MCD) is a major regulator of fatty acid oxidation catalyzing the decarboxylation of malonyl coenzyme A . Although its involvement in human diseases has been well studied, its role in intramuscular fat (IMF) deposition remains unknown. In this present study, 1726 bp of MCD cDNA was cloned (OM937122) from goat liver, including 5′UTR of 27 bp, 3′UTR of 199 bp, and CDS of 1500 bp, encoding 499 amino acids. In this present study, although the overexpression of MCD increased the mRNA expression of FASN and DGAT2, the expression of ATGL and ACOX1 was also activated significantly and resulted in a decrease in cellular lipid deposition in goat intramuscular preadipocytes. Meanwhile, the silencing of MCD increased the cellular lipid deposition and was accompanied by the expression activation of DGAT2 and the expression suppression of ATGL and HSL, despite the expression suppression of genes related to fatty acid synthesis, including ACC and FASN. However, the expression of DGAT1 was not affected significantly (p > 0.05) by the expression alteration of MCD in this present study. Furthermore, 2025 bp of MCD promoter was obtained and predicted to be regulated by C/EBPα, SP1, SREBP1, and PPARG. In summary, although different pathways may respond to the expression alteration of MCD, the expression of MCD was negatively correlated with the cellular lipid deposition in goat intramuscular preadipocytes. These data may be beneficial for enhancing our understanding of the regulation of IMF deposition in goats.Malonyl-CoA decarboxylase ( Goat meat is increasingly popular because of its high protein, high calcium, and low cholesterol. Thus, the quality of goat meat is in higher demand. Intramuscular fat (IMF) content affects meat tenderness, flavor, etc. Increasing IMF is an important way to improve goat meat quality ,2.Many adipose-related genes and substances are known to affect lipid deposition . MalonylMCD in the liver reversed high-fat-diet-induced hepatic and muscle insulin resistance in rats [Malonyl-CoA decarboxylase, which catalyzes the decarboxylation of malonyl-CoA, was first characterized from the goose uropygial gland  and is h in rats . In addi in rats , and isc in rats ,18. MCD  in rats  and lung in rats .MCD knockdown and overexpression in goats. These data may provide important evidence for the role of MCD in intramuscular fat deposition.Although current studies suggest that MCD is an essential regulator of lipid metabolism in humans or rats, little is known about its role in ruminants. In addition, no studies on the MCD promoter have been reported. Our hypothesis was that MCD played an important role in regulating intramuscular fat deposition. In this present study, we elucidated the function of MCD in regulating cellular lipid metabolism using the treatment of Two two-day-old male goats were purchased from Sichuan Jianyang Dageda Aminal Husbandry Co., Ltd. . The animal studies were approved by the Southwest Minzu University Animal Care and Use Committee. Goat intramuscular precursor adipocytes were isolated as previously described . BrieflyMCD.The third-generation cells were treated with 50 μmol/L oleic acid  and harvested on days 0, 1, 3, and 6. These samples were used to explore the lipid deposition in goat intramuscular preadipocytes during differentiation and the mRNA expression levels of MCD gene in GenBank (XM_018061772) using Primer Premier 5.0 software . PCR was performed using the touchdown PCR method. PCR reaction system: PrimeSTAR® Max DNA Ploymerase  12.5 μL, cDNA 1 μL, upstream primer 1 μL, downstream primer 1 μL, and ddH2O 8.5 μL. The procedure is as follows: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 s, 68 °C annealing for 30 s, 72 °C extension for 90 s, each cycle minus 1 °C, 13 cycles; 98 °C denaturation for 10 s, 55 °C annealing for 30 s, 72 °C extension for 90 s, extension at 72 °C for 5 min, storage at 4 °C. Then, PCR product was added to 2 μL 2x Taq 70 °C extension for 30 min. The PCR products were ligated with pMD® 19-T  vector and sequenced by Beijing Qingke Biotechnology Co. We used Blastn (NCBI) for homology analysis and MEGA5.0 software for constructing phylogenetic trees.Goat liver tissue preserved in the laboratory was removed from liquid nitrogen. After grinding with liquid nitrogen, 1 mL Trizol  was added for RNA extraction. cDNA was obtained according to the reverse transcription kit’s  instructions, and the primers were designed according to the predicted sequence of the goat MCD gene sequence obtained from the previous cloning, subclonal primers were designed . The upstream and downstream primers were added to the Hind III and BamH I sites (underlined), respectively, and synthesized by Shanghai Bioengineering Co. PCR amplification was performed using pMD19-T-MCD plasmid as a template. PCR reaction system: LA Taq enzyme  12.5 μL, pMD19-T-MCD plasmid 1 μL, upstream primer 1 μL, downstream primer 1 μL, and ddH2O 8.5 μL. The procedure is as follows: 94 °C pre-denaturation for 4 min; 98 °C denaturation for 15 s, 64 °C annealing for 30 s, 72 °C extension for 70 s, each cycle minus 1 °C, 13 cycles; 98 °C denaturation for 15 s, 51 °C annealing for 30 s, 72 °C extension for 70 s, extension at 72 °C for 5 min, and storage at 12 °C. PCR products and pcDNA3.1 plasmids were double-digested. Digestion reaction system: pcDNA3.1 plasmids or PCR products 1 ug, K buffer 2 μL, Hind III 1 μL, BamH I  1 μL, and supplemented with ddH2O to 20 μL. The double-enzyme digestion reaction system was placed in a 37 °C water bath for 12 h. The digested products were then purified, and the gene fragment and pcDNA3.1 vector were ligated using T4 ligase  in a metal bath at 16 °C for 12 h. Finally, the recombinant plasmids were double-digested and sequenced for identification. The siRNA was synthesized by GenePharma . The siRNA-MCD sequence is as follows, sense: 5′-GCAGCAUCCAGACAAUCAUTT-3′, anti-sense: 5′-AUGAUUGUCUGGAUGCUGCTT-3′. Negative control siRNA is as follows, sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, anti-sense: 5′-ACGUGACACGUUCGGAGAATT-3′.Based on the When the third-generation cells grew to cover 70–80% of the bottom, the cells were transfected with pcDNA3.1-MCD (OMCD), pcDNA3.1 (ONC), siRNA-MCD (siMCD), and negative control siRNA (siNC), respectively, according to the instructions of Lipofetamine 3000 Transfection Kit . In this study, transfer into pcDNA3.1-MCD plasmid (OMCD) or siRNA-MCD (siMCD) was used as the experimental group, and transfer into pcDNA3.1 plasmid (ONC) or negative control siRNA (siNC) was used as the control group. The cells were first starved, the medium was discarded, washed three times with pre-cooled PBS, 900 μL Opti medium was added to each well of the 6-well plate, and the cells were placed in a constant temperature incubator at 37 °C for 4 h. After the starvation treatment, premixes were prepared for transfection. Premix preparation for overexpression experiment: Premix A was prepared by adding 3 μL lip3000 to 50 μL Opti medium. Premix B was prepared with 50 μL Opti medium, 2.5 μL P3000, and 1 μg plasmid (pcDNA3.1-MCD or pcDNA3.1). Premix A and Premix B were mixed thoroughly and let to stand at room temperature for 20 min. Premix preparation for interference experiment: 100 μL Opti medium, 6 μL lip3000, and 4 μL siRNA were mixed well and allowed to stand at room temperature for 10 min. After the premix was finished standing, it was suspended and added dropwise to complete the cell transfection for overexpression and interference experiments. After 6 h of transfection, cells were incubated with a final concentration of 50 μmol/L oleic acid (Sigma) induction solution. Following 48 h of culture, cells were harvested.−ΔΔCt method.Extraction of total RNA from cells was completed using Trizol reagent . The concentration and integrity of total RNA were determined using a Nanodrop 2000  and 2% agarose gel electrophoresis, respectively. Then, 1 μg of RNA was taken to synthesize cDNA by Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). RT-qPCR primers are listed in Cells were first washed three times with pre-chilled PBS and then fixed with 10% formaldehyde solution for 30 min. Formaldehyde was discarded, PBS was washed 3 times, and Oil Red O staining solution was added and rested at room temperature for 20 min. Observation of lipid droplet deposition in cells was completed using microscope. Finally, Oil Red O was dissolved in 60% isopropanol, aliquoted into 96-well plates, and quantified at 510 nm using a colorimeter.The triglyceride concentration of the samples was measured according to the instructions of Tissue Triglyceride (TAG) Content Assay Kit . After treating the cells for 48 h, the cells were washed three times with pre-chilled PBS, and 200 μL of cell lysate was added to each well of the 6-well plate and placed on ice for 10 min. The cells were scraped into a centrifuge tube, centrifuged at 2000 r/min for 5 min, and the supernatant was removed and placed in a water bath at 70 °C for 10 min. After cooling to room temperature, the samples to be measured were mixed with the working solution and incubated at 37 °C for 15 min. The OD value was measured at 550 nm, and the standard curve was plotted to calculate the triglyceride concentration of each sample.ELISA experiments were performed according to the instructions of Goat malonyl-CoA (MCA) ELISA Kit  to detect the concentration of malonyl-CoA in each sample. First, the appropriate amount of lysate was added to the cell samples, placed on ice for 10 min, and aspirated into a centrifuge tube. Subsequently, the samples were centrifuged at 2500 r/min for 5 min, and the supernatant was aspirated as the sample to be tested. After washing 5 times with washing solution, 50 μL each of color developers A and B was added and incubated for 15 min at 37 °C. A total of 50 μL of termination solution was added after equilibration to room temperature, the OD value was measured at 450 nm, and the standard curve was plotted to calculate the malonyl-CoA concentration of each sample.The total protein was measured with the BCA protein concentration assay kit , which was used for triglyceride and malonyl-CoA level standardization.2O 11.6 μL, and back genomic DNA template 1 μL. PCR amplification procedure: pre-denaturation at 94 °C for 4 s, denaturation at 94 °C for 10 s, annealing at 67 °C for 30 s, extension at 72 °C for 100 s, each cycle minus 1 °C, 12 cycles; denaturation at 94 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 100 s, extension at 72 °C for 10 min, storage at 12 °C. The products were finally identified by sequencing. JASPARA (https://jaspar.genereg.net/) (accessed on 12 October 2022) and gene-regulation  (accessed on 12 October 2022) were used to predict transcription factor binding sites of MCD promoter.To obtain the MCD promoter, the primers  were designed after referring to the MCD-genomic DNA sequence in NCBI. Goat longissimus dorsi tissue kept in the laboratory was removed from liquid nitrogen, placed in a mortar containing liquid nitrogen, and ground to a powder. It was scraped into a centrifuge tube