Bouea macrophylla Griffith) seeds of 3 Thai varieties of this plant were studied in terms of nutrition, phytochemicals, chemical antioxidants and the bioactivity of their extracts. Maprang seeds revealed high levels of carbohydrates, dietary fiber, energy, potassium, phosphorus, magnesium, and calcium. The Maprang seed extracts possessed a high polyphenolic content and exhibited antioxidant properties against DPPH˙, ABTS˙+, and ferric reduction. Additionally, 18-compounds were charaterized by RP-HPLC-DAD with two being recognized as gallic acid and ellagic acid and 16-unknown gallotannins. The HPLC fingerprint was composed of 4 major compounds. The extract showed active growth inhibition against leukemia, lung cancer cell lines and for 15 strains of bacteria. It is known to be particularly effective in drug resistant cells. Our results indicated that maprang seeds are a new natural source of nutrition, minerals and phytochemicals that may be applicable for use as a food supplement and as an effective drug in the treatment of certain diseases.In this study, the Maprang ( The Bouea macrophylla Griffith) , but the taste is notably different. Maprang tastes a bit like a mango if one eats the fruit with the rind, and a bit like a plum due to its pulp-like texture. However, the rind of the fruit is also edible. There are three varieties of maprang found in Thailand that are clasified according to taste Marian plums, plum mangos, gandaria or maprang (riffith)  is a troA growing body of evidence suggests that fruit by-products (seeds and peels) are potentially rich sources of natural antioxidants and other bioactive compounds , 11. The22.1trans-ferulic acid (FA), pyrocatechol (PyC), protocatechuic acid (PA), vanillic acid (VA), resveratrol (RV), kaempferol (KF), quercetin (QT), apigenin (AN), delphinidin chloride, pelargonidin chloride, procyanidin B1, cyanidin chloride, malvin chloride, and ascorbic acid (Vit C), which were all purchased from Sigma-Aldrich. Additionally, Kesselguhr was also purchased from Sigma-Aldrich (Singapore).Type I water was purified with PURELAB Option-Q system . The standard compounds included gallic acid (GA), caffeine (CF), (+)-catechin hydrate (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), (-)-gallocatechin (GC), (-)-gallocatechin gallate (GCG), tannic acid (Tannins), ellagic acid (EA), 2.2Fifty kilograms of maprang fruit, both the unripe  and the ripened  specimens in three varieties including maprang wan (sweet maprang), mayong chid, and maprang prieyo (sour maprang) were harvested during the period of March–April 2015 from the Marian Plum Plantation located in Nakhon Nayok Province (latitude and longitude: 14˚12′N and 101˚13′E). The fruit was triple-washed with tap water and transverse sectioned. Seeds were removed, weighed, and minced. Seeds were then dried with hot air at 60 °C for further extraction. Subsequently, 350 g of minced seeds were immersed in 3.5 L of various hydro-ethanolic systems  in order to macerate them for 7 days with daily shaking. The extraction solutions were filtered through Kesselguhr and were dried by evaporation at 40 °C at an approximate rotation speed of 200 rpm using a rotary evaporator . The crude extract of the maprang seeds was collected and stored at room temperature in a desiccator for further study. The extraction yield was then determined.2.3st edition source that was published in Thailand in 2003. Moisture content was determined according to AOAC (2012) 925.10 and 950.46 methods. Protein contents were determined by AOAC (2012) 991.20 which is commonly referred to as the Kjeldhal Method. Lipid content was determined by AOAC (2012) 9948.15 method. Ash was determined according to AOAC (2012) 923.03 and 920.153 methods. The dietary fiber content was determined by the AOAC (2010) 985.29 method. Minerals were determined by using an ICP-OES technique based on the in-house method of TE–CH–170 listed in the 18th edition of 2005, Ch.50 (984.27) and Ch.9 (999.10). Only zinc could be determined by the ICP-MS technique, which was employed to determine mineral content using an in-house method based on AOAC 2005.999.10.Fifty kilograms of maprang fruit were transported to the Central Laboratory (Thailand) Company Limited, Chiangmai Branch and were certified (accreditation number 1079/48) by the Bureau of Laboratory Quality Standards, Ministry of Public Health, Thailand for further proximate and mineral analysis. The fresh maprang seeds were analyzed for their energy levels, moisture, protein, lipid, starch, dietary fiber, ash, and mineral content. Briefly, carbohydrate and energy contents were determined according to a method of food analysis based on a contendum of a 12.4The stock solution of MPSEs was freshly prepared at 1 mg/mL volume in purified water and was filtrated with a 0.45 μm syringe filter (Whatman's). HPLC profiles of MPSEs were performed using a Shimadzu LC-20AD Prominence Liquid Chromatograph system equipped with a SPD-M20A Prominence Diode Array Detector and with a DGU-20A3 Prominence Degasser . The wavelength scanning was done between 190 and 800 nm, while any wavelength occurring at 270 nm was carefully monitored. Notably, 20 μL of each extract (300 μg/mL) was injected into the ZORBAX Eclipse Plus C18 Analytical column  with an Eclipse Plus-C18 Analytical Guard Column . The total phenolic content was determined from the standard curve of gallic acid and expressed as a gallic acid equivalent, and listed as mg GAE/mg of maprang Maprang seed extracts were prepared at a concentration of 1 mg/mL in water. 125 μL of each MPSE solution was mixed together with 500 μL of water and 125 μL of 2M-Folin-Ciocalteu (Sigma) in a 24-well plate. The reaction was allowed to progress for 6 minutes before 1.25 mL of 7% Na2.63. Next, a FRAP solution was prepared by mixing all three of the solutions by means of a volume ratio between solutions 1, 2, and 3 in order to obtain a 10:1:1 ratio; with a final volume of 10 mL. Additionally, 3000 μL of FRAP solution was mixed together with 100 μL of 1 mg/mL-MPSEs to keep the reaction stable at room temperature in the dark for 8 minutes. Afterward, the absorbance at 593 nm for each reaction was measured using a UV-Vis spectrophotometer. Ferrous sulfate (FeSO4.7H2O) was used as a standard anti-oxidation agent for the FRAP 2+ equivalent, i.e. μg Fe2+E/μg of maprang.Fresh solutions were prepared of: (1) 300 mM-Acetate buffer (pH = 3.6), (2) 10 mM-TPTZ  by dissolving 0.031 g TPTZ in 10 mL of 40 mM-HCl at 50 °C, and (3) 20 mM-FeCl2.72S2O8) into 7 mM-ABTS ). This solution was then incubated at room temperature in the dark for 12–16 hours to produce ABTS˙+ radicals. Afterward, these free radicals were diluted using ethanol to give an absorbance of 752 nm equalling 0.7 ± 0.02 AU as ABTS˙+ stock solution + stock solution (990 μL) ocurred at room temperature in the dark for 4 minutes, and an absorbance of 752 nm was measured by UV-Vis spectrophotometer. Trolox  was used as a standard to scavenge ABTS˙+. The scavenging activity at 50% (IC50) of the test compound was expressed as Trolox equivalent, i.e. μg TE/μg of the maprang extract.This was done by preparing 2.45 mM-potassium persulfate  of MPSEs was evaluated from the plot of %IC and the concentration relationship. Vitamin C was used as a positive control.A free radical solution of 0.2 mM-DPPH (di-(phenyl)- iminoazanium) was prepared in ethanol. A solution of 0.5 mL of MPSEs was mixed with 2.5 mL of 0.2 mM-DPPH to obtain a final concentration of MPSEs at 0, 12.5, 25, 50, and 100 μg/mL. Time was allowed for the reaction to proceed at room temperature in the dark for 30 minutes. Afterward, the absorbance at 517 nm of each reaction was measured using a UV-Vis spectrophotometer 2.9adr) and small cell lung carcinoma cells (GLC4 and GLC4/adr) were used in this study. They were grown in a RPMI1640 medium combined with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C with 5% CO2 in a humidified CO2-incubator. The concentrations of MPSEs were prepared at 10 mg/mL in water. Various concentrations of MPSEs  were incubated with cancer cells that had a beginning quantity at 1 × 104 cells in 1 mL of completed RPMI1640 medium. They were then placed in a humidified CO2-incubator for 72 hours. Afterward, the number of cells were counted by flow cytometer (Beckman Coulter Epics XL), and the percentage of inhibition cancer cell growth (%IC) was calculated using the following All green maprang seed extracts were obtained by various extraction systems and were selected to be tested for anticancer properties. Drug sensitive and drug resistant cancer cell lines of erythromyelogenous lukemic cells . In addition, the resistance factor (RF) between drug resistant and drug sensitive cell lines was determined by the ratio of IC50 (of resistant cell)/IC50 (of sensitive cell).N2.102.10.1Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus ATCC 43300, Enterococus faecalis, vancomycin-resistant Enterococcus faecalis, Escherichia coli ATCC 25922, extended-spectrum beta-lactamase producing E. coli, Proteus mirabilis, Salmonella enteritidis, Shigella boydii, Vibrio cholerae, Vibrio parahemolyticus, Klebsiella pneumoniae, Enterobacter aerogenes, Pseudumonas aeruginosa, and Acinetobecter buamannii were examined. One yeast strain, Candida albicans ATCC 90028, was also examined in this study. All bacteria were subcultured in Mueller-Hinton agar (MHA) at 37 °C with shaking at 200 rpm