In total, data for 432 TCM active ingredients with clear pharmacological effects were obtained. Five critical RA-related genes were identified; CCL5 and CXCL10 were selected for molecular docking. Target prediction and network-based proximity analysis showed that dioscin could modulate 22 known RA clinical targets. Dioscin, asiaticoside, and ginsenoside Re could effectively inhibit in vitro cell proliferation and secretion of TNF-α and IL-1β in RA rat synovial cells. Using bioinformatics and computer-aided drug design, the potential small anti-RA molecules and their mechanisms of action were comprehensively identified. Dioscin could significantly inhibit proliferation and induce apoptosis in RA rat synovial cells by reducing TNF-α and IL-1β secretion and inhibiting abnormal CCL5, CXCL10, CXCR2, and IL2 expression.This study is aimed at screening potential therapeutic ingredients in traditional Chinese medicine (TCM) and identifying the key rheumatoid arthritis (RA) targets using computational simulations. Data for TCM-active ingredients with clear pharmacological effects were collected. Absorption, distribution, metabolism, excretion, and toxicity were evaluated. Potential RA targets were identified using the Gene Expression Omnibus (GEO) database, protein–protein interaction network, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and potential TCM ingredients using AutoDock Vina. To examine the mechanisms underlying small molecules, target prediction, Gene Ontology, KEGG, and network modeling analyses were conducted; the effects were verified in rat synovial cells using cell proliferation assay. The activities of tumor necrosis factor TNF- Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory, and progressive autoimmune disease that affects synovial joints and other organ systems . To dateCurrently, the available treatment of RA includes nonsteroidal anti-inflammatory drugs, glucocorticoid drugs, and biological macromolecular therapy; however, they are costly and often exert toxicity and cause side effects , 7. TherTraditional Chinese medicine (TCM) has a long history of RA treatment based on rich experiences in clinical applications. RA, a common type of arthritis, belongs to “Bi syndrome” category in Chinese medicine . Currentin vitro cellular experiments, in combination with existing literature analysis, to explore important targets for the treatment of RA and further screen out the active ingredients of TCM with potential therapeutic effects. In vitro experiments were performed to confirm these findings, with the aim of providing powerful methods and technical support for the treatment of RA with TCM. The workflow of this study is shown in Although several TCM compounds have been reported to exert good curative effects and minor side effects, the discovery of additional potential anti-RA ingredients from a large number of common small TCM molecules with clear pharmacological effects has important practical significance. In this view, we integrated bioinformatics, computer-aided drug design, and https://pubchem.ncbi.nlm.nih.gov/) was used to confirm the chemoinformatics data for each ingredient, including molecular name CAS, PubChem CID, molecular formula, canonical SMILES, and SDF files. A more authoritative and reliable database of active ingredients in commonly used Chinese medicines has been constructed.The active ingredients of common TCMs that have been proven to have clear pharmacological effects were collected from published literature in Chinese , 11. Thehttp://www.swissadme.ch/) [P, solubility, blood–brain-barrier (BBB) permeant, Pgp substrate, GI absorption, bioavailability score, synthetic accessibility, metabolic stability, Ames test, and hERG.The ACD/Labs software and SwissADME online system (dme.ch/)  were usehttps://www.ncbi.nlm.nih.gov/geo/), “rheumatoid arthritis” was queried to download the gene expression profile chip data related to RA. Perl language (version 5.32.0) was used to perform preprocessing of the original data, such as standardization, correction, and gene name annotation. The R language-based limma package [p.value < 0.05.In the GEO database ( package  (versionhttps://string-db.org/) to obtain the PPIs of the DEGs. The parameters were set as follows: organism, Homo sapiens; combined score threshold, 0.7. In addition, Gephi software  was used to visualize the PPI network, and cytoHubba in Cytoscape  [All selected genes were imported into the STRING database  , in whicThe GO database constructed by the GO Consortium in 2000 contains information for the biological process (BP), molecular function (MF), and cellular component (CC) of genes . A biolop value < 0.05 and q value < 0.05.In this study, clusterProfiler (version 3.14.3)  was usedThe key protein targets in the PPI network were identified, and the targets in the reported RA-related pathways were obtained from the KEGG pathway enrichment analysis as described in Sections http://www.rcsb.org/). PyMOL  was used to evaluate key target proteins to remove water molecules, impurities due to the presence of ions, and perform other changes. AutoDockTools  was used to hydrogenate and charge the processed protein-ligand; the processed PDB files were converted and saved in the PDBQT file formatThe PDB file for the potential key target proteins was downloaded from the PDB database (http://openbabel.org/) was used to convert the 2D SDF file into a 3D structure file. The original 3D structure was directly converted into a PDB file, and all small molecules were processed with minimum energy; finally, the PDB file was converted into a PDBQT fileThe information of small molecules was downloaded from PubChem in two SDF format files, 2D and 3D. OpenBabel  was used to perform a semiflexible molecular docking and calculate the affinity values  of all small molecules with potential key targets. Generally, the lower the affinity value, the greater the possibility that the small molecule binds to the receptor; therefore, according to the affinity value, the small molecules were sorted from small to large, and small molecules of TCM with potential anti-RA activity were screened outTo determine the docking center parameters, we referred to the binding site (region) of the protein receptor and the original ligand. The box size was defined as 30 × 30 × 30, and AutoDock Vina  [http://sea.bkslab.org/) [http://www.swisstargetprediction.ch/) [http://stitch.embl.de/), to predict their potential targets. In particular, the screening threshold precision for HitPick was set at 50%, the threshold MaxTc for SEA was set at 0.7, and the threshold probability for SwissTargetPrediction was set at 0.15. Molecular docking verification, GO function annotation, and KEGG pathway enrichment analyses were performed on the predicted potential targets.Corresponding canonical SMILES of the obtained small molecules were imported into online prediction systems, such as HitPick (hitpick) , SEA (htab.org/) , SwissTaion.ch/) , and STIA is the target set of small molecules of TCM, ‖A‖ is the number of the targets of A, B is the clinically known set of RA treatment targets, ‖B‖ is the number of the targets of B, d is the shortest path distance between two nodes in the human PPI background network, 〈dAA〉 represents the average distance between the target points of the component action, 〈dBB〉 represents the average distance between the key genes, and 〈dAB〉 represents the average distance between the small Chinese medicine molecular target set and the clinically known RA treatment target set on the background network.Next, we determined whether the small molecules of TCM screened have direct or indirect regulatory effects on the clinically known RA treatment targets. Given that some of these molecules may not directly act on certain RA targets but regulate the disease via the intervention of its neighboring targets, we further analyzed the small molecules of TCM using the calculation method of network proximity, based on the human PPI background network , using tSAB < 0, the small molecule target A of TCM and the clinically known RA treatment target set B are close in network topology, indicating that the small molecule of TCM can interfere with the target set A and regulate the clinically known RA treatment target set B. When SAB ≥ 0, A and B are separated in the network topology, indicating that the small molecule of TCM has no significant regulatory intervention on B. Therefore, we can calculate the value of SAB to further judge whether the small molecules of TCM can interfere with the known clinical disease target set by regulating some other target proteins to further improve the treatment of the disease.Generally, when http://db.idrblab.net/ttd/) and DrugBank (https://www.drugbank.ca/). Thereafter, the intersection of the RA targets collected using the two databases was used to construct the final known RA clinical treatment target set.For identifying the clinically known RA treatment targets, we queried across two databases: Therapeutic Target Database  and igraph (version 1.2.5) to complete the aforementioned programming, calculations, and analyses.Materials: rat synovial cells were induced using type II collagen and isolated in our laboratory.Reagents: PBS and RPMI 1640 medium were purchased from Gibco ; fetal bovine serum (FBS) was purchased from Biological Industries ; antibiotic-antimycotic, bovine serum albumin, and trypsin-EDTA (0.05%) were purchased from Life Technologies ; rat TNF-α and interleukin IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from LinkTech .Instruments: EPOCH type multifunctional microplate reader , 3111 CO2 incubator , CX23 microscope , and HFsafe-1200LC biological safety cabinet  were used for the in vitro studies.