However, nifS and nifU are not found in N2-fixing Paenibacillus strains, and the mechanisms involved in Fe–S cluster biosynthesis of nitrogenase is not clear. Here, we found that the genome of Paenibacillus polymyxa WLY78 contains the complete sufCDSUB operon, a partial sufC2D2B2 operon, a nifS-like gene, two nifU-like genes (nfuA-like and yutI), and two iscS genes. Deletion and complementation studies showed that the sufC, sufD, and sufB genes of the sufCDSUB operon, and nifS-like and yutI genes were involved in the Fe–S cluster biosynthesis of nitrogenase. Heterologous complementation studies demonstrated that the nifS-like gene of P. polymyxa WLY78 is interchangeable with Klebsiella oxytoca nifS, but P. polymyxa WLY78 SufCDB cannot be functionally replaced by K. oxytoca NifU. In addition, K. oxytoca nifU and Escherichia coli nfuA are able to complement the P. polymyxa WLY78 yutI mutant. Our findings thus indicate that the NifS-like and SufCDB proteins are the specific sulfur donor and the molecular scaffold, respectively, for the Fe–S cluster formation of nitrogenase in P. polymyxa WLY78. YutI can be an Fe–S cluster carrier involved in nitrogenase maturation in P. polymyxa WLY78.NifS and NifU (encoded by  S) system . The Nifnelandii . Nitrogeproteins ,8. Subseproteins ,10,11.Escherichia coli and closely related enterobacteria possess both Isc and Suf systems, encoded by the iscRSUA-hscBA-fdx-iscX operon and the sufABCDSE operon, respectively. The Isc system functions under normal growth conditions, whereas the Suf system operates under stress conditions such as iron starvation and oxygen limitation [In addition to the Nif system that is specifically responsible for the synthesis of the Fe–S clusters of nitrogenase, Isc and Suf are required for a broad range of cell functions . Escherimitation ,13,14. Imitation ,16,17. Imitation ,19,20,21Bacillus subtilis, Fe–S cluster assembly is mediated by the Suf system consisting of a sufCDSUB operon and a distant sufA [B. subtilis SufS functions as a cysteine desulfurase, and SufBCD is a scaffold, both of which are functionally similar to the corresponding E. coli Suf components [B. subtilis SufU interacts with SufS and is involved in sulfur transfer from SufS to SufBCD, just as SufE is involved in the E. coli Suf system. B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU [In the Gram-positive model of the bacterium ant sufA . B. subtmponents . B. subtth IscSU ,24.E. coli NfuA binds a 4Fe–4S cluster and transfers this cluster to the target protein and acts as a scaffold/chaperone for damaged Fe–S proteins under oxidative stress and iron starvation conditions [E. coli CsdA is the third cysteine desulfurase except for IscS and SufS, and it is also engaged in two separate sulfur transfer pathways by recruiting Suf components and by interacting with CsdE and CsdL participates [In addition to the three distinct Nif, Suf, and Isc systems, some Fe–S cluster biosynthetic genes have been identified. For example, nditions ,26,27. Eicipates .Paenibacillus polymyxa WLY78 is a Gram-positive, facultative anaerobic, endospore-forming, N2-fixing bacterium. The genome of this bacterium contains a minimal and compact nif gene cluster composed of nine genes (nifB nifH nifD nifK nifE nifN nifX hesA nifV) [nif cluster has great potential use in engineering nitrogen fixation into non-N2-fixing organisms [nif gene cluster enables E. coli to synthesize the catalytically active nitrogenase, but the specific activity of the enzyme expressed in E. coli was only approximately 10% of that observed in Paenibacillus [nif gene clusters of A. vinelandii and Klebsiella oxytoca, the P. polymyxa WLY78 nif cluster lacks nifS and nifU. Thus, we attempted to identify whether nifS-like and nifU-like or other Fe–S cluster biosynthetic genes are responsible for the Fe–S cluster assembly of nitrogenase.sA nifV) . This mirganisms . Our recbacillus . CompareP. polymyxa WLY78 and found that there are an entire sufCDSUB operon, a partial sufC2D2B2 operon, a partial isc system (iscSR), and a single iscS (iscS2), one nifS-like, and two nifU-like genes (nfuA-like and yutI). Mutation and complementation analysis of these putative Fe–S assembly genes revealed that SufCDB and NifS-like proteins are essential for biosynthesis of nitrogenase. Furthermore, heterologous complementation studies demonstrated that K. oxytoca nifS is able to restore the nitrogenase activity of the nifS-like mutant of P. polymyxa WLY78, but K. oxytoca nifU cannot complement any of the sufC, sufD, and sufB mutants. We also demonstrated that yutI is involved in nitrogen fixation, but other genes are not. Our study not only reveals the distribution and functions of the Fe–S cluster biosynthetic genes involved in nitrogen fixation in P. polymyxa WLY78, but also provides insight into the mechanisms of Fe–S cluster assembly.Here, we searched the genome of P. polymyxa WLY78 by using MetalPredator  and identified 118 genes encoding putative Fe–S cluster-containing proteins and Fe–S cluster biosynthetic genes (P. polymyxa WLY78 include an entire Suf system (sufCDSUB), a sufA gene, a partial Suf system (sufC2D2B2), a partial Isc system (iscSR), a single iscS2 gene, and a nifS-like and two nifU-like (nfuA-like and yutI) genes.We searched the genome of ic genes . As showP. polymyxa WLY78, sufC, sufD, sufS, sufU, and sufB (sufCDSUB) are tightly arranged with the same transcriptional direction. However, sufA is not in the sufCDSUB cluster of P. polymyxa WLY78, which differs from the suf operon arrangement in E. coli. The current analysis using BLAST alignment showed that the SufA protein of P. polymyxa WLY78 shares 26.23% and 48.33% identities with E. coli and B. subtilis SufA proteins, respectively. The complete sufCDSUB operon of P. polymyxa WLY78 shows a similar arrangement as that of B. subtilis. The SufCDSUB proteins showed 18.11–46.96% identity with their corresponding components from E. coli Suf and 47.84–76.82% identity with their corresponding components from B. subtilis Suf at the amino acid level  whose predicted products SufC2, SufD2, and SufB2 showed 84.88%, 62.27%, and 84.73% identities with their corresponding components of the entire Suf system, respectively.In the genome of id level A. In addP. polymyxa WLY78 contains an iscR gene and two iscS genes. Of the two iscS genes, one is linked to iscR as a dicistronic iscSR operon and the other (here designated as iscS2) is located elsewhere. The iscRS operon of P. polymyxa WLY78 has a similar organization as that of B. subtilis. However, compared to the isc operon (iscRSUA-hscBA-fdx) of A. vinelandii and E. coli, the P. polymyxa WLY78 isc operon (iscRS) lacks iscUA and other genes  that are not associated with the nif gene cluster (nifB nifH nifD nifK nifE nifN nifX hesA nifV). P. polymyxa WLY78 has a nifS-like gene encoding a 397-amino-acid protein, which showed 28.77% and 28.36% identity with NifS of A. vinelandii and K. oxytoca, respectively  binding and invariable residues Cys that may be involved in substrate binding. A protein alignment revealed that the amino acid sequences derived from two NifU-like proteins with a single domain (NfuA-like and YutI) have high similarity with those of the C-terminal domain of NifU from A. vinelandii and K. oxytoca and the C-terminal domain of NfuA from E. coli and A. vinelandii  and a complementation strain (ΔnifS-like/nifS-like). In comparison with wild-type P. polymyxa WLY78, the ΔnifS-like mutant exhibited nearly no activity under the nitrogen-limited condition, indicating that nifS-like is required for nitrogen fixation. Complementation of ΔnifS-like with the nifS-like gene carried in the plasmid was able to restore nitrogenase activity, suggesting that a change in nitrogenase activity was due solely to the deletion of nifS-like , encoding for 85 and 81 amino acids, respectively, that share similarity with the C-terminus of the Fe–S scaffold protein NifU and the Fe–S carrier NfuA in A. vinelandii  did not result in a further decline in nitrogenase activity, suggesting that NfuA-like has a limited ability to serve the function of YutI in nitrogen fixation. Furthermore, heterologous complementation studies showed that K. oxytoca nifU and the E. coli nfuA gene were able to restore the nitrogenase activity of the ΔyutI mutant, suggesting that P. polymyxa yutI performs similar functions as K. oxytoca nifU and E. coli nfuA.The activity of the Δxa WLY78 B. CompleyutI mutant was complemented with the mutated yutI gene whose coding product Cys-49 or Cys-52 or both cysteine residues were replaced with alanine residue(s). As shown in yutI mutant as well as the wild-type yutI gene did. These results indicate that the conserved Cys-49 and Cys-52 in the YutI protein are responsible for the Fe–S cluster binding of nitrogenase.To investigate whether the two conserved cysteine residues (Cys-49 and Cys-52) in the YutI protein play roles in Fe–S assembly, the ΔP. polymyxa WLY78 contains two iscS genes (iscS and iscS2), no IscU homologues have been identified. Amino acid sequence analysis reveals that IscS and IscS2 of P. polymyxa have 35% identity. However, only IscS has conserved the SSGSACTS sequence, which represents the feature of the IscS protein  is lethal [P. polymyxa WLY78 contains another partial suf operon (sufC2D2B2) due to which the inactivation of sufCDB does not lead to lethality. Attempts to knock out sufSU in P. polymyxa WLY78 were unsuccessful, suggesting that sufSU might exist to maintain basic levels of Fe–S assembly for survival. In this study, the deletion mutants, including ΔsufC, ΔsufD, ΔsufB, ΔsufCDB, ΔsufC2D2B2, and ΔsufA, were successfully disrupted. qRT-PCR further revealed that the messenger RNA (mRNA) levels of other suf genes in the sufCDSUB operon were not affected by one suf gene mutation  showed a significant decrease in nitrogenase activity compared to the wild-type strain, while ΔsufB2C2D2 and ΔsufA mutants exhibited almost similar activity as the wild-type strain. Furthermore, complementation of ΔsufC, ΔsufD, and ΔsufB mutants with the corresponding P. polymyxa WLY78 suf gene restored nitrogenase activity to the wild-type level, suggesting that sufCDB genes are essential for the Fe–S cluster assembly of nitrogenase in P. polymyxa WLY78. We also tried to complement with K. oxytoca nifU and found that nifU could not restore the nitrogenase activity of any suf mutants , and sufCDB genes are involved in the Fe–S cluster assembly of nitrogenase in P. polymyxa