Numerous studies have examined the relationship between overweight, central obesity, and plasma levels of DHEA and DHEAS. The sodium-dependent organic anion transporter Soat (Slc10a6) is a plasma membrane uptake transporter for sulfated steroids. Significantly increased expression of Slc10a6 mRNA has been previously described in organs and tissues of lipopolysaccharide (LPS)-treated mice, including white adipose tissue. These findings suggest that Soat plays a role in the supply of steroids in peripheral target tissues. The present study aimed to investigate the expression of Soat in adipocytes and its role in adipogenesis. Soat expression was analyzed in mouse white intra-abdominal (WAT), subcutaneous (SAT), and brown (BAT) adipose tissue samples and in murine 3T3-L1 adipocytes. In addition, adipose tissue mass and size of the adipocytes were analyzed in wild-type and Slc10a6−/− knockout mice. Soat expression was detected in mouse WAT, SAT, and BAT using immunofluorescence. The expression of Slc10a6 mRNA was significantly higher in 3T3-L1 adipocytes than that of preadipocytes and was significantly upregulated by exposure to lipopolysaccharide (LPS). Slc10a6 mRNA levels were also upregulated in the adipose tissue of LPS-treated mice. In Slc10a6−/− knockout mice, adipocytes increased in size in the WAT and SAT of female mice and in the BAT of male mice, suggesting adipocyte hypertrophy. The serum levels of adiponectin, resistin, and leptin were comparable in wild-type and Slc10a6−/− knockout mice. The treatment of 3T3-L1 adipocytes with DHEA significantly reduced lipid accumulation, while DHEAS did not have a significant effect. However, following LPS-induced Soat upregulation, DHEAS also significantly inhibited lipid accumulation in adipocytes. In conclusion, Soat-mediated import of DHEAS and other sulfated steroids could contribute to the complex pathways of sex steroid intracrinology in adipose tissues. Although in cell cultures the Soat-mediated uptake of DHEAS appears to reduce lipid accumulation, in Slc10a6−/− knockout mice, the Soat deletion induced adipocyte hyperplasia through hitherto unknown mechanisms.In addition to the endocrine and paracrine systems, peripheral tissues such as gonads, skin, and adipose tissue are involved in the intracrine mechanisms responsible for the formation of sex steroids  DHEAS, 16α-hydroxy-DHEAS, pregnenolone sulfate, 17α-hydroxypregnenolone sulfate, androstenediol-3-sulfate, epiandrosterone-3β-sulfate, androsterone-3α-sulfate, testosterone-17β-sulfate, epitestosterone-17α-sulfate, 5α-dihydrotestosterone sulfate, estrone-3-sulfate, and 17β-estradiol-17-sulfate, representing the physiologically most relevant sulfated steroids, all have been identified as SOAT substrates , suggesting that Slc10a6 represents an inflammation-responsive gene  and organic anion transporters (OATs) all represent multispecific carriers . Howeverbstrates . After tbstrates . SLC10A6n humans . Accordin humans . In mice bladder . In a reive gene .Slc10a6/Soat in adipocytes. The experiments were performed in vivo in Slc10a6−/− knockout and wild-type mice and in LPS-treated mice and in vitro using the 3T3-L1 adipocyte cell line.Based on these previous findings, the present study aimed to extend our knowledge of the role of Soat in adipogenesis by investigating the mRNA and protein expression pattern of All chemicals, unless otherwise stated, were obtained from Sigma-Aldrich .2 in DMEM  supplemented with 10% newborn calf serum  and 1% penicillin/streptomycin . At confluence, the cells were differentiated into mature adipocytes by culturing in DMEM/F12/glutamate medium  supplemented with 20 μM 3-Isobutylmethylxanthine , 1 μM corticosterone, 100 nM insulin, 200 μM ascorbate, 2 μg/ml apo-transferrin, 5% fetal calf serum , 1 μM biotin, 17 μM pantothenate, 1% penicillin/streptomycin, and 300 μg/ml Pedersen fetuin  (Murine 3T3-L1 preadipocytes were cultured at 37 °C and 5% CO France)  for 9 da France) . The celSlc10a6, Slco1a1, Slco1a4, and Slco1b2 mRNA transcripts, gene-specific TaqMan Gene Expression Assays Mm00512730_m1, Mm01267415_m1, Mm00453136_m1, and Mm00451510_m1  were used, respectively. The Mm99999915-g1 assay (Thermo Fisher Scientific) was used for the control amplification of glyceraldehyde-3-phosphate dehydrogenase (Gapdh). In the case of Slco2b1 mRNA, real-time PCR was performed using the fluorescent dye SYBR Green PCR Master Mix  and the following primers: Slco2b1 forward 5′-AGT TTG AGC AGG GCT TCT ACC-3′ and Slco2b1 reverse 5′-CTG TGA CAT AGG ACA AAG AAC TTG A-3’. The results were normalized to the mRNA expression of Gapdh (forward primer 5′-ACG GGA AGC TCA CTG GCA TG-3′ and reverse primer 5′-CCA CCA CCC TGT TGC TGT AG-3′). A standard curve analysis with these primer pairs revealed r2 of 0.999 and PCR efficiency of 106.6% for Slco2b1 and r2 of 0.999 and PCR efficiency of 99.6% for Gapdh. The TaqMan expression assay efficiencies were as follows: r2 of 0.998 and PCR efficiency of 97.2% for Slc10a6, r2 of 0.994 and PCR efficiency of 96.5% for Slco1a1, r2 of 0.996 and PCR efficiency of 103.6% for Slco1a4, r2 of 0.991 and PCR efficiency of 98.7% for Slco1b2, and r2 of 0.997 and PCR efficiency of 98.5% for Gapdh. Real-time amplification was performed on an Applied Biosystems 7300 Real-Time PCR System in 96-well optical plates using 5 µL cDNA, 1.25 µL TaqMan Gene Expression Assay, 12.5 µL TaqMan Universal PCR Master Mix, and 6.25 µL water in each 25 µL reaction. The plates were heated for 10 min at 95 °C, and then 40 cycles of 15 s at 95 °C and 60 s at 60 °C were applied. All data were expressed as fold changes using the 2−ΔΔCt method with the calibrator indicated in the Figure legends. In the case of carrier expression profiling, ΔCt values are presented.The cells were stimulated with 10 ng/ml LPS (Sigma-Aldrich) for different periods of time. The cells were harvested and total mRNA was extracted using the Maxwell RSC simplyRNA Tissue Kit (Promega). Complementary DNA (cDNA) was synthesized from 1 μg total RNA using 8 μL of RT-mix SuperScript III Reverse Transcriptase . For quantitative analysis of the expression of the l-lysine–coated 8-well μ-slides . After 48 h, the cells were incubated with or without 10 ng/ml LPS for 24 h and, afterward, were fixed with 2% paraformaldehyde (PFA) and blocked with blocking buffer containing 1% bovine serum albumin and 4% goat serum in phosphate-buffered saline , 7.3 mM Na2HPO4 (Roth), pH 7.4) for 30 min at room temperature. Then, 3T3-L1 cells were incubated at 4°C overnight with an antibody against murine Soat329-344 , followed by labeling with the secondary antibody Alexa Fluor 488 goat anti-rabbit  and the nuclear marker Hoechst 33342 . Cell imaging was performed at room temperature on an inverted Leica DM5500 fluorescence microscope .The cells were grown on poly-During adipocyte differentiation (days 0–9), the cells were treated with 1 µM DHEA, 100 nM testosterone, 10 µM DHEAS, 10 µM STX64, or 1 µM flutamide in the presence or absence of LPS (10 ng/ml) in 6-well plates. The cells were then washed with cold PBS and fixed with 4% formalin (v/v) for 30 min. After Oil Red O staining, the cells were photographed using a phase-contrast microscope  at ×10 magnification. For quantitative measurement of lipid accumulation, the stained cells were dried, Oil Red O was extracted in isopropanol, and absorbance was determined at 520 nm using a microplate reader.2O instead of LPS. The mice were euthanized for organ and tissue resection 2 h after injections. Epididymal  and subcutaneous adipose tissue specimens were resected and shock-frozen in liquid nitrogen for subsequent analysis of mRNA expression. Animal experiments were performed at the University of Regensburg, Germany, and all the animal studies were approved by the local government agency . Furthermore, the organ samples were obtained from wild-type (WT) and Slc10a6−/− knockout mice after anesthesia with ketamine (120 mg/kg) and xylazine (16 mg/kg) and exsanguination by heart puncture. These experiments, including euthanasia and tissue preparations, were approved by the local regulatory authority (Regierungspräsidium Giessen) with the reference number V54-19 c 20 15 h 02 GI 20/23 Nr. A8/2013.Male C57BL/6 wild-type mice  received an intraperitoneal injection of 1 μg LPS per animal after overnight fasting. The control animals received intraperitoneal injections of the corresponding volume of H−/−Slc10a6 knockout mice were fixed for 10 min in 4% PFA and embedded in Tissue-Tek O.C.T. . The fixed sections were permeabilized, blocked with 5% goat serum in PBS-TritonX-100 (0.1%), and incubated with the primary Soat antibody  overnight at 4°C. Subsequently, the tissue sections were incubated with the secondary antibody Alexa Fluor 488 goat anti-rabbit . The slides were counterstained with Hoechst 33342 and analyzed on a Leica DM5500 B microscope. The captured images were analyzed with Leica Fluorescence Workstation LAS AF 6000 software (Leica Microsystems).For histological analyses, the tissue samples from WT and −/−Slc10a6 knockout mice were fixed in 10% formalin and processed for paraffin embedding. Paraffin tissue sections were cut into 3.5-µm slices and processed for hematoxylin and eosin (H&E) staining. For quantification of the size of the adipocytes, six tissue sections of each group were selected and photographed using the DMi1 microscope (Leica Microsystems) at ×10 magnification. Finally, the adipocyte size was quantified using Fiji which is ImageJ software with the add-on Adiposoft plugin .The adipose tissues from WT and −/−Slc10a6 knockout mice in duplicate by ELISA . The measurements were repeated for samples exceeding an intra-duplicate variation of 20%.Concentrations of the adipokines adiponectin, resistin, and leptin were measured in the serum of WT and t-test and one-way ANOVA with Tukey’s multiple comparison tests were performed to determine statistical significance. All data were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.All graphs and calculations were prepared using GraphPad Prism software 6.07 . Student’s unpaired Slc10a6−/− knockout mouse model was established in a previous study by our group  mice. All were derived from het × het breeding and were genotyped as previously reported . Interestingly, WAT and SAT in female mice and BAT in male mice showed significantly larger adipocyte size in the Slc10a6−/− knockout mice than that in the WT mice.A closer analysis of body weight, adipose tissue weight, and adipocyte size was then carried out in groups of 10 WT  mice and 10 reported . All micMaterials and Methods section. To examine whether Soat is expressed in 3T3-L1 cells, we first measured Slc10a6 mRNA levels using qPCR. Slc10a6 mRNA was detected in 3T3-L1 preadipocytes and showed a significant 46.4-fold upregulation during differentiation into 3T3-L1 adipocytes  is involved in the uptake of sulfated steroids by hepatocytes , OATP-D (SLCO3A1), OATP-E (SLCO4A1), and of STS, could be detected in human adipose tissue and in cultured preadipocytes and adipocytes, whereas OATP-A (SLCO1A2), OATP-C (SLCO1B1), OATP-8 (SLCO1C3), OAT3 (SLC22A8), and OAT4 (SLC22A11) were not detected. In addition, de novo biosynthesis of steroid hormones from cholesterol. They showed that none of these enzymes could be detected at the mRNA level, while STS mRNA was expressed in samples from cultured preadipocytes, adipocytes, and adipose tissues. The absence of these essential enzymes required for cholesterol-mediated estrogen and androgen biosynthesis suggests that uptake carriers for sulfated steroid precursors, indeed, might be essential for the regulation of the adipose tissue by steroids.The expression of ipocytes . In addiSlc10a6 mRNA expression was upregulated in the liver of mice treated with the cytokine IL-1β. The inflammation-mediated upregulation of Soat expression was likely modulated by the nuclear receptor farnesoid X receptor (FXR), the retinoid X receptor (RXR), and glucocorticoid receptor (GR), which were expressed by all tissues, including white and brown adipose tissues. Since the testes have higher Soat expression than other tissues, the testes were used in this study as the control tissue. However, Soat shows higher expression in the BAT and is less expressed in WAT of untreated mice when compared to that in the testes. In contrast, Slc10a6 mRNA levels are upregulated in WAT and downregulated in BAT specimens of LPS-induced mice (Slc10a6 mRNA expression in WAT, SAT, and PAT specimens of mice in vivo and in mature 3T3-L1 adipocytes in vitro (Slco2b1 and Slco1a4 were downregulated in LPS-treated mature 3T3-L1 adipocytes.A global gene expression analysis in mice demonstrated that Soat is among the most strongly induced genes by LPS in the liver and macrophages . In addiced mice . We founin vitro . In contvia the AR. via the AR, as depicted in DHEA treatment suppresses both proliferation and adipocyte differentiation of 3T3-L1 cells and inhibits AIM-induced lipid accumulation in 3T3-L1 adipocytes . This fiin vivo, Slc10a6−/− knockout mice were analyzed. We observed that LPS-induced inflammation increased the expression of Soat in adipocytes and adipose tissues, contributing to lipolysis by increased uptake of the Soat substrate DHEAS, while deletion of Soat caused adipocyte hyperplasia in mice. We are aware that our study has some limitations. The most important is that the steroid profiles in the adipocytes could not be analyzed, so it was not possible to determine how DHEAS was exactly converted to active androgens.In conclusion, Soat is a specific uptake carrier for sulfated steroids with significant expression levels in adipose tissues and a proposed role in obesity. To clarify this role


