Gordonia cholesterolivorans. Strain A16 exhibited superior tetramethrin degradation activity, and utilized tetramethrin as the sole carbon source for growth in a mineral salt medium (MSM). High-performance liquid chromatography (HPLC) analysis revealed that the A16 strain was able to completely degrade 25 mg·L−1 of tetramethrin after 9 days of incubation. Strain A16 effectively degraded tetramethrin at temperature 20–40 °C, pH 5–9, and initial tetramethrin 25–800 mg·L−1. The maximum specific degradation rate (qmax), half-saturation constant (Ks), and inhibition constant (Ki) were determined to be 0.4561 day−1, 7.3 mg·L−1, and 75.2 mg·L−1, respectively. The Box–Behnken design was used to optimize degradation conditions, and maximum degradation was observed at pH 8.5 and a temperature of 38 °C. Five intermediate metabolites were identified after analyzing the degradation products through gas chromatography–mass spectrometry (GC-MS), which suggested that tetramethrin could be degraded first by cleavage of its carboxylester bond, followed by degradation of the five-carbon ring and its subsequent metabolism. This is the first report of a metabolic pathway of tetramethrin in a microorganism. Furthermore, bioaugmentation of tetramethrin-contaminated soils (50 mg·kg−1) with strain A16 (1.0 × 107 cells g−1 of soil) significantly accelerated the degradation rate of tetramethrin, and 74.1% and 82.9% of tetramethrin was removed from sterile and non-sterile soils within 11 days, respectively. The strain A16 was also capable of efficiently degrading a broad spectrum of synthetic pyrethroids including D-cyphenothrin, chlorempenthrin, prallethrin, and allethrin, with a degradation efficiency of 68.3%, 60.7%, 91.6%, and 94.7%, respectively, after being cultured under the same conditions for 11 days. The results of the present study confirmed the bioremediation potential of strain A16 from a contaminated environment.Tetramethrin is a pyrethroid insecticide that is commonly used worldwide. The toxicity of this insecticide into the living system is an important concern. In this study, a novel tetramethrin-degrading bacterial strain named A16 was isolated from the activated sludge and identified as  Tanacetum cinerariifolium [Natural pyrethrin insecticides were produced by iifolium . Naturaliifolium . Their miifolium . Synthetiifolium . The syniifolium . The cheiifolium .−1, with a hatching rate of only 9% [However, pyrethroid insecticides also have some side effects on the environment. Previous studies on their metabolic pathways and hazards in the environment have mostly focused on soil. With the widespread use of pyrethroid insecticides, they can enter the natural water cycle through surface water flow, infiltration through soil, and via other routes, causing pollution to domestic water and riverbed sediments, and leaving serious indoor residues . As a sy only 9% . As a ch only 9% . Differe only 9% . For exa only 9% . Previou only 9% .H-isoindol-2-yl)methyl ester) is a popular type I pyrethroid. The continuous use of tetramethrin and its environmental residues have caused serious concern. Therefore, it is urgent to develop effective strategies to solve the problems related to tetramethrin residues. Microbial degradation of organic pollutants in the environment has attracted widespread attention [Bacillus subtilis 1D, Photobacterium ganghwense 6046, Acinetobacter baumannii ZH-14, and Paracoccus acridae SCU-M53, among others [Environmental problems caused by the large-scale use of pyrethroid insecticides have become increasingly serious. Tetramethrin cyclopropanecarboxylic acid  to isolate and characterize the tetramethrin-degrading strain from activated sludge, (ii) to optimize the process parameters for tetramethrin degradation using response surface methodology, (iii) to test the degradation kinetics of tetramethrin and other pyrethroids, (iv) to identify the intermediate metabolites formed during tetramethrin degradation, and (v) to estimate the bioremediation potential of strain A16 in water and soil environments.Gordonia cholesterolivorans. Thus, strain A16 was identified as G. cholesterolivorans strain A16. The phylogenetic tree of strain A16 is represented in A bacterial strain that was able to degrade the tetramethrin and other pyrethroids was isolated and characterized using physical, chemical, and microbiological methods. The potential strain was designated as A16. Strain A16 not only degraded the tetramethrin, but was also found to effectively degrade other pyrethroids such as prallethrin, fenvalerate, bifenthrin, beta-cypermethrin, allethrin, and permethrin. The colonies of the degrading strain A16 on beef extract peptone solid medium were white and yellow, and the single colonies were circular. The color from the middle to the edge became lighter, and its edges were irregular. The colony surface was translucent. Strain A16 cells were rod-shaped, without spores and flagella . The genGordonia species showed potential to degrade other organic pollutants [Gordonella has not been reported to degrade pyrethroid pesticides, so there is still room for exploration and development of the biodegradation ability of pyrethroid pesticides by Gordonella. In addition, Bacillus, Pseudomonas, Raoultella, Brevibacterium, Aspergillus, Acinetobacter, Candida, and Trichoderma have been characterized on the basis of biochemical and molecular tools for their pyrethroid degradation potential in soil and water environments [Gordonella bacterial strains participate in highly effective degradation of tetramethrin and other pyrethroids.Previous researchers reported that the llutants ,26,27. Hronments ,33,34,35The growth-linked degradation study clearly showed that as the growth of bacterial strain A16 increased over time, the overall concentration of tetramethrin present in the medium decreased . CompareBacillus sp. FA4 degraded fipronil pesticide (50 mg·L−1), as the bacterial cell density increased in the pesticide supplemented medium under optimized conditions [A growth-linked biodegradation study was also conducted previously and reported that bacterial strain nditions . In a minditions . The mecnditions . Pesticinditions .−1) by strain A16 showed that it can tolerate high concentrations and rapidly degrade tetramethrin. As shown in −1, 50 mg·L−1, 100 mg·L−1, 200 mg·L−1, 400 mg·L−1, and 800 mg·L−1 of tetramethrin, the degradation efficiency was 100%, 95.1%, 89.6%, 72.6%, 65.0%, and 56.7%, respectively. When the concentration of tetramethrin was 25 mg·L−1, the degradation efficiency reached 95.7% after 7 days of incubation, and it was completely degraded after 9 days of incubation. The results showed that strain A16 had a good degradation effect at low concentrations (<100 mg·L−1). However, with the increase of the concentration of tetramethrin, the degradation rate decreased, which might be due to the strong toxicity of high concentrations of tetramethrin.The degradation kinetics of different concentrations of tetramethrin (25–800 mg·LR2 = 0.9424), and the maximum specific degradation rate (qmax) of tetramethrin was 0.4561 day−1. The half rate constant (Ks) was 7.3 mg·L−1 and the inhibition coefficient (Ki) was 75.2 mg·L−1. The Andrews equation was then further derived, and the corresponding concentration (Smax) of qmax was 23.5 mg·L−1, which was the best predicted concentration for the degradation of tetramethrin by strain A16.The results showed that the degradation efficiency of strain A16 decreased with the increase of the concentration of tetramethrin, indicating that tetramethrin could be used as a substrate for the growth of strain A16 during the biodegradation process. At high concentrations, it could also inhibit the growth of strain A16. Thus, the Andrews equation was used to simulate the kinetic analysis of the biodegradation process of tetramethrin at different concentrations . The degKi = 479.4 mg·L−1; Ks = 12.1 mg·L−1; qmax = 0.6980 day−1) of fipronil degradation in a mineral salt medium with bacterial strain Bacillus sp. FA3 were performed, which significantly reduced the half-life of fipronil [Enzymatic kinetics , pH (X2), and inoculation amount (X3)—to predict the optimal degradation parameters for strain A16. The residues of tetramethrin (Y1) obtained by HPLC analysis represented the effects of three variables on strain A16 at different levels. Y1 is the predicted tetramethrin degradation (%); and X1, X2, and X3 are coded values of temperature, pH, and inoculum size, respectively.The Box–Behnken design based on RSM was used to optimize the degradation conditions of tetramethrin in strain A16. Based on the preliminary study of single factor experiment, three independent variables were selected—temperature  suggested that the model was accurate and reliable. The regression analysis showed that the single factor and square terms of temperature (X1) and pH (X2), namely X1, X2, X12 and X22, had significant effects on the degradation of tetramethrin by strain A16 (p < 0.05), while X32 had no significant effects on the degradation of tetramethrin by strain A16 (p > 0.05). The interaction term of inoculation size (X3) and pH (X2) had no significant effect on the metabolic activity of strain A16 (p > 0.05).The analysis of variance (ANOVA) of the quadratic polynomial model is presented in X1), pH (X2), and inoculum size (X3) on tetramethrin degradation with different independent variables as a constant that indicate the maximum degradation that could be achieved inside the design boundaries  and pH values (5–9). This is an important feature of bioremediation by organisms in a variable environment . The resradation .Our results showed effective degradation with various pyrethroids by strain A16 . The deg−1. Meanwhile, strain A16 showed no obvious substrate specificity for various typical pyrethroid pesticides, suggesting that strain A16 may be suitable for bioremediation of various polluted environments. A single microbial strain can degrade many pyrethroids through its wider metabolism [Bacillus subtilis BSF01 has been investigated for its degradation of cypermethrin, cyfluthrin and cyhalothrin, and broader substrate specificity was reported due to the expression of the carboxylesterase CesB [Previous studies showed that the degradation activity of pyrethroid-degrading strains was significantly inhibited at high pyrethroid concentrations ,50,51. Ttabolism ,53,54. Base CesB .This experiment simulated the degradation ability of strain A16 in soil slurry and the results are shown in −1) in unsterilized soil-containing slurry reached 82.9%, while for tetramethrin sterilized slurry it reached 74.1% after 11 days of experiment. The results showed that there was no non-degradation or delayed degradation by strain A16 after the sludge was inoculated, and the degradation ability in the unsterilized soil slurry was always higher than in the sterilized soil slurry. Similarly, the degradation efficiency of tetramethrin in unsterilized soil slurry was higher than in sterilized soil slurry when strain A16 was not added in this experiment, suggesting that the indigenous soil microflora play a role in the degradation of tetramethrin. The degradation process of tetramethrin in soil slurry followed the first-order kinetics equation. The degradation constant (k) of tetramethrin with strain A16 was 0.0048 and 0.0054 day−1 in sterilized and unsterilized soil slurry treatment groups, respectively, whereas the k was 0.0373 and 0.0396 day−1 for tetramethrin in sterilized and non-sterilized soil slurries without the strain A16, respectively (−1) with strain A16 (1.0 × 107 cells g−1 of soil) tremendously accelerated the degradation rate of tetramethrin, and its half-life (t1/2) was reduced by 125.8 and 110.8 days in sterilized and non-sterilized soil slurries, respectively, in comparison with soil slurries without the strain A16. In addition, no obvious lag period was observed during the degradation experiment in soil slurry with strain A16. The observation of soil slurry suggested that strain A16 has a strong tetramethrin degradation ability in contaminated