T, AW1T, GTP1T, and HM2T, were isolated from various environments in Busan and Jeju Island, Republic of Korea. The 16S rRNA sequencing results indicated that the four novel strains belong to the genus Ramlibacter. All four strains were tested for their potential cellulolytic properties, where strain USB13T was identified as the only novel bacterium and the first within its genus to show cellulolytic activity. When tested, the highest activities of endoglucanase, exoglucanase, β-glucosidase, and filter paper cellulase (FPCase) were 1.91 IU/mL, 1.77 IU/mL, 0.76 IU/mL, and 1.12 IU/mL, respectively at pH 6.0. Comparisons of draft whole genome sequences (WGS) were also made using average nucleotide identity, digital DNA-DNA hybridization values, and average amino acid identity values, while whole genome comparison was visualized using the BLAST Ring Image Generator. The G + C contents of the strains ranged from 67.9 to 69.9%, while genome sizes ranged from 4.31 to 6.15 Mbp. Based on polyphasic evidence, the novel strains represent four new species within the genus Ramlibacter, for which the names Ramlibacter cellulosilyticus sp. nov.  Ramlibacter aurantiacus sp. nov. , Ramlibacter albus sp. nov. , and Ramlibacter pallidus sp. nov.  are proposed.In this study, four novel bacterial strains, USB13 Ramlibacter, which belongs to the family Comamonadaceae, within the order Burkholderiales, and class Betaproteobacteria, was first proposed in 2003 by Heulin et al. with Ramlibacter tataouinensis TTB310T introduced as its type species1. As of this writing, a total of 11 species have been validly published according to the List of Prokaryotic names with Standing in Nomenclature . 2 These species have been isolated from various soils including subdesert soil1, ginseng soil4, rhizosphere soil5, forest soil8, and garden soil9. Members of the genus Ramlibacter are Gram-negative, asporogenous, and rod- or coccoid-shaped bacteria. The DNA G + C content of members of the genus ranges from 68.5% to 70.0%. Additionally, they are known to have ubiquinone-8 (Q-8) as their major respiratory quinone, as well as palmitic acid (C16:0), summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c), and summed feature 8 (consisting of C18:1 ω7c and/or C18:1 ω6c) as their major fatty acids. Currently, there have not been any reports of Ramlibacter species with cellulolytic activity, making Ramlibacter cellylosilyticus sp. nov. the first within the genus.The genus β-1,4-glycosidic bonds. Despite being Earth’s most abundant biomass component, its recalcitrant nature has made cellulose one of the least utilized bioresources10. Generally, cellulose is converted to glucose either chemically or enzymatically. Inorganic acids are used during chemical hydrolysis, resulting in degradation products that are toxic to microbes which are used in following fermentation steps. Thus, detoxification steps are necessary in order to remove the harmful byproducts. Opposed to chemical hydrolysis, enzymatic hydrolysis does not require these additional detoxifications steps, making enzymatic hydrolysis an eco-friendlier method to obtain glucose11. Hydrolysis of cellulose commonly requires three enzymes to degrade it into glucose monomers: endoglucanases , exoglucanases , and β-glucosidases12. Endoglucanases cut random amorphous sites within the cellulose polymer, generating cellulose ends that exoglucanases in turn act on to cleave from the end. This results in producing products such as tetrasaccharides or cellobiose, a disaccharide, which is then further hydrolyzed by β-glucosidases to generate glucose13. Additionally, the synergistic action of multiple carbohydrate-active enzymes (CAZymes) such as glycosyl hydrolases, also known as glycoside hydrolases (GH), is required to degrade cellulose14. While the complete mechanics are not certain, the main three cellulase enzymes and CAZymes work together through coordinated and complementary oxidative, hydrolytic, and non-hydrolytic activities, which in turn contribute to the total cellulase activity15.Cellulose is a homopolymer of D-glucose units linked by 17. Some examples are Fibrobacter17, Ruminococcus18, and Cellulomonas19, where many species have been isolated from the rumen, intestinal tract, and feces of herbivores.Due to the abundance of cellulosic biomass and its potential as biofuel, there is currently an unprecedented demand for cellulolytic microbes. Although fungi were once thought to be the only viable solution for cellulose degradation, the focus has now shifted to cellulolytic bacteria due to their faster growth rates and ability to withstand extreme environmentsT, and non-cellulolytic strains AW1T, GTP1T, and HM2T. Additionally, cellulolytic activity was assessed by measuring endoglucanase, exoglucanase, and β-glucosidase activity, as well as total cellulase activity, which was assessed by measuring the activity of filter paper hydrolysis.In this study, we present the chemotaxonomic, physiological, and genomic comparisons between the cellulolytic strain USB13T was isolated from a sea water sample collected from a shallow coastal region in Haeundae Beach, Busan . Strains AW1T, GTP1T, and HM2T were isolated from soil samples collected, respectively in the Jeju Island regions of Aewol , Seogwipo , and Seopjikoji Beach . The sea water sample was subsequently diluted with a sterile 0.85% (w/v) NaCl solution using the standard ten-fold serial dilution method (10−1 to 10−4). The diluted sample was spread onto Reasoner’s 2A agar  and incubated for 5 days at 28 °C. Soil samples were submerged in 25 mL of sterile 0.85% (w/v) NaCl solution and rotated for 1 h by using an orbital shaker. The supernatant was then diluted using the standard ten-fold serial dilution method (10−1 to 10−4) and spread onto R2A agar, which was incubated for 5 days at 28 °C. Colonies were repeatedly selected and subcultured onto new R2A agar plates to ensure the purity of the strain. The purified colonies were then sent to Solgent Co., Ltd.  for 16S rRNA gene sequence analysis. The novel strains were designated USB13T, AW1T, GTP1T, and HM2T and were stored at −80 °C in R2A broth containing 50% glycerol (w/v) for future experiments. For strain conservation, the four novel strains were deposited in the Korean Agricultural Culture Collection , the Korean Collection for Type Cultures , and the NITE Biological Resource Center .During an expedition investigating indigenous bacterial communities in South Korea, strain USB1320. Fatty acids exceeding 10% of the total fatty acid content were considered the major fatty acids of the strain. Isoprenoid quinones were extracted according to the methods described by Collins and Jones21 and were analyzed using high-performance liquid chromatography as reported by Komagata and Suzuki22. Polar lipids of the novel strains were extracted according to a previously published method23.For chemotaxonomic characterization, cells of the novel strains and the reference strains were collected from R2A agar plates that were incubated for 5 days at 30 °C. Fatty acids were extracted and analyzed as reported previouslyT, AW1T, GTP1T, and HM2T, the non-staining KOH lysis method was employed24. Oxidase activity was detected with 1% (w/v) tetramethyl-p-phenylenediamine  and catalase activity was assessed by observing production of oxygen bubbles after dropping 3% (v/v) hydrogen peroxide (H2O2) solution. Cell morphology was observed through transmission electron microscopy (TEM)  using cells grown in R2A broth for 3 days at 30 °C. Cells were stained with 2% (w/v) uranyl acetate for 2 sec25. Growth temperature ranges were assessed by observing colony growth at 4, 7, 10, 15, 20, 25, 28, 30, 35, 37, 40, 42, 45, 50, and 55°C26. NaCl tolerance was measured by inoculating strains on R2A agar plates supplemented with various NaCl concentrations (1.0–12.0% w/v at 1.0% increments) and incubating them for 7 days at 30 °C. The pH ranges were examined by cultivating strains at 30 °C in R2A broth adjusted to pH 4.0–10.0  using citrate/NaH2PO4 buffer (pH 4.0–5.0), phosphate buffer (pH 6.0–8.0), and Tris buffer (pH 9.0–10.0), as described in a previous study27. Anaerobic growth was assessed by incubating strains for 10 days on R2A plates at 30 °C in a GasPak jar . Oxygen absorber strips  were used to ensure the absence of oxygen. Hydrolysis of carboxymethyl cellulose , Tween 20 , Tween 80 , casein , starch , chitin , and DNA  was tested as described previously by Smibert and Krieg28. Siderophore detection was measured through the blue agar chrome azurol S (CAS) assay29. Biochemical characteristics such as enzyme production and substrate assimilation were determined using the API ZYM and API 20NE test systems (bioMérieux).To determine the Gram reaction of strains USB1330. Degradation of CMC was indicated by the formation of clear zones surrounding the colonies. Additionally, for evaluation of their cellulose-degrading ability, strains were inoculated in 100 mL of basal salt medium 2SO4, 1.3 g/L; MgCl2, 0.1 g/L; CaCl2, 0.02 g/L; FeSO4⋅7H2O, 0.001 g/L; yeast extract, 5.0 g/L; final pH adjusted to 7.4 with 8 N NaOH)31 containing Whatman Grade 1 qualitative filter paper  and incubated in a shaking incubator (160 rpm) at 30 °C for 7 days. All experiments were conducted in triplicate to ensure reproducibility. In addition, only strains showing positive results for both CMC hydrolysis and filter paper degradation were examined in further experiments.To detect their potential cellulolytic activities, the novel strains were inoculated on R2A agar plates supplemented with 1% (w/v) CMC sodium salt and incubated for 7 days at 30 °C. Following incubation, agar plates were stained with 1% (w/v) Congo red dye for 15 min and washed twice with 1 M NaCl solution to visualize the hydrolysis zoneT, AW1T, GTP1T, and HM2T were sequenced through Sanger sequencing using the universal bacterial primer sets 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) as well as 518F (5′-CCAGCAGCCGCGGTAATAC-3′) and 805R (5′-GACTACCAGGGTATCTAATC-3′), while the sequences were obtained through Sanger sequencing, as previously described by Kim et al.32. The nearly complete 16S rRNA genes were assembled using SeqMan software , and their similarities to each other, and also to closely related type strains were calculated using National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) searches33 and the EzBioCloud server (https://www.ezbiocloud.net/)34.The 16S rRNA genes of strains USB1335, and CLUSTAL_W36. Using MEGA 7 software, phylogenetic trees were reconstructed according to the neighbor-joining (NJ)37, maximum-likelihood (ML)38, and maximum-parsimony (MP)39 methods. Tree topology confidence levels were estimated by bootstrap analyses40 based on 1,000 replications while evolutionary distances were calculated using Kimura’s two-parameter model41. To obtain further taxonomic evidence, a UBCG phylogenomic tree based on the core gene set was reconstructed using the publicly available genomes of closely related taxa42. Based on 16S rRNA sequence similarity and phylogenetic analyses, a total of seven type strains of the genus Ramlibacter were selected for further chemotaxonomic tests and comparative analyses. R. monticola G-3-2T, R. alkalitolerans CJ661T, R. ginsenosidimutans BXN5-27T, R. henchirensis TMB834T, and R. tataouinensis TTB310T were obtained from the KACC and R. humi 18 × 22-1T and R. rhizophilus YS3.2.7T were obtained from the KCTC.Multiple sequences were aligned using the MEGA 7 softwareT, AW1T, GTP1T, and HM2T were prepared using the TruSeq Nano DNA Library Prep kit  with an insert size of 350 bp. The draft genomes were sequenced by Macrogen Co., Ltd.  using the HiSeq X platform  and assembled using the SOAPdenovo version 3.10.1 de novo assembler43. DNA G + C content of the novel strains was calculated from genome data while genome contamination and completeness were assessed using the CheckM bioinformatics tool (https://ecogenomics.github.io/CheckM). 