Casuarina equisetifolia is widely used in agroforestry plantations for soil stabilization, ecosystem rehabilitation, reclamation, and coastal protection. Moreover, C. equisetifolia has remarkable resistance to typhoons, desert, low soil fertility, drought, and salinity, but not cold. Therefore, it is significant to breed high-quality Casuarina varieties to improve the tolerance and adaptability to cold weather by molecular techniques. The establishment of a rapid and efficient callus induction and regeneration system via tissue culture is pre-requisite for the genetic transformation of C. equisetifolia, which is so far lacking. In this study, we reported an efficient and rapid regeneration system using stem segment explants, in which callus induction was found to be optimal in a basal medium supplemented with 0.1 mg⋅L–1 TDZ and 0.1 mg⋅L–1 NAA, and proliferation in a basal medium containing 0.1 mg⋅L–1 TDZ and 0.5 mg⋅L–1 6-BA. For bud regeneration and rooting, the preferred plant growth regulator (PGR) in basal medium was 0.5 mg⋅L–1 6-BA, and a combination of 0.02 mg⋅L–1 IBA and 0.4 mg⋅L–1 IAA, respectively. We also optimized genetic a transformation protocol using Agrobacterium tumefaciens harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) as a reporter gene. Consequently, 5 mg L–1 hygromycin, 20 mg L–1 acetosyringone (As), and 2 days of co-cultivation duration were optimized to improve the transformation efficiency. With these optimized parameters, transgenic plants were obtained in about 4 months. Besides that, Agrobacterium rhizogenes-mediated transformation involving adventitious root induction was also optimized. Our findings will not only increase the transformation efficiency but also shorten the time for developing transgenic C. equisetifolia plants. Taken together, this pioneer study on tissue culturing and genetic transformation of C. equisetifolia will pave the way for further genetic manipulation and functional genomics of C. equisetifolia. Casuarina equisetifolia, a symbiotic nitrogen-fixing tree, plays an important role in economic and environmental improvements in tropical and subtropical littoral zones of Asia, the Pacific, and Africa  for 30 s, followed by rinsing with sterile water five times. Then, the shoots were further disinfected with 6–8% (v/v) sodium hypochlorite for 8 min and rinsed with sterile water five times. Finally, the sterile young shoots were cut into small pieces of about 1 cm in length and placed in a basal medium  supplemented with different phytohormone combinations. The pH of media was adjusted to 5.7. For each hormone treatment, three replicates were performed, each with 14 explants.Young shoots were cut from 30- to 90-day-old seedlings of A. rhizogenes, seeds of C. equisetifolia were sown in pots filled with soil and grown in the greenhouse at 25°C in a 16-h/8-h day/night photoperiod. After 30 days, healthy seedlings of about 15 cm height were selected for agro-infiltration.For transformation using –1 6-benzylaminopurine (6-BA) and 0.05 mg L–1 kinetin (KT); C2: 1.5 mg⋅L–1 6-BA and 0.03 mg⋅L–1 naphthylacetic acid (NAA); C3: 1.5 mg⋅L–1 6-BA and 0.02 IBA; C4: 0.1 mg⋅L–1 NAA and 0.1 mg⋅L–1 thidiazuron (TDZ); and C5: 0.5 mg⋅L–1 6-BA and 0.1 mg⋅L–1 TDZ. Pieces of sterile young shoots were placed in the medium at 28°C in the dark. After about 15 days, the effect of each hormone treatment on callus induction was observed.For callus induction, five combinations of plant growth regulators (PGRs) were used as follows: C1: 0.5 mg⋅L–1 6-BA; B2: 0.5 mg⋅L–1 6-BA and 0.05 mg⋅L–1 KT; B3: 0.75 mg⋅L–1 6-BA and 0.03 NAA; B4: 1.5 mg⋅L–1 6-BA and 0.02 mg⋅L–1 IBA; and B5: 0.5 mg⋅L–1 6-BA and 0.1 mg⋅L–1 TDZ. To establish the preferred conditions, the rate of bud regeneration was recorded after growth for 30 days at 28°C under a 16-h light (60–80 μmol photons m–2 s–1)/8-h dark photoperiod. The buds (about 1 cm in length) were shifted to a rooting medium for root induction. The rate of root induction was calculated after 20-day cultivation in three different rooting media as follows: R1: 0.02 mg⋅L–1 IBA and 0.04 mg⋅L–1 IAA; R2: 0.4 mg⋅L–1 IAA; and R3: 1.5 mg⋅L–1 IAA.For bud regeneration, good-quality calluses were shifted to the media with the following growth regulator combinations: B1: 0.5 mg⋅L–1 carbenicillin (Car) and six different concentrations of hygromycin . Each treatment consisted of 20 calluses, and 3 replicates were performed. The status of the calluses was observed, and the rate of callus induction was determined after 15 days of incubation in the dark.Sterile young stems were placed in an optimized callus induction medium with 250 mg⋅LAgrobacterium tumefaciens strain GV3101 was used for genetic transformation of C. equisetifolia. The binary expression vector pCAMBIA1301, with a β-glucuronidase (GUS) reporter gene under cauliflower mosaic virus (CaMV) 35S promoter, was mobilized into strain GV3101 using the freeze-thaw method . The culture was incubated at 28°C until an OD600 of 0.6–0.8 was attained, and then centrifugated immediately at 5,000 rpm for 10 min. The pellet was resuspended in an approximately 50-mL ½ MS liquid containing 30 g L–1 sucrose and 20 mg L–1 As. The final concentration of the A. tumefaciens suspension was adjusted to an OD600 of 0.6 for transformation.w method . A singl–1) in an Agrobacterium infective suspension were evaluated. Stem segments of approximately 1 cm in length each were excised from the sterile young shoots and grown in an optimized callus induction medium. After 15 days, induced calluses were submerged into the Agrobacteria infective suspension and shaken at 28°C and 150 rpm for 2 h. Excess bacterial suspensions were removed from the calluses by drying on sterile filter papers. Subsequently, the calluses were placed in a basal medium with 0.1 mg L–1 NAA, 0.1 mg L–1 TDZ, and different concentration of As  for co-cultivation at 28°C. After co-cultivation, the calluses were washed 6–8 times with 250 mg L–1 carbenicillin water (each wash was 3 min) and submerged in 250 mg L–1 carbenicillin water for 8 min to decontaminate Agrobacteria. The induced calluses were then transferred into an optimized selection medium containing PGRs, hygromycin with different concentrations , and carbenicillin (250 mg L–1). Each treatment consisted of 15 calluses in 3 replicates. All hygromycin-resistant shoots were further assayed by GUS histochemical staining.For transformation, three co-cultivation durations  and four As concentrations  at 28°C for about 8–12 h. The inoculum was then transferred to a 20-ml LB broth with the same antibiotics as 1:20. After the OD600 reached around 0.8, the cultures were centrifuged at 5,000 × g for 10 min at room temperature and then resuspended in 25 ml MES buffer containing 10 mM MES-KOH (pH 5.2), 10 mM MgCl2, and 100 μM As.A single colony of For transformation, seedlings of 5–8 cm height were gently picked from the pots and the residual soil on the roots was carefully removed. The stem between the cotyledons and roots was gently scratched using a clean blade, and then the entire plant was soaked into the infection solution for 2–4 h. After infection, the seedlings were replanted in the pots for growth. Scratched parts were exposed necessarily above the soil, so callus induction could easily be observed. Callus growth was observed about 15 days after infection in the wounded part of the stem. After about a month, when the hairy roots had developed well, the transgenic rate for the hairy roots was detected using LUYOR-3415RG (Hand-held Lamp). Transgenic efficiency was calculated as:Transgenic root induction efficiency =  × 100%;Transformation efficiency =  × 100%.C. equisetifolia, we tested 38 different combinations of plant growth regulators  in a basal medium  (–1 6-BA + 0.05 mg⋅L–1 KT), C2 (NO. 6: 1.5 mg⋅L–1 6-BA + 0.03 mg⋅L–1 NAA), C3 (NO. 26: 1.5 mg⋅L–1 6-BA + 0.02 mg⋅L–1 IBA), C4 (NO. 37: 0.1 mg⋅L–1 TDZ + 0.1 mg⋅L–1 NAA), and C5 (NO. 32: 0.1 mg⋅L–1 TDZ + 0.5 mg⋅L–1 6-BA). It was later observed that the calluses turned brown in the C2, C3, and C4 media  . Stem sehytagel) . After 1C4 media . The staC4 media . On the C4 media . Thus, tC4 media .–1 6-BA), B2 (0.5 mg⋅L–1 6-BA + 0.05 mg⋅L–1 KT), B3 (0.75 mg⋅L–1 6-BA + 0.03 mg⋅L–1 NAA), B4 (1.5 mg⋅L–1 6-BA + 0.02 mg⋅L–1 IBA), and B5 (0.5 mg⋅L–1 6-BA + 0.1 mg⋅L–1 TDZ). Besides B1, which only contained 6-BA, the other media had 6-BA with KT, NAA, IBA, and TDZ. It took about 1–2 months for the calluses to develop adventitious buds  was 80%, while in R1 (NO. 20: 0.02 mg⋅L–1 IBA + 0.04 mg⋅L–1 IAA), and R3 (NO. 22: 0.02 mg⋅L–1 IBA + 1.5 mg⋅L–1 IAA) it was around 55 and 10%, respectively  significantly promoted multiple root organogenesis. The statistical analysis of root induction rate showed that the rate in R2 . It took about 7–10 days to generate calluses and 15 days to develop roots . Then, t–1). The results showed that the rate of callus induction declined with increasing in concentration of hygromycin, and reached the steady state at hygromycin a concentration of 5 mg L–1  were supplemented into the Agrobacterium liquid medium for callus growth. We found that 100 mg L–1 carbenicillin was effective in inhibiting Agrobacterium growth , which was required to remove residual m growth . In the Agrobacterium-mediated plant transformation  was used for transformation. We found that up to 80% of the calluses were surrounded by Agrobacterium after 3 days of co-cultivation, and that most of the calluses grew well after 1 or 2 days , was also optimized to improve the frequency of transformation. r 2 days . TherefoC. equisetifolia.To confirm the positive transgenic plants, hygromycin resistant tissues in different stages, such as callus, bud, root, and seedling, were stained using a GUS buffer solution. A strong GUS staining signal was detected in different tissues of transgenic plants, but not in those of control plants . FurtherAgrobacterium (GV3101) carrying the CaMV 35S:GUS binary vector at OD600 ≈0.8 for 2 h and co-cultivated in a co-cultivation medium for 2 days. Next, the infected calluses were transferred to a medium containing 0.5 mg L–1 6-BA, 0.1 mg L–1 TDZ, 250 mg L–1 Car, and 5 mg L–1 Hyg for resistant callus proliferation. After 2–4 weeks, a selection medium with 0.5 mg L–1 6-BA, 250 mg L–1 Car, and 5 mg L−1 Hyg was used for adventitious bud growth, which may last for 1–2 month. Afterward, buds with more than 1-cm length were shifted to the rooting medium supplemented with 0.02 mg L–1 IBA, 0.4 mg L–1 IAA, 250 mg L–1 Car, and 5 mg L–1 Hyg. Roots appeared in about 1–2 weeks after shifting the buds to the rooting medium. For better growth, the seedlings were planted into the basal medium supplemented with 0.5 g L–1 activated carbon, 5 mg L–1 Hyg and 250 mg L–1 Car to improve root growth  and transformation (95.9%) than strain C58C1 (0%). The schematic diagram of the transgenic system for C. equisetifolia by A. rhizogenes-mediated transformation is shown in C. equisetifolia seedlings of 5–8 cm height were scratched and infected using an Agrobacterium suspension  method, it took about 4.5 months in total. On the other hand, plant transformation using the Agrobacterium rhizogenes-mediated (K599) hairy root development system took around 45 days to generate transgenic plants, which fairly shortened the time to produce transgenic plants and saved time for studying tree biology at the molecular level.Due to its potential for revegetation under arid conditions, equenced . Therefoessfully , differeessfully . In thisC. equisetifolia, different kinds of auxin or cytokinin, such as NAA, IAA, IBA, 6-BA, TDZ, and KT, and various combinations of those were used in the basal