for 18–24 hours, while the yeast cells were grown in Sabouraud dextrose agar (SDA) at 37 °C for 24–48 hours.A total of 15 bacterial strains consisting of 2.10.2Maparang seed extracts (100 mg) of both the unripe (MYCG95) and ripe (MYCO95) fruit were prepared by putting them into a 50 mg/mL solution using 5% DMSO in order to dilute the specimens to a desired concentration of 5 mg/mL using Mueller-Hinton broth (MHB). Seed extracts were filtered with a 0.22 μm syringe filter, aliquoted, and stored at -70 °C until it was time for analysis.2.10.3Escherichia coli ATCC 25922 was selected as a microorganism candidate in this study. Briefly, an overnight culture of bacteria was subcultured into MHB and incubated at 37 °C with shaking at 200 rpm until the OD600 reached 0.6–0.8. The bacterial suspension was adjusted to approximately 1 × 108 CFU/mL with McFarland standard No. 0.5 and the suspension was spread over the surface of MHA plates using a sterile cotton swab. The media was punched aseptically with a sterile cork borer and 100 μL of 5 mg/mL seed extracts were individually added. Gentamicin (40 μg/mL) was introduced into the well as a positive antibiotic control. The plate was then incubated at 37 °C for 18–24 hours, and the inhibition zone was measured in millimeters (mm). The experiments were performed independently in triplicate and the mean value was calculated.Prior to determination of antimicrobial properites, an agar well diffusion assay was performed to evaluate the antimicrobial aspects. 2.10.48 CFU/mL with McFarland standard turbidity No. 0.5. Subsequently, 50 μL of diluted bacteria was mixed with 50 μL of 2-fold serially diluted seed extract. The final bacterial inoculum was recorded at approximately 5 × 105 CFU/mL. Gentamicin and 5% DMSO were used as the antibiotic and solvent control, respectively. Plates were incubated at 37 °C for 20 hours. The minimum inhibitory concentration (MIC) was observed after the addition of 0.2 mg/mL p-iodonitrotetrazolium chloride  in all wells and was further incubated at 37 °C for 30 minutes. The color change from a clear solution to pinkish-red formazan indicated bacterial growth. The MIC interpretation was identified by the dilution of the seed extract that displayed no visible pink color The broth microdilution method was performed according to the Clinical and Laboratory Standard Institute (CLSI) standard procedure. The procedures followed M07-A9 guidelines for bacteria (2012) and M27-A3 for yeast (2008). Briefly, the bacteria were cultured in MHB and incubated at 37 °C with agitation at 200 rpm overnight. The culture was adjusted to approximately 1 × 10C. albicans was cultured in SDA and incubated at 37 °C overnight. The culture was adjusted to the No. 0.5 McFarland standard turbidity (1 × 106 CFU/mL) using RPMI 1640-MOPS medium, followed by dilution 1:100 and 1:20 with fresh medium. One hundred microlitres of yeast suspension (approximately 5.0×102–2.5 × 103 CFU/mL) was mixed with 100 μL of 2-fold serially diluted seed extract. Fluconazole and 5% DMSO were used as the antifungal drug and solvent control, respectively. The microtiter plate was incubated at 37 °C for 48 hours. These experiments were separately performed in triplicate and both the mean and SD of the MIC, and the minimum fungicidal concentration (MFC) value, were calculated.In order to determine the level of antifungal activity, an isolated colony of 2.11p < 0.05. Statistical analysis was performed using OriginPro2018 software.The data are presented as mean ± standard deviation (SD) of triplicate measurements. The data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's test to detect significanct differences among means of each factor with the level of significance being 33.12 flow rate of 3.4 mL/min), maprang seed fat was obtained by 11% yield Maprang seed kernels were the portion of the fruit used to extract maprang flavonoids and for the purposes of further studying their chemical, antioxidant, and biological properties. The percentage (%w/w) of fresh seed kernels to fresh maprang fruit was 14.04% (MPWG), 12.82% (MPPG), and 6.49% (MYCG) for unripe seeds. For ripe seeds, it was 14.71% (MPWO), 13.62% (MPPO), and 5.7% (MYCO). We found a slight increase in the number of seed kernels from the unripe to the ripe fruit being about 0.7% in maprang wan and 0.8% in maprang prieyo, but the opposite tendency was found in mayong chid with a 0.7% decrease being observed. In comparison to mango fruit where the seed kernel represents approximately 20% of the whole fruit 3.2p < 0.05). The TPC values obtainedof the Bouea macrophylla Griffith seed extract were obtained based on the TPC of 0.689 mg GAE/mg SEs; using hot water extraction results that were provided by Zainah et al Mangifera indica L.) seed kernel extracts at 0.082–0.744 mg GAE/mg MSKEs; these findings have been well documented by Torres-León et al Amygdalus persica Linn, Annona squamosa L., Citrullus lanatus (Thunb), Matsum and Nakai, Dimocarpus longgana Lour, Durio zibethinus L., Eriobotrya japonica (Thunb.) Lindl, Litchi chinensis Sonn, Prunus salicina Lindl, Prunus salicina Lindl (san hua), Vitis vinifera Linn (red), and Vitis vinifera Linn (black) Bouea macrophylla Griffith is a rich source of polyphenolic compounds that can be extracted from many parts of the plant including the seed kernels, leaves, cortex, and fruit. Rajan and Baht found that the TPC of unripe kundang  fruit to be in the range of 0.023–0.050 mg GAE/mg MPFEs Bouea macrophylla Griffith was obtained from seed kernels followed by the cortex, leaves, and lastly the fruit itself.This is the first time that the total phenolic content (TPC) of maprang seed kernel extracts is being reported using various percentages of ethanolic solvent extraction, as is indicated in 3.3p < 0.05) of mean values were found regarding the percentage of ethanol of all unripe fruit specimens and nearly all ripened fruit specimens, with the exception of maprang wan MPWO75 which showed significant differences from MPWO95 and MPWO25. We found no significant differences (p < 0.05) of mean values within unripened and ripened fruits of each maprang variety within the solvent extraction systems, except for MPWG75 and MPWO75 which were significantly different. Within the maprang variety the antioxidant capacity of the extracts obtained from maprang wan, maprang prieyo, and mayong chid were not significantly different (p < 0.05) among both the unripe and ripened fruit.The antioxidant activities of maprang seed kernel extracts were evaluated using TEAC, FRAP, and DPPH assays, and the results are shown in 2+E/μg MPSEs (MPWO25) to 139.86 ± 18.36 μg Fe2+E/μg MPSEs (MPWO75). We found no significant differences (p < 0.05) in the mean values between the unripe and ripened fruit. There were only two significant differences evident among the maprang varieties, with the unripened fruit containing 95% ethanol (MPPG95-MYCG95) and the ripened fruit containing 25% ethanol (MPWO25-MYCO25). For maprang wan, the ethanol percentages in the ripened fruit MPWO50 (89.54 ± 11.67 μg Fe2+E/μg MPSEs) were significantly different from MPWO75 and MPWO25. For maprang prieyo, we found that only the unripened fruit MPPG25 (116.46 ± 17.93 μg Fe2+E/μg MPSEs) was significantly different to the other varieties, such as the MPPG50, MPPG75, and MPPG95. In the mayong chid, we found that only three pairs of the unripened fruit revealed significant differences; these being MYCG25-MYCG50, MYCG25-MYCG75, and MYCG50-MYCG75 based on the results of the Tukey's test  in a comparison of any mean pairs within maprang varieties, maturation level of maprang fruit, or percentage of ethanol in any particular extraction system. These mean values (IC50) presented a minimum of 20.87 ± 1.90 μg/mL (MYCO95) and a maximum of 46.34 ± 22.64 μg/mL (MYCO95). Vitamin C, being the positive control IC50, was 13.80 ± 2.26 μg/mL. Nithitanakool et al determined that the IC50 of Vitamin C (2.20 ± 2.26 μg/mL) on DPPH radical scavenging was lower than in our results, and they also found that IC50 was 1.95 ± 0.04 μg/mL for mango seed kernel extracts Bouea macrophylla Griffith seed extract by hot water extraction was highly active in scavenging DPPH radicals with IC50 4.73 ± 0.51 μg/mL, and this activity was greater than among MPSEs 50 at 20.03 μg/mL and 55.83 μg/mL, respectively ·+ scavenging activity with (EC50) being 0.24 ± 0.08, 0.73 ± 0.07, and 0.70 ± 0.06, respectively et al. found that the seed extracts of the Anisophyllea laurina R. Br. ex Sabine fruit revealed the highest activity in the ethanol solvent; followed by methanol, ethyl acetate, and water, respectively We found no significant differences by DPPH assay 3.4Notably, this is the first time that the proximate composition of fresh seed kernels obtained from both unripe and ripe maprang fruit has been determined. These compositions are presented in 3.51–18) being indicated as numeric labelled-peaks. Most of them (1–16: Pk1-Pk16) were presented at 30 minutes of retention time. In particular, the phytochemicals and the four major compounds of the maprang seed kernels were compounded at the peak numbers 1, 4, 12, and 13. Their concentrations were determined by the percentage of area in the peak as is shown in 12, followed by 4, and either 13 or 1 based on the extraction solvent used. The concentrations using 75% Ethanol as an extraction solvent gave the highest sum of the four peak areas in all maprang