Moreover, amoebic forms were observed to present morphological changes such as loss of complexity in the cytoplasm and lack of cellular structure as well as cell volume Fig. , at 1 h 90. Treated amoebae showed the typical bright-blue stained nuclei at 24 h post incubation with the molecule as shown in Figs. Laurinterol induced chromatic condensation of treated amoebae at a concentration of IC90 of laurinterol experienced plasmatic membrane damage after 24 h of incubation.As shown in Figs. Naegleria fowleri treated trophozoites since the JC-1 dye remained in the cytoplasm in its monomeric form, shown as green fluorescence  than its counterparts in the Pacific coast25.The marine environment is an interesting source of bioactive compounds which could be potential novel therapeutic agents against cancer and parasites, among others, as it has been previously describedNaegleria fowleri species, a Free-Living Amoebae which is able to act as an opportunistic pathogen, is a fulminant CNS disease which needs novel, more effective and faster therapeutic options. Furthermore, the current options are not fully effective and lack of availability worldwide creates the need to find alternative options and sources13.Primary amoebic encephalitis (PAM) caused by L. johnstonii against Naegleria fowleri in vitro. Regarding the obtained activities against the trophozoite stage, it is important to highlight that the most active tested compound, laurinterol was active at low concentrations although not as active as the commonly used therapeutic agent against Naegleria which is amphotericin B (IC50 = 0.12 ± 0.03 and CC50 ≥ 200 μM)9. Nevertheless, laurinterol is a small molecule which could be even synthetized in the laboratory as well as its derivatives26. Hence, revisiting the potential use of known small molecules such as this one, at least as anti-amoebic agents, seems to be a good option in this field for drug discovery purposes.In this study, we aimed to investigate the potential of some natural sesquiterpenes isolated from 27. The phenomenom of PCD involves morphological and cellular events being the most commonly described ones: chromatin condensation, nuclear DNA fragmentation, cell shrinkage, blebbing, loss of mitochondrial membrane potential, the formation of apoptotic bodies, and the exposure of phosphatidylserine among others29.Programmed Cell Death (PCD) and apoptosis-like processes have been previously described in protozoa and in multicellular organismsNaegleria genus, PCD events in treated cells with amphotericin B were described by Cardenas-Zuñiga and colleagues for the first time30. In this study, the effect of a non-lytic dose of amphotericin B (10 µg/ml) was evaluated in cultures of N. fowleri and N. gruberi. Among the observed events indicative of PCD, authors reported the presence of blebs, DNA condensation, ROS presence and electron-dense granules30.In Naegleria through the intrinsic pathway since the mitochondrial potential was collapsed even at 24 h, as well as ATP level . Hence, the observed PCD inductions effects are exclusively induced by this compound in N. fowleri.For the development of novel therapeutic agents, our laboratory has focused on the evaluation of compounds of diverse origin but which are also inducers of PCD/apoptosis-like events, avoiding those molecules that induce necrosis and, hence, inflammation which is a non desirable side effect in the search of antiamoebic agents. In the present study, laurinterol was shown to induce chromatin condensation, plasma membrane damage, mitochondrial disfunction as well as increased levels of ROS. Since these effects were observed even after 24 h time of incubation with laurinterol, we postulate that this molecule could induce apoptosis in el Figs. , 8. PCD Naegleria fowleri still needs further studies to be fully elucidated. Nevertheless, laurinterol has been reported as an inhibitor of Acetylcholinesterases (AChE)31. These enzymes play a key role in the human body since they catalyze the breakdown of acetylcholine and of some other choline esters that function as neurotransmitters32. In protists, AchE have been postulated to act as biomediators and their action is used by protozoa in processes such as cell to cell communications, reproduction, adhesion or motility. The recently reported genome project data for Naegleria fowleri and Naegleria gruberi species34 have made available sequences in the databases (i.e. https://www.uniprot.org/uniprot/D2V1M1) which are compatible to AchE-like enzymes. Therefore, the inactivation of these enzymes could trigger a cascade of events which could be the key to understand the observed morphological changes and PCD related events.In addition, the exact mechanism of action of laurinterol in 2) showed inhibitory activity of ouabanin-sensitive dog kidney P Type Na+,K+-ATPase at IC50 of 40 and 400 µM36. In addition, laurinterol exhibited induction of apoptosis by DNA fragmentation and activation of several caspases in melanoma cells. It was also demonstrated the transcriptional activation of the tumor suppressor gene p53 and the activation of p21 promoter. These studies indicate that laurinterol can induce apoptosis in melanoma cells through a p53-dependent pathway37.Previous reports indicate that phenolic sequiterpenes laurinterol and debromolaurinterol  , whereas cellular evaluations showed reasonable cytotoxicity against lung cancer A549 and breast cancer MDA-MA-468 cell lines. The compound also showed potent inhibition of helicase activity consistent with its ATPase inhibitory activity with complete inhibition