μl of fresh culture medium and 10 μl of Cell Counting Kit-8 (CCK-8) reagent were added to the wells. After incubation at 37°C for 4 h, the absorbance at 450 nm was measured with a microplate reader to calculate the IC50 values using Graph Pad (Version: 8.0) software.Synovial cells were cultured in RPMI-1640 medium supplemented with 10% FBS. After counting, the cells were seeded in 96-well culture plates at a density of 2000 cells/well. The cells were incubated with different concentrations of the small molecules of TCM. After 72 h, the culture medium was discarded; 90 5 cells/well. A total of 1800 μl of RPMI-1640 serum-containing culture medium per well was added to the culture, with an additional 200 μl drug solution. The final concentration of each sample was calculated as the IC50 concentration of the cells. The drug-free group was used as the arthritis model cell control group and cultured at 37°C in a 5% CO2 incubator for 72 h. The culture medium was collected, centrifuged at 400 × g for 5 min, and the supernatant was collected. ELISA was used for the detection of TNF-α and IL-1β, according to the manufacturer's instructions.The collagen II-induced arthritis (CIA) rat synovial cells were maintained and cultured in RPMI-1640 medium containing 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin and were seeded in a 6-well culture plate at a density of 4 × 10The sample proteins collected as described in After collection of published literature data and confirmation from the PubChem database, chemoinformatic data of 432 effective small molecules of TCM and their corresponding 3D structure SDF files were obtained  evaluation showed that 330 small molecules exhibited optimal lipophilicity . The average value for the synthetic accessibility of small molecules was 4.69, suggesting that most compounds were easy to synthesize in the laboratory. The prediction of cardiac inhibitory toxicity (hERG) suggested that 19 small molecules had cardiotoxic properties , leukocyte migration (GO:0050900), immune response activation of the cell surface receptor signaling pathway (GO:0002429), and antigen receptor-mediated signaling (GO:0050851). Thirteen CC functions mainly include the outer side of the plasma membrane (GO:0009897), extracellular matrix containing collagen (GO:0062023), and membrane rafts (GO:0045121). In addition, 19 MFs involved receptor ligand activity (GO:0048018), cytokine activity (GO:0005125), G-protein-coupled receptor binding (GO:0001664), and endopeptidase activity (GO:0004175), among several others.KEGG pathway enrichment analysis revealed that RA-related DEGs mainly involved 31 related pathways. Among them, Th17 cell differentiation (hsa04659), rheumatoid arthritis (hsa05323), NF-kappa B signaling pathway (hsa04064), TNF signaling pathway (hsa04668), Th1 and Th2 cell differentiation (hsa04658), and Toll-like receptor signaling pathway (hsa04620) have been reported to be closely related to RA. A total of 30 targets enriched in these pathways were screened out, namely, BCL2A1, BIRC3, BLNK, CCL5, CD247, CD3D, CXCL10, CXCL11, CXCL6, CXCL9, GADD45B, HLA-DMB, HLA-DOB, HLA-DPB1, IL15, IL1R1, IL21R, IL2RG, ITGB2, JUN, JUNB, LCK, MMP9, PLCG2, PRKCB, SOCS3, SPP1, TLR7, TLR8, and TNFSF11, as shown in We further examined the intersection shown in AutoDock Vina was used to perform bulk molecular docking on the two protein targets CCL5 and CXCL10, and the affinity values of 432 small molecules of TCM to act on the targets were obtained, except for the very few unsuccessful dockings. The distribution of the molecular docking scores of all small molecules of TCM on CCL5 and CXCL10 is shown in In this study, we only listed the top 10 small molecules of TCM with 15 molecular docking scores, as shown in Among the small molecules, two saponins, polyphyllin II and dioscin, had affinities of less than -9.0. Based on molecular docking conformation analysis of these two small molecules, we hypothesized that a combination of dioscin with CCL5 and CXCL10 was most stable. Therefore, we selected dioscin for subsequent in-depth analysis, and the binding conformations of CCL5 and CXCL10 are shown in Figures To comprehensively analyze the potential anti-RA mechanism of dioscin, we used HitPick, SEA, SwissTargetPrediction, and STITCH to predict and screen the targets of dioscin. As a result, we found that dioscin had two potential targets, CXCR2 and IL2. In addition to the results of molecular docking screening presented in Next, we verified the molecular docking of dioscin for CXCR2 (PDB ID: 6LFO) and IL2 (PDB ID: 4NEM). The results showed that the docking score of dioscin and CXCR2 was -9.1, which was identical to the docking score of dioscin and IL2 (-9.1). The molecular binding conformation also indicated that dioscin could form stable interactions with these two protein targets, as shown in Figures Furthermore, GO function annotation and KEGG pathway enrichment analysis revealed that the four targets CXCR2, CCL5, CXCL10, and IL2 were mainly involved in GO functions, such as cellular calcium homeostasis (GO:0006874), calcium homeostasis (GO:0055074), cellular divalent inorganic cation homeostasis (GO:0072503), kinase regulator activity (GO:0019207), cytokine activity (GO:0005125), G-protein-coupled receptor binding (GO:0001664), and external side of the plasma membrane (GO:0009897). The KEGG pathway was mainly enriched in 10 pathways. The interaction pathways between viral proteins and cytokines and cytokine receptors (hsa04061), the interaction pathway between cytokines and cytokine receptors (hsa04060), chemokine signaling pathway (hsa04062), cytoplasmic DNA sensing pathway (hsa04623), epithelial cell signaling pathway in Helicobacter pylori infection (hsa05120), Chagas disease pathway (hsa05142), Toll-like receptor signaling pathway (hsa04620), TNF signaling pathway (hsa04668), influenza A (hsa05164), and human cytomegalovirus infection pathway (hsa05163) are shown in A total of 140 and 193 known RA targets were collected from the TTD and DrugBank databases, respectively. After the two sets were intersected, 23 common RA treatment targets were obtained: ABCG2, AKR1B1, ALOX5, CASP1, DHFR, DHODH, HRH4, IL2, JAK1, JAK2, JAK3, MMP9, NR3C1, PDE4A, PDE4B, PDE4D, PLA2G1B, PPARA, PTGS1, PTGS2, TLR7, TLR9, and TNF. Among them, IL2 is also a potential direct target of dioscin.SAB = −1.16 < 0), where 〈dAB〉 = 2.85, 〈dAA〉 = 4.62, 〈dBB〉 = 3.40. Therefore, we speculated that dioscin could indirectly regulate and intervene in other 22 known RA targets by directly acting on the four targets, i.e., CCL5, CXCL10, CXCR2, and IL2, in treating RA. Based on further analysis of the human PPI network, we found that CCL5, CXCL10, CXCR2, and IL2 interacted with 54 proteins in the human PPI network to regulate other therapeutic targets of RA, as shown in Using the algorithm, we obtained the proximity values of dioscin to the 23 clinically known RA treatment targets on the human PPI network (https://tcmspw.com/) and obtained 42 small molecules of TCM with anti-RA effects and corresponding therapeutic targets , asiaticoside (300 μg/ml), neferine (400 μg/ml), ginsenoside Re (300 μg/ml), polyphyllin II (800 μg/ml), Bupleurum saikosaponin A (750 μg/ml), and alpha-crocin I (1000 μg/ml), for in vitro experiments. Tripterygium glycosides (150 μg/ml) were used as a control for small molecules of TCM. Therefore, a total of eight small molecules of TCM were included in this experiment.Based on the results of molecular docking, we selected seven small molecules, namely, dioscin  were selected for follow-up research. Target prediction and κB ligands [Reportedly, chemokines play an important role in inflammatory cell infiltration in the joints of patients with RA , and che ligands . Studies ligands , and an  ligands . Similar ligands . Therape ligands . Studies ligands .Dioscoreanigra, D. zingiberensis, and D. fuzhouensis. Dioscin exerts many therapeutic effects on various diseases [Dioscin is found in medicinal plants such as diseases , 33, sucdiseases . In an adiseases . Moreovediseases . Other sdiseases  and exerdiseases . In addidiseases . In our In this study, we used an algorithm based on network proximity to innovatively detect the regulation and intervention ability of dioscin on the known clinically therapeutic targets of RA. The results clearly showed that dioscin, by acting directly on four targets, indirectly intervenes and affects 54 related proteins and eventually regulates the main therapeutic targets of RA. We found that, in the network of dioscin-regulated RA targets, UBC (degree = 18), ARRB1 (degree = 14), APC (degree = 8), VCAN (degree = 7), GRB2 (degree = 6), IL2RB (degree = 6), FGR (degree = 6), and GNA15 (degree = 5) had higher degree values. The discovery of these target proteins in the network provides new insights for further related research and could serve as an important reference for the development of other RA drugs.in vitro experiments confirmed that dioscin exerted significant regulatory effects on CCL5, CXCL10, CXCR2, and IL2. The series of integrated analysis methods performed in this study are expected to provide powerful technical support for the treatment of RA and development of novel molecules of TCM.In summary, this study comprehensively used bioinformatics, molecular docking-based virtual screening, network modeling, and other computational methods; collected 432 commonly used TCM active ingredients; and analyzed the overall ADMET characteristics of these small molecules. The GEO and PPI network analyses helped to identify several key target genes associated with RA, and molecular docking technology was used to screen the small molecules of TCM for the treatment of RA. Furthermore, we analyzed the potential treatment mechanism of RA through network proximity-based prediction, and


Capsicum annuum L.) contain various nutrients and phytochemicals that enhance human health and prevent the pathogenesis of certain diseases. Here, we report that oral administration of orange sweet pepper juices prepared by a high-speed blender and low-speed masticating juicer reduces UVB-induced skin damage in SKH-1 hairless mice. Sweet pepper juices reduced UVB-induced skin photoaging by the regulation of genes involved in dermal matrix production and maintenance such as collagen type I α 1 and matrix metalloproteinase-2, 3, 9. Administration of sweet pepper juices also restored total collagen levels in UVB-exposed mice. In addition, sweet pepper juices downregulated the expression of pro-inflammatory proteins such as cyclooxygenase-2, interleukin (IL)-1β, IL-17, and IL-23, which was likely via inhibiting the NF-κB pathway. Moreover, primary antioxidant enzymes in the skin were enhanced by oral supplementation of sweet pepper juices, as evidenced by increased expression of catalase, glutathione peroxidase, and superoxide dismutase-2. Immunohistochemical staining showed that sweet pepper juices reduced UVB-induced DNA damage by preventing 8-OHdG formation. These results suggest that sweet pepper juices may offer a protective effect against photoaging by inhibiting the breakdown of dermal matrix, inflammatory response, and DNA damage as well as enhancing antioxidant defense, which leads to an overall reduction in skin damage.Sweet pepper fruits ( The skin, the largest organ of the human body, is composed of the epidermis, dermis, and subcutis, which provide the first line of defense against external environmental factors, such as physical damage, temperature shock, pathogens, and ultraviolet (UV) radiation . In addiSunlight is one of the most crucial causes of skin aging, also known as photoaging, and accounts for up to 90% of visible skin aging . Skin phCapsicum with more than 200 varieties, are considered natural sources of bioactive compounds such as carotenoids, vitamin C and E, flavonoids, phenolic compounds, and dietary fiber [Lactobacillus plantarum have been indicated to exert protective effects against oxidative stress-mediated retinal damage [Numerous epidemiological and laboratory studies suggest regular consumption of fruits and vegetables can enhance skin health and delay skin aging . A wide ry fiber . Previoury fiber ,10,11. Fry fiber , anti-inry fiber , cancer ry fiber , and prery fiber . Yellow/ry fiber . Using il damage . Four col damage . In addil damage  and imprFresh vegetable/fruit juice has been receiving great interest from consumers because of a simple household juicing preparation, retaining the major health-promoting components of whole vegetables/fruit, and the absence of additives and preservatives ,22. ConsThe consumption of fresh vegetable/fruit juice has recently been attracting great interest in the improvement of skin health. Fresh juice contains a wide range of bioactive compounds, such as carotenoids and