Leclercia, of which both species are of clinical importance, but the type species Leclercia adecarboxylata also displays plant growth-promoting properties in the rhizosphere.Acute Oak Decline (AOD) is a decline disease first reported on native oaks in the UK, but in recent years reports from further afield such as Europe and the Middle East, indicate that the distribution and host range is increasing at an alarming rate. The stem weeping symptoms of the disease partially develop due to polymicrobial-host interaction, caused by several members of the order Enterobacterales. While investigating the rhizosphere soil of AOD-unaffected trees, termed ‘healthy’ trees, and diseased oaks suffering from Acute Oak Decline (AOD), an enrichment method designed for enhanced recovery of Enterobacterales led to the recovery of several isolates that could not be classified as any existing species. These isolates showed a close relationship to the genus Leclercia with varying degrees of relatedness. As such a complete polyphasic approach was used to determine the true taxonomic position of these isolates. This involved whole genome sequencing, phylogenomic analysis, phylogenetic analysis of both the 16S rRNA and four housekeeping gene sequences, combined with phenotypic testing and fatty acid analysis. Both the phylogenomic and phylogenetic analyses separated the isolates into four clusters, two of which were contained in the Leclercia clade. The remaining two clusters formed a separate lineage far removed from any currently defined species. Further investigation into the role of the isolates as plant growth-promoting bacteria as well as plant pathogens was investigated computationally, revealing a number of plant growth-promoting traits as well as virulence genes related to motility, adhesion and immune modulation.Partial sequencing of four housekeeping genes revealed similarity to the genus Leclercia tamurae sp. nov. (type strain H6S3T = LMG 32609T = CCUG 76176T), Silvania gen. nov., Silvania hatchlandensis sp. nov. (type strain H19S6T = LMG 32608T = CCUG 76185T) and Silvania confinis sp. nov. (type strain H4N4T = LMG 32607T = CCUG 76175T). Due to their interesting protein annotations and alignments, these species warrant further investigation for their role in relation to plant health.Based on the genotypic and phenotypic data presented here, these isolates could be differentiated from each other and their closest neighbours. As such we propose the description of The online version contains supplementary material available at 10.1186/s12866-022-02711-x. Escherichia adecarboxylata for a novel bacterial species in the family Enterobacteriaceae [. The majority of strains originally isolated by Leclerc, were from food and notably produced a yellow pigment, when tested by IMViC the isolates resembled Escherichia coli [E. adecarboxylata as it was first described as another synonym of Enterobacter agglomerans syn. Erwinia herbicola (now Pantoea agglomerans) [E. agglomerans [E. adecarboxylata to a novel genus as Leclercia adecarboxylata [In 1962 H. Leclerc, a key figure in the understanding of enteric bacteria, proposed the name omerans) . This promerans)  and Farmomerans)  who usedlomerans . FinallyLeclercia remained uncommon. However, publications relating to L. adecarboxylata infections and risks to human health have risen in recent years with multidrug resistant strains being isolated from both bovine samples and humans suffering from respiratory disease [Leclercia is a generalist and regularly isolated from soil and water [L. adecarboxylata on an injury gained while surfing [Leclercia may also play more beneficial roles to plants as members of the rhizosphere, where it has been repeatedly isolated and shown to exhibit plant growth-promoting qualities [Leclercia pneumoniae, was described in the genus [L. adecarboxylata and other closely related species.Since its description in the late 1980s, literature on the genus  disease –9. Infecnd water , 7. This surfing . Howeverualities . Most rehe genus . The strLeclercia, identified by partial gyrB sequencing, were isolated from rhizosphere soil collected from Hatchlands Park, Guildford, UK. AOD is a decline disease that was first reported on native oak in the UK but is now seen to have a wider range of hosts and locations. AOD has recently been reported in Spain, Switzerland, Poland, Portugal, Latvia and Iran, with symptoms observed on other species of oak aside from Quercus petraea and Quercus robur [Brenneria goodwinii and Gibbsiella quercinecans have been identified as the causative agents [While investigating the role of the rhizosphere in the cause and development of Acute Oak Decline (AOD) in the present study, several potential novel species of us robur –17. The e agents . Declinee agents . The mode agents . The rhie agents . Recent e agents . These de agents . As suchLeclercia and a novel genus Silvania gen. nov. containing two novel species, Silvania hatchlandensis sp. nov. and Silvania confinis sp. nov.Using a polyphasic taxonomic approach, we performed a comprehensive classification of isolates collected from the rhizosphere soil surrounding the roots of healthy and symptomatic oak trees. The results gained in this study support the proposal of a novel species of Q. petraea and Q. robur) found at Hatchlands Park, Guildford, UK. The list of strains and isolation sources can be found in Table S1  and four strains assigned to Leclercia based on their whole genome sequences. Due to the lack of sequence variation  and phylogenetic distance observed between the MLSA genes of these strains and the type strain of L. adecarboxylata, we concluded that they belong to this species. Cluster 2, situated proximal to the L. adecarboxylata cluster, contained strains isolated from three cardinal points around two healthy oaks, one in the parkland and another in the woodland, and was strongly supported by a bootstrap value of 99%, suggesting the strains belong to a novel Leclercia species. A higher degree of sequence variation was observed within Cluster 2 with strains exhibiting 96–100% sequence similarity across the four housekeeping genes, and the gyrB gene displaying the most heterogeneity. The gyrB sequence similarity between Cluster 2 strains and the type strain of L. adecarboxylata (LMG 2650T) was > 94.3%, and > 98.5% for the other three genes. Clusters 3 and 4 were contained in a clade with 99% bootstrap support and consisted of one and two strains, respectively, isolated from both healthy parkland oak and diseased woodland oak rhizosphere soil. This clade was situated on a separate lineage on the border of the Leclercia clade with a greater phylogenetic distance, suggesting the strains could belong to a potential novel genus with two novel species. An additional six strains, identified as Leclercia sp. in GenBank, clustered on three separate lineages in the Leclercia clade , suggesting they belong to several further potential novel Leclercia species. Of the six strains, Z96–1 has been incorrectly assigned to L. adecarboxylata [Leclercia pneumoniae 49125T was unclear, as it clustered on the border of the Enterobacter clade, far removed from Leclercia.Bacterial strains were isolated from rhizosphere soil surrounding five healthy and two diseased native British oaks . Strains H6S3T and H6W5 (Cluster 2) showed 99.40–99.55% 16S rRNA gene pairwise sequence similarity to several Enterobacter species including the type strains of E. huaxiensis, E. cancerogenus, E. sichuanensis and E. chengduensis as well as 99.33% to L. adecarboxylata. The strains suggested as belonging to a potential novel genus by MLSA, H4N4T (Cluster 3) and H19S6T (Cluster 4), displayed highest pairwise similarity to Lelliottia jeotgali PFL01T with 99.48 and 99.45% to L. adecarboxylata NBRC 102595T, respectively and a generally high similarity to Lelliottia and Enterobacter species. These results are not unusual for members of the order Enterobacterales which are known for being highly homogenous, meaning their taxonomic position at the species level cannot reliably be determined based on their 16S rRNA gene sequences [Leclercia species cluster within the Enterobacter clade, and species of the potential novel genus are situated on separate lineages in proximity to the Lelliottia clade.Nearly complete 16S rRNA gene sequences (1344 bp) were obtained for the strains of the potential novel species . The genomes showed little variation, with the size and G + C DNA content ranging from 4.71–4.87 Mbp and 55.6–56.4 mol%, respectively. The genomes were submitted to GenBank under the BioProject numbers PRNJA837588 and PRNJA837589, and the genome features and accession numbers are listed in Table S2  demonstrated 89.9% dDDH, 98.6% ANI and 98% AAI values to each other but < 70% dDDH, < 95% ANI and 94–95% AAI values to L. adecarboxylata, confirming they belong to a single novel taxon. Finally, H4N4T (Cluster 3) and H19S6T (Cluster 4) were 45.6% similar based on dDDH, and 92.1 and 95% similar based on ANI and AAI, respectively. Both strains demonstrated lower values of < 35% dDDH, < 88% ANI and 90–91% AAI to strains of Leclercia, providing further support for their classification of a novel genus. Therefore, we propose Leclercia tamurae sp. nov. for strains in Cluster 2, and Silvania gen. nov. with Silvania hatchlandensis sp. nov. and Silvania confinis sp. nov. for strains in Clusters 3 and 4.To complement the phylogenomic comparison, a DNA similarity matrix was created through Average Nucleotide Identity (ANI), Average Amino Identity (AAI) and digital DNA-DNA hybridisation (dDDH) comparisons. The ANI and dDDH values are presented in Table  and AAI –29. LikeLeclercia strains support the phylogenies of the MLSA and phylogenomic trees. Strains GL3 and 119,287 demonstrated similarity values indicating they belong to a novel species closely related to L. adecarboxylata and L. tamurae sp. nov. Species of Leclercia exhibited 94–95% AAI similarity, while the two novel species of Silvania gen. Nov. were 95% similar based on AAI. In contrast, strains Z96–1, W6, Colony 189, LSNIH1 and L. pneumoniae 49125T were less related to species of Leclercia with AAI values ranging from 88 to 94%), suggesting these strains most likely belong novel genera, although further work would be required to fully understand their taxonomic position. It is worth noting that L. pneumoniae 49125T was least related to all strains of Leclercia species displaying AAI values of 88–89%. There is no currently accepted AAI cut-off for delineating genera, although several values have been suggested [Enterobacteriaceae and a comprehensive study of the family is needed before a genus delineation cut-off can be proposed.The dDDH, ANI and AAI values for the additional uggested –32. HoweL. adecarboxylata, L. tamurae sp. nov. and species of Silvania gen. nov. as plant growth-promoting bacteria (PGPB) playing a positive role in the soil, their plant growth-promoting traits (PGPT) were investigated computationally. The results from the DIAMOND MEGAN pipeline comparison against the PLant-associated BActeria web resource (PLaBAse) database revealed larger numbers of important plant interaction proteins through the PGPT viewer and KEGG orthology viewer. The resulting PGPT data showed that each submitted annotated genome had between 5500 and 5638 PGPTs aligned to known proteins. The majority produced indirect effects such as stress relief and biocontrol, competitive exclusion and genes involved in colonising the plant system. Of the direct effects, the main categories of the genes were involved in bioremediation, phytohormone production and biofertilisation. Figures L. adecarboxylata H10E4. Traits of interest included potassium and phosphate solubilisation, nitrogen and iron acquisition, sulphur assimilation and carbon dioxide fixation, features which all directly aid plant growth by increasing nutrient availability. 