Chronic heat stress has a negative impact on poultry meat quality. Although this has been extensively investigated, previous studies have primarily focused on metabolic alterations and oxidative stress in the pectoralis major (PM) muscle under chronic heat stress, and not all of the underlying molecular mechanisms are completely understood.A total of 144 male Arbor Acres broilers (28 d old) were randomly allocated into 3 treatment groups: (1) the normal control (NC) group, with broilers raised at 22 °C and fed a basal diet; (2) the heat stress (HS) group, with birds raised at 32 °C and fed a basal diet; and (3) the pair-fed (PF) group, with birds raised at 22 °C and fed the amount of feed equal to the feed consumed on the previous day by the HS group. The experiment lasted for 14 d.P < 0.05); and increased drip loss, cooking loss, shear force, hardness, and decreased pH, redness (a*); and springiness of PM muscle (P < 0.05). Furthermore, chronic heat stress decreased muscle fiber density, increased connective tissue, and led to intracellular vacuolation. The transcriptome analyses indicated that the effect of chronic heat stress on meat quality was not only related to metabolism and oxidative stress, but also to signal transduction, immune system, transport and catabolism, cell growth and death, and muscle structure.Chronic heat stress decreased the average daily feed intake and average daily gain, increased feed:gain ratio (Chronic heat stress has a negative impact on the growth performance, meat quality, and the PM muscle structure of broilers. Transcriptome analysis revealed a comprehensive understanding of the mechanism of the chronic heat stress-induced deterioration of broiler meat quality at the transcriptional level.The online version contains supplementary material available at 10.1186/s40104-022-00759-3. Poultry meat has highly digestible proteins and low-fat content . Modern Heat stress can cause sharp changes in the physiology and metabolism of birds, which negatively affect a variety of production parameters including feed intake and conversion efficiency, growth, egg production and quality, carcass traits, immune function, meat production, and quality , 4, 5. IThe transcriptome is the complete set of transcripts and their quantity in cell or tissue. It can quantify the changing expression levels of each transcript during development and under different conditions . TranscrOne hundred and forty-four chickens at 28 days of age (1460 ± 20 g) were randomly divided into three environmental chambers : normal control group , heat stress group  and pair-fed group . Each environmental chamber included six replicates (cages) (length × width × height: 1.2 m × 0.8 m × 0.45 m), and each cage included eight chickens. All birds had free access to water and received a common basal feed. The feed formula is shown in Table th and 14th d of the experiment. Average daily gain (ADG), average daily feed intake (ADFI), and feed:gain ratio (F:G) were calculated at the end of the experiment.Daily feed intake was measured at 06:00 every day. The live weights of broilers were recorded on the 7st, 3rd, 7th, and 14th d of the experiment, the body surface temperature (BST) of the birds was measured at 10:00 by using a Fluke Ti400 thermal imaging camera   and 14th (42 days of age) d of the experiment, 12 chickens (2 chickens per cage) were selected, electrically stunned, and slaughtered. The entire right PM muscle was stripped for meat quality determination. A sample of muscle (0.5 cm × 0.5 cm × 2 cm) was collected and fixed with 4% paraformaldehyde fixative solution. Additional PM muscle samples were collected in 3 cryovials and stored in liquid nitrogen.At the 7A pH/ORP meter  was used to measure the muscle pH at 45 min and 24 h postmortem. The results for each meat sample are the average of 3 measurements at different locations.A chroma meter  was used to measure meat color at 24 h postmortem. The results for each meat sample are the average of 3 measurements at different locations.The drip losses were measured as previously described . BrieflyAt 24 h postmortem, PM muscle samples (3 cm × 5 cm × 5 cm) were weighed and placed into valve bags and cooked until the central temperature reached 70 °C in a 72 °C water bath. When the samples were chilled to room temperature, they were wiped and reweighed. Then the percentage of lost weight was calculated.The samples after cooking loss determination were cut into 3 strips (1 cm × 1 cm × 3 cm) parallel to the muscle fibers. Then, the shear force was measured 3 times using a digital meat tenderizer . The results for each meat sample are the average of 3 measurements at different locations.A cylindrical sample from cooked meat samples was taken with a sampler and then the textural characteristics were assessed by using a TMS-PRO texture analyzer . The texture test parameters were as previously described .After dehydrated in a graded series of ethanol, paraformaldehyde-fixed muscle samples were embedded in paraffin and cut into 6 µm thick sections, and stained with hematoxylin and eosin. Images were captured using a light microscope .A total of 6 broilers in each group, were mixed in pairs to make 3 samples. Then, total RNA was extracted with 1 mL TRIzol reagent  according to the manufacturer's instructions. RNA quality was assessed using an Agilent 2100 Bioanalyzer . Sequencing libraries were constructed using an RNA sample preparation kit  according to the manufacturer's instructions. Then, the libraries were sequenced on the Illumina Hiseq Novaseq6000 Platform.The fastp (version 0.18.0) was used to obtain high-quality, clean reads. The rRNA mapped reads were removed by using the Bowtie2 (version 2.2.8) tool. The software Stringtie and HISAT2 were used to align the clean reads to the reference genome.The fragments per kilobase of transcript per million mapped fragments (FPKM) of each transcription region were calculated by using the RSEM software to quantify its expression abundance and variations. The R package gmodels  was used P < 0.05 and an absolute fold change >1.5 were considered differentially expressed. Then differentially expressed genes (DEGs) were carried on the GO functional and KEGG pathway enrichment analyses by using GOseq [P-values (P < 0.05) were used to determine the significantly enriched GO terms and KEGG pathways.DESeq2 software was used for differential expression analysis between two groups, and the genes withng GOseq  and KOBAng GOseq , respect−ΔΔCt method and β-actin was selected as reference gene for normalization.Twelve genes were selected for qRT-PCR analysis to verify the reliability of RNA sequencing results (RNA-Seq). Specific primer pairs were designed by using the Primer3 software  Table 22. ReversP < 0.05. The data of growth performance were analyzed with the cage as the experimental replicate and other indices were analyzed by the mean of two sampled chickens per cage as a replicate (n = 6).One-way ANOVA was used to analyze the data using SPSS Statistics . The results are presented as the mean ± standard deviation (SD), and the significant difference was set at P < 0.05).As shown in Fig. P < 0.05, Table P < 0.05, Table P < 0.05, Table Compared with the NC group, heat stress decreased the ADG and ADFI and increased the F:G ratio . Furthermore, the springiness of the PM muscle in the HS group was declined compared with that in the NC group (P < 0.05). At the 14th d of the experiment, the hardness of the PM muscle from the HS group increased, while springiness decreased (P < 0.05).As shown in Table th d of the experiment, PM muscle fibers from the HS group were scattered, the connective tissue increased, and vacuoles appeared in some cells compared with the NC and PF groups. At the 14th d of the experiment, PM muscle damage in the HS group was further aggravated. At both time points, the morphology of the muscle fibers in the NC and PF groups were relatively intact and there was no damage. Heat stress had no significant effect on the fiber diameter of the PM muscle. Compared with the NC group, the muscle fiber density of the PM muscle in the HS and PF groups showed a decreasing trend , acetyl-CoA carboxylase alpha (ACACA), carnitine palmitoyl transferase 2 (CPT2), acyl-CoA thioesterase 9 (ACOT9), diacylglycerol kinase zeta (DGKZ), glycerol-3-phosphate acyltransferase 3 (GPAT3), 3-hydroxyacyl-CoA dehydratase 3 (HACD3), lipoprotein lipase (LPL), lipase A (LIPA), lipin 2 (LPIN2), palmitoyl-protein thioesterase 1 (PPT1), and peroxisome proliferator-activated receptor gamma (PPARG). Glucose metabolic processes related genes such as fructose-bisphosphatase 2 (FBP2), bisphosphoglycerate mutase (BPGM), glucose-6-phosphate isomerase (GPI), glycogen synthase (GYS1), pyruvate dehydrogenase kinase 4 (PDK4), lactate dehydrogenase A (LDHA), probable D-lactate dehydrogenase mitochondrial isoform X2 (LDHD), pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1), fructose-2,6-biphosphatase 3 (PFKFB3), phosphoglucomutase 1 (PGM1), phosphoglycerate mutase 1 (PGAM1), phosphoglucomutase 2 like 1 (PGM2L1), phosphoglycerate kinase 2 (PGK2), pyruvate kinase (PKM), succinate-CoA ligase ADP-forming beta subunit (SUCLA2) and triosephosphate isomerase 1 (TPI1) were down-regulated in PM muscle from HS group compared with that from the NC and PF groups. Aconitase 1 (ACO1), aldolase fructose-bisphosphate C (ALDOC), aconitate decarboxylase 1 (ACOD1), enolase family members , fructose-bisphosphatase 1 (FBP1), aldolase fructose-bisphosphate C (GPD1L2), lactate dehydrogenase B (LDHB), phosphogluconate dehydrogenase (PGD), phosphoglucomutase 2 (PGM2), and solute carrier family 2 members  were up-regulated in birds from the HS group. In the HS group, muscle structure related genes such as actin alpha1 (ACTA1), myotubularin related protein 7 (MTMR7), myosin family members , tropomodulin 4 (TMOD4), troponinI 2 (TNNI2), troponin I3 (TNNT3), were down-regulated, while actin beta (ACTB), actinin alpha1 (ACTN1), actin filament associated protein 1 (AFAP1), ARP2 actin related protein 2 family members , anillin actin binding protein (ANLN), actin related protein 2/3 complex subunit family members , coactosin like F-actin binding protein 1 (COTL1), calponin 2 (CNN2), destrin actin depolymerizing factor (DSTN), Fascin actin-bundling protein 1(FSCN1), leiomodin 2 (LMOD2), myosin family members , spire type actin nucleation factor 1 (SPIRE1), tropomodulin 1 (TMOD1) were up-regulated. Compared with the NC and PF groups, all genes related to apoptosis listed in Table SESN1), solute carrier family 25 member 4 (SLC25A4), apoptosis-associated tyrosine kinase (AATK), heat shock protein 90 alpha family class B member 1 (HSP90AB1), mitofusin 2 (MFN2), and programmed cell death 2-like (PDCD2L).Heat stress significantly up-regulated genes related to lipid metabolism and synthesis processes such as acyl-CoA synthetase family members  increased after a cyclic high-temperature treatment [Previous studies reported that chronic heat stress reduces growth performance by impairing the gut integrity and appetite of broilers , 4. The M muscle . In the reatment . SimilarIn the current study, the transcriptome of PM muscle was used to explore the mechanisms of inferior meat quality induced by chronic heat stress. The PCA result demonstrated that heat stress was the main factor responsible for the difference in the transcriptional profiles of the HS group (98.3%), indicating that chronic heat stress was the major factor causing the deterioration of growth performance and meat quality. Confirmed the previous hypothesis that heat stress reduces growth performance has another mechanism, besides the reduction in feed intake. In order to investigate the effects of chronic heat stress and eliminate