With the increase in life expectancy and consequent aging of the world’s population, the prevalence of many neurodegenerative diseases is increasing, without concomitant improvement in diagnostics and therapeutics. These diseases share neuropathological hallmarks, including mitochondrial dysfunction. In fact, as mitochondrial alterations appear prior to neuronal cell death at an early phase of a disease’s onset, the study and modulation of mitochondrial alterations have emerged as promising strategies to predict and prevent neurotoxicity and neuronal cell death before the onset of cell viability alterations. In this work, differentiated SH-SY5Y cells were treated with the mitochondrial-targeted neurotoxicants 6-hydroxydopamine and rotenone. These compounds were used at different concentrations and for different time points to understand the similarities and differences in their mechanisms of action. To accomplish this, data on mitochondrial parameters were acquired and analyzed using unsupervised  and supervised (decision tree) machine learning methods. Both biochemical and computational analyses resulted in an evident distinction between the neurotoxic effects of 6-hydroxydopamine and rotenone, specifically for the highest concentrations of both compounds. It is predicted that this number will rise to around 135 million in 2050, with a projected economic burden of up to 20 billion US dollars and rising . A key cuffering ,7. Manifll death ,9.No useful diagnostic tools exist for the early detection of neurodegenerative diseases, with the neuropathological evaluation at autopsy being the actual golden diagnostic standard . MoreoveDifferentiated SH-SY5Y cells are useful in vitro models to investigate cellular and mitochondrial morphological and bioenergetic alterations in mitochondrial dysfunction-related pathologies such as neurodegenerative diseases . MoreoveOur first objective was to choose an interval of 6-OHDA and rotenone sub-toxic concentrations, i.e., that were not cytotoxic when incubated for 24 h c. HoweveRegarding rotenone, and especially when cells were incubated with increasing concentrations  for 96 h, the resulting dose–response curves were different d. EffectFor finishing the cytotoxicity profile evaluation, caspase 3/7 activity was measured with Caspase-Glo 3/7 Assay. For this, cells were treated for 24 h with the selected concentrations of both compounds or with 1 µM doxorubicin (Dox), as a positive control. The highest non-lethal concentrations of both compounds increased caspase 3/7 activity. The increase in caspase 3/7 activity was statistically significant at the highest 6-OHDA and rotenone concentrations, 50 µM and 0.25 µM, respectively .We next investigated whether the mitochondrial network was affected by non-lethal concentrations of 6-OHDA or rotenone. Firstly, we aimed to assess alterations in the polarized mitochondrial network by high-throughput microscopy, upon labeling mitochondria with the mitochondrial membrane potential-dependent dye Mitotracker red CMXRos a,b. In tSince there were alterations in the polarized mitochondrial network, we next studied mitochondria membrane potential (ΔΨm), since changes in this parameter can indicate mitochondrial dysfunction. In order to indirectly address alterations in ΔΨm, we studied changes in mitochondrial network polarization by high-throughput microscopy, recurring to the previously described labeling approach. Cells were incubated with the selected concentrations of 6-OHDA or rotenone for 3 h c or 24 hConfocal microscopy was used to assess more specific details regarding mitochondrial network morphology and content when cells were treated with 6-OHDA (50 µM) and rotenone (0.25 µM) for 24 h j–l, whilMIEF2) (MIEF2 (MFN1) (FIS1) transcripts compared to control cells  . Rotenon) (MIEF2  and mito2 (MFN1) , respectol cells .In order to study mitochondrial bioenergetics, the Seahorse/Agilent Mito Stress Test was used with a Seahorse XFe96 Extracellular Flux Analyzer. Here, prior to the beginning of the standard assay, cells were acutely treated with 6-OHDA (50 µM) and rotenone (0.25 µM). Unsurprisingly, basal respiration OCR was similar between the three groups a. HoweveThis assay also records variations in ECAR h. As expNDUFA9) (COX4I1) (ATP5G) (CYB) (p = 0.012) (ATP6) (SDHA) mRNA levels in cells treated with 6-OHDA, compared to untreated controls (COX4I1 transcripts (CYB (ATP6 (UQCRC2)  complexes and ATP synthase subunits encoded by nuclear DNA  and by mNDUFA9) , cytochr(COX4I1)  and ATP  (ATP5G) -related = 0.012)  and ATP ) (ATP6) , when cocontrols . In rotenscripts  and an apts (CYB  and ATP6YB (ATP6  mRNA lev(UQCRC2) , when coNext, we wanted to understand the behavior of mitochondrial and glycolytic components of real-time cellular ATP production rate. We used the Seahorse/Agilent ATP Rate assay, since it can distinguish the site of ATP production, either from glycolysis (glycoATP) or from mitochondria (mitoATP), as well as the total ATP production rate. Control cells had a substantially higher ATP production rate than treated cells after the acute treatments . Under c2O2), 500 µM, was used as a positive control. No significant differences were observed in these assays with either of the test compounds. Nevertheless, a 26% and 30% increase, on average, in DCF fluorescence was measured when cells were incubated, respectively, with 0.125 µM and 0.25 µM rotenone for 3 h compared to control counterparts . SH-SY5Y cells were incubated for 3 h  and 24 hterparts . The samterparts .For further clarification of the data obtained, DCF oxidation rate was measured when cells were acutely incubated with 6-OHDA and rotenone. Remarkably, in this experiment, 6-OHDA treated cells exhibited significantly higher dye oxidation rate than their control counterparts a. RegardNext, we wanted to uncover if 6-OHDA and rotenone increased mitochondrial superoxide anion, contributing to the cellular oxidative stress. For this purpose, we used the Mitosox Red Mitochondrial Superoxide Indicator. All 6-OHDA concentrations induced a 20% increase, on average, of Mitosox oxidation rate. Rotenone concentrations 0.125 µM and 0.25 µM induced an evident increase in Mitosox oxidation rate c. FurtheSOD1) (CAT) were, on average, 85% decreased compared to control cells (GPX1) and glutathione peroxidase 4 (GPX4) (SOD2) mRNA  . More prol cells . On the D2) mRNA .Since both 6-OHDA and rotenone induced alterations in mitochondria morphology and bioenergetics, we next aimed to assess whether these treatments affected mtDNA copy number and mRNA levels of mitochondrial biogenesis-associated genes. Although we did not find any statistical difference in mtDNA copy number induced by any compound, a 36% and 43% average decrease in mtDNA copy number was measured when cells were treated with 50 µM 6-OHDA or 0.25 µM rotenone for 24 h, respectively a.NRF1) (TFAM) (SIRT1) (AMPK)  (p = 0.08)  b, mitoch) (TFAM) c and GA ) (TFAM) d, and th (SIRT1) e and ade) (AMPK) f, shown K) (NRF1 b and TFAF1 (TFAM c mRNA le = 0.06) b and a 6 = 0.08) c. Furthen factor d. On theor  or with the four concentrations of rotenone . This includes 36 samples, each described by 11 numeric features and one target (a specific cell treatment). These 11 numeric features encompass experimental assays done before or at 24 h of compound incubation, being considered early predictors of later neurotoxic manifestations.To understand which subset of features measured in this work contributed to a better discrimination between the experimental groups, we determined the mutual information (information gain) between each individual feature and the experimental class. A subset of 8 out of 11 features  had information gains higher than 0.5 a. HierarHierarchical clustering perfectly separated control cells from cells treated with different concentrations of both treatments (6-OHDA and rotenone). After the clustering step, leaves were labelled with the corresponding concentrations b. When tTo corroborate that (i) the unsupervised analysis and the biological findings showing that 6-OHDA and rotenone have different mechanisms of action and degrees of toxicity, and (ii) that this toxicity was time- and dose-dependent, a supervised decision tree model was trained to predict the real class from the data. The test results obtained using the decision tree are shown in a confusion matrix in Cells treated with 0.25 µM rotenone were entirely correctly classified, while the classification of cells treated with 0.125 µM was only 50% correct. The remaining samples treated with this rotenone concentration were predicted to be either 0.25 µM (25%) or 0.06 µM (25%) rotenone, which makes sense since those were the two closest rotenone concentrations.The remaining class predictions had low values (25%). In the case of 6-OHDA-treated cells, the misclassified samples appeared in the closest 6-OHDA concentrations. Interestingly, cells treated with the lowest rotenone concentrations (0.03 and 0.06 µM) both had 25% of samples predicted to belong to the 6.25 µM 6-OHDA. This could be because the lowest concentration of both compounds could cause similar effects on SH-SY5Y cells.The use of computational applied statistical methodologies in biology, i.e., combining classical statistics with ML algorithms, is a continuously evolving field. In this interdisciplinary work, we applied ML methodologies in original biological data to predict which experimental features could inform us about the mechanism and degree of toxicity of 6-OHDA and rotenone related with neurodegeneration before cell death. Concerning the ML approaches, we used unsupervised and supervised methods. Finally, we performed an interdisciplinary analysis of both biological and computational results retrieving the most important conclusions from the combination of both fields.Different studies revealed similar results regarding the effects of 6-OHDA and rotenone on SH-SY5Y cells. Firstly, both compounds were previously shown to induce a mitochondrial fragmentation phenotype ,22,23 anAdditionally, both compounds have been reported to induce autophagy. A study by Chu and collaborators, using both primary cortical neurons and undifferentiated SH-SY5Y treated with sublethal concentrations of rotenone and 6-OHDA, showed an evident increase in LC3 levels and LC3 puncta co-localized with mitochondria. A decrease in p62 levels was also present, suggesting an increased autophagic flux .2O2, O2• − and •OH production [Nonetheless, 6-OHDA and rotenone are mitochondrial toxicants known to have different mechanisms of action. Firstly, while rotenone is a highly lipophilic and hydrophobic molecule that can easily cross cell membranes without the need for specific membrane transport mechanisms , 6-OHDA oduction .These differences could help to explain why, in our work, despite inducing similar effects in caspase activity, mitochondrial-encoded ETC subunits mRNA levels, mitochondrial morphology, Mitosox Red oxidation, mtDNA copy number, and lysosomal protease activity; in the remaining parameters, 6-OHDA and rotenone promoted distinct effects. 