Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO2 + H2, although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution.Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon (cydA2B2), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions.Conclusion:C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes. Cupriavidus necator H16 is a particularly well-suited organism for C1 fermentation due to its ability to grow on CO2 + H2 to produce valuable bioproducts, including polymers and potentially chemicals and fuels , which under specific conditions enhanced PHB synthesis (Escherichia coli-produced anaerobic CODH from Acetobacterium woodii on its cell surface (C. necator show low affinity towards CO and appear to be unaffected by its presence (C. necator show low affinity for CO (C. necator (C. necator growth is likely to be significantly inhibited.Synthesis gas (syngas) is a promising energy rich feedstock for microbial fermentation , which ctabolise . C. necaynthesis . There h surface . The , with the addition of 15 g/L agar  if for solid agar medium. The pH value was adjusted to pH 6.9 using 1 M HCl. For counter selection during conjugation with E. coli S17-1, 10 μg/mL gentamicin was added to agar plates.All species and strains used in this study are listed in S medium  was usedE. coli was grown aerobically in LB at 37°C and liquid cultures shaking at 200 revolutions per minute (RPM) in 50 mL Falcon tubes. C. necator was grown in LB or MM at 30°C and liquid cultures shaking at 200 revolutions per minute (RPM) in 50 mL Falcon tubes.C. necator was cultured in serum bottles, which were sealed with butyl rubber stoppers and aluminium clamps. Under these conditions, the respective culture and serum bottle volumes were 20 mL F-MM and 150 mL, respectively, for heterotrophic growth, and 25 mL MM and 250 mL, respectively, for autotrophic growth. Different volumes of CO, N2, CO2 and H2 were injected into the serum bottles through a hypodermic needle and 0.2 µm nitrocellulose filter using a commercial gas exchange system (GR Instruments). Gas mixtures were injected in addition to the existing 1 bar atmospheric pressure to give a total of 2 bars (heterotrophic growth) or 2.4 bars (autotrophic growth).For growth under a defined headspace, 2/50% air (v/v). Heterotrophic growth under a CO atmosphere was initiated with a starting OD600 of 0.05 and heterotrophic growth under an N2 atmosphere was initiated with a starting OD600 of 0.02. For autotrophic growth, strains were first cultured in 5 mL F-MM to mid exponential phase, then washed with PBS. Autotrophic growth comparisons were performed with headspaces containing either 15% CO/65% H2/10% CO2/10% air or 30% CO/50% H2/10% CO2/10% air or 50% CO/30% H2/10% CO2/10% air. Control growth comparisons were performed with equivalent headspaces where CO had been replaced with N2, i.e., either 15% N2/65% H2/10% CO2/10% air or 30% N2/50% H2/10% CO2/10% air or 50% N2/30% H2/10% CO2/10% air. All autotrophic experiments were started using an OD600 of 0.01.For heterotrophic growth comparisons, three colonies of each strain were individually grown in 5 mL F-MM to mid exponential phase. These were then subcultured in 20 mL F-MM in serum bottles with either 50% CO/50% air (v/v) or 50% NE. coli were prepared and transformed by heat shock as previously described . Oligonucleotide primers from Sigma-Alrich were used for PCR amplifications, performed using either the DreamTaq PCR Master Mix (×2) (Thermofisher Scientific) or Q5 polymerase (New England Biolabs). The former was used for screening of clones by colony PCR whereas Q5 polymerase was used for amplification of sequences for cloning. Restriction enzymes were purchased from NEB and “FastDigest” enzymes from Fermentas (Thermofisher Scientific). The Qiagen DNA Gel Extraction Kit was used to extract gel purified DNA for subsequent cloning. Chemically competent escribed . PlasmidC. necator H16 as previously described  upstream and downstream of the six deletion sites were PCR-amplified from the C. necator genome with primers containing overlapping regions (All plasmids were constructed using NEBuilder HiFi DNA assembly (New England Biolabs) following the manufacturer’s instructions. Derivatives of pLO3  were useescribed . The tet regions . The lefC. necator H16, a single colony was used to inoculate 5 mL F-MM and grown to an OD600 of around 2. This culture was used to inoculate ten separate 150 mL serum bottles containing 20 mL F-MM under a 2 bar atm of 50% CO/50% air (v/v). These cultures were shaken at 200 RPM in a 30°C incubator for at least 48 h to reach an OD600 of around 2. This culture was used to inoculate 20 mL F-MM to an OD600 of 0.01 in a fresh serum bottle and this was incubated under the same conditions. This subculturing cycle was repeated until cultures showed a clear improvement in growth. The cultures were monitored 18 h after each inoculation to observe whether the OD600 had dramatically increased. After this length of time wild type cultures would typically yield an OD600 of around 0.1, whereas evolved cultures yielded an OD600 of around 1, which was visually distinguishable. The purity of these cultures was checked taking a 1 mL sample of OD600 cells and making serial dilutions, plating out 10−5 on F-MM agar plates. Single colonies were screened by colony PCR using primers for the hsdRN gene (H16_A0006) to ensure they did represent H16 strain derivatives. Samples were taken every three subcultures and preserved in case they needed reanalysing or as a backup if cultures became contaminated. Single colonies of CO tolerant evolved cultures where than obtained by plating. To evolve the H16 strain under autotrophic conditions, a similar growth regime was used. A single H16 colony was grown in 5 mL F-MM pre-culture, then diluted to an OD600 of 0.01 in 250 mL serum bottles containing 25 mL MM and a headspace of 15% CO/65% H2/10% CO2/10% air (v/v) under 2.4 bar pressure. Following 18 days of incubation at 200 RPM in a 30°C incubator, cultures were used to reinoculate fresh serum bottles to an OD600 of 0.01 with higher CO headspace concentrations of 30% and 50%, respectively (represented by gas mixtures consisting of 30% CO/50% H2/10% CO2/10% air (v/v) and 50% CO/30% H2/10% CO2/10% air (v/v), respectively, under 2.4 bar pressure). The subculturing cycle was then repeated under the 50% CO atmosphere until cultures took only 3 days to reach stationary phase. Single colonies of CO tolerant evolved cultures were then obtained by plating.For the heterotrophic evolution of CO-tolerant ® M1000 PRO micro plate reader . 100 μL of sample was pipetted into a well of a black 96-well plate . Fluorescence was measured using 585 nm as excitation wavelength and an emission wavelength of 620 nm. The gain factor for fluorescence was set manually to 80%. The OD600 of each sample was measured using the micro plate reader to normalise fluorescence by optical density. To correct for background fluorescence, the normalised fluorescence of the empty vector control -encoding biosensor plasmid pEH006  and an Ihttps://www.ncbi.nlm.nih.gov/sra) and can be found under BioProject ID: PRJNA945825. The accession numbers for the strains used or created in this study are as follows: parental H16 wild type, SRX19749121; EA, SRX19746595; EB, SRX19746596; EC, SRX19746603; E1, SRX19746604; E2, SRX19746605; E3, SRX19746606; E4, SRX19746607; E5, SRX19746608; E6, SRX19746609; E7, SRX19746610; E8, SRX19746597; E9, SRX19746598; E10, SRX19746599; SynE1, SRX19746600; SynE2, SRX19746601; SynE3, SRX19746602.All raw NGS data used in this study is deposited in the NCBI Sequence Read Archive , where their in-house software was used to trim sequencing reads and assess their quality. The “Standard Whole Genome Service” included Illumina next-generation sequencing with a guaranteed minimum coverage of 30x. The published genomes of ator H16  was usedThe mapped reads were used to call single nucleotide polymorphisms (SNPs) by using CLC Genomics Workbench 22 (Qiagen). The following parameters were used for the Mapping and Variants workflow: Ploidy = 1; Ignore positions with coverage above = 100,000; Restrict calling to target regions = not set; Ignore broken pairs = yes; Ignore non-specific matches = reads; Minimum coverage = 10; Minimum count = 2; Minimum frequency (%) = 80.0; Base quality filter = yes; Neighbourhood radius = 5; Minimum central quality = 20; Minimum neighbourhood quality = 15; Read direction filter = no; Relative read direction filter = yes; Significance (%) = 1.0; Read position filter = no; Remove pyro-error variants = yes; In homopolymer regions with minimum length = 5; With frequency below = 0.7.t-test for side-by-side comparisons of the wild type and a given mutant under a particular condition, or for side-by-side comparisons of the same strain under two different conditions, with p values below 0.05 considered to be statistically significant.All data are presented as mean ± standard deviation (SD) with the number of independent replicas given in the respective figure legends. Statistical analyses were performed using GraphPad Prism software version 9 , employing a paired C. necator H16 is significantly inhibited by high concentrations (90%) of CO  together with air in the headspace.When grown in a complex medium with pyruvate, %) of CO , howeverC. necator growth. Pre-cultures were grown in fructose minimal medium (F-MM) and used to inoculate serum bottles containing F-MM and headspaces comprised of 50% air with different CO and N2 mixtures (C. necator reached stationary phase after 24 h but with 15% (v/v) CO, the lag phase was increased, with cultures taking 30 h to reach stationary phase   without