the influence of feed intake, we deleted the DEGs between the NC and PF groups. Then, selected 1239 DEGs as the target DEGs from those remaining genes in the HS groups, which were significantly compared with the NC and PF groups. The target genes were related to metabolic process, response to stimulus, catalytic activity, cell part, cell growth and death, HIF-1 signaling pathway, lysosome, regulation of actin cytoskeleton, and sphingolipid metabolism. Among the target DEGs, the genes involved in cell part, membrane, and sphingolipid metabolism are related to cell membrane integrity and apoptosis under heat stress , and we 3 exposure induced up-regulation of the CYPS and oxidative stress in the jejunum of broilers [CYP1C1, CYP2J21, CYP2J23, CYP39A1, CYP4A22) were up-regulated in birds from the HS group  are a group of heme-containing proteins that catalyze the activation of molecular oxygen, resulting in the generation of reactive oxygen species (ROS) , 26. In broilers . In thisACACA, GPAT3, and PPARG. This might lead to the deposition of intramuscular fat, which is consistent with the previous study [LPIN2, LPL, and LIPA were up-regulated while the carnitine palmitoyl transferase1 (CPT1), one of the rate-limiting enzymes of fat oxidation, was down-regulated in HS group compared with the NC (log2(FC): −0.35) and PF (log2(FC): −1.04) groups. These results may be the reason for the decrease in beta-oxidation in heat-stressed birds.Chronic heat stress has been shown to cause lipid metabolism changes . In the us study . Zhao etus study . In thisSLC2A14, SLC2A6, and ATPase family members were up-regulated ) beta, a rate-limiting enzyme of the tricarboxylic acid cycle, was down-regulated of broilers experienced heat stress, which might result in a reduction in ATP production. These findings were consistent with previous results which showed that chronic heat stress damaged the mitochondrial structure and affected muscle energy metabolism , 32. At ACTA1, were up-regulated in the HS group. The expression of myosin family members MYH1E, MYH1F, MYL1, MYLK2, and MYO18B were down-regulated, while MYH1B, MYO1E, MYO1F, MYO3AL, and MYO7A were up-regulated in the HS group. Moreover, the expression of TNNT3 and TNNI2 in the HS group were decreased. Actin, myosin, and troponin are structural proteins of muscle cells and they play a pivotal role in the mechanism of muscular contraction [Several studies have reported that heat stress causes muscle damage. Chronic heat stress causes histopathological damage in broilers . Sula ettraction , 39. Thetraction . GO and CHOP) and subsequently activated cell apoptosis [TGFB1), v-myc avian myelocytomatosis viral oncogene homolog (MYC), Janus kinase 1 (JAK1), and programmed cell death 1 (PDCD1) were up-regulated. In the present study, the histological observation found that vacuoles were observed inside the cell and some muscle fibers were degraded in the HS groups. These results demonstrate that chronic heat stress causes cell apoptosis. Our previous study showed that oxidative stress led to apoptosis and autophagy and resulted in a decline in meat quality by decreasing muscle pH24h and increasing shear force value [Several studies have demonstrated that heat stress affects apoptosis. Heat stress activates the FasL/Fas and TNF-α systems and induces apoptosis of follicular cells in laying hens . Heat expoptosis , 45. Chepoptosis . In thisce value . This maIn conclusion heat stress has a negative impact on growth performance and meat quality. The transcriptome analyses indicated that the effect of chronic heat stress on meat quality was not only related to metabolism and oxidative stress, but also to signal transduction, immune system, transport and catabolism, cell growth and death and muscle structure. The results provide a comprehensive understanding of the mechanism of chronic heat stress-induced deterioration of broiler meat quality at the transcriptional level.Additional file 1: Table S1. List of some differentially expressed genes related to meat quality.


Acox1, Pparα, and Ucp2) and lipophagy (Atg7). Moreover, we confirm this effect on 3D spheroids and organoids. Conclusion: Incubation with bergamot PF reduces intracellular lipid neutral fat potentially by increasing intracellular pathways related to beta-oxidation.Background: Bergamot polyphenolic fraction (PF) extract exerts a beneficial against liver steatosis. However, the fundamental processes underlying this beneficial effect of bergamot PF remain elusive. In this work, we examined the effect of bergamot PF extract on 2D and 3D hepatocyte cultures. Material and Methods: We evaluated the effect of bergamot PF in 2D and 3D cultures from rat, human hepatoma cells, and human primary hepatocytes. Results: In 2D cell culture, we demonstrated that incubation with bergamot PF decreases intracellular lipid content and is associated with an increase in expression levels of ß-oxidation genes ( Non-alcoholic or metabolic associated fatty liver disease (NAFLD) is the most common cause of chronic liver disease, and it is set to become the leading cause of liver transplants ,2. SpeciCitrus bergamia risso et poiteau (Bergamot), the fruit from a tree growing in Calabria, a southwest region of Italy, has a high content of flavonoids and glycosides [ycosides ,8. Moreoycosides ,10,11, aycosides  and rodeycosides . However2. Sample were harvested by trypsinization, and they were subcultured twice weekly.Rat hepatoma cells, McA Rh7777, were obtained from the American type culture collection (ATCC). The cell was maintained in DMEM , supplemented with 10% FBS, 1% penicillin streptomycin , and 1% sodium pyruvate , at 37 °C in 5% CO2 and grown for a total of 96 h.The HepG2 + LX-2 spheroids were generated as previously described . Brieflyg for 5 min. Once the cells collect at the bottom, self-aggregation causes formation of organoids. At day 1 after seeding, 100 µL of serum-free maintenance medium was added to make a total of 200 µL per well. Next, every 48 h, 50% of the media was replenished for fresh serum-free medium until day 7. The volume of spheroids and organoids were determined using the following formula: 4/3 π r3, where “r” was the mean of the long and short diameter of the spheroid divided by 2.Briefly, for the generation a liver organoid model composed of primary human hepatocytes (PHH), cryopreserved primary human hepatocytes  were used ,16. CellBergamot PF extract as previously prepared  and charWe calculated the bergamot PF extract amount to be use in our experiments based on Parafati et al.  by usingFor the 2D culture, McA Rh-7777 cells were treated for 24 h with 50 µM of oleic acid  conjugated to fatty acid-free bovine serum albumin (BSA) and bergamot PF extract dissolved in the cell media at concentrations of 0.001, 0.001, 0.1, and 1 µg/mL.Instead, HepG2 + LX-2 spheroids, 24 h after seeding, were treated only with bergamot PF extract dissolved in the cell media at a concentration of 1 µg/mL for 72 h. The treatment was refreshed every 48 h.Human liver organoids, 24 h after seeding, were treated only with bergamot PF extract dissolved in the cell media at a concentration of 1 µg/mL for 6 days. The treatment was refreshed every 48 h.4 cells/well in 96-well plates. Cell viability was determined by 3--2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, MTT  solution (5 mg/mL) was added to each well and incubated at 37 °C for 4 h. The supernatants were then removed and replaced by 100 mL of DMSO. The optical density (OD) was measured at a wavelength of 570 nm.For the 2D culture, McA Rh7777 cells were seeded at a density of 1 × 10® 3D cell viability assay  according to the manufacturer’s instructions. Briefly, single spheroids were transferred into a white 96-well assay plate  with 50 µL PBS. Then, 50 µL of assay reagent was added to each sample well and vigorously mixed to allow the reagent to penetrate the spheroids, after which the plate was incubated at room temperature for 20 min in darkness. Then, the plate was placed in the SpectraMax i3 (Molecular Devices) counter and luminescence was measured using the SoftMax Pro 6.3 software .For HepG2 + LX-2 spheroids and human liver organoid, total cellular adenosine triphosphate (ATP) was measured in the Cell-Titer-Glo4 cells/well in 24-well plates. After treatment, the cells were washed with PBS, and fixed with 2% paraformaldehyde for 5 min. Intracellular lipids were stained with Oil Red O solution  for 20 min. Cell nuclei were stained with Mayer for 5 min. All the staining procedure was carried out at room temperature by protecting the samples from the direct light. Images were acquired with an Olympus microscope (BX53) and high-resolution Olympus digital image camera (XC50). The images were quantified using ImageJ software .For evaluate intracellular lipid content, McA Rh-7777 cells were seeded in a coverslip at a density of 5 × 10w/v paraformaldehyde (Sigma-Aldrich) for 2 h, then incubated with 20% w/v sucrose in phosphate buffered saline (PBS) overnight, washed three times with PBS and embedded with OCT Cryomount . Then, 8-µm-thick sections were made using a cryostat  and transferred into glass slides and stored at −80 °C for 1 h, after which ORO staining was performed. Nuclei were stained using DAPI. Images were acquired using an Axio KS 400 Imaging System and AxioVision 4.8 software (Zeiss) at 20X. The ORO-stained area was normalized to the number of DAPI-stained nuclei and quantified using ImageJ .Instead, HepG2 + LX-2 spheroids and human liver organoid after treatment were fixed with 10% 4 cells/well in 24-well plates. Cells were then washed with PBS, and fixed with 2% paraformaldehyde for 5 min. Cell nuclei were stained with DAPI for 5 min. Triglyceride content was stained with AdipoRed lipid assay  for 10 min at 37 °C. All of the staining procedure was carried out by protecting the samples from the direct light. Images were acquired using an Axio KS 400 Imaging System and AxioVision 4.8 software (Zeiss) at 20X magnification. The images were quantified using ImageJ software .For evaluate intracellular triglyceride content, McA Rh-7777 cells were seeded in a coverslip at a density of 5 × 10Srebp-1c, Acox1, and Pparα were quantified by real time-PCR using SYBR® Green dye   according to the manufacturer’s instructions. cDNA was synthesized from 1 µg total RNA, using a high-capacity cDNA Reverse Transcription Kit . The mRNA expression of aly) See .Ucp2, Atg7, ApoB, Mttp, and β-Actin according to the manufacturer’s protocol. All reactions were performed in triplicate. Data were analyzed using the 2−ΔΔCt method normalized to β-Actin  and reverse transcribed using a high-capacity cDNA reverse transcription kit  according to the manufacturer’s instructions. Gene expression was assessed by real-time qPCR using master mix (Life Technologies) and TaqMan probes for ctin See .SREBP-1C, ACOX1, PPARα, UCP2, ATG7, and β-ACTIN according to the manufacturer’s protocol. All reactions were performed in triplicate. Data were analyzed using the 2−ΔΔCt method normalized to β-Actin  and reverse transcribed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Gene expression was assessed by real-time qPCR using master mix (Life Technologies) and TaqMan probes for ctin See .p-values less than 0.05 were considered significant. Statistical analysis was performed with GraphPad Prism 5.0.Data are represented as mean ± standard deviation (SD) of at least three independent experiments and analyzed using a two-tailed Student’s t-test, linear regression, and nonparametric Mann–Whitney test. The To ensure that bergamot PF extract does not affect cell viability, McA Rh7777 cells were incubated with 50 µM of oleic acid and an increasing amount of bergamot PF extract  for 24 h. Given that bergamot