6-OHDA induced a decrease in mitochondrial area and ΔΨm, mRNA levels of nuclear-encoded ETC subunits, fusion/fission proteins, and antioxidant enzymes, and ATP production rate. Moreover, 6-OHDA also prompted an acute increase in mitochondrial bioenergetic parameters. Rotenone promoted a decrease in mitochondrial bioenergetic parameters and ATP production rate while inducing an increase in ΔΨm and mitochondrial biogenesis-associated genes.To understand the differences and similarities of non-toxic concentrations of 6-OHDA and rotenone on differentiated SH-SY5Y cells and to complement our biological data analysis, ML methodologies, such as hierarchical clustering and supervised classification algorithms, were used to clarify two main questions. Firstly, was it possible to distinguish between control and treated cells? And if so, is it also possible to distinguish between control cells and cells treated with different 6-OHDA or rotenone concentrations? Hierarchical clustering is an unsupervised approach that estimates the dissimilarities (based on distances) between any pair of observations. Being an unsupervised method, hierarchical clustering aimed to discover hidden patterns in the data without using the respective class labels for each observation. This methodology allowed us to have a broader idea of how the different observations were organized. Information gain revealed that the features MitoTracker area and intensity (3 and 24 h), mitochondrial (Mitosox oxidation 3 h and oxidation rate) and cellular (DCFDA fluorescence 3 h)-derived oxidative stress, and Caspase activity, seem to have an enhanced ability to signal neurodegeneration induction, prior to cell death, caused by 6-OHDA and rotenone. Using this subset of eight features with higher information gain, hierarchical clustering automatically grouped control cells in one cluster and cells treated with higher concentrations of 6-OHDA and higher rotenone concentrations in distinct clusters. This occurred because the higher the concentration of each compound, the more evidently different biologic effects on SH-SY5Y cells were demonstrated. On the other hand, milder rotenone and 6-OHDA concentrations were clustered together since they caused smaller and similar effects. Biological results were also used to train a decision tree predictive model. In this methodology, the output labels are known during model training . The decision tree algorithm could correctly classify a high percentage (>75%) of cells treated with the highest 6-OHDA and rotenone concentrations , but only 50% of control cells. Interestingly, this model was able to predict correctly 75% of cells treated with 6.25 µM 6-OHDA, being an improvement from the results derived from the hierarchical clustering analysis. Regarding cells treated with the lowest rotenone concentration, the hierarchical clustering results already suggest that classification methods may be unable to classify samples in their respective class correctly. Misclassified samples were predicted to be in classes corresponding to the remaining rotenone concentrations or the class of cells treated with 6.25 µM 6-OHDA. Similar to what happened in the hierarchical clustering analysis, the predictive model was unable to distinguish between cells treated with a milder concentration of both compounds (except on cells treated with 6.25 µM 6-OHDA), since the biological effects caused by these conditions only induced smaller and similar effects.2, 95% air at 37 °C. The media used for undifferentiated cells was Dulbecco’s Modified Eagle’s Medium  supplemented with 4.5 g/L D-(+)-glucose , 0.8 g/L L-glutamine , 1.2 g/L HEPES , 3.7 g/L sodium bicarbonate , 0.1 g/L sodium pyruvate , 10% fetal bovine serum  and 1% penicillin/streptomycin . Cell media was changed every 2 to 3 days and cells were subcultured, until the 23rd passage, when reaching 90–100% of confluence.SH-SY5Y human cell line  was cultured in a humidified atmosphere with 5% CO2 in a low D-(+)-glucose (0.9 g/L) media supplemented with 1% FBS and 10 µM retinoic acid  for 3 days. Exceptionally, cells were plated at a density of 120,000 cells/cm2 in the Seahorse experiments  due to equipment sensitivity.We previously described the protocol used for SH-SY5Y cell differentiation in ref. . Brieflyv/v). Cell treatment strategies are specified in the respective results subsection and in the respective figure legend.Two mitochondrial poisons were used, 6-OHDA  and rotenone . Rotenone was dissolved in dimethyl sulfoxide  while 6-OHDA is water-soluble. The final concentration of DMSO used was never higher than 0.1% (w/v) SRB  for 1 h at 37 °C. Subsequently, SRB was removed, and wells were washed with 1% acetic acid to remove the unbound stain. Dye bound to cell proteins was extracted with 10 mM Tris-base solution, pH 10, and absorbance was read at 510 nm and 620 nm (reference wavelength) in a Cytation 3 reader . Then, we subtracted the 620 nm absorbance value from the 510 nm absorbance value for each condition. Data were normalized to the average control (100%) condition.To evaluate the cytotoxic effects of 6-OHDA and rotenone on cell mass, the sulforhodamine B (SRB) assay  was used2, at 37 °C for 3 h. The fluorescent signal was monitored using a 540 nm excitation wavelength and 590 nm emission wavelength in a Cytation 3 microplate reader. A blank condition (wells without cells) was used as a fluorescent reference signal and subtracted from the remaining conditions. Data were normalized to the average control (100%) condition.The effect of 6-OHDA and rotenone toxicity was also evaluated by measuring the cellular metabolic activity using the resazurin reduction assay. The resazurin assay is based on the reduction of resazurin  to resorufin by dehydrogenases present in viable cells . After cIntracellular ATP levels were assessed using CellTiter-Glo Luminescent Cell Viability Assay . Cell differentiation and treatments were undertaken in 96-well white opaque-bottom plates  and, at the end of the cell treatments, the medium was removed from the wells, replaced by 50 µL of fresh medium plus 50 µL of the Cell Titer-Glo reagent, and mixed for 2 min on an orbital shaker to promote cell lysis. The plate was incubated for 10 min and the luminescent signal was recorded using a Cytation 3 microplate reader . Data were normalized to the average control (100%) condition.Cell membrane integrity and cell death induction were evaluated using propidium iodide (PI) and Hoechst 33,342 staining. After the differentiation protocol and 6-OHDA and rotenone incubations, cell nuclei were stained with 4 µM PI  and 1 µg/mL of Hoechst 33342 . For being a cell-impermeant stain, only cells without an intact membrane were labeled with PI, while Hoechst 33342 labeled all cell nuclei. Both PI and Hoechst 33,342 were added directly to the media without aspirating it in order to avoid removing dead, PI-positive, cells. Cells were incubated with both stains for 5 min and visualized using an INCell Analyzer 2200  cell imaging system with 10× magnification . The image analysis was performed using IN Cell Analyzer 1000 analysis Software-Developer Toolbox . The PI negative cells were divided by the total number of cells to calculate the live cell population. Data were normalized to the average control (100%) condition.Caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay . Cell treatments were performed in 96-well white opaque-bottom plates. Some cells were treated with 1 µM Doxorubicin  for 24 h as a positive control. At the time of the assay, the cell medium was removed from the wells and replaced by 50 µL of fresh medium. Then, 50 µL of the Caspase-Glo 3/7 reagent was added and mixed for 2 min on an orbital shaker to promote cell lysis. The plate was incubated for 30 min and the luminescent signal was recorded using a Cytation 3 microplate reader. A blank condition (wells without cells) was used as a luminescent reference signal and subtracted from the remaining conditions.2, at 37 °C. For positive control, 10 µM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone  was used and incubated at the same time as the dye incubation. Cells were also stained with 1 µg/mL Hoechst 33,342 for normalization purposes. An INCell Analyzer 2200  cell imaging system was used for visualization and image acquisition using a 20× objective . The image analysis was performed using the IN Cell Analyzer 1000 analysis software-Developer Toolbox. A protocol was created to quantify a specific target’s fluorescence in IN Cell Developer Toolbox for image analysis. The image stacks were uploaded to the program to identify our target set and establish the respective parameters: area, intensity, and number. The representative images were visualized using ImageJ 1.52a .Live cell high-throughput microscopy was used to detect alterations in mitochondrial polarization and network distribution in differentiated SH-SY5Y cells exposed to increasing (non-toxic) concentrations of 6-OHDA and rotenone. Cells were differentiated on Corning 96-well black polystyrene microplates  and after the treatment with either compound, mitochondria were stained for 30 min with 50 nM of the mitochondrial fluorescent dye Mitotracker Red CMXRos  in a humidified atmosphere, 5% CO2, at 37 °C. Subsequently, cells were washed 3 times with phosphate buffer saline (PBS) 1× and fixed with 4% paraformaldehyde in PBS 1× overnight at 4 °C. Afterwards, cells were washed 3 times with PBS 1x and incubated with 1 μg/mL Hoechst 33342 in PBS 1× to label cell nuclei. Regarding the study of lysosomal parameters, after cell differentiation and treatment, cells were incubated for 30 min with 75 nM LysoTracker Green DND-26 and 1 μg/μL Hoechst 33,342 in a humidified atmosphere, 5% CO2, at 37 °C. Finally, cells were imaged under a Carl Zeiss Laser scanning confocal microscope  using an oil Plan-Apochromat 63× DIC M27  objective with a numerical aperture of 1.4. A BP 545/25, FT 570, BP 605/70 filter set was used to measure the red fluorescence from Mitotracker Red CMXRos, a BP 470/40, FT 495, BP 525/50 for the measurement of the green fluorescence from LysoTracker Green DND-26 and a G 365, FT 395, BP 445/50 filter set was used to assess the blue fluorescence from Hoechst 33342. Excitation was achieved with an HXP 120V light source. For fixed cells mitochondrial analysis, Z-stack series were made and consisted of 0.2–0.4 µm slice intervals and rendered into a single 2D images using the “maximum intensity projection” processing tool using Zeiss Zen 2 software .Confocal microscopy was used to accomplish a more detailed definition of the mitochondrial network, to be able to apply the ImageJ macros Mitochondrial Network Analysis (MiNA) toolset  and MitoBy using ImageJ 1.52a, it was possible to apply the MiNA toolset that provided us with data related to the mean of mitochondrial branch length, summed mitochondrial branch length and mitochondrial network branches . Moreove2. Firstly, a XFe96 sensor cartridge was placed in a 96-well calibration plate containing 200 µL/well of calibration buffer and left to hydrate overnight at 37 °C. Next, the medium in the 96-well plate containing the cells was replaced with 175 µL/well of prewarmed low-buffered serum-free minimal DMEM  medium (pH 7.4) and incubated at 37 °C for 1 h to allow the temperature and pH of the medium to reach