G3KL, an AMP with a multibranched dendritic topology of the peptide chain, permeabilizes the inner and outer membranes of Gram-negative bacteria including multidrug-resistant strains, leading to efficient bacterial killing. Here, we show that permeabilization of the outer and inner membranes of Pseudomonas aeruginosa by G3KL, initially detected using the DNA-binding fluorogenic dye propidium iodide (PI), also leads to a synergistic effect between G3KL and PI in this bacterium. We also identify a synergistic effect between G3KL and six different antibiotics against the Gram-negative Klebsiella pneumoniae, against which G3KL is inactive.There is an urgent need to develop new antibiotics against multidrug-resistant bacteria. Many antimicrobial peptides (AMPs) are active against such bacteria and often act by destabilizing membranes, a mechanism that can also be used to permeabilize bacteria to other antibiotics, resulting in synergistic effects. We recently showed that  There is an urgent need to develop new antibiotics due to the increasing resistance of bacteria to the current antibiotics. Antibiotic resistance is particularly problematic with Gram-negative bacteria due to the presence of an additional outer membrane, which, in combination with multidrug efflux pumps, represents an efficient barrier against most antimicrobial compounds . Many anG3KL as a membrane permeabilizing agent  [G3KL selectively disrupts bacterial versus mammalian membrane models as evidenced by vesicle leakage assays [50 ~1000 μg/mL) [G3KL acts as a rapid membrane permeabilizer and strong membrane disruptor of bacterial cells. In this study, we show that G3KL destabilizes the LPS (lipopolysaccharide) layer, disrupts the outer and the inner membranes, interacts with DNA, and accumulates in Gram-negative bacteria up to an amount of dendrimer corresponding to 10% of the bacterial weight [Herein, we report our investigation of possible synergistic effects between antibiotics and peptide dendrimer ng agent . G3KL israry hit  by seques (MRSA) ,33. G3KLe assays , displaye assays , anti-bie assays ,36, low 0 μg/mL) , and low0 μg/mL) . Our stul weight .G3KL permeabilizes the outer and inner membranes of P. aeruginosa cells using fluorescence microscopy and propidium iodide (PI), a fluorogenic DNA-binding dye, which is impermeable to intact cell membranes [G3KL might enable entry of potent antibacterial compounds at sub-inhibitory concentrations of G3KL and the cytotoxic compound. To test the feasibility of this approach and possible synergy between antimicrobials and G3KL, we used the classical checkerboard assay and stained live bacteria with 3--2,5-diphenyltetrazolium (MTT) (i) was followed as described by Park et al. [i < 0.5; partial synergy for 0.5 ≤ FICi < 1; additive for FICi = 1; indifferent for 1 < FICi < 4; antagonism for FICi ≥ 4.We previously showed that embranes . Permeabum (MTT)  [32,39,4k et al. . We consP. aeruginosa, A. baumannii, E. coli, and K. pneumoniae as Gram-negative bacteria and MRSA as Gram-positive bacterium. We first tested whether the permeabilization effect of G3KL observed with PI might result in a synergistic effect with this dye, which binds to DNA and therefore should interfere with bacterial growth [PI was essentially inactive against Gram-negative bacteria but showed moderate activity against MRSA (methicillin-resistant Staphylococcus aureus)  . In the K. pneumoniae, neither PI nor G3KL nor their combination showed any activity (G3KL permeabilizes K. pneumoniae toward other antibiotics (see below), we interpret these data as an indication that PI is inactive against this bacterium at the highest concentration used, even in the presence of membrane permeabilization. The effect between PI and G3KL was indifferent in the case of MRSA, indicating that G3KL, which is inactive against this bacterium, also does not significantly increase the activity of PI against this bacterium (In the case of activity . As G3KLacterium .PMB), a well-known membrane-disrupting natural antimicrobial cyclic peptide [PI in P. aeruginosa , chloramphenicol (FICi = 0.6), and azithromycin (FICi = 0.5), in line with the partial synergy observed with PI  is very close to its MIC value when used alone (4–8 μg/mL). Nevertheless, G3KL was shown to kill P. aeruginosa within two hours by disrupting the outer membrane, the inner membrane, and ultimately by complexing with some negatively charged intracellular components, which probably includes DNA and proteins [G3KL impairs the binding of small molecule drugs to such targets, which would also contribute to the absence of synergistic effects.In  synergy , as expe with PI . Combinaethoprim . In geneproteins . Thus, iK. pneumoniae, we observed a strong synergy between G3KL and vancomycin , novobiocin (FICi < 0.2), chloramphenicol (FICi < 0.5), azithromycin (FICi < 0.4), and trimethoprim (FICi < 0.5), and partial synergy with the broad-spectrum antibiotic gentamicin (FICi < 0.6)  , Table 2eumoniae ,59,60. Seumoniae ,14,30,61eumoniae ,62.G3KL did not increase the activity of ampicillin and sulfamethoxazole, which were inactive against K. pneumoniae when used alone , ampicillin (FICi < 1), and azithromycin (FICi < 0.8)   and an ii < 0.8) , suggestG3KL with vancomycin (FICi < 0.3), erythromycin (FICi < 0.3), novobiocin (FICi < 0.2), and trimethoprim (FICi < 0.5). Killing kinetics on K. pneumoniae at an initial inoculum of ~106 CFU/mL showed that the pairs G3KL/vancomycin (32 μg/mL/32 μg/mL) and G3KL/trimethoprim (32 μg/mL/16 μg/mL) effectively killed the bacteria after 4 h, and G3KL/erythromycin (32 μg/mL/16 μg/mL) after 8 h, all below the MIC level  against K. pneumoniae , 1% acetic acid (ciprofloxacin), and DMSO .Peptide dendrimer  earlier . VancomyP. aeruginosa (PAO1), A. baumannii (ATCC19606), E. coli W3110 (TE823), K. pneumoniae (NCTC418), and methicillin-resistant S. aureus (COL). The minimal inhibitory concentration (MIC) was determined by using the broth microdilution method. A colony of bacteria was picked and grown in a Luria-Bertani  medium overnight at 37 °C. Stock solutions of 1 mg/mL of the samples were prepared in sterilized Milli-Q water and diluted to the starting concentration of 128 μg/mL in 300 μL Mueller Hinton (MH) medium. The diluted samples were added to the first well of the 96-well microtiter plate  and diluted serially by ½. Bacteria were quantified by measuring the optical density at 600 nm and diluted to OD600 of 0.022 in MH medium. We used 4 μL of the diluted bacterial solution to inoculate the sample solutions (150 μL) with a final inoculation of about 5 × 105 CFU/mL. The plates