WLY78.Nitrogenase is a complex metalloenzyme, and the Fe–S clusters of nitrogenase play a critical function in electron transfer and in the reduction of substrates driven by the free energy liberated from Mg-ATP hydrolysis ,11,31. Iif genes ,33. Howeand nifU . In thisnifS-like gene of P. polymyxa WLY78 is involved in nitrogen fixation. The NifS-like protein from P. polymyxa WLY78 and NifS from K. oxytoca and A. vinelandii have common characteristics, which contain conserved residues known to be essential for activity, substrate recognition, and PLP binding. However, P. polymyxa WLY78 nifS-like exhibits two features different from A. vinelandii nifS and K. oxytoca nifS. One is that each of A. vinelandii nifS and K. oxytoca nifS is linked together with nifU as a nifSU operon that is located in a large nif gene cluster [P. polymyxa WLY78 nifS-like is not linked together with any nif genes. The other feature is that there is a C-terminal extension consisting of 20-21 amino acids, including the consensus sequence SPL(W/Y)(E/D)(M/L)X(K/Q)XG(I/V)D(L/I)XX(/V)XWXXX in A. vinelandii NifS and K. oxytoca NifS [P. polymyxa WLY78. Deletion of the A. vinelandii nifS gene did not lead to complete loss of nitrogenase activity, suggesting that the housekeeping Fe–S cluster biosynthetic system Isc could weakly replace NifS function [P. polymyxa WLY78, since loss of NifS-like function does not completely eliminate the capacity for nitrogen fixation. K. oxytoca nifS could complement the P. polymyxa WLY78 nifS-like mutant, which further confirmed that the NifS-like protein is the specific S donor for the assembly of Fe–S clusters for nitrogenase in P. polymyxa WLY78.The NifS protein was involved in providing sulfur to the iron–sulfur clusters of nitrogenase . In this cluster ,33, whiloca NifS , while tfunction ,37. We dP. polymyxa WLY78 possess two NifU proteins (NfuA-like and YutI) that share significant sequence homology to the C-terminus domain of the NifU family and include the conserved CXXC motif. Such sequence conservation indicates a likely role for NfuA-like and YutI in Fe–S protein maturation in P. polymyxa WLY78. In A. vinelandii, the NfuA protein contains the N-terminal A-type domain and the C-terminal Nfu-type domain, and functional loss of the Nfu-type domain has no obvious effect on nitrogenase maturation [P. polymyxa WLY78, and deletion of yutI results in lower nitrogenase activity. The conserved cysteine residues in the CXXC motif were shown to be functionally important for NfuA in E. coli, A. vinelandii, and Pseudomonas aeruginosa [A. vinelandii [K. oxytoca and the NfuA of E. coli are functionally interchangeable with the YutI of P. polymyxa WLY78. The NfuA as an Fe–S cluster carrier was able to accept the Fe–S cluster from scaffold proteins IscU or SufBCD in vitro [P. polymyxa WLY78 could potentially be a stand-alone carrier protein, playing a role similar to NfuA to accept the Fe–S cluster from the scaffold protein, as described in other microorganisms.NifU was shown as an Fe–S scaffold protein constructed of several domains for maturation of the nitrogenase . The Nfuturation . In thisruginosa ,39,40,41in vitro . TherefoiscS and iscS2 of P. polymyxa WLY78 are not required for nitrogen fixation. The result is in line with our previous work that showed that the iscSR system cannot increase any activity of E. coli 78-7 carrying a Paenibacillus nif gene operon [A. vinelandii [iscS led to no defects in Fe–S metabolism, suggesting that IscS is not used for housekeeping Fe–S protein maturation in P. polymyxa WLY78. However, in B. subtilis, the iscS gene could not be deleted, indicating that IscS participates in essential cellular processes [The cysteine desulfurase IscS is a highly conserved and essential component of the Isc system that serves as a sulfur donor for Fe–S cluster biogenesis . In the e operon . In addinelandii ,43. Inacrocesses .sufCDSUB operon) is highly conserved in N2-fixing Paenibacillus strains [E. coli [B. subtilis [Staphylococcus aureus [Synechocystis [suf operon can have severe consequences: for instance, disruption of sufCDSUB or its individual gene is lethal for B. subtilis [P. polymyxa suf genes had not been characterized before; therefore, it was not clear what impact the disruption of suf would have on the metabolism and viability. Directly obtaining the sufCDSUB operon mutant of P. polymyxa WLY78 was unsuccessful, which places the Suf component as key proteins in Fe–S cluster metabolism. This is in accordance with the fact the Suf Fe–S cluster biosynthetic system is essential for Gram-positive bacteria viability. However, in this study, we disrupted single sufC, sufD, and sufB genes of the suf operon successfully, showing that they are required for nitrogen fixation. This is in line with our previous work that the transcript abundances of sufCDSUB was up-regulated in N2-fixing condition [suf (sufCDSUB) operon can increase nitrogenase activity of E. coli 78-7 from 10% to 20% [sufS and sufU could not be deleted, suggesting a critical role of these proteins for the survival of P. polymyxa WLY78. Our finding that the sufCDB mutant phenotype could not be rescued by the nifU of K. oxytoca demonstrated that SufCDB is not interchangeable with the NifU of P. polymyxa WLY78. RT-PCR analysis indicated that nifU was transcribed in these complementation strains  or LD medium  at 30 °C with shaking. For assays of nitrogenase activity, P. polymyxa strains were grown in nitrogen-limited media under anaerobic conditions. The nitrogen-limited media contained (per liter) 0.4 g of Na2HPO4, 3.4 g of KH2PO4, 26 mg of CaCl2·2H2O, 30 mg of MgSO4, 0.3 mg of MnSO4, 36 mg of ferric citrate, 7.6 mg of Na2MoO4·2H2O, 10 mg of p-aminobenzoic acid, 5 µg of biotin, 2 mM glutamate, and 4 g of glucose as the carbon source. E. coli strains JM109 were used for routine cloning. Thermo-sensitive vector pRN5101 [P. polymyxa WLY78. The shuttle vector pHY300PLK was used for the complementation experiment. When appropriate, antibiotics were added in the following concentrations for maintenance of plasmids: 100 μg/mL of ampicillin, 12.5 μg/mL of tetracycline, and 5 μg/mL of erythromycin.Bacterial strains and plasmids used in this study are summarized in  pRN5101  was usednifS-like, ΔnfuA-like, ΔyutI, ΔnfuA-like/ΔyutI, ΔiscS, ΔiscS2, ΔsufC, ΔsufD, ΔsufB, ΔsufA, and ΔsufC2D2B2 were constructed through homologous recombination with pRN5101. The upstream (ca. 1 kb) and downstream fragments (ca.1.0 kb) flanking the coding region of iscS, iscS2, nifS-like, nfuA-like, yutI, sufC, sufD, sufB, sufCD, sufA, and sufC2D2B2 were separately PCR-amplified from the genomic DNA of P. polymyxa WLY78. The primers used for these PCR amplifications are listed in iscS, iscS2, nifS-like, nfuA-like, yutI, sufC, sufD, sufB, sufCD, sufA, and sufC2D2B2 were then fused with the Hind III/BamH I-digested pRN5101 vector using the Gibson assembly master mix , generating the recombinant plasmids pRDiscS, pRDiscS2, pRDnifS, pRDnfuA, pRDyutI, pRDsufC, pRDsufD, pRDsufB, pRDsufCD, pRDsufA, and pRDsufC2D2B2. Then, each of these recombinant plasmids was transformed into P. polymyxa WLY78, and the double-crossover transformants ΔiscS, ΔiscS2, ΔnifS-like, ΔnfuA-like, ΔyutI, ΔsufC, ΔsufD, ΔsufB, ΔsufCD, ΔsufA, and ΔsufC2D2B2 were selected from the initial Emr transformants after several rounds of nonselective growth at 39 °C and confirmed by PCR amplification and sequencing analysis. Further, pRDnfuA, pRDiscS2, and pRDsufCD were transformed into ΔnfuA-like, ΔiscS, and ΔsufB, respectively, to generate double mutants ΔnfuA-like/ΔyutI, ΔiscS/ΔiscS2, and ΔsufCDB.The in-frame-deletion mutants ΔnifS-like, ΔyutI, ΔsufC, ΔsufD, ΔsufB, and ΔsufCDB was performed. For complementation of ΔnifS-like, a 1578 bp DNA fragment containing the coding region of nifS-like and its own promoter was PCR-amplified from the genomic DNA of P. polymyxa WLY78. For complementation of the ΔyutI mutant, a 626 bp DNA fragment containing the coding region of yutI and its own promoter was PCR-amplified. For complementation of the ΔsufC mutant, a 1173 bp DNA fragment carrying the sufC coding region and its own promoter was PCR-amplified. For complementation of ΔsufD and ΔsufB mutants, a 390 bp promoter region of the sufCDSUB operon and their respective coding region were PCR-amplified. For complementation of the ΔsufCDB mutant, a 2501 bp DNA fragment containing the coding region of sufCD and its promoter, and a 1398 bp nifB coding sequence was PCR-amplified. These fragments were digested with BamHI/HindIII and ligated into the vector pHY300PLK, generating vectors pHYnifS, pHYyutI, pHYsufC, pHYsufD, pHYsufB, and pHYsufCDB. Each of these recombinant plasmids was correspondingly transformed into its mutants, and tetracycline-resistant (Tcr) transformants were selected and confirmed by PCR and sequencing. Complementation of ΔP. polymyxa WLY78 as the template and by using the primer sets P-yutI-F/YutIC49A-R and YutIC49A-F/C-yutI-R. The two PCR products were then fused with the Hind III/BamHI-digested pHY300PLK vector, yielding the complementary plasmid pHYyutI-C49A, and then the plasmid was transformed into ΔyutI. The same procedure was to obtain pHYyutI-C52A and pHYyutI-C49/52A. The accuracy of mutagenesis was verified by DNA sequencing.The substituted forms of YutI were constructed by the PCR-based mutagenesis method. The pairs of mutagenic primer were designed to be complementary to each other and to span the substituted site. To produce the YutIC49A variant, two separate PCR reactions were conducted with nifS gene was PCR-amplified from the genomic DNA of K. oxytoca M5a1, being under the control of the nifS-like promoter of P. polymyxa WLY78, and cloned to the plasmid pHY300PLK, generating the vector pHYnifS (K.o). This vector was transformed into a ΔnifS-like mutant, yielding the heterologous complementation strain ΔnifS-like/nifS-K.o. An 829 bp DNA fragment containing the coding region of nifU from K. oxytoca M5a1 and a 365 bp promoter region of yutI were PCR-amplified and assembled, generating the vector pHYnifU. Then, the constructed vector pHYnifU was transformed into the ΔyutI, ΔsufC, ΔsufD, ΔsufB, and ΔsufCDB mutants, yielding the heterologous complementation strain ΔyutI/nifU-K.o, ΔsufC/nifU-K.o, ΔsufD/nifU-K.o, ΔsufB/nifU-K.o, and ΔsufCDB/nifU-K.o, respectively. Similarly, a 591 bp DNA fragment containing the coding region of nfuA from E. coli and a 365 bp promoter region of yutI were PCR-amplified and assembled, generating the