equilibrium before the first measurement. For the Seahorse XF Cell Mito Stress Test assay measurement of OCR and ECAR, 2 µM oligomycin  were injected through port B, 2 µM FCCP were injected through port C and port D was loaded with 1 µM rotenone  plus 1 µM antimycin A . Both the calibration plate and the sensor cartridge were loaded into the XFe96 Extracellular Flux Analyzer for calibration. The calibration plate was then replaced with the study plate. Three baseline rate measurements of SH-SY5Y cell line OCR were made using a 3 min mix, 3 min measure for 3 cycles. Rotenone (0.25 µM), 6-OHDA (50 µM) or medium (control) were then pneumatically injected through port A into the corresponding wells and mixed, with OCR measurements made using a 3 min mix, 3 min measuring time for 5 cycles. Lastly, oligomycin, FCCP, rotenone and antimycin A were sequentially pneumatically injected and mixed, with OCR measurements made using a 3 min mix, 3 min measuring time for 3 cycles. Several parameters were evaluated, such as the acute response, ATP production, proton leak, maximal respiration, spare respiratory capacity and non-mitochondrial respiration-linked OCR. Acute responses were measured as the last OCR measurement before oligomycin injection minus the last measurement before port A injection. After adding 2 µM oligomycin, it was possible to calculate ATP production-linked OCR by subtracting the last measurement before oligomycin injection and the minimum rate measurement after the oligomycin injection. Moreover, it was also possible to calculate the proton leak-linked OCR by subtracting the non-mitochondrial respiration-linked OCR from the minimum rate measurement after oligomycin injection. When cells were treated with 2 µM FCCP it was possible to evaluate the maximal respiration-linked OCR and spare respiratory capacity. Maximal respiration-linked OCR corresponds to the maximum rate measurement after FCCP injection minus the non-mitochondrial respiration-linked OCR, while the spare respiratory capacity is calculated by subtracting the basal from the maximal respiration. Lastly, non-mitochondrial respiration-linked OCR is the minimum rate measurement after rotenone and antimycin A injection.Oxygen consumption and extracellular acidification rates were measured at 37 °C using a Seahorse XFe96 Extracellular Flux Analyzer  following the Seahorse XF Cell Mito Stress Test  and Real-Time ATP Rate  Assay protocols. To be in the range of the equipment sensitivity, cells were differentiated at a density of 120,000 cells/cmRegarding the Real-Time ATP Rate Assay, the procedure was very similar. However, in this assay, the cells were not injected with FCCP. The cells were firstly acutely treated with rotenone (0.25 µM), 6-OHDA (50 µM) or medium (control) through port A. Subsequently, cells were treated with 2 µM oligomycin through port B followed by 1 µM rotenone plus 1 µM antimycin A through port C. The cycles of mix and measuring were the same used in the Mito Stress Test assays. Oligomycin, FCCP, rotenone, and antimycin A were prepared in DMSO and diluted to the desired final concentration in serum-free minimal DMEM.For both assays, results were normalized by cell nuclei number in each well and analyzed using the Software Version Wave Desktop 2.6 .2O2, cat. BCBT5298 Panreac AppliChem ITW Reagents, Darmstadt, Germany) as positive control for 3 h or 24 h. At the end of these incubation periods, cells were incubated with 5 µM of H2DCFDA for 30 min at 37 °C, 5% CO2, and the fluorescent signal was monitored using a 485 nm excitation wavelength and 528 nm emission wavelength in a Cytation 3 reader. Regarding the kinetic assay, the differentiation process was the same. Then, cells were incubated with 5 µM H2DCFDA for 30 min at 37 °C, 5% CO2. After this, the medium containing the dye was removed, and cells were treated with increasing concentrations of 6-OHDA, rotenone or 1 mM H2O2 as a positive control. Lastly, the fluorescent signal was monitored using a 485 nm excitation wavelength and 528 nm emission wavelength in a Cytation 3 reader. A kinetic assay was made measuring the fluorescent signal every minute for 5 h in order to determine where dye oxidation was linear. This read always occurs in the first 50 min. Then, a linear regression analysis was calculated, and the slope was used to estimate the H2DCFDA oxidation rate. In these experiments, low D-(+)-glucose medium supplemented with 1% FBS and 10 µM RA but without sodium bicarbonate was used.For studying the effect of 6-OHDA and rotenone on intracellular oxidative stress, the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate  dye was used. Two types of assays were carried out, endpoint and kinetic assays. For the endpoint assay, cells were differentiated in Corning 96-well black polystyrene microplates and subsequently treated with increasing concentrations of 6-OHDA, rotenone or 500 µM of hydrogen peroxide -glucose medium supplemented with 1% FBS and 10 µM RA but without sodium bicarbonate was used.In order to determine if oxidative stress had a mitochondrial contribution, the fluorogenic dye Mitosox Red Mitochondrial Superoxide indicator  was utilized. This dye is specifically targeted to mitochondria in live cells . After cg for 5 min. The pellets were washed in 5 mL of PBS 1× and the suspension was again centrifuged at 2000 g for 5 min. The resulting pellets were stored at −80 °C until total DNA extraction. Total DNA was extracted from cell pellets using the QIAamp DNA mini kit , following the manufacturer’s protocol. The samples were sonicated for 10 min to avoid dilution bias [After cell differentiation and treatments, trypsinized-collected cells were centrifuged at 2000× ion bias  and thenPureZOL Reagent  was used to isolate RNA fractions from whole-cell samples. In order to do this, cells were differentiated in 6-well plates and then treated either with 50 µM 6-OHDA or 0.25 µM rotenone for 24 h. After this, the medium was removed and 1 mL of PureZOL was added to each well. The reagent was mixed by pipetting up and down and incubated at room temperature for 5 min to allow nucleoprotein complexes disruption. Total RNA was extracted with RNeasy mini kit , following the manufacturer’s protocol. Total RNA concentration and purity were evaluated using a Nanodrop 2000 . Subsequently, RNA was converted into cDNA using the iScript cDNA synthesis kit , following the manufacturer’s instructions. Quantitative reverse-transcriptase PCR (qRT-PCR) was performed using the SsoFast Eva Green Supermix, in a CFX96 real-time PCR system , with the primers defined in Lysosomal protease activity in cells was measured using DQ Green BSA . When this quenched fluorogenic substrate suffers hydrolysis by cellular proteases the quenching disappears, and a bright product is produced. After cell differentiation in Corning 96-well black polystyrene microplates, the medium was replaced with new medium containing 10 µg/mL DQ Green BSA and incubated for 1 h at 37 °C. After dye incubation, cells were treated with increasing concentration of 6-OHDA, rotenone, or 2 µM trichostatin A  as positive control in Earle’s Balanced Salt Solution (EBSS) medium. Lysosomal protease activity was measured as fluorescence in a Cytation 3 reader at 495 nm excitation, 525 nm emission each min for 4.5 h in order to find out where DQ Green BSA hydrolysis was linear. This linearity occurred always in the last 170 min. Then, a linear regression analysis was calculated, and the slope was used to estimate the DQ Green BSA hydrolysis rate.The complete dataset used in the analysis comprises 36 samples, each described by 11 numeric features and 1 target. The attributes considered were caspase 3/7 activity, Mitotracker Red CMXRos area and intensity (3 h and 24 h incubations with both compounds), Mitosox oxidation (3 h incubation with the referred compounds) and oxidation rate, DCFDA fluorescence (3 h and 24 h incubations with either compound) and oxidation rate, and DQ BSA hydrolysis. The target of each instance corresponds to one of the 9 possible classes (4 samples per class): Control, 6.25, 12.5, 25 and 50 µM for 6-OHDA and 0.03, 0.06, 0.125 and 0.25 µM for rotenone. The dataset is balanced, it does not contain any missing values and data were standardized across features. The small number of samples prevented a full and strong statistical analysis of the results. Nevertheless, it allowed the identification of relevant hidden patterns and trends.Exploratory data analysis, information gain, hierarchical clustering, and supervised predictive modeling were performed using Orange Data Mining version 3.25.1 . Hierarcp < 0.05, (**) p < 0.01, (***) p < 0.001 and (****) p < 0.0001. Seahorse experiment Real-Time ATP Rate Assay data are present in columns and statistical relevance was evaluated using Kruskal–Wallis test (one-way ANOVA on ranks) pairwise (control vs. 6-OHDA or control vs. rotenone) to assess statistical significance of total ATP levels, (*) p < 0.05, (**) p < 0.01. Additionally, two-way ANOVA with Sidak’s multiple comparisons test was used to assess statistical significance of mitoATP production rate (control vs. 6-OHDA or control vs. rotenone) ($$) p < 0.01, ($$$$) p < 0.0001) and glycoATP production rate (control vs. 6-OHDA or control vs. rotenone), (###) p < 0.001. The remaining results are presented in box-and-whisker plots where the middle line represents the median. The applied method—Min to max—shows all points, plots the whiskers down to the minimal value and up to the maximum value and represents each individual value as a point. Kruskal–Wallis test followed by Dunn’s post hoc analysis was used for multiple conditions comparison. The statistical significance was set at p < 0.05 . The number of experiments carried out is represented in the legend of the figures, as well as the number of replicates (at least 2) and the statistical tests applied.Regarding classical statistics, the data were analyzed using the Version 8.0.1/2 of GraphPad Software, Inc. . The cytotoxicity analysis data are presented as points with a connecting line, Mean ± SEM and statistical relevance was evaluated using Kruskal–Wallis test (one-way ANOVA on ranks) pairwise (control vs. 6-OHDA or control vs. rotenone), (*) Together, biological assays and computational methodologies allowed for a distinction between the neurotoxic effects of 6-OHDA and rotenone. For this, features related to mitochondrial physiology, mitochondrial and cellular-derived oxidative stress, and caspase activation, seem to have an enhanced ability to signal neurodegeneration induction, prior to cell death, caused by 6-OHDA and rotenone. This distinction was more evident for the higher concentrations of both compounds that caused more severe and different effects between 6-OHDA and rotenone and between these and control cells. Computational methods also corroborated biological assays in the case of cells treated with lower concentrations of rotenone and 6-OHDA. However, the decision tree algorithm correctly classified 75% of cells treated with 6.25 µM 6-OHDA, which is an improvement concerning the clustering analysis and a finding not foreseen when the biological assays were exclusively taken into consideration.


