Millions suffer from skin diseases. Functional interfollicular epidermal stem cells are needed in skin therapy or drug screening in vitro. We obtained functional interfollicular epidermal stem cells with intact stemness and cell junctions by treating them with Wnt3a. Moreover, epidermal stem cell-derived extracellular vesicles were useful in epidermal cell growth. Finally, functional epidermal 3D organoids with polarity were cultured using Wnt3a and the supernatant derived from interfollicular epidermal stem cells and fresh medium in a 1:1 ratio. These results provide novel directions for the improvement of skin organoids and their potential in clinical application. In mammals, several distinct cell populations of the skin comprise three basic layers—epidermal layer, dermal layer, and hypodermis or subcutaneous fat layer. The epidermis is the outermost layer, composed of stratified cell layers maintained by keratinocytes, including both stem cells and the mature cells in abundance. The basal layer of the epidermis has undifferentiated proliferative progenitor cells expressing keratins, including keratin 5 (K5) and keratin 14 (K14) . Skin hoVarious junctional and cytoskeletal proteins, including cytoskeletal building blocks and associated cell-matrix proteins or intercellular junctions, control the regenerative capacity of the epidermis in maintaining the EpSCs, which is useful for epidermal morphogenesis and growth . For exaMillions suffer from skin diseases caused due to mutations or thermal and pressure injuries, such as epidermolysis bullosa (DB) or chronic diabetic ulcers . With thIn this study, we established both 2D and 3D EpSC culture system using Wnt3a protein. In addition, we first described the proteomic characteristics of EpSC-derived extracellular vesicles and their important roles on epidermal development that may benefit clinical application or drug discovery in the future.g for 3 min, and washed three times with PBS at 4 °C. Subsequently, the cells were seeded onto 6-well plates, pre-coated with Matrigel . Advanced DMEM/F12  medium containing NEAA , B-27 , GlutaMAX , HEPES , N-Ace , hEGF , A83-01 , fosklin , and penicillin/streptomycin  with or without Wnt3a  was prepared. The cells were incubated overnight and filmed using the Real-Time Cell History Recorder  inside the incubator. Images were taken every day using the bright channels. EpSCs (treated with or without Wnt3a or XAV939) were cultured in 96-well plates and counted daily for 5 days to perform the proliferation curve.Human skin tissue samples were obtained from Chinese PLA General Hospital, and ethical approval and informed consent were obtained. The interfollicular epidermal stem cells were isolated as described , 20 withFor 3D cultures, the cells obtained from the epidermis were cultured on ultra-low attachment culture dishes, in advanced DMEM/F12 medium with sodium hyaluronate  and Wnt3a for 5 days. The culture medium was partially replaced every alternate day, and XAV939  was added, starting from day 5. The organoids were harvested after 5 days of culture for the next experiment.Cells were fixed in 4% formaldehyde for 20 min and washed in PBS. Thereafter, the samples were treated with 0.25% Triton X-100 for 20 min, blocked in 10% serum for 1 h at 25 °C, and incubated with primary antibodies, such as COL17A1 , K14 , K10 , PLEC , ITGB1 , ITGA6 , DSC2 , DSG1 , β-catenin , P63 , CLDN1 , and Ki67  overnight at 4 °C. Thereafter, the cells were incubated with secondary antibodies for 1 h at 25 °C, counterstained with DAPI, and the samples were sealed with Fluoro-Gel for Photography. The pictures were taken at × 20 or × 40 magnification and analyzed using Volocity Demo (× 64).Total RNA was isolated using the RNeasy Mini Kit (Qiangen). Thereafter, the cDNA was synthesized using reverse transcriptase , according to the manufacturer’s instructions. qRT-PCR was performed using the SYBR Green master mix (TOYOBO) on the Bio-Rad iQ5 Real-Time PCR detection system. Data were collected using the Bio-Rad CFX Manager software. Gene expression within a sample was normalized to GAPDH expression by the 2−ΔΔCt method. The primer sequences for PCR are available in Supplementary Table S2 flasks under normal culture conditions in media supplemented with 10–15% fetal bovine serum until they reached a confluency of 70–80%. Thereafter, the cells were cultured for > 48 h in media supplemented with extracellular vesicle-free FBS . The culture medium was collected and centrifuged at 300 g at 4 °C for 10 min and 2000 g at 4 °C for 20 min to remove dead cells. The supernatants were further centrifuged at 10,000 g for 30 min at 4 °C to eliminate contaminating cellular debris. The extracellular vesicles were pelleted from the final supernatants by ultracentrifugation for 70 min at 4 °C and 110,000 g (Beckman SW32Ti rotor) using an SW32 Ti swinging bucket rotor . The pellets were washed in PBS to eliminate contaminating proteins. Another round of centrifugation was performed at high speed and the supernatants were discarded. The extracellular vesicles samples were used for transmission electron microscopy (TEM) and western blot analysis.The extracellular vesicles were isolated from the human epithelial stem cell (EpSC) culture supernatant using a differential centrifugation protocol , 22 withThe samples were washed in PBS and fixed overnight in 0.1% formalin and for 2 h in 2.5% glutaraldehyde. Thereafter, the samples were washed in PBS, dehydrated in ascending concentrations of ethanol, and air-dried. The ultrastructure of artificial skins was gold sputtered and mounted for imaging on a transmission electron microscope.The extracellular vesicles were collected and homogenized on ice in RIPA buffer, supplemented with a protease inhibitor cocktail . For immunoblotting, equal amounts (20 μg per lane) of extracellular vesicle lysates were separated on a 10% SDS polyacrylamide gel. The samples were blocked with 5% skim milk for 2 h and incubated with anti-CD9 , K1 , K14 , ITGB1 , ITGA6 , ERK1 , ECAD , and ICAM1  overnight at 4 °C. Thereafter, the blots were washed three times and incubated with secondary antibodies for 50 min at 37 °C. Subsequently, the blots were washed three times and the signals were detected using the Pierce™ ECL Western Blotting Substrate  and Millipore Immobilon™ Western Chemiluminescent HRP Substrate  at 25 °C.https://www.uniprot.org/, downloaded on April 2020). Protein quantification was performed according to the intensity-based absolute quantification method [https://david.ncifcrf.gov/) [http://matrisomeproject.mit.edu/) was used to annotate the extracellular matrix (ECM) proteins. Protein–protein interactions were retrieved from the online tool STRING [The EpSC-derived extracellular vesicles were collected and digested using the protein lysis buffer. Thereafter, the protein mixtures were extracted, processed, and digested as described previously . The pepn method  as implerf.gov/)  was usedrf.gov/)   software for Apple Mac.For all results, the statistical data are shown as means ± S.E.M. Student’s The epidermis was separated from human skin tissue with Dispase II digestion. Thereafter, the epidermal cells were isolated from the epidermis by trypsin digestion for the 2D culture on matrix gel Fig. a. The adMMP1, MMP2, MMP3, MMP7, MMP8, and MMP19, compared to the control group , heparan sulfate proteoglycan 2 (HSPG2), alpha-2-macroglobulin (A2M), and alpha-1-microglobulin/bikunin precursor (AMBP), of the BM, an important EpSC niche, were found in Wnt-treated EpSCs compared to the control group  and zinc finger E-box binding homeobox 2 (ZEB2), were downregulated in Wnt3a-treated EpSCs; however, they were upregulated in the XAV939 group  is important for its stemness in the natural state. We found that EpSCs isolated from the human skin and cultured with Wnt3a can express more components of the BM, including COL4A1, FBN1, HEPG2, A2M, and AMBP , we obtained 3D organoids which were close to the epidermis, including basal and mature layers after 10 days.In this study, we demonstrated the role of the Wnt pathway in enhancing the EpSCs’ function through 2D/3D systems, as well as the potential role of EpSC-derived extracellular vesicles on skin development. These results provide novel directions for the improvement of skin organoids and tissue-engineered skin and stem cells for clinical transplantation and drug screening.Additional file 1: Supplementary Table S1. Details of primer sequences used for PCR in this study.Additional file 2: Supplementary Table S2. Proteome profile of epidermal stem cell (EpSC)-derived extracellular vesicles.Additional file 3: Supplementary Table S3. List of extracellular matrix (ECM) proteins identified in epidermal stem cell (EpSC)-derived extracellular vesicles.