and treated with ultrasound to form a cell suspension. Then, the instructions of the blood/cell/tissue genomic DNA extraction kit  were followed to finally obtain DNA. PCR system: LA Taqase  0.2 μL, 10x Buffer 2 μL, dNTP Mix 3.2 μL, upstream and downstream primers 1 μL each, ddHt-test in SPSS 20.0 software . A difference of p < 0.05 is considered significant, while a difference of p < 0.01 is considered extremely significant.All results were expressed as “mean ± SD” and analyzed based on a MCD (MLYCD) gene was 1726 bp (OM937122), including 5′UTR of 27 bp, 3′UTR of 199 bp, and CDS of 1500 bp, encoding 499 amino acids. Blastn analysis showed that the sequence (CDS) similarity between goat MCD and homo sapiens (AF090834.1), Ovis aries (XM_004015497.5), Bos indicus (XM_019979237.1), and Sus scrofa (XM_021093741.1) was 86.33%, 99.20%, 96.93%, and 88.53%, respectively. The phylogenetic tree showed that goat MCD was most closely related to sheep, followed by bovine, pig, and human, and most distantly related to rat and mouse , ATGL (p < 0.01), FASN (p < 0.01), and ACOX1 (p < 0.01) were significantly up-regulated, while the expressions of HSL (p < 0.05), CPT1A (p < 0.01), and CPT1B (p < 0.01) were significantly down-regulated. DGAT1 and ACC were not affected by MCD overexpression  compared to the control group , ACC (p < 0.01), FASN (p < 0.01), ACOX1 (p < 0.05), and CPT1A (p < 0.05), while the expression of DGAT2 (p < 0.01) was increased. The expression of DGAT1, ATGL, and CPT1B was not affected by MCD silencing . Bioinformatics analysis showed that the MCD promoter has transcription factor binding sites, such as specificity protein 1 (SP1), sterol regulatory element binding protein 1 (SREBP1), CCAAT/enhancer binding protein α (CEBPα), peroxisome proliferator-activated receptor γ (PPARG), and Nuclear Factor Y (NF-Y). In addition, the analysis revealed the presence of GC-box and CCAT-box on the MCD promoter , which may release much more free fatty acids [ACOX1 and decreased expression of CPT1s may illustrate that it was the peroxisome, but not the mitochondria, that responded to the increased release of exact free fatty acids induced by MCD overexpression. Meanwhile, much more free fatty acids may also provide ligands for the regulators of lipid synthesis [MCD by directly binding to the promoter according to the promoter analysis results. This may explain the increasement in FASN and DGAT2. However, the lower concentration of malonyl-CoA (the substrate for lipid synthesis) by MCD overexpression hampers lipid synthesis [To explore the mechanism by which MCD inhibits intramuscular fat deposition in goats, we next performed an expression analysis of genes related to lipid metabolism. The balance between lipid synthesis and lipolysis contributes to cellular lipid deposition. For lipid synthesis, the synthesis of new lipids begins with ACC-catalyzed carboxylation of acetyl-CoA to form malonyl-CoA, which is used as a substrate for FSNA-catalyzed production of fatty acids and finally for DGAT-catalyzed production of TAG . For lipxidation  or oxidaxidation . Inhibitatocytes . In thisty acids . The incynthesis . These rynthesis ,30, may ynthesis  despite MCD activated the lipogenesis (increased expression of DGAT2 [ATGL and HSL), resulting in the lower concentration of cellular free fatty acids and downregulated expression of fatty acid synthesis by FASN and ACC. Meanwhile, the increased concentration of malonyl-CoA may explain the inhibition of fatty acid oxidation mediated by CPT1s [DGAT2, these data underlined the positive role of lipid deposition induced by MCD suppression in goats.We also observed that consistent with the increased cellular lipid accumulation, the silencing of of DGAT2 ) and supby CPT1s . AlthougMCD gene. The expression alteration of MCD may negatively regulate cellular lipid deposition, with the involvement of C/EBPα, SP1, SREBP1, and PPARG. Understanding the regulatory role of MCD in intramuscular fat deposition in goats may help improve goat meat quality.In conclusion, we cloned 1726 bp of the CDS sequence and 2025 bp of the promoter sequence of the goat


The efficacy of scavenging alkyloxy- and alkylperoxy-type free radicals via hydrogen-atom-transferring property was in the following order: gelatin > gelatin–vanillin > gelatin–syringaldehyde > gelatin–anisaldehyde. Electron-donor properties determined using the ABTS assay revealed the following order in one-electron reduction of ABTS•+: gelatin > gelatin–anisaldehyde > gelatin–vanillin > gelatin–syringaldehyde.The present study aimed at preparing novel free-radical scavenging and water-soluble compounds derived from gelatin. Specifically, gelatin–syringaldehyde, gelatin–anisaldehyde, and gelatin–vanillin were synthesized and thoroughly studied for their physicochemical properties. In particular, the compounds were characterized by UV-Vis spectroscopy, Fourier-transform infrared spectroscopy, and scanning electron microscopy. Notably, as demonstrated by thermogravimetry and differential scanning calorimetry, all three derivatives exhibited higher thermal stability than gelatin itself. Free-radical scavenging activities of the examined compounds were explored by (i) a standard spectrophotometric ABTS assay and (ii) an assay of oxidative degradation of hyaluronic acid monitored by rotational viscometry. We found that gelatin and gelatin–syringaldehyde demonstrated the highest efficacy in scavenging  Collagen is a water-insoluble protein presented in animals’ connective tissues such as tendons, cartilage, and bones . Gelatin•OH groups in polyvinyl alcohol [Gelatin is a low-cost, commercially available material . Compare alcohol ,12. Furt2 groups of gelatin. These modifications resulted in an improvement in the antibacterial properties of the given gelatin derivatives [There are various ways to modify native gelatin, including modifications by phenolic aldehydes, among others, and those performed with p-anisaldehyde, syringaldehyde, or vanillin. The modification reaction is based on the immobilization of the reactant to the matrix of biopolymers by combining the phenolic aldehyde group with free –NHivatives ,9,13.p-Anisaldehyde , one of the isomers of anisaldehyde, is an organic compound commonly found in synthetic and natural fragrances . It is aSyringaldehyde , belonging to the group of flavonoids, is a naturally occurring compound with various bioactive characteristics. Major natural sources of syringaldehyde are lignin and the cell wall of such plants as Manihot esculenta and Magnolia officinalis ,18. SyriVanillin, a natural phenolic compound extracted from the pods of Vanilla planifolia orchid, is one of the most extensively used flavoring agents in the food, beverage, and cosmetics industries . ImportaHyaluronan or hyaluronic acid (HA) is a prominent high-molar-mass linear glycosaminoglycan found in ECM, reaching a size of up to 8 MDa. The adult human body contains about 12–15 g of HA, most of which occurs in connective tissue, including skin, the vitreous body of the eye, synovial fluid of articular joints, intervertebral disks, embryonic mesenchymal tissues, and umbilical cord. Hyaluronan is involved in a number of processes, including skin wound healing, tissue repair and regeneration, organization of ECM, joint lubrication, regulation of cell adhesion and motility through receptors that interact with the cytoskeleton, angiogenesis via promoting cell proliferation, cell differentiation, and cell migration. HA also possesses immunomodulatory, anticancer, and antiproliferative properties .Both gelatin and HA are appropriate candidates for pharmaceutical and medical applications, including tissue engineering and regenerative medicine. Their ability to mimic the architecture of ECM has been reported for respective hybrid hydrogels of gelatin and HA. Scaffolds consisting of both polymers were prepared and used for culturing a variety of cells, including vascular endothelial cells, myoblasts, osteoblasts, and stem cells .•OH, alkyloxy- and, alkylperoxy-type radicals using the so-called hydrogen-atom-transferring (HAT) assay and free-electron donating assay via quenching the ABTS•+ probe.The aim of the present study was: (i) to synthesize derivatives of gelatin by coupling it with naturally occurring aldehyde compounds, namely, p-anisaldehyde, vanillin, and syringaldehyde, and (ii) to investigate their properties in scavenging reactive oxygen species such as −1 for gelatin and 3282 cm−1 for the new gelatin derivatives correspond to the intermolecular hydrogen-bonded –OH stretching and –NH stretching in secondary amides (gelatin amide A) [−1 confirmed the presence of –C=O stretching in amides (amide I band). Further, distinct absorption bands obtained around 1430 cm−1 in the range of 1560–1335 cm−1 corresponded to the N–H bending in secondary amides (amide II bands) [−1 was shifted to 1630 cm−1, and a new peak for –C=N formation was generated due to the interaction of amino groups with aldehyde groups of vanillin (at 1527 cm−1), p-anisaldehyde (1526 cm−1), or syringaldehyde (1527 cm−1). In particular, characteristic aromatic bands C=C were observed in the range of 1400–1440 cm−1 in combination with the N–H bending in secondary amides (amide II bands). In addition, the stretching vibration of =C–H in the aromatic ring was observed at 3062 cm−1 for gelatin–anisaldehyde, at 3075 cm−1 for gelatin–syringaldehyde, and at 3080 and 3059 cm−1 for gelatin–vanillin. The bands in the range of 1240–670 cm−1 were attributed to the amide III region of gelatin [−1 for all derivatives. Furthermore, the peaks around 1440 cm−1 confirmed the formation of the gelatin Schiff base [The results of FT-IR depicted in amide A) . For purI bands) . For the gelatin ,26. The lin at 15 cm−1, p-The UV-visible spectra of gelatin A and thrThe thermal stability of gelatin and three selected gelatin derivatives, characterized in detail by SEM cf. , was speDifferential scanning calorimetry DSC, B analysi1H NMR analysis of gelatin, gelatin–anisaldehyde, gelatin–syringaldehyde, and gelatin–vanillin was performed 2) Arg at δ 1.79 ppm, δCH2 (Arg) at δ 2.65 ppm, βCH2 Glut at δ 2.06 ppm, γCH2 (Glu) at δ 2.33 ppm, and γCH2 (Pro) at δ 2.2–2.3 ppm. Hydroxyl protons, αCH2 (Ala) and δCH (Pro), were at δ 3.5–3.7 ppm. The signal at δ 4.09 referred to αCH2 (Gly). The signal at δ 4.4 ppm referred to αCH2 (Pro) [A H2 (Pro) . The couH2 (Pro) ,39.•OH radical-induced HA degradation. The decrease in dynamic viscosity of the HA solution was 5.2 mPa · s within 5 h. When the gelatin solution was added in the volume of 100 µL, minimal prevention of HA oxidative degradation was seen (red curve). The addition of 1000 µL of the gelatin solution (green curve) resulted in a more significant inhibition of HA degradation. The dynamic viscosity of the HA solution dropped by only 2.22 mPa · s. Similar results are shown in Results in •OH radicals, gelatin and gelatin–syringaldehyde were shown to be the most potent: they scavenged 57% of these radicals. A slightly weaker effect was shown for the derivatives gelatin–vanillin (52%) and gelatin–anisaldehyde (45%). As for the scavenging of alkyloxy- and alkylperoxy-type radicals, the most potent of these was gelatin, followed by gelatin–vanillin, gelatin–syringaldehyde, and gelatin–anisaldehyde. These results indicate that gelatin and its derivatives exerted their H-atom-transferring properties.