were then incubated at 37 °C for 18 h. For each assay, sterility (broth only) and growth control  were checked with two columns in the plate. The next day, 15 μL of MTT (1 mg/mL stock solution)  was added to each well of the plate. The MIC was defined as the lowest concentration of the peptide dendrimer with a colorless well indicating no bacterial growth.Antimicrobial activity was assayed against G3KL [PI with G3KL, PMB, and ciprofloxacin, PI was diluted across the rows and G3KL, PMB, and ciprofloxacin across the columns. For checkerboard assay testing the combinations of G3KL with small-molecule antibiotics, G3KL was diluted across the rows and the small-molecule antibiotics across the columns. We then added 75 μL containing 2× the final concentration of G3KL, PMB, and ciprofloxacin to each well containing the respective drug  to be tested in combination, across the columns and down the rows, resulting in a checkerboard of 150 μL final volume with the final wells containing only G3KL or the antibiotics.To verify the activity of two drugs in combination, the checkerboard method was used to determine the MICs for each antibiotic alone and in combination. Both G3KL  and a pa600 of 0.022 in an MH medium. We used 4 μL of the diluted bacterial solution to inoculate the sample solutions (150 μL) with a final inoculation of about 5 × 105 CFU/mL. The plates were then incubated at 37 °C for 18 h. The next day, 15 μL of MTT (1 mg/mL stock solution) was added to each well of the plate so that MIC and MICcomb were defined as the lowest concentration of the peptide dendrimer and/or antibiotics with a colorless well indicating no bacterial growth. The FIC of each antibiotic was calculated as follows:Similar to broth microdilution, bacteria were quantified by measuring the optical density at 600 nm and diluted to ODA synergistic interaction is defined by an FIC index of <0.5, partial synergy is defined by an FIC index of ≥0.5 and <1, an additive interaction by an FIC index of 1.0, indifferent by an FIC of >1 and <4, and antagonism is defined by an FIC of ≥4 .K. pneumoniae (NCTC418) were performed on G3KL (32 µg/mL), vancomycin (32 μg/mL), erythromycin (16 μg/mL), novobiocin (2 μg/mL), G3KL/vancomycin (32/32 μg/mL), G3KL/trimethoprim (32/16 μg/mL), and G3KL/erythromycin (32/16 μg/mL) and using 2× the concentrations indicated by the checkerboard assay. Untreated bacteria at 1 × 106 CFU/mL were used as a growth control.Time-kill kinetics against K. pneumoniae (NCTC418) was picked and grown overnight with shaking (180 rpm) in a 5 mL LB medium  at 37 °C. The overnight bacterial culture was diluted to OD600 0.002 (2 × 106 CFU/mL) in fresh MH  medium. Stock solutions of G3KL and antibiotics (8 mg/mL) were prepared in sterilized Milli-Q water and diluted to two-times more concentrated than the required concentration in the fresh MH  medium. G3KL and antibiotic were pre-mixed when needed. We mixed 100 µL of the adjusted bacteria and 100 µL samples in a 96-well microtiter plate  and the time-kill kinetics started at the moment of mixing. Ninety-six-well microtiter plates were incubated at 37 °C under shaking (180 rpm). Bacterial growth was quantified at 0, 0.5, 1, 2, 3, 4, 5, and 6 h, G3KL/erythromycin (32/16 μg/mL) were also quantified at 7 and 8 h. The quantification was performed by plating 10-fold dilutions of a sample in sterilized 0.9% NaCl on LB agar plates. LB agar plates were incubated at 37 °C for 10 h and the number of individual colonies was counted at each time-point. The assay was performed in triplicate.A single colony of G3KL can be exploited to obtain synergistic effects with other substances. G3KL showed only weak or no synergy with small-molecule antibiotics in the case of P. aeruginosa, a bacterium against which the dendrimer is very active, probably because the concentration of G3KL necessary to permeabilize the membrane is very close to the MIC value. G3KL showed very significant synergistic effects when tested against K. pneumoniae, against which the dendrimer is inactive when used alone, revealing that G3KL is capable of permeabilizing the membrane even if it does not show activity. The effect is particularly striking in combination with vancomycin, erythromycin, or trimethoprim because these antibiotics are inactive against K. pneumoniae when used alone. However, our synergistic study showed no effects against MRSA, indicating that G3KL is not only inactive against this Gram-positive bacterium, but also does not significantly permeabilize its membrane.The experiments above showed that the membrane permeabilizing effects of antimicrobial peptide dendrimer


Phyllostachys edulis (moso bamboo) seedlings to nitrogen (N) and potassium (K) to benefit nutrient management practices and the design of proper fertilizer in nursery cultivation.This study aimed to gain an understanding of the growth response of 8(4×24) was used to study the effects of N forms , N concentrations , and K+concentrations  on seedling height, leaf number, chlorophyll content , biomass, root systems, and N content of P. edulis seedlings. Plants were grown in vermiculite under controlled greenhouse conditions.An orthogonal array LP. edulis seedlings, followed by the effect of N and K+ concentrations. Among the N forms, NH4+ significantly improved the growth of P. edulis seedlings compared with NO3−. Seedling height, leaf number, chlorophyll SPAD value, biomass, and root system architecture  were greater with 8 mmol/L of NH4+ treatments than with 32 mmol/L of NH4+treatments, whereas root diameter and N content of P. edulis seedlings were higher with 32 mmol/L of NH4+ than with 8 mmol/L of NH4+. K displayed inconsistent effects on the growth of P. edulis seedlings. Specifically, seedling height, leaf number, biomass and root volume increased when the K+ concentration was increased from 0 to 0.5 mmol/L, followed by a decrease when the K+ concentration was further increased from 0.5 to 3 mmol/L. Root average diameter of the seedlings was the highest with a K+ concentration of 1.5 mmol/L, and K had some inhibitory effects on the chlorophyll SPAD value of the seedlings. P. edulis seedlings performed well with 8 mmol/L NH4+and further tolerated a higher concentration of both NH4+ and NO3−, although excessive N could inhibit seedling growth. A lower concertation of K (≤ 0.5 mmol/L) promoted seedling growth and increasing K+ concentration in the nutrient solution did not alleviate the inhibitory effect of high N on the growth of P. edulis seedlings. Therefore, NH4+nitrogen as the main form of N fertilizer, together with a low concertation of K+, should be