Cryptosporidium parvum (C. parvum) is a cosmopolitan parasite that infects various livestock animals including cattle. Microsatellite typing tools for identification of C. parvum subtypes are currently employed to better understand the species-specific epidemiology of cattle cryptosporidiosis. The aim of this study was to analyse the population genetics of C. parvum strains infecting cattle and recognise geographical distribution and time-span correlations in subtype prevalence in Poland. In total, 1601 faecal samples were collected from 2014 to 2018 from healthy cattle from dairy, meat and mixed breeds at the age of 1 week to 4 months. The 267 farms visited were randomly selected and represented all Polish provinces. PCR–RFLP based identification of C. parvum at the 18 small subunit ribosomal RNA (SSU rRNA) locus was performed, followed by strain subtyping by GP60-PCR. C. parvum in Polish cattle was estimated at 6.2% (100/1601). Animals below the age of 1 month were the major host for this parasite. Excluding one breed, that of dairy-meat mixed, there were no significant differences observed between breed and presence of C. parvum infections . Infected animals were detected in 15 out of 16 Polish provinces, with significant regional prevalence diffrences . When the population genetics of C. parvum strains were analysed, 11 parasite subtypes from the IIa and IId genetic families were identified. Compared to other parasite strains, IIaA17G1R1 and IIaA17G2R1 appeared at statistically significantly higher frequency . The prevalence of C. parvum subtypes in cattle was breed-related .The overall prevalence of C. parvum subtypes showed that strains from the IIa subtype family predominated in the tested cattle population. However, relations in changes of subtype prevalence and circulation over time were observed. They were associated with the disappearance of some strains and emergence of new variants from the same genetic family in different geographical locations.The analysis of the population genetics of  C. parvum subtypes from the IIa allele family predominated in the tested cattle. The prevalence of C. parvum subtypes in cattle was breed-related. Dynamicity in the population C. parvum strains circulating in cattle was shown. C. parvum infections [C. parvum infections in cattle herds in Europe has been found to range from 1.2 to 100% and has more often been observed in diarrhoeic animals [Among the parasitic diseases of cattle, cryptosporidiosis is of major importance , 2. Sympfections –6. The p animals , 8. Outs animals , 10, 3.6 animals , 12 and  animals , 14.Cryptosporidium infection in cattle was undertaken by Rzeżutka et al. in 2013 [Cryptosporidium infection in the tested animal population was estimated at 17%; however, C. parvum was not the most frequently detected parasite species, being discovered in 5.1% of cases. The analysis of the microsatellite GP60 region of the C. parvum genome has revealed a various number of circulating subtypes belonging to three subtype families, which were IIa, IId, and IIl before 2014 [C. parvum subtype in European cattle [C. parvum strains of IIa subtype predominating compared to the sporadically appearing IId and IIl strains. In research up to the time of writing, the C. parvum IIaA17G1R1, IIaA17G2R1 and IIaA15G2R1 strains have been most often identified. Of note is that IIaA15G2R1 has also appeared as the dominant subtype in several European cattle populations [C. parvum can cause infections in various hosts including humans [C. parvum infections in humans are more common in areas of intensive livestock farming [In Poland, the first attempt to assess the prevalence of  in 2013 . The oveore 2014 . Other in cattle . Reportsn cattle , and then cattle , C. parvulations –27. C. pg humans . A majorg humans –32. It hC. parvum subtypes of bovine origin, our knowledge of population genetics of strains of this parasite and their geographical prevalence is still not complete. Furthermore, the population structure and subtype distribution in livestock as they relate to host age also require analysis. The aim of this study was to analyse the population and geographical distribution of C. parvum strains infecting cattle in Poland. Additionally, changes of subtype prevalence and circulation over time in the population of Polish cattle were investigated.Although numerous studies have been conducted that aimed at identification of NdeI digestion of all Cryptosporidium-positive samples, a restriction pattern of 18 SSU rRNA amplicons characteristic of C. parvum was shown for 100 DNA samples. Two samples contained a mixture of different parasite sequences including that of C. parvum. Their subsequent treatment with MboII confirmed identification of C. parvum and revealed the presence of C. bovis. The overall prevalence of C. parvum in Polish cattle was estimated at 6.2% (100/1601). C. parvum infections were detected in all age groups of cattle at 11.9% (63/527) frequency at 1–4 weeks, 4.9% (23/464) at > 4–8 weeks and 2.3% (14/610) at > 8–16 weeks. A relationship was observed between animal age and frequency of infection . Animals below the age of 1 month were the major host for this parasite and the number of C. parvum-positive cattle decreased with animal age.The 18 SSU rRNA gene fragment was successfully amplified in 725 out of 1601 cattle faeces samples. After C. parvum DNA, the majority of infections (74/1601) being detected in dairy cattle of HO breed  in appearance of C. parvum infections, with one exception: animals of MDM breed differed from the other breeds . Mixed C. parvum and C. bovis infections were not associated with any particular breed (95% TPIAll breeds: < 0.0001–42.54%).The sampled animals mainly represented dairy breeds (74.8%), meat and mixed dairy–meat breeds being far smaller proportions (respectively 14.5% and 10.7%). Animals of Polish Black and White Holstein Friesian (HO), Limousie (LM), mixed exclusively meat breed (MM), Simentaler (SM), mixed exclusively dairy breed (MS) and mixed dairy–meat breed (MDM) breeds were found positive for C. parvum positive. None of the tested cattle from Śląskie province (SL) herds was C. parvum positive. Infections with this parasite were detected in farms located across 15 Polish provinces with varied prevalences  in Poland were shown . Pomorskie (PM), ZP, Łódźkie (LD), Podkarpackie (PK) and Mazowieckie (MZ) provinces were characterised by higher prevalence of infections (16.7 ≥ 9.3%) and in descending order of prevalence the remaining provinces were Wielkopolskie (WP), Podlaskie (PL), Kujawsko-Pomorskie (KP), Lubelskie (LB), Warmińsko-Mazurskie (WM)  and Lubuskie (LS), Opolskie (OP), Świętokrzyskie (SK), Małopolskie (MP), DS and SL . The highest frequency of parasite infections was in PM at 16.7% and the lowest in DS province at 1.1%, where only 1 out of 15 farms kept infected animals  out of the 267 monitored farms and were located over the whole country. All evaluated farms in the Zachodniopomorskie (ZP) and Dolnośląskie (DS) provinces appeared to be 6.5% out C. parvum strains. Strains from the IIa subtype family dominated with 80 affiliations and strains from the IId subtype were the other 2 successful identifications. The IIaA17G1R1 (n = 26) and IIaA17G2R1 (n = 24) strains were detected in the greatest numbers. Three subtypes, IIaA21G1R1 (n = 1), IIaA18G3R1 (n = 1) and IIaA21R1 (n = 1), were only sporadically found. The other detected strains were IIaA15G2R1 (n = 10), IIaA16G1R1b (n = 9), IIaA22G1R1 (n = 4), IIaA10G1R1 (n = 2), IIaA14G2R1 (n = 2) and IIdA24G1 (n = 2). None of the tested samples contained more than one C. parvum GP60 subtype. The ANOVA showed that only C. parvum IIaA17G2R1 and IIaA17G1R1 infections occurred at significantly higher frequency than other parasite subtypes . The infections caused by IIaA17G2R1  and IIaA17G1R1  dominated in the studied animal population. In the case of the remaining C. parvum subtypes  there were no significant differences observed in the frequency of their prevalence. Mixed infections caused by two or more parasite subtypes were detected neither at the farm nor at animal level.The subtype was successfully identified by PCR amplification with GP60 primers for 82 out of 100 detected parasite strains but was not for the other 18 C. parvum strains within the following IIa subtype groups . C. parvum IIaA14G2R1 strains revealed 96.4% mutual nucleotide sequence identity. A higher genetic resemblance (99.6–100%) was shown for IIaA22G1R1 strains. The phylogenetic relationships between strains representing different parasite subtypes were presented on Fig. The nucleotide sequence analysis of the GP60 gene fragment gene showed 100% sequence identity for C. parvum subtype and cattle age group . Nevertheless, particular subtypes were only shown in animals at a certain age. For instance, the IIaA15G2R1, IIaA21G1R1, IIaA10G1R1, IIaA18G3R1, IIaA21R1 and IIaA24G1 subtypes were detected in cattle below 4 weeks of age, while IIaA14G2R1 only occurred in animals between the ages of 4 and 8 weeks , whereby 10 out of 11 C. parvum subtypes were found in HO calves, C. parvum IIaA22G1R1 strains were only detected in LM cattle and IIaA24G1 only in MM animals (Table There was no relationship observed between infections caused by a particular C. parvum were detected on 44 (16.48%) of the 267 farms. Two commonly detected IIa subtypes, i.e., IIaA17G1R1 and IIaA17G2R1, were found respectively in 8 and 6 out of the 16 Polish administrative provinces. In comparison to IId subtypes, the two aforementioned strains from the IIa allele families occurred frequently over almost the entire country except in the LS, SL and DS provinces . The IIaA17G1R1 strains prevailed in PK and SK, while the IIaA17G2R1 did in MP, MZ and WM. On cattle farms in ZP and LS, IIaA16G1R1b and IIdA24G1 infections comprised the majorities of observed infections, respectively.Animals infected with ed on 44 6.48% of C. parvum subtypes in cattle in Poland, the disappearance of the strains IIaA14G1R1, IIaA18G1R1c and IIaA19G1R1 was observed with time. In their place, the new variants IIaA18G3R1 and IIaA14G2R1 representing the same genetic family appeared; however, they did so in different geographical locations. Based on the current results, it can be assumed that some subtypes from the IIa (A19G1R1) and IIl (A19R3) genetic families are not currently present in Poland. In the place of the parasite strains IIdA22G1b, IIdA23G1, and IIdA24G1c, which before 2014 had been detected in cattle herds in western Poland (ZP and PM provinces) as well as in LB province in the east of the country, a single subtype IIdA24G1 was found at the time of this investigation. Its appearance was limited to only LS in western Poland. Strains of the IId classification did not spread to other geographical locations in the country, which may indicate their endemic prevalence. For the IIa strains prevalent in Polish cattle, a change in their population dynamics related to the time and place of their detection was observed.Analysing