organic carbon source and headspaces comprised of syngas-like mixtures with CO concentrations ranging from 0% to 50% (v/v) (p = 0.00001)  was chosen as the maximum CO concentration as it is within the range typically observed for syngas mixtures and because increasing above this threshold would have severely reduced the volume of air (and thus oxygen) available in the headspace, with further negative effects on mixtures . In the ry phase  and dispor both) . For gro0% (v/v) . A signi0.00001) , with ce0.00001) . When 30C. necator can evolve to tolerate high concentrations of CO adaptive laboratory evolution was carried out under both heterotrophic and autotrophic conditions as described below.To establish whether 600 of approximately 3, then reinoculated into a fresh serum bottle diluted to an OD600 of 0.01 every 3 days. The purpose of this pilot experiment was to establish whether CO tolerance of the H16 strain could be improved in principle and also the degree of growth improvement that might be expected within a reasonable timeframe. After eighty-one subcultures equivalent to an estimated 665 generations and carried out over 8 months, three isolates designated EA, EB, and EC were obtained from the evolved culture, and shown to display a clear growth advantage over the H16 parent strain under the employed CO growth regime, with a significantly higher growth rate and shorter lag phase but similar final OD  (Since growth was still observed for heterotrophic H16 cultures in the presence of 50% (v/v) headspace CO , this cosolates) .C. necator H16 could be evolved to a faster growing more CO tolerant phenotype, the experiment was repeated on a larger scale, with ten independently evolving lineages. For this, a single colony of C. necator H16 was cultured in F-MM. The genomic DNA of this culture was extracted and the remaining part used to inoculate ten separate pre-cultures in F-MM which were then independently subcultured in the presence of CO as described above, resulting in ten separate cultural lineages designated Evo1 to Evo10. This continued until a significant visible change in OD appeared within 18 h of re-inoculation, which would signify that the cells had adapted to the presence of CO. This was observed around the 15th round of re-inoculation, at which point stocks were generated for all cultures and preserved at −80°C. The evolved cultures were plated out and single colonies were isolated to represent defined strains designated isolate E1 to E10, according to their culture origin . A growth comparison between the unevolved H16 parent strain and E1 to 10 was carried out under the same subculturing conditions in the presence and absence of 50% CO , hereafter referred to as 50% N2) both H16 parent strain and E1 to E10 isolates reached peak OD600 after 28 h , therefore it was assumed that further ALE of these strains would not lead to further improvements in CO tolerance.The growth rate of the 8-month pilot isolates EA to EC was not significantly different to any of the obtained growth rates for E1 to E10 (Illumina next-generation sequencing (NGS) was performed as described in the Materials & Methods for the evolved isolates E1 to E10, as well as isolates EA, EB and EC from the 8-month pilot evolution experiment . For thibd ubiquinol oxidase operon (hereafter referred to as cytochrome bd) was detected in all ten isolates E1 to E10 and in all three isolates EA to EC from the initial pilot culture (phcA gene itself (T→G) for isolate E8, changing a leucine to an arginine codon. Two point mutations were found within the RNA polymerase sigma factor 54 (rpoN) gene. A C→A transversion converted a serine codon into a stop codon and was found in two of the isolates (E6 and E7) and a A→C transversion changed a glutamine codon into a proline codon and was only seen on one occasion (E1)  upstream of the cytochrome  culture . A 12 bp culture . The delion (E1) .bd ubiquinol oxidase II (encoded by cydA2B2) is one of two cytochrome bd terminal oxidases  of the C. necator electron transport chain (cydA2B2 (G→A) may alter expression of the cydA2B2 gene. To test this hypothesis, BPROM . The slightly improved growth of these mutants as seen in 600 and/or somewhat faster growth compared to the wild type in the later stages of growth. Under the N2 atmosphere, the wild type reached peak OD600 after 28 h . Similar to what was observed in the presence of CO, the improved growth of these mutants as seen in cydA2B2 had no direct effect on CO resistance but generally improved in vitro growth performance under serum bottle conditions.Heterotrophic growth of the single knock-in mutants was compared with the H16 wild type in the presence and absence of 50% CO . Under tter 48 h . In contter 28 h . Interesbd operon cydA2B2 appeared to be the only mutation that gave C. necator significantly increased tolerance to CO. Since the mutation was not in a protein-encoding region, it may be altering operon expression; therefore, further investigation into this operon was carried out, using three approaches. First, the upstream intergenic region of the operon was tested for promoter activity using an RFP-based reporter plasmid to elucidate whether introduction of the point mutation increased the level of expression as previously hypothesised. Second, the operon was overexpressed in an otherwise wild type background, again to determine the resulting effect on CO tolerance. Additionally, the two subunit-encoding genes of the operon were knocked out to determine how this impacted on CO tolerance.The single point mutation (G→A) 210 bp upstream of the cytochrome cydA2B2, two RFP-based reporter plasmids were constructed containing the entire 616 bp upstream intergenic region of cydA2B2 and the backbone of pEH006  and an equivalent negative control vector lacking the pBAD promoter (pEH006E) , so that the strength of the predicted promoter on pCWS13 could be compared to the output given by different inducer concentrations.To ascertain whether the G→A mutation influenced gene expression of f pEH006 . One plapEH006E) . The lat2, C. necator pCWS13 produced approximately 1000-fold more fluorescence than C. necator pCWS12 and following 48 h around 500-fold more (p < 0.00001 for each time point)  . LikewiscydA2B2 would positively influence CO tolerance, an expression plasmid containing these genes was constructed and introduced into C. necator. This plasmid, pCWS14, was comparable to the arabinose inducible RFP reporter pEH006, but contained the cydA2B2 operon in place of rfp. A derivative of pEH006, lacking the RFP gene (pCWS15) was also created and used as a control plasmid. The level of fluorescence produced by pCWS13 after 24 h was comparable to pEH006 induced with 0.1 mM and after 48 h to pEH006 induced with 0.2 mM arabinose   (C. necator pCWS14 (p = 0.006), which reached stationary phase after 45 h, 18 h earlier than C. necator pCWS15, presumably due to increased expression of cydA2B2. In the presence of 0.2 mM arabinose, C. necator pCWS15 displayed no further improvement in growth; however, C. necator pCWS14 exhibited reduced growth, reaching a lower peak OD600 than with 0 or 0.1 mM arabinose and exhibiting a reduced growth rate  . Additio > 0.05) . Howeverwth rate . Taken t2, both C. necator pCWS15 and pCWS14 displayed no significant difference in growth rate with 0 mM, 0.1 mM or 0.2 mM arabinose , all reaching stationary phase after 26 h .When grown under 50% Nter 26 h . Howeverce of CO . A reduca pCWS17 , encodinbd impacts on the capacity of C. necator to tolerate CO, a knock-out plasmid derived from pLO3 was constructed (pCWS11) to delete the cydA2B2 operon as described in the Methods. The obtained knock-out mutant was also genetically complemented using the knock-in plasmid pCWS16 to reintegrate cydA2B2 into the original deletion site. Growth of the generated cydA2B2 knock-out mutant (C. necator cydA2B2 KO) was compared to that of the H16 wild type, complemented mutant (C. necator cydA2B2 KI) and the generated, defined SNP knock-in strain cydA2B2 SNP. Under a 50% CO atmosphere the wild type strain as well as cydA2B2 KI reached peak OD600 after 47 h and the cydA2B2 SNP strain after 27 h . The complemented mutant strain cydA2B2 KI did not show a significant difference in growth rate to the wild type H16 strain (p = 0.469). Under a 50% N2 atmosphere, the wild type, cydA2B2 SNP, cydA2B2 KO and cydA2B2 KI strains reached their peak OD600 after 28 h (p > 0.05); bd did not noticeably impact on the growth of C. necator in the absence of CO.To investigate whether the lack of cytochrome ter 27 h . In contth rates , which wter 28 h . There wcydA2B2 plays a role in CO resistance when C. necator is grown autotrophically, a growth comparison between the H16 wild type, cydA2B2 SNP and E1 strains was performed with a syngas-like mixture containing 15% CO/65% H2/10% CO2/10% air (v/v). Neither cydA2B2 SNP nor E1 displayed a significantly increased growth rate compared to the wild type (p > 0.05 for both strains) (bd is limited to heterotrophic growth.To investigate whether strains)  (Supplemstrains) . These rC. necator H16 can adapt to autotrophic growth in the presence of high CO concentrations, a single colony was grown in an F-MM pre-culture, then diluted to an OD600 of 0.01 in a serum bottle containing MM and a headspace of 15% CO/65% H2/10% CO2/10% air (v/v), based on the established tolerance threshold shown in To observe whether 600 of approximately 0.7 over 18 days, then reinoculated to an OD600 of 0.01 into a fresh serum bottle containing higher CO headspace concentrations of either 30% and 50%, respectively (represented by gas mixtures consisting of 30% CO/50% H2/10% CO2/10% air (v/v) and 50% CO/30% H2/10% CO2/10% air (v/v), respectively). These cultures were now able to reach stationary phase after 4 days and therefore continued subculturing with 50% CO was carried out as described above until a shift occurred following the eighth round of reinoculation, whereby the culture took 3 days to reach stationary phase. When compared to the H16 parent strain as previously described, following plating and testing of single colonies, the selected evolved isolates, designated synE1, synE2 and synE3, displayed a significantly increased growth rate (p < 0.0001 for each isolate) when grown in the presence of 15% CO  (These cultures were grown to an ODf 15% CO . The evof 15% CO . In the  > 0.05) .cydA2B2 SNP strain was subjected to the same autotrophic evolution regime described above. The autotrophic growth of three isolates from the evolved culture, designated cydA2B2 Evo1, cydA2B2 Evo2 and cydA2B2 Evo3, was compared against the synE1, cydA2B2 SNP and wild type H16 strains on MM under a syngas-like atmosphere containing 15% CO as described above (cydA2B2 Evo isolates and synE1 reached peak OD600 after 52 h and both the wild type and cydA2B2 SNP strains after 20 days. There was a significant difference in growth rate between the cydA2B2 Evo isolates and cydA2B2 (p < 0.01 for each isolate) as well as the wild type (p < 0.01 for each isolate), indicating clear adaptation to the syngas atmosphere (cydA2B2 Evo and synE1 (p = 0.781).It was also desirable to obtain a strain that exhibited increased CO tolerance under both autotrophic and heterotrophic conditions, and hence the ed above . The cydmosphere . There wcydA2B2 SNP and cydA2B2 Evo isolates (p = 0.445), reaching stationary phase at the same time (cydA2B2 SNP and the cydA2B2 Evo isolates grew significantly faster than the wild type and synE1 (p < 0.01 each comparison).Additionally, to confirm whether the synE1 strain had also developed heterotrophic CO resistance, the growth of this strain on F-MM and 50% CO/50% air (v/v) was compared to the wild type, isolates . SynE1 dame time . WhereashoxH, one of the two large subunits of the soluble hydrogenases in C. necator  to an OD600 of 0.01. In the presence of CO, hoxH SNP displayed a significantly faster growth rate than the wild type and was able to reach peak OD600 after 72 h (p = 0.30). In contrast, as might be expected due to its soluble hydrogenase deficiency, hoxH KO was not able to grow within the time frame of the experiment. In the absence of CO, only hoxH KO displayed a significantly different growth rate to the wild type (p = 0.00001)  , taking C. necator H16 that have adapted to growth in the presence of high CO concentrations, to gain insights into potential molecular and physiological resistance mechanisms as well as strategies to engineer better performing strains.A key issue when considering syngas and similar industrial waste gases as feedstocks for industrial fermentations is their high content of CO and associated toxicity. Bacteria show varying degrees of sensitivity to CO, with respiration and growth being inhibited in the presence of the gas , althougC. necator H16 was already remarkably tolerant to the gas under the tested heterotrophic conditions, being able to grow with 50% CO in the headspace albeit with extended lag phases and reduced growth rates, in agreement with previous reports   fits into this picture  , but als (CORMS) , consistg CORM-3 . How the picture  has yet C. necator, E. coli and Aquifex aeolicus which are not significantly affected in their activity by CO (C. necator H16 this includes both membrane-bound (MBH) and soluble hydrogenase (SH). Hence, it was intriguing to find that a single base change in hoxH, encoding the larger, NiFe active site carrying subunit of the heterodimeric hydrogenase moiety of SH, had such a marked effect on autotrophic CO tolerance. This mutation, which was present in both evolved lines , changed proline 288 to a serine and this change was independently confirmed to confer increased CO tolerance by engineering it into the H16 wild type. This suggests that at least under in vivo conditions SH tolerance to CO can be further improved. In future studies it would be interesting to establish how the proline to serine mutation impacts on HoxH’s structure and specifically its catalytic NiFe center.Bacterial “standard” [FeFe] and [NiFe] hydrogenases are usually strongly inhibited by CO . A notabty by CO . For C. During heterotrophic ALE, several loci including those encoding the putative quorum sensing system Phc and the alternative sigma factor RpoN (sigma 54) accumulated mutations in several of the independently evolved lines, suggesting that these changes conferred a fitness benefit under the employed conditions. Since the tested SNPs and deletions did not increase CO tolerance when introduced into the unevolved H16 parent strain, it seems reasonable to assume that they represented adaptations to the specific serum bottle conditions used for the ALE experiments.C. necator H16 can be metabolically engineered to utilise CO derived carbon by introducing and expressing a heterologous CO dehydrogenase. Employing a similar approach for our evolved strains, it would be interesting to test whether this improves growth and CO incorporation of syngas-fed cultures.Previous work by


This study investigates the early effects of PGF2α signaling on mitochondrial dynamics and mitophagy in bovine corpora lutea. Luteolytic mediator PGF2α, via PKC/ERK and AMPK signaling, activates mitochondrial fission and promotes PINK–Parkin mitophagy, placing mitochondria as novel targets in response to PGF2α. Prostaglandins are arachidonic acid-derived lipid mediators involved in numerous physiological and pathological processes. PGF2α analogues are therapeutically used for regulating mammalian reproductive cycles and blood pressure, inducing term labor, and treating ocular disorders. PGF2α exerts effects via activation of calcium and PKC signaling, however, little is known about the cellular events imposed by PGF2α signaling. Here, we explored the early effects of PGF2α on mitochondrial dynamics and mitophagy in the bovine corpus luteum employing relevant and well characterized in vivo and in vitro approaches. We identified PKC/ERK and AMPK as critical protein kinases essential for activation of mitochondrial fission proteins, DRP1 and MFF. Furthermore, we report that PGF2α elicits increased intracellular reactive oxygen species and promotes receptor-mediated activation of PINK–Parkin mitophagy. These findings place the mitochondrium as a novel target in response to luteolytic mediator, PGF2α. Understanding intracellular processes occurring during early luteolysis may serve as a target for improving fertility.  The corpus luteum is a transient endocrine gland that secretes the steroid hormone progesterone to maintain pregnancy , 2. ThisDNM1L; commonly referred to as DRP1) is a key mitochondrial GTPase responsible for controlling mitochondrial fission and is a major contributor to the manifestation and pathogenesis of various diseases  is a protein kinase that works cooperatively with the E3 ubiquitin ligase, Parkin, to monitor the mitochondrial state and tag damaged mitochondria for degradation . In healProper control of the life span of the corpus luteum is essential for the establishment and maintenance of pregnancy in mammals . In the P < 0.01; P < 0.05; P > 0.05; P > 0.05; To evaluate the early temporal effects of PGF2α on progesterone production, cows were administered a single dose of saline or PGF2α (i.m.) and corpora lutea were collected at zero time, 1, 2, and 4 h posttreatment. Serum progesterone was decreased 2 h postinjection of PGF2α  in phosphorylation of DRP1 at Ser616 1 h posttreatment with PGF2α and a 3.7- and 3.6-fold increase 2 and 4 h post-PGF2α, respectively . Moreover, PGF2α stimulated a 1.7-fold increase in phosphorylation of MFF at Ser146 1 h posttreatment with PGF2α and a 1.5- and 1.4-fold increase 2 and 4 h post-PGF2α, respectively . Levels of total DRP1 and MFF protein expression were unchanged in response to PGF2α . Immunohistochemistry of luteal tissue revealed an observed increase in the phosphorylation of DRP1  in phosphorylation of AMPK at Thr172 1 h posttreatment with PGF2α and a 6.5- and 6.8-fold increase 2 and 4 h post-PGF2α, respectively  to analyze expression of transcripts for bovine granulosa, large, and small luteal cells , 43DNM1LP < 0.05; P < 0.05, P < 0.05; P < 0.05; To determine the influence of luteotropic and luteolytic agents on the differential phosphorylation of DRP1, enriched populations of large luteal cells were treated for 30 min with luteotropic agents (LH or the adenylyl cyclase activator forskolin [FSK]) or luteolytic agents  . TreatmeP < 0.05; P < 0.05; P < 0.05; Because our in vivo results showed that the localization of phospho-DRP1 (Ser616) appears to ensue in large luteal cells, enriched populations of large luteal cells were prepared and treated with luteolytic hormones, PGF2α 100 nM;  or TNFα P < 0.05; During the early stages of luteolysis, PGF2α stimulates an increase in inflammatory cytokines, such as TNFα, interleukins , and cytokine signaling intermediates , all of which may contribute to luteal regression . Large lP < 0.05; P > 0.05; P < 0.05; As alluded to in previous section, the luteolytic actions of PGF2α manifest through receptor-mediated stimulation via the PKC signaling pathway and activation of downstream protein kinases. Large luteal cells were treated with PMA 20 nM;  for up tP < 0.05; P < 0.05; To determine the role of PKC/MAPK signaling on the phosphorylation of DRP1 at Ser616, enriched populations of large luteal cells were treated up to 24 h with PGF2α (100 nM) in