13% of the PGPT involved abiotic stress responses to neutralise salinity, osmotic, nitrosative/oxidative, herbicidal, and acidic stress, which are predisposing environmental factors in decline disease [L. adecarboxylata MO1s plant growth-promoting association [Leclercia and Silvania gen. nov. species although H4N4T had more alignments and the largest number of PGPTs identified. However, given their highly conserved AAI values of 90–91%, this is unsurprising and a further implication of their phylogenetic relatedness. The conclusive statement for each strain annotated genome comparison against plant bacterial only interaction factors (proteins) or PIFAR, suggested that the novel species were all capable of interaction with plants, but the identified interaction factors were related to virulence. 31–32% of factors were toxins (syringomycin and toxoflavin), 17–19% were exopolysaccharides (namely amylovoran), 8–9% of Silvania gen. nov. and 11–12% of Leclercia factors were for detoxification (of plant compounds such as isothiocyanate), and ~ 15% were adhesion and metabolism genes. Between 0.6–0.9% (Leclercia) and 2% (Silvania gen. nov.) of the identified bacterial plant interaction markers were plant cell wall degrading enzymes which are key markers of phytopathogens. The features identified through PIFAR such as EPS, toxins and PCWDE implicate the novel isolates as having pathogenic potential. These genes are associated with the invasion, colonisation and degradation of plant tissue [To investigate the potential of  disease . It has  disease , especiaociation  were idet tissue . Howevert tissue .Fig. 3KrResults from the comparison to the virulence factor database (VFDB), however showed 126–140 proteins from the novel strains were aligned to known virulence proteins from other pathogens with the vast majority related to motility, immunomodulation and adhesion. Some Type VI Secretion System (T6SS) effector delivery system proteins were identified, although no complete set of the assembly proteins and no secreted effector proteins were identified in the alignments. These results imply that the novel isolates have low pathogenic potential, although experimental pathogenicity trials with the type strains should be performed for conclusive understanding of their pathogenic potential.L. adecarboxylata MO1 that the novel strains isolated in the present study promote plant growth through their action in the rhizosphere, especially in relation to heavy metal detoxification [Overall, we can speculate on the isolates’ potentially positive role in the rhizosphere through several important direct PGPT genes such as heavy metal detoxification, biofertilisation and phytochemical signalling which all aid plant growth and resilience. Alongside the direct effects are other indirectly positive PGPTs such as stress relief for osmotic, heat, salinity and competitive exclusion genes. However, based on the alignments made in both the VFDB and through the PIFAR database the novel isolates here all contain genes related to virulence such as motility, adhesion, and Immune modulation genes. These genes could implicate a potential for pathogenicity, although all genes identified could also be utilised by PGPB for colonisation of the plant endosphere. While we cannot conclude on the role these isolates play in this niche, it is probable based on comparison to fication .Leclercia were straight rods averaging 1.38 × 2.26 μm, while Silvania gen. nov. strains were short straight rods averaging 1.31 × 1.81 μm. Cells are motile with peritrichous flagella and appear singly or in pairs . The fatty acid profiles for each strain can be seen in Table 18:1 ω7c which is higher in Leclercia species and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) which is higher in Silvania gen. nov. species.Based on the results generated by the Sherlock Microbial Identification System Version 6.4 (MIDI Inc.), the major fatty acids (above 10% relative abundance) were CLeclercia was a monospecific member of the Enterobacteriaceae since being defined in the 1960s until 2022, it has remained an interesting genus with clinical and environmental importance including plant-growth promoting abilities when added to the rhizosphere. The work presented here provides evidence that both the type species of the genus and a number of phylogenetically related species are associated with the rhizosphere of both healthy and diseased oaks suffering from AOD.Even though Leclercia tamurae sp. nov. (type strain = H6S3T = LMG 32617T = CCUG 76176T), Silvania gen. nov. with the type species as Silvania hatchlandensis sp. nov (type strain = H19S6T = LMG 32608 T = CCUG 76185 T) and Silvania confinis sp. nov (type strain = H4N4T = LMG 32607 T = CCUG 76175 T) and the amendment of the genus and type species descriptions for Leclercia. The addition of a new species to the genus Leclercia furthers our understanding of this clinically and environmentally important genus. Moreover, the taxonomic position of Leclercia has always been distantly removed from other genera of enteric bacteria within phylogenomic and phylogenetic trees. Through the addition of the closely related genus Silvania gen. nov. composed of two species, the wider taxonomic relationship of both genera within the family Enterobacteriaceae can be further understood.The genomic, genotypic, chemotaxonomic and phenotypic data suggests the strains investigated in this study represent three novel species, two of which belong to a novel genus. As such the following descriptions are proposed; Leclercia .2S production, urease, tryptophan deaminase, acetoin production (VP) and gelatinase. Nitrite is reduced to nitrate. Production of β-glucosidase and α-galactosidase, acidification of galacturonate and phenol red, (ID 32). Resistant to 1% sodium lactate, fusidic acid, D-serine, rifamycin, lincomycin, guanidine HCl, niaproof 4, vancomycin, tetrazolium violet, tetrazolium blue, lithium chloride, aztreonam and sodium butyrate (Biolog Gen III).Gram-negative rods, ranging from 1.39–1.54 μm wide and 2.01–3.06 μm long. All strains possess fimbriae and are motile by peritrichous flagella, and are oxidase negative, catalase positive, facultative anaerobes. After 48 h on TSA, all species appear as cream-coloured, circular, convex colonies between 2 and 3 mm in diameter with entire, slightly undulate margins. After longer periods of incubation some strains may develop a yellow diffusible pigment, although the conditions required are not consistent. Growth is observed from 10 to 41 °C for all strains, although some strains can grow at 4 °C, with optimal growth observed between 30 and 35 °C. The majority of strains grow at pH 6–9 and at supplemented salt concentrations of 1–8%, with some strains only able to grow up to 7%. Positive for β-galactosidase and indole production. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H16:0, C18:1 ω7c and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The DNA G + C content ranges from 55.8–56.4 mol%.The major fatty acids are CLeclercia adecarboxylata.The type species is The description is as given above for the genus with the following additional characteristics.In addition to the carbon sources listed in Table SThe DNA G + C content of the type strain is 55.8 mol%.Leclercia adecarboxylata .The type strain is Leclercia tamurae .The description is as given above for the genus with the following additional characteristics.Leclercia, although not within a set timeframe.After 48 h on TSA, colonies are circular, matte, brittle and cream-coloured with slightly undulate margins with an average diameter of 3 mm. All strains are capable of forming the yellow pigmentation associated with N-acetyl-β-glucosaminidase. Utilisation of the following carbon sources is variable: sucrose, D-salicin, 3-methyl glucose, D-serine, L-pyroglutamic acid, citric acid, D-malic acid, tween 40, α-hydroxy-butyric acid, β-hydroxy-D, L-butyric acid and formic acid. Variable resistance to troleandomycin and potassium tellurite is observed.In addition to the carbon sources listed in Table SThe DNA G + C content of the type strain is 56.4 mol%.T (= LMG 32609T = CCUG 76176T) and was isolated from healthy Quercus robur rhizosphere soil in Hatchlands, Guildford, UK.The type strain is H6S3Silvania .2S production, urease, tryptophan deaminase, acetoin production and gelatinase. Nitrite is reduced to nitrate. Positive for the acidification of galacturonate and production of β-glucosidase and α-galactosidase (ID 32). Resistance to 1% sodium lactate, rifamycin, lincomycin, guanidine HCl, niaproof 4, vancomycin, tetrazolium violet, tetrazolium blue, lithium chloride, aztreonam and sodium butyrate is observed.Gram-negative, straight rods (1.2–1.4 × 1.6–2.0 μm) and motile by peritrichous flagella. Cells appear singly or in pairs. Oxidase negative, catalase positive facultative anaerobes. Colonies appear as cream-coloured, convex circles with raised entire margins and a diameter of 3–4 mm on TSA. Growth is observed between 4 and 37 °C with an optimum growth temperature of 30 °C. Positive for β-galactosidase, negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, HN-acetyl-D-galactosamine, fusidic acid, D-serine, L-pyroglutamic acid, pectin, quinic acid, α-keto-glutaric acid and D-malic acid. Variable resistance to troleandomycin, nalidixic acid and potassium tellurite is observed. The major fatty acids are C16:0, C18:1 ω7c and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c).Variable features of the genus include indole production; fermentation of rhamnose, saccharose, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, D-lyxose, D-tagatose; acidification of phenol red and production of β-glucuronidase and malonate. Utilisation of the following carbon sources is variable: stachyose, The DNA G + C content ranges from 55.7 - 55.9 mol%.Silvania hatchlandensis.The type species is Silvania hatchlandensis .The description is as given above for the genus with the following additional characteristics.N-acetyl-D-galactosamine, L-pyroglutamic acid, quinic acid, α-keto-glutaric acid and D-malic acid are utilised.Cells are on average 1.25 × 1.94 μm in size. After 48 h on TSA, the colonies appear as slightly raised circles with raised entire margins and an average diameter of 4 mm. Positive for indole production (API 20 and API 50 CHB/E), the acidification of phenol red and the production of β-glucuronidase (ID 32). Variable features of the species include the fermentation of methyl-α-D-mannopyranoside and methyl-α-D-glucopyranoside; the production of malonate. In addition to the carbon sources listed in Table SThe DNA G + C content of the type strain is 55.9 mol%.T (= LMG 32608T = CCUG 76185T) and was isolated from diseased Quercus robur rhizosphere soil in Hatchlands, Guildford, UK.The type strain is H19S6Silvania confinis .The description is as given above for the genus with the following additional characteristics.Cells are on average 1.37 × 1.68 μm in size. After 48 h on TSA, the colonies appear as slightly raised circles with raised entire margins and an average diameter of 3 mm. In addition to the carbon sources listed in Table SThe DNA G + C content of the type strain is 55.7 mol%.T (= LMG 32607T = CCUG 76175T) and was isolated from healthy Quercus robur rhizosphere soil in Hatchlands, Guildford, UK.The type strain is H4N4Q. robur and Q. petraea) from both the parkland and woodland. Twenty oak trees were selected in a paired fashion . Rhizosphere soil samples were collected from the cardinal points around each of the 20 trees comprising a total of 80 samples. Following collection, soil was placed in sterile sample bags and transported directly to the University of the West of England, where they were stored at − 20 °C until processed.Samples were collected from Hatchlands Park, Guildford, UK from asymptomatic  and AOD symptomatic oaks , disrupted at 1150 RPM by a magnetic stirring rod for 10 minutes and the resulting suspension was placed in a shaking incubator at 250 RPM at 28 °C for 48 hours. Suspensions were removed from the incubator, allowing the sediment to settle before being diluted four-fold in ¼ strength Ringers (Oxoid). 