vector pHYnfuA (E. coli). Then it was transformed into the ΔyutI mutant, yielding the heterologous complementation strain ΔyutI/nfuA-E. coli.For heterologous complementation assays, the P. polymyxa WLY78 and its mutant strains were grown overnight in LD medium. The cultures were collected by centrifugation, washed three times with sterilized water, and then resuspended in a nitrogen-limited medium containing 2 mM glutamate as a nitrogen source to a final OD600 of 0.2–0.4. Then, 4 mL of the culture was transferred to a 25 mL test tube, and the test tube was sealed with a rubber stopper. The headspace (21 mL) in the tube was then evacuated and replaced with argon gas. Then, 2.1 mL of C2H2 (10% of the headspace volume) was injected into the test tube. Cultures were incubated at 30 °C. C2H4 production was analyzed by gas chromatography. The nitrogenase activity was expressed in nmol C2H4/mg protein/h. The nitrogenase activity assays were measured at least three times, and the error bars were evaluated as the standard deviation. Statistical analyses were performed using SPSS software version 20 . One-way ANOVA was employed to check the significant differences between strains. Means of different strains were compared using the least significant difference (LSD) at the 0.05, 0.01, or 0.001 level of probability.Acetylene reduction assays were performed, as described previously, to measure nitrogenase activity . P. polyP. polymyxa WLY78, ∆nifS-like, ∆yutI, ∆sufCDB, ∆sufC, and ∆sufD were grown under N2-fixing conditions (2 mM glutamate and without O2) and harvested after 8 h of incubation. To detect the expression of the nifU gene, total RNA was extracted from ∆sufC, ∆sufD, ∆sufB, ∆sufCDB, ∆sufC/nifU-K.o, ∆sufD/nifU-K.o, ∆sufB/nifU-K.o, and ∆sufCDB/nifU-K.o. Total RNA was isolated using TRIzol . The possibility of contamination of genomic DNA was eliminated by digestion with RNase-free DNase I . The integrity and size distribution of the RNA were verified by agarose gel electrophoresis, and the concentrations were determined spectrophotometrically. Synthesis of cDNA was carried out using RT Prime Mix according to the manufacturer’s specifications . cDNA (0.4 µg) was used for qRT-PCR. The relative transcript levels of sufD, sufS, sufU, and sufB were determined with 16S rDNA as a control by the SYBR Premix Ex Taq (Tli RNaseH Plus) kit . Primers for sufC, sufD, sufS, sufU, sufB, nifH, nifD, nifU, and 16S rDNA used for RT-PCR or qRT-PCR are listed in For quantitative real-time-PCR (qRT-PCR), cultures of P. polymyxa WLY78, ∆nifS-like, ∆sufCDB, and ∆yutI were grown under N2-fixing conditions and harvested after 20 h of incubation. Cells were collected and disrupted in lysis buffer  by sonication on ice. Cell debris were removed by centrifugation, and 40 µg of total proteins was analyzed by SDS-PAGE (10% acrylamide) and immunoblotting. Proteins were blotted onto a nitrocellulose (NC) membrane, and the Fe and MoFe proteins were detected using polyclonal anti-NifH and anti-NifD, respectively. Binding of antibodies was visualizedby enhanced chemiluminescence (ECL) .Cultures of P. polymyxa WLY78, the sufC, sufD, and sufB genes of the suf (sufCDSUB) operon, the nifS-like gene, and the yutI gene are required for the Fe–S cluster biosynthesis of nitrogenase. The cysteine desulfurase NifS-like catalyzes the production of sulfur, and SufCDB proteins provide a molecular scaffold for the assembly of the Fe–S cluster. YutI is an Fe–S carrier that is involved in nitrogen fixation.In


Natural environments can be used to promote health through facilitatingrecreational walking. However, efforts to encourage this often neglect messagesidentified in psychological research that are effective at influencingintentions to walk. This is despite the National Institute for Health and CareExcellence stating that promotional efforts should utilize theoreticalframeworks of behaviour change and be targeted towards less active adults. As anillustrative example, this experiment compared a prototypical recreationalwalking brochure with an “enhanced” version including suchpersuasive messages on people’s intentions to walk for recreation innatural environments. The enhanced brochure heightened intentions forinexperienced recreational walkers through our hypothesized mechanisms, butappeared to dissuade already-experienced walkers. Optimal messaging strategiesin recreational walking brochures require tailoring to more and less activereaderships. Guidelines are provided for authors of recreational walkingbrochures, though the principles and techniques could easily be extended toother means of outdoor walking promotion. Physical inactivity is a key public health challenge, contributing tonon-communicable diseases and premature mortality . WalkingThe use of natural environments such as green spaces  or bluespaces  for recreational walking is one such place-basedstrategy. Natural environments support brisk levels of walking . There iNatural environments, therefore, appear to be a promising setting for promotinghealth-enhancing physical activity, in particular recreational walking, whichmay be sustainable in the longer-term. However, interventions to promotephysical activity in natural environments have had limited success . IncontRegarding recreational walking in natural environments, there is policy-levelprecedent for its promotion. The National Institute for Health and CareExcellence (NICE) encourage local authority directors for countrysidemanagement, the environment, parks, public health, and leisure services, tocollaborate to ‘develop walking programmes for adults who are not activeenough, based on an accepted theoretical framework for behaviourchange’, and ‘ensure groups that are likely to be the leastactive are encouraged to participate, by addressing issues that may act as abarrier’ , p. 18. Although paper or digital brochures are commonly used in interventions . TheiriOne way of helping local authorities produce theory-informed messages is throughproviding guidance on which persuasive messages are effective at encouragingless active adults to form stronger intentions to undertake recreationalwalking. These messages should target mechanisms (processes by which behaviourchange occurs) and corresponding techniques (ways of affecting mechanisms)proposed by psychological theories  have beenot have comparableeffects for people who already regularly undertook recreational walking.Ultimately, we aimed to provide guidance to local authority directors on howsimple modifications could be made to existing recreational walking brochures in order to adhereto NICE guidelines and thus more optimally promote recreational walking innatural environments for those who would usually be less likely to do this.This study therefore hypothesized that ‘enhancing’ a recreationalwalking brochure with messages targeting attitudes, normative beliefs andperceived behavioural control could encourage so-called‘non-walkers’ to form stronger intentions for recreationalwalking in natural environments compared with an existing brochure. We furtherhypothesized that such enhancements would https://www.cint.com/consumer-insights-network/) were invited byemail in September 2015 to participate. Cint participants earn small financialrewards for completing online surveys. Participants who exhibit systematicresponses biases are removed .Guidance on behaviour change techniques that can be incorporated intowritten materials was consulted . PotentiMessages operationalizing these techniques were written into the enhancedbrochure being mindful not to interrupt the route directions whichconstituted the main narrative of the brochure. Piloting these brochures helped clarify messages targeting injunctivenormative beliefs , as too artificial. Such messageswere deleted.Recreational walking intentions were operationalized in two ways. The primaryoutcome analysed was a binary response to whether or not a participantrequested further walking information about outdoor recreational walking innatural environments at the end of the survey. Requesting furtherinformation was interpreted as reflective of greater intentions to engage insuch walks in the future. Two 7-point Likert-scale items measuringbehavioural intentions  were colTo distinguish people who do not regularly undertake recreational walks innatural environments from those who do, an item which classifiedparticipants into five stages of readiness to change their recreationalwalking behaviour  was creaα = 0.89).Items were created to assess whether the enhanced brochure impacted‘non-walkers’ intentions through the proposed psychologicalchange mechanisms of changing attitudes, raising normative beliefs, andheightening perceived behavioural control . Items mThe experiment also collected a series of demographic details which wereoperationalized in analyses as follows: sex , age, ethnicity , long-standing illness  and annual pre-taxhousehold income (five quintiles or ‘don’t know’).Ethnicity , long-stParticipants were randomized to one of the conditions based on a hyperlink sentto them in an invitation email. After giving consent, participants responded toquestions concerning recreational walking status, short- and long-termpropensity for visiting natural environments, ethnicity, long-standing illnessand income on successive pages. Prior to seeing the brochure, they read textthat was adapted from a study concerning immersion in natural environments. Participantswho indicated that they had not read the leaflet(n = 22), completed the experiment in underthree minutes (n = 96) or over20.26 min (n = 18), or had missingdata (n = 4) were excluded. This left atotal of n = 395, with 202 (51%) inthe original brochure condition and 193 (49%) in the enhanced brochurecondition. Females comprised 54% of the sample and the mean age was 42.