Plasmodium falciparum has a complex life cycle during which it needs to differentiate into multiple morphologically distinct life forms. A key process for transmission of the disease is the development of male and female gametocytes in the human blood, yet the mechanisms determining sexual dimorphism in these haploid, genetically identical sexual precursor cells remain largely unknown. To understand the epigenetic program underlying the differentiation of male and female gametocytes, we separated the two sexual forms by flow cytometry and performed RNAseq as well as comprehensive ChIPseq profiling of several histone variants and modifications.The protozoan malaria parasite We show that in female gametocytes the chromatin landscape is globally remodelled with respect to genome-wide patterns and combinatorial usage of histone variants and histone modifications. We identified sex specific differences in heterochromatin distribution, implicating exported proteins and ncRNAs in sex determination. Specifically in female gametocytes, the histone variants H2A.Z/H2B.Z were highly enriched in H3K9me3-associated heterochromatin. H3K27ac occupancy correlated with stage-specific gene expression, but in contrast to asexual parasites this was unlinked to H3K4me3 co-occupancy at promoters in female gametocytes.P. falciparum.Collectively, we defined novel combinatorial chromatin states differentially organising the genome in gametocytes and asexual parasites and unravelled fundamental, sex-specific differences in the epigenetic code. Our chromatin maps represent an important resource for future understanding of the mechanisms driving sexual differentiation in The online version contains supplementary material available at 10.1186/s12915-023-01568-4. Plasmodium parasites, the infectious agents causing malaria, have a complex life cycle, in which they undergo asexual replication in the human host and sexual replication in the Anopheles mosquito. For successful transmission from the human to the vector, the parasites need to differentiate into male and female gametocytes in a process referred to as gametocytogenesis. During this process that takes around 10 to 12 days in Plasmodium falciparum, the parasites sequester in the bone marrow [Malaria poses a global health burden with more than 400,000 deaths each year, even though progress towards the elimination of malaria has been made in the past decades . The Plae marrow  and drame marrow . Upon mae marrow . On the e marrow . Therefoe marrow .P. berghei and the human malaria parasite P. falciparum showed that the majority of gametocyte specific proteins are found pre-dominantly either in male or in female gametocytes, reflecting the fundamentally different biological functions of the two sexes [Male and female gametocytes can be morphologically distinguished from each other around day seven of development, when they are classified as stage IV gametocytes. Female gametocytes are characterised by condensed hemozoin, an accumulation of osmiophilic bodies which are involved in gamete emergence, a high density of ribosomes and a relatively small nucleus with a nucleolus, consistent with their need to engage in protein synthesis important for the emerging macrogamete and zygote . mRNAs cwo sexes , 12.P. falciparum is regulated. It has been shown that epigenetic regulation plays a critical role for commitment to gametocytogenesis by de-repression of the transcription factor AP2-G, which is silenced by H3K9me3 and its associated factor heterochromatin protein 1 (HP1) in asexual parasites [Little is known about how the sexual dimorphism in arasites –16. Furtarasites –21. Howevar gene family encoding the major virulence factor PfEMP1 [var gene silencing [Plasmodium parasites H2A.Z occupies intergenic regions across the euchromatic genome irrespective of their activity, however H2A.Z enrichment levels positively correlate with transcription levels suggesting it may define promoter strength [var genes, as it is deposited in the promoter of the transcribed var gene together with the histone variant H2B.Z [P. falciparum co-occurs in the same nucleosomes with H2A.Z throughout the genome [P. falciparum [Plasmodium [P. falciparum [T. brucei, acetylated H2A.Z is enriched at TSS nucleosomes of polycistronic transcription units where its presence correlates with gene expression, possibly by being involved in RNA PolII recruitment [In asexual parasites, the chromatin is principally divided into the transcriptionally permissive euchromatin and the transcriptionally silent heterochromatin. The latter is marked by H3K9me3 and its associated protein HP1 and restricted to subtelomeric regions and internal islands, where it is involved in the repression of virulence-associated multigene families, such as genes of the r PfEMP1 , and othr PfEMP1 –28. Thisr PfEMP1 . Anotherilencing .Converseilencing  and is iilencing . In yeasilencing . In asexstrength –36. H2A.nt H2B.Z , 37. Thent H2B.Z  and in Pe genome . The hise genome , 40, 41.e genome . H3K4me3lciparum . In addilciparum . Acetylaasmodium , also poasmodium  and is blciparum . In the ruitment .P. falciparum gametocytes characterised by H3K9me3 and HP1 indicated that several gametocyte specific genes as well as the erythrocyte remodelling machinery are differentially marked in gametocytes, implicating these processes in gametocyte maturation [P. berghei investigated the differences of HP1 and H3K9ac coverage separately in mature male and female gametocytes, revealing differences in their heterochromatin and euchromatin landscape pointing to a complex epigenetic regulation underlying the different gene expression patterns observed [P. falciparum, no data is available on how the sex-specific transcriptomes change over gametocyte differentiation and how the chromatin landscape organises the genome to regulate sex-specific gene expression patterns.Few studies have addressed how chromatin structure impacts gene regulation during sexual development. It has been demonstrated using bulk gametocyte cultures that the transcriptome changes dynamically during the 10 day gametocyte maturation process , 47, 48 turation , 50. Morturation . Importaturation , 52 and turation , 54. Howobserved . In P. fP. falciparum gametocytes during their maturation and to investigate the underlying epigenetic landscape. To this end we profiled several histone modifications using chromatin immunoprecipitation followed by high-throughput sequencing (ChIPseq) in asexual parasites, female gametocytes, and for some modifications also in male gametocytes, and correlated these with the transcriptional data. Finally, we also analysed the combinatorial patterns of these modifications in female gametocytes and ring stage parasites, generating comprehensive chromatin state maps for the two parasite stages.To close some of the gaps in our knowledge of sexual differentiation in malaria parasites, we set out to study the transcriptome of separated male and female ABCG2 gene was used [R2 = 0.83; D6 vs D10 R2 = 0.84; D4 vs D10 R2 = 0.64) than the female gametocytes , suggesting that the male gametocytes transcriptome is more dynamic during sexual maturation  and later (day 6 and day 10) female gametocytes, respectively. The 969 genes (290 (30%) of unknown function) with peak expression in early female gametocytes (Cluster 3) contained mostly genes related to “mitochondrion”, which is maternally inherited during fertilisation , and “TCarasites , 66. Thietocytes .In the male gametocytes, three clusters could be clearly distinguished (Cluster 5–7). Early male gametocytes (day 4 – stage II/III) showed highest expression of 579 genes (118 (20%) of unknown function). The predominant processes were related to proteolysis and proteasome complex, consistent with the critical function of proteostasis during metazoan spermatogenesis , 68. In It is known that the transcriptome of mature gametocytes is fundamentally different from asexual parasites , 5, 12 aPlasmodium [Toxoplasma gondii, TgCARM1 mediated H3R17me2 is implicated in parasite differentiation and is located in promoters of stage specifically expressed genes [Tri-methylation of histone 3 lysine 4 (H3K4me3) is well-studied in asexual parasites, and is typically associated with promoters , 35. Di-asmodium  but has ed genes .R2 = 0.93 and R2 = 0.97 in female day 6 gametocytes and ring stage parasites, respectively), suggesting that H3K4me3 and H3R17me2 globally co-occur , indicating global remodelling of the H3K4me3/H3R17me2 modification landscape in sexual stages , which has upstream peaks in ring stage parasites as well as female day 6 gametocytes and is expressed in both stages . Peaks that would intersect both features were called “overlapping” peaks. In asexual parasites, most peaks were found to be overlapping , or located in intergenic regions , with a smaller proportion localising in coding regions . In contrast in gametocytes the majority of peaks were intragenic  or overlapping , with only 1.2% accumulating in intergenic regions  , Pf3D7_1478000 (gexp17)) [geco did not correlate with H3K9me3 patterns, as highest transcription was observed in ring stage parasites, followed by male and then female gametocytes. As geco transcription was reported to peak in rings and early gametocytes [geco expression later during the life cycle in salivary gland sporozoites [Generally, H3K9me3 was highly conserved at telomeres and subtelomeres and over intrachromosomal islands covering variant surface antigen genes as described previously , 50, 55.ly) Fig. A. Howevely) Fig. B, especily) Fig. C. Intrigly) Fig. B. This wgexp17)) , which hgexp17)) , 50. Of Fig. Pfg1-748, Pf3ap2-g gene is sufficient for its activation [Consistent with a function during commitment but not during gametocyte maturation, the gene coding for the sex-specific transcription factor AP2-G was covered with H3K9me3 in all stages. However, in gametocytes only the gene body was heterochromatic, whereas in ring stage parasites H3K9me3 stretched over the upstream region as well  Fig. A. To do To interrogate how global chromatin indexing differs between ring stage parasites and female gametocytes, the bins in the female day 6 gametocyte UMAP were coloured according to their corresponding state in ring stage parasites Fig. B. The revar gene introns [To better understand the epigenetic code in asexual and sexual parasites, we analysed the distribution of the chromatin states in relation to different genetic elements including exons, introns, and intergenic regions Fig. C. To cap introns , 87, 88.p-value < 0.01)  of at least 300 bp in length (representing one to two nucleosomes) within 2 kb upstream of their ATG gene start  in their promoter in gametocytes or ring stage parasites showed significantly higher expression in gametocytes than in ring stage parasites, which may indicate the presence of an activating mark that was not studied in our set of histone modifications, or lower nucleosomes occupancy, which was previously linked to