Objective: To investigate the effect of Mingmu Xiaomeng tablets (MMXM) on the expression of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)-related proteins in a diabetic rat model.Methods: Thirty-two male Sprague Dawley rats were randomly divided into four groups: normal control (NC), diabetic model (DM) control, MMXM, and calcium dobesilate (CD) Rats injected with streptozotocin (STZ) were used as an experimental diabetes model. After 14 weeks, autophagy and PI3K/Akt/mTOR signaling pathway proteins were detected by western blot. Glial fibrillary acidic protein (GFAP) expression in Müller cells was examined by immunohistochemistry. Retinal function was evaluated with electroretinography, and retinal ultrastructure was observed by transmission electron microscopy. Serum cytokine levels were detected with protein chip technology.Results: MMXM restored autophagy by decreasing the protein expression of LC3-II and p62 and reducing the phosphorylation of PI3K, Akt, and mTOR, thus promoting autophagy. MMXM decreased GFAP expression in retinal Müller cells; restored electrophysiology indexes and retinal ultrastructures; and reduced serum levels of interleukin (IL)-1β, IL-4, IL-6, tumor necrosis factor-α, and vascular endothelial growth factor.Conclusion: MMXM may protect the diabetic retina by inhibiting PI3K/Akt/mTOR signaling and enhancing autophagy. Diabetic retinopathy (DR) is one of the main retinal vascular complications of diabetes and a major cause of visual impairment and blindness in working age people worldwide. According to the 9th edition of the global diabetes map released by the International Diabetes Federation in 2019, global adult population (20–79 years) prevalence is estimated to be 9.3% (463 million people) . The numHyperglycemia-induced DR pathogenesis is related to four main biochemical changes: polyol pathway activation, increased advanced glycation end-products (AGEs), activation of protein kinase C, and induction of the hexosamine pathway . These pAutophagy is an evolutionarily conserved catabolic pathway that controls cell homeostasis by disposing of cytoplasmic cargo, denatured proteins, damaged organelles, and invasive pathogens through lysosomal degradation . BalanceIlex pubescens and Concha arcae can dissipate and disperse phlegm. Therefore, MMXM can be used to treat DR and improve the quality of life of affected patients.Mingmu Xiaomeng tablets (MMXM) are a hospital preparation developed in the early 1990s based on the clinical experience of Professor Meifang Zhang, a famous ophthalmologist at the Guangdong Provincial Hospital of traditional Chinese medicine (TCM), after several generations of clinical verification in ophthalmology. MMXM are mainly composed of modified Shengmai Powder and Wendan Decoction, and are widely used in patients with difficult ophthalmic diseases with qi-yin deficiency and phlegm-stasis syndrome . ShengmaCalcium dobesilate  was registered by the European Pharmacopoeia and British Pharmacopoeia in 1997 and 1998, respectively . It is cBased on preliminary experiments and the existing literature , we estaIlex pubescens Hook. & Arn.), Wa Leng Zi (Concha arcae), Mai Dong [Ophiopogon japonicus (Thunb.) Ker Gawl], Chen Pi , Zhi Shi (Citrus aurantium L. Aurantii Fructus Immaturus), San Qi [Panax notoginseng (Burkill) F.H. Chen], Ji Li (Tribulus terrestris L.), Mi Meng Hua , Wu Wei Zi [Schisandra chinensis (Turcz.) Baill.] at a dry-weight ratio of 6:6:6:3:2:2:2:2:2:1. MMXM was provided by the Guangdong Provincial Hospital of TCM . Calcium dobesilate capsules were purchased from Ebewe Pharma GmbH . Betaine, caffeic acid, hyperoside, notoginsenoside R1, hesperidin, ginsenoside Rb1, luteolin, quercetin, and ferulic acid were obtained from the National Institutes for Food and Drug Control , chlorogenic acid was obtained from Solarbio Science and Technology Co., Ltd. , and berberine was obtained from Aladdin Bio-Chem Technology Co., Ltd. .MMXM was composed of Dang Shen [Codonopsis pilosula (Franch.) Nannf.], Mao Dong Qing (Ground MMXM powder (200 mg) and 1 ml methanol:water  were vortexed to mix, centrifuged at 4°C for 10 min  and the supernatant was passed through a 0.22 μm filter membrane. The 11 standards were diluted to 2.00 mg/ml with methanol. All analyses were performed on Ultimate 3000 UPLC system  and Q Exactive mass spectrometer (Thermo Fisher). Both the positive and negative ion modes were applied in parallel reaction monitoring at a resolving power of 17,500 (scan range: 50–500 m/z). Mobile phases 0.1% formic acid aqueous solution (A) and acetonitrile (B) were used to separate the analytes on a Welch RP-C18 column . The gradient elution was as follows: 0–0.5 min, 2% B; 0.5–6.5 min, 2%–98% B; 6.5–9.0 min, 98% B; 9.0–9.3 min, 98%–2% B; 9.3–10.0 min, 2% B. The flow rate was 0.3 ml/min, the injection volume was 5 μL, and the ion spray voltage was 3.2 kV. The aux gas heater and capillary temperatures were 350 and 300°C, respectively.The procedure of animal experiment is illustrated in n = 8/group) as follows: 1) normal control rats (NC) treated with distilled water (10 ml/kg); 2) STZ-induced diabetic rats (DM) treated with distilled water (10 ml/kg); 3) STZ-induced diabetic rats treated with MMXM at 1 g/kg (MMXM)  injection. DM was induced by intraperitoneal injection of STZ dissolved in 0.1 mol/L of citrate buffer (pH 4.5) at a dose of 60 mg/kg body weight. Age-matched control rats received an equal volume of vehicle. 3 days after STZ injection, a random glucose level >16.7 mmol/L was considered diabetic . The aniRetinal tissues were prepared with radioimmunoprecipitation assay lysis buffer containing phenylmethanesulfonyl fluoride and phosphate inhibitor (100:1:1). Lysates were centrifuged at 12,000 r/min for 30 min at 4°C, and the supernatants were collected. Equal amounts of protein (20 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes . After blocking with 5% non-fat dry milk at room temperature for 2 h, the PVDF membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) three times and incubated overnight at 4°C with different primary antibodies . The above-mentioned primary antibodies were obtained from Cell Signaling Technology  except p-PI3K which was from Bioworld . Next, the PVDF membranes were washed with TBST three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (CST) at room temperature for approximately 1 h. Finally, the PVDF membranes were washed with TBST three times, and the blots were developed with chemiluminescence reagent using an enhanced chemiluminescence kit .2O2 to block endogenous peroxidase activity, and blocked with 3% bovine serum albumin. They were then incubated with GFAP antibody  at a dilution of 1:1,500 overnight at 4°C. The next day, the sections were washed with PBS three times for 5 min each and incubated with an HRP-conjugated secondary antibody  for 50 min at room temperature. After washing with PBS, the sections were stained with 3,3-diaminobenzidine tetrahydrochloride for approximately 1 min, counterstained in hematoxylin, dehydrated in absolute alcohol, cleared in xylene, and finally mounted in synthetic resin for microscopic examination.The eyeballs were immersed in Formaldehyde, Aceticacid, Alcohol, Saline , dehydrated in alcohol, and then embedded in paraffin. Paraffin-embedded tissue blocks were cut into serial 4 μm sections. Paraffin-embedded retina sections were dewaxed, rehydrated, washed with phosphate-buffered saline (PBS), incubated with 3% HAfter 12 weeks of treatment, ERG was performed according to the International Society for Clinical Electrophysiology guidelines . Before 2 pieces, then fixed in 2.5% glutaraldehyde solution overnight at 4°C. The next day, the retinas were dehydrated in a graded series of ethanol baths and embedded in Epon 812. The site of the retina studied with TEM was selected by toluidine blue staining. Ultrathin sections were cut with a Leica UC7 microtome , stained with uranyl acetate and lead citrate, and examined under a Hitachi HT7700 electron microscope .The eyes were enucleated, fixed in 2.5% glutaraldehyde for 1 h at room temperature, trimmed into 1 mmSerum was collected from the upper aorta and centrifuged. Samples were analyzed and quantified with multianalyte arrays .p < 0.05 was considered statistically significant. Graphical results were analyzed using GraphPad Prism 7 .All data are presented as mean ± standard error of the mean. Differences between groups were analyzed using Statistical Package for the Social Sciences version 22.0 . Results were analyzed using one-way analyses of variance for comparisons of more than three groups, followed by Least Significant Difference post hoc multiple comparisons. The peaks in the UPLC profile were identified as betaine, chlorogenic acid, caffeic acid, berberine, hyperoside, notoginsenoside R1, hesperidin, ginsenoside Rb1, luteolin, quercetin, and ferulic acid . The conp < 0.05, p < 0.05). The expression levels of p-PI3K, p-Akt and p-mTOR were significantly upregulated in the MMXM and CD groups versus the NC group , while only p-Akt and p-mTOR protein levels were significantly lower in the CD group (p < 0.05).Compared with the NC group, LC3-II and p62 expression were significantly increased in the retina of the DM group . The amplitudes of all three waves increased in the CD group, but only the b wave amplitude difference was statistically significant (p < 0.05). Collectively, these results indicate that MMXM restored retinal function in diabetic rats.Compared with the NC group, the amplitudes of the a, b, and the second wave of oscillatory potentials (OPs2) decreased in DM rats; the amplitude of the b wave was more affected than that of the a wave, but the differences for both were statistically significant , as did serum IL-1β, IL-4, IL-6, TNF-α, and VEGF levels in the CD group .Compared with the NC group, interleukin (IL)-1β, IL-4, IL-6, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF) expression in the DM group were significantly increased , which indicated that they were damaged due to abnormal glucose metabolism. MMXM inhibited GFAP overexpression in retinal Müller cells in diabetic rats and protected against early diabetic retinal neurodegeneration.We found that the amplitudes of a, b, and OPs waves decreased in DM rats, which indicated that they had developed DR . The a wA growing body of evidence suggests that local inflammatory processes in the retina are indispensable for early DR development . Müller The PI3K/Akt/mTOR pathway is a convergence of proinflammatory mediators, growth factors, and downstream substrates that regulate cell survival processes. This makes inhibition of PI3K/Akt/mTOR signaling an attractive therapeutic target for DR. mTOR inhibitors could suppress the pro-inflammatory phenotype. Dampening mTOR signaling could also decrease NF-κB expression, thus reducing the expression of downstream proinflammatory mediators such as TNF-α, monocyte chemoattractant protein-1, VEGF, IL-1β, RAGE, vascular cellular adhesion molecule-1, and ICAM-1 . These pin-vitro study is required to explore the effects of MMXM or its various components on retinal cells induced by high glucose, and will help to elucidate the mechanisms underlying treatment of DR. It will be desirable to assess the preparation at a lower dose in order to assess the therapeutic relevance of the results further.In this study, activation of the PI3K/Akt/mTOR signaling cascade and decreased autophagy activity in diabetic rats promoted the expression of various inflammatory cytokines and diabetic retinal neurodegeneration. After intragastric MMXM administration, inflammatory factor expression decreased, autophagy was restored and retinal function was improved, which may be related to inhibition of the PI3K/Akt/mTOR pathway. This study provided the first evidence that MMXM directly contribute to the prevention of the early DR. Future