44For genome comparison, the known whole genome sequences of the Ramlibacter reference strains were obtained from the NCBI database while whole genomes of R. monticola KACC 19175T, R. alkalitolerans KACC 19305T, and R. ginsenosidimutans KACC 17527T were sequenced and assembled using the same methods as those for the novel strains. Genome similarities of nucleotide sequences among the novel strains and their reference strains were determined by measuring the digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) values. ANI values were calculated using the OrthoANI calculator from EzBioCloud (https://www.ezbiocloud.net/tools/ani) through pairwise comparisons between genome sequences based on the sequence analysis tool, USEARCH45, while dDDH values were obtained using the Genome-to-Genome Distance Calculator  based on calculations from Formula 246, and AAI values were calculated using the online tool developed by the Kostas lab (http://enve-omics.ce.gatech.edu/aai/index). The draft genome of USB13T, AW1T, GTP1T, and HM2T was annotated using the NCBI Prokaryote Genome Automatic Annotation Pipeline (PGAP), the Rapid Annotation using Subsystems Technology (RAST) server47, and the Cluster of Orthologous Groups of proteins (COG) database48. Identification of the CAZymes was done using the carbohydrate-active enzymes database 49. The antiSMASH version 5.0 database was used to predict and identify existing secondary metabolism genes and biosynthetic gene clusters within the genome50. Additionally, a visual genomic comparison of the genomes of strains USB13T, AW1T, GTP1T, and HM2T was generated using the BLAST Ring Image Generator (BRIG) under default parameters  with USB13T as the reference genome51.Genomic DNA libraries of strains USB132HPO4, 1.44 g/L; KH2PO4, 0.24 g/L; pH 7.4), according to the methods described by Ji et al.52. Subsequently, samples were washed three times with PBS buffer, followed by dehydration using a graded series of ethanol (30–100%). Samples were coated with platinum  and examined under field emission-scanning electron microscopy (FE-SEM) analysis.Samples were prepared to visualize morphological interactions between cellulose-degrading bacteria cells and cellulose fiber strands. Strains were first inoculated in 100 mL of basal salt medium containing Whatman Grade 1 qualitative filter paper  and incubated for 14 days at 30 °C. Following incubation, the degraded filter paper and bacterial cell mixture was loaded on round glass cover slips  and fixed with 2.5% glutaraldehyde in 1X phosphate buffered saline  as the carbon source. Strains were incubated for a total of 7 days where cellulase activity was to be measured at 1, 3, 5, and 7 days following initial inoculation. On the days of testing, supernatant was collected and centrifuged at 5000 rpm for 15 min at 4 °C for enzyme activity analysis.β-glucosidase, and total cellulase were determined by examining the amount of reducing sugar (glucose) released from CMC sodium salt, avicel, salicin, and filter paper, respectively. Enzyme activities were assessed in buffer solutions with different pH values  according to the dinitrosalicylic acid (DNS) method described by Ghose53. Endoglucanase, exoglucanase, and β-glucosidase activities were measured by incubating a mixture of 0.05 M citrate buffer, 0.5 mL of crude enzyme supernatant, and the corresponding substrates for 30 min at 50 °C. Total cellulase activity was measured by incubating 0.5 mL of crude enzyme supernatant with 1.0 mL of 0.05 M citrate buffer and one strip of Whatman Grade 1 qualitative filter paper  for 60 min at 50 °C. After incubation, the substrate hydrolysis reaction was terminated by adding 3 mL of DNS reagent. Results were measured spectrophotometrically where glucose standards were used to approximate the amount of glucose produced. All spectrophotometric data were measured using a UV–Vis spectrophotometer . Enzyme activity was defined in international units (IU or U); one unit of enzymatic activity was defined as the amount of enzyme that releases 1 μmol of glucose per mL per 1 min of reaction. All experiments were conducted in triplicate and the data obtained were presented as mean values ± standard deviation.The activities of endoglucanase, exoglucanase, Ramlibacter species. C16:0 and summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c) were identified as the common major fatty acids (> 10%) of the novel strains USB13T, AW1T, GTP1T, and HM2T. Other than the aforementioned fatty acids, strain USB13T had C10:0 3-OH additionally as its major fatty acid, whereas strains AW1T and HM2T shared C17:0 cyclo and summed feature 8 (consisting of C18:1 ω7c and/or C18: 1 ω6c) as its additional fatty acids. Detailed comparisons of the fatty acid profiles of the novel strains and their reference strains are summarized in Table The predominant respiratory quinone for all novel strains was ubiquinone 8 (Q-8), consistent with other T, AW1T, GTP1T, and HM2T shared major polar lipids diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), which was consistent with the major polar lipids of the reference strains. Additionally, the polar lipid profile of USB13T consisted of one unidentified phosphoaminolipid, two unidentified phosphoglycoaminolipids, and six unidentified polar lipids while the polar lipid profile of AW1T had one unidentified lipid, one unidentified phosphoglycolipid, and three unidentified glycolipids in addition. The polar lipid profile of strain GTP1T additionally consisted of two unidentified phosphoaminolipids, and that of strain HM2T additionally had one unidentified phosphoaminolipid, one unidentified phosphoglycolipid, one unidentified phosphoglycoaminolipid, and two unidentified phospholipids. Polar lipid profiles of the novel strains USB13T, AW1T, GTP1T, and HM2T are shown in Figure Strains USB13T produced reddish white and flat colonies while strain AW1T produced orange, convex colonies, strain GTP1T produced white, convex colonies, and strain HM2T produced cream-colored, flat, transparent colonies. Under TEM, monotrichous flagella were observed only in strain HM2T, and when tested for motility, strain USB13T and AW1T showed gliding motility, whereas strain GTP1T was non-motile. Strains USB13T and HM2T showed positive results for both catalase and oxidase activities; strain AW1T showed positive results for catalase and negative results for oxidase activity, and strain GTP1T showed negative results for catalase and positive results for oxidase activity. All strains were identified to be strictly aerobic, while showing negative results for urea, gelatin, starch, chitin, and DNA hydrolysis and positive results for hydrolysis of Tween 80. In addition, strain USB13T was the only strain to produce iron-chelating siderophores. When tested for NaCl tolerance, growth of strain USB13T was observed in NaCl concentrations of 0–7% (w/v), possibly due to the fact the strain was isolated from a marine environment. A detailed comparison of physiological and morphological characteristics between the novel species and its closely related Ramlibacter strains is presented in Table 8.When grown on R2A agar, strain USB13T; 2, AW1T; 3, GTP1T; 4, HM2T; R. monticola KACC 19175T; 6, R. alkalitolerans KACC 19305T; 7, R. ginsenosidimutans KACC 17527T; 8, R. humi KCTC 52922T; 9, R. henchirensis KACC 11925T; 10, R. tataouinensis KACC 11924T; 11, R. rhizophilus KCTC 52083T. All strains are positive for esterase lipase (C8), while all strains are negative for chitin hydrolysis. All data were obtained from this study unless indicated otherwise. + , Positive; w + , weakly positive; -, negative.Strains: 1, USB13T, indicating that strain USB13T solely possessed CMC-hydrolyzing activity among the four novel strains. When inoculated in basal salt medium, filter paper from the USB13T sample underwent degradation, whereas samples containing strains AW1T, GTP1T, and HM2T did not show any signs of degradation.R2A agar plates supplemented with 1% (w/v) CMC were stained with Congo red dye after 7 days of incubation. Clear zones only formed around colonies of strain USB13Comamonadaceae and genus Ramlibacter. Using BLASTn, 16S rRNA gene sequence similarities were determined where strain USB13T was closest to strain GTP1T (98.5%), followed by strain HM2T (98.1%) and strain AW1T (97.1%). Strain AW1T shared the highest similarity with strain GTP1T (97.3%), followed by strain HM2T (97.1%), while strain GTP1T shared a similarity of 98.2% with strain HM2T. Phylogenetic analysis based on the MP method , exoglucanase , and β-glucosidase  were not present in the genome11.Based on NCBI PGAP annotation and CAZyme prediction results, strain USB13T (258), followed by GTP1T (230), HM2T (212), and AW1T (181). The high number of genes in strain USB13T may be a contributing factor in the strain’s cellulolytic activity. A comparison of COG gene count distribution of the novel strains is presented in Table COG predictions Fig.  revealedT: ribosomally synthesized and post-translationally modified peptides (RIPP)-like cluster , terpene synthesis , RIPP precursor peptide recognition element (RRE)-containing cluster , and redox-cofactor . Among