supplied in the cultivation and nutrient management practices of moso bamboo.Our study showed that N form played a significant role in the overall performance of  Phyllostachys edulis) is a species of large monopodial bamboo that is native to China and extensively cultivated throughout China, Japan, Korea, and Vietnam (Moso bamboo ( Vietnam . Moso ba Vietnam . Moso ba Vietnam . However Vietnam . In cont Vietnam . This pr Vietnam . However Vietnam .4+) and nitrate (NO3−) are the two primary forms of inorganic N available to plants in soil , there is no apparent N form preference, but the growth of bamboo seedlings especially the aboveground parts is improved with elevated NH4+ available from 2 to 40 mmol/L  , the growth of P. edulis seedlings is inhibited, and all of the seedlings eventually are died when NH4+ is supplied as a sole N source. However, 4+ concentrations (16∼40 mmol/L) are beneficial for the growth of aboveground organs, although root growth is suppressed to some extent with NH4+ levels ranging from 24 to 40 mmol/L. The contradictions in these studies indicate that N form preference and NH4+ tolerance of moso bamboo seedlings require further study.Nitrogen (N) is often an important factor for plant growth and productivity in terrestrial ecosystems. Under natural conditions, ammonium (NH in soil . In wellN source . Due to N source . For exafer NO3− . A previ0 mmol/L . Althougsium (K) . A previ (K)  further clarify the N form preference of P. edulis seedlings and (2) determine the effects of K on the response of moso bamboo to different N forms. This study presents the appropriate fertilization requirements of moso bamboo cultivation based on the experimental data.Orthogonal arrays (often referred to as Taguchi methods) can be used to examine large numbers of factors in a much smaller number of experiments, allowing for the exploration of a unique subset of factor combinations. Therefore, it is a sophisticated time- and cost-efficient testing strategy . Orthogo bicolor , Dendroctiflorus , and Ipooea spp. . Using oP. edulis were collected from GuanYang City, Guangxi Province, China and stored at 4 °C before sowing. The moso bamboo seeds were soaked overnight in water at 40 °C, shelled, sterilized by soaking in 20% NaClO for 20 min, rinsed in sterile water at least five times, and then germinated in plastic pots  filled with vermiculite. One month later, approximately five cm tall, three foliate seedlings were selected for different treatments.Seeds of 8 (4 × 24) orthogonal arrays were employed to study K+ concentrations , N forms , and N concentrations  on seedling growth and N uptake 2SO4, and NaNO3, respectively. A nitrification inhibitor C2H4N4 (7 µM) was added to all treatments to prevent nitrification. One replicate consisted of six pots with two seedlings planted per pot, there were three replicates per treatment. Plants were grown in a greenhouse at 25/18 °C ± 3 °C day/night temperature, 65∼70% relative humidity, and 14/10 h day/night photoperiod. Pots were rotated every week to eliminate location effects. After treatment for 2 months, aboveground growth traits, root systems, and N content were analyzed.LN uptake . The nutHeight was measured with a ruler. The number of unfurled leaves above the cotyledonary node were counted. The chlorophyll content  of the leaves was determined with a chlorophyll meter . Root morphology, including root total length (RL), average diameter (AD), root surface area (RS), root volume (RV) and root tips were determined using an automatic scanning apparatus  equipped with WinRHIZO 2012 software .2SO4-H2O2, and the total N content was determined according to the Kjeldahl method . Using the same software, an analysis of variance (ANOVA) was conducted and post hoc comparisons were conducted using Duncan’s multiple comparison test with differences considered significant at p < 0.05.The data were analyzed to determine the range (R and R’) of orthogonal tests using DPS7.05 statistical software  than the treatments of NO3−  , and seedlings showed greener, heathier leaves and less necrosis in the NH4+ treatments than in the NO3− treatments  showed significantly better root architecture parameters than seedlings cultured in the other combinations/treatments  compared with the other treatments, except for treatment 1 (32 mmol/L NH4+ + 0 mmol/L K+)  .The growth indices of seedling height, leaf number, SPAD value and biomass were positively correlated with each other, and the correlations were also positive for root system architecture parameters such as root length, root surface area, root volume and root tips . Growth 4+ treatments compared to the NO3− treatments, suggesting that the seedlings of moso bamboo prefer NH4+ 2SO4) strongly promotes the production of new culms in the first year after fertilization of P. edulis and P. reticulata groves. However, such results contradict the findings presented by 3−. Considering the cultivation medium, pH and nutrient supply are different among these experiments , suppress aboveground growth, biomass accumulation and root system, while the total N content is increased by 35.77% compared to treatments with 8 mmol/L NH4+(treatments 1 and 7) (P. edulis seedlings suffer from the toxicity due to excessive NH4+.Although NH1 and 7) , suggest4+ and NO3− by the root system is determined by different affinities. In the low concentration range, the uptake is mediated by high-affinity transport systems (HATS), while under high concentrations , the activity of low affinity transport systems (LATS) takes over from HATS , which may also contribute to NH4+ toxicity, especially at low K+ levels  caused a further inhibition of plant growth and increased NH4+ accumulation in moso bamboo seedlings. The external K+ may not be high enough to alleviate NH4+ toxicity, as described previously for Arabidopsis treated with 5∼20 mmol/L KNO3  with 8 mmol/L NO3− treatment enhances the N content of moso bamboo seedlings by 53.85% . Therefore, it is recommended that moso bamboo seedlings should be fed 8 mmol/L NH4+ fertilizer with a moderate K+ concentration.N form and concentration play a significant role in the growth and N content of moso bamboo seedlings, while K10.7717/peerj.9938/supp-1Supplemental Information 1The seedlings showed greener, heathier leaves and less necrosis in the NHClick here for additional data file.10.7717/peerj.9938/supp-2Supplemental Information 2The raw data were used for range, variance and correlation analysis to compare the importance of N form, N and K concentrations on the growth of bamboo seedlings.Click here for additional data file.