Actinidia chinensis) is a nutrient-dense fruit abundant in vitamin C and phenolic compounds, and it exhibits strong antioxidant capacity. However, the antioxidants in ‘Jinfeng’ kiwifruit have seldom been extracted and analyzed, and the conditions for the extraction of kiwifruit antioxidants by ultrasound-assisted extraction (UAE) have seldom been investigated. In this study, response surface methodology (RSM) was used to optimize UAE conditions to extract antioxidants from ‘Jinfeng’ kiwifruit. In addition, the antioxidant capacity, contents of total phenolics and total flavonoids, ascorbic acid, and the profiles of antioxidants were also analyzed. The results showed that the optimal UAE conditions included 68% ethanol, liquid/solid ratio at 20 mL/g, extraction time at 30 min, extraction temperature at 42 °C, and ultrasonic power at 420 W. Under these conditions, the ABTS value of kiwifruit was 70.38 ± 1.38 μM TE/g DW, which was 18.5% higher than that of the extract obtained by conventional solvent extraction. The total phenolic and flavonoid contents were 15.50 ± 0.08 mg GAE/g DW and 5.10 ± 0.09 mg CE/g DW, respectively. Moreover, 20 compounds were tentatively identified by UPLC–MS/MS, and the content of main compounds, such as procyanidin B2, neochlorogenic acid, and epicatechin, were determined by HPLC–DAD. This research revealed the profiles of antioxidant phytochemicals in ‘Jinfeng’ kiwifruit, which can be a good dietary source of natural antioxidants with potential health functions.Kiwifruit ( Actinidia) is an edible berry reputed for its remarkable nutritional value and attractive taste −) was generated from the precursor ion at m/z 218. Thus, it was tentatively identified as D-pantothenic acid, which is also known as vitamin B5, after comparing it with the database and literature − ions at m/z 115, 129, and 117, respectively. The fragment ions of these three compounds were generated by losing the –CO2 group. Therefore, they were tentatively identified as fumaric, itaconic, and succinic acids [3 lost an –H2O group from the precursor ion m/z 133 and was tentatively identified as malic acid [5 produced the [M–H]− at m/z 191 and generated a secondary fragment at m/z 111 by losing –2H2O–CO2. Compared with the database and previous literature, this compound was tentatively identified as citric acid − at m/z 179 and generated a secondary fragment at m/z 135 by losing a carboxyl group. Compared with the existing literature, this compound was identified as caffeic acid [12 had the [M–H]− at m/z 341 on the MS with two fragment ions at m/z 179 ([M–H–hexoside]−) and m/z 135 ([M–H–hexoside–COOH]−) in the MS2 spectrum. Two daughter ions were the same as the fragments produced in caffeic acid; thus, compound 12 was regarded as a caffeic acid derivative and tentatively identified as caffeic acid-O-hexoside [O-hexosides were reported previously and exhibited relatively higher contents than other determined phenolic compounds [9 and 15 shared the same [M–H]− ion at m/z 353 and similar MS2 fragment ions at m/z 191, 179, and 135 but varied in retention time. Analysis on the MS2 spectrum, the generation of m/z 191 ([C7H11O6]−) fragment was caused by losing the caffeoyl, while the generation of m/z 179 ([C9H8O4]−) fragment was caused by losing the quinoyl group. The generation of m/z 135 contributed to the separation of –COOH from the m/z 179 fragments. When compared to previous findings, two compounds were identified as neochlorogenic acid and chlorogenic acid −) was generated by losing the hexoside from the precursor ion, and the ion m/z 109 ([C6H5O2]−) was generated by losing the –COOH from ion m/z 153. According to previous findings, compound 8 was tentatively identified as protocatechuic acid-O-hexoside, which could also be called 3,4-dihydroxybenzoic acid-O-hexoside − ion at m/z 289 and similar MS2 fragment ions at m/z 245, m/z 179, and 271 but varied in retention time. The ion m/z 245 ([C14H13O4]−) fragment was generated through losing the CO2, while the generation of the m/z 179 ([C9H8O4]−) fragment was caused by losing hydroquinone (–C6H6O2). The fragment at m/z 271 was obtained by dehydrating from the [M–H]− ion. According to previous findings, compound 11 was catechin, and compound 18 was epicatechin since the retention time of compound 11 was shorter than that of compound 18 − ion. Quinone–methide cleavage from the deprotonated molecular ion produced fragment ion at m/z 289 and then derived the ion m/z 245 by losing a carboxyl group. Thus, compounds 16 and 17 were identified as procyanidin dimer B-type isomers [Flavan-3-ol is a large group of flavonoids with a symbolic ion fragment at m/z 289, comprising catechin and epicatechin and their polymers and glycosides . Four flpound 18 ,46,55. C isomers .8H6O3. Compared with the database and previous literature, compound 19 was identified as quercetin 3-O-glucoside, and compound 20 was determined as quercetin 3-O-rhamnoside [Flavonol is another significant group of flavonoids, consisting of kaempferol, quercetin, isorhamnetin, and their glycosides . Ion m/zamnoside ,55. Howeamnoside .Actinidia deliciosa) (5.63 mg/g DW), and 40% higher than that of other Actinidia chinensis cultivars (4.27–4.34 mg/g dry mass) [Actinidia chinensis, while Actinidia arguta had the lowest level of ascorbic acid [The determination of ascorbic acid and major phenolic compounds in kiwifruit was conducted using HPLC–DAD by comparing the retention time and the calibration curves with those of standard compounds. The results are displayed in ry mass) ,62. The ry mass) . For exabic acid .O-glucoside and quercetin 3-O-rhamnoside). Among these nine compounds, procyanidin B2, neochlorogenic acid, and epicatechin were the three most abundant phenolic compounds with contents over 100 μg/g DW. Similar phenolic profiles were also reported in other yellow-fleshed kiwifruit cultivars [O-glucoside and quercetin 3-O-rhamnoside. ‘Jinfeng’ kiwifruit had a lower level of quercetin 3-O-rhamnoside than ‘Jinshi’ (4.59 µg/g DW) and ‘Jinyan’ (4.73 µg/g DW), while it had a higher level of quercetin 3-O-glucoside than ‘Hort 16A’ (0.45 µg/g DW) [In total, nine major phenolic compounds were detected and quantified, including three phenolic acids , four flavanols , and two flavonols (quercetin 3-ultivars . Neochloultivars . Four flultivars . Though ultivars ,63. In tUltrasound-assisted extraction has been optimized for recovering phytochemicals from ‘Jinfeng’ kiwifruit, and it resulted in an 18.5% increment in antioxidant activity compared with conventional extraction. Twenty phytochemicals were tentatively identified by UPLC–MS/MS, and one of them was identified for the first time and needed further confirmative study. In addition, nine phenolic compounds were quantified by HPLC–DAD, and procyanidin B2, neochlorogenic acid, and epicatechin were the three most abundant phenolic compounds. Though ‘Jinfeng’ kiwifruit showed antioxidant profiles similar to those of other kiwifruit cultivars, it exhibited a relatively high level of TPC, TFC, and ascorbic acid content. This work reveals the high antioxidant capacity of ‘Jinfeng’ kiwifruit and its major antioxidants. However, evaluations of the nutritional composition and other health benefits of this kiwifruit cultivar are limited, which require further study. In addition, the effects of different developmental stages and processing techniques on its nutritional value should be studied. Collectively, ‘Jinfeng’ kiwifruit is rich in antioxidant phytochemicals, can be a good source of dietary antioxidants, and can also be developed into functional foods with potential health benefits, which should be further investigated in the future.


Serial blood collections were performed during chemotherapy for measuring the amount and the global methylation pattern of cell-free DNA (cfDNA), the promoter methylation of SFRP2 and SDC2 genes, and the plasma homocysteine level. The average cfDNA amount was higher (p < 0.05) in nmCRC patients with recurrent cancer (30.4 ± 17.6 ng) and mCRC patients with progressive disease (PD) (44.3 ± 34.5 ng) compared to individuals with remission (13.2 ± 10.0 ng) or stable disease (12.5 ± 3.4 ng). More than 10% elevation of cfDNA from first to last sample collection was detected in all recurrent cases and 92% of PD patients, while a decrease was observed in most patients with remission. Global methylation level changes indicated a decline (75.5 ± 3.4% vs. 68.2 ± 8.4%), while the promoter methylation of SFRP2 and SDC2 and homocysteine level (10.9 ± 3.4 µmol/L vs. 13.7 ± 4.3 µmol/L) presented an increase in PD patients. In contrast, we found exact opposite changes in remission cases. Our study offers a more precise blood-based approach to monitor the treatment response to different chemotherapies than the currently used markers.Monitoring the therapeutic response of colorectal cancer (CRC) patients is crucial to determine treatment strategies; therefore, we constructed a liquid biopsy-based approach for tracking tumor dynamics in non-metastatic (nmCRC) and metastatic (mCRC) patients ( KRAS, BRAF, APC, or TP53, that can occur in the tumor tissue  .SFRP2 and SDC2 genes were selected. Duplex ddPCR assays were designed for the two genes using PyroMark Assay Design 2.0 software (Qiagen). Primer pairs were tested in silico by BiSearch software .Based on the previous results of our research group , two DNAsoftware . Two hydKRAS mutation status was determined from formalin-fixed, paraffin-embedded tissue by a pathologist as part of the treatment protocol. To monitor the alteration of the mutated KRAS copy number during the therapy, ddPCR analysis was performed on the plasma samples using the QX200 Droplet Digital PCR system (Bio-Rad). As negative controls, cfDNA samples of patients with wild-type KRAS tumors were also examined. KRAS G12/G13 Screening Kit (Bio-Rad) was applied that contains assays for detecting seven KRAS mutations , providing precise detection of the mutated copies with a sensitivity of 0.2%. The PCR was carried out in 22 µL reaction volume containing 11 µL ddPCR Supermix for Probes  (Bio-Rad), 1.1 µL multiplex primers/probes  (Bio-Rad), and 9.9 µL cfDNA template. The PCR protocol was the same as described above, except the annealing temperature was 55 °C. After reading the droplets, QuantaSoft Software (Bio-Rad) was used for data analysis, and the rate of mutated and wild-type copies was determined.in vitro assays at the Department of Laboratory Medicine, Semmelweis University. Chemiluminescent microparticle immunoassays were performed using Architect i2000SR . The normal reference values for CEA and CA 19–9 were set as 0–5 ng/mL and 0–37 U/mL, respectively. The amount of homocysteine was quantified in all blood samples with enzyme cycling assay using Roche Cobas C311 analyzer . The normal reference range differs between men (5.4–16.0 µmol/L) and women (4.4–13.5 µmol/L).The serum CEA and CA 19-9 markers were measured with p < 0.05) were assessed by the Kruskal–Wallis test, followed by Dunn’s test or one-way ANOVA followed by Tukey’s multiple comparisons test using Prism8 software . In paired comparisons, Wilcoxon matched-pairs signed rank tests or Student’s t-test with a significance criterion (p < 0.05) were performed with Prism8 software depending on non-normal or normal data distribution, respectively (GraphPad). In the case of unpaired comparisons, Mann–Whitney test or unpaired t-test was applied. Prism8 software (GraphPad) was used for ROC curve analysis and determination of sensitivity and specificity. After calculating Youden’s index, the cut-off value for the highest sensitivity was selected. Multivariable logistic regression was performed to predict the effect of mean percentage changes in the selected parameters  on disease outcome. Global DNA methylation was excluded from this analysis due to missing values. Additionally, as the KRAS mutant allele occurred in only 58% of the patients, the mutant allele frequency was also excluded from this calculation. To evaluate the goodness of fit of the statistical model, McFadden pseudo R2 was calculated, and the predictive performance of the parameters was measured by plotting ROC curves and calculating AUC.For the normality test, the Shapiro–Wilk test was used. Statistical significances (The present study offers the possibility to monitor the therapeutic response during chemotherapy with a minimally invasive blood-based method applying the alterations of cfDNA amount, global and local DNA methylation pattern, and HCY level. Digital PCR technique provides a highly sensitive method for examining genetic and epigenetic patterns; however, as KRAS mutation frequency is relatively low in CRC cases, its detection has limited diagnostic applicability. On the other hand, gene-specific methylation may become a more promising, widely used marker. This study also highlighted a possible connection between the elevated homocysteine concentration, the reduced global DNA methylation level, and the increased cell-free DNA amount in CRC patients with progressive disease.