high transcriptional activity in gametocytes , 90. GenState 7 (220 genes) and state 8 (78 genes) only existed in gametocytes and were predominantly characterized by H2A.Z, either unmodified (state 8) or featuring low levels of acetylation (state 7). Interestingly, these states were also enriched upstream of many genes related to parasite invasion and associated with significantly lower expression levels in female gametocytes than in ring and schizont parasites  was prevalent upstream of 778 genes including genes associated with cell cycle, IMC, and parasite invasion. Consistent with their function in later developmental stages, these genes showed significantly lower expression in ring stage parasites compared to schizonts and gametocytes , showed a broad range of expression. Interestingly, the most highly expressed genes in this group were associated with protein export and include the major genes coding for the nd Pf113 –96. Of nIn conclusion, we identified chromatin states that are stage specifically enriched in genes linked to particular biological functions and may act as the anchor for recruiting specific chromatin remodelling complexes which fine tune the expression of these genes. In particular we found that H3K27ac occupancy was highly correlated with stage specific gene transcription. In gametocytes, the presence of unacetylated H2A.Z seems to be associated with transcriptional repression.In this study, we reveal fundamental differences in the transcriptional regulation and epigenetic code of developing male and female gametocytes in comparison to asexual parasites. When analysing genes with peak expression in each stage  in its C-terminal tail, which suggests it may also fulfil an architectural role in heterochromatin formation and maintenance. The heterochromatin association of H2A.Z could be facilitated by post-translational modifications that would distinguish heterochromatin associated H2A.Z from euchromatin associated H2A.Z, and indeed we observed that H2A.Z was less acetylated in heterochromatic than in euchromatic nucleosomes. In differentiated mouse cells, H2A.Z is not associated with heterochromatin, however its monoubiquitinated form is enriched on the silenced X-chromosome, which is also marked with the repressive mark H3K27me3 [P. falciparum [In terminus . InteresH3K27me3 . Ubiquitlciparum , but itsT. brucei, H2A.Zac localizes to nucleosomes around the TSS and may be involved in recruiting RNA-Pol II to activate transcription [P. berghei H3K9ac correlated with transcription in asexual parasites as well as male gametocytes and ookinetes but not female gametocytes [P. falciparum sporozoites but not in oocysts [Of note, acetylated H2A.Z co-occurs with H3K4me3 at active promoters and with H3K27ac at enhancers, while being depleted from bivalent, silent enhancers in embryonic stem cells . In T. bcription . This isetocytes . Convers oocysts . These o oocysts , and ali oocysts . UltimatP. falciparum and reveal fundamental differences not only between sexual and asexual parasites, but in particular also between male and female gametocytes. Clearly, chromatin modifications regulating transcription in gametocytes extend beyond the functionally known combinatorial patterns of histone modifications, and we need to take other, recently identified posttranslational modifications into consideration to better understand the processes regulating sexual differentiation. Asexual and sexual parasites differ significantly in their cellular metabolism, and recent publications increasingly demonstrate how changes in metabolites can govern differential gene expression programs by causing alternative chromatin modifications [P. falciparum.In conclusion, our data demonstrate the complexity of the histone code in genome organization of the malaria parasite ications . SeveralTo isolate male and female gametocytes, the previously described 3D7::ABCG2-GFP parasite line was used . For det2, 5% CO2, and 94% N2 at 37 °C.Parasites were cultivated as described previously , at 5% hTo induce gametocytogenesis, a tightly synchronised ring stage culture at about 6–8% parasitemia was stressed by adding 50% fresh medium to the partially spent medium, thereby decreasing the haematocrit (day -2) . At abou6 parasites per µl), heated for 5 min at 95 °C and subsequently spun at 14,000 × g for 10 min. Finally, the supernatant was loaded on a 4 – 12% Bis–Tris gel (Invitrogen) in NuPAGE MES SDS running buffer (Thermo Fisher Scientific) and run for 1.25 h at 120 V. Thereafter, proteins were transferred onto Nitrocellulose membranes . The blotting was conducted at 400 mA for 1 h in transfer buffer  in a Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Biorad) blotting chamber. After blotting, the membranes were blocked for 2 h in blocking solution (5% non-fat milk powder in TBS with 0.05% Tween). The first antibody was applied in blocking solution with 0.02% sodium azide at 4 °C overnight. After washing the membrane in TBS-T, horse radish peroxidase (HRP) conjugated secondary antibodies in blocking solution were applied to the membrane for 2 h at RT. Detection was done using chemiluminescent HRP substrate (Immobilon ® Western Millipore) and imaging with a ChemiLux Imager (Intas Science Imaging).For the preparation of complete cell lysates, the erythrocytes were lysed on ice by resuspension in 0.075% Saponin (Sigma Aldrich) in PBS. Upon lysis, the erythrocytes were centrifuged at 2300 × g for 10 min at 4 °C. The pellet was washed twice with PBS, resuspended with 2 × Laemmli-Buffer  at least two days before sorting and then further cultivated in complete culture medium. As outlined in Additional File For RNA extraction, FACS sorted gametocytes or culture pellets of ring and schizont stage infected erythrocytes were resuspended in 10 times the pellet volume TRIzol® Reagent (Life Technologies). RNA was extracted by addition of 0.2 volumes chloroform followed by 30 min centrifugation at full speed and 4 °C. The aqueous layer containing the RNA was purified using the RNeasy Mini Kit . DNA was removed by on-column digest and an additional in solution digest with DNase I . The concentration of the resulting RNA was measured by NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific) and the purity was determined by qPCR with primers specific for SBP1.8 parasites. The mix was transferred to a prechilled 2 ml tissue grinder (Kimble) and incubated on ice for 30 min. Subsequently, IGEPAL CA-630 was added to a final concentration of 0.25% and the parasites were lysed with 100 strokes. The lysed parasites were then centrifuged for 10 min at 20,000 × g, 4 °C to pellet the nuclei.Sorbitol synchronized cultures of ring stage parasites (4–14 hpi), schizont stage parasites (38–48 hpi) or purified gametocytes were crosslinked at a final concentration of 1% PFA (16% PFA Thermo Fisher Scientific) for 10 min at 37 °C. The reaction was quenched for 5 min on ice with 125 mM glycine. The infected erythrocytes were then pelleted (5 min at 800 × g at 4 °C) and permeabilized in 0.075% saponin in PBS on ice to release haemoglobin. The parasite pellets were then washed twice with PBS (2300 × g at 4 °C for 5 min) and resuspended in 2 ml cold lysis buffer ) per 108 nuclei and sonication for 2 times 8 cycles of 30 s ON and OFF in a chilled Bioruptor® Sonication System (Diagenode). The sheared material was spun  and the supernatant was then transferred and diluted 1:10 with ChIP Dilution Buffer . The diluted chromatin was then pre-cleared with 30 µl Protein G Sepharose 4 Fast Flow  for 1 h at 4 °C under agitation. The beads were prepared for the immunoprecipitation (IP) by blocking in 0.1% BSA (Sigma Aldrich) in ChIP Dilution Buffer for 10 min. After two washing steps with ChIP Dilution Buffer, the beads were preincubated with the respective antibodies  per 10Libraries were prepared from the ChIP and RNA samples. For ChIP sequencing the Accel-NGS™ 2S Plus DNA Library Kit for Illumina platforms (SWI Swift Bioscience) was used according to the manufacturer’s guidelines, including the Accel-NGS™ 2S Indexing Kit (SWI Swift Bioscience) until the PCR amplification step. The RNA sequencing libraries were prepared using the NEBNext Ultra II directional RNA Library Prep Kit for Illumina (NEB) with the NEBNext Poly(A) mRNA Magnetic Isolation module (NEB) and the NEBNext Multiplex Oligos for Illumina (NEB). For both library types, Ampure XP beads (Beckman Coulter) were used for the purification steps and the library amplification step was performed with 12–15 cycles using KAPA Polymerase  with the dsDNA HS Assay Kit (Thermo Fisher Scientific Q32854) or the RNA HS Assay Kit (Thermo Fisher Scientific Q32855) to determine the concentration, and subsequently on a TapeStation (Agilent Technologies) using the D5000 kit according to the manufacturers protocol to determine molarity from the average fragment size. The analysis was done using the TapeStation Analysis Software 3.1.1., with a size range of 150 bp to 2000 bp. The libraries were subsequently pooled to a final concentration of 5 nM and sequenced as 100–150 bp paired end reads for RNAseq and 100–150 bp paired end reads for ChIP-Seq on an Illumina HiSeq 2500 instrument . The files were then trimmed to remove remaining adaptors and low quality reads using TrimGalore 0.6.4 . If necessary, the files were further trimmed using Cutadapt 2.8 [Plasmodium falciparum (PlasmoDB 3D7 version 28) using Bowtie2 version 2.3.4.3 with default settings for paired-end reads [p-value < 0.01. The list of ookinete specific genes was retrieved from PlasmoDB using the following settings: > fivefold overexpressed in ookinetes compared to all other stages on a floor of 100 reads [The raw data was processed on the Spartan HPC (University of Melbourne). ChIPseq data in fastq format was first quality checked using FastQC 0.11.8  Parasites expressing GFP-tagged ABCG2 [TM Deep Red and FACS-sorted according to their GFP signal. The mitotracker/GFP double positive (G+M+) female gametocyte population and the mitotracker positive, GFP negative (G-M+) male gametocyte population were then cultivated and reanalysed after two days by A) live microscopy and B) FACS. The GFP fluorescence intensity of the mitotracker positive cells was determined and significant differences between the populations determined by t-test. C) Heatmap showing the z-score transformed sense expression of several previously described stage specific marker genes (colour coded) determined by RNAseq to validate the cellular identity of the harvested parasite populations. D4 = Day 4, D6 = Day 6, D10 = Day 10, M = male gametocyte, F = female gametocyte. Male gametocytes: day 4: n=3, day 6: n=2, day 10: n=1; female gametocytes: day 4: n=2, day 6: n=4, day 10: n=4; ring stage parasites: n=2, schizont stage parasites: n=1. E) Heatmap of the spearman correlation coefficients between the RNAseq data sets from the different stages. Male gametocytes: day 4: n=3, day 6: n=2, day 10: n=1; female gametocytes: day 4: n=2, day 6: n=4, day 