the clusters, the RRE-containing cluster showed 11% similarity to streptobactin, a tricatechol-type siderophore isolated from Streptomyces sp. YM5-79956. Strain AW1T had a total of eight gene clusters which encoded for: arylpolyene , terpene , RIPP-like proteins , non-ribosomal peptide synthetase cluster (NRPS)-like , betalactone , N-acetylglutaminylglutamine amide , type I polyketide synthase , and heterocyst glycolipid synthase-like polyketide synthase . Strain GTP1T possessed four gene clusters that encoded for RRE-containing cluster , homoserine lactone , a signaling molecule known for its involvement in bacterial quorum sensing, the RIPP-like cluster , and terpene synthesis . Strain HM2T had two gene clusters that encoded for resorcinol , an organic compound known for its antiseptic properties, and terpene , which showed 100% similarity for carotenoid synthesis. BRIG analysis results showed that a majority of the regions within the four analyzed genomes were conserved with at least 70% similarity  activities of strain USB13T, wherein activities for endoglucanase was the highest and β-glucosidase was the lowest in all experiments. As seen in Fig. β-glucosidase activity was relatively resistant to pH change as its activity decreased less than 50%. On day 7, enzyme activities were measured as 1.91 IU/mL for endoglucanase, 1.77 IU/mL for exoglucanase, 0.76 IU/mL for β-glucosidase, and 1.12 IU/mL for FPCase at pH 6.0. When measured at pH 8.0, where enzyme activity was the lowest, enzyme activities were measured as 0.51 IU/mL for endoglucanase, 0.25 IU/mL for exoglucanase, 0.45 IU/mL for β-glucosidase, and 0.23 IU/mL for FPCase; all values were less than half of the measured activity at pH 6.0. The results of strain USB13T are comparable to FPCase results of other species such as Mucilaginibacter polytrichastri RG4-7T (0.98 U/mL) isolated from the moss Polytrichastrum formosum14, Paenibacillus lautus BHU3 (2.9 U/mL) isolated from a landfill site57, and Serratia rubidaea DBT4 (0.5 U/mL) isolated from the gastrointestinal tract of a black Bengal goat58.The enzymatic assay results showed endoglucanase, exoglucanase, β-glucosidase, the cellulolytic activity of strain USB13T was confirmed through SEM images, CMC agar screening, and enzymatic assay results. However, because PGAP annotation results showed that other non-cellulolytic strains also possessed CAZymes, in some cases more than strain USB13T, further research is necessary to understand the mechanics of how CAZymes and other cellulases interact to degrade cellulose, and how these genes are expressed under certain conditions. Furthermore, the cellulolytic activity of strain USB13T can be further optimized for commercial use by adjusting growth conditions such as pH, temperature, and growth media.Despite the absence of the main three cellulolytic enzymes, endoglucanase, exoglucanase, and 59. In these habitats, cellulolytic bacteria utilize cellulose while cohabiting with non-cellulolytic bacteria. There have been many studies suggesting the synergistic role non-cellulolytic bacteria play in cellulose degradation, where non-cellulolytic bacteria aid cellulose degradation by neutralizing pH or removing harmful metabolites62.While cellulolytic bacteria are known to inhabit animal intestinal tracts, the rumen, and soil, they can be found almost everywhere, such as ocean floors, municipal landfills, and even extreme environments such as hot springs11. Due to the versatile uses of bacterial cellulases, the cellulolytic strain USB13T has the potential to become an invaluable resource. However, further research of the novel strain’s cellulose-degradation mechanisms is necessary to develop and commercially make use of its bacterial cellulases in the future. In addition, research regarding co-culturing non-cellulolytic bacteria and strain USB13T may also help in developing effective methods to use an otherwise underutilized bioresource.Bacterial cellulases have shown immense value in various industries such as animal feed processing, food and brewery production, and agriculture, not to mention biofuel synthesis through biomass utilizationT, AW1T, GTP1T, and HM2T should be assigned to the genus Ramlibacter, differences in fatty acid compositions, polar lipid profiles, and physiological characteristics suggested that the four novel strains are noticeably distinct from other validly published species of the genus. Additionally, genomic characteristics such as ANI, dDDH, and AAI values further supported the novel strains’ position as a distinct species within the genus Ramlibacter. Therefore, we propose that the strains USB13T, AW1T, GTP1T, and HM2T represent novel species within the genus Ramlibacter.While phylogenetic analyses indicated that the novel strains USB1363.The descriptions of the novel species are given according to the standards of the Judicial Commission of the International Committee on Systematic BacteriologyRamlibacter cellulosilyticus .T are Gram-negative, rod-shaped, non-flagellated and motile by gliding. The strain is positive for both oxidase and catalase activity, while cells have a width of 0.3–0.5 μm and length of 2.0–2.4 μm. When observed on R2A agar, colonies are reddish white, flat with entire margins, and have a diameter of 1–2 mm. Growth of strain USB13T is observed at 7–50 °C , at pH 5.0–10.0 , and at NaCl concentrations of 0–7% . The strain is unable to grow in anaerobic conditions. Produces siderophores and hydrolyzes Tween 20, Tween 80, CMC, and esculin. According to the API ZYM results, the strain showed positive results for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, acid phosphatase, β-galactosidase, α-glucosidase, and β-glucosidase. In the API 20NE assay, strain USB13T showed positive results only for β-galactosidase. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C16:0, C10:0 3-OH, and summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoaminolipid, two unidentified phosphoglycoaminolipids, and six unidentified polar lipids. The G + C content is 69.7%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain USB13T are MN603953 and JACORT000000000, respectively.Cells of strain USB13T (= KACC 21656T = NBRC 114839T) was isolated from shallow coastal water at Haeundae Beach, Busan, Republic of Korea.The type strain USB13Ramlibacter aurantiacus .T are Gram-negative, coccoid to short rod-shaped, non-flagellated, and motile by gliding. The strain is negative for oxidase activity, and positive for catalase activity. When observed on R2A agar, colonies are orange, convex, with entire margins, and 0.5–1.0 mm in diameter. Under TEM cells have and approximate width of 0.3–0.5 μm and length of 0.6–0.8 μm. Growth of strain AW1T can be observed at 7–45 °C , at pH 7.0–10.0 , and at NaCl concentrations of 0–3% . The strain does not grow under anaerobic conditions but is able to hydrolyze Tween 80. In addition, AW1T is not able to produce siderophores. In the API ZYM assay, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, and β-glucosidase. In the API 20NE assay, positive for esculin hydrolysis. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C16:0, C17:0 cyclo, summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c), and summed feature 8 (consisting of C18:1 ω7c and/or C18:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoglycolipid, one unidentified lipid, and three unidentified glycolipids. The G + C content is 68.6%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain AW1T are MN498045 and JAEQNA000000000, respectively.Cells of strain AW1T (= KACC 21544T = NBRC 114862T) was isolated from soil at Aewol, Jeju Island, Republic of Korea.The type strain AW1Ramlibacter albus .T is non-motile, Gram-negative, strictly aerobic, positive for oxidase activity, and negative for catalase activity. When observed on R2A, colonies are white, convex, with entire margins, and 1–2 mm in diameter. Under TEM, cells lack flagella, are rod-shaped, and have a width of 0.7–0.8 μm and length of 1.6–1.9 μm. Growth of strain GTP1T can be observed at 10–45 °C , at pH 5.0–8.0 , and at NaCl concentrations of 0–2% . The strain shows positive results for Tween 20 and Tween 80 hydrolysis. GTP1T does not produce siderophores when tested on CAS-blue agar. According to API ZYM results, strain GTP1T is positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), and leucine arylamidase, while the API 20NE assay results show negative results for all substrates. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C16:0 and summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and two unidentified phosphoaminolipids. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C16:0, C17:0 cyclo, summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c), and summed feature 8 (consisting of C18:1 ω7c and/or C18:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoaminolipid, one unidentified phosphoglycolipid, one unidentified phosphoglycoaminolipid, and two unidentified polar lipids. The G + C content is 67.9%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain GTP1T are MN498046 and JACORU000000000, respectively.Strain GTP1T (= KACC 21702T = NBRC 114488T) was isolated from soil at Seogwipo, Jeju Island, Republic of Korea.The type strain GTP1Ramlibacter pallidus .T are Gram-negative, and positive for both oxidase and catalase activities. When observed on R2A agar, colonies are cream-colored, transparent, 1.0–2.5 mm in diameter, and flat with entire margins. Under TEM, monotrichous flagella are observed, and cells are rod-shaped with a width of 0.4–0.78 μm and length of 1.7–1.8 μm. The strain shows the fastest growth at a temperature range of 25–35 °C and at pH values between 8.0 and 9.0. When NaCl is present, growth is observed at concentrations of 0–3% (w/v), with optimal growth was observed at concentrations of 0–1% (w/v). The strain is not able to tolerate anaerobic conditions. Strain HM2T hydrolyzes Tween 80 and weakly hydrolyzes casein. However, siderophore production cannot be observed when tested on CAS-blue agar. According to API ZYM tests, strain HM2T shows positive results for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. In addition, API 20NE tests show positive results for nitrate (NO3) to nitrite (NO2-) reduction and esculin hydrolysis. The G + C content is 69.9%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain HM2T are MN498047 and JADDIV000000000, respectively.Cells of strain HM2T (= KCTC 82557T = NBRC 114489T) was isolated from soil at Seopjikoji, Jeju Island, Republic of Korea.The type strain HM2Supplementary Information.