polyphenols that contribute to maintaining skin functions and preventing skin injury and carcinogenesis. Carotenoids have been demonstrated to exert various benefits on the skin, including protection against sunlight-induced erythema, pigmentation and collagen degradation, the inhibition of UV-induced oxidative stress and inflammation, and the prevention of photoaging and photocarcinogenesis . In addiProtective effects of HSB and LSM sweet pepper juices against photoaging were examined using UVB-irradiated hairless mice and animal experimental design was illustrated in Skin photoaging is characterized by the atrophy of the dermal connective tissue that is associated with the destruction of extracellular matrix (ECM) components, particularly collagen fibers. UV radiation stimulates the synthesis of matrix metalloproteinase (MMP) members, such as MMP-1, -2, -3, -9, and -13 in the skin keratinocytes and fibroblasts, which in turn degrade collagen and other ECM proteins, thereby leading to wrinkle formation and skin aging . IncreasChronic UVB irradiation is reported to cause skin inflammation, as characterized by accelerating the production of pro-inflammatory mediators and cytokines. Cyclooxygenase-2 (COX2) catalyzes the rate-limiting step in the conversion of arachidonic acid into prostaglandins . UV-induAberrant expression of COX-2 was observed in the models of human and murine skins acutely and chronically irradiated with UVB ,49,50. INuclear factor kappa (NF-κB) is a redox-sensitive transcription factor responsible for regulating the transcriptional expression of a wide range of pro-inflammatory factors, such as COX-2, cytokines, chemokines, and adhesion molecules . ActivatThe skin is one of a few organs that come into direct contact with exogenous oxidative sources, such as sunlight. Solar UV radiation can promote the formation of reactive oxygen species (ROS) such as superoxide anion radical, hydrogen peroxides, hydroxyl radicals, and other oxidants that directly and/or indirectly damage to biomolecules, such as proteins, lipids, and nucleic acids and interrupts cellular signaling pathways, eventually leading to skin damage. Approximately 80% of ROS in the skin are thought to be produced by UVA and UVB irradiation .Oxidative stress is thought to play a central role in triggering and driving the signaling events that lead to cellular responses, such as cell death, cellular senescence, and inflammation, following UVB radiation. ROS can activate redox-sensitive transcription factors, such as NF-κB and AP-1 that upregulate the production of inflammatory mediators and cytokines. In addition, UVB-induced ROS also stimulates the release of MMP enzymes, such as MMP-1, -2, -3, -9, and -13, in keratinocytes and fibroblasts, which in turn degrade collagen and other ECM proteins, and simultaneously attenuate collagen synthesis in the skin . TherefoSkin cells are equipped with an antioxidant defense system, consisting of enzymatic and non-enzymatic antioxidants, which maintain the pro-oxidant/antioxidant balance by ROS elimination. Nevertheless, flooding of ROS can overwhelm the cellular antioxidant defense capacity and further generate reactive oxidants, resulting in oxidative stress and consequently oxidative photodamaging of the skin . TherefoA battery of antioxidant enzymes plays a vital role in counteracting excessive ROS accumulation, thereby maintaining cellular redox homeostasis. Primary antioxidant enzymes, including catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) are considered to be the most important antioxidant defense in the skin . SOD actTo further investigate whether the protective effects of HSB and LSM sweet pepper juices were linked to the enhancement of the antioxidant defense system, we examined the expression of antioxidant enzymes in the mouse skin tissues. UVB irradiation abolished the expression of primary antioxidant enzymes, including CAT, GPx, and SOD-2, suggesting provoked oxidative stress in UVB-exposed mouse skin a. In conDNA damage induced by UVB irradiation occurs mainly via oxidative processes and 8-OHdG is the major mutagenic form of oxidative DNA damage . The preIn conclusion, the regular intake of fresh vegetable/fruit juice containing various intact phytonutrients without preservatives is believed to bring about health benefits, including skin health improvement. Sweet orange pepper fruit with high levels of carotenoids, vitamin C, and other bioactive components have the potential for use in a dietary supplement for skin health. In this study, oral administration of sweet pepper juice exerted a protective effect against UVB-induced skin aging in a hairless mouse model by inhibiting MMP expression and restoring collagen synthesis, thereby preventing skin wrinkles. UVB-induced skin inflammation was suppressed by the consumption of sweet pepper juice. Simultaneously, sweet pepper juice also maintained the antioxidant defense capacity of the skin and thus prevented UVB-induced oxidative damage. In addition, juicing methods were considered in the present study and sweet pepper juice prepared by an LSM juicer may exhibit pronounced efficacies, compared to the juice by HSB. However, different experimental models with long-term consumption are required to compare the biological effects of HSB and LSM juices.Formalin, hematoxylin, and eosin were purchased from Sigma-Aldrich . Anti-Col1A1, p-p65, p65, COX-2, MMP-2, MMP-3, TNF-α, and peroxidase-conjugated secondary anti-rabbit antibodies were obtained from Cell Signaling Technology . Anti-catalase, GPx, SOD-2, IL-1β, Il-17, Il-23, MMP-9, and peroxidase-conjugated secondary anti-mouse antibodies were purchased from Santa Cruz Biotechnology . Anti-8-OHdG antibody was procured from Thermo Scientific . All other chemicals used in this study were of analytic grade.Capsicum annuum L.) were purchased from Hoengseong Rainbow farm  and processed right after arrival. For purpose of this study, fresh orange sweet pepper juices were prepared daily using a high-speed blender  and a low-speed masticating juicer . The major bioactive constituents in orange sweet pepper juices were analyzed. Total phenolic content in LSM and HSN juices was 111.77 ± 1.88 mg/100 mL and 96.45 ± 3.56 mg/100 mL, respectively, while total carotenoid content was 3.49 ± 0.01 mg/100 mL and 4.18 ± 0.1 mg/100 mL, respectively. The level of total ascorbic acid was 119.80 ± 3.19 mg/100 mL for LSM juice and 100.63 ± 1.10 mg/100 mL for HSB juice.Orange sweet pepper fruits  and housed in plastic cages at 25 ± 2 °C and 50 ± 5% relative humidity under a 12–12 h light–dark cycle. All animals were allowed free access to standard food and water ad libitum. All experiment protocols were approved by the Institutional Animal Care and Use Committee of Kyungpook National University (No. KNU 2021-182). After a week of acclimation, mice were randomly divided into seven groups (n = 10): Normal control, HSB juice (10 mL/kg), LSM juice (10 mL/kg), UVB, UVB + HSB juice (10 mL/kg), UVB + LSM juice (10 mL/kg), and UVB + Vitamin C . HSB juice, LSM juice, or Vitamin C were orally administered to the animals in each group daily for 7 weeks, while the mice in the normal control and UVB groups were given an equal amount of distilled water. Vitamin C is well-known to protect against UVB-induced skin damage ,69 and vDorsal skin tissues of each group were fixed in 10% neutral formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) for general histopathology and Masson’s trichrome to examine epidermal thickness and collagen fiber, respectively. For immunostaining, the sections were incubated with specific mouse anti-8-OHdG primary antibody in a humidified chamber overnight at 4 °C and followed by incubation with horseradish-conjugated secondary antibody and 3,3’-diaminobenzidine. Representative H&E, Masson’s trichrome, and immunostained images of sections were captured using a digital microscope .Dorsal skin tissues were homogenized in distilled water and total collagen content was measured by total collagen assay kit  according to the manufacturer’s instructions.g for 15 min at 4 °C to obtain supernatant. Protein samples were aliquoted out and stored at −80 °C. The protein concentrations were measured using a PierceTM BAC protein assay kit (Thermo Fisher Scientific). An equal amount of protein samples was resolved on SDS-PAGE gel and then transferred to polyvinylidene fluoride membrane using a semidry transfer system . After blocking with 5% non-fat skim milk, the membranes were incubated with specific primary antibodies overnight at 4 °C and followed by hybridization with proper secondary antibodies for 3 h at 4 °C. Eventually, protein bands were visualized using Western Blotting Luminol reagent (Santa Cruz Biotechnology).Dorsal skin tissues were homogenized in RIPA buffer containing protease and phosphatase inhibitors  using Precellys 14 tissue homogenizer . The lysates were then centrifuged at 13,000× p-value < 0.05 was considered to be statistically significant.Data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Statistical analysis was performed using analysis of variance followed by the Tukey’s post hoc test. The


The loss and/or dysregulation of several cellular and mitochondrial antioxidants’ expression or enzymatic activity, which leads to the aberrant physiological function of these proteins, has been shown to result in oxidative damage to cellular macromolecules. In this regard, it has been surmised that the disruption of mitochondrial networks responsible for maintaining normal metabolism is an established hallmark of cancer and a novel mechanism of therapy resistance. This altered metabolism leads to aberrant accumulation of reactive oxygen species (ROS), which, under specific physiological conditions, leads to a potential tumor-permissive cellular environment. In this regard, it is becoming increasingly clear that the loss or disruption of mitochondrial oxidant scavenging enzymes may be, in specific tumors, either an early event in transformation or exhibit tumor-promoting properties. One example of such an antioxidant enzyme is manganese superoxide dismutase , which detoxifies superoxide, a ROS that has been shown, when its normal physiological levels are disrupted, to lead to oncogenicity and therapy resistance. Here, we will also discuss how the acetylation of MnSOD leads to a change in detoxification function that leads to a cellular environment permissive for the development of lineage plasticity-like properties that may be one mechanism leading to tumorigenic and therapy-resistant phenotypes. A fundamental theme in precision, or personalized, cancer medicine is identifying tumors that are vulnerable to specific therapies, based on well-defined molecular biomarkers or tumor signatures ,2,3,4. ICancer Discovery) [The estrogen receptor pathway has a critical role in breast cancer and, therefore, endocrine therapy, which blocks ER signaling, is highly effective; however, over time, a small subset of ER+ tumors recur due to the development of endocrine resistance ,8. Multiscovery)  stated lWe define lineage plasticity as a reversible or irreversible reprogramming of cellular systems, where a mature somatic cell can display plasticity, via a change in cell “identity” by