varieties by 78.4% (MPWG75), 78.68% (MPPG75), 72.89% (MYCG75), 73.43% (MPWO75), and 60.11% (MYCO75), and 62.94% in MPPO75 (64.8% MPPO95 highest). It is remarkable that 12 (24–40%) followed by 4 (19–26%) was the most abundant compound of maprang seeds in all three varieties for both the unripe and ripe fruit. HPLC chromatograms of each ethanolic solvent is represented in 17 was not only found in the unripe maprang fruit , but that it also appeared later in the ripe maprang fruits , as well. In addition, intensity of peaks 13, 14, 15 and 16 decreased in the ripened fruit specimens when compared to the unripe fruit specimens. It could be possible that these compounds (13 to 17) are related metabolites that form during the maturation process of the maprang fruit. Whereas, peak 18 might represent a highly polymerized form of gallotannins. We also found that compound 1 increased in concentration relative to an increase in water content, or by a decrease in ethanol in the extraction solvent (95% < 75% < 50% < 25). Compound 1 (Rt 5.05 minutes) and compound 11 (Rt 21.73 minutes) were considered to be gallic acid (GA) and ellagic acid (EA), respectively. Others, such as compound 4 (Rt 13.68 min), 12 (Rt 22.82 min), 13 (Rt 26.22 min), did not correspond to any standard polyphenol that was found in this study (see 4 showed the same retention time as (-)-epigallocatechin gallate (EGCG) (Rt 13.68 minutes), but was not the identical compound, as is shown in 4 and EGCG. The HPLC corresponding UV-Vis absoption spectral of these compounds, 1, 4, 12, 13, GA, and EGCG are shown in 4 was 223 and 271 nm, whereas for the EGCG they were 229 and 273 nm. We observed a similar shape of the spectrum with very similar characteristic peak absorptions  for the compound peaks 3, 5–10, 12–17. These compounds possibly belong to catechins and/or hydrolysable tannins  based on their electronic absorption characteristics Mangifera indica L.) seed kernels, and that they belong to the same Anacardicaeae family as the maprang fruit [Vitis vinifera L.) seed extract Dimocarpus longan Lour) [Litchi chinensisSonnnerat, Sapindaceae) [Tamarindus indica L.) The HPLC fingerprints at a wavelength of 270 nm for the maprang seed kernel extracts of three varities, maprang wan (MPW), maprang prieyo (MPP), and mayong chid (MYC), in both the unripe (G) and ripe (O) forms of the fruit were investigated and the results are presented in tudy see . Interesng fruit , 37, 38.at 12 24–% followendaceae) , 42, and3.6adr) and lung cancer (GLC4 and GLC4/adr) cells. Data regarding how unripe maprang seed kernel extracts can inhibit cancer cell growth by 50 percent (IC50) using various ethanolic extraction solvents is presented in 50 varied between 3 and 45 μg/mL. The maprang variety extracts that displayed the highest toxicity toward all four cancer cells was MPWG75 (IC50: 4–16 μg/mL), followed by MPPG50 (IC50: 2–5 μg/mL), and lastly MYCG75 (IC50: 6–16 μg/mL). To clarify, these concentrations were for maprang wan, maprang prieyo, and mayong chid, respectively. We found the IC50 of MPPG75 (2–14 μg/mL) to be at the same level as MPWG75 and MYCG75. All maprang seed extracts except MPWG75, MPWG95, and MYCG95 were more toxic  to doxorubicin-resistant cells  than to doxorubicin-sensitive cells , as the resistance factor was less than 1 (see adr) and lung cancer (GLC4 and GLC4/adr) cells 4, 12, 13, and 1; see adr, GLC4, and GLC4/adr with their IC50 values being equal to 8.9 ± 2.6, 5.8 ± 2.2, 10.9 ± 2.2, and 6.9 ± 1.0 μg/mL, respectively. Conversely, the IC50 of doxorubicin was found to be 0.01 ± 0.00, 0.72 ± 0.72, 0.02 ± 0.01, and 0.33 ± 0.21 μg/mL for K562, K562/adr, GLC4 and GLC4/adr, respectively. The screening antiprolifertive activity test results of Bouae macrophylla Griffith seed extracts on human squamous cell carcinoma (HTB-43), breast cancer (MCF7), and (MDA-MB-231) cell lines using MTT assay have already been reported 50 values being 29.32 ± 5.80, 18.65 ± 2.94, 21.14 ± 6.97 and 34.36 ± 16.50 μg/mL, respectively. Only the hexane extractdisplayed inhibition against MCF7 (59.07 ± 5.76) and MDA-MB-231 (123.35 ± 28.65). Ethanolic seed extracts of mangos (Mangifera indica), which belong to the same Anacardiaceae family as the maprang fruit, exhibited anticancer properties against human breast cancer MCF-7 and MDA-MB-231 cells with IC50 values of 30 and 15 μg/mL, respectively. However, these extracts showed low toxicity to normal breast cancer MCF-10A cells 4) Previous HPLC results indicate that the maprang seed extracts obtained from unripened fruit revealed higher levels of maprang polyphenols than the ripe fruit. Consequently, they have been selected for testing of their toxicity in doxorubicin-sensitive and -resistant leukemic  and ripe (MYCO95) fruit specimens was pre-screened in E. coli using an agar well diffusion assay, and the results indicated a clear zone of inhibition taking place in both MYCG95 and MYCO95 with an average diameter of 21 and 19 mm, respectively. A broth microdilution method was performed and this process indicated that both MYCG95 and MYCO95 had an inhibitory effect against pathogenic gram-positive, gram-negative bacteria (15 strains), and yeast  with the MICs ranging from 39.0-625.0 μg/mL. Notably, V. parahemolyticus, S. boydii and vancomycin resistant E. faecalis strains were particularly inhibited by ripe maprang seed kernel extracts with MICs at 52.0 ± 22.6, 78.1 ± 0 and 78.1 ± 0 μg/mL, respectively. This was the case for ripened seed extracts, but was also found to be the same for the unripened seed extracts. Notably, they also displayed active inhibition against V. parahemolyticus (MIC 39.0 ± 0 μg/mL), S. boydii (MIC 104.1 ± 45.1 μg/mL), and vancomycin resistant E. faecalis (MIC 104.1 ± 45.1 μg/mL). The degree of growth inhibition of drug resistant bacteria including methicillin resistant S. Aureus , vancomycin resistant E. Faecalis  and ESBL producing E. coli  was observed by these seed extracts. Surprisingly, most of the microorganisms tested could be inhibited, but were unable to be killed with the MBC/MFC values being over 2,500 μg/mL. The MIC and MBC/MFC values of the maprang seed extracts against several microorganisms are summarized in Bouea macrophylla Griffith) seed kernels exhibited similar antimicrobial properties to mango (Magnifera indica L.) seed kernels. Again, they both belong to the Anacardiaceae family and their properties have been well documented. Kabuki et al reported that mango seed kernel extracts (MSKE) that were obtained using ethnanolic solvents exhibited antimicrobial activity against 43 strains (18 species) in both gram-positive and gram-negative bacteria. The MSKE showed remarkable antimicrobial activity against gram-positive strains that included S. aureus, Bacillis sp., Clostidium sp., and Listeria monocytogenes with the MICs being in a range of 50–500 μg/mL Campylbacter jejuni and Yersinia enterocolitica with the MICs for both specimens equaling 100 μg/mL. Jiamboonsri et al also reported that the ethanolic extraction obtained from the Thai mango “fahlun” seed kernel extract showed antimicrobial activity against S. aureus (ATCC 25923) and 19 clinically isolated MRSA strains. Both of these strains MICs were 470 μg/mL and the MBCs varied from 940 to 3,750 μg/mL S. Aureus. In addition, Mutua et al reported that the MSKE methanolic extraction of four mango varieties of Kenya exhibited antimicrobial activity against E. coli, S. aureus, and C. albican based on an agar disk diffusion assay Gall chinesis exhibited antimicrobial activity against both gram-positive  and gram-negative  specimens with the MICs varying between 250 and 1,500 μg/mL Schinopsis brasiliensis Engl. exhibited antimicrobial activity against both gram-positive and gram-negative bacteria with the MICs ranging from 390 to 1,250 μg/mL This study represents the first time that the antimicrobial properties of maprang seed extracts 4Bouea macrophylla Griffith) seed kernels are a new natural source of polyphenols, and that these polyphenols are more abundant than in any of the other parts of the maprang plant including the leaves, fruit, and the cortex of the fruit. Seed kernels also contain carbohydrates and dietary fiber. The mineral content includes potassium, phosphorus, magnesium, and calcium. Surprisingly, maprang seed extracts demonstrate greater anticancer and antibacterial activity in drug resistant cells than in drug sensitive cells. These results suggest that maprang seed polyphenols might have great potential in overcoming the multidrug resistance that typically occurs with regard to cancer and other harmful microorganisms. In conclusion, it should be noted that maprang (Bouea macrophylla Griffith) seeds are also a viable natural source of nutrition. The minerals and phytochemicals found in these seeds demonstrate bioactivity against cancer and bacteria, and there may be further applications for these seeds as a food source in addition to their use in medicines. This in-depth study may help to promote the cultivation of maprang trees in Thailand and help find other new natural sources of antioxidants that can be obtained from similar fruit by-products, which can potentially be useful in terms of health promotion among the general population and economic development.Our results indicate that maprang .This work was supported by the The authors declare no conflict of interest.No additional information is available for this paper.