In this study, we aimed to evaluate the role of ALMS1 in the morphology of primary cilia and regulation of cellular signaling using a knockdown model of the hTERT-RPE1 cell line. ALMS1 depletion resulted in the formation of longer cilia, which often displayed altered morphology as evidenced by extensive twisting and bending of the axoneme. Transforming growth factor beta/bone morphogenetic protein (TGF-β/BMP) signaling, which is regulated by primary cilia, was similarly affected by ALMS1 depletion as judged by reduced levels of TGFβ-1-mediated activation of SMAD2/3. These results provide novel information on the role of ALMS1 in the function of primary cilia and processing of cellular signaling, which when aberrantly regulated may underlie Alström syndrome. ALMS1 gene (HGNC:428). This gene encodes a protein of 4,169 amino acids localized to centrosomes and basal bodies of primary cilia  is a rare, autosomal recessive disorder included in the group of ciliopathies, which share a common etiology: defects in the structure and function of cilia  signaling  at ciliary pocket, an invagination of the plasma membrane at the base of the cilium that represents a major site for exo- and endocytosis  primary cilia, assessing ciliary length, and morphology, and (ii) TGF-β/BMP signaling, performing stimulation assays to find a possible molecular link between ALMS and the TGF-β/BMP pathway.Therefore, it is necessary to further explore the molecular basis of ALMS to decipher the pathophysiological mechanisms underlying ALMS phenotypes and also to better define the involvement of ALMS1 in the ciliary activity. In this study, we aimed to establish an 2 atmosphere in a humidified incubator. Cultures were passaged once or twice a week. For ALMS1 silencing, RPE-1 cells were transiently transfected using a commercial pool of four small interfering RNAs (siRNAs) [ON-TARGET plus Human ALMS1 (7840) SMARTpool (5 nmol) from Dharmacon]. A negative control siRNA  was assayed in parallel for each experiment. We followed a reverse transfection protocol using Lipofectamine®RNAiMAX (Invitrogen) according to the manufacturer's instructions and maintaining a final concentration of 30 nM siRNA. For that, cells were plated on coverslips or cultured dishes from a 90 to 95% confluent flask to reach 30–50% confluency at 24 h after plating. Then, the culture medium was changed to a serum-free medium, and cells were incubated for additional 48 h before harvesting to promote primary cilia formation.hTERT RPE-1 (RPE-1) and HeLa cells  were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum , 1% penicillin-streptomycin (Gibco), and 2 mM L-Glutamine (Lonza) at 37°C and 5% COALMS1. Relative quantification was carried out in 20 μL reactions using 2 μL of diluted cDNA [1:50 for the reference gene beta-actin (ACTB) and 1:10 for ALMS1 gene] and 1 μL of commercial TaqMan® Gene Expression Assays . All samples were run in triplicate on the StepOne™ Real-Time PCR System (Applied Biosystems) for 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C. The relative amount of ALMS1 mRNA normalized to ACTB was calculated in each sample using the Pfaffl model  according to the manufacturer's protocol. A total of 790–850 ng of RNA was reverse transcribed into cDNA using SuperScript® IV Reverse Transcriptase (Invitrogen) with an incubation step at 50°C during 50 min as recommended for long transcripts such as  Pfaffl, . BiologiCells grown on glass coverslips were washed twice in cold PBS and fixed in a cold 4% (w/v) paraformaldehyde (PFA) solution for 15 min, followed by two washes with PBS and a permeabilization step using PBS/0.2% (v/v) Triton-X100/1% (w/v) bovine serum albumin (BSA) buffer for 12 min. After one PBS wash, cells were subjected to double immunostaining starting with a block step in a PBS/2% (w/v) BSA solution for 30 min at room temperature (RT), then incubation with primary antibodies in blocking solution for 90 min at RT, three washes with PBS for 5 min each, incubation with secondary antibodies and DAPI (1 μg/mL) in blocking solution for 45 min at RT in the dark, and finally, three washes with PBS for 5 min each. Once finished, coverslips were mounted in Prolong® Diamond Antifade Mountant (Molecular Probes) according to manufacturer's instructions and imaged at 100× magnification using an Olympus BX-83 upright microscope with a DP71 color camera with DP controller software. Images were processed for publication using Adobe Photoshop (v. CS6) or Fiji/ImageJ software (v.1.51d). Measurement of cilia length was carried out on imaged cells in at least 50 cells per condition across three-independent experiments. The following primary antibodies and dilutions were used: rabbit anti-ALMS1  and mouse anti-acetylated tubulin  as a ciliary marker. Secondary antibodies were used at 1:600: Alexa Fluor® 488-conjugated goat anti-rabbit  and Alexa Fluor® 594-conjugated goat anti-mouse . Nuclei were stained with DAPI.g for 10 min. Then, pellets were incubated on ice for 10 min in 40–50 μL of ice-cold RIPA lysis buffer with 0.1% (v/v) protease inhibitor cocktail  and 1 mM sodium orthovanadate as phosphatase inhibitor (Sigma-Aldrich). Lysates were centrifuged at 4°C, 14,500×g for 25 min, and the supernatant was collected and conserved at −20°C until used. Protein concentration was measured by Bradford assay  and incubated in fresh DMEM with 2 mM L-Glutamine for 30 min before stimulation. Then, TGFβ and BMP signaling were independently stimulated by adding specific ligands for 0, 10, 30, and 90 min: recombinant human TGFβ1  at 2 ng/mL  and recombinant human/mouse/rat BMP-2  at 50 ng/mL. After stimulation, cells were washed twice in ice-cold PBS, harvested using a scraper, and centrifuged in a Sigma® 1–14 K at 4°C, 7,400×ALMS1 TGF-β/BMP stimulation assay, we starved HeLa cells for 24 h, and then added 2/4 ng/mL TGF-β or 50/100 ng/mL BMP. We extracted mRNA before stimulation and 24 h after stimulation.For the Samples for SDS-PAGE were prepared by mixing 20 μg of each lysate with Laemmli sample buffer (Bio-Rad) and 5% (v/v) β-mercaptoethanol, then boiled at 95°C for 5 min and run on a 12% Mini-PROTEAN® TGX™ precast gel (Bio-Rad) for 40–50 min. Proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane using the Trans-Blot® Turbo™ Transfer System (Bio-Rad) at 25 V for 30 min. Membranes were blocked in 5% (w/v) non-fat dry milk in TBST [TBS/0.1% (v/v) Tween-20] for 1.5 h at RT, then incubated overnight at 4°C with primary antibodies diluted in blocking solution, washed three times for 5 min each with blocking solution, incubated with horseradish peroxidase-conjugated secondary antibodies diluted in blocking solution for 45 min at RT and finally washed three times for 5 min each with TBS.Blots were revealed using Clarity™ Western ECL Substrate (Bio-Rad) in a ChemiDoc™ XRS+ System and Image Lab™ software . Blots were probed for detection of phosphorylated SMAD2 , phosphorylated SMAD1/5 ,and phosphorylated ERK1/2 . Immunoblots signals were quantified by densitometry analysis using Image Lab™: each intensity value was normalized to total protein staining as a loading control (following a standard protocol for Coomassie Brilliant Blue R-250 staining) and then fold-change ratios were calculated using the corresponding normalized intensity value of the control sample stimulated for 30 min as a reference in each blot. At least three biological replicates from separate experiments were used for protein level calculations. The following primary antibodies and dilutions were used: rabbit anti-phospho-SMAD2 , rabbit anti-phospho-Smad1/5 , and rabbit anti-phospho-p44/42 MAPK (ERK1/2)  from Cell Signaling Technology. Anti-rabbit IgG HRP-linked  was used as secondary antibody with 1:15,000 dilution,).t-test for two independent samples, while for protein levels we used a two-way ANOVA with Sidak post-hoc correction. All tests were considered statistically significant if p ≤ 0.05. Statistical analyses were accomplished with the SPSS software  or GraphPad Prism .All data are presented as mean ± standard deviation (SD). Initially, a Shapiro test was carried out to confirm the normality of the data. To compare the calculated means for ciliary length between control and siRNA-treated cells, we used the Student's ALMS1 gene and 72 h after transfection, which included 24 h of serum starvation, depletion efficiency was assessed by RT-qPCR and IFM analyses. A mean reduction of 67%  in ALMS1 expression was achieved . As shown in achieved , and as achieved .p = 0.041) (p = 0.002) and >90° . A significantly lower number of cells subjected to ALSM1 knockdown displayed cilia with no or little bending (0–24°) .While depletion of ALMS1 did not affect the ability of cells to form primary cilia , we obse= 0.041) . Further= 0.041) . The num= 0.041) , ALMS1-dp < 0.0001, 30 min: p = 0.0149 and 90 min: p = 0.0028) and ERK1/2 at 30 min (p = 0.027)  . In cont= 0.027) .ALMS1 promoter  and BMP2   .The study of ALMS has generated much interest in the last years since it represents a monogenic model of obesity and metabolic syndrome. Nevertheless, the molecular basis of ALMS has not yet been elucidated. ALMS1 protein plays a wide range of biological roles, encompassing both ciliary and extraciliary functions, what has raised some controversy about ALMS should be considered a ciliopathy caused by cilia dysfunction or if, however, it is associated with a loss of ALMS1 functions unrelated to primary cilia  ALMS fibroblasts show greater responsiveness to TGF-β stimulation, which produces an excessive deposition of extracellular matrix components. Therefore, we propose that ALMS1 silencing leads to the inhibition of SMAD2 phosphorilation, which may prevent the protector effect against fibrosis associated with this protein and also trigger the systemic fibrosis displayed by ALMS patients. On the other hand, downregulation of TGF-β signaling could also explain other hallmark features related to ALMS such as dilated cardiomyopathy. Thus, several patients with ALMS1 mutations show mitogenic cardiomyopathy, a rare form of dilated cardiomyopathy defined by an uncontrolled proliferation of cardiomyocytes  disturbing the signaling through canonical and non-canonical pathways. ALMS1 may therefore be a key regulator of TGF-β signaling, which could explain some of the most severe features developed by ALMS patients. Fibrosis, one of the major causes of morbidity and mortality in ALMS, is primarily driven by TGF-β ligands, especially activators of the SMAD2/3 axis, which is upregulated in many human fibrotic conditions (Walton et al., ALMS1 promoter (ALMS1 expression is enhanced (ALMS1 sequence harbors nuclear localization signals (Collin et al., Although ALMS1 seems to regulate both canonical and non-canonical cascades in response to TGF-β stimulation, the presence of binding sites for SMAD3/4 in promoter  may poinenhanced . In addiALMS1 in ciliary assembly and also in the inhibition of TGF-β signaling in cultured human cells, which may represent novel pathophysiological mechanisms underlying ALMS that could explain some of the most severe phenotypes related to this syndrome, such as fibrosis or cardiomyopathy. Despite our results are based on an in vitro system of cultured cells, the finding of TGF-β alterations related to ALMS1 might open new therapeutic options that should be explored for ALMS patients.In conclusion, we report a role for The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MÁ-S and DV designed the study. MÁ-S, ML-D, and BB-M performed the experiments. MÁ-S, ML-D, BB-M, CS, SC-S, and DV analyzed the data. MÁ-S, ML-D, BB-M, SC-S, and DV written and revised the manuscript. STC designed the study, analyzed the data and written, and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sprouts under a combination of selenium and LED light quality treatment exhibited no remarkable change fresh weight, but had a shorter hypocotyl length, lower moisture content and heavier dry weight, especially with 1R2B+Se treatment. The contents of carotenoid, soluble protein, soluble sugar, vitamin C, total flavonoids, total polyphenol and contents of total glucosinolates and organic Se were dramatically improved through the combination of Se and LED light quality. Moreover, heat map and principal component analysis showed that broccoli sprouts under 1R2B+Se treatment had higher nutritional quality and health-promoting compound contents than other treatments. This suggests that the Se supplement under suitable LED lights might be beneficial to selenium-biofortified