medium. In this study, we found that cytokinin TDZ was more effective because of its higher callus induction efficiency  (data not shown). Co-culture duration is also an important factor altering transformation efficiency by influencing Agrobacterium-plant cell interactions, and transformation efficiency for different species benefit from different co-cultivation (Following genetic transformation with the strain GV3101 harboring vector pCAMBIA1301, several factors were evaluated and optimized in our study. In previous studies, 200–250 mg/L cef has been used . Here, 2tivation . In thistivation .vir gene expression and promotes the efficiency of plant genetic transformation (Agrobacterium suspension and co-cultivation medium obviously improved the rate of callus and bud induction (C. equisetifolia. Thus, it is important to shorten the time required for the development of C. equisetifolia transgenic plants. A. rhizogenes-mediated root transformation not only saves time for transgenic plant production, but also provides an efficient way to assess gene function. As each transgenic root originates from a single cell in A. rhizogenes-mediated root transformation, a great number of transformants can be obtained (Agrobacterium strain is one of the factors that influence the process of T-DNA delivery from Agrobacterium into plant cells (C. equisetifolia plants in a short time.Acetosyringone, a phenolic compound, induces ormation . Our resnduction . The devobtained  and analobtained . The Agrnt cells . Again, nt cells . MoreoveC. equisetifolia. Because of higher efficiency and simple propagation, these transformation systems can be used to produce C. equisetifolia transgenic lines with a variety of genes, which may be conductive to study its unique traits, especially tolerance to drought, salinity, and saline-alkali. Moreover, the establishment of these systems could make it possible to improve its cold stress tolerance by genetic engineering.In summary, we established and optimized the protocol for regeneration and two different systems for genetic transformation of The original contributions presented in the study are included in the article/HR: conceptualization, methodology, formal analysis, investigation, writing—original draft, review and editing, and funding acquisition. YX: methodology, formal analysis, investigation, and writing—review and editing. XZ: methodology, formal analysis, and investigation. YZ: methodology and formal analysis. JH: writing—review and editing. FC: investigation. GQ and SL: conceptualization, writing—review and editing, and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


BCL9L, is an oncogene and interacts with m6A patterns in the Wnt signaling pathway. In conclusion, m6A regulators and their interactive genes impact the outcome of various cancers. Evaluating the m6A subtype and the signature of individual tumors may inform the design of adjuvant treatments.N6-Methyladenosine (m6A) is an RNA modification that interacts with numerous coding and non-coding RNAs and plays important roles in the development of cancers. Nonetheless, the clinical impacts of m6A interactive genes on these cancers largely remain unclear since most studies focus only on a single cancer type. We comprehensively evaluated m6A modification patterns, including 23 m6A regulators and 83 interactive coding and non-coding RNAs among 9,804 pan-cancer samples. We used clustering analysis to identify m6A subtypes and constructed the m6A signature based on an unsupervised approach. We used the signatures to identify potential m6A modification targets across the genome. The prognostic value of one target was further validated in 3,444 samples from six external datasets. We developed three distinct m6A modification subtypes with different tumor microenvironment cell infiltration degrees: immunological, intermediate, and tumor proliferative. They were significantly associated with overall survival in 24 of 27 cancer types. Our constructed individual-level m6A signature was associated with survival, tumor mutation burden, and classical pathways. With the signature, we identified 114 novel genes as potential m6A targets. The gene shared most commonly between cancer types, The online version contains supplementary material available at 10.1186/s12943-021-01362-2. N6-Methyladenosine (m6A) is a eukaryotic mRNA modification that modulates gene expression , 2 and ar > 0.3)   Fig. b. In the.3) Fig. c.Fig. 1ASB2, P2RX6, AXL, ID2, and SOCS2. Four miRNAs including miR-143, miR-29a, miR-125b-1, and miR-145 showed lower expression in tumor tissues. All of the lncRNAs had higher expression in tumor tissues. Using the first two principal components (PCs) from tumor and normal tissues, we found that the m6A patterns had good differential diagnostic value , PTEN (5.7%), NOTCH1 (3.6%), CTNNB1 (3.6%), XIST (3.4%), and MALAT1 (3.4%). We also observed a relatively high mutation frequency in uterine corpus endometrial carcinoma (UCEC) (7.6%).The somatic mutation status of these genes is shown in Fig. Ptrend < 0.05 in Cox proportional-hazards model)  , including TP53, NOTCH1, CTNNB1, and PTEN  within each cancer type, separately. Three subtypes were identified by the Elbow method Figures -S5. The el) Fig. a. In addel) Fig. b or diseel) Fig. c. In theFig. P <  × 10− 16N Figure .Fig. 2aIn the analysis of single sample gene set enrichment analysis (ssGSEA) of tumor microenvironment (TME) cell infiltration, the beta coefficients (95% CIs) of clusters 2 and 3 were shown in Fig. P = 8.66 × 10− 44)   or high tumor grade (P = 8.16 × 10− 23)   Fig. g. Higher) Figure . Higher r = 0.53 in the overall population  with the TMB scores in 16 cancer types  score in the overall pan-cancer population. Unsurprisingly, they had a strong positive correlation with Pearson n Figure A. The m6s Figure B.We applied the ssGSEA to compute the enrichment score of 2236 canonical pathways across the genome. A total of 949 pathways (42.4%) were significantly associated with the m6A signature after FDR corrections, indicating that m6A modifications are linked to a broad range of biological processes Table , with coPFDR < 0.05   Table .BCL9L . BCL9L was significantly up-regulated in tumor tissues in 10 cancer types  and m6A subtypes (Ptrend = 2.81 × 10− 66) in the overall population  , gastric cancer , breast cancer , liver cancer , and ovarian cancer  and a protein dataset of breast cancer    that also participate in the Wnt signaling progress, BCL9L had strong correlations with MYC (r = 0.34) and CTNNB1 (r = 0.36)  Figure . BCL9L i) Figure .PFDR < 0.05, indicating they played important roles in cancers  were associated with survival in the meta-analysis with rs Table .In summary, this study demonstrates that m6A regulators and interactive genes may play an important role in cancer outcomes. Our systemic evaluation of m6A patterns improves the understanding of the dysregulation of RNA methylation in tumor microenvironments. The predicted interactive target genes may provide additional insight into clinical therapeutic targets.Additional file 1. Supplementary materials and methods, and supplementary Table S1-S7, Figure S1-S15. Table S1. Summary of genes that interact with m6A modification. Table S2. Top 200 canonical pathways associated with the m6A signature. Table S3. Potential m6A modification targets associated with the m6A signature. Table S4. Independent validation of the potential m6A targets. Table S5. Study characteristics of the pan-cancer datasets. Table S6. Sample size of tumor-normal pairs in each cancer type. Table S7. Gene list used to generate the m6A subtypes in each cancer. Figure S1. Heatmap of the fold change (FC) values in pan-cancer. Figure S2. PCA plots of m6A related genes in tumors and adjacent normal tissues. Figure S3. Receiver-operating characteristic curves of predicted performance to discriminate from tumor and normal tissues. Figure S4. Average cost of different clusters using the Elbow method. Figure S5. Distribution of the m6A subtype across each cancer type. Figure S6. Kaplan-Meier plots of m6A subtypes and overall survival in pan-cancer. Figure S7. Associations between somatic mutations and m6A subtypes. Figure S8. Heatmap of the genes associated with overall survival in at least five cancer types and used to generate an m6A signature. Figure S9. Distribution of m6A signature in low-grade (grade 1&2) and high-grade (grade 3&4) patients. Figure S10. Associations between tumor mutation burden score and m6A signature in pan-cancer. Figure S11. The top 20 biological pathways that are associated with the m6A signature. Figure S12. Differential comparison of BCL9L in TCGA tumor and adjacent-normal tissues. Figure S13. BCL9L and survival outcome in pan-cancer. Figure S14. External validation of BCL9L prognostic value in public GEO datasets. Figure S15. Correlations between BCL9L and m6A interactive genes in the Wnt signaling pathway. (DOCX 7598kb)


Essential amino acid quantification indicated that threonine, leucine and phenylalanine were the most abundant amino acids found in the investigated infant formulas. In conclusion, infant formulas are still unconforming to nutritional breast milk quality and must be improved in order to follow current global health authority guidelines.Infant formulas, designed to provide similar nutritional composition and performance to human milk, are recommended when breastfeeding is not enough to provide for the nutritional needs of children under 12 months of age. In this context, the present study aimed to assess the protein quality and essential amino acid content of both starting (phase 1) and follow-up (phase 2) formulas from different manufacturers. The chemical amino acid score and protein digestibility corrected by the amino acid score were calculated. The determined protein contents in most formulas were above the maximum limit recommended by FAO and WHO guidelines and at odds with the protein contents declared in the label. All infant formulas contained lactoferrin (0.06 to 0.44 g·100 g Infant nutrition during the first two years of life is essential for adequate development and crucial for survival and long-term health and well-being. Nourishment deficiencies caused by inadequate nutrition during this development stage may cause immediate damage, increasing infant morbidity and mortality, while also potentiating growth delays and low school achievements and increasing the risks for chronic and degenerative adulthood diseases ,2. In thIFs are available in both liquid or powdered form and contain proteins isolated from cow milk or other the milk of animal species, as well as from vegetables, in both intact or hydrolyzed forms, although other nutrients may be added in the amounts and ratios recommended for each child age to ensure adequate growth, optimal development and immune and metabolic system maturation ,8,9. ResIFs formulated from cow milk are produced at low-costs and widely marketed, and processed to adjust macro and micronutrient contents to obtain nutritional characteristics as similar as possible to human breast milk, which is safer for neonates or infants under one year old . Cow milDespite the attempts of manufacturers to mimic the composition and/or performance of human milk, it is not yet possible to state that IFs nutrients display the same bioavailability as human milk components since differences between the development and long-term health of infants fed IFs compared to exclusively breastfed individuals are still significant . FurtherIFs were purchased in commercial establishments located in the metropolitan region of the municipality of Rio de Janeiro, Brazil. All