PF extract was dissolved directly in the media, cell media was used as a negative control. Cell viability was measured by 3--2,5-diphenyltetrazolium bromide (MTT) assay. This assay showed that bergamot PF extract did not affect cell viability at 24 h as compared to the control .p = 0.001)  and after 24 h the intracellular lipid content was examined. Bergamot PF extract resulted in a dose-dependent lowering of intracellular neutral lipids as compared to the control (= 0.001) A,B. At a.1 µg/mL B.p = 0.001) (p < 0.05)  and, after 24 h, the intracellular triglycerides were measured. Bergamot PF extract incubation resulted in a dose-dependent reduction in the intracellular triglyceride content (= 0.001) . Further < 0.05) .Acox1, Pparα, and Ucp2 in McA Rh7777 cells incubated with increasing concentrations of bergamot PF extract . After 24 h of incubation, we observed a dose-dependent increase in Acox1 (p = 0.0001), Pparα (p = 0.001), and Ucp2 (p = 0.009)  A–C.p = 0.01) . After 24 h incubation, we observed a dose-dependent increase in Srebp-1c ( = 0.01) D.ApoB and Mttp in McA-Rh7777 cells incubated with increasing concentrations of bergamot PF extract . After 24 h incubation, we observed that bergamot PF extract does not influence the levels expression of ApoB and Mttp . After 24 h incubation, we observed a dose-dependent increase in Atg7 (p = 0.01)  E.p = 0.03, To confirm our results in a different cell type and in a more physiological system, we tested incubation with bergamot PF extract in 3D spheroids generated by immortalized hepatocytes (HEPG2) and hepatic stellate cells (LX2) in a physiological ratio of 24:1 (14). Consistently, incubation with bergamot PF extract resulted in a reduction in the intracellular neutral lipid content  and PPARα (p = 0.03)  A,B.SREBP-1C in immortalized spheroids incubated with 1µg/mL bergamot PF extract for 72 h. After 96 h incubation, we observed that bergamot PF extract did not influence the levels of expression of SREBP-1C (Next, we examined mRNA levels of genes involved in triglyceride synthesis, namely SREBP-1C D.p = 0.003, To further confirm the effect of bergamot PF extract incubation, we generated a human liver organoid by using primary human hepatocytes from a single donor. Briefly, a total of 3000 cryopreserved primary human hepatocytes were seeded on ultra-low attachment 96-well plates. The day after seeding, cells were incubated with 1 µg/mL bergamot PF extract for 6 days. Consistent with the previous experiments, bergamot PF extract incubation resulted in a reduction in the intracellular neutral lipid content (The main finding of this study is that incubation with bergamot PF extract results in a reduction in intracellular lipid content in human hepatocytes that may potentially be due to an increase in beta-oxidation.Our bergamot PF extract contained 40% of neoeriocitrin, naringin, and neohesperidin. Administration of these polyphenols resulted in a reduction in liver fat content in humans and rats. To replicate these data, we incubated rat hepatic cells with bergamot PF extract and examined the intracellular lipid content. Incubation with bergamot PF extract resulted in a dose-dependent lowering of intracellular neutral lipids and triglycerides, except for the 0.1 µg/mL treatment B.Atg7, a gene involved in autophagy/lipophagy, a fundamental process involved in human liver disease [Next, we examined the metabolic pathways responsible for the intracellular homeostasis of triglycerides, and we observed an increase in genes involved in beta-oxidation. In the McA-rh7777 cell culture, consistent with the previous study in rats, we observed an increase in  disease . HoweverTo replicate our data in human cells, we used 3D spheroids composed of immortalized human hepatic and hepatic stellate cells. Bergamot PF extract resulted in a reduction in intracellular neutral lipids and an increase in beta-oxidation. However, in this model there was no difference in the synthesis of triglycerides and lipophagy. These data may suggest a species-specific effect of bergamot PF on autophagy in rats as opposed to humans. Species-specific studies are warranted to clarify this observation.To further confirm the beneficial effect of bergamot PF, we used a liver organoid model composed by human primary hepatocytes. Consistent with previous results, in human and rat hepatoma cells, incubation of bergamot PF extract resulted in a reduction in intracellular neutral lipids. These data show a robust and reproducible effect across hepatocytes derived from three different donors.Bergamot extract contains compounds with a statin-like effect ,10,11. POur study has some limitations. Polyphenols contained in bergamot PF extract are chemically unstable at neutral pH values ,21. As aIn conclusion, treatment with bergamot PF results in a reduction in hepatocyte intracellular neutral lipids. This effect may potentially be mediated by an increase in beta-oxidation.


Dermacentor marginatus (28.08%), Hyalomma asiaticum (21.28%), Hyalomma anatolicum (17.18%), Dermacentor reticulatus (2.01%), Ixodes ricinus (3.35%), Ixodes persulcatus (0.33%), Hyalomma scupense (12.87%) and Hyalomma marginatum (14.90%). Ticks collected from livestock animals were examined for the pathogen spectrum of transmissible infections to determine the degree of their infection. Four pathogen DNAs (lumpy skin disease virus (LSDV), Coxiella burnetti, Teileria annulata, and Babesia caballi) were detected by PCR in Dermacentor marginatus, Hyalomma asiaticum, Hyalomma scupense, Hyalomma anatolicum. The infection of ticks Dermacentor marginatus and Hyalomma asiaticum collected on cattle in the West Kazakhstan region with LSDV was 14.28% and 5.71%, respectively. Coxiella burnetti was found in the ticks Dermacentor marginatus (31.91%) in the Turkestan region and Hyalomma anatolicum (52.63%) in the Zhambyl region. Theileria annulata was found in ticks Hyalomma scupense (7.32%) and Dermacentor marginatus (6.10%) from cattle in the Turkestan region. Babesia caballi was isolated only from the species Hyalomma scupense (17.14%) in the Turkestan region. There were no PCR-positive tick samples collected from sheep. RNA/DNAs of tick-borne encephalitis virus (TBEV), African swine fever virus (ASFV), Hantavirus hemorrhagic fever with renal syndrome (HFRS), and chlamydia pathogens were not found in ticks. The new data give a better understanding of the epidemiology of tick-borne pathogens and the possibility of the emergence of tick-borne animal diseases in Kazakhstan.Ticks carry and transmit a wide variety of pathogens  that pose a threat to humans and animals worldwide. The purpose of this work was to study ticks collected in different regions of Kazakhstan for the carriage of various pathogens. The collected ticks were examined by PCR for the carriage of various pathogens. A total of 3341 tick samples parasitizing three animal species  were collected at eight regions of Kazakhstan. Eight tick species were found infesting animals:  Ticks are significant vectors of various diseases that pose serious public health threats and significant economic losses, affecting the health and productivity of animals ,2. Ticks2. The relief of Kazakhstan is mostly flat—more than 80% of the country is dry steppes. The diversity of landscape and climatic conditions and the animal world of the country creates the prerequisites for the existence of foci of various pathogens, primarily associated with ticks.The territory of Kazakhstan occupies from the eastern outskirts of the Volga delta in the west to the Altai Mountains in the east, from the West Siberian Plain in the north to the Tien Shan mountain system in the south of the country. The total area is 2724.9 thousand kmDermacentor marginatus, Hyalomma marginatum, Hyalomma anatolicum, Hyalomma asiaticum, Hyalomma scupense have epidemiological significance in the territory of Kazakhstan [The tick fauna of Kazakhstan includes more than 30 species of ixodid ticks, recognized as carriers of a number of dangerous infectious pathogens. zakhstan . In Kazazakhstan , tick-bozakhstan , babesiozakhstan , Q feverzakhstan ,9 and Lyzakhstan .Over the past few decades, an increase in both the number of ticks in Kazakhstan and the number of cases of tick-borne diseases (TBD) has been recorded ,11,12,13Research on tick-borne diseases in Kazakhstan is rare. All research findings concerning tick-borne diseases are the results of studies performed within the framework of small scientific projects. These studies are insufficient to better understand the risk of tick-borne infections in different ecological zones. There is no state program to study the prevalence of pathogens among ticks in Kazakhstan. Therefore, many issues of the current state of tick populations and their epidemiological significance in the territory remain insufficiently studied. The current situation on the spread of ixodid ticks—potential carriers of infections—in the Republic of Kazakhstan requires further study of the ranges, abundance and infection of these arthropods, as well as the establishment of their epidemiological significance. Eliminating this deficiency is the goal of this study, studying ticks collected in different regions of Kazakhstan for the carriage of various pathogens.Ixodidae family from 3 genera were identified: Dermacentor , Ixodes , Hyalomma . The most abundant species found were Dermacentor marginatus (28.08%) and Hyalomma siaticum (21.28%) and Hyalomma anatolicum (17.18%). Other five species, i.e., Dermacentor reticulatus (2.01%), Ixodes ricinus (3.35%), Ixodes persulcatus (0.33%), Hyalomma scupense (12.87%) and Hyalomma marginatum (14.90%), were rarer  was detected using PCR in Dermacentor marginatus, Hyalomma asiaticum, Hyalomma scupense, and Hyalomma anatolicum  and Hyalomma asiaticum (5.71%) collected from cattle in the Bokey Orda district of the West Kazakhstan region. Coxiella burnetti DNA was detected in Dermacentor marginatus (31.91%) collected from cattle in the Otrar district of Turkestan region and Hyalomma anatolicum (52.63%) in Taraz city of Zhambyl region. Teileria annulata DNA was found in Hyalomma scupense (7.32%) and Dermacentor marginatus (6.10%) collected from cattle in the Tolebi district of Turkestan region. Babesia caballi DNA was isolated only in Hyalomma scupense (17.14%) in the Tolebi district of Turkestan region , which encode the small ribosomal RNA subunit, are identical to strains from Pakistan , Turkey (MG569892), China (KU554731), India (MF287925), and Italy (MT341858).The nucleotide sequences of Babesia caballi (GenBank accession number OP077204), which encodes a small ribosomal RNA subunit, is 100% identical to isolates from Israel  and South Africa .The nucleotide sequence of The nucleotide sequence of LSDV , which encodes the ankyrin-repeat protein gene, was highly identical with LSDV strain Kubash/KAZ/16 (MN642592), LSDV Kenya isolate Kenya (MN072619), LSDV strain LSDV/Russia/Dagestan/2015 (MH893760).Coxiella burnetii , it was found that the sequenced region of the IS1111A gene, was highly identical with Coxiella burnetii strain AuQ31 (KT954146), Coxiella burnetii isolate TW-1 (EU000273), Coxiella burnetii isolate ICMR 9 (MT920358)  .Ticks play a substantial role as vectors of pathogens. Pathogens transmitted by ticks are responsible for the majority of the vector-borne diseases in temperate regions of America, Europe and Asia. According to some researchers, more than 100,000 cases per year of tick-borne diseases are recorded in the world . Tick-boThe ticks collected in this study reinforce previously recorded distributions of tick species in Kazakhstan ,18,19,20Dermacentor marginatus (938/28.08%), Hyalomma asiaticum (711/21.28%), Hyalomma anatolicum (574/17.18%), Hyalomma marginatum (498/14.90%), Hyalomma scupense (430/12.87%), Ixodes ricinus (112/3.35%), Dermacentor reticulatus (67/2.01%), Ixodes persulcatus (11/0.33%). On 175 cattle 2127 (63.66%) ticks belonging to 8 species , on 247 sheep 1085 (32.48%) ticks belonging to 7 tick species  and on 76 horses 129 (3.86%) ticks belonging to 5 tick species  were identified.In the present study, 3341 ticks encompassing 8 species were collected from 8 regions of Kazakhstan: Dermacentor marginatus, Hyalomma asiaticum, Hyalomma anatolicum, Hyalomma scupense) out of eight tested were PCR-positive and considered as vectors of pathogens among domestic animals. Lumpy skin disease, Q fever, babesiosis, and theileriosis viral DNAs were detected in ticks from various regions of Kazakhstan.In our study, four species of ticks (Lumpy skin disease (LSD) is an infectious disease that is transmitted by various arthropods . Almost Ixodes ricinus , Boophilus annulatus (14.3%), Dermacentor marginatus (13.8%), Hyalomma marginatum (11.6%), and Haemaphysalis scupense (8.1%). This led to the conclusion that ixodid ticks may have acted as LSDV reservoirs during the 2015 outbreaks, but more detailed studies are required to confirm these preliminary results [Dermacentor marginatus (14.28%) and Hyalomma asiaticum (5.71%) collected from cattle in the West Kazakhstan region. These data are consistent with the results of our previous studies, where it was shown that Dermacentor marginatus and Hyalomma asiaticum ticks taken from sick animals during the LSD epizootics in the Atyrau region were involved in LSDV transmission [Until 2015, the disease had not been reported in northern temperate regions and there were many gaps in the knowledge about potential carriers of LSDV. The first report on the transmission of LSDV by ticks in northern latitudes was confirmed after LSD outbreaks in Russia. Viral DNA has been detected at least in 13 species of ixodid ticks belonging to six genera. LSDV genome has been detected in  results . In our natus 14.% and HyaCoxiella burnetii that is prevalent throughout the world and has a significant impact on animal welfare and human health [Coxiella burnetii infection in China is 10% in humans, 15% in cattle, and 12% in goats.Q fever is an infectious disease caused by n health ,30. The n health . Betweenn health . In Chinn health . The oveCoxiella burnetii remains limited to this day. Besides the main route of transmission via inhalation of infectious aerosols, ticks are still under debate as potential vectors for Coxiella burnetii. The importance of ticks in the epidemiology of Q fever remains controversial, although a sufficient number of studies have shown the presence of Coxiella burnetii in ticks [Coxiella burnetii infection among 25 different tick species in 23 European countries. Significantly higher prevalence was observed in southern European countries [However, knowledge of in ticks . Recent ountries .Coxiella burnetii specific DNA in eight tick species  from four provinces in Northwest China. Recent studies have shown that the prevalence of Coxiella burnetii in ticks in four provinces in Northwest China averages 10%, 11.9% in Xinjiang and 2% in three northeastern provinces  and Inner Mongolia. Coxiella burnetii DNA was found in Dermacentor silvarum, Ixodes persulcatus, Hyalomma conicinna, Hyalomma japonica, Boophilus microplus, Dermacentor nuttalli, Dermacentor niveus, Hyalomma detritum, Hyalomma scupense, and Hyalomma qinghaiensis [In China, only one study detected haiensis .Coxiella burnetii in saigas [Coxiella burnetii in various regions of the country. Our studies showed PCR-positive results for Coxiella burnetii in Dermacentor marginatus (31.91%) collected in Turkestan region and Hyalomma anatolicum (52.63%) in the Zhambyl region. These data are consistent with the results of our previous studies [Q fever has been reported in Kazakhstan since the early 1950s . There an saigas . There iCoxiella burnetii in Hyalomma asiaticum and Dermacentor marginatus was 4% and 2.3%, respectively. In the present study, we did not detect positive samples in other regions of Kazakhstan. However, according to some researchers, Coxiella burnetii was previously detected in the northern and western regions [Coxiella burnetii in various regions of Kazakhstan, it is necessary to continue monitoring studies with a larger number of samples.The authors have shown that in 2013, in the Kyzylorda region, the tick infestation of  regions . To obtaHyalomma anatolicum, Hyalomma lusitanicum, Hyalomma scupense, Hyalomma detritum detritum, and Hyalomma dromedarii [Theileriosis is distributed throughout the world, from Asia, the Middle East and South Europe to North Africa ,39 and iomedarii ,41,42. Aomedarii .The prevalence of diseases caused by blood parasites in Kazakhstan is poorly understood. There are isolated reports indicating the presence of these diseases among animals and ticks in Kazakhstan ,44.Theileria annulata are present among ticks Rhipicephalus turanicus in the Almaty region, Dermacentor marginatus in the Turkestan region and Rhipicephalus turanicus in the Zhambyl region. The prevalence among ticks ranged from 2.30% to 33.33% [Theileria annulata DNA was detected in Hyalomma scupense (7.32%) and Dermacentor marginatus (6.10%) tick species collected from cattle. All PCR-positive ticks were collected in Turkestan region. These results are consistent with the results of our previous studies [Dermacentor marginatus is the main carrier of Theileria annulata in Turkestan region. The detection of theileria in Hyalomma scupense indicates that this tick species is a potential vector for this disease.Studies conducted by a group of scientists from Kazakhstan, China and Hungary showed that o 33.33% . In our scupense .32% and Babesia caballi and Theileria equi. Equine babesiosis is endemic in most countries of the tropical and subtropical regions of the world [Babesia caballi and Theileria equi. Scoles and Ueti described 33 Ixodid tick species belonging to six genera as competent vectors responsible for equine piroplasmosis [Hyalomma, Dermacentor, Rhipicephalus are the biological vectors of Theileria equi and Babesia caballi, while other genera of ticks, including Amblyomma, Haemaphysalis and Ixodes, are suspected but not confirmed. The list is probably not exhaustive since most research studies are focused on only a small subset of ticks closely related to horses.Equine Piroplasmosis (EP) is widespread in almost all countries of the world and cause enormous harm to agriculture ,46. Animhe world ,48,49. Hhe world ,51. The lasmosis . The autTheileria species in livestock and wild animals or their ticks is not well studied in our country. Recent studies have suggested that causative agents of piroplasmosis in horses are present in ticks in the territory of Almaty and Turkestan regions [Babesia caballi DNA in one Dermacentor marginatus tick and Theileria equi DNA in two Hyalomma asiaticum ticks in the Almaty region. The same study also revealed that Babesia caballi DNA was found in 2 ticks and Theileria equi DNA in 6 Dermacentor marginatus ticks in Turkestan region.The presence of  regions . In theiBabesia caballi was isolated only from the Hyalomma scupense (17.14%) specie collected from horses. All PCR-positive ticks were collected in Turkestan region. Based on the data obtained, it can be assumed that Hyalomma scupense is one of the likely vectors of this disease. To confirm these data, it is necessary to continue studies on a larger number of samples.In our studies, the DNA of Our results have increased awareness about the distribution of tick-borne pathogens in Kazakhstan. However, study results on tick distribution, population and disease presence are insufficient in Kazakhstan to better understand the risk of tick-borne infections within various ecologic zones. Further research on ticks in different regions is needed to assess the current situation in the country. Further research on ticks in different regions is required to assess the current situation in the country.Dermacentor marginatus, Dermacentor reticulatus, Ixodes ricinus, Ixodes persulcatus, Hyalomma asiaticum, Hyalomma scupense, Hyalomma marginatum, and Hyalomma anatolicum) were collected from animals . Species identification of ticks was confirmed morphologically ,54.While sampling and accounting, sampling staff had to follow special precautions as wearing a personal protective clothing with a high neckline and cuffs, also periodic self- and mutual inspections for the presence of ticks.After morphological evaluation, ticks were disinfected with 70% ethanol, then rinsed in ultrapure water, again treated with 70% ethanol, and homogenised in PBS . The resEach tick was processed separately, according to the principle “one tick—one sample”. DNA/RNA was isolated from each tick.Dermacentor marginatus, Dermacentor reticulatus, Ixodes ricinus, Ixodes persulcatus, Hyalomma asiaticum, Hyalomma scupense, Hyalomma marginatum, Hyalomma anatolicum) was carried out using commercial TRizol reagent  in accordance with manufacturer’s instructions. DNA/RNA concentrations were measured using a NanoDrop 2000 spectrophotometer . To assess the purity and quality of nucleic acids in spectrophotometric measurement, the purity of the sample was determined based on the ratio of optical densities at A260/A280.Extraction of nucleic acids from tick suspensions . RT-PCR was performed using a Rotor-Gene Q thermal cycler .PCR to detect theileria. Theileria species verification was performed using the AccuPrime™ Taq DNA Polymerase System PCR kit . Species-specific primers targeting the 18S rRNA gene fragment ThEq F1 (AGACAGAGG AAG GAT TGACA) and ThEqR (CTGATGACTTGCGCATACTA), which can amplify a product of 386 bp, were used. Plasmid DNA containing inserts corresponding to the Theileria spp. 18S rRNA gene was used as a positive control.2 (50 mM), 1 µL of 20 pmol F primer, 1 µL of 20 pmol R primer, 0.5 µL of Taq DNA Polymerase, 16 µL of deionized water, 3 µL of DNA. Reactions were performed under the following conditions: initial denaturation 94 °C for 2 min, followed by 35 cycles of denaturation 94 °C for 20 s, annealing at 55 °C for 30 s, extension at 68 °C for 60 s, followed by a final extension at 68 °C for 7 min.Each PCR reaction was carried out in a 25 μL reaction volume, which contained 2.5 µL 10× PCR buffer, 1 µL of MgClPCR to detect babesia. PCR analysis was performed in a final volume of 25 µL which contained 2.5 µL of 10× PCR buffer, 1 µL of MgCl2 (50 mM), 1 µL of 20 pmol F primer, 1 µL of 20 pmol R primer, 0.5 µL of Taq DNA Polymerase, 16 µL of deionized water, 3 µL of DNA. AccuPrime™ Taq DNA Polymerase System PCR Kit . Detection of Babesia caballi was carried out using the primers B.Cab_F (TCAGCACCTTGAGAGAAATC) and B.Cab_R (ACAGATTACCCACACCTTTC), which amplify a product of 451 bp. Plasmid DNA containing inserts corresponding to the gene for the small subunit of ribosomal RNA of Babesia spp. was used as a positive control. Amplification conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 20 s, annealing at 55 °C for 30 s, extension at 72 °C for 40 s, and then a final extension at 72 °C for 7 min.PCR to detect the LSDV. The reaction mixture consists of 25 µL total volume: 2.5 µL of 10× PCR buffer, 1 µL of dNTP (10 mM), 2 µL of MgCl2 (25 mM), 1 µL of 20 pmol F primer, 1 µL of 20 pmol R primer, 0.5 µL of 5 units Taq DNA Polymerase, 14 µL of deionized water, 3 µL of DNA. The AccuPrime™ Taq DNA Polymerase System kit  was used to formulate the PCR mixture. Thermal cycling conditions were as follows: 3 min at 94 °C 35 cycle, 20 s at 94 °C, 30 s at 55 °C, 40 s at 72 °C and 7 min at 72 °C. Primers LSDV F-CTGCAAAGGCGGATAATTATGATG and LSDV R- CCATGGTGTCTTATGACCCCAAT were used for PCR. The size of the PCR product