•+ percentages were 64.7% and 63.3%, respectively, when gelatin and gelatin–anisaldehyde were added at their highest concentration, i.e., 20 mg/mL. In comparison, gelatin–syringaldehyde (panel C) revealed a higher potency for scavenging ABTS•+. The amounts of unscavenged ABTS•+ were 68%, 50%, and 40% when adding this compound at concentrations of 5, 10, and 20 mg/mL, respectively. Finally, gelatin–vanillin was found to be the most effective derivative for scavenging ABTS•+ (panel D); the amounts of unscavenged ABTS•+ were the lowest among the examined compounds. Thus, gelatin–vanillin was revealed to be the most effective electron donor compound of all the tested compounds in the study.As depicted in 1H NMR, thermoanalytical methods), thermogravimetry, differential scanning calorimetry, and the microscopic method (SEM), an original rotational viscometric method has been employed to examine the free-radical properties of all four compounds. The latter method has been applied to compare free-radical inhibition properties between native gelatin and novel gelatin–aldehyde derivatives.The novelty of the present study is the design, preparation, and essential physicochemical characterization of the novel gelatin-based aldehydes. Along with spectrophotometric methods  [•OH generation in Fe2+-mediated Fenton reaction. In contrast, gelatin in the same concentration range hardly inhibited formed •OH radicals. He et al. (2002) [•OH-induced apoptosis of HeLa cells more effectively than gelatin.The results of the present study demonstrated that gelatin and its derivatives were effective at given concentrations in inhibiting reactive-oxygen-species-mediated HA degradation. Few papers report the efficacy of gelatin for scavenging . (2002)  and Xiao. (2002) , using e. (2002)  showed tw = 1.69 MDa, Mw/Mn = 1.63) was purchased from Lifecore Biomedical Inc., Chaska, MN, USA. Gelatin powder  was purchased from Fluka, Buchs, Switzerland. Syringaldehyde (98%), vanillin (98%), and p-anisaldehyde (98%) were supplied from Chemical Engineering Co., Beijing, China. CuCl2·H2O p.a., NaCl p.a., ethanol p.a. (96%), and acetone p.a. were purchased from Slavus Ltd., Košice, Slovakia. Ascorbic acid was from Merck KGaA, Germany. 2,2′-Azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) diammonium salt was obtained from Sigma-Aldrich Chemie GmbH, Steinheim, Germany. Deionized high-purity-grade water, with a conductivity of ≤0.055 µS/cm, was made using the TKA water purification system .High-molar-mass hyaluronan . Further, 0.3 g of the given aldehyde was dissolved in 10 mL of ethanol at ambient temperature, followed by dropwise addition to gelatin solution under stirring. The mixture was heated to 70 °C and stirred for 6 h, and the solution converted from colorless or faintly yellow to yellow due to the formation of a Schiff base. Finally, acetone was added to form a precipitate that was subsequently isolated from the reaction mixture by filtration. The final product was washed several times with ethanol to remove the excess aldehyde reagents and was then dried at 60 °C in an oven. The product yields ranged from 94% to 99%. −1 with a resolution of 2 cm−1 in absorbance mode.A Fourier-transform infrared spectrophotometer  was used to analyze gelatin and its novel derivatives. All samples were freeze-dried using liquid nitrogen, crushed to a fine powder (KBr: sample = 140:2 mg/mg), and pressed into a transparent disk with a diameter of 13 mm by applying a 105 N force. The FT-IR spectra were obtained by recording 64 scans from 4000 to 400 cmThe UV-Vis spectra of the gelatin and Schiff base derivatives were determined using a spectrophotometer . The samples of gelatin and gelatin derivatives (0.05 g) were dissolved in 10 mL of distilled water under heating. Absorbances of the solutions in a quartz cell were scanned from 200 to 600 nm.Scanning electron microscopy (SEM) was applied to examine gelatin, gelatin–anisaldehyde, and gelatin–syringaldehyde after coating them with a thin layer of gold under vacuum. Morphological changes of the sample’s surface were followed using a secondary electron detector of SEM  was carried out using a thermogravimetric analyzer device  at a temperature range of 25–500 °C with a heating rate of 10 °C/min under nitrogen flow (20 mL/min).Differential scanning calorimetric analysis of gelatin and its derivatives  was carried out using a differential scanning calorimeter device  at a temperature range of 25–400 °C with a heating rate of 10 °C/min under nitrogen flow (30 mL/min).1H NMR spectra of gelatin and novel gelatin derivative molecules were obtained using 500 MH Jeol . 1H NMR spectra were also obtained separately in 0.5 wt. % D2O to characterize the structure of the molecules.The Hyaluronan (HA) was dissolved overnight in 0.15 M aqueous NaCl as follows: First, 4.0 mL of solvent was added to HA (16 mg). Then, after 6 h, an additional 3.9, 3.8, or 2.9 mL of 0.15 M NaCl was added. Stock solutions of ascorbic acid (16 mM) and cupric chloride (160 µM) were prepared in 0.15 M NaCl. To prepare stock solutions of gelatin and its derivatives, the compounds (0.1 g) were dissolved in 10 mL of deionized water under heating at 50 °C.2 (1 µM) and ascorbic acid (100 µM). The procedure was as follows: CuCl2 stock solution (50 µL) was added to the HA solution (7.90 mL) and stirred for 30 s. After 7.5 min without stirring the reaction mixture, the stock solution of ascorbic acid (50 µL) was added to the HA solution, stirred for 30 s, and the mixture was immediately transferred into the viscometer Teflon® vessel.HA degradation was induced by the oxidative system consisting of CuCl(a)2 (50 µL) was added to the HA solution (7.8 or 6.9 mL) and stirred for 30 s. After 7.5 min without stirring, 100 or 1000 µL of gelatin or its derivatives was added and stirred for 30 s. Finally, the stock solution of ascorbic acid (50 µL) was added, stirred for 30 s, and the reaction mixture was immediately transferred into the viscometer Teflon® vessel. Under these conditions, the compounds were examined for their ability to inhibit the initiation phase of HA degradation.In the first set of experiments, the stock solution of CuCl(b)® vessel. Under these conditions, the compounds were tested for their ability to inhibit the propagation phase of HA degradation.In the second set of experiments, a similar procedure as described in (a) was applied. However, after 7.5 min, 50 µL of stock solution of ascorbic acid was added, followed by stirring the HA mixture for 1 h. Then, 50 µL of the stock solution of gelatin or its derivatives was added and stirred for 30 s. The HA solution was immediately transferred into the viscometer TeflonProcedures to explore gelatin and its derivatives as inhibitors of HA degradation were as follows:−1, and a temperature of 25 °C. The data were recorded in 3 min intervals using a rotational viscometer .The changes in the dynamic viscosity value were monitored for 5 h. The parameters of the measurements were 180 rpm, a shear rate of 237 s●+ probe was prepared at room temperature 24 h before the measurements as follows: ABTS aqueous stock solution (7 mM) was mixed with K2S2O8 aqueous solution (2.45 mM) in the equivolume ratio. On the next day, 1 mL of the generated ABTS●+ solution was diluted with deionized high-purity grade water to the volume of 60 mL [ABTSof 60 mL ,43.●+ solution was mixed with 50 µL of the stock and working solutions of gelatin and its derivatives . The light absorbance was recorded in triplicate within 15 min at the wavelength of 734 nm using UV-Vis 1800 spectrophotometer .Gelatin and its derivatives were dissolved in water yielding the stock concentrations of 20 mg/mL. These solutions were diluted with water to reduced concentrations of 10 and 5 mg/mL. For the measurements, 2 mL of the ABTS


SLC29A3, in 2012 the first gene associated with DSS, a unique homozygous duplication . Patient 2 presented similarly with fractures and metaphyseal osteosclerosis but with no platyspondyly at age 2 months. She was homozygous for a novel nonsense mutation in SLC29A3 . Patient 3 had ocular disease at age 2 years, presented for short stature at age 11 years, and did not begin to fracture until age 16 years. Radiographs showed mild platyspondyly and focal metaphyseal and femoral osteosclerosis. She was homozygous for a unique splice site mutation in TNFRSF11A (c.616+3A>G). Patient 4 at age 2 years manifested developmental delay and frequent infections but did not fracture. He had unique metadiaphyseal splaying and osteosclerosis, vertebral end‐plate osteosclerosis, and cortical thinning of long bones but no mutation was detected of SLC29A3, TNFRSF11A, TCIRG1, LRRK1, or CSF1R associated with DSS. We find that DSS from defective SLC29A3 presents earliest and with fractures. DSS from compromised TNFRSF11A can lead to optic atrophy as an early finding. Negative mutation analysis in patient 4 suggests further genetic heterogeneity underlying the skeletal phenotype of DSS. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Dysosteosclerosis (DSS), the term coined in 1968 for ultrarare dysplasia of the skeleton featuring platyspondyly with focal appendicular osteosclerosis, has become generic by encompassing the genetic heterogeneity recently reported for this phenotype. We studied four unrelated Turkish patients with DSS to advance understanding of the new nosology. Patient 1 suffered femur fractures beginning at age 1 year. DSS was suspected from marked metaphyseal osteosclerosis in early childhood and subsequently platyspondyly accompanying patchy osteosclerosis of her appendicular skeleton. She harbored in  During infancy and early childhood the osteosclerosis of DSS is widely distributed but especially pronounced in expanded metaphyses, whereas diaphyses are broad and radiodense or radiolucent. Then, these features of DSS evolve. Expanded osteosclerotic metaphyses become osteopenic, sometimes including portions of the diaphyses where the cortex may be thin. Cranial nerve palsies and developmental delay can occur. Skin changes are sometimes noted. By middle age, there can be diffuse osteosclerosis of cranial bones, mandible, maxilla, mastoid, and skull base. Diagnosing DSS may be delayed because its radiographic hallmarks can go undocumented or misunderstood. In 2010, we reported that DSS in early childhoodrepresents an “osteoclast‐poor” form of OPT wherein some heritable defect transiently abrogates osteoclastogenesis.Dysosteosclerosis (DSS or DOS), considered an osteopetrosis (OPT) when first described in 1934 by Ellis,1 was SLC29A3). The following year, Sule and colleagues found no SLC29A3 defect in one patient, thereby suggesting genetic heterogeneity for DSS. In 2016, Iida and colleagues associated “leucine rich repeat kinase 