dedifferentiation to a progenitor-like state or by transdifferentiation to an alternative differentiated cell type, leading to the emergence of new phenotypes ,15,16,17+ dependent class III histone deacetylases that are found in various genomes from bacteria to humans [Saccharomyces cerevisiae Sir2 that have been shown to regulate life span and aging in model organisms, including Saccharomyces cerevisiae, C. elegans, and Drosophila melanogaster [+-dependent enzymes. These seven proteins are localized to the nucleus , mitochondria , and cytoplasm (Sirt2) and share a 275-amino-acid catalytic deacetylase domain [Sirtuins are NADo humans ,21,22, wo humans ,24,25,26o humans ,28. Sirtnogaster ,29,30,31Sirtuins, including the primary mitochondrial sirtuin, Sirt3 ,22, appe2+ atom [Thus, an important theme that has emerged in the last several years is that SIRT3, and its most important downstream targets, direct multiple mitochondrial processes. In this regard, it appears that the non-physiological levels of MnSOD acetylation (Ac), at least in part, connect metabolic and bioenergetic balance and tumor cell growth and survival. This is based on a recently identified novel mitochondrial signaling axis centered on MnSOD-Ac, which, when dysregulated, disrupts cell metabolism, leading to aberrant ROS levels ,48. MnSO2+ atom ,50. The 2+ atom ,51, K68 2+ atom ,52,53, a2+ atom ,35,37,382+ atom ,55,56,572+ atom ,45.Sirt3, and thus containing MnSOD-Ac, develop estrogen-receptor-positive (ER+), poorly differentiated, high Ki-67 mammary gland tumors that exhibit very similar characteristics to human luminal B breast malignancies [+/−) exhibit decreased MnSOD activity, increased oxidative stress, decreased life span, as well as aging-related phenotypes, especially carcinogenesis, which includes breast malignancies [Recently, we have been focusing on MnSOD-K68-Ac as one of the key SIRT3 targets that may play an important role in the oncogenicity observed in cells lacking SIRT3. In this regard, mice lacking gnancies ,36,44,56gnancies . In thisgnancies .2•− are oxidized to oxygen O2 and H2O2 through the catalytic reduction of Mn3+ to Mn2+ and restoration of Mn2+, respectively [2•− produced during energy production in mitochondria, MnSOD detoxification/ROS scavenging enzymic activity is critical to ATP production, maintaining mitochondrial homeostasis, and cell survival.The mitochondria electron transport chain (ETC) directs the flow of electrons in normal cellular metabolism, resulting in the production of superoxide radicals at less than 1% of the total rate of electron transport from NADH to oxygen ,60,61. Aectively ,67. Due In order to regulate the activity of proteins, cellular signaling networks employ different mechanisms, including post-translational modifications (PTMs), such as acetylation ,69,70,71−/− MEFs infected with lenti-MnSODK68Q decreased activity, while infection with lenti-MnSODK68R showed a significant increase in MnSOD activity, as compared to MEFs infected with wild-type (WT) lenti-MnSODWT [K68Q acetylation mimic mutant, as well as MnSOD-K68-Ac, to determine any differences in charge distribution, compared to the MnSOD wild-type.We recently published data, using site-directed MnSOD mutants, where K68 was substituted with either a glutamine (Q) ,79, an a-MnSODWT ,35,38. I-MnSODWT  deacetylK68QMnSOD and MnSOD-K68-Ac have nearly identical physiological and biochemical properties using tissue culture, molecular biology, and codon substitution experiments in bacteria [WT and MnSOD-K68-Ac, which clearly showed that K68-Ac increases the negative surface charge distribution along the α1/α2 helices adjacent to the Mn2+ binding site/tetramerization interface of MnSOD-K68-Ac [We have previously published that bacteria . In addiD-K68-Ac . ImportaD-K68-Ac  and Lu eD-K68-Ac . The tetD-K68-Ac . For conMnSOD constructs  were immunoprecipitated (IPed) and tested for enzymatic activity. These experiments showed that MnSOD from MEFs lacking MnSOD (MnSOD−/−) expressing K68QMnSOD exhibited a 40-fold increase in peroxidase activity, compared to IPed MnSOD-K68R or MnSOD-WT [Experiments with the MnSOD-K68-Ac mimic mutant, as well as the deacetylation mimic mutant, have indicated that MnSOD-K68Q loosens the homotetramer and dimeric/monomeric forms are favored. Based on these results it was surmised that MnSOD-K68-Ac may have a different function than the detoxification SOD activity observed for the homotetrameric complex. In this regard, the Flag-tagged MnSOD-WT . IdenticMnSOD-WT ,53.Sirt3 develop ER+ luminal B-like tumor properties, suggesting that MnSOD, perhaps when acetylated, may function as a tumor promoter, instead of its more traditional function as a detoxification enzyme and tumor suppressor (TS) [c-Myc or Ras [−/− primary mouse embryo fibroblasts (pMEFs) infected with lenti-MnSODK68Q, and c-Myc or Ras, became immortalized  but not pMEFs infected with c-Myc or Ras alone. In contrast, co-infection of lenti-MnSODK68R with c-Myc or Ras failed to immortalize pMEFs [c-Myc and Ras were immortalized, as has been previously shown [K68R prevented immortalization of pMEFs with c-Myc and Ras, implying that MnSODK68R is a tumor suppressor. Lastly, pMEFs infected with lenti-MnSODK68Q, as compared to cells infected with MnSODK68R, exhibited increased: (1) soft agar growth, a measure of anchorage independence; (2) growth when plated at low density (250 cells/60 mm plate), a measure of proliferative capacity; (3) doubling time, a measure of proliferative rate; and (4) xenograft growth, a measure of tumorigenicity [We previously showed that mice lacking sor (TS) ,84. For c or Ras , are reqze pMEFs ,52. As aly shown ,86. Integenicity .K68QMnSOD, which is proposed to model MnSOD-K68-Ac, can function as an in vitro oncogene as opposed to its more established role as a detoxification enzyme that can, under specific conditions, function as a tumor suppressor. These results may explain why initial clinical data suggest that MnSOD acts as a tumor suppressor [These established in vitro tissue culture transformation experiments support the premise that MnSOD-K68-Ac can function, under specific conditions, as a tumor promoter ,87. Thusppressor ; howeverppressor ,90. ThesThus, MnSOD may switch from a tetrameric dismutase to a monomeric peroxidase in response to specific cellular stress conditions, such as nutrient status and/or transformation . In this+/− heterozygous mice had a 50% reduction in SOD activity and increased 8-oxo-2-deoxyguanosine (8oxodG) in nuclear DNA [+/− tissues. Tumor formation was observed in all MnSOD+/− mice with increased oxidative DNA damage, which indicates the correlation of MnSOD activity and carcinogenesis [−/−Sirt3 MEFs also showed an elevation of oxidative stress induction, which is associated with abnormal physiology changes in mitochondria. Additionally, ER/PR-positive mammary tumor development was observed in mice lacking Sirt3 (34). Other studies also showed an increase in Ki-67 in knockout Sirt3 female mice ER+ tumors, a marker for human luminal B breast malignancies [K68Q, the MnSOD acetylation mimic mutant. Together, these results show a correlation of Sirt3, MnSOD acetylation status, and ROS detoxification activity, as well as mitochondrial metabolic stress with carcinogenesis, implicating Sirt3 as a tumor suppressor.Considering the important role of MnSOD in detoxification, it is worthwhile to mention that the role of MnSOD goes beyond this activity. Van Remmen et al. revealed that MnSODlear DNA ,55,58, sogenesis ,91. Integnancies ,35,45. Agnancies ,87. Furt2•−) into hydrogen peroxide (H2O2); however, it has been shown to demonstrate peroxidase activity under certain conditions [2•−, it has been suggested that MnSOD’s second important function is as a signaling hub, as well as a superoxide dismutase [2•− and H2O2 oxidize targets [2O2 has a long half-life in aqueous solutions and can easily permeate mitochondria membranes; however, due to varied reduction potentials of amino acids, it has been shown that H2O2 selectively binds and oxidizes the active site of several phosphatases, rendering them inactive [As discussed above, it is proposed that there may be a mitochondrial equilibrium shift in the MnSOD tetrameric/monomeric ratio when K68 is acetylated. MnSOD catalyzes the dismutation of superoxide -derived cancer stem-like cells, overexpression of MnSOD and FOXM1 mitigated the effects of genistein, decreasing stemness/lineage-plasticity-like markers and the profound migratory/invasive activity [MnSOD and FOXM1 enhanced the effect of genistein [2O2 concentrations, acting as a signaling molecule that increases expression of stemness/lineage-plasticity-like markers; however, the actual MnSOD activity in cells is not addressed in these experiments.MnSOD has also been linked to signaling in cancer cells through the epithelial to mesenchymal transition (EMT), also referred to as a lineage-plasticity-like phenotypic switch, leading to more aggressive phenotypes, pan anticancer resistance properties, and a metastasis permissive tumor cell phenotype ,110,111.activity , whereasenistein . These s2O2 signaling with overexpression of catalase reduces the expression of HIF2α, as well as stemness biomarkers, including Oct4 and Nanog [2O2 levels, which adds intriguing possibilities when compared to previous reports [K68QMnSOD mutant, which is a biochemically validated acetylation mimic, also showed an increase in stemness markers [2O2 levels than the parental cell line (A431-P) [MnSOD led to an increase in H2O2 and invasiveness of the cells in both the parental and metastatic cell line [2O2 signaling and reversed the effects of the siRNA [2O2 concentrations, which in turn leads to a potential lineage-plasticity-like switch to cells that exhibit stemness or EMT biomarkers.In a quite interesting study, it was shown that the forced overexpression of MnSOD also led to an increase in acetylation of lysine 68 in breast cancer cells . He et and Nanog . Based o reports ,112,113. markers  and prov(A431-P) . Using sell line . Treatmehe siRNA . These tK68QMnSOD or made in bacteria using a codon expansion method, the concentration of monomeric MnSOD also increased as a proportion and/or change in the tetrameric to monomeric ratio [K68QMnSOD acetylation mimic mutant or silencing SIRT3 also shows a higher proportion of monomer and peroxidase activity [2O2 concentration increases. The conditional nature of MnSOD all falls back to whether it is acetylated or not, and on the functionality of SIRT3.Shedding light on the seemingly opposing nature of MnSOD, as it was previously discussed when cells are manipulated to express MnSOD-K68-Ac experimentally by using the acetylation mimic ic ratio . In addiic ratio ,92. Instactivity . A potenactivity . Therefoactivity , H2O2 co2+ cofactor, whereas bacteria can utilize both MnSOD and a distinct but similar FeSOD using Fe3+ as a cofactor. The essential structure of these enzymes is remarkably similar, especially in the active site region. In both, the active site region contains the metal cofactor ligated by three histidine residues, one aspartate residue, and one hydroxide ion that is stabilized by a highly conserved glutamine residue. In MnSOD, the glutamine arises from the C-terminal domain (Q146 E. coli), but