Spodoptera litura (Fabricius) is an agriculturally significant polyphagous insect pest that has evolved a high level of resistance to conventional insecticides. A dietary assay was used in this work to assess the resilience of field populations of S. litura to λ-cyhalothrin. Analysis of the function and expression of the cytochrome P450 gene was used to test the sensitivity of S. litura larvae to sub-lethal concentrations of the insecticidal plant chemical Precocene 1, both by itself and in combination with λ-cyhalothrin. The activity of esterase enzymes (α and β) was found to decrease 48 h post treatment with Precocene 1. The activity of GST enzyme and cytochrome P450 increased with Precocene 1 treatment post 48 h, however. Expression studies revealed the modulation by Precocene 1 of cytochrome P450 genes, CYP4M16, CYP4M15, CYP4S8V4, CYP4G31, and CYP4L10. While CYP4M16 expression was stimulated the most by the synergistic Precocene 1 + λ–cyhalothrin treatment, expression of CYP4G31 was the most down-regulated by Precocene 1 exposure. Hence, it is evident that λ–cyhalothrin-resistant pest populations are still sensitive to Precocene 1 at a sublethal concentration that is nevertheless capable of hindering their development. Precocene 1 can therefore be considered a potent candidate for the effective management of insecticide-resilient S. litura. Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is a cosmopolitan insect pest, widely distributed throughout tropical and subtropical regions  and deltamethrin  family, with a varied functional group of hemoproteins . In inseS. litura for detoxifying host plant allelochemicals and other xenobiotics has not been much explored.The mechanism of the detoxifying P450 enzyme in insects helps them resist phytoconstituents and chemical insecticides . VariousS. litura, in particular, develops resistance when exposed to synthetic pesticides , upon exposure to λ-cyhalothrin.The major damage to agricultural crops results mainly from attack by lepidopteran insects . S. litusticides , 2021b. S. litura were obtained from agricultural land in Kadayam , Tenkasi District, India, and cultured in the Biopesticides and Ecotoxicology Laboratory (BET Lab), SPKCEES, Manonmaniam Sundaranar University, Alwarkurichi. To maintain generation, the larvae were kept in an insectary at 27 ± 1°C, with relative humidity (RD) of 85%, under a 12:12 L:D photoperiod schedule. Castor leaves were given as feed, and the adults that emerged were placed in a container (10 × 10 × 7 cm), with castor leaves for pairing . A 10% honey solution was given to adults for oviposition, and a sanitary black cloth was used to cover the cage.The larvae of Precocene 1, phenyl methylsulfonyl fluoride (PMSF), and NADPH were purchased from Sigma-Aldrich . The λ-cyhalothrin was purchased commercially from a local chemical company in India. Dithiothreitol (DTT), glycerol, and Tris were obtained from Himedia Chemicals, Hyderabad, India, while 7-ethoxycoumarin, 7-hydroxycoumarin, EDTA, and bovine serum albumin were obtained from Sigma-Aldrich, Mumbai, India. Reagent grade chemicals and solvents were used.Preparation of an artificial and chemically supplemented diet followed previously published protocols . The PreThird-instar larvae were used to find the effects of Precocene 1 uptake and tolerance to λ-cyhalothrin for all instances, as this developmental stage allows for observation of weight gain and mortality. 0.2% Precocene 1 was added to the artificial diet for 48 h before bioassay, and the control larvae were given the artificial diet containing the 0.1% DMSO solution. In total, twenty-five third-instar larvae were used for the experiment with five replications. In addition, in order to study the toxicity of λ-cyhalothrin, a diet merger methodology was followed using third-instar larvae . A standS. litura larvae were fed with an artificial diet with or without 0.2% Precocene 1 for 48 h. To obtain the concentration of 25 mg/L, the PBO was dissolved in acetone. Using a micro-syringe, 10 µg of PBO/larva was applied topically in the dorsal prothorax region of individual larvae of S. litura. After sterilization, plastic cups were used to place the 2 h PBO-exposed larvae of S. litura, to which were added different concentrations of insecticide solutions, namely, 150, 180, 210, 240, and 270 of λ-cyhalothrin mg/L, with or without 0.2% Precocene 1, to assess the toxicity of λ-cyhalothrin. The diet with Precocene 1 was fed to the larvae for 48 h, without the pre-treated PBO of λ-cyhalothrin, and the larvae were kept in cups with perforated lids. Every bioassay was conducted in triplicate.Piperonyl butoxide (PBO) was used as the synergist, and its presence or absence was evaluated as described earlier. Ten fourth-instar larvae were treated with Precocene 1 and then homogenized on ice with 0.1 M phosphate buffer (7.2 pH), containing 1 mM EDTA, 1 mM DTT, 1 mM PTU, 1 mM PMSF, and 20% glycerol. Tissues were collected after 24 and 48 h periods of homogenization in 2 ml of the buffer, and centrifugation was performed at 4°C, 10,000 g for 15 min. Solid debris and cellular materials were separated and the supernatant was transferred into Eppendorf tubes. These were placed on ice for the assay of carboxyl esterase, glutathione-S-transferase, and cytochrome P450. The total content of protein was then estimated using the procedure of The α and β-carboxylesterase activity was determined  in the lGlutathione S-transferase assays were carried out, following the protocol of Determination of Cyt P450 activity was carried out by peroxidation of a TMBZ assay following the protocol of S. litura larvae was extracted by TRIzol (Invitrogen). Synthesis of cDNA was performed in 20 µl of reactant, having 1 µl of total RNA, 0.6 µl of forward primer (10 pmol), 0.6 µl of reverse primer (10 pmol), and 20 units of RNase inhibitor, 1 µl of dNTP mixture (10 mM each), and 1 µl oligo (dT)18 primer (50 µM) at 42°C , for 1 h by reverse transcription. The concentration of RNA was quantified at 260–280 nm absorbance. The quality was assessed by agarose gel electrophoresis, and staining was performed with ethidium bromide (EB).The total content of the RNA in ® RT Reagent Kit with gDNA Eraser   according to the manufacturer’s protocol. To study the RT-PCR, primers  , 0.8 µl of each primer (10 pmol/µl), and 6.4 µl ddH2O. The RT-PCR program was carried out using the melting curve dissociation methodology (from 55°C to 95°C), and by following the thermal conditions: initial denaturation 95°C for 30 s, subsequently 40 cycles of 95°C for 5 s, and 60°C for 30 s. A melting curve analysis was then performed to assess the specificity and consistency of the PCR products. The expression levels of target genes were calculated with the 2−ΔΔCT method and normalized to the internal housekeeping gene β-actin.To estimate the amount of RNA using agarose gel electrophoresis, the reverse transcription to cDNA from 1 µg of total RNA was conducted with a PrimeScript primers  were plap < 0.05) for different groups by using the Tukey test. Statistical analysis was carried out with SPSS 11.5 software.Data are represented in mean ± SD. Resulting pairs were compared using the Student’s t-test. One-way ANOVA was applied to determine the significant differences , a considerable decrease due to the synergistic effect of PBO with Precocene 1 (61.06 mg ai/L).The impact of Precocene 1 and its synergistic effect with PBO against the tolerance and sensitivity of S. litura at 24 and 48 h after exposure is reported in S. litura, due to the various treatments, is shown in the order of Precocene 1 + λ-cyhalothrin > Precocene 1>λ-cyhalothrin > control and was 1.95, 1.5, 1.1, and 0.8, respectively, in the said order at 24 h of exposure. At 48 h of exposure, profound activity was observed in Precocene 1 + λ-cyhalothrin (2.7), followed by λ-cyhalothrin, Precocene 1, and the control .The activity of the α-esterase enzyme in the control, the effect of Precocene 1 only, of λ-cyhalothrin, and of the synergistic effect of Precocene 1 with λ-cyhalothrin in the larva of S. litura at 24 and 48 h of exposure in the control, Precocene 1, λ-cyhalothrin, and the combined effect of Precocene 1 + λ-cyhalothrin is shown in S. litura as in the case of alpha esterase was observed, and the values were 2.01, 1.7, 1.4, and 1.15 at 24 h of exposure. Similarly, in the larva of S. litura, the enzyme activity at 48 h of exposure was also found to be of the same order as reported in the alpha esterase activity in S. litura at 48 h, with values of 2.89, 2.6, 2.10, and 1.33.The β-esterase enzyme activity in the larvae of -cyhalothrin, the activity of the enzymes was found to be greater, being 1.89 and 2.11 in the larva of S. litura. The enzyme activity of S. litura was followed in the order of Precocene 1, λ-cyhalothrin, and the control as 1.29, 1.13, and 0.98 at 24 h of exposure, while a similar rate of enzyme activity was seen in S. litura larva at 48 h of exposure . A similar increasing pattern of gene expression was noticed in CYP4L10, where the values were 1.01, 1.77 1.89, and 1.98-fold. When compared with the control in all other families, the gene expression was found to increase, whereas a changing pattern of gene expression was observed among the three groups. Among the five families studied, the CYP4M16 showed higher gene expression due to the combined effect of Precocene 1 with λ-cyhalothrin at 24 h of exposure.The expressions of the genes CYP4M16, CYP4M15, CYP4S8V4, CYP4G31, and CYP4L10 expressions of S. litura after an exposure period of 48 h in the control, Precocene 1, λ-cyhalothrin, and Precocene 1 + λ-cyhalothrin groups are reported in CYP4M16, with Precocene 1 + λ-cyhalothrin (2.65); the lowest level of gene expression (1.20) was observed in the Precocene 1-treated CYP4G31 of S. litura; and no variation in the gene expression pattern was noticed in the control.The S. litura larvae in response to λ-cyhalothrin. Furthermore, the impact of Precocene 1 on the activity of P450, esterases, and glutathione S-transferases, and the relative gene expression levels of cytochrome genes  were also assessed.Plants produce a variety of secondary metabolites, or allelochemicals, which play a defensive role against plant eaters and pathogens. Furthermore, the natural predators of herbivores are attracted by these allelochemicals . In the Prosopis juliflora, and petroleum benzene leaf extract