broccoli sprout production.Selenium (Se) supplement was combined with different LED light qualities to investigate mutual effects on the growth, nutritional quality, contents of glucosinolates and mineral elements in broccoli sprouts. There were five treatments: CK:1R1B1G, 1R1B1G+Se (100 μmol L Brassica oleracea L. var. italica) has received considerable attention for its rich bioactive composition, especially with respect to glucosinolates (GLs), which can reduce the risk of different diseases, including cancer, inflammation, cardiovascular and neurodegenerative diseases [Broccoli  and red (R) light-emitting diodes (LEDs) are commonly used for plant growth in view of their roles as essential energy sources for photosynthetic carbon assimilation . CombinePrimary and secondary metabolites in specialty vegetable crops will be higher under narrow-band LED lights, due to plants only receiving B and R wavelengths compared with other wavelengths found in the spectra emitted from fluorescent/incandescent bulbs. It is possible to reduce possible light stress that plants are subjected to in the growing environment by using narrow-band wavelengths . Stem elSelenium supplementation combined with different LED light qualitites had a significant impact on the growth of broccoli sprouts . SproutsA combination of Se and LED light quality treatment significantly affected broccoli sprouts’ pigment content . The conAs shown in The contents of Vc, TPC, and TF in broccoli sprouts were affected by the combination of Se and LED light quality treatment . CompareSelenium supplementation combined with different LED light quality treatments significantly affected the contents of organic Se, K, Ca, Mg, S and Zn in broccoli sprouts . The higNine GLs were identified by HPLC in broccoli sprouts , which cA heat map was used to analyze the response among the investigated parameters and showed a broad view of the effects of the combination of selenium and LED light quality on broccoli sprouts . The 1R1The most significant overall effects of the combination of selenium and LED light quality on growth, phytochemical composition and mineral element of broccoli sprouts were assessed by principal component analysis (PCA) . The comThe combination of selenium and LED light quality had a negative effect on the growth of broccoli sprouts, resulting in a reduction in hypocotyl elongation and fresh weight, as well as moisture content . Low Se −1) reduced chlorophyll content in basil, leading to higher contents of carotenoid [Previous studies have shown that a significant increase in chlorophyll content  can be observed in lettuce  and brocrotenoid . In thisrotenoid . Many plrotenoid . In thisrotenoid .Soluble sugars and soluble proteins are important parameters in evaluating the nutritional quality of vegetables, because they provide essential proteins and energy for the human body. Selenium treatment plays a pivotal role in increasing quality parameters of tomato fruit, such as soluble solid content , contentVitamin C (Vc) provides vital antioxidants in plants, and plays a significant role in plant stress response and cell wall metabolism and expansion . VitaminPAL , CHS , and DFR (dihydroflavono l-4-reductase), which are key elements in the biosynthetic pathways of anthocyanin and flavonoid compounds [Phytochemicals, including anthocyanins, polyphenols, and flavonoids, play an important role in the protection of cardiovascular health and prevention of certain cancers . A combiompounds . Thus, S−1), more S may be required for appropriate translocation, and thus broccoli takes up more S to meet this demand [−1 [Mineral nutrients play a crucial role in plant physiology and metabolism. Organic selenium is more bio-available to the human body than inorganic selenium . The cons demand . The conmand [−1 . This mimand [−1 . The accmand [−1 . Howevermand [−1 . In genemand [−1 . Hence, BCAT4, MAM1, CYP79B2 and FMO2, were the cause of inhibition of glucosinolate accumulation in the Se-treated broccoli [MYB28 and MYB34, which play crucial roles in regulating aliphatic- and indole-glucosinolate biosynthesis, respectively, were significantly down-regulated by selenate in the young leaves and florets of broccoli [−2 s−1 B LED (470 nm) compared with the darkness control, white LED light and R LED light treatments, while there was no obvious effect on those of the roots [Glucosinolates, which contain nitrogen and sulfur in their structures, are the major class of secondary metabolites found in Brassica crops, and play important roles in human body nutrition and health . Seleniubroccoli . In addibroccoli . Interesbroccoli . The acche roots . The canhe roots . TherefoBrassica oleracea var. italica cv. lvhua) were obtained from SANHERB Company . Surface-sterilized seeds were soaked in 5% sodium hypochlorite for 15 min, drained and soaked with distilled water at 30 °C for 5 h, germinated and grown in plastic trays (32.5 × 24 × 4.5 cm) in the growth chamber for 4 days in dark at 25 °C with 75% relative humidity. The broccoli sprouts were transferred into a growth chamber equipped with blue (B)/red (R)/green (G) LED light. One liter of 100 μmol L−1 sodium selenite solution or deionized water was added to the containers, and then 10 mL of 100 μmol L−1 sodium selenite solution was sprayed twice per day; the control sprouts were sprayed with deionized water. There were five treatments: CK:1R1B1G LED (red 660 ± 10 nm:blue 460 ± 10 nm:green 520 ± 10 nm = 1:1:1), 1R1B1G+Se (100 μmol L−1 Na2SeO3), 1R1B+Se (red:blue = 1:1), 1R2B+Se (red:blue = 1:2), 2R1B+Se (red:blue = 2:1) , 25 ± 1 °C, and relative humidity (RH) of 70 ± 10%, 500 ± 100 μmol·mol−1 CO2. On the seventh day, the sprouts were sampled. The sampled sprouts were frozen in liquid nitrogen, freeze-dried, milled, and stored at −40 °C for further analysis. For each treatment, three replicates were taken for analysis.The study was carried out in an artificial light plant factory at South China Agricultural University. Seeds of broccoli (e = 2:1) . The envBroccoli sprouts’ hypocotyl length (HL) was measured using a ruler. Fresh sprouts were weighed to obtain the fresh weight (FW), then dried at 75 °C for 72 h to obtain the dry weight (DW). Thirty broccoli sprouts were used as a sample group for each measurement.v:v) until they turned white at ambient temperature for 24 h in darkness. The absorbance of the extract solution was measured by UV-spectrophotometer  at 645 nm (A645), 663 nm (A663) and 440 nm (A440). Chlorophyll and carotenoid concentrations were calculated as follows [−1) = 12.7 ×A663 − 2.69 × A645Chlorophyll a (mg·L−1) = 22.9 × A645 − 4.86 × A663Chlorophyll b (mg·L−1) = 8.02 × A663 + 20.20 × A645Total Chlorophyll (mg·L−1) = 4.7 × A440 − 0.27 × Total ChlorophyllCarotenoids (mg·L−1) = Photosynthetic pigment (mg·L−1) × 8 mL × 10−3/0.2 gPhotosynthetic pigment (mg·gFresh broccoli sprouts samples (0.2 g) were dipped in 8 mL acetone ethanol mixture  was measured according to the method described by Xu . A grounSoluble sugar (SS) content was performed using the anthronesulfuric acid colorimetry method . 0.5 g fg for 10 min at 4 °C, and 0.2 mL supernatant was combined with 0.8 mL distilled water and 5 mL Coomassie brilliant blue G-250 solution (0.1 g·L−1). After 5 min, the soluble protein content was detected at 595 nm by UV-spectrophotometer.Total soluble protein (SP) contents in broccoli sprouts were determined according to the Coomassie brilliant blue G-250 dye method . 0.5 g fw/v). 10 mL extracted solution was mixed with 1 mL phosphate-acetic acid, 2 mL 5% vitriol and 4 mL ammonium molybdate. After 15 min, the mixed solution was determined at 705 nm by UV-spectrophotometer.Vitamin C (Vc) was assayed by the molybdenum blue spectrophotometry . 0.5 g fg for 10 min at 4 °C. 1 mL supernatant were mixed with 0.5 mL foline-phenol and 11.5 mL 26.7% sodium carbonate for 2 h in darkness. Total phenolic content was determined by reading the absorbance at 765 nm.Total phenolic compound content (TPC) was determined by the method of Vernon  with min2. After 5 min, the reaction solution was mixed with 0.7 mL 10% Al(NO3)3, and 6 min later, the mixture was added to 5 mL 5% NaOH. 10 min later, absorbance was read at 760 nm by UV-spectrophotometer.The samples for total flavonoid contents (TF) assay were extracted as the method for total phenolics. The total flavonoid contents were determined by the aluminum nitrate method . 1 mL ex−1 barium acetate was then added, and the mixture was centrifuged at 4000× g for 10 min (ambient temperature). The extraction was repeated twice more. Then, the supernates were combined and loaded onto a mini column containing 500 μL of DEAE-Sephadex A-25 that had been conditioned with 2 mol L−1 acetic acid and washed with 6 mol L−1 imidazole formate. After loading, the column was washed with 0.02 M sodium acetate buffer. 500 μL of a sulfatase solution  was added, and the preparation was incubated overnight. The latter was dissolved in 2 mL of distilled water and filtered through a 0.22 μm membrane filter. HPLC analysis of desulphoglucosinolates was carried out by a Waters e2695 liquid chromatograph  equipped with a model 2489 UV/Vis detector and controlled by Waters Empower3 software. Samples (20 μL) were separated at 30 °C on a reversed-phase C18 column . The mobile phase consisted of acetonitrile (A) and distilled water (B) and flow rate was 1.0 mL·min−1. Chromatograms were recorded at 229 nm. The glucosinolates were identified by their retention times and spectral data as compared by standards.Glucosinolates (GLs) were extracted and analyzed as previously described with slight modifications . The freDried broccoli sprouts were weighed and ground to powder. The content of nitrogen (N) in sprouts was determined by Ojeda . Phosphop < 0.05. Multivariate principal component analysis (PCA) was performed by and XLStat 2019 software . A heat map was generated using TBtools (http://cj-chen.github.io/TBtools/).Data were analyzed by a one-way analysis of variance (ANOVA), using SPSS 22.0 software . Means comparison was performed using Duncan’s test at The combination of selenium and different ratios of blue/red/green narrow-band LED light quality was able to significantly increase the nutritional quality and health-promoting compounds of broccoli sprouts, including carotenoids, soluble sugars, soluble proteins, Vc, TA, TPC, TF, GLs and organic Se, in general without negatively affecting the fresh weight of sprouts. Moreover, PCA and cluster heat map showed that broccoli sprouts with 1R2B+Se treatment had higher nutritional quality and content of health-promoting compounds than other treatments. The combination of Se and 1R2B (1R2B+Se) might be beneficial to the production of selenium-biofortified high-quality broccoli sprouts.