agricultural fields.The degradation efficiency of strain A16 for tetramethrin  was 331.2 -dione, acrylamide, and N-ethylacetamide, respectively. The corresponding compound structure is shown in The results of GC-MS showed that the retention time (RT) of compound A was 26.0 min and the mass/charge ratio (as 331.2 . Compounas 331.2 . Based oN-(hydroxymethyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione and chrysanthemol acid. N-(hydroxymethyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione was then converted to 2-buty-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione. Under the action of A16 hydrolase, 2-buty-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione was degraded into acrylamide and N-ethylacetamide. At the same time, chrysanthemol acid was transformed into chrysanthemol, which was formed into cyclopropyl methanol. The intermediate products of tetramethrin were eventually reduced to non-toxic small molecules, such as water and carbon dioxide, and this was observed after 11 days of the experiment.Based on the results of metabolite identification of tetramethrin by GC-MS, a novel metabolic pathway for tetramethrin in strain A16 was proposed . Strain The study of metabolic pathways is crucial to the analysis of the biodegradation potential of microorganisms. Sometimes, the intermediate metabolite is more toxic than the parent compound ,62,63. T−1; these were used for the degradation analysis of tetramethrin, which was stored in a dark bottle in a refrigerator at 4 °C. The mineral salt medium (MSM) had the following composition: 2 g (NH4)2SO4, 0.2 g MgSO4·7H2O, 0.01 g CaCl2·2H2O, 0.001 g FeSO4·7H2O, 1.5 g Na2HPO4·12H2O, and 1.5 g KH2PO4 per liter of distilled water were used for the cultivation of tetramethrin-degrading soil bacterial strains in this study. The incubation of strain A16 was carried out in Luria Bertani medium (LB) containing 10 g tryptone, 5 g yeast extract and 10 g NaCl per liter, respectively. The pH values of the two media were adjusted to 7.0, then the media were autoclaved at 121 °C for 20 min.Tetramethrin (99% purity), D-cyphenothrin (98% purity), chlorempenthrin (94% purity), prallethrin (95% purity), and allethrin (93% purity) were obtained from Wuhan Yuancheng Pharm Co., Ltd. . Chromatographic-grade acetone and acetonitrile was obtained from Fisher Scientific . All other chemicals and solvents used in this study were of analytical grade. Stock solutions were prepared by dissolving tetramethrin in chromatographic-grade acetone with a concentration of 10 g·L−1. After 7 days of incubation at 30 °C and 200 revolutions per minute on a rotary shaker, the concentration of tetramethrin was increased from 50 mg·L−1 to 100 mg·L−1, 200 mg·L−1, 400 mg·L−1, and 800 mg·L−1, respectively, according to a 5% inoculation amount [2, 103, and 104 times dilution, and placed in a 30 °C incubator for inverted culture for 2 days. After the LB plate grew out of single colonies, different single colonies were picked for streak purification at least three times. The degradation potential of the isolates was confirmed by high-performance liquid chromatography (HPLC)  [Sewage sludge samples were collected from the wastewater treatment tank of a pesticide factory located in Guangzhou, Guangdong Province, China. Enrichment was carried out in MSM to isolate bacteria from obtained soil samples. A total of 5 g of activated sludge sample was added to 100 mL of MSM liquid medium containing tetramethrin stock solution (acetone as solvent), and the final concentration of tetramethrin was 50 mg·Ln amount . Each trThe bacterial strain was characterized and identified by its morphology and genetic analysis based on 16S rDNA gene sequence. The physico-morphological characterization of the bacterial strain was examined by a scanning electron microscope (SEM) .NCBI, accessed on 15 July 2020). The related sequences with high homology were selected for multi-sequence alignment. The phylogenetic analysis and phylogenetic tree construction were performed using MAGA X software (Version 10.0) [Total genomic DNA was extracted with a Master Pure DNA Purification Kit  as per the manufacturer instructions. The 16S rDNA gene was PCR-amplified with universal primer pairs, forward primer (27F: 5′-TGACGAGTGGCGGACGGGTG-3′) and reverse primer (1492R: 5′-CCATGGTGTGACGGGCGGTGTG-3′). The 1405 bp 16S rDNA sequences measured by strain A16 were subjected to BLAST comparative analysis in the GenBank Nucleotide Library through a BLAST search in the National Center for Biotechnology Information (on 10.0) .g) and washed twice with sterile saline (0.9% NaCl) during the late-exponential growth before inoculation. Unless otherwise stated, the densities of strain A16 were adjusted with sterile N-saline to approximately 1.0 × 107 colony-forming units (CFU) per milliliter.The bacterial strains were stored in 30% glycerol at −80 °C. Before each experiment, strain A16 was thawed and inoculated in LB medium at 30 °C in a 200 rpm rotary shaker for 24 h. Bacterial cells were harvested by centrifugation  value at 600 nm by UV-spectrophotometer , and the amount of residual tetramethrin was determined by HPLC as described below.For the growth and degradation experiments, triplicate cultures were grown in MSM supplemented with 50 mg·L−1). In each concentration group, 1 mL of bacterial suspension was inoculated in MSM medium of each sample. The samples were incubated at 30 °C and 200 revolutions per minute on a rotary shaker for 11 days. The degradation of different concentrations of tetramethrin by strain A16 was determined by HPLC. Each test was performed in triplicate with non-inoculated samples as the control. The residual pesticide concentration was determined by HPLC.To investigate the effect of different initial concentrations on the degradation of tetramethrin by strain A16, a single-factor test was designed using different concentrations. Samples were set at six concentration gradients  is a common method to optimize the growth and degradation conditions of microorganisms. RSM has great advantages, as it requires fewer experimental runs to efficiently estimate quadratic surfaces for the optimization of responses . Therefosoftware . The exp−1 permethrin, bifenthrin, fenvalerate, beta-cypermethrin, allethrin, prallethrin, chlorempenthrin, and D-cyphenothrin. All cultures were conducted in triplicate with a 50 mL conical flask and incubated at the optimum cultural conditions for 11 days with non-inoculated cultures that served as controls. Triplicate cultures were incubated in MSM at 30 °C and 200 revolutions per minute on a rotary shaker. Sampling was carried out at regular intervals for 11 days and the number of residual pesticides was measured by HPLC as described below.To explore its ability to degrade various synthetic pyrethroids, strain A16 was inoculated into sterilized MSM supplemented with 50 mg·L−1 tetramethrin incubated at 30 °C and 200 revolutions per minute on a rotary shaker to yield a final bacterial count of approximately 1.0 × 107 cells g−1 of soil. Tetramethrin residues were extracted at 1, 3, 5, 7, 9, and 11 days of culture, and flasks without the bacterial strain served as the control. Extracted tetramethrin was quantified through HPLC.The soil slurry method was used to investigate the degradation of tetramethrin in the field by strain A16. Soil samples were collected from the top layer of soil (3–10 cm) to examine the tetramethrin degradation pattern, taken from the agricultural experimental field of South China Agricultural University, Guangdong Province, China. No pesticide was applied for more than five years. The sample soil was placed in a cool ventilated place and naturally dried. After drying, the soil was milled and passed through a 2 mm sieve. A small amount of soil was sterilized at 121 °C for 120 min. About 10 g of autoclaved soil was transferred into a 250 mL flask that contained 50 mL of MSM. Then, 1 mL active bacterial inoculum was inoculated into each flask containing 50 mg·L−1 of tetramethrin, and samples were collected from MSM on 1, 3, 5, 7, 9, and 11 days of incubation. Non-inoculated samples containing the same amount of tetramethrin served as the control. Metabolites were detected by gas chromatography–mass spectrometry (GC-MS) . Specially, the derivatization of metabolites before GC-MS was not needed in this study. To identify the metabolites, mass spectrometry analyses were matched with authentic standard compounds from the National Institute of Standards and Technology  library database.To identify tetramethrin and its metabolic products of biodegradation, strain A16 was grown in MSM media containing 50 mg∙LC0 is the initial concentration of tetramethrin (mg·L−1), Ct is the content of tetramethrin at time t, k is the degradation constant (day−1), and t is the degradation time (days).To confirm the effect of strain A16 on tetramethrin degradation, a first-order kinetic model was followed to describe the biodegradation process :(4)Ct=C0t1/2) of tetramethrin:k is the degradation constant.Equation (5) was used to calculate the theoretical half-life  was used to determine the degradation products of tetramethrin.18 reverse phase column  and UV detector. The mobile phase was composed of acetonitrile and deionized water (75:25) at a flow rate of 1.0 mL·min−1. The injection volume and detection wavelength were 10 μL and 235 nm, respectively [Tetramethrin was quantified with a Waters 2690 HPLC system equipped with a Phenomenex Cectively ,81.−1. The injection volume was 1 μL without split injection. The full scan range was 30–500 nm to analyze diverse metabolites. The detector MSD ion source temperature was 230 °C; the quadrupole temperature was 150 °C; and the inlet temperature was 250 °C. The initial column temperature was maintained at 90 °C for 2 min, and was then increased to 150 °C incrementally by 6 °C per minute for 1 min. Then, it was raised to 180 °C at an increment of 10 °C per minute and maintained for 4 min. Finally, the temperature was increased to 260 °C by 20 °C per minute for 10 min [The metabolites of tetramethrin were detected by Agilent 5977B/79800B GC-MS. GC-MS analysis was performed on a DB-5MS quartz capillary column (30.0 cm × 250 μm × 0.25 μm). The carrier gas used was helium (99.999% purity) and its flow rate was 1.5 mL·minr 10 min ,82.G. cholesterolivorans A16 with strong tetramethrin degradation activity. Strain A16 utilized tetramethrin as the sole carbon source for its growth over a wide range of temperatures (20–40 °C) and pH values (5–9), indicating that the strain has excellent applicability in different environments. It is noteworthy that strain A16 tolerated and degraded tetramethrin up to a concentration of 800 mg·L−1, thus giving it an exceptional ability to colonize ecological niches where pesticide concentrations are high. This study first demonstrated that the bacterium had complete detoxification and metabolic pathways of tetramethrin. Moreover, in experiments conducted with soil, strain A16-based bioaugmentation efficiently degraded tetramethrin at a significantly reduced half-life, demonstrating that it has great advantages in bioremediation of tetramethrin-contaminated water and soil due to its adaptability in different environments. Furthermore, strain A16 was capable of degrading a wide range of synthetic pyrethroids, suggesting that it is a potent and effective candidate for the bioremediation of pyrethroid-contaminated terrestrial and aquatic environments. However, further in-depth studies of its interaction with the environment and the degradation genes that encode for key enzymes are needed to develop a safe and efficient strategy to clean up tetramethrin contamination.The present study identified a novel bacterial isolate