‘Non-walkers’ comprised 46% of the sample.Originally, 535 participants were randomized to the two conditions  = 0.00, p = 0.99,pη = 0.00], sex, ethnicity , household income, illness/disability  or propensity for visiting naturalenvironments in the short term , or long term . Recreational walking status also did notdiffer with experimental condition . Descriptive statistics for the outcomesand mediators as a function of recreational walking status can be seen in Participants did not differ between experimental conditions in terms of age[not prompt more requests for further recreationalwalking information than the original brochure [odds ratio(OR) = 0.82;95% CI: 0.54, 1.25; Supplementary Table SC), but did prompt strongerstated intentions. As expected, people classified as‘walkers’ reported stronger revealed and stated intentions than ‘non-walkers’overall.Analysing all participants and controlling for potential confounds, the enhancedbrochure did  = 2.56;95% CI: 1.33, 5.07;  = 5.77;95% CI: 3.00, 11.51). Last, and unexpectedly, ‘walkers’who read the enhanced brochure made significantly fewerrequests than ‘non-walkers’ who read the original brochure. The pattern was the same for stated intentions,though effects were slightly weaker, and in the case of the latter unexpectedfinding, not significant  and all effects wereweaker , justifyb = 0.26;95% CI: 0.02, 0.50), explaining ≈45% of the variance inthe total effect; but attitudes and subjective norms did not mediate the effect, explaining ≈73% of the variance inthe total effect.As coefficients did not change substantially following the inclusion ofcovariates, the mediation models excluded covariates in favour of just theexperimental conditions, outcomes, and mediators. For‘non-walkers’, perceived behavioural control significantlymediated the effects of the enhanced brochure on stated intentions, explaining ≈25% of the variance inthe total effect. As a comparison, the same models were performed for‘walkers’  and as predicted, neitherdifferences in attitudes, descriptive norms, perceived behavioural control, northeir sum, mediated the relationship between the experimental brochure conditionand either outcome variable.None of the three constructs significantly mediated the effects of the enhancedbrochure on the revealed intentions for ‘non-walkers’ on theirown; but the combination of all three did, but not‘walkers’. This study further justifies the need for behaviourchange theory when designing recreational walking brochures, and indeed physicalactivity interventions more generally  and affective attitudes. This studycannot deconstruct which type of message may be more persuasive, but studieshave previously suggested that affective attitudes may be more important forpredicting the uptake of physical activity  which maThere are multiple ways in which brochure designers can promote perceivedbehavioural control . In this study, we targeted thischange mechanism in a number of ways (Supplementary Table SA): (i) promptingreattribution of past failures ; (ii) prompting barrier identification and planning inrelation to anticipated barriers (e.g. difficulty in climbing hills); (iii)setting graded tasks/goals ;(iv) providing feedback on performance (e.g. commending the recipient onsuccessful completion of a stage); (v) using arguments to bolster confidence(e.g. arguing against self-doubt and asserting that they can succeed in changingtheir behaviour); and (vi) prompting organization of social support . This change mechanism (enhancing confidence) may beprompted by a variety of behaviour change techniques  and was it is avery positive leaflet that made me feel comfortable in taking it on despitehaving no experience’; and ‘it was veryencouraging and felt like it was addressing me as an individual and not justgiving the route directions, which is the norm…it made me want tostart walking again’.Previous research has further demonstrated that confidence-building aspects ofperceived behavioural control are particularly important for forming intentionsto take up physical activity more generally  maybe most effective at encouraging ‘non-walkers’ to contemplatefuture outdoor recreational walking, our mediation models also suggest that thecombination of our three key change mechanisms is also important. Brochuredesigners are encouraged to consult practical guidance e.g.  on how tless likely to request further walking information afterreading the enhanced brochure, suggesting that the changes to the originalbrochure actually dissuaded these individuals from walking innatural environments.Brochure designers may also wish to design persuasive messages for‘walkers’ who are more familiar with walking trails. In thisstudy, these were classified as people who were already undertaking outdoorrecreational walking in natural environments. These people were substantiallyThe tone of the leaflet was quitecondescending’, and ‘leave the motivationalstuff to a separate section…it is annoying andpatronizing’. This could be seen as analogous to the notionof ‘baby talk’ in health psychology research where a health careprovider underestimates the patient’s knowledge and uses languageperceived as patronizing, thus leading to disengagement . Thus,‘walkers’, even more so than ‘non-walkers’,could be particularly responsive to the written content of recreational walkingbrochures.The original brochure may appeal more to ‘walkers’ because italready contained the sort of information that was more persuasive for thisgroup  those appealing to ‘non-walkers’ which attemptto increase intentions to engage in outdoor recreational walking in naturalenvironments by targeting determinants such as perceived behavioural control; and,(ii) those aimed at already-motivated ‘walkers’ which can assumemotivation, avoid the use of patronizing language, and focus on extrinsic featuresof a recreational walking route with clear instructions, thus supporting walkingmaintenance. Brochure authors are encouraged to make use of these guidelines andother existing practical guidance on how to construct messages which targetevidence-based antecedents of physical activity behaviour change and to be vigilantto the variability in effective communication strategies for different targetaudiences. Provision of supportive natural environments for physical activity isnecessary, but it is not a sufficient means of altering community- orpopulation-level physical activity behaviour. Individualized approaches, such asthose presented in this article remain fundamental to altering physical activitybehaviours.Health Promotion International online.This study was approved by the University of Exeter’s Sport and HealthSciences Research Ethics Committee.daaa150_Supplementary MaterialsClick here for additional data file.


Physician Associates (PAs) are healthcare professionals who have a general medical education background, having completed a two-year postgraduate degree. Whilst the number of PAs employed in healthcare trusts continues to increase, the number working in mental health settings remains small.Mersey Care NHS Foundation Trust employed two PAs two years ago. In August 2019, a third PA was recruited to work at Clock View Hospital, a general adult inpatient unit.This analysis aimed to establish the views of different members of the team across the three general adult wards and the Psychiatric Care Unit (PICU) at Clock View Hospital on the role of the PA.A sample of members of staff was identified from across the three general adult inpatient wards at and the PICU, comprising: senior doctors , junior trainees (Core Trainee and Foundation Trainees), Ward Manager, Deputy Ward Manager, Band 5 nurse and Assistant Practitioner. Each member of staff was asked to answer the question “On a scale of 1 to 10 , how happy are you to have a PA working on your ward?” Each staff member was then asked to provide comments on their views on the role of the PA.Twenty-three members of staff participated – 3 x senior doctors, 4 x junior trainees, 4 Ward Managers, 4 Deputy Ward Managers, 4 x Band 5 nurses and 4 x Assistant Practitioners. The respondents were distributed equally across the three general adult wards and the PICU. All 23 members of staff provided a score of 10 out 10 to the question about how happy they were to have a PA working on the ward. Many of the staff members provided some very positive comments on their respective views about the role of the PA at Clock View Hospital. No negative comments were provided by any members of staff.It is clear from the large sample of members of staff of different grade at Clock View Hospital that were surveyed that the PA has been a warmly received and welcome addition to the inpatient team and that the PA is viewed as having become an important and valued member of the inpatient team. This provides a strong argument for both Mersey Care NHS Foundation Trust, and other mental health trusts across the U.K., to consider employing more PAs to work in their inpatient units.


For JPUA-TDI, the presence of a signal at 7.94 ppm evidenced the possible side reactions between urethane linkages with secondary amine that resulted in an aryl-urea group (Ar-NH-COO-). Meanwhile, the peak of 2.89 ppm was assigned to the α-position of methylene to the carbamate (-CH2NHCOO) group in the JPUA-IPDI. From the rheological study, JO and JPUA-IPDI in pure form were classified as Newtonian fluids, while JPUA-TDI showed non-Newtonian behaviour with pseudoplastic or shear thinning behaviour at room temperature. At elevated temperatures, the JO, JPUA-IPDI mixture and JPUA-TDI mixture exhibited reductions in viscosity and shear stress as the shear rate increased. The JO and JPUA-IPDI mixture maintained Newtonian fluid behaviour at all temperature ranges. Meanwhile, the JPUA-TDI mixture showed shear thickening at 25 °C and shear thinning at 40 °C, 60 °C and 80 °C. The master curve graph based on the shear rate for the JO, JPUA-TDI mixture and JPUA-IPDI mixture at 25 °C, 40 °C, 60 °C and 80 °C was developed as a fluid behaviour reference for future storage and processing conditions during the encapsulation process. The encapsulation process can be conducted to fabricate a self-healing coating based on a microcapsule triggered either by air or ultra-violet (UV) radiation.Jatropha oil-based polyol (JOL) was prepared from crude Jatropha oil via an epoxidation and hydroxylation reaction. During the isocyanation step, two different types of diisocyanates; 2,4-toluene diisocyanate  and isophorone diisocyanate (IPDI), were introduced to produce Jatropha oil-based polyurethane acrylates (JPUA). The products were named JPUA-TDI and JPUA-IPDI, respectively. The success of the stepwise reactions of the resins was confirmed using  Polyurethane acrylate (PUA) is a subclass of polyurethane (PU) where the polyol and diisocyanate have been reacted and end-capped with an acrylate functional group during the termination reaction . The preIn the coating industry, the utilisation of vegetable oil as a precursor and binder has begun over the last few decades. This is because vegetable oils are cost-efficient and environmentally friendly compared to petroleum-based chemicals. This solves the issues related to petrochemical resin such as price fluctuation and the release of hazardous volatile organic compounds (VOCs) into the environment . VegetabJatropha curcas, which has currently has been widely planted in Latin America, Africa, India and South East Asia [Jatropha oil (JO) has emerged as one of the most suitable candidates for producing polyol for PUA applications as it is classified as a nonedible oil. It contains a phorbic ester compound that is poisonous for oral consumption ,16. JO iast Asia . It contast Asia . Apart fast Asia ,18, whicast Asia  but highast Asia  and olivast Asia .In the previous study, we reported the synthesis of jatropha oil-based polyol (JOL) via the epoxidation and hydroxylation route. JOL was then reacted with two types of diisocyanate known as 2,4-toluene diisocyanate  and isophorone diisocyanate (IPDI). As a result, two series of JPUA were produced and identified as JPUA-TDI and JPUA-IPDI, respectively. The mechanical properties for both the UV-curable JPUA-TDI and JPUA-IPDI-based films such as hardness, contact angle, haze and transmittance was discussed. The ratio of 35:65 (wt.