at 100 µM. Thus, this data proved that allolaurienterol is an ATP-competitive molecule most likely binding to the ATP-binding pocket at the interface between the N-terminal and C-terminal domains39.On the other hand, allolaurinterol (4) Fig. , another+,K+-ATPase and H+-ATPase, two P-type ATPases, are essential to control homeostatic processes. A third P-type ATPase, the K + or Na + efflux ATPase (ENA ATPase), was discovered and, for many years, it was considered to be exclusively a fungal enzyme. ENA ATPase is now known to be also present in bryophytes and protozoa43. In protozoa, the presence of a ouabain-insensitive Na+-ATPase activity was found in Trypanosoma cruzi epimastigotes, stimulated by Na+, and inhibited by furosemide41. The fact that the ENA ATPase is found in almost all fungi, as well as in bryophytes and protozoa has been suggested to be an adaptive mechanism required in these organisms to prevail in certain life conditions. ENA genes found in the genome of the amoeba-flagellate Naegleria gruberi also point out the presence of ouabain-insensitive Na+-ATPase in this species. The structural characteristics of the ENA ATPase revealed that it maintains highly conserved sequences that are characteristic of P-type ATPases, such as the catalytic site, sequences involved in the nucleotide and Mg2+ binding, and others that are specific to ENA ATPases. N. gruberi shows the presence of the P type ATPase ouabain-insensitive and sensitive to furosemide identifying an ENA ATPase in the control of Na+43. The obtained results revealed that the Na+-ATPase ion pump of N. fowleri was ouabain-insensitive but sensitive to furosemide at concentrations of 0.71 ± 0.04 mM as mentioned in the “50 of 13,42 ± 2.57 µM) was able to inhibit this pump at concentrations 100 times lower than furosemide.In cells, NaN. fowleri  , possibly due to the increasement in volume of the molecule, which may limit the capacity to interact with the ATPases nucleotide binding pocket43. Nevertheless, further studies to evaluate this drug target as well as SARs approaches should be developed in the near future.Thus, an inhibition of sodium efflux ATPase would cause an increase in intracellular sodium concentration, which will trigger ROS, DNA damage and, hence, apoptosis(5) Fig.  does notNaegleria fowleri trophozoites. Moreover, laurinterol-treated amoebae showed signs of PCD as shown by DNA condensation, damages in cell membrane and mitochondria and ROS generation triggered by a possible inhibition of ENA ATPase ion pump. Therefore, laurinterol is presented as a novel, highly active, PCD inducer and low toxic anti-Naegleria compound which should be exploited for the development of novel therapeutic agents against PAM.In conclusion, laurinterol showed a high activity against Laurencia johnstonii was collected by hand off the coast of Baja California Sur, Mexico (24° 21′ 10.8″ north (N), 110° 16′ 58.8″ west (W)). A voucher specimen (code 13-003) was deposited at the Herbarium of the Laboratory of Marine Algae of the Interdisciplinary Center of Marine Science (CICIMAR) and it was identified by Dr. R. Riosmena Rodríguez from the Autonomous University of Baja California Sur (UABCS).L. johnstonii were crushed and extracted with EtOH for three days at 25 °C. Dissolvent was replaced three times, the combined extracts were filtered through a Whatman no. 4 filter paper and concentrated under vacuum. The resulting extract was chromatographed in Sephadex LH-20  to obtain five fractions. The active fraction SF3 was separated in Flash silica gel (130 × 70 mm) using a stepwise gradient from n-hexane to ethyl acetate to obtain seven fractions. The active fraction SF3.2 (95% n-hexane) was chromatographed on a normal phase open column (300 × 50 mm) using a stepwise gradient from n-hexane to ethyl acetate to yield pure compounds 1, 2 and 3.Washed and dried specimens of 25D + 17 ; HRESIMS m/z 293.0531 [M−H]− , 295.0518 [M−H]−  1H NMR  δ 0.55 , 0.58 , 1.15 , 1.28 , 1.32 , 1.41 , 1.66 , 1.95 , 2.09 , 2.29 , 5.26 , 6,61 , 7.61 ; 13C NMR  δ 16.2 (C-12), 18.6 (C-13), 22.2 (C-14), 23.5 (C-15), 24.4 (C-3), 25.3 (C-4), 29.6 (C-2), 35.9 (C-5), 114.9 (C-10), 118.8 (C-8), 132.3 (C-11), 134.0 (C-6), 135.9 (C-9), 153.3 (C-7).White crystal; [α]25D -6.35 ; HRESIMS m/z 215.1432 [M−H]−  1H NMR  δ 0.52 , 0.59 , 0.91 , 1.13 , 1.33 , 1.43 , 1.66 , 1.96 , 2.13 , 2.28 , 5.17 , 6.56 , 6.70 , 7.39 ; 13C NMR  δ 16.3 (C-12), 18.8 (C-13), 20.6 (C-14), 23.6 (C-15), 24.3 (C-3), 25.3 (C-4), 27.7 (C-2), 36.0 (C-5), 48.0 (C-1), 117.3 (C-10), 120.8 (C-8), 128.8 (C-11), 131.4 (C-6), 136.7 (C-9), 154.0 (C-7).Colorless amorphous solid; [α]25D -46 ; HRESIMS m/z 293.0536 [M−H]− , 295.0528 [M—H]-  1H NMR  δ 1.21 , 1.42 , 1.46 , 1.59 , 2.05 , 2.20 , 2.31 , 2.85 , 4.94 , 5.11 , 5.56 , 6.73 , 7.45 ; 13C NMR  δ 21.2 (C-14), 22.2 (C-15), 27.8 (C-12), 31.2 (C-4), 37.6 (C-3), 39.1 (C-5), 49.8 (C-1), 106.9 (C-13), 115.5 (C-10), 120.4 (C-8), 131.2 (C-11), 132.7 (C-6), 137.2 (C-9), 153.0 (C-7), 165.4 (C-2).Colorless amorphous solid; [α]5) was obtained by oxidation of laurinterol according to Ichiba and Higa (1986) with some modifications. Briefly, 1 (50 mg) was dissolved in 80% acetic acid (1 mL) and 200 μL of 25% chromium trioxide solution in acetic acid was added. The reaction was left under magnetic stirring for 2 h at 4 °C and extracted with dichloromethane after addition of water. The reaction mixture was separated on a normal phase open column  eluted with n-Hex/EtOAc (99:1) to obtain 7 fractions. Fraction 2 was purified by HPLC on a normal phase column  eluted with n-Hex/EtOAc (99:1) to