Defining transcriptional profiles of substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) dopamine neurons is critical to understanding their differential vulnerability in Parkinson’s Disease (PD). Here, we determine transcriptomes of human SNc and VTA dopamine neurons using LCM-seq on a large sample cohort. We apply a bootstrapping strategy as sample input to DESeq2 and identify 33 stably differentially expressed genes (DEGs) between these two subpopulations. We also compute a minimal sample size for identification of stable DEGs, which highlights why previous reported profiles from small sample sizes display extensive variability. Network analysis reveal gene interactions unique to each subpopulation and highlight differences in regulation of mitochondrial stability, apoptosis, neuronal survival, cytoskeleton regulation, extracellular matrix modulation as well as synapse integrity, which could explain the relative resilience of VTA dopamine neurons. Analysis of PD tissues showed that while identified stable DEGs can distinguish the subpopulations also in disease, the SNc markers SLIT1 and ATP2A3 were down-regulated and thus appears to be biomarkers of disease. In summary, our study identifies human SNc and VTA marker profiles, which will be instrumental for studies aiming to modulate dopamine neuron resilience and to validate cell identity of stem cell-derived dopamine neurons. Aldh1a7, Sox6, Cbln1, Vav3, Atp2a3, and VTA e.g., Calb1, Otx2, Crym, Cadm1, and Marcks  and the ventral tegmental area (VTA) . SNc dopd Marcks . Transcrd Marcks  except fd Marcks . These ain vitro derivation of SNc dopamine neurons, which represent the ideal cell type for transplantation in PD . The slides with sections were kept at −20°C during the sectioning and subsequently stored at −80°C until further processed. The laser capture procedure followed by sequencing library preparation (LCM-seq) was carried out as described (Sample preparation prior LCM-seq was carried out as follows. Frozen midbrain tissues (controls and PD), of rostral to intermediate midbrain level , obtaineescribed , 2018. Described . To limiescribed , we trieescribed .Samples were sequenced using an Illumina HiSeq2000, HiSeq2500, or NovaSeq platforms (reads of 43 or 50 bp in length). The uniquely mapped reads were obtained by mapping to the human reference genome hg38/GRCh38 using STAR with default settings. The reads per kilobase of transcript per million mapped reads (RPKM) were estimated using “rpkmforgenes”  to 10.88P-value of 0.05. Identified DEGs  are shown in Differentially expressed genes were identified using the R package “DESeq2” (version: 1.16.1)  where thTo counteract the variability among human subjects and identify the most reliable DEGs between SNc and VTA neurons across datasets we developed a bootstrapping approach coupled with DESeq2 . The staIn detail:(1)Define ¨N¨ and ¨M¨ as the number of samples in Groups 1 and 2, respectively. Choose ¨I¨ as a reference representing a given number of samples from ¨N¨ and ¨M¨.(2)Define ¨i ¨ as the number of randomly selected samples from Groups 1 and 2, where i ∈{3, 4, 5, …, ¨I-1¨}. In the human dataset, as we have 12 paired samples, the I ∈{3, 4, 5, 6, 7, 8, 9, 10, 11}.(3)Pool ¨i¨ samples (temporary considered a ¨new data set¨) and calculate DEGs with DESeq2.(4)Repeat steps (2) and (3) for ¨j¨ times .(5)For every round of random selection and DESeq2, save the full list of DEGs, compute and rank their frequency.(6)Set a threshold (30% ratio in this study) to consider DEGs with higher frequency as stable genes.Reliable genes appear when frequencies are above: Total times of bootstrapping × ratio (300 in this study). A stringent, but fair ratio can be defined by comparing the percentage of identified stable genes overlapping with the top (most significant) 10%, 20%, 30%, …, DEGs identified by DESeq2 alone.To reliable identify DEGs between human SNc and VTA samples, while minimizing subject variability, we selected 12 control individuals  where both neuronal populations were available and sequenced. Hence, the number of randomly selected samples  was from three to 11 and the algorithm repeated 1000 times .N = 73 cells) and VTA . To compensate the unbalance in cell number and adjust dataset representation compared to the human analysis (66%), we first randomly collected a subset of 73 VTA cells, pairing both SNc and VTA. Similarly, the number of randomly selected samples was 20, 25, 30, …, 70 and the algorithm repeated again 1,000 times.For this adult mouse dataset  we definBased on the DE genes between SNc and VTA , two STRData visualization was achieved using Principal Component Analysis (PCA) and Hierarchical Clustering (H-cluster). PCA was calculated with the function “prcomp” in R with default parameters. Then samples are projected onto the first two dimensions, PC1 and PC2. For H-cluster we used the R function “pheatmap” (version 1.0.12) with the clustering method of “ward.D2.”SEZ6) and one VTA-preferential gene (CDH13) based on the sequencing data. In brief, midbrain sections of human fresh frozen tissue (dapB-C1) and a positive control probe against tyrosine hydroxylase  (SEZ6 (Cat. 411351-C1) or CDH13 probes (Cat. 470011-C1). Slides were counterstained with fresh 50% Gill Solution  for 2 min, washed in water and dried for 15 min at 60°C before mounting with Pertex . For every sample (n = 5), we imaged 5–6 random fields within the SNc and VTA regions. On average 194.25 ± 43.02 cells were imaged per region and staining. Pictures were made at 40X magnification using the bright-field of a Leica microscope (DM6000/CTR6500 and DFC310 FX camera). After randomization and coding of all the images, the number of dots within melanised cells (dopamine neurons) were counted using ImageJ (version 1.48) and later the average number of dots per cells determined for each region. Cells were classified as having either 0, 1, 2, 3, 4, 5 dots/cell. If cells had >5 dots they were classified within the 5 dots/cell category.RNAscope  was usedn tissue  were quin tissue , we firs, TH-C1) . Once weInvestigators performing the quantification were blinded to the sample, target region (SNc and VTA) and probe staining.t-test  was used to compare the mean average dots per cell (for SEZ6 or CDH13 staining) between the SNc and VTA. Where applicable, individual statistical tests are detailed in the figure legends where significance is marked by P < 0.05. The number of subjects/cells used for each experiment is listed in the figure or figure legends. Results are expressed as mean ± SD or SEM as specified in the figure legend.For this study, statistical analyses were performed using ¨R¨. For the RNAscope analysis a paired 1 (GEO) under accession number GSE114918. Human samples re-analyzed from the Nichterwitz study , sample handling and preservation. Therefore, prior to conducting differential gene expression analysis we performed extensive quality control analysis to rule out undesired influences from sample processing (P = 0.515) , and the general neuronal marker NEFH, while they lack glial markers MFGE8, CX3CR1, or GPR17. This clearly demonstrates the selective enrichment of dopamine neurons using the LCM-seq methodology (KCNJ6 (GIRK2) and CALB1, two genes often used to distinguish between SNc or VTA dopamine neurons, were also expressed  . However= 0.515)  and thushodology . KCNJ6  (N = 18), even though the same experimental method was used. In fact, only seven SNc and 21 VTA DEGs overlapped across the current and the previous cohorts (N = 3)  . However cohorts . Notably (N = 3) , failed  (N = 3) . This suP < 0.05. This random sampling of three subjects, followed by DESeq2 analysis, was performed a total of 1,000 times, and the DE frequency over these 1,000 comparisons was recorded for this iteration (i = 3 subjects). Subsequently, this process was repeated using subsets of four subjects, then five, up to a maximum of 11 of the 12 subjects. For each subset size (i3–i11)the DEG frequency was calculated by the 1,000x comparisons of that iteration  and 16 of the VTA stable genes  were among the aforementioned seven and 21 overlapping DEGs, that we identified across the two studies.To evaluate how sample size may affect DEG detection, we used a bootstrapping algorithm in combination with DESeq2. To reduce the biological variability, we considered only those subjects for which both SNc and VTA samples were available . To begin with this approach, a subset of three subjects were randomly chosen from the pool of total 12 subjects. Differential expression analysis was then performed between the SNc and VTA samples of these subjects (DESeq2), and DEGs were selected with adj teration . By consation , separated genes into SNc or VTA enriched lists, and ranked the lists from most to least frequently DEs   as the output of the overall bootstrapping approach showed genes with the 30% threshold  represent SNc and VTA stable genes  . To idenle genes . We alsole genes . All eigP < 0.05  or a mor01, SEZ6  and the 6 , and a strict selection-criteria  we identify 33 of the most stable DEGs. Among these, 25 of the genes define VTA identity, while eight define SNc identity, which together accurately classify LCM-seq samples from our previous , and current  subject cohorts. We confirm the utility of our bootstrapping approach on a larger published mouse single cell data set and show that identified DEGs there could correctly classify SNc and VTA dopamine neurons.Using an iterative bootstrapping without replacement coupled with DESeq2  is a serine protease inhibitor which can promote neurite extension by inhibiting thrombin, and which appears down-regulated in Alzheimer’s disease . Interestingly, mice lacking Rgs6, a related family member of the human SNc stable gene RGS16, develop specific degeneration and cell loss of SNc dopamine neurons at the age of 12 months  can be found in the article/EH, QD, and JA: conceptualization and funding acquisition. JA, MC, SC, MW, NK, QD, and EH: methodology, investigation, writing—review, and editing. SC, JA, MW, and NK: software, formal analysis, and visualization. JA, SC, QD, and EH: writing-original draft. EH and QD: supervision and project administration. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococcus aureus, FtsH, a membrane-bound metalloprotease, plays a critical role in bacterial virulence and stress resistance. This protease is also known to sensitize methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics; however, the molecular mechanism is not known. Here, by the analysis of FtsH substrate mutants, we found that FtsH sensitizes MRSA specifically to β-lactams by degrading YpfP, the enzyme synthesizing the anchor molecule for lipoteichoic acid (LTA). Both the overexpression of FtsH and the disruption of ypfP-sensitized MRSA to β-lactams were observed. The knockout mutation in ftsH and ypfP increased the thickness of the cell wall. The β-lactam sensitization coincided with the production of aberrantly large LTA molecules. The combination of three mutations in the rpoC, vraB, and SAUSA300_2133 genes blocked the β-lactam-sensitizing effect of FtsH. Murine infection with the ypfP mutant could be treated by oxacillin, a β-lactam antibiotic ineffective against MRSA; however, the effective concentration of oxacillin differed depending on the S. aureus strain. Our study demonstrated that the β-lactam sensitizing effect of FtsH is due to its digestion of YpfP. It also suggests that the larger LTA molecules are responsible for the β-lactam sensitization phenotype, and YpfP is a viable target for developing novel anti-MRSA drugs.In the Gram-positive pathogen  Staphylococcus aureus is a Gram-positive pathogenic bacterium causing multiple diseases ranging from soft-tissue infections to life-threatening diseases such as endocarditis, toxic shock syndrome, and necrotizing pneumonia [S. aureus (MRSA) infections cannot be effectively treated with most β-lactam antibiotics because it produces PBP2a, a transpeptidase whose active site is not accessible to most β-lactam antibiotics [neumonia ,2. The mibiotics ,4,5,6,7.Although PBP2a has been ascribed to the main resistance determinant for β-lactam antibiotics in MRSA, the overall β-lactam resistance of MRSA requires additional molecular factors. For example, since PBP2a can carry out only a transpeptidation reaction, the transglycosylase activity of PBP2 is