1” (LRRK1) with osteosclerotic metaphyseal dysplasia  featuring characteristics of DSS. In 2018, two genes underlying autosomal recessive OPT became associated with DSS: (i) “tumor necrosis factor receptor superfamily member 11A” (TNFRSF11A) that encodes receptor activator of NF‐κB (RANK) (OMIM # 612301), and (ii) “T‐cell immune regulator 1” (TCIRG1) that encodes a component of the vacuolar proton (H+) pump necessary for osteoclasts (OCs) to produce hydrochloric acid (OMIM # 259700). Most recently, in 2019, biallelic mutations of “colony‐stimulating factor 1 receptor” (CSF1R) were associated with dysosteosclerosis‐Pyle disease (DSS‐PD), also called “brain abnormalities, neurodegeneration, and dysosteosclerosis” (BANDDOS: OMIM # 618476).Delineation of the etiology of DSS began in 2012 when we reported two girls who harbored biallelic mutations of the nucleoside transporter gene “solute carrier family 29 member 3” (SLC29A3).5 The SLC29A3 defects, one a novel homozygous TNFRSF11A change, and one for whom no mutation was found.Herein, we characterize the phenotype of four young unrelated Turkish patients with DSS. Two carried novel homozygous The principal clinical and radiological features and results from mutation analyses of our four patients are detailed below and summarized in Table SLC29A3. In 2015, we briefly reported her features of DSS and unique mutation. Her parents were first‐degree cousins. At age 4 months, severe anemia (hemoglobin [Hb] 3 mg/dL) was attributed to OPT despite no hemolysis or extramedullary hematopoiesis. Subsequently, folic acid supplementation  corrected this problem. Fracturing began at about age 1 year. Shedding of her deciduous teeth was delayed. Neurodevelopmental milestones were normal with good grades at school. Four of at least seven femoral breaks required surgery, including one to remove metal plates. A supracondylar specimen of femur reportedly showed focal, irregular, lytic, and dysplastic enchondral bone adjacent to mature compact bone. Skeletal scintigraphy at age 12 years revealed symmetrically increased radionuclide uptake attributed to increased osteoblastic activity at the proximal and distal femurs and at the proximal humeral and tibial metadiaphyses. Sequential dual‐energy X‐ray absorptiometry (DXA) demonstrated  markedly elevated but decreasing areal bone mineral density (aBMD) at the L1–L4 spine; +10.8 at age 7 years, +6.3 at age 10 years, +6.4 at age 15 years, +8.7 at age 17 years, +7 at age 22 years, and +5.6 at age 23 years. Bone turnover markers (BTMs) spanning ages 11 to 23 years included elevated urinary deoxypyridinoline , but normal serum alkaline phosphatase , C‐terminal telopeptide , and procollagen type 1 N‐terminal propeptide (P1NP) . Serum osteocalcin (OCN) levels  ranged from low to high , arm span 159 cm; and arm span‐height difference 12.7 cm (Nl −6.3 to +6.5). She weighed 53 kg (−0.7 SD). Mid‐parenteral height was 156 cm, and her healthy 30‐year‐old and 28‐year‐old sisters were 158 cm and 161 cm tall, respectively. Disproportionate short stature included a short trunk, but prior femoral fractures explained her nearly normal upper‐lower segment ratio of 0.8 (Nl 0.88–1.1). Mild prognathism, prominent nose, short philtrum, small maxillary lateral incisors, and broad shoulders were apparent. At age 18 years, hypertrichosis/hirsutism  was without hyperandrogenemia or menstrual irregularity. Audiology and fundus and visual field examinations were normal. DSS became suspected when her radiographs revealed marked metaphyseal sclerosis that became increasingly generalized from age 1–15 years Fig. . Patchy SLC29A3 mutation. Her parents were second‐degree cousins. Fetal ultrasound indicated no antenatal fractures or deformities. Pregnancy and delivery were uneventful and birth weight was 3270 g. At age 2 months, a fibular fracture prompted referral for “osteogenesis imperfecta.” Height, weight, and head circumference were 57.5 cm (−0.22 SD), 4.7 kg (−0.97 SD), and 38.5 cm (−0.64 SD), respectively. Frontal bossing, open anterior (3 × 4 cm) and posterior (0.5 × 0.5 cm) fontanelles, gray‐blue sclera, mild hypertrichosis, and restricted elbow extension were present. Growth was good with appropriate neurodevelopmental milestones at age 6 months. Audiology, ophthalmology, metabolic and heavy metal screening, and BTMs were normal except for elevated urinary DPD  and weight 23.6 kg (−2.2 SD). Body proportions were normal . Family members were not short, and reportedly without heritable diseases. Pycnodysostosis was suspected because her facial dysmorphism included proptosis, beaked nose, malar hypoplasia, and short philtrum. Nystagmus had been first noticed at age 2 months, and was present at age 18 years when cranial and orbital tomography showed bilateral proptosis and thickening of optic nerve sheet indicating optic canal narrowing. Poor oral hygiene, crowded teeth, and multiple caries were present. Immunoglobulin levels were not measured because there had been no recurrent infections. Femoral fractures, three requiring surgery, began at age 16 years. Adult height was 138 cm (−4.2 SD) with mid‐parenteral height 154 cm (−1.55 SD). Arm span was 151 cm, arm span/height difference 12.2 cm (Nl −6.3 to +6.5 cm), and upper‐lower segment ratio 0.88 (Nl 0.88–1.1) showing that her short stature principally reflected her short trunk. Cranial bones and ribs were osteosclerotic . Then, in 2018, Guo and colleagues reported her the first example of DSS from TNFRSF11A mutation.Patient 3, 21 years old, harbored a unique homozygous mutation in tic Fig. . Platysptic Fig. . Long botic Fig. . Pycnodytic Fig. . DXA of olleagues20 repSLC29A3, TNFRSF11A, LRRK1, TCIRG1, and CSF1R) that had come to be associated with DSS (see Discussion). His parents were unrelated. He weighed 2.2 kg when delivered at 36 weeks gestation to a 25‐year‐old woman who had noticed diminished fetal movement during the last week of her pregnancy. Intensive care for 10 days included respiratory support for 3 days. At age 6 months, slow motor development was reported. At age 14 months, inborn‐error‐of‐metabolism studies were negative, and OMD was considered when radiographs showed metaphyseal osteosclerosis. Remarkably, at age 27 months, fever, cough, and vomiting accompanied unexplained severe hypercalcemia (19.8 mg/dL [Nl 8.8–10.6]) with physiologically suppressed serum parathyroid hormone (PTH), hypophosphatemia, low‐normal ALP, and normal 25‐hydroxyvitamin D . Skeletal deformity was absent, but spasticity and atrophic muscles were present. Skin changes of DSS, such as red rashes, were present. Oral candidiasis was detected although serum immunoglobulin levels, immune phenotyping, nitroblue tetrazolium (NBT) testing, metabolic screening (including phosphoethanolamine for hypophosphatasia), blood gases, and lead level were normal. Adenoidectomy was performed at age 3.5 years. Scoliosis persisted from age 4.5 years. BTMs were unremarkable except for low OCN, but assayed after pamidronate treatment , Ion Torrent (Thermo Fisher Scientific) next‐generation sequencing (NGS) examined 35 genes that: (i) cause osteosclerotic disorders, (ii) condition skeletal remodeling, or (iii) reflect mouse models featuring elevated bone mass: ie, TNFRSF11A (RANK), TNFRSF11B (OPG), TNFSF11 (RANKL), VCP, SQSTM1, TGFB1, IFITM5, MAFB, CSF1, CSF1R, TRAF6, RELA, RELB, REL, NFKB1, NFKB2, TFEB, CA2, CLCN7, CTSK (CATHEPSIN K), OSTM1, PLEKHM1, TCIRG1, SOST, SLC29A3, LRP4, LRP5, LRP6, SNX10, FAM20C, FAM123B (AMER1), TYROBP, LEMD3, DLX3, and PTDSS1. Variants were then verified by Sanger sequencing. For patient 4, LRRK1 was Sanger sequenced using primers we designed (sequences available on request). At least 10 bases of intronic DNA sequence at each exon/intron boundary were sequenced to identify potential messenger RNA (mRNA) splice site mutations. Patient 2 was studied at Marmara University School of Medicine where clinical exome sequencing was performed using SOPHIA Clinical Exome Solution V2 via Illumina Nextseq 550 platform . Twenty‐seven genes: TNFRSF11A (RANK), TNFRSF11B (OPG), VCP, SQSTM1, TGFB1, IFITM5, MAFB, CSF1, CSF1R, TRAF6, NFKB2, CA2, CLCN7, CTSK (CATHEPSIN K), OSTM1, TCIRG1, SOST, SLC29A3, LRP4, LRP5, LRP6, SNX10, FAM20C, FAM123B (AMER1), TYROBP, LEMD3, and DLX3 were filtered. SOPHIA DDM‐V4 analyzed the data. For segregation analysis, we used the Illumina MiSeq platform.Mutation analyses were performed for patients 1, 3, and 4 in our research laboratory at Washington University School of Medicine, St. Louis, MO, USA. Written consent was obtained as approved by the Marmara University Medical Faculty Research Ethics Committee (MAR‐Y‐09.2021‐623) Istanbul, Turkey in accordance with the 1964 Helsinki declaration and its later amendments. Coding exons and exon‐intron boundaries of eveloped.4, 5SLC29A3 revealed a novel homozygous 18‐basepair (bp) duplication in exon 3  predicting in‐frame tandem duplication of six amino acid residues  that was heterozygous in her consanguineous parents  in intron 6 of TNFRSF11A .All exons and adjacent mRNA splice sites of 11A Fig. . This va1A  could not be undertaken for this patient.Our mutation analysis was negative for olleagues22 in Our four patients' clinical, radiographic, and genetic findings indicating DSS Table  and asseThe genotype/phenotype spectrum of DSS was assessed from the literature using Medline, Embase, Scobus, Web of Science, bone abstracts and Mendeley and was then summarized Table . until evidence of genetic heterogeneity in 2013, DSS seemed a distinctive Mendelian OPT. In 2010, we described in a girl with classic features of DSS absence of OCs and unresorbed calcified primary spongiosa emblematic of OPT. In 2012, we reported that she and a similarly affected girl manifested impaired osteoclastogenesis and OC action and discovered they harbored biallelic SLC29A3 mutations. In 2015, The International Skeletal Dysplasia Registry regarded DSS as an OPT‐like disorder.From its first reporting in 19341 unti DSS features acquired platyspondyly and osteosclerosis , but patchy osteosclerosis later on, and osteopenia in adulthood. Hence, DSS has a particularly complex and enigmatic pathogenesis. The changing skeletal phenotype can go undocumented or unappreciated and therefore undiagnosed. By 2022, among 23 individuals with mutational analysis from 45 with the DSS phenotype, six, four, two, and 11 had defects of SLC29A3, TNFRSF11A, TCIRG1, or CSF1R, respectively. The heterogeneity among the clinical and radiological presentations, complications, and prognoses for each etiology is as follows.Although osteosclerosis and impaired bone modeling  characterize the OPTs including DSS, the platyspondyly and spontaneous resolution of the osteosclerosis of DSS do not.10 DSS whereas arm length was unaffected. Disproportionate dwarfism featuring a short trunk became a constant finding by adulthood despite recurrent femoral fracturing. The body proportions of patient 3 changed from age 10 to 17 years, highlighting her progressive trunk shortening. However, platyspondyly with a shortened trunk was not evident