the presence or absence of commercially available small molecule inhibitors . PKC inhP < 0.01) 1 h posttreatment and remains elevated for at least 6 h when compared with control  to promote fission of mitochondria. Large luteal cells were treated with PGF2α 100 nM;  or TNFα To determine whether phosphorylated DRP1 (Ser616) translocates to the mitochondria after PGF2α treatment, mixed luteal cell cultures were stimulated with PGF2α (100 nM) or PMA (20 nM) for 1 h and mitochondria were immediately isolated. Cultures of dispersed mixed luteal cells were used for mitochondrial isolation because of low cell yield after enrichment of large luteal cells. After incubation, the cells were fractionated, and aliquots of total cell lysate, cytosolic, and mitochondrial fractions were subject to Western blotting. We observed abundant nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitors, alpha (IκBα) and alpha-tubulin (TUBA) in the cytosolic fractions but not mitochondrial fractions . We alsoP < 0.05; P < 0.05; P < 0.05; The mitochondrial receptor MFF was recently was identified as a downstream substrate of AMPK signaling . To deteP < 0.0001; P < 0.0001; P < 0.001; P < 0.0001; Because our data strongly support the hypothesis that PGF2α acutely influences mitochondrial dynamics through phosphorylation of DRP1 and MFF, we set out to determine the effects of PGF2α on mitochondrial morphology using confocal microscopy . MitochoP < 0.0001; Transmission electron microscopy was employed to observe PGF2α-induced morphological changes to mitochondrial size and cristae organization . Under bP > 0.05; P < 0.0001; Mdivi-1 is a highly efficient small molecule inhibitor of mitochondrial fission. Mdivi-1 binds to an allosteric site blocking conformational change necessary for DRP1 self-assembly and GTP hydrolysis required for mitochondrial division . To inveP < 0.0001; ROS are highly reactive molecules that, if unchecked, can cause intracellular damage . PreviouP < 0.05; P > 0.05; P < 0.05; Mitochondrial fission and mitophagy are two cellular mechanisms that synchronously regulate mitochondrial quality control systems to protect cells from cytotoxic ROS production . AMPK isP < 0.05; P < 0.05; P < 0.05; P < 0.05; We and others have reported that MFF is a downstream substrate of AMPK signaling . MoreoveP < 0.01) and remained elevated 4 h posttreatment in large luteal cells  within 30 min of treatment and 1.8-fold for 1 and 4 h posttreatment when compared with control  for up to 4 h and subject to Western blotting . PGF2α iP < 0.01; P < 0.0001; To determine the effects of PGF2α on mitophagy activation, we stimulated enriched populations of large luteal cells with PGF2α (100 nM) for 6 h and visualized mitophagy by confocal microscopy using Mtphagy Dye . Under bIn the present study, we delineate the effects of the luteolytic hormone, PGF2α, on mitochondrial dynamics and activation of mitophagy in bovine luteal cells. To our knowledge, the present study provides the first demonstration in any tissue that PGF2α signaling (1) stimulates the phosphorylation of DRP1 and MFF, (2) stimulates mitochondrial fission, (3) promotes an increase in intracellular ROS production, and (4) activates mitophagy of damaged mitochondria. These findings indicate that DRP1 is a PGF2α/PKC-sensitive molecule, and both mitochondrial dynamics and mitophagy are targets of PKC and AMPK signaling in large luteal cells during the initiation of luteolysis.The fate of the corpus luteum is governed by luteotrophic  or luteolytic hormones . We recently reported that LH, via the PKA signaling pathway, reduces DRP1 activity and association with mitochondria, thus stabilizing mitochondria to steroidogenesis, a process required for progesterone synthesis in luteal cells . Here, wMitochondria undergo continual cycles of fusion and fission to regulate morphology and meet energy demands. Steroidogenesis is a complex process that requires the fusion of mitochondria to precede . MitochoMitochondria represent a major source of intracellular ROS generation. ROS are highly reactive molecules that, if unchecked, can cause intracellular damage . PKC actMitochondrial fission and mitophagy are two cellular mechanisms that synchronously regulate mitochondrial quality control pathways and protect cells from cytotoxic ROS production . PINK1 aThe mechanisms involved in luteolysis are highly complex, species-specific, and not well understood. Here, in vivo administration of PGF2α results in a precipitous decline (52%) in systemic progesterone concentration 2 h after treatment. This is consistent with the current notion that serum progesterone concentrations fall in parallel with luteal blood flow during functional regression of the gland. Interestingly, intraluteal tissue progesterone concentrations were also decreased (54%) 4 h after PGF2α treatment, yet, no changes in steroidogenic enzymes, STAR, CYP11A1 or HSD3B, protein expression  or mRNA A limitation of the current study is that this study examined the luteal response to a single luteolytic dose of a potent PGF2α analogue commonly used to regulate the reproductive cycle. However, physiological luteolysis occurs in response to multiple sequential pulses of uterine-derived PGF2α . In addiLuteolysis is a natural event necessary to regulate the female estrous cycle. An adequate corpus luteum function is, however, essential for the establishment and maintenance of pregnancy. Defects in luteal function are associated with implantation failure and premature termination of pregnancy. In the present study, we provide evidence that luteolytic hormones modulate mitochondrial dynamics by increasing the phosphorylation and activity of DRP1 and MFF. Furthermore, we bring forward the notion that PGF2α induces mitochondrial fission and uncoupling of cristae junctions, a process that requires proper integration of PKC and AMPK signaling pathways. We highlight PKC/ERK as a key upstream regulator of DRP1 phosphorylation and AMPK as a crucial regulator associated with phosphorylation of MFF. In conjunction with shifts in mitochondrial dynamics, PGF2α triggers intracellular rises in ROS production and activation of mitophagy. Together, these findings signify that PGF2α de-stabilizes luteal mitochondria as a proximal event during luteolysis. Taken together, our findings place the mitochondria as a novel target downstream of PKC and AMPK signaling in response to the luteolytic lipid mediator, PGF2α. Understanding the cellular processes involved with early luteal regression may serve as a target for improving fertility.The luteal phase is often overlooked in fertility research and the present findings may be relevant to human reproduction. Although the initial signal for luteolysis in non-human primates and women is a loss of gonadotropic support , studies4O2O7, Na3VO4, Triton X-100, glycerol, dodecyl sodium sulfate, β-mercaptoethanol, bromophenol blue, Tween-20, paraformaldehyde, phorbol 12-myristate 13-acetate (PMA), SP600125 (JNK inhibitor), and SB207580 (p38 MAPK inhibitor) were purchased from Sigma-Aldrich. The phosphate buffer solution, DMEM , penicillin–streptomycin solution, Trypan Blue, Halt Protease, and Phosphatase Inhibitor Cocktail were purchased from Invitrogen Corporation (Thermo Fisher Scientific). The opti-MEM, M199 culture medium, and gentamicin sulfate were purchased from Gibco (Thermo Fisher Scientific). Collagenase was purchased from Atlanta Biologicals (Flowery Branch). Prostaglandin F2α was purchased from Cayman Chemical and bovine LH was purchased from Tucker Endocrine Research Institute. Recombinant TNFα was purchased from R&D systems. Go6983, PKC inhibitor was purchased from Abcam. No. 1 glass coverslips, microscope slides, and chemiluminescent substrate  were from Thermo Fisher Scientific. Fluoromount-G and clear nail polish were purchased from Electron Microscopy Sciences. FSK and compound C was purchased from EMD Millipore. BCA protein assay and 4–20% Mini-PROTEAN TGX precast protein gels were purchased from Bio-Rad and the nonfat milk was from a local Kroger. Mitochondrial isolation kit was purchased from QIAGEN . Mdivi-1 and AICAR were purchased from Tocris. An ELISA kit for progesterone was purchased from DRG International, Inc. ImmuChemTM Coated Tube Progesterone 125I RIA kit was purchased from ICN Pharmaceuticals, Inc. Penicillin G-sodium, streptomycin sulfate, HEPES, BSA, deoxyribonuclease l, FBS, Tris–HCl, sodium chloride, EDTA, EGTA, sodium fluoride, Na3 sections were snap-frozen in liquid N2 for subsequent protein analysis or fixed in 10% formalin for immunohistochemistry. The University of Nebraska–Lincoln Institutional Animal Care and Use Committee approved all procedures and facilities used in this animal experiment and animal procedures were performed at the University of Nebraska—Lincoln, Animal Science Department.Post-pubertal, non-lactating multiparous female cattle (n = 6) of composite breeding  beef cows from the beef physiology herd at the Eastern Nebraska Research and Extension Center (ENREC), were used in this study. Cows were synchronized using two intramuscular injections of PGF2α  11 d apart. At mid-cycle (days 9–10), the cows were treated with an intra-muscular injection of saline (n = 3) or PGF2α . At each of four time-points postinjection , three cows per treatment were subjected to a bilateral ovariectomy through a right flank approach under local anesthesia as previously described , 73, 74.2 gas. The samples were reconstituted in 1 × ELISA buffer at a 1:200 dilution.Progesterone was extracted from luteal tissue homogenate using a double-extraction procedure before ELISA assay as described . BrieflyPlasma progesterone concentrations were determined using