essential for cell-wall synthesis in MRSA . PBP4 isftsH [S. aureus, FtsH directly degrades the following 12 proteins: LrgB, SAUSA300_0310, SAUSA300_0579, SaeQ, YpfP, CydA, CyoE, Ffh, SAUSA300_1351, HemA, HtrA, and MbtS [S. aureus. HrtA and HrtB form a heme-regulated transport system and are involved in heme metabolism [Various mutations are known to increase MRSA β-lactam resistance. By the genome sequencing of a highly resistant subpopulation of four historically early MRSA isolates , the Tomasz group identified 27 mutations . Of the ftsH . FtsH isftsH ,21. In Sand MbtS ,23. Althand MbtS ,25. SaeQand MbtS . YpfP isand MbtS . As cytoand MbtS . HemA isand MbtS . HslO istabolism . Finallytabolism .ftsH increases the β-lactam resistance of S. aureus USA300, the predominant MRSA in the US [ftsH increases the MRSA β-lactam resistance, we hypothesized that FtsH overexpression would sensitize MRSA to β-lactams and found that it does. Therefore, in this study, we further sought to identify the underlying molecular mechanism.Independently of the previous study , we founn the US . Since tftsH gene in S. aureus USA300, the oxacillin MIC of the strain increased eightfold from 8 µg/mL to 64 µg/mL. The MIC increase was abolished by the further deletion of mecA (the gene encoding PBP2a) -inducible promoter, the oxacillin MIC of USA300 decreased 16-fold from 8 µg/mL to 0.5 µg/mL , which inhibit the transpeptidase activity of PBPs . PBP2a was detected by Western blotting, and PBP2 was visualized by the Bocillin-FL assay, where the fluorescent penicillin is used as a probe to detect PBPs [ftsH-deletion increased the cellular levels of the two FtsH substrate proteins, SaeQ and YpfP  , the 5 µg/mL) , indicat2-DAG), a glycolipid serving as the membrane anchor for LTA [2-DAG, the disruption of ypfP abolishes the production of the glycolipid. When that happens, diacylglycerol (DAG), not Glc2-DAG, serves as the anchor molecule, resulting in the synthesis of altered LTA [ypfP mutation increased the size of LTA  in 6 +ATc in ypfP mutation reduced the MIC of β-lactams  by 16 to 2048 times   . The disP mutant , and incftsH, and ypfP mutants; however, no significant difference was observed  whose oxacillin MIC is not decreased by FtsH overexpression. The USA300 (pYJ-ftsH-His6) cells were spread on the TSA plate containing 8, 16, and 32 µg/mL oxacillin with ATc (100 ng/mL) and incubated until colonies arose. Colonies grew only on TSA with 8 or 16 µg/mL oxacillin, and the mutant frequency was ~1.4 × 10−6 on TSA with 16 µg/mL oxacillin. We sequenced the genomes of 18 suppressor mutants (12 from oxacillin 8 µg/mL and 6 from 16 µg/mL), and the results are shown in rpoC, vraB, and SAUSA300_2133 . The rpoC gene encodes an RNA polymerase β’ subunit, whereas the vraB gene encodes an acetyl-CoA c-acetyltransferase. The role of the 2133 gene is not known. We named the three mutations ‘core mutations’ . To test this possibility, we examined whether the murine infection by the ypfP mutant can be treated with oxacillin. Although LTA is essential for growth in most S. aureus, including USA300, it is not in the MW2 strain [ypfP-mutant of USA300 showed reduced virulence, compared with WT, whereas such a reduction in virulence was not observed with the ypfP-mutant of MW2  is 8 times lower than that of the 8-9 strain (MIC = 32 µg/mL); therefore, it is safe to conclude that the RpoC mutation is the major contributing factor of the increased MIC of the suppressor mutant 8-9.The suppressor mutant study showed that the β-lactam-sensitizing effect of FtsH could be blocked by the combination of three core mutations: RpoC S723T, VraB A144S, and SAUSA300_2133 V220L. RpoC is the β’ subunit of the RNA polymerase; therefore, the mutation in RpoC is expected to give a pleiotropic effect on staphylococcal gene transcription and expression. Although the mechanism is not clear, mutations in RNA polymerase subunits are associated with the increase in β-lactam resistance of . aureus . Since wmutation b. Unlikecholerae . IntriguypfP-knockout mutation increased the LD50 of S. aureus RN4220 approximately threefold in murine and silkworm infection models, respectively [ypfP mutation decreased the bacterial virulence in USA300 but not in MW2 , respectively. For transduction of mutations and plasmids, heart infusion broth (HIB) with 5 mM CaCl2 was used. For MIC measurement, cells were grown in Mueller–Hinton Broth with 2% NaCl. When necessary, antibiotics were added to the growth media at the following concentrations: ampicillin, 100 µg/mL; erythromycin, 10 µg/mL; and chloramphenicol, 5 µg/mL.The bacterial strains and plasmids used in this study are listed in n strain  and was vraB and SAUSA300_2133, a 2 kb DNA fragment was PCR-amplified with the primer sets P4203/P4204 (for vraB) or with P4205/P4206 (for SAUSA300_2133)  at 30 °C overnight, and the resulting cultures (1–2 µL) were inoculated into 2 mL TSBcm5. After overnight growth at 42 °C, the cultures were diluted 105 folds in TSBcm5, and the diluted cultures (100 µL each) were spread on TSAcm5 and incubated at 42 °C overnight. Five colonies on the plates were grown in TSBcm5, and chromosomal DNAs were purified. The co-integration of the plasmid was confirmed by PCR-amplification of the purified DNA with the primer sets P4246/P4248 (for pKOR1-vraB) and P4246/P4249 (for pKOR1-2133). The cointegrate strains were grown in TSB at 30 °C overnight. Then the overnight cultures were diluted 100 times in TSB and incubated at 30 °C for 10 h. Finally, the resulting culture was diluted 100 times in TSB and incubate at 30 °C overnight. The cells were diluted 105 times in sterile water, and 100 µL was spread on TSA containing 0.2 µg/mL anhydrotetracycline and incubated at 30 °C overnight. Fifty colonies on the plate were examined for growth in the presence of chloramphenicol (10 µg/mL), and eight colonies, which failed to grow in the presence of chloramphenicol, were selected and grown in TSB at 30 °C. From the chromosomal DNA of the vraB and SAUSA300_2133 strains were PCR amplified with the primer sets P4043/P4044 and P4045/P4046, respectively (For reproducing the mutations in 00_2133) . Primersn method . The assectively .2  was added to the media. The MICs of the strains harboring pYJ335 or pYJ-ftsH-His6 were assessed in the absence or presence of anhydrotetracycline (100 ng/mL).Minimum inhibitory concentration (MIC) for various antibiotics was determined with a serial dilution of antibiotics according to the recommendations proposed by the Clinical and Laboratory Standards Institute (CLSI) using the microdilution method . Cells wftsH carrying pYJ-ftsH-His6 was diluted and spread onto TSA containing erythromycin (10 µg/mL), oxacillin , and anhydrotetracycline (100 ng/mL). The plates were incubated at 37 °C until colonies appeared. The colonies were streaked on the same TSA plate to confirm their oxacillin resistance. Then, the FtsH expression was confirmed by Western blotting with an anti-His6 antibody. Finally, the suppressor mutants were grown in TSBerm10, and genomic DNA was purified with UltraClean Microbial Kit  and sent to the Center for Genomics and Bioinformatics at Indiana University, where the mutant genomes were sequenced with Illumina NextSeq 500.Overnight culture of USA300Δ6 and the anti-PBP2a antibody were purchased from Invitrogen and Abnova, respectively.Western blot analysis of proteins was carried out as described previously . The Sae6, the overnight culture was diluted 1:100 into TSBerm10 without or with anhydrotetracycline (100 ng/mL). Cultures were normalized for their optical density (OD600 of 4). Cells in the normalized cultures (0.8 mL) were pelleted by centrifugation, suspended in 50 μL of a lysostaphin solution , and incubated at 37 °C for 30 min. DNaseⅠ  was added to the cell lysates, and the samples were incubated at 37 °C for 15 min. Then 0.5 μL of proteinase K  was added, and the samples were further incubated at 50 °C for 2 h and mixed with 50 μL of 2 × SDS- PAGE sample buffer. Each sample (10 µL) was subjected to 15% SDS-PAGE. Western blotting was performed with anti-LTA monoclonal antibody  at a 1:1000 or 1:2000 dilution as primary antibody and the horseradish peroxidase-linked anti-mouse antibody  at a 1:5000 dilution as the secondary antibody.Western blot analysis of LTA was carried out as previously described , with moThe Western blots were visualized by the Supersignal West Pico PLUS Chemiluminescent Substrate , and the images were taken and processed with LAS-4000 . All Western blots were repeated at least three times with similar results.ftsH were transferred into fresh TSB and incubated for 6 h at 37 °C with shaking. The strain USA300ΔftsH harboring pYJ-ftsH -His6 was cultured in TSBerm10 without or with anhydrotetracycline (100 ng/mL). Cells were collected by centrifugation, suspended in TSM buffer  containing lysostaphin (50 μg/mL), and incubated at 37 °C for 5 min. After brief sonication of the cell lysates, cell debris was removed by centrifugation at 7000 rpm, 4 °C for 10 min. The membrane fraction was collected by ultracentrifugation . The membranes were washed twice in TKM buffer  and collected by ultracentrifugation . Finally, the membranes were suspended in TKMG buffer  and stored at −80 °C until use. The membrane vesicles (100 µg) were labeled with 100 µM Bocillin-FL  for 10 min at 30 °C. The reaction was stopped by adding 5 × SDS-PAGE sample buffer. Labeled membrane proteins were separated on a 7.5% SDS-PAGE gel and detected with a LAS-4000  followed by quantification with Multi Gauge software .Membrane vesicles were prepared as described before . BrieflyThe autolysis assay was carried out as described by Bose et al.  without The test strains were grown in 3 mL TSB at 37 °C with shaking (200 rpm) to exponential growth phase (OD600 = 1). Cells were collected by centrifugation and suspended in 3% glutaraldehyde , 0.1 M sodium phosphate, pH 7.2. After overnight fixation at 4 °C, the samples were sent to the Electron Microscopy Core Facility at Indiana University. At the facility, the specimens were post-fixed in 1% Osmium tetroxide, dehydrated, infiltrated, and embedded in Embed 812 . Thin sections were cut (80–90 nm), heavy metal stained, then viewed on a Tecnai Spirit . Digital images, including all measurements, were taken with an AMT  CCD camera.n = 10, 8-week-old, Jackson Laboratory) were used. For the virulence test, mice were infected via retro-orbital injection with 108 CFU (USA300 strain) or ~7.5 × 107 CFU (MW2 strain) of either WT or ypfP and observed for nine days. For the oxacillin-treatment test for USA300 strain, 108 CFU (USA300 WT) or 2 × 108 CFU  of bacteria were administered into mice via retro-orbital injection. Then, 50 µL of various concentrations of oxacillin (1-28 mg/mL) were administered intramuscularly 1 h after the bacterial infection and daily for two more days. The infected mice were observed for two weeks.The murine blood infection experiment was carried out as described previously with minor modifications . In the S. aureus, the ATP-dependent protease FtsH sensitizes the bacterium to β-lactam antibiotics by degrading YpfP, an enzyme synthesizing the glycolipid anchor molecule for LTA synthesis. Since the lack of YpfP increases the size of LTA molecules as well as the thickness of the cell wall, we hypothesize that the large LTA molecules affect the cell-wall synthesis process directly. Finally, the in vivo efficacy of oxacillin on the ypfP mutants in the murine infection model demonstrates that YpfP is a viable target for the development of β-lactam potentiator against MRSA. In