Marantodes pumilum (Primulaceae) has been used in Malaysian folk medicine to help women regain strength after delivery and for “sickness in the bones.” It was previously revealed that its extracts inhibited xanthine oxidase (XO) activity in vitro. The leaves and roots of M. pumilum var. alata (MPA), var. pumila (MPP), and var. lanceolata (MPL) were individually extracted in ethanol (80%). The anti-hyperuricemic activity was initially assessed by XO inhibition with a spectrophotometric in vitro assay. The most active extract was further investigated on hyperuricemic rat model induced by potassium oxonate to determine serum uric acid levels and liver XO effect. The in vitro anti-inflammatory activity was carried out on monosodium urate (MSU) crystal-induced pro-inflammatory cytokines 1α, IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α) secretion using human peripheral blood mononuclear cells and ELISA technique, and prostaglandin E2 (PGE2)secretion using radioimmunoassay. The active extract was then investigated on gout-induced inflammation with MSU crystals to determine pro-inflammatory cytokines and PGE2 secretion levels in the synovial fluid of rat knee joint. Quantitative analysis using validated HPLC was performed on the extracts to determine presence of bioactive flavonoids. The findings revealed that extract of MPP leaves gave the highest inhibitory activity on XO (IC50 130.5 μg/mL) compared to other extracts tested. However, all extracts possessed significantly lower activity compared to allopurinol (IC50 0.13 μg/mL). Oral administration of MPP leaf extract (200 mg/kg) significantly reduced serum uric acid level in hyperuricemic rats in time-dependent manner to the baseline level and it was as effective as allopurinol (5 mg/kg). The extract also inhibited liver XO activity (25%) compared to allopurinol (45%). In vitro anti-inflammatory assay showed that extract of MPP roots inhibited MSU crystals-induced secretion of IL-1α, IL-1β, IL-8, TNF-α, and PGE2 with IC50 values of 36, 25, 38, 18, and 46 μg/mL, respectively. Oral administration of the MPP root extract (200 mg/kg) significantly decreased IL-1α, IL-1β, IL-6, TNF-α, and PGE2 levels in rat’s synovial fluid as effective as indomethacin. There were no significant body weight changes of all experimental animals. MPP extracts showed presence of myricetin, quercetin and kaempferol. Myricetin was detected with values of 0.2 and 0.6 mg/g for root and leaf extracts, respectively. The anti-hyperuricemic of MPP leaf and anti-inflammatory of MPP root indicated that MPP may be promising for complementary therapy of gout. Gout is the most common inflammatory arthritis with an increasing incidence and prevalence worldwide . AccordiGout occurs due to the deposition of uric acid crystals in tissues, tendons and joints which causes inflammatory response . TherefoNatural products, including minerals, plants, animals, microorganisms, and marine organisms, have been used as drugs since prehistoric times. There are so many plants used for traditional medicine, such as in Ayurvedic Medicine, Chinese Traditional Medicine, Unani Medicine, Traditional Korean Medicine, Kampo, etc., which probably have been used for hundreds or thousands of years for therapeutic purposes and need to be proven for its efficacy and safety to fulfill the modern standards of therapeutic agents . Considein vitro and in vivo. The most famous example of natural product as treatment for gout is colchicine, an alkaloid from plant Colchicum autumnale. It has been used clinically for treatment of gouty inflammation  Kuntze (Primulaceae) is previously known as Labisia pumila (Blume) Fern.-Vill under the family of Myrsinaceae -5-(pentadec-4′-enyl)-resorcinol, (Z)-5-(pentadec-8′-enyl)-resorcinol and (Z)-5-(pentadec-10′-enyl)-resorcinol were isolated from MPA roots . Xanthine oxidase from bovine milk (20 U/mL) was purchased from Roche Diagnostic GmbH . IL-8 ELISA kits were purchased from Abnova, Germany. Kits for determination of xanthine oxidase activity and plasma uric were purchased from BioVision , while all of the other kits were purchased from Cayman, United States. Radiolabelled PGE2  and liquid scintillation cocktail were purchased from Perkin Elmer .HPLC-grade MeOH and acetonitrile were obtained from Merck , HPLC-grade orthophosphoric acid was obtained from Fisher Scientific  while pure water for HPLC was obtained from ultrapure water system machine . Dexamethasone phosphate was obtained from Duopharma (M) Sdn. Bhd., Malaysia. Lymphoprep was obtained from Axis-Shield PoC AS , while indomethacin, allopurinol, potassium oxonate, uric acid, xanthine substrate, unlabelled PGEM. pumilum, were collected from Hutan Gunung Bujang Melaka, Kampar, Perak, Malaysia. The plants were authenticated by Emeritus Professor Dato’ Dr. Abdul Latiff Mohamad and voucher specimens of MPA (UKMB 30006/SM 2622), MPP (UKMB 30007/SM s.n), and MPL (UKMB 30008/SM s.n) were deposited in the Herbarium of Universiti Kebangsaan Malaysia. Fresh plant materials were washed, garbled, air-dried and ground. Each dried powder was individually extracted with 80% ethanol in ratio of 1:10 by exhaustive maceration for 3 days at room temperature and was repeated for 10 times. The organic filtrate was collected and concentrated under reduced vacuum pressure to remove the residual organic solvent. The solvent-free extracts were combined and then freeze-dried to obtain crude. The crude extract was stored at 4°C until further use.The leaves and roots of three varieties of n = 3, ≥18 years old) who fulfilled the following inclusion criteria of non-smoker, fasted overnight, and had not taken any medicines or supplements. The experimental protocol for in vitro cytokines and PGE2 assays was approved by the Human Ethical Committee of Universiti Kebangsaan Malaysia with approval number of UKM 1.5.3.5/244/NF- 040-2011 and UKM 1.5.3.5/244/NF- 016-2013, respectively. The protocol was in accordance to the principles outlined in the Declaration of Helsinki  with approval number FF/2013/JAMIA/25-SEPT/537-OCT.2013-FEB.2015  were obtained from Laboratory Animal Resource Unit, Universiti Kebangsaan Malaysia (LARU-UKM), Malaysia. The animals were housed in plastic cages and maintained on a 12 h/12 h light/dark cycle and the temperature and humidity were kept at 25 ± 2°C and 50%, respectively. They were fed with a commercial laboratory diet and allowed food and water FEB.2015 .TM GO Microplate spectrophotometer. The percentage of XO inhibitory activity was calculated as Eq. 1:Xanthine oxidase (XO) inhibitory activity was measured using a slightly modifying spectrophotometric method as described by A is the enzyme activity without test extract; B is the control of A without test solution and enzyme, C and D are the activities of the test extract with and without enzyme, respectively. Using this method, the activity of six samples were screened and the active extracts with activity ≥50% were further assayed using varying concentrations ranging from 25 to 400 μg/mL to obtain its half maximal inhibitory concentration (IC50) values which calculated using GraphPad Prism® .where in vitro XO inhibitory with varying concentrations of xanthine substrate solution  .Lineweaver-Burk plot was analyzed in order to determine the mode of enzyme inhibition of the most potent extract and allopurinol. The xanthine oxidase enzyme kinetic study was performed in a similar way to assay procedure for nd 0 μM) . The Linn = 6).Animals were divided into six groups  on 1st, 7th, and 14th day as a negative control.Group III:animals treated with 5 mg/kg of allopurinol, orally, for 14 days and received 250 mg/kg of potassium oxonate, i.p on 1st, 7th, and 14th day as a positive control.Group IV:animals treated with 50 mg/kg of MPP leaves extract, orally, for 14 days and received 250 mg/kg of potassium oxonate, i.p on 1st, 7th, and 14th day.Group V:animals treated with 100 mg/kg of MPP leaves extract, orally, for 14 days and received 250 mg/kg of potassium oxonate, i.p on 1st, 7th, and 14th day.Group VI:animals treated with 200 mg/kg of MPP leaves extract, orally, for 14 days and received 250 mg/kg of potassium oxonate, i.p on 1st, 7th, and 14th day.Potassium oxonate (125 mg/mL) dissolved in 0.9% saline solution was administered intraperitoneally 1 h before oral administration of test samples or allopurinol on the 1st, 7th, and 14th day of experiment. Samples (100 mg/mL) and allopurinol (10 mg/mL) were individually suspended homogeneously in 3% Tween 20. Animals were fasted 2 h before drug administration. Treatments were administered once a day by oral gavage for 14 consecutive days.g for 10 min at 4°C. Serum was separated and stored at −20°C until assay for uric acid quantification. At the end of experiment, rats were sacrificed by cervical dislocation after blood collection and under mild anesthesia. The organs  were immediately excised, washed in cold saline (0.9%), dried with paper towel, weighed and rapidly stored at −80°C.Rats were anesthetised 1 h after drug/extract administration on 1st, 7th and 14th days in order to collect blood from retro-orbital plexus. The blood was allowed to clot for approximately 1 h at room temperature and then centrifuged at 2500 × The serum uric acid levels were determined by enzymatic-colorimetric method using a uric acid assay kit . The protocol was in accordance of kit manufacturer’s instruction. Briefly, in each well, 5 μL of serum, 45 μL of uric acid assay buffer and 50 μL reaction mixture  were mixed. The same relative volume was used for the calibration curve and blank. The reaction mixture was incubated for 30 min and the absorbance was measured using a microplate reader at a wavelength of 570 nm.g for 10 min at 4°C. Lipid layer was carefully removed and the supernatant was further centrifuged at 10,000 × g for 60 min at 4°C. The final supernatant was used for liver XO assay determined using the XO colorimetric assay kit from BioVision  and measured at a wavelength of 570 nm. One unit of XO is the amount of enzyme which catalyzes the oxidation of xanthine, yielding 1 μmol of uric acid and H2O2 per minute at 25°C. Protein concentration was determined spectrophotometrically based on the method of Crude enzyme extract was prepared according to the method by 2 for 27 h. The plates were incubated again for 4 h with 20 μL of MTT (5 mg/mL). The supernatant was carefully discarded and the formazan blue crystals produced by cells were dissolved in 100 μL of DMSO (100%) and the absorbance was measured at a wavelength of 570 nm.The fresh blood collected from healthy volunteer was obtained in heparin-containing tube. Then, PBMCs were isolated by Lymphoprep gradient separation method as describe by us previously . Cell vi2O with NaOH (9 mL/0.5 N); adjusted to pH 8.9 at 60°C by adding HCl, cooled for 3 days in cold room, then washed and dried. Then, the MSU crystals were sterilized by heating at 180°C for 2 h before experiments. Needle-shaped crystals were suspended in sterile saline to give a suspension of 200 μg/mL.Monosodium urate (MSU) crystals were prepared according to previously described method with slight modification . Briefly2. After pre-incubation, cells were incubated with 20 μL of MSU crystal suspension (200 μg/mL) for 24 h. After incubation, cells were centrifuged for 10 min at 300 × g and 4°C. The supernatant was carefully transferred into a sterile tube and the concentration of cytokines in the supernatant was measured using appropriate ELISA kits for human. The cytokine secretion levels were compared with the negative control that was considered as 100% cytokine secretion. The percentage inhibition (% I) was calculated using Eq. 2:The PBMCs (100 μL) were pre-incubated with equal volume of extracts or dexamethasone as a positive control or complete medium with 0.5% DMSO as a negative control for 3 h at 37°C in 5% CO2 for 24 h with 10 μL MSU crystals suspension (200 μg/mL). The plasma was obtained by centrifugation at 3000 × g for 15 min at 4°C (Blood (1 mL) was obtained in heparin-containing tube and pre-incubated for 30 min in the presence or absence of extracts (50 μg/mL) or indomethacin (10 μg/mL) as a positive control or 0.5% DMSO in RIA buffer as negative control. Then, the mixture was incubated at 37°C containing 5% COn at 4°C .g for 15 min at 4°C, 300 μL of supernatant was added to 3 mL liquid scintillation cocktail in Pico Pro Vial . The radioactivity was measured using a liquid scintillation analyzer . The normalized percentage bound (%B/Bo) was then calculated using Eq. 3.The assay was carried out according to our method as previously reported . The pla2 in picogram (ρg). The %B/Bo values of serial dilutions of standard PGE2 with concentrations ranging from 2.45 to 400 ρg/0.1 mL were used to obtain a standard curve plot. The concentration of PGE2 (ρg/0.1 mL) in each sample were determined by interpolating the %B/Bo values. Percentage inhibition was calculated using Eq. 2.The %B/Bo values were plotted using semi-logarithmic graph against the corresponding concentration of standard PGEn = 6).An experimental model of gouty inflammation using monosodium urate (MSU) crystals as inducer was used in order to evaluate the anti-inflammatory effect of MPP roots extract, as described previously  with sliGroup I:normal control group.Group II:MSU control group , intra-articular in the right knee joint on 11th day) as a negative control.Group III:animals treated with 3 mg/kg of indomethacin, orally, for 14 days and received 50 μL of MSU crystals suspension (100 mg/mL), intra-articular in the right knee joint on 11th day as a positive control.Group IV:animals treated with 50 mg/kg of MPP roots extract, orally, for 14 days and received 50 μL of MSU crystals suspension (100 mg/mL), intra-articular in the right knee joint on 11th day.Group V:animals treated with 100 mg/kg of MPP roots extract, orally, for 14 days and received 50 μL of MSU crystals suspension (100 mg/mL), intra-articular in the right knee joint on 11th day.Group VI:animals treated with 200 mg/kg of MPP roots extract, orally, for 14 days and received 50 μL of MSU crystals suspension (100 mg/mL), intra-articular in the right knee joint on 11th day.Monosodium urate crystals (100 mg/mL) suspended in 0.9% sterile saline solution was administered