Paeonia qiui is a wild tree peony native to China. Its leaves show a clear purple-red color from the germination to the flowering stage, and it has high leaf-viewing value. A MYB transcription factor gene, designated as PqMYB4, was isolated from leaves of P. qiui based on transcriptome datas. The full-length cDNA of PqMYB4 was 693 bp, encoding 230 amino acids. Sequence alignment and phylogenetic analysis revealed that PqMYB4 was a R2R3-MYB transcription factor clustered with AtMYB4 in Arabidopsis thaliana. Moreover, it contained a C1 motif, an EAR repression motif and a TLLLFR motif in the C-terminal domains, which were unique in transcription repression MYB. Subcellular location analysis showed that PqMYB4 was located in the cell nucleus. PqMYB4 was highly expressed in the late stage of leaf development, and was negatively correlated with the anthocyanin content. The petiole of wild-type Arabidopsis seedlings was deeper in color than the transgenic lines of PqMYB4 and showed a little purple-red color. The seed coat color of Arabidopsis seeds that overexpressed PqMYB4 gene was significantly lighter than that of wild-type seeds. In transgenic Arabidopsis, the expression level of AtCHS, AtCHI, AtDFR and AtANS were down-regulated significantly. These results showed that PqMYB4 was involved in the negative regulation of anthocyanin biosynthesis in tree peony leaves, which can control the anthocyanin pathway genes. Together, these findings provide a valuable resource with which to further study the regulatory mechanism of anthocyanin biosynthesis in the leaf of P. qiui. They also benefit the molecular breeding of tree peony cultivars with colored leaf. Anthocyanins are a class of flavonoids derived from phenylalanine. They are water-soluble pigment that can make plants exhibit colors ranging from orange-red to blue-purple. Anthocyanins are synthesized in the cytosol and transported to the vacuole for storage. In plants, anthocyanins are often present in pollen, petals and fruits, attracting pollinators and spreading seeds; moreover, they have a protective effect against a series of biological or abiotic stress, such as pathogen infection, strong light, low temperature and phosphate stress . At presNicotiana tabacum) x3Lx6Lx3R, which is important for interaction with a basic helix-loop-helix (bHLH) protein , when NtMYB5 was overexpressed in tobacco, the flower color became lighter, and the expression levels of most structural genes in the anthocyanin biosynthesis pathway were reduced [PqMYB4 transgenic Arabidopsis seedlings, while a little accumulation of anthocyanins was observed in wild type , a C3 motif (CX1-2CX7-12CX2C) and a C4 motif (FLGLx4-7V/LLD/GF/YR/Sx1LEMK) at the C-terminus in transcriptional repression factors; some also had a TLLLFR motif ,37. Subs mutated . The TLL mutated . In Arab mutated , but in  mutated . In addi mutated ,34. ThesP. qiui plants were grown under field conditions in Northwest A&F University, Yangling Shaanxi, China. The samples were collected in the morning during March and April in 2018, including sepals, petals, pistils, stamens and the leaves at six different leaf color stages   A. In ordThe seeds of Arabidopsis were germinated in a growth chamber at 26/22 °C day/night temperature, with a 70% relative humidity and light/dark cycle of 15/9 h. Arabidopsis ecotype Columbia (Col) was used for ectopic overexpression experiment.® RT reagent Kit with gDNA Eraser , according to the manufacturer’s instructions. First, the reaction mixture contained 2 μL of 5× gDNA Eraser Buffer, 1 μL of gDNA Eraser and corresponding volume RNA samples, ddH2O added up to 10 μL, 42 °C 2 min to remove genomic DNA; Next, reverse transcription reaction: add 1 μL of PrimeScript RT Enzyme Mix I, 1 μL of RT Primer Mix, 4 μL of 5× PrimeScript Buffer 2 and 4 μL of RNase-Free ddH2O to the first reaction solution, total reaction volume was 20 μL. The PCR program was carried out with an initial step of 37 °C for 15 min; then 85 °C for 5 s and 4 °C for storage.The total RNA of tissue and leaf samples was isolated using the TIANGEN RNA Prep Pure Plant kit, according to the manufacturer’s instructions . The quality and concentration of RNA samples were tested by Goldview-stained agarose gel electrophoresis and spectrophotometric analysis, respectively. The first-strand cDNA was synthesized from 1 μg of DNA-free RNA samples using a PrimeScriptPqMYB4, was screened. According to the transcriptome sequence of PqMYB4, gene specific primers PqMYB4-F and PqMYB4-R were designed by Oligo7.0 software . Then, the expected products were ligated into the pMD19-T vector , placed at 4 °C overnight, and transformed the ligation products into Escherichia coli competent cell. After positive screening and colony PCR identification, the correct bacterial solution was sent to the company for sequencing.Based on previous transcriptomic datas of tree peony leaves, a MYB transcription factor gene related to leaf anthocyanin biosynthesis, software , and thehttps://www.ncbi.nlm.nih.gov/orffinder/). The amino acid composition, protein molecular weight, theoretical isoelectric point (pI) and stability were predicted by using online software ProtParam (http://web.expasy.org/protparam/). The hydrophobic property and charge distribution were analyzed by using online software ProtScale . The transmembrane domain of PqMYB4 protein was analyzed by using TMHMM server 2.0 software (http://www.cbs.dtu.dk/services/TMHMM). The signal peptide of PqMYB4 was predicted by SignalP 4.1 Server . The secondary and tertiary protein structure predictions were conducted by using SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) and SWISS MODEL (https://swissmodel.expasy.org/), respectively. The conserved domain was identified by SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1). The homologous sequences alignment of PqMYB4 with other species was performed by DNAMAN version 8 software, and the phylogenetic tree was constructed using Neighbor-Joining method (NJ) of MEGA7.0 software.Open reading frame and deduced amino acid sequence of PqMYB4 was searched by ORF Finder online tool in NCBI . After incubation at 25 °C for at least 16 h in the dark, samples were observed under a confocal laser scanning microscope.For subcellular localization analysis, the AtMY75 and PqMYB4 cDNA fragments were inserted into the EcoR I site of p35S-GAL4-DB plasmids. The preparation and transformation of Arabidopsis protoplasts were carried according to the method described previously [® Assay System , using Promega GloMax 20/20 Microplate luminometer .The p35S-GAL4-BD, the effector plasmid, the reporter plasmid containing firefly luciferase and internal plasmids containing Renilla luciferase, respectively, were prepared as described previously . For theeviously . The effPqMYB4 in A. thaliana, the full-length cDNA of PqMYB4 was subcloned from the pMD19-T vector digested with Kpn I and Sal I to the modified binary vector pCAMBIA1300, digested with Kpn I and Sal I, generating the pCAMBIA1300-PqMYB4 overexpression construct. Thus, PqMYB4 was expressed under the control of the CaMV 35S promoter. This overexpression construct was introduced into Agrobacterium tumefaciens strain GV3101 for Arabidopsis transformation. The generated overexpression construct pCAMBIA1300-PqMYB4 in Agrobacterium strain GV3101 was transformed into a wild-type Arabidopsis plant using the floral dip method, as previously described [A. tumefaciens infection solution (OD600 = 1.0 − 1.8), containing 5% sucrose and 0.02% Silwet L-77, was prepared to infect inflorescences, and the infection time per inflorescence was 2 min. Subsequently, these plants were transferred to a dark treatment for 24 h. These steps were repeated twice according to the growth state of the plant. The harvested seeds were planted on 1/2 MS plates containing 20 mg·L−1 hygromycin in growth chamber at 22 ± 2 °C, under a 15/9 h light/dark (120 µmol·m−2·s−1) cycle. Hygromycin resistant seedling with green leaves and well-established roots were selected as transformants, and then transferred from the plates to moistened potting soil. The positive transformants was confirmed via the PCR method. The transgenic plants were used for further analysis, and the wild-type non-transformed lines grown in the same conditions.For ectopic expression of escribed . An A. t530 − 0.25 × A657) × FW−1). Three replicates were analyzed for each sample.The total anthocyanin concentration in tree peony leaves was determined according to the method described previously . MoreovePqMYB4 gene at different leaf stages in tree peony and the anthocyanin biosynthetic pathway genes in transgenic Arabidopsis. The sequence of anthocyanin biosynthesis related genes in Arabidopsis was obtained from TAIR (https://www.arabidopsis.org/). According to the sequence of PqMYB4 and related structure genes, qRT-PCR specific primers were designed , 0.8 μL of diluted cDNA, 0.4 μL of ROX and 7.2 μL of ddH2O. The PCR program was carried out with an initial step of 95 °C for 30 s, and 40 cycles of 95 °C for 5 s, 55 °C for 30 s, 72 °C for 30 s; then 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s for the dissociation stage. Pqubiquitin and Atactin were used as reference genes to normalize the expression data [−ΔΔCT comparative threshold cycle (Ct) method. Three biological replicates were performed for each gene.Quantitative real time PCR (qRT-PCR) assay was carried out to determine the expression level of structure genes and designed . The qRTion data . The melPqMYB4, the R2R3-MYB transcription factor gene, was isolated from P. qiui, had a C1 conserved motif, an EAR inhibitory motif and a TLLLFR inhibitory motif at the C-terminus. PqMYB4 expression negatively correlates with anthocyanin biosynthetic gene expression and anthocyanin accumulation in P. qiui. Moreover, it was dominantly expressed in green tissues. PqMYB4 has the function of inhibiting the synthesis of flavonoids (anthocyanins).