Hedera helix (ivy) leaves at six locations exposed to different ambient air pollution conditions. Daily, we monitored ambient black carbon (BC), PM2.5, PM10, nitrogen dioxide, and ozone concentrations and found that ambient air pollution led to a 2–7-fold BC increase on leaves, the phylloplane BC load. Our results further indicated that the phylloplane BC load correlates with the diversity of bacterial and fungal leaf communities, impacting diversity more than seasonal effects. The bacterial genera Novosphingobium, Hymenobacter, and Methylorubrum, and the fungal genus Ampelomyces were indicators for communities exposed to the highest phylloplane BC load. Parallel to this, we present one fungal and two bacterial phylloplane strains isolated from an air-polluted environment able to degrade benzene, toluene, and/or xylene, including a genomics-based description of the degradation pathways involved. The findings of this study suggest that ambient air pollution shapes microbial leaf communities, by affecting diversity and supporting members able to degrade airborne pollutants.Ambient air pollution exerts deleterious effects on our environment. Continuously exposed to the atmosphere, diverse communities of microorganisms thrive on leaf surfaces, the phylloplane. The composition of these communities is dynamic, responding to many environmental factors including ambient air pollution. In this field study, over a 2 year period, we sampled  Ninety-one percent of the world’s human population is currently living in places where air pollution parameters exceed World Health Organization (WHO) guideline limits . It is m2 [6–107 cells·cm−2 [Always in contact with the surrounding air, abundant and diverse communities of microorganisms naturally exist on the surface of above-ground parts of plants, known as the phyllosphere . The phy2 , the phylls·cm−2 . Most stlls·cm−2 .Hedera helix (ivy), an evergreen plant known for its hardiness and climbing ability [H. helix has a widespread distribution in the northern hemisphere in diverse environments such as private gardens, city centers, municipal parks, nature reserves, and forests, and it shows a high capacity to take up and filter pollutants out of the air [H. helix leaves at six locations exposed to different ambient air pollution conditions. We characterized these locations daily by monitoring ambient BC, PM2.5, PM10, nitrogen dioxide, and ozone concentrations. Furthermore, we measured phylloplane BC load and carried out extensive biennial metabarcoding surveys. In addition, we here present three phylloplane microorganisms isolated from an air-polluted environment that are able to degrade benzene, toluene, and xylene, including a genomics-based description of the degradation pathways involved.It is evident that ambient air pollution not only poses hazards to humans, animals, and plants, but also influences phylloplane microbial communities, as leaf surfaces are continuously exposed to the atmosphere. However, to date, there has been little research done on the extent and nature of this influence. Here, we deployed metabarcoding to study the effects of ambient air pollution in the field on bacterial and fungal communities in the phylloplane of  ability . H. heli the air . Over eiH. helix plants. WGS84 coordinates were as follows: 50.936546, 5.317226 (HGa); 50.917344, 5.326918 (HGb); 50.934242, 5.337944 (HKa); 50.928680, 5.332674 (HKb); 50.940104, 5.438675 (DM); 50.921694, 5.433951 (DW). HGa and HGb are located on the outer city ring road of Hasselt, HKa and HKb are located on the inner city ring road, and DM and DW are located in two close-by nature reserves . Permission for sampling was obtained, and it was performed in accordance with institutional and international guidelines. Black carbon (BC), PM2.5, PM10, nitrogen dioxide, and ozone concentrations were monitored daily by the Belgian Interregional Environment Agency from 1 August 2017 until 31 August 2019 (n = 761) and calculated for every sampling site using the RIO–IFDM model [2.5 and PM10, 78% for nitrogen dioxide, and 65% for ozone [t-tests with Bonferroni correction. Temperature and precipitation were also monitored daily for this period by an automated weather station owned by the Royal Meteorological Institute of Belgium .We selected six sampling sites around Hasselt, Belgium harboring stable and healthy populations of reserves . The soin = 3840; 80 per site, per sampling round) from H. helix plants ranging in age from 3–6 months were collected during eight sampling events, one per season, over the course of two seasonal cycles (years): 14 November 2017 (I); 5 February 2018 (II); 14 May 2018 (III); 1 August 2018 (IV); 5 November 2018 (V); 19 February 2019 (VI); 21 May 2019 (VII); 23 August 2019 (VIII). Plant leaves were identified as specimens belonging to H. helix by the first author; voucher specimens are available from Hasselt University. Leaves were cut from the plants at shoulder height using sterile forceps, put into sterile tubes (five leaves per tube) filled with phosphate buffer , and immediately transferred to the laboratory. Leaf weight was determined gravimetrically, and microbial cells were detached from the leaf surface by sonication , followed by shaking on an orbital shaker . Next, resulting leaf wash suspensions  were centrifuged , and the resuspended pellets were pooled into four samples per site, per sampling round, resulting in a total of 192 samples. Samples were immediately stored at −80 °C until DNA isolation.Leaves  was collected as previously described, except the resuspended pellets were not pooled, resulting in a total of 96 samples . Leaf wash suspensions were immediately stored at −20 °C until BC detection.An additional set of n = 96) were detected using a specific and sensitive detection technique based on the non-incandescence-related white light (WL) generation of the particles under femtosecond-pulsed illumination, as previously described [3 and a pixel dwell time of 1.54 µs. In total, 175 images per perfusate sample were obtained by recording z-stacks with a 1 µm slice thickness at five different locations in the aliquot (n = 480). The images were acquired using ZEN Black 2.0 software .BC particles present in leaf wash suspensions . First, a peak-searching algorithm counted pixels above a threshold value. Here, threshold values of 0.1% lower than the highest pixel intensity value of the narrow second-harmonic generation channel (405/10) and two-photon-excited autofluorescence channel (550/200) were chosen. Next, the thresholded pixels of both images were compared, and only the overlapping ones were counted as BC particles. The average number of particles detected in the washing sample z-stacks was normalized to the imaging volume, and the results were expressed as the number of detected BC particles per g leaf material. Differences in mean values between sampling sites were analyzed by performing ANOVA and multiple n = 192) were centrifuged , and genomic DNA was isolated using a NucleoSpin Soil kit . Initially, two different sequencing libraries were prepared, one for bacterial and one for fungal phylloplane metabarcoding. The bacterial sequencing library was constructed by PCR amplification of the V3–V4 hypervariable region of the 16S rRNA gene using 341F (5′–TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG–3′) and 785R (5′–GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC–3′) primers [TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGARTCATCGARTCTTTGAA–3′) and ITS4R (5′–GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCTCCGCTTATTGATATGC–3′) primers [2, 0.2 µM dNTP mix, 0.05 U·µL−1 enzyme blend , 0.2 µM of each primer, 5% DMSO, and 1 µL of DNA template. Amplification conditions were as follows: 95 °C for 2 min; 25 cycles of 95 °C for 30 s, 54/58 °C  for 30 s, 72 °C for 1 min; held at 72 °C for 6 min. PCR products were purified using AMPure XP beads  and subsequently indexed (n = 384) using the Nextera XT index kit . The 25 µL index PCR reaction consisted of 1× reaction buffer with 1.8 mM MgCl2, 0.2 µM dNTP mix, 0.05 U·µL−1 enzyme blend , 0.2 µM of each index primer, and 1 µL of purified PCR product. Amplification conditions were as follows: 95 °C for 2 min; 14 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min; held at 72 °C for 6 min. After another round of purification, the DNA concentration of every indexed sample was quantified with a Qubit dsDNA HS assay kit and the Qubit 2.0 fluorometer  prior to equimolar pooling of the samples. Correct amplicon size and integrity were checked on an Agilent 2100 Bioanalyzer system , followed by sequencing using a MiSeq Reagent Kit v3 on a MiSeq system  at Hasselt University, Belgium. When preparing the sequencing libraries, a mock community containing eight bacterial and two fungal strains was included to assess sequencing biases, errors, and other artefacts .Leaf wash suspensions ( primers  containi primers  includint-tests with Benjamini–Hochberg correction. Before inter-sample (beta) diversity was measured, counts in every sample were scaled by n = 190/192 (bacteria/fungi). Principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) and was performed on Bray–Curtis dissimilarity matrices, and different outcomes were tested by geometric partitioning of variation with analysis of similarities (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA), using vegan 2.5–7 (R package). Differentially abundant taxa between designated categories were identified by linear discriminative analysis (LDA) with effect size [p < 0.05) differential abundance between the indicated categories. Next, LDA was used to estimate the effect size of each differentially abundant taxon; only LDA log10 scores ≥2 were considered. All mentioned data handling, statistics, and creation of plots  were done within R 4.1.0 [Demultiplexed metabarcoding data were received in FASTQ format with all additional nucleotides removed except the 341F/785R (bacteria) or gITS86F/ITS4R (fungi) primers, and they were further processed with DADA2 1.18.0 . Primer ect size , where t R 4.1.0 .n = 300) of H. helix phylloplane isolates for their potential to metabolize diesel fuel (DF). Individual cultures were grown for 24 h in LB medium [600 nm) was reached between 0.8 and 1.2, pelleted by centrifugation, washed three times, and resuspended in 0.01 M phosphate-buffered saline (PBS). Culture suspensions were left overnight at 30 °C on a shaker (150 rpm). The next day, 80 µL of the suspensions were transferred to 750 µL of Bushnell–Haas (BH) medium [−1 2,6-dichlorophenolindophenol (DCPIP) and 5 µL of polytetrafluoroethylene (PTFE) filter-sterilized 0.45 µM DF as the sole carbon source [n = 200) of this collection containing isolates sampled from HGa, HKb, DM, and DW was previously described [n = 100), comprising isolates sampled only from HKb (isolated in the same way as described before [v/v) glycerol and 0.15 M NaCl, and stored at −45 °C.First, we screened a collection (B medium  at 30 °C) medium  with then source . After iescribed  and resuescribed ,38, bothd before , but usi600 nm) was reached between 0.8 and 1.2, pelleted by centrifugation, washed three times, and resuspended with BH medium. Using 20 mL headspace GC–MS vials, benzene, toluene, or ortho-, meta-, para-xylene was added to one series of BH medium and one series of BH medium with additional macronutrients  resulting in a final concentration of 20 mg·L−1 benzene, toluene, or xylene in 8 mL of medium including 800 