in FeSOD, the glutamine arises from the N-terminal domain (Q69 E. coli) [MnSOD is an old, evolutionally conserved protein and is required for organisms that make ATP through respiration, primarily coming in two varieties that are categorized based on the cofactor identity. Eukaryotic MnSOD utilizes a MnE. coli) . Despite2+(Mn)SOD is able to catalyze the reduction of O2•−, but the Fe3+(Mn)SOD generated from Fe2+(Mn)SOD during the previous reaction is not able to oxidize O2•−, and the catalytic cycle cannot be completed [2+ cofactor under normal conditions. Studies with S. cerevisiae have suggested that the exclusivity of MnSOD within mitochondria arises because the metalation process is coupled with the import of the unfolded polypeptide MnSOD into the mitochondria [It has been well established that substitution of iron into MnSOD, (Fe(Mn)SOD), leads to a virtually complete loss of dismutase activity for the enzyme. It is also well understood that the structure of the enzyme is virtually unchanged upon incorporation of the “wrong” metal and the loss of activity most likely arises from a deficiency in the reduction potential of the Fe-substituted enzyme. Vance et al. suggested that Feompleted . A similompleted . Howeverchondria , and oncS. cerevisiae by disruption of manganese trafficking factor (mtm1) [ssq1 or grx5 [Mis-metalation can be induced in r (mtm1) . Mtm1 pl or grx5 , has als or grx5 . This is or grx5 ,115,118.2O2 [∙+ [2O2 and supported protein radical formation. The cultivated Mn-bound MnSOD was unable to oxidize Amplex Red and the formation of protein radicals was not observed [Recent developments suggest that Fe(Mn)SOD has deleterious effects, not only because of its loss of dismutase function, but also because of a gained pro-oxidant/peroxidase function . In earl2O2 . However2O2  and seve2O2 [∙+  or Ample2O2 [∙+ , whereas2O2 [∙+ . Further2O2 [∙+ . Cells gobserved . Thus, tIn conclusion, the data in this review article suggest that MnSOD-Ac, including K68-Ac, may function as an oncoprotein in an advanced stage of cancer, in contrast to the more traditional premise that MnSOD is a detoxification TS , and thi


RUNX2mutations. Although numerous patients have been described, a direct genotype–phenotype correlation forRUNX2has been difficult to establish. Further cases must be studied to understand the clinical and genetic spectra of CCD.Cleidocranial dysplasia (CCD) is a skeletal disorder affecting cranial sutures, teeth, and clavicles, and is associated with theTo characterize detailed phenotypes and identify variants causing CCD in five unrelated patients and their family members.Clinical and radiographic examinations were performed. Genetic variants were identified by exome and Sanger sequencing, data were analyzed by bioinformatics tools.RUNX2variants in all affected individuals. Three were novel, comprising a frameshift c.739delA ) in exon 6 (Patient-1), a nonsense c.901C>T (p.(Gln301*)) in exon 7 (Patient-2 and affected mother), and a nonsense c.1081C>T (p.(Gln361*)) in exon 8 (Patient-3). Two previously reported variants were missense: the c.673C>T (p.(Arg225Trp)) (Patient-4) and c.674G>A (p.(Arg225Gln)) (Patient-5) in exon 5 within the Runt homology domain. Patient-1, Patient-2, and Patient-4 with permanent dentition had thirty, nineteen, and twenty unerupted teeth, respectively; whereas Patient-3 and Patient-5, with deciduous dentition, had normally developed teeth. All patients exhibited typical CCD features, but the following uncommon/unreported phenotypes were observed: left fourth ray brachymetatarsia (Patient-1), normal clavicles (Patient-2 and affected mother), phalangeal malformations (Patient-3), and normal primary dentition .Three cases were sporadic and two were familial. Exome sequencing successfully detected the heterozygous pathogenicRUNX2function. Here, we report a new CCD feature, unilateral brachymetatarsia, and three novel truncating variants, expanding the phenotypic and genotypic spectra ofRUNX2, as well as show that the CCD patients can have normal deciduous teeth, but must be monitored for permanent teeth anomalies.The study shows that exome sequencing is effective to detect mutation across ethnics. The two p.Arg225 variants confirm that the Runt homology domain is vital for The incidence of CCD is about one in a million.  CCD is also known as Scheuthauer-Marie-Sainton syndrome, cleidocranial dysostosis, oste-odental dysplasia, generalized dysostosis, mutational dysostosis, and cleidocranial-pubic dysostosis.  The main clinical features of CCD are hypoplastic or aplastic clavicles, defective ossification of anterior fontanelle, and dental anomalies .  The unique facial appearances of CCD are prominent forehead, midfacial hypoplasia, and hypertelorism. Other bone deformities include wide pelvic joint, coxa vara , coxa valga , genu valgum (knock-knee deformity), delayed growth of the pubic bone, merging failure of the lower jaw, scoliosis, and brachydactyly.  Treatment for CCD depends on the symptoms. The patient may undergo several orthopedic and facial reconstructive surgery to increase satisfaction and function.  Moreover, Orthodontics and oral surgery treatment are necessary to remove extra teeth and guide the eruption of affected teeth. Cleidocranial dysplasia  is a congenital disorder that affects bone growth and teeth formation. RUNX2gene.  The human runt-related transcription factor 2 gene  consists of 9 exons, but only 8 exons encode the 521-amino acid RUNX2 protein. The RUNX2 comprises a poly-glutamine and poly-alanine  repeat region, runt homologous domain (RHD amino acids 101-226), nuclear localization signal , proline–serine–threonine (PST)-rich region (amino acids 236-516), nuclear matrix targeting signal , and VWRPY pentapeptide sequence (amino acids 517-521).  Both the QA and PST regions act as the transactivation regions. The RHD is highly conserved and plays a role in DNA binding.  The VWRPY pentapeptide sequence functions as a transcriptional repression region. Previous studies have reported that the human CCD is caused by various variants in the In humans, the heterozygous loss-of-function variants inRUNX2are the major cause of CCD (60-70% of patients). However, the genetic conditions in the remaining 30-40% of the cases are still unknown. Direct genotype–phenotype correlation for RUNX2 has been difficult to establish due to variable phenotypic penetrance of the variants. Most variants in the RHD domain result in a classical CCD phenotype.  However, some exceptions like the families with p.Thr200Ala exhibited significant intrafamilial variability: two of four children showed classic CCD, while the father, who also possessed the variant, only had dental anomalies.  Diverse intra- and inter-familial genotype-phenotype correlations have been reported. For example, in a family with c.203delAinsCG, mild intra-familial genotype-phenotype were observed, including bell-shaped rib cage, hypoplastic iliac wing, and hypoplastic femoral head; in a family with c.614C>G, significant intra-familial correlation were observed, including head and neck findings, delayed tooth eruption, hypoplastic clavicle, and short stature; and in patients with c.1281delC, no inter- or intra-familial correlation was found.  In a study of 42 unrelated CCD patients, there was no phenotypic difference between patients with deletions or frameshifts and those with other intragenic variants in RUNX2,  whereas another study, with 24 unrelated Japanese patients, observed short stature to be much milder in the patients with the intact RHD domain than in those without.  Despite having more than 180 different pathogenic variants identified, the genotype-phenotype correlation of the syndrome is still unclear.  The identification of novel variants and phenotypes can provide more understanding about CCD and how each variant contributes to different expressivity.RUNX2 is a transcription factor that either activates or inhibits the gene expression by regulating transcription through binding specific DNA sequences or interacting with transcriptional co-inducers and co-depressors to regulate osteogenesis. RUNX2in all five cases. Three were novel pathogenic variants, expanding the genotypic spectrum ofRUNX2.Although numerous patients have been described, more patients need to be reported for a better understanding of its clinical and genetic spectra. Herein, we describe the clinical and molecular characteristics of five unrelated Thai CCD patients. Exome sequencing successfully identified causative variants in Tooth number was used according to the FDI World Dental Federation notation system or ISO 3950 Dentistry – Designation system for teeth and areas of the oral cavity (https://www.iso.org/standard/68292.html).Five unrelated Thai patients clinically diagnosed with CCD (Patient-1 to Patient-5) and six additional family members of Patients-2, -3, and -4 were recruited. Thorough investigation and blood collection were performed after receiving the written informed consent from all subjects or their legal guardians. The study was approved by the Human Research Ethics Committee  and complied with the Declaration of Helsinki. The clinical diagnostic criteria of CCD involve (a) hypoplasia or aplasia of the clavicles, (b) prominent forehead, enlargement of the fontanelles or delayed closure of the anterior fontanelle, and/or (c) retention of primary teeth, failure of secondary teeth, and supernumerary teeth. RUNX2 were confirmed by Sanger sequencing (Supplementary Table 1). Prediction software, PolyPhen-2, SIFT, and MutationTaster, were used to analyze the potential pathogenicity of the variants based on the possible impact structure and function of the protein. The American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guideline was used to classify the variants.  The variants were identified as novel if they were not published in the ClinVar-NCBI (https://www.ncbi.nlm.nih.gov/clinvar) and the gnomAD (https://gnomad.broadinstitute.org/).Genomic DNA extracted from 3 ml of peripheral blood leukocytes of the probands, their available family members, and from healthy individuals (as control) was subjected to variant analysis using exome or Sanger sequencing. For exome sequencing, genomic DNA was captured using a SureSelect Human All Exon version 4 kit  and sequenced using Hiseq 2000 Sequencer . The sequence reads were aligned to the human genome reference sequence  hg19) using the Burrows-Wheeler Alignment software (http://bio-bwa.sourceforge.net/). Downstream processing was performed with SAMtools (http://samtools.sourceforge.net/) and annotated by the dbSNP and 1000 Genomes. Subsequently, the variants were filtered out if their frequency were higher than 1% in the 1000 Genomes Project, Genome Aggregation Database (gnomAD), and our in-house database of 2,166 Thai exomes. The causal variants Identified inRUNX2(SCV001763563) (Patient-1 (1-I:2), a 28-year-old female, complained of missing teeth and chewing difficulty. She had short stature, narrow and down-sloping shoulders, metopic depression, hypertelorism, prominent orbital ridges, and protruding chin. Brachydactyly and short fourth toe of the left foot were also observed . Skull 763563) ,F.RUNX2(SCV001763562) (Patient-2 (2-II:1), a 21-year-old male, was referred for orthognathic surgery due to chewing difficulty. Clinical examination showed that the patient had a dolichofacial shape, facial asymmetry (chin shift to the right side), metopic depression, and protruding chin . Skull 763562) ,F.rd percentile), macrocephaly , metopic depression, saddle nose, hypertelorism, small shoulders that could be opposed at the midline, and lordosis . A novel heterozygous nonsense variant, c.1081C>T (p.