intra-articularly in the right knee joint of each animal, except those of normal control group on the 11th day of experiment. Samples (100 mg/mL) and indomethacin (10 mg/mL) were individually suspended homogeneously in 3% Tween 20. Animals were fasted 2 h before drug administration. Treatments were administered once a day by oral gavage for 14 consecutive days. At the end of experiment, rats were sacrificed after anesthesia in order to collect synovial fluid. After skin shaving, a 27 G needle was inserted into the synovial cavity to inject 100 μL sterile saline, while the second needle was inserted next to the first needle to collect synovial fluid and immediately stored at −80°C until assay. The organs  were immediately excised, washed in cold saline (0.9%), dried with paper towel and weighed.2 in synovial fluid. TNF-α, IL-1α, IL-1β, and IL-6 were measured using a multi-cytokine bead array detection system  according to the manufacturer’s instruction. Meanwhile, PGE2  were measured using single ELISA assay according to the manufacturer’s instruction.The inflammation was evaluated by measuring TNF-α, IL-1α, IL-1β, IL-6, and PGEin vitro and in vivo assays, i.e., leaf and root extracts of MPP, using RP-HPLC based on the method described by Phytochemical analysis was performed for the most active extracts from HPLC analysis was performed using a Waters system  with C-18 column , photodiode array detector at a wavelength of 360 nm and flow rate of 0.6 mL/min. The mobile phase consisted of methanol (A) and acidified water with 0.1% orthophosphoric acid (B) and eluted by a linear gradient of 40–100% A (0–10 min), an isocratic composition of 100% A (12–15 min), and a linear gradient of 100–40% A (15–20 min). The column was equilibrated for 20 min before next injection. Injection volume of each solution was 10 μL. The RP-HPLC method was validated based on the determination of linearity, precision, limits of quantification (LOQ), and detection (LOD) for three detected reference standards, i.e., quercetin, myricetin and kaempferol.versus the areas under the peaks. The standard curve equation obtained from reference standards was used to quantify the concentration of flavonoids in extracts.The extracts of MPP leaves and roots (100 mg/mL) and a mixture of reference standards of kaempferol, myricetin and quercetin (1 mg/mL) were prepared in HPLC-grade MeOH, sonicated for 5 min and filtered using 0.45 μm membrane prior to analysis using the validated HPLC conditions. Stock solution of mixture of reference standards was further diluted into a series of two-fold dilutions . The calibration curve was plotted with five concentrations of reference standards solution n = 3) and the data presented as mean ± standard error of mean (SEM). The IC50 values were calculated with a non-linear regression analysis using GraphPad Prism 5 software. The values were obtained from at least three determinations (n = 3). Data were statistically analyzed using one-way analysis of variance (ANOVA) and post hoc Tukey’s test for multiple comparisons and p ≤ 0.05 was considered to be statistically significant. Pearson’s correlation coefficient was used to analyze the degree of association between xanthine oxidase activities and uric acid levels in vivo.All data were analyzed using GraphPad Prism 5 software. Each experiment was carried out in triplicate (p ≤ 0.01). Allopurinol (100 μg/mL) strongly attenuated XO activity with percentage of inhibition value of 99.8% as shown in 50 values were determined for those active extracts and all active extracts were found to possess a dose-dependent XO inhibitory effect as shown in In this study, the highest inhibition of XO activity was found from MPP leaves extract (400 μg/mL) with value of 87%. All extracts showed a lower activity compared to allopurinol as positive control .The results revealed that MPP leave extract possessed the strongest XO inhibitory activity with ICType of inhibitory activity of XO by MPP leaf extract was established from Lineweaver-Burk plots . The Micp ≤ 0.05). However, only treatment with 200 mg/kg/day of extract was able to reduce serum uric acid levels to the baseline value after 14 days of experiment (p > 0.05). Allopurinol (5 mg/kg) reduced serum uric acid levels compared to hyperuricemic control group (p ≤ 0.05). This reduction was observed even after 1 day of treatment with allopurinol and the normal serum uric acid levels were maintained throughout the 14 days of experiment. The data showed that MPP leaf extract elicited a slower onset of action compared to allopurinol. However, there was no significant difference between the effect of allopurinol and extract at the dose of 200 mg/kg at day 14 (p > 0.05). This demonstrated that the effectiveness of extract at the dose of 200 mg/kg was comparable to allopurinol after 14 days of treatment.In this study, the baseline values of serum uric acid levels of each group, measured before starting MPP leaf extract and potassium oxonate administration (Day 0), ranged from 1.6 to 1.7 mg/dL as shown in p ≤ 0.05) as shown in p ≤ 0.05).Liver XO inhibitory activity in rats was evaluated in order to further investigate the anti-hyperuricemic effect of the extract. The results revealed that potassium oxonate, beside producing hyperuricemia, cause a slight but significant induction of liver XO activity compared to normal control group .Treatment with MPP leaf extract showed a dose-dependent effect on both serum uric acid levels and liver XO inhibitory activity . A PearsM. pumilum extracts at 50 and 100 μg/mL. Per cent (%) viability of all M. pumilum extracts at concentration of 50 μg/mL and dexamethasone at 5 μg/mL was greater than 90% after 27 h of exposure  to dexamethasone. Only MPP roots showed significantly higher (p ≤ 0.05) inhibitory activity against TNF-α compared to dexamethasone, whilst that of the other active extracts were comparable (p > 0.05) to dexamethasone. However, all active extracts showed significantly lower (p ≤ 0.05) inhibitory activity against IL-1α than dexamethasone.The inhibitory activity on the secretion of five cytokines  of 50 value of active extracts demonstrated that the inhibitory activity was in a concentration dependent manner. The IC50 values for all active extracts were significantly higher than dexamethasone as positive control (p ≤ 0.001). The results showed that extract of MPP roots possessed the lowest IC50 values for IL-1α (35.71 μg/mL), IL-1β (25.06 μg/mL), and IL-8 (38.4 μg/mL), whilst MPL roots showed the lowest IC50 value for TNF-α (15.38 μg/mL) as seen in Determination of IC2 secretion (p ≤ 0.05) compared to indomethacin (10 μg/mL). The result showed that out of the two active extracts, the IC50 value of extracts of MPP leaves (IC50 42.33 μg/mL) was found to be more potent than the root part (IC50 45.52 μg/mL). However, the IC50 values of active extracts were significantly higher (p ≤ 0.001) than indomethacin with IC50 value of 0.35 μg/mL.Out of six extracts (50 μg/mL), roots and leaves of MPP were found to actively inhibit PGEecretion . However2 in synovial fluid of MSU control rats when compared with normal control rats (p ≤ 0.05). However, treatment with the extract reduced levels of these inflammatory mediators in a dose-dependent manner. Both indomethacin and 200 mg/kg of MPP roots extract significantly reduced all cytokines and PGE2 to similar levels. It demonstrated that treatment with extract of MPP roots at dose of 200 mg/kg for 14 days was as effective as indomethacin at 3 mg/kg.Articular inflammation was quantified by examining the changes in markers attenuated by the treatment. Body weight did not show significant differences among groups during the 14 experimental days , 5 althoBased on the results obtained for linearity, LOD and LOQ, the method was giving a linear response in the selected range. LOD was acceptable and the method was able to detect and quantify the analyte below the minimum concentration of flavonoids used in the calibration curve . The preIn this study, three flavonoids, reportedly present in MPP roots and leaves, have been identified using HPLC, i.e., myricetin, quercetin and kaempferol, with retention time at 14.95, 17.13, and 18.72 min, respectively . The peaGout is caused by increased level of uric acid in blood (hyperuricemia) due to metabolism disorders of purine. Uric acid is the end product ofseveral deamination and oxidation processes of adenin and guanine, with xanthine oxidase (XO) widely distributed in the body, including liver, intestine, kidney, lungs, myocardium, brain, plasma and other tissues . A valuein vivo and XO inhibitory activity in vitro. In vitro study showed that MPP leaves inhibited XO, although it was ten-times less potent than allopurinol. Phenolics, such as flavonoids, have been reported to possess XO inhibitory activity  to allopurinol, but the effect on liver XO inhibitory activity was significantly lower than allopurinol (p ≤ 0.05). It has to be considered that the reduction of serum uric acid levels could be accomplished mainly through other mechanism of action such as uricosuric activity probably by modulating human urate transporter 1 (URAT1), glucose transporter 9 (GLUT9) and organic anion transporter 1(OAT1)  . Flavono effects . Hence, 50 values were significantly lower than dexamethasone. Out of the three active extracts, two were root part while only one was leaf part of M. pumilum varieties. It demonstrated that the root part maybe contained more active components compared to the leaf part and it affected their biological activity.From this study, only MPP root, MPL root and leaf extracts gave inhibitory activities in MSU-induced inflammation of cytokines and PGE2 secretion. However, the IC2. These cytokines and PGE2 biologically act to cause the clinical features of inflammation include severe pain, edema, and erythema in the joint  and quercetin (100 mg/kg) significantly (p ≤ 0.05) reduced serum uric acid in potassium oxonate-induced hyperuricemic mice .in vivo assay. AR provided the technical assistance for in vitro anti-inflammatory assay.ER was the master candidate who conducted the experimental works and drafted the manuscript. JAJ was the project leader responsible for the research design and editing of the manuscript. EK was a project team member who contributed ideas for the research design and reviewed the manuscript. JJ and KH are project team members who contributed ideas for the research design. FB was veterinarian who helped with the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The genes down-regulated in white skin were associated with pigmentation, melanocyte–keratinocyte interaction, and keratin, while up-regulated ones were mainly associated with cellular energy metabolisms. Furthermore, those DE lncRNAs were predicted to be implicated in pigmentation, keratin synthesis and cellular energy metabolism. In general, this study provides insight into the transcriptional difference involved in melanocyte-loss-induced keratinocyte changes and promotes the Bama pig as a biomedical model in skin research.Skin is the body’s largest organ, and the main function of skin is to protect underlying organs from possible external damage. Melanocytes play an important role in skin pigmentation. The Bama pig has a “two-end-black” phenotype with different coat colors across skin regions, e.g., white skin (without melanocytes) and black skin (with melanocytes), which could be a model to investigate skin-related disorders, specifically loss of melanocytes. Here, we generated expression profiles of mRNAs and long noncoding RNAs in Bama pig skins with different coat colors. In total, 14,900 mRNAs and 7549 lncRNAs were expressed. Overall, 2338 mRNAs/113 lncRNAs with FDR-adjusted  Skin is the largest organ for mammals, exhibiting a complex heterogeneous and multilayered structure and containing various components and more than 10 types of cells [Much research has been performed to investigate or demonstrate the structural components and physiological mechanisms of skin in humans and mice. For example, pigmentation, which depends on the production of eumelanin and pheomelanin in melanocytes, provides protection for the skin of humans and other animals, as well as directly affecting appearance . ResearcThe Bama pig is an indigenous breed in China, with a “two-end-black” coat color phenotype . The white skin of the Bama pig lacks melanocytes . With thLncRNAs are commonly defined as non-coding RNA with a length more than 200 bp, which regulate some interesting gene expression ,11. To eSix two-year-old female Bama pigs of a similar-weight and raised under the same feeding and rearing conditions were chosen for the experiments. The pigs were humanely killed, after being stunned by an electric shock to ameliorate their suffering. Then, white skin from the back and black skin from the buttocks with a depth of 8 mm  was collA total of 3 μg of RNA (per skin sample) was extracted using Trizol reagent , in accordance with the manufacturer’s instructions, as was RNA of eight tissues from 3 Bama pigs . The integrity of RNA was checked by gel electrophoresis on 1.0% agarose gel with GoldView staining using an Agilent 2100 bioanalyzer. A NanoDrop spectrophotometer (Thermo Scientific) was used to measure the RNA concentrations. The RNAs with a ratio of absorbance at 260/280 nm of over 1.8 were selected for further study.Six samples from three different pigs  were selected for sequencing. Approximately 1 µg of total RNA (per sample) and oligo (dT) magnetic heads were used for enriching poly (A) RNAs. The resulting fragments were used as a template for reverse transcription. Random hexamer primers, buffer, dNTPs, DNA polymerase I, and RNase H were used for generating RNA-Seq complementary DNA (cDNA) libraries; next, RNA-seq was conducted following the manufacturer’s standard procedures. High-quality strand-specific libraries were sequenced on the HiSeq X platform  and the bases were called using the software CASAVA v.1.8.2 (Illumina); then, 150-bp paired-end reads were obtained.30 scores and GC content of the clean data were calculated. Clean data were mapped to the pig genome (Sus scrofa 11.1 of release 90 from Ensembl) using TopHat (version 2.1.0) [High-quality data were controlled by removing poly-N and low-quality reads from the raw data. As a consequence, a total of 44 GB of clean data was acquired. The Qn 2.1.0) .  