the structure of the population genetics of C. parvum strains of this subtype continued to affect the cattle population in Poland, although their prevalence was limited to only PL and ZP. Also in the same period of time, in the population of IIaA16 strains, two groups of variants were found, i.e., IIaA16G1R1b and IIaA16G3R1. The IIaA16G1R1b subtype prevailed and it was only present in the WP province. Currently, its spread to other regions such as ZP, PK, and PL is observed, which means transmission from the western to the eastern part of Poland. Of note is that infections in cattle caused by this subtype are endemic in the WP province. Over a period of 8 years the frequency of IIaA17G1R1 and IIaA17G2R1 infections was high and they are still considered dominant C. parvum subtypes in cattle in Poland. In many cases, they co-circulated in a given geographical area. Furthermore, the constant presence of the IIaA17G1R1 strains in WM, PM and ZP and of the IIaA17G2R1 strains in MZ, PL and WM may indicate the endemic nature of these infections.Before 2014, the IIaA15G2R1 subtype was circulating in livestock almost nationwide, also being the only parasite subtype detected in DS, SL, LD, and SK provinces . From 20Cryptosporidium species have been detected in cattle, symptomatic cryptosporidiosis is mainly associated with C. parvum infections. The frequency of infections is varied and depends on the animals’ age and the place of breeding [C. parvum infections in the cattle population in Poland was estimated at 6.2%. As seen in our previous study the overall prevalence of C. parvum infections in cattle from Poland at the age of 1 day to 6 years was estimated at 5.1% but only animals below the age of 1 month were the major host for this parasite. In fact, C. bovis was the most frequently detected parasite species [C. parvum in cattle have been reported outside Europe in Argentina (25.5%), Brazil (42.2%) and China (30–60%) [Although different breeding , 33, 34.breeding , 35, 36;breeding . The pre species . Further species , 36, 37  species . Higher (30–60%) –41.C. parvum was more often detected in dairy Polish Black and White Holstein Friesian cattle than in meat or mixed dairy–meat animal breeds. This observation is consistent with previous findings that C. parvum infections occur more frequently in dairy cattle than in other breeds [C. parvum subtypes was found.It is believed that the animal production system (beef or dairy cattle) could have an impact on the frequency of infection , 43. It r breeds , 44. IntC. parvum strains isolated from Polish cattle, 11 subtypes representing the IIa and IId genetic families were detected. The six subtypes IIaA10G1R1, IIaA15G2R1, IIaA16G1R1b, IIaA17G1R1, IIaA17G2R1 and IIdA24G1c were previously identified in a bovine host in Poland [C. parvum subtypes in European cattle herds [C. parvum as well as other closely related strains such as IIaA16G3R1 and IIaA16G2R1 have also been identified in livestock in Europe [C. parvum strains [Based on the microsatellite sequence analysis of the GP60 gene of n Poland  to 14.5%le herds . In contle herds , 43, 45.n Europe , 34. In n Europe  and in S strains . In Euro strains , 49.C. parvum subtypes  from the IIa genetic family were detected. They had not been identified in calves in Poland prior to this. The strain IIaA18G3R1 has been previously found in livestock elsewhere in Europe [C. parvum IIaA14G2R1, its prevalence in cattle in Europe has not exceeded 3.8% [In this study, five new n Europe , 35. Itsn Europe , 22. Thin Europe , 51. In ded 3.8% , 23, 43.ded 3.8% . The othded 3.8% , 53, 54.Cryptosporidium infections in other species of farm ruminants in Poland, IIaA17G1R1 strain has also been occasionally detected in sheep [C. parvum IIaA16G1R1, IIaA19G1R1 and IIaA23G1R1 subtypes limited to one farm has been described by Del Coco et al. [C. parvum subtypes in cattle. Nevertheless, herd size, animal movement, farm management practices, or husbandry system could have an influence on regional subtype prevalence. However, based on the existing data, it would be difficult to determine their significance in the epidemiology of bovine cryptosporidiosis at subtype level.There was no relationship observed between infections caused by a specific parasite subtype and cattle age, and this observation is consistant with our previous finding . Howeverin sheep . Furthero et al. . The autC. parvum strains in cattle from Poland has been shown, as exemplified by the emergence of the IIlA19R3 subtype in the bovine host for the first time. In fact, the strains from the genetic family IIl rarely appear in Europe [C. parvum strains which are disappearing from Poland are IIaA14G1R1, IIaA18G1R1c and IIaA19G1R1. Hitherto, the IIaA14G1R1 and IIaA19G1R1 subtypes have been identified in cattle in Estonia [C. parvum strains from the IId genetic family are the majority of those disappearing or found to occur endemically in particular geographical areas. Out of three IId subtypes circulating before 2014 in cattle, only one remained, IIdA24G1. It had not been identified in cattle in Europe, although it was present in humans [C. parvum subtypes from the IId genetic family were found in Europe with a frequency ranging from 1.1 to 15.3% [Population dynamicity in n Europe , 7, 47.  Estonia  and the  Estonia  with fre Estonia , 27, 54. Estonia . In Polan humans –59. Howen humans . Neverthto 15.3% .C. parvum strains. Although the sampling scheme covered all Polish provinces, the number of sampled animals may not reflect their actual population size perfectly in each sampled region. Therefore, the findings for the variability of detected subtypes in a particular region may be biased and may not mirror the subtypes’ real transmission dynamics in cattle. A further shortcoming is that PCR amplification with GP60 primers was unsuccessful for 18 C. parvum strains, probably because of either the low quantity of Cryptosporidium DNA or low specificity of the primer’s sequences to the DNA sequence of the detected subtype. A greater number of identified strains could have had influence on the frequency of those strains’ detection and on the interpretation of subtype prevalence results. In particular this could be important for sporadically detected subtypes as it might have changed our perception of their prevalence.As a final observation, the research findings of this study were limited by sample representativeness and lack of subtype identification for all detected C. parvum strains circulating over a period of 8 years in Polish cattle herds. Two strain subtypes predominated in cattle, i.e., IIaA17G1R1 and IIaA17G2R1, which over time were not displaced by other subtypes. Nevertheless, a certain strain population dynamicity was observed, which was associated with the disappearance of some strains  and emergence of new variants  from the same genetic family in different geographical locations. The geographical change of strain prevalence from the western to the eastern part of Poland was evident for IIaA16G1R1b subtype. In the case of IIaA15G2R1 its prevalence range was geographically limited although it was circulating in livestock almost nationwide. Strains of the IId subtypes did not spread over time to other geographical locations suggesting their endemic prevalence. No age-dependent distribution of C. parvum subtypes was shown among the various cattle breeds being reared in Poland. Breed-dependent diversity of C. parvum subtypes was  found, however, with the majority of subtypes being detected in HO cattle. The results of this study will also help in better understanding the subtype-related epidemiology of C. parvum infections in cattle and humans that could have originated from livestock, although they do not provide evidence for their possible transmission between hosts.The results of this study have enhanced our knowledge of the population genetics of n = 527), > 4–8 weeks old (n = 464), and > 8–16 weeks old (n = 610) and were dairy varieties (Polish Black and White Holstein Friesian (HO), Jersey (JE), Polish Red and White Holstein Friesian (RW), Brown Swiss (BS), and Mixed exclusively dairy breed (MS)), meat varieties (Mixed exclusively meat breed (MM), Aberdeen Angus (AN), Charolaise (CH), Salers (SL), Limousie (LM), and Belgian Blue (BB)) and dairy–meat varieties (Mixed dairy–meat breed (MDM), Black and White lowland (NCB), Polish Red (RP), Montbeliarde (MO), Polish Black and White (ZB), and Simentaler (SM)). Freshly voided faecal samples of 10–15 g were placed individually into plastic containers, labelled and sent to the laboratory. For comparison studies analysing the time-related changes of geographical distribution of C. parvum subtypes in cattle herds in Poland, archive data on the occurrence in the Polish cattle population of specific subtypes were accessed. Those subtypes were A17G1R1, A17G2R1, A15G2R1, A16G1R1b, A10G1R1, A14G1R1, A16G3R1, A18G1R1c and A19G1R1 from IIa; A23G1, A24G1c and A22G1b from IId; and A19R3 from IIl genetic families. These strains were detected between 2010 and 2014 in different administrative provinces across Poland [In total, 1601 faecal samples were collected from healthy cattle at the age of 1 week to 4 months from 2014 to 2018 , 20 µg BSA (Thermo ScientificTM), and molecular grade water up to 50 µl reaction volume. Primers were used at the concentration of 0.1 µM (GP60 assays) or 0.5 µM each (18 SSU rRNA assay). Five or two µl of sample DNA or PCR products were the templates for primary and secondary amplifications respectively. Depending on the assay, the temperature profile was consisting of initial denaturation performed at 94 °C for 3–5 min, then 35 cycles of denaturation at 94 °C for 45 s., annealing at 55 °C (18SSU assay) or 50 °C /52 °C (GP60 assays) for 45 s., and extension at 72 °C for 1 min. A final extension step (72 °C for 7–10 min. depending on the assay) was included followed by a soak at 4 °C. All reactions were performed in a Biometra thermocycler . For each set of samples, the appropriate positive and negative controls were included during the nucleic acid extraction and PCR analyses. For restriction fragment analysis, 4 μl of nested-PCR product was digested in a total reaction volume of 10 µl consisting of 1 µl NdeI or MboII (Thermo ScientificTM) and the appropriate FastDigest reaction buffer at 37 °C for 1 h followed by 65 °C for 5 min. PCR products and their restriction fragments were analysed in 2.5% agarose gels stained with SimplySafe (Eurx).Samples were analysed using molecular methods according to previously described protocols . BrieflyNA locus  followedNA locus , 63. ExtR assays , 65 geneThe GP60 amplicons were excised from the agarose gel, purified and directly sequenced in both directions using the ABI Prism BigDye Terminator v3.1 Cycle sequencing kit on an ABI 3730XL DNA sequencer  at the Genomed S.A. sequencing service. Particular subtypes were identified based on trinucleotide repeats present in the analysed sequences. A single representative sequences for each subtype group sharing mutual 100% sequence similarity as well as all sequences that did not show 100% genetic resemblance within particular subtype group were deposited in the GenBank under the accession numbers OM836436—OM836449. The sequence similarity of detected strains was assessed by the maximum likelihood method with a Tamura-Nei parameter model using MEGA version 7.0.9 . The relC. parvum infections between age groups of animals was determined by a chi-square (χ2) test. The frequency of parasite occurrence in cattle in different Polish regions (provinces) was analysed using a Kruskal–Wallis Chi-squared test. The relationship between the occurrence of C. parvum infection and breed expressed as true prevalence estimate (TPI) was evaluated based on Bayesian posterior probability (POPR) [The significance of differences in the frequency of y (POPR) . Estimaty (POPR) .2 test was also employed to show the relationships between C. parvum subtypes and their occurrence in calves of different breeds and ages by group and between subtypes and their geographical distribution in Poland. The frequency of occurrence of C. parvum subtypes in the studied cattle population was assessed by ANOVA. The calculations were performed with Statgraphics Centurion v. XVI  and R software v. 4.1.1 [Concluding the statistical work, the χv. 4.1.1 .