selected IFs are marketed in several commercial establishments, conveniently available to be purchased by consumers in the most populated part of the city. Although no formal inquiry was performed, as the selected formulas are commonly supplied in the largest supermarkets in town, they are considered a high demand by consumers and were, thus, evaluated in the present study.All products are registered by the Brazilian regulatory agency ANVISA, marketed as powdered formulas packed in aluminum cans and labeled by each manufacturer. Inclusion criteria consisted of the following: formulas should be prepared exclusively from non-hydrolyzed cow milk proteins, for children aged 0 to 6 months (phase 1 starting IFs) and for children aged 6 to 12 months (phase 2 follow-up IFs). IFs produced using protein sources other than cow milk, such as soy or wheat protein, as well those designed for specific needs, such as lactose-free or hydrolyzed protein conditions, were not selected. A total of ten formulas marketed in Brazil produced by three different manufacturers filled the inclusion criteria. Thus, five phase 1 and five phase 2 formulas, and three distinct batches of each brand were thus selected, totaling thirty samples (N = 30). The three different batches of each brand were identified using the same two capital letters and a number, where the number indicates different batches, from 1 to 3. Manufacturers and brands were not disclosed for ethical reasons, and samples were identified by codes Total protein contents were assessed according to the Kjeldahl method proposed by the Association of Official Analytical Chemists . Nitroge® at 595 nm and 450 nm using a UV-1800 spectrophotometer . After establishing the appropriate dilution factor, the samples were diluted in the staining solution  and a total of 20 μL of each sample (5 mg·mL−1 of protein) were loaded in each well of 4% and 12% stacking and resolution acrylamide gels, respectively, and the runs were performed at 200 V and 25 mA for about 2 h. Apparent molecular weights were estimated using electrophoresis molecular marker weights  applied to the central well. After the runs, the gels were stained with Coomassie Blue G-250 for 24 h, destained with 20% acetic acid, 20% methanol, and 60% distilled water to visualize the protein bands and photo documented using a Gel Doc XR + Gel Documentation System. The relative abundance of the gel bands was estimated by TotalLab Quant® software , through staining intensity and thickness.A protein electrophoretic analysis was carried out in six stages, namely extraction, quantification, preparation and SDS-PAGE resolving followed by gel staining and destaining, and, finally, photo documentation . Protein® water  and 500 μL were mixed to 500 μL of a solution containing 0.1 M bi-tris-HCL/buffer (pH 6.8), 6 M guanidine hydrochloride, 5.37 mM sodium citrate and 19.5 mM d-dithiothreitol (pH 7.0). The resulting suspensions were incubated for 1 h at room temperature and then centrifuged for 20 min at 20,000× g at 4 °C. The small lipid layer formed during this step was removed, and the remaining solubilized solution was diluted 1: 3 (v:v) with 4.5 M guanidine hydrochloride and solvent A, and maintained at −20 °C until analysis.The identification and quantification of the main target proteins in the investigated IFs, α-lactalbumin (α-LA), β-lactoglobulin (β-LG), Κ-casein (Κ-CN), α-casein (α-CN), β-casein (β-CN) and lactoferrin (Lf), were performed by HPLC, following protein extraction according to Bobe et al. . Briefly®, KYO, JPN), coupled to an analytical column C18 , and a photodiode array detector model SPD-20A (Shimadzu®) managed by the LabSolutions System software (Shimadzu®), employed in two distinct chromatographic conditions: (i) Κ-CN, α-CN, α-La, β-CN and β-Lg separation and identification according to Bonfatti et al. [−1 using a 0.1% trifluoroacetic acid (TFA) in water, and 0.1% TFA in acetonitrile mixture. An injection volume of 20 µL was applied, the total analysis time for each sample was 45 min and the column temperature was maintained at 45 °C; and (ii) lactoferrin separation and identification as described by Duchén et al. [−1 and the previously described mobile phase. An injection volume of 10 µL was applied, the total analysis time for each sample was 25 min and the column temperature was set at 45 °C.The HPLC system comprised a quaternary pump LC-20AD  ratio. Calibration curves were constructed by plotting increasing protein standard concentrations, and all samples and standards were run in triplicate.® water (Merck Millipore) and 10 mL of each sample were mixed with 10 mL of 6N HCl and heated in an oven at 110 °C for 24 h. Hydrolyzed samples were then transferred to 50 mL volumetric flasks, made up with Milli-Q® water and triplicate aliquots were filtered through non-sterile 0.22 μm MF-Millipore® hydrophilic membranes . The filtered solutions were maintained at −20 °C until analysis.Nine essential amino acids threonine, lysine, histidine, valine, methionine, isoleucine, leucine, phenylalanine and tryptophan were identified and quantified in the IFs samples according to Furota et al. , with moTM UltiMateTM 3000 RSLC nano System  liquid chromatograph equipped with a Corona Ultra Charged Aerosol Detector (CAD)  was used. Orthogonal chromatography was performed using cyan columns  connected to a C8 column . Data were acquired with the Chromeleon 7.2 software (Thermo Fisher Scientific®).A Thermo Scientific−1 flow rate using A 0.1% formic acid (FOA) in water and solvent B 100% acetonitrile (ACN) mixture. The gradient elution (solvent B) was increased from 0% to 25% from 0 to 30 min; 25% to 70% from 31 to 46 min and 70% to 0% from 47 to 60 min. A 20 µL injection volume was applied, the column temperature was maintained at 30 °C and the total analysis time for each sample was 60 min. Amino acid standards were purchased from Sigma  ranging from 50 to 63 mg·100 mL−1.Essential amino acids were eluted at a 0.5 mL·minAn in vitro gastrointestinal digestion mimicking physiological conditions during the gastric and intestinal phases was performed according to Oomen et al.  and Sagr® water and 2.5 mL of artificial gastric fluid, containing 2.75 g NaCl; 0.27 g NaH2PO4; 0.82 g KCl; 0.42 g CaCl2; 0.31 g NH4Cl; 0.65 g glucose; 0.085 g urea; 3 g mucine; 2.64 g swine gastric pepsin; 1 g bovine albumin and 8.3 mL HCl. After adjusting the volume to 500 mL, the pH was adjusted to 2.0 with 5 M HCl, the vials were sealed with a rubber septum and the atmosphere was charged with N2. The vials were then transferred to an orbital shaker at 260 rpm and 37 °C for 2 h to complete the gastric phase digestion (GPD), followed by the removal of 5 mL aliquots for further analysis.In a glass vial, 5 g of each sample were mixed with 2 mL of Milli-Q3 were added to the GPD mixture to adjust the pH to 6.0, followed by 2 mL of an artificial intestinal fluid containing 6.75 g NaCl; 0.517 g KCl; 0.205 g CaCl2; 3.99 g NaHCO3; 0.06 g KH2PO4; 0.0375 g MgCl2; 0.1375 g urea; 25 g swine bile; 4 g swine pancreatin; 1.2 g albumin bovine, 0.185 mL HCl and volume adjustment to 500 mL. The glass vials were then resealed under an N2 atmosphere and maintained in the same conditions as the GDP followed by the collection of 5 mL aliquots for further analysis.In this stage, 0.9 M NaHCOThe supernatants collected at the end of representative in vitro digestions  were analyzed regarding nitrogen content employing the Kjeldahl AOAC 930.29 method . The IVPTo calculate the AAS, the essential amino acid concentrations in the IFs were estimated and compared with daily amino acid infant requirements using the amino acid content in breast milk proteins as reference to define amino acid scores for infant foods . The PDCF was found. Results were considered significant when p < 0.05. Data were expressed as the means ± standard deviations (SD). All statistical analyses were carried out using the Graphpad Prism software version 5 for Windows® . All measures were acquired in triplicate.Significant differences in IFs protein contents were assessed through a One-way analysis of variance (ANOVA) with repeated measures. Differences in protein fractions and amino acids between IFs and their respective batches were evaluated by a two-way analysis of variance (ANOVA) with repeated measures. Differences in phase 1 and phase 2 IFs IVPD, AAS and PDCAAS were estimated by a one-way analysis of variance (ANOVA) with repeated measures. An additional post hoc analysis (Bonferroni correction) was performed when a significant −1, higher than all phase 1 and phase 2 IFs. Phase 2 formulas, termed ME2, DM2 and DA2, contained the highest total protein levels, 22.64 ± 0.56 g·100 g−1, 19.95 ± 0.71 g·100 g−1, 19.33 ± 1.20 g·100 g−1, respectively, when compared to the remaining phase 2 and all phase 1 formulas. NC1, NN1 and ME1 contained the lowest total protein contents, of 9.67 ± 0.03 g·100 g−1, 11.94 ± 0.61 g·100 g−1 and 11.81 ± 0.35 g·100 g−1, respectively, compared to the other IFs (p < 0.05).The SDS-PAGE profiles presented in ® software, the relative percentage of the protein fractions identified in the analyzed gels, they exhibited different patterns among IFs brands and between batches , β-CN (57.95 ± 0.50 mg·g−1), κ-CN (125.14 ± 0.39 mg·g−1) and β-Lg (56.62 ± 0.41 mg·g−1) contents compared to all IFs, but the CM the lowest Lf (0.31 ± 0.02 mg·g−1) compared to phase 1 and phase 2 IFs.Whole powdered bovine milk samples were analyzed to compare phase 1 and phase 2 IFs CM protein fractions. CM contained the highest α-CN (43.26 ± 0.30 mg·gp < 0.001) between phase 1 and phase 2 formula brands was noted concerning the average value of each protein fraction. Phase 1 mean values ranged from 86.07 to 129.85 mg·g−1 for κ-CN, from 7.51 to 22.83 mg·g−1 for α-CN, from 0.31 to 13.76 mg·g−1 for α-La, from 7.54 to 29.19 mg·g−1 for β-CN, from 13.25 to 41.94 mg·g−1 for β-Lg and from 0.22 to 1.51 mg·g−1 for Lf, while, the means of phase 2 IFs ranged from 82.97 to 129.59 mg·g−1 for κ-CN, from 7.09 to 19.22 mg·g−1 for α-CN, from 0.51 to 12.57 mg·g−1 for α-La, from 4.72 to 33.10 mg·g−1 for β-CN, from 15.59 to 36.32 mg·g−1 for β-Lg and from 0.28 to 8.55 mg·g−1 for Lf. Thus, κ-CN was the most abundant protein, and α-La and Lf, the less abundant ones.Variation of major protein fractions between the three batches of the same phase 1 and phase 2 IFs brands. −1 and tryptophan, from 0.10 to 0.14 mg·g−1, and similar contents for both amino acids were observed in both phase 1 and phase 2 samples. DM1, however, presented the highest valine contents, of 1.34 ± 0.02 mg·g−1, as well as me threonine, 1.99 ± 0.01 mg·g−1; isoleucine, 1.69 ± 0.01 mg·g−1 and phenylalanine, 3.24 ± 0.01 mg·g−1, when compared to other phase 1 IFs. DA1 and DM1 presented the highest threonine and leucine contents, of 4.34 ± 0.01 and 4.19 ± 0.02 mg·g−1 and 4.14 ± 0.02 and 3.96 ± 0.02 mg·g−1, respectively, when compared to other phase 1 IFs. Furthermore, the highest lysine contents were found in NN1, NC1 and ME1, of 0.26 ± 0.03, 0.20 ± 0.03 and 0.20 ± 0.04 mg·g−1, respectively. Concerning phase 2 formulas, DM2 contained the highest threonine content, 4.70 ± 0.02 mg·g−1, as well as methionine, 2.13 ± 0.02 mg·g−1; isoleucine, 1.99 ± 0.01 mg·g−1; leucine, 4.56 ± 0.01 mg·g−1 and phenylalanine 3.28 ± 0.02 mg·g−1 compared to other phase 2 IFs. The highest lysine contents were observed in DM2 and NC2, 0.19 ± 0.01 and 0.19 ± 0.01 mg·g−1, respectively, and the highest valine values, of 1.29 ± 0.03, 1.27 ± 0.01 and 1.25 ± 0.01 mg·g−1 were detected in NC2, NN2 and DM2, respectively.The average amino acid content in phase 1 and phase 2 IFs are presented in When comparing phase 1 vs. phase 2 IFs amino acid contents, a significant difference in phase 1 lysine, valine and isoleucine contents was observed, higher in phase 1 compared to phase 2. On the other hand, higher threonine, methionine, leucine and phenylalanine were detected in phase 2 IFs. Finally, histidine and tryptophan contents were similar in both types of formula .Phase 1 and phase 2 IFs %IVPD are displayed in −1, 1.019 ± 0.010 mg·g−1, 1.008 ± 0.007 mg·g−1 and 1.169 ± 0.002 mg·g−1, respectively. Almost all amino acids presented higher AAS values in phase 1 compared to phase 2 IFs (p < 0.01).Chemical amino acid scores (AAS) were calculated as recommended by FAO/WHO (2007), considering the ratio between essential amino acid contents and those recommended for children aged 0 to 12 months old, used to identify infant formulas that could limit essential infant amino acid supplies . The AASp < 0.01).Protein digestibility values are displayed in −1, varying throughout the lactation stage, while whole cow milk contains about 3.33 g·100 mL−1, about three-fold higher than human milk [−1 and a maximum of 3.0 g·100 kcal−1, while protein concentrations in phase 2 formulas should range between 1.8 g·100 kcal−1 to 3.5 g·100 kcal−1 [−1, not exceeding the maximum limit [IFs aimed for children under the age of one are formulated from cow milk or from the milk of other animal species or their mixture, and/or eventually, other ingredients suitable for infant feeding . Herein,man milk ,25,26. T0 kcal−1 ,28. On tum limit ,23.