yield 5, (2 mg).Laurinterol dimer (5). White crystal; [α]25D—32.5 ; HRESIMS m/z 585.1000 [M−H]− , 587.0981 [M−H]− , 589.0951 [M−H]− ; 1H NMR  δ 0.54 , 0.57 , 1.11 , 1.26 , 1.33 , 1.38 , 1.63 , 1.92 , 2.02 , 2.18 , 4.77 , 7.83 ; 13C NMR  δ 16.3 (C-13), 18.9 (C-12), 19.9 (C-15), 22.5 (C-14), 24.3 (C-3), 25.4 (C-4), 29.7 (C-2), 35.5 (C-5), 48.7 (C-1), 115.9 (C-10), 122.0 (C-8), 133.3 (C-11), 135.1 (C-9), 135.6 (C-6), 152.1 (C-7). NMR data are in accordance with those previously reported for the natural compound49. X-Ray crystallographic data of 5 is reported for the first time  and furosemide (CAS no. 54-31-9) were acquired from Sigma-Aldrich.Naegleria fowleri with reference number ATCC 30808 from the American Type Culture Collection  was used in this study. The strain was axenically cultured at 37 ºC in 2% (w/v) Bactocasitone medium  supplemented with 10% (v/v) foetal bovine serum (FBS), containing 0,5 mg/ml of Streptomycin sulfate  and 0.3 µg/ml of Penicillin G Sodium Salt . The strains were cultured in a biological security facility of level 3 at our institution following Spanish biosafety guidelines for this pathogen.A type strain of Naegleria fowleri trophozoites were determined using a colorimetric assay as previously described9. Briefly, Naegleria trophozites were seeded in duplicate on a 96-well microtiter plate with 50 μL from a stock solution of 2 × 105 cells/ml. After that, a serial dilution of the evaluated compounds (in the same culture medium as the amoeba) was added to the plate. A negative control in the plates, consisted in amoebae in medium alone. Finally, the AlamarBlue reagent  was placed in each well (10% of medium volume) and plates were incubated with slight agitation for 48 h at 37 °C. After that, plates were analysed with an EnSpire Multimode Plate Reader  using a wavelength of excitation of 570 nm and a wavelength of emission of 585 nm. To calculate the percentages of growth inhibition and the 50% and 90% inhibitory concentrations (IC50 and IC90) a no linear regression analysis was performed with a 95% confidence limit using the SigmaPlot 12.0 software . Experiments were performed in triplicated and the mean values were also calculated. A paired two-tailed t-test was used for the analysis of the data and the values of P < 0.05 were considered statistically significant. In all assays, 1% DMSO was used to dissolve the highest dose of the compounds. When 1% DMSO was added to medium alone (without any investigated compound) no effect was observed on the amoebae.The activities of the tested compounds against 2 humidified incubator as previously described51.Cytotoxicity of laurinterol (the most active tested compound) was evaluated after 24 h incubation of a murine macrophage J774.A1 cell line (ATCC # TIB-67) with different concentration of the tested compound at 37 ˚C in a 5% CO5 cells/ml well were incubated in a 96-well plate for 24 h with the previously calculated IC90. As it has been previously reported52, the double-staining pattern allows the identification of three groups in a cellular population: live cells will show only a low level of fluorescence, cells undergoing PCD will show a higher level of blue fluorescence (as chromatin condenses), and dead cells will show low-blue and high-red fluorescence (as the Propidium Iodide stain enters the nucleus).A double-stain apoptosis detection kit (Hoechst 33342/PI)  and an EVOS FL Cell Imaging System AMF4300,  were used in this assay. The experiment was carried out by following the manufacturer’s recommendations, and 5 × 10Naegleria fowleri trophozoites were treated with the IC90 of laurinterol for 24 h and exposed to CellROX Deep Red  at 37 °C in the dark. Cells were observed in an EVOS FL Cell Imaging System AMF4300 .The generation of intracellular ROS was evaluated by using the CellROX Deep Red fluorescent probe . 90 of laurinterol for 24 h and the experiment was carried out by following the manufacturer’s recommendations. Images were captured using an EVOS FL Cell Imaging System AMF4300, Life Technologies, Madrid, Spain. The obtained staining pattern allowed the identification of two groups in a cellular population: live cells will show only red fluorescence; cells with low mitochondrial potential, (undergoing PCD) will show a higher level of green and red fluorescence.The collapse of an electrochemical gradient across the mitochondrial membrane during PCD induction in treated amoebae was detected with the JC-1 mitochondrial membrane potential detection kit . Amoebae were incubated with the IC5 cells/ml with the previously calculated IC90 of laurinterol.ATP levels were measured using a CellTiter-Glo Luminescent Cell Viability Assay . The effect of the drug on the ATP production was evaluated by incubating 5 × 105 trophozoites were incubated with the previously calculated IC90 of laurinterol. After 24 h of incubation, the SYTOX Green was added at a final concentration of 1 μM (Molecular Probes). Cells were observed after 15 min in an EVOS FL Cell Imaging System AMF4300 .The SYTOX Green assay was performed to detect alterations of the membrane permeability in treated cells. Briefly, 5 × 10Supplementary file 1