%) between the monomer and JPUA was determined as the best ratio based on the performance of the mechanical properties .For fabrication of a self-healing coating, the pure JO, and the selected uncured coating formulations of JPUA-TDI and JPUA-IPDI were used as a healing agent or core content of the microcapsule in the next stage of the experiment. The microcapsules act as a reservoir that contains an active healing agent and is distributed evenly in the coating material. As the coating is exposed to scratches and cracks, the microcapsules will rupture and release the active agent into the crack volume. The active agent will react with the catalyst or natural condition such as atmospheric moisture, oxygen and sunlight to activate the polymerisation reaction in order to repair the crack surfaces .Several studies have been conducted on other types of oil such as linseed ,24, tungAs a solution, a rheology study was proposed to predict the fluid behaviour of JO and the selected uncured formulation mixture of JPUA-TDI and JPUA-IPDI in designing the microcapsule. Currently, no rheology study has been conducted to understand the fluid behaviour of the healing agent before the encapsulation process is carried out. The rheological properties provide information regarding fluid behaviour such as viscosity and shear stress at different temperatures and shear rates. This will also involve the correlation between the rheological properties with the chemical structure . In addi1H NMR analysis. Next, the chemical structure of the pure JO and uncured formulation mixture of JPUA-TDI and JPUA-IPDI were studied by relating them to the rheological properties and experimental parameters such as the variation in shear rate and temperature. Finally, the shear rate master curve graph for JO and both formulation resins of the JPUA-TDI mixture and JPUA-IPDI mixture was developed.In this paper, the aim was to further investigate the chemical structure of JO-based resin during the synthesis reaction of epoxidation, hydroxylation and isocyanation via Crude Jatropha oil was supplied by Biofuel Bionas Malaysia Sdn. Bhd., Kuala Lumpur, Malaysia. The chemical composition of the jatropha oil is listed in −1 under FTIR analysis.Details of the synthesis procedure of JOL and JPUA have been reported in the previous study of the same authors . In brie−1 and the appearance of a new peak at 824 cm−1 of the FTIR spectra were reported. This evidenced that the carbon double bond (C=C) had been converted to the epoxy group (C–O–C) to produce epoxidised Jatropha oil (EJO) [During the epoxidation step, 1 mol of JO was mixed with 0.6 mol of formic acid at 40 °C with a stirring rate of 300 rpm for 15 min. Then, 1.7 mol of hydrogen peroxide was added in a dropwise manner and the temperature was increased gradually to 60 °C. The mixture was allowed to complete the reaction over a total duration of 4.5 h. The mixture of epoxidised jatropha oil (EJO) was cooled and moved into a separating funnel. The unwanted aqueous layer was then carefully discarded. The remaining EJO was washed with distilled water until it was free from acid. The excess water was removed using a rotary evaporator with a temperature of 60 °C. The disappearance of the peak at 3012 cmil (EJO) .−1, which indicated that EJO was converted into Jatropha oil-based polyol (JOL) [For the hydroxylation step, EJO was reacted with methanol in the presence of an acid catalyst. Initially, 9 mol of methanol was mixed with 1 mol of 0.07% of sulphuric acid at 40 °C and 300 rpm for 15 min. Next, 10 mol of EJO was added into the solution and the reaction was mixed at 65 °C and 300 rpm for 30 min. The mixture was cooled and then transferred into a separating funnel to remove the aqueous layer. The JOL was washed with distilled water until it reached neutral pH. To purify the JOL, the solution was run under a vacuum distillator at 60 °C to remove the excess water and methanol. For FTIR analysis, the broad peak of the hydroxyl (OH) group appeared at 3435 cmol (JOL) .To produce JPUA, JOL was reacted with two types of diisocyanates, namely 2,4-TDI and IPDI via an isocyanation reaction. The reaction was performed based on a stoichiometric calculation of (1:1:1) for JOL:IPDI:HEMA, respectively. As isocyanates are sensitive to moisture, the reaction was conducted in a four-neck flask in a nitrogen-purged system.w/w) of DBTDL was mixed with JOL at 350 rpm and 60 °C for 15 min. Then, IPDI was added slowly via a dropping funnel to start the isocyanation reaction. Upon completion of the dropping of IPDI, the mixture was stirred continuously for another 2 h. From FTIR monitoring, the isocyanate peak (N=C=O) at 2270 cm−1 was observed during the initial addition of IPDI. The disappearance of the peak at 2270 cm−1 after 2 h indicated that all isocyanates had been consumed. Moreover, the peak of (–OH) at 3435 cm−1 was shifted to 3350 cm−1, which corresponds to (–NH) amide bending. New peaks of (–NH) amide stretching and carbonyl (C=O) were observed at 1532 at 1720 cm−1, respectively [Next, 1% (ectively .2) was observed at 1663 cm−1 and the acrylate vinyl functionality of (CH2=CH–COO–) appeared at 815 cm−1 under FTIR analysis, which proved that the acrylation reaction had occurred [The reaction was terminated via an acrylation reaction with HEMA. Before HEMA was added, the temperature was set to 40 °C and HEMA was introduced into the mixture in a dropwise manner. Then, the temperature was raised up to 70 °C and the reaction was continued for 1 h to complete the reaction of JPUA-IPDI. The peak of the acrylate double bond . DMSO solvent was used to dilute the sample and also as an internal standard during the analysis.The chemical structures of JO, EJO, JOL, JPUA-TDI and JPUA-IPDI were predicted using 1H NMR spectrum, as shown in Equation (1) [f is integration area of the functional group of either the carbon double bond, epoxy or hydroxyl group, and Atotal is the total integration area of the triglyceride.For synthesis of the polyol, the presence of the functional groups such as the carbon double bond, epoxy and hydroxyl groups during the stepwise reactions was determined as the function to one mole of triglyceride in JO, EJO and JOL, respectively. The percentages of the functional groups were calculated based on the integration of the peak areas of the tion (1) .(1)Funcw/w) of benzophenone was added based on the total amount of either JPUA-TDI or JPUA-IPDI and TMPTA. The benzophenone produced a radical species only when it was exposed to UV light to start the radical polymerisation reaction. Therefore, all samples were put in amber bottles to avoid exposure to UV sunlight. All components were mixed using a mechanical stirrer at 300 rpm for 15 min at room temperature until it reached a homogenous condition.The rheological properties of the JO, JPUA-TDI and JPUA-IPDI were compared between their original form and the formulation mixture. The components of each sample were prepared as listed in −1. The rheological properties of the resins were determined using a Haake Mars Rheometer . Then, 1 g of resin was laid on a flat sample platform for a plate–plate geometry mode. The gap between the spindle and platform was set at 1 mm. The spindle (PP60) was then rotated on the sample. First, the viscosity and shear stress of pure JO, JPUA-TDI and JPUA-IPDI were measured at 25 °C with the shear rate range set between 0 and 100 sViscosity measures the liquid flow resistance and is defined as force (F) divided by area (A); (F/A). The speed of a moving liquid or (dv/dr) is known as the shear rate, whilst shear stress is the force that acts parallel to the cross-section of the material. The correlation between viscosity (η), shear rate (γ) and shear stress (σ) is expressed in Equation (2) below :(2)η=σ˙γ−1 for all samples. For a greater understanding of the relationship between shear stress in the range of tested shear rates, the power law equation was used. The equation is expressed as Equation (3) below.