µL of bacterial suspension. Vials were incubated in the dark for 10 days at 30 °C on a shaker (100 rpm). With regard to GC–MS analysis, samples were incubated at 80 °C for 20 min, and 1 mL of the headspace was injected into a TRACE 1310 gas chromatograph coupled to an ISQ single quadrupole GC–MS system .Our screening effort resulted in six DF metabolizers which were further analyzed for their potential to degrade benzene, toluene, and xylene (BTX) using headspace gas chromatography–mass spectrometry (GC–MS). Individual cultures were grown for 24 h in LB medium at 30 °C on a shaker (150 rpm) until a culture density ; then, genomic DNA was isolated from the pellets using the DNeasy Blood & Tissue kit . The DNA concentration was quantified with a Qubit dsDNA HS assay kit and the Qubit 2.0 fluorometer . The Nextera DNA Flex library preparation kit along with the Nextera DNA CD indices  were used to tagment the DNA libraries for sequencing following the manufacturer’s instructions. Tagmentation was performed with 100 ng of genomic DNA, and amplification conditions were as follows: 68 °C for 3 min; 98 °C for 3 min; five cycles of 98 °C for 45 s, 62 °C for 30 s, 68 °C for 2 min; 68 °C for 1 min. The DNA concentration of every indexed sample was quantified as previously described prior to equimolar pooling of the samples. Library size distribution was checked on an Agilent 2100 Bioanalyzer system , followed by sequencing on a HiSeq X Ten system  by Macrogen .We sequenced the genomes of two bacteria and one yeast that were able to degrade benzene, toluene, and/or xylene. Individual cultures were grown for 24 h in LB medium at 30 °C on a shaker (150 rpm) and centrifuged  at six sampling sites  differences were observed for the six sampling sites for a number of parameters  at HGa, followed by HGb and HKa,b. Conversely, the lowest concentrations for these parameters were detected at DM and DW, sites where O3 concentrations were highest. We also measured phylloplane BC load during each of the eight sampling events (2 (and lower O3 concentrations) and, to a lesser extent, PM2.5 and PM10 correlate with a 2–7-fold increase in phylloplane BC load, other characteristics of the sampling sites appeared to have an influence. Notably, ambient air pollution was highest at HGa,b, while HKa,b had the highest phylloplane BC load.We monitored ambient BC, PMng sites a–e. We ang sites . A cycliectively . Overallrameters . For exag events . Here, wH. helix plants, growing at six locations with different phylloplane BC loads due to differences in ambient air pollution, resulted in a dataset of 190/192 (bacteria/fungi) samples including 1,888,912 16S rRNA V3–V4/5,167,679 ITS2 sequences covering 15,610/1685 amplicon sequence variants (ASVs). Rarefaction analysis indicated all samples were sufficiently sequenced to represent their true biological identity  diversity of bacteria and fungi by calculating two metrics: the observed number of ASVs and the Shannon diversity index. Bacterial alpha diversity was lower in samples with low phylloplane BC load (bcL), compared to samples with medium and high BC load  (p < 0.001) distinctive groupings depending on phylloplane BC load for both bacterial . Moreover, the calculated effect sizes show that the BC load-correlating shift on fungal diversity was greater than on bacterial diversity. Such distinctive groupings were absent for seasonal categorizations (p < 0.001), albeit with much lower effect sizes than those of phylloplane BC load . Our results show that phylloplane BC load, which was increased by ambient air pollution by 2–7-fold, strongly correlated with the diversity of bacterial and fungal H. helix leaf communities, and had more impact than seasonal effects, over a 2 year investigation period.Our metabarcoding effort of the bacterial and fungal phylloplane of identity . To testctively) a. On thectively) d. Next, acterial b,c and facterial e,f H. heizations . Howeverp < 0.05) differentially abundant taxa . Subtercola, Diplorickettsia, Kaistia, Caulobacter, Terriglobus, Apiognomonia, Lalaria, Gibellulopsis, Melanconiella, and Phyllactinia were associated with communities with medium phylloplane BC load (bcM). Rahnella, Micropruina, Commensalibacter, Pseudocercosporella, Hypholoma, Heterobasidion, Gymnopilus, Herpotrichia, Boletus, Dicarpella, and Cryptosporella were indicators for communities with low phylloplane BC load (bcL). Indicators at higher taxonomic levels  were also identified. Class Cytophagia was an indicator for bcH phylloplane communities. Families Coxiellaceae, Kaistiaceae, and Plectosphaerellaceae, orders Streptosporangiales, Legionellales, Opitutales, and Taphrinales, and classes Clostridia, Opitutae, and Taphrinomycetes were associated with bcM phylloplane communities. Lastly, families Sphingomonadaceae, Strophariaceae, Bondarzewiaceae, Cortinariaceae, Gnomoniaceae, Lophiostomataceae, Boletaceae, and Valsaceae, orders Caulobacterales, Russulales, and Boletales, and class Agaricomycetes were indicators for bcL phylloplane communities.To better understand the nature of these observed diversity shifts, we identified indicators by analyzing for significantly . Our screening effort resulted in six DF metabolizers that were all isolated from bcH samples (HKb). We further analyzed these isolates for their potential to degrade benzene, toluene, and xylene (BTX). Two bacteria, Bacillus licheniformis VSD4 and Pseudomonas sp. VS38, were each able to completely degrade 20 mg·L−1 benzene and toluene in 10 days  xylene. Three of the diesel fuel metabolizers, Rhodococcus erythropolis VSD3, Pseudomonas sp. VS40, and Pseudomonas sp. VS59, were not able to notably degrade BTX. To better understand which degradation pathways are involved in the observed BTX degradation, we sequenced the genomes of B. licheniformis VSD4, Pseudomonas sp. VS38, and Rhodotorula sp. VS67  related to these BTX degradation pathways were found in the genome of Rhodotorula sp. VS67.Given the impact of ambient air pollution on bacterial and fungal ivy leaf communities, it is reasonable to suggest that phylloplane microorganisms may be able to degrade common airborne pollutants. Therefore, we screened a collection of 300  10 days a,b. Howe 10 days c. Rhodotsp. VS67 . We assieactions . The pre3 and NOx, the levels of O3 and NO2 are inextricably linked, and any resultant reduction in the level of NO2 is invariably accompanied by an increase in the level of O3, and vice versa [Due to the chemical coupling of Oce versa . HKa,b ace versa . Previouce versa ,51,52, wce versa .H. helix, we assessed the intra-  and inter-sample (beta) diversity. While fungal alpha diversity was similar under all three phylloplane BC load conditions, alpha diversity was highest for bacterial communities present on leaves with medium and high phylloplane BC loads exposed to greater ambient air pollution. This corroborates with a study on bacterial phylloplane communities of Platanus × acerifolia trees in Antwerp, Belgium, which showed that alpha diversity was twice as low in the phylloplane sampled in urban green and waterfront locations than adjacent to busy roads, urban residential areas, and industrial sites [Carpinus betulus trees across three locations, (i) the city center of Warsaw, Poland, (ii) a forest in a UNESCO World Heritage Site (Białowieża), (iii) and a forest in one of the world’s oldest operational oil fields (Bóbrka), alpha diversity was again highest in the most urban sampling location, the city center of Warsaw [H. helix leaf communities. Other studies have shown that plants often have microbial leaf communities exhibiting high seasonal variability [H. helix in Antwerp, Belgium also found that leaf communities differed greatly between urban and nonurban locations, where traffic-generated PM was lower [Platanus × hispanica trees suggested that a discriminating environmental effect is related to ultrafine particulate matter (UFP) and BC deposited on leaves [Novosphingobium, Hymenobacter, and Methylorubrum, and the fungal genus Ampelomyces were indicators for communities with the highest phylloplane BC load. Hymenobacter was shown to dominate the urban phylloplane in two other studies [Ampelomyces quisqualis was noted, with various intensity, in north-eastern Poland on different species of Erysiphales selected as potential indicators of urban pollution [To test if phylloplane BC load influences the bacterial and fungal leaf microbiome of al sites . Anotheral sites . In a stf Warsaw . While tiability ,59,60,61as lower , and anon leaves . With ren leaves . Here, w studies ,62, and ollution .B. licheniformis VSD4 and Pseudomonas sp. VS38, and one yeast, Rhodotorula sp. VS67, isolated from an air-polluted environment that are effectively able to degrade benzene, toluene, and/or xylene. Genome analyses revealed genes for the meta-cleavage of catechol for B. licheniformis VSD4 and for the ortho-cleavage of catechol for Pseudomonas sp. VS38. Degradation pathways involving catechol meta-cleavage are well-documented for several Bacillus strains [B. licheniformis [Bacillus strains are reported as BTX degraders, e.g., B. subtilis DM-04 [B. amyloliquefaciens W1 [Pseudomonas strains are famous for their BTX degradation potential, including P. putida F1 [P. putida YNS1 [P. stutzeri OX1 [Pseudomonas spp. VI4.1 and VI4T1 [Rhodotorula sp. VS67, a red yeast, does not have the genetic basis to facilitate degradation mechanisms related to the meta- and ortho-cleavage pathways of catechol. It remains unclear via what mechanism it was able to degrade xylene as the sole carbon source. Since it was able to completely degrade benzene  only when other carbon sources where present, co-metabolization by monooxygenases is most likely involved [Cladophialophora sp. T1 was able to degrade toluene and xylene via co-metabolization [Cladophialophora immunda CBS 110551 associated the initial oxidation of the methyl-group of toluene with catalyzation by a membrane-bound cytochrome P450 [From a three-stage experiment comprising (i) isolation and (ii) screening of phylloplane microorganisms for diesel fuel metabolization, and (iii) measurements of BTX degradation, we here present two bacteria,  strains ,67,68, iniformis . Howeveris DM-04  and B. aciens W1 . On the utida F1 , P. putiida YNS1 , P. stutzeri OX1 , and Psend VI4T1 . Our anainvolved . The bioinvolved . For exalization . Transcrome P450 ,80.H. helix leaves at six locations exposed to different ambient air pollution conditions. We showed that ambient air pollution increases the phylloplane black carbon (BC) load 2–7-fold, and that this BC load strongly correlates with the diversity of bacterial and fungal H. helix leaf communities, impacting this diversity even more than seasonal effects. We further identified indicators for communities with high, medium, and low phylloplane BC load. Parallel to this, we present one fungal and two bacterial phylloplane strains isolated from an air-polluted environment able to degrade BTX, common airborne pollutants, including a genomics-based description of the degradation pathways involved. The findings of this study indicate that ambient air pollution contributes to shaping bacterial and fungal ivy leaf communities by impacting the diversity and supporting a community which includes members able to degrade airborne pollutants.Ambient air pollution has wide-ranging and deleterious effects on our environment and is a major issue for the global society. It is evident that air pollution also influences phylloplane microbial communities, as leaf surfaces are continuously exposed to the atmosphere, but the extent and nature of this influence remains unclear. In this field study, over a 2 year period, we sampled