The inter- and intra-day precision were 89.3%–109.2%, RSD was less than 15%, and the repeatability was good. The recoveries of both metabolites and IS were over 89%, and matrix effect was within 15%. The validated analytical method was further applied to study the pharmacokinetic profiles of GG-1 and GG-2 in vivo. The pharmacokinetic parameters suggested that Tmax of GG-1 was equivalent to that of GG-2, and MRT0-t, t1/2 of GG-2 were a little higher than those of GG-1. Importantly, AUC0-t and Cmax of GG-2 were almost twice as those of GG-1. In short, the validated UFLCMS/MS analytical method was feasible to simultaneously determine two galangin metabolites GG-1 and GG-2 in rat plasma and further analyze in vivo metabolism of galangin.Galangin has multiple pharmacological efficacies, such as anti-cancer, anti-inflammation and anti-oxidation. Galangin can be rapidly converted into glucuronidated metabolites in vivo. This study aimed to establish an UFLC-MS/MS analytical method to simultaneously determine the concentrations of two glucuronidated metabolites of galangin, galangin-3-O-β-D-glucuronic acid (GG-1) and galangin-7-O-β-D-glucuronic acid (GG-2) in rat plasma. After oral administration of galangal extract (0.3 g/kg), blood samples were collected from the orbital sinus, then treated by methanol precipitation and further gradient-eluted with Phenomenex Kinetex 2.6 µm XB-C18 column. The mass spectrometer was manipulated in the negative electrospray ionization (ESI) and selected multiple reaction monitoring (MRM) mode for the analytes. The precursor-to-product ion pairs applied for GG-1, GG-2 and chrysin  were m/z 445.2→269.0, 445.2→268.9 and 253.0→142.9, respectively. The results showed that the linear ranges for both GG-1 and GG-2 were 2.0–2000.0 ng/mL ( Alpinia officinarum Hance, known as galangal, is a traditional Chinese medicine, the rhizome part of which is often applied to treat epigastric cold and pain, stomach cold, vomiting and other diseases. Phytochemical studies have shown that galangal mainly contain flavonoids, diphenylheptane, phenylpropanol, volatile oil and other chemical components  was established to determine galangin concentration in rat plasma by adopting diazepam as IS , there aThis work firstly established and validated an UFLC-MS/MS analytical method to simultaneously determine two galangin metabolites GG-1 and GG-2 in rat plasma, which was further successfully applied in pharmacokinetic study for GG-1 and GG-2 after oral administration of galangal extract, and provided a feasible in vivo analytical method for galangin metabolites.A. oxyphylla fruits by our laboratory and confirmed through MS and NMR analysis. HPLC-grade formic acid and methanol were bought from Aladdin Industrial Inc. . Double distilled water was obtained by the LabTower EDI15 system . Anesthetics isoflurane was purchased from Gene&I Corporation . Galangal extract was prepared by our laboratory as the reported method (GG-1 and GG-2 standards (the purity of both standards was over 97%) were separated from rat urine after oral administration of galangin according to previous reports and quantified by HPLC–PDA–MS . As IS, d method . HPLC anSprague Dawley (SD) rats  were obtained from Changsha TianQing Biotechnology Co., Ltd. . The rats were fed with free access to fodder and water at room temperature. The rats were adapted to the environment for a short time and experienced an overnight fasting period before oral administration. The experiment was approved by the animal ethics committee of Hainan Medical University  and implemented according to the Guidance for Ethical Treatment of Laboratory Animals .Galangal extract was weighed precisely and suspended in water (with co-solvent Tween-80) to 30 mg/mL. Serial blood samples of approximately 0.2 mL were collected from orbital venous at 0, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h in five rats after a single oral administration of galangal extract (0.3 g/kg). The dose selection was referred to the previous study . After b2 via a TechneTM Sample Concentrator. Methanol (30 μL) was added to the concentrate, vortexed and centrifuged ditto, then 10 μL of the supernatant was used for LC-MS/MS analysis.IS-spiked methanol solution  was added to plasma samples (60 μL). The mixture was vortexed vigorously for 10 min and centrifuged at 13,000 for 10 min. The upper supernatant was enriched under NTM triple quadrupole mass spectrometer  equipped with an ESI source . Data collection was operated with AB-SCIEX Analyst software 3.0. Chromatographic separation was accomplished at 40 °C on a Phenomenex Kinetex 2.6 μm XB-C18 column  with a guard column . The mobile phase was composed of water -methanol , flow rate was 0.35 mL/min with a gradient elution: 0–0.01 min (1%B), 0.01–0.50 min (1%B), 0.51–4.0 min (40%B→50%B), 4.01–8.00 min (80%B→100%B), 8.01–10.00 min (1%B).LC-MS/MS system included a Shimadzu Prominence UFLC chromatographic system  and API 4000+Mass spectrometer was manipulated in the negative ion and selected multiple reaction monitoring (MRM) mode for the analytes. MS/MS operating conditions were optimized by infusion of the standard solution (1 μg/mL) of each analyte and IS into the ESI source via a syringe pump . The precursor-to-product ion pair and collision energy (CE) for quantification of the analytes as follows: m/z 445.2→269.0 , 445.2→268.9 , 253.0→142.9  for GG-1, GG-2 and IS, respectively.The method validation was operated referring to the Guidance for Industry Bio-analytical Method Validation . StandarThe specificity of the method was investigated through comparing the chromatograms of six different blank plasma samples, blank plasma spiked with the analytes and IS at lower limit of quantification (LLOQ) concentration and plasma samples collected after oral administration of galangal extract (0.3 g/kg) in rats. The selectivity was evaluated by the peak interference of the analytes to IS.Seven standard calibration concentration levels  of plasma samples were prepared