The Ni/Ce1−xZrxO2 catalysts were characterized via XRD analysis, SEM-EDX, TEM-EDX, and CO chemisorption and tested in carbon dioxide methanation. The obtained Ce1−xZrxO2 materials were single-phase solid solutions. The increase in Zr content intensified crystal structure strains and favored the reducibility of the Ce1−xZrxO2 oxides but strongly affected their microstructure. The catalytic activity of the Ni/Ce1−xZrxO2 catalysts was found to depend on the composition of the Ce1−xZrxO2 supports. The detected negative effect of Zr content on the catalytic activity was attributed to the decrease in the dispersion of the Ni0 nanoparticles and the length of metal–support contacts due to the worsening microstructure of Ce1−xZrxO2 oxides. The improvement of the redox properties of the Ce1−xZrxO2 oxide supports through cation modification can be negated by changes in their microstructure and textural characteristics.Ce Nicapproach ,5. Becauapproach ,7,8. CeOaterials ,14,15,16acancies . Oxygen olecules ,20,21,221−xZrxO2 oxides are promising systems in terms of catalytic activity and stability [1−xZrxO2 catalysts in the methanation of carbon oxides. The reducibility of Ce1−xZrxO2 oxides was experimentally shown to increase with the Zr content [1−xZrxO2 catalysts in CO2 methanation. Ocampo et al. [2 methanation over 5 wt.%Ni/Ce1−xZrxO2 catalysts differing in Zr content . The 5 wt.%Ni/Ce0.5Zr0.5O2 catalyst showed the highest activity. Nie et al. [2-ZrO2 mixed oxides containing 40 wt.% Ni. The catalyst with a Ce/Zr molar ratio of 9:1 (Ce0.9Zr0.1O2) exhibited the best catalytic properties. Atzori et al. [2-ZrO2 catalysts (30 wt.% Ni—Ce1−xZrxO2) in CO2 and CO co-methanation. The activity was the same for the catalysts with Zr content in x = 0-0.5 and decreased at a higher content. To summarize, a correlation between the activity of Ni/Ce1−xZrxO2 catalysts and the Ce/Zr ratio has not been fully understood. All the reported studies indicated that nickel catalysts based on highly doped Ce1−xZrxO2 oxides (x > 0.5) are less effective. The data concerning systems based on Ce1−xZrxO2 oxides with a lower Zr content (x ≤ 0.5) are somewhat controversial.The modification of ceria by doping with foreign cations is a well-known strategy to improve its redox properties . Nickel tability ,24,25,26tability ,28,29. O content . The rego et al.  studied e et al.  studied i et al.  tested N1−xZrxO2 support materials and the performance of Ni/Ce1−xZrxO2 catalysts in CO2 methanation. A series of Ce1−xZrxO2 supports with different Zr contents  were prepared via the Pechini method. The supported catalysts Ni/Ce1−xZrxO2 containing 10 wt.% Ni were synthesized via the impregnation method. DFT calculations and TPR studies were employed to evaluate the impact of Zr content on the reducibility of the Ce1−xZrxO2 oxides. A wide range of physical methods was used to reveal the structure features of the Ce1−xZrxO2 support materials and Ni/Ce1−xZrxO2 catalysts. The catalytic properties of the Ni/Ce1−xZrxO2 samples were analyzed with regard to the structural features of both the support materials and catalysts.The present work aims to provide further insight into the relation between the composition of the Ceeff = 5 eV) [The DFT+U calculations were performed using the Vienna Ab initio Simulation Package (VASP) program . The gen = 5 eV)  was used = 5 eV)  to take 1−xZrxO2 supercells  were built by replacing Ce atoms with Zr ones (1−xZrxO2 (100) and (111) surfaces were modeled as p (2 × 2) and p (3 × 3) slab cells. The (100) and (111) Ce1−xZrxO2 supercells contained four and three layers, respectively. A vacuum space of 15° Å was set between neighboring slabs to keep the spurious interaction.The face-centered cubic unit cell of a fluorite-type structure  was computed as:1−xZrxO2−δ) and E(Ce1−xZrxO2) are the energies for surfaces with and without oxygen vacancy, and E(O2) is the energy of gas-phase O2.The energy of oxygen vacancy formation  mixed oxides was prepared via the Pechini method [3)3*6H2O (99.4%) and ZrO(NO3)2*8H2O (99.98%) salts were used as precursors. Aqueous solutions of the salts with required Ce:Zr molar ratios  were prepared. Citric acid (CA) was added to the aqueous solutions at 80 °C and vigorously stirred for 30 min. The CA:metal molar ratio was 1:1. Subsequently, ethylene glycol C2H4(OH)2 (EG) was added. The CA:EG molar ratio was 3:2. Next, the solution was heated at 100 °C to promote the polyesterification reaction and water evaporation with the formation of a polymeric resin. The obtained solids were mechanically milled to powders and calcined at 450 °C for 8 h.A series of Cei method . The Ce2·4H2O (99.0%) and ethylene glycol (99.5%) were dissolved in the distilled water under stirring at 70°C for 20 min. Then, the support material was added into the solution. The EG:Ni molar ratio was set to 5.3. The suspension of the support and impregnating solution was stirred for 2 h at 70 °C and dried at 120 °C for 12 h in air. The dried samples were heated at a rate of 2 °C/min and calcined in air at 400 °C for 2 h. The catalysts were designated as Ni/Ce1−xZrxO2, with x indicating Zr content.The catalysts containing 10 wt.% Ni were prepared by the impregnation of the obtained CeElemental compositions of the catalysts were determined via X-ray fluorescent spectroscopy (XRF) using an ARL-Advant’x device . Measurements were carried out in a helium atmosphere using the Rh X-Ray tube. UniQuant software was used to calculate the element percentages.BET, m2/g) of the Ce1−xZrxO2 oxides were determined by N2-physisorption at −196 °C. The experiments were carried out using an ASAP 2400 instrument .The BET specific surface areas  and microstrain (Δd/d) effects to the line broadening was performed by means of Williamson–Hall plots [XRD values, the crystallite surface area (SXRD) values were calculated assuming the crystallites were quasi-spherical:3).X-ray diffraction (XRD) measurements were carried out using a D8 Advance diffractometer  equipped with a Lynxeye linear detector. The measurements were carried out using the Cu Kα radiation (λ = 1.5418 Å) in the 2θ range of 10–83° with a step of 0.05°. XRD phase analysis was performed using the ICDD PDF-4+ database. The evaluation of substructure parameters of the Cell plots . From th1−xZrxO2 crystallites, an agglomeration coefficient (ξ) was calculated on the basis of the ratio of SBET and SXRD values:To estimate the degree of agglomeration of the CeThe average crystallite size of nickel-containing phases was estimated by the line broadening analysis according to the Scherrer equation . The Rie0 nanoparticles in catalysts after catalytic experiments were determined using the pulse chemisorption of CO on the assumption that each surface Ni atom adsorbed one CO molecule. The measurements were carried out using a Chemosorb analyzer . An amount of 50 mg of each sample was placed inside a U-shape quartz reactor and reduced at 350 °C in H2 flow (100 mL/min) for 30 min. The treated sample was subsequently cooled down to room temperature, followed by Ar purge. After that, pulses of CO were fed to the reactor (100 μL) until the amount of CO in the outlet stopped changing according to the thermal conductivity detector. The amount of chemisorbed CO was estimated. CO adsorption over the pure supports was negligible.The average sizes of metallic NiTransmission electron microscopy (TEM) studies were carried out using a JEM-2200FS  and a Themis Z electron microscope  operated at 200 kV. Images in Scanning-TEM (STEM) mode were acquired using a high-angle annular dark field (HAADF) detector. The local elemental composition of the samples was studied using a Thermo Fisher Scientific Super-X EDX spectrometer. The samples were ground, suspended in ethanol, and placed on a copper grid coated with a holey carbon film.Scanning electron microscopy–energy-dispersive X-ray analysis (SEM-EDX) studies were carried out using a dual-beam scanning electron microscope, Tescan Solaris FE/SEM . The experiments were performed in secondary electron mode at an accelerating voltage of 20 kV. The microscope is equipped with an AztecLive EDX spectrometer  with a Silicon Drift Detector and energy resolution of 128 eV. The cross-sections of catalyst granules in epoxy resin were prepared for the examination. The cross-sections with a given flatness of 0.25 μm were covered with a conductive carbon layer of 10–20 nm thickness.2 in Ar was fed at 40 mL/min. The rate of heating from 25 to 800 °C was 10 °C/min. The TPR curves were normalized per sample mass.The temperature-programmed reduction (TPR) via hydrogen was performed with 40–60 mg of sample in a quartz reactor using a flow setup with a thermal conductivity detector. The gas mixture containing 10 vol.% of H2, 16 vol.% H2, and Ar as balance. The catalyst load was 150 mg, 0.2–0.5 mm fraction, and the flow rate—75 mL/min. Prior to the experiment, each catalyst was reduced in 10 vol.% H2 in Ar flow (50 mL/min) at 400 °C for 1 h. The compositions of the inlet and outlet gas mixtures were determined using a gas chromatograph, KHROMOS-1000 , equipped with a thermal conductivity detector (CaA molecular sieves column) and flame ionization detector (Porapak Q column) with a methanator characterized by sensitivity to CO, CH4, and CO2 of ~1 ppm. The separation of CO, CH4, and CO2 on the column followed by the methanation of carbon oxides allowed the flame ionization detector to be used to analyze their concentration. The equilibrium compositions were calculated using equilibrium software HSC 7.0. It was assumed that equilibrium mixtures only contained gaseous substances , i.e., no carbon deposition processes were taken into account. The catalytic properties of Ni/Ce1−xZrxO2 catalysts were also compared with those of industrial catalyst NIAP-07-05 for the methanation of carbon oxides, which contains 38 wt.% NiO, 12 wt.% Cr2O3, and 50 wt.% Al2O3 (further denoted as 38Ni−Cr−Al). The catalyst has a Ni0-specific surface area of 3.3 m2/g in a reduced state [The catalytic tests were performed in a U-shaped tubular continuous-flow reactor (i.d. = 3 mm) at ambient pressure and the temperature range from 200 to 400 °C. The temperature was controlled using a K-type thermocouple placed in the middle of the catalyst bed. The feed gas contained 4 vol.