The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion  increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation. Our data suggest that loss of arpin is a new hallmark of acute inflammation in ulcerative colitis and that the Arp2/3 complex may serve as a therapeutic target to treat inflammatory bowel diseases.Intestinal epithelial barrier dysfunction is a hallmark of inflammatory bowel diseases, but the underlying mechanisms remain poorly understood. Interepithelial junctions that are connected to the actin cytoskeleton stabilize cell contacts. While the role of junctional transmembrane and scaffolding proteins for barrier integrity has been extensively studied, the role of actin-binding proteins in this context is still poorly understood. Thus, it is critical to improve our understanding of how actin-binding proteins regulate both TJ and cytoskeleton architecture during inflammatory disorders. Here, we provide in vitro and in vivo , adherens junctions (AJ), and desmosomes . Under i in vivo . Arp2/3 lization . Thus, flization . The moslization , but notlization . While tlization , nothingWe hypothesized that Arp2/3 is locally inhibited by arpin to regulate barrier integrity by controlling actin cytoskeleton and TJ architecture. To test this hypothesis, we employed two different strategies: (1) depleting the endogenous PIA arpin and (2) using the well-established specific pharmacological Arp2/3 inhibitor CK666.2 at 37°C to mimic inflammation.The colorectal adenocarcinoma epithelial Caco-2 (clone C2BBE1) cell line was obtained from ATCC  and cultured according to their instructions. Confluent Caco-2 monolayers were treated with 50 ng/ml tumor necrosis factor (TNF)α  and 10 ng/ml interferon (IFN)γ  and incubated for 48 h in a humidified atmosphere with 5% COEnd-point PCR was performed using Platinum®, PCR SuperMix, 0.15 μM forward primers, 0.15 μM reverse primers . Conditions were as follows: activation at 95°C for 10 min, 40 cycles including denaturation phase at 95°C for 15 s, and data acquisition during the annealing/extension step at 60°C for 60 s. Following the last cycle, the melting curve was generated by heating from 60°C to 95°C in increments of 0.6°C/s. Relative expression was quantified using the 2CT–ΔΔ method , 0.15 μM forward primer, 0.15 μM reverse primer, and 100 ng cDNA using a StepOneT method .7SL (Actb were used as housekeeping genes.Human 7SL  and mousRadioimmunoprecipitation assay (RIPA) buffer cell and tissue lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.45-μm-pore nitrocellulose membranes , blocked with Tris-buffered saline (TBS) containing 0.05% Tween (TBS-T) and 5% skim milk, or 5% bovine serum albumin (BSA) for myosin-II light chain (MLC) and phosphorylated-MLC (p-MLC), for 1 h at room temperature (RT), and incubated overnight in primary antibodies at 4°C with gentle agitation. Membranes were washed with TBS-T three times for 10 min each and incubated with species-specific secondary antibodies conjugated to horseradish peroxidase for 1 h at RT with gentle agitation. After another three washes for 10 min each, bands were visualized using SuperSignal West Pico substrate (Thermo Fisher Scientific) and a ChemiDoc device . Pixel intensity was quantified using ImageJ software . Primary and secondary antibodies are listed in TM protease cocktail and 2 × PhosSTOPTM) and shaken gently for 4 h at 4°C. Cell extracts (1 mg protein) were pre-cleared with equilibrated beads for 2 h at 4°C. Then, beads were pelleted at 3,500 rpm for 5 min at 4°C, and pre-cleared extracts were incubated overnight at 4°C under gentle rotation with primary antibodies. Unrelated serum was used as a negative control. The next day, 10 μl of equilibrated G-agarose beads blocked with lysis buffer containing 4% BSA were added to the antibody–antigen complex and incubated overnight at 4°C with gentle rotation. The antibody–antigen–beads conjugates were separated by centrifugation at 3,500 rpm for 5 min at 4°C, washed three times with 1 × phosphate-buffered saline (PBS) and eluted with Laemmli buffer. Samples were denatured by boiling for 5 min at 95°C and analyzed by Western blot.10 μl of Protein G-sepharose beads (Sigma-Aldrich) were equilibrated in 500 μl of lysis buffer  for 10 min at RT and permeabilized with 0.2% Triton X-100 in PBS for 5 min at RT or in absolute ethanol for 30 min at −20°C. Coverslips were then blocked for 20 min in PBS containing 3% BSA. Primary antibodies were incubated overnight at 4°C. Then, coverslips were washed and incubated with species-specific fluorescently labeled secondary antibodies for 1 h at RT. Coverslips were mounted in Prolong medium containing 4’,6-diamidino-2-phenylindole  and analyzed using a confocal laser microscope .4 at 4°C. Subsequently, cells were dehydrated with increasing concentrations of ethanol, embedded in Spurr’s resin , and polymerized at 60°C for 48 h. Ultrathin sections were cut using an Ultracut E ultramicrotome , stained with uranyl acetate and lead citrate, and analyzed using a JEOL 1400 electron microscope . Quantification of microvilli and vesicle numbers and TJ length was done in at least six cells of each tested condition from two independent experiments. To this end, frames were drawn at intercellular junctions (5 × 2 μm) and along the entire apical membrane of single cells in order to delimit zones for counting of junction-related vesicles and microvilli, respectively.Cells were processed as previously reported . Briefly5 Caco-2 cells were seeded on 0.4-μm pore size Transwell filters . Transepithelial electrical resistance (TER) was measured using a Milicell-Electrical Resistance System (Milicell-ERS) MERS00001. TER is presented as Ω × cm2 or as relative TER normalized to control cells. In the experiments using CK666 during the formation of monolayers, culture medium containing CK666 or dimethylsulfoxide (DMSO) alone was exchanged every 2 days.Here, 6 × 102+/Mg2+. Then, 100 μg of fluorescein isothiocyanate (FITC)-dextran  were added to the apical side and incubated for 2 h at 37°C. Medium from the bottom chamber was collected, and the amount of diffused dextran was measured using a fluorometer. Emission values were normalized to control cells (set to 100%).At the end of each TER assay, monolayers were rinsed three times with pre-warmed Hanks’ balanced salt solution (HBSS) supplemented with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glucose, and CaA stable arpin-depleted Caco-2 cell line was generated by lentiviral transduction using the trans-lentiviral packaging kit (Thermo Fisher Scientific) and the pLKO.1 plasmid  according to the manufacturer’s instructions. The following shRNA sequences were used: shRNA scrambled: 5′-CGGAGAAGTGGAGAAGCATAC-3′ and shRNA arpin: 5′-GGAGAACTGATCGATGTATCT-3′ . shRNA iCorrected density = ∗ mean gray of background)integrated density – . Briefly, maximum projections of confocal stacks were converted to 8-bit format. Then, five cells from each of four independent experiments were delineated with the “free selection tool.” Area, mean gray value, and integrated density were measured for each cell and for three random background fields. Corrected density was calculated using the following formula: ad libitum access to standard pellet diet and water over the entire experimental period of 7 days. The colitis group received 2.5% dextran sulphate sodium  in drinking water. To assess the severity of colitis, the disease activity index (DAI) was scored daily from 0 to 4 based on body weight loss, diarrhea, and intestinal bleeding  were used in colitis experiments. All animals were given bleeding . For CK6bleeding  at days Cross sections of colon Swiss rolls  embeddedascendens. The colon was flushed with PBS followed by instillation of 1.5% Evans blue solution. The dye was incubated for 15 min, then mice were euthanized, and colons were removed, rinsed with abundant PBS, and washed with 1 ml of 6 mM N-acetylcysteine in PBS to eliminate excess dye. Colon weight was recorded, the dye was extracted overnight at RT in 2 ml of N, N dimethylformamide and measured spectrophotometrically at 610 nm  in 0.9% saline solution. After laparotomy, colons were exposed, and a G22-polyethylene tube was inserted into the colon t 610 nm .Human tissue samples were obtained from UC patients who underwent surgical resection. The study was approved by the ethics committee of the University of Würzburg  , and writ-test, two-tailed t-test followed by a Mann–Whitney test, or ANOVA with Bonferroni’s correction or Tukey’s post hoc test, as indicated in the figure legends. Statistical analyses were performed using GraphPad Prism software v5.0. Values of probability p < 0.05 were considered statistically significant. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.Data are presented as mean ± standard deviation (SD) and are representative of at least three independent experiments. Significance between groups was assessed by two-tailed Student’s in silico analysis of the arpin protein sequence using “The Eukaryotic Linear Motif” and “PhosphoSitePlus” resources  in control and TNFα/IFNγ-treated Caco-2 cells by qRT-PCR and found that only arpin was significantly downregulated during inflammation  was confirmed by confocal microscopy . Moreovein vitro. We emphasize that this protective effect of CK666 was only observed in well-established cell monolayers because treatment of sparse Caco-2 cells with CK666 significantly delayed formation of monolayers  leads to alteration of the IEB, as shown by permeability assays , we testnolayers , confirmnolayers . Next, wnolayers , suggestnolayers .While total junction protein levels are usually unchanged in epithelial monolayers challenged with TNFα/IFNγ, they are redistributed from junctions to the cytosol by endocytotic processes, causing barrier disruption and hyperpermeability . In agrein vitro also occurs in vivo. To this end, we administered CK666 in mice with DSS-induced colitis. The DAI consisting of body weight loss, diarrhea, and intestinal bleeding was evaluated daily during an experimental period of 7 days. Either 5 mg/kg of CK666 or DMSO alone (vehicle control) were i.p. injected daily starting on day 3 of DSS treatment when colitis symptoms started to manifest. The dose of 5 mg/kg of CK666 showed beneficial effects in previous studies in vivo  induced IEB dysfunction, whereas Arp2/3 inhibition improved it. Pharmacological inhibition of the Arp2/3 complex has been used as a tool to dissect biological functions of this complex. CK666 is a small molecule that binds to the Arp2/3 complex, stabilizes its inactive conformation, and blocks its activation without promoting disassembly of preformed actin branches . Arp2 anvia CK666 during inflammation prevented barrier dysfunction triggered by pro-inflammatory cytokines. Considering that arpin depletion would contribute to Arp2/3 activation, all our data coincide well and point to arpin-mediated Arp2/3 inhibition to prevent junction protein internalization. However, the fact that inhibition of Arp2/3 by CK666 reinforced the epithelial barrier was surprising because other studies demonstrated that inhibition of Arp2/3 had detrimental effects on several functions such as migration, lamellipodia formation, and cytokinesis in vitro  might be contributing to the observed effect on the intestinal epithelium due to the systemic delivery of CK666, our in vitro data support that indeed Arp2/3 inhibition in the epithelial cells is at least partly responsible for the IEB protection under inflammatory conditions. The extent of contribution of Arp2/3 inhibition in other cell types remains to be determined.The physiological relevance of our findings was proven Arp2/3 is critical for proper organ functionality and development. For example, mice with specific deletion of Arp2/3 in the intestinal epithelium and the epidermis presented impaired transcytosis and dehydration (increased permeability), respectively, both resulting in lethality , 2015. TThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethical Committee of the University of Würzburg  (61). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Institutional Animal Care and Use Committee, CINVESTAVIPN.SC-P performed the research and statistical analysis, analyzed and interpreted the data, and wrote the manuscript. AM-G, KC-O, JG-C, and HV-R performed the research and analyzed the data. RM-F performed the research and analyzed and interpreted the data. LC-B, MSh, and PN analyzed and interpreted the data. SF and NS analyzed human tissue samples and interpreted the data. AG provided essential reagents and interpreted the data. MSc conceived and supervised the study, analyzed and interpreted the data, and wrote the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Paeonia ostii were determined, and the fatty acid (FA) distributions in triacylglycerol and phospholipids were characterized. The lipids in the kernel were mainly phospholipids (43%), neutral glycerides (24%), fatty acyls (26%), and sphingolipids (4.5%). The dominant neutral glycerides were TAG and diacylglycerol. The PL components included phosphatidic acid, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, and phosphatidyl ethanolamine. As the kernel developed, the profiles of the molecular species comprising TAG and PL changed, especially during the earlier phases of oil accumulation. During rapid oil accumulation, the abundances of sphingosine-1-phosphate, pyruvic acid, stearic acid, and alpha-linolenic acid changed significantly; the sphingolipid metabolism and unsaturated FAs biosynthesis pathways were significantly enriched in these differentially abundant metabolites. Our results improve our understanding of lipid accumulation in tree peony seeds, and provide a framework for the analysis of lipid metabolisms in other oil crops.Lipid components in the developing kernel of  Paeonia section Moutan DC.), a historically important royal flower, has been cultivated in China for nearly 2000 years1. Nine wild species have been described in section Moutan, and all are native to China2. Tree peonies, which are woody, oleaginous plants, have become increasingly commonly cultivated due to the high-quality oil that is produced in their seeds; this oil has attracted significant economic and academic interest. Tree peony oil is characterized by high levels of unsaturated fatty acids , including alpha linolenic acid , linoleic acid (LA), and oleic acid (OA)4. The seeds of Paeonia ostii and P. rockii were identified as a novel source of high-quality edible oils in 2011 by the National Health Commission of the People’s Republic of China5. In particular, P. ostii is a prospective industrial crop, as ALA levels in P. ostii seeds are high, and this plant is already widely cultivated in many parts of China6.The tree peony (8. The synthetic process that leads to the accumulation of triacylglycerol (TAG), in seed oil bodies begins with the de novo synthesis of FAs in plastids12. The synthesized FAs are exported to the endoplasmic reticulum (ER) for acyl editing or to the plastid for desaturation13. Modified FAs are then assembled into TAG via the Kennedy pathway in the ER, dependent on the acetyl CoA pool14, or via the phospholipid:diacylglycerol acyltransferase (PDAT) pathway, independent of the acetyl CoA pool15. The ALA biosynthesis and TAG assembly pathways in the tree peony have been investigated using transcriptomics, proteomics, and microRNA20. Several such studies have focused on the cloning, expression, and function of key genes in the ALA metabolism of P. suffruticosa (including wild species and cultivated varieties), including stearoyl-ACP desaturase (SAD), fatty acid desaturase 2 (FAD2), FAD3, FAD8, lysophosphatidic acid acyltransferase (LPAAT), diacylglycerol acyltransferase, (DGAT), PDAT, and oleosin24.Previous studies of peony seed oil have mainly focused on the types and contents of FAs in tree peonies, including wild species and cultivated varieties of a variety of provenances; other studies have investigated the influence of paternal inheritance on FA content25. Although studies of FA biosynthesis in the tree peony have explored FA-associated metabolic pathways and gene function, little is known about the intracellular lipid species. In oil seeds, the formation of ALA TAG from LA under the action of delta-15 FA desaturase is only the most direct step in the process of ALA synthesis in cells; this process also involves acyl editing, the Lands cycle, and TAG assembly13. Thus, differences in the relative abundances of lipid species, such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), diacylglycerol (DAG), and TAG, among oil crops is of substantial importance.Polyunsaturated fatty acids (PUFAs) like ALA are stored in seeds as oil droplets; this storage mechanism involves many complex metabolic pathways, which require various lipid molecules as ligands or donors, as well as other lipid molecules as signal factors to regulate metabolic pathwaysP. ostii kernel. To address this knowledge gap in the present study, we identified the lipid species in the developing kernel and investigated the distributions of FAs in the TAGs and major phospholipids (PLs). The results of this study will provide a basis from which to elucidate further details of the lipid metabolism and will also improve our understanding of mechanisms regulating tree-peony oil metabolism.To the best of our knowledge, no previous studies have characterized the lipid molecular species in the P. ostii seed development lasted about 120 days. During the early stage of seed development (7–35 DAF), total lipid accumulation was limited (5.71% at 35 DAF), but by the end of kernel cell stage (35–42 DAF), total lipid content had increased (7.66% at 42 DAF). Between 42 and 49 DAF, total lipid content increased significantly to 11.84%, nearly double the accumulation at 35 DAF , Progenesis QI qualitative analysis identified 1963 lipids: 1455 in positive ion mode and 508 in negative ion mode , PCs (11 molecular species), PEs (10 molecular species), PGs (5 molecular species), PIs (4 molecular species), and PSs 26: the greatest PL losses were observed in PE, PC, and PI in peanut seeds.Lipid species were screened a second time, and those with fragmentation scores > 60 and scores > 45 were selected. Using this scoring system, we identified 56 TAGs Table  and 64 P27. Alves et al.28 also detected odd FAs, including 13:0, 15:0, 15:1, 17:0, 17:1, 19:0, and 21:0, on TAG and polar lipids of olive (Olea europaea L.) by LC–MS/MS.Several FAs not commonly detected in tree peony seeds were identified. LC–MS/MS data analysis identified odd FAs (saturated and unsaturated), including 13:0, 15:0, 15:1, 17:0, 17:1 and 21:0, on TAG and polar lipids Table . UnusualWe selected the lipid components with the 50 highest VIP values, corresponding to those that were the most differentially abundant between pairs of sample sets (T1–4). We then used hierarchal-clustering heatmaps, which clearly and intuitively show differences in lipid abundance among groups, to visualize differences in lipid abundance among sample sets. The heat maps showed that the abundance patterns of the lipid components with VIP values > 50 changed significantly between adjacent kernel-developmental stages Fig. . The difTo better understand the variations in the FA composition of lipids during seed development, we characterized differences in the molecular species comprising TAG, DAG, PA, PC, and PE among the four developmental stages.3. The most abundant acyl chains in TAG at all time points were 18:1/18:2/18:3, 18:1/18:1/18:3, 18:1/18:1/18:2, 16:0/18:3/18:3, 16:0/18:1/18:3, 16:0/18:2/18:3, and 16:0/18:1/18:3 , decreased in abundance during the period 35–49 DAF. Indeed, there was a general shift toward more unsaturated TAG molecular species during the oil accumulation period 29 and rape (Brassica napus L.)25.Our FA composition analysis showed that UFAs were the main molecular species comprising TAGs. This was consistent with a previous report, which stated that UFAs accounted for more than 90% of all FAs in seedsP. ostii kernel also changed greatly over the four stages of kernel development  via PDAT in P. ostii. The results of the tree-peony lipidomic analysis were consistent with the results of the tree-peony transcriptome analysis. A previous study, which used lipidomics to reveal the details of lipid accumulation in the developing seeds of oilseed rape, suggested that DGAT may have important effects on the molecular composition of TAG25.Our analysis showed that the composition of PAs, PCs, and PEs in the P < 0.05; Table P < 0.05; Fig. Several lipid components were identified as significantly differentially abundant between adjacent developmental stages  and ALA were significantly up-regulated at 77–119 DAF as compared to 35 DAF, while palmitoylcarnitine and sphinganine-phosphate were slightly less abundant. Conversely, pyruvic acid, sphingosine-1-phosphate, and sulfonate were down-regulated at 77–119 DAF as compared to 35 DAF Table ; in part32. Pyruvate is pivotal for the metabolic connections among these nutrients34. During rapid oil accumulation in P. ostii (77–119 DAF), pyruvic acid abundance decreased significantly, possibly because it was used in multiple metabolic pathways, including the citrate cycle, carbon fixation in photosynthetic organisms, and glycolysis/gluconeogenesis  as compared to the period of rapid growth before oil accumulation (before 49 DAF); during the oil accumulation period, sphinganine-phosphate content increased slightly . The abundances of 18:1, 18:2, and 18:3 in TAG acyl chains increased significantly during the rapid oil-accumulation stage in the kernel. Fewer different FAs were identified in PA and PC than in TAG, although more odd-carbon FAs were identified in PA. During seed development, monounsaturated FAs in PC and PE increased, while PUFAs decreased. The types and contents of lipid metabolites differed significantly among the different stages of kernel development; lipid distributions seemed to be specific to the developmental stage. Lipid-related metabolites, such as sphingosine-1-phosphate, pyruvic acid, stearic acid, and ALA, were significantly differentially abundant during the critical period of oil accumulation (49–119 DAF), and the corresponding metabolic pathways were significantly enriched. Notably, the sphingolipid metabolic pathways were significantly enriched during rapid seed-oil accumulation, but not significantly enriched in other periods. Our results provide a basis for future studies of the lipid metabolism and its regulation in tree peonies.The lipids extracted from P. ostii trees in Shanghai Chenshan Botanical Garden, Shanghai, China . These P. ostii were introduced to the garden more than 10 years ago, and have been grown under the same environmental and cultivation conditions since that time. Now, there are thousands of living plants of P. ostii growing in Shanghai Chenshan Botanical Garden. We observed the development of the seeds produced by P. ostii trees. The budded flowers of four selected plants  were hand-pollinated with pollen collected from a fifth P. ostii tree (CS10). The pollination date was designated 0 days after fertilization (DAF). Between 0 and 119 DAF, seeds were collected every seven days from each plant . After seed collection, the kernels were manually extracted, frozen in liquid nitrogen, and stored at − 80 °C. In addition, the voucher specimens of P. ostii was identified by Yonghong Hu and deposited in Shanghai Chenshan Herbarium numbered CSH0184197.The seeds were collected from mature 3. Based on preliminary results, four stages of kernel development that differed significantly with respect to lipid accumulation were selected for further analysis: 35 DAF (sample set T1), 49 DAF (sample set T2), 77 DAF (sample set T3), and 119 DAF (sample set T4). Each sample set included eight replicates . Two samples from each of the four trees listed above were taken.To explore how total lipids changed throughout kernel development, we measured the lipids contents in kernel samples taken from the four selected plants between 35 and 119 DAF . Total lipids were extracted from the dried biomass as previously describedWe transferred 50 mg of each sample from sample sets T1–4 (accurately weighted) to separate 1.5 mL Eppendorf tubes. Two small steel balls were added to each tube, along with 20 μL of 2-chloro-1-phenylalanine (0.3 mg/mL), 20 μL of heptadecanoic acid (0.01 mg/mL) dissolved in methanol , and 500 μL of water. All samples were incubated at − 20 °C for 2 min, and then ground at 60 Hz for 2 min. Each homogenate was transferred to a 10 mL glass