−1); and n is the flow behaviour index.The viscosity and shear stress of the JO, JPUA-TDI mixture and JPUA-IPDI mixture were compared at four different temperatures, namely 25 °C, 40 °C, 60 °C and 80 °C. The shear rate values were set in the range from 0 to 100 sBased on the calculation, the consistency index (k) and flow behaviour index (n) for JO, the JPUA-TDI mixture and JPUA-IPDI mixture were determined from the graph of ln shear stress versus ln shear rate.Next, the master curve of shear stress–shear rate was constructed with the aim to simultaneously fit the data of different temperatures and to predict the flow behaviour. It offers accurate and practical results rather than fitting the data separately . Based oThe shift factor, aT, was calculated based on the calculated shear rate divided by the reference shear rate (at 60 °C). The compilations of the relationship between the shear stress and shear strain of JO, the JPUA-TDI mixture and JPUA-IPDI mixture at 25 °C, 40 °C, 60 °C and 80 °C were analysed.1H NMR analysis. The 1H NMR can predict the molecular structure based on hydrogen shifting. The spectra of 1H NMR of JO, EJO and JOL are shown in In the previous study by the same authors, the FTIR analysis was conducted at each step of the chemical reaction to produce JPUA-TDI and JPUA-IPDI. However, FTIR only detected the existence of the functional group where both JPUA-TDI and JPUA-IPDI produced a similar trend due to owning a similar functional group . TherefoH2), (R–OCH2–CH(OR’)–CH2OH) and (ROCH2–CH(OR’)–CH2OR), respectively [1H NMR spectra, those moieties were identified accordingly as a, b and c. However, during the isocyanation process to produce the JPUA-TDI and JPUA-IPDI, the chemical structures became longer and more complex. Therefore, as mentioned by Abril-Milán and co-workers, the analysis of the proton NMR spectra for PU was conducted on the basis of a certain part of the fatty acid. This involved the existence of active functional groups such as hydroxyl of the fatty acid chain. However, the analysis was relatively valid to give important information for an average chemical structure of the whole PU [1H NMR analysis, as it is too small compared to the whole chemical structure of the jatropha oil-based PUA.In general, JO, EJO, JOL, JPUA-TDI and JPUA-IPDI inherit similar features of the Jatropha oil triglyceride backbone. For example, the signals around 2.2 to 2.3 ppm, 3.5 to 3.7 ppm and 4.00 to 4.30 ppm indicated glycerol moieties of (OCO–Cectively . In the whole PU . In thisHC=CH–) that was detected at 5.35 ppm under 1H NMR. Apart from that, the signal of the methylene group adjacent to the proton of the carbon double bond (RCH2CH=CH–) was spotted at 2.02 ppm. Meanwhile, the peak of 2.75 ppm was attributed to the methylene protons between the two carbon double bonds (=CH–CH2–CH=), which proved the existence of linoleic fatty acid in the triglyceride of JO molecules. This result was comparable with other studies on pure JO [In  pure JO ,44.HC=CH–) at 5.35 ppm was diminished. However, there was a trace of an unsaturated part of the carbon double bond in the EJO sample, which is shown by a small peak of 2.8 ppm that possibly belongs to (=CH–CH2–CH=). Nevertheless, the existence of epoxy features was shown by the presence of the methine of the epoxy group in EJO (–HC–O–CH–) at the 2.90 to 3.00 ppm region. Apart from that, the signal at 1.41 to 1.57 ppm was recognised as a methylene proton adjacent to the epoxy group (–HC–O–CH–CH2–), whereas the chemical shift at 1.65 ppm was attributed to the methylene proton adjacent in between two epoxy groups (HCOCH–CH2–HCOCH). Similar observations have been revealed by other researchers on palm and jatropha oil-based epoxy resin [In the epoxidation step, the carbon double bond (C=C) in the JO sample was converted into an epoxy (C–O–C) group. In xy resin ,43. Morexy resin .H–OH) of the hydroxyl group [Next, the EJO underwent hydroxylation where the ring opening reaction of epoxy occurred to form the hydroxyl group. Based on yl group ,45. Furtyl group . This ev1H NMR diagram of JPUA-TDI in H3) of 2,4-TDI was shown at the peak of 2.73 ppm. The presence of the carbamate group (–NH–COO) at 4.80 ppm evidenced the reaction between the isocyanate and hydroxyl groups. The formation of a peak of 7.94 ppm explained the possible side reactions between urethane linkages with secondary amine that resulted in the aryl-urea group (Ar–NH–COO–). This result has good agreement, as mentioned by Yildiz et al., [For isocyanation, JPUA-TDI was formed by the reaction of JOL with aromatic TDI, while JPUA-IPDI was synthesised from cycloaliphatic IPDI-based diisocyanate. The chemical reactions were terminated with an acrylation reaction with HEMA. The proton NMR spectra of JPUA-TDI and JPUA-IPDI are depicted in  et al., , on stru et al., .1H NMR diagram of JPUA-IPDI in H2–) were mostly attributed by peaks of 1.23 ppm. The peak of 7.94 ppm corresponded to the proton on the carbamate group (–NHCOO–) in the urethane linkage after reaction between the isocyanate (–NCO) of IPDI with the hydroxyl (–OH) group. Moreover, the proton signal of (R2CHNCOO–) that attached to the cycloaliphatic group appeared at 4.11 ppm, as reported by Patil et al. [H2NHCOO), which possibly connected to the cycloaliphatic moeities [H3) appeared at 1.90 ppm [Based on the l et al. . As revemoeities . Similar1.90 ppm .−1 at 25 °C. The rheogram is displayed in A rheological study was performed to provide information about how the processing condition such as shear rate and temperature affects the fluid behaviour. Initially, the viscosity and shear stress of the JO, JPUA-TDI and JPUA-IPDI in their virgin form were determined over a shear rate range from 0 to 100 sAs seen in the graph in the inset of A liquid can flow due to the ability of the molecules to slide or move between each other. Generally, as the shear rate increased, a higher force would be required to deform the intermolecular force among the molecules in the JO, JPUA-TDI and JPUA-IPDI in order to move in between each other. Thus, the shear stress of JO, JPUA-TDI and JPUA-IPDI increased with the shear rate . FurtherJPUA-TDI had the highest viscosity followed by JPUA-IPDI and JO. Based on the structure investigation as illustrated in In addition, the viscosity of JPUA-TDI and JPUA-IPDI related to the shape of their molecules is described in 2–) in between the cycloaliphatic ring and urethane bond (–NHCOO–) on the acrylate side chain produced more dangling chains on the JPUA-IPDI structure [Meanwhile, the presence of the methylene group  and shift factor values are tabulated in −1. This indicated that the information regarding the JO and JPUA-IPDI mixture fluid behaviour at different temperatures was successfully combined in a single data sheet for feasibility studies and for future reference. However, as the JPUA-TDI mixture was a non-Newtonian fluid with shear thinning and shear thickening, the data obeyed a perfect single line only until the shear rate of 70 s−1. For this reason, it was challenging to utilise the non-Newtonian fluid for the encapsulation process as the trend was sometimes unpredictable. The JPUA-TDI mixture was discarded due to the possibility of considerable difficulty in the processing, as reported by other researchers, such as agglomeration issues, sticky microcapsules [Based on capsules  and low capsules . Therefo1H NMR analysis via the presence of urethane linkage features. The chemical structure was predicted based on proton shifting. The chemical structure of the JO, JPUA-TDI and JPUA-IPDI affected their rheological behaviour. The viscosity of the resin was determined in the manner of JPUA-TDI > JPUA-IPDI > JO related to their structural conformation. For the JPUA-TDI mixture and JPUA-IPDI mixture, the addition of a TMPTA monomer effectively reduced the fluid viscosity. At 25 °C, 40 °C, 60 °C and 80 °C, the JO and JPUA-IPDI mixture exhibited Newtonian behaviour and showed single overlapping data when presented in the master curve graph over the whole shear rate range. The JPUA-TDI mixture showed a shear thickening fluid at 25 °C and shear thinning behaviour at 40 °C, 60 °C and 80 °C. Apart from that, the JPUA-TDI mixture revealed an unstable fluid behaviour as it only conformed in a single line until a shear rate of 70 s−1 in the master curve graph. In order to minimise problems such as agglomeration and sticky microcapsules, the JPUA-TDI mixture was discarded and only the JO and JPUA-IPDI mixture were chosen to proceed for the encapsulation experiment, which targeted self-healing coating applications using air or the UV mode of repairing.Success of the chemical reaction in producing JPUA-TDI and JPUA-IPDI was confirmed via


The aim of the present study was to describe in detail an approach to proctectomy in ulcerative colitis (UC), which can be standardized; near-total mesorectal excision (near-TME), to prevent injuries to autonomic pelvic nerves and subsequent sexual dysfunction.We demonstrate the technique ex vivo on a cadaver from a male patient in lithotomy position and on a sagittal section of a male pelvis. We also demonstrate the technique in vivo in two male patients diagnosed with UC, with no history of sexual dysfunction or bowel neoplasia. The study was performed at the Human Embryology and Anatomy Department. University of Valencia, Spain.nervi erigentes, the inferior hypogastric plexus, the pelvic plexus and the cavernous nerves.The posterolateral dissection during a near-TME is similar to that of an oncologic TME, whereas the anterolateral is similar to that of a close rectal dissection. The near-TME technique preserves the superior hypogastric plexus, the hypogastric nerves, the The near-TME technique is the standardisation of the technique for proctectomy in male patients with ulcerative colitis. Near-TME requires experience in pelvic surgery and an exhaustive knowledge of the embryological development and of the surgical anatomy of the pelvis.The online version contains supplementary material available at 10.1007/s10151-022-02579-z. Among the proposed techniques for proctectomy in patients with ulcerative colitis (UC), the most frequently described is total mesorectal excision (TME) , commonlAuthors of a recent position statement of the Italian Society of Colorectal Surgery (SICCR) advised that a plane of dissection close to the rectum or an incomplete mesorectal excision is preferred to preserve fertility and avoid nerve injuries, if no rectal cancer or dysplasia of any degree is present in the rectum . Some auThe aim of the present study was to describe in detail an approach to proctectomy in UC, which can be standardised, the near-total mesorectal excision (near-TME), to prevent injuries to autonomic pelvic nerves and subsequent sexual dysfunction.In video 1, we demonstrate the technique on a cadaver from a male patient in lithotomy position and on a sagittal section of a male pelvis. In video 2, we demonstrate near-TME in two male patients diagnosed with UC, with no history of bowel neoplasia.nervi erigentes can be injured during posterior dissection, at the level of S3-S4; the inferior hypogastric plexus is threatened during the lateral mesorectal dissection, the cavernous nerves during anterolateral dissection, and the connections of cavernous nerves of both sides during the anterior dissection [The injury of the autonomic nerves during TME can occur at five sites: the superior hypogastric plexus and the hypogastric nerves can be damaged during the posterior dissection of the mesorectum, at the level of the promontory; the ssection  Fig. 1)nervi erilevator ani, as no other nerve structures are encountered following this route Supplementary file2 (MP4 416217 kb)Supplementary file3 (DOCX 11 kb)Below is the link to the electronic supplementary material.