of Manihot esculenta, in S. litura. In the present study, the diet incorporating Precocene 1 led to an increased level of tolerance to the insecticide, λ-cyhalothrin, in S. litura. A similar kind of resistance to λ-cyhalothrin was noticed in S. exigua (H. armigera (H. zea forα-cypermethrin when exposed to xanthotoxin (S. exigua fed with gossypol (litura, is corroborated by the results of H. armigera when fed with gossypol on exposure to pyrethroid, and quercetin to λ-cyhalothrin, respectively. In the result of RT-PCR at 24 and 48 h, the transcriptional levels of the CYP4M16, CYP4M15, CYP4S8V4, CYP4G31, and CYP4L10 enzymes of S. litura increased more markedly in all treatments other than in the control, and such activity resulted in the enhancement of P450 gene expression. The results of the present study accord with those of Concerning the synergistic ratio, findings on the individual effect of Precocene1 in an enhanced state, as opposed to the synergistic and lone effect of PBO, deviate from the results of . exigua  and H. aarmigera , due to thotoxin . Likewisgossypol . The enhS. litura, the role of plant allelochemicals in the activity of the detoxification enzymes viz., esterase, GST, and Cytochrome P450, and also the gene expression levels of CYP4M16, CYP4M15, CYP4S8V4, CYP4G31, and CYP4L10 in response to λ-cyhalothrin. It is obvious from the results that the S. litura showed different degrees of resistance, and after Precocene 1 treatment, particularly, the P450 gene showed a low level of expression, as in the control. Research is needed to discover the candidate genes that respond specifically to the natural toxins of plants, and also the impact of insecticides against such pests, in order to improve pest management strategies.The impact of active compounds isolated from a natural plant could elevate sensitivity to insecticides by enhancing the activity of detoxification enzymes in agricultural insects. Furthermore, the cytochrome P450 enzyme system certainly plays a vital role in ways insects resist the plant’s chemical defense mechanisms. The current investigation assessed the impact of Precocene 1 alone, PBO, and their combined effect on the synergistic potential of


Glutamine (Gln) is an immunomodulatory protein that mediates oxidative stress, inflammation, and apoptosis, but has not been reported in the treatment of hyperoxia (Hyp)-induced brain injury. The aim of this study was to determine whether Gln could improve hyp-induced brain injury in neonatal rats to and later learning and memory dysfunction, and to explore its possible mechanisms. We prepared a model of neonatal rat brain injury caused by normobaric hyperoxia while administered with Gln for 7 days for evaluation. Learning memory function was assessed with the Morris water maze test. Histological analysis, protein expression analysis, oxidative stress and inflammation level analysis were performed using hippocampal tissue. Gln treatment significantly reduced brain tissue water content, oxidative stress levels, microglia activation and inflammatory factor expression, and attenuated tissue damage and apoptosis in the hippocampal region. Gln ameliorates hyp-induced learning, memory impairment in neonatal rats in water maze test. It also increased MKP-1 protein expression and decreased p-p38, p-ERK and p-JNK. Therefore, it is hypothesized that Gln may exert neuroprotective effects by increasing MKP-1 expression to negatively regulate MAPK signaling, with potential cognitive improvement in hyp-induced brain injury. Goat anti-rabbit IgG/horseradish enzyme labeling (#16K22C) was purchased from Boster Biological Technology , and goat anti-mouse IgG/horseradish enzyme labeling (#ZB-2305) was purchased from Zhongshanjinqiao biotechnology . β-actin antibody (bsm-33036m) (1:1000) was purchased from Beijing Boaosen Biotechnology Co., Ltd. . L-glutamine (G8540) was purchased from Sigma-Aldrich (United States). Rat IL-1β (SBJ-H0417), IL-6 (SBJ-H0465), TNF-α (SBJ-H0038) ELISA kit reagent kits were purchased from Nanjing Sempega Biotechnology Co. . ROS (MM-43700M2), GSH-PX (MM-20251R2), SOD (MM-20387R2), MDA (MM-0385R1) ELISA kits were purchased from Jiangsu Meimian Industrial Co., Ltd. . TUNEL kit was purchased from Promega (United States). H-E staining kit was purchased from Beijing Solarbio Science and Technology Co., Ltd. . Iba-1 antibody was purchased from Santa cruz biotechnology (United States). DAB kit was purchased from Beijing Boaosen Biotechnology Co., Ltd. .SD neonatal rats, weighing 6.7–8.2 g, were used in this study, provided by the Animal Section of Yanbian University. All animals were kept together with their dams in per cage on a 12 h light/dark cycle in a temperature-controlled room (22°C ± 1°C) with free access to water and food.n = 15) 1 day after birth: control group (Con: 21% O2), normal drug group (Con + Gln: 21% O2), Hyp group (Hyp: 85% O2) and drug group (Hyp + Gln: 85% O2). The Hyp and Hyp + Gln groups were placed in glass chambers and oxygen concentrations were measured twice daily with a digital oximeter, and nitrogen dioxide and water were absorbed with soda lime. The dams were rotated between the hyperoxia- and normoxia-exposed pups every day to avoid undersupply of the young. Gln was dissolved in 0.9% Nacl to be used. Gln (1 μg/g B.W) was administered intraperitoneally to newborn rats in the Con + Gln and Hyp + Gln groups at the same time each day, and the same volume of 0.9% NaCl was administered intraperitoneally to newborn rats in the Con and Hyp groups at the same time for 7 days.60 newborn rats were randomly divided into four groups (n = 3) was anesthetized, fixed on the operating table, and the thoracic cavity was exposed. After injection of chilled PBS solution from the apex of heart, the tissue was fixed by injection of 4% paraformaldehyde. The brain tissue was then quickly removed, immersed in 4% paraformaldehyde solution for 24 h and fixed, and subjected to histopathological analysis  after tissue pruning, dehydration, transparency, paraffin embedding and conventional sectioning. In each group, newborn rats (n = 3) were randomly selected and executed after anesthesia, and a pair of hippocampal tissues were rapidly isolated on ice, treated with liquid nitrogen and placed in an ultra-low temperature refrigerator at −80°C for backup . And each group of newborn rats (n = 3) was then randomly selected and executed after anesthesia, and brain tissue was removed to determine the water content of brain tissue. At P30, each group of neonatal rats (n = 6) began the Morris water maze experiment , and following ether anesthesia, the brain tissues were immediately removed and weighed. The wet weight (W) of the brain tissues was marked and baked in a 60°C oven. The dry brain tissue weight (D) is measured when there is no weight change for two consecutive days. The brain tissue water content (%) was determined using the formula [(W-D)/W] 100%.Newborn rats were selected at random from each group , ROS, superoxide dismutase (SOD) and glutathione (GSH) Elisa kits. The MDA level was measured by the absorbance of the sample at 532 nm, expressed as nmol/mg protein. The SOD level was measured by the absorbance of the sample at 550 nm, expressed as unit/mg protein. The ROS level was measured by the absorbance of the sample at 525 nm, expressed as RFU/mg protein. The GSH level expressed as ng/mg protein. All results were normalized by the total protein concentration in each sample.Hippocampal tissues were removed and washed with pre-cooled saline, dried on filter paper. The hippocampal samples with saline solution were homogenized at a ratio of 1:9 (g/mL) in a homogenizer, and the supernatant was collected by centrifugation at 3,000 rpm for 10 min. The hippocampal samples with saline solution were homogenized at a ratio of 1:9 (g/mL) in a homogenizer, and the supernatant was collected by centrifugation at 3,000 rpm for 10 min. The expression levels of TNF-α, IL-1β and IL-6 in hippocampus tissue were determined using quantitative ELISA kits according to the manufacturer’s instructions.n = 6) were placed in the water facing the wall of the tube from quadrants 1 to 4 sequentially, swam for a maximum of 2 min to find the platform, and remained on the platform for 20 s. If the platform was not found within 2 min, they were guided to the platform for 20 s and the escape latency was recorded as 2 min. In the second phase, spatial memory was examined : the platform was removed and the rats were all placed in the water along the pool wall from the same quadrant and the swimming time and distance in the target quadrant and the number of times they crossed the platform in 2 min were recorded. The experimental process was recorded and analyzed by the data acquisition system.To observe the ability to reflect spatial learning and memory, the MWM test was conducted at P30-P34. The Morris water maze apparatus consisted mainly of a 160 cm-wide, 60 cm-high circular pool with a black interior and a water level 30 cm deep, which was kept at 20°C ± 1°C. A cylindrical platform with a diameter of 12 cm wide was set 1 cm below the water surface of the third quadrant. In the first phase, spatial learning ability was tested : each rat was tested four times a day (with a 2-min break in between) for 4 days. In each test, newborn rats   solution. The apoptotic cells were quantitatively assessed, with three animals examined per group and three slices per hippocampal sample. TUNEL-positive hippocampal neurons of the CA1 zone were identified and counted under ×200 magnification, and the average number of positive apoptotic hippocampal neurons in the CA1 of three brains in a group was calculated.Brain tissue sections were dewaxed and treated with proteinase K working solution to permeabilize the cell and nuclear membranes and 3% HThe slides underwent dewaxing, dehydration, rehydration, antigen repair in boiling sodium citrate buffer, cold confirmed to room temperature and then rinsed in PBS. After 15 min of endogenous peroxide blocker blockade, the slides incubated with primary antibody Iba-1 (1:500) overnight at 4°C. After rewarming at room temperature for 1 h, PBS rinsing, they were then incubated with secondary antibodies  at room temperature for 30 min. PBS rinsing them again and DAB color development for 5 min. The CA1 and CA3 regions of the hippocampus were analyzed. The images  were captured by the microscope