−1), our findings indicate that ME1C, DM1A and DM1B phase 1 IFs contained protein contents above recommendations, but not below the minimum limit. In addition, all phase 2 IFs contained protein concentrations within the limits established by Brazilian current legislation. However, when considering the limits established by the Codex Alimentarius, more than one batch of each brand of some phase 1 and phase 2 IFs exceeded the maximum protein recommendation of 3.0 g·100 kcal−1, namely ME1C, DM1A, DM1B phase 1 IFs and ME2B, ME2C, NC2C, NN2B, DM2A, DM2C, DA2A and DA2C phase 2 IFs  guidelines, the maximum protein content in IFs is established as 3.5 g·100 kcal0 kcal−1 .Nutritional information on food labels aids consumers and professionals to select a balanced diet, contributing to reducing the incidence of unhealthy conditions associated with inadequate eating habits . Non-comBreast milk or IFs are the recommended exclusive protein sources for newborns and infants up to six months of age. Thus, the quality, amounts, and conformation of IFs proteins should be strictly controlled in order to sustain adequate and safe infant growth, ensuring short and long-term health and proper development. The amount and quality of protein intake during the first two years of life have important effects on muscle protein development and linear growth, supporting optimal brain development and, consequently, better cognitive evolution, and ensuring the development of healthy and mature immune and digestive systems ,12. Prot−1 or low protein concentrations varying from 1.77 to 2.2 g·100 kcal−1 before and after the 5th month of life, respectively, were compared to a group of exclusively breastfed babies. The ingestion of high protein formulas resulted in greater weight in the first two years of life, although no growth effect was observed [A broad multi-center randomized and controlled clinical trial conducted in several countries investigated the effects of protein intake on infant growth and adiposity, where healthy infants fed cow milk-formulas containing high protein concentrations ranging from 2.9 to 4.4 g·100 kcalobserved . Weight observed ,36,37. Fobserved .−1), DM1A  and DM1B  IFs investigated herein, containing the maximum recommended protein content by children under the age of one may increase the risk for obesity. Protein IFs content should preferentially be kept closer to the minimum limit, in order to manage childhood risk for overweight and obesity.Considering protein IFs content and the Koletzko trials , the conPoor nutrition during pregnancy is related to low nephron endowment, which may comprise a potential driver for hypertension and renal disease later in life . PrematuEnsuring nutrient consistency and standardization in distinct batches of the same brand of IFs should be a major goal concerning the nutritional quality of these formulas, as these formulas may comprise the exclusive food source offered to some infants for over six months, and marketing IFs with unrecommended nutrient composition/concentration can lead to inadequate child development. In the present study, consistency among batches from the same manufacturer was observed for DM1/DM2, DA1/DA2, ME2, NC2 and NN2, while brands ME1, NC1 and NN1, exhibited a non-uniform composition with statistical protein content differences in at least one of the three tested lots . This mas1-CN, αs2-CN, β-CN and κ-CN, while whey proteins, i.e., β-Lg, α-La, Lf, lysozyme, bovine serum albumin, immunoglobulins (Igs) and other smaller soluble proteins, present in colloidal form and in their glycosylated forms, compose the MFGM [In general, mammal milk contains insoluble proteins and soluble whey proteins, which include smaller floating proteins associated with milk fat globule membranes (MFGM). Caseins comprise the majority of insoluble milk proteins and are found in micellar form as αthe MFGM ,45,46,47the MFGM . Caseinsthe MFGM . In addithe MFGM ,50. Heres1-CN, β-CN κ-CN and γ-CN, except for the αs2-CN variant, and main caseins correspond to β-CN, a highly phosphorylated protein, which aids in calcium absorption and contributes to zinc absorption. CM, however, contains main casein subunits αs1-CN, αs2-CN, β-CN, κ-CN and γ-CN, with higher concentrations of α-CN and κ-CN [Whey or casein CM protein profiles differ from human milk. Human milk contains most protein subunits, such as αand κ-CN ,52,53,54and κ-CN ,56. Lf cand κ-CN ,58. IFs The HPLC analyses carried out herein, an analytical method more sensitive and accurate than the SDS-PAGE technique, demonstrated that casein IFs contribution to total protein contents was higher compared to whey proteins. κ-CN was detected by HPLC in higher concentrations compared to SDS-PAGE, where it was apparently the least abundant protein. κ-CN may undergo thiol disulfide linkages with proteins containing disulfide bonds, such as α-La, β-Lg, BSA, IGs and Lf, when subjected to high temperatures, resulting in ҡ-CN aggregates that move slowly or remain stacked in the stacking gel during the electrophoresis run, altering correct concentration estimations . ConsequSDS-PAGE can simultaneously identify caseins and whey proteins, although information obtained following IFs sample SDS-PAGE seems to be less accurate compared to urea-PAGE or native-PAGE for caseins and whey proteins, respectively. Caseins display similar molecular weights, differing only slightly from each other, resulting in inadequate SDS-PAG discrimination . ReverseThe protein concentrations described herein indicate significant differences between IFs brands and batches. Cow milk differences can be explained by protein milk content and the relative concentrations of individual proteins present during the lactation period, cattle feeding or formula manufacturing, which can spoil heat-sensitive nutrients ,65. WheyThe supply of essential amino acids in the first month of a child’s life is provided by the set of proteins found in breast milk and/or IFs that must meet physiological newborn needs . Whey pr−1) reveal that most of the investigated IFs contain amino acid concentrations under established guidelines, except for threonine in ME1, ME2, DM1, DM2, DA1 and DA2, which were above 77 mg·kcal−1 , and methionine in NC1, NC2, NN1, NN2, DM1, DM2 and DA1, detected at 24 mg·kcal−1, higher than the reference limit. It is important to note that a lower supply of essential amino acids and non-compliance to recommendations in the starting IFs (phase 1) can be more harmful when compared to the follow-up IFs (phase 2), as IF phase 1 is the only feeding recommended in the first six months of age when breast milk is not available. Unlike the starting IFs, follow-up IFs are offered to complement the introduction of new proteins from vegetable and animal origins. Therefore, even if the follow-up IFs are below the recommended intake, other protein sources can compensate and fulfill recommended essential amino acids concentrations.Recommendation of essential and semi-essential amino acid contents per 100 kcal of IFs have been established considering breast milk proteins as reference ,28. The Most essential amino acids were detected in all IFs, although at lower concentrations compared to established guidelines, probably due to reduced concentration of α-La and Lf whey proteins, the best source of essential amino acids, as indicated by the HPLC analysis . EssentiEssential and semi-essential amino acids supplies, digestibility, absorption and transport through the gastrointestinal tract influence infant growth, and are important for cell and tissue maintenance . The absAssessing the protein quality of foodstuffs is important when considering nutritional infant benefits. In this regard, the AAS and PDCAAS are commonly employed to assess the protein quality of food matrices . The AASAccording to IFs composition protocols, essential and semi-essential amino acids can be added to improve formula protein quality, but in amounts just enough to fill this purpose ,28. The It is important to note that studies concerning the amino acid scores of CM-based IFs are scarce. In the present study, protein composition and essential amino acid ratios were variable, and total protein contents reported on the investigated IFs labels are not adequate and do not reflect IFs quality, making it difficult for pediatricians and nutritionists to accurately compare between available commercial products.Despite attempts to mimic the composition or performance of breast milk by IFs manufacturers, proteins present in IFs cannot be proven as exhibiting the same bioavailability as those found in human milk. All formulas displayed adequate protein bioaccessibility, with proteins almost entirely digested in the intestine, generating free amino acid, dipeptides and tripeptides available for absorption. Only some IFs brands exhibited total protein content in accordance with product labels. Total protein content varied between batches from the same manufacturer, which may result in negative effects in children that consume the same brand regularly over six months. In addition, most IFs displayed protein contents above the maximum limits established by Brazilian regulation, as well as the European Commission Directive 2006/141/EC and Codex Alimentarius (FAO and WHO).Casein contents were higher than whey proteins in all IFs, presenting three different subunits and with K-CN as the most abundant. Concerning whey protein contents, Lf amounts were lower than in human milk, jeopardizing newborn defenses, as this protein exhibits strong antimicrobial activity. High β-Lg concentrations comprised a non-desirable component, as this protein, which is not present in human breast milk, can induce allergy in susceptible individuals.Therefore, despite the advances noted in technological IFs processing in recent years leading to safer and more adequate IFs for children under the age of one, the investigated brands still displayed marked nutritional quality differences when compared to human breast milk, requiring continuous improvements to follow current international guidelines due to the major health effects associated to poor nutrition or obesity.