BCS1L encodes a homolog of the Saccharomyces cerevisiae bcs1 protein, which has a known role in the assembly of Complex III of the mitochondrial respiratory chain. Phenotypes reported in association with pathogenic BCS1L variants include growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis and early death (GRACILE syndrome), and Björnstad syndrome, characterized by abnormal flattening and twisting of hair shafts (pili torti) and hearing problems. Here we describe two patients harbouring biallelic variants in BCS1L; the first with a heterozygous variant c.166C>T, p.(Arg56*) together with a novel heterozygous variant c.205C>T, p.(Arg69Cys) and a second patient with a novel homozygous c.325C>T, p.(Arg109Trp) variant. The two patients presented with different phenotypes; the first patient presented as an adult with aminoaciduria, seizures, bilateral sensorineural deafness and learning difficulties. The second patient was an infant who presented with a classical GRACILE syndrome leading to death at 4 months of age. A decrease in BCS1L protein levels was seen in both patients, and biochemical analysis of Complex III revealed normal respiratory chain enzyme activities in the muscle of both patients. A decrease in Complex III assembly was detected in the adult patient’s muscle, whilst the paediatric patient displayed a combined mitochondrial respiratory chain defect in cultured fibroblasts. Yeast complementation studies indicate that the two missense variants, c.205C>T, p.(Arg69Cys) and c.325C>T, p.(Arg109Trp), impair the respiratory capacity of the cell. Together, these data support the pathogenicity of the novel BCS1L variants identified in our patients. Mitochondrial diseases encompass a heterogeneous group of genetic defects characterized by a dysfunction in mitochondrial energy metabolism. Clinically, defects in the mitochondrial oxidative phosphorylation (OXPHOS) system that consists of four respiratory chain enzymes (CI-CIV) and the ATP synthase (CV) manifest either within single organs or as multiorgan syndromes.The involvement of both the nuclear and mitochondrial genome in the build of the OXPHOS system underpins the varied biochemical response and clinical heterogeneity seen in patients with inherited mitochondrial diseases. OXPHOS deficiencies can be recognized as isolated events affecting individual respiratory chain complexes, or more commonly occurring as combined defects, usually affecting complexes containing mitochondrially encoded OXPHOS subunits .1bc or Complex III is a central enzyme of the respiratory chain, catalyzing the transfers of electrons from coenzyme Q to cytochrome c and subsequently forcing protons to mitochondrial intermembrane space via the Q cycle mechanism (2) that consists of 11 polypeptide subunits, one of which is encoded by the mitochondrial genome and remaining 10 are nuclear encoded. Mutations in the mitochondrially encoded structural subunit of Complex III, the cytochrome b gene (MT-CYB), have been largely associated with isolated mitochondrial myopathy and exercise intolerance. However, in recent years, the wide use of next-generation sequencing (NGS) technologies enabled the discovery of a number of variants in nuclear encoded mitochondrial Complex III structural subunits  and assembly factors  . The BCS1 homolog ubiquinol-cytochrome c reductase complex chaperone (BCS1L) encodes for a highly conserved yeast homolog of bcs1 that facilitates the last step of Complex III assembly    to multiy death)  and mildBCS1L gene. The individual carrying the compound heterozygous variant c.166C>T, p.(Arg56*) and c.205C>T, p.(Arg69Cys) in BCS1L manifested with an adult-onset aminoaciduria and phosphaturia consistent with a renal Fanconi syndrome, seizures, bilateral sensorineural deafness and learning difficulties. In contrast, the individual carrying a homozygous c.325C>T, p.(Arg109Trp) variant presented with developmental delay, persistent lactic acidosis, nephrocalcinosis, liver dysfunction and early death. BCS1L protein levels were decreased in both patients, and the activities of respiratory chain complexes were normal in muscle. A negative OXPHOS phenotype in the yeast ∆bcs1L null mutant was completely rescued by expressing the wild-type human bcs1L gene, but not with the bcs1LR109W or bcs1LR69C mutant genes mimicking the patients’ variants. These data provide functional evidence that supports the pathogenic effects of the BCS1L variants identified in our patients.Here, we report two very different clinical cases of two unrelated patients with underlying pathogenic variants in the 2) (CKD-EPI) at age 49 years  and evidence of oligohydramnios. She was born at 36 weeks’ gestation by spontaneous vaginal delivery. APGAR scores were 9 at 1 minute and 9 at 5 minutes, and the birth weight was 1.3 kg. There was a notable family history of paternal twins (aunt and uncle) dying within the first few weeks of life of unknown cause and two paternal male cousins who had also died at 2 weeks and 14 weeks of age of unknown cause. At 2 days after birth, the child was noted to be hypoglycaemic with a raised anion-gap metabolic acidosis, with an elevated serum lactate level (5.8 mmol/l) and a raised serum ammonia level (173 μmol/l). Biochemical investigations revealed normal acylcarnitine profile (excluding disorders of fatty acid and branched-chain amino acid catabolism). Liver function was abnormal with a conjugated hyperbilirubinemia. Treatment was commenced, including sodium bicarbonate, Dalivit, vitamin E and vitamin K. Thiamine, carnitine and alphacalcidol were also given and the child stabilized, allowing discharge from the neonatal unit at 43 days old. Genetic investigations were initiated, and karyotyping showed chromosome 46XX with a del (This child was born from consanguineous Bangladeshi parents (first cousin marriage). Antenatally, fetal ultrasound scans had identified both poor fetal growth . Subsequently, the patient underwent whole genome sequencing, and biallelic compound heterozygous variants in BCS1L c.166C>T, p.(Arg56*) (previously reported as pathogenic in *) and p.(Arg69Cys) is less than 0.01, and in silico pathogenicity tools predicted the c.205C>T, p.(Arg69Cys) variant to be pathogenic  (RefSeq NM_004328.4) in the BCS1L gene. The affected male sibling of P2 also carried the c.325C>T, p.(Arg109Trp) variant. The minor allele frequency (MAF) was less than 0.01, and the c.325C>T, p.(Arg109Trp) variant was predicted to by highly pathogenic  that appeared to have hallmarks of the GRACILE syndrome associated with defects in thogenic .BCS1L variants were confirmed by Sanger sequencing  showed that both parents were heterozygous carriers for c.325C>T, p.(Arg109Trp) variant (quencing  and mappquencing . The par variant .c oxidase (COX) activity around the periphery of some fibres  and cytochrome e fibres . Gomori e fibres . Respirae fibres . Mitochoe fibres .BCS1L variants present in P1 and P2. The steady-state levels of BCS1L protein were diminished in lysates isolated from skeletal muscle derived from P1 and fibroblasts of P2 when compared to age-matched controls , W303Δbcs1, which display an OXPHOS-negative phenotype characterized by failure to grow in media containing obligatory aerobic compounds as the only carbon sources   and p.(Arg109Trp) indicating that they severely impaired the cells’ respiratory capacity.To better define the oxidative growth deficiency, we measured oxygen consumption, NADH-cytochrome c oxidoreductase (NCCR)and COX activity. The respiratory activity of ∆/hBCS1L) . Moreove/hBCS1L) . TogetheBCS1L with markedly different phenotypes.We present two patients with biallelic variants in BCS1L variants, given his similar phenotype of chronic kidney disease, epilepsy and learning difficulties. The renal failure seen in P1 (and perhaps his brother) is likely to be multifactorial, but will include progressive nephrocalcinosis and likely tubulointerstitial damage secondary to mitochondrial defects  and weighing only 40 kg, 40 kg and 45 kg, respectively, with profound to severe hair loss   . There aBCS1L presenting with a GRACILE syndrome had no detectable Complex III defect and no neurological findings; however, these patients had increased iron overload  in overload .Here, we demonstrated that whilst BCS1L protein levels were greatly decreased in muscle from P1, there was only a mild decrease in the Complex III subunit UQCRC2 and the amount of Complex III . UQCRC2 *) and p.(Arg69Cys) affect different functions of BCS1L. The p.(Arg56*) variant has been previously identified in a British patient that presented with Complex III deficiency and GRACILE syndrome  accompanied by an early death at the age of 2 days. The second variant p. identified in this patients resides in the AAA ATPase domain of BCS1L and is likely to be causative alone or in combination with the p.(Arg56*)  and p.(Arg109Trp) missense variants are located (within the import auxiliary sequence) . AlthougIt is noteworthy that tissue-specific expression of BCS1L may play a role in determining phenotypes . BCS1L iBCS1L variants, including a patient with a BCS1L-related mitochondrial disease with progressive renal failure and a patient with a GRACILE syndrome harbouring a novel BCS1L variant. Detailed mitochondrial studies were able to reveal defects that were absent on muscle biopsy and confirm the pathogenicity of the BCS1L variants. Our data support the role of BCS1L in the incorporation of the Fe-S protein into Complex III, affirming BCS1L as a candidate disease gene in patients presenting without an obvious mitochondrial defect in muscle. Patients presenting with GRACILE syndrome or BCS1L-related mitochondrial disease phenotypes should be directly screened for the presence of BCS1L variants, or BCS1L variants identified by next-generation sequencing should be prioritized for further investigation.In conclusion, we have discovered and characterized new clinical phenotypes associated with Written informed consent for diagnostic and research based studies was obtained from the patient 1 (P1) and the family of patient 2 (P2) in accordance with the Declaration of Helsinki protocols. Ethical approvals for P1 were obtained from the Northern and Yorkshire Research Ethics Committee (09/H0903/36) and for Genomics England from the HRA East of England—Cambridge South Research Ethics Committee.Genomic DNA/protein was extracted from peripheral-blood lymphocytes, skeletal muscle and fibroblasts according to standard protocols.CLCN5, OCRL1 and CTNS was performed by UKGTN. Whole exome sequencing was performed by Genomics England via the 100 000 genomes project. Confirmation of mutations was performed using Sanger sequencing, using exon-specific primers. For P2, candidate gene Sanger sequencing was performed for BCS1L and MPV17.Following informed and written consent, blood samples were obtained from P1 and P2, and DNA was extracted using standard methods. All methods were performed in accordance with the relevant ethical guidelines and regulations. For P1, sequencing of https://gnomad.broadinstitute.org/) and ExAC (http://exac.broadinstitute.org/) external variant databases were used for MAF analysis. PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/) and CADD (https://cadd.gs.washington.edu/) in silico pathogenicity tools predicting missense variants were used. Candidate variants were confirmed by Sanger sequencing.GnomAD (BCS1L patient fibroblast cell lines were grown in high glucose Dulbecco’s modified Eagle’s medium (Sigma) containing 10% fetal calf serum, 1× non-essential amino acids, 50 U/ml penicillin, 50 μg/ml streptomycin and 50 μg/ml uridine at 37°C and 5% CO2 in a humidified incubator.Age-matched primary control and Cryo-sections of skeletal muscle biopsies (10 μm) undergone standard diagnostic histochemical analyses according to previously established procedures . The enzCellular protein extracts were prepared from control and patient fibroblasts or skeletal muscle samples and analyzed by western blotting as previously described in . BrieflyBN-PAGE analysis was performed to assess the steady-state levels of individual OXPHOS complexes. Mitochondrial extracts isolated from fibroblasts or skeletal muscle samples were solubilized with n-dodecyl β-d-maltoside (DDM) as described previously . A totalImmunoblotting was carried using primary antibodies against: NDUFB8 (Abcam ab110242), SDHA (Abcam ab14715), UQCRC2 (Abcam ab14745), COXI (Abcam ab14705), ATP5A (Abcam ab14748), BCS1L (Abnova H00000617-M01), UQCRFS1 (Abcam 14 746) and Total OXPHOS Human WB Antibody Cocktail (ab110411), followed by species appropriate HRP-conjugated secondary antibodies (Dako). ECL-prime  and BioRad ChemiDoc with Image Lab software were used for detection.Approximately 80 000 cells were seeded into 35 mm μ-Dishes  overnight. For mitochondrial network analysis, cells were incubated for 30 minutes in 5 nm TMRM . Facilitated by a 100× magnification oil immersion objective, an inverted multipoint scanning iSim (VisiTech) with a Ti-E microscope (Nikon) was used to capture Z-stack images of individual cells (using a 590 l filter). Triplicate experiments were undertaking using consistent microscope and laser settings (n ⩾30 cells). Images underwent Richardson-Lucy deconvolution (NIS Elements) before being processed on Fiji. Maximum intensity projections were generated and analyzed using MiNA . Total nMATa ade2–1 leu2–3112 ura3–1 his3–1 trp1–1 BCS1 and its isogenic bcs1::HIS3 (∆bcs1) , using BCS1L cDNA cloned in the pYEX plasmid (Yeast strains used in this work were W303–1A:  (∆bcs1) . Strainsal. 1994 , except  plasmid  as templtgccacagtacccgtactcag-3′.BCS1R69CFw: 5′-ggttgcttagctggctcaccaggtgagccagctaagcaacc-3′.BCS1R69CRw: 5′-ctgagtacgggtactgtggcgggggaaatggattcgggtagaacg-3′.BCS1R109WFw: 5′-ccattttatctggtattcaataccagataaaatgg-3′.BCS1R109WRv: 5′-cgttctacccgaatccatttccccbcs1LR69C and pYEX-BX/bcs1LR109W mutant constructs were used to transform the ∆bcs1 yeast strain, using the lithium acetate method  and the COX (cIV) were measured spectrophotometrically on a mitochondrial enriched fraction prepared as previously described  according to , the Medical Research Council (MRC) International Centre for Genomic Medicine in Neuromuscular Disease, Mitochondrial Disease Patient Cohort (UK) (G0800674), the UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust, the MRC/EPSRC Molecular Pathology Node, The Lily Foundation and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children. P.F.C. is a Wellcome Trust Principal Research Fellow (212219/Z/18/Z), and a UK NIHR Senior Investigator, who receives support from the Medical Research Council Mitochondrial Biology Unit (MC_UU_00015/9), the Evelyn Trust and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. P.G. and C.C.B. is funded by Telethon GGP15041.Olahova_et_al_HMG_Supplementary_data_ddz202Click here for additional data file.