early on in our youngest patients 2 and 4. Patient 3's fractures began relatively late; ie, in adolescence.Patient 1, our oldest at 23 years old, provided almost a 20‐year follow‐up, whereas patient 2 is the youngest reported with DSS , perhaps because its unique metabolic acidosis leaches mineral from bone. Patient 4 manifested unexplained marked hypercalcemia once, but was without a CA2 mutation or acidosis. Although no fractures had been detected, perhaps his hypercalcemia reflected his immobility. Hypercalcemia is not a feature of the OPTs unless following restoration of OC action by marrow cell transplantation. In our initial patient harboring SLC29A3 mutations, serum ionized calcium was high‐normal at approximately 1 year old, but decreased after excessive dietary calcium was corrected. In DSS, low serum PTH levels can occur, suggesting mineral homeostasis is impacted. Hypocalcemia from decreased bone resorption and diminished gastrointestinal absorption of calcium underlies “osteopetrorickets” from TCIRG1 deactivation.Herein, we found metaphyseal osteosclerosis in early childhood. However, patient 2 demonstrated it might not be present neonatally, yet appear by age 2 months. For patient 1, followed to age 22 years, metaphyseal osteosclerosis extended as she grew, but mid‐diaphyseal radiolucency persisted up to age 11 years. Then, patchy osteosclerosis with sparse radiolucency involved the diaphyses. At older ages, radiolucent areas predominated, especially after physeal fusion and cessation of growth. Axial osteosclerosis, including of the ribs, pelvis, and vertebrae in patients 1, 3, and 4 was a more constant finding. However, in patient 1 it was decreased during adulthood. In patient 3, osteosclerosis increased steadily with better demarcated focal osteosclerosis, especially mid‐diaphyseal, and fracture‐prone dense bone within osteopenic long bones. Perhaps absence of apparent metaphyseal sclerosis together with unaffected hand and forearm bones indicates DSS due to  type 3),3 perh as in other OPTs. Indeed, quiescent bone remodeling was suggested in our four patients by low serum OCN levels and low‐normal ALP activity. Patients 1 and 2 had elevated urinary DPD, a marker of bone resorption, whereas serum CTX levels were normal. However, BTMs were inconsistent among our four patients, perhaps reflecting their genetic heterogeneity for DSS as well as evolving skeletal phenotype.Slow fracture healing can occur in DSS,4 as iSLC29A3 mutation. The former had Turkish heritage. Their few OCs formed from peripheral blood weakly demineralized a crystalline calcium‐phosphate surface. Low serum tartrate‐resistant acid phosphatase (TRAP) matched a paucity of OCs. Therefore, OC formation and function in DSS can become impaired and increase bone mass, but then apparently recover sufficiently to resorb calcified primary spongiosa and osteosclerosis. Currently, however, OMIM does not mention our 2010 report of OC‐poor OPT in DSS and considers the two patients examples of “H syndrome” or “histiocytosis‐lymphadenopathy plus syndrome” (602782), which features biallelic SLC29A3 mutation causing short stature (but not fracturing), histiocytosis, and lymphadenopathy with or without cutaneous, cardiac, and/or endocrine features , joint contractures, and/or deafness. Now, six individuals have been reported to have DSS from SLC29A3 mutation.The two unrelated girls we reported in 2012 with DSS were compound heterozygous  and homozygous  for mutation.5 The  homozygous mutation of TNFRSF11A encoding RANK was reported to cause “OC‐poor” OPT. About 20 examples are published. However, in 2018 Guo and colleagues specified that our patient 3 had DSS. Now, they attribute the DSS phenotype to four individuals with biallelic loss‐of‐function defects in TNFRSF11A. Splice site mutations of TNFRSF11A, exemplified by our patient 3, that truncate or extend the encoded RANK protein can cause DSS whereas “DSS” from TNFRSF11A missense mutation p.R129C featured diffuse osteosclerosis and extramedullary hematopoiesis consistent with a severe OPT. TNFRSF11A mutations that alter N‐terminal folding of the encoded RANK and compromise its interaction with RANKL cause OPT, whereas some functional RANK seems to cause DSS. Nevertheless, the skeletal phenotype of the severe OPT, can also improve with aging. The eldest person (age 59 years) reported with biallelic TNFRSF11A mutations had severely osteopenic long bones.Beginning in 2008,18 homTCIRG1  reportedly caused metaphyseal sclerosis typical of DSS.TCIRG1 encodes a component of the vacuolar proton (H+) pump, and biallelic mutations thereby cause “osteopetrorickets” from an abundance of nonfunctional OCs together with impaired gastrointestinal absorption of calcium due to hypochlorhydria. The OPT from biallelic TCIRG1 defects is “OC‐rich” and treatable by hematopoietic stem cell transplantation that generates functional OCs. The TCIRG1 splice site mutation at the beginning of this paragraph (c.117+4A>C), is homologous to splice site mutation c.117+4A>T reported in 2000 to cause OPT in a Turkish patient. When this mutation accompanied p.A796fs*34 above, the phenotype was milder.In 2018, in two siblings, compound heterozygosity of l of DSS.21TCI harbored homozygous defects of LRRK1. Their clinical and radiographic features of DSS resemble those attributable to SLC29A3 or TCIRG1 mutation, including fractures without severe extramedullary hematopoiesis or short stature. Radiological features include metaphyseal and vertebral sclerosis with mildly under‐modeled long bones that may evolve, including diminishing metaphyseal sclerosis. However, there is no prominent platyspondyly.The eight individuals reported to date with “osteosclerotic metaphyseal dysplasia” (OMIM % 615198)3 harb In 2017, in first‐cousin carrier parents, heterozygous mutation (p.Y540*) of CSF1R was detected. Biallelic CSF1R mutations were identified by Guo and colleagues and Kındış and colleagues in 2019 and 2021, respectively. The 2019 report concerned a homozygous splice acceptor site mutation (c.1754−1G>C), published back‐to‐back by Guo and colleagues. However, these two reports describe an osteosclerotic phenotype atypical for DSS. In fact, biallelic CSF1R defects do not always have a bone phenotype; eg, the second case, harboring p.H643Q, reported in 2019 by Oosterhof and colleagues, and two siblings with the p.T833M mutation. Monoallelic CSF1R mutation underlies hereditary diffuse leukoencephalopathy with spheroids (HDLS)—a rapidly progressive lethal neurodegenerative disease of adults that features cerebral white matter, behavioral, cognitive, and motor changes as well as dementia. However, no bone changes were reported in 2021 by Guo and Ikegawa from a relatively large experience. Typical HDLS has occurred with “sclerosing skeletal dysplasia”, with undertubulation and flaring of the metaphyses of most of the tubular long bones and fish‐shaped vertebrate. An osteosclerotic adolescent boy with HDLS reported in 2020 by Breningstall and Asis was heterozygous for CSF1R p.Q481*. His radiographs, kindly provided to us by Dr. G.N. Breningstall (unpublished), showed platyspondyly and osteopenic long bones with Erlenmeyer flask deformity and focal osteosclerosis. His CSF1R defect was compound heterozygous in a patient with “brain abnormalities, neurodegeneration, and dysosteosclerosis” (BANDDOS: OMIM # 618476). The father, who was heterozygous, had mild cortical hyperostosis and normal cranial tomography, yet short‐term memory loss from age 70 years, and parenchymal calcification were detected in the 76‐year‐old paternal grandfather, who was also heterozygous.The “DSS‐Pyle disease spectrum” features generalized osteosclerosis and irregular dysplastic metaphyses associated with neonatal and infant lethality from leukoencephalopathy, intracranial calcification, Dandy‐Walker malformation, cystic dilation of the posterior fossa and ventricles, and agenesis of the corpus callosum.49 In  Neurodevelopmental delay, macrocephaly, seizures, intracranial calcification, and delayed myelination are features of the DSS‐Pyle disease spectrum. His cranial MRI showed a myelinization defect, but no leukoencephaly or findings of Pyle disease. Microcephaly, not macrocephaly, was present.Our patient 4, lacking identification of a causal gene, uniquely suffered severe mental/motor retardation, infections, and one episode of severe unexplained hypercalcemia. In DSS, infections have been frequent without any detectable immune defect.4, 5SLC29A3 and TNFRSF11A mutations features short stature. However, fracturing sometimes with slow healing , begins earlier and seems more common with SLC29A3 mutations . Metaphyseal sclerosis characteristic of DSS was not observed at different ages in three of four patients harboring TNFRSF11A mutations. The exception differed from DSS because diffuse diaphyseal sclerosis ended with a small area of osteopenia and continued with the osteosclerotic metaphyses.TNFRSF11A‐associated DSS features particularly severe osteosclerosis of the axial skeleton together with optic atrophy, craniosynostosis, and extramedullary hematopoiesis. Contrary to DSS from defective SLC29A3 and TCIRG1, mild TNFRSF11A mutations likely leave hand bones unaltered , whereas severely compromised RANK protein can cause marked osteosclerosis of the entire skeleton including hand bones.The DSS phenotype associated with tient 1),5 begiSLC29A3, TNFRSF11A, TCIRG1, LRRK1, and CSF1R. Further genetic heterogeneity, including an X‐linked recessive form, seems likely. In clinical practice, establishing the genetic basis for DSS will help understand the complications, prognosis, and treatment.Our experience improves understanding of the clinical, radiographic, and genetic heterogeneity of the DSS phenotype. The changing radiographic hallmarks can be important to suspect and diagnose DSS, now associated with mutations of ive form,24 seeSerap Turan: Conceptualization; data curation; investigation; methodology; supervision; visualization; writing – original draft; writing – review and editing. Steven Mumm: Formal analysis; investigation; methodology; validation; writing – review and editing. Ceren Alavanda: Formal analysis; investigation; methodology. Betul Sare Kaygusuz: Data curation; investigation; visualization. Busra Gurpinar Tosun: Data curation; resources; visualization. Ahmet Arman: Data curation; investigation; methodology; validation. Margaret Huskey: Formal analysis; methodology; validation; visualization. Tulay Guran: Data curation; investigation; visualization. Shenghui Duan: Data curation; resources; supervision; visualization. Abdullah Bereket: Methodology; project administration; resources; supervision; writing – review and editing. Michael P. Whyte: Investigation; methodology; project administration; resources; supervision; writing – original draft; writing – review and editing.None.https://publons.com/publon/10.1002/jbm4.10663.The peer review history for this article is available at Appendix S1: Supplementary InformationFigs. S1–S3Table S1Click here for additional data file.