% COed state .1−xZrxO2 lattice decreases with an increase in Zr content (4+ cation (0.84 Å) compared to the Ce4+ cation (0.97 Å). Doping CeO2 with Zr leads to a slight decrease in Ce–O distances. The calculated Ce–O and Zr–O distances are in the range of 2.314–2.375 Å and 2.205–2.258 Å, respectively.According to DFT+U calculations, the parameter of the Ce content  due to a1−xZrxO2 (111) surface is more stable than the (100) surface. The CeO2 (111) surface is known to be the most stable surface among the low-index surfaces (100), (110), and (111) [The calculated values of the surface energy are presented in nd (111) ,45. Zirc1−xZrxO2 are also listed in f values decrease considerably in the range of compositions 0 < x < 0.5, while at x > 0.5, the Ef values increase. The Ce0.5Zr0.5O2 surfaces are characterized by the lowest energies of oxygen formation. It was found that Zr incorporation facilitates the generation of oxygen vacancy. The obtained result agrees with data of interatomic potential simulations reported by G. Balducci et al. [4+/Ce3+ reduction energy is reduced even by small amounts of zirconium incorporated into ceria. Based on DFT+U calculations, Yang et al. [4+ cations counterbalance steric strains arising at formation Ce3+ cations, which are larger than Ce4+ cations [The calculated energies of oxygen vacancy formation in Cei et al. ,47, whicg et al. ,49 also  cations ,49.1−xZrxO2 samples are shown in The powder XRD patterns of the Ce2 . The lattice shrinkage indicates the formation of substitutional solid solution Ce1−xZrxO2. The gradual decrease in the lattice parameter with the increase in Zr content is observed. The composition of the Ce1−xZrxO2 solid solutions was evaluated with use of Vegard’s rule. The linear dependence of the lattice parameter on the Zr content is provided elsewhere [1−xZrxO2 solid solutions. As can be seen from lsewhere . Estimat1−xZrxO2 oxides  was determined from particle size distribution histograms shown in insets in 1−xZrxO2 crystallite size to decrease as the Zr content increases in accordance with the XRD results.All the oxides are highly dispersed; the DBET ones . Such a ficients  showed tM images d–f, the tallites , dHRTEM 1−xZrxO2 oxides was studied. The H2-TPR profiles are reported in 2-TPR profile of the low-doped Ce0.9Zr0.1O2 oxide exhibits two broad peaks at 500 °C and 820 °C. Such a two-peak pattern is characteristic of CeO2 oxide and reflects the stepwise reduction in the surface and bulk [0.75Zr0.25O2 and Ce0.5Zr0.5O2 oxides exhibit asymmetric peaks with significantly increased intensities. The second high-temperature reduction peak is not observed. These data indicate that surface and bulk reduction processes occur almost simultaneously. The observed intensive asymmetric peak reflects the co-reduction in surface and bulk. This suggests the improvement of the reducibility of the mixed Ce1−xZrxO2 oxides with an increase in Zr content in full agreement with previous TPR studies of Ce1−xZrxO2 oxides [The reduction behavior of the Ceand bulk . The TPR2 oxides ,29,30.1−xZrxO2 catalysts. The broad peaks from the oxide NiO phase (PDF#00-047-1049) are detected in the XRD patterns of the as-prepared catalysts, while the narrow peaks from the metallic Ni0 phase (PDF #00-004-0085) are observed in the XRD patterns of the catalysts aged under reductive conditions of CO2 methanation.1−xZrxO2 catalysts according to the XRD and CO chemisorption data. The Ni/Ce0.9Zr0.1O2 catalyst is characterized by a higher dispersion of initial NiO crystallites as well as Ni0 crystallites formed upon reduction. Reaction conditions provoke the sintering of nickel species. The average size of Ni0 crystallites in the aged catalysts is larger than the size of NiO crystallites in the as-prepared catalysts. The Ni/Ce0.9Zr0.1O2 catalyst is characterized by a higher resistance of Ni0 crystallites to sintering. The average size of Ni0 crystallites in the Ni/Ce0.9Zr0.1O2 catalyst is about two times smaller than in other catalysts. CO chemisorption results . In a previous work, we reported that the synergism of redox properties of the Ni/Ce1−xZrxO2 system enhances the catalytic performance in the methanation of carbon oxides [The catalytic performance of the Ni/Cen oxides .0.9Zr0.1O2 catalyst exhibited the highest catalytic activity. The CO2 half-conversion temperature (T50) was 244, 266, and 280°C for Ni/Ce0.9Zr0.1O2, Ni/Ce0.75Zr0.25O2, and Ni/Ce0.5Zr0.5O2, respectively. The observed decrease in activity in the sequence Ni/Ce0.9Zr0.1O2 >> Ni/Ce0.75Zr0.25O2 > Ni/Ce0.5Zr0.5O2 correlates with a decrease in Ni0-specific surface area: 6 >> 2.9 > 2 mNi2/gcat. The revealed higher dispersion of Ni0 particles and more developed contacts between them and support surface can explain the higher catalytic activity of the Ni/Ce0.9Zr0.1O2 catalyst. As was shown above, the Ni/Ce0.9Zr0.1O2 catalyst contained Ni0 nanoparticles with dNichem sizes of 11.2 nm, and the least active Ni/Ce0.5Zr0.5O2 catalyst contained Ni0 nanoparticles with dNichem sizes of 33.7 nm (0.9Zr0.1O2 catalyst is characterized by more developed contacts between nickel particles and the support (0 dispersion and a developed metal–support interface area are highly important for the catalytic activity for CO2 methanation [The Ni/Ce 33.7 nm . It was  support b. It is hanation ,54,55.1−xZrxO2 catalysts as well as the degree of metal–support interaction [1−xZrxO2 oxides can affect their microstructure and texture features and, consequently, the dispersion of supported nickel nanoparticles. This influence is likely related to the synthesis technique used. The Ce1−xZrxO2 oxides under study were prepared via the Pechini method. A similar effect was observed by Iglesias et al. [1−xZrxO2 catalysts with supports prepared using the coprecipitation method. It was shown that an increase in Zr content diminishes the specific surface area of the Ce1−xZrxO2 supports with a decrease in the dispersion of supported Ni0 nanoparticles. It was also reported that the increase in Zr content reduces the specific surface area of the Ce1−xZrxO2 oxides prepared via the sol–gel technique [It was recently suggested that zirconium addition impacts the dispersion of nickel nanoparticles in Ni/Ceeraction ,31. The s et al.  in the sechnique  and the echnique .1−xZrxO2 mixed oxides with different compositions were prepared using the Pechini method and used as the supports for Ni/Ce1−xZrxO2 catalysts. It was demonstrated that an increase in Zr content enhances the distortion of the crystal structure of Ce1−xZrxO2 oxides and leads to improvements in their redox properties. It was also found that microstructure features of Ce1−xZrxO2 oxides change with cation composition.In this study, Ce1−xZrxO2 composition on the structure and catalytic activity of the Ni/Ce1−xZrxO2 catalysts in CO2 methanation was investigated. The activity of the Ni/Ce1−xZrxO2 catalysts decreased in the order: Ni/Ce0.9Zr0.1O2 >> Ni/Ce0.75Zr0.25O2 > Ni/Ce0.5Zr0.5O2. The drop in the activity correlated with the decrease in the dispersion of metallic Ni0 nanoparticles. It was revealed that differences in the microstructural characteristics of the Ce1−xZrxO2 supports are responsible for differences in the dispersion of supported Ni0 nanoparticles and the length of the metal–support interface.The effect of the Ce1−xZrxO2 oxides, which are important for catalysis, through cation modification can be counterbalanced by worsening their microstructure characteristics, which determine the dispersion of supported Ni0 nanoparticles.It was shown that improving the redox properties of Ce


Objectives: .Asthma is a highly heterogeneous disease, and T-helper cell type 17 (Th17) cells play a pathogenic role in the development of non-T2 severe asthma. Misshapen like kinase 1 (MINK1) is involved in the regulation of Th17 cell differentiation, but its effect on severe asthma remains unclear. Our previous studies showed that methyl-CpG binding domain protein 2 (MBD2) expression was significantly increased in patients with Th17 severe asthma and could regulate Th17 cell differentiation. The aim of this study was to investigate how MBD2 interacts with MINK1 to regulate Th17 cell differentiation in Th17-dominant asthma.Materials and methods: Female C57BL/6 mice and bronchial epithelial cells (BECs) were used to establish mouse and cell models of Th17-dominant asthma, respectively. Flow cytometry was used to detect Th17 cell differentiation, and the level of IL-17 was detected by enzyme-linked immunosorbent assay (ELISA). Western blot and quantitative real-time PCR (qRT-PCR) were used to detect MBD2 and MINK1 expression. To investigate the role of MBD2 and MINK1 in Th17 cell differentiation in Th17-dominant asthma, the MBD2 and MINK1 genes were silenced or overexpressed by small interfering RNA and plasmid transfection.Results: Mouse and BEC models of Th17-dominant asthma were established successfully. The main manifestations were increased neutrophils in BALF, airway hyperresponsiveness (AHR), activated Th17 cell differentiation, and high IL-17 levels. The expression of MBD2 in lung tissues and BECs from the Th17-dominant asthma group was significantly increased, while the corresponding expression of MINK1 was significantly impaired. Through overexpression or silencing of MBD2 and MINK1 genes, we have concluded that MBD2 and MINK1 regulate Th17 cell differentiation and IL-17 release. Interestingly, MBD2 was also found to negatively regulate the expression of MINK1.Conclusion: Our findings have revealed new roles for MBD2 and MINK1, and provide new insights into epigenetic regulation of Th17-dominant asthma, which is dominated by neutrophils and Th17 cells. This study could lead to new therapeutic targets for patients with Th17-dominant asthma. These risk factors induce an immune response to the initial initiation of T cells by antigen presenting cells (APCs), mainly dendritic cells and bronchial epithelial cells (BECs) . SeveralThe germinal center kinase family regulates a variety of cellular processes, including cell growth and differentiation, gene transcription, and immune and stress responses, through the mitogen-activated protein kinase pathways . As a meIn addition to genetics, environmental influences also play a role in asthma development, which may be regulated by epigenetic mechanisms . The epiin vivo and in vitro, while MINK1 was decreased, and there was a reverse expression between MBD2 and MINK1. In addition, we also investigated the effect of MBD2 intervention on MINK1 expression and Th17 cell differentiation and found that MBD2 deletion promoted MINK1 expression and inhibited Th17 cell differentiation and IL-17 release. Taken together, these data suggested that MBD2 might promote Th17 cell differentiation by inhibiting MINK1, thereby exacerbating the severity of asthma.A previous study showed that MBD2 was significantly increased in patients with Th17 severe asthma compared to healthy controls and patients with asthma . In thisFemale C57BL/6 mice  were provided by the animal center of the Second Xiangya Hospital of Central South University  and maintained under specific pathogen-free conditions. All experimental protocols were approved by the Animal Care and Use Committee of the Second Xiangya Hospital of Central South University and were conducted in accordance with the guiding principles of the institution. The establishment of a Th17-dominant asthma mouse model was consistent with previous reports . AnimalsThe T2 asthma group was sensitized by