in quadruplicate by adding 6 uL of 20, 50, 100, 200, 1,000, 10,000 and 20,000 ng/mL series working concentration of standard solution to 54 uL of blank plasma and oscillation blend. The calibration curves were established by plotting peak area ratios (y) of each analyte to IS versus nominal concentrations (x) of least square linear regression analysis with a weighting factor 1/x. LLOQ was ruled as the lowest concentration that can be quantified on the condition of precision not exceed 20% and accuracy within ±  20%.n = 5) within the same day. The inter-day precision and accuracy were assessed with three batches QC samples on three consecutive days. The accuracy and precision must meet the criterions: the intra- and inter-day precision (RSD) must not more than 15% and accuracy should be less than ±  15%.The intra-day precision and accuracy were analyzed with three levels of quality control (QC) samples  Three freeze–thaw cycles; (2) Short-term stability of plasma samples pre-extracted at room temperature; (3) After being prepared 8 h at auto-sampler; (4) 15 days at −20 °C. The stability (RSD) must be less than 15%.The quantitative upper limit sample  was injected for analysis immediately after the blank sample was analyzed, and the residue of the analytes and IS on the blank sample atlas was investigated. Six samples with the analyte concentration exceeding the quantitative upper limit were prepared with blank plasma and the dilution factor of 10 times was investigated.max and Tmax were observed values with no interpolation. The area under concentration-time curve up to the last measured time point (AUCt0→) was calculated by the trapezoidal rule method. All data were indicated as the mean ±  SD.Plasma samples were prepared from 5 male SD rats according to the methods as described in section “3. Drug Administration and Blood Collection”, and plasma concentrations of GG-1 and GG-2 were determined by the established method at each time point. To acquire pharmacokinetic parameters, the concentration-time curve was obtained by a non-compartmental method using DAS software package . The CThe molecular formula, molecular weight and MS/MS spectra of GG-1, GG-2 and IS (chrysin) were shown in As shown in The intra- and inter-day precision and accuracy for QC samples are shown in Matrix effect and extraction recovery data were shown in In this study, stability confirmation was carried out under various experimental conditions, i.e., short-term storage (4 h at room temperature), autosampler (8 h at room temperature), freeze-thaw (3 cycles) and 15-day stability. As shown in The residual peak area of the quantitative upper limit sample was less than 20% of the quantitative lower limit, and the residual peak area of IS was not more than 5% of IS peak area. The accuracy of diluted sample was within ± 15%, RSD <15%.max for GG-1 and GG-2 was 6069.6 ± 1140.6 and 10,596.0 ± 2395.7 ng/mL, respectively, and both achieved at 0.2 ± 0.1 h. The AUCt0−, MRTt0−, the elimination half-life (t1∕2) for GG-1 and GG-2 was found to be 2,390.9 ± 678.0 and 4,554.9 ± 884.9 h ⋅ ug/L, 1.4 ± 0.8 and 1.6 ± 0.7 h, 2.2 ± 0.7 and 3.3 ± 0.2 h, respectively. It should be pointed out that because the plasma concentrations of GG-1 and GG-2 at the blood collection points (12 h and 24 h) could not be detected, we only included the plasma concentrations from 0 h to 8 h after oral administration of galangal extract in the plasma concentration-time curves of GG-1 and GG-2.After validation, UFLC-MS/MS analytical method was successfully applied in pharmacokinetic study of two galangin metabolites GG-1 and GG-2 in SD rats. The plasma concentration-time curves of GG-1 and GG-2 in rats after oral administration of galangal extract (0.3 g/kg) have been shown in P450 in rats  were much higher than those of galangin-3-O- β-D-glucuronic acid (GG-1).To preliminary study the bioavailability of two galangin metabolites GG-1 and GG-2, this work firstly developed an UFLC-MS/MS analytical method to simultaneously determine the two galangin metabolites in rat plasma in which calibration curve, LLOQ, precision, accuracy, matrix effect, extraction recovery, stability, residual effect and dilution reliability were all up to the FDA standard. Furthermore, we applied the validated method to acquire pharmacokinetic information for two galangin metabolitesGG-1 and GG-2 after oral administration of galangal extract in rats.As a result, we found that Tposition . Additioposition . TherefoOn the other hand, in view that galangin possesses multiple biological activity, such as anti-cancer , anti-oxt0−, MRTt0−, t1∕2, Tmax, Cmax for GG-1 and GG-2after oral administration of galangal extract in rats. In short, this work provided a feasible analytical means and certain foundation for in vivo bioanalysis for galangin metabolites.This study developed an UFLC-MS/MS analytical method in order to simultaneously determine two galangin metabolitesGG-1 and GG-2 in rat plasma. Moreover, after validation we applied this analytical method to in vivo metabolism of galangin and acquired pharmacokinetic information including AUC10.7717/peerj.11041/supp-1Supplemental Information 1All measured values in method validation.Click here for additional data file.10.7717/peerj.11041/supp-2Supplemental Information 2The measured values of concentration-time and the calculated pharmacokinetic parameters of GG-1.Click here for additional data file.10.7717/peerj.11041/supp-3Supplemental Information 3The measured values of concentration-time and the calculated pharmacokinetic parameters of GG-2.Click here for additional data file.10.7717/peerj.11041/supp-4Supplemental Information 4Click here for additional data file.