Gardenia florida has been widely used for its antioxidative, hypoglycemic, and anti-inflammatory properties. However, whether G. florida fruit extract (GFFE) regulates hair growth has been rarely studied. This study was the first application of GFFE on hair growth both in vitro  and in vivo (C57BL/6 mice). The effects of GFFE on cell proliferation and hair growth-associated gene expression in hDPCs were examined. Moreover, GFFE was applied topically on the hair-shaved skin of male C57BL/6 mice, the hair length was measured, and the skin histological profile was investigated. GFFE promoted the proliferation of hDPCs and significantly stimulated hair growth-promoting genes, including vascular endothelial growth factor (VEGF) and Wnt/β-catenin signals, but suppressed the expression of the hair loss-related gene transforming growth factor-β1 (TGF-β). Furthermore, GFFE treatment resulted in a significant increase in the number, size, and depth of cultured hair follicles and stimulated the growth of hair with local effects in mice. In summary, the results provided the preclinical data to support the much potential use of the natural product GFFE as a promising agent for hair growth.As a herbal medicine, the extract from the fruits of  Recently, the number of hair loss patients has continued to increase, with a trend in younger people and females , 2. Hairβ-catenin signaling pathway [α-reductase [β [HDPCs play key roles in the proliferation, differentiation, and hair cycle control of hair follicles . Several pathway –20, pros pathway , and pro pathway  stimulateductase –26, TGF-ctase [β , FGF-5 [ctase [β , and proctase [β  are knowα-reductase and is not valid for women [Hair loss is recognized as a common dermatological problem, which significantly influences the patient quality of life and mental state, and the development of treatment agents has been markedly growing. Current drugs available for hair loss treatment include medications such as Minoxidil® and Finasteride® , 30. Minor women , 34.Therefore, the development of new ways to enhance hair growth and prevent hair loss is urgently needed in the hair care industry , 36. RecGardenia florida fruits have been used in antioxidative, anti-inflammatory, and hypoglycemic purposes [β1 and DKK1 decreased; however, the LEF1, Wnt5a, β-catenin, and VEGF increased; we also verified the effects of GFFE treatment on protein expression of TGF-β1, Wnt5a, and VEGF in hDPCs by western blot examination. Then, we investigated GFFE for its potential effect in the C57BL/6 mouse model showing the hair length difference, and histological findings verified that retardation of entry into catagen occurred in response to GFFE application. Taken together, these data considered that GFFE may be a new ingredient for hair growth-promoting and hair loss treatment potential.As a traditional herbal medicine that is widely distributed in most parts of China, purposes . This stG. florida fruit extract (GFFE) was purchased from the Nanjing DASF Biotechnology Co., Ltd . Human dermal papilla cells (hDPCs) were purchased from Sciencell Research Laboratories, Inc.2 at 37°C. Cultured cells were then treated with various concentrations of GFFE for 24 hours according to the kit protocol. CCK-8 solution was added, followed by a 4-hour incubation, and absorbance at 450 nm was measured on a TECAN Enzyme standard instrument Spark 10 M. The cell proliferation rates were calculated from OD and represented as percentages of negative control (NC).Cell proliferation was measured using Cell Counting Kit-8 . Human DP cells were seeded in 96-well plates at a density of 3,000 cells/well and incubated in a humidified atmosphere with air and 5% CO5) and incubated until grown to 80–90% confluence in a 10% FBS DMEM medium. After culture with serum-free DMEM medium for 24 h, the cells were added to each treatment and incubated for 24 h in aserum-free DMEM (NC), with 19.5 μg/mL of GFFE. Total RNA was extracted using Monzol™ Reagent  according to the manufacturer's instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference. For RT-PCR, 1 µl each of the forward primer (10 ng/µl) and reverse primer (10 ng/µl) per well, iTaq universal Green supermix (2x) 10 µl, nuclease-free H2O 6 µl, and cDNA 2 µl were mixed to a final volume of 20 µl. The PCR procedure software recorded the average fluorescence value of each cycle of the reaction. The relative expression levels of different genes in the cells were obtained by comparing the Ct values. The experiment was repeated three times with three replicates seated per reaction.In vitro model, hDPCs were plated on a 6-well plate  with the following setting condition: 95°C for 10 min; 40 cycles including 95°C for 10 sec, 60°C for 30 sec, and 72°C for 30 sec. cDNA was amplified using MonScript™ RTIII All-in-One Mix with dsDNase .As for qRT-PCR, MonAmpTM ChemoHS qPCR Mix  was used following the instructions of the manufacturer. The qRT-PCR was performed using a BIO-RAD CFX96β1, 5′-GCAGAAGGATCACCACAACC-3′ (forward), TGF-β1, 5′-TGTCTGCACTGCGGAGGTAT-3′ (reverse) [β1-catenin, 5′-AACTTGCTCAGGACAAGGAA-3′ (forward), β1-catenin, 5′-TCCTAAAGGATGATTTACAGGTC-3′ (reverse) [Primers were used as follows: GAPDH, 5′-TGTTGCCATCAATGACCCCTT-3′ (forward), GAPDH, 5′-CTCCACGACGTACTCAGCG-3′ (reverse) ; VEGF, 5reverse) ; DKK1, 5reverse) ; β1-catereverse) ; LEF1, 5reverse) .β1  and rabbit anti-Tubulin . We used BeyoECL Star (Beyotime) reagent to detect protein.The cultured cells were collected and lysed in RIPA buffer (Beyotime) with protease inhibitors (Beyotime) after rinsing twice with PBS (precooled at 4°C). Protein was quantified with a BCA protein concentration determination kit (Beyotime), and then separated by SDS- polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. After blocking in 5% nonfat milk for 2 h at 25°C, the membranes were probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies . Primary antibodies used were rabbit anti-VEGF protein , rabbit anti-Wnt5a , rabbit anti-TGF-n = 5) were used for this study. The dorsal area hairs (approximately 2 cm in width and 3 cm in length) on the back of the mouse in the telogen phase were shaved 24 h before applying the treatments. Animals in group 1 served as NC (water), and group 2 received 2% minoxidil as a positive control group. The animals in group 3, group 4, and group 5 received different doses  of GFFE, respectively. The 5 groups were applied topically to the test area with 0.2 mL once a day, for 30 days. The animals were isolated for an hour and then kept back in their respective cages. During the application period, differences between treatment and nontreatment groups in average body weight or abnormalities in the mice were not observed. The hair regrowth activities of GFFE were evaluated by observing and photographing the back skin of mice. On day 14, day 21, and day 30, 10 hairs were plucked from the mice's shaved area randomly from all groups and the average length of each treatment was measured.The animals were housed in a temperature-controlled room with a 12-h light/dark cycle and were allowed free access to food and water in the course of experiments. Twenty-five male five-week-old C57BL/6 mice  in 5 randomized groups (μm tissue section was stained with the standard hematoxylin and eosin (H&E staining) protocol [Dorsal skin tissues from each mouse were excised and fixed in 4% neutral formalin for 24 h. They were embedded in paraffin after routine tissue processing. For the hair follicle's histopathologic evaluation, a 4 protocol . HematoxOn 14, 21, and 30 days after beginning the animal experiment, 10 hairs were plucked from the mice's shaved area randomly from all groups and the average length of each treatment was measured.t-test for the comparison among groups using SPSS WIN (v25.0). The results were considered statistically significant if the p values were less than 0.05.All treatments were repeated at least three times. Data represent biological replicates. The data were statistically analyzed by Student's ∗), 110.67% (∗), and 112.92% (∗∗), respectively. Particularly, the 0.032% GFFE exhibited the highest cell proliferation rates than the NC group. Collectively, these results suggest that GFFE facilitates the proliferation of DPCs and improves the hair-inducing abilities of these cells.Dose-dependent proliferative effects of GFFE  tested on hDPCS were first examined by CCK-8 assay following 4-h culture . After iβ-Catenin, the transducer of Wnt signaling, has been reported to contribute to the maintenance of HF structures and is translocated to the nucleus which is important for the development and growth of hair follicles [a, which has been classified as a noncanonical Wnt family member, can also activate β-catenin signaling in the presence of the appropriate Frizzled receptor 4 [β-catenin signaling including DKK1, LEF1, Wnt5a, and β-catenin mRNA were investigated by means of RT-PCR.Wnt signals play a crucial role in the activation of bulge stem cells to progress toward hair formation and the induction of the anagen phase in hair growth which is required for the establishment of the hair follicle . The Wntollicles . It was ollicles . As a spollicles . DKK1 waollicles , 48. Wntceptor 4 . Genes rβ-catenin signaling, was reduced by 21%, 54%, and 67% in hDPCs treated with 0.008%, 0.016%, and 0.032% GFFE, respectively; the mRNA level of LEF1, a positive regulator of Wnt/β-catenin signaling, was increased by 66% (∗∗), 22% (∗), and 25% (∗) in hDPCs treated with 0.008%, 0.016%, and 0.032% GFFE, respectively; the mRNA level of wnt5a, a positive regulator of Wnt/β-catenin signaling, was increased by 162% (∗∗), 36% (∗∗), and 5%  in hDPCs treated with 0.008%, 0.016%, and 0.032% GFFE, respectively; the mRNA level of β-catenin, a positive regulator of Wnt/β-catenin signaling, was increased by 32% and 91% in hDPCs treated with 0.16% and 0.032% GFFE, respectively, but 0.008% GFFE decreased the expression of β-catenin (∗∗)  .β2 in DPCs, and these sequential events contributed to the shortening of the human hair cycle [β1 was regarded to regulate hair growth suppression by dose-dependently repressed hDPCs growth [β antagonists were effective in preventing catagen-like morphological changes and promoting elongation of hair follicles. In this study, GFFE decreased the expression of TGF-β1 in hDPCs. 0.008%, 0.016%, and 0.032% GFFE treatments showed 57%, 41%, and 64% decrease in TGF-β1 mRNA expression compared to NC, respectively (∗∗)  .VEGF mRNA is in the anagen phase and it is less expressed in the catagen and telogen phases in DPCs during the hair growth cycle [∗∗) and 19% (∗) greater VEGF mRNA expression compared to NC, respectively. However, 0.032% GFFE showed 6%  decreased VEGF mRNA expression compared to NC  to determine whether GFFE affects the hair-inducing ability of hDPCs. The above results suggested that GFFE could promote the hair-inducing ability of hDPCs. Next, we evaluated the effects of the GFFE on the protein expression and activity of Wnt5a protein level increased with all GFFE treatment groups. 