10: n=4; ring stage parasites: n=2, schizont stage parasites: n=1. Figure S2. Western blot of histone variants and modifications in bulk gametocyte cultures. H3 served as a loading control and Pfs16 as gametocyte specific marker. HP1 = heterochromatin protein 1.Figure S3. Overview over log2-transformed ChIP/Input coverage across all 14 P. falciparum chromosomes in female gametocytes and ring stage parasites. H3K4me3 , H3R17me2 , H3K27ac , H2A.Z , H3K9me3 . Figure S4. Heatmaps showing the log2-transformed ChIP/Input coverage of all genes and their 2 kb up/downstream region for each modification sorted into groups according to their expression. The black bar in the silent genes represents silent mitochondrial and apicoplast genes, that carry no histone modifications. A) Day 6 female gametocytes. B) Day 6 male gametocytes. C) Ring stage parasites. H3K4me3 , H3R17me2 , H3K27ac , H2A.Z , H2A.Zac , H3K9me3 . Figure S5. Global ChIPseq and RNAseq profiles for regions of particular interest in female gametocytes and ring stage parasites. Genes are marked depending on their genomic orientation in either green (+ genes) or orange (- genes). Introns are shaded in light green or dark red. The position of the ATG is marked by a dashed line. Transcripts (FPKM) are shown in black. Regions shown: SBP1 from 65.5 kbp to 72 kbp, AP2-G from 904.7 kbp to 918 kbp, Pfs230 from 368.4 kbp to 381.2 kbp, PUF1 from 773.2 kbp to 783.5 kbp. H3K4me3 , H3R17me2 , H3K27ac , H2A.Z , H3K9me3 . Figure S6. H3K4me3 and H3R17me2 coverage in genes with peak expression in asexual or sexual parasites. Average log2-transformed ChIP/Input coverage plots from two biological replicates for H3K4me3 and H3R17me2 in day 6 female gametocytes and ring stage parasites over genes with peak expression in early (cluster 3) and late (cluster 4) female gametocytes and rings (cluster 1). Figure S7. H3K4me3/H3R17me2 coverage in gametocytes is significantly increased in ookinete and oocyst specific genes. Log2-transformed ChIP/Input coverage plots from two biological replicates for H3K4me3 and H3R17me2 in day 6 female gametocytes and ring stage parasites over ookinete genes upregulated after JIB-04 treatment , region shown from 2167 kbp to 2170 kbp. RNAseq data: Male gametocytes: day 4: n=3, day 6: n=2, day 10: n=1; female gametocytes: day 4: n=2, day 6: n=4, day 10: n=4; ring stage parasites: n=2; ChIPseq data: H3K9me3 . Figure S9. H2A.Z is associated with heterochromatic areas in day 6 female gametocytes. Overview over chromosome 7 showing log2-transformed ChIP/Input coverage for H2A.Z  and H3K9me3  in all stages, H2A.Zac in male (n=1) and female gametocytes (n=2), and H2B.Z in female gametocytes (n=1). Zoom into internal heterochromatic cluster on chromosome 7. Genes are marked in either green (+ genes) or orange (- genes). Introns are marked either in light green or dark red, depending on gene orientation. Figure S10. H3K27ac and H2A.Zac coverage is unchanged in translationally repressed genes. Average log2-transformed ChIP/Input coverage plots for H2A.Zac  in day 6 male and female gametocytes and H3K27ac in female gametocytes (n=3) over all genes in comparison to genes ranking in the top expressed tercile (excluding DOZI-repressed genes) and DOZI-repressed genes in day 6 female gametocytes. Figure S11. Heat maps of chromatin states discovered in female day 6 gametocyte and ring stage parasites. Heatmap showing the log2-transformed ChIP/Input coverage of the studied histone modifications over all 50 bp bins represented in each of the A) female day 6 gametocyte states and B) ring stage parasite states. Figure S12. Length distribution of chromatin states. Neighbouring bins with identical chromatin states were fused and the lengths of the continuous regions with the same chromatin state were plotted as violin plots for female day 6 gametocytes and ring stage parasites.ed ABCG2  were tigeatment  and gene clusters including GO analyses.Additional file 4: Table S3. Comparison of heterochromatic loci with published data from Bunnik et al. 2018 [al. 2018  and Frasal. 2018 .Additional file 5: Table S4. List of genes associated with different chromatin states upstream in female gametocytes and ring stage parasites including Gene Ontology analyses.Additional file 6: Table S5. Antibodies used for western blot (WB), immunofluorescence analysis (IFA), and chromatin immunoprecipitation (ChIP).Additional file 7. Uncropped western blots shown in Figure S


The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs  differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors The online version contains supplementary material available at 10.1007/s00204-023-03591-4. Pyrrolizidine alkaloids (PAs) are phytotoxins with a high structural diversity formed in 3% of all flowering plant species worldwide  are involved in the hepatocellular uptake of retrorsine and senescionine, which are both cyclic PA diesters  and ATP-binding cassette (ABC) superfamily, respectively Nigam . The SLCd-tetrahydropalmatine (d-THP) and quinidine. First, the cytotoxicity of the three PAs was investigated using the resazurin reduction assay in the absence or presence of OCT1 inhibitors. Subsequently, the genotoxic effects of the three selected PAs were determined with and without OCT1 inhibition using western blot analysis of the DNA damage markers γH2AX and p53. Furthermore, the DNA damage response markers pCHK1 and pCHK2 were assessed upon PA exposure in the absence or presence of OCT1 inhibitors.Up to now little information is available on the impact of the chemical structure (PA monoester vs. open-chained diester vs. cyclic diester) on the OCT1-mediated uptake into hepatocytes. Therefore, we selected three structurally diverse PAs  and analyzed their toxicity in human HepG2-CYP3A4 liver cells, primary human hepatocytes (PHH) and V79-CYP3A4 Chinese hamster fibroblasts. The impact of OCT1 was studied using the pharmacological inhibitors d-THP as established OCT1 inhibitors  on cell viability, both inhibitors displayed strong cytotoxicity at 500 µM , we used quinidine and  SI Fig. A. Therefd-THP and quinidine prevented the cytotoxic effects of lasiocarpine in HepG2-CYP3A4 cells  for 24 h in the absence or presence of the OCT inhibitors (100 µM each). As expected, lasiocarpine caused a concentration-dependent decrease in cell viability, with a reduction below 50% at a concentration of 40 µM  are the gold standard for in vitro toxicokinetic studies of the liver, since they display full metabolic competence and express a plethora of efflux and influx transporters  in HepG2-CYP3A4 cells using the well-established DNA damage markers γH2AX and p53  triggered by the three selected PAs in HepG2-CYP3A4 cells and studied the impact of OCT inhibition. The DDR is coordinated by apical DDR kinases including ATM and ATR, which phosphorylate a plethora of downstream substrates such as the checkpoint kinases CHK1 and CHK2  blocked PA triggered DDR activation as evidenced by reduced pCHK1 and pCHK2 levels.Then we addressed the question whether the reduced cytotoxicity upon OCT1 inhibition is associated with an attenuated genotoxicity. Our results provided evidence that OCT1 inhibition also prevents the genotoxic effects of all three selected PAs as attested by reduced DNA damage levels (γH2AX and p53). However, quinidine only partially blocked γH2AX formation triggered by lasiocarpine, whereas it strongly attenuated γH2AX formation caused by heliotrine and riddelliine. This finding might indicate that low intracellular levels of lasiocarpine are sufficient to cause substantial γH2AX formation, bearing in mind that this is an open-chained PA diester with a higher genotoxic potency than the cyclic PA diester riddelliine and the PA monoester heliotrine  cells in case of an acute intoxication by using i.v. administered OCT1 inhibitors.2. Cell culture medium and supplements were obtained from Gibco Life Technologies  and Pan Biotech . All cell lines were mycoplasma negative, as demonstrated by routine PCR testing using Venor®GeM OneStep . Cryopreserved PHH pooled from five Caucasian donors were from Thermo Fisher Scientific  and were maintained as described recently  and quinidine, were purchased from Hycultec  and dissolved in DMSO to obtain 100 mM stock solutions stored at − 20 °C. Stock solutions were diluted in cell culture medium to reach final concentrations in the experiments as indicated . For the inhibitor studies, cells were pre-incubated for 1.5 h with 100 µM d-THP or quinidine. The medium was then aspirated and fresh medium was added, which contained the PAs under investigation with or without 100 µM of the respective OCT1-inhibitor for 24 h. Cells were then analyzed as described below.The PAs used  were of highest purity and were from Phytolab . All PAs were dissolved in DMSO to prepare stock solutions (50–150 mM), which were stored at − 20 °C. Two OCT1 inhibitors, namely 2PO4, 154 mM NaCl, 3.7 mM Na2HPO4) at 37 °C in humidified atmosphere of 5% CO2. Cell viability was determined using a microplate reader  with 544 nm for excitation and 590 nm for emission. In addition, phase-contrast microscopy was performed using a Leica microscope (HI PLAN I 20x/0.30 PH1 or HI PLAN I 10x/0.22 PH1) equipped with Leica MC170 HD camera.HepG2 and HepG2-CYP3A4 cells  as well as V79 and V79-CYP3A4 cells  were seeded on 96-well plates, grown overnight and incubated with increasing PA concentrations without or with 100 µM of each OCT1-inhibitor. PHH were seeded on collagen type I coated 96-well plates at density of 62,500 cells/well and treated as described above. Saponine (0.1%) served as positive control and solvent (DMSO) as negative control. Cell viability was determined using the resazurin reduction assay as described previously  and proteins were visualized with a c300 chemiluminescence imager . The primary antibodies directed against CYP3A4 , p53  and HSP90α/β (sc-13119) were obtained from Santa Cruz Biotechnology . The primary antibody raised against γH2AX  was from Abcam . The primary antibodies p-CHK1  and p-CHK2  as well as CHK1 (#2360) and CHK2 (#2662) were obtained from Cell Signaling Technology . The primary antibody directed against OCT-1/SLC22A1  was obtained from Novus Biologicals LLC . HRP-conjugated secondary antibodies conjugated were purchased from Santa Cruz Biotechnology  and Cell Signaling Technology .HepG2-CYP3A4 cells  were grown on 3.5 cm plates, incubated with increasing PA concentrations for 24 h and directly harvested with 1 × Laemmli loading buffer as described  and allowed to grow for 24 h. Immunofluorescence staining and confocal microscopy were essentially performed as reported  from representative experiments. Statistics were carried out by Graphpad Prism software (Version 9). Statistical significance was defined as P < 0.05 and statistical analyses were performed using unpaired two-tailed Students t-test with respect to the negative control.Supplementary file1 (DOCX 4390 KB)Below is the link to the electronic supplementary material.