The aim of this study was to assess the nutritional status and risk factors for malnutrition in Behçet’s disease (BD) and neuro-Behçet’s disease (NBD) patients. Medical recordings of 74 patients with BD without neurological involvement (BDWoNI), 72 patients with NBD, and 50 patients with other diseases  were analyzed retrospectively. The serum analyses were performed in the inactive period of disease. Chronic malnutrition was defined as low levels of serum albumin (<3.5 g/dL) with normal sedimentation rate and normal serum CRP levels. Six (8.3%) of the patients in NBD group, 1 (1.4%) of the patients in BDWoNI group, and none of the patients in control group had chronic malnutrition (p = 0.029). Malnutrition rate was higher in NBD than control group (p = 0.036). The mean expanded disability status scale score was 2.92 ± 3.35 (range: 0–8) in patients with malnutrition and 1.44 ± 1.76 (range: 0–9) in patients without malnutrition in NBD group (p = 0.457). The ratio of patients having a progressive disease course was 33.3% and 7.6% in patients with and without malnutrition in NBD group, respectively. The median value of the duration of neurological involvement was 2 years (0–16) in patients with malnutrition and 6.5 years (0–18) in patients without malnutrition in NBD group (p = 0.046). There was no association between malnutrition and medications, disability scores, functional system involvement or findings on brain MRI.Malnutrition was higher in patients with NBD. Higher disability level and progressive disease course may be risk factors for malnutrition in NBD. Malnutrition may be seen more frequently in earlier phases of neurological involvement. Behçet’s disease (BD) is a multisystem immune mediated disorder of unknown etiology. Nervous system involvement in BD is referred to as neuro-Behçet’s disease (NBD) and classified as parenchymal and nonparenchymal [1]. Brainstem, thalamus, basal ganglia, spinal cord, and cerebral hemispheres are main regions affected in parenchymal NBD. Manifestations of nonparenchymal NBD include cerebral venous thrombosis, intracranial hypertension syndrome (pseudotumour cerebri), and aseptic meningitis .Malnutrition can result from starvation, diseases or advanced ageing, alone or in combination [3]. Malnutrition in chronic inflammatory diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and systemic sclerosis and in which gastrointestinal system is predominantly affected such as Crohn’s disease or ulcerative colitis has been investigated in several studies [4–8]. However, malnutrition rate and risk factors for malnutrition and impact of nutritional status on the course of disease have not been well studied in chronic inflammatory disorders affecting nervous system. To our knowledge there is no study in English based literature investigating nutritional issues in NBD, a relatively rare inflammatory disease of nervous system. The aim of this study is to assess the nutritional status and risk factors for malnutrition in NBD patients. Approval was obtained from the research ethics committee of Ankara University. Files of the patients with a diagnosis of Behçet’s disease and neuro-Behçet’s disease between January 2007 and December 2018 at our hospital were reviewed. As a control group, patients with carpal tunnel syndrome or lumbar discopathy who had serum albumin, C-reactive protein (CRP) levels and erythrocyte sedimentation rate in their files were included in the same period. Date of diagnosis with BD and NBD, type of NBD , medications, functional system involvements according to the Kurtzke functional system score, expanded disability status scale (EDSS) scores, modified Rankin scale score (mRS), findings on brain magnetic resonance imaging (MRI), serum albumin, C-reactive protein (CRP) levels, and erythrocyte sedimentation rate that were performed in the inactive period of BD and NBD were recorded. Just serum albumin, C-reactive protein (CRP) levels and erythrocyte sedimentation rate were documented in the control group. Chronic malnutrition was defined as low levels of serum albumin (<3.5 g/dL) with normal sedimentation rate and normal serum CRP levels [3]. Statistical analysis was performed using the Statistical Package for the Social Sciences v: 11.5 . Data are presented as mean ± standard deviation and median (minimum-maximum) for quantitative variables, and as percentages for qualitative variables. Chi-Square test was used for comparing qualitative variables between groups. When normal distribution is not the case, Mann–Whitney U and Kruskal–Wallis tests were used for comparison of quantitative variables in two and more than two groups, respectively. When normal distribution was provided, the differences of quantitative variables between more than two groups were analyzed using a one way ANOVA test. A value of p < 0.05 was considered statistically significant. When Kruskal–Wallis was applied, Bonferroni correction was applied for multiple comparisons, and a p value < 0.016 (0.05/3) was considered significant as error correction. Seventy-two patients with NBD, 74 patients with BD without neurological involvement (BDWoNI), and 50 patients with other diseases as control group  were included in the study (Table 1). The mean ages of patients in the groups were similar . As the result of the ANOVA test was not significant, multiple comparisons were not done as further analysis. The ratio of male patients was higher in the NBD group ; whereas male/female ratio was similar in other two groups (p = 0.115). Duration of Behçet’s disease was longer in NBD group (p = 0.002). Data about neuro-Behçet’s disease duration, NBD type, EDSS score and mRS scores were available for only NBD patients. Demographic and clinical features of the patients are summarized in Table 1.The disease was inactive in all patients in BDWoNI and NBD groups when blood analysis was performed. Mean serum albumin levels were 4.1(2.5–5.7), 4.3(3.3–5.0), 4.1(3.5–5.1) g/dL in NBD, BDWoNI and control group, respectively. Mean serum albumin level was higher in the BDWoNI group than the other two groups (p < 0.001). Six (8.3%) patients in the NBD group, 1 (1.4%) patient in the BDWoNI group had chronic malnutrition whereas there was not any patient with chronic malnutrition in the control group; the malnutrition rate was different statistically between three groups (p = 0.029); the difference was between NBD and control group (p = 0.036); malnutrition rate in NBD was similar to the BDWoNI group (p = 0.061). In the NBD group, the mean EDSS score was 2.92 ± 3.35 (range: 0–8) in patients with malnutrition and 1.44 ± 1.76 (range: 0–9) in patients without malnutrition; the difference was not statistically significant (p = 0.457). The NBD type was parenchymal in 43 (65.2%) of patients without malnutrition and 4 (66.7%) of patients with malnutrition; the difference was not statistically significant (p = 0.843). Two of six patients with malnutrition in NBD group (33.3%) had a progressive course; this ratio was 7.6% (5 of 66 patients) in the group of NBD patients without malnutrition; the difference was not significant (p = 0.101). Eight patients in NBD group and 6 patients in BDWoNI group had gastrointestinal system (GIS) involvement; interestingly, none of them had malnutrition.The median value of BD duration was 20.5 years (min-max: 0–25) in patients with malnutrition and, 14 years (min-max: 1–42) in patients without malnutrition in NBD group; the difference was not significant (p = 0.909). Due to presence of only one patient with malnutrition in BDWoNI group, statistical analysis was not possible for evaluation the relationship between disease duration and malnutrition in that group. So, we collected all patients with (6 patients in NBD group and one patient in BDWoNI group) and without malnutrition (66 patients in NBD group and 73 patients in BDWoNI group) in two groups, and assessed whether BD duration had any effect on the presence of malnutrition; there was not any relationship between them (p = 0.804). The median value of the duration of neurological involvement was 2 years (0–16) in patients with malnutrition and 6.5 years (0–18) in patients without malnutrition in NBD group; malnutrition rate was higher in patients with shorter duration of neurological involvement (p = 0.046). EDSS score, mRS score, functional system involvement  and lesion localization on brain MRI were not related to the presence of malnutrition in NBD group (Table 2). Except one patient in BDWoNI, all patients in BDWoNI and NBD group were treated with azatiopyrin and/or kolsisin (Table 2). Oral steroid plus azatiopyrin and/or kolsisin were given in three and 14 patients in BDWoNI group and NBD group, respectively; there was not any significant difference between patients with and without malnutrition in terms of treatment . It has been well known that neuro-Behçet’s disease is more frequent in males [1]. A similar finding was found in our NBD group, too. Behçet’s disease may involve gastrointestinal system which may have a major effect on malnutrition. There are some data on malnutrition in inflammatory bowel diseases (IBD). In a large prospective multicenter study, the prevalence of malnutrition was 20% in outpatients with IBD [8]. In that study, the risk factors for malnutrition were active disease, history of abdominal surgery and self-imposed food restrictions. The authors emphasized the high frequency of food avoidance in patients with IBD based on their beliefs. There were 12 patients with gastrointestinal system involvement in our study, the disease was inactive at the time that serum samples were obtained, and none of these patients had chronic malnutrition. This finding may indicate that gastrointestinal involvement unless it is active, has no major contributing effect on chronic malnutrition in BD patients. The mechanisms in disease related malnutrition without inflammation include dysphagia, psychiatric disorders and malabsorption. Inflammation is known to have an important role in malnutrition [9–11]. The pathways triggered by inflammation are different, so the term ‘disease-related malnutrition with inflammation (DRMWI)’ is used [9]. Inflammation leads to catabolic condition and results in anorexia and tissue breakdown. Disease related malnutrition with inflammation is classified as chronic DRMWI and acute disease- or injury-related malnutrition. There is a more prominent inflammatory response in acute DRMWI. On the other hand, chronic DRMWI may be seen in patients with a chronic disorder including cancer, chronic kidney and pulmonary diseases or congestive heart disease and is characterized by malnutrition and an ongoing mild inflammatory activity which is indicated by biochemical markers including high levels of serum CRP and/or low levels of serum albumin [9]. According to this definition, reduced levels of albumin, even in the case of normal levels of serum CRP is considered as an indicator of inflammation. In other words, inflammation is regarded as a cause of malnutrition [10]. In the last ESPEN guideline, the authors mentioned that there are no good biochemical markers of nutritional status, and plasma albumin levels may be used to monitor catabolic activity [9]. The levels of serum albumin, prealbumin, transferrin, retinol binding protein, complement C3 fraction and immunoglobulin M have been widely used as indicators of nutritional status [12–14]. Among these serum proteins, albumin has been considered as a better indicator for chronic malnutrition because of its long half-life (21 days) . Measurement of serum albumin level is easy and has a low cost. Based on some previous studies, we defined chronic malnutrition as low levels of serum albumin with normal sedimentation rate and normal serum CRP levels .Chronic malnutrition rate was higher in patients with NBD (8.3%) than control group (0), in the study. Moreover, although the difference was not statistically significant, chronic malnutrition rate was higher in NBD group than BDWoNI group (1.4%). We thought the involvement of nervous system may be a major risk factor for malnutrition. On the other hand, Baron M et al. demonstrated that although more than 20% of their systemic sclerosis cohort was at high risk for malnutrition, only 2% of them had low albumin levels; they suggested that low serum albumin level may not be a useful marker for malnutrition because patients with malnutrition may have normal albumin levels in chronic diseases [6]. This issue should be clarified through further research. The nutritional profile of patients with chronic inflammatory disorders including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and systemic sclerosis may be affected by the inflammation with altering the metabolism and by drugs used to control the disease which causes appetite and gastrointestinal changes . Limited physical activity due to joint involvement also may contribute to malnutrition in these patients. A study assessing the nutritional status of 72 women with rheumatoid arthritis using anthropometric and food consumption measurements showed that, despite half of the patients were overweight or obese, their calorie and microelements intake were low [4]. Moreover, the frequency of low-calorie intake was higher among patients treated with combined leflunomide+methotrexate and leflunomide+prednisone. Fukuda et al. showed that the mean arm muscle area (which expresses muscle protein) was significantly lower and serum albumin level  was negatively correlated with disease activity in patients with RA [5]. Although we did not find any effect of BD duration on the presence of malnutrition, the patients with malnutrition had longer disease duration than without it in all BDWoNI and NBD groups. However, we found that the duration of neurological involvement was shorter in patients with malnutrition than others in NBD group. We thought that malnutrition may be more frequent in earlier years of neurological involvement and with good control of the disease the rate of malnutrition may decrease with time. Moreover, the treatment modalities were similar in patients with and without malnutrition. Therefore, we thought the medications have no any effect on malnutrition in NBD patients.Malnutrition rate and risk factors for malnutrition have been reported in patients with stroke, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Parkinson’s disease (PD). For instance, a meta-analysis revealed the risk factors for malnutrition in stroke as malnutrition on admission, dysphagia, previous stroke, diabetes mellitus, tube feeding and reduced level of consciousness [15]. Among these risk factors, dysphagia is considered to have a major contributing effect on malnutrition. Recently, the European Society for Clinical Nutrition and Metabolism (ESPEN) reported a guideline for nutrition in neurology [16]. This guideline offers recommendations for malnutrition in patients with stroke, ALS, MS and PD, and is generally focused on dysphagia. However, malnutrition may not always be caused by dysphagia. It has been shown that there was no association between dysphagia and malnutrition in MS patients . In our study, too, none of our patients had dysphagia and there was not any association between brainstem involvement and malnutrition in NBD patients. Despite the difference was insignificant, the ratio of patients having a progressive neurological disease course was higher in patients with malnutrition than in patients without malnutrition  in NBD group in the study. The underlying inflammatory mechanisms may differ between relapsing remitting and progressive disease course. It has been shown that olfactory function may be impaired in BD patients [19]. Olfactory dysfunction may affect appetite and cause malnutrition. However, we did not evaluate olfactory functions in the study; this issue should be investigated in future researches. Mean EDSS was almost 3 and 1.5 in patients with and without malnutrition in NBD group, respectively. Although the difference was not significant statistically, we thought higher disability level may negatively affect malnutrition frequency; this issue should be evaluated in future studies including larger sample sizes. Another aspect of malnutrition in NBD may be mood disorders. It has been shown that total energy intake was significantly higher in patients with stroke that not having anxiety/depression than in patients having anxiety/depression [20]. Mood disorders are common in BD patients and may alter food intake [21].Six (8.3%) of patients in NBD group, 1 (1.4%) of patients in BDWoNI group, and none of the patient in control group had chronic malnutrition (p = 0.029). Malnutrition rate was higher in NBD than control group (p = 0.036). The mean EDSS score was 2.92 ± 3.35 (range: 0–8) in patients with malnutrition and 1.44 ± 1.76 (range: 0–9) in patients without malnutrition in NBD group (p = 0.457). The ratio of patients having a progressive neurological disease course was 33.3% and 7.6% in patients with and without malnutrition in NBD group, respectively. The median value of the duration of neurological involvement was 2 years (0–16) in patients with malnutrition and 6,5 years (0–18) in patients without malnutrition in NBD group (p = 0.046). There was no association between malnutrition and medications, disability scores, functional system involvement or findings on brain MRI.In summary, the rate of chronic malnutrition was higher in NBD than control group in this study. Moreover, despite it has not been found statistically significant, chronic malnutrition rate was higher in NBD group. Higher disability level and progressive disease course may be risk factors for malnutrition in NBD. Furthermore, malnutrition may be seen more frequently in earlier phases of neurological involvement. Our study was retrospective in nature. NBD is a relatively rare neurological disorder, so, the number of patients was small. Chronic malnutrition was defined with only serum albumin and CRP levels and sedimentation rate in the study. A detailed nutritional assessment was not performed. For these reasons the results of this study should be interpreted cautiously. However, findings of this study may draw attention to the higher incidence and potential importance of nutritional status in patients with NBD. There seems to be a number of possible risk factors needed to be investigated including possible taste and odor changes, depression/anxiety presence, dietary habits, and their effect on nutritional status in patients with NBD. Future prospective studies including larger sample sizes may provide a better understanding of nutritional status and risk factors of malnutrition in NBD patients. The study was a clinical study and retrospective in nature. So, we did not provide informed consent.