Abdominal aortic aneurysm (AAA) involves the degradation of vascular fibres, and dilation and rupture of the abdominal aorta. Hypoperfusion in the vascular walls due to stenosis of the vasa vasorum is reportedly a cause of AAA onset and involves the induction of adventitial ectopic adipocytes. Recent studies have reported that ectopic adipocytes are associated with AAA rupture in both human and hypoperfusion-induced animal models, highlighting the pathological importance of hypoperfusion and adipocytes in AAA. However, the relationship between hypoperfusion and AAA remains unknown. In this study, we investigated the changes in inflammation-related factors in adipocytes at low glucose and serum levels. Low glucose and serum levels enhanced the production of AAA-related factors in 3T3-L1 cells. Low glucose and serum levels increased the activation of protein kinase B , extracellular signal-regulated protein kinase 1/2, p38, c-Jun N-terminal kinase, and nuclear factor (NF) кB at the protein level. The inflammatory factors and related signalling pathways were markedly decreased following the return of the cells to normal culture conditions. These data suggest that low glucose and serum levels increase the levels of inflammatory factors through the activation of Akt, mitogen activated protein kinase, and NF-κB signalling pathways. The pathophysiology of abdominal aortic aneurysm (AAA) is characterized by the degradation of the vascular structure due to chronic inflammation and aortic dilation . AAA is VV penetrates the outer layer of the tunica media of larger vessels and supplies blood and nutrition. VV stenosis causes low nutritional supply to the adventitia and outer media of the parent vessel. Under healthy conditions, the adipocytes function normally . AdiposeTo determine the effect of low glucose and serum levels on cell viability, we cultured 3T3-L1 cells in different culture media: control (CT), 4.5 g/L glucose and 10% foetal bovine serum (FBS); Low 1, 1.0 g/L glucose and 10% FBS; Low 2, 1.0 g/L glucose and 5% FBS; Low 3, 1.0 g/L glucose and 1% FBS; Low 4, 4.5 g/L glucose and 1% FBS; Low 5, 4.5 g/L glucose and 0% FBS. The viability of 3T3-L1 cells without induction of differentiation was significantly decreased in Low 3, −4, and −5 conditions compared to that in CT medium ). There were very few viable cells in Low 5 condition; therefore, this condition was eliminated from the subsequent experiments. Next, we investigated the effect of different culture media on the viability of cells following induction of differentiation, as shown in . Cell viNext, we evaluated the effect of low glucose and serum levels on the generation of ROS and NO, and expression of iNOS. Intracellular ROS production by 3T3-L1 cells increased significantly to 128.31 ± 1.70% under Low 3 condition compared to that in CT condition on day 4, whereas it was significantly suppressed in Low 3/CT conditions on day 9 ). ComparNext, we assessed the effect of low glucose and serum levels on TNF-α and IL-1β secretion by 3T3-L1 cells. Compared to the CT condition, levels of TNF-α and IL-1β secreted in the culture media were significantly increased under Low 3 conditions on day 4 . CompareTo examine the effects of low glucose and serum levels on the expression of MMP-2 and MMP-9, mRNA and protein levels of MMP-2 and MMP-9 were determined by real-time PCR and western blotting, respectively . MMP-2 mThe activity of MMPs in the conditioned medium was determined using gelatin zymography . The actNext we examined whether MAPK activation was involved in the oxidative stress and inflammation induced by low glucose and serum levels. Low glucose and serum levels significantly stimulated p38, ERK1/2, and JNK phosphorylation compared to CT in 3T3-L1 cells after 4 days . MoreoveThe expression of pAkt/Akt was significantly increased in cells under low glucose and serum levels compared to that under CT condition, and the phosphorylation of Akt was downregulated in Low 3/CT condition compared to that in Low 3 condition . NF-κB aA previous study found that hypoperfusion of adventitial VV due to stenosis causes the onset of AAA by inducing ectopic adipocytes in the AAA wall ,5. AbnorOxidative stress is associated with multiple mechanisms in AAA, including vascular inflammation and increased metalloproteinase activity . ROS andPrevious studies have shown that ROS induces the expression of matrix-degrading enzymes, such as MMP-2 and MMP-9, and proinflammatory cytokines such as TNF-α and IL-1β ,24. In aNF-кB is closely associated with the regulation of inflammation and immune responses in the physiological or pathophysiological processes of AAA . SeveralIncreased levels of phosphorylated ERK and JNK have been observed in both human and rodent AAA tissues . MAPKs aRecently, Akt signalling was reported to be increased during AAA development, and suppression of Akt inhibited AAA formation . Our resIn conclusion, glucose and serum deprivation may induce inflammation in adipose tissue through the activation of MAPKs, Akt, and NF-кB signalling pathways. Nutritional supplementation may attenuate glucose- and serum deprivation-induced inflammation in the AAA wall. A limitation of this study is the lack of information concerning the interactional response of different cell populations in the AAA wall as we used only one type of cell culture to determine the effect of low glucose and serum levels on adipocytes. Further investigations are required to clarify the relationship between our experimental conditions and inflammation in hypoperfused AAA wall.2. Induction of adipocyte differentiation was induced using Adipo Inducer Reagent  containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. Cell viability assay was performed using the CellTiter96 Aqueous One Solution Cell Proliferation Assay kit .Mouse 3T3-L1 adipocytes were purchased from the American Type Culture Collection . The cells were plated in a 24-well plate and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% foetal bovine serum , 1% penicillin-streptomycin, 2 mM L-glutamic acid, 100 mM pyruvic acid, 4.5 g/L glucose, and 7.5% sodium carbonate at 37°C under an atmosphere of 5% COTo estimate the effect of nutrient stress, we cultured cells in the following condition mediums for 4 days: control, 4.5 g/L glucose and 10% FBS; Low 1, 1.0 g/L glucose and 10% FBS; Low 2, 1.0 g/L glucose and 5% FBS; Low 3, 1.0 g/L glucose and 1% FBS; Low 4, 4.5 g/L glucose and 1% FBS; Low 5, 4.5 g/L glucose and 0% FBS. To estimate the effect of restoration by returning normal nutritional condition, we cultured cells for 9 days in Low/CT groups.The oxidation of 2ʹ,7ʹ-dichlorofluorescin diacetate  was used to determine intracellular ROS production in the cells. Cells were treated with DCFH-DA (50 µM) for 1 h in the dark. The intensity of the fluorescent product, dichlorofluorescein (DCF), was determined at 485 (excitation wavelength) and 530 nm (emission wavelength) using a fluorescence microplate reader .Equal volumes of Griess reagent and culture media were mixed and incubated in the dark for 10 min at room temperature. The optical density of the nitrite-containing samples was measured using a microplate reader (Bio-Tex Instruments) at 540 nm. A standard calibration curve was generated using sodium nitrite.The level of TNF-α and IL-1β was analysed using an enzyme-linked immunosorbent assay .2. The gels stained with 0.1% amido black 10B .The MMPs were separated on a 7.5% polyacrylamide resolving gel containing 2.5 mg/mL gelatin, and the gel was incubated for 30 min at room temperature in a 2.5% Triton X-100 solution and incubated at 37°C for 18 h in 50 mM Tris–HCl buffer, pH 8.0, containing 10 mM CaClTM Kit . cDNA was synthesized using PrimeScriptTM II 1st strand cDNA Synthesis Kit (Takara Bio. Inc.). Real-time PCR was conducted using SYBR Premix Ex Taq II (Takara Bio. Inc.). Primers used for the real-time PCR experiments are shown in Table S1. The amplification conditions used were 5 s at 95°C and 30 s at 65°C for 40 cycles. β-actin served as the internal control.Total RNA was isolated using Sepasol-RNA II Super  and TURBO DNA-freeProtein extracts were boiled for 5 min in SDS sample buffer, separated using SDS-PAGE, and transferred onto polyvinylidene difluoride membranes . The membranes were probed with the following primary antibodies: rabbit anti-MMP-2 , mouse anti-MMP-9 , Akt , pAkt , p65 NF-κB , IкB-α , ERK1/2 , pERK1/2 , p38 , p-p38 , JNK , pJNK , c-Fos , c-Jun , iNOS , and mouse anti-β-actin . All immunoreactive proteins were detected using ECL™ Western Blotting Detection Reagent .P < 0.05. Statistical analyses were performed using Stat View 5.0 software .Statistical differences were determined using the Tukey-Kramer test. Differences were considered significant at Click here for additional data file.


E. coli derived recombinant human bone morphogenetic protein-2 (rhBMP-2) delivery systems . After the trial, we completed implant therapy and observed over an average of 3 years. This follow-up study was performed retrospectively to compare result of implant treatment at the preserved alveolar ridge site.We previously reported similar efficacies of alveolar ridge preservation (ARP) on single extraction socket with two differentPatients who underwent extraction of single tooth and received ARP with one of two rhBMP-2 delivery systems from October 2015 to October 2016 were enrolled. Twenty-eight patients  who underwent implant therapy and prosthetic treatment were included in study. Stability and marginal bone loss (MBL) of each implant were collected from medical charts and radiographs, and analyzed. The survival and success rates of implants were calculated.P = 0.316 and 0.185, respectively). MBL at the latest follow-up was 0.014 mm in Group 1 over 33.76 ± 14.31 months and 0.021 mm in Group 2 over 40.20 ± 9.64 months, with no significant difference (P = 0.670). In addition, the success rate of implants was 100% (14/14) in Group 1 and 92.9% (13/14) in Group 2, with survival rate of 100% (14/14) in Group 1 and 92.9% (13/14) in Group 2.The primary implant stability represented by implant stability quotient (ISQ) for Groups 1 and 2 was 69.71 and 72.86, respectively. The secondary implant stability for Groups 1 and 2 was 78.86 and 81.64, respectively. Primary and secondary stabilities were not statistically different (We confirmed good prognosis in both groups as a result of implant therapy after ARP with each of two rhBMP-2 carriers. Maintenance and preservation of alveolar width and height are important for proper dental implant placement. However, it is difficult to place implants in areas of previous tooth extraction since alveolar bone is prone to horizontal and vertical absorption , 2. TherE. coli-derived rhBMP-2 (Cowell BMP) and newly developed absorbable collagen sponge delivery system with E. coli derived rhBMP-2 (O-BMP) showed similar efficiency for the alveolar ridge preservation (ARP). [We previously compared the efficacy of two rhBMP-2 delivery systems  on alveolar bone preservation. And we concluded from the 12-week clinical trial that the β-tricalcium phosphate and hydroxyapatite particle delivery system with n (ARP). .This retrospective follow-up study was conducted to compare the outcomes of implant therapy at the preserved alveolar bone sites.This study was performed in accordance with the Helsinki guidelines and was approved by the Institutional Review Board at Seoul National University Bundang Hospital (B-2010-645-103). Patients who underwent single-tooth extraction and ARP with one of two rhBMP-2 delivery systems from October 29th, 2015 to October 6th, 2016 were chosen. Informed consent was obtained from all subjects. Among them, 28 patients whose medical records indicates implant therapy and prosthetic treatment at a preserved alveolar bone site were selected, with 14 patient in each group .Patient demographics, treated site, characteristics of the implant fixture, implant treatment procedures, primary and secondary stability values, and amount of alveolar bone loss were collected using medical chart and radiographs . If additional surgeries were performed at the time of implant placement, the type of and reason for surgery were noted.The stability of each implant was measured as implant stability quotient (ISQ) according to an Osstell Mentor . Primary stability was measured immediately after implant placement, and secondary stability was measured at the time of the second surgery or during the impression for the prosthesis.To determine the amount of marginal bone loss (MBL), radiographic analyses were performed. For measuring it, intraoral periapical radiographs produced with the paralleling technique on a digital intraoral radiographic device  and measurement system  were used. MBL was defined as the mean differences of the shortest vertical distances from the mesial and distal aspect of implant shoulder to the implant-bone contact points on the aspect measured between the radiographs taken immediately after functional loading, 1 year after loading, and final observation that exceeded 1 year after loading .P = 0.836 and 0.693, respectively)  years. Group 2 contained 6 males and 8 females with a mean age of 59.14 ± 10.77 (30–70) years. The surfaces of all implants were sandblasted, large grit, acid-etched (SLA). The distribution of implant types was 8 and 6 Dentium implants , 5 and 2 Neobiotech implants , and 1 and 6 Osstem implants  in Groups 1 and 2, respectively. The mean diameter and length of the implant fixtures were 4.89 ± 0.40 (4.5−6.0) mm and 9.21 ± 1.64 (7.0−1.5) mm in Group 1, respectively, and 4.86 ± 0.50 (4.0–6.0) mm and 9.43 ± 1.16 (7.0–10.0) mm in Group 2. There were no statistical differences in diameter and length of the implants  procedure were completed for bone height compensation in Group 1. Of the 4 GBR procedures conducted in Group 2, half were conducted for bone height and other the half for bone width compensation  (Table Primary stability (ISQ) was 69.71 ± 6.01 and 72.86 ± 9.81 for Groups 1 and 2, respectively, and secondary stability (ISQ) was 78.86 ± 5.49 and 81.64 ± 5.34, neither of which was statistically different (P = 0.210). The mean total treatment period was 14.56 ± 9.03 months for Group 1 and 9.45 ± 2.56 months for Group 2 (P = 0.052). The mean healing period was 5.65 ± 1.66 and 4.91 ± 1.88 months for Groups 1 and 2 (P = 0.286), respectively. The average observation period after implant placement was 33.76 ± 14.31 months in Group 1 and 40.20 ± 9.64 months in Group 2 . MBL was 0.013 ± 0.039 mm and 0.001 ± 0.022 mm for Groups 1 and 2, respectively, at 1 year after loading (P = 0.551), and no implant showed annual vertical bone loss greater than 0.2 mm. MBL was 0.014 ± 0.039 mm in Group 1 and 0.021 ± 0.056 mm in Group 2 at the last follow-up (P = 0.670)  in Group 1 and 92.9% (13/14) in Group 2. Only one implant in Group 2 was removed, at 31.31 months after functioning due to progressive alveolar bone loss and subsequent mobility due to peri-implantitis. The survival rate was 100% (14/14) in Group 1 and 92.9% (13/14) in Group 2.Many studies have reported the efficacy of horizontal and vertical bone volume preservation of ARP after tooth extraction –26. ThisThe ISQ quantifies implant stability as a value from 1 to 100; a value less than 45 means implant failure, while a value between 60 and 70 indicates success of the implant . Nedir eThe success rate of single-tooth implants varies according to placement protocol. Success rates of single-tooth implants over 1–2 years are reported as 66.7–92.0% for immediate placement after extraction and 83.3–100% for placement at 3–5 months after extraction –32. In tSince there was no significant difference in groups for MBL, success rate, or survival rate, we propose that adequate-quality alveolar bone was formed with implant treatment after ARP with the two rhBMP-2 delivery systems. These results can be seen as continuation and extension of our previous study showing well-preserved extraction sockets in both groups after ARP. The change of alveolar height and width at the level of the alveolar crest at 12 weeks after ARP with Cowell BMP and O-BMP were − 0.68 ± 1.42 mm and − 0.26 ± 2.58 mm, respectively . HoweverOne limitation of this study is that we used implants from three companies. However, all the implants used in this study were surface-treated with SLA, bone level implant with internal connections, and possessed the same taper type. In addition, even though we used CBCT for evaluation of changes of alveolar bone in our previous study, use of periapical radiographs for this analysis in the present study is another limitation.The outcome of implant treatment after ARP with each of two different rhBMP-2 delivery systems was favorable. Therefore, we suggest that ARP using either of these systems is appropriate pre-treatment for later implant therapy.