100 ml of dilution was spread-plated on Eosin Methyl Blue agar  and incubated at 28 °C for 48 hours, both aerobically and anaerobically for the isolation of single colonies. All strains were stored in 50% glycerol at − 80 °C and subsequently cultured on Luria-Bertani  agar and nutrient agar  or in LB and nutrient broth incubated at 28 °C. Table S1  in which 1.5 cm of fine root (≤ 2 mm) were ground, incubated in 100 μL of extraction buffer at 95 °C for 10 minutes and then diluted in 100 μL of dilution buffer. Extracted root DNA was used in the amplification of the actin gene in a Loop-Mediated Isothermal Amplification reaction (LAMP) to confirm the identity of root samples as originating from oak . An isolation strategy originally designed for the recovery of enteric bacteria from food was utilised for the isolation of rhizosphere soil bacteria . 10 g of− 1 NaOH / 0.25% SDS for 15 min, followed by 10-fold dilution of the lysate and centrifugation to pellet cell debris. The isolated genomic DNA was used in subsequent PCR reactions, and was stored at − 20 °C.Alkalic lysis  was usedgyrB, rpoB, infB and atpD, as described by Brady et al [B and atpD, see Additional file PCR amplification and sequencing was performed on housekeeping genes, dy et al  and the dy et al . HoweverLeclercia, were downloaded from GenBank via BLAST [gyrB – 742 bp, rpoB – 637 bp, infB – 615 bp, atpD – 642 bp and 16S rRNA gene – 1344 bp. Sequences for the housekeeping genes were conceptually translated in MEGA to ensure they were in the correct reading frame and that no errors were made from alignment gaps. 16S rRNA gene pairwise similarity for the potential novel species was calculated using the EZBioCloud server [To ensure coverage in both directions for the 16S rRNA and housekeeping genes sequenced for isolates, consensus sequences were generated in UGENE V 38.1 . Sequencia BLAST  and addeia BLAST  to the fd server . Finallyd server  was appld server . MaximumERIC and BOX PCR using the primers and protocol from Versalovic, Koeuth and Lupski,  were useT, H6W5, H10E4, H19S6T and H4N4T) by enzymatic cell lysis with lysozyme and RNase A, purified on Solid Phase Reversible Immobilisation beads, followed by sequencing on the Illumina HiSeq platform by Microbes-NG . Trimmomatic 3.0 was used to trim adapters at a sliding window quality cut-off of Q15 [DNA was extracted from (H6S3f of Q15  and SPAdf of Q15 .d5 and 100 bootstrap replicates [d5 [. Subtree Pruning and Regrafting (SPR) were used to ensure the best topology for the final tree, which was rooted at the midpoint [Pairwise comparisons of the genomes were calculated using Genome Blast Distance Phylogeny (GBDP) with the Type Strain Genome Server  and the plicates . The resates [d5 . Subtreemidpoint .Average Nucleotide Identity (ANI) were calculated in FastANI , AverageT, H6W5, H10E4, H19S6T and H4N4T were queried against the PLant-associated BActeria web resource (PLaBAse) database using the DIAMOND MEGAN pipeline [The protein annotations produced from PGAP  for H6S3pipeline . First, pipeline . Each anpipeline . The alipipeline .https://plabase.informatik.uni-tuebingen.de/pb/form.php?var=PGPT-Pred) and queried against the BlastP+HMMER Aligner/Mapper. Finally, to determine if novel isolates are plant-associated bacteria the PIFAR-BASE was used to identify ‘plant bacterial only interaction factors’ from the annotated protein files for each isolate using the BlastP+HMMER Aligner/Mapper.Krona plots were created to visualise the PGPT genes identified as groupings defined by their interaction with plants (direct/indirect) and further specific roles . The annTo further understand the potential of these bacteria as pathogens further comparisons were made against the Virulence Factor Database  downloadLight microscopy was used to assess cell length and width, as well as strain motility and morphology. An Olympus SC180 camera linked with CellSens v1.11 microscopy imaging software was used to record all results . Negative staining of isolates followed by transmission electron microscopy  was used to observe the flagella arrangement. Negative staining was performed as previously published . The mor1 beef extract, 5 g l− 1 peptone). Both pH and salt tolerance broths were inoculated in triplicate with individual colonies for each strain and incubated overnight at 30 °C, shaking at 180 RPM.The full range of temperatures at which growth was assessed was 4, 10, 25, 28, 30, 37 and 41 °C on TSA from 24 h to 7 days. To test the pH survival range, the pH of tryptone soy broth  was adjusted using 1 M sodium acetate/acetic acid and 5 M carbonate/bicarbonate buffers to create a set of broths ranging from 4 to 10 pH in increments of 1. Survival in a range of salts concentrations from 1 to 7%, in increments of 1%, was tested by the addition of 1% w/v NaCl to saline-free nutrient broth .Antibiotic resistance against penicillin V 10 μg, penicillin G 10 μg, tetracycline 30 μg, ampicillin 10 μg, chloramphenicol 30 μg, colistin sulphate 10 μg, streptomycin 25 μg, cefotaxime 5 μg, ciprofloxacin 10 μg, cefepime 30 μg, gentamycin 10 μg and kanamycin 30 μg was tested. Mid-log range bacterial lawns were made on TSA by spread-plating 100 μL of mid-log phase overnight culture and six antibiotic discs were applied at equal distances using a disc dispenser (Oxoid). Plates were incubated at 30 °C for 24 h after which the zone of clearance was checked to determine if the strains were sensitive. Resistance was concluded in no zone of clearance was recorded. Included in all tests were the type strain of T, H6W8, H6W5, H6S9, H4N4T, H18E8 and H19S6T which covered a range of strains from each of the four clusters shown in the MLSA phylogenetic tree. All API galleries were read after 24 h incubation at 37 °C with the API 50 CH/B galleries read again at 48 h. The GEN III microplates were incubated at 30 °C and scored at 16 h and again at 24 h before false positives could occur. Both the type strain of L. adecarboxylata LMG 2803T and LMG 2650, the strain isolated from mango were included as reference strains. Oxidase and catalase activity were tested using Kovács reagent (1% tetramethyl-p-phenylenediamine dihydrochloride) and 3% v/v H2O2, respectively.Phenotypic tests were performed using the commercial assays API 20E, API50CHB/E, API 32 (bioMérieux) and GEN III GN/GP microplates (Biolog) which were used according to the manufacturer’s instructions. The strains tested were H10E4, H20N5, H6S3T and H10E4 (Cluster 1); H6S3T, H6S9, H6W5 andH20N5 (Cluster 2); H4N4T (Cluster 3) and H19S6T (Cluster 4). FERA Science Ltd. performed the service after strains were grown on TSA at 30 °C for 24 h. The Sherlock Microbial Identification System Version 6.4 (MIDI Inc.) protocol was followed, and results were compared against the RTSBA6 6.21 library.Fatty Acid Methyl Ester (FAME) profiles were determined for strains LMG 2803Additional file 1: Table S1. List of strains included in this study along with location, year of isolation, source and GenBank accession numbers for MLSA sequences. Table S2. Genome features of the strains sequenced in this study including accession numbers, size, G + C content etc. Table S3. Average amino acid identity (AAI) values between Leclercia and Silvania species. Table S4. Positive phenotypic characteristics shared by members of the genera Leclercia and Silvania.Table S5. Alternative MLSA sequencing primers used in this study.Additional file 2: Fig. S1. ML phylogenetic tree based on 16S rRNA gene sequences for novel species and genus described in this study as well as the closest phylogenetic relatives. Fig. S2. ERIC PCR patterns generated for strains of Leclercia adecarboxylata, Leclercia tamurae sp. nov., Silvania hatchlandensis gen. nov. sp. nov. and Silvania confinis sp. nov. Fig. S3. TEM images of Leclercia tamurae sp. nov. H6S3T, Silvania hatchlandensis gen. nov. sp. nov. H19S6T and Silvania confinis sp. nov. H4N4T.


Neutrophil extracellular trap (NET)-formation represents an important defence mechanism for the rapid clearance of infections. However, exaggerated NET formation has been shown to negatively affect tissue-regeneration after injury. As our previous studies revealed the strong tissue-protective and regenerative properties of the secretome of stressed peripheral blood mononuclear cells (PBMCsec), we here investigated the influence of PBMCsec on the formation of NETs. The effect of PBMCsec on NET formation was assessed ex vivo in ionomycin stimulated neutrophils derived from healthy donors using flow cytometry, image stream analysis, and quantification of released extracellular DNA. The effect of PBMCsec on molecular mechanisms involved in NET formation, including Ca-flux, protein kinase C activity, reactive oxygen species production, and protein arginine deiminase 4 activity, were analysed. Our results showed that PBMCsec significantly inhibited NET formation. Investigation of the different biological substance classes found in PBMCsec revealed only a partial reduction in NET formation, suggesting a synergistic effect. Mechanistically, PBMCsec treatment did not interfere with calcium signalling and PKC-activation, but exerted anti-oxidant activity, as evidenced by reduced levels of reactive oxygen species and upregulation of heme oxygenase 1 and hypoxia inducible-factor 1 in PBMCsec-treated neutrophils. In addition, PBMCsec strongly inhibited the activation of protein arginine deiminase 4 (PAD4), ultimately leading to the inhibition of NET formation. As therapeutics antagonizing excessive NET formation are not currently available, our study provides a promising novel treatment option for a variety of conditions resulting from exaggerated NET formation. They exGp91phox . Further4 (PAD4) , which p4 (PAD4) . In addi4 (PAD4) . Ultimat4 (PAD4) ,13.Neutrophil functions, specifically the extrusion of NETs, are considered beneficial during infection . HoweverRecent advances in cell-derived, yet cell-free medicinal products have increasingly gained attention in regenerative medicine ,28. AlthAlthough PBMCsec possesses compelling immunomodulatory effects , potentiThis study was conducted in accordance with the Declaration of Helsinki and applicable local regulations. Use of human neutrophils was approved by the institutional ethical review board of the Medical University of Vienna  (protocol code 1539/2017). Written informed consent was obtained from all donors.7 cells/mL and exposed to 60 Gy γ-irradiation . Subsequently, cells were cultured in phenol red-free CellGenix GMP DC medium  for 24 h. Cells, as well as cellular debris, were removed by centrifugation. The conditioned supernatants containing the secretome were filtered through 0.22 µm filters followed by viral clearance using Theraflex methylene blue technology . The secretomes were lyophilized and sterilized by high-dose γ-irradiation . CellGenix GMP DC medium without cells was used as vehicle control. The GMP batches A000918399131, A00918399136, and A000918399132 were used in this study. The stock concentration of one vial lyophilized secretome equals to 25 units/mL.PBMCsec was produced in compliance with good manufacturing practice by the Austrian Red Cross, Blood Transfusion Service for Upper Austria  as previously described ,34. Brievol/vol) chloroform–methanol and, subsequently, excessively vortexed. Then, 0.7 M of formic acid was used to acidify the emulsion (a one-fourth volume of the chloroform–methanol mix) and homogenized by thorough shaking. Phase separation was obtained by leaving the samples on ice for 30 min. The lower, organic phase was further applied to rotary vacuum evaporation  in order to eliminate solvents. The protein fraction was isolated by combining four times the volume of ice-cold acetone  to one volume of reconstituted PBMCsec, followed by thorough vortexing and incubation at −20 °C for 60 min, to obtain a protein precipitate. After centrifuging the sample at 18,000× g for 10 min, ice-cold acetone was added to the protein pellet, briefly vortexed and centrifuged at 18,000× g for 10 min. Acetone was discarded and remaining acetone