Breast cancer is the most common malignant tumor in women and is a big challenge to clinical treatment due to the high morbidity and mortality. The pH/ROS dual-responsive nanoplatforms may be an effective way to significantly improve the therapeutic efficacy of breast cancer. Herein, we report a docetaxel (DTX)-loaded pH/ROS-responsive NP that could achieve active targeting of cancer cells and selective and complete drug release for effective drug delivery. The pH/ROS-responsive NPs were fabricated using nanocarriers that consist of an ROS-responsive moiety , cinnamaldehyde  and cyclodextrin (α-CD). The NPs were loaded with DTX, modified with a tumor-penetration peptide  and named DTX/RGD NPs. The cRGD could promote DTX/RGD NPs penetration into deep tumor tissue and specifically target cancer cells. After internalization by cancer cells through receptor-mediated endocytosis, the pH-responsive acetal was cleaved to release CA in the lysosomal acidic environment. Meanwhile, the high ROS in tumor cells induced the disassembly of NPs with complete release of DTX. In vitro cellular assays verified that DTX/RGD NPs could be effectively internalized by 4T1 cells, obviously inducing apoptosis, blocking the cell cycle of 4T1 cells and consequently, killing tumor cells. In vivo animal experiments demonstrated that the NPs could target to the tumor sites and significantly inhibit the tumor growth in 4T1 breast cancer mice. Both in vitro and in vivo investigations demonstrated that DTX/RGD NPs could significantly improve the antitumor effect compared to free DTX. Thus, the DTX/RGD NPs provide a promising strategy for enhancing drug delivery and cancer therapy. Breast cancer, as the most commonly malignant cancer in the world, remains the greatest challenge in clinical cancer research ,2,3. Cli+ exhausted without adequate supplementation. Taken together, pH/ROS dual-responsive nanoplatforms may be an effective way to significantly enhance selective and complete drug release, as well as improve the therapeutic efficacy against breast cancer.As one of the characteristics of the tumor microenvironment, a high concentration of ROS has been widely used in the design of nanomedicines . AlthougHowever, this passive targeting strategy cannot ensure an optimal accumulation of drugs in the tumor and prevent undesirable side effects simultaneously ,30. The In this study, we developed a cRGD-functionalized pH/ROS dual-responsive nanocarrier to load DTX, a famous chemotherapeutic agent against breast cancer. The nanocarrier consists of an ROS-responsive moiety (HPAP), CA   and α-CD2000, DSPE-PEG-cRGD (cyclic Arg-Gly-Asp) and polystyrene nanoparticles (PS NPs) were obtained from Xi’an Ruixi Biotechnology Co., Ltd. . The Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were provided by HyClone Inc. . The streptomycin–penicillin solution and FITC-phalloidin were obtained from Solarbio Life Sciences Co., Ltd. . The 4′,6-Diamidino-2-phenylindole (DAPI), Enhanced Cell Counting Kit-8 (CCK-8), Hydrogen Peroxide Assay Kit and Cell Cycle Kit were supplied by Beyotime Biotechnology Co., Ltd. . The Matrigel was purchased from Corning Inc. . An Annexin V-FITC Apoptosis Detection Kit was provided by Becton, Dickinson and Company .Pluronic F-127 (polyethylene-polypropylene glycol) were purchased from Sigma–Aldrich Co. . Cy5 free acid was provided by Lumiprobe, LLC. . DSPE-PEG2. The cell culture medium was changed every two days and cells were passaged in a 1:3 ratio.The mouse breast cancer cell line 4T1 and human breast cancer cell line MDA-MB-231 were provided by the Cell Bank of the Chinese Academy of Sciences . The cells were cultured in a DMEM cell culture medium supplemented with 10% fetal bovine serum, 100 µg/mL streptomycin and 100 IU penicillin at 37 °C in a humidified atmosphere containing 5% COSix- or eight-week-old female BAlB/c mice weighing approximately 20 g were supplied from the experimental animal center of Army Medical University  and kept in an SPF-level sterile animal room. All animal experiments were performed in accordance with the guidelines approved by the ethics committee of the Army Medical University .2000 were dispersed in 400 µL of anhydrous ethanol and 7.0 mL of deionized water. The dispersed solution was heated at 65 °C for 0.5 h. Meanwhile, 50 mg of CA-Oxi-αCD and 10 mg of DTX were dissolved in 400 µL of methanol and 200 µL of DMSO, then the drug-containing solution was added dropwise to the lipid-dispersed solution with gentle stirring followed by fast stirring for 3 min. After self-assembly for 2 h under slow stirring at room temperature, the DTX-loaded NPs were harvested and centrifuged at 15,000× g for 10 min with a high-speed freezing centrifuge . After being washed with 5% F127 twice, the NPs were then suspended in ultrapure water. Additionally, the RGD-modified DTX-loaded NPs were fabricated using a similar procedure as the NPs with slight changes: 4 mg of DSPE-PEG2000 and 4 mg of DSPE-PEG-cRGD were added to the lipid dispersion to fabricate the NPs. Furthermore, 5 mg of Cy5-conjugated α-CD was added to fabricate the Cy5-labeled NPs. The particle size, polydispersity index (PDI) and zeta potential of the NPs were determined with dynamic light scattering (DLS) and laser Doppler anemometry using a Malvern Zetasizer . The morphology of the NPs was observed with a transmission electron microscope . The drug loading and encapsulation efficiency were determined as previously reported /[A450 (1% Triton X-100) − A450 (PBS)] × 100%.The biocompatibility of the NPs with sheep blood cells was evaluated with a hemolysis assay. Briefly, NPs were incubated with 3% red blood cell suspensions at different concentrations  for 1 h at 37 °C in 5% CO5 cells per well in 12-well plates and allowed to grow for 24 h at 37 °C in 5% CO2. Then the cells were treated with DTX, PS NPs, DTX/NPs and DTX/RGD NPs with an equivalent concentration of 5 ng/mL of DTX in cell culture medium for 48 h at 37 °C in 5% CO2. The control was treated with cell culture medium. After 48 h of incubation, the intracellular concentrations of H2O2 in 4T1 cells were determined according to the manufacturer’s protocols of a Hydrogen Peroxide Assay Kit.4T1 cells were seeded at a density of 2 × 102. The cells were then treated with free Cy5, Cy5-labeled NPs or Cy5-labeled RGD NPs with an equivalent concentration of 2 µg/mL of Cy5 for 4 h at 37 °C in 5% CO2. Then the cells were washed and fixed with 4% paraformaldehyde for 20 min. After being permeabilized with 0.5% Triton-X for 5 min at room temperature, the cells were incubated with FITC-phalloidin (100 nM) for 30 min at 37 °C in the dark. Then the cell nuclei were stained with DAPI (5 μg/mL) for 10 min at room temperature, and the intracellular uptake of the NPs was obtained with confocal laser scanning microscopy .4T1 cells were cultured in confocal dishes for 24 h at 37 °C in 5% CO5 cells per well in 12-well plates and cultured for 24 h at 37 °C in 5% CO2. Then the cells were incubated with free Cy5, Cy5-labeled NPs or Cy5-labeled RGD NPs with an equivalent concentration of 2 µg/mL of Cy5 for 4 h at 37 °C in 5% CO2. After incubation, the cells were collected and the fluorescence intensity of intracellular Cy5 was detected using flow cytometry .We also quantitatively analyzed the uptake of NPs in 4T1 cells with flow cytometry. Briefly, 4T1 cells were seeded at a density of 2 × 102. After the cells were washed with cold PBS three times and fixed in 4% paraformaldehyde for 20 min at room temperature, the cell nuclei were stained with DAPI (5 μg/mL) for 10 min at room temperature. Finally, the penetration of the NPs in the 3D tumor spheroids was observed using CLSM  in layer-scanning mode.4T1 cells were seeded on confocal dishes precoated with a thin layer of Matrigel. Then the cells in the tumor spheroids were incubated with Cy5, Cy5-labeled NPs or Cy5-labeled RGD NPs with an equivalent concentration of 2 µg/mL of Cy5 for 4 h at 37 °C in 5% CO4 cells per well for 24 h at 37 °C in 5% CO2. Then the cells were treated with DTX, blank NPs or DTX-loaded NPs at a concentration of 0.31, 1.25, 5.00, 20.00 or 80.00 ng/mL for 24 h or 48 h at 37 °C in 5% CO2. After treatment, the cells were washed with PBS three times and incubated with CCK-8 solution for another 0.5 h at 37 °C in 5% CO2. Finally, the absorbance of the cultures was measured at 450 nm using a Thermo Multiskan Spectrum spectrophotometer . The 50% inhibitory concentration (IC50) was calculated by plotting the dose–response curve using GraphPad Prism 8.0.The in vitro cytotoxicity of NPs against 4T1 cells was evaluated using the CCK-8 assay. 