system . The experiment was repeated three times. The average number of activated microglia in the CA1 and CA3 area of three hippocampal in a group was calculated under ×400 magnification.To investigate the expression of proteins by western blot analyses, hippocampal tissue samples were lysed in a protein cell lysis buffer : PMSF : phosphatase inhibitor  = 100:1:1). The protein concentration of the samples was determined using a bicinchoninic acid (BCA) protein assay kit . The samples were boiled for 10 min, and proteins were separated by electrophoresis using a 10% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Then, proteins transferred to PVDF membranes . The membrane was blocked with 5% skim milk (diluted with TBST), incubated with primary antibody overnight at 4°C. The membrane was washed three times with 0.1% TBST, and incubated with secondary antibody for 1.5 h and then washed again with TBST. Then, the membranes were detected with an enhanced chemiluminescence kit . The samples were wrapped in plastic wrap and transferred into the dark room. The processes of developing and fixing were done after the exposure of X-ray film. The results were scanned by a gel imaging system and analyzed by using ImageJ software. The grayscale value of each band was normalized to the grayscale value of the β-actin band, and the relative expression of the proteins was determined. The primary antibody included MKP-1 (1:1000), p-p38 (1:1000), p38 (1:1000), p-JNK (1:1000), JNK (1:1000), p-ERK (1:1000), ERK (1:1000), BDNF (1:1000), Synapsin Ia (1:1000), MBP (1:1000), Bax (1:1000), Bcl-2 (1:1000). The secondary antibodies include Goat anti-rabbit IgG/horseradish enzyme labeling (1:5000) and goat anti-mouse IgG/horseradish enzyme labeling (1:5000).p < 0.05 was considered statistically significant.SPSS26.0 software was used for statistical analysis, and GraphPad Prism 8.0.1 software was used to crate figures. Values are presented as mean ± standard deviation, and statistical significance was determined by one-way ANOVA followed by Tukey and Bonferroni tests. Univariate repeated measures ANOVA was used to analyze escape latency (MWM) for independent group comparisons. p < 0.001) than that in the Con group and that in the Hyp + Gln group was significantly lower (p < 0.01) than that in the Hyp group. Con (72.11 ± 0.12), Con + Gln (71.94 ± 0.32), Hyp (77.41 ± 0.37), Hyp + Gln (74.66 ± 0.29) (n = 3).In this study, brain edema was observed in each group by measuring brain tissue water content. As shown in In addition, H&E staining was used to evaluate the success of the model preparation and to determine whether Gln ameliorates hyp-induced brain tissue damage. As shown in p < 0.05) (p < 0.01) (p < 0.01) (p < 0.01) (p < 0.05) and MDA (p < 0.01) content and considerably higher GSH (p < 0.05) and SOD (p < 0.01) content than the Hyp group. These results suggest that Gln reduces the level of oxidative stress induced by hyp. ROS: Con (5851.05 ± 90.01), Con + Gln (5880.49 ± 303.02), Hyp (8160.46 ± 380.24), Hyp + Gln (6899.07 ± 184.10); MDA: Con (3.50 ± 0.21), Con + Gln (3.30 ± 0.23), Hyp (13.23 ± 0.78), Hyp + Gln (8.13 ± 0.29); GSH: Con (44.03 ± 2.54), Con + Gln (43.47 ± 2.58), Hyp (27.60 ± 0.44), Hyp + Gln (32.43 ± 1.53); SOD: Con (133.37 ± 2.49), Con + Gln (132.87 ± 1.00), Hyp (98.77 ± 1.78), Hyp + Gln (110.70 ± 1.36) (n = 3).The supernatant of the hippocampal tissue homogenate from each group of neonatal rats was tested using relevant kits to observe changes in oxidative stress levels. The results showed that total ROS levels ( < 0.05)  and MDA  < 0.01)  were sig < 0.01)  and SOD  < 0.01)  were conp < 0.01, CA3: p < 0.01), suggesting microglial activation. However, Gln administration significantly reduced microglial activation after hyp . CA1: Con (3.00 ± 0.58), Con + Gln (3.33 ± 0.33), Hyp (9.33 ± 0.88), Hyp + Gln (5.67 ± 0.67). CA3: Con (3.33 ± 0.33), Con + Gln (3.67 ± 0.33), Hyp (9.00 ± 0.58), Hyp + Gln (5.00 ± 0.58) (n = 3).Microglia were marked out with Iba-1, and microglia activation in CA1  and CA3 p < 0.0001), IL-1β (p < 0.0001), and IL-6 (p < 0.0001), showing that hyp induces an inflammatory response. However, these changes were significantly reversed after Gln application . TNF-α: Con (2.73 ± 0.84), Con + Gln (2.69 ± 0.41), Hyp (9.70 ± 0.15), Hyp + Gln (5.67 ± 0.22). IL-1β: Con (2.65 ± 0.08), Con + Gln (2.68 ± 0.08), Hyp (11.16 ± 0.44), Hyp + Gln (7.33 ± 0.60). IL-6: Con (2.38 ± 0.05), Con + Gln (2.42 ± 0.02), Hyp (7.71 ± 0.30), Hyp + Gln (4.94 ± 0.16) (n = 3).To verify whether Gln could inhibit the inflammatory response caused by hyp, we examined the expression of TNF-α , IL-1β , and IL-p < 0.01), while Gln administration exerted an inhibitory effect on hyp-induced apoptosis (p < 0.05) (n = 3).TUNEL staining was performed to evaluate the inhibitory effect of Gln on apoptosis . The res < 0.05) . Con (0.p < 0.05) and caspase-3 content (p < 0.01) were significantly increased in the Hyp group; in contrast, it considerably improved in the Hyp + Gln group  (n = 3).To further clarify the apoptosis of hippocampal tissues in each group, caspase-3, Bax and Bcl-2 was determined by western blotting . The res < 0.01) . Bax/Bclp < 0.05), MBP (p < 0.05) and BDNF (p < 0.05) proteins decreased significantin the Hyp group. In the Hyp + Gln group, the expression of these proteins was significantly higher than that in the Hyp group . This indicated that Gln increased synapsin I (n = 3).To evaluate whether the neuroprotective effect of Gln is related to synaptic plasticity, impaired myelin formation, and the absence of neurotrophic factors, the expression of brain-derived neurotrophic factor (BDNF), synapsin I, and myelin basic protein (MBP) was examined after homogenizing hippocampal tissue obtained from each group of rats . The resnapsin I , MBP (Finapsin I  and BDNFnapsin I  expressip < 0.05) (p < 0.05) (p < 0.01) (p < 0.01) (p < 0.01) and significantly decreased p-p38/p38 (p < 0.05), p-ERK/ERK (p < 0.05), and p-JNK/JNK (p < 0.01) expression. MKP-1: Con (0.28 ± 0.01), Con + Gln (0.28 ± 0.01), Hyp (0.32 ± 0.01), Hyp + Gln (0.84 ± 0.11); p-p38/p38: Con (0.71 ± 0.00), Con + Gln (0.73 ± 0.05), Hyp (1.29 ± 0.17), Hyp + Gln (0.77 ± 0.02); p-ERK/ERK: Con (0.53 ± 0.04), Con + Gln (0.53 ± 0.03), Hyp (0.83 ± 0.04), Hyp + Gln (0.66 ± 0.04); p-JNK/JNK: Con (0.93 ± 0.03), Con + Gln (0.91 ± 0.04), Hyp (1.23 ± 0.01), Hyp + Gln (1.03 ± 0.02) (n = 3).The MAPK signaling pathway is closely associated with oxidative stress, inflammation, and apoptosis. To assess the role of Gln in a model of hyp-induced brain injury and its association with the MKP-1/MAPK signaling pathway, we examined the expression of MKP-1/MAPK signaling pathway-related proteins . The res < 0.05)  was slig < 0.05) , p-ERK/E < 0.01) , and p-J < 0.01)  expressip < 0.05; p < 0.01) in the Hyp + Gln group from Days three to four. These results suggest that rats in all groups had the ability to learn and remember. These abilities were impaired to varying degrees by hyp-induced brain injury; however, Gln improved the spatial learning abilities of rats with hyp-induced brain injury. In the observation of the swimming path on Day 5, it was found that the Hyp group rats crossed the platform position (p < 0.001) often than the Con group did, and the percentage of distance traveled in the target quadrant (p < 0.01) than that of the Con group. In the Hyp + Gln group, the number of platform crossings increased (p < 0.01) compared to that in the Hyp group, and the percentage of distance traveled in the target quadrant increased (p < 0.01) compared to that in the Hyp group. These results suggest that hyp-induced brain damage during the neonatal period affects memory capacity in adolescent rats. However, administration of Gln significantly improved the duration spent in the plateau quadrant, suggesting that Gln effectively alleviated memory impairment induced by hyp. No significant difference was observed in the mean swimming speed between groups . The escape latency  showed aposition  less (p quadrant  was lessn groups . Latencyin vivo antioxidant precursor substance with multiple regulatory mechanisms and known antioxidant potential (Long-term hyperoxia treatment can lead to behavioral abnormalities and neurocognitive and learning deficits in preterm infants, which is thought to be related to damage to hippocampal neurons caused by oxidative stress, inflammation, apoptosis, and reduced expression of neurotrophic factors . Spatialotential , and itsotential . Thus, GOxidative stress is a key factor in the complex cascade of brain injury mechanisms. In preterm infants, whose antioxidant systems are not yet well-developed, the excessive ROS generated in a hyperoxic environment trigger an inflammatory response that induces prostaglandin production and leads to the formation of intercellular interstitial edema . MicroglMany proteins also play important roles in the development and treatment of brain injury. BDNF is an important protein that can influence neuroplasticity, affecting cognition, brain structure and function . SynapsiMAPK mediates cellular responses to external stimuli and regulates a variety of cellular activities such as cell proliferation, differentiation, apoptosis, and neuronal plasticity . EvidencThis study shows that in newborn rats with hyp-induced brain injury, Gln has potent anti-inflammatory and antioxidant activities, reduces hippocampal damage and neurological dysfunction, ameliorates hyp-induced brain injury, and decreases apoptosis, thus alleviating learning difficulties and memory dysfunction in newborn rats with hyp-induced brain injury. Our results suggest that the molecular mechanism underlying these effects may be related to the MKP-1/MAPK signaling pathway . This st