Gene Expression Omnibus (GEO) and the accession number is GSE125517.p-value < 10−4 by Hmmscan [p-value < 10−10 by BLASTX (https://blast.ncbi.nlm.nih.gov/); and (4) prediction and calculation of the coding potential of the remaining transcripts by CPC [Mapped reads were assembled by StringTie and then merged with Cuffmerge (part of Cufflinks version 2.2.1) . Then, c Hmmscan ; (3) coms by CPC . Transcrs by CPC .p values < 0.05 and |log2(FC)| > 1 were considered to be differentially expressed.The mRNA and lncRNA expression level of fragments per kilobase per million mapped reads (FPKM) of each sample was calculated by StringTie (version 1.3.3) ; mRNAs wFPKM values of the six samples  were used for principal component analysis (PCA) analysis, as well as clustering analysis.The RNA-seq data of melanocytes, keratinocytes and fibroblasts were downloaded from reference . The CIBThe R package WGCNA was used to detect interactions between lncRNA and mRNA . The Cythttp://david.abcc.ncifcrf.gov/). To predict the functions of the DE lncRNAs, the differentially expressed genes that were within 100 kb of the lncRNAs or showed a high correlation with the lncRNA were collected and performed functional enrichment analysis as well. The KEGG pathways or GO terms with Benjamini-corrected p-value < 0.05 were significant.Gene ontology (GO) functional enrichment analysis and KEGG pathway functional enrichment analysis were performed with DE genes at the DAVID web server (Hmisc (an R package from https://cran.r-project.org/), with the aim of identifying correlations between expression of functional lncRNAs and DE mRNAs [Pearson’s correlation coefficients between the expression of DE lncRNAs and the DE mRNAs were calculated with DE mRNAs . All theTCONS_00019024 and that of CYGB (n = 3). To investigate the correlation between expression of TCONS_00019024 and CYGB, RNAs from the skin of six Bama pigs were used for the experiment (n = 6). The genomic DNA in RNA samples was removed by gDNA Eraser  at 42 °C for 5 min. Five micrograms of RNA was reverse-transcribed into cDNA using RT Reagent Kit . The primers for the genes and lncRNAs were designed using Primer 5.0 and tested by NCBI Primer-Blast. The volume of the reaction mixture was 10 µL, with 1 µL of cDNA, 0.5 µL of primers, 5 µL of SYBR , and 3 µL of RNA-free water. The following RT-PCR reaction was performed for all genes and lncRNAs: 95 °C for 3 min; followed by 40 cycles of 95 °C for 10 s and amplification at the optimal temperature for each sample for 30 s; 95 °C for 30 s; and then a melting curve analysis (65 °C to 95 °C). The expression of β-actin was used to correct the gene expression data. The 2−ΔΔCT method was used to analyze the RT-PCR data and calculate relative expression. If a gene was up-regulated in black skin, its expression relative to that in white skin was calculated. If a gene was up-regulated in white skin, its expression relative to that in black skin was also calculated. The t-test was used to test the significance of differences in gene expression.Twenty-one genes (3 lncRNA included)  were selApproximately 49.03 million raw reads were generated for each sample, and after quality control, 48.81 million clean reads per each were obtained for further analysis. A total of 93–97% of the clean reads were mapped to the pig genome reference . FinallyA total of 2338 mRNAs were identified to be differentially expressed between two groups . Among tp = 4.77 × 10−4), WNT signaling pathway (p = 9.86 × 10−3), PI3K-Akt signaling pathway (p = 7.49 × 10−9), cAMP signaling pathway (p = 9.53 × 10−3), and ECM-receptor interaction (p = 2.51 × 10−5)   [27,28,2ite skin  , which were proved to be less expressed in white skin [ETS1, TCF7L1, and SOX18; (2) genes that regulate melanocyte development, differentiation, or migration, such as KIT, HGF, EDN1 and WNT11 [DCT, TYRP1, PMEL and MYRIP [SOX transcript factor family is required for melanocyte development, and mutation of the SOX gene family could result in Waardenburg syndrome [KIT (c-kit)  plays a crucial role in melanocyte development, melanocyte differentiation, and melanocyte migration [KIT gene may cause melanocyte defects in European domestic pigs [TYRP1 or PMEL caused defects in eumelanin synthesis instead of melanocyte loss [KRT) gene [We also noticed 31 coat color genes down-regulated in white skin  gene ,52,53.p = 2.99 × 10−15), external side of the plasma membrane (41 genes) (p = 1.19 × 10−9), calcium ion binding (89 genes) (p = 7.60 × 10−13), and extracellular exosome (251 genes) (p = 7.33 × 10−8) (p = 2.07 × 10−3), hair follicle development (p = 3.64 × 10−2), and skin development (p = 2.21 × 10−2)  ( × 10−8) , as well × 10−2)   C.p = 6.78 × 10−28), carbon metabolism (p = 6.51 × 10−13), TCA cycle (p = 1.33 × 10−5), and propanoate metabolism (p = 7.37 × 10−7) (p = 2.87 × 10−7), oxidoreductase activity (p = 1.04 × 10−6), NADP binding (p = 3.04 × 10−3), fatty acid metabolic process (p = 5.11× 10−5), and mitochondrial membrane (p = 8.70 × 10−4)  . These oPI3K-Alt signaling pathway (p = 4.0 × 10−2) which was involved in pigmentation  might play roles in keratin synthesis, response to insulin, β-oxidation of fat and melanocyte survival. For example, there were four keratin genes  oriented nearby TCONS_00077733 within 100 kb, particularly KRT80 which is convergent and 17,754 bp downstream of TCONS_00077733 , which assumed that TCONS_00077733 might be related to keratin synthesis. Moreover, TCONS_00042201 is located 336 bp upstream of IRS1 . IRS1 encodes insulin receptor substrate 1, which is associated with response to insulin [TCONS_00060772 is a lncRNA that is located overlapping HSD17B4 and their expression is correlated . HSD17B4 is involved in the β-oxidation of fat [TCONS_00019024 is a divergent lncRNA [CYGB (cytoglobin), located 3888 bp upstream of CYGB. This lncRNA/mRNA pair was found to be up-regulated in black skin, showing a Pearson’s correlation coefficient of 0.932 (p = 1 × 10−2)  C. CYGB isurvival . These rp < 0.05) in expression level with 113 DE lncRNAs. These 474 DE mRNAs were significantly enriched in oxidative phosphorylation (p = 4.68 × 10−8), mitochondrial inner membrane (p = 6.46 × 10−8) and plasma membrane (p = 2.20 × 10−2)  . In addiby trans . These rTRPM1, TYRP1, PMEL, MLANA and DCT) were significantly highly expressed in black skin and 21 genes above were indeed differentially expressed   D. We als in skin E. In conSkin is both important and the largest organ of the body, and provides protection for underlying organs. Melanocyte inhabits the skin and its major function is the pigmentation of skin and hair. Loss of melanocyte can cause pigmentation deficiency. Mechanisms of melanocyte deficiency are still not fully understood and transcriptome profiles studies related to the lack of melanocytes are insufficient. Even the mechanism associated with the cross-talk between melanocytes and other types of cells in the skin is still not well-understood in the pig. Therefore, we performed transcriptomic analysis of Bama pig skin to reveal the mechanism potentially associated with the loss of melanocytes or interaction networks related to melanocytes in the pig.Even with a great similarity between white and black skins, the Pearson correlation coefficient was 0.92  which waITSN1, BTK, FYN and CCR1, were associated with calcium release of the plasma membrane  which might be involved in keratin synthesis and cellular energy metabolism. The black skin was different from the white skin in the expression of keratin genes and genes related to cellular energy metabolism. lncRNA was shown to play critical roles in the skin, such as melanin synthesis in goats [H19 regulates Dsg1 expression and the consequent keratinocyte differentiation through acting as an endogenous “sponge” of miR-130b-3p in humans [TCONS_00077733 and TCONS_00060772 might be one of causes responsible for the difference between black and white skin, but more evidence is needed to support this assumption. Taken together, the results of this study provide evidence of the interactions involved in pigmentation. This research provides insight into the complex mechanisms associated with the “two-end-black” phenotype.In this study, we identified two DE lncRNAs (in goats , psoriasin goats  and exprin goats . In addin humans . Thus, wIn conclusion, we had the opportunity to work on the same individual to systematically reveal transcriptome differences. Our results suggested that the loss of melanocytes could contribute to the expression of melanogenesis genes, and lack of melanocytes might be one of the causes of the white skin differing from the black skin in keratinocyte function and the development of hypertrophic scars. In addition, the present research implies that lncRNAs play roles in skin. Our research could promote the application of the Bama pig in research on melanocyte deficiency.