4T1 cells were seeded in 96-well plates at 1 × 105 cells per well for 24 h at 37 °C in 5% CO2. The cells were treated with DTX, blank NPs, DTX/NPs or DTX/RGD NPs with an equivalent concentration of 20 ng/mL of DTX in cell medium at 37 °C in 5% CO2 for 48 h. After 48 h of incubation, the cells were centrifuged and washed with cold PBS three times. Then the cells were fixed with cold 70% ethanol for 24 h at 4 °C. Finally, the cells were stained with PI according to the manufacturer’s instructions and determined with flow cytometry .To investigate the effect of NPs on the tumor cell cycle, 4T1 cells were cultured in 12-well plates at a density of 2 × 105 cells per well and allowed to grow for 24 h at 37 °C in 5% CO2. The cells were then incubated with DTX, blank NPs, DTX/NPs or DTX/RGD NPs with an equivalent concentration of 20 ng/mL of DTX in cell medium at 37 °C in 5% CO2 for 48 h. After 48 h of incubation, the cells were collected and the apoptosis percentage was determined with an Annexin V-FITC apoptosis-detection kit according to the manufacturer’s instructions.4T1 cells were seeded in 12-well plates at a density of 2 × 106 4T1 cells into the fourth mammary fat pad (right). When the tumor volume grew to approximately 300 mm3, the mice were injected with Cy5, Cy5-labeled NPs or Cy5-labeled RGD NPs at a Cy5 dosage of 1 mg/kg via vein. After administration for 2 h, 8 h, 24 h or 48 h, the mice were anesthetized and imaged with an in vivo imaging system . At 48 h post-injection, the mice were sacrificed and the tumors and major tissues were harvested and imaged. The semi-quantitative analysis of fluorescence intensity in tumors was obtained through an in vivo imaging system software. In addition, the cryosectioned tumor tissues were fixed with 4% paraformaldehyde for 20 min, and the nuclei were then stained with DAPI (5 μg/mL) for 10 min at room temperature and finally imaged with CLSM .We investigated the in vivo distribution of NPs in mice bearing a 4T1 breast cancer model. Briefly, the mice were injected with 1 × 105 4T1 cells into the fourth mammary fat pad. Five days after inoculation, tumor-bearing mice were randomly divided into 5 groups (n = 6) and treated with saline, blank NPs, DTX, PLGA NPs or DTX/RGD NPs via vein at a dose of 5 mg/kg DTX every four days for five times. The tumor volumes and body weights were measured every 2 days. The tumor volumes of the mice were measured and calculated using the following formula: V = 1/2 (L × W2), in which L (length) is the longest diameter and W (width) is the shortest diameter perpendicular to length. After the termination of the therapy, all mice were sacrificed, and the tumors and lungs were collected for photo imaging, weighing and histopathologic examination. The remaining tumors were homogenized, and the DTX concentration in the tumors was determined using LC-MS/MS .To investigate the antitumor efficacy of DTX/RGD NPs in vivo, the in vivo 4T1 breast tumor model was established. The female BALB/c mice were subcutaneously injected with 5 × 10t-test for two groups and one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test for multiple-group comparisons. Statistical significance was defined as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.All data are reported as the mean ± standard deviation (SD) with at least three independent experiments. Statistical analysis was performed using the unpaired two-tailed Student’s The detailed synthetic route and the characterization of the pH/ROS dual-responsive materials (CA-Oxi-αCD) are displayed in the 2O2 for 2 h, the size of NPs was changed from 261.10 ± 6.72 nm to 151.97 ± 1.76 nm, and the PDI of NPs was changed from 0.18 ± 0.03 to 0.06 ± 0.02, owing to ROS-triggered degradation of HPAP in the carrier and subsequent dissociation of the NPs, resulting in the promotion of drug release. Moreover, the changes in size of the NPs in the pH 5.0/1.0 mM H2O2 medium was similar to the 1.0 mM H2O2 medium. Additionally, the size of the NPs at pH 5.0 medium decreased about 50 nm compared to the NPs distributed in water, implying that an acidic pH might cause the disintegration of the NPs construction, which helps to release the drug and diffuse it towards the tumors’ core [The size changes of DTX-loaded NPs in acidic and/or ROS conditions were determined using DLS to evaluate the capability of the pH/ROS dual-responsiveness. As shown in rs’ core . However2O2 conditions, implying that the carrier was seriously disrupted under pH 5.0/H2O2 conditions, allowing the complete release of the DTX. Meanwhile, approximately 80% of the DTX was released from the NPs in pH 5.0 or 1.0 mM H2O2 medium within 48 h. However, only approximately 50% of the DTX was released from the NPs under pH 7.4. In addition, under the pH 5.0 and pH 5.0/H2O2 conditions, the CA was released from the NPs and completely released under the pH 5.0/1.0 mM H2O2 condition at 48 h. Incubation of NPs in PBS or 1.0 mM H2O2 resulted in minimal release of CA, suggesting that the pH-responsiveness of NPs comes from acetal cleavage between CA and α-CD , 6/6 (blank NPs), 4/6 (DTX), 3/6 (DTX/PLGA NPs) and 0/6 (DTX/RGD NPs), respectively. Accordingly, the proportion of tumors exceeding 600 mm3 in the group of DTX/RGD NPs remained the lowest in all groups, showing the optimal therapeutic efficacy of DTX/RGD NPs (Our previous study demonstrated that RGD-modified NPs had the optimal antitumor effect in vitro and tumor-cell targeting in vivo; thus, the in vivo antitumor efficacy of cRGD-modified NPs was further evaluated in 4T1 tumor-bearing mice. As shown in /RGD NPs E. The re/RGD NPs F. To furVβ3-mediated active-targeting effect and efficiently inhibited 4T1 tumor growth and lung metastasis. Overall, our study provides a great potential strategy for the treatment of metastatic breast cancer and clinical translation.In summary, we reported a cyclic RGD-functionalized pH/ROS-responsive NP for enhanced cancer therapy. The NPs displayed pH/ROS dual-responsiveness and complete drug release in vitro. Moreover, the NPs could effectively target 4T1 cells and enter inside tumor spheroids. In addition, the NPs greatly inhibited the proliferation and induced the apoptosis of tumor cells in vitro. In vivo, DTX/RGD NPs actively accumulated at the tumor site via an α


Diabetic nephropathy is a one of the risk factor for end stage renal disease in patients with type 2 diabetes mellitus. Urinary microalbumin currently marker using for diagnosis of nephropathy in type 2 diabetes mellitus due to its larger variability and lessspecificity. Urinary nephrin is podocyte protein can be act as a early predictable and prognostic marker than micro albumin fornephropathy in type 2 diabetes mellitus. This case-control prospective observational study included 60 type 2 diabetes mellitus and30 controls after applying criteria. Basic biochemical, clinical and experimental data were measured and recorded. There was asignificantly elevated level of urinary nephrin in type 2 diabetes mellitus patients when compared to controls. The urinary nephrinsignificantly elevated in normo albuminuria group only when compared to urinary ACR and it is positive association with kidney damage.This study concluded significantly elevated levels of urinary nephrin might be used for early diagnosis and prognostic marker fornephropathy in type 2 diabetes mellitus. Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder characterized by hyperglycemia due to insulin resistance in targettissues including the liver and skeletal muscles. This leads to multisystem failure .The preTo investigate urine indicators in the early diagnosis of diabetic nephropathy in patients with type 2 diabetes mellitus, acase-control prospective observational study was conducted. The study included a total of 90 participants, 30 healthy controls,and 60 patients who were diagnosed with T2DM according to American Diabetes Association (ADA) criteria between March 2017 andFebruary 2018 and who visited the endocrinology, metabolism, and nephrology outpatient departments at Akash Institute of MedicalSciences and Research Centre, Bangalore Rural, Karnataka. Thirty wholesome controls made constituted Group 1. Based on thealbuminuria levels, the patients were split into two groups of thirty each: type 2 diabetes mellitus patients with normo albuminuria(Group 2) and type 2 diabetic mellitus patients with micro albuminuria (Group3). After presenting the study to the subjects in theirnative tongue, their informed consent was obtained. After receiving approval from the institutional thesis protocol approval committeeand institutional ethics committee, the study was carried out.Random spot urine samples were taken to check for albuminuria in all the groups of study subjects. The patient was placed inGroup 2, or type 2 DM with normoalbuminuria, if the UACR levels were less than 30 mg/g creatinine and type 2 DM with microalbuminuria,if the UACR levels above 30 but fell short of 299 mg/g creatinine were considered.Kolmogorov-Smirnov test was used to assess the distribution of the data. For categorical data, continuous variables areexpressed as mean and standard deviation. ANOVA was used to assess differences between the three groups under investigation,and it was followed by post hoc multiple testing using Tamhane's or Bonferroni's tests, if necessary. The correlations betweenthe markers were examined using Pearson's correlation analysis. Microsoft Excel spreadsheets, SPSS software for Windows version 16 was used. Statistics were deemed significant at P 0.05. Table 1(see PDF) displays the demographic and medicaltraits of the T2DM cases and controls. The age of the study participants was, respectively, 43.90 ± 8.05 and 46.05 ±4.75 years for controls and T2DM cases (p 0.001*). The mean FBS concentration was observed significantly elevated in T2DM patients(150.89 ± 53.08) when compared to controls (89.98 ± 9.84) respectively; P value 0.001**. The serum creatinine(0.79 ± 0.15 and 3.90 ± 3.12) and serum urea (20.65 ± 6.01 and 49.45 ± 28.71) were significantlyincreased in patients with T2DM when compared to controls respectively; P value 0.001**. Urinary ACR also significantly elevated inT2DM patients (45.19 ± 24.89) when compared to controls (4.96 ± 3.01) respectively; P value 0.001**. The T2DM patients(2.01 ± 1.51) showed a significantly elevated levels of urinary nephrin when compared to controls (0.68 ± 0.12)respectively; P value 0.001**. Table 2(see PDF) displays the