(Gln361*)), in exon 8 ofRUNX2(SCV001763561) was detected (Patient-3 (3-II:1), a 6-year-old girl, presented short stature ), in exon 5 ofRUNX2 , a 20-year-old female, had a chief complaint of prominent chin and tooth missing. The patient presented a square-shaped face, metopic depression, and a slant and narrow shoulder that can be opposed at the midline . Skull 893992) ,F. 12 within normal range (0.25+0.04 g/cm 2 ); total body less head BMD was 0.267 g/cm 2 ; lumbar spine BMD was 0.142 g/cm 2 ; and whole-body mineral content was 163.14 g. At age 3, his height was 86.2 cm (<3 rd percentile). Examinations showed that 5-II:1 had macrocephaly , metopic depression, narrow sloping shoulders opposing at the midline, and lordosis ), in exon 5 ofRUNX2 , a 3-year-old boy, was referred for genetic analysis due to congenital malformations of skull and face bones, deformed clavicles, and a history of bilateral clavicular fractures. At 4 months old, his whole-body bone mineral density (BMD) was 0.267 g/cm ordosis . Skull ordosis . The paordosis . His bo893991) ,F. 1Phenotypically, all patients in this study shared typical features of CCD consisting of frontal and parietal bossing, metopic depression, unclosed fontanelle, and Wormian bones. Except for 2-II:1 and 2-I:2, all patients had hypoplastic/aplastic clavicles and abnormal facility in opposing the shoulders. 1-I:2, 2-II:1, 2-I:2, and 4-II:2 who had the permanent dentition showed multiple dental anomalies including clinically missing teeth, unerupted teeth, supernumerary teeth, and skeletal class III malocclusion. In contrast, 3-II:1 and 5-II:1 showed all deciduous teeth erupted and only crossbite.RUNX2and with heterogeneous phenotypic penetrance.  In this study, we reported five index patients with five differentRUNX2variants who had classic craniofacial features of CCD with clinical heterogeneity.Approximately two-thirds of CCD cases have been found to harbor pathogenic variants in Most of the missense variants have been observed in the RHD domain.  Consistently, the other two missense variants (c.673C>T and c.674G>A) were detected in the hotspot RHD domain and previously reported in many cases with typical CCD features.  Although these two variants alter the same amino acid p.Arg225, different clinical features were observed in patients 4-II:2 and 5-II:1. Patient 4-II:2 had aplastic clavicles and no parietal bossing, whereas 5-II:1 had deformity and pseudarthrosis of clavicles and lordosis. Functional analyses showed that the variants at Arg225 markedly reduced transactivation activities and interfered with nuclear accumulation of the RUNX2 protein, resulting in haploinsufficiency of the protein. Three truncating variants identified in the PST region were novel. The variants in the PST were shown to produce non-functional truncated proteins and impair the transactivation activities of RUNX2 on osteocalcin promoter, indicating that loss of function in RUNX2 is responsible for CCD. RUNX2variants, including the missense in the RHD domain and the truncating variants in the N-terminal, QA, and PST regions.  A correlation between dental abnormalities and location ofRUNX2variants was demonstrated in a group of five patients having normal clavicles. The nonsense variants in the QA region presented milder dental phenotypes than those with missense variants in the RHD domain.  With variants in the PST region and normal clavicles, previous CCD cases were reported with multiple supernumerary teeth.  Similarly, supernumerary teeth were found in patients 2-II:1 and 2-I:2. Further studies are needed to confirm this genotype-phenotype correlation.Normal clavicles were observed in Patients 2-II:1 and 2-I:2, who possessed the novel p.Gln301* variant in the PST region. They also shared similar craniofacial features, including facial asymmetry, class III malocclusion, and unerupted teeth, suggesting intra-familial genotype-phenotype correlation. Normal clavicles were previously reported in CCD cases with differentRUNX2is expressed from early stage to the formation of roots and periodontium.  It plays a role in osteoblast and odontoblast differentiation, alveolar bone remodeling essential for tooth eruption, and the maintenance of the periodontal ligament.  InRUNX2knockout mice, molar tooth development arrested at the late bud stage.  Interestingly, bothRunx2–/– andRunx2- mice developed lingual buds in front of the upper molars more than in wild-type mice, representing the extension of dental lamina for successional teeth.  It was suggested thatRunx2may have different effects at different stages of tooth development. Accordingly, the development of permanent dentition in CCD patients is severely disturbed while the primary dentition is rarely affected.  Patients 1-I:2, 2-II:1, 2-I:2, and 4-II:2 with permanent dentition, showed several dental anomalies, such as malocclusion, clinically missing of permanent teeth, unerupted teeth, and supernumerary teeth; whereas patients 3-II:2 and 5-II:1, with primary dentition, showed no remarkable tooth abnormalities. It may also be assumed that the effects of RUNX2 haploinsufficiency on the development of the primary dentition may be too subtle to be detected clinically. During tooth development,nd and 5 th proximal metacarpal bone, shortening of the 2 nd and 5 th middle phalanges, and fusion of the 1 st distal phalanx. Hand abnormalities, including brachydactyly, tapering fingers, and short and broad thumbs, are frequently observed in CCD patients.  A decrease or loss-of-function ofRUNX2is associated with CCD; while gain-of-function due to intragenic duplications is related to metaphyseal dysplasia with maxillary hypoplasia and brachydactyly .  These suggest that fine-tuning the expression level ofRUNX2is important for normal development of the phalanges or metacarpals. There have been only few clinical case reports describing brachymetatarsia in CCD cases, but without genetic variant identification.  Our findings could lead to genotype-phenotype correlation in the future.Apart from typical CCD features, 1-I:2 also presented with brachydactyly and fourth-ray brachymetatarsia of the left foot and 3-II:1 had partial pseudoepiphysis of the 2 RUNX2is the most common causative gene of CCD in this small sample of five patients. Here, we report three novelRUNX2variants expanding the genotypic spectrum of CCD and confirm that theRUNX2missense variants associated with typical CCD features are commonly observed in the RHD domain. Additionally, we demonstrated that a patient with the novel frameshift variant, c.739delA ), inRUNX2had brachymetatarsia, substantiating it as a clinical feature of CCD.This study showed that


Background: Immunotherapy has been demonstrated favorable in head and neck squamous cell carcinoma (HNSCC). Studies indicated that immune-related gene prognostic index (IRGPI) was a robust signature, and N6-methyladenosine (m6A) methylation had a significant impact on the tumor immune microenvironment (TIME) and immunotherapy of head and neck squamous cell carcinoma. Thus, combining indicated that immune-related gene prognostic index with m6A status should offer a better predictive power for immune responses.Methods: Head and neck squamous cell carcinoma samples from the cancer genome atlas  and gene expression omnibus database  were used in this study. Cox regression analysis was used to construct the indicated that immune-related gene prognostic index through immune-related hub genes which were identified by weighted gene co-expression network analysis (WGCNA). The m6A risk score was constructed by least absolute shrinkage and selection operator (LASSO) regression analysis. Principal component analysis was used to construct a composite score, and systematically correlate subgroups according to tumor immune microenvironment cell-infiltrating characteristics.Results: A composite score was determined based on indicated that immune-related gene prognostic index and m6A risk score. Head and neck squamous cell carcinoma patients in the cancer genome atlas were divided into four subgroups: A , B , C , and D , and overall survival (OS) was significantly different between subgroups (p < 0.001). The characteristics of tumor immune microenvironment cell infiltration in the four subgroups were significantly different in subgroups (p < 0.05). The receiver operating characteristic (ROC) curves show the predictive value of composite score for overall survival was superior to other scores.Conclusion: The composite score is a promising prognostic signature which might distinguish immune and molecular characteristics, predict prognosis, and guide more effective immunotherapeutic strategies for head and neck squamous cell carcinoma. Immunote solved . Consequ6A risk score, and explore the molecular and immunological characteristics of diverse subgroups of IRGPI, m6A risk score and composite score. They then were compared with microsatellite instability (MSI), tumor immune dysfunction and exclusion (TIDE), tumor inflammation signature (TIS), and other signatures.In this study, we develop a composite HNSCC prognostic signature based on IRGPI and mn = 498, 455 tumor samples and 43 para-cancer samples) of HNSCC patients (n = 454) with RNA-seq data , miRNA-seq data, copy number variation (CNV) and well-defined clinical stages were screened from The Cancer Genome Atlas (TCGA) . We downloaded simple nucleotide mutations in tumor samples from 451 patients. RNA-seq data and survival information of 270 HNSCC samples (GSE65858) were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo). Lists of immune-related genes were downloaded from the InnateDB (https://www.innatedb.ca/) and ImmPort (https://www.immport.org/shared/home) databases.Samples (The RNA-seq of 498 samples (454 tumors vs. 43 para-cancer samples) obtained from TCGA was analyzed for genetic differences by the Wilcoxon test, and miRNA-seq data was analyzed using the edgeR package of R. Those genes with |log2FC| > 1 and false discovery rate (FDR) < 0.05 were considered as DEGs. After extracting the DEGs of immune-related genes obtained from ImmPort and InnateDB databases, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on immune-related DEGs by using the clusterProfiler package of R.