Low mineralization activity by human osteoblast cells (HOBs) indicates abnormal bone remodeling that potentially leads to osteoporosis. Oxidation, the most prominent form of high-density lipoprotein (HDL) modification, is suggested to affect bone mineralization through the inflammatory pathway. Adiponectin, which possesses anti-inflammatory activity, is postulated to have the ability to suppress the detrimental effects of oxidized HDL (oxHDL). This study aimed to investigate the effects of HDL before and after oxidation on markers of mineralization and inflammation. The protective effects of adiponectin on demineralization and inflammation induced by oxHDL were also investigated. OxHDL at 100 µg/mL protein had the highest inhibitory effect on mineralization, followed by lower calcium incorporation. OxHDL also had significantly lower expression of a mineralization marker (COL1A2) and higher expression of inflammatory markers ) compared to the unstimulated control group. These findings suggest that oxHDL reduces the mineralization activity of HOBs by increasing the expression of inflammatory markers. Interestingly, co-incubation of adiponectin and oxHDL in HOBs resulted in higher expression of mineralization markers  and significantly reduced all targeted inflammatory markers compared to the oxHDL groups. On the contrary, HDL increased the expression of mineralization markers (COL1A2 and STAT-3) and exhibited lower expression of inflammatory cytokines (IL-6 and TNF-α), proving the protective effect of HDL beyond the reverse cholesterol transport activity. Bone mineralization is an important process during bone construction and hardening. During the bone mineralization process, calcium and phosphate are accumulated in the mineral vesicle that buds off the osteoblast cells to produce hydroxyapatite crystals. This hydroxyapatite crystal is deposited onto the organic extracellular matrix in an organized manner . Bone miA histomorphometry study by Byers et al. in 1997 found that most osteoporotic patients had a significantly lower number of osteoblast cells, and these osteoblasts have a lower mineralization rate, indicating a lower percentage of active osteoblasts in patients and leading to reduced efficiency of the active osteoblasts .Pro-inflammatory processes are suggested to play an important role in inhibiting osteogenic differentiation of mesenchymal stem cells (MSCs) and osteoblast differentiation, leading to disruption of bone-forming activities . OsteoblPrevious studies reported the presence of oxidized lipids and lipoproteins that induce inflammation in osteoblasts, causing these cells to malfunction . The accHDL (high-density lipoprotein) is another lipoprotein that is also prone to oxidation, especially when exposed to oxidative stress, inflammation, hyperglycemia-induced advanced glycation end-products, and the presence of reactive oxygen species. HDL is known to have many beneficial activities in cardioprotection. However, oxidation causes the components of HDL to change in terms of the quantity and quality, and this HDL later becomes dysfunctional ,15. ThisOsteoporosis is also associated with obesity. The secretion of myokines, osteokines, and adipokines is the main bridge of the crosstalk between bone tissue and fat tissue . For insTherefore, the aims of this study were to investigate the effects of HDL before and after oxidation on bone mineralization to examine whether oxHDL can induce inflammation in primary human osteoblasts and elucidate its underlying mechanism and, finally, investigate the ability of adiponectin in suppressing the detrimental effects of oxHDL.p < 0.01), 35.7 ± 2.1% (p < 0.01), 59.7 ± 8.2% (p < 0.0001), respectively, vs. 0.0 ± 1.6%) (p < 0.0001) vs. 58.2 ± 3.1% (p < 0.0001)). Only 50 µg/mL protein oxHDL did not significantly inhibit the formation of mineral nodules (26.5 ± 18%), but it did show a decremental trend. A trend of a reduction in calcium deposition was also observed in the HOBs cells treated with 10, 25, 50, and 100 µg/mL protein oxHDL   B. Furthectively) C. Microsp < 0.05, p < 0.0001)) and co-incubation of oxHDL with 5 µg/mL adiponectin (0.1 ± 0.006 relative mRNA expression (p < 0.0001)) showed an increment in RUNX2 gene expression when compared to the unstimulated (0.002 ± 0.000 relative mRNA expression) and oxHDL group (0.002 ± 6.7 × 10−5 relative mRNA expression)  A. In gen−5 relative mRNA expression) and adiponectin alone, 5–15 µg/mL , did not have any effect on the mRNA expression of ALPL when compared to the unstimulated group (0.0005 ± 0.0001 relative mRNA expression) (p < 0.0001), oxHDL + 10 µg/mL adiponectin: 0.0017 ± 0.0001 relative mRNA expression (p < 0.0001), and oxHDL + 15 µg/mL adiponectin: 0.0016 ± 5.5 × 10−5 relative mRNA expression (p < 0.0001)) compared to oxHDL alone. Cells treated with HDL had a neutral effect on HOBs in terms of ALPL mRNA expression compared to the unstimulated group (0.0012 ± 0.0002 relative mRNA expression vs. 0.0005 ± 0.0001 relative mRNA expression), but it was significantly higher than cells treated with oxHDL.One of the markers is ALPL, a gene coding for alkaline phosphatase protein. In this study, the cells treated with oxHDL (0.0006 ± 5.1 × 10ression) B. Unexpep < 0.0001)). In addition, cells stimulated with 10 and 15 µg/mL adiponectin also showed significantly higher mRNA secretion of COL1A2 when compared to the unstimulated group (10 µg/mL adiponectin: 2.8 ± 0.1 relative mRNA expression (p < 0.0001), 15 µg/mL adiponectin: 3.9 ± 0.3 relative mRNA expression, respectively (p < 0.0001), and unstimulated: 0.28 ± 0.007 relative mRNA expression). Incubation of cells with HDL also showed a significantly higher mRNA expression of COL1A2 compared to the unstimulated group and the oxHDL group (HDL: 3.2 ± 0.1 relative mRNA expression (p < 0.0001) vs. unstimulated: 0.28 ± 0.007 relative mRNA expression and oxHDL: 0.499 ± 0.025 relative mRNA expression).Moreover, this study also measured the mRNA expression of COL1A2, which codes for the synthesis of the α2 chain, one of the collagen chains intertwined with the other two α1 chains to form collagen 1 C. In thi−5 relative mRNA expression (p < 0.01)) showed higher mRNA expression of bone gamma-carboxyglutamate protein (BGLAP), which encodes for osteocalcin, compared to the unstimulated group. Moreover, cells co-incubated with oxHDL and 5 µg/mL adiponectin (0.0025 ± 0.0003 relative mRNA expression (p < 0.0001)) showed significantly higher mRNA secretion of BGLAP compared to the oxHDL group and unstimulated group ), and 10 µg/mL of adiponectin (0.042 ± 0.001 relative mRNA expression (p < 0.01)) resulted in a significantly higher mRNA expression of STAT-3 compared to the unstimulated group (0.017 ± 0.0006 relative mRNA expression) (p < 0.0001)). These results showed that HDL and adiponectin might have the ability to increase the secretion of STAT-3 in HOBs. However, oxHDL did not have any effects on the expression of STAT-3 in HOBs.STAT-3 is one of the important transcription factors involved in stimulating the bone formation activity of osteoblast cells . Interesression) A. The cop < 0.0001)) (p < 0.0001)). In addition, cells incubated with the highest concentration of adiponectin alone (15 µg/mL) also showed significantly higher mRNA expression of PPAR-α compared to the unstimulated group (0.015 ± 0.0007 relative mRNA expression vs. 0.0063 ± 0.0002 relative mRNA expression (p < 0.0001)). Cells treated with HDL only showed a trend of increment in the mRNA expression of PPAR-α when compared to the unstimulated group (0.0079 ± 0.0003 relative mRNA expression vs. 0.0063 ± 0.0002 relative mRNA expression (p < 0.0001)).The expression of PPAR-α was also measured as a previous study demonstrated the involvement of PPAR-α in pre-osteoblast cell maturation . Similar0.0001)) B. The prp < 0.0001)) (p < 0.0001)). This suggests that adiponectin has a protective effect against the upregulation of IL-6 heightened by oxHDL. Furthermore, a high concentration of adiponectin (15 µg/mL) showed significantly lower IL-6 mRNA expression compared to the unstimulated group (0.12 ± 0.008 relative mRNA expression vs. 0.32 ± 0.008 relative mRNA expression (p < 0.0001)). Incubation of cells with HDL alone showed a significantly lower mRNA expression of IL-6 (0.2 ± 0.01 relative mRNA expression) compared to the unstimulated group (p < 0.05) and oxHDL group (p < 0.0001).Inhibition of osteoblast differentiation and maturation by pro-inflammatory cytokines is one of the crucial factors that disturbs a balanced bone remodeling process, leading to bone loss . In this0.0001)) A. The prp < 0.0001) vs. unstimulated: 0.0007 ± 0.0001 and HDL: 0.00012 ± 2.27 × 10−5) (−5 relative mRNA expression (p < 0.0001), oxHDL + 10 μg/mL adiponectin: 0.00018 ± 1.9 × 10−5 relative mRNA expression (p < 0.0001) and oxHDL + 15 μg/mL adiponectin: 0.0001 ± 3.4 × 10−5 relative mRNA expression (p < 0.0001), respectively, vs. oxHDL: 0.0034 ± 0.0002 relative mRNA expression). The highest concentration of adiponectin (15 µg/mL) also showed significantly lower TNF-α mRNA expression compared to the unstimulated group (0.0001 ± 3.4−5 vs. 0.0007 ± 0.0001 (p < 0.05)). Cells treated with HDL also showed significantly lower TNF-α mRNA expression (0.00012 ± 2.27 × 10−5) compared to the unstimulated group, suggesting adiponectin and HDL could have a protective effect in terms of TNF-α mRNA expression. TNF-α is another inflammatory cytokine measured in this study. The presence of oxHDL significantly induced higher mRNA expression of TNF-α compared to the unstimulated and HDL groups (oxHDL: 0.003 ± 0.0001 relative mRNA expression ( × 10−5) B. This ep < 0.0001) vs. unstimulated: 0.01 ± 0.0004 relative mRNA expression and HDL: 0.005 ± 0.0001 relative mRNA expression). This effect of oxHDL might be suppressed by the presence of adiponectin because significantly lower mRNA expression of the RELA proto-oncogene NF-ĸβ subunit was observed during co-incubation of 5 to 15 µg/mL adiponectin with oxHDL compared to the cells treated with oxHDL alone (oxHDL + 5 µg/mL adiponectin: 0.02 ± 0.0007 relative mRNA expression (p < 0.0001), oxHDL + 10 µg/mL adiponectin: 0.04 ± 0.003 relative mRNA expression (p < 0.0001) and oxHDL + 15 µg/mL adiponectin: 0.03 ± 0.0025 relative mRNA expression, respectively (p < 0.0001), vs. oxHDL: 0.11 ± 0.0054 relative mRNA expression). However, cells incubated with adiponectin alone (5–15 µg/mL) showed higher RELA proto-oncogene NF-ĸβ subunit mRNA expression compared to the unstimulated group (5 µg/mL adiponectin: 0.03 ± 0.0008 relative mRNA expression (p < 0.01), 10 µg/mL adiponectin: 0.05 ± 0.002 relative mRNA expression (p < 0.0001), and 15 µg/mL adiponectin: 0.03 ± 0.002 relative mRNA expression (p < 0.05), respectively, vs. unstimulated: 0.01 ± 0.0004 relative mRNA expression), and the values were significantly lower than cells treated with oxHDL (0.11 ± 0.0055 relative mRNA expression). The finding from this study suggests that the increment in inflammatory cytokines by oxHDL is via RELA proto-oncogene NF-ĸβ subunit (P65) activation C. the REThis study