Nifedipine (NF) is reported to have many beneficial effects in antihypertensive therapy. Recently, we found that NF induced lipid accumulation in renal tubular cells. Palmitic acid-induced renal lipotoxicity was found to be partially mediated by endoplasmic reticular (ER) stress, while it can also be elicited by NF in kidney cells; we examined the induction of suspected pathways in both in vitro and in vivo models. NRK52E cells cultured in high-glucose medium were treated with NF (30 µM) for 24–48 h. ER stress-induced lipotoxicity was explored by staining with thioflavin T and Nile red, transmission electron microscopy, terminal uridine nick-end labeling, and Western blotting. ER stress was also investigated in rats with induced chronic kidney disease (CKD) fed NF for four weeks. NF induced the production of unfolded protein aggregates, resulting in ER stress, as evidenced by the upregulation of glucose-regulated protein, 78 kDa (GRP78), activating transcription factor 6α (ATF6α), C/EBP-homologous protein (CHOP), and caspases-12, -3, and -7. In vitro early apoptosis was more predominant than late apoptosis. Most importantly, ATF6α was confirmed to play a unique role in NF-induced ER stress in both models. CKD patients with hypertension should not undergo NF therapy. In cases where it is required, alleviation of ER stress should be considered to avoid further damaging the kidneys. The endoplasmic reticulum (ER) is a cellular organelle involved in protein folding and secretion, calcium homeostasis, and lipid biosynthesis . When EREvidence implies that ER stress-induced cellular dysfunction and cell death are major contributors to many diseases, including neurodegenerative diseases, diabetes, metabolic syndromes, cancers, hepatic dyslipidemia, steatosis, and chronic kidney disease (CKD) ,7,8. Upr2+ release to protect β-cells from high-glucose-induced ER stress and apoptosis . S. S33]. SResults in We previously demonstrated that NF induced ectopic lipid accumulation in rat NRK52E proximal tubular cells , and we grp) genes through activation of the ER stress element (ERSE) [db/db mice via decreasing ER stress-related apoptotic markers [ATF6, a member of the leucine zipper protein family which acts as a target of p38 mitogen-activated protein kinase (MAPK) phosphorylation , can cont (ERSE) . It was t (ERSE) . SREBPs t (ERSE) . To furt markers ,47. As m markers ; thus ATAnnexin V is commonly used to detect apoptotic cells due to its ability to bind to phosphatidylserine, a marker of apoptosis when it is on the outer leaflet of plasma membranes. Elevated responses to Annexin V were found in both acute and chronic renal conditions . ApoptosResults shown in To the present, however, one problem that has remained to be solved is whether NF induces lipotoxicity prior to ER stress or vice versa; obviously, further investigation is required.NF , thapsigargin (TPS) , tauroursodeoxycholic acid , thioflavin T (ThT) : 2′,7′-dichlorofluorescin diacetate (DCFDA) , doxorubicin (DR) , and a high-fat diet  were purchased from various vendors. The protein extraction solution was from iNtRON Biotechnology . All other chemicals unless otherwise stated were purchased from Sigma-Aldrich.2. Upon reaching 80% confluence, cells were trypsinized with 0.25% trypsin-0.02% ethylenediaminetetraacetic acid (EDTA) for 5 min at 37 °C and then re-passaged at a ratio of 1:3–1:4. NRK52E cells were treated with 30 µM NF for 24–48 h and then harvested for further analysis. The selected dose of NF corresponded to clinical use in humans [Ca(L)I, 20 µM NF is required [The normal rat kidney epithelial-derived NRK52E cell line CRL-1571) was purchased from the Bioresource Collection and Research Center (BCRC), Food Industry Research Development Institute . Cells were cultured in 5% bovine calf serum-supplemented Dulbecco’s modified Eagle medium (DMEM) with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose  at 37 °C in a humidified atmosphere with 5% COn humans ,55,56,571 was purn humans  corresporequired . So, thirequired ,61.This experiment was approved by the Institutional Animal Care and Ethics Committee of Taipei Medical University . Briefly, 32 male Sprague-Dawley rats, aged 4 weeks and weighing 200–250 g, purchased from BioLasco , were housed at three rats per cage and acclimated in the first week on basic chow in an animal room at 24 ± 2 °C, a relative humidity of 70–75%, and a 12/12-h light/night cycle. These rats were divided into four groups. Group 1 rats served as the normal control . Each rat in group 2, the DR control (DRCKD), received a single shot of DR (7.5 mg/kg) intraperitoneally (IP) at the beginning or at week 2, and then rats in groups 1 and 2 were fed regular chow until the end of the experiment (week 9) with no further treatment. Each rat in group 3 (DRCKD + NF) received a single shot of DR as in group 2, and then also received an NF injection (0.5 mg/kg NF IP) beginning at the start of week 3, once daily from Monday to Friday each week and fed regular chow until week 9. To compare the lipidemic status in the kidneys , the choNRK52E cells were treated with 30 µM NF as indicated. The extraction of cytoplasmic and nuclear proteins was carried out as instructed by the cytoplasmic and nuclear protein extraction kits . The proteins obtained were transferred to new Eppendorf flasks and stored at −80 °C. A standard protocol for Western blotting was used as described previously . The speNRK52E cells were treated with NF were cultured on coverslips for the indicated times. Then, cells were stained with 3 µM of filtered ThT for 10 min at room temperature to detect protein aggregates. Coverslip slides were washed in 80% ethanol, >95% ethanol, and >distilled water and mounted in aqueous mounting media. Before analyzing the stained image under a fluorescent microscope , the slides were dried in the dark overnight.4 cells/well of NRK52E cells treated with or without NF were seeded in a 96-well plate and allowed to attach overnight. Cells were washed once in 1× PBS buffer, and then cells were stained with 25 µM DCFDA in 1× PBS buffer for 45 min at 37 °C. After removing all of the staining solution, the signal was monitored, and the sample was photographed at Ex/Em 485/535 nm with a fluorescent microscope (Olympus).In total, 2.5 × 105 NRK52E cells were seeded into two-well chamber slides and treated with 30 µM of NF for 16 h. Cells were fixed in 0.1 M cacodylate solution containing 2% paraformaldehyde and 2.5% glutaraldehyde for 1 h. After removing the fixative solution, cells were rinsed thrice with a buffer solution containing 0.1 M cacodylate and 7% sucrose, each time for 15 min. Specimens were stained with 1% OsO4 contrasting solution (in 0.1 M cacodylate buffer) for 1–2 h, and then subjected to a gradient ethanol dehydration method . Dehydrated specimens were treated thrice with propylene oxide (PO), each time for 10 min. The resin had previously been evacuated under a vacuum for 8 h, and the PO/resin (1:1) was mixed and evacuated under a vacuum overnight. Specimens were embedded and heated to 62 °C in the oven for 48–72 h until completely set, then reshaped, sliced, and subjected to TEM HT7700  analyses to access the intracellular ultrastructures.In total, 3 × 10A TUNEL assay was carried out with an In Situ Cell Death Detection Kit . 