was allowed to evaporate at room temperature. Finally, the protein pellet was resuspended in 0.9% NaCl in the initially used volume. DNA was isolated by adding equal amounts of isopropanol  as PBMCsec and 1/10 volume of 7.5 M sodium acetate (Merck Millipore) and incubated at −20 °C for 1 h. After centrifugation for 5 min at 18 000× g the DNA pellet was washed twice with 1 mL 70% Ethanol (Merck Millipore) followed centrifugation at 18,000× g for 5 min. The DNA pellet was allowed to dry for 10 min at room temperature prior to resuspension in double distilled, nuclease-free H2O. Extracellular vesicles were obtained by ultracentrifugation at 110,000× g for 2 h at 4 °C, as previously described [The lipid fraction was purified according to Folch et al. (PMID: 13428781) with minor modifications. Briefly, one part reconstituted PBMCsec was mixed with 9 parts 2:1  according to manufacturer’s instructions. In brief, magnetic beads were resuspended in 2 mL of buffer A. One-fourth of the total amount of processed blood of magnetic beads and buffer B were added to the blood and incubated at room temperature for 5 min under constant gentle rotation. Blood and isolation cocktail mix were placed in the MACSxpress Separator (Miltenyi Biotec) and allowed to separate for 15 min. Clear, neutrophil-containing top phase was transferred into a fresh tube and washed with basal RPMI 1640 without phenol red . If required, a red blood cell lysis was performed using a Red Blood Cell Lysis Buffer  for 10 to 15 min at room temperature. Neutrophils were resuspended in basal RPMI 1640 in an assay dependent concentration without phenol red for further use.Either isolated neutrophils or whole blood samples after red blood cell lysis were pre-treated with 2 units/mL PBMCsec or equivalent vehicle medium for 20 min at 37 °C. Cells were then stimulated with 5 µM ionomycin  or 100 nm phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich), or 10 µM Thapsigargin (Abcam) for 2 h at 37 °C unless indicated otherwise.After stimulation with indicated compounds, cells were centrifuged and stained with anti-citrullinated Histone H3 antibody  to detect NETs, anti-CD66b antibody  and anti-CD15 antibody  to identify neutrophils. Flow cytometric analysis was performed using BD FACSCanto II and BD FACSDiva software (version 6.1.3) .6 cells/mL condition in a 96-well plate. Cell death was assessed over the indicated time periods in a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Microplate reader  and the BMG Labtech Optima software .Incucyte Cytotox Dye for Counting Dead Cells  was used according to the manufacturer’s instructions. In brief, cells were treated as indicated, followed by the addition of 250 nM cytotox green dye for staining 100 µL cell suspension in a concentration of 4 × 106 cells per condition in a 96-well plate and incubated for 2 h. Continuous absorbance measurements at 450 nm were performed using a microplate reader  and the BMG Labtech Optima software (software version 2.20Rs).EZ4U  assay was performed according to manufacturer’s instructions. Briefly, the substrate was dissolved in 2.5 mL activator solution and pre-warmed to 37 °C. Then, a 20 µL substrate was added to 200 µL cell suspension at a concentration of 4 × 10ROS production was measured using the DCFD/H2DCFDA cellular ROS assay kit (Abcam). Cells were treated as indicated and the assay was performed as recommended by the manufacturer.6 cells per condition, either isolated neutrophils or whole blood, pre-treated with PBMCsec or vehicle for 20 min as indicated, were washed, resuspended in 400 µL full medium containing 1 µM Fura Red , and incubated for 30 min at 37 °C. Cells were washed once with medium, resuspended in 4 mL medium and incubated for another 30 min at 37 °C. Subsequently, cells were rested on ice for up to 30 min. Data were acquired on a FACSAria III flow cytometer . Before intracellular calcium flux measurement, 1 mL of Fura Red-loaded cells was transferred to a FACS tube and pre-warmed for 5 min at 37 °C in a water bath. The cells were kept at 37 °C during the whole measurement. The baseline response was recorded for 30 s prior to stimulation with 5 µM ionomycin. Changes in calcium mobilization were recorded for a total of 120 s. Fura Red was excited using a 405 nm violet laser and a 561 nm green laser and changes in emission were detected with a 635 LP, 660/20 BP, and a 655 LP, 795/40 BP filter set, respectively. The ‘Fura Red Ratio’ over time was calculated using the Kinetics tool in FlowJo software  as follows:Ratiometric calcium flux measurements with Fura Red were performed as described by Wendt et al., with minor modifications . In brie2(Merck Millipore), 50 mM MgCl2 , 40 mM 2-mercaptoethanol (Merck Millipore) and either 2 units/mL PBMCsec or equivalent vehicle control or DNase I (Thermo Fisher Scientific) as positive control for 1 h at 37 °C. 10 µL of each sample were loaded into a 1% agarose gel with gel red  together with 2 µL 6x loading dye (Thermo Fisher Scientific). Electrophoresis was performed at 200 V for 35 min. DNase activity was determined by absence of λ-DNA.DNase activity was measured by incubating 0.25 µg/µL Lambda DNA with 0.5 M acetate/NaOH at pH 4.8 (Merck Millipore), 50 mM CaCl6 cells per condition of 4 individual donors were pooled.The Human Apoptosis Array kit  was used in accordance with the manufacturer’s instructions with no modifications. Isolated neutrophils were treated as indicated and cell lysates of 4 × 10The inhibitory capacity of PBMCsec was measured using the PAD4 inhibitor Screening Assay kit  and performed according to manufacturer’s instructions. For Western blot analysis, cells were lysed in 1x Laemmli sample Buffer  supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich), After sonication SDS-PAGE electrophorese was performed on 4–20% gradient gels . Proteins were electrotransferred onto 0.2 µM nitrocellulose membranes  and immunodetected using primary antibodies against pan phospho-PKC (βII Ser660) antibody . This antibody detects endogenous PKC α, β I, β II, δ, ε, η and θ isoforms phosphorylated at carboxy-terminal residue homologous to serine 660 of PKC βII. Peroxidase-conjugated secondary antibody were detected with the SuperSignal West Dura Extended Duration Substrate , according to manufacturer’s instructions. A Ponceau S solution (Sigma-Aldrich) staining served as equal loading control.p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p < 0.0001.Statistical analyses were performed using Prism 8.0.1 (Graph Pad Prism). Data were shown ± standard deviation (SD). One-way ANOVA and Sidak’s multiple comparisons test were performed and * +CD66b+citH3+ staining  pumps, which prevents the storage of excess intracellular calcium in the endoplasmic reticulum [Induction of NETs requires an increase in intracellular calcium levels ,9,11. Weeticulum . The addSince activation of the NET signalling pathway down-stream of NADPH leads to the production of ROS and activation of PAD4 ,10,11, wThe activation of PAD4 with subsequent histone-citrullination represents one of the final steps in the NETs signalling pathway . Thus, wThe formation of NETs is a highly effective first line defence mechanism against invading pathogens . HoweverStaphylococcus aureus infection were more efficiently cleared by macrophages after treatment with reslovins [The tissue regenerative and anti-inflammatory action spectrum of PBMCsec is multifaceted ,38,53,54eslovins . These deslovins . HoweverIn addition to the lipid fraction, PBMCsec-derived proteins also showed a strong inhibitory action on NET formation. Previous studies demonstrated that addition of either bovine or human serum albumin to ionomycin-treated cells almost completely blocked NET formation by chelating calcium . HoweverOur data suggest the inhibition of ROS production and PAD4 activation as the two major modes of action for the reduction in NET formation by PBMCsec. Oxidative stress, especially the generation of ROS, is a hallmark of NET formation ,61 and HPAD4 is one of the most prominently investigated factors critical for NET formation, and PAD inhibitors have been extensively studied in the context of a broad variety of diseases, including multiple sclerosis , myocardIn summary, we have demonstrated a strong NETs-inhibitory activity of PBMCsec via a dual mechanism. Specifically the identification of a PAD4 inhibitor, produced naturally in the human body, as well as the prevention of ROS production might strongly improve the treatment of diseases associated with excessive NET formation, such as rheumatoid arthritis , multipl


Both mimetic complexes produced ammonia from urea, (1) and (2), were ten- and four-fold slower than urease, respectively. The presence and importance of several reaction intermediates were evaluated both experimentally and theoretically, indicating the aquo intermediate as a key intermediate, coordinating urea in an outer-sphere manner. Both complexes produced isocyanate, revealing an activated water molecule acting as a base. In addition, the reaction with different substrates indicated the biomimetic complexes were able to hydrolyze isocyanate. Thus, our results indicate that the formation of an outer-sphere complex in the urease analogues might be the reason urease performs a different mechanism.Urease is an enzyme containing a dinuclear nickel active center responsible for the hydrolysis of urea into carbon dioxide and ammonia. Interestingly, inorganic models of urease are unable to mimic its mechanism despite their similarities to the enzyme active site. The reason behind the discrepancy in urea decomposition mechanisms between inorganic models and urease is still unknown. To evaluate this factor, we synthesized two  Urease is a nickel-containing enzyme responsible for urea hydrolysis into carbon dioxide and ammonia A. The acSynthetic models of enzymes are designed based on the enzymatic active site and on its catalytic mechanism. The design of a biomimetic complex focuses on the recognition of specific interactions to obtain an adequate parallel between natural and synthetic catalysts . This pabis-nickel complexes, (1) and (2), were based on -Trost-bis-ProPhenol(L) as a ligand -Bis-ProPhenol ligand in acetonitrile in the presence of nickel acetate or nickel chloride, respectively. Consistent data were obtained from microanalysis, UV-Vis spectroscopy (−1), whereas (2) formed a 1:1 electrolyte (108.5 μS cm−1 in the conductivity). Therefore, the color change of (2) in addition to the value of its solution conductivity indicate a fast ligand substitution between chloride and solvent, confirming the different reaction rates by changing the labile ligand.Complexes (1) and (2) were synthesized from Trost  and 680 nm (ε = 3.2 dm3 mol−1 cm−1), which could be ascribed to 3A2→3T1g (F) and 3A2→3T1g (P) based on comparisons to other nickel complexes of the literature. Complex (2) exhibited bands at 463 nm (ε = 9 dm3 mol−1 cm−1), 563 nm (ε = 6.7 dm3 mol−1 cm−1), 606 nm (ε = 7 dm3 mol−1 cm−1), 655 (ε = 6 dm3 mol−1 cm−1), and 688 nm (ε = 5.5 dm3 mol−1 cm−1). The observed shoulders at the 600–700 nm region for both complexes could be ascribed as a spin flip transition 3A2→1Eg, which is consistent to other octahedral nickel complexes of the literature +complex [2L(Urea)]+ (intermediate 1b or 1c) would be a possible intermediate during urea hydrolysis. However, a stepwise reaction between (1) and urea followed by addition of stoichiometric amounts of water in anhydrous acetonitrile resulted in no detection of ammonia, suggesting urea hydrolysis in these complexes most probably proceeds via complexes 1e or 1g.Considering ligand substitution is important for catalysis, we decided to explore in more detail the reaction mechanism of urea decomposition by (1) and (2) complexes. Seven possible intermediates were proposed to be formed during the urea hydrolysis reaction  . This is+complex ,14. Ther−1 to 132 μS cm−1 was observed upon water titration, indicating the apparent formation of complexes 1e or 1g. Solid IR analysis of the