Kaempferia parviflora against oxidative stress and inflammation-induced damage in human dermal fibroblasts (HDFs) stimulated by tumor necrosis factor-α (TNF-α). The experimental data identified 5,7,4′ trimethoxyflavone (TMF) as the most potent constituent in preventing TNF-α-induced HDF damage among the tested compounds and it was not only effective in inhibiting matrix metalloproteinase-1 (MMP-1) production but also in stimulating collagen, type I, and alpha 1 (COLIA1) expression. TMF suppressed TNF-α-stimulated generation of ROS and pro-inflammatory mediators, such as cyclooxygenase-2 (COX-2), interleukin (IL)-1β, and IL-6 in HDFs. TMF also inhibited the pathways regulating fibroblast damage, including mitogen-activated protein kinase (MAPK), activator protein 1 (AP-1), and nuclear factor-kappa B (NF-κB). In conclusion, TMF may be a potential agent for preventing skin aging and other dermatological disorders associated with oxidative stress and inflammation.Similar to other organs, the skin undergoes a natural aging process. Moreover, constant direct exposure to environmental stresses, including ultraviolet irradiation, causes the signs of skin aging to appear rather early. Reactive oxygen species (ROS) and inflammatory responses accelerate skin damage in extrinsic aging. In this study, we aimed to investigate the skin protective effects of polymethoxyflavones found in  Aging is a natural process involving gradual changes that begin in early adulthood. Many bodily functions begin to deteriorate gradually during the early middle age, including skin structure and function. The skin is a true reflection of the changes that accumulate over time. The natural aging process (intrinsic aging) normally begins in the mid-twenties and is characterized by fine wrinkles as well as thin and transparent, sagging, and dry skin. Although these changes usually begin in the twenties, the signs of intrinsic aging are typically not visible for decades. However, recently, the signs of skin aging tend to appear earlier with more severe symptoms, such as deep wrinkles or leathery skin, as the skin is frequently exposed to extreme environmental factors, including ultraviolet (UV) radiation, smoking, pollutants, and toxins (extrinsic aging) . These eThe ECM, including elastins, collagen fibrils, and ground substance, is mainly synthesized by dermal fibroblasts located in the dermal layer of the skin, which maintains the structural integrity of most tissues . Hence, TNF-α, a pro-inflammatory cytokine secreted from keratinocytes and fibroblasts following UV, has a vital part in mediating aging and inflammatory diseases in the skin. In terms of cutaneous aging, TNF-α regulates various signaling pathway-induced production cases of collagenase as well as inhibites collagen bio-synthesis in dermal fibroblasts . In detaAdditionally, TNF-α is also considered an important mediator in inflammatory skin disorders, including psoriasis, a popular chronic inflammatory ailment marked by aberrant keratinocyte proliferation, increased cutaneous vascularity, and numerous infiltrating inflammatory cells. TNF-α provoke the production of pro-inflammatory cytokines associated with psoriatic pathogenesis, such as IL-1 and IL-6. IL-1 promotes the generation of additional cytokines as well as the expression of cytokeratin 6 (CK6), a hallmark of hyperproliferative and activated keratinocytes, while IL-6 causes T-cell proliferation and keratinocyte hyperproliferation ,6. FurthPandanus utilis and the root of Lithospermum erythrorhizon to prevent skin aging [Thus, modulation of TNF-α activity could be a potent approach for the development of new therapeutic agents in preventing skin aging and inflammatory skin diseases. Natural products represent a diverse and abundant source of antioxidant and anti-inflammation agents that are not only effective in preventing signs of age but are also safe and less irritating to the skin. Therefore, natural plants and herbs have been used in traditional practices to preserve youthfulness of the skin. For example, the Naxi people, an ethnic group that lives in Southwest China, traditionally apply an oil prepared from the seed of in aging .Zingiber officinale) has long been used as a food seasoning or as a medicinal agent for treatment [Kaempferia parviflora (KP) rhizome, also called black ginger, which has been traditionally used in Thailand and Laos in recent years, has been explored recently. K. parviflora belongs to the ginger family (Zingiberaceae) and is cultivated mainly in Thailand, Vietnam, and Laos [Ginger , 5,7,4′ trimethoxyflavone (TMF), and 5,7-dimethoxyflavone (DMF) , which aWith regard to the protective effects on skin, black ginger, together with its main constituents, has been shown to be a potential antioxidant source, which protects the skin against UVB-induced oxidative stress. Park et al.  reportedIn this study, we aimed to investigate protective effects of DMF, TMF, and PMF from KP against TNF-α-induced HDF damage via expression of MMP-1 and collagen type I alpha 1 (COLIA1), and other relevant signaling pathways.KP obtained from the Gyeongdong market  was ground to a powder form using a blender . A sample of the powder (50 g) was extracted with 500 mL of ethanol solvent at room temperature for 2 h and filtered before drying in a rotary evaporator . After evaporation of the solvent, the extract was dried overnight in a vacuum oven . The KP extract (0.1 g) was dissolved in 1 mL of methanol to constitute a 0.1 g/mL stock solution.18 column . Standard samples  were dissolved in 1 mL of methanol to make a 0.001 g/mL stock solution. All the samples were filtered using a 0.45 μm filter  before injection into the HPLC system.HPLC  analysis was performed. Compounds were separated on an Aegispak CThe conditions of HPLC analysis were as follows: column temperature, 50 °C; injection volume, 10 μL. The mobile phase consisted of 0.1% phosphoric acid aqueous solution (solvent A) and 0.1% phosphoric acid in acetonitrile (solvent B) at 1 mL/min flow rate. The gradient elution program used for the sample was as follows: 30–40% of solvent A from 0–5 min; 40–43% from 5–20 min; 43–80% from 20–21 min; 80% from 21–30 min; 80–30% from 30–31 min; and 30% from 31–40 min. The UV detection wavelength was 254 nm.The content was calculated using the area of the peak in the HPLC analysis. The formula used to calculate the content was as follows: content (g/100) = (sample area/STD area) × (sample dilution volume × dilution factor/STD dilution volume × dilution factor) × (STD amount taken/sample amount taken) × 100.2 at 37 °C. 5,7-Dimethoxyflavone , 5,7,4′-Trimethoxyflavone , 3,5,7,3′,4′-Pentamethoxyflavone , and Quercetin  were dissolved in dimethyl sulfoxide  to produce stock solutions at a concentration of 100 mM. TNF-α  stock solution (20 µg/mL) was prepared in 1% filtered bovine serum albumin . The final concentration of DMSO was maintained under 1‰, in which the cytotoxicity of the vehicle (DMSO) could not be detected compared to the control cells. For cell experiments, 24 h after plating the cells, HDFs were starved in serum-free DMEM overnight and treated with TNF-α and other compounds.Human dermal fibroblasts (HDFs) juvenile foreskin  were grown in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  and 100 U/mL of penicillin–streptomycin  in a humid atmosphere containing 5% CO4 cells/well) and allowed to adhere for 24 h. After overnight starvation in serum-free medium, the cells were treated with a range of concentrations of samples and incubated for 24 h. Next, 10 μL of EZ-Cytox solution  was added to each well, incubated for 30 min, and OD450 was measured using a microplate reader . The viability of the HDFs was assessed using the following formula:HDFs were plated in 96-well plates (1 × 104 cells/well) and incubated for 24 h. The cells were starved overnight in serum-free DMEM. Following this, the HDFs were exposed to 6.25 and 12.5 µM TMF for 1 h and subsequently with a mixture of 20 ng/mL TNF-α and 10 µM DCFDA (Sigma-Aldrich) for 15 min. After washing with phosphate-buffered saline , the fluorescence intensity of DCFDA was analyzed using a microplate reader  at excitation and emission wavelengths of 485 nm and 535 nm, respectively. Fluorescent images were captured using an IX51 fluorescence microscope  equipped with a CCD camera.HDFs were plated in 96-well plates  and 24 h (to investigate MMP-1 and COLIA1 mRNA expression). Total cellular RNA was isolated using the RNeasy Mini Kit . RNA was reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit . PCR was performed using the AccuPower® 2X GreenStar™ qPCR Master Mix  using sense and antisense primers as listed in HDFs were plated in 6-well plates at a density of 3 × 104 cells/well) and incubated for 24 h. Next, the medium was discarded, replaced by serum-free DMEM, and the plates were incubated overnight. The cells were then treated with the indicated concentrations of the samples for 1 h and subsequently with 20 ng/mL TNF-α for 12 h (to test the secretion of IL-1β and IL-6) and 24 h (to investigate MMP-1 and COLIA1 secretion). The concentration of IL-1β, IL-6, MMP-1, and COLIA1 in the supernatant were determined using sandwich ELISA kits .HDFs were plated in 48-well plates . To assess p65 translocation and COX-2 expression, the cells were incubated with 20 ng/mL TNF-α for 4 and 6 h, respectively. Next, the cells were washed with PBS and lysed in 1 × radioimmunoprecipitation assay (RIPA) buffer supplemented with 1× protease inhibitor cocktail , 1 mM Na3VO4, and 5 mM NaF to obtain whole-cell lysates. In addition, the cells were washed with PBS and lysed in the cytoplasmic lysis buffer containing 10 mM KCl, 0.1 mM EDTA, 10 mM HEPES (pH 7.5), 1 mM DTT, 0.5% Nonidet−40, and 0.5 mM PMSF, along with the 1× protease inhibitor cocktail. After 10 min incubation, the pallets obtained after centrifuging were subsequently pipetted in nuclear extraction buffer containing 400 mM NaCl, 1 mM EDTA, 20 mM HEPES (pH 7.5), 1 mM DTT, and 1 mM PMSF with 1× protease inhibitor cocktail, and incubated in ice for 30 min. After centrifuging, the supernatants were collected and used as nuclear lysates. Protein concentration in the lysate was determined using the BCA Protein Assay Kit . Equal amounts of protein samples were separated on a polyacrylamide gel and transferred to a polyvinylidene difluoride membrane  activated by methanol. After blocking with 5% non-fat milk in tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h, the membranes were probed with the primary antibodies  of interest diluted in 1% BSA overnight at 4 °C and with suitable secondary antibodies  diluted in 1% BSA for 1 h. Next, the membranes were treated with the EZ–Western Lumi Femto Kit  and the immunoreactive bands were captured using the Fusion Solo Chemiluminescence System .HDFs were plated in 60 mm culture dishes at a density of 4 × 104 cells/well) were plated in an 8-well chamber slide and allowed to adhere for 24 h. Next, the medium in the chamber slide was replaced with serum-free DMEM. After 24 h of incubation, the cells were treated with 6.25 and 12.5 μM TMF for 1 h and subsequently with 20 ng/mL TNF-α for 4 h. The cells were fixed with 4% paraformaldehyde in PBS  for 10 min and