intraperitoneal injection of 100 μg OVA (1 mg/ml) and 2 mg aluminum hydroxide dissolved in 200 μL saline on days 0–7, followed by atomization with 6% OVA solution on days 14–20 for 30 min . Mice weMethacholine (Mch)-induced airway resistance was measured with direct plethysmography  on day 21 . First, Bronchoalveolar lavage fluid (BALF) was collected by injecting 0.5 ml of saline into the lungs 3 times through an endotracheal tube (37°C). BALF inflammatory cells were centrifuged and resuspended in cold phosphate-buffered saline , then the cells were fixed and stained with Wright-Giemsa, and 200 cells were counted and differentially counted under a light microscope using a counting chamber.via the trachea, then removed and stored in 10% formalin. Fixed lung tissues were paraffinized and sectioned (5 μm) for hematoxylin and eosin (H&E) staining and immunohistochemistry . Select stained sections per group were collected and assessed for MBD2, MINK1, eosinophil, and neutrophil protein expression.The lungs were fixed with 10% formalin g, the supernatant was discarded and the cells were resuspended with MEM containing 10% FBS. The cells were then inoculated in culture bottles and left standing at 37°C with 5% CO2 for 2 h to remove contaminated non-epithelial cells. Subsequently, the culture medium (including suspended cells) was collected, and the supernatant was removed after centrifugation at 800 g for 5 min. BECs were cultured with bronchial epithelial growth medium  in a humidified incubator at 37°C with 5% CO2.Bronchial epithelial cells (BECs) were isolated using an improved version of the protocol previously reported . BrieflyGenerally, studies have shown that 95% of epithelial cells are positive for cytokeratin, an epithelial marker that is not expressed by lymphocytes . The BECTo induce Th17-dominant asthma, BECs were treated with 100 μg/ml HDM and 100 ng/ml LPS for 24 h. BECs were treated with 100 μg/ml HDM for 24 h to establish T2 asthma or PBS to establish normal controls .Small interfering RNA targeting MBD2, MINK1 , and negative control (siR-NC) were purchased from RiboBo . The MBD2 interfering sequence was 5′-GCAAGATGATGCCTAGTAA-3′ and the MINK1 interfering sequence was 5′-GCAAGTACAAGAAGCGATT-3′. Mouse MBD2, MINK1 over-expression plasmids , and negative control (OE-NC) were purchased from HonorGene . Small interfering RNA and plasmid were transfected in BECs for 48 h using Lipofectamine 3000  according to the manufacturer’s protocols. After 2 days, the cells were treated with 100 μg/ml HDM and 100 ng/ml LPS for 24 h.+ T cells were isolated by magnetic bead separation . The established asthma model and transfected BECs were cocultured with CD4+ T cells (TCs) at a ratio of 10:1 (TCs: BECs) for 24 h, respectively, in complete RPMI 1640 culture medium  supplemented with 10% FBS, 1% penicillin and streptomycin, soluble anti-CD28 , soluble anti-CD3e , and IL-2 . For analysis of T cell subsets, suspended cells were collected 24 h later, and the concentration of IL-4 and IL-17 A was determined by flow cytometry to obtain the ratio of Th2 to Th17 cells. Total protein of BECs was extracted for western blotting.Mouse spleen CD4+ T cells were stimulated with 2 μL/ml (1 × 106 cells/ml) leukocyte activation cocktail , and cultured at 37°C with 5% CO2 for 6 h, then collected for flow cytometry analysis. After a 6-h incubation, cells were stained with a marker of cell viability  for 15 min at room temperature in the dark. Then, cells were stained for surface markers with FITC-anti-CD4 antibody (Biolegend) followed by fixation and permeabilization using the Cytofix/Cytoperm Soln Kit (BD Pharmingen) for 30 min at 4°C in the dark. After washing with permeabilization buffer, cells were stained for intracellular markers with APC-anti-IL-17 A (Biolegend) and PE-anti-IL-4 (BD Pharmingen) antibodies in permeabilization buffer for 30 min at 4°C in the dark. Isotype controls were employed in the control group. Flow cytometry was performed, and data were analyzed using FACS CantoⅡ(Becton Dickinson) and FlowJo version X software.CD4After dissection, the right lung lobes were flash frozen for protein analysis. The lungs were crushed and lysed in a radio immunoprecipitation (RIPA) buffer containing 1% protease inhibitors . Proteins in cells were prepared in the same buffer. The protein concentration was measured by using the bicinchoninic acid (BCA) assay  according to the manufacturer’s instructions. A 30-μg sample of protein was transferred to the membrane after 1–1.5 h of sodium salt dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The membrane was washed with Tris-buffered saline-Tween 20 (TBST) for 5 min and sealed with 5% skim milk powder at room temperature for 1.5 h. After that, the membrane was incubated with appropriately diluted MBD2, GAPDH , and MINK1 antibodies at 4°C overnight. The membrane was incubated with the secondary antibody at room temperature for 1 h. Images were obtained using a chemiluminescence gel imaging system, and the band intensities were measured using ImageJ software . The relative protein expression level was calculated as the ratio of the gray value of target bands to that of GAPDH.β-actin was used as the internal reference. According to the cycle threshold (Ct) value of the samples, the expression volume of related genes was calculated by the 2−ΔΔCt method. Primers for target genes were generated by Sangon Biotechnology . The primer sequences were: MBD2-forward, 5′-AGTGCTGGCAAGAGCGA-3′ and MBD2-reverse, 5′-GCCGGTCCTGAAGTCAAA-3′;Total RNA from lung tissues and BECs was extracted using TRIzol regent (Invitrogen). The first-strand cDNA was synthesized with the PrimeScript RT Reverse transcriptase Reagent Kit . Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Premix Ex Taq kit  in the CFX96 Real-Time PCR Detection System (Bio-Rad). β-actin-forward, 5′-GTGCTATGTTGCTCTAGACTTCG-3′ and β-actin-reverse, 5′-ATGCCACAGGATTCCATACC-3′.MINK1-forward, 5′-CCACCTACTATGGGGCCTTTA-3′ and MINK1-reverse, 5′-AGCACCGCAGAACTCCATC-3′; The levels of IL-17 and IL-4 in mouse serum and cell supernatant were determined by enzyme-linked immunosorbent assay (ELISA) using the Mouse IL-17 ELISA Kit  and Mouse IL-4 ELISA Kit . All experiments were performed according to the manufacturers’ instructions.g for 1 min, and then the supernatant was stored at 4°C. Next, we added anti-MBD2 antibody and rabbit IgG to the remaining supernatant and incubated it overnight at 4°C with rotation to pull down the immunoprecipitation (IP) products. To collect antibody/antigen/DNA complexes, 60 μL protein G agarose was added to each IP and incubated for 1 h at 4°C by rotation. Then, 100 μL of elution buffer was added to each tube containing the antibody/agarose complex (including the input) for elution; this was incubated at room temperature for 15 min. Subsequently, crosslinking of the eluted protein/DNA complex was reversed by the addition of 5 M NaCl and incubated overnight at 65°C. Then 1 ml of binding reagent A was added, and the DNA flow-through was collected with a spin filter, separated, and purified. The isolated DNA was detected by ChIP-PCR using the following primers: F, 5′-ACGGCGGCAGCGGAGT-3′ and R, 5′-AGGTCGATGTCGTCCAGGCT-3′. The value of quantitative PCR was normalized with input DNA for comparison.Chromatin immunoprecipitation (ChIP) assay was conducted by using the ChIP assay kit  according to the manufacturer’s instructions. First, 37% formaldehyde was added to the medium to fix the cross-linking of protein and DNA. The cells were then collected and washed three times in cold PBS, centrifuged and resuspended in SDS lysis buffer. The cell lysates were then sonicated to crosslink DNA fragments between 200 and 1000 base pairs in length. To obtain input, 10 μL of supernatant was prewashed with protein G agarose and centrifuged at 4000 × p-value < 0.05 indicated a statistically significant difference.All experiments were performed at least 3 times, and all data were expressed as mean ± standard deviation (M ± SD). The differences among different groups were analyzed by one-way analysis of variance (ANOVA) or Kruskal–Wallis test followed by Dunn’s multiple comparisons test. All statistical analyses and graph generation were performed using GraphPad Prism 8.0.1 software (GraphPad Software Inc.). A To assess whether Th17-dominant asthma was established, airway resistance and BALF cells induced by Mch were measured on day 21. Compared to the normal control and T2 asthma mice, mice with Th17-dominant asthma had higher baseline RL. After Mch challenge, the RL of these mice increased significantly, especially in the mice with Th17-dominant asthma . In BALF+ T cells were detected by flow cytometry. Compared to the normal and T2 asthma groups, both Th2 and Th17 cells were increased in the Th17-dominant asthma group, especially Th17 cells, while Th2 cells were mainly increased in the T2 asthma group  to analyze the promoter region of MINK1. The prediction of CpG islands is shown in Next, to further understand the molecular mechanism of MBD2 involvement in Th17-dominant asthma, we attempted to determine whether there was any relationship between MBD2 levels and MINK1 expression in BECs exposed or not exposed to HDM + LPS. First, we confirmed by using western blot and qRT-PCR analysis that MINK1 gene silencing or overexpression in BECs with or without HDM + LPS exposure was successful. Then, we detected the expression of MBD2 in each group. There was no significant difference in MBD2 mRNA and protein expression when the MINK1 gene was silenced or overexpressed . We then+ T cells. Then the differentiation of Th17 cells was analyzed by flow cytometry, and the level of IL-17 was detected by ELISA. The results showed that Th17 cells and the cytokine IL-17 expressed the same trend when MBD2 was silenced or overexpressed. However, when MINK1 was silenced or overexpressed, Th17 cell differentiation and IL-17 showed the opposite trend, increasing or decreasing, respectively. Together, these data indicated that MBD2 induced Th17 cell differentiation by maintaining MINK1 silencing, thereby contributing to the development of Th17-dominant asthma  patients were positively correlated with the SLE disease activity index  inflammasome plays a pathogenic role in inflammatory diseases, and MINK1 positively regulates NLRP3 inflammasome. In the mouse model of acute sepsis, MINK1 deficiency reduces the NLRP3 activation and inhibits the inflammatory response . MINK1 cIn conclusion, the data from this study demonstrated that MBD2-mediated Th17 cell differentiation is associated with reduced MINK1 expression in Th17-dominant asthma. Our findings have revealed new roles for MBD2 and MINK1 and provide new insights into epigenetic regulation of Th17-dominant asthma, which is dominated by neutrophils and Th17 cells. This study could lead to new therapeutic targets for patients with severe asthma.