soluble fraction (SI8) presented bands at 1698 cm−1 and 1477 cm−1 that could be from a protonated carboxylate + before the urea interaction. Complex 1d, obtained from the water titration experiments, enabled us to analyze the importance of this complex in the reaction since upon addition of an anhydrous urea solution to an anhydrous solution of complex 1d, stoichiometric amounts of ammonia were produced (0.23 μmol). A control reaction, using an anhydrous solution of (1) complex, resulted in no ammonia formation, strengthening the idea of an aquo/hydroxo complex acting as the active species in catalysis.Thus, to support the hypothesis of complex 1e and/or 1g formation, water titration of a (1) solution, followed by conductivity measurements was performed. The presence of intermediates 1e and/or 1g in solution would imply the production of a 1:1 electrolyte. Beyond precipitation, an increase in conductivity from 40 μS cmboxylate  or from 15N urea solution indicate the complexes perform a different mechanism than urease. Surprisingly, the ammonia/ammonium signal could not be detected, even when urease was the catalyst, despite the presence of bubbles in the NMR tube assumed to be ammonia. Upon (1) addition, the signal corresponding to urea (73 ppm) disappeared, and a new signal at 243 ppm could be detected, which was assigned to isocyanate (In the jack bean urease active site (PDB: 4GY7), two of the three coordinated water molecules are possibly aquo-species, whereas the bridging water is suggested to be a hydroxo-form at the optimum pH . The briocyanate B. Isocyaocyanate B. The stocyanate A. Isocyaocyanate C. The eqa of the water molecule bond to nickel + 2]+ complex ]+ complex due to its higher stability over the monodentate coordination, which has a binding energy near 292 kJ.mol−1. Contrastingly, the acetate monodentate is slightly more stable than the corresponding bidentate mode. In general, the urea complex is 441 kJ.mol−1 less stable than the acetate, indicating a difficulty in substituting acetate by urea.To corroborate our hypothesis, theoretical calculations on the transition states of a self-elimination mechanism and a catalyzed elimination mechanism with [Ni2L(Urea)]+ complex  of the first water molecule ]+ complex plus one isolated water molecule ]+-hydroxo-complex is possible with the addition of the second water molecule ]+ ]+ complex seems to inhibit any hydrolytic reaction. Ammonia self-elimination from the [Ni2L(Urea)]+ ]+ complex and ammonia resulted in the elimination of water to obtain coordinated isocyanate. These results are in agreement to our experimental analysis in which a [Ni2L(Urea)] was unable to produce ammonia, suggesting that if urea coordinates before water, then a well of energy stability is reached to suppress any hydrolytic reaction.Sequential addition of two water molecules to the + 2]+ complex results in a net stabilization energy of −102 kJ mol−1 2]+-urea) (support information video). Moreover, the interaction between coordinated waters and urea seems to have a deep influence on the reaction transition state. If the interaction takes place through one of the nitrogen atoms, then the transition state is only 90 kJmol−1 (2L(H2O)2]+ complex, which stabilizes urea by secondary interactions. This forces it to undergo an elimination reaction to form isocyanate, which is further hydrolyzed into ammonia and carbon dioxide.Interestingly, the aquo complex 1d , was the−1 (δsNH), 1656 cm−1 (δasNH), and 1595 cm−1 (νCO) (SI11). Therefore, these experiments indicate urea coordination to the nickel centers might not suffice to activate it to hydrolysis in which an outer-sphere encounter complex should be determined for these reactions. Kryatov et al. .3H2O.ACN): theor./exper.: 62.35/62.3, 6.21/5.98, 4.64/4.40 and 12.97/12.90.C/H/N/Ni , 1719 cm−1, 1671 cm−1, 1579 cm−1 (C = O asymmetric and symmetric stretching).IR: 3200–2800 cm45H47N2Ni2O7+ = 843.2090) and 797.2119 (C44H45N2Ni2O5+ = 797.2035). HRMS (m/z): 843.2178  was then added to the flask, followed by addition of 0.2 mmol of Trost -Bis-ProPhenol Ligand  and 28 μL of triethylamine (1 eq.). The reaction proceeded under stirring for 24 h at 40 °C. After this period, the solution was filtered, and the filtrate concentrated to 5 mL. Cold diethyl ether was then added to the reaction mixture. After filtration, any insoluble material was removed and the solvent evaporated to dryness, yielding 35 mg (36% yield) of the desired complex. All attempts to isolate suitable crystals for X-ray analysis have not been successful so far.To a Schlenk flask, 97 mg of NiCl55H74ClN4Ni2O3].) theor./exper.:66.59/66.52; 7.52/7.69; 5.65/5.35C/H/N , 2493 cm−1 (C = N)IR: 3063–2667 cm51H70N3Ni2O6), 671.22 (z = −1) (C31H41NNi2O8)LRMS (m/z): 468.22 (z = 2+) (C−1), water aliquots were added (100 μL each) until reaching a stable conductivity of 132 μS cm−1. Any residual solid was filtered off, and the filtrate was dried for three days at high vacuum for a further reaction with urea.To 5 mL of a 0.1 mM solution of (1) in acetonitrile (conductivity: 40.2 μS cmComplex (3)  was dissolved in 900 μL of anhydrous acetonitrile in a Schlenk flask under a nitrogen atmosphere. To this solution, 100 μL of a 60 mM urea solution (in anhydrous acetonitrile) was added, and the reaction mixture was kept at 20 °C for 10 minutes. After this period, a 100 μL aliquot of the reaction was analyzed for ammonia via the Berthelot method. Two control reactions were composed of: (a) 100 μL of a 60 mM urea solution (in anhydrous acetonitrile) and (b) addition of 100 μL of a 60 mM urea solution (in anhydrous acetonitrile) to a 900 μL solution of (1) (2 mM in anhydrous acetonitrile).2 = 0.99). Control reactions without the catalysts were also performed. All analyses were performed in triplicate.For the determination of ammonia formation, 100 μL of the complex dissolved in acetonitrile (2.8 mM) and 900 μL of urea solution (60 mM) were mixed in a 5 mL flask with stirring. The reaction was kept at 20 °C. Aliquots of 100 μL of the reaction were taken at different reaction times  and were mixed with 250 μL of hypochlorite solution (2.5%) and 250 μL of sodium citrate/NaOH solution (0.38 M and 0.46 M). After mixing this solution, 300μL of sodium salicylate (2.75 M)/sodium nitroprussiate (9.4 μM) solution was added. After incubation of the solution at 20 °C for 15 minutes, the solution was analyzed by UV-Vis spectroscopy, monitoring the band absorption at 650 nm. Quantification was performed by means of a calibration curve with seven points . After temperature stabilization at 25 °C, 100 L of the catalyst solution (urease (16 mM) or mimetic (2.2 mM)) was added and the reaction initiated. A 50 μL sample was taken from the reaction mixture after 10, 20, 30, 60, 120, and 180 s of reaction and analyzed by the indophenol method to determine the quantity of ammonia formed during the reaction. Amines or alcohols expected to form in the reaction were quantified by gas-chromatography analysis. All the hydrolysis reactions were performed in triplicate.N-methyl urea, n-butylcarbamate, and acetamide hydrolysis: These reactions were also tested in 12 h of reaction at 25 °C and 50 °C.N-phenyl urea, N-allylurea hydrolysis: These reactions were also tested in 12 h of reaction at 50 °C.Benzyl benzylcarbamate and benzyl p-toluylcarbamate hydrolysis: These reactions were also tested at different pHs , temperatures , and solvents .15N-urea (10 mg) was dissolved in 900 L of a 1:1 mixture of CD3CN/water and was analyzed in a 400 MHz Bruker spectrometer. After addition of 100 μL of a 1 mM acetonitrile solution of the complexes to the urea solution, the 15N NMR experiment was immediately reset. The 15N chemical shifts were referenced to the nitromethane.The dc magnetic susceptibility was measured over 2.0–300 K with a superconducting quantum interference device magnetometer (Quantum Design MPMS).2, 100 kPa, injector 220 °C, detector 220 °C. A method composed of an isotherm at 180 °C was employed.After the reaction from 4.6 was finished, it was analyzed by GC analysis using an achiral capillary column. The GC-MS conditions were carrier gas-HCalculations of full geometry optimization with the Density Functional Theory (DFT), using B3LYP exchange-correlation functional , and SVP15N experiments, but the fast ligand exchange in (2) complex resulted in a stationary equilibrium between urea and products, which could be easily shifted by pH decrease or bicarbonate addition. The equilibrium shift indicated isocyanate could suffer hydrolysis in the reaction conditions. When N-phenylurea was employed as a substrate, phenyl isocyanide and aniline could be detected by gas chromatography, corroborating the isocyanate hydrolysis hypothesis. Isocyanate hydrolysis by a urease analogue was first described in this work, revealing the geometry between coordinated water and urea is possibly different from the ones obtained in urease enzyme. Therefore, urea hydrolysis performed by complexes (1) and (2) seems to involve a different mechanism than the one from urease enzyme. Further refinements in ligand secondary coordination sphere could have an influence to mimic urease enzyme.Two new dinuclear nickel complexes were synthesized and characterized. Both complexes were able to produce ammonia from urea solution. We have observed ligand substitution from acetate to chloride had a deep impact in the inorganic urease models, enabling a faster reaction rate with chloride as the more labile ligand. Both complexes were shown to produce isocyanate in the


This study investigated these relationships using the data from The Cancer Genome Atlas database. We calculated risk scores using a Lasso regression analysis and divided the patient samples into two risk groups . Furthermore, we used univariate and multivariate Cox analyses to determine independent prognostic indicators and explore correlations between the regulatory factors and immune infiltrating cell characteristics. Finally, quantitative reverse transcriptase–polymerase chain reaction (PCR) and The Human Protein Atlas were used to verify signature-related gene expression in clinical samples. We identified expression differences in 35 regulatory factors between the tumor and normal tissue groups. Next, we constructed a five-gene risk score signature  using the screening criteria (p < 0.01), and then divided the cases into high- and low-risk groups based on their median risk score. We also screened for independent prognostic factors related to age, tumor grade, and risk score. Furthermore, we constructed a Norman diagram prognostic model by combining two clinicopathological characteristics, which demonstrated good prediction efficiency with prognostic markers. Then, we used a single-sample gene set enrichment analysis and the cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method to evaluate the tumor microenvironment of the regulatory factor prognostic characteristics. Moreover, we evaluated five risk subgroups with different genetic signatures for personalized prognoses. Finally, we analyzed the immunotherapy and immune infiltration response and demonstrated that the high-risk group was more sensitive to immunotherapy than the low-risk group. The PCR results showed that NSUN5 and HNRNPA2B1 expression was higher in tumor tissues than in normal tissues. In conclusion, we identified five m6A and m5C regulatory factors that might be promising biomarkers for future research.In recent years, methylation modification regulators have been found to have essential roles in various tumor mechanisms. However, the relationships between N Renal cell carcinoma (RCC) is the most common renal malignancy, resulting in more than 14,000 deaths annually in the United States . Clear cN-Methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications are newly discovered gene expression regulation mechanisms . These mm5C modifications are another prevalent RNA modification type occurring in mRNA, transfer RNA, ribosomal RNA, and some noncoding RNAs . StudiesNSUN2 expression in breast cancer  