demographic and medical traits of the study groups. The age of the studyparticipants was, respectively, 43.90 ± 8.05, 45.38 ± 5.57 and 46.05 ± 4.75 years for group 1,2 and 3 (p 0.05*).The mean FBS concentration was observed significantly elevated in group 2 and 3 (140.30 ± 50.11 and 161.48 ± 54.47)when compared to group 1 (89.98 ± 9.84) respectively; P value 0.001**. The serum creatinine  and serum urea  were significantlyincreased group 3 and 2 when compared to group 1 respectively; P value 0.001**. Urinary ACR also significantly elevated in group 2and 3 (10.56 ± 7.92 and 83.76 ± 39.03) when compared to group 1 (4.96 ± 3.01) respectively; P value 0.001**.The group 2 and 3 subjects (0.68 ± 0.12) showed a significantly elevated when compared to group 1 (2.01 ± 1.51)respectively; P value 0.001**. Table 3(see PDF) shows the pearson correlation urinary ACR with other parameters of the study, there was asignificantly positive correlation between the urinary ACR and FBS, urea, creatinine and urinary nephrin  respectively P value is 0.001**.Table 4(see PDF) shows the pearson correlation urinary nephrin with other parameters of the study,there was a significantly positive correlation between the urinary nephrin and FBS, urea, creatinine and urinary ACR respectively P value is 0.001**. Figure 1(see PDF) represents the urinary ACR levels in T2DM cases and controls.The T2DM with microalbuminuria patients showed significantly elevated levels of urinary ACR when comparedto T2DM with normoalbuminuria and controls. Figure 2(see PDF) represents the urinary nephrin levels in T2DM cases and controls.The T2DM with micro and normoalbuminuria patients showed significantly elevated levels of urinary nephrin when comparedto controls. Figure 3(see PDF) represents scatter plots of urinary nephrin and urinary ACR. There was a significantly positiveassociation between urinary nephrin and urinary ACR levels.Diabetic nephropathy is one of the most common conditions worldwide in T2DM. However, it has become increasingly evident over theyears that hyperglycemia by itself is not the sole cause of DN. Several pathophysiological pathways including hemodynamic pathway,metabolic pathways, inflammation pathway and other alternation pathway are involved in the development of DN. Persistent microalbuminuria (30-299 mg/day), which is validated on at least two times three to six months apart, is currently the gold standard testfor the early diagnosis of diabetic nephropathy. However, it has drawbacks, including low sensitivity, variable plasma concentrationcaused by a number of variables, and a significant inter-individual variation of 47% . TolessThis study concludes significantly elevated levels of urinary nephrin levels in type 2 diabetes mellitus patients might be used forearly diagnosis and progression of nephropathy.


The direct bioconversion of crude glycerol, a byproduct of biodiesel production, into 1,3-propanediol by microbial fermentation constitutes a remarkably promising value-added applications. However, the low activity of glycerol dehydratase, which is the key and rate-limiting enzyme in the 1,3-propanediol synthetic pathway, caused by crude glycerol impurities is one of the main factors affecting the 1,3-propanediol yield. Hence, the exploration of glycerol dehydratase resources suitable for crude glycerol bioconversion is required for the development of 1,3-propanediol-producing engineered strains.Klebsiella pneumoniae 2e, was characterized. The 2eGDHt exhibited the highest activity toward glycerol, with Km and Vm values of 3.42 mM and 58.15 nkat mg−1, respectively. The optimum pH and temperature for 2eGDHt were 7.0 and 37 °C, respectively. 2eGDHt displayed broader pH stability than other reported glycerol dehydratases. Its enzymatic activity was increased by Fe2+ and Tween-20, with 294% and 290% relative activities, respectively. The presence of various concentrations of the crude glycerol impurities, including NaCl, methanol, oleic acid, and linoleic acid, showed limited impact on the 2eGDHt activity. In addition, the enzyme activity was almost unaffected by the presence of an impurity mixture that mimicked the crude glycerol environment. Structural analyses revealed that 2eGDHt possesses more coil structures than reported glycerol dehydratases. Moreover, molecular dynamics simulations and site-directed mutagenesis analyses implied that the existence of unique Val744 from one of the increased coil regions played a key role in the tolerance characteristic by increasing the protein flexibility.In this study, the novel glycerol dehydratase 2eGDHt, which has a tolerance against crude glycerol impurities from This study provides insight into the mechanism for enzymatic action and the tolerance against crude glycerol impurities, of a novel glycerol dehydratase 2eGDHt, which is a promising glycerol dehydratase candidate for biotechnological conversion of crude glycerol into 1,3-PDO.The online version contains supplementary material available at 10.1186/s13068-023-02427-8. Klebsiella genus strains, which show high tolerance to the crude glycerol environment, are promising candidates for 1,3-PDO production from crude glycerol [Crude glycerol is the main byproduct from biodiesel production, and it is generated in enormous quantities in the biodiesel industry , 2. The glycerol , 10. Howglycerol , 12.Klebsiella species, glycerol dehydrogenase, glycerol dehydratase, and 1,3-PDO oxidoreductase are the major enzymes associated with 1,3-PDO production [Klebsiella genus strain, glycerol dehydratase catalyzes the coenzyme B12-dependent dehydration reaction of glycerol generating 3-hydroxypropionaldehyde (3-HPA), which is further reduced to the fermentation product 1,3-PDO by 1,3-propanediol oxidoreductase [Klebsiella genus strains have been used for crude glycerol fermentation, and the main focus is typically on the tolerance of the strain toward impurities and optimization of the fermentation conditions, while there was less concern about glycerol dehydratase activity during fermentation [12-dependent glycerol dehydratase have been extensively explored [In the glycerol metabolism pathway of oduction . Among toduction . In the eductase . A numbeeductase –17. Durieductase . Numbersentation –21. The explored , 17, 22.explored . NeverthK. pneumoniae 2e, which has the capacity to produce 1,3-PDO efficiently from crude glycerol [Klebsiella species with high tolerance against crude glycerol impurities.In our previous study, we characterized the newly isolated strain glycerol . The enzglycerol . HoweverK. pneumonia 2e revealed that its glycerol dehydratase (2eGDHt) enzyme consisted of three subunits  encoded as dhaB1 (AYO66109.1), dhaB2 (AYO66110.1), and dhaB3 (AYO66111.1), respectively, with a total length of 2693 bp [12-dependent and B12-independent families were selected for protein sequence-based phylogenetic tree construction. As shown in Fig. Klebsiella genus strains, which belong to the B12-dependent glycerol dehydratase family. The common feature in the enzymatic reaction of B12-dependent glycerol dehydratase is that it proceeds by a radical mechanism that requires coenzyme B12 as a cofactor for generating highly active primary carbon radicals for catalysis [In our previous study, the whole genomic analysis of  2693 bp . To inveatalysis .Fig. 1BiKlebsiella genus strains, a multiple sequence homologous alignment analysis was conducted. As shown in Fig. Klebsiella genus strains. The sequence similarities of the α, β, and γ subunits reached 90.2%, 92.7%, and 88.6%, respectively, among those glycerol dehydratases. The catalytic amino acid residues, including Glu171, Gln142, Glu222, Gln297, and Ser363, were absolutely conserved in all seven glycerol dehydratase protein sequences compared, which was consistent with a previous study [K. pneumoniae strains were very similar, showing only a few different amino acids. Notably, an isoleucine was located at position 744 in 2eGDHt instead of the valine found in the other two compared glycerol dehydratase protein sequences from K. pneumonia.To study the sequence similarity of 2eGDHt with other glycerol dehydratases from us study . The glydhaB gene-encoding 2eGDHt was amplified from the genomic DNA of K. pneumoniae 2e by PCR and cloned and inserted into the pET-28b vector. The recombinant 2eGDHt was overexpressed as a soluble protein after induction with 0.8 mM IPTG and purified to homogeneity by affinity chromatography on a Ni–NTA resin column. As shown in Fig. 12-dependent glycerol dehydratase from bacterial strains exists as a dimer of heterotrimers (αβγ)2 in the natural state [The full-length al state , 27. NatKm and Vmax values for glycerol were 3.42 mM and 58.15 nkat mg−1, respectively. The catalytic efficiency (Km/Kcat) of 2eGDHt for glycerol reached 53.17 s−1 mM−1, which was higher than that for 1,2-PDO (46.48 s−1 mM−1).To explore the substrate specificity of 2eGDHt, a number of different alcohol substrates, including glycerol, 1,2-propanediol , methanol, butanol, isopropanol, and ethanol, were selected for enzymatic assays. As listed in Table K. pneumoniae strains [K. pneumoniae strain [To investigate the effect of temperature on the activity of 2eGDHt, the enzymatic activity was determined at different temperatures (4–65 °C). As shown in Fig.  strains . The teme strain .Fig. 3BiK. pneumoniae strain showed maximum activity at pH 8.5, and it lost more than 60% of its activity when the pH was below 7.5 [K. pneumoniae 10,018, which retained less than 20% relative activity after preincubated at pH 6.0 