centrifuge tube, 1.5 mL of a mixture of dichloromethane and methanol (v:v = 2:1) was added, and the tube was vortexed for 1 min. Each tube was chilled at − 20 °C for 20 min and then centrifuged at 1500 g and 4 °C for 15 min. The bottom layer of the stratified liquid was transferred to a new LC–MS injection vial, and evaporated in a vacuum. Next, this process was repeated using the remaining stratified liquid in the glass centrifuge tube: 1.5 mL of a mixture of dichloromethane and methanol (v:v = 2:1) was added to the stratified liquid in each tube, and the tubes were vortexed for 1 min. The tubes were then chilled at − 20 °C for 20 min. Chilled samples were centrifuged at 1500 g and 4 °C for 15 min. The bottom layer of the stratified liquid in each was transferred to the LC–MS vial containing the first extraction and allowed to dry out. After volatilization, the lipid residue in each LC–MS vial was redissolved with 400 μL isopropanol acetonitrile (v:v = 1:1) by vortexing for 30 s, followed by ultrasonic extraction for 3 min. This solution was then transferred to a 1.5 mL tube and centrifuged for 10 min at 9000 g and 4 °C. After centrifugation, 100 μL of the supernatant was removed, diluted with 150 μL isopropanol-acetonitrile (v:v = 1:1), filtered through a 0.22 μm microfilter, and transferred to a LC–MS injection vial. All injection vials were stored at − 80 °C until LC–MS analysis.Samples from each group were mixed in equal amounts to generate a quality control (QC) sample. One QC sample was analyzed every eight samples, and the results of these analyses were used to evaluate the stability of the system throughout the experiment.A Nexera UPLC  system, fitted with a Q-Exactive quadrupole-Orbitrap mass spectrometer equipped with a heated electrospray ionization (HESI) source , was used to analyze the metabolic profiles in both ESI-positive and ESI-negative ion modes. ACQUITY UPLC BEH C18 columns  were used in both positive and negative modes. The binary gradient elution system consisted of a 6:4 solution of acetonitrile–water  and a 1:9 solution of acetonitrile-isopropanol . Separations were performed using the following gradient: 30% B for 3 min, 30–62% B from 3 to 5 min, 62–82% B from 5 to 15 min, 82–99% B from 15–16.5 min, hold at 99% B for 1.5 min, 99–30% B from 18 to 18.1 min, and hold at 30% B from 18.1 to 22 min. The flow rate was 0.35 mL/min, and the column temperature was 45 °C. All samples were kept at 4 °C throughout the analysis. The injection volume was 5 μL. The mass range of the instrument was set to m/z 120–1800. The ion spray voltage was set to 3500 V in the positive ion mode and to -3100 V in the negative ion mode. The heater temperature was set to 300 °C, and the capillary temperature was set to 320 °C. The sheath gas flow rate was 45 arb, the aux gas flow rate was 15 arb, and the sweep gas flow rate was 1 arb. The S-Lens RF Level was 50%.37. The Hotelling’s T2 region, shown as an ellipse in the score plot of each model, defines the 95% confidence interval of the modeled variation. Variable importance in the projection (VIP) ranks the overall contribution of each variable to the OPLS-DA model, and those variables with VIP > 1 are considered relevant for group discrimination [CITE]. We used the default eight-round cross-validation, with one-eighth of the samples excluded from the mathematical model in each round, to guard against overfitting.The acquired LC–MS raw data were analyzed using progenesis QI software  with the following parameters: precursor tolerance set to 5 ppm; fragment tolerance set to 10 ppm, and retention time (RT) tolerance set to 0.02 min. Internal standard detection parameters were deselected for peak RT alignments, isotopic peaks were excluded from the analysis, the noise elimination level was set to 10.00, and the minimum intensity was set to 15% of base peak intensity. The matrix data were obtained using three dimensional datasets , and an RT-m/z pair was used as the identifier for each ion. The matrix was further reduced by removing any peaks missing values (ion intensity = 0) in more than 60% of the samples. An internal standard was used for data QC. Principal component analysis (PCA) and  partial least-squares-discriminant analysis (O)PLS-DA were performed to visualize metabolic alterations among experimental groups, after mean centering (Ctr) and Pareto variance (Par) scaling, respectivelyhttp://www.lipidmaps.org/). Metabolites were considered to differ significantly among groups if the OPLS-DA-obtained variable influence on projection (VIP) values were > 1.0 and the p-values generated by two-tailed Student’s tests of the normalized peak areas were < 0.05.Lipid identification was performed using progenesis QI Data Processing Software, based on public database Lipidmaps (v2.3)  metabolic database (https://www.kegg.jp/kegg/pathway.html)38using the MBRole (http://csbg.cnb.csic.es/mbrole2/) for KEGG pathway analysis. Metabolites were considered significantly enriched in a KEGG pathway if P < 0.05. KEGG enrichment pathways and heat maps were constructed using OmicShare, which is a free online platform for data analysis (www.omicshare.com/tools). Visual analysis of the metabolite-associated metabolic pathways was performed using Mapman (version 3.6.0 RC1)40.Metabolites were identified as differentially abundant between developmental stages (sample sets T1–4) when absolute value of log2 (FC) > 1.5 and All charts were drawn using Excel 2017, OmicShare, and GraphPad Prism 7.0. Image layout was performed using Adobe Illustrator CS6. Parameter data were analyzed using one-way ANOVAs in GraphPad Prism 7.0.The experiments involving plants performed in this study complied with the laws of China.Supplementary Information 1.Supplementary Information 2.


Melittin effectively protected against HT22 cell viability and significantly deregulated the Aβ25–35-induced overproduction of intracellular reactive oxygen species. Western blot analysis showed that melittin suppressed cell apoptosis and regulated Bax/Bcl-2 ratio, as well as the expression of proapoptotic related factors: Apoptosis-inducing factor (AIF), Calpain, Cytochrome c (CytoC), Cleaved caspase-3 (Cleacas3). Additionally, melittin enhanced the antioxidant defense pathway by regulating the nuclear translocation of nuclear factor erythroid 2-like 2 (Nrf2) thus upregulated the production of the heme oxygenase-1 (HO-1), a major cellular antioxidant enzyme combating neuronal oxidative stress. Furthermore, melittin treatment activated the Tropomyosin-related kinase receptor B (TrkB)/cAMP Response Element-Binding (CREB)/Brain-derived neurotrophic factor (BDNF), contributing to neuronal neurogenesis, and regulating the normal function of synapses in the brain. In our in vivo experiment, melittin was shown to enhance the depleted learning and memory ability, a novel finding. A mouse model with cognitive deficits induced by Aβ25–35 intracerebroventricular injection was used. Melittin had dose-dependently enhanced neural-disrupted animal behavior and enhanced neurogenesis in the dentate gyrus hippocampal region. Further analysis of mouse brain tissue and serum confirmed that melittin enhanced oxidant–antioxidant balance, cholinergic system activity, and intercellular neurotrophic factors regulation, which were all negatively altered by Aβ25–35. Our study shows that melittin exerts antioxidant and neuroprotective actions against neural oxidative stress. Melittin can be a potential therapeutic agent for neurodegenerative disorders.Melittin, a 26-amino acid peptide, is the main component of the venom of four honeybee species and exhibits neuroprotective actions. However, it is unclear how melittin ameliorates neuronal cells in oxidative stress and how it affects memory impairment in an in vivo model. We evaluated the neuroprotective effect of melittin on Aβ Highlights25–35; For the first time, melittin has been reported to enhance cognitive function in a learning memory-deficit model; Melittin ameliorating mechanisms were observed via the nuclear translocation of Nrf2, stimulating 1-HO production and the regulation of the TrkB/CREB/BDNF pathway.Melittin alleviates HT22 in vivo and in vitro oxidative stress injury induced by AβApis dorsata, Apis mellifera, Apis florea, and Apis cerena, which contain 95.8 ± 3.2%, 76.5 ± 1.9%, 66.3 ± 8.6%, and 56.8 ± 1.8% melittin, respectively [For centuries, bee venom therapy has been used to manage acute and chronic human diseases; recently, this focus has steadily gained more interest from researchers and practitioners for bee venom’s anti-neurodegeneration ability . Melitti2O2 [Regarding neuroprotective effects, as evidenced by previous studies, melittin exhibits suppressive effects on the proinflammatory, and potentially pro-oxidative, response of BV2 microglia including the ability to reduce nitric oxide (NO) and inducible nitric oxide synthase (iNOS) by blocking LPS-induced activation of NF-κB7, suppressing the expression of COX-2/PGE2, resulting in anti-inflammatory properties . Another2O2 .As the primary component of honeybee venoms, melittin potential is high, and more research needs to be conducted on this compound to further explore its role in combating neurodegeneration. Neuro-oxidative stress ignited by beta amyloid (Aβ) in the human brain is a main trigger for the progression of neural disorder ,7, and mIn previous cardiology and renal studies, melittin was reported to regulate the nuclear translocation of Nrf2, a key transcription factor that upregulates HO-1 expression ,9. HO-1 25–35 fragment is the shortest segment that was proven to have similar oxidative neurotoxicity output compared with the full-length Aβ1–42 [25–35 has become a common neurodegeneration igniter agent that simulates in vitro intercellular damage caused by excessive redox impacts [Regarding the establishment of a neurodegenerative model for this study: Aβ aggregated species in neurodegenerative brains is considered a major cause of the onset of cognitive dysfunction disease because of its ability to alter intercellular redox balance ,12,13. Ah Aβ1–42 . With goh Aβ1–42 , in rece impacts ,17,18,19 impacts ,20,21,2225–35, and Fluoromount™ Aqueous Mounting Medium were purchased from Sigma–Aldrich , Aβ25–35 aggregation was performed  before being introduced to the experiments [TM WST-8 Cell Viability Assay Kit was obtained from BIOMAX . The specific mouse primary antibodies  and the secondary anti-mouse IgG (HRP-linked antibody) were purchased from Cell Signaling Technology, Inc. . Specific BDNF, lamin B, and mAChR1 antibodies were purchased from Merck KGaA . Pro-PREP protein extraction solution was purchased from iNtRON Biotechnology . First doublecortin antibody  and Alexa Fluor 488 secondary antibody  were used. Cellular Reactive oxygen species (ROS), SOD (Superoxide Dismutase), Lipid Peroxidation (MDA), Lactate Dehydrogenase (LDH), and protein carbonyl activity required acetylcholine esterase (AchE) assay kits, AIF first antibody, CytoC first antibody, and CleaCas3 first antibody . A nuclear extraction kit was purchased from Cayman Chemical Company, Inc. . An Ach level assay kit was obtained .We purchased 1 × DMEM, 1 × PBS, and 1 × TBS from Welgene, Inc. . Trypsin-EDTA (0.25%), FBS, 2,7-dichlorofluorescein diacetate (DCFDA), and penicillin–streptomycin were obtained from Invitrogen . Melittin, Aβeriments . The Qua2 and 37 °C. Cells were cultured at a density of 1 × 104 cells per well (with 0.1 mL media) in a 96-well plate, and 3 × 105 cells per well (with 3 mL media) in a 6-well plate. Twenty-four hours after seeding, drugs were administrated into the wells as explain in the next parts.The HT22 mouse hippocampal cell line was kindly gifted by Professor Na Chang Su and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with FBS (10%) and penicillin–streptomycin (1%) in an incubator at 5% CO25–35 was added. Cells were incubated for another 24 h, followed by incubation with WST-8 for 1 h. Absorbance was measured at 450 nm using a VersaMax Microplate Reader . The experiments were repeated three times.To assess cell