is 742 bp. When setting up PCR, the DNA of the Dermatitis nodulares/2016/Atyrau/KZ LSDV strain was used as a positive control.PCR to detect Coxiella burnetti. The amplification was performed in a total volume of 25 µL, containing 3 µL of DNA sample, 2.5 µL of 10× Buffer, 1 µL of 50 mM MgCl2, 1 µL of primer Trans1, 1 µL of primer Trans2 and 0.5 µL of Taq DNA polymerase . The Trans-PCR thermal program using primers Trans 1: 5′-TGG TAT TCT TGC CGA TGA C-3′; Trans 2: 5′-GAT CGT AAC TGC TTA ATA AAC CG-3′ was carried out under the following conditions: initial denaturation at 95 °C for 5 min followed by 40 cycles consisting of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 1 min, then a final extension at 72 °C for 7 min [Coxiella burnetii was used as a positive control.or 7 min . When sePCR to detect chlamydia. The reaction was performed in a final volume of 25 µL containing 2.5 µL of 10× PCR buffer, 1 µL of MgCl2 (50 mM), 1 µL of 20 pmol F primer, 1 µL of 20 pmol R primer, 0.5 µL of Taq DNA Polymerase, 16 µL of deionized water, 3 µL of DNA. AccuPrime™ Taq DNA Polymerase System kit  and primers hi1f (GCA GTC GAG AAT CTT TCG CAA TG), hi1r (AGC TGC TGG CAC GGA GTT AG) were used to compile the PCR mixture. When setting up PCR, the DNA of Chlamydia trachomatis serovar L2 strain 434 was used as a positive control.The thermal profile comprised 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 45 s at 58 °C, 60 s at 68 °C, and 7 min at 68 °C.PCR to detect TBEV. TBEV-RNA was detected by a set of reagents “OM-Screen-TBE-RT” . RT-PCR was performed by sequentially adding 15 µL of the diluent to the reaction mixture in stranded microtubes, 20 µL of negative control, 20 µL of samples in the required repetition, and then 20 µL of TBE positive control. PCR amplification program: 50 °C for 15 min, 95 °C for 5 min, 50 cycles , fluorescence signal reading at 60 °C.PCR for the detection of ASFV. PCR was performed using the African swine fever (ASF) test system for the detection of African swine fever virus by polymerase chain reaction . The reaction mixture consists of 25 µL total volume: 3.5 µL of PCR-c-1 ASFV, 10 µL of 2.5× PCR blue buffer, 0.5 µL of TaqF polymerase, 1 µL of UdG-TL enzyme, 10 µL of DNA, 10 µL of NC- negative control, 10 µL of ASFV DNA FRP positive control. PCR amplification program: 1 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 25 s at 62 °C, 25 s at 72 °C and 1 min at 72 °C.PCR for the detection of HFRS. For the detection of HFRS, a set of reagents “OM-Screen-HFRS-RT”  was used. Real-time PCR is performed by serially adding 15 µL of diluent (RB) to the reaction mixture in stranded microtubes (RM-HFRS), then adding 20 µL of negative control, 20 µL of test samples, and then 20 µL of positive control. PCR amplification program: 1st stage: 50 °C, 15 min; 2nd stage: 95 °C–5 min; 3rd stage: 50 cycles , fluorescence signal reading at 58 °C.For sequencing, obtained PCR products were used as the template. Sequencing was performed on an Applied Biosystems 3130 automated DNA sequencer  using the BigDye Terminator v3.1 Cycle Sequencing kit . The resulting nucleotide sequences were analyzed in Sequencer v. 4.5 .The identities and similarities of sequenced isolates were analyzed using the basic local alignment search tool (BLASTn) of the National Center for Biotechnology Information (NCBI) GenBank database and MEGA 11.0 program .Theileria annulata , Babesia caballi , Coxiella burnetii .The analysis used available nucleotide sequences of strains in GenBank:


They also exhibit outstanding lightabsorption properties suitable for solar energy applications. Here,we describe the synthesis of RbPbI3 by mechanochemicalprocedures with green credentials, avoiding toxic or expensive organicsolvents; this specimen exhibits excellent crystallinity. We reportneutron powder diffraction data, essential to revisit some subtlestructural features around room temperature (200–400 K). Inall these regimes, the orthorhombic Pnma crystalstructure is characterized by the presence along the b direction of the crystal of double rows of edge-sharing PbI6 octahedra. The lone electron pairs of Pb2+ ionshave a strong stereochemical effect on the PbI6 octahedraldistortion. The relative covalency of Rb–I versus Pb–Ibonds shows that the Pb–I-related motions are more rigid thanRb–I-related vibrations, as seen in the Debye temperaturesfrom the evolution of the anisotropic displacements. The optical gap,measured by diffuse reflectance UV–vis spectroscopy, is ∼2.51eV and agrees well with abinitio calculations. The thermoelectric Seebeck coefficient is 3 ordersof magnitude larger than that of other halide perovskites, with avalue of ∼117,000 μV·K–1 at 460K.All-inorganic leadhalide perovskites like CsPbBr 3 perovskiteshave been grownfrom a mechanochemical method under mild milling conditions. Theyyield a better-crystallized sample with appealing thermoelectric propertiesand a suitable band gap for solar cell applications.All-inorganic RbPbI Strong semiconducting band gap absorption is observedin nanocrystalline perovskites self-assembled on mesoporous TiO2 films. Significant power conversion efficiencies are measuredunder full sunlight in single-junction devices based on highly crystallineperovskite absorbers due to their intense visible to near-infraredabsorptivity. In fact, the electron–hole diffusion length ina MAPbI3 absorber is ∼100 nm,3 enabling highly efficient planar heterojunction PSCs. Amongother advantages, flat PSCs present an inherent suitability for flexiblesubstrates, besides their ability to form hybrid silicon/perovskitetandems. Unfortunately, the paradigmatic MAPbI3 is unstablein humid air. An alternative is to combine it in a bilayer solar-cellarchitecture with either the narrower band gap, but also unstable,formamidinium lead iodide (FAPbI3) or with the more stablemethylammonium lead bromide (MAPbBr3).4 Formamidinium-based perovskites also suffer from problemslike the “yellow-phase impurity” (∂-FAPbI3), which can be suppressed via the addition of RbI, whichin turn forms RbPbI3 impurities that turn out not to havedetrimental effects and contribute to a higher mobility of the chargecarriers with a longer useful lifetime, culminating in excellent 20.3% power output.5Perovskitesolar cells (PSCs) are extremely appealing technologiesfor providing inexpensive solar electricity.3 (α-CsPbI3), can only be stabilizedat high temperatures. However, it can form nanoscale quantum dots(QDs) that work well in efficient optoelectronic devices.6 Crucially, α-CsPbI3 QD filmsdo not deteriorate in ambient air. Colloidal perovskite QD photovoltaiccells form light-emitting diodes with an open-circuit voltage of 1.23V. Together with other all-inorganic perovskite lead halides , the entire visible spectral region (410–700nm) is accessible with bright photoluminescence (PL),7 and for photodetector devices that can be tuned in nanocrystals(NCs), by simple halide ion exchange reactions.8 Furthermore, it has been recently demonstrated that thepresence of methylammonium in hybrid perovskites adds a significantsource of nonradiative loss,9 stemmingfrom the removal of a hydrogen atom from the organic molecule, whichcan be triggered by photons incident on the cell. This effect, reallydetrimental for efficiency, is not present in all-inorganic perovskites.All-inorganic perovskitescan provide an alternative to hybridorganic–inorganic perovskites, but they are not without hindrances.The compound with the most suitable band gap, the cubic phase of bulkCsPbI3  and RbPbI3, agree very well with experimentalvalues.13 The band gap of CsPbI3 makes it ideally suited for usein tandem solar cells.15 However, the ambient instabilityof CsPbI3 hampers its application in thin film form. Forthis reason, the much enhanced phase stability in environmental conditionsof the Rb counterpart makes it a good light absorber candidate foruse in solar cells and photodetectors, justifying further attention.Both CsPbI3 and RbPbI3 compounds share commonfeatures, such as the orthorhombic Pnma symmetryof the crystal structure and isotropic thermal expansion with nearlyidentical relative change of the lattice parameters.16 Yet, their structural evolutions differ strikingly near600 K: at 602 K, CsPbI3 has a first-order reversible phasetransformation Pnma → Pm3̅m, although RbPbI3 maintains its Pnma structureuntil it melts.15 PL studies show a PLcenter value of 2.1 eV at 300 K.19 The RbPbI3 perovskite-related halide has even been introduced into solarcells, taking advantage of the superior stability in environmentalconditions, including a TiO2/RbPbI3 configuration.17 Even though the device performance with RbPbI3 is inferior compared to that with the cesium counterpart, this approach establishesthe realization of highly stable perovskite films, achieving an incipientphotovoltaic performance for real applications.Promisingly, band gaps from first-principles calculations of leadhalide perovskite semiconductors, including CsPbXn polyhedra in three(3D), two (2D), and zero (0D) dimensions: 3D CsPbBr3, 2DCsPb2Br5, 0D Cs4PbBr6,3D CsPbCl3, 2D CsPb2Cl5, 0D Cs4PbCl6, 3D CsPbI3, and 3D RbPbI3.20 In the latter case, nevertheless,the sample was not structurally characterized; moreover, the RbPbI3 halide exhibits great potential for quantum dots applications,21 making necessary a profound study on its crystalstructure. Transport properties, such as the Seebeck coefficient andthermal conductivity, remain barely known for RbPbI3. Assimilar families of halides are being studied for possible thermoelectricapplications,25 it is of great importance to shed light on the mentioned properties.Recently, all-inorganicperovskite-type halides were synthesizedby all-solid-state mechanochemical synthesis with various dimensionalities,as defined by the PbX3 prepared by mechanochemistry.We confirm the existence of an orthorhombic Pnma phasethat persists in the whole temperature range. The NPD data allow determininghighly accurate values of the displacement factors. The analysis ofthese displacement factors unveils that Pb–I bonds are morerigid than the Rb–Cl bonds, while theoretical topochemicalevaluations disclose a relevant covalent contribution for the Pb–Ipair bond. Furthermore, scanning electron microscopy (SEM), differentialscanning calorimetry (DSC), optical spectroscopy, and thermoelectricand thermal conductivity characterization complement the structuralstudy. The Seebeck coefficient above room temperature (RT) is exceptionallyhigh, around 44,000 μV·K–1 at 400 K.For this reason, here, we present temperature-dependent (200–400K) neutron powder diffraction (NPD) data to study the structural evolutionof highly crystalline RbPbI23 was obtained as a microcrystalline powder frommechanosynthesis in a planetary ball mill, from stoichiometric amountsof RbI (Strem) and PbI2 (Alfa-Aesar), working in N2 atmosphere. 