β = 7 and signed network type, and topological overlap measure (TOM) was used to calculate the degree of association between genes and the adjacency matrix was converted into TOM similarity matrix. After that, the TOM similarity matrix was transformed into a dissimilarity matrix based on 1-TOM, and a dynamic pruning tree was constructed to identify the modules. Finally, we set the minimum cluster size to 25 and the merge threshold function to 0.25 to separate the six modules. The igraph package of R was used to construct a gene co-expression network for genes with edge weights greater than 0.2 in the module of interest (turquoise module). After batch adjustment for biotechnological biases, using the ComBat algorithm of the sva package in TCGA and GEO cohorts, univariate Cox regression analysis was used to identify immune-related hub genes that were associated with overall survival (OS).WGCNA package of R was used to perform weighted gene co-expression network analysis (WGCNA) for immune-related DEGs. In order to identify hub genes, 498 HNSCC samples were clustered to detect outliers based on the immune-related DEG expression matrix, and the similarity matrix was constructed after excluding outlier data . Then, the similarity matrix was converted into the adjacency matrix with soft threshold Genes with significant impact on OS were selected from immune-related hub genes by multivariate Cox regression analysis. The IRGPI of each sample was calculated by multiplying the expression values of specific genes by their weighting in the Cox model, and then summed. Survival analysis was used to evaluate the predicting prognostic ability of IRGPI in TCGA and GEO cohorts. Patients were divided into IRGPI-high and IRGPI-low groups based on the median.6A RNA methylation, including 16 readers “YTHDC1, YTHDC2, IGF2BP1, IGF2BP2, IGF2BP3, YTHDF1, YTHDF2, YTHDF3, HNRNPA2B1, HNRNPC, RBMX, FMR1, LRPPRC, ELAVL1, EIF3A, and PRRC2A”, 10 writers “METTL3, METTL5, METTL14, METTL16, WTAP, VIRMA, RBM15, RBM15B, ZC3H13, and CBLL1” and three erasers “FTO, ALKBH3, and ALKBH5”.According to published studies, we collected 29 regulators of m6A RNA methylation, the survival package of R was used for univariate Cox regression analysis to construct a prognostic network map. The glmnet package of R was used for the least absolute shrinkage and selection operator (LASSO) regression analysis. After simulation 1,000 times and cross-validation, the m6A regulators that strongly correlated with prognosis and their coefficients were obtained. Then, the m6A risk score was calculated by multiplying each obtained coefficient by the corresponding m6A regulator expression and summing the total values. HNSCC patients were divided into m6A-risk-high and m6A-risk-low groups according to the median value, and the differences in OS between the two groups were assessed.Based on the gene expression of 29 regulators of m6A risk score. IRGPI and m6A risk score were standardized by using the descriptives function of SPSS, and their weights were calculated by the principal component method in factor analysis. The composite score equaled the sum of the normalized IRGPI and m6A risk score multiplied by their corresponding weights. Univariate and multivariate Cox regression analyses were performed to verify the independent prognostic value of IRGPI, m6A risk score and composite score, and their relevant gene signatures were constructed by principal component analysis (PCA), with principal components 1, 2, and 3 selected as signature scores. The chi-square test  was applied to test the correlation between clinicopathological features and composite score subgroups. Based on the interaction between mRNA and miRNA in the ENCORI database (http://starbase.sysu.edu.cn/) (https://www.string-db.org/) to construct the protein-protein interaction (PPI) network.Four subgroups were classified by pairwise combination of IRGPI and medu.cn/) , we usedhttp://www.gsea-msigdb.org/gsea/index.jsp) using the clusterProfiler package of R. The number and quality of genetic mutations in subgroup tumor samples was analyzed by using the maftools package of R. Correlation analysis of the composite score with PD-1/L1, CTLA-4 and tumor mutation burden (TMB) were performed. To identify the immune characteristics of 454 HNSCC samples, we analyzed their RNA-seq data by using the CIBERSORT tool (https://cibersort.stanford.edu/) and performed 1,000 iterations to estimate the relative proportions of 22 kinds of immune cells among subgroups. We analyzed the expression differences of 22 kinds of immune cells among subgroups, and selected differentially-expressed immune cells (p < 0.05) to analyze their influence on OS, after using the survminer package of R to calculate the optimal cutoff value of the corresponding immune cells for dividing tumor samples into two groups depending on the degree of infiltration. To further clarify immunological and molecular functions among subgroups, we performed single sample GSEA (ssGSEA) by using the GSVA package of R, and compared normalized ssGSEA scores among subgroups. Survival analysis was conducted for items with statistical differences (p < 0.05) according to the optimal cutoff value, which also calculated by the survminer package of R, of the items for dividing tumor samples into two groups. We evaluated the percentages of the three scores in immunized subtypes and final survival states. By using the time ROC package of R, we performed time-dependent receiver operating characteristic (ROC) curve analyses to obtain the area under the curve (AUC) and compared the prognostic value among composite score, m6A risk score, IRGPI, TIS, TIDE, interferon gamma (IFNG), MSI, Merck18, CD274, CD8, Dysfunction, Exclusion, myeloid-derived suppressor cells (MDSC), tumor-associated fibroblast (CAF), and TAM (M2) scores. The TIS score was calculated as the mean of normalized expression on a log2-scale of 18 characteristic genes (http://tide.dfci.harvard.edu/).Based on the DEGs, gene set enrichment analysis (GSEA) was applied to the composite score subgroups with the GO, KEGG and HALLMARK gene sets, which were downloaded from the GSEA database , 4,855 DEGs were identified, which included 3,550 upregulated genes and 1,305 downregulated genes , among wp < 0.05, p < 0.05, To obtain immune-related hub genes, 498 samples were clustered to detect outliers  based on the 638 immune-related DEGs matrix, and the data was analyzed using WGCNA after deleting two outlier samples . The logp < 0.001, n = 270) . A prognostic index for tumor samples could be calculated by the formula: IRGPI = (HTN3 expression * 0.096) + (CTSG expression * −0.152) + (MAPT expression * 0.122) + (CCL28 expression * −0.094) + (PTX3 expression * 0.138) + (SEMA3G expression * −0.140) + (USP2 expression * 0.107). With the median IRGPI as the threshold, patients with high IRGPI had worse OS than those with low IRGPI  among the 29 regulators. The CNV frequency showed it was very common among regulators, with 15 undergoing copy number amplification, and 14 undergoing copy number deletion  + (0.119 * HNRNPC expression) + (0.487 * METTL5 expression). According to the median risk score, HNSCC patients with a low m6A risk score had better OS than those with a high one , subgroup B , subgroup C  and subgroup D . IRGPI and m6A risk score were standardized and multiplied by the weight coefficient obtained from PCA to obtain the composite score, using the formula: Composite score = 0.5*Z-score (IRGPI) + 0.5*Z-score (m6A risk score). Survival analysis showed from subgroup A to D, the OS was better than that of the previous subgroup . Univariate Cox regression analysis showed that age, stage, radiotherapy, IRGPI, m6A risk score and composite score were significantly correlated with the prognosis of HNSCC , resting natural killer (NK) cells, M0 macrophages, resting dendritic cells, resting mast cells, eosinophils and neutrophils were significantly different in subgroups , Exclusion, CAF and MDSC scores were higher, while CD8 and IFNG scores were higher in the IRGPI-low subgroup , IFNG, CD8, CAF, MDSC, and Merck18 scores demonstrated significant differences between subgroup A and subgroup D, but their differences among other subgroups were not uniform  scores to evaluate the potential clinical efficacy of immunotherapy in subgroups categorized by IRGPI, msubgroup . TAM . TP53 mutation is not only the most common genetic event in cancer, but is also associated with more aggressive disease and poorer patient outcomes in many cancers, especially in HNSCC . The Not6A risk score subgroups, which is consistent with the results of our survival analysis. In addition, tumor drug resistance is the main cause of chemotherapy failure, and studies have shown that TAMs are closely related to the drug resistance of tumors  scores on the HNSCC cohort in TCGA. The results showed that the composite score was better than other scores in predicting both short-term and long-term survival. Although the AUC value of composite score declined slightly at 5 years, this might due to the limited number of patients with a 5-year OS.Finally, we compared the predictive prognostic ability of composite score with IRGPI, m6A risk score, is an independent prognostic signature, which is more useful in identifying immune and molecular characteristics, predicting patient prognosis and guiding therapy as compared with single prognostic signatures. Further studies are still needed to clarify that the composite score is potential prognostic indicator for immunotherapy.In conclusion, the composite score, constructed based on IRGPI and m


Heterotopic ossification (HO) histologically refers to extraskeletal bone formation in non-ossifying tissues, most commonly noted in the extremities, buttocks, abdominal wall, and hip joints. HO developing in the mesentery  is very rare, with fewer than 100 cases reported in the literature. It usually occurs in adult male patients with a history of repeated abdominal trauma. So far, only two cases of HMO have been reported with the development of hematopoietic bone marrow. Here, we report the third case of HMO with true trilineage hematopoiesis in a 66-year-old female with suspicious mesenteric-retained foreign material from prior surgical procedures, including hysterectomy for endometrial adenocarcinoma and multiple repairs for incisional hernia. Heterotopic ossification (HO) refers histologically to extraskeletal bone formation in non-ossifying tissues with a yet to be determined incidence, exact etiology, and pathogenesis -4. It wa6/µL; hemoglobin, 7.6 g/dL; hematocrit, 23.5%; mean corpuscular volume, 81.6 fL; mean corpuscular hemoglobin, 26.3 pg; mean corpuscular hemoglobin concentration, 32.2 g/dL; red cell distribution width, 15.4%; platelet count, 145 k/µL, mean platelet volume, 8.2 fL). Computed tomography of the abdomen reported a linear opacity adjacent to the gastric body and antrum, as well as a high-density mass in the mesentery suspicious for possibly retained foreign body. After five abdominal surgeries for a complicated postoperative course, she fully healed and underwent an ileostomy take-down one month later. During the surgery of ileostomy take-down, the mesentery appeared markedly congested and slightly adhered to the anterior abdominal wall. An intra-abdominal mass was palpated in the mesentery. Pathological gross examination showed that the excised mass was well-circumscribed and firm, located in fibrous and adipose tissue, and measured 1.5 cm in the greatest dimension with a whitish trabeculated cut surface. Microscopic examination showed mature cortical lamellar bone with osteocytes, cement line and rimming osteoblasts, and true trilineage hematopoiesis with erythropoiesis, granulocytopoiesis, and megakaryocytopoiesis  for both HMO and ectopic marrow development remain to be elucidated.