also measured the expression of the cAMP-responsive element-binding protein 1 (CREB) gene coding for the CREB proteins, a nuclear factor involved in the cyclic AMP signaling pathway. Activation of the cAMP signaling pathway usually correlates with a decline in mineralization and osteogenesis activity in differentiated osteoblasts . However2+ incorporation into matrix vesicles by the annexin calcium channel and other calcium-binding phospholipids is a crucial step in the formation of hydroxyapatite crystals. A reduction in calcium incorporation was also observed in UMR106 rat osteoblasts during exogenous induction with Cu2+-induced oxLDL, which later disturbed the osteoblast protein markers, RUNX2 and OPN [A reduction in mineralization activity in HOBs indicates disruption of bone formation activity. It is one of the crucial factors contributing to the unbalanced bone remodeling process as when lost bone fails to be replaced, it may lead to osteoporosis . Instead and OPN . In this study, the reduction in the mineralization activity in HOBs by oxHDL might be due to the decrease in COL1A2 expression. COL1A2 is the gene that codes for collagen type 1 protein. This protein is the most dominant constituent of the collagenous organic matrix in bone, which provides the site for hydroxyapatite crystal deposition. Therefore, with the presence of oxHDL, it might reduce the collagen cross-linking and repress the deposition of the crystallized matrix. Subsequently, it might lead to bone mineralization activity impairment, which is commonly observed in patients diagnosed with senile osteoporosis and osteogenesis imperfecta . The findings form this study suggest that the inhibitory activity of oxHDL towards the mineralization of HOBs might be via the inflammatory pathway. Inflammation is a well-known culprit that disturbs the balanced bone remodeling process by inducing excessive osteoclast differentiation and inhibiting osteoblast maturation, leading to osteoporosis ,36. PatiInterestingly, this study found that adiponectin showed protective effects by suppressing the activation of NF-ĸβ during its co-incubation with oxHDL, which then reduced the secretion of pro-inflammatory cytokines (IL-6 and TNF-α). The suppression of NF-ĸβ inflammatory pathways may be due to the high mRNA expression of PPAR-α induced by adiponectin when incubated alone and during co-incubation with oxHDL. PPAR-α has been known to have inhibitory effects on NF-ĸβ activity, resulting in decreased secretion of IL-6 . In addi−/− deficiency mice proved that the elimination of ApoA-1, the prominent component of HDL, leads to severe osteoporosis [−/− deficiency mice showed that this cell prefers to differentiate into adipocytes rather than osteoblast cells. However, further analysis needs to be carried out to understand HDL’s beneficial activity on HOBs fully.This study also found that before undergoing oxidation, HDL could have some beneficial effects on HOBs. The mRNA expression of COL1A2 and STAT-3 is increasing. There is no solid explanation regarding the protective activity of HDL in preventing osteoporosis due to the lack of laboratory research on this topic. However, a low level of HDL is commonly linked to osteoporosis. A study carried out by Blair’s research groups in ApoA-1oporosis . In thesPrimary human osteoblast cells (HOBs) were purchased from Promocell  at passage number 2. The cell tested positive for alkaline phosphates using the 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) assay and positive for mineralization ability using Alizarin Red S. HOBs were grown and maintained in osteoblast growth medium containing 0.1 mL/mL (10%), fetal calf serum (FBS), and 1% Antibiotic-Antimycotic Solution, as recommended by the Promocell. Phosphate-buffered saline (PBS)  was used as the washing solution and Accutase  was used as the detachment solution during the sub-culturing. Cells in passages 6–7 were used for this study. Upon stimulation, the culturing medium was replaced with a treatment medium composed of DMEM media  containing 15% FBS , 10 mM β-glycerophosphate , 0.1 mM ascorbic acid , and 1% Antibiotic-Antimycotic Solution .4)  for 4 h at 37 °C. The mixture of HDL and CuSO4 was placed in a 15 mL centrifuge tube wrapped with aluminum foil and the incubation was performed in an incubator set at 37 °C. The oxidation was stopped by the addition of 2.5 mM EDTA . The oxidized HDL was dialyzed again with PBS for 24 h in three buffer exchanges stored at 4 °C and used within 2 weeks. The value of TBARs in the oxHDL was measured using an OxiSelect™ TBARS Assay Kit  to evaluate the degree of oxidation. The average value of TBARs in this experiment was 119.9 ± 21 nmol/L/mg protein of MDA. Human high-density lipoprotein (HDL) was purchased from Merck Millipore . The HDL is certified to be from a healthy individual and the human plasma tested negative for HBsAg and HIV-I, HIV-II, HBc, and hepatitis C antibodies. Firstly, the HDL was dialyzed against phosphate-buffered saline (PBS)  for 24 h in the dark at 4 °C with three buffer exchanges to remove any preservative agents. Then, about 1 mg protein/mL HDL was incubated with 50 µM copper sulphate  at 4 °C for 45 min. Next, the fixative reagent was removed, and the cells were washed with distilled water three times. Then, the cells were incubated with 1 Ml of 2% alizarin red (PH 4.2) for 20 min at room temperature with gentle shaking. The dye was removed, and the cells were gently washed twice with distilled water. Pictures of the cells were taken using an inverted microscope .g. About 500 µL of the supernatant was transferred into a new micro-centrifuge tube. Then, about 200 µL of 10% ammonium hydroxide was added into each tube to neutralize the acidic solution. The standard curve of alizarin red was prepared by diluting the 4 Mm alizarin red in serial dilution. The Ph of each dilution was between 4.1 and 4.3. In total, 150 µL of the samples and standard curve dilution was transferred into a 96-well plate. The optical density (OD) was measured using a microplate reader  at a wavelength of 450 nm. The concentration of alizarin red obtained for each sample was calculated based on the standard curve.In total, 800 µL of 10% acetic acid was added to each well and incubated at room temperature for 30 min with gentle shaking. The cells in each well were collected using a cell scraper, transferred into a 1.5 mL micro-centrifuge tube, and vortexed for 30 min. Then, the tubes were heated at 85 °C using a digital block heater for 10 min. The tubes were put on ice for 5 min. The tubes were then centrifuged for 15 min at 20,000× About 10,000 cells/well HOBs were seeded overnight in a 96-well plate in the treatment medium. Then, the cells were induced with a treatment medium containing (i) media only as the negative control; (ii) 100 µM inorganic pyrophosphate (positive control); and (iii) 10, 25, 50, and 100 µg/mL protein oxHDL in 100 µL of media  for 14 days. The media was changed every 4 days. Then, on the 14th day of incubation, the cells were washed twice with PBS . About 0.6 N HCL (100 µL) was added to each well to decalcify the cells. After 24 h, the supernatants were collected to quantify the calcium content using a Calcium Colorimetric Assay Kit  while the protein content inside the cells was collected by solubilizing the cells with 0.1 N NaOH. The amount of calcium was normalized with the cells’ total protein content and quantified using a BCA protein assay kit .® RNA/Protein kit . The concentration and purity of the extracted protein and RNA were measured using a Nanodrop ND-100 Spectrophotometer. About 300,000 cells/well HOBs were seeded overnight in a 6-well micro-plate containing treatment medium. On the next day, the cells were incubated with (i) inorganic pyrophosphate (positive control); (ii) culture medium ; (iii) HDL (100 μg/mL protein); (iv) oxHDL (100 μg/mL protein); (v) adiponectin ; and (vi) the combination of oxHDL (100 μg/mL) with adiponectin . Total RNA was extracted using the AllPrep® Green Supermix , and 2 µL of DNase free water in a 10 µL volume and loaded into 96-well clear microtiter plates . Then, the reaction plates were sealed with optically clear adhesive films  before insertion into the CFX96TM Real-Time PCR Detection System  to perform PCR at 95 °C for 30 s (polymerase activation and DNA denaturation), 95 °C for 5 s (denaturation), 60 °C for 30 s , and repeated for another 49 cycles. Normal template controls (NTCs) and a reverse transcription control (RTC) were included in every reaction plate. A melt curve was obtained for each run to ensure the purity of the primer. The PCR efficiency was obtained by analyzing a series of cDNA (standard curve). Relative gene expression, ΔCT, was calculated using the formula: Ratio (references/target) = 2Ct(references)−Ct(target) .The total RNA extract samples were reverse transcribed to complementary DNA (cDNA) using an iScript™ gDNA Clear cDNA Synthesis Kit  before the quantitative real-time PCR assay (rt-qPCR). Then, the cDNA was either stored at −20 °C or directly used as the template for the qPCR assay. SYBR-green was used as a detection method in the rt-qPCR assay. GAPDH and HPRT-1 were selected as the reference genes. The primer sequence for each measured gene is presented in p < 0.05. The data were analyzed using Statistical Package for the Social Sciences (SPSS) version 21.0. The data were expressed as the mean ± SEM obtained from three experiments, where each assay was performed in triplicates (n = 3). A one-way ANOVA test was performed followed by post-hoc analysis (Bonferroni). The level of significance was set at From this study, it can be concluded that inflammation is the link responsible for the occurrence of osteoblast demineralization induced by oxHDL. Even though HDL might not exhibit solid protective activity, it shows a better effect than oxHDL. Oxidation causes HDL to lose its anti-inflammatory properties, impairing its protective functions, where dysfunctional HDL starts to behave like a pro-inflammatory molecule. Interestingly, the detrimental effects of oxHDL could be suppressed by the presence of adiponectin. This shows that adiponectin may possess some beneficial activities. These findings could be fundamental for future development of drugs or treatments that could either protect HDL from being oxidized in the human body or drugs that could elevate the secretion of adiponectin.