blocked with 5% normal goat serum  containing 0.3% Triton™ X-100  in PBS for 1 h. Next, the cells were probed with primary antibody  at 4 °C overnight and with secondary antibodies conjugated with Alexa Fluor® 488  for 2 h. Subsequently, the cells were stained with 10 µM Hoechst (Sigma-Aldrich) and incubated in the dark for 10 min. Fluorescent images were obtained using an IX51 fluorescence microscope (Olympus) coupled to a CCD camera.HDFs . One-way analysis of variance (ANOVA) with Tukey’s honest significance test was applied to assess statistical differences. Statistical significance was set at The amounts of PMF, DMF, and TMF in the KP extract was determined by HPLC analysis . Contentp = 0.05) and 73.02 ± 2.37% (p = 0.05), respectively. Similarly, exposure to 50 and 100 µM TMF reduced cell viability to 84.47 ± 1.14% (p = 0.05) and 44.65 ± 0.07% (p = 0.05), respectively. PMF did not show considerable cytotoxicity effects on HDFs, whereas the viability of HDFs treated with 25, 50, and 100 µM of Quercetin significantly reduced to 92.18 ± 0.87% (p = 0.05), 84.70 ± 0.47% (p = 0.05), and 81.75 ± 0.72% (p = 0.05), respectively. Hence, the maximum dose used for to subsequent experiments was set at 12.5 µM.Prior to analyzing the anti-aging effects of KP-derived polymethoxyflavones, including DMF, TMF, and PMF, we investigated their cytotoxicity on HDFs to determine a suitable concentration range for further experiments. Quercetin, a strong antioxidant that exerts a protective effect on the skin as evidenced in previous anti-photoaging studies ,26 and up = 0.05) compared to the control group. Among the tested samples, TMF exerted the strongest inhibitory effect on mRNA expression of MMP-1. Treatment with 6.25 and 12.5 µM TMF diminished mRNA expression of MMP-1 to 1.98 ± 0.03-folds (p = 0.05) and 1.76 ± 0.06-folds (p = 0.05), respectively, while DMF considerably mitigated mRNA expression of MMP-1 to 2.10 ± 0.29-folds (p = 0.05) at the dose of 12.5 µM. PMF and Quercetin did not significantly inhibit the effect on mRNA expression of MMP-1 in TNF-α-stimulated HDFs (p = 0.05), whereas treatment with most of the samples significantly decreased MMP-1 secretion in a concentration-dependent manner. Among the studied samples, the compound most effective in inhibiting MMP-1 secretion was DMF when it attenuated the secretion of MMP-1 to 1.53 ± 0.03-folds (p = 0.05) and 0.88 ± 0.06-folds (p = 0.05) at the concentration of 6.25 and 12.5 µM, respectively. In addition, exposure to 12.5 µM TMF 3 clearly reduced the secretion of MMP-1 by 1.45 ± 0.03-folds (p = 0.05) and treatment of cells with 6.25 and 12.5 µM PMF mitigated MMP-1 secretion by 1.50 ± 0.01-folds (p = 0.05) and 1.52 ± 0.02-folds (p = 0.05), respectively. Quercetin (used as a positive control) also suppressed MMP-1 secretion by 1.16 ± 0.03 (p = 0.05) and 1.19 ± 0.05-folds (p = 0.05) at concentrations of 6.25 and 12.5 µM, respectively  and 0.80 ± 0.00-folds (p = 0.05), respectively. Among the polymethoxyflavones isolated from KP, including DMF, TMF, and PMF, only TMF showed a positive effect in restoring the COLIA1 level. TMF significantly restored the mRNA expression of COLIA1 to 0.89 ± 0.01-folds (p = 0.05) at the concentration of 6.25 µM and enhanced COLIA1 secretion to 0.90 ± 0.00-folds (p = 0.05) and 0.94 ± 0.00-folds (p = 0.05) at the concentrations of 6.25 and 12.5 µM, respectively. Exposure to 12.5 µM Quercetin increased mRNA expression and secretion of COLIA1 to 0.99 ± 0.01-folds (p = 0.05) and 0.86 ± 0.00-folds (p = 0.05), respectively . In the TNF-α-treated group, the mRNA expression and secretion of COLIA1 decreased to 0.70 ± 0.03-folds  on five factors associated with HDF damage including inhibition of MMP-1 mRNA expression, inhibition of MMP-1 secretion of MMP-1, stimulation of COLIA1 mRNA expression, and stimulation of COLIA1 secretion indicated that TMF showed the most potent compound in preventing TNF-α-induced fibroblast damage among the tested polymethoxyflavones . Therefop = 0.05) and 1.76 ± 0.07-folds (p = 0.05), respectively. However, exposure to 12.5 µM TMF suppressed mRNA expression and secretion of IL-1β to 3.37 ± 0.13-folds (p = 0.05) and 1.03 ± 0.02-folds (p = 0.05), respectively (p = 0.05) and 1.61 ± 0.01-folds (p = 0.05), respectively. However, treatment with 12.5 µM TMF significantly attenuated mRNA expression and secretion of IL-6 to 1.12 ± 0.13-folds (p = 0.05) and 1.51 ± 0.02-folds (p = 0.05), respectively  and decreased by 1.07 ± 0.01-folds (p = 0.05) and 1.01 ± 0.01- folds (p = 0.05) in cells treated with 6.25 and 12.5 µM TMF, respectively. In addition, the fluorescent images clearly visualized the inhibitory effect of TMF in ROS formation when the DCFDA intensity significantly enhanced in the TNF-α-treated group and reduced in the groups exposed to TMF in a dose-dependent manner  proteins are produced by resident fibroblasts in the dermis and are responsible for the skin’s resilience and strength. As a result of the disorganization of the ECM in the dermis, the skin begins to age. Although there are other kinds of collagen in the dermal ECM , Type I collagen is the most significant structural protein in the dermal ECM. The major cause of aged skin, which appears thin, smooth, dry, and lacks elasticity, is diminished collagen fibrils and decreased Type I collagen production. During skin aging, MMP-1, an interstitial collagenase, is responsible for the destruction of collagen fibrils. Varani et al. found that as age increases, MMP-1 levels rise and collagen expression falls in human skin; moreover, excessive ECM breakdown in the dermis by MMP-1 contributes significantly to connective tissue inflammation. As a result, in skin aging, the balance between MMP 1 and Type I collagen expression is critical ,29.Skin may age either intrinsically, via the chronological aging process that affects all bodily organs, or extrinsically, as a result of environmental stresses such as UV radiation, cigarette smoke, stress, and pollution. Both extrinsic and intrinsic skin aging processes are linked to skin inflammation via the generation of inflammatory cytokines. During intrinsic and extrinsic aging, different skin cells produce TNF-α, a 17-kDa inflammatory cytokine that functions as an important modulator of the metabolic response and cell activities. UV irradiation stimulates TNF-α receptors on the cell surface, which is one of the most important causes of skin aging. TNF-α reduces collagen expression and increases MMP expression, resulting in ECM deposition. Apart from the context of skin aging, TNF-α is also considered a vital regulator in inflammatory skin diseases such as psoriasis. TNF-α increases the production of pro-inflammatory cytokines including IL-1 and IL-6, and activates the NF-κB-signaling pathway, which promotes the development of psoriasis ,6,7. ThuKaempferia parviflora (KP) rhizome including polymethoxyflavones such as 3,5,7,3′,4′-pentamethoxyflavone (PMF), 5,7,4′ trimethoxyflavone (TMF), and 5,7-dimethoxyflavone (DMF) against TNF-α-induced human dermal fibroblast (HDF) damage via expression of MMP-1 and COLIA1. The experimental data indicated that the dose-dependent manner could not be observed in the effect of PMF and quercetin in inhibiting mRNA expression and secretion of MMP-1, and neither in the effect of TMF in stimulating COLIA1 mRNA expression. Additionally, among the tested samples, TMF was the most potent constituent compound of KP that prevented fibroblast damage induced by TNF-α, wherein it not only clearly inhibited MMP-1 expression but also stimulated COLIA1 expression. The skin protective effect of TMF was stronger than that of quercetin. Subsequently, a series of mechanistic assessments were performed to determine the skin protective efficacy of this compound.In this study, we investigated the protective effects of polymethoxyflavones from the Pro-inflammatory mediators such as COX-2, IL-1, and IL-6 play a key role in promoting the signs of cutaneous aging and inflammatory skin conditions. In skin aging, IL-6 is a regulator correlated with the generation of wrinkles on the skin, whereas COX-2 is well known to mediate the synthesis of prostaglandin E2 (PGE2), a lipid-derived signaling molecule involved in the production of MMP-1 and the suppression of collagen biosynthesis in HDFs . In psorApart from inflammation responses, oxidative stress is a major factor associated with the aging process of the skin. ROS arising from both intrinsic  and extrinsic aging (photoaging) stimulate the expression of MMPs and suppress transforming growth factor beta (TGF-β) signaling, which causes collagen breakdown and inhibition of collagen fibril biosynthesis . Thus, iMAPK, together with two downstream pathways, namely AP-1 and NF-κB, associated with oxidative stress and inflammatory responses, play a central role in regulating skin damage ,37,38. RIn summary, among the polymethoxyflavones found in KP, TMF constitutes the most potent compound in preventing TNF-α-induced HDF damage by not only inhibiting MMP-1 expression but also by restoring COLIA1 expression. The skin protective mechanism of TMF in TNF-α-stimulated in HDFs is mediated through the suppression of oxidative stress and inflammatory response via inhibition of ROS and pro-inflammatory mediators, including COX-2, IL-1β, and IL-6 production. Moreover, TMF also mitigates phosphorylation of MAPKs, phosphorylation and translocation of NF-κB, as well as the expression and phosphorylation of c-Fos and c-Jun, which inhibit MMP-1 generation and breakdown of the collagen fibrils downstream.Nonetheless, there may be some possible limitations in this study. For example, the potential of TMF in preventing skin aging was only studied in HDFs. To fully elucidate the anti-skin aging effect of TMF, the study should be extended to investigating on many different cell lines including keratinocytes, melanocytes, and organotypic 3D skin models. In addition, the protective mechanisms of polymethoxyflavones against TNF-α-induced senescence in dermal fibroblasts have not been presented in this study, although it should be considered in upcoming studies . FurtherThe results of this study indicate that among the major bioactive compounds found in KP, TMF represents the most potent constituent in preventing TNF-α-induced HDF damage, where it exerts a dual efficacy in inhibiting MMP-1 production and upregulating COLIA1 expression in TNF-α-stimulated fibroblasts. The protective effect of TMF may be due to the suppression of ROS and pro-inflammatory regulators, such as COX-2, IL-1β, and IL-6 at upstream, that lead to the reversal of the signal transduction pathways associated with MMP-1 and pro-inflammatory cytokine production, as well as with collagen degradation at downstream, including MAPK, AP-1, and NF-κB pathways. Collectively, our data suggest that TMF may be a potential antioxidant and anti-inflammatory agent in preventing skin aging and other dermatological disorders associated with oxidative stress and inflammation. In addition, to fully elucidate the anti-skin aging effect of TMF, further studies on the skin protective efficacy of TMF on many different types of skin cells, such as keratinocytes, melanocytes, and organotypic 3D skin models, must be conducted.