1.5 g of the reactants was milled using 30 zirconiaballs of 5 mm diameter, with a final 8.6:1 mass ratio, for 4 h at450 rpm in a Retsch PM100 mill. A laboratory X-ray diffraction (XRD)pattern was collected on a Bruker D5 diffractometer with Cu Kα (λ = 1.5418 Å) radiation. In order to investigatethe crystallographic structure, a NPD study was carried out at 200,250, 300, 350, and 400 K in the D20 instrument  with a wavelength of 1.540 Å. The sample, containedin a V cylinder, was introduced in a cryo-furnace; the patterns werecollected for 30 min each. The refinement of the crystal structurewas performed by the Rietveld method using the Fullprof software.27 A pseudo-Voigt function was chosento generate the line shape of the diffraction peaks. The backgroundwas interpolated between regions devoid of reflections. The followingparameters were refined in the final run: scale factor, backgroundcoefficients, zero-point error, pseudo-Voigt corrected for asymmetryparameters, positional coordinates, anisotropic displacement factors,and occupancy factors. For the neutron refinements, the coherent scatteringlengths for Rb, Pb, and I were 7.090, 9.405, and 5.280 fm, respectively;these distinct values guarantee a precise determination of the atomicpositions. Moreover, as the scattering length values do not decaywith the diffraction angle, intense peaks are obtained at high angles,thus improving the accuracy of the displacement factors. DSC measurementswere carried out in the range 100–300 K with a heat pulse method.Field-effect SEM (FE-SEM) images were obtained in a FEI-Nova microscope,with an acceleration potential of 5 kV, coupled to an energy-dispersiveX-ray spectrometry (EDXS) device, working with an acceleration voltageof 18 kV and 60 s of acquisition time. The optical diffuse reflectancespectrum of the RbPbI3 powder was measured at RT usinga UV–vis spectrophotometer Varian Cary 5000.RbPbI–3 mbar). The electrical resistivity was measured using an AgilentE4980A LCR meter. The total thermal conductivity was calculated fromthe thermal diffusivity (α) using a Linseis LFA 1000 equipment,by the laser-flash technique. The thermal conductivity (κ) wasdetermined using κ = α × cp × d, where cp isthe specific heat and d is the sample density.In orderto measure the transport properties, the powder was pressedto a pellet shape with no applied heat, using a cold press. The thermoelectricproperties were measured in the resulting pellet, with neither sintering/annealingnor any other step in between. Seebeck coefficient was obtained bymeasuring simultaneously the drop voltages across the sample and aconstant reference wire with an electrometer (Keithley 6517B) anda nanovoltmeter (Keithley 2182A) under vacuum (10328 using CRYSTAL17 package.29 The basisset of rubidium (Rb), lead (Pb), and iodine (I) was defined usingthe POB-TZVP basis developed byLaun and co-workers.30 The Coulomb andexchange series thresholds  ofthe package were set as 10–8, 10–8, 10–8, 10–8, and 10–16, respectively. The shirking factors (Pack–Monkhorst and Gilatnet) were set as 6 and 6, respectively. In the structure optimizationstep, the gradient components and nuclear displacements were adjustedwith tolerances on their root-mean-square of 0.0003 and 0.0012 a.u.,respectively. The main bond critical points (BCPs) of the structureswere evaluated, according to “quantum theory: atoms in molecules”(QTAIM), in order to assist in understanding the electronic characteristicsof the chemical bonds. The QTAIM was carried out with the TOPOND program within the CRYSTAL17 package.31 The crystal representation was carried out by VESTA software.32The quantum models were elaborated according to density functionaltheory (DFT) with PBE functional44.13 was obtained as a yellowish polycrystalline powder.LaboratoryXRD was used for initial crystallographic identification at RT, withorthorhombic tetragonal symmetry, in agreement with previous reports,indexable in the space group Pnma (SG: #62). It belongsto the NH4CdCl3 structural type.16 The crystal structure was preliminarily refinedin this structural model from laboratory XRD data, as displayed in a = 10.2924(6) Å, b = 4.7795(2) Å, c = 17.4049(9) Å, and V = 856.19(8) Å3. These parameters are slightlylarger than those reported, of a = 10.2761(9) Å, b = 4.7793(4) Å, c = 17.3933(12) Å,and V = 854.23 Å3.RbPbI4.2Pnma space group. TheRb+, Pb2+ cations, and the three types of I– anions  are all allocated at 4c  Wyckoffsites. 6 octahedra sharing edges,directed along the b direction of the crystal, withthe Rb+ ions in the interstices in between the octahedra,in ninefold coordination, with the Rb–I bond lengths spanningfrom 3.079(4) to 4.091(5) Å, at 300 K. Each PbI6 octahedronis composed of a Pb–I1 bond length (3.036(4) Å), two Pb–I2distances (2 × 3.222(3) Å), and three Pb–I3 bondlengths (3.397(4) Å, 2 × 3.248(3) Å), at 300 K.The structural data published in the literature correspond to refinementsfrom the XRD data [ref ], but thdn and <d> arePb–In and <Pb–I> distances,respectively.33 The value obtained in thiscase is Δd = 10.6 × 10–4, defining aconsiderable distortion that evidences the effect of the lone electronpair of Pb2+ ions.The octahedral distortionwas calculated using the “distortionindex” which is defined as4.3a, b, c, and V unit cellparameters. All parameters regularly increase upon warming up, asexpected from the thermal expansion. From the volume evolution, athermal expansion coefficient of 39.1 × 10–6 is determined in the 200–400 K temperature range. The Rietveldplots and the crystallographic data at 200 and 400 K are displayedin Figures S1 and Tables S1 and S2 of the Supporting Information, respectively.Temperature-dependentNPD patterns were measured at 200, 250, 350,and 400 K, showing that the orthorhombic unit cell is maintained inall the temperature ranges. 4.4Ueq, in units of Å2), as derived from theanisotropic coefficients U11, U22, U33, U12, U13, and U23 for each atom within the Pnma crystal structure. The Debye model is supposed to describe the temperatureevolution of the MSDs, as summarized belowds2 is the intrinsic disorder, θD is the Debyetemperature, m is the atom’s mass, and T, kB, and ℏ keep theirusual meaning.35 As low-temperature points (<100K) were not taken, the uncertainty associated with ds2 is high, when it is considered as a fittingparameter. In order to avoid this, such a parameter was kept equalto zero for all the atoms .The moderateabsorption of neutrons by the heavy Rb and Pb atoms was suitable toprobe the thermal variation of the mean-square displacement factors(MSDs) in the temperature range 200–400 K. Here, the MSDs wereanalyzed in their equivalent displacement parameters , in halide perovskites.36 The bonding stiffness may be evaluated fromthe Debye temperature by considering the harmonic one-particle potential(OPP).34 Here, the harmonic potential iswritten as Vopp(dD2) = 1/2KdD2, whereK , while the |V|/G values of the Rb–I bonds are less than1. These different behaviors of the BCPs of the Pb–I bondsindicate that such bonds fall into a transient class showing a relevantcovalent contribution,38 as estimated from the Debye analysisabove. From a relative comparison, it is observed that the Rb–Ibond has a greater ionic character than the Pb–I bond, resultsthat corroborate the values indicated in the obtained results forthe Debye temperature. The Laplacian of electronic density isolineswith the atomic site’s information is depicted in The topochemical analysis of the mainbond critical points (BCPs)was performed to evaluate the chemical environment of RbPbI4.5–1) (S)  7a, Seebe–1) (S) 7b, and p–1) (S) 7c, defin39 At 400 K, the Seebeck coefficient is ∼44,000μV·K–1. This value increases up to 117,000μV·K–1 at 460 K, and immediately afterward,decreases again down to 53,000 μV·K–1 at 520 K. Even with this high Seebeck coefficient, which could predicta good thermoelectric performance, the resistivity is high enoughto hamper the power factor, which shows values not higher than 2 ×10–4 mW·m–1·K–2. These types of materials can be appropriately doped to modify theircarrier density,22 altering their resistivityand Seebeck coefficient conveniently.The Seebeck coefficientstrongly varies with temperature, showingremarkably high values, rarely seen in thermoelectric materials.25 remaining always below 0.2 W·m–1·K–1 at all the measured temperatures, from 323 K up to573 K. This nearly constant evolution with temperature has been seenbefore in hybrid organic–inorganic perovskite single crystals25 and in the RbPb2Br5 perovskiteprepared by mechanochemistry.On the other hand, thethermal conductivity κ 8a is lowZT (S2σT/κ), yields the result displayed in –4, which is 2 orders of magnitude higherthan the figure of merit reported for other halide perovskites, suchas Bi-doped MAPbBr3,25 MASnBr3,40 and RbPb2Br5.The combination of theseparameters in the thermoelectric figureof merit, 4.62 ballsimpact against the reactants, a highly disaggregated product of smallparticles is expected. However, SEM images of the as-prepared RbPbI3 polycrystals show a heterogeneous mixture of large agglomerates(10–20 μm) along with smaller fragments .In a mechanosynthesis process, as high-energy ZrOragments 9a. It cafication 9b, much 42 or spin-coatingtechnique.43 As the stability of the powdersample is higher than that compared to the solvent-based method, asreported previously by our group,44 thereis a good chance that the thin films could also show an enhanced stability.These powders resulting from ball milling can be dispersed in aparticular medium  in order to be used fordrop-coating-based thin films4.73 powder were determined by diffuse reflectanceUV–vis spectroscopy. The Kubelka–Munk function  is closely related to the optical absorption coefficient is in closeagreement with that obtained by ab initio calculationsfor the orthorhombic structure, Pnma, called δ-RbPbI3, of 2.663 eV.11 Using the previouslydescribed DFT model, we have also determined the density of states(DOS) with the contribution of each element of RbPbI3,as well as the electronic band gap, as depicted in The opticalabsorption characteristics, in particular the band gap,of RbPbIfficient10. The e3 model indicates anindirect band gap transition, as seen in the band structure .As shown in 53 material was obtained bymechanosynthesis using mild ball-milling. The unit cell parametersat RT are slightly larger than those described for samples synthesizedby conventional solid-state reaction methods. The detailed crystalstructure was refined from NPD in the 200–400 K temperaturerange. In this whole stability range, the crystal structure is orthorhombic,as defined in the Pnma space group; the lattice parametersand volume linearly increase with temperature. The bonding stiffnesswas estimated using the harmonic OPP from the Debye temperature analysisof the anisotropic MSD parameters, showing that the Pb–I bondsare more rigid than the Rb–Cl bonds. This result was corroboratedby theoretical topochemical evaluations, in which the transient characterof the Pb–I pair bonds was foreseen with a relevant covalentcontribution. The thermoelectric properties are appealing, consideringthe large Seebeck coefficient and low thermal conductivity, and mayexpand the possibilities for future thermoelectric applications, althoughthe large electrical resistivity limits the thermoelectric figureof merit. A band gap of ∼2.51 eV (indirect transition) is suitablefor solar cell applications.A well-crystallized RbPbI