a RIA to detect progesterone concentrations as previously described . Progestg at 4°C for 15 min. Protein was collected and concentrations were determined using BCA protein assay. Samples were suspended in 6× Laemmli buffer and placed on a dry heat bath at 100°C for 6 min.Approximately, 100 mg of tissue was homogenized in RIPA buffer supplemented with 1× Halt Protease and Phosphatase Inhibitor Cocktail and sonicated at 40% power setting  as previously described . After sProteins (30 μg/sample) were resolved using 10% SDS–PAGE or 4–20% Mini-PROTEAN TGX precast protein gel and then transferred to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline + 0.1% Tween-20 (TBS-T) containing 5% nonfat milk solution at room temperature for 1 h. The membranes were incubated in a primary antibody  for 24 hTotal proteins were normalized to ACTB before calculation of fold induction. The ratio of phosphorylated DRP1 to total DRP1 was determined for each treatment and time point. Fold increases because of the treatment (control versus PGF2α) were then calculated.Portions of ovaries containing corpora lutea obtained from cows treated with an intramuscular injection of saline (n = 3) or PGF2α  were fixed in 10% formalin for 24 h and then changed into 70% ethanol until embedded in paraffin. Tissues were cut into 4 μm sections and mounted onto polylysine-coated slides. Slides were deparaffinized through three changes of xylene and through graded alcohols to water and microwaved in unmasking solution (Vector H-3300) for antigen retrieval. Endogenous peroxidase was quenched with 0.3% hydrogen peroxide in methanol for 30 min. Sections were incubated with anti-DRP1, anti-phospho-DRP1 (Ser616 or Ser637), anti-phospho-MFF (Ser146) or anti-MFF as indicated in GSE83524) to analyze the expression of steroidogenic machinery in freshly isolated bovine granulosa , large luteal , and small luteal  cells from mature corpora lutea. Details of the isolation and analysis were previously published  in basal medium  for 45 min in spinner flasks at 35°C. The supernatant was transferred to a sterile 15 ml culture tube, washed three times with sterile PBS, and re-suspended in 10 ml of elutriation medium  on ice. Fresh dissociation medium was added to the remaining undigested tissue and incubated with agitation for an additional 45 min and processed as described above. Viability of cells was determined using Trypan Blue and cell concentration was estimated using a hemocytometer before cell elutriation.Freshly dissociated cells were resuspended in 30 ml elutriation medium. Dispersed luteal cells were enriched for small and large luteal cells via centrifugal elutriation as previously described . Cells w5 cells/well and 2 × 105 cells/well, respectively. Cells were cultured in culture media  at 37°C in an atmosphere of 95% humidified air and 5% CO2 as described above.Enriched populations of small and large luteal cell cultures were plated in 12-well culture dishes at 5 × 102 for 2 h.Before treatments, cells were rinsed with PBS and fresh culture medium was placed on cells and equilibrated at 37°C in atmosphere of 95% air and 5% CO2.To determine the influence of luteotrophic and luteolytic hormones on the differential phosphorylation of DRP1, enriched populations of small and large luteal cells were treated with the culture medium alone, FSK , PGF2α (100 nM), phorbol 12-myristate 13-acetate , TNFα  or luteinizing hormone  for 30 min at 37°C in atmosphere of 95% air and 5% CO2For time response experiments with drug inhibitors , cells were pretreated for 1 h and then subsequently treated with culture medium alone or PGF2α (100 nM) for 0, 0.5, 1, 4, 6 or 24 h at 37°C in an atmosphere of 95% air and 5% COAfter incubation, cells were immediately placed on ice and rinsed three times with 1 ml of ice-cold PBS. The cells were lysed with 50 μl cell lysis buffer and removed from the culture dish using a cell scraper for sonication at 40% power setting  as previously described . SamplesProteins (20 μg/sample) were resolved and visualized as described in Part 1. Total proteins were normalized to ACTB or tubulin before calculation of fold induction. The ratio of phosphorylated DRP1 to total DRP1 was determined for each treatment and time point. Fold increases because of the treatment  were then calculated.4, resin-embedded, and subject to ultra-thin sectioning for electron microscopy. Transmission electron microscopy images were captured using a FEI Tecnai G2 Spirit transmission electron microscope at the University of Nebraska Medical Center.To observe differences in mitochondrial networks, enriched small and large luteal cells were fixed in 3% (wt/vol) paraformaldehyde and 0.2% glutaraldehyde in PBS, pH 7.4, post-fixed in 2% OsOTo evaluate the effects of PGF2α on mitochondrial morphology, enriched large luteal cells were equilibrated in fresh culture medium enriched with 1% BSA for 2 h before treatment with control media or PGF2α (100 nM) for 4 h. Following PGF2α stimulation, enriched large luteal cells were fixed and processed as described above and subject to ultra-thin sectioning for electron microscopy. Transmission electron microscopy images were captured using a FEI Tecnai G2 Spirit transmission electron microscope at the University of Nebraska Medical Center. 10–15 images  from large luteal cells were used for quantification of the mitochondrial area using ImageJ.5 cells/well and enriched large luteal cell cultures were seeded at 2.5 × 105 cells/well.For all confocal experiments, sterile No. 1 glass coverslips (22 × 22 mm) were individually placed in each well of a six-well culture dish. Mixed luteal cells were seeded at 3 × 10Biotin was added to phospho-DRP1 (Ser616) and phospho-MFF (Ser146) using a commercially available kit per the manufacturer’s protocol  as previously described .2 for 30 min before termination of the experiment.In brief, luteal cells were equilibrated in fresh culture medium enriched with 1% BSA for 2 h before treatment with PGF2α (100 nM) or PMA (20 nM) for 30 min. Cells were maintained at 37°C in an atmosphere of 95% humidified air and 5% COCells were fixed for 30 min with 200 μl 4% paraformaldehyde at 4°C and rinsed three times with PBS. To evaluate the effects of PGF2α on the phosphorylation of DRP1 and MFF, cell membranes were permeabilized with 200 μl 0.1% Triton-X in PBS-T (0.1% tween-20) at room temperature for 10 min. The permeabilized cells were then washed with PBS and blocked in 5% BSA for 24 h at 4°C. The cells were then rinsed and appropriate antibodies for co-localization  were add2. Mitotracker DeepRed (250 nM) was added to culture media 30-min before imaging. The cells were washed fresh culture media and subject to live-cell imaging.Luteal cell cultures were equilibrated in fresh culture medium enriched as described above and stimulated with PGF2α (100 nM) for 4 h and maintained at 37°C in an atmosphere of 95% humidified air and 5% CO2. CellROX reagent (5 μM) was added to cultures 30 min before live-cell imaging.We used CellROX, a weakly fluorescent cell-permeant dye that exhibits bright green photostable-fluorescence after oxidation by ROS production. In brief, luteal cell cultures were stimulated with PGF2α for 4 h and maintained at 37°C in an atmosphere of 95% humidified air and 5% CO2 for 6 h. To observe the co-localization of Mitophagy Dye and lysosome, the cells were incubated with Lyso dye 30 min before imaging at 37°C. The cells were washed 3× with Hanks’ HEPES buffer and subject to live-cell imaging.We used Mitophagy Dye, a weakly fluorescent mitochondrial dye that exhibits bright photostable fluorescence after fusion with lysosomes. In brief, large luteal cells were washed with PBS and placed in a fresh serum-free medium containing 100 μmol/l Mitophagy Dye. Cells were stimulated with PGF2α and maintained at 37°C in an atmosphere of 95% humidified air and 5% COImages were collected using a Zeiss LSM800 confocal microscope with Airyscan equipped with a 63× oil immersion objective (1.4 N.A) and acquisition image size of 1,584 × 1,584 pixel (77.96 μm × 77.96 μm), and 1,024 × 1,024 pixel (101.31 μm × 101.31 μm). The appropriate filters were used to excite each fluorophore and emission of light was collected between 450 to 1,000 nm. Cells were randomly selected from each slide and 30–45 z-stacked (0.15 μm) images were generated from the bottom to the top of each experiment. To determine the effects of PGF2α on mean fluorescence intensity of phospho-DRP1, and phospho-MFF, images were converted to maximum intensity projections and processed utilizing ImageJ  analysis software. Mean fluorescence intensity was determined as previously described . The JAC6 cells/dish. Cells were treated with culture medium alone, PGF2α (100 nM) or PMA (20 nM) for 1 h at 37°C in an atmosphere of 95% air and 5% CO2. After incubation, the cells were immediately rinsed with ice-cold PBS and mitochondria were isolated per the manufacturer’s protocol.Mixed luteal cell cultures were plated in 150 × 22 mm culture dishes at 10 × 10t tests to evaluate paired responses. Two-way ANOVA was used to evaluate repeated measures with Dunnett’s posttests to compare means. All statistical analysis was performed using GraphPad Prism software from GraphPad Software, Inc. All data are presented as the means ± SEM.Each experiment was performed at least three times, each using cell preparations from separate cows and dates of collection. The differences in means were analyzed by one-way ANOVA followed by Tukey's multiple comparison tests to evaluate multiple responses, one-way ANOVA followed by Dunnett’s post test to compare means or by All data are available in the main text or the supplementary materials.