Y) and both chroma  components, respectively, under random access profile.With the rapid advancement in many multimedia applications, such as video gaming, computer vision applications, and video streaming and surveillance, video quality remains an open challenge. Despite the existence of the standardized video quality as well as high definition (HD) and ultrahigh definition (UHD), enhancing the quality for the video compression standard will improve the video streaming resolution and satisfy end user's quality of service (QoS). Versatile video coding (VVC) is the latest video coding standard that achieves significant coding efficiency. VVC will help spread high-quality video services and emerging applications, such as high dynamic range (HDR), high frame rate (HFR), and omnidirectional 360-degree multimedia compared to its predecessor high efficiency video coding (HEVC). Given its valuable results, the emerging field of deep learning is attracting the attention of scientists and prompts them to solve many contributions. In this study, we investigate the deep learning efficiency to the new VVC standard in order to improve video quality. However, in this work, we propose a wide-activated squeeze-and-excitation deep convolutional neural network (WSE-DCNN) technique-based video quality enhancement for VVC. Thus, the VVC conventional in-loop filtering will be replaced by the suggested WSE-DCNN technique that is expected to eliminate the compression artifacts in order to improve visual quality. Numerical results demonstrate the efficacy of the proposed model achieving approximately −2.85%, −8.89%, and −10.05% BD-rate reduction of the luma ( With emerging technologies that have rapidly evolved, multimedia services and video applications have significantly increased. Therefore, higher resolution (4K and 8K), especially for video games, e-learning, video conferencing, and surveillance tasks, is required to meet end-users viewing quality specifications. A next generation video encoding, established by the Joint Video Experts Team (JVET) in July 2020 , was theIn the same way, as for HEVC, in order to remove video compression artifacts and improve reconstructed video quality, VVC standard adopts the loop filtering technique, including the deblocking filter (DBF), sample adaptive offset (SAO), and adaptive loop filter (ALF). The DBF is designed to eliminate artifacts along block borders using discontinuity-based smoothing filters , 4. ThenALF is a modern VVC function that removes distortions between restored and original images that are the most current loop filters . AlthougIn order to increase the video quality, many CNN filtering methods have been suggested for HEVC and VVC standards –12. ThesThe remainder of this study is organized as follows: In recent years, artificial intelligence has seen tremendous progress in computer vision topics, in particular in image and video compression –15. DeepIn order to enhance loop filtering and postprocessing, Ma et al. in  have devThe suggested model provides a compromise between the compression and encoding complexity. In this study, we propose a wide-activated squeeze-and-excitation deep CNN- (WSE-DCNN-) based in-loop filtering approach for VVC video quality enhancement and achieve coding gains.The VVC standard  still emD, the coding bits needed represents by R and the Lagrange multiplier controlling the trade-off between D and R is λ. The coding tree unit (CTU) level on/off control is adopted to avoid a reduction in RDO performance. The frame-level filtering would be shut off to prevent oversignal, if the enhancement quality is not worth to cost the signaled bits. Specifically, the control flags at the CTU-level and frame-level are designed as follows. For each CTU, if the RD performance of the filtered CTU achieves better quality, the corresponding CTU control flag is enabled; otherwise, the flag is disabled. After all the CTUs in one frame are determined, the frame-level RD cost before and after filtering are calculated in equation , as shown in J=D+λR,whU and V) and luma (Y), would filter out the three components simultaneously. The WSE-DCNN model proposed consists of six inputs; three are YUV reconstructed and the other three include the QP quantization parameter and the luma and chroma coding unit. These inputs are first normalized to provide better convergence in the training process and then fed to the proposed model. Hence, the three (Y/U/V) reconstructions are normalized to  based on the highest bit depth value. This means that the normalized values ) are achieved by the following equation.B denotes the bit depth, P″ is the normalized value in normalized Y/U/V at , and W and H are the width and the height of the reconstructed frame, respectively.Various quantization parameters (QPs) contribute to a variety of reconstructed video quality. This makes it easier to use a single set of parameters to fit reconstructions with different qualities. QP should be normalized to QPmap .(3)QPmapY) and chroma  components also represents the inputs. Since the blocking artifacts are mainly caused by CU block partition, the division information of CU is converted into coding unit maps (CUmaps) and normalized. For example, for each CU in each frame, the boundary position is filled with two and the other positions are filled with one. However, the normalization factor is two, and two CUmaps can be obtained, one as Y − CUmap and the other denoted by UV − CUmap.The CU partition of the luma .In the second step, each channel obtains a value according to the squeeze operation using global average pooling (GAP) σ) activation functions, respectively. Y4 is the first fully connected layer followed by ReLU, which is refined by a certain ratio r. Then, the second fully connected layer followed by the sigmoid activation function is denoted by Y5, and it gives each channel a smoothing gating ratio in the range of .The excitation operation is described by two fully connected layers followed by ReLU and sigmoid  is exploited to train the deep video compression techniques . The BVIThe proposed deep learning framework is trained offline in a supervised learning manner. The deep framework used during the training phase is the TensorFlow-GPU . In the Xi is the ground truth of the proposed model, where i ∈ {1,…, N}. F(.) is the output of the WSE-DCNN model, where Yi represents the compressed images, i ∈ {1,…, N}, and θ is the parameter set of the proposed framework.To train the proposed WSE-DCNN model, we assume that the mean square error (MSE)  is appliThe loss function evaluation is the way to judge whether the model is well trained or not. It indicates, as shown in The proposed WSE-DCNN technique is implemented in the VVC standard in order to replace the conventionally applied filtering system during the testing process. All experiments are evaluated using a random access configuration at four QP values 22, 27, 32, and 37) under the VVC JVET common test conditions (CTC) . The RD , and 37 Y, U, and V in the table show the BD-rate of Y, U, and V components, respectively.Compared to the original VVC standard, Y component and 8.89% and 10.05% for both chroma U and V components under random access profile, as given in U and V chrominance. It is also apparent that, for some sequences, the compression performance varies widely, such that video sequence content affects the proposed model. In addition, the suggested model performs better in terms of RD performance for high motion or rich texture video sequences, such as Campfire, CatRobot1, Kimono2, RaceHorses, and BQSquare. Consequently, the suggested CNN-based loop filtering outperforms VVC with the conventional loop filtering algorithm in terms of RD performance.The proposed technique achieves better mean coding gains when integrated into VVC standard. It can achieve 2.85% BD-rate savings for luma PSNR is also used as a quality metric to test the performance of our proposed filtering technique integrated into the VVC standard, which is defined by the following equation .(10)PSNRIn order to show the subjective visual quality and to further verify the effectiveness of the suggested model, the RaceHorses video sequence for class D was encoded by QP 22 under random access profile. A comparative performance of the proposed approach was made with other CNN-based filtering methods, as given in Compared to other previous approaches, for all test sequences from class A1 to class D, the proposed WSE-DCNN system implemented in VVC better performed in terms of compression performance for both luma and chroma components, as given in We presented a RD performance curves for the suggested model-based in-loop filtering, compared to other approaches with QPs values under random access scenario for class A1 to class D. The RD performance curves comparisons are given in Y, U, and V components, respectively. The comparative results reveal that the proposed in-loop filtering framework proves its effectiveness in improving video quality. In future work, two deep learning algorithms will be developed, one to improve the VVC CU partition at interprediction in order to reduce VVC complexity reduction and the other to replace original filters to enhance visual quality.In this article, we have introduced a deep learning technique to improve VVC video quality while enhancing the user's services. To alleviate the coding artifacts as well as ringing, blocking, and blurring, the proposed WSE-DCNN technique is integrated into VVC standard to replace the traditional in-loop filtering. Compared to original VVC filters, simulation results show that the proposed system offers best objective and subjective compression efficiency, with a BD-rate reduction of approximately −2.85%, −8.89%, and −10.05% for