Moringa oleifera Lam. (M. oleifera) is valuable plant distributed in many tropical and subtropical countries. It has a number of medicinal uses and is highly nutritious. M. oleifera has been shown to inhibit tumor cell growth, but this effect has not been demonstrated on prostate cancer cells. In this study, we evaluated the inhibitory effect of M. oleifera alkaloids (MOA) on proliferation and migration of PC3 human prostate cancer cells in vitro and in vivo. Furthermore, we elucidated the mechanism of these effects. The results showed that MOA inhibited proliferation of PC3 cells and induced apoptosis and cell cycle arrest. Furthermore, MOA suppressed PC3 cell migration and inhibited the expression of matrix metalloproteinases (MMP)-9. In addition, MOA significantly downregulated the expression of cyclooxygenase 2 (COX-2), β-catenin, phosphorylated glycogen synthase 3β, and vascular endothelial growth factor, and suppressed production of prostaglandin E2 (PGE2). Furthermore, FH535 (β-catenin inhibitor) and MOA reversed PGE2-induced PC3 cell proliferation and migration, and the effects of MOA and FH535 were not additive. In vivo experiments showed that MOA (150 mg/kg) significantly inhibited growth of xenograft tumors in mice, and significantly reduced the protein expression levels of COX-2 and β-catenin in tumor tissues. These results indicate that MOA inhibits the proliferation and migration, and induces apoptosis and cell cycle arrest of PC3 cells. Additionally, MOA inhibits the proliferation and migration of PC3 cells through suppression of the COX-2 mediated Wnt/β-catenin signaling pathway. Prostate cancer is the second most common malignant tumor in men worldwide, and the fifth leading cause of cancer-related death . In more2 synthesis (2 can activate the GSK3β/β-catenin pathway via G-protein coupled receptors (EP2 and EP4), resulting in transcription of oncogenes such as c-myc, cyclin D1, and vascular endothelial growth factor (VEGF), and growth and migration of tumor cells. In addition, a number of studies have reported that COX-2 was highly expressed in prostate cancer and stimulated prostate cancer cell proliferation (Cyclooxygenase (COX) is a key rate-limiting enzyme involved in conversion of arachidonic acid to prostaglandins (PG). There are three COX subtypes, including COX-1, COX-2, and COX-3. COX two plays an important role in tumor cell growth, invasion, and metastasis through regulation of PGEynthesis . Moreoveferation . TherefoMoringa oleifera Lam. (M. oleifera) is one of the Moringaceae, and it is a valuable plant distributed in many tropical and subtropical countries -induced inhibition of growth and migration of prostate cancer cells have not been characterized. The present study investigated the role of MOA in inhibition of growth and migration of PC3 prostate cancer, and explored the potential mechanisms underlying these effects.Development of novel anti-tumor drugs from natural sources has received increased interest in recent years. ountries . A largema cells , cervicama cells , human oma cells , breast ma cells . Alkaloi effects . HoweverM. oleifera was obtained from Yunnan Tianyou Technology Development Co., Ltd. in Dehong Prefecture, Yunnan Province, China (Batch No. 20190001S), and identified by Professor Jiang-miao Hu . A voucher specimen (No. YSTY-14) was deposited in the Engineering Research Center of development and utilization of Food and Drug Homologous Resources, Ministry of Education, Yunnan Agricultural University, Kunming, China. M. oleifera leaf powder (10 kg) was extracted three times with 50% ethanol for 24 h each time. The extracts were filtered, combined, concentrated, and the ethanol was evaporated. The aqueous solution obtained following concentration was adjusted to pH 2 with 10% HCl, then extracted three times with ethyl acetate. The acidified water solution was adjusted to pH 10 using a sodium hydroxide solution and extracted three times with chloroform. The chloroform extracts were combined, and the chloroform was evaporated to yield 30 g of alkaloids .The leaves of 2 incubator maintained at 37°C.Ten cancer cell lines  and human normal prostate epithelial RWPE-1 cells were purchased from the Chinese Academy of Science . The cells were cultured in DMEM High Glucose, 1:1 DMEM:F12 or RPMI 1640 medium  supplemented with 10% fetal bovine serum  and penicillin-streptomycin  in a 5% CO4 cells/well) were cultured in 96-well plates. When cells reached 90% confluence, they were treated with MOA  for 48 h, and untreated cells served as controls (0 μg/ml). Twenty microliters of MTT  solution was added to each well to a final concentration of 5 mg/ml, and the cells were incubated for 4 h. Following incubation, the media was discarded. Two hundred microliters of DMSO was added to each well, and the plates were shaken for 10 min. Absorbance was measured at 492 nm. The half maximal inhibitory concentration (IC50) values were calculated as the drug concentrations necessary to inhibit 50% proliferation as compared to untreated control cells.Ten cancer cell lines  were seeded in 96-well plates for 24 h and treated with different concentrations of MOA  for 24, 48, or 72 h. After 24, 48, and 72 h, the cell viability was evaluated by the MTT assay according to the manual.The PC3 cells and human normal prostate epithelial RWPE-1 cells (1 × 104 cells/well) were seeded in 96-well plates for 24 h. PC3 cells were pretreated with PGE2 (20 μM) for 1 h, then with