Process 2 results in converting high-density quiescent cells into low-density quiescent cells. Processes 3 and 4 cause a fast or slow decline in the quiescence of low- or high-density quiescent cells, respectively. Here, we tested our hypothesis by assessing how four different geroprotectors influence the four processes that could link cellular aging to cellular quiescence. We found that these geroprotectors differently affect processes 1 and 2 and decelerate processes 3 and 4. We also found that a rise in trehalose within quiescent yeast contributes to chronological aging and quiescence maintenance. These data collectively provide conclusive evidence for a mechanistic link between cellular aging and cellular quiescence.We propose a hypothesis of a mechanism linking cellular aging to cellular quiescence in chronologically aging budding yeast. Our hypothesis posits that this mechanism integrates four different processes, all of which are initiated after yeast cells cultured in a medium initially containing glucose consume it. Quiescent cells that develop in these cultures can be separated into the high- and low-density sub-populations of different buoyant densities. Process 1 of the proposed mechanism consists of a cell-cycle arrest in the G A. A110]. The present study had the following two objectives aimed at testing our hypothesis.tor1Δ and ras2Δ on these processes. Each of these geroprotectors delays chronological aging and extends the longevity of S. cerevisiae . T. T109]. 1 phase . In yeas Q cells . In yeastor1Δ and ras2Δ. Each of them was assessed in yeast cultured under non-CR conditions on 2% glucose. Of note, all these interventions exhibit strong geroprotective effects under non-CR conditions of culturing , 1 mM ATP , 0.2 mM NADP+ , and mixture of hexokinase  and glucose-6-phosphate dehydrogenase  were added, and the mixture was incubated for 30 min at 25°C. The NADPH generated from NADP+ was measured fluorimetrically .To measure glycogen concentration, 50 μl of alkali extract were added to 500 μl of glycogen reagent (50 mM sodium acetate (pH 4.6) and 0.02% bovine serum albumin (BSA)  with and without of 10 μg/ml amyloglucosidase 6 U/mg ). The mixture was incubated for 30 min at 25°C. 500 μl of glucose reagent (100 mM Tris/HCl (pH 8.1), 2 mM MgCl2PO4/KOH (pH 7.5) and 0.02% BSA  with or without 15 mU trehalase ). The mixture was incubated for 60 min at 37°C. 800 μl of glucose reagent (100 mM Tris/HCl (pH 8.1), 2 mM MgCl2, 1 mM DTT , 1 mM ATP , 0.2 mM NADP+ , and mixture of hexokinase  and glucose-6-phosphate dehydrogenase (8 U) ) were added, and the mixture was incubated for 30 min at 25°C. The NADPH generated from NADP+ was measured fluorimetrically .To measure trehalose concentration, 50 μl of alkali extract were added to 150 μl of trehalose reagent  Analysis ToolPak - VBA. All data are presented as mean ± SEM. The


Stevia rebaudiana L. (SRLe) extract to in situ biosynthesize nanoscale alpha hematite (α-Fe2O3) nanoparticles (NPs) with potent antioxidant, antimicrobial, and anticancer properties. SRLe-α-Fe2O3 was characterized using physiochemical analyses, including UV/Vis, FTIR, XRD, DLS, EDX, SEM, and TEM studies. Among tested solvents, CHCl3/MeOH (2:1 v/v) SRL extract (least polar solvent) contained the highest EY, TPC, and antioxidant capacity of ~3.5%, ~75 mg GAE/g extract, and IC50 = 9.87 ± 0.7 mg/mL, respectively. FTIR confirmed the engagement of coating operation to the colloidal α-Fe2O3 NPs. TEM, SEM, and DLS revealed that SRLe-α-Fe2O3 has a spherical shape, uniform size distribution with aggregation for an average size of ~18.34 nm, and ζ = −19.4 mV, forming a repulsive barrier that helped to improve stability. The synthesized nanoparticles displayed considerable antibacterial activity against E. coli and S. aureus bacterial growth, and exhibited superior activity against the A549 lung cancer cell lines. These findings indicate that the increased availability of bioactive substances with antioxidant properties of SRLe makes it a potentially interesting material for the preparation of biologically active compounds and green synthesis of nanoparticles.In the present study, we utilized  Stevia rebaudiana L. (S. rebaudiana) is a perennial herb [Stevia leaves, indicating the importance of the nutritive structure of S. rebaudiana  and its application as an antimicrobial and anticancer agent. Hence, this study aims to investigate the size-controlled green synthesis process using SRLe and the surface functionalization synthesis of nanoscale alpha hematite (α-Fe2O3) NPs via in situ oxidation–precipitation methods, along with the prediction of biological activities, antimicrobial activities, and anticancer. Furthermore, this is the ever first report to elucidate the phytochemicals in S. rebaudiana plant extract and to synthesize S. rebaudiana leaf extract-mediated α-Fe2O3 NPs with provden antibacterial and anticancer efficacy. A schematic representation of the present study is clearly depicted in Furthermore, the antioxidant activity of either single or bimetallic combination synthesized nanocomposites via chemical or green techniques utilizing various phytochemicals (leaf extracts) was also evaluated . NanoparSRLe parts . The results are shown in 3/MeOH (2:1 v/v) leaf extract (least polar solvent) contained the highest EY and TPC of ~3.5% and ~75 mg GAE/g extract. AcOH root extract (most polar solvent) contained the lowest EY and TPC of ~2.2% and ~55.6 mg GAE/g extract. EtOAc stem extract exhibited an EY and TPC of ~2.7% and ~64 mg GAE/g extract, respectively. These outcomes are consistent with previously reported results by Kim et al. [Stevia extract confirmed that the chemical polarity properties of the solvent influenced the qualitative structure and physicochemical activity of the extracts, as confirmed in a previous report by Bęben et al. [It is well known that the EY of bioactive chemical compounds relies on various factors, including the types of solvents with varying polarities, pH, time, and extraction temperature, in addition to the chemical installation of the basic samples. Under the same time and temperature conditions, the solvent and the chemical properties of the sample are the two most important factors . In thism et al. . Howevern et al. .SRLe were indexed according to the DPPH radical-scavenging activity (3/MeOH (2:1 v/v) leaf extract had significantly higher radical-scavenging activity with an IC50 value of 12.87 ± 0.7mg/mL as compared to the EtOAc leaf extract, while AcOH root extract exhibited significantly lower radical-scavenging activity with IC50 values of 20.07 ± 1.2 mg/mL and 36.54 ± 1.1 mg/mL, respectively, compared to the control . This behavior is similar to that reported by Criado et al. [S. rebaudiana extracts at various doses. S. rebaudiana extract’s radical-scavenging efficacy improved with increasing dose (25–625 mg/mL). The IC50 value was determined to be 335.94 g/mL. These findings suggest that the CHCl3/MeOH (2:1 v/v) extract of S. rebaudiana is a potentially strong antioxidant agent for the improvement of additional drugs. According to Kähkönen et al., S. rebaudiana extract may have higher antioxidant activities since it has a larger TPC [Extraction solvents affect the EY and the TPC, thus significantly affecting the biological activity of the extract . The antactivity C. The ino et al. , Furthero et al.  investigrger TPC .3/MeOH (2:1 v/v) of SRLe had a slightly higher phytochemical content accumulation than stems and roots. The peaks in the HPLC chromatogram of CHCl3/MeOH (2:1 v/v) leaf extract were identified by comparing the retention time and UV spectra of bioactive phenolic compounds in the sample with standards within 10 min. The HPLC results of bioactive phenolic content are listed in 3/MeOH (2:1 v/v) extract according to HPLC chromatograms: ferulic acid, syringic acid, protocatechuic, catechin, coumaric acid, caffeic acid, gallic acid, and chlorogenic acid. As indicated in S. rebaudiana (Bertoni) [HPLC currently represents the most popular and reliable technique for the analysis of phenolic compounds. Various supports and mobile phases are available for the analysis of phenolics including anthocyanin, proanthocyanins, hydrolysable tannins, flavonols, flavan-3-ols, flavanones, flavones, and phenolic acids in different plant extracts and food samples . Under tBertoni) . Many biBertoni) ,30, presBertoni) , catechiBertoni) .2O3. The mean values recorded for all the systems showed a PS distribution in nanometers as shown in SRLe-αFe2O3 was about ~19.60 ± 3.8 nm, while the PDI was 0.237  was used to measure the PS, PDI, and ζ-potential of bare SRLe-αFeas 0.237 A. Furthee-αFe2O3 B. A ζ-poaline pH , indicataline pH . The hig−1, C–H stretching in the frequency range 2800–3000 cm−1, C=O stretching at the frequency of 1628 cm−1, and C–O stretching in the frequency range of 1050–1400 cm−1. The presence of the –OH band and C=O stretching suggests that the constituents in the extract had –OH and C=O groups as functional groups. Phenolic and flavonoid compounds are compounds containing hydroxyl and carbonyl groups as functional groups [−1, indicating the presence of polyphenols such as terpenoids and flavonoids, which may also operate as bio-reducing agents. As a result, proteins can serve as both stabilizing and reducing agents. Fe–O nanoparticles were responsible for the peak at 770 cm−1. These functional groups were observed on the surface of SRLe-αFe2O3 NPs produced from leaf extracts of stevia plants. These results are in line with previous studies on green nanoparticle synthesis [The FTIR showed a different vibration according to functional groups with a characteristic absorption in the IR region. FTIR spectroscopy is useful for detecting the characteristic peaks and functional groups of compounds. The FTIR spectrum of the extract is shown in l groups . These aynthesis .SRLe-αFe2O3 NPs  [The coordination complex was validated as the absorption peak was identified at 390 nm using the UV/visible spectral analysis of the synthesized e2O3 NPs A. Our re. (2022) .SRLe-αFe2O3 NPs was determined via powder XRD analysis. SRLe-αFe2O3 NPs with a number of distinctive peaks (2θ) at diffraction angles of 29.42°, 34.04°, 43.95°, 55.12°, 57.46°, and 64.83°, corresponding to indices (210), (104), (113), (024), (112), and (541) on JCPDS card numbers 33-0664 and 341266, matching nicely with the crystal structure of α-Fe2O3 NPs [The degree of crystallinity of the synthesized e2O3 NPs ,41.SRLe-αFe2O3 was analyzed using TEM  and iron (65.56%) were both detected through EDX analysis, as shown in 2O3 [Using energy0dispersive X-ray spectroscopy (EDX), the elemental mapping of the biogenic xygen 34.% and iroE. coli and S. aureus using the well diffusion method. The ability of the antibacterial agent (NPs) is shown in S. aureus, while the minimum effect was noted for E. coli in a dose-dependent manner. For S. aureus, the zones of inhibition were 13 ± 0.4 mm and 18 ± 0.5 mm for the 10 mg/mL and 15 mg/mL doses, respectively, which almost doubled to an inhibition zone of 21.5 ± 0.8 mm for 30 mg/mL. No inhibition was detected at the 5 mg/mL dose. It has been reported that iron nanoparticles possess antibacterial properties due to their nanoscale size, which enables them to accumulate and deposit on the surface of bacteria during testing [2O3) NPs can be explained by several possible mechanisms such as stability in an ambient environment, generation of ROS , hydroxyl radicals (OH−), etc.), oxidative stress, and release of ions by nanoparticles reacting with the bacteria’s thiol groups (–SH), which can alter the cellular structure of microorganisms, thus interrupting DNA reproduction and inhibiting enzyme and protein synthesis [E. coli strains with inhibition zones of approximately 11 ± 1.3 mm and 16.5 ± 1.4 mm, respectively, at doses of 15 mg/mL and 30 mg/mL. At the same time, no inhibitions were detected at doses of 5 mg/mL and 10 mg/mL. These results were significantly similar to those of Bhuiyan et al. (2020), who reported no antibacterial efficiency for α-Fe2O3 synthesized using a leaf extract of Carica papaya against E. coli strains at a concentration of 5 (mg/disc) [Green synthesized nanoparticle suspensions of various concentrations were tested for antibacterial activity against  testing ,46. Addi testing . The actynthesis ,49. Furtmg/disc) . A negatmg/disc) . As a reSRLe-αFe2O3 nanoparticles at 31.25 to 250 μg/mL was observed against A549 cells and Vero-derived kidney epithelia isolated from green monkeys; the results are shown in SRLe-αFe2O3NPs synthesized from the green species, 100% of the cell lines survived the damage. The treatment of A549 lung cancer cell lines with SRLe-αFe2O3 NPs inhibited the proliferation of cell lines in a time-dependent and dose-dependent manner. It was observed that the morphology was not effectively destroyed when SRLe-αFe2O3 NPs were used at low concentrations, with a complete loss of cells occurring at higher concentrations [50 = 51.2 μg/mL) of the cells surviving after 48 h. Approximately similar results of synthesized αFe2O3 nanoparticles using L-ascorbic acid as a reducing agent were reported by Kumar et al. (2022), with an IC50 ≤30 µg/mL [SRLe-αFe2O3 NPs, leading to excessive production of ROS-mediated