In this study, the amino acid arginine (ARG) and P-glycoprotein (P-gp) inhibitors verapamil hydrochloride (VER), piperine (PIP) and quercetin (QRT) were used as co-formers for co-amorphous mixtures of a Biopharmaceutics classification system (BCS) class IV drug, furosemide (FUR). FUR mixtures with VER, PIP and QRT were prepared by solvent evaporation, and mixtures with ARG were prepared by spray drying in 1:1 and 1:2 molar ratios. The solid-state properties of the mixtures were characterized with X-ray powder diffraction (XRPD), Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) in stability studies under different storage conditions. Simultaneous dissolution/permeation studies were conducted in side-by-side diffusion cells with a PAMPA  membrane as a permeation barrier. It was observed with XRPD that ARG, VER and PIP formed co-amorphous mixtures with FUR at both molar ratios. DSC and FTIR revealed single glass transition values for the mixtures (except for FUR:VER 1:2), with the formation of intermolecular interactions between the components, especially salt formation between FUR and ARG. The co-amorphous mixtures were found to be stable for at least two months under an elevated temperature/humidity, except FUR:ARG 1:2, which was sensitive to humidity. The dissolution/permeation studies showed that only the co-amorphous FUR:ARG mixtures were able to enhance both the dissolution and permeation of FUR. Thus, it is concluded that formulating co-amorphous salts with ARG may be a promising option for poorly soluble/permeable FUR. During drug development, molecules categorized under the Biopharmaceutics classification system (BCS) as class IV, i.e., those compounds that show both insufficient solubility and problems in membrane permeability and/or active cellular efflux across intestinal membranes, are the most difficult from the oral formulation point of view . The curCo-amorphous systems are combinations of two or more low-molecular-weight components that form homogeneous, amorphous single-phase systems ,3. Co-amIn this study, we used furosemide (FUR) as a model drug. FUR is a problematic drug, as it belongs to BCS class IV but also with respect to preparation methods; it experiences thermal decomposition upon melting (a problem for melting methods) , it is pFurosemide  was used as a model drug in combination with the co-formers l-arginine , piperine , quercetin  and verapamil hydrochloride .In the preliminary studies, the co-amorphous mixtures were prepared in situ in a differential scanning calorimeter , with a RCS90 cooler  and nitrogen flow of 50 mL/min from physical mixtures (PMs). The PMs were prepared in an oscillatory mill . The drug and a conformer with a total mass of 500 mg were transferred into 25 mL milling jars. Mixing was performed at 30 Hz for 5 min. The mixtures were stored at ambient temperature and humidity. Samples were weighed  into hermetic aluminium pans pierced with a pin . The samples were equilibrated at 40 °C and heated at 10 °C/min until the melting points were reached . The temperature was held for 5 min at melting, after which fast cooling to −50 °C was performed. After this, the samples were heated at 10 °C/min to 215 °C . The results were obtained using TRIOS Software . The co-melting of FUR–PIP and FUR–VER was also attempted on a hot plate . The mixtures (100–500 mg) were placed on an aluminium plate, mixed and melted at 170 °C for one minute, after which the melt was cooled on an ice bath.2O5 . Co-amorphous FUR–VER and FUR–PIP were prepared by dissolving 850 mg of the PM in 50 mL of MeOH on a Petri dish. The solvent was evaporated on a hot plate  at 120 °C for eight minutes. The remaining solid was collected and stored in a refrigerator at 0% RH.Larger-scale preparation was performed by a solvent evaporation approach. Spray drying was conducted with a Buchi Mini Spray Dryer B-191 . The PMs were dissolved in MilliQ water with overnight stirring at 50 °C (500 mg in 20 mL for the 1:1 mixture or in 10 mL for the 1:2 mixture). The spray drying was performed with an outlet temperature of 160 °C. The other settings can be found in XRPD measurements were performed with a Bruker D8 DISCOVER system  using Cu Kα radiation with λ = 1.5418 Å and a motorized slit. The samples were analysed at 40 kV and 40 mA from 5 to 35° 2 θ using a scanning speed of 0.120 s/step and a step size of 0.013°. The scattered radiation was collected with a 1D LYNXEYE detector fully open.g, midpoint), possible recrystallization  and melting  temperatures were measured in duplicate DSC thermograms with a TA DSC 2500 as described in The glass transition (Tg s for the mixtures (Equation (1)).1 and W2 are the mass fractions of the components and Tg1 and Tg2 are the glass transition temperatures (K) of the components. The constant K can be obtained with Equation (2):1 and ρ2 are the densities of the components (g/cm3). The densities were ρFUR = 1.61 g/cm3 [3 [3 [3 [The Gordon–Taylor equation was used to calculate the theoretical T61 g/cm3 , ρARG = g/cm3 [3 , ρVER = m3 [3 [3  and ρPIP[3 [3 [3 .−1 using a resolution of 4 cm−1 and taking an average of 64 scans per sample. The OMNIC 9 software  was used to collect the data.The FTIR spectra were obtained using a Thermo Nicolet iS50 FTIR spectrophotometer  equipped with an attenuated total reflectance (ATR) accessory. Spectra were collected over the range 650–4000 cm2O5. The samples were analysed regularly with XRPD and FTIR, as described above.The amorphous samples were stored under the following conditions: 4 °C/0% RH, ambient temperature (approximately 22 °C)/60% RH and 40 °C/0% RH. An RH of 60% was obtained with a saturated NaBr solution, and RH of 0%, with PThe equilibrium solubility for FUR was measured in pH 5.2 buffer. A buffer solution of pH 5.2 was prepared according to  using Na2) using PAMPA membranes, according to the protocol previously described in [The dissolution and permeation of FUR were tested simultaneously in side-by-side diffusion cells , and the dissolution samples were diluted further with an ACN/H2O 70/30 solution (1:4 v/v) and analysed with HPLC as described below.Powder samples corresponding to 10 mg of FUR (except for 25 mg of FUR with ARG) were placed in the donor cells, from which dissolution samples (300 µL) were withdrawn at 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 min and filtered immediately through a 30 mm polyethersulfone (PES) 0.22 µm membrane filter . Simultaneously, the acceptor cell was emptied (and discarded). The volumes removed from the cells were replaced with the buffer. The permeation test was conducted similarly but only taking samples from the acceptor cells. All the experiments were run in triplicate. The samples were diluted with acetonitrile (ACN) ) (30%) and trifluoroacetic acid  (0.1%), with a flow rate of 1.2 mL/min. FUR was detected at a wavelength of 234 nm, and its retention time was 2.6 ± 0.02 min. A FUR standard solution of 100 µg/mL was prepared in ACN/H2O (70/30), from which dilutions of 0.1, 0.5, 5, 10, 25 and 50 µg/mL were prepared in ACN/H2O. The standard curve was linear (r2 = 0.9997), and the repeatability of the method (RSD) was 2.6% (25 µg/mL) and 7.6 (1 µg/mL). The results were analysed using the Gilson Unipoint software .Gilson HPLC equipment consisting of a Gilson 321 pump, a Gilson UV–vis 151 detector , a Gilson 234 autoinjector  and a reversed-phase column  with a Phenomenex precolumn was used. The mobile phase was acetonitrile  (70%), Hp-values < 0.05 (95% confidence level) in t-tests (ANOVA single-factor).The area under curve (AUC) values for dissolution and permeation were calculated from the dissolution and permeation curves (between 0 and 360 min) by linear trapezoidal integration using Origin Pro 2015 64-bit . Differences were considered significant with Piperine , verapamil hydrochloride  and quercetin  were selected as co-formers for FUR, as they can inhibit P-gp-mediated efflux and thus potentially improve a drug’s bioavailabilty when co-administered orally with P-gp substrates ,22,26. AThe FUR mixtures with ARG (prepared by spray drying), PIP and VER (prepared by evaporation) at 1:1 and 1:2 molar ratios were amorphous according to XRPD . Insteadg values, theoretical Tg values obtained with the Gordon–Taylor equation, and possible recrystallization (Trc) and melting (Tm) temperatures observed for the materials are shown in g values that were higher than expected based on the Gordon–Taylor equation, except for FUR:VER 1:2. This indicates that the mixtures were homogenous single-phase systems and, furthermore, that intermolecular interactions may have been formed between the components [g value and a FUR-rich phase with a higher Tg. The co-amorphous mixtures with ARG as co-formers displayed considerably higher Tg values than theoretically predicted, i.e., indicative of salt formation between the components (1:1 molar ratio), as expected [g of the FUR:ARG system has previously been observed to have the highest value at a 1:1 molar ratio due to salt formation [g than the 1:1 system  and 1139 cm−1 (symmetric SO2 stretching) in FUR [−1, and to 1143 and 1145 cm−1 in the co-amorphous FUR:VER 1:1 and 1:2, respectively.In the FTIR spectra of the crystalline FUR and the physical mixtures with VER a, the NH) in FUR  had shif−1 (C-H of the methoxy groups); 2700–2400 cm−1 (N-H of protonated amine); 1608, 1591 and 1516 cm−1 (aromatic C=C); and 1256 and 1239 cm−1 (C-O-C of aromatic esters) [The spectrum of VER shows the characteristic absorptions at approx. 3000–2800 cm esters) . The dis−1 (aromatic C-H stretching), 2854 cm−1 (C-H stretching of methylenedioxy group), 1633 cm−1 (C=0 stretching), 1610 cm−1 , 1446 cm−1 (aromatic C=C stretching) and 1250 cm−1 (asymmetric =C-O-C stretching) [−1. In addition, the FUR peak (amino) at 1668 cm−1 shifted to 1681 cm−1, which is an even larger shift than that observed with the co-amorphous mixtures with VER. The sulphonyl peak of FUR at 1315 cm−1 shifted to 1335 cm−1 (1:1) and 1340 cm−1 (1:2) in the co-amorphous mixtures. The changes in PIP absorptions were more modest, but the aromatic C=C stretching peak at 1446 cm−1 shifted to 1442 cm−1 in both co-amorphous mixtures, and the aromatic C-H stretching at 2942 cm−1 broadened and reduced in intensity. These changes may indicate intermolecular interactions between the components.The spectrum of PIP b shows tetching) . When co−1 in crystalline FUR) together with changes in the guanidine structure of ARG  have previously been reported as confirmation of salt formation between the components [In the case of the ARG mixtures c, salt fmponents ,28.g values of the samples and intermolecular interactions between the components [The X-ray diffractograms of the co-amorphous mixtures stored at 4 °C/0% RH, 40 °C/0% RH and 25 °C/60% RH are shown in mponents ,38.The equilibrium solubility of FUR was found to be 0.504 ± 0.05 mg/mL at a pH of 5.2 , and it The dissolution test showed clearly that in the case of the PMs, the dissolution of FUR from the FUR:PIP and FUR:VER mixtures was lower when compared to that with crystalline FUR alone a. This iWith the amorphous mixtures, the behaviour of the FUR:PIP and FUR:VER mixtures was similar to that of the corresponding PMs, and supersaturation was not reached b. This mAmorphization provided a further increase in FUR dissolution for the amorphous mixtures with ARG b. More sWhen examining the permeating amounts, the lowest total permeated amounts of FUR from crystalline materials were observed for FUR:VER 1:1 (0.15 mg), which correlates well with the dissolved amounts and the concentration gradient driving the passive permeation a. With tThe AUC values for the dissolution and permeation of the different mixtures are shown in In general, the correlation between the AUC values for dissolution and permeation was linear ; thus, tAs PIP and VER are known P-gp inhibitors, it would be essential to also study permeation in relevant cell models in order to reveal their possible permeation-enhancing effects ,44,45. Hgs that were higher than the values predicted by the Gordon–Taylor equation, except for FUR:VER 1:2, indicative of the formation of homogenous single-phase systems and intermolecular interactions between the components. Instead, FUR:VER 1:2 most probably consisted of two phases (VER-rich and FUR-rich). The co-amorphous mixtures with ARG showed considerably higher Tg values than theoretically predicted, indicative of the presence of salt formation between the components (1:1 molar ratio). However, for FUR:ARG 1:2, the Tg was significantly lower than for the 1:1 mixture due to ARG being present as an excess amorphous component. Intermolecular interactions between the co-amorphous components, particularly salt formation between FUR and ARG, were verified by FTIR measurements. Increased Tg values and stabilizing intermolecular interactions improved the physical stability of the co-amorphous mixtures at storage conditions of 4 °C/0% RH, 40 °C/0% RH and 25 °C/60% RH for at least two months, except for FUR:ARG 1:2, which became sticky at 25 °C/60% RH.Co-amorphous mixtures of FUR were prepared with the amino acid ARG (by spray drying) and P-gp inhibitors VER and PIP (by solvent evaporation) at 1:1 and 1:2 molar ratios. All the co-amorphous mixtures showed single TIn the simultaneous dissolution/permeation studies, the co-amorphous mixtures prepared by solvent evaporation (VER and PIP as co-formers) did not increase the dissolved amounts or AUC values for dissolution and, furthermore, did not elevate the permeating amounts of FUR or its AUC value for permeation, when compared to the case for the crystalline drug, possibly due to the poor powder properties when the powder came into contact with the dissolution medium. Instead, when a co-amorphous mixture was prepared with ARG, all the above-mentioned properties were enhanced in comparison to those of crystalline FUR. However, when examining the ratio between the dissolution and permeation of FUR, permeation occurred more efficiently in relation to dissolution with the co-amorphous mixtures with VER and ARG. This may indicate that different permeation-enhancing factors other than an increased concentration gradient may be involved, and thus, the further clarification of the dissolution/permeation interplay for these formulations would be enlightening.