β-catenin inhibitor FH535 (2.5 μM) for 1 h, and finally treated with MOA (40 μg/ml) for 48 h. After 48 h, the cell viability was evaluated by the MTT assay according to the manual.The PC3 cells  were cultured in 6-well plates , then treated with MOA  for 24, 48, or 72 h. After culturing for 15 days, the cells were fixed in methanol and stained with 0.1% crystal violet for 15–30 min. After washing multiple times, the plates were allowed to dry, the cells were photographed, and crystal  violet was dissolved using 10% glacial acetic acid . Absorbance was measured at 560 nm, and statistical analysis was performed.5 cells/well, then treated with MOA  for 48 h. The cells were harvested, washed twice with PBS, and centrifuged. Then, 100 μl of Annexin-V/FITC and PI dye  was added, and the cells were incubated for 20 min at room temperature in the dark. The cells were analyzed using flow cytometry . The percentage of apoptotic cells was determined using FlowJo software .The PC3 cells and human normal prostate epithelial RWPE-1 cells were seeded in 6-well plates at a density of 2 × 105 cells/well, then treated with MOA  for 48 h. The cells were fixed in 70% ethanol overnight at 4°C in a refrigerator, then incubated with RNase-containing PI dye  for 30 min at 37°C. Cell cycle was evaluated using flow cytometry . The percentage of cells in each cell cycle stage was determined using FlowJo software .The PC3 cells were seeded in 6-well plates at a density of 2 × 106 cells/plate. The cells were allowed to adhere, and a sterilized micro-pipette tip was used to scratch across the cell layer. The cells were then treated with MOA  for 48 h. The scratches were photographed using an inverted microscope at the beginning and end of the experiment, and the results were analyzed using Image-pro and GraphPad Prism 5. Each experiment was repeated independently in triplicate.PC3 cells were seeded in 60 mm plates at a density of 1 × 105 cells) containing different concentrations of MOA  were added to the upper chamber of a transwell apparatus. Eight hundred microliters media containing 10% FBS with different concentrations of MOA was added to the lower chambers. The cells were incubated for 48 h, and the cells were washed with PBS three times, fixed in methanol for 30 min, stained with 0.1% crystal violet  for 30 min, then visualized and photographed. The cells were then incubated in 33% glacial acetic acid for 10 min, and absorbance was measured at 570 nm.Two hundred microliters of PC3 cells suspensions (serum-free medium containing 3 × 105 cells) containing PGE2 (20 μM), β-catenin inhibitor FH535 (2.5 μM), or different concentrations of MOA  were added to the upper chamber of a transwell apparatus. Eight hundred microliters media containing 10% FBS with different concentrations of MOA was added to the lower chambers. The cells were incubated for 48 h, the cell migration rate was evaluated by the cell migration assay according to the manual.Two hundred microliters of PC3 cells suspensions , and total protein was extracted using RIPA buffer. Protein concentration was measured using a BCA protein assay kit . Proteins were separated on 10% SDS polyacrylamide gels and transferred to 0.45 μm polyvinylidene fluoride membranes . The membranes were blocked with 5% skim milk  for 60 min, then incubated with primary antibodies against Bax, Bcl-2, CyclinE, p21, MMP9 , COX-2, β-catenin , GSK-3β, phosphorylated glycogen synthase 3β (P-GSK-3β), VEGF , and β-actin  overnight at 4°C. The membranes were washed with PBST three times, then incubated for 1 h with HRP-conjugated secondary IgG antibody , and the signal was detected using ECL western blotting substrate .Tumor tissues were collected and total protein was extracted using RIPA buffer, the protein levels of COX-2 and β-catenin were determined by Western blotting.6) were seeded in confocal dishes , and incubated with MOA  for 48 h. The cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed three times with PBS, and permeabilized with PBS containing 0.5% Triton-X-100 for 15 min at room temperature. The cells were then washed three times with PBS. Five percent BSA was added to the cells, and they were incubated overnight at 4°C. The cells were then incubated with a β-catenin antibody  overnight at 4°C, washed three times, then incubated with a secondary antibody  for 1 h at room temperature. Then, DAPI was added and the cells were incubated at room temperature for 15 min to stain the cell nucleus. Cells were visualized using a confocal fluorescence microscope .Human PC3 cells (2 × 106/plates) were seeded in 60-mm plates and treated with MOA  for 48 h. The cells were collected, and total protein was extracted using RIPA lysis buffer and quantified. The levels of PGE2 in the cell lysates were measured using enzyme linked immunosorbent assay (ELISA)  according to the manufacturer’s instructions.PC3 cells (1 × 106 cells per mouse). After 2 weeks, the mice were randomly divided into three groups of six mice each, including a control group , an MOA-150 mg/kg group, and an MOA-300 mg/kg group. All mice underwent oral gavage for 26 days, and tumor size was measured every 2 days using a caliper (volume mm3 = length × width × width/2). The mice were then euthanized with CO2, and tumor tissues were excised, and stored in formalin at −80°C until use. The animal experiment protocols were reviewed and approved by the Institutional Animal Ethical Committee of Yunnan Agricultural University (IACUC-20190410-26).Eighteen male BALB/C nude mice (5–6 weeks old) were purchased from Changzhou Cavans Laboratory