oxidative stress in the cell and, thus, DNA damage, as presented in previous investigations [50 = 117.5 μg/mL) of the Vero cells survived when given the same SRLe-αFe2O3 NPs up to a dose of 250 μg/mL. These results are similar to those of Bhuiyan et al. [Stevia rebaudiana L. to synthesize SRLe-αFe2O3 NPs is an ecofriendly and high-efficiency approach.The cell viability and cytotoxic effect of different concentrations of trations . Neverth30 µg/mL . These on et al. . Because2O3 from Stevia leaf extract can exhibit synergism and disruptive effects on biofilm-based infection as a result of its effective antibacterial activity against established in this study. In addition, we recommend undertaking in vivo studies to demonstrate the efficacy of our designed formulations.The present study had some limitations that need to be addressed in the future. Firstly, the in vitro effect of the designed formulations needs to be evaluated on pre-established biofilms. Secondly, a lack of funding precluded the completion of an antibiotic resistance experiment on both bacteria tested. Future investigations will reveal whether the biosynthesized alpha hematite FeS. rebaudiana was obtained from Sugar Crops Research Institute . Methanol (MeOH), ethyl acetate (EtOAc), chloroform (CHCl3), and acetone (AcOH) solvents were purchased from Merck . FeCl3·6H2O and FeCl2·4H2O were obtained from Aladdin Chemical Reagent Company . Folin–Ciocâlteu reagent, 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radicals, and all HPLC standards were purchased from Sigma–Aldrich (Germany). E. coli and S. aureus were isolated and identified at the Microbiology Unit in the National Cancer Institute Faculty of Medicine Cairo University, Cairo, Egypt.S. rebaudiana materials were washed several times using ddH2O, and all parts  were dried in the open air at room temperature. Lastly, a powder was obtained by grinding all parts mechanically before storing in polyethylene bags in a freezer at −4 °C until use.The 3/MeOH (2:1 v/v) in conical flasks for 72 h at room temperature with shaking . Then, all samples were filtered through Whitman filter paper No 42 and concentrated under reduced pressure using a rotary evaporator in vacuo at 45 °C. Finally, the extracts were preserved in sterilized airtight, labeled bottles in a refrigerator at 4 °C until required for analysis.TPCs were extracted following the method of Anokwuru et al.  with somThe extraction yield (EY) was calculated as using the following formula:S. rebaudiana extracts was defined using the Folin–Ciocâlteu assay [2CO3 (2% w/v), 0.1 mL of CuSO4, and 0.1 mL of sodium and potassium tartrate, before mixing. Then, 0.4 mL of NaOH (0.5 M) was added after 4 min to the mixture, while 0.2 mL of Folin-Ciocâlteu reagent (1:1 v/v) was added after 10 min. Next, the solution was left for 30 min, and its absorbance was estimated using a UV/Vis spectrophotometer at 750 nm. The TPC was calculated as mM GAE using a GA standard curve.The TPC of the eu assay . In brieStevia extract was mixed with 3.6 mL of a methanol solution of DPPH (0.1 mM). An equal amount of 0.4 mL of methanol (0.004% w/v) was used as a blank with 3 mL of DPPH solution. All samples were evaluated in triplicate, vortexed for 3 min, and incubated in the dark for 35 min at 37 °C. The reduction in absorbance of each sample was measured against methanol as a blank on a UV spectrophotometer  at 515 nm, and the data were recorded every minute for 16 min. The PI of the DPPH antioxidant activity was calculated using the following formula:50 value, with a lower value representing stronger DPPH scavenging capacity. An ascorbic acid standard curve was used as a positive control.The 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical-scavenging assay was carried out according to a previously reported method . Briefly3/MeOH (2:1 v/v) extract.HPLC was carried out using a GBC 1100 Series HPLC system equipped with a UV detector . BioactiSRLe-αFe2O3 NPs were synthesized using ferric chloride (FeCl3·6H2O), as described in [SRLe-αFe2O3. Then, 1 M of NaoH was added until attaining pH 11. The solution eventually turned cloudy black. Subsequently, the solution was centrifuged at 12,000 rpm for 10 min and washed with dH2O to remove any impurities or absorbed ions. Finally, the product was dried at 60–70 °C for 48 h using a fan-assisted oven . For size estimation, 3 mL of bare SRLe-αFe2O3 NPs samples were imaged using transmission electron microscopy . Thin SRLe-αFe2O3 NPs films were created on a carbon-coated copper grid by simply dropping a small quantity of sample on the grid, before blotting away any excess solution using blotting paper [®, JEOL DATUM Ltd., Tokyo, Japan). A drop of SRLe-αFe2O3 NPs was dried onto an aluminum grid under a mercury lamp for 5 min to obtain a coating thickness of 400 Å.The ng paper . The optThe UV/Vis spectra of diluted samples were measured using a Varian Cary-100 Konc spectrophotometer  at 230 V/50 Hz, in the wavelength range of 300–600 nm .SRLe-αFe2O3 NPs were determined using a Rigaku D/Max-lllC X-ray diffractometer  at a voltage of 40 kV and a current of 40 kA. The patterns were recorded as a function of the 2θ angle in the range of 10°−80° with a step size of 0.01° at a scanning rate of 0.02 steps/s with the help of a monochromatized X-ray beam with a copper filter .XRD patterns of the as-synthesized SRLe-αFe2O3 NPs were characterized using an FTIR spectrometer  to inspect the functional groups contained in the prepared samples. Potassium bromide (KBr) was mixed with the prepared samples. A disc was loaded at high pressure and measured at a wavelength of 400–4000 cm−1 with a resolution of 4.0 cm−1.The optical properties of SRLe-αFe2O3 NPs were added to each pit at successive concentrations , before incubating for 24 h at 37 °C. After incubation, zones of growth inhibition were measured to the nearest millimeter to determine the antimicrobial potency of the screened antimicrobial substances [The well diffusion method was performed in triplicate according to the method in . Brieflybstances . The resSRLe-αFe2O3 NPs (autoclaved). The cells were dissolved in 200 µL of DMSO (dimethyl sulfoxide). The absorbance spectra of the specimens were distinguished by recording the optical density at 560 nm and subtracting the background at 620 nm using a microplate reader [SRLe-αFe2O3 NP sample, control denotes the control sample, and blank denotes the wells without Vero cells and A549 cells [The cytotoxic assessment of A459 cells derived from lung cancer cells (ATCC CCL-185) and Vero cells isolated from kidney epithelia extracted from African green monkeys (ATCC CCL-81) were evaluated using the MTT assay (5 mg/mL in PBS). The medium from the wells was evacuated after incubation. Then, MTT (20 µL) was incorporated into each well along with 25 μL of e reader .(3)% Ce49 cells .p < 0.05). All analysis was conducted using SAS 9.4 for Windows x64 from the SAS Institute , and graphical outputs were generated using GraphPad Prism software .Each experiment was carried out at least in triplicate, and all data were presented as the mean ± SD. Analysis of statistical significance was performed using one-way ANOVA and the post-hoc Tukey test  (least polar solvent) contained the highest EY and TPC of ~3.5% and ~75 mg GAE/g extract, respectively, along with an antioxidant radical-scavenging activity of IC50 = 12.87 ± 0.7 mg/mL. These phenolic compounds played an important role in increasing the stability of SRLe-αFe2O3 NPs. The antibacterial efficacy of synthesized NPs toward isolated Gram-negative and Gram-positive bacteria was moderate. Although nanoparticles were toxic at high concentrations, they demonstrated remarkable effectiveness  against the A549 lung cancer cell line, indicating that they might be a viable choice for eradicating tumor cells at optimal doses. However, further research is needed to discover the exact doses and reaction conditions needed to employ nanoparticles for these purposes.This study focused on the green synthesis of alpha hematite αFe


Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic β-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.The rising antimicrobial resistance is particularly alarming for  Acinetobacter baumannii is a notorious opportunistic pathogen responsible for up to 10% of the nosocomial infections caused by Gram-negative pathogens, including wound, blood, and urinary tract infections, and ventilator-associated pneumonia . This highlights the role of serum complement to kill DpoMK34-treated A. baumannii MK34.CPS serves as a virulence factor that helps the pathogen to escape from phagocytosis and complement-mediated killing. Hence, removal of the bacterial capsule of strain MK34 may sensitize bacteria to immune components of human serum. In this regard, we investigated the effect of different serum concentrations on DpoMK34-treated 34 cells . PretreaEmergence of phage resistant mutants is a common observation upon phage exposure, and we made a similar observation upon incubation of phage PMK34 with its host strain . SpottinA. baumannii strain MK34  at 37 °C while shaking (250 rpm), or on LB agar plates (LB supplemented with 2% agar). Kanamycin (50 µg/mL), chloramphenicol (25 µg/mL), and sucrose 5% (w/v) were added to LB broth or agar for selection of the transformed E. coli cells. Acinetobacter phage vB_AbaP_PMK34 (phage PMK34) was previously isolated, characterized, and sequenced. A double layer assay  as described before  and then spread over the surface of plain LB agar [2% (w/v)]. After solidification, 10 µL of a two-fold serial dilution of DpoMK34  was spotted on a bacterial lawn, left for air drying and incubated for 16 h at 35 °C. A positive result was visible as a halo zone of reduced turbidity. PBS pH 7.4 (10 µL) was spotted as a control. The minimum halo-forming concentration (MHFC) was defined as the lowest concentration that causes a zone of reduced turbidity visible with the naked eye. Bacterial cell counts for zones spotted with 100 µg/mL and 6.25 µg/mL of purified DpoMK34 were performed by suspending excised agar blocks (4 × 4 mm) from the treated surfaces, or PBS as control, in 2 mL of PBS pH 7.4 followed by vigorous vortexing for 1 min. Afterwards, the bacterial suspensions were serially diluted in the same buffer and plated for counting.The enzymatic activity of DpoMK34 was analyzed using a spot-on-lawn assay as described before . Hundred3PO4, 0.04 M H3BO3, 0.04 M CH3COOH, and 0.15 M NaCl) adjusted to the pH test range (4–11). After 30 min incubation, dilutions were prepared using the same universal buffer with the respective pH and spotted on a bacterial lawn. A drop (10 µL) of the universal buffer with the respective pH was used as control.Thermal stability was assessed by incubating 2.64 µM (200 µg/mL) DpoMK34 at different temperatures  using a water bath for 30 min, followed by cooling down on ice during 10 min. A two-fold dilution series of the incubated protein solutions were then spotted as described above. Similarly, pH stability was evaluated by dialyzing stock solution of DpoMK34 (2.64 µM) against Britton Robinson universal buffer  A. baumannii MK34 cells were resuspended in either PBS pH 7.4 or DpoMK34 . After two hours incubation at 35 °C, cells were pelleted  and washed twice using LB broth. The cells were then diluted to 106 CFU/mL with LB broth and incubated with phage PMK34 at multiplicity of infection (MOI) of 0.01 and incubated at 35 °C. At specific time intervals  100 µL samples were added to 900 µL cold LB and spun down . The number of non-adsorbed phage particles was counted using a double layer assay . These mixtures were then incubated at 37 °C for additional 2 h, serially diluted in 100% human serum and plated on LB agar plates for bacterial cell counting. PBS pH 7.4 treated A. baumannii MK34 cells were included as control. In parallel, a similar experiment was conducted using heat-inactivated serum (56 °C for 30 min).The ability of DpoMK34 to render serum-resistant lsewhere . Briefly8 PFU/mL) to assess their sensitivity. In addition, the possibility of resistance development after repeated exposures to DpoMK34 and phage PMK34 was investigated as described previously [A. baumannii MK34 cells picked up with a sterile loop from a zone of reduced turbidity in soft agar spotted with 10 µL of 1.32 µM DpoMK34 equivalent to 100 µg/mL). Subsequently, this overnight culture was 100-fold diluted in LB supplemented with either DpoMK34  or PBS and incubated for 24 h at 35 °C. Afterwards, the cells were spun down , washed twice with LB and sub-cultured two more times in LB with either DpoMK34 or PBS for 24 h at 35 °C. After the three rounds of exposure, the treated cells were spun down , washed twice and allowed to grow in LB till reaching OD655nm = 0.6. Subsequently, the sensitivity of the cells to DpoMK34 and phage PMK34 was evaluated using the same spot-on-lawn assay as described above.Three phage PMK34 resistant mutants (colonies growing within plaques) were subcultured in LB for 24 h at 35 °C then dropped with 10 µL of 1.32 µM DpoMK34 or phage PMK34  Unless otherwise stated, statistical significance was considered when p < 0.05.Statistical significance between means of different groups was assessed by two-tailed Student’s