In vivo results of a mouse model of breast cancer-transplanted tumor showed that tumor volume reduced significantly, Ki-67-positive tumor cells decreased, and TUNEL-positive tumor cells increased in tumors after treatment with activated platelet membrane nanovesicles (aPNs). After treatment with aPNs, not only the number of neutrophils, CD8+, CD4+ T cells, and B cells increased, but also IL-12, TNF-α, and IFN-γ levels elevated significantly in tumor tissues.Platelets play a crucial role in the recruitment of neutrophils, mediated by P-selectin, CCL5, and ICAM-2. In this study, we prepared platelet membrane nanovesicles from activated platelets. Whether activated platelet membrane nanovesicles can recruit neutrophils has not been reported, nor has their role in antitumor immunity. The results of SDS-PAGE showed that the platelet membrane nanovesicles retained almost all the proteins of platelets. Western blotting showed that both the activated platelets and the platelet membrane nanovesicles expressed P-selectin, ICAM-2, and CCL5.  Solarbio Biotechnology Co., Ltd. provided RPMI-1640, phosphate buffer saline (PBS), trypsin, Cy5, and non-pre-stained protein marker. Bovine serum albumin (BSA) was procured from Yeasen Biological Technology Co., Ltd. BCA Protein Quantitation Kit and Coomassie Blue Fast Staining Solution were from Beyotime Biology Co., Ltd. Polyvinylidene fluoride (PVDF) membrane was produced by Cell Signaling Technology, Co., Ltd. P-selectin rabbit primary antibody was provided by Shanghai Lianshuo Biological Technology Co., Ltd. ICAM-2 (F-5) mouse primary antibody was purchased from Santa Cruz Biotechnology Co., Ltd. CCL5 rabbit primary antibody was provided by Solarbio Biotechnology Co., Ltd. β-Actin (60008-1-Ig) mouse primary antibody was provided by Proteintech Group, Inc. Servicebio Biological Technology Co., Ltd. provided hematoxylin and eosin (HE), Ki-67 immunofluorescence detection kit, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) immunofluorescence detection kit. Mouse IFN-γ ELISA kit (E-EL-M0048c) and IL-12 ELISA Kit (E-EL-M0726c) were provided by Elabscience Biological Technology Co., Ltd. Mouse TNF-α ELISA kit (ab208348) was purchased from Abcam Trading Co., Ltd. Ly6G (1900–02) mouse primary antibody was provided by SouthernBiotech Co., Ltd. CD4 (CL647-65104) and CD8 (CL488-65069) fluorescent antibodies were produced by Proteintech Group, Inc. CD19 (ab245235) mouse primary antibody and CD11b (ab24874) mouse primary antibody were produced by Abcam Trading Co., Ltd.2. The Hunan Slake Jingda Laboratory Animal Co., Ltd. provided the female ICR mice of 6-week-old.4T1 cells were provided by the Cancer Institute of Central South University and cultured in RPMI-I640 medium containing 10% FBS under the condition of 37°C and 5% COA sample of whole blood from female ICR mice was collected in tubes containing heparin anticoagulant. The whole blood was centrifuged and washed to obtain platelets. The platelets were repeatedly frozen and thawed to obtain PLTm, and then PLTm nanovesicles were generated by ultrasonic treatment .SDS-PAGE was applied to display the protein expression profile of PLTs and PLTm nanovesicles. Western blotting was used for the detection of the expression of specific proteins  on PLTs and PLTm nanovesicles. RIPA lysis buffer containing protease inhibitor cocktail was used to lyse PLTs and PLTm nanovesicles. After that, the lysates were centrifuged at 4°C  and the total protein in the supernatant was measured using the BCA protein detection kit. SDS loading buffer was added to the supernatant, and the total protein was degenerated by heating them at 100°C for 5 min. A quantity of 40 μg total protein was loaded into each gelatin well, and electrophoresis was performed. The gelatin was quickly dyed with Coomassie blue staining solution and photographed. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes and sealed with 5% skimmed milk in TBST at room temperature for 1 h. After that, these blots were incubated with P-selectin, ICAM-2, CCL5, and β-actin primary antibody for 2 h; washed with TBST; incubated with corresponding horseradish peroxidase-linked secondary antibody; washed with TBST; and added with chemiluminescent solution for development with Gel imaging system .6/100 μl). When the tumor volume exceeded 100mm3, dilute solution of Cy5 in PBS and Cy5 (red fluorescence)-labeled PLTm nanovesicles were intravenously injected, respectively. After 48 h, we gleaned the vital organs, including the hearts, livers, spleens, lungs, kidneys, brains, and tumors of the mice, and evaluated the fluorescence intensity by animal imaging . Tumor tissue sections were photographed by using a confocal laser scanning microscope (CLSM) .Female ICR mice were subcutaneously inoculated with 4T1 cells (1.0×106/100 μl). When the tumor volume exceeded 100 mm3, PBS and aPNs were injected into the tail vein, respectively, once a day for four times. The length and width of the tumors were measured every 4 days to calculate tumor volume and assess antitumor activity. Tumor volume was calculated as: length × width2/2. At day 20, the mice were killed, and the collected tumor samples were stored in 4% paraformaldehyde and then made into tissue sections.Female ICR mouse were subcutaneously inoculated with 4T1 cells  and Reagent 2 (dUTP) were added, and the sections were incubated at 37°C for 2 h and washed with PBS. Following this, the PBS was removed and DAPI was added, and the slides were incubated for 10 min to avoid light. The slides were sealed with anti-fluorescence quencher and observed, and its images were collected by using a confocal laser scanning microscope (CLSM) .+ T lymphocytes (CD8), neutrophils , B lymphocytes (CD19), and CD4+ T lymphocytes (CD4) primary immunofluorescence antibody for 1 h in dark, washed with PBST, incubated with secondary antibody, washed with PBST for 3 times, and re-dyed with DAPI. The cells were observed, and images were obtained under CLSM.After fixation, embedding, and sectioning, tumor tissue sections were stained with CD8A standard and sample diluent was added to the tissue samples according to the instruction. The wells were sealed and incubated at 37°C for 90 min, and the plate was washed. Biotinylated antibody diluent or biotinylated antibody working solution was added, the wells were sealed and incubated at 37°C for 60 min and then washed. An enzyme-conjugated diluent or enzyme-conjugated working solution was added, and the wells were sealed and incubated at 37°C for 30 min, and washed. A color substrate was added and the wells were incubated at 37°C for 15 min. A reaction-stopping solution was added, and OD450 was measured immediately. The best fitting curve was drawn, and the concentration of the samples was calculated.Observed under transmission electron microscope (TEM) , the diameter of activated PLTm nanovesicles was about 150 nm , which win vivo. PBS-diluted Cy5 solution served as control. At 48 h after intravenous injection, retention of aPNs-Cy5 (red fluorescence) in tumor was more obvious than that of Cy5 alone. Moreover, there was no significant accumulation in the two groups in visceral tissues, including heart, spleen, lung, and kidney, except for a small amount of fluorescence in the liver  induce apoptosis of tumor cells directly by secreting granzyme B and perforin; or act on tumor cells indirectly by releasing cytokines, such as IFN-γ and TNF, resulting in consecutive appearance of antigens and the proliferation of T cells (+ CTL in tumor tissues increased significantly in the aPN group (Activated CD8 T cells . ComparePN group , suggestIFN-γ, produced by adaptive and innate immunocytes, including T cells and NK cells, serves as a potent immunomodulatory cytokine . IFN-γ mTNF-α secreted by tumor cells promotes cell apoptosis and inhibits survival in cancer  and contIL-12, secreted from various immunocytes, such as neutrophils and B cells, stimulates the production of IFN-γ by T cells , playing+, CD4+T cells, and B cells in the tumor and promote the release of IFN-γ, TNF-α, and IL-12; thus, the antitumor immunity and anticancer effect was enhanced (Activated platelet membrane nanovesicles, which retain almost all of the platelet-proteins, target tumor tissues through P-selectin and CD44, and mediate neutrophil recruitment through P-selectin, ICAM-2, and CCL5. Subsequently, neutrophils increase the number of CD8enhanced . Based o


Glioblastoma (GB) is a tumor of the central nervous system characterized by high proliferation and invasiveness. The standard treatment for GB includes radiotherapy and chemotherapy; however, new therapies are needed. Particular attention was given to the role of histone methyltransferase enhancer of zeste-homolog-2 (EZH2) in GB. Recently, several EZH2-inhibitors have been developed, particularly GSK343 is well-known to regulate apoptosis and autophagy processes; however, its abilities to modulate canonical/non-canonical NF-κB/IκBα pathways or an immune response in GB have not yet been investigated. Therefore, this study investigated for the first time the effect of GSK343 on canonical/non-canonical NF-κB/IκBα pathways and the immune response, by an in vitro, in vivo and ex vivo model of GB. In vitro results demonstrated that GSK343 treatments 1, 10 and 25 μM significantly reduced GB cell viability, showing the modulation of canonical/non-canonical NF-κB/IκBα pathway activation. In vivo GSK343 reduced subcutaneous tumor mass, regulating canonical/non-canonical NF-κB/IκBα pathway activation and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Ex vivo results confirmed the anti-proliferative effect of GSK343 and also demonstrated its ability to regulate immune response through CXCL9, CXCL10 and CXCL11 expression in GB. Thus, GSK343 could represent a therapeutic strategy to counteract GB progression, thanks to its ability to modulate canonical/non-canonical NF-κB/IκBα pathways and immune response. Glioblastoma (GB) is the most common and aggressive cancer of the central nervous system (CNS) with a global incidence of 10 cases per 100,000 people per year ; whereas50 values for the U87 cell line are 4.06 μM at 24 h and 4.68 μM at 48 h.GB cell viability was assessed following 24 h and 48 h of treatment with GSK343 at increasing concentrations . GSK343 treatment significantly reduced viability in all GB cell lines in the same way and in a concentration-dependent manner both at 24 h and 48 h compared to the control groups A,B. HoweSince GSK343 showed similar effects on cell viability in all GB cell cultures, we decided to continue to analyze the effect of GSK343 only on the U87 cell line, because it represented one of the most frequently used cell lines in the field of GB .The effect of GSK343 on the NF-κB/IκBα pathway was evaluated in U87 cell lysates. Our results demonstrated that the treatment with GSK343 for 24 h at the concentrations of 10 and 25 μM was able to significantly reduce NF-κB expression and also at the lower concentration of 1 μM, restored IκB-α expression and decreased IKKβ in a concentration-dependent manner compared to the control group A–C. AddiThe effect of GSK343 on the apoptotic pathway was investigated in U87 cell lysates. After 24 h of treatment, our data demonstrated that GSK343 at the concentrations of 1, 10 and 25 μM was able to increase the expression of pro-apoptotic protein as Bax, while p53 expression was notably increased only at the concentrations of 10 and 25 μM A,B; instConsidering the involvement of EZH2 in epithelial–mesenchymal transition (EMT) , we deciAdditionally, we investigated the effect of GSK343 treatment on matrix metalloproteinases (MMPs), particularly MMP2 and MMP9 expression, showing that the treatment with GSK343 for 24 h at the concentrations of 1, 10 and 25 μM was able to notably reduce their expression compared to the control group C,D. To cThe histological evaluation demonstrated that the control group was characterized by nuclear pleomorphism and high cellular density A,A1,A2. Based on the in vitro results, we evaluated the effect of GSK343 treatment on canonical and non-canonical NF-κB/IκBα pathways also in the U87-xenograft model. Our results demonstrated that the control group was characterized by an increase in nuclear NF-κB expression and a decrease in cytosolic IκBα expression A,B. MoreApoptosis plays a key role in GB progression . TherefoConsidering the in vitro results, we investigated the effect of GSK343 on the EMT process also in the U87-xenograft model by evaluating E-cadherin and N-cadherin expression. Immunohistochemical localization demonstrated that the control group was characterized by a decrease in E-cadherin expression and consequently an increase in N-cadherin expression A,F. HoweOxidative stress (OS) has been considered as one of many contributors in developing risk of cancer, including GB ; therefoGSK343 cytotoxicity was evaluated in primary GB cell culture obtained from