Tetracarpidium conophorum was administered daily by oral gavage at a dose of 100, 200 and 400 mg/kg BW of extract for three weeks, along with the TP injections and 5 mg/kg of finasteride for comparison. On day 21, the animals were sacrificed after anesthesia. Prostate were excised, weighed and used to determine relative prostate weight. Quantitative and qualitative phytochemical screening was also done and it showed the presence of flavonoids (0.370 mg/ml), tannins (0.458 mg/ml), phenols (0.508 mg/ml) and steroids (0.257 mg/ml). The prostate specific antigen level was evaluated, the result showed the data for extract group 200 mg/kg, 400 mg/kg, finasteride control group and BPH control group to be 0.186 ± 0.0023 ng/ml, 0.153 ± 0.005 ng/ml, 0.119 ± 0.0125 ng/ml and 0.332 ± 0.004 ng/ml respectively.Benign prostatic hyperplasia (BPH) is a common urological disorder of men, characterized by prostatic enlargement and urethral obstruction. In this study, BPH was induced in experimental groups by daily subcutaneous injections of testosterone propionate (TP) for 3 weeks.  Specifications TableValue of the data•Tetracarpidium conophorum being used as an anti-hyperplastic agent.The data presented gives the quantitative assessment of certain phytochemicals responsible for the beneficial property of •The data given may correlate with the results obtained using same or different plant in other regions.•Data given can influence the development of a new drug against benign prostatic hyperplasia without extravagant side unlike finasteride.•The data obtained from prostate specific antigen and relative prostate weight may correlate with data obtained in other regions with a different plant or similar plant.11.1See 1.2See 1.2.1Relative prostate weight is used to evaluate the growth of benign prostatic hyperplasia in males .Table 3R1.2.2Prostate specific antigen (PSA) is found in blood serum when it is increased due to the presence of prostate diseases. The disruption of the epithelium leads to diffusion of PSA into the blood .Table 4P2Tetracarpidium conophorum were collected within Sango-Ota, Ogun State, Nigeria and was identified at the Botany Department of Covenant University, Ota, Ogun State, Nigeria. The leaves were picked from the branch, air dried and ground to powdered form before use. The pulverized leaves were weighed (600 g), soaked for 3 days in 80% ethanol, sieved and extracted (32 g) using a rotary evaporator.Fresh 2.1Qualitative and quantitative phytochemical screening was carried out according to the method described by Tiwari 2.2A total of thirty male Wistar rats (Rattus norvegicus) weighing 170 g to 230 g were purchased from the Federal University of Agriculture, Abeokuta, Nigeria. The rats were housed in the Animal house of Covenant University at room temperature and alternating light cycle 12 h light and dark cycle. All experimental procedures were carried out in compliance with Guidelines for the Care and Use of Laboratory Animals prescribed and approved by Covenant University Health Research Ethics Committee. After one week of acclimatization, the rats were randomly divided into six (6) groups of five (5) rats each.Animal treatment:Normal control group: subcutaneous injection of olive oil.BPH control group: no treatment. 3 mg/kg body weight TP (BPH control group).100 mg/kgTetracarpidium conophorumgroup: 100 mg/kg body weight Tetracarpidium conophorumorally and 3 mg/kg body weight TP.200 mg/kgTetracarpidium conophorumgroup: 200 mg/kg body weight Tetracarpidium conophorumorally and 3 mg/kg body weight TP.400 mg/kgTetracarpidium conophorumgroup: 400 mg/kg body weight Tetracarpidium conophorumorally and 3 mg/kg body weight TP.Finasteride control: 5 mg/kg body weight finasteride and 3 mg/kg body weight TP.Finasteride was used as the standard drug against BPH and as a positive control. The subcutaneous injection of TP, finasteride and oral administration of the extracts by oral gavage were done daily concurrently Shin . The rat2.3Blood was collected by cardiac puncture into plain bottles, spun for 15 minutes at 3500 rpm to obtain the serum  significance by one way analysis of variance (ANOVA), then Fischer׳s Test for post hoc comparison. Data were expressed as mean ± SEM (standard error of mean) of five replicates. Microsoft Excel 2013 was also used in plotting standard curves. Values of


SH3BP2 gene. The bone is replaced by a fibrous granuloma containing multinucleated giant cells. Cells of the cherubism granuloma have never been systematically analyzed. Hence, the aim of this study was to characterize the cells in human cherubism granulomas, to determine the osteoclastic characteristics of the multinucleated giant cells and to investigate the potential role of TNF-α in human cherubism.Cherubism is a rare autosomal dominant disorder of the jaws caused by mutation of the Seven granulomas were analyzed in pathology, molecular biology and immunohistochemistry. Granulomas were composed mainly of macrophages or osteoclasts within a fibroblastic tissue, with few lymphoid cells. Myeloid differentiation and nuclear NFATc1 localization were both associated with disease aggressiveness. OPG and RANKL immunohistochemical expression was unexpected in our specimens. Five granuloma cells were cultured in standard and osteoclastogenic media. In culture, cherubism cells were able to differentiate into active osteoclasts, in both osteoclastogenic and standard media. IL-6 was the major cytokine present in the culture supernatants.Multinucleated giant cells from cherubism granulomas are CD68 positive cells, which differentiate into macrophages in non-aggressive cherubism and into osteoclasts in aggressive cherubism, stimulated by the NFATc1 pathway. This latter differentiation appears to involve a disturbed RANK-L/RANK/OPG pathway and be less TNF-α dependent than the cherubism mouse model.The online version of this article (10.1186/s13023-018-0907-2) contains supplementary material, which is available to authorized users. Cherubism is a rare, autosomal dominant, benign fibro-osseous disease of the jaws with incomplete penetrance and variable expressivity . The firSH3BP2 gene (SH3 domain-binding protein 2), located on chromosome 4p16.3 [SH3BP2 mutations [SH3BP2 heterozygotes, heterozygous mice do not exhibit any cherubism phenotype, and homozygous mutants develop severe bone loss due to osteoclast hyperactivity. Despite this important difference in genetic expression, Sh3bp2 KI mice are considered a cherubism model [Sh3bp2 KI myeloid progenitor cells induce the activation of the NFATc1 signaling pathway, leading to hyperactive osteoclasts [Sh3bp2 KI mice develop systemic inflammation as a result of systemic infiltration by macrophages into tissues, as well as bone loss [At the molecular level, cherubism is caused by mutation of the e 4p16.3 . SH3BP2 e 4p16.3 –13. Moste 4p16.3 . Recent utations . Howeversm model . Accordism model . In vitreoclasts , 15. In one loss , defininone loss .The main objective of the present study was to determine if this auto-inflammatory bone disease paradigm could also be applied to human cherubism. To do so, we systematically examined the types of cells present in granulomas from 7 cherubism patients to look for evidence of chronic inflammation. We then characterized the osteoclastic features of the MGC both in vivo and in vitro. We also explored the potential role of TNF-α in the pathogenesis of human cherubism, and searched for potential biomarkers of the disease. Thus, we showed that in human cherubism, osteoclasts are the major myeloid cell type embedded within a fibrous stroma. The characteristics of these CD68-positive cells (macrophage vs. osteoclast) may predict the aggressiveness of the disease. Moreover, we demonstrated that first human cherubism granuloma is heterogeneous according to the patient and second the mechanism underlying human cherubism appeared to be different from that of mice.SH3BP2 gene was performed for 7 patients. All patients underwent intraosseous cherubism granuloma curettage.This study included 7 patients (5 children and 2 adults) treated and followed in the maxillo-facial surgery department of Necker Hospital, Paris, through the MAFACE reference center for rare facial malformations. The 5 children were already part of our previously published cohort . All patThe cherubism cases were classified according to patient age at the time of surgery  and sub-classified according to their aggressiveness based on the usual radiologic classification  and diseGranuloma samples were obtained at the time of surgery and divided into 2 parts, one for pathological examination and the second for the biochemical and biomolecular studies outlined below. Tissues for pathological examination were fixed in 10% formalin and embedded in paraffin. Four samples of normal alveolar bone, collected during third molar extraction, were included after written informed consent as controls for biomolecular analysis.4-μm thick sections were cut from each paraffin block. Section staining with hematoxylin/eosin/safranin (HES) was automated by using a Leica Autostainer : after deparaffinization , staining was performed by successive baths in hematoxylin GILL2 , 1% eosin , 0.12% safranin in 1% hydrochloric acid solution  and 95% ethanol. The sections were then mounted in synthetic resin . Two pathologists blinded to clinical history scored the slides, according to the previous description of cherubism , 22. For2O2 to quench endogenous peroxidase activity. Staining was visualized in brown color using a diaminobenzidine tetrahydrochloride chromogen substrate . Sections were counterstained with hematoxylin. Slides were scored by two pathologists blinded to clinical history and the primary antibody used. The number of positive cells was evaluated for each assay from five randomly selected areas (200 HPF), except for vimentin. Vimentin expression was semi-quantitatively evaluated .Immunohistochemical staining was performed using a Ventana Benchmark® XT automated slide preparation system . 