The Fe3O4@C@MCM41-guanidine was prepared via surfactant directed hydrolysis and condensation of tetraethyl orthosilicate around Fe3O4@C NPs followed by treatment with guanidinium chloride. This nanocomposite was characterized by using Fourier transform infrared spectroscopy, vibrating sample magnetometry, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, thermal gravimetric analysis, wide-angle X-ray diffraction and low-angle X-ray diffraction techniques. This nanocomposite have high thermal, chemical stability, and uniform size. Fe3O4@C@MCM41-guanidine catalyst demonstrated high yield (91–98%) to prepare of Knoevenagel derivatives under the solvent free conditions at room temperature in the shortest time. Also, this catalyst was recovered and reused 10 times without significant decrease in efficiency and stability. Fortunately, an excellent level of yield (98–82%) was observed in the 10 consecutive catalyst cycles.In this study, preparation, characterization and catalytic application of a novel core–shell structured magnetic with carbon and mesoporous silica shells supported guanidine (Fe In addition, core–shell NPs are planed so that the shell material can improve the oxidative stability, thermal stability or reactivity of the core material or a cheap core material is used to transport the costly shell material5. Core–shell NPs have many usages in biomedical research6, MRI9, catalysis12, drug delivery13, energy harvesting15, plasmonic17, sensors20, etc. Much progress has been chance in the development of rational synthetic methods for the production of various core–shell NPs. Among the types of core–shell NPs, mSiO2-coated magnetite (Fe3O4) NPs have attracted the attention of researchers in various fields such as biomedical, sensor, catalyst, etc., due to their illustrious attributes such as unique magnetic response, low cytotoxicity, high colloidal stability, high adsorption capacity, high chemical and thermal stability, high surface area, high colloidal stability and high availability of silanol groups on its surface for any modification22. Some of recently developed mSiO2-coated magnetite NPs are Fe3O4@MCM-41-SB/Pd3, Fe3O4@SiO2@MCM41-IL/WO42−23, Fe3O4@nSiO2@mSiO2–Fe24, Fe3O4@MCM-41/Melamine25, Fe3O4@SiO2@mSiO2@TiO226, Fe3O4@mSiO2@BiOBr27 and Fe3O4@mSiO2@mLDH28. Meanwhile, carbon and polymer-coated NPs have also attracted a lot of attention due to their unusual attributes. Specifically, magnetite NPs coated by carbon show a high conductivity and are very engrossing for energy-storage and catalysis applications32. Some recently developed examples are Fe3O4@CN@HM33, Fe@C@Mo6O1834, Au-Fe3O4@Carbon35, Fe3O4@C@Au36 and Fe3O4@Carbon@MnO237. Given the positive properties mentioned for magnetite NPs coated with silica or carbon, the development of effective synthetic methods for the production of core–shell NPs containing magnetite core and both carbon and silica shells will be very valuable29.In recent years, core–shell nanoparticles (NPs) have attracted much attention. The combination of core and shell material, their design and geometry lead to the creation of unique properties in them44. Some of the recently developed heterogeneous catalysts for this reaction are MS/Ag2CO345, LDH-ILs-C1246, CoFe2O447, MgO/ZrO248 and Fe3O4@OS-NH249.On the other hand, the Knoevenagel reaction is one of the most important processes for the formation of carbon–carbon double bonds in synthetic organic chemistry and allows the production of low-electron olefins. Generally, Knoevenagel reactions are performed by the condensation of carbonyl compounds with active methylene. In recent years, many homogeneous and heterogeneous catalysts have been introduced to perform this reaction, that heterogeneous catalysts have received much attention due to their recoverability, reusability, structural deterioration resistance and easy separation of the products55. In the continuance of the above studies and considering the importance of mSiO2 and carbon-coated magnetite NPs in the catalysis process, our motive in this study is the design and synthesis of a novel core@ double-shell structured Fe3O4@C@MCM-41 nanocomposite. In this reaserch, the guanidine was selected as an important organic group for functionalization of our designed nanocomposite due to it is a strong base for a variety of base-catalyzed organic reactions as well as its easy chemical immobilization on Fe3O4@C@MCM-41. The Fe3O4@C@MCM41-guanidine catalyst showed high performance to prepare of Knoevenagel derivatives in the solvent free conditions with highest yield (91–98%) in the shortest time at room temperature. Also, the Fe3O4@C@MCM41-guanidine can recovered and reused at least 10 times without the significant loss in its activity and productivity.Guanidines are important categoris of organic compound. In the synthetic organic chemistry, including the Knoevenagel reaction, guanidines have been shown to act as organic bases and nucleophilic catalysts. But, the main problem of catalysts based on guanidines is their separation from the product, which needs solid liquid or liquid–liquid techniques in many reactions. Therefore, supported guanidine catalysts have been more investigated. Supported guanidine catalysts are prepared via the physical or chemical immobilization of guanidines on various supports such as alumina, magnetic NPs, metal oxides, carbon nanotubes and silica3 (28–30% wt) were purchased from Merck. Ethanol (≥ 99.8%) was purchased from Fluka. FT-IR spectra were recorded in the range of 400–4000 cm−1 on Alpha Centaur FT-IR spectrophotometer using KBr pellets. The morphology of the synthesized NPs was investigated by using the EM3200 scanning electron microscopy (SEM) device. Transmission electron microscopy (TEM) image was recorded using a FEI TECNAI 12 BioTWIN microscope. TGA analysis was performed by a Netzsch STA 409 PC/PG apparatus in the temperature range of 25–900 °C. Powder X-ray diffraction (PXRD) analysis were carried out using D8 ADVANCE XRD and Philips XPert Pro XRD equipment, respectively. X-ray distribution analysis (EDS) was performed by the means of an EDS Sirius SD device. The magnetic effect of the synthesized NPs was investigated using vibrating sample magnetometer (VSM) of Meghnatis Daghigh Kavir Co. The uniform dispersion of the reactants was obtained using a KMM1-120WE301ultrasonication device. The TLC-Grade-silica gel-G/UV 254 Thin-layer chromatography (TLC) device was used to evaluate the reaction advancement and determine the reaction completion. The melting point was measured using a Barnstead Electro-Thermal device.All chemicals were purchased from Sigma-Aldrich Chemical Co. , Merck Chemical Co.  and Fluka Chemical Co. . Cetyltrimethylammonium bromide (99%), Tetraethyl orthosilicate (≥ 99%), 3-chloropropyltrimethoxysilane (≥ 97%), Resorcinol (≥ 99%), Formaldehyde solution (37 wt. %), Toluene dried (≥ 99.5%), Guanidinium chloride (≥ 99%), Triethylamine (≥ 99.5%) and Ethyl cyanoacetate (≥ 98%) were purchased from SigmaAldrich and used without further purification. Benzaldehydes (97–99%) and NH3O4 NPs were firstly prepared according to reported procedures56. To prepare the Fe3O4@RF NPs, 0.7 g of Fe3O4 NPs were added to the reaction vessel containing 200 mL of water and 100 mL of ethanol. Then the resulted mixture was placed in to the ultrasonic bath for 30 min at room temperature. In the following, 1.5 mL of NH3 (28–30% wt) was added in the reaction vessel at room temperature and obtained mixture was again placed in the ultrasonic bath for 15 min. After that, 1 g of resorcinol and 0.1 mL of formaldehyde solution were added in the reaction vessel, and the resulting mixture was stirred at room temperature for 24 h. Then, the resulting precipitate was separated by using a magnetic field, washed several times with water and ethanol, dried at 60 °C for 6 h and was named Fe3O4@RF57.The magnetic Fe3O4@C@MCM41, first 0.6 g of Fe3O4@RF NPs were firstly added into the reaction vessel containing 60 mL of distilled water and was placed into the ultrasonic bath at 40 °C for 30 min. Subsequently, 3 mL of NH3 (28–30% wt) and 1 g of cetyltrimethylammonium bromide (CTAB) were added into the reaction vessel. Then, 0.7 mL of Tetraethyl orthosilicate (TEOS) was added dropwise, and the resulting mixture was stirred for 2 h. In the next step, the reaction mixture was stirred for 48 h at a temperature of 100 °C. After the end of the reaction, the obtained precipitate was separated by a magnetic field and dried. The acquired material was poured into a crucible and placed in a furnace with a temperature of 350 °C for 5 h for the remove of CTAB surfactant. Finally, the resulting material was poured into a crucible, carbonized at 600 °C for 6 h under argon atmosphere and named Fe3O4@C@MCM4158.To preparation of Fe3O4@C@MCM41 NPs, 0.2 g of Fe3O4@C@MCM-41 was added into the flask reaction containing 26 mL of dry toluene and dispersed under ultrasonic waves. After that, 0.1 mL of 3-chloropropyltrimethoxysilane was added into the reaction mixture and refluxed for 24 h under argon. Then, the product was collected using a magnetic field, washed with 20 mL of toluene and 30 mL of ethanol and dried at 60 °C for 12 h.To immobilize 3-chloropropyltrimethoxysilane