mRNA methylation and stability, inducing tumor cell proliferation and survival by inhibiting the tumor suppressor, let −7 g (ERCC1) expression, which is associated with worse prognosis (MZF1) expression, resulting in a poor prognosis  transcriptome and clinical data from The Cancer Genome Atlas (TCGA) to analyze the differentially expressed regulatory factors in ccRCC. Next, we constructed a prognostic risk model using Lasso regression and Cox analyses. Finally, we screened five prognostic regulatory factors as a model signature and combined the independent prognostic factors to construct a nomogram diagram.Immune cell infiltration plays a decisive role in tumorigenesis and development. Furthermore, cancer cells shape their microenvironment by secreting various cytokines, chemokines, and other factors, leading to the reprogramming of surrounding cells. Therefore, they play a decisive role in tumor survival and progression . This stOverall, we combined m6A and m5C to explore the influence of regulatory factors on ccRCC prognoses to provide new ccRCC biomarkers and construct a reliable prognostic model suitable for use in the clinic.https://portal.gdc.cancer.gov/). We included 611 TCGA samples; 539 were tumor tissue (ccRCC) samples, and 72 were normal tissues samples. We removed all samples with missing data.We downloaded the ccRCC transcriptome (HTseq-FPKM) and clinical data from the TCGA-KIRC database  and validation (n = 262) groups using a 1:1 ratio and screened the prognostic adjustment factors. We calculated the risk score as follows:We randomly divided the 525 ccRCC samples into training  curves. This process was performed in the training and validation groups.p-value of ≤0.001 in the multivariate Cox regression analysis and the risk score to construct the prognostic nomogram model. The calibration curve and C-index were used to predict the model’s performance. We also created 1-, 3-, and 5-year ROC curves to verify the model’s predictive ability and performed a Kaplan–Meier analysis to evaluate the OS of the low- and high-risk groups .We selected clinical indicators with a http://bioninformatics.mdanderson.org/estimate/) to assess correlations between regulatory factors and immune and matrix scores.We used the single-sample gene set enrichment analysis (ssGSEA) algorithm to obtain the relative abundance of TME-infiltrated cells per sample. Next, we used the CIBERSORT algorithm to analyze differences in immune cell infiltration between the high- and low-risk groups. The CIBERSORT software deconvolves the matrix of immune cell subtypes according to linear support vector regression rules . We downWe purchased human ccRCC (769-P) and immortalized proximal tubule epithelial (HK2) cell lines from the Cell Bank of the Chinese Academy of Sciences. 769-P and HK2 cells were cultured in Roswell Park Memorial Institute -1,640 medium  and Dulbecco’s Modified Eagle Medium  , respectively, containing 10% fetal bovine serum  at 37°C and 5% carbon dioxide.–ΔΔCt method to analyze the relative expression of the prognostic genes based on the normalized relative expression of the β-actin gene. The primers were as follows: NSUN5: Forward, TGCCTCGATTTGTGCGTGTG, Reverse, GACAGCTGGCCCTGTCCT; GAPDH: Forward, TGACTTCAACAGCGACACCCA, Reverse, CACCC-TGTTGCTGTAGCCAAA; ZC3H13: Forward, TGGTGCTGGAGAAGGATACGA, Reverse, CTATCACATCTAAGGGATCTGGCA; HNRNPA2B1: Forward, GCTTTGGGGATTCACGTGGT, Reverse, CCACTGCCATATCCATCAGATCC. We used The Human Protein Atlas database to analyze NSUN5 and HNRNPA2B1 protein expression in clinical specimens.We verified the expression levels of the prognostic genes by qRT-PCR analysis. Total RNA was extracted using Trizol reagent , and complementary DNA was synthesized using PrimeScript RT reagent Kit . qRT-PCR was performed on 7,500 Real-Time PCR Systems  using SYBR GreenER Supermix . The PCR conditions comprised an initial melting step at 95°C for 1 min, followed by 35 cycles of 95°C for 90 s, 60°C for 30 s, 72°C for 30 s, and then 72°C for 10 min. We used the 2p-values of <0.05 were considered statistically significant. We indicated the various levels of statistical significance as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.All statistical analyses were performed using R . We created box plots to visualize differential gene expression using the “reshape2” and “ggpubr” packages. We screened the prognostic adjustment factors using a Lasso regression analysis and the “glmnet” package and generated ROC curves using the “survivalROC” package. The survival curve was obtained using the “survminer” package, and we used the “rms” package to calculate the C-index and generate calibration curves. All p < 0.01; NSUN4, FMR1, LRPPRC, HNRNPA2B1, ZC3H13, RBM15B, METTL14, YTHDF3, and IGFBP2 were significantly higher in the normal tissue samples than in the tumor tissue samples. The expressions of YTHDC2, NSUN6, RBM15, NSUN2, NOP2, IGFBP3, ALYREF, TET3, FTO, ALKBH5, RBMX, NSUN5, METTL3, and WTAP were significantly higher in the tumor tissue samples than in the normal tissue samples. TET1 expression did not differ among the groups , and METTL14 and ZC3H13 were associated with a protective effect (HR <1). We used these five factors to generate the prognostic risk model.We further screened the methylation regulatory factors to explore their prognostic value. We selected 22 regulatory factors as the analysis object . Univarid ZC3H13 . NSUN5, NOP2, HNRNPA2B1, and NSUN5 were highly expressed in the high-risk group, and the low-expressed genes METTL14 and ZC3H13 were highly expressed in the low-risk group . Therefore, age, grade, and risk score are independent prognostic factors for KIRC OS (The areas under the curve (AUCs) for the 1-, 3-, and 5-year ROC curves were 0.717, 0.701, and 0.723, respectively, using the training set data and the prognostic risk model . We perf KIRC OS .p < 0.001). Furthermore, OS was better in the low-risk group than in the high-risk group  to construct a Norman diagram model . Next, w+ T cells, activated dendritic cells, CD56 dim natural killer (NK) cells, and myeloid-derived suppressor cells (MDSCs) in the high-risk group.Tumor infiltration is crucial for tumor development. Therefore, we performed a ssGSEA analysis to evaluate correlation patterns between immune infiltrating cells and the risk score . There wNext, we used the estimation of stromal and immune cells in malignant tumor tissues using the expression data (ESTIMATE) algorithm to identify associations between the risk groups and the immune and stromal scores. The immune score was higher in the high-risk group than in the low-risk group, and the stromal score was higher in the low-risk group than in the high-risk group .The CIBERSORT algorithm identified the types of immune cells in ccRCC , and we p < 0.001; Immunotherapy is an emerging ccRCC therapy, and the current first-line treatment involves immune checkpoint inhibitors. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1  are the two most important immune checkpoint molecules (ICMs) regarding improved OS . Other iNSUN5, ZC3H13, METL14, NOP2, and HNRNPA2B1 in ccRCC using the HK2  and 769-P (ccRCC) cell lines. METL14 is under-expressed in ccRCC tissues  to verify the effectiveness of the risk model and introduced clinicopathological factors to improve the model’s reliability.+ T cells promote renal cancer cell proliferation by regulating YBX1  , and theFinally, we used the CIBERSORT and ESTIMATE algorithms to generate immune and stromal scores per risk group. The immune score was higher in the high-risk group than in the low-risk group, and the stromal score was higher in the low-risk group than in the high-risk group. Furthermore, the high-risk group was sensitive to anti-CTLA-4. Liu et al. reported that CTLA-4 as an oncogene accelerates ccRCC development with high prognostic value .However, our research has limitations. First, we only have internal verification and lack external platform verification. Therefore, the predictive power of the risk model may be limited. Also, additional basic experimental research is needed to determine the detailed mechanisms of these five regulators.Our study evaluated the combined role of m6A and m5C regulatory factors in regulating the KIRC TME. First, we constructed a new gene signature with five relevant regulatory factors. Then, using this signature, we created a novel prognostic model to stratify KIRC and normal tissue samples based on risk. Finally, we explored the link between the new gene signature and immune infiltration and obtained new potential immune checkpoints. These tools may help clinicians make more personalized and accurate prognosis predictions.


According to multivariate analyses, five soybean varieties were selected. These soybean varieties produced different peptide profiles and differently reduced cholesterol concentration (43–55%) by inhibiting HMGCR in fatty-acid-stimulated HepG2 hepatocytes. Selected digested soybean varieties inhibited cholesterol esterification, triglyceride production, VLDL secretion, and LDL recycling by reducing ANGPTL3 and PCSK9 and synchronously increasing LDLR expression. In addition, selected soybean varieties hindered LDL oxidation, reducing the formation of lipid peroxidation early (conjugated dienes) and end products . The changes in HMGCR expression, cholesterol esterification, triglyceride accumulation, ANGPTL3 release, and malondialdehyde formation during LDL oxidation were significantly (p < 0.05) correlated with the glycinin:β-conglycinin ratio. Soybean varieties with lower glycinin:β-conglycinin exhibited a better potential in regulating cholesterol and LDL homeostasis in vitro. Consumption of soybean flour with a greater proportion of β-conglycinin may, consequently, improve the potential of the food ingredient to maintain healthy liver cholesterol homeostasis and cardiovascular function.Clinical studies indicate that the consumption of soybean protein might reduce cholesterol and LDL levels preventing the development of atherosclerotic cardiovascular diseases. However, soybean variety can influence soybean protein profile and therefore affect soybean protein health-promoting properties. This study investigated the composition and effects of nineteen soybean varieties digested under simulated gastrointestinal conditions on hepatic cholesterol metabolism and LDL oxidation in vitro. Soybean varieties exhibited a differential protein hydrolysis during gastrointestinal digestion. Soybean varieties could be classified according to their composition (high/low glycinin:β-conglycinin ratio) and capacity to inhibit HMGCR (IC Over the last few decades, the prevalence of overweight and obesity among adults has increased worldwide. Over 2 billion adults are overweight, while over 650 million are obese . MetabolClinical evidence has proven the benefits of soybean protein in regulating lipid metabolism in the liver and other tissues. Clinical trials have demonstrated that soybean intake may offset several markers associated with MAFLD and lipid peroxidation . Human sVarying protein profiles in different soybean varieties, primarily characterized by distinct glycinin:β-conglycinin ratios, have been associated with differential peptide release under simulated gastrointestinal conditions, different antioxidant properties in intestinal epithelial cells , and dis® TGX™ gels , and Precision Plus Protein™ Dual Xtra standard were purchased from Bio-Rad . Simply Blue Safe Stain was obtained from Invitrogen . Bovine serum albumin (BSA), D-serine (98% purity), and simvastatin (97% purity) were used as standards and obtained from Sigma-Aldrich . Unless otherwise specified, all additional reagents were obtained from Sigma-Aldrich.Nineteen different soybean varieties  were supplied by Benson Hill  and stored at 4 °C until use. DC protein assay, 2× Laemmli buffer, 10× tris/glycine/SDS buffers, mini-PROTEANSoybean grains were processed in a coffee grinder. The milled soybean was sieved (1.16 mm) to achieve uniform particle size materials, and then preserved at −20 °C until required. The