Methodology. The present study analyzed the transcriptome of seedlings from two asparagus bean cultivars—Dubai bean and Ningjiang 3—using Illumina RNA sequencing (RNA-seq). Correlations between samples were determined by calculating Pearson correlation coefficients (PCC) and principal component analysis (PCA). Differentially expressed genes (DEGs) between two samples were identified using the DESeq package. Transcription factors (TF) prediction, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs were also performed.Low temperature is a type of abiotic stress that threatens the growth and yield of asparagus bean. However, the key genes and regulatory pathways involved in low temperature response in this legume are still poorly understood. Phenotypes and physiological indices indicated that Ningjiang 3 seedlings tolerated cold better than Dubai bean seedlings, in contrast to adult stage. The transcriptome dynamics of the two cultivars were closely compared using Illumina RNA-seq following 0, 3, 12, and 24 h of cold stress at 5 °C and recovery for 3 h at 25 °C room temperature. Global gene expression patterns displayed relatively high correlation between the two cultivars (>0.88), decreasing to 0.79 and 0.81, respectively, at 12 and 24 h of recovery, consistent with the results of principal component analysis. The major transcription factor families identified from differentially expressed genes between the two cultivars included bHLH, NAC, C2H2, MYB, WRKY, and AP2/ERF. The representative GO enrichment terms were protein phosphorylation, photosynthesis, oxidation-reduction process, and cellular glucan metabolic process. Moreover, KEGG analysis of DEGs within each cultivar revealed 36 transcription factors enriched in Dubai bean and Ningjiang 3 seedlings under cold stress.These results reveal new information that will improve our understanding of the molecular mechanisms underlying the cold stress response of asparagus bean and provide genetic resources for breeding cold-tolerant asparagus bean cultivars. Low temperature is an essential environmental factor that limits plant distribution, growth, development, and yield . AccordiThe transcriptome is the complete set of transcripts in a cell, both in terms of type and quantity, for a specific developmental stage or physiological condition . To charVigna unguiculata L. Walp.) is one of the most important grain legumes worldwide and is cultivated in the warm to hot regions of Africa, Asia, and the Americas  is a subspecies of cowpea that is mainly grown in eastern and southern Asia to produce immature green pods (Cowpea (Americas . As a waAmericas . Asparageen pods . Low temunguiculata) conferred cold tolerance during germination  1 in the seeds of cowpea  using Illumina RNA-seq and uncover the genes and mechanisms of different asparagus bean genotypes associated with cold stress. Our results provide updated information and genetic resources for improving the low-temperature tolerance of asparagus bean.We selected the asparagus bean cultivars Dubai bean and Ningjiang 3 that are respectively cold-tolerant and -sensitive to chilling temperatures when adults . Seeds wHalf of the leaf samples were frozen in liquid nitrogen and stored at −80 °C. Fresh leaves were weighed; proline and superoxide dismutase (SOD) contents in the remaining samples were evaluated using respective assay kits  as described by the manufacturer.Total RNA was extracted using the RNAiso Plus kit  as described by the manufacturer. The integrity and quality of the RNA was confirmed by 1.5% agarose gel electrophoresis and using a NanoDrop ND-1000 spectrophotometer .® Ultra™ RNA Library Prep Kit for Illumina  as described by the manufacturer. Libraries were sequenced on an Illumina Hiseq X platform, and 125–150 bp paired-end reads were generated.Since 21 RNA samples were extracted from each cultivar, we constructed a total of 42 sequencing libraries using NEBNextv. 0.6.1. The fragments per kilobase of transcript per million mapped reads (FPKM) of each gene was calculated according to their length and the number of reads mapped to them . Genes with adjusted P < 0.05, and log2 fold change ≥ 2 were considered differentially expressed.Differentially expressed genes (DEGs) between Dubai bean and Ningjiang 3 under cold stress and recovery were analyzed using the DESeq package in R (ver. 1.18.0). Resulting v. 3.6.0RC1 tool. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment was analyzed using TBtools (Gene Ontology (GO) enrichment of DEG sets was analyzed using the BiNGO plugin of Cytoscape . Transcr TBtools .http://bioinfo.ut.ee/primer3-0.4.0/) and synthesized . Quantitative RT-PCR was performed using three technical replicates and SYBR Premix Ex Taq TM II (Takara Bio Inc.) on a Bio-Rad CFX96 Real-Time PCR system . The PCR amplification conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The relative expression of the selected genes was normalized to that of the cowpea actin gene (GenBank ID: LOC114185033) and calculated using the 2CT−ΔΔ method.We validated the RNA-seq results using 15 cold-responsive genes, including 10 AP2/ERF transcription factors, four circadian rhythm-related genes, and ICE1. One symptom of chilling injury is a water deficit in shoots due to imbalanced water transport and transpiration . Under cThe fresh weight of Dubai bean and Ningjiang 3 leaves progressively decreased under cold stress at 5 °C . After 3P < 0.01).Antioxidant enzymes, such as SOD, catalase (CAT), and ascorbate peroxidase (APX), help plants clear excessive reactive oxygen species (ROS) produced at low temperatures . In addiTaken together, we initially inferred that cold tolerance is better for Ningjiang 3 than Dubai bean at the seedling stage based on the phenotype, leaf fresh weight and proline and SOD contents.We used Illumina RNA-seq to investigate transcriptome dynamics at seven time points during cold stress and recovery using total RNA isolated from leaves of Dubai bean and Ningjiang 3. Three independent biological replicates of leaves at each time point were analyzed .Over 2 billion high-quality reads (average ∼49 million reads per sample) were generated from each plant at different time points  and mappv. 0.6.1, and the FPKM of each gene was calculated  of all samples from seven time points based on the mean FPKM of three biological repeats for all expressed genes . The PCCWe conducted PCA using the factoextra and FactoMineR packages in R . SamplesTranscription factors (TFs) play a vital role in regulating biotic and abiotic stress response networks . We inveWe investigated the potential functions of DEGs by analyzing GO enrichment . A few Gv. 3.0 to further determine functional groups . The correlation was lower at 12 and 24 h of recovery . The results of a subsequent PCA analysis were consistent, indicating that the two cultivars have similar physiological and molecular responses to stress at 5 °C. However, at 12 and 24 h of recovery at 25 °C, the phenotypes and transcriptomes of the two cultivars considerably differed, indicating that the difference in cold tolerance between them was mainly reflected during recovery. Both cold-tolerant and cold-sensitive rice and maize varieties are impaired under low temperature, but cold-tolerant varieties recover better . A tempe2O2 and reducing the risk of hydroxyl radical formation from superoxide  obtained from RNA-seq (left) and qRT-PCR (right) analysis. The color scale at the bottom represents Z-score. The values between the two heatmaps represent the correlation between expression profiles of selected genes obtained from RNA-seq and qRT-PCR analysis.Click here for additional data file.10.7717/peerj.13167/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.13167/supp-3Supplemental Information 3C0, C3, C12, C24 represent 5 ° C cold stress for 0, 3, 12, 24 h, respectively. R3, R12, R24 indicate 25 ° C recovery for 3, 12, 24 h, respectively.Click here for additional data file.10.7717/peerj.13167/supp-4Supplemental Information 4C0, C3, C12, C24 represent 5 ° C cold stress for 0, 3, 12, 24 h, respectively. R3, R12, R24 indicate 25 ° C recovery for 3, 12, 24 h, respectively.Click here for additional data file.10.7717/peerj.13167/supp-5Supplemental Information 5C0, C3, C12, C24 represent 5 ° C cold stress for 0, 3, 12, 24 h, respectively. R3, R12, R24 indicate 25 ° C recovery for 3, 12, 24 h, respectively.Click here for additional data file.10.7717/peerj.13167/supp-6Supplemental Information 6N stands for Ningjiang 3 and D stands for Dubai bean. C0, C3, C12, C24 represent 5 ° C cold stress for 0, 3, 12, 24 h, respectively. R3, R12, R24 indicate 25 ° C recovery for 3, 12, 24 h, respectively.Click here for additional data file.10.7717/peerj.13167/supp-7Supplemental Information 7N stands for Ningjiang 3 and D stands for Dubai bean. C0, C3, C12, C24 represent 5 ° C cold stress for 0, 3, 12, 24 h, respectively. R3, R12, R24 indicate 25 ° C recovery for 3, 12, 24 h, respectively.Click here for additional data file.10.7717/peerj.13167/supp-8Supplemental Information 8N stands for Ningjiang 3 and D stands for Dubai bean. C0, C3, C12, C24 represent 5 ° C cold stress for 0, 3, 12, 24 h, respectively. R3, R12, R24 indicate 25 ° C recovery for 3, 12, 24 h, respectively.Click here for additional data file.10.7717/peerj.13167/supp-9Supplemental Information 9N stands for Ningjiang 3 and D stands for Dubai bean. C0, C3, C12, C24 represent 5 ° C cold stress for 0, 3, 12, 24 h, respectively. R3, R12, R24 indicate 25 ° C recovery for 3, 12, 24 h, respectively.Click here for additional data file.