0.008%, 0.016%, and 0.32% GFFE groups showed 66% (∗∗), 79% (∗∗), and 25% (∗) greater Wnt5a protein expression than NC, respectively , 67% (∗∗), and 81% (∗∗) attenuated TGF-β1 protein expression compared to NC, respectively (∗∗) increased VEGF protein level compared to NC, but VEGF protein level with 0.008% and 0.016% GFFE treatments showed 24% (∗∗) and 18% (∗) decreased level compared to NC, respectively  were shaved on the back when dorsal skin hair follicles are in telogen. GFFE or NC treatment was applied topically every day. In NC mice, only a few scattered pigmented spots were apparent at least until day 21. In contrast, both the GFEE  and 2% Minoxidil groups achieved hair regrowth and became remarkably grey after 7 days . FormatiThe morphology of the hair follicles was evaluated histologically to define the effect of GFFE on hair growth. The comparison and difference between NC, MXD, and GFFE groups in number, size, and depth of follicle were observed by histological photos , 3.68 mm (∗), 4.18 mm (∗∗), and 3.96 mm (∗), respectively. In day 21, the hair length of NC, MXD, 0.5% GFFE, 1% GFFE, and 2% GFFE was 4.93 mm, 7.86 mm (∗∗∗), 6.74 mm (∗∗), 7.91 mm (∗∗∗), and 7.60 mm (∗∗∗), respectively. In day 30, the hair length of NC, MXD, 0.5% GFFE, 1% GFFE, and 2% GFFE was 8.11 mm, 11.05 mm (∗∗), 10.21 mm (∗), 11.13 mm (∗∗), and 10.83 mm (∗), respectively (10 hair follicles were plucked from the mice's shaved area randomly from all groups, and the average length of each treatment was measured on day 14, day 21, and day 30. In day 14, the hair length of NC, MXD, 0.5% GFFE, 1% GFFE, and 2% GFFE was 2.43 mm, 4.12 mm (ectively .β-catenin signaling pathway activity while attenuating TGF-β1 synthesis activity in hDPCs and also found that GFFE up-regulated the protein expressions of VEGF and β-catenin and downregulated TGF-β. Moreover, GFFE promoted hair regeneration and repair in mouse models. Collectively, our results unveil a novel functional role of GFFE and strongly imply that GFFE can be developed as a herbal medicine for the treatment of hair loss diseases. In a future study, we will explore the specific constituents of antiremoval factors in GFFE as well as conduct GFFE in vivo and clinical trials to elucidate their application potential.In this study, GFFE showed strong hair growth-promoting activity. GFFE a was natural compound and has many pharmacological and physiological activities . The hai


Background and objectiveGiven the enormous public health burden posed by acute ischemic stroke (AIS), it becomes imperative to identify associated risk factors such as serum uric acid (SUA) and lipid profile that can be tangibly assessed. This could potentially provide useful markers for disease risk or progression, facilitating timely diagnosis and management of AIS. This study aimed to evaluate the SUA and lipid levels in patients with AIS.MethodThis cross-sectional study enrolled 66 AIS patients aged >18 years, from both genders. After a thorough medical history and clinical examination, each patient was subjected to SUA analysis and lipid profiling using an auto-analyzer with dedicated reagents. Results were statistically analyzed using Chi square test, and p-value ≤0.05 indicated statistical significance.Results and interpretationThe study cohort showed a mean age of 61.17 ± 14.01 years and male to female (M:F) ratio of 1.7:1, with mean blood levels of SUA, triglyceride, and high-density lipoprotein (HDL) reaching 5.68 ± 1.71 g/dL, 205.42 ± 105.08 g/dL, and 29.80 ± 8.45 g/dL, respectively. Most patients suffered from hypertension (81.82%), diabetes (77.27%), and alcoholism (24.24%). Cerebrovascular Doppler findings revealed the combined presence of plaque and stenosis (24.24%). Male AIS patients showed a significantly greater association with alcohol and smoking/nicotine use (p<0.001). Gender showed no significant association with SUA, lipid profile, hypertension, and diabetes (p>0.05).ConclusionAIS is associated with hyperuricemia and dyslipidemia, with no significant gender differences. Acute ischemic stroke (AIS), also called a cerebrovascular accident, is defined as an abrupt onset of a neurologic deficit that is attributable to a focal vascular cause that leads to cerebral ischemia. Its diagnosis depends on medical history, physical examination, and clinical and laboratory studies, including brain imaging . It is oGiven the high prevalence and significant morbidity/mortality associated with AIS, it becomes important to identify its risk factors and related disease indicators in order to provide timely management. Diabetes, hypertension, insulin resistance, smoking, obesity, hypercholesterolemia, and physical inactivity have been found to elevate the risk of suffering AIS. These risk factors are known to be associated with increased serum uric acid (SUA) level, which has also been reported as an independent predictor of stroke -6. HyperHowever, there is a paucity of scientific literature studying both, hyperuricemia and impaired lipid profile, in AIS patients. Identifying the changes in these profiles could aid in the timely diagnosis and management of such patients and help formulate targeted efforts to mitigate the risk of AIS. Therefore, the present study was conducted to evaluate the SUA and lipid profile in patients with AIS.This cross-sectional study was conducted at a tertiary care hospital in Bengaluru, India, from October 2018 to September 2020, after obtaining ethical clearance from the institutional review board. The present study included patients aged >18 years, irrespective of their gender, diagnosed with AIS based on clinical and radiological evaluation, including computed tomography (CT) and magnetic resonance imaging (MRI) of the brain, after obtaining written informed consent from them. The study also excluded patients with the chronic intake of drugs causing hyperuricemia, patients on treatment for hyperuricemia, and patients with conditions that alter serum uric acid levels .The sample size was calculated based on a previous study conducted by Arora et al. . Conside2p {z(1-α/2) + z(1-β)}]2/[µ2d],n = [2S2p = (S12 + S22)/2,wherein S2 = standard deviation in the first group, S22 = standard deviation in the second group, µ2d = mean difference between the samples, α = significance level, 1-β = power.where n = minimal sample size, S1Accordingly, 66 patients were included in this study. After thorough history taking, a routine clinical examination of all systems, as well as measurements of blood pressure, pulse rate, respiratory rate, body temperature, and oxygen saturation, was performed. Following this, a fasting blood sample (3-5 mL) was taken from each patient using a sterile syringe and needle and subjected to uric acid analysis and lipid profiling employing Cobas 6000 auto-analyzer  with dedicated reagents. Serum uric acid (SUA) levels were assessed by enzymatic (uricase) calorimetric method , triglyceride (TG) levels by enzymatic (lipase) calorimetric method, total cholesterol (TC) by enzymatic (cholesterol esterase) calorimetric method, high-density lipoprotein (HDL) by direct measure, low-density lipoprotein (LDL) by direct measure, and very low-density lipoprotein (VLDL) by triglyceride percentage.For SUA, the lower limits of detection were 0.2 mg/dL (range: 0.2-25.0 mg/dL), and intra-assay and inter-assay coefficients of variation were equal to 0.5% and 1.7%, respectively. Hyperuricemia was defined as a serum urate concentration >7.5 mg/dL (450 µmol/L) in men and >6.2 mg/dL (372 µmol/L) in women, in agreement with the normal value provided by the clinical laboratory.Statistical analysisData were compiled and analyzed using statistical software R version 4.0.3 and Microsoft Excel . Continuous variables are represented in mean ± standard deviation (SD) form and categorical variables by a frequency table. QQ plot/Shapiro-Wilk’s test was used to check the normality of variables. Independent sample t test/Mann-Whitney U test was done to compare two groups. Chi square test has been used to assess the association between two categorical variables. A p-value ≤ 0.05 indicates statistical significance.The study consisted of 66 patients out of which 40 (60.6%) were males and 26 (39.4%) were females with a mean age of 61.17 ± 14.01 years and a male to female (M:F) ratio of 1.7:1. Tables Table Table Given the enormous personal and public health burden posed by AIS, it becomes imperative to identify associated risk factors that can be tangibly assessed. SUA and lipid levels are a few of such measurable factors that have been found to be associated with AIS and could provide useful markers for disease risk or progression. With this vision, the present study was conducted to evaluate the SUA and lipid levels in patients with AIS. This study showed that dyslipidemia was seen in all 66 (100%) patients and all of them had more than one lipid parameter deranged. Mean serum uric acid levels in the studied patient were 5.67 ± 1.68, and 21 patients (31.81%) had hyperuricemia.This study resonates with the findings of Arora et al., who studied a similar population with a mean age of 63.2 ± 14.8 years and found the mean SUA levels to be 5.5 ± 01.7 g/dL . AnotherDyslipidemia observed in the present study was also mirrored in the work by Yuan et al. who noted a significant rise in TG, TC, and low-density lipid cholesterol (LDL-C) with a significant fall in high-density lipid cholesterol (HDL-C) levels in AIS patients and suggested that an adverse lipid profile, especially elevated LDL and TC, is an independent risk factor for large artery atherosclerotic cerebrovascular accident . MoreoveThe presence of other risk factors for stroke like smoking, alcohol, hyperhomocysteinemia, diabetes, and hypertension, as seen in the present study, has also been confirmed by other researchers -21. In oConclusively, the present study establishes the association of hyperuricemia and dyslipidemia with AIS. This can potentially be utilized to assess at-risk individuals and intervene early at various systemic levels to minimize AIS-related morbidity and mortality. However, the present research was conducted at a single medical center with a small sample size and as a cross-sectional design with no control group. Further multicentric longitudinal studies are encouraged with a larger sample size to confirm these parameters as markers of AIS. This could revolutionize policy-making, resource allocation, and therapeutic options in managing patients at risk of AIS.