for 240 min [K. pneumoniae strains. The above discrepancy in the biochemical characteristics of 2eGDHt compared with those of other Klebsiella glycerol dehydratases might be caused by the differences in their sequences and protein structures.The effect of pH on enzyme activity was assayed at various pH values ranging from 4.2 to 9.0. As shown in Fig. elow 7.5 , 31. The 240 min . These r2+, Cu2+, Co2+, Fe3+, Fe2+, Mn2+, Zn2+ Ca2+, and Na+) present at a concentration of 10 mM together with the glycerol substrate at 37 °C and pH 7.0. As shown in Fig. 2+ which decreased the relative activity to 31.1%. However, the presence of Fe2+ increased the activity of 2eGDHt by 294%, but Fe3+ showed nearly no effect on 2eGDHt activity. These results implied that none of the detected metal ions were essential for the catalytic activity of 2eGDHt. Glycerol dehydratase is a metalloenzyme that requires K+ as the essential cofactor for catalysis. The introduction of Fe2+ might have caused changes in K+ coordination by 2eGDHt that maintained the proper position and orientation of the substrate during the reaction, while other metal ions might have had the opposite effect [To understand the effects of metal ions on the enzymatic activity of 2eGDHt, the enzyme activity was investigated with different metals  with 2eGDHt, was used as the template for the model predictions. Compared with 1iwp-GDHt, the predicted 2eGDHt structure showed a very similar αβγ heterotrimer and its heterohexamer (αβγ)2 , which is a common feature of B12-dependent glycerol hydratases [αβγ heterotrimers were connected only by the interactions of two α subunits in the (αβγ)2 heterohexamer. Meanwhile, the α subunit constitutes a (β/α)8 barrel, known as the triose-phosphate isomerase (TIM) barrel, located at the central part in both 1iwp-GDHt and 2eGDHt, which binds the substrate and the essential cofactor K+ [12-dependent glycerol dehydratases from the Klebsiella genus, it is not surprising that the general biochemical characteristics, such as the substrate specificity, optimum catalytic temperature, and catalytic efficiency, of 2eGDHt were so similar to those of other reported B12-dependent glycerol dehydratases.To provide the protein structural basis of 2eGDHt, the three-dimensional (3D) structure of the protein was predicted with AlphaFold 2.1. As shown in Fig. dratases . The twoactor K+ . Moreoveactor K+ . ConsideK. pneumoniae 2e, which was isolated from a biodiesel-derived waste-contaminated soil environment and showed excellent performance in converting crude glycerol into 1,3-PDO. This may explain why the temperature and pH stabilities and the crude glycerol impurity tolerance of 2eGDHt were different from those of other glycerol dehydratases, albeit with highly similar sequences.However, as shown in Fig. K. pneumonia strains. This suggested that the unique residue Iso744 from one of the increased coil regions might play a critical role in the flexibility of 2eGDHt in the crude glycerol environment.To study the effects of the key coil regions on the activity of 2eGDHt in the presence of crude glycerol impurities, a comparison of the root-mean-square fluctuations (RMSFs) of 2eGDHt in water and a mixture of impurities from crude glycerol was performed with molecular dynamic (MD) simulations. As shown in Fig. To investigate the role of residue Iso744 in the tolerance of 2eGDHt against crude glycerol impurities, site-directed mutagenesis of Iso744 was conducted. As shown in Fig. Furthermore, the enzymatic activities of the I744V mutant were determined in pure and crude glycerol. As shown in Fig. K. pneumoniae 2e with high tolerance against the main impurities in crude glycerol was characterized. Among various alcohol substrates, 2eGDHt showed the highest activity with glycerol, and Km and Vm values were 3.42 mM and 58.15 nkat mg−1, respectively. It exhibited optimum activity at pH 7.0 and 37 °C. The enzyme was substantially activated by Fe2+ and Tween-20 with 294% and 290% relative activities, respectively. It was highly stable in the presence of various concentrations of the crude glycerol impurities and their mixtures, which mimicked the crude glycerol environment. The protein structure predicted for 2eGDHt contained more coil structures than those of reported glycerol dehydratases. The presence of a unique Val744 in one of the increased coil regions of 2eGDHt played a key role in its tolerance toward crude glycerol impurities by increasing the protein flexibility. This work furnishes insight into the enzymatic properties and molecular mechanism for tolerance of the novel glycerol dehydratase toward crude glycerol impurities, which is of great significance for the use of industrial glycerol dehydratase for 1,3-PDO production from crude glycerol.In this study, the novel glycerol dehydratase 2eGDHt from d-1-thiogalactopyranoside (IPTG) and 3-methyl-2-benzothiazolinone hydrazine (MBTH) were purchased from Macklin . A plasmid extraction kit and gel extraction kit were purchased from Tiangen Biotech and DongShengbio , respectively. All restriction enzymes and DNA polymerases were purchased from Takara Biotechnology . Polymerase chain reaction (PCR) primers were synthesized by Qinkebio . All other reagents used were of molecular biology grade.Isopropyl-β-K. pneumoniae 2e was isolated from a soil sample collected from a biodiesel-derived waste-contaminated region in our previous study [Escherichia coli DH5α and BL21 (DE3) cells purchased from Transgene  were used for cloning and recombinant protein overexpression, respectively. The plasmid vector pET-28b purchased from Vazyme  was used for recombinant protein expression. K. pneumoniae 2e was cultured in nutrient broth (NB) medium at 37 °C and 170 rpm. E. coli was incubated in Luria–Bertani (LB) broth medium supplemented with 50 μg/mL kanamycin at 37 °C and 200 rpm.us study . EscheriKlebsiella genus. Homology modeling of glycerol dehydratase from K. pneumoniae 2e was performed with a version of AlphaFold 2.1 [K. pneumoniae 2e was predicted with Gramm-X and PDBePISA software [Sequence similarities were detected with the NCBI BLAST algorithm. Phylogenetic analyses was carried out with MEGA 5.1 using the neighbor-joining algorithm with bootstrap values. Multiple sequence alignment was conducted with Clustalx and GeneDoc software based on the protein sequence of glycerol dehydratase from the Fold 2.1 . The homsoftware , 46. AllK. pneumoniae 2e used as a template for amplification of the gene-encoding glycerol dehydratase was extracted as previously described [dhaB amplification are listed in Additional file E. coli BL21 (DE3) cells for the overexpression of 2eGDHt.The genomic DNA of escribed . The priE. coli BL21 (DE3) cells for recombinant protein overexpression.Site-directed mutation of 2eGDHt was conducted with the Site-directed Mutagenesis Kit  according to the manufacturer’s instructions. The primers used for site-directed mutagenesis are listed in Additional file E. coli BL21 (DE3) were inoculated into 100 mL of LB liquid medium with 50 μg/mL kanamycin at 37 °C until the OD600 of the cell reached 0.6. The protein was induced by the addition of 0.8 mM IPTG and was further incubated for 4 h at 37 °C. Cells were collected by centrifugation at 4 °C and 12,000g for 10 min and washed twice with PBS buffer . The cells were resuspended in lysis buffer  and disrupted by sonication for 5 min at 4 °C. The supernatant was harvested after centrifugation at 12,000g and 4 °C for 15 min. The recombinant protein containing a His-tag in the collected supernatant was purified by Ni–NTA resin  according to the manufacturer’s instructions. The purified proteins were analyzed by SDS-PAGE, and the protein concentration was measured with a BCA protein assay kit .The recombinant 12, and an appropriate amount of the enzyme samples. After incubation at 37 °C for 10 min, the enzyme reaction was terminated by the addition of 500 μL of 0.1% MBTH and incubation at 37 °C for 15 min. Subsequently, 1 mL of double-distilled H2O was added, and the amount of aldehyde formed was determined from the absorbance at 305 nm. The enzyme activity needed to produce 1 μM aldehyde under the assay conditions was defined as one unit of enzyme activity.The glycerol dehydratase activity was determined with the 3-methyl-2-benzothiazolinone hydrazine (MBTH) method . BrieflyThe substrate specificity of the enzyme was investigated with 0.2 M glycerol, 1,2-propanediol, 1,2-butanediol, butanol, methanol, ethanol, and isopropanol used as the substrates. The enzyme activities against different substrates were determined at 37 °C and pH 7.0.Km), maximum velocity (Vmax), and turnover number (Kcat) were obtained from the Michaelis–Menten equation in Lineweaver–Burk plots. The catalytic efficiencies were calculated with Kcat/Vmax.To assay the kinetic parameters of 2eGDHt for glycerol and 1,2-PDO, the enzyme was incubated with various substrate concentrations (2 to 200 mM) at the optimum temperature (37 °C) and pH (7.0). The Michaelis constant  and chemical reagents  to give final concentrations of 10 mM in the reaction mixture.The effects of metal ions on the enzymatic activity of 2eGDHt were tested by adding various metal ions : NaCl was added at 2%, 4%, 6%, and 8%; methanol was added at 7.5%, 10%, 12.5%, and 15%; and oleic acid and linoleic acid were added at 0.6%, 0.8%, 1.0%, and 1.2% . The mixThe MD simulations were conducted with the GROMACS 2022.2 software and the GROMOS CHARMM36m force field . The proAdditional file 1: Table S1. Primers used for PCR in this study.