4-μm thick sections were incubated with primary antibody Table . After w2O2 followed by a wash in PBS. Sections were blocked in a 5% normal human serum for 30 min before incubation with the primary antibodies or isotype control for 60 min at room temperature. After a PBS wash, the sections were then incubated with the secondary antibody for 30 min, then with streptavidin/horseradish peroxidase for 30 min . Staining was visualized as a brown color by using a diaminobenzidine tetrahydrochloride chromogen substrate . Slides were scored by two pathologists blinded to clinical history and the primary antibody used. The signal for each antibody was evaluated for MGCs and stromal cells. For NFATc1, the numbers of nuclear and cytoplasmic positive cells were evaluated.For immunohistochemistry staining, 4-μm thick sections were first deparaffinized with xylene for 30 min, post-fixed with 90% ethanol for 10 min and then washed in distilled water for 5 min. For antigen retrieval, the sections were incubated in a 78 °C water bath for 30 min in pH 6 or pH 9 buffer, cooled at room temperature for 20 min, washed in PBS for 10 min and finally incubated with reagents from an avidin/biotin kit . Endogenous perodixase activity was blocked by incubating the sections with 3% HA TRAP activity assay was performed on paraffin-embedded tissue sections and cell cultures from granulomas on Lab-Tek™ chamber slides (Dutscher). Paraffin sections were deparaffinized and rehydrated, stained in pH 5.2 acetate buffer containing 2.5 mM Naphthol AS-TR phosphate, 0.36 M N–N dimethylformamide, 0.1 M sodium tartrate and 4 mM Fast Red TR Salt (Sigma-Aldrich). Hematoxylin was used for nuclear staining. TRAP-positive giant multinucleated (> 3 nuclei) cells were considered as osteoclasts. The number of osteoclasts (MGC > 3 nuclei) and number of nuclei per osteoclast were evaluated from five randomly selected areas (200 HPF).-Cq as previously described [GAPDH, SDHA, TBP and HPRT were used as reference genes.Total RNA was extracted from the granuloma samples, bone controls or cell cultures using Trizol Reagent  according to the manufacturer’s instructions. The total RNA yield (ng) was determined fluorometrically using a Qubit fluorometer . Total RNA (1 μg) was reverse transcribed using SuperScript II™ reverse transcriptase  according to the manufacturer’s instructions. Real-time quantitative PCR was carried out using SYBR-green master mix  in a BCR Bio-Rad Opticon thermocycler . PCR conditions were: 98 °C for 30 s followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 20 s. Primer sequences of all the analyzed genes are shown in  for the resorption assay, Lab-Tek II chamber slides 1 well   for the ELISA supernatant study, or Lab-Tek II chamber slides 8 wells  for the TRAP activity assay with standard medium. After 12 h of incubation at 37 °C in a 5% CO2 humidified atmosphere, half of the cell culture was incubated in standard medium and the other half in osteoclastogenic medium. Standard medium contained minimal essential medium  and 1% Penicillin-streptomycin, 1% L-glutamine  and 10% fetal calf serum . For the Lab-Tek cultures, osteoclastogenic medium contained standard medium supplemented with RANKL at 30 ng/ml and M-CSF at 25 ng/ml . For the dentin slices cultures, osteoclastogenic medium contained standard medium supplemented with RANKL at 60 ng/ml and M-CSF at 25 ng/ml . Half of the cell culture was maintained for 3 days, and the other half was maintained for 7 days. In the 7-day cultures, the culture medium was changed at day 3. Supernatants were collected at day 3 and day 7 for ELISA. Cultures in Lab-Tek 8 chambers were fixed in 4% paraformaldehyde for TRAP activity measurement, and cultures in Lab-Tek 1 chambers were treated with Trizol Reagent for RNA extraction. Cultures on dentin slices were washed in distilled water and sonicated to remove cells. The slides were then stained with 0.5% toluidine blue to reveal lacunar resorption areas by light microscopy.Samples from only 5 cherubism cases yielded cell cultures. Tumor tissues were washed in PBS. Fragments of the tumors were incubated at 37 °C for 60 min in a hyaluronidase and collagenase solution mix (1 mg/ml for each enzyme) in PBS. The number of cells was counted manually using a Mallassez counting chamber. Cells were cultured either on a dentin slice  (at a density of 0.5 × 106 cells/cm2), in standard medium and osteoclastogenic medium for 14 days.Whole blood (15 ml) was obtained from the French Transfusion Establishment (EFS). Blood was diluted with 1X PBS, layered on 12 ml of Ficoll , and then centrifuged . The PBMC layer was collected and washed in PBS, and then counted in a Malassez counting chamber. The cells were plated in Lab-Tek 8 chambers . The lower and higher detection limits for each cytokine are shown in Additional file p < 0.05 being considered significant.Results are expressed as mean ± 2 SEM. Statistical comparisons were made using ANOVA, with SH3BP2 mutation , and one had the most common SH3BP2 mutation , previously described 7. Noteworthy was the wide variation in cherubism aggressiveness in this patient sample.Seven patients with cherubism, five children and two adults, were investigated in this study. Patient ages ranged from 6 to 45 years old at the time of surgery (intraosseous granuloma curettage). None of the patient presented intercurrent disease or medical therapy. The children were categorized as group 1 and the adults as group 2. They were further classified according to granuloma radiological grade and evolution after surgery , bone formation , and inflammation   and 1-C1 (p = 0.0003). RANK-L mRNA was expressed by all samples, and significantly (p = 0.0121) more expressed in sample 1-B2. OPG mRNA was expressed by all samples, and significantly (p = 0.002) more expressed in sample 2-A. The RANKL/OPG ratio was positive in all cases except sample 2-A. NFATc1 mRNA was expressed by all samples, and significantly (p = 0.0001) increased in 1-C2. Osteocalcin and ALP mRNA were expressed by all samples, and significantly more expressed in 2-A (p = 0.00001). IL6-R mRNA was expressed by control bone, 1-C1 and 2-B, with a significant increase in 1-C1 (p = 0.0287). Granulomas and control bone did not express IL-6 and IL17 mRNAs.All granulomas (except 1-B1) and the bone controls provided suitable material for RNA extraction and RT-qPCR analysis. The granulomas and bone expressed M-CSF, TNF-α, TNF-R1, and TNF-R2 mRNAs with no significant differences between the granuloma and bone. RANK mRNA was expressed by all samples, and significantly more expressed in samples 1-B2 , and a RANK-L/OPG ratio greater than 1. However, the overall results of the biomolecular analysis reflected extensive inter-tumor heterogeneity Fig. -d and diWe first began with a simple histologic analysis. Hematoxylin-eosin-safranin-stained sections of six of the cherubism granulomas  showed multinucleated giant cells within a collagen-rich and well-vascularized fibrous stroma Fig. -d. The g+. Among these lymphocytes, most were CD8+ T lymphocytes, some were CD4+ cells, and fewer still were CD20+ cells (Table + (antigen expressed by myeloid cells) could be mononucleated or multinucleated   Fig. , Table 3ei) Fig. . We founei) Fig. . In areaei) Fig. . In the ei) Fig.  , most of which were CD8+ lymphocytes; however, immature macrophages (CD68+ mononuclear cells), mature macrophages  and osteoclasts  were observed. TRAP activity, NFATc1 staining and the number of nuclei in CD68+ cells, strongly suggesting an osteoclastic phenotype, seem to be associated with the aggressiveness of cherubism. In non-aggressive cherubism, CD68+ cells displayed a macrophage phenotype, whereas in aggressive cases, CD68+ cells displayed osteoclast characteristics .Overall, the histopathological analysis revealed great heterogeneity of cherubism granulomas, in part due to the aggressiveness of the disease; and the unexpected and atypical expression of the RANK/RANKL/OPG triad in cherubism granulomas  Table . ConsistTo analyze the osteoclastic characteristics of the granuloma MGC and determine if the cells are hypersensitive to RANK-L and M-CSF, as demonstrated in the cherubism mouse model , we enzyFive of the seven cases  yielded material suitable for granuloma cell culture. Cases 1-B2 and 1-C2 were only cultured in osteoclastic medium, and analyzed at day 7. In culture, TRAP-positive MGC with greater than 3 nuclei were considered as osteoclasts Fig. . At day p = 0.053) and size (p = 0.09) to those in the control osteoclast culture , but showed a greater number of nuclei (p = 0.002) versus the control osteoclast culture  , the granuloma osteoclasts (day 7) were comparable in terms of both number/HPF p = 0.03 and size p = 0.0 to thosep = 0.75) compared to the control osteoclast culture (day 14), but they were larger (p = 0.02) and tended to have more nuclei (p = 0.03). In addition, from day 3 to day 7, granuloma osteoclast number/HPF, number of nuclei per osteoclast, and osteoclast size increased  were comparable in terms of number/HPF , TRAP-positive, and able to resorb bone. Granuloma cells can differentiate into osteoclasts and maturate without RANK-L and M-CSF medium supplementation . However, these cells are still sensitive to RANK-L and M-CSF, because in osteoclastogenic medium the granuloma cells differentiate further into osteoclasts and the osteoclasts maturate from day 3 to day 7, and they generate more pits than when cultured in standard medium.It must be noted that these cell cultures were not pure MGC cultures. Thus, the results of standard medium cultures suggest that granuloma cells might secrete cytokines which stimulate osteoclast differentiation, maturation and fusion. In addition, we cannot rule out that some MGC might have been present at the start of the culture.The main cytokines involved in auto-inflammatory bone diseases  are TNF-TNF-α was detected in the supernatant of all cultures . IL-6 was detected in the supernatant of all cultures at higher levels than for TNF-α . IL-17 was not detected. OPG was detected in the supernatant of all cultures , whereas unbound RANK-L levels were generally lower than OPG levels (from barely detectable to 151 ng/ml), leading to a RANK-L/OPG ratio of less than 1 in most cases , the number of osteoclasts increased. Instead, IL-6 might stimulate osteoclast differentiation and maturation.Cherubism is a rare disease described as an auto-inflammatory disease from the mouse model point of view. However human cherubism, and specially cherubism intraosseous lesion is poorly described in the literature. In the present study, we extensively (in situ and in vitro) explored cherubism intra-osseous lesions in order to determine if human cherubism could be considered as an auto-inflammatory bone disease. For this purpose, we explored cells in presence, and characterized the osteoclastic characteristics of MGC. Moreover, we examine the expression of inflammatory cytokines by granuloma cell cultures. Thus, we showed that human cherubism granuloma is composed mainly of osteoclasts or macrophages and relatively few immune cells, mainly CD68-positive cells, within a fibroblastic environment. The characteristics of these CD68-positive cells (macrophage vs. osteoclast) may predict the aggressiveness of the disease. Moreover, we demonstrated that the mechanism underlying human cherubism is different from that of mice; since, contrary to mice, it does not seem that TNF-α underlies the physiological mechanism of human cherubism, and that RANK-RANKL-OPG triad is not playing their usual role.This study is the first large cellular and molecular analysis carried out on human cherubism granuloma samples. Our results show that MGC are derived from a monocyte lineage within a fibrous stroma with few inflammatory cells. The myeloid cells present in cherubism granuloma are mainly macrophages and osteoclasts, and the osteoclast phenotype may determine the aggressiveness of the disease. In addition, this study demonstrated that these osteoclast-like MGC are truly functional osteoclasts, able to resorb bone. Moreover, we showed a low expression of TNF-α pathway protein both in granuloma and in culture, while IL-6 is more widely expressed. In addition, it seems that the RANK/RANKL/OPG triad is disturbed in cherubism.In this study, the seven unrelated patients presented an array of phenotypes, including both adult and childhood cases and non-aggressive to highly aggressive granulomas. Moreover, the study included a wide variety of analyses not previously performed in any study of human cherubism: ex vivo and in vitro studies, and pathological, immunohistological, biomolecular and cellular analyses.+, TRAP− mononucleated cells), multinucleated macrophages , osteoclast precursors , and osteoclasts . First, these results confirmed that MGC are derived from a monocyte lineage, as previously described [The immunohistochemical analysis permitted us to characterize the types of cells present within the cherubism granuloma. Fibroblast cells and monocyte lineage cells are the most prevalent cells, with some rare lymphoid cells, principally CD8+ cells. These results support the definition of cherubism as an auto-inflammatory disease . Auto-inescribed . In thisescribed . Moreoveescribed .RANKL mRNA expression was low in the granulomas; and in cultured cells, RANKL protein expression was also low. Furthermore, it seems that a negative feedback process is activated, with increased secretion of OPG. By immunohistochemistry, we showed numerous OPG-positive cells around MGC, in aggressive cherubism . Liu et al. [Sh3bp2 KI models, the observed osteopenia is due to an osteoblast functional defect and a reduction in OPG synthesis [Here, we discovered that RANKL has an unexpected expression in cherubism. We showed that the cherubism granulomas contained very few RANKL-positive cells (by immunohistochemistry), and these RANKL-positive cells were localized in regions poor in MGC. Moreover, u et al.  showed synthesis , 28. In In the present study, one adult patient (2-A) in the remodeling phase was included. Interestingly, the patient’s granuloma displayed characteristics of osteogenesis: a RANK-L/OPG ratio less than 1, and high expression of ALP and OC (osteoblast markers).Sh3bp2KI/+ bone marrow-derived M-CSF-dependent macrophages are highly sensitive to TNF-α and can differentiate into osteoclasts independently of RANK-L. Moreover, Wang et al. [Interestingly, cherubism cells differentiate into functional osteoclasts upon exogenous stimulation by RANK-L Fig. , but areg et al.  suggesteTNFR1, TNFR2 and TNF-α expression comparable to that in normal alveolar bone. Moreover, in vitro, the level of TNF-α in the culture supernatant was low. This result is in line with the dubious role of TNF-α in human cherubism. Indeed, Amaral et al. [TNF-α transcript was low in cherubism, and others reported that anti-TNF-α therapy failed to improve cherubism lesions [These results suggest that cherubism cells produce a cytokine(s) that stimulates osteoclast differentiation (reflected in the increased number of osteoclasts from day 3 to day 7) and fusion (increased osteoclast size and nuclei from day 3 to day 7) in standard medium. Studies of mouse models of cherubism , 29, 31 l et al.  showed t lesions , 34. Thu lesions –37.Sh3bp2 KI models, NFATc1 is implicated in bone loss but not in inflammation [This study is a first step to understand the molecular pathways leading to human cherubism. Our results suggest that osteoclast activation is mediated by translocation of NFATc1 into the nucleus, leading to the transcription of osteoclastogenic effectors such as TRAP. The role of NFATc1 had already been identified in the mouse model. However, in the ammation . In humaammation , 33, 38,ammation . In the ammation .Many challenges remain. First, because of the rarity of cherubism, a large prospective study is difficult to undertake. Second, the granulomas are highly variable not only between patients, but also within each granuloma itself. Thus, it is difficult to draw firm conclusions based on a single biopsy, and serial coring may be necessary. Finally, since MGC are osteoclasts , secondary cultures are impossible; indeed, serial analyses in cherubism cultures are difficult. Addressing these challenges will require investigation of a much larger series of patients, probably international in scope owing to the rarity of the disease, and creation of a human in vitro model.In summary, we showed that MGC are CD68-positive cells. These cells may differentiate into macrophages and osteoclasts, and their differentiation profile determines the aggressiveness of the disease. However, we do not know what triggers the choice between the osteoclast and macrophage lineage fates and hence the cherubism severity. In addition, we demonstrated that MGC differentiate through NFATc1-RANK-L-dependent and RANK-L-independent pathways whose activation is more likely due to IL-6 than to TNF-α. Our study brings new insights in the cellular mechanisms of human cherubism but leaves some important questions unresolved, such as the exclusive jaw bone localization, the absence of systemic disorders, and the natural history of the disease with its usually spontaneous resolution in adulthood.Additional file 1:Primary antibodies used for automated immunohistochemistry. AE1-AE3 (pan cytokeratin), CD (cluster differentiation). (DOCX 14 kb)Additional file 2:Primary, control and secondary antibodies for manual immunohistochemistry. NFATc1 (nuclear factor of activated T cells cytoplasmic 1), RANK-L (receptor of activated nuclear factor kappa B ligand), OPG (osteoprotegerin), RANK (receptor of activated nuclear factor kappa B), IL (interleukin), TNF-R1 (Tumor necrosis factor receptor 1). (DOCX 15 kb)Additional file 3:List of the primers used for qPCR analysis. GAPDH , HPRT (hypoxanthine-guanine phosphoribosyltransferase), SDHA (succinate dehydrogenase complex subunit A), TBP (TATA-binding protein), RANK-L (receptor of activated nuclear factor kappa B ligand), OPG (osteoprotegerin), M-CSF (macrophage colony stimulating factor), NFATc1 (nuclear factor of activated T cells 1), RANK (receptor of activated nuclear factor kappa B), IL6-R (interleukin 6 receptor), IL6 (interleukin 6), TNF-R (tumor necrosis factor receptor), TNF-α (tumor necrosis factor α), ALP  (DOCX 16 kb)Additional file 4:ELISA cytokine detection kit characteristics. RANK-L (receptor of activated nuclear factor kappa B ligand), OPG (osteoprotegerin), M-CSF (macrophage colony stimulating factor), IL (interleukin), TNF (tumor necrosis factor). (DOCX 15 kb)Additional file 5:p = 0.003). RANK-L mRNA was significantly more expressed in 1-B2 (p = 0.012). OPG mRNA was significantly more expressed in 2-A (p = 0.0002). RANKL/OPG ratio was positive in all cases but 2-A. NFATc1 mRNA was significantly increased in 1-B2 (p = 0.0001). Osteocalcin and ALP mRNA were significantly more expressed in 2-A (p < 0.0001). (TIF 26330 kb)Biomolecular characteristics of cherubism granulomas. Results show the relative expression levels of RANKL, OPG, RANK, M-CSF, RANKL/OPG ratio, NFATc1, TNF-α, TNFr1, TNFr2, alkaline phosphatase (ALP), osteocalcin and OPG mRNA obtained by RT-qPCR on the surgical specimens. Tumors and bone expressed M-CSF, TNF-α, TNF-R1, TNF-R2 mRNA without significant differences. RANK mRNA was more expressed in cases 1-B2 and 1-C1 . A: Case 1-C2; RANK control . B: Case 1-C1: OPG control . C: Case 1-C1: RANKL control . D: Case 1-C2:NFATc1 control . (TIF 26330 kb)Additional file 7:Patient 1B2 HES and Immunohistochemistry photographs. . Granuloma did not expressed AE1/AE2. Few stromal cells expressed CD3 and CD5. No cells expressed CD20. Few stromal cells expressed CD4 and CD8. Stromal cells and MGC expressed CD68. All cells expressed vimentine. Granuloma cells did not expressed RANK, OPG and NFATc1. Some stromal cells expressed RANKL. TRAP assay were negative. In HES, the granuloma showed collagen-rich fibrous area with few MGC. (TIF 18313 kb)Additional file 8:Patient 1C1 HES and Immunohistochemistry photographs. . Granuloma did not expressed AE1/AE2. Some stromal cells expressed CD3 and CD5. Few cells expressed CD20 and CD4. Some stromal cells expressed CD8. Stromal cells and MGC expressed CD68. All cells expressed vimentine. Stromal and MGC cells expressed RANK. Some stromal cells expressed RANKL and OPG. TRAP assay were positive for MGC. NFATc1 was expressed in the nuclei and cytoplasm of MCG. In HES, the granuloma showed intralesional hemorrhage with many MCG. (TIF 18313 kb)Additional file 9:Cherubism giant multinucleated cells (MGC) A-C: Standard medium cultures: MGC differentiate into osteoclasts . A. Osteoclast number increased from day 3 to day 7. B. Nuclei number per osteoclast increased from day 3 to day7. C: size of osteoclasts increased from day 3 to day 7. D-F: Osteoclastogenic medium cultures: MGC are sensitive to RANKL and M-CSF. D: Osteoclast number increased from day 3 to day 7 in culture with osteoclastogenic medium. E: Nuclei number per osteoclast increased from day 3 to day 7 in culture with osteoclastogenic medium. F. Size of osteoclasts increased from day 3 to day 7 in culture with osteoclastogenic medium. RANK-L (receptor of activated nuclear factor kappa B ligand), M-CSF (macrophage colony stimulating factor), TRAP: tartrate resistant acid phosphatase (TIF 1304 kb)