Since glioblastoma stem cells (GSCs) have paramount roles in tumor initiation, progression, recurrence, and therapy resistance, innovative approaches to specifically identify and isolate GSCs in a straightforward manner would be invaluable both for clinical and scientific applications. We demonstrate here that glioblastoma tumors have a subpopulation of cells with intracellular autofluorescence that display all the hallmark features of GSCs, and establish this biomarker as a new, simple, rapid and inexpensive way to identify and isolate this highly aggressive subpopulation of cells. Our findings are a great contribution to the neuro-oncology field, as they allow further studies on the molecular basis of GSCs, which may ultimately contribute to the identification of novel therapeutic targets and the development of smarter treatments to eliminate these cells. +) cells in GBM that bear all the functional characteristics of GSCs, including higher capacity to grow as neurospheres, long-term self-renewal ability, increased expression of stem cell markers, and enhanced in vivo tumorigenicity. Mechanistically, the autofluorescent phenotype is largely due to the intracellular accumulation of riboflavin, mediated by the ABC transporter ABCG2. In summary, our work identifies an intrinsic cellular autofluorescent phenotype enriched in GBM cells with functional stem cells features that can be used as a novel, simple and reliable biomarker to target these highly malignant tumors, with implications for GBM biological and clinical research.The identification of cancer stem cells (CSCs), which are implicated in tumor initiation, progression, therapy resistance, and relapse, is of great biological and clinical relevance. In glioblastoma (GBM), this is still a challenge, as no single marker is able to universally identify populations of GBM cancer stem cells (GSCs). Indeed, there is still controversy on whether biomarker-expressing cells fulfill the functional criteria of bona fide GSCs, despite being widely used. Here, we describe a novel subpopulation of autofluorescent (Fluo Gliomas are the most common primary tumors of the central nervous system, accounting for almost 80% of primary brain malignancies, of which glioblastoma (GBM) is the most aggressive subtype ,2,3. Des+–tumor cells were considered to represent CSCs due to their self-renewal ability as evidenced by the formation of anchorage-independent neurospheres, their high proliferation potential, and their differentiation capacity [+–BTSCs also displayed enhanced in vivo tumorigenicity in immunocompromised mice, providing strong evidence for the functional distinctness of CSCs in brain tumors [Recent evidence suggests that intratumor heterogeneity and poor response to current therapies are at least in part related to the existence of cancer stem cells (CSCs) in GBM ,10,11,12capacity . CD133+–n tumors . Severaln tumors ,10,11,12Due to the pronounced heterogeneity of GBMs, many additional cell surface markers besides CD133 ,13,14 haRecently, an intrinsic autofluorescent phenotype has been described for CSCs derived from various human epithelial solid tumors, such as pancreatic ductal adenocarcinoma, colorectal carcinoma, hepatocellular carcinoma and non–small–cell lung carcinoma . While GIDH–wildtype, MGMT promoter partially methylated, and TERT promoter mutation C228T); obtained from ATCC, Manassas, VA, USA–STR analyses were performed to confirm their authenticity), and 5 human patient-derived primary GBM cultures , GBML19, and GBML42 ), established in our lab as previously described [Six different GBM culture models were used: one established human GBM cell line (U251 (v/v) CO2.GBML1, GBML12, GBML19, and GBML42 cultures were maintained in Roswell Park Memorial Institute (RPMI) 1640 , and GBML18 and U251 cultures were maintained in Dulbecco’s Modified Eagle Medium . All cultures were supplemented with 10% fetal bovine serum  and 1% penicillin and streptomycin . Neurospheres were cultured in NeuroCult NS–A Proliferation Kit  supplemented with 20 ng/mL epidermal growth factor , 20 ng/mL basic fibroblast growth factor  and 1% B27 (Invitrogen). In both conditions, adherent and neurospheres cultures were incubated at 37 °C in a humidified atmosphere containing 5% (+) cells, GBM cells were excited with a 488 nm blue laser and selected as the intersection with filters 530/40 and 580/30  with DAPI  before flow cytometry analysis using FACS Canto II (BD Biosciences). In order to identify autofluorescent , anti–CD15 , and anti–CXCR4 . All antibodies were APC–conjugated. Cells were resuspended in 200 µL of FACS flow buffer (BD Biosciences) with DAPI and analyzed by FACS Canto II. Data was analyzed with FlowJo 10.0 software.To characterize autofluorescent cells, human primary GBM cultures  were analyzed by flow cytometry for the expression of CSCs surface markers. Briefly, 1 × 10v/v) CO2.Before sorting, GBM cultures were incubated overnight with 40 µM of Riboflavin  in a humidified atmosphere at 37 °C and 5% (−) and autofluorescent cells (Fluo+) of human GBM cultures were sorted using a FACS Aria III equipment (BD Biosciences) and correspondent data was analyzed by FACS Diva 7 software (BD Biosciences). Before cell sorting, cell lines were resuspended at a concentration of 5 × 106 cells/mL in sorting buffer ; 3 mM EDTA (v/v)) and filtered through a 40 μM strainer (BD Biosciences) to eliminate cell clumps. Cells were then sorted through a 100 μM nozzle at a sheath pressure of 20 psi. A yield sorting modality (Yield mask sorting for FACS Aria III) was chosen. Gating strategy for sorting was performed as indicated in − subpopulation during sorting procedures an appropriate distance between gates for Fluo+ and Fluo− cells is required. Sorted cells (Fluo− and Fluo+ subpopulations) were collected in 5 mL polypropylene tubes (BD Biosciences) containing 1 mL collection medium (DMEM or RPMI supplemented with 20% FBS) and transferred to cell culture flasks with prewarmed media (DMEM or RPMI supplemented with 10% FBS and 2% Pen/Strep).Nonautofluorescent cells (Fluo3 human primary GBM cells in Neurospheres media in 24–multiwell plates (0.5 mL/well). Cells were incubated in a humidified atmosphere at 37 °C and 5% (v/v) CO2. Neurospheres were supplemented with fresh media every 4 days (250 µL/well). After 21 days, the number of neurospheres was counted and pictures were taken. For serial passaging, neurospheres were harvested and dissociated with accutase  every 21 days. The content of Fluo+ cells in neurospheres was evaluated by flow cytometry as described above.Neurospheres were generated by culturing 1.5 × 104 cells/mL of both Fluo− and Fluo+ GBM cells were performed, in 96-well plates. At the end, in each well, cell densities ranged from 1000 to 1 cells in 100 µL of neurospheres media [v/v) CO2, and were supplemented with fresh media every 4 days. After 21 days, the fraction of wells not containing neurospheres was obtained for each condition and plotted against the initially plated cellular density. Stem cell frequencies and statistical significance was calculated using the ELDA software .Multiple serial dilutions from 4 × 10es media . Culture2 flasks at an initial density of 1.5 × 105 cells and treated for 9 days with TMZ  or vehicle (1% DMSO). Culture medium containing TMZ or vehicle was renewed every 3 days. At each timepoint , total cells were trypsinized and the percentage of Fluo+ cells was evaluated by flow cytometry as described above.For TMZ (Sigma-Aldrich) treatment, primary GBM cultures  were plated in T25 cm5 cells of GBML1, GBML18, GBML19 and GBML42 cells were plated in 35 mm diameter plates, and were irradiated with 2, 4, 6, 8 and 10 Gy at 1.94 Gy/min, at room temperature in a 137Cs irradiator (Shepherd Mark-I (model SN1068); J. L. Shepherd and Assoc., San Fernando, CA, USA). Subsequently, cells were washed once with PBS and fresh media was added to the plates that were maintained in a humidified atmosphere at 37 °C and 5% (v/v) CO2. After three days, total cells were trypsinized and the percentage of Fluo+ cells was evaluated by flow cytometry as described above.For irradiation treatment, 1.5 × 10ΔΔCt method. The list of primers used can be found in Total RNA from FACS–sorted human primary GBM cultures was extracted with Trizol (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was performed using 1 µg of total RNA with High Capacity cDNA Reverse Transcription Kit . Gene-specific mRNA levels were assessed by quantitative real-time PCR (qRT–PCR) in a real-time thermocycler  using Fast SYBR Green  according to the manufacturer’s instructions, by the 24 cells/well, in 6-multiwell plates, in duplicate, and incubated in a humidified atmosphere at 37 °C and 5% (v/v) CO2. After 24 h, fresh media  or fresh media supplemented either with RBF (40 µM) or FTC (5 µg/mL) was added to the respective wells.For riboflavin (RBF) and fumitremorgin C  treatments, GBM cultures were plated at an initial density of 1 × 104 GBM cells were plated in 6–well plates, in duplicate, and incubated in a humidified atmosphere at 37 °C and 5% (v/v) CO2. After 24 h, cells were washed twice with PBS, and fresh media (control condition), basal media  or basal media containing RBF (40 µM) was added to the respective wells.For basal medium treatment, 4 × 10+ cells was evaluated by flow cytometry as described above.In all assays, after 3 days of incubation, GBM cells were trypsinized and washed twice with PBS, and the content of Fluo5, 1.0 × 105, 1.5 × 104, 5.0 × 103, or 1.0 × 103 FACS-sorted U251 Fluo− and Fluo+ cells were subcutaneously injected in 1:1 (v/v) with Matrigel  and serum-free DMEM media into the right flank of 12-18-weeks-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ  male mice (3–7 mice per group). A positive response was scored when tumor was palpable. Tumor size and body weight were measured at least one time per week. Tumor volume was assessed by measuring the two largest sides and calculated with the formula “v = (3.14 × L1 × L1 × L2)/6”. Humane endpoint for sacrifice was applied when one of the mice presented a tumor with ≥2 cm on the larger side. After euthanasia, tumors were collected and weighted.For the in vivo LDA subcutaneous assay, five groups receiving 3.0 × 104 cells (U251 Fluo− or Fluo+) were stereotactically injected into the brain striatum  of 12–weeks–old NSG male mice (6 per group). Mice were anesthetized with a mixture of ketamine (75 mg/kg) and medetomidine (1 mg/kg). Butorphanol (5 mg/kg) was used as analgesia. Cells were resuspended in 5 µL of cold PBS 1× and injected using a 10 µL Hamilton syringe  at the rate of 1666 µL/min. Animals’ body weight was evaluated ~3 times per week, and general behavior and symptomatology daily. Humane endpoint for sacrifice was established as severe weight loss . All brains were collected for histological and molecular analyses. For the intracranial orthotopic model, a total of 5 × 10All animals were maintained under standard laboratory conditions, including 12 h light/dark artificial cycle, controlled ambient temperature (21 ± 1 °C), and relative humidity of 50–60%. Sentinel mice housed in the same room were used to confirm specific pathogen-free health status, according to FELASA guidelines. During experiments, mice were always manipulated in a flow hood chamber, except during surgery.2O2 in TBS for 10 min. Ki-67 , Nestin  and Sox2  immunohistochemical staining was performed based on the streptavidin–biotin-–peroxidase complex principle using the LabVision kit  according to the manufacturer’s instructions. Regarding Ki–67 staining, tissues were permeabilized using TBS-Tween 0.5%, for 10 min, before antigen retrieval. For all staining DAB substrate  was used as chromogen, followed by counterstaining with hematoxylin.Tissues sections were deparaffinized and rehydrated by xylene and ethanol series. Sodium citrate buffer  was used for antigen retrieval. Endogenous peroxidase activity was blocked with 3% Ht-test analysis was used. Overall survival of orthotopic GBM xenografted mice was compared between groups (Fluo− vs. Fluo+) by the log-rank test and plotted as Kaplan–Meier curves. Results are presented as normalized means ± standard deviations (SD), and statistical significance was defined as p < 0.05 for a 95% confidence interval.All statistical analyses were performed using GraphPad Prism 6.0 . To assess the statistical differences between groups in the in vitro assays, unpaired Student’s +) cells in two human primary GBM cultures (GBML1 and GBML18) expanded in two–dimensional adherent conditions. Using confocal microscopy, both cultures presented a rare fraction of cells displaying intrinsic green fluorescence , suggesting that these autofluorescent cells may have increased stemness properties.First, we analyzed the putative presence of autofluorescent , a substrate for the ABCG2 transporter, has been linked to the autofluorescent phenotype in CSCs from various carcinomas . Therefo+ cells . Concord+ cells . TogetheABCG2 mRNA in Fluo− and Fluo+ primary GBM cultures. Indeed, ABCG2 was significantly and consistently overexpressed in the Fluo+ subpopulations as compared to their respective Fluo−-counterparts . Interestingly, mice injected with Fluo+ cells developed tumors more rapidly than mice injected with Fluo− cells , demonstrating different kinetics of tumor growth  as compared to mice injected with Fluo− cells .GSCs are defined by particular characteristics, including self–renewal capacity, unlimited proliferation, stem cell marker expression, and ability to differentiate into multiple cell lineages. As such, GSCs play a decisive role during tumor initiation and progression . Importa markers ; increas markers ; and enh markers . Consistin vitro . Togethe+ subpopulation overexpresses the ABCG2 transporter  cells could be a promising and rational approach for these very aggressive tumors. For example, GBM patients may also potentially benefit from therapies targeting ABCG2; indeed, some anti-ABCG2 therapies have been recently tested in preclinical and clinical contexts [+ GSCs. In addition, the levels of autofluorescence, reflecting the GSC content, may be useful for the follow-up evaluation of tumor response to therapy. Finally, and considering the widely known heterogeneity of GBMs, a deeper molecular analysis of all our primary cultures should be performed.Mechanistically, we demonstrate that the accumulation of riboflavin inside GBM cells significantly contributes to the autofluorescent phenotype , consistnsporter E, a find(+ cells F,G, demo in vivo . This sa in vivo . In the  in vivo ,45. Addicontexts ,51,52,53In conclusion, our study demonstrates that intracellular autofluorescence can be used as a reliable biomarker to identify, track, and isolate GBM cells with GSC features, impacting on biological research and clinical monitoring and interventions.