4′,6-Diamidino-2-phenylindole (DAPI) counterstaining was conducted according to instructions given by the supplier, Thermo Fisher Scientific . Results were examined under a fluorescent microscope (Olympus).Nile red  is an excellent vital stain for detecting intracellular lipid droplets by fluorescence microscopy and flow cytometry . In thisAnnexin V/PI staining of NRK52E cells treated with or without NF was carried out under a Muse Cell Analyzer following protocols given by the Muse Annexin V and Dead Cell Kit .t-test using SPSS 10.0 computer statistical software . An analysis of variance (ANOVA) was also used with Tukey’s test to analyze variances and the significance of the difference between paired means. The significance of the difference was judged by confidence levels of *,#p < 0.1, **,##p < 0.05, and ***,###p < 0.01.Statistical analyses were performed with Student’s NF induces lipotoxicity in kidney cells, which can further damage kidneys that are progressing to CKD. In the present work, we found that NF induces extensive production of unfolded protein aggregates, resulting in ER stress and lipogenesis, while the main ER stress- and apoptosis-related marker proteins were all upregulated, including GRP78, ATF6α, cleaved caspase-12, CHOP, cleaved caspase-3, and cleaved caspase-7. These markers, in turn, led to more-significant early apoptosis than late apoptosis in NRK52E cells.It is suggested that CKD patients with hypertension not be prescribed NF therapy. In cases where it is required, alleviating ER stress should be considered to avoid further damaging hypertensive kidneys.


To identify a primary cause of CA, we analyzed The Cancer Genome Atlas (TCGA) genomic and transcriptomic data for genes encoding the 367 proteins that localize to the centrosome (the “centrosome-ome”). We identified the following candidates for primary causes of CA: gain-of-function alterations of CEP19, CEP72, CTNNB1, PTK2, NDRG1, SPATC1, TBCCD1; and loss-of-function alterations of CEP76, MCPH1, NEURL4, and NPM1. In cellulo analysis of these candidates revealed that loss of MCPH1/microcephalin caused the most robust increase in centriole number. MCPH1 deep gene deletions are seen in 5–15% of human cancers, depending on the anatomic site of the tumor. Mechanistic experiments demonstrated that loss of MCPH1 caused a CDK2-dependent increase in STIL levels at the centrosome to drive CA. We conclude that loss of MCPH1 is common in human cancer and is likely to be a cause of CA.The centrosome is the microtubule organizing center of human cells and facilitates a myriad of cellular functions including organization of the mitotic spindle to ensure faithful chromosome segregation during mitosis, cell polarization and migration, and primary cilia formation. A numerical increase in centrosomes, or centrosome amplification (CA), is common in cancer and correlates with more aggressive clinical features and worse patient outcomes. However, the causes of CA in human cancer are unclear. Many previous studies have identified mechanisms of CA  Previous studies have established in cellulo models of CA by overexpression of PLK43 or modulating other centrosome genes4. Although this identifies many genes as candidates for underlying causes of CA, the actual clinically-relevant molecular mechanisms driving CA in human cancer have not been determined.The centrosome is the microtubule-organizing center of human cells. A numerical increase in centrosomes, or centrosome amplification (CA), is common in cancer and correlates with more aggressive clinical features and worse patient outcomes5. Additional evidence comes from centriole analysis of human cancer cell lines, demonstrating that only a subpopulation of cell lines with CA have an increase in ploidy, suggesting different origins of ploidy and CA6. We therefore sought to identify potential primary genetic mechanisms leading to centriole overduplication in human cancer. To address this question, we analyzed genomic and transcriptomic alterations in 367 centrosome proteins using TCGA data from 10,207 independent cancers representing 22 of the most common tumor sites. We identified a list of candidate centrosome genes that are most frequently altered in cancer and tested them in cellulo to determine the predominant causes of centriole overduplication in human cancer.CA is thought to arise by two major mechanisms: (1) centriole overduplication and (2) cell doubling events, each of which could be further subdivided into those with and without a primary genetic cause. We previously determined that the former, centriole overduplication, is primarily responsible for CA in human melanoma7. Data were extracted from the following 22 TCGA datasets using cBioPortal8, selected based on their size and availability of associated clinical data: acute myeloid leukemia, 200 patients; breast carcinoma, 1,105; melanoma, 478; colorectal carcinoma, 633; glioblastoma, 607; low grade glioma, 532; ovarian serous adenocarcinoma, 607; lung adenocarcinoma, 522; lung squamous cell carcinoma, 504; prostate adenocarcinoma, 499; head and neck squamous cell carcinoma, 530; renal clear cell carcinoma, 538; stomach adenocarcinoma, 478; bladder urothelial carcinoma, 413 patients; hepatocellular carcinoma, 442; cervical carcinoma, 309; endometrial carcinoma, 373; esophageal adenocarcinoma, 186; pancreatic adenocarcinoma, 186; pheochromocytoma and paraganglioma, 184; sarcoma, 365; and thyroid carcinoma, 516. This yielded a total of 10,207 cancers. We analyzed the percent of cancers with mutations, copy number variations, and altered gene expression (mRNA Z score < 2 or > 2) for each of the 367 centrosome genes. Each data set  was weighted equally during analyses in order to be able to detect certain tissue-specific