soybean flour was defatted using a Soxhlet extraction apparatus, as previously described .w/v) ratio to generate an oral bolus. The oral bolus was then merged in a 1:1 (w/v) ratio with the gastric mastermix at pH 3, which contained simulated gastric fluid and pepsin , and digested for 2 h at 37 °C. The gastric phase was then mixed with the intestinal mastermix, including bile solution (10 mmol L−1), and pancreatin (100 U mL−1), and digested for 2 h at 37 °C. The duodenal phase was subsequently digested using a previously described method [Streptomyces griseus, 4 mg mL−1, 3.5 U mg−1) was added, and the colonic phase was incubated at pH 8 and 37 °C for 1 h. Simultaneously, blank digestions were performed. The digestion was terminated by heating the digestion mixtures for 5 min at 100 °C. The bioaccessible fractions (soluble components) were retrieved by centrifugation at 4 °C, 3200× g, 40 min. The digestion supernatants were recovered, frozen at –80 °C, and freeze-dried.The simulated digestion was performed in accordance with the standardized INFOGEST protocol . Defatted method . Pronase−1 Tris-HCl buffer, pH 8.2). The suspension was homogenized and incubated in an ultrasonic bath at 40 °C for 70 min. The protein concentration of non-digested and digested soybean varieties flours was determined using the DC protein assay, as specified by the manufacturer (BioRad). The degree of hydrolysis (DH) was assessed as described [o-phthaldialdehyde (OPA) reagent was obtained by combining 10 mg of OPA 250 μL of ethanol, 9.8 mL of phosphate-buffered saline (PBS), and 20 μL of β-mercaptoethanol. Digested soybean  was combined with 100 μL of OPA reagent and 140 μL of water. Serine was used as standard. The degree of hydrolysis was calculated using Equations (1) and (2).hot = 7.8, Serine-NH2 was the serine concentration (mmol g protein−1) in digested soybean, β = 0.342, α = 0.970. Soluble protein was extracted from the non-digested defatted flours using the method previously described . In brieescribed . In brieThe protein profile of non-digested and digested soybean flours was examined using SDS-PAGE. In brief, samples (20 μg of protein) were loaded in 4–20% Tris-Glycine gels. After that, electrophoresis was run for 35 min at 200 V and 400 mA. The gels were then rinsed three times with distilled water before being stained with SimplyBlue at room temperature for 1 h. The gels were subsequently destained with distilled water, and images were acquired using ImageQuant 800 Fluor colorimetric imaging. ImageJ was used to conduct densitometric measurements . Proteins were tentatively identified by comparing their molecular weights to those of known proteins and their proportion in relation to the total protein concentration measured using the densitogram area under the curve . The gly−1) and HMG-CoA substrate (0.3 mg mL−1) were combined with digested soybean varieties (10–3000 μg protein mL−1) or simvastatin (0.1–30 μg mL−1) in a UV compatible 96-well plate. To complete a final volume of 200 μL per well, PBS pH 7.4 was added. The analyses were initiated (time 0) by adding HMG-CoA reductase  and incubated at 37 °C. The rates of NADPH consumed were monitored every 30 s for up to 20 min by reading the decrease in absorbance at 340 nm.HMGCR was evaluated in plate, using an in vitro biochemical cell-free assay. The HMGCR (EC 1.1.1.34) assay kit was used according to the manufacturer’s instructions . In summary, NADPH (400 μmol L−1) and analyzed by LC-QTOF-MS/MS using Alliance 2795 HPLC system coupled to an Ultima mass spectrometer on the positive ion electrospray mode (+ESI) . The gradient mobile phase A contained 95% water, 5% acetonitrile, and 0.01% formic acid, while the gradient mobile phase B contained 95% acetonitrile, 5% water, and 0.1% formic acid. The flow rate was 400 µL min−1 and the PDA detector recorded the signal at 280 nm. All peaks with an intensity higher than 20% were examined using the MassLynx V4.1 program . After obtaining the fragmentation pattern of each peak and determining the charge, MaxEnt3 was used to deconvolute representative mass spectra, which were then exported in the peptide sequencing tool to identify potential peptide sequences embedded within the soybean protein. Since distinguishing isoleucine (I) and leucine (L) is not possible, only isoforms with L were recovered, while peptide sequences having I instead of L are also possible. After that, I/L combinations were evaluated, and peptides were chosen based on the best parental protein prediction. The BLAST database  was used to compare the sequences obtained from digested soybean varieties to previously published protein sequences. Mass, isoelectric point (pI), net charge, and hydrophobicity were analyzed using the PepDraw  database. Potential biological activity was calculated using PeptideRanker .Peptides released from the selected soybean digested under gastrointestinal conditions were diluted in water .The HepG2 (HB-8065) human hepatocytes were obtained from the American Type Culture Collection  and cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin at 37 °C, and 5% CO5 cells cm−2. Then, they were incubated in EMEM supplemented with 10% FBS in the absence  or presence of FFAs  conjugated in BSA (1%), and the digested soybean varieties  or simvastatin . After 24 h treatment, supernatants were collected, centrifuged, and stored at −80 °C until being used as described earlier [50) for cholesterol concentration increase was determined, cells were cultivated in the absence  or presence of FFAs  and the digested soybean varieties  or simvastatin (IC50 = 160 ng mL−1). The treated cells were rinsed twice with ice-cold PBS, and then RIPA Lysis Buffer System  was added to lyse the cells. The cell suspension was sonicated and centrifuged at 10,000× g for 10 min at 4 °C to eliminate cell debris. Simultaneously, different aliquots were mixed with 4× Laemmli buffer, boiled for 5 min, and then either frozen or directly stored at −80 °C until further analysis. The protein concentration in cell lysates was quantified with the DC protein assay (BioRad).Hepatocytes were cultured for 24 h seeded in flasks at a density of 5 × 10 earlier . Once thIntracellular HMGCR activity was measured in cell lysates of hepatocytes treated under the conditions described in Intracellular hepatic total cholesterol, free, and esterified cholesterol, and triglyceride levels were determined in HepG2 cell lysates by enzymatic colorimetric kits . A blank containing cell lysates buffer was included in all assays. Cholesterol increase was defined as the relative increase in total intracellular cholesterol concentration upon FFA stimulation.https://www.rcsb.org/pdb/home/home.do, accessed on 16 December 2022). Instant MarvinSketch was used to create peptide structures . AutoDock Tools was used to combine non-polar hydrogen atoms, add Gasteiger partial charges, and specify the root of each structure’s rotatable bonds. Furthermore, AutoDock Tools was used to determine the docking space dimensions , center point, and flexible torsions. The docking space center was selected based on the location of the co-crystallized inhibitor . One hundred runs were performed for each ligand, and the conformation with the best binding mode was used to calculate the ligand binding energy (ΔG) using PRODIGY [ΔG is the peptide binding energy, R is the gas constant , and T is the absolute temperature (K).Peptides produced during the simulated gastrointestinal digestion of the five selected varieties were analyzed as potential ligands for HMGCR and PCSK9 through molecular docking. The 3D crystal structures of HMGCR (2Q1L) and PCSK9 (7S5H) were obtained from the Protein Data Bank website ( PRODIGY . Each pew/v) nonfat dry milk in Tris-buffered saline + 0.1% Tween 20 for 1 h at room temperature. Membranes were incubated overnight at 4 °C with primary mouse antibodies for human p-AMPKT172 , AMPK , SREBP-2 , HMGCR , SIRT1 , p-ACCS78/S80 (sc-271965 epitope in amino acids S78 and S80), ACC , SREBP-1c , FASN , LDLR , and PCSK9  purchased in Santa Cruz Biotechnology, unless otherwise stated. All the membranes were then washed and probed with secondary sheep anti-mouse antibodies . SuperSignal™ West Femto maximum sensitivity chemiluminescent (ECL) substrate (Invitrogen) was used to reveal the protein bands, and then pictures were obtained on an ImageQuant 800 System . Protein loading controls  were used to calculate the relative expression of each protein.Similar quantities of cell lysate protein (15 μg) were separated by electrophoresis using 4–20% gradient SDS-PAGE gels. Separated proteins were transferred onto PVDF membranes , which were then blocked with 5%  in 96-well ELISA plates  with 3,3′,5,5′ tetramethylbenzidine substrate . The ApoB concentration was calculated with the ApoB standard provided by the manufacturer in parallel on the same plate.−1 oleic:palmitic acid, 2:1) and digested soybeans at their IC50 for cholesterol concentration increase  or simvastatin (160 ng mL−1) for another 24 h. After treatments, cells were washed twice with PBS, fixed with formalin (4%), rinsed again with PBS, and incubated with Nile Red (1 μg mL−1) and NucBlue™ reagent  for 20 min. Residual staining was eliminated with PBS, and the fluorescent signal was measured under the following conditions (excitation/emission): 530⁄590 nm for the lipid content and 360⁄460 nm for the nuclei content. Lipid accumulation levels were normalized with the number of cells according to the nuclei signal. Then, representative images were collected using a Cytation5 Cell Imaging Multi-Mode Reader .Intracellular lipid accumulation was investigated in HepG2 cells seeded in 96-well plates, kept in complete growth medium for 24 h, and then treated with FFAs  according to the manufacturer’s instructions. The absorbance was measured at 450 nm.−1), the concentrated cell supernatants (50 μg of protein), and fluorescent substrates were incubated at 37 °C for 90 min on a 96-well plate (black bottom). The fluorescence intensities were measured at 485/528 nm, excitation/emission, respectively.The extracellular proteins in the culture medium of the treated cells were concentrated with a centrifugal concentrator MWCO 10 kDa . LPL activity was measured using a fluorometric assay according to the manufacturer’s instructions kit . Briefly, the LPL  and digested soybean varieties at their IC50 for cholesterol concentration increase  and simvastatin (160 ng mL−1) for another 24 h. At the end of the treatment, the culture medium was replaced with 10 μg mL−1 well LDL-Dil  solution . The cells were additionally incubated for 2 h at 37  °C. Finally, the culture medium was aspirated and replaced with PBS. Cells were stained with Hoechst 33342 (Invitrogen) for 20 min and washed with PBS. The degree of LDL uptake was measured using a fluorescent plate reader . The nuclei fluorescence intensity  was used to normalize the values. Representative images were collected using a Cytation5 Cell Imaging Multi-Mode Reader (BioTek).To evaluate LDL uptake, HepG2 cells were seeded in 96-well plates, kept in complete growth medium for 24 h and then treated with FFAs  were incubated in a medium containing phosphate buffer  and different concentrations of digested soybean (0.3–1000 µg protein mL−1) for 5 min at 37 °C. Afterward, the oxidation was initiated by the addition of CuSO4  in the reaction medium. Early and end products from lipid peroxidation chain reactions were monitored by the formation of conjugated dienes (CD) or malondialdehyde (MDA) and 4-hydroxynonenal (HNE), respectively.LDL oxidation was evaluated in plate, using an in vitro biochemical cell-free assay. Human LDL (50 µg protein mLAUC) and then normalizing with the area of the non-treated control (∫CAUC) following Equation (4).CD formation was monitored spectrophotometrically by changes in the absorbance at 234 nm. Absorbance was measured each 2 min (0–240 min). Kinetic curves were fitted to nonlinear regression curves using the Gompertz growth equation . The max50) was calculated.Since