The structural characteristics of materials after conventional sintering at 500–1300 °C were studied. It is demonstrated that the formation of a pure pyrochlore phase, Bi1.6Y0.4Ti2O7, occurs after high-temperature calcination. Complex oxides Bi2Ce2O7 and Bi1.6Y0.4Ce2O7 have a pyrochlore structure formed at low temperatures. Yttrium doping of bismuth cerate lowers the formation temperature of the pyrochlore phase. As a result of calcination at high temperatures, the pyrochlore phase transforms into the CeO2-like fluorite phase enriched by bismuth oxide. The influence of radiation-thermal sintering (RTS) conditions using e-beams was studied as well. In this case, dense ceramics are formed even at sufficiently low temperatures and short processing times. The transport characteristics of the obtained materials were studied. It has been shown that bismuth cerates have high oxygen conductivity. Conclusions are drawn about the oxygen diffusion mechanism for these systems. The materials studied are promising for use as oxygen-conducting layers in composite membranes.Synthesis and study of materials based on bismuth cerates and titanates were carried out. Complex oxides Bi Ts oxygen ,11,19,202B2O6O’ structure, O (48f Wyckoff positions) and O’ (8a Wyckoff positions) are non-equivalent and belong to two different sublattices ; however, while studying oxide ionic transport, these forms are sometimes undistinguishable, thus suggesting some kind of cooperative migration involving both O and O’ anions [The transport characteristics of pyrochlores are affected by their structural and defect features. In the real pyrochlore structure, various defects are present, including antistructural cation disordering:ts, etc. ,23,24,25’ anions ,26,27. F’ anions . The fea’ anions ,29 or bl’ anions  oxygen t2Ti2O7 at temperatures above 612 °C [As is known, the thermal instability of Bie 612 °C  is due te 612 °C ,33. Dopee 612 °C ,26. Henc4+/Ce3+ cations and a high oxygen vacancy content [Bismuth cerates were previously studied as photocatalysts  and inor content ,35,36. H0.3Bi0.7MnOx–Bi1.5Y0.3Sm0.2O3, etc.) using unique equipment of the Budker Institute of Nuclear Physics [2 as fuel and air as oxidant up to 500 mV/cm2 at 700 °C, which is promising for practical application. For an asymmetric membrane comprised of LaBiMnO-YSmBiO layers supported on a binary Ni-Al foam substrate, the oxygen flux under the air/He gradient was up to 5 mL O2 at 950 °C, which is really high [Obtaining functional ceramics (for solid oxide fuel cells and permselective membranes) with the required morphological and transport properties is a separate problem in materials science, where sintering is the most important procedure. With traditional thermal sintering in a furnace, long-term processing, and high temperatures are required to obtain ceramics with desired properties, such as gas tightness, homogeneous phase composition, etc. To solve this problem, radiation-thermal sintering (e-beam processing) is proposed ,39,40,41 Physics ,39,40,41 Physics ,39,40,41 Physics , etc.), 18O2 in the flow reactor to acquire data on the oxygen diffusivity required for these applications.This work aimed at studying the structural and oxide ionic transport properties of bismuth titanates and cerates, including Y-doped ones, and the effects of such processing as radiation-thermal sintering for pyrochlores. Its realization will provide the basis for manufacturing materials for solid oxide fuel cells or oxygen-conducting membranes. The effect of doping with Y on the structural stability and phase composition of these materials was studied. The sinterability of samples was investigated in order to check the adaptability of these materials to obtain gas-tight ceramics for electrochemical applications, including radiation-thermal sintering by e-beams. The oxygen transport properties were studied by the temperature-programmed isotope exchange of oxygen with C2Ce2O7 and Bi1.6Y0.4Ce2O7 were synthesized via the modified Pechini technique, as described elsewhere [1.6Y0.4Ti2O7 sample synthesized by the citrate method, corresponding salts in the required ratios were added to a solution of citric acid in water , while the Me:citric acid ratio was equal to 1:2. Bi(NO3)3·5H2O (>99%), Ce(NO3)3·6H2O (>99%), Ti (OCH(CH3)2)4 (>99%), and Y(NO3)3·6H2O (>99%) were used as initial reagents. The xerogels obtained were dried in a drying oven at 110 °C for 12 h, then calcined at 500 °C for 3 h. As-prepared powders were pressed into pellets and sintered at 700 °C for 3 h, at 900 °C for 3 h, at 1100 °C for 10 h, and at 1300 °C for 8 h using conventional sintering.Bilsewhere . For the−1, and after achieving 1100 °C, samples were sintered for 30 min.Radiation-thermal sintering was carried out using an accelerator, ILU-6 . Electron pulses with 2.4 MeV energy, 328 mA pulse beam current, a pulse duration ~600 s, a narrow scan, and up to 25 Hz pulse frequency were used. The temperature of the samples was controlled using a Pt-Pt-Rh thermocouple and FildPoint  controlling module. Power adjustment was carried out by changing the frequency of pulses. The heating rate was 50 C minθ range of 15–95° with a step of 0.05°. Rietveld refinement for quantitative analysis and calculation of lattice parameters was carried out using the software package Topas V.4.2.X-ray diffraction (XRD) studies were performed using a Bruker D8 Advance diffractometer with the Lynx-Eye detector using CuKα radiation. XRD patterns were recorded in the 2−1, 32 scans, resolution 4 cm−1) were acquired using a Cary 660  spectrometer with the GladiATR PIKE Technologies console.Infrared (IR) spectra . The experiments were performed in the secondary electron mode at an accelerating voltage of 20 kV.18O2 in the flow reactor. Samples (0.25–0.5 mm fraction) were loaded into quartz tube reactors (with an inner diameter of 3 mm). Pretreatment was carried out in He + 1% O2 flow (25 mL min−1) at 700 °C for 30 min. The isotope exchange was carried out in He + 1% C18O2 + 1% Ar flow (25 mL min−1) while heating from 50 to 800 °C with a constant ramp of 5 °C min−1. 18O atomic fraction (α) and C16O18O atomic fraction (f16–18) responses were analyzed in order to estimate isotope exchange kinetic parameters.Oxygen mobility and surface reactivity were studied by the temperature-programmed isotope exchange (TPIE) of oxygen with CCompositions and lattice parameters of all prepared samples are presented in 3+ into Bi2Ce2O7 decreases the lattice constant from 5.413 Å to 5.407 Å, thus evidencing substitution of ions in the lattice with contraction as expected, since the ionic radius of Y3+  is smaller than that of Bi3+   . With in−1 and 3401 cm−1 observed in IR spectra correspond to –OH symmetric vibrations appearing due to water adsorption (−1 corresponds to Bi–O bond vibrations [−1 is shown in −1 presents the stretching vibration in the structure of the cerium oxide (Ce-O-Bi) mode [According to IR spectroscopy data for both Bi cerate samples, the bands at 1631 cmsorption . The incbrations ,44,45. TBi) mode .1.6Y0.4Ti2O7 samples after sintering at 500 and 900 °C are not single-phased , sintering temperature, and duration, which affect the particles’ aggregation and growth. Hence, it was demonstrated that RTS allows for carrying out sintering processes for shorter times and at lower temperatures compared to those for conventional sintering. Such a difference in sintering temperature and duration required to obtain the desired gas-tightness for radiation–thermal sintering and conventional sintering is apparently related to the dissipation of radiation energy in heterogeneous structures, thermal-diffusional stimulation of mass transfer, and other features of the RTS process [ 1100 °C . A simil visible c,d. The  process .18O2 data for the bismuth cerate sample, a few peaks in the TPIE curve are visible, providing evidence of strong nonuniformity of oxygen mobility (−1. The most clearly expressed peak (150 °C) is determined by fast oxygen diffusion, which involves about 1/3 of the total oxygen content. This is probably oxygen-bound cerium. The rate of substitution of the rest of the oxygen is characterized by less-expressed peaks. For the description, the model including a single diffusion channel across the most mobile oxygen of Ce–O–Ce chains with subsequent exchange with neighboring more strongly bound oxide anions in the lattice was used [β) is 0.012 min−1 for bismuth cerate. The calculated parameters of the isotope exchange are given in According to the TPIE with Cmobility . Accordiwas used . The meaβ) is 0.017 min−1 for Y-doped bismuth cerate H2O +While compared with other Bi titanate-based pyrochlores, the oxygen tracer diffusion coefficient values of Y-doped Bi titanate are slightly lower than those for Sc-doped Bi titanate and significantly lower than those for Mg- and Zn-doped Bi titanates (It is difficult to compare the Bi cerate-based pyrochlores studied in this work with similar materials since there is a lack of information on the oxygen mobility of Bi cerates in the literature. However, it is comparable to or higher than that for Mg- and Zn-doped Bi titanates (As mentioned in the Introduction, pyrochlore-like oxide materials can be utilized in catalytic reactors based on oxygen ,2 and hyHence, undoped and Y-doped Bi cerates studied in the current work possessing a high oxygen mobility  meet the1.6Ti0.4Ce2O7, Bi2Ce2O7, and Bi1.6Y0.4Ce2O7 were synthesized using Pechini and citrate methods, and the structural characteristics after calcination using conventional and e-beam sintering were studied. It was shown that the formation of the pure pyrochlore phase Bi1.6Y0.4Ti2O7 occurs after high-temperature calcination at 1100 °C. In addition, the complex oxides Bi2Ce2O7 and Bi1.6Y0.4Ce2O7 have a fluorite structure with negligible amounts of Bi2O3 formed at low temperatures. As a result of calcination at high temperatures, the pyrochlore phase turns into a fluorite CeO2 phase enriched with bismuth oxide. RTS of bismuth titanate made it possible to obtain fine-grained ceramics in a minimum processing time and at a much lower temperature. The transport characteristics of pyrochlore samples were studied. It has been shown that bismuth cerates have high oxygen conductivity. Conclusions are drawn about the mechanism of oxygen diffusion in these systems. The obtained materials can potentially be used as oxygen-conducting layers of catalytic membranes.Complex oxides Bi