Vitamin D deficiency is common, but no data have been reported on vitamin D levels in light chain (AL) amyloidosis.2D] and vitamin D binding protein (DBP). Measurements were made by liquid chromatography-tandem mass spectrometry. Kidney survival and overall survival (OS) were assessed in association to vitamin D status.In this exploratory study, stored serum samples from 173 patients with newly diagnosed AL amyloidosis were analyzed for vitamin studies which included 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D deficiency was noted in 37.6% of patients and was independently associated with low eGFR and hypoalbuminemia. Progression to ESRD occurred in 23.7% of evaluable patients. Patients who progressed to ESRD had lower serum 25(OH)D and 1,25(OH)2D levels compared to those who did not progress to ESRD. On a multivariate analysis, severe 25(OH)D deficiency was an independent predictor of progression to ESRD as was renal stage, while 1,25(OH)2D deficiency was not.Cardiac and kidney involvement occurred in 69% and 63% of patients, respectively. 25(OH)D deficiency (<20 ng/mL) was seen in 56.6% of the patients and was notably found among patients with heavy proteinuria (96%), hypoalbuminemia (84.3%) and morbidly obese patients (68.3%). Heavy proteinuria (>5 gr/24-h) and vitamin D supplementation were independent predictors of 25(OH)D level on nominal multivariate regression analysis. 1,25(0H)Hypovitaminosis D is common in AL amyloidosis, particularly among patients with heavy proteinuria. Severe 25(OH)D deficiency at time of diagnosis predicts progression to ESRD. Light chain (AL) amyloidosis is a rare B-cell secreting clonal disorder characterized by circulating immunoglobulin light chains with amyloidogenic properties . The clo2D]. 1,25(OH)2D reflects 1-alpha hydroxylation of 25(OH)D, a process that occurs primarily within the kidney. Circulating 1,25(OH)2D levels are approximately 1000-fold lower than those of 25(OH)D.Vitamin D deficiency is assessed by measurement of the storage form of vitamin D, namely 25-hydroxyvitamin D [25(OH)D]. The prevalence of vitamin D deficiency, defined as a 25(OH)D level below the optimal range of <20 ng/ml, has been reported to range from 15-50% within the general United States (US) population –8. IdentAside from its roles in maintaining serum calcium levels and skeletal homeostasis, vitamin D has been shown to play an important role in regulation of differentiation, proliferation, apoptosis, metastatic potential and angiogenesis in a variety of malignancies –11. Low No studies have explored vitamin D levels in light chain (AL) amyloidosis. We therefore performed a study to examine vitamin D levels among patients with AL and to explore the impact vitamin D has in this rare disease.2D] and vitamin D binding protein (DBP). All vitamin D measurements were made by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Total 25(OH)D and 1,25(OH)2D was assessed by the additive sums of the 25(OH)D2 + 25(OH)D3 and 1,25(OH)2D2 + 1,25(OH)2D3 components, respectively. Samples obtained for vitamin D studies were collected within 90 days of diagnosis (median 20 days) and were stored at -20°C.In this study patients with biopsy-proven AL amyloidosis diagnosed between January 1, 2000 and September 30, 2013 were included if they had stored serum samples. The stored serum samples were obtained for vitamin D studies, which included 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D  was ascertained for 162 patients (94% of the study population). Ten patients presented or progressed to ESRD within 3 months of AL diagnosis and were excluded from this analysis. Of the remaining patients, 36 patients developed ESRD, representing 23.7% of patients assessed for progression to ESRD. Patients who developed ESRD had lower baseline 25(OH)D levels compared to patients who did not progress to ESRD  are provided in vs 49.2 months; P=0.03; 2D ≥18 pg/mL and those with lower levels D levels >20 ng/mL had shorter overall survival (OS) compared to patients with lower 25(OH)D values D level <20 ng/mL showed a trend towards longer OS , whereas a 1,25(OH) did not Table 5.In this study we explored, for the first time, the prevalence and risk factors for vitamin D deficiency in patients with AL amyloidosis. We found that more than half of our study population had vitamin D deficiency (defined as 25(OH)D <20 ng/mL), while over a fifth of the study cohort had severe vitamin D deficiency [25(OH)D <10 ng/mL]. Vitamin D deficiency was particularly common among patients with significant proteinuria, an established risk factor for vitamin D deficiency. We have also found that severe vitamin D deficiency was associated with increased risk for progression to end-stage renal disease. Overall, the findings in this study support measurement of baseline vitamin 25(OH)D in patients with AL amyloidosis, particularly among those with significant proteinuria.The proportion of patients with vitamin D deficiency in this study (56.6%) is higher compared to the general US population. In the National Health and Nutrition Examination Survey (NHANES), conducted in a similar time period to our study (2001 to 2010), the prevalence of vitamin D deficiency among US adults age 18 or above ranged between 27.1% and 30.8% (depending on age group)  and popuWe identified several risk factors for hypovitaminosis D in AL amyloidosis. The most significant risk factor for low 25(OH)D levels was significant proteinuria. Patients with nephrotic syndrome are known to be at risk for vitamin D deficiency . Kidney via modulation of several pathways including the renin-angiotensin, NF-kB and Wnt/β-catenin signaling pathways D deficiency is an independent predictor of progression to ESRD. This finding has been reported in previous studies with patients with chronic kidney disease –34. The pathways . In addipathways . HoweverIrrespective of the mechanisms by which vitamin D deficiency increases the risk of ESRD, correction of vitamin D deficiency in patients with CKD , should be assessed in prospective studies given the potential benefit of such treatment. A large, randomized trial among type II diabetes mellitus participants did not demonstrate a renoprotective effect for vitamin D supplementation versus placebo . However2D deficiency, was associated with improved survival compared to patients with normal vitamin D levels. This observation stands in contrast to observations among cancer patients (25(OH)D deficiency, but not 1,25(OH)patients , 18–22. patients . As survpatients , 40 and patients , vitamin2D levels to be normal in subjects with t(11:14) as compared to those with non-t. t is the most common genetic abnormality in patients with AL amyloidosis  (In this study there was a trend for 25(OH)D and 1,25(OH)poptosis –43. As a (BCL-2) , an antivia the gold standard method of liquid chromatography-tandem mass spectrometry. We were also powered for several observations including the positive effect of concurrent vitamin D supplementation on serum 25(OH)D and the expected seasonal variation on serum 25(OH)D levels. The study follow-up period was long (median >16 years), allowing sufficient time of observation for events . The primary limitation to our study was its retrospective design. We did not preselect number of samples based on predefined hypothesis, which may limit the study power. These limitations negatively affect the generalizability of the above findings and require further studies.Our study has several strengths. These include that this is the first report on the incidence of vitamin D deficiency in patients with AL amyloidosis, and the ability to measure vitamin D metabolites and vitamin D binding protein In conclusion, we have shown that patients with renal AL amyloidosis have a high incidence of vitamin D deficiency. We anticipate our findings will serve as the basis for future prospective clinical efforts to improve vitamin D levels in these patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Mayo Clinic Institutional review Board. The patients/participants provided their written informed consent to participate in this study.EM, MTD, and MG designed the study, analyzed the data, wrote the first draft and approved the final version of the manuscript; NL, AD, ML, FB, DD, SH, PK, YH, AF, MH, WG, TK, RW, SR, RG, MB, SR, and SK provided care for patients, revised the manuscript critically, and approved the final version of the manuscript. RK performed patients’ follow-up, revised the manuscript critically, and approved final version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by CA90628-21 Pal Calabresi K12 Career Development Award.EM: honorarium from Janssen and consultation fee from Protego (fee paid to institution). NL served on advisory board for Takeda Pharmaceuticals. AD: research funding from Celgene, Millennium Pharmaceuticals, Pfizer, and Janssen and received a travel grant from Pfizer. ML: Research Funding from Celgene. PK is a principal investigator of research studies for which Mayo Clinic has received funding from AbbVie, Takeda, Sanofi, Janssen, Karyopharm, Glaxo SmithKline, Regeneron Pharmaceuticals, Ichnos Sciences and Amgen. He has served on the Medical advisory board meetings of Sanofi, Pharmacyclics, BeiGene, Cellectar, GSK, X4 and Karyopharm. SK received research funding for clinical trials to the institution from Abbvie, Amgen, BMS, Carsgen, Janssen, Astra-Zeneca, Novartis, Roche-Genentech, Takeda, Tenebio, Molecular Templates, received consulting/Advisory Board participation  from Abbvie, Amgen, BMS, Janssen, Roche-Genentech, Takeda, Astra-Zeneca, Bluebird Bio, Epizyme, Secure Biotherapeutics and  Oncopeptides, Beigene, Antengene. MG served as a consultant for Millennium Pharmaceuticals and received honoraria from Celgene, Millennium Pharmaceuticals, Onyx Pharmaceuticals, Novartis, GlaxoSmithKline, Prothena, Ionis Pharmaceuticals, and Amgen.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Quantitative susceptibility mapping (QSM) facilitates mapping of the bulk magnetic susceptibility of tissue from the phase of complex gradient echo (GRE) MRI data. QSM phase processing combined with an  Multiple sclerosis (MS) is an autoimmune disease characterized by the presence of demyelinated lesions in the central nervous system. Progressing demyelination can be monitored using noninvasive MRI methods such as myelin water fraction mapping ,2,3,4,5,Both myelin and iron can be quantified in MRI on the basis of their magnetic susceptibility. Quantitative susceptibility mapping (QSM) ,13,14,15n in QSM .Recently, we proposed an ocessing . We demotibility . In thisThe complex signal exponent of the gradient echo MRI signal is approximated as ,22,23:=.The Institutional Review Board determined that activities involved in the present study did not constitute human subjects research as the project did not involve identifiable private information from or about living subjects. As a result, neither IRB approval nor a notice of exemption was required.3, first TE = 5.9 ms, ΔTE = 5.9 ms, #TE = 8, TR = 52.2 ms, flip angle = 12 degrees, receiver bandwidth (rBW) = 244 Hz/pixel, acquisition time 69 min. For lesion identification, T2 FSE  = 12, acquisition time 36 min) was acquired.Data acquired in four formalin-fixed postmortem brain slabs were analyzed. Formalin-fixed postmortem brain specimens were embedded in 1% agarose gel to minimize motion and provide MR-visible medium to measure the magnetic field generated by the tissue. The tissue blocks were not washed prior to embedding. The tissue was scanned on a 3T clinical MRI scanner  using an 8-channel head coil. A three-dimensional multi-echo gradient echo (GRE) sequence with unipolar readout gradient was acquired for susceptibility mapping and http://itksnap.org/, accessed on 10 June 2022). Tracings were reviewed by an experienced neuroradiologist, and average Prior to source separation, s (ARLO) . To estis (ARLO) . Then, ts (ARLO) , after ws (ARLO) . QSM wass (ARLO) . Co-regilgorithm ,31. EachAll processing was performed on a desktop PC .Lesions of interest were excised, embedded in paraffin, and cut into 5 μm sections. Sections were deparaffinized in xylene, rehydrated, and antigen retrieval was performed with 10 mM sodium citrate buffer (pH 6) for 20 min. Sections were quenched, blocked, and incubated overnight with a primary antibody against myelin basic protein , CD68 , followed by the appropriate biotinylated secondary antibodies and avidin/biotin staining kit with diaminobenzidine (DAB) as the chromogen (Vector Laboratories ABC Elite Kit and DAB Kit). Negative controls included isotype-controls and the absence of immunolabeling in tissues that do not express MBP or CD68. DAB-enhanced Perls’ Prussian blue was used to detect ferric iron. Slides were immersed in 4% ferrocyanide/4% hydrochloric acid for 30 min in the dark, and staining was enhanced through incubation with DAB for 30 min at room temperature. After staining, all sections were rinsed, dehydrated, cover-slipped, and digitized using a Mirax digital slide scanner.2) were manually drawn within the lesion and adjacent normal appearing white matter by a reader with 5 years of experience (KG). NAWM ROIs were sampled 0.57 ± 0.24 mm from the outer rim edge in iron− lesions and 1.24 ± 0.52 mm from the outer rim edge in iron+ lesions. For each lesion, we placed 1 ROI in the NAWM, 1 ROI in the rim (lesion perimeter for rim-negative lesions), and 1 ROI in the center. Lesions were defined histologically by the absence of MBP staining in the center. Histology ROIs were captured in Panoramic Viewer (version 1.15.4). Each image was processed in FIJI [Regions of interest   .In the comparison of Our data demonstrate the validity of the recently proposed The main idea in the is small ,35,36,37is small  used in is small ,20. Theris small ; howevery source ,39. Thisominates ,41, QSM isotropy ,43,44, wisotropy ,17,19,21y source .The optical density used in the present work is only semi-quantitative, which may also contribute to the large spread in the correlation between histology and susceptibility source separation in f myelin ,47 and lIn conclusion,