Animal Co., Ltd. and all mice were subcutaneously injected with PC3 cells ] overnight. On the next day, the sections were incubated with horseradish peroxidase-linked secondary antibodies for 30 min, stained with 3,3′-diaminobenzidine (DAB) for 2 min, then counterstained with hematoxylin for 1 min. Photographs were taken using an Olympus microscope .Tumor tissues were embedded in paraffin and sectioned (3 μm). The sections were dehydrated and subjected to antigen retrieval using 0.01 M sodium citrate. The sections were then placed in 3% Hp < 0.05 was considered statistically significant.All data were presented as the mean ± standard error of the mean (SEM) of at least three replicate experiments. Statistical analysis was performed using GraphPad Prism 5 . Differences between two groups were analyzed using Student’s t test and differences among three or more groups were analyzed using one-way ANOVA. 50 of the cell lines were 459.3, 564.4, 876.2, 125.5, 95.95, 283, 413, 237.7, 276.9, and 379 μg/ml, respectively  were treated with MOA  for 48 h. ICectively . These rp < 0.001)  for 24, 48, and 72 h, then analyzed using the MTT and colony formation assays. The results showed that MOA decreased viability of PC3 cells in a dose-dependent manner . Colony < 0.001) . However< 0.001) . These rp < 0.05) and 69.24 ± 2.76% (p < 0.001) apoptotic cells, respectively, which was significantly greater than that in control cells (10.00 ± 2.98%). However, MOA has no effect on the apoptosis rate of RWPE-1 cells (p < 0.01), 32.57 ± 1.36% (p < 0.001), and 39.82 ± 1.23% (p < 0.001) in response to treatment with 20, 40, and 80 μg/ml MOA for 48 h, and the percentage of cells in G1 phase decreased from 48.47 ± 1.76% in control cells to 35.41 ± 0.33% (p < 0.01), 37.88 ± 5.03%, and 28.96 ± 0.37% (p < 0.001) in response to treatment with 20, 40, and 80 μg/ml MOA for 48 h  in PC3 cells compared with those in control cells  and 382.00 ± 24.01 pg/mg protein (p < 0.01), respectively  and FH535 (2.5 μM) (an inhibitor of β-catenin) for 48 h, and measured cell proliferation and migration using the MTT and transwell assays. The results showed that PGE2 significantly enhanced proliferation (p < 0.05) and migration (p < 0.05) of PC3 cells, and FH535 significantly inhibited PGE2-mediated proliferation (p < 0.001) and migration (p < 0.001) of PC3 cells (2 (20 μM) and MOA (40 μg/ml), with or without FH535 (2.5 μM), for 48 h. The results showed that MOA significantly inhibited PGE2-mediated proliferation (p < 0.001) and migration (p < 0.001) of PC3 cells, and the inhibitory effects of MOA and FH535 on PGE2-mediated proliferation and migration were not additive , which was a tumor inhibition rate of 25.16%  and β-catenin (p < 0.01) as compared to those in the control group  inhibit growth of tumor cells. Water extracts of M. oleifera leaves have been shown to inhibit growth of lung cancer, liver cancer ; Papaverine and colchicine inhibited the growth of HT29, T47D, and HT1080 cell lines by inducing apoptosis, but they are not toxic to normal mouse embryonic fibroblast NIH3T3 cell lines. In addition, the alkaloids of evodiamine and rutaecarpine, which isolated from ar cells . In thisStudies have shown that alkaloids such as atrine , tetrandCell migration plays an important role in many physiological and pathological processes, such as tissue repair and tumor cell metastasis. In this study, MOA significantly inhibited PC3 cell migration by using the wound healing and transwell assays. Matrix metalloproteinases (MMPs) promote tumor cell invasion and metastasis by breaking down the basement membrane and extracellular matrix. MMP2 and MMP9 are members of the MMPs family, and they have the function of decomposing the collagen of basement membrane. It is known that the malignant degree of different types of tumors is positively correlated with the excessive expression of MMP2 and MMP9. In this study, MOA significantly decreased the expression of MMP9. The results suggest that MOA suppressed PC3 cell migration and inhibited the expression of MMP9.2, an arachidonic acid metabolite produced by COX-2, is a mitogen and contributes to development of prostate cancer , GSK3β, and casein kinase 1 (CK1). This prevents β-catenin from entering the nucleus, which prevents transcription of downstream target genes (in vitro and in vivo. In addition, MOA significantly inhibited phosphorylation of GSK3β in a dose-dependent manner, which indicated that MOA inhibited the Wnt/β-catenin signaling pathway.The Wnt signaling pathway plays an important role in cell proliferation, differentiation, migration, apoptosis, and tumor development. Disruption of the classical Wnt/β-catenin pathway is one of the most common abnormalities associated with human malignancies . Abnormaet genes . In the et genes . A previet genes . Anotheret genes . In our 2 and Wnt/β-catenin signaling in development of many cancers, including lung cancer (2/β-catenin signaling pathway may be a potential target to inhibit cancer cell growth and migration. For example, tanshinone IIA has been shown to downregulate VEGF levels by inhibiting the expression of COX-2 and Wnt/β-catenin signaling, which resulted in inhibition of colon cancer cell growth (2 to regulate activation of the β-catenin signaling pathway (2 to regulate activation of the β-catenin signaling pathway (2/β-catenin signaling pathway.Numerous studies have shown a link between COX-2/PGEg cancer , skin cag cancer , breast g cancer , colorecg cancer , and neug cancer . In addil growth . Total fl growth . Further pathway . In addi pathway . In our