on the surface of Fe3O4@C@MCM-41/Pr-Cl was added into a flask reaction containing 20 mL of dry toluene under ultrasonic waves. Subsequently, 0.08 g of guanidinium chloride and 0.003 of Triethylamine were added into the flask, and the obtained mixture was refluxed condition for 28 h. After, the acquired precipitate was separated by using a magnetic field, washed with 20 mL of ethanol and dried at 50 °C for 8 h. and named Fe3O4@C@MCM-41-guanidine  polymer layer. Then, the Fe3O4@RF was covered with the MCM-41 shell by using the Sol–gel method in the presence of CTAB surfactant under alkaline conditions. After calcination to remove CTAB and carbonization, the Fe3O4@C@MCM41 was treated with 3-chloropropyltrimethoxysilane and Guanidine (Fe3O4@C@MCM41-guanidine)  polymer layer, respectively  analysis of Fe3O4@C@MCM41-guanidine was showed in Fig. 3O4@C with MCM-41 and functionalization with guanidine, the same LXRD pattern is observed  analysis of MCM-41 and Fe3O4@C@MCM41-guanidine nanocatalyst is composed of Iron, Silicon, Oxygen, Carbon, and Nitrogen elements confirming the successful incorporation/immobilization of expected species in the material framework  technique was used for determine the appearance and particle size of the Feize Fig. .Figure 63O4@C@MCM41-guanidine nanocatalyst. The TEM image showed that the designed nanocatalyst has a core–shell structure with a black core (magnetite particles) and gray shells  analysis was employed to validate the appearance and shell-core structure of the Fells Fig. .Figure 73O4@C@MCM41-guanidine catalyst was investigated using thermal gravimetric analysis. This analysis was performed in the temperature range of 25 to 900 °C. The weight loss in the first stage (up to 100 °C) indicates the extraction of water and existing organic solvents in the synthesis stages of the catalyst. In the second stage, the weight loss in the temperature range of 100 to 300 °C indicates the extraction of residual surfactant. The weight loss in the next stage which occurs in the temperature range of 300 to 480 °C shows the elimination of organic groups. In the last stage, the weight loss at the temperature range of 400 to 800 °C shows the extraction of the remaining organic groups. This analysis shows the presence and thermal stability of the groups that covered the surface of the Fe3O4 magnetic NPs  analysis. The results of this analysis showed that Fe3O4 and Fe3O4@C@MCM41-guanidine have a superparamagnetic behavior. The magnetic saturation of Fe3O4 and Fe3O4@C@MCM41-guanidine nanomaterials were 60 and 47 emu/g, respectively. The decrease in saturation magnetization confirms successful chemical immobilization of Carbon, MCM41 and guanidine moieties on the surface of the Fe3O4 NPs (Fig. 3O4 and Fe3O4@C@MCM41-guanidine nanomaterials, which are very important for their easy separation in the chemical processes.The magnetic properties of FeNPs Fig. . Also, t3O4@C@MCM41-guanidine nanostructure, its catalytic activity was investigated in the Knoevenagel reaction. For this purpose, the reaction conditions were firstly optimized. To optimize the reaction condition, the condensation between benzaldehyde and ethyl cyanoaceteat was selected as the model reaction. The effects of temperature, catalyst loading, and solvent were investigated under ultrasonic conditions. Examination of different solvents showed that the reaction yield was very low in the presence solvents such as toluene and acetonitrile, and it was slightly better in polar solvents such as ethanol and water. The best result was obtained under the solvent-free conditions. Subsequently, the catalyst loading was optimized with 0.5, 1.5, and 2.5 mol% of catalyst. The best yield was obtained with 1.5 mol%. It should be noted that the increase of catalyst loading did not affect the reaction progress because the amount of raw materials corresponds to 1.5% of catalyst and the excess amount of catalyst will be practically useless. The catalytic effect of Fe3O4@C@MCM41-guanidine can be proved by noting that the catalyst-free reaction did not make much progress after a long time (Table After the synthesis of Fe3O4@C@MCM41-guanidine nanocatalyst in solvent-free conditions and at room temperature under ultrasonic waves, ethyl 2-Cyano-3-phenyl acrylate products were obtained with high yields. As shown in Table 3O4@C@MCM41-guanidine nanocatalysts in the Knoevenagel reaction (Table 45.From the reaction of different aldehydes in the presence of 1.5 mol% of Fe3O4@C@MCM41-guanidine nanocatalyst in the Knoevenagel reaction, 1 mmol of benzaldehyde, 1 mmol of Ethyl cyanoacetate, and 1.5 mol% of Fe3O4@C@MCM41-guanidine nanocatalyst at room temperature were selected as model reaction under solvent-free condition and ultrasound waves. Upon completion of the process, the magnetic nanocatalyst was separated using an external magnet, washed completely with ethanol, and dried. Then the recovered catalyst was reused in the next reaction cycle under the same conditions as the first run. The results show that the catalyst can be reused at least 10 times without significantly reducing the reaction time and efficiency (Fig. To investigate the recyclability and reusability of FeIn the mechanism of the Knoevenagel reaction, one of the active hydrogens of methylene is separated by a base. Then, a nucleophilic attack is performed on carbon aldehyde or ketones. As can be observed in the Fig. 3O4@C@MCM41-guanidine nanocatalyst was successfully prepared and its catalytic performance was studied. FT-IR, EDX and TGA are shown successful stabilization of the guanidine groups on the surface of the Fe3O4@C@MCM41 nanostructure. SEM and TEM analysis confirms the spherical structure of the Fe3O4@C@MCM41-guanidine nanostructure. The WXRD proved the high stability of crystalline structure of Fe3O4 NPs during the material preparation. The LXRD also confirmed the formation of mesoporous silica shell around Fe3O4@C NPs. The VSM analysis demonstrated good magnetic properties of this nanocomposite. The catalyst showed excellent activity in the Knoevenagel reaction. Catalyst was simply recycled and reused 10 times without significant reduction in activity.In summary, in this study, Fe


The estimated crystallite size (Ds) for Gd2O3, CdS/Gd2O3, and CdS/Gd2O3@GO was 29.6, 11.6, and 11.5 nm, respectively. The degradation of methylene blue (MB) reaches the highest values after 60 min under visible light irradiation with a dye concentration of (0.25 ppm). Whereas in powdered composition the efficiency of dye removal has been enhanced under UV irradiation, it reduced by increasing the MB concentration to 0.50 ppm with visible light irradiation. In addition, the CdS with/without Gd2O3 and GO were integrated into electrospun nanofibrous cellulose acetate (CA) through the electrospinning technique. The compounds of Gd2O3, CdS/Gd2O3, and CdS/Gd2O3/GO were encapsulated into CA nanofibers for the degradation of MB under visible and UV irradiation. The apparent rate constant (k) achieves a value of 0.006, 0.007, and 0.0013 min−1 while the removal efficiency reaches 41.02%, 54.71%, and 71.42% for Gd2O3@CA, CdS/Gd2O3@CA, and CdS/Gd2O3/GO@CA, respectively, after 60 min under UV irradiation.Efficient cleaning of contaminated water by photocatalysis has become an effective strategy in recent years due to its environmental and ecological designation. Cadmium sulfate (CdS) is an excellent photocatalyst in the visible region but has low quantum efficiency. In order to increase the photocatalytic efficiency, CdS was modified with gadolinium oxide (Gd Graphene oxide (GO) (99.5%) was purchased from Sigma Aldrich and ammonium solution (25%), cellulose acetate (CA), and deionized water were used to prepare the solutions.Cadmium chloride (CdCl2·2H2O (0.25 mol) and Na2S (0.09 mol) were dissolved in 50 mL of deionized (DI) water, separately. The CdCl2·2H2O solution was then dropped wisely into the Na2S container, while the pH value was kept at 10 using drops of NaOH. After 1 h of stirring, the solution was maintained for 24 h for precipitation. Equal weights of both gadolinium oxide (Gd2O3) and cadmium sulfide (CdS) were combined with and mixed in 50 mL of DI water under highly powerful sonication for 15 min. The solution was then left for precipitation and drying (CdS/Gd2O3). Also, 0.05 g of graphene oxide (GO) was combined with cadmium sulfide (CdS) and gadolinium oxide (Gd2O3) then sonicated, precipitated, and dried to get the sample of CdS/Gd2O3@GO as shown in Fig. The CdS was prepared using the co-precipitation method. CdCl2O3@CA, CdS/Gd2O3@CA, and CdS/Gd2O3/GO@CA nanofibers, 0.165 g of the acquired powder was added into 15 mL of CA (10 wt.