Euglena gracilis, such as polysaccharide paramylon, exhibit antimicrobial, anti-viral, antitumor, and anti-inflammatory activities. Whether live E. gracilis cells and its aqueous extract accelerate burn wound healing remains to be investigated. In this study, live E. gracilis cells and its aqueous extract were mixed with chitosan-hyaluronic acid hydrogel (CS/HA) to form cell + CS/HA and extract + CS/HA, which were then smeared onto the deeply burned skin of mice. The efficacy of these mixtures in accelerating wound healing was assessed through wound size reduction measurement, histological and immunofluorescence analyses, and serum pro-inflammatory cytokine level  determination. The live E. gracilis cells and its aqueous extract were found to facilitate wound healing by enhancing re-epithelization and reducing fibroplasia without stimulating excessive inflammatory response. In conclusion, live E. gracilis cells and its aqueous extract can be potentially used to treat cutaneous wounds.Naturally occurring compounds isolated from the microalga  Synechococcus elongatus PCC7942, accelerates wound healing by promoting angiogenesis  and Th 2 cell responses  in lymphomonocytes and has an immune-activating effect (Saccharomyces cerevisiae) are sensed by sophisticated cooperating pathways through both membrane-bound and cytosolic pattern recognition receptors (PRRs), resulting in robust activation of IL-1β-mediated inflammatory response in human primary macrophages. In this study, immunosuppressive effect of live Euglena gracilis cells and its aqueous extract for facilitating skin wound healing was investigated in an animal model. The expression of three important pro-inflammatory mediators of antibody responses along different pathways such as IL-6, IFN-γ, and IL-1β was evaluated.Previous studies have reported that β-1,3-D-glucan modulated the Th1 and/or the Th2 cell response in experimental animals and human patients of allergic rhinitis and digestive cancers , 2019. Lesponses . Serum lg effect . KankkunE. gracilis is a unicellular green microalga with flagellar motility  possess antimicrobial, antiviral, and antitumor properties (Euglena activates cellular metabolism and reduces the signs of aging and cutaneous fatigue (US patent US8741357B2)  . The aute health . Hence, E. gracilis cells, and its aqueous extract into CS/HA in a 1:1 volume ratio. Sonicated and alkalized paramylon, extracellular vesicle, live E. gracilis cells, and its aqueous extract were uniformly distributed in CS/HA. Cells survived in CS/HA after a 24-h incubation in an illuminating incubator without shaking at 37°C (data not shown). Keeping CS/HA as the control group, the aforementioned four groups were smeared onto the deeply burned skin of mice. Wound size reduction was calculated as follows: wound size reduction (%) = (A0 − At)/A0 × 100, where A0 is the initial wound area and At is the wound area at day 14 after wounding  has good biocompatibility, and therefore, it can be used as a delivery device not only for mobilizing stem cells to the injection site, but also for sustainable release of bioactive molecules or growth factors . CS/HA cwounding . ResultsE. gracilis cells were grown in the EM medium under a light intensity of 100 μmol/m2/s in an illuminating incubator without shaking at 26°C until the cells reached the stationary phase 3, 60 μl HCl, 0.01 mg/L Vb1, 0.0005 mg/L Vb12, 20 mg/L CuSO4·5H2O, 0.4 g/L ZnSO4·7H2O, 1.3 g/L Co(NH3)·H2O, and 1.6 g/L MnCl2·4H2O.ry phase . The medE. gracilis culture medium was incubated for 1 week, and then, 1 L of this medium was centrifuged at 1,000 × g for 4 min and collected. Cell precipitates were rinsed three times with distilled water and resuspended in 1× phosphate buffered saline (PBS) at 37°C. After cultivating the cells for 1 week, the aqueous extract was isolated from 1 L E. gracilis culture medium. Briefly, the culture was centrifuged at 7,000 × g for 3 min, and the cell pellets were rinsed three times with distilled water and then ultracentrifuged at 10,000 × g for 5 min three times. An ultrasonic cell pulverizer  was used as an emulsified dispersion device (70% power). The precipitated cells with 20 volumes of 1× PBS were ultrasonically treated for 8 min. The device adopts a working time of 48 s and intermittent 12 s, which can effectively prevent temperature increase and improve emulsion efficiency. The cellular debris was removed using a 0.22-μm Millipore filter.The Chitosan-hyaluronic acid hydrogel was prepared according to the method of The Animal Research Committee of the Health Science Center of Shenzhen University approved all experimental procedures. Successful skin wound healing involves a series of events with complex cell signaling cascades that coordinate several fundamental biological processes . Re-epitIn total, 22 female 8-week-old BALB/c mice (weight: 16–20 g) were anesthetized through intraperitoneal injection of 4% chloral hydrate (1 ml/100 g). After shaving the mice, two dorsal wounds were symmetrically clipped out using a copper bar (diameter: 1 cm) punch, which was heated using a water bath at 95°C.6 live E. gracilis cells mixed with CS/HA in 1:1 volume ratio), extract + CS/HA (0.05 g/ml aqueous extract from E. gracilis mixed with CS/HA in 1:1 volume ratio), and CS/HA (control group). A total of 42 wound sites (14 wound sites/group) were analyzed.The mice were randomly assigned to three groups: cell + CS/HA  was fixed in a 4% paraformaldehyde fix (PFA) solution, dehydrated with a graded alcohol series, embedded in paraffin, sectioned (section thickness: 4 μm) perpendicularly to the wound surface, and stained with hematoxylin and eosin (H&E). Masson’s trichrome staining was used to determine the degree of collagen maturity.To identify vascular structures, immunofluorescence histochemistry was performed for an endothelial cell marker, CD31. For immunofluorescence staining, skin excised from the dorsal wound sites was fixed in 4% PFA , dehydrated in a 30% sucrose solution, embedded in optimal cutting temperature compound (OCT), and sectioned (section thickness: 4 μm) perpendicularly to the wound surface. Tissue sections were blocked in 5% BSA for 30 min at room temperature and incubated with rabbit CD31 monoclonal antibody  overnight at 4°C . Images E. gracilis cells and its aqueous extract was examined by investigating serum pro-inflammatory cytokine levels through enzyme-linked immunosorbent assay (ELISA). In total, 15 blood samples (5 samples/group) were obtained from the inferior vena cava of the mice under anesthesia on day 14. IL-1β (cat no. 88-7013) and IL-6 (cat no. 88-7064) levels were measured using the ELISA Development Kit, according to the manufacturer’s protocols . Each sample was measured in duplicate, and cytokine concentration was calculated on the basis of standard curves provided with the kits. The results are expressed in pg/ml. IFN-γ (cat no. JM-02465M1) was measured using the ELISA Development Kit, according to the manufacturer’s protocols . Because the IFN-γ level is excessively high, each sample was diluted 5-fold, and the results were multiplied by the dilution factor (5-fold). As serum available was insufficient, IL-6 concentration was detected for one sample in each group.The effect of live p value less than 0.01 (p < 0.01) was considered extremely significantly different, p < 0.05 was statistically significant.Statistical significance was evaluated using GraphPad Prism , and statistical analysis was performed using analysis of variance (ANOVA) followed by the Tukey’s test for post-hoc analysis. Gross observations revealed an increase in size reduction of wounds treated with cell + CS/HA and extract + CS/HA compared with those treated with CS/HA at 14 days post-wounding . Wounds Reduced scar length and increased collagen maturity were used to assess the wound healing degree . Both ceDermal fibroblasts could secrete collagen and promote collagen deposition in the healing process . In the Masson’s trichrome staining showed that wounds treated with cell + CS/HA  and extrThe effect of cell + CS/HA, extract + CS/HA, and CS/HA applications on serum pro-inflammatory cytokines in the mice was investigated through ELISA. Cell + CS/HA and extract + CS/HA did not induce notable changes in IFN-γ and IL-1β  levels. E. gracilis cells and its aqueous extract accelerated wound healing in the mice without inducing excessive inflammatory response. The cell + CS/HA and extract + CS/HA treatments led to greater wound size reduction and re-epithelialization, lesser degree of fibroplasia, and enhancement of new blood vessel formation in the wound bed. Both cell + CS/HA and extract + CS/HA treatments did not induce excessive inflammatory response, as revealed by no notable changes in IFN-γ and IL-1β concentrations. Moreover, inflammatory cells were not present in the dermis. Our findings that live E. gracilis cells and its aqueous extract facilitate wound healing without excessive immune responses are inconsistent with those obtained with paramylon of E. gracilis, which accelerates wound healing through its anti-inflammatory effect  , and it also triggers the release of intracellular calcium from its storage site (showing the switch of a cell from the resting state to the activated state) (US patent US8741357B2)  . Short peptides have also been used as active elements for the detection of their own receptors (Euglena extract and their functions are being preceded to verify this hypothesis.Although no study has reported the application of live 41357B2) . The aqu41357B2) . The aut41357B2) . Furthervitality . A high is cells . In our eceptors . For exain vivo and in vitro. Pharmacological inhibition of STAT3 also inhibited expression of RANKL in osteoblastic cells induced by IL-1β, TNF-α, and IL-6 in vitro as well as in the joints of a collagen-induced arthritis (CIA) model in vivo  is the key mediator of both chronic inflammation and joint destruction in rheumatoid arthritis (RA). The major pro-inflammatory cytokines in RA, TNF-α, IL-6, and IL-1 induced STAT3 activation either directly or indirectly and stimulated expression of IL-6 family cytokines and receptor activator of nuclear factor kappa B ligand (RANKL) in an autocrine/paracrine manner  in vivo . In thisE. gracilis cells and its aqueous extract. The molecules that mediate the different effects of live E. gracilis cells and its aqueous extract on the proliferation and migration of endothelial cells, keratinocytes, and fibroblasts also remain unknown. Thus, live E. gracilis cells and its aqueous extract may also be selectively enriched in some functional molecules that mediate their regulatory effects, although this approach requires further exploration.A limitation of our study is that we did not identify the molecular mechanism underlying the pro-angiogenic and pro-wound healing effects of live E. gracilis cells and its aqueous extract can facilitate wound healing without stimulating excessive inflammatory response during the healing process. This study is the first to provide evidence for the potential of live E. gracilis cells and its aqueous extract in treating cutaneous wounds. The successful wound healing in mice suggests the potential use of E. gracilis in humans for wound care.Our findings suggest that live