Paramuricea macrospina resulted to be a gonochoric internal brooding species with a 1:1 population sex ratio. Oogenesis lasted ~12–14 months, whereas spermatogenesis was significantly shorter, only lasting 6 months. Fertilization occurred during late summer (August) and larval release occurred during autumn (September–October). The organic matter and total lipid content showed a slight seasonal variability. Stable isotopic composition remained constant throughout the year, reflecting a general stability in gorgonian food sources. Conversely, the free fatty acid composition varied seasonally, reflecting changes in P. macrospina energetic demands probably related to gametogenesis and larval brooding. The reproductive ecology and biochemical composition of P. macrospina significantly differ from shallow coastal gorgonian species, reflecting the higher environmental stability of deeper environments.This study examined the sexual reproductive cycle, energy storage and metabolic requirements of a Mediterranean gorgonian in a mesophotic ecosystem (~70 m depth).  In the past few years there has been a substantial increase in the ecological characterization of coral-dominated ecosystems located at 30–150 m depth . In tropSexual reproduction is a crucial process in the maintenance of Mediterranean shallow gorgonian populations , 20 as wIn a temperate sea such as the Mediterranean, shallow-water gorgonians exhibit a marked seasonality of activity and secondary production as a consequence of the strong seasonal environmental variability . Food caParamuricea macrospina  has been recently reported as one of the most frequent and abundant gorgonian in Mediterranean mesophotic ecosystems, dominating maërl beds on the outer continental shelf at 65–100 m depth (δ13C and δ15N) composition were assessed over an annual cycle to address the following questions: (1) Are there differences in the reproductive timing and reproductive output compared to shallow gorgonians? (2) Are there differences in the annual dynamic of energy storage and metabolic requirements compared to shallow gorgonians? (3) How are the reproductive cycle, energy storage and metabolic demands in a mesophotic temperate gorgonian population on the continental shelf? m depth  9]. The. TheParaParamuricea macrospina colonies were sampled monthly on the outer continental shelf of the Menorca Channel at 60–75 m depth  were converted to diameters d) were then transformed to volume Sex identification was performed under optical microscope and according to the color and appearance of sexual products , 20, 52.Colonies collected in June, July, and August  were used to quantify the population sex ratio. Samples in which no sexual products were found inside 10 polyps were not considered . A totalOrganic matter (OM) in the coenenchyme was monthly quantified in four colonies. Approximately 10 mg (± 0.1 mg) of coenenchyme dry weight from each sample was reduced to ash during 4 h at 500 °C in a muffle (Relp 2H-M9), and the OM was calculated as the difference between the coenenchyme dry weight and ash weight , 53. Res-1 of OM.Total lipid content in the tissue was quantified in five colonies per sampling event. Approximately 10 mg (± 0.1 mg) of coenenchyme dry weight from each sample were homogenized in 3 ml of chloroform:methanol 2:1, and total lipids were quantified colorimetrically further details in , 54 with-1 of OM, and in percentage of saturated free fatty acids (SFFA), monounsaturated free fatty acids (MUFFA), and polyunsaturated free fatty acids (PUFFA).Five colonies for each sampling event were used to determine the FFA content and composition, following previously used methodology . Approxiδ13C and δ15N) composition of the gorgonian tissue was assessed from monthly samples of three colonies. Approximately 2 mg (± 0.001 mg) of coenenchyme dry weight from each sample was acidified with HCl 1 M during 48 h to eliminate carbonates, and the δ13C composition was determined with Thermo Finnigan EA1108 analyzer and a Thermo Finnigan MAT253 spectrometer. Finally, approximately 2 mg (± 0.001 mg) of coenenchyme dry weight from each sample was directly analyzed with the Thermo Flash EA112 analyzer and the Thermo Delta V advantage spectrometer to determine the δ15N composition.The stable isotope (SI)  were ordinated by means of a principal component analysis (PCA) performed on transformed data .The recorded ratio of male to female colonies was 1.41 (36/51) and did not significantly deviate from 1:1  of the coenenchyme dry weight, with seasonal fluctuation ranging from 20.7 ± 2.5% in autumn to 34.1 ± 3.4% in summer . Summer -1 OM (mean ± SD), with significantly higher values  in summer (1778 ± 504 μg lipid mg-1 OM)  .(n-3) and 18:4(n-3).FFA concentration progressively increased from mid autumn (October) to late summer (August) . PUFFA aδ13C  and δ15N  (δ13C ranged between -21.6 ± 0.3% (mean ± SD) in winter to -21.9 ± 0.1% in summer. The δ15N ranged between 5.2 ± 0.5% (mean ± SD) in winter to 4.7 ± 0.4% (mean ± SD) in summer.SI composition showed no significant differences amongst seasons in both = 0.242) . The δ13Paramuricea macrospina to sexually reproduce annually, with larval development inside the female polyps , and larval release occurring in autumn. Gonochorism of P. macrospina colonies, and 1:1 population sex ratio, agree with the general pattern previously observed in shallow Mediterranean gorgonian species , [, [Paramuerranean  and TablP. macrospina is delayed 2–3 months with respect to shallow Mediterranean gorgonian species which generally spawn during late spring—early summer [L. sarmentosa, a common inhabitant of the Mediterranean continental shelf [Spawning of male gametes and larval fertilization in the studied mesophotic population of y summer , 20, 61.al shelf , also pral shelf .P. macrospina spawning. This might support that timing in P. macrospina reproductive cycle is conditioned by this late increase in seawater temperature occurring on the Mediterranean continental shelf. Reproductive timing was also related with the increase in seawater temperature at 50 m depth in two mesophotic coral species in the Red Sea, with spawning occurring in late summer [P. macrospina, it is also interesting that larval release in September and October also coincides with the beginning of the autumn phytoplankton bloom in the study area [Reproductive timing has been suggested to be conditioned by seawater temperature , 84, sine summer . In the udy area , which cP. macrospina showed smaller oocytes , in highly hydrodynamic environments such as P. clavata [P. macrospina, which has smaller colonies mainly distributed in lower densities (3.2 ± 5 colonies m-2) over maërl beds of the outer continental shelf where hydrodynamism is less intense [P. macrospina reproductive output in the Marmara Sea, where it occurs in shallow environments with similar temperature conditions but stronger hydrodynamism [When compared to other internal brooding species,  oocytes , only exAcabaria , 74. The species , which g brooder . Differeorallium , 80, 90,logorgia . Howeverlogorgia , 91. Thu clavata , 89. Con intense . Future dynamism .P. macrospina showed little seasonal variation with higher values during summer, coinciding with the progressive increase of sexual product volume  colonies of P. clavata [Eunicella singularis [13C and ∂15N composition of P. macrospina tissue  from the heterotrophic E. singularis (60 m depth), P. clavata and P. macrospina   . This fa.9 ± 0.6 ) could rërl beds , such asP. macrospina presented a marked seasonal change. Indeed, the progressive increase of FFA content from mid autumn to summer , which is mainly characterized by SFFA markers during winter, and by PUFFA markers during spring and summer  ((n-3), 20:4(n-3) and 22:6(n-3) (ESM1) during gamete development could be directly related to the increased fecundity, fertility and egg quality [(n-3) and 24:0 predominate during late summer and mid autumn (ESM1), when larvae are present inside the female polyps. The 18:3(n-3) is an essential FA that can be converted into the high energy and biologically active FFA 20:5(n-3) and 22:6(n-3) [(n-3) to fulfil their metabolic demands [Unlike the lipid content, the energetic requirements (FFA content and composition) of mer Figs  and 9, imer Figs , suggestcreases) . Besides quality , 101. On2:6(n-3) . In this demands .P. macrospina from a mäerl bed at ~70 m depth occurs 2–3 months later than in shallow coastal gorgonian species [P. clavata. The differences in the habitats where the two species occur, poses the question about the possible adaptive advantage of their respective reproductive strategy.Reproduction of  species , 20, pro species . The sexP. macrospina’s mesophotic population. This contrasts with the strong seasonality observed in shallow gorgonian species [Dampening of environmental variability with depth  is refle species .P. macrospina’s metabolic requirements (with a mobilization of high-energy PUFFA in spring and early summer). Conversely, larval development in the maternal polyp does not appear to require high metabolic demands.Gametogenesis increased the S1 Table(PDF)Click here for additional data file.