%) in a glass bottle then sonicated for 30 min using an ultrasonic probe  to improve the dispersibility of nanofiber materials. The prepared electrospinning solution was transferred to a 20-mL syringe and proceeded with the flowing rate of injection: 1 mL h−1 via a syringe pump. The applied voltage was kept at 18 ± 0.1 kV, the distance from the needle tip to a collector was set as 16 cm, and the syringe needle was 22 φ. The electrospinning experiments were carried out at room temperature. The electrospun Gd2O3@CA, CdS/Gd2O3@CA, and CdS/Gd2O3/GO@CA were raised and accurately peeled off from the aluminum foil . Thus, the particle size and distribution were investigated using a transmission electron microscope. The optical properties were studied using UV–visible spectroscopy. In addition, XPS has characterized the chemical composition which was collected on K-ALPHA. Fourier-transmission infrared was used to characterize the functional groups of the matter surface. The surface area of the powdered compounds was checked using the Brunauer–Emmett–Teller (BET) technique.The crystal structure was analyzed by the XRD technique: Cu, kC0) was defined as an initial concentration. A UV lamp of 15 W was placed about 12 cm above the dye solution and illuminated the sample for 60 min. At every interval time, 3 mL of the dye solution was taken by syringe to be investigated using UV–Vis spectrophotometer. In the degradation of dye under visible light, the experiment was carried out by halogen lamp (from 400 up to 800 nm at a power ~ 500 W) in a locked box at room temperature via a container of 30 mL with MB (0.25 and 0.5 ppm) and 100 mg of powdered compounds. The solution was kept in a dark place for 60 min and the distance between the halogen lamp and the sample was 18 cm. Every 10 min, the MB solution was detected by a spectrophotometer. The degradation efficiency (η %) was calculated via the following equation  was utilized as an example of an organic pollutant. The photocatalytic activity was analyzed under both UV and visible light irradiation. To investigate dye degradation induced by UV irradiation, experiments were performed in 30 mL containers with MB concentrations of 0.25 and 0.5 ppm and 120 mg of nanofiber composition. These very low concentrations are used to prevent the rate of degradation from decreasing with increasing dye concentration , (110), (103), and (201) with reference card (00–006-0314) belong to the hexagonal system of CdS. The position of peaks seems to be with no significant shift, but the intensity decreased with CdS/Gd2O3. The crystallite size is calculated via Scherrer’s formula  could be applied  was used to identify the morphology of the compounds. Although the images are not so clear due to some accidental problems, Fig. 2O3 and GO NPs with the CdS compounds. Furthermore, it confirmed the presence of C, S, Cd, and Gd amounts of CdS/Gd2O3@GO compound with 7.8, 16.7, 15.4, 35.9, and 24.0%, respectively. The atomic percentages are abbreviated in Table Elemental analysis was carried out by energy-dispersive X-ray (EDX) as shown in Fig. 2O3/GO powdered is illustrated in Fig. 2O3 could be assigned to the cumulative influence of continual fragmentation , C = O (532.3 eV), Gd–O (531.3 eV), and OH group or a water molecule on the surface of nanocompositions (532.0 eV). The convoluted high-resolution spectrum elucidates that the Cd3d spectrum includes peaks at 412.3 eV (Cd3d3/2) and 405.6 eV (Cd3d5/2) which was symmetric with the particular binding energy of Cd2+ in CdS in Fig. 2O3 and CdS/Gd2O3@GO photocatalyst. The peaks at 143.0 and 149.4 eV coincide with Gd4d5/2 and Gd4d3/2 transitions of Gd2O3. At the same time, the two single peaks of 163.3 eV (S2p1/2) and 161.7 eV (S2p3/2) estimated the particular peaks of the S2− in the composition  is used to analyze the chemical composition and elements status of CdS/Gd–1 is ascribed to Gd–O stretching in Gd2O3. The spectra of GO that manifest a peak at 1042 cm−1 can be assigned to the C–O stretching. The broadband of 3271 cm−1 could be attributed to the stretching vibration mode of O–H. The stretching vibrations of hydroxyl (OH) groups of water adsorbed by the samples were ascribed to the broad peak shown at 3100–3600 cm−1. The peaks at 2921 and 2854 cm−1 indicate the existence of stretching vibration of C–H. The band of 1635 cm−1 is attributed to O–H–O bending oscillations because the molecules of water are adsorbed on the composite’s surface. The exposed band of 1594 cm−1 is assigned to the asymmetric vibrational mode belonging to the carboxyl group (C = O). The bands concerning 1414 and 1326 cm−1 manifest the existence of CH2 and C–O bending, respectively. The bands at 1048 to 843 cm−1 are referred to the stretching of C–O and the bending mode of O–H vibration, respectively. In Fig. −1 in the spectra is attributed to the symmetric and anti-symmetric stretching of O–H vibrations. In addition, the band at 1625 cm−1 is concerned with the bending vibrations of absorbed water on the surface of nanofibers. The band 1633 cm−1 is assigned to the bending mode of H–O–H. The peak at 1102 to 1180 cm−1 is attributed to C–O–C stretching.The FT-IR spectra are shown in Fig. 2) adsorption/desorption isotherms using the Brunauer–Emmett–Teller (BET) method at 77 K  was about 24 m2/g as demonstrated in Table 3/g), whereas the average pore diameter reached 9.95 nm. The BET surface area of CdS loaded on activated carbon AC/CdS and pure CdS were 69.4 and 27.5 m2/g  progressively decline slowly with increasing irradiation time. It is emphasized that embossing CdS with Gd2O3 and Gd2O3@GO develops photocatalytic activity in the visible and UV region. It is depicted in Fig. η) reaches the highest values at 65%, 77%, and 91.07% for Gd2O3, CdS/Gd2O3, and CdS/Gd2O3@GO under UV light after (60 min) of irradiation time. However, the degradation efficiency under visible irradiation tends to be lower than its analogue under UV illumination. The efficiency (η) is ranged from 42.85%, 60.37%, and 82.35% for Gd2O3, CdS/Gd2O3, and CdS/Gd2O3@GO under visible light, respectively. As shown in Fig. k. Notably, it is well assured that the photocatalytic degradation of MB is obeyed as the first-order kinetics defined by the following equation  and irradiation time which expands the correlation coefficient R2 and rate constant k for the MB dye degradation. The degradation ratio of MB is changed with the use of several photocatalysts under visible and UV irradiation as extended in Fig. R2 and k were calculated by plotting t/qt versus t, where qt represents the amount of MB absorbed at time t. The maximum adsorption quantity (qmax) was calculated to determine the conversion capacity value using the following equation , V is the volume of working solution (L), and m is the mass of the catalyst. The pseudo-first-order kinetic model is used to estimate the interaction between MB molecules and catalysts. Table kapp for all samples under visible and UV irradiation, while Table k1, qmax according to L–H model, in visible and UV illumination for all samples, with R2 ranging between 0.877 and 0.999. The similarity between calculated qmax and those determined experimentally indicates that MB adsorption obeys pseudo-first-order kinetics for all adsorbents  of the photocatalytic degradation for the Gd2O3, CdS/Gd2O3, and CdS/Gd2O3/GO were 0.0056, 0.0074, and 0.0014 min−1, respectively.Figure 2O3/GO@CA photocatalysts deteriorate more rapidly with increasing time. Figure 2O3/GO@CA manifests better degradation efficiency around 61.13% in comparison to Gd2O3@CA and CdS/Gd2O3@CA under visible light. In contrast, the degradation efficiency of CdS/Gd2O3/GO@CA composite (71.42%) is gradually increased as compared to that of Gd2O3@CA and CdS/Gd2O3@CA under UV irradiation. The nanofibers were left in the samples for 60 min to obtain the adsorption equilibrium state. The plot C/C0 varied with time as clarified in Fig. C/C0) and degradation time (t) under UV irradiation. The apparent rate constant (k1) and R2 are reported in Table The efficiency of nanofibers based on CA comprising the three inorganic composites degenerate MB from aqueous solutions under visible and UV light. Figure omposite 1.42% is 2O3@CA, CdS/Gd2O3@CA, and CdS/Gd2O3/GO@CA under visible light, respectively. Also, the efficiency (η) is 37.5%, 46.37%, and 63.15% for the nanofiber membranes under UV irradiation as displayed in Fig. C/C0 with varied time (up to 60 min) are shown in Fig. kapp) is matched with Ln (C/C0), which donates a straight line with a slope coinciding with kapp value. The values of kapp, k1, and qmax are listed in Tables Plot adsorbance versus wavelength as a function of time reveals a distinguished adsorbance crest at 664 nm see Fig. a, b. Mor2O3, and GO nanostructures under visible and UV irradiation as shown in Fig. +) at the VB    reacts with hydroxyl ion (OH−) and generates active OH•  reaches the highest values at 42.85%, 60.37%, and 82.35% in visible light, while it extends the values to 65%, 77%, and 91.07% for Gd2O3, CdS/Gd2O3, and CdS/Gd2O3@GO under UV light. On the other hand, in case of concentration 0.5 ppm, η reaches the values of 28.15%, 35.11%, and 60% for Gd2O3, CdS/Gd2O3, and CdS/Gd2O3@GO under visible light. Regarding the nanofiber compositions with a concentration 0.25 ppm, the photocatalytic features of the Gd2O3@CA, CdS/Gd2O3@CA, and CdS/Gd2O3/GO@CA are characterized under visible and UV light irradiation. The highest photodegradation activity (71.42%) was found for the nanofiber composite CdS/Gd2O3/GO@CA under UV irradiation, in comparison to an activity (61.13%) under visible light. This nanofiber composite could be employed in abundant applications and particularly for the degradation of organic pollutants.The powdered compositions Gd