patients, demonstrating that GSK343 treatment for 24 h at the concentrations of 1, 10 and 25 μM was able to significantly reduce primary GB cells viability compared to the control group, confirming its antiproliferative effect .Considering the role of EZH2 in immune response, we decided to evaluate the levels of chemokines CXCL9, CXCL10 and CXCL11, key mediators for the trafficking of antitumor immune cells, on primary GB cell culture by ELISA assay. Our data demonstrated that GSK343 treatment at the concentrations of 10 and 25 μM was able to increase CXCL9, CXCL10 and CXCL11 levels compared to the control group A–C.GB is the most common and aggressive malignant tumor of the central nervous system (CNS) . In the 2−), hydroxyl radical (·OH), and hydrogen peroxide (H2O2) are involved in carcinogenesis, particularly in the tumor initiation and promotion phases [Various studies revealed that EZH2 is able to modulate many cellular processes, including inflammation and cell adhesion by targeting genes such as IL-1β and CDH13 . Particun phases . Despiten phases ,47, studn phases ,47. In tn phases ,49. Inden phases . In thisn phases . Therefon phases , we decin phases ,37. EMT n phases . It is cn phases . Due to n phases ,51. Our n phases ,53. The n phases ,32, not n phases . It has n phases ,54. In t® HTB-14™ Homo sapiens brain likely glioblastomas), U138MG (U138 MG ATCC® HTB-16™ Homo sapiens brain glioblastoma IV grade), and A172 (A172\ATCC® CRL-1620™ Homo sapiens brain glioblastoma) were obtained from ATCC . The GB human cell lines U87, U138 and A172 were cultured in 75 cm2 flask with, respectively, ATCC-formulated Eagle’s Minimum Essential Medium  for U138 and Dulbecco’s modified Eagle’s medium  for U87 and A-172, both supplemented with antibiotics , L-glutamine  and 10% (v/v) Fetal bovine serum . Normal human astrocytes (NHA)  were used as normal counterparts [2 at 37 °C.Human GB cell lines: U87 MG  at increasing concentrations of 1 μM, 10 μM, 25 μM and 50 μM dissolved in culture medium with 0.001% of DMSO for 24 h and 48 h.NHA, U87, U138 and A172 cells were cultured at a density of 4 × 10Control groups: U87, U138 and A172 cell lines were treated with culture medium.Vehicle groups: U87, U138 and A172 cell lines were treated with 0.001% of DMSO dissolved in culture medium.GSK343 1 μM group: U87, U138 and A172 cell lines were treated with GSK343 1μM dissolved in culture medium with 0.001% of DMSO.GSK343 10 μM group: U87, U138 and A172 cell lines were treated with GSK343 10 μM dissolved in culture medium with 0.001% of DMSO.GSK343 25 μM group: U87, U138 and A172 cell lines were treated with GSK343 25 μM dissolved in culture medium with 0.001% of DMSO.GSK343 50 μM group: U87, U138 and A172 cell lines were treated with GSK343 50 μM dissolved in culture medium with 0.001% of DMSO.Furthermore, we evaluated the cytotoxicity of GSK343 at the concentrations of 1, 10, 25 and 50 μM in NHA cells as a control for 24 h and 48 h .50) of GSK343 from the percentage viability of U87 cells was calculated using Graph Pad prism 7.04 software by the interpolation of the values in dose–response curves.Cell viability was measured using a mitochondria-dependent dye for live cells , as previously described by Campolo et al. . Cells ww/v nonfat dried milk/0.1% Tween-20 (PMT) and membranes incubated overnight at 4 °C. Membranes were then incubated with secondary antibody  for 1 h at room temperature. To ascertain the blots, they were loaded with equal amounts of protein lysate, they were also incubated with anti-βactin for cytosolic fraction  and anti-lamin A/C for nuclear fraction . Signals were detected with enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer’s instructions . The relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDocTMXRS + software. The densitometric values are normalized with Lamin A/C and β-actin and expressed as % of the control.Western blot analysis in U87 cell lysates was performed as previously described . The folThe levels of NIK and IKKβ were measured by ELISA kit according to the manufacturer’s instructions in U87 cell culture supernatant .Cell migration in U87 cells was evaluated using Cell Migration Assay Kit  according to the manufacturer’s instructions.p-value of less than 0.05 was considered significant.All values are expressed as mean ± standard error of the mean (SEM) of “n” observations. Each analysis was performed three times with three samples replicates for each one. The results were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. A ® HTB-14™ Homo sapiens brain Likely glioblastomas) was obtained from ATCC . U87 cells were cultured in 75 cm2 flask with Dulbecco’s modified Eagle’s medium  supplemented with antibiotics , L-glutamine  and 10% (v/v) Fetal bovine serum  in a humidified atmosphere containing 5% CO2 at 37 °C.The human GB cell line U87  and housed in microisolator cages under pathogen-free conditions on a 12 h light/12 h dark schedule for a week. Animals were fed a standard diet and water ad libitum. Animal experiments followed Italian regulations on protection of animals used for experimental and other scientific purposes (DM 116192) as well as EU regulations (European Directive 2010/63/EU amended by Regulation 2019/1010).6 human U87 cells in 0.2 mL of Phosphate-Buffered Saline (PBS) and 0.1 mL Matrigel . Once tumors were palpable (100 mm3), the animals were randomly divided into groups to receive vehicle or GSK343 treatment. GSK343 was administered at doses of 5 mg/kg and 10 mg/kg once daily via intraperitoneal (ip) injection every day for 21 days, as described by Bownes et al. [Xenograft tumor model was performed, as previously described . Mice wes et al. . The aniControl group (vehicle): intraperitoneal (ip) administration of PBS with 0.001% of DMSO.Control group+ GSK343 5 mg/kg: intraperitoneal (ip) administration of GSK343 5 mg/kg dissolved in PBS with 0.001% of DMSO.Control group+ GSK343 10 mg/kg: intraperitoneal (ip) administration of GSK343 10 mg/kg dissolved in PBS with 0.001% of DMSO.Animals were randomly divided into 3 groups, as described below:Histological evaluation was performed, as previously described . Tumor sw/v non-fat dried milk/0.1% Tween-20 (PMT) and membranes incubated overnight at 4 °C. Membranes were then incubated with secondary antibody  for 1 h at room temperature. To ascertain those blots were loaded with equal amounts of protein lysate, they were also incubated with β-actin antibody  or lamin A/C . Signals were detected with enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer’s instructions . The relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDocTMXRS + software. The densitometric values are normalized with Lamin A/C and β-actin and expressed as % of the control.Tumor samples from each mouse were suspended in extraction Buffer A , homogenized at the highest setting for 2 min, and centrifuged at 12,000 rpm for 4 min at 4 °C, as previously described . Supernav/v; sc-8426) and anti-N-cadherin . At the end of the incubation with the primary antibodies, the sections were washed with PBS and incubated with a secondary antibody  for 1 h at room temperature. The reaction was revealed by a chromogenic substrate (brown DAB), and counterstaining with nuclear fast-red. Immunohistochemical images were obtained and observed using a Zeiss microscope with Axio Vision software. The percentage area of immunoreactivity  is expressed as % of the total tissue area (red staining) of five random fields with objective lens at 20 ×  and 40× ; the analysis was performed using ImageJ.Immunohistochemical localization was performed, as previously described . Slides The levels of NIK, IL-1β, TNF-α and SOD were measured by ELISA kit according to the manufacturer’s instructions  ELISA kit cat. No MBS034842 MyBiosource; San Diego, CA, USA). Samples were thawed on ice, homogenized in 300 μL lysis buffer and centrifuged at 14,000× g for 10 min at 4 °C; then, supernatants were collected and stored at −20 °C.Malondialdehyde (MDA) content was evaluated in the homogenate supernatant of the tumor samples by colorimetric reaction with thiobarbituric acid (TBA). The MDA content was measured at a wavelength of 532 nm, as previously described ,62.ROS evaluation was performed in tumor samples using Reactive Oxygen Species (ROS) Assay Kit  according to manufacturer’s instructions.p-value of less than 0.05 was considered significant.All values are expressed as mean ± standard error of the mean (SEM) of “n” observations. Each analysis was performed three times with three samples replicates for each one. The results were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. A Primary tumor GB cells from patients were acquired according to protocol approved by the Regional Ethical Board at the University of Messina. All subjects gave their informed consent for inclusion before they participated in the study. The study was performed in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of AOU “G. Martino,” Hospital of Messina (No. 47/19 of 05/02/2019). Tumor samples were processed aseptically, and primary cell cultures were initiated using DMEM  with 15% heat-inactivated fetal calf serum (FCS) , 2 mM GlutaMAX-I , 1% insulin-transferrin-selenium-X supplement , and 1% penicillin-streptomycin mixture . Cells were used within 7 days of plating or established as primary cell lines, as previously described .Control group: primary GB cells obtained from patients were treated with culture medium.Vehicle group: primary GB cells obtained from patients were treated with 0.001% of DMSO dissolved in culture medium.GSK343 1 μM: GB cells from patients were treated with GSK343 at concentration of 1 μM dissolved in culture medium with 0.001% of DMSO.GSK343 10 μM: GB cells from patients were treated with GSK343 at concentration of 10 μM dissolved in culture medium with 0.001% of DMSO.GSK343 25 μM: GB cells from patients were treated with GSK343 at concentration of 25 μM dissolved in culture medium with 0.001% of DMSO.Primary GB cells culture obtained from patients were treated with GSK343 at the concentrations of 1, 10 and 25 μM dissolved in culture medium with 0.001% of DMSO for 24 h. After 24 h, primary GB cells were incubated at 37 °C with MTT  for 1 h. The medium was removed by aspiration, and the cells were lysed with 100 μL of DMSO . The extent of reduction of MTT to formazan was quantified by measurement of optical density at 550 nm (OD550) with a microplate reader as described .The levels of CXCL9, CXCL10 and CXCL11 were measured in primary GB cells supernatant by ELISA assay according to manufacturer instructions .GSK343 and other chemical reagents are purchased by Sigma-Aldrich . All stock solutions were prepared in non-pyrogenic saline .p-value of less than 0.05 was considered significant.All values are expressed as mean ± standard error of the mean (SEM) of “n” observations. Each analysis was performed three times with three samples replicates for each one. The results were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. A In conclusion, our data demonstrated, for the first time, that GSK343 treatment was able to modulate canonical and non-canonical NF-κB/IκBα pathway activation in GB. Moreover, in the field of cancer research, our preliminary data obtained about the capacity of GSK343 to modulate OS and immune response in GB appears very interesting. Therefore, based on the obtained results, GSK343, thanks to its numerous abilities, could represent a possible therapeutic strategy to contrast or reduce GB growth, which has a extremely high mortality due to its resistance to currently used therapies. However, considering the limitations of preclinical models, further studies are needed for a fuller understanding of the mechanism of action of GSK343 in GB pathology.