Phaseolus vulgaris) at a field site in its centre of domestication to characterise rhizosphere and endosphere bacterial communities at the vegetative, flowering, and pod filling stage. 16S r RNA gene amplicon sequencing of ten samples yielded 9,401,757 reads, of which 8,344,070 were assigned to 17,352 operational taxonomic units (OTUs). Rhizosphere communities were four times more diverse than in the endosphere and dominated by Actinobacteria, Bacteroidetes, Crenarchaeota, and Proteobacteria (endosphere: 99% Proteobacteria). We also detected high abundances of Gemmatimonadetes (6%), Chloroflexi (4%), and the archaeal phylum Thaumarchaeota (Candidatus Nitrososphaera: 11.5%): taxa less frequently reported from common bean rhizosphere. Among 154 OTUs with different abundances between vegetative and flowering stage, we detected increased read numbers of Chryseobacterium in the endosphere and a 40-fold increase in the abundances of OTUs classified as Rhizobium and Aeromonas . Our results indicate that bean recruits specific taxa into its microbiome when growing ‘at home’.Plant roots recruit most prokaryotic members of their root microbiota from the locally available inoculum, but knowledge on the contribution of native microorganisms to the root microbiota of crops in native versus non-native areas remains scarce. We grew common bean ( Arabidopsis thaliana to crops, or from one crop species to another. Therefore, a successful application of microbiomes will depend on a significant diversification of model systems , mixed by inverting the tube several times and incubated for 10 min at 65 °C and for further 10–20 min at room temperature. After adding 300 µL of chloroform and mixing vigorously, the suspension was centrifuged for 5 min at 10,000× g, the aqueous phase was transferred to a new sterile 1 mL Eppendorf tube, mixed with 300 µL of isopropanol and vigorously shaken on a vortex. Total DNA was precipitated via centrifugation , the supernatant was discarded, and the pellet was washed with 500 µL of 70% (v/v) ethanol. Finally, the ethanol was discarded, and the pellet was air-dried and suspended in 100 µL of distilled sterile water. The purity and quality of the total DNA extracted from both compartments was checked individually for each sample by electrophoresis on a 1.1% (w/v) agarose gel and using a Nanodrop 2000 .Total DNA was extracted from 0.2–0.3 g rhizosphere soil per plant using the MoBio PowerSoilreported  but withE. coli DH5α cells as described by Sambrook and Russel [2 2 mM, 10 pmol of each primer (pJET1.2 forward 5′-CGA CTC ACT ATA GGG AGA GCG GC-3′ and pJET1.2 reverse 5′-AAG AAC ATC GAT TTT CCA TGG CAG-3′), 100 µM of each dNTP, and 1.5U of Taq DNA Polymerase (Invitrogen). After initial denaturation at 95 °C for 3 min, each thermal cycle was performed as follows: denaturation at 94 °C for 30 sec, annealing at 60 °C for 30 s, and elongation at 72 °C for 1 min. At the end of 30 cycles, the final extension step was at 72 °C for 8 min. Twenty-eight positive clones were sequences by the Sanger method at the National Laboratory of Genomics for Biodiversity (LANGEBIO), at Centro de Investigación y de Estudios Avanzados (CINVESTAV)—Unidad de Genómica Avanzada, Irapuato, GTO, México (https://langebio.cinvestav.mx/).As mentioned in the Results d Russel . Ligatiod Russel  and usedPhaseolus mitochondrial genes, we used the beforementioned primers to generate amplicons from all ten samples. The 25 µL PCR reaction mixture contained approximately 1 ng of total DNA for rhizosphere samples and 10 ng for root endosphere samples, 10× PCR reaction buffer, MgCl2 2 mM, 10 pmol of each primer, 100 µM each dNTP, and 1.5U of Taq DNA Polymerase (Invitrogen). After initial denaturation at 94 °C for 5 min, each thermal cycle was performed as follows: denaturation at 94 °C for 1 min, annealing at 52 °C for 45 sec, and elongation at 72 °C for 1 min. At the end of 30 cycles, the final extension step was at 72 °C for 8 min. For rhizosphere samples, six parallel reactions per sample were performed, PCR products were purified and concentrated, using the Illustra GFX DNA and Gel Band Purification Kit  and pooled. In the case of the endosphere samples, PCR products from six parallel reactions per sample were pooled, separated by electrophoresis on a 1.1% agarose gel, and the 750 bp band was excised and purified using the Illustra GFX DNA and Gel Band Purification Kit. High-throughput sequencing of the resulting 16S rRNA gene amplicon pools was performed on the MiSeq 2 × 300 Illumina platform.Since BLAST analyses unambiguously confirmed that the sequences of all 750 bp products matched to bacterial 16S rRNA genes and those of the 1300 bp products to Bioinformatic processing and data analysis followed general protocols , includihttps://github.com/joey711/phyloseq/issues/143, accessed on 6 August 2015), using the following parameters: lowest rarefaction depth in the series of depths (m), highest rarefaction depth in the series of depths (x), highest rarefaction depth in the series of depths (s), step size to increment from the low to high depths and the number of replicates to perform at each depth (n); where m = 1000 reads/sample, x = 1,100,000 reads/sample, s = 1000 reads/sample and n = 3 replicates. The mean values resulting from the three replicates at each step were used for constructing the rarefaction curves of every sample. To visualize the diversity structure of all sample communities, Principal Component Analysis was performed. The Bray-Curtis dissimilarity (distance) metric was calculated with the phyloseq R package [Rarefaction curves were constructed using an adaptation of the MacQIIME (version 1.9.0) command multiple_rarefactions.py in the R phyloseq 1.16.2 version package  from  to be the enriched community. All p-values were adjusted for multiple comparisons with the FDR correction using the Benjamin-Hochberg method.To determine OTUs appearing at different abundances between ontogenetic stages, the OTU abundance table generated by QIIME was analysed using the Statistical Analysis of Metagenomic Profiles (STAMP) software package was used . P-value v3.30.3 . We conshttps://www.genome.jp/brite/ko00001, accessed on 1 April 2021) which appeared particularly enriched in each of the compartments.We used PICRUST  and the To validate these predictions, we selected five genes from different functional groups for PCR-based amplification from the original total DNA used for amplicon generation. Published degenerate primers were used for niH  and nodC


The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL−1, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6–112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 μL saliva samples.Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17β-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC–MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01–2 ng mL Steroid hormones include glucocorticoids, which regulate carbohydrate and lipid metabolism and have inflammatory and immunosuppressive effects; mineralocorticoids, which regulate blood pressure through ionic equilibrium in body fluids; and sex hormones, which control reproductive functions and secondary sexual characteristics ,2,3. StaMost steroids are sulfated by sulfotransferases (SULTs), which increase their solubility in aqueous solution and their excretion into urine. In addition, some of these sulfated steroids are desulfated by steroid sulfatases (STSs), with these steroids returning to their free forms ,3. This The sulfated steroid metabolite dehydroepiandrosterone-3-sulfate (DHEAS) ,9 has beSulfated steroid metabolites can be analyzed by immunoassays or by mass spectrometry coupled to chromatography. Immunoassays, however, are not suitable for simultaneous analysis of a series of steroids due to their cross-reactivity, difficulties distinguishing among steroids with similar structures, and the need to generate specific antibodies against each compound ,3,10,13.Sulfated steroids have been measured in plasma, serum, and urine samples. However, blood collection is invasive and may itself induce stress, whereas urine collection is simple, but urinary concentrations of compounds are affected by the volume of urine excreted. In contrast, saliva can be easily collected non-invasively from subjects of all ages, from children to the elderly, and collection devices are relatively inexpensive. Furthermore, the concentrations of sulfated steroids in saliva are highly correlated with their concentrations in plasma or serum ,23. BecaIn-tube solid-phase microextraction (IT-SPME) is a method by which samples can be easily extracted and concentrated using open-tube fused silica capillaries with coated inner surfaces as extraction devices, followed by online coupling to LC and LC–MS online using column switching technique . The entThe aim of this study was to establish a fully automated online simultaneous analysis system, consisting of IT-SPME coupled with stable isotope dilution LC–MS/MS, for four sulfated steroid metabolites , pregnen−1 each of PREGS, DHEAS, and CRTS and 5 ng mL−1 of E2S. As PLOT columns including Supel-Q and Carboxen 1006 have a larger adsorption surface area and thicker film layer, the amounts extracted were greater than those for other liquid-phase columns  of PREGS, DHEAS, CRTS, and E2S onto the Supel-Q PLOT capillary column were found to be 56.6 ± 3.5%, 45.0 ± 2.2%, 47.3 ± 2.4%, and 39.7 ± 0.8%, respectively. The sulfated steroid metabolites extracted into the capillary column were dynamically desorbed and introduced directly into the LC column by online mobile phase flow using a column switching system. After analysis, the capillary column was cleaned and conditioned with methanol and mobile phase flow prior to the next analysis, thereby allowing its repeated use without carryover. All of these operations were programmed and automated  in the ESI-negative ionization mode. The [HSO4]− ion (m/z 97) and [SO3]− ion (m/z 80) formed by cleavage of the [M−H]− of each compound were detected as fragment ions (Q3 mass). For each precursor ion [M−H]−, the m/z 97 for PREGS, DHEAS, CRTS, and their stable isotope-labeled compounds and the m/z 80 for E2S and E2S-d4 were selected as product ions, and the MS/MS operating parameters were optimized. The MRM transitions and MS/MS parameters set for each compound are shown in Four sulfated steroid metabolites, along with their stable isotope-labeled compounds as internal standards (ISs), exhibited abundant deprotonated ions [M−H]v/v), with a flow rate of 0.2 mL min−1 resulting in good peak shapes and selective detection in MRM mode with a runtime of 6 min , in the presence of 0.2 ng mL−1 each of PREGS-13C2-d2, DHEAS-d5, and CRTS-d4, respectively, whereas linearity for E2S was validated at concentrations of 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 ng mL−1 in the presence of 1.0 ng mL−1 E2S-d4. Calibration curves for each compound were linear, with correlation coefficients above 0.9998 (n = 3).The analytical method was validated based on generally accepted validation criteria recommended in the ICH guidelines . Linearie 0.9998 . The CVsS/N = 3) of each sulfated steroid metabolite in standard solutions ranging from 0.30 to 3.20 pg mL−1 , was validated by performing five independent analyses on the same day and on five different days. The intraday and interday variations of these analyses were found to be 2.1–7.7% and 4.0–11.1%, respectively  of these sulfated steroid metabolites ranged from 16 to 172 pg mL−1 saliva , DHEAS-d5 , CRTS-d4 , and E2S-d4 , were used as internal standards (IS). These standard and IS compounds ; all other chemicals were of analytical reagent grade.PREGS sodium salt and E2S sodium salt were purchased from Sigma-Aldrich Japan , DHEAS hydrate was from Tokyo Kasei Kogyo , and CRTS potassium salt was from Toronto Research Chemicals Inc. . Their stable isotope-labeled compounds, PREGS-ompounds  were disg for 1 min to elute the saliva solution. If not immediately used for analysis, the samples were stored frozen at −20 °C and thawed spontaneously just before analysis. A 0.1 mL aliquot of mixed IS solution was added to 0.05 mL of each saliva sample, followed by ultrafiltration using an Amicon Ultra 0.5 mL 3K  regenerated cellulose 3000 molecular weight cutoff centrifugal filter device at 15,000 rpm for 20 min. Each filtrate was pipetted into a 2.0 mL autosampler vial with septa, to which was added 0.05 mL of 0.2 M potassium hydrogen phthalate–HCl buffer (pH 3). The total volume was made up to 1.0 mL with distilled water, and the vials were set into the autosampler for IT-SPME LC−MS/MS analysis. The concentrations of the sulfated steroid metabolites in saliva were calculated using calibration curves constructed from the ratios of the peak heights of each sulfated steroid metabolite to the peak height of their IS compounds.Saliva samples were obtained from 20 healthy volunteers (10 men and 10 women). The experimental protocol was approved by the ethics committee of Shujitsu University , and all volunteers provided written informed consent. Saliva samples were collected in Salisoft tubes . The tubes were centrifuged at 2500× v/v), and a flow rate of 0.2 mL min−1. Electrospray ionization (ESI)–MS/MS conditions included: a turbo ion spray voltage of –4500 V; a turbo ion spray temperature of 450 °C; ion source gas GS1 and GS2 flows of 20 and 11 L min−1, respectively; a curtain gas (CUR) flow of 10 L mL−1; and a collision gas (CAD) flow of 4.0 L min−1. Multiple reaction monitoring (MRM) transitions in negative ion mode and other parameters, including dwell time, declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP), are shown in − and the related product ions for each compound. Quadrupoles Q1 and Q3 were set at unit resolution  Model 1100 series LC system and an Applied Biosystems  API 4000 triple quadrupole mass spectrometer, with LC separation on an InertSustain swift C18 column . The LC conditions included a column temperature of 30 °C, a mobile phase consisting of distilled water/acetonitrile (55/45, solution . AnalystIT-SPME was essentially performed as described in our previous works ,32. A GCIn this study, we succeeded for the first time in efficiently extracting and concentrating highly polar sulfate conjugates by IT-SPME at acidic pH, and we constructed an automated analysis system coupled online with LC–MS/MS to enable selective and sensitive simultaneous analysis of four sulfated steroid metabolites. The method is easy to apply to the analysis of small volumes of saliva samples without tedious pretreatment except for ultrafiltration. This method may be a useful tool in analyzing the regulation of steroid metabolism and in determining the diagnosis of related diseases.