viability in 96-well plates, after 24 h, melittin was added to the wells (0.1 to 30 μM for a safety concentration test and 0.1 to 3 μM for cellular protection activity), and 1 h later, 7 μM of Aβ25–35. For apoptosis assay, cells were washed with kit assay buffer twice, then to each well we added 200 µL assay buffer + 1 µL kit solution of CytoCalcein 450 (for live cell detection) + 1 µL kit solution of 7-AAD (for detecting necrotic cells) + 2 µL kit solution of Apopxin Green Indicator (for detecting apoptosis cells), followed by incubated at room temperature, in the dark, for 45 min. Subsequently, cells were washed once and replaced with 100 µL assay buffer. Live cells were detected using a 4′,6-diamidino-2-phenylindole (DAPI) channel (Ex/Em = 405/450 nm), necrotic cells were detected using a Red Fluorescent Protein (RFP) channel (Ex/Em = 550/650 nm), and apoptosis cells were detected using a Green Fluorescent Protein (GFP) channel (Ex/Em = 490/525 nm) with a 30× objective. In total, three images were taken at the center of three replicated wells (containing a sum of 270–330 cells), and the apoptosis cell percentage was calculated relative to the total number of observed cells in each image. For ROS assay, cells were washed with kit assay buffer twice, and then each well was filled with 200 µL of 25 μM DCFDA solution and incubated at 37 °C, in the dark, for 45 min. The DCFDA solution was then removed and replaced with 100 µL assay buffer. ROS fluorescence levels were detected via the GFP channel (Ex/Em = 490/525 nm) with a 30× objective. In total, three images were taken at the center of three replicate wells and analyzed with ImageJ software . An Invitrogen EVOS FL Auto Imaging System  fluorescence microscope was used.For apoptosis and ROS immunofluorescent experiments: In black 96-well, clear bottom plates, experiment started 7 h after treatment with Aβ25–35; cell lysates were performed as per the kit instructions, with SOD, MDA, LDH, and protein carbonyl levels according to the manufacturer’s instructions. The data were collected using a VersaMax Microplate Reader .For SOD, MDA, LDH, and protein carbonyl assays: In 6-well plates, the experiment started 7 h after treatment with Aβ25–35 for 7 h, cells were harvested. A part of the cell pellet was lysed to obtain whole-cell protein. The other part was mixed with hypotonic buffer containing phosphatase and protease inhibitors. After 10 min of incubation at 4 °C, 10% Nonidet P-40 Assay Reagent was added to stimulate phase separation. After centrifugation , the supernatant as the cytosolic extract was stored at −80 °C until use. The remaining pellets were added to the nuclear extraction buffer for 30 min at 4 °C. After centrifugation , the supernatant was collected as the nuclear extract.To examine the nuclear translocation of Nrf2, we fractionated nuclear and cytosolic proteins from the whole cell content . Cells wICR mice  were obtained from Dehan Biolink Co.  and housed with two mice per cage, with specific pathogen-free conditions  under a 12-h light/dark cycle and with free access to standard mouse food  and water. The mice were acclimatized for five days before the experiments were initiated. All behavioral experiments were conducted under the same ambient conditions according to the Guide for Care and Use of Laboratory Animals of the National Research Council  and were approved by the Committee of Animal Care and Experiment of Dongshin University, Korea (DSU2019-04-02).25–35 intracerebroventricular (ICV.) injections were performed as described in previous studies: The prepared Aβ25–35 solution or PBS was injected (5 µL) into the right cerebral ventricle using a 28-gauge stainless steel needle via stereotaxic coordinates (in mm) from the bregma—A: -0.22, L: 1.0, V: 2.5, with flow rate at 5 μL/min [25–35. A melittin maximum safety dosage of approximately 1.5 mg/kg was previously determined [25–35 ICV. + PBS SC.; Mel 1.5 group, 5 μg Aβ25–35 ICV. + melittin 1.5 mg/kg SC.; and Mel 0.15 group, 5 μg Aβ25–35 ICV. + melittin 0.15 mg/kg SC. Aβ5 μL/min ,24. On dtermined ,27. TherThe Morris water maze (MWM) test was applied to evaluate the effects of melittin on mice spatial learning and memory as previously described with minor modifications . The MWMOn day 11, all mice were anesthetized, blood and brains samples were collected. In each group, the brains of a subset of animals were used for immunofluorescence analysis  and the others brain’s hippocampus were collected immediately for biochemical or western blot analysis. 2 ice powder and cut into 25-μm sagittal sections using a cryostat and transferred to gelatin-coated slides [Brains were post-fixed overnight before being incubated with 30% sucrose in sodium phosphate buffer  for 24 h at 4 °C. The brains were then frozen by dry COd slides . At room25–35 treatment, the cell pellet was collected and lysed. Mice hippocampus samples were homogenized with lysate buffer immediately after collection. Western blotting was performed as described previously [Cells were grown in 6-well plates. After 7 h of Aβeviously . ProteinHippocampus lysate, or blood serum samples were prepared according to the manufacturer’s instructions. After adjusting the concentrations to normalize total protein levels, ROS, NO, MDA, AchE, and Ach assays were carried out according to the manufacturer’s instructions. Data were collected using a VersaMax Microplate Reader .Statistical evaluation was performed using SPSS software, version 18.0, and the data are expressed as the mean ± standard deviation. The presented escape latency and path length data in the MWM test were analyzed by repeated-measures two-way analysis of variance (ANOVA). The other behavioral data and biomarker changes in vitro were tested using one-way ANOVA for multiple comparisons.25–35-induced stress, melittin (0.3–3 μM) exhibited observable dose-dependent effects; therefore, these concentrations were selected for further study  pretreatment dose-dependently enhanced Nrf2 nuclear translocation. At 3 μM, melittin treatment markedly increased Nrf2 nuclear translocation by up to 4-fold and HO-1 production by 2-fold compared to Aβ25–35 treatment only  and analyzed the alteration of melittin (0.15–1.5 mg/kg) on the escape latency time and the number of platform area crossings by mice. Melittin treatment (1.5 mg/kg) significantly reduced the escape latency of mice on the 10th day  increased it by 2.5-fold  in both hippocampal tissue and mouse serum. Melittin ameliorated ROS levels in a dose-dependent manner. In addition, NO and MDA levels were significantly reduced in the melittin-treated groups   and an ove model .Neurodegenerative diseases, including Alzheimer which results in a decrease of human cognitive function, have become the main obstacles that degrade human life span and the overall wellbeing .25–35 fragment is the shortest segment of full-length Aβ1–42that can form a beta-sheet; this fragment was proven to have similar oxidative neurotoxicity output compared with the full-length Aβ1–42 [25–35 has grown over the last decade [Through aging, Aβ is a product that naturally emerges and accumulates in the brain. Accumulated Aβ gradually forms aggregates with beta-sheet structures and has the capacity to alter the intercellular redox balance of neurons cells. This is considered a major cause of the onset of cognitive dysfunction ,13,39. Ah Aβ1–42 . With got decade .2+ influx, destabilize intercellular balance, and produce cellular oxidative stress [25–35 and Aβ1–42were also both found to cause mitochondrial abnormalities via the deactivation of mitochondrial complex IV [25–35 and Aβ1-42 is still a topic of debate, but the single methionine-35 located in both Aβs was mentioned as its redox states are significantly important for Aβ-correlated free radical oxidative stress and neurotoxicity causes [In detail, both full-length and short Aβs were proven to form ion-like channels in cell membranes that promote Cae stress ,41,42. Amplex IV ,44,45,46mplex IV ,47,48. Ey causes ,50,51.25–35 induced injury in HT-22 cells, including massive ROS release, resulting in MDA and LDH leakage and increased protein carbonyl levels, which substantially reduced HT-22 cell viability. Melittin dramatically mitigated Aβ25–35-induced oxidative stress injury by reducing ROS, MDA, SOD, and eventually, the protein carbonyl levels. To further explain these phenomena, we examined the melittin effect on the generation of HO-1, an important component of the cellular antioxidant system. This protein expression is inducible and has been demonstrated to shield cells against oxidative damage [25–35-induced HT22 cell injury was attributed to its antioxidant properties. This is in line with the findings of previous studies indicating that melittin enhances Nrf2 nuclear translocation and subsequently upregulates the expression of important antioxidant genes such as HO-1 [In the in vitro study, our results demonstrated that Aβe damage . HO-1 exe damage ,33. Our  as HO-1 ,9.25–35 presence significantly depleted the TrkB/CREB/BDNF pathway. Remarkably, melittin induced p-TrkB activation, increased the amount of p-CREB transcription factor, and in turn upregulated the expression of BDNF [25–35, an protective effect which was confirmed by the normalization of Bax/Bcl-2 ration, apoptosis-inducing factor, Calpain, CytoC, and CleaCas3.One other mechanism of melittin can be related to the TrkB/CREB/BDNF pathway, which is a commonly studied direction along with antioxidant aspects ,31,52,53 of BDNF . Moreove of BDNF ,55. The 25–35 into mice brains was a utilized as a mean to induce oxidative stress, initiating synaptic loss, suppressing neurogenesis, and resulting in cognitive impairments in animal experiments [For in vivo research, the injection of Aβeriments ,20,21,22In this study, melittin significantly enhanced the memory and learning abilities of cognitive impairment-induced animals, a novel finding. The neuroprocessing ability of mice is closely related to hippocampal physiology . Among t25–35 ICV., and melittin treatment clearly decreased these oxidative stress markers. Our results showed melittin reduced the amount of NO accumulation and iNOS protein expression in the hippocampus, which indicates melittin’s effect in lowering neuronal-derived nitric oxide—a key element stimulating neural diseases [To further explore the antioxidant property of melittin, we measured ROS and MDA levels in the hippocampus and serum; the levels of these parameters were significantly increase due to Aβdiseases ,59.25–35 in hippocampal tissues was significantly normalized by melittin pretreatment. Excessive AChE activity leads to a decrease in the Ach level in hippocampal cholinergic synapses. Aβ25–35 ICV. injection augmented AChE activity by 1.5-fold in hippocampal tissue, whereas pretreatment with melittin completely attenuated the excessive activation of AChE and increased ACh levels. Regarding intercellular neurotrophic factors, the transcription factor CREB, which plays a key role in BDNF synthesis, is essential for memory and synaptic plasticity [Maintenance of the balance of the cholinergic system is necessary for normal memory function . Previouasticity . In lineTherefore, through both in vivo and in vitro experiments, melittin had proved itself to be a drug candidate combating neurovegetative disease.The drug administration route of melittin is often subcutaneous, which can cause adverse effects if over-dosed . AlthougMelittin is the dominant component of four most common honeybee species’ venoms. The present study demonstrates that melittin exerts neuroprotective effects by regulating the activation of Nrf2/HO-1 and the TrkB/CREB/BDNF pathways in HT22 cells. Also, for the first time to our knowledge, melittin was found to recover the depleted learning and memory ability in a neurodegenerative in vivo model. These findings contribute to clarify the underlying ability and mechanism of melittin to protect neuronal cells from neuronal-oxidative stress and promote the role of melittin as a medication combating cognitive impairment disorders.