values obtained from tested digests were relative to that of the control from the same experimental run, variations caused by LDL susceptibility difference, instrument sensitivity, or reagents can be minimized. Finally, the dose required to cause a 50% inhibition of LDL oxidation . Results were expressed as µmol equivalents of 1,1,3,3-tetramethoxypropane Lp < 0.05. Univariate and bivariate analysis  and post hoc Tukey’s test. Differences were considered significant at −1 soybean flour (−1 digested soybean) was not associated with protein concentration in the defatted flour  among samples, from 20% in V11 to 37% in V16 (The degree of hydrolysis varied (% in V16 D. Protei% in V16 . Glycini50 for the nineteen soybean varieties and glycinin:β-conglycinin ratios are presented in 50 varied from 59 to 229 µg protein mL−1. The inhibitory properties of digested soybean varieties were 199–774-fold lower than those of simvastatin (IC50 = 296 ng mL−1). Previous reports also described that peptic/tryptic soybean hydrolysates might inhibit HMGCR [The nineteen soybean flour varieties digested under simulated gastrointestinal conditions were tested for their inhibition of the activity of HMGCR. it HMGCR . Other lit HMGCR ,45.50 (lower inhibition). Group 2 was characterized by a higher degree of hydrolysis and higher enzyme inhibition. Both groups exhibited similar levels of glycinin and β-conglycinin. On the contrary, group 3 was characterized by the highest concentration of β-conglycinin. Accordingly, five different representative varieties were selected for the following experiments . V1 and V17, having similar HMGCR inhibitory properties , differed in their glycinin:β-conglycinin ratio . V3 and V9 exhibited higher HMGCR inhibition  but varied on their glycinin:β-conglycinin ratio . V18 showed a potential to inhibit HMGCR (IC50 = 135 µg protein mL−1) similar to V1 and V17 but was characterized by the lowest glycinin:β-conglycinin ratio (0.4).Multivariate analysis, PCA, and hierarchical cluster analysis classified the nineteen soybean varieties according to their chemical composition and HMGCR inhibitory properties G–I. PC1 The digests from the selected five varieties were further characterized by LC-MS/MS, and de novo peptide sequencing was used to identify the peptides found in each digested variety. TIC chromatograms show the peptide profiles in the selected digested soybeans A. Repres−1. Peptides were also ranked according to their potential bioactivity. NKLGK exhibited the lowest bioactivity (15.0%) and GPA the highest (75.5%).Thirteen different peptides were tentatively identified (seventeen when considering their L/I and flipped variants). Peptides found in the different varieties contained between two and eight amino residues . Most ofmaxC) after food/hydrolysate/peptide consumption, concluding that the peptide’s structure affects its intestinal absorption and metabolic fate. Zhang et al. [maxC was 130 µg mL−1. Considering that 20–200-fold maxC doses produced accurate results when measuring compounds’ effects in vitro [−1.Although soybean peptides have been detected in plasma, quantitative assays are not available. Sato et al.  reviewedg et al.  investigin vitro , in our −1) and the drug control  in human HepG2 liver cells  (p < 0.05), the digested soybean fraction counteracted FFA’s detrimental effects, at least at 100 µg mL−1. Comparably, simvastatin (0.1–10 µg mL−1) prevented (p < 0.05) the loss in cell viability  A. Althouiability B–G.50 values for the five selected soybean varieties differed significantly (p < 0.05). Varieties V1, V17, and V18 inhibited better HMGCR (lower IC50), followed by V17 and V3.The HMGCR activity was evaluated in FFA-treated cells to confirm the hypocholesterolemic effects observed in the cell-free model. In addition, the concentration of total intracellular cholesterol was measured . The sel50 values for HMGCR inhibition significantly correlated with the concentration of glycinin  and negatively with the β-conglycinin levels . Then, the presence of β-conglycinin seemed to benefit the inhibition of HMGCR. The cholesterol concentration increase was inversely correlated to the inhibition of HMGCR . The higher hypocholesterolemic and HMGCR inhibitory effects of β-conglycinin in comparison to glycinin were previously demonstrated in vivo [−1), with the active site of HMGCR. The active site of HMGCR, where HMG-CoA is reduced, is located in a cis-loop where the main catalytic residues are Lys691, Glu559, Asp767, and His866 [560, Cys561, Arg590, Asn655, Ser661, Lys691, His752, Asn755), attractive charges and salt bridges , π-type interactions , and van del Waals interactions. Similarly, simvastatin interacted with HMGCR’s active site thought hydrogen bonds  and alkyl interactions . Both molecules shared some hydrogen bonds, indicating that their interactions might be similarly effective. All the other peptides released from selected soybean varieties under gastrointestinal conditions exhibited binding energies between −6.9 and −9.2 kcal mol −1 and interaction constants between 0.02 and 8.75 µmol L−1  as a transcriptional regulator and the rate-limiting enzyme of cholesterol biosynthesis, HMGCR . In turnr = −0.942, p < 0.001). Other reports have proven that the FFA-triggered SREBP-2 precursor expression would be associated with an increased expression of the SREBP-2 mature and active form, tightly linked to the subsequent expression of HMGCR and the enhancement in cholesterol synthesis [r = 0.947, p < 0.001). However, the HMGCR expression and activity determined in cells did not correlate with the in-plate HMGCR activity inhibition, presumably due to the changes caused by absorption and metabolism in the cell culture model.SREBP-2 precursor expression increase (1.3-fold) was also counterbalanced by soybean varieties (63–100%) C. SREBP-ynthesis ,55. The p < 0.05), but some digested soybean varieties  reduced it (39–73%) as well as simvastatin (84%) . Similarly, esterified cholesterol correlated with the glycinin:β-conglycinin ratio . Adams et al. [r = 0.711, p < 0.001). The enzyme acyl-CoA cholesterol acyltransferase (ACAT) catalyzes intracellular cholesterol esterification, which is a crucial mechanism for preventing excessive cellular amounts of free cholesterol, which can be harmful to cells [Upon the FFA treatment, liver cells viewed their intracellular concentration of free cholesterol increase 2.4-fold F. Selectin (84%) G. Esteris et al.  reportedto cells . After eto cells . In our to cells  reportedto cells  demonstrto cells  observedp < 0.05). FFA-treated HepG2 hepatocytes exhibited 3.7-fold higher intracellular triglycerides than non-treated cells (p < 0.05) prevented the accumulation of triglycerides. Lipid homeostasis is controlled through multiple nutrient sensors, such as sirtuin 1 (SIRT1), AMPK, acetyl-CoA carboxylase (ACC), sterol regulatory element-binding protein 1c (SREBP-1c), and fatty acid synthase (FASN) [r = −0.831, p < 0.001). Comparably, FFA-triggered and exacerbated (2.8-fold) FASN expression was reduced  by digested soybean varieties  . MAFLD is characterized by excessive fat deposition in the form of triglycerides in the liver (steatosis) . As obseed cells C. Soybeae (FASN) . We obsee (FASN) . AMPK ine (FASN) . FFA auge (FASN) ,67. Here17, V18) H. FASN ePrevious research also indicated that β-conglycinin in soybeans might be associated with reduced FASN expression in adipocytes . Interesp < 0.05). ANGPTL3 release correlated with the glycinin:β-conglycinin ratio . Concurrently, the activity of LPL, reduced (33%) by the presence of FFA, was protected by selected soybean varieties V1 and V18  , however, other extracellular factors, including ANGPTL8, secreted from FFA-stimulated HepG2 cells might also contribute to LPL inhibition [r = −0.766, p < 0.01). There is scarce information on the effects of dietary bioactive compounds on the regulation of ANGPTL3. Some phenolic compounds, flavones, and xanthones have demonstrated their ability to reduce the expression of ANGPTL3 and increase LPL activity [ANGPTL3 is a hepatokine primarily produced in the liver and then released into the bloodstream in response to agonists of the liver X receptor (LXR) . Since Actively) B. The inhibition . LPL actactivity ,74. Inteactivity ,76. Findactivity .r = −0.739, p < 0.01). Similarly, LDLR expression inversely correlated with HMGCR activity  and ANGPTL-3 . Elevated HMGCR activity and ANGPTL-3 release are associated with diminished LDLR expression and LDL uptake in the liver. Since those proteins are overexpressed under MAFLD conditions, regulating them using food compounds may represent a nutritional strategy to prevent atherosclerotic cardiovascular diseases derived from high cholesterol and LDL levels [r = −0.829, p < 0.001). Previous reports demonstrated that peptides from major soybean proteins could stimulate LDLR and inhibit PCSK9 [−1, whereas the constant for PCSK9–peptide interaction ranged 0.35–12.27 µmol L−1. 194 and Asp238, Asp374, and Phe379 [222, Ser225, Cys255, Asp374, Phe379, Ser381, Gln382, Ser383), carbon–hydrogen bonds (Ser372), π-π staked and van del Waals interactions. Comparably, the synthetic inhibitor (a cyclic peptide) interacted though hydrogen bonds , carbon–hydrogen bonds , halogen interactions , alkyl (Ile369), and π-type interactions. Then, the similarities between the interacting residues profiles indicate the potential of this FEEINKVL to behave as a PCSK9 inhibitor. Consistently, FFA elicited a reduction (39%) in LDL hepatic uptake  and MDA+HNE synthesis . Previous studies demonstrated that the 3-month intake of high β-conglycinin soybean milk reduced the levels of oxidized LDL in overweight men to a greater extent than in regular soybean milk [Oxidized LDL is the most prominent risk factor in atherosclerotic cardiovascular diseases . Soybeanean milk .Multivariate analysis investigated the association among all the parameters analyzed A–C. PCA Despite the potential in vitro biological activity of the peptides released from the selected soybean varieties under simulated gastrointestinal conditions, their limited bioavailability constrains their effectiveness . PeptideThis study found that the soybean variety affects the protein composition and peptide release under simulated gastrointestinal conditions. In turn, protein composition influenced the hypocholesterolemic properties of soybean varieties digested under gastrointestinal conditions. We present, for the first time, the hypolipidemic potential of the digested soybeans in a cell model of MAFLD. Digested soybean varieties reduced cholesterol synthesis in human liver cells by inhibiting HMGCR activity, decreased lipogenesis by reducing ACC and FASN expression, reduced VLDL release, promoted LDL clearance by reducing ANGPTL3 and PCSK9 expression and by prompting LDLR expression, and finally inhibited LDL oxidation. These effects were associated with the differential proportion of glycinin and β-conglycinin in soybean proteins, i.e., their glycinin:β-conglycinin ratio. Remarkably, the greater the proportion of β-conglycinin in soybean varieties digested under gastrointestinal conditions, the more the resulting peptides reduced HMGCR expression, the concentration of esterified cholesterol and triglycerides, the release of ANGPTL3, and the production of MDA during LDL oxidation. Then, soybean digested under gastrointestinal conditions may modulate cholesterol and lipid metabolism in hepatocytes based on the glycinin:β-conglycinin ratio. The outcomes of this study, based on results from an in vitro model of MAFLD using liver cells, provide new insight into the potential beneficial effects of soybean on cardiovascular health. Soybean intake may help to regulate cholesterol homeostasis in the liver and LDL oxidation, improving the potential for cardiovascular health. Soybean ingredients made from soybeans with greater proportions of β-conglycinin may be useful to inspire foods and meals containing synergistic components that together can improve the potential for healthful outcomes.