alterations that may give rise to CA. Data were not available for the following centrosome genes: BOD1L2, PTPN20, ROSSF10, STARD9, TSSK2, WASH1.A list of all 367 centrosome genes . We then calculated the fold enrichment of alterations in each centrosome gene in p53 mutated/deleted cancers versus p53 wild-type cancers. Genes were excluded if their alterations were not enriched in p53 mutated/deleted cancers. We also supplemented our list of hits with the following three centrosome genes identified by MutSigCV2 and grown in basal medium supplemented with 10% fetal bovine serum and 100 units/mL penicillin–streptomycin. The basal medium used for each cell line is as follows: high glucose DMEM for HeLa, PC3, Phoenix, and 293T; RPMI-1640 for DLD-1; and McCoy’s 5a for HCT116. MCF10A cells were grown in 1:1 DMEM and Ham’s F-12 supplemented with horse serum, EGF, hydrocortisone, cholera toxin, and 100 units/mL penicillin–streptomycin.CAL-51, DLD-1, HCT116, HeLa, MCF10A, MDA-MB-231, MDA-MB-453, MDA-MB-468, PC3, Phoenix, RPE, and 293T cell lines were obtained from ATCC. All cell lines were authenticated by STR analysis using the Promega PowerPlex 16 HS System Kit (DC2101) at the University of Wisconsin Translational Research Initiatives in Pathology (TRIP) laboratory. Cells were propagated at 37 °C in 5% COLentivirus encoding shRNAs were produced by transfecting a T25 of 293T cells with 2 µg pLKO.1 vector, 1 µg pVSV-G, and 1 µg psPAX2, and Fugene (Promega). For stable retroviral transduction, a T25 of Phoenix cells was transfected with 2 µg pSuperior.retro.puro construct and 1 µg pVSV-G. For both lentivirus and retrovirus, fresh medium was applied at 24 h post-transfection, and virus was harvested 24 h later, clarified by centrifugation and filtration through a 0.45 μm membrane to remove cell debris, and diluted 1:1 with complete medium containing 10 μg/mL polybrene. Target cells (HeLa) at 40–60% confluence were transduced for 24 h, then given fresh media for 24 h, then 1 µg/mL puromycin was added for 4 days. Fresh media was added for 24 h before using the cells for subsequent experiments.To overexpress cDNAs of centrosome candidate genes, HeLa cells were plated in 6-well plates, grown to 80% confluence, and transfected with 2 µg cDNA in pcDNA5 using Fugene (Promega). Cells were given fresh media 12 h after the transfection. 24 h after the transfection, cells were either transferred to coverslips for fixation and immunofluorescence or harvested for qRT-PCR to assess transfection efficiency.Chemicals used in this study include aphidicolin , doxycycline , puromycin , CHIR-124 , milciclib , centrinone B , thymidine , and RO-3306 .The following cDNAs were acquired: NPM1 was cloned from HeLa cDNA library, MCPH1 was from Shiaw-Yih Lin (Addgene plasmid #16205), STIL was from transOMIC (BC16223), PTK2 was from Michael Davidson (Addgene plasmid #55122), NDRG1 was from Ben Park (Vanderbilt University), CTNNB1 wild-type and S33Y were from Eric Fearon (Addgene plasmids #16828 and #19286), and CEP19 (GE MHS6278-202828648), SPATC1 (GE MHS6278-202802271), and TBCCD1 (GE MHS6278-202801247) were from GE Healthcare. These cDNAs were cloned into pcDNA5/FRT/TO with C-terminal mNeonGreen tag . Sequencing was performed to verify cloning fidelity (Protein CT and Quintara Biosciences). All constructs were full-length cDNAs unless otherwise noted.Mutant NPM1  was cloned from a HeLa cDNA library using the following primers: forward 5′CCCCGGATCCATGGAAGATTCGATGGACATGG, reverse: 5′ CCCCGAATTCCTATTTTCTTAAAGAGACTTCCTCCACTGCCAGACAGAGATCTTGAATAGCCTCTTGGT.To make shRNA vectors, shRNAs targeting sequences were cloned into pLKO.1-TRC, a kind gift from David Root (Addgene plasmid #10878). The following sequences were used for initial screening: CEP76, 5′ ACGACTTTAGTAGCATCATAT; MCPH1, AGGAAGTTGGAAGGATCCA; NEURL4, 5′ CCATCATGCAAGACGGTAATT; NPM1, 5′ TTACGAAGGCAGTCCAATTAA; non-targeting control, 5′ CCGCAGGTATGCACGCGT . Lentivirus was produced as described above. Efficiency of gene depletion was assessed by qRT-PCR. Primer sequences are available in Supplemental Table To make doxycycline-inducible shRNA cell lines, the MCPH1 targeting sequences were cloned into pSuperior.retro.puro following the manufacturer’s protocol (OligoEngine). Two different shRNAs were used for MCPH1: 5′ AGGAAGTTGGAAGGATCCA and 5′ CTCTCTGTGTGAAGCACCT. Retrovirus was made and used to transduce HeLa cells stably expressing TetR , as described above.To genetically inhibit CDK2, pcDNA3-myc3-p27  was obtained and transfected into HeLa cells using Fugene (Promega).5. Cells were seeded on glass coverslips in 24-well plates and fixed with 100% ice-cold methanol for 15 min. Fixed cells were then blocked for 30 min in 3% bovine serum albumin (BSA) and 0.1% triton X-100 in PBS (PBSTx + BSA). Primary antibodies were incubated in PBSTx + BSA for 1 h at room temperature and washed three times in PBSTx, followed by secondary antibody incubation in PBSTx + BSA for 30 min at room temperature and two washes with PBSTx. Cells were counterstained with DAPI and mounted on glass slides with Prolong Gold antifade medium (Invitrogen). Image acquisition was performed on a Nikon Eclipse Ti inverted microscope with 60 × and 100 × objectives and Hamamatsu ORCA Flash 4.0 camera. For images displayed in the figures, optical sections were taken at 0.2-μm intervals and deconvolved using Nikon Elements. Where appropriate, the observer was blinded to treatment condition during image acquisition and analysis. Images were processed and analyzed using Nikon Elements.IF and imaging were carried out as previously describedPrimary antibodies used were: centrin , γ-tubulin , pericentrin , CEP164 (gift from Erich Nigg), β-actin , α-tubulin , FLAG , human autoantibody against centromere , MCPH1 (kind gift from Hui Dai and Shiaw-Yih Lin), PLK4 , STIL , and CDK2 . Alexa fluor-conjugated secondary antibodies were used .T value of three housekeeping genes  was subtracted from each experimental CT, then T method was employed to calculate the fold change in gene expression. Primer sequences are provided in Supplemental Table RNA was isolated from cells using TRI Reagent (Molecular Research Center). DNase treatment was used to remove DNA contamination. RNA was then converted to cDNA using the Quantitect Reverse Transcription Kit (Qiagen). A StepOne Plus (Applied Biosystems) real-time PCR thermal cycler was used for amplification. The average C3VO4), 1 mM PMSF, protease inhibitor cocktail (Thermo Scientific), and 1 mm dithiothreitol. Samples were sonicated and heated in SDS buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membrane (Millipore), and blocked for at least 30 min in 5% milk and 0.1% Tween 20 in Tris-buffered saline, pH 7.4 (TBST + milk). Membranes were incubated 1 h at room temperature or overnight at 4 °C with primary antibodies diluted in TBST + 5% milk, washed three times with TBST, and incubated for 1 h at room temperature in secondary antibodies conjugated to horseradish peroxidase in TBST + 5% milk. Membranes were washed and developed with luminol/peroxide (Millipore) and visualized with film.Cells were lysed in lysis buffer  containing phosphatase inhibitors , CEP131 , FLAG , GFP , and alpha tubulin . HRP-conjugated secondary antibodies were used . Relative intensities of bands were calculated using ImageJ from scanned images and normalized to their respective loading control intensity.2, 0.1 mM NaF, 10 μM Na3VO4) with 1 mM DTT, 1 μM cold ATP, 2 μCi [γ-32P] ATP, and 4 μg histone H1 . Reactions were resolved by SDS-PAGE. Dried gels were exposed to a storage phosphor screen , and γ-32P incorporation was visualized by Typhoon TRIO imager .Cell extracts from a nearly confluent T25 flask were lysed in buffer (see above) and incubated with 1 µg of either anti-CDK2 antibody  or anti-GFP antibody  overnight. Antibody–antigen complexes were precipitated using protein A and G sepharose beads . Bead complexes were washed three times with lysis buffer. Kinase assay reactions were incubated at 30 °C for 30 min in buffer  or Microsoft Excel. Two-tailed 11 or p2113, we ranked alterations in centrosome genes by the fold enrichment in p53/p21 mutant versus p53/p21 wild-type tumors , which is found exclusively in acute myeloid leukemia (AML), and causes a frameshift mutation at the C-terminus. Patients with this mutation have worse outcomes  E3 ubiquitin ligase. The F-box proteins in human cells is βTrCP (BTRC). We examined these three genes, but again find a relative dearth of alterations . CDK2 inhibition reduced CA in the context of MCPH1 depletion . However, inhibition of CHK1 did not impair CA caused by MCPH1 depletion 14. Therefore, we asked whether loss of p53 was permissive of CA in the setting of MCPH1 depletion. We used the following cell lines: HeLa, which lack functional p53 due to infection with HPV and expression of the E6 oncogene; DLD1 and HCT116 which have wild-type 53, and HCT116 p53−/−, in which p53 has been deleted. MCPH1 was depleted in these cell lines  but not in the lines with functional p53  the use of cell lines from different species; (2) the quantification of γ-tubulin foci instead of centrin foci (centrioles), a method that can be insensitive to centriole amplification; or (3) the use of acute (e.g. RNAi) versus chronic (e.g. knockout) depletion of MCPH1, as chronic depletion of MCPH1 may allow time for compensation by other centriole duplication machinery. MCPH1 deletion as a cause of CA will need to be validated in additional cohorts of primary human cancers.However, we did identify MCPH1 deletion as one genetic cause of CA, with deep deletions present in up to 10% of cancers depending on disease site, and validated that this deletion is sufficient to cause CA in model systems. MCPH1 was first identified as a transcriptional repressor of human telomerase reverse transcriptase (hTERT)45. Our study also revealed MCPH1 deletion as a cause of chromosomal instability in human cancer, as demonstrated by frequent aberrant mitoses. This is consistent with the following previous reports: (1) Mcph1 knockout mouse neuroprogenitor cells exhibit more mitotic spindle defects, including multipolar spindles45; (2) Mcph1 knockout mouse embryonic fibroblasts (MEFs) exhibit more metaphase aberrations in response to ionizing radiation compared to wild-type MEFs46; (3) U2OS cells treated with MCPH1 siRNA also exhibit more mitotic aberrations, namely chromosome missegregation, disorganized spindles, and cytokinesis failure29; and (4) HeLa cells treated with MCPH1 siRNA exhibit premature chromatin condensation prior to mitosis and longer time from nuclear envelope breakdown to chromosome alignment in metaphase. In addition to chromosomal instability, MCPH1 is critical for protecting genome stability, as it helps mediate intra-S and G2 arrest after DNA damage47. Mechanistically, MCPH1 is important for recruiting RAD51/BRCA2 to sites of DNA damage46. MCPH1 may also be important for gametogenesis, as Mcph1-knockout mice are infertile and exhibit meiotic defects46.MCPH1 has also been implicated in regulating the cell cycle. Mcph1 knockout murine neural progenitor cells demonstrate uncoupling of the centrosome cycle from the cell cycle49. Most of the deletions we observed in MCPH1 are coincident with larger 8p deletions. This begs the question: deletion of which gene or genes on 8p confers the tumorigenicity of 8p deletion? Herein we provide evidence that one mediator of the phenotype observed in 8p-deleted cancers is MCPH1, which may cause CA and chromosomal instability in 8p-deleted cancers. However, one caveat to our study is that MCPH1 deletion may be a passenger event rather than a driving event in human cancer. Another question raised is whether loss of one copy of MCPH1 is sufficient to cause CA. Previous work has shown that Mcph1+/− MEFs exhibit CA, and Mcph1−/− MEFs have even greater CA43. Furthermore, our data showing that overexpression of MCPH1 reduces CA in cell lines with heterozygous MCPH1 deletion suggest that deletion of one copy of MCPH1 is indeed sufficient to cause CA, albeit to a lesser extent than homozygous MCPH1 deletion.MCPH1 is on chromosome 8p23.1, a region of the genome that is commonly deleted in human cancer, is occasionally associated with gains of chromosome 8q, and portends worse outcomes11, p2113, or the PIDDosome genes14. In tumors with MCPH1 deletion, we observed that approximately 55% also had one of the above compensatory alterations compared to approximately 40% of tumors without MCPH1 deletion. Therefore, there are likely other potential compensatory alterations that are permissive of cellular proliferation in the context of CA. Previous work has also shown that p53 knockout is more permissive of CA with Mcph1 depletion in MEFs43. Mechanistically, MCPH1 may contribute to p53 stability by inhibiting MDM2-mediated ubiquitination and degradation of p5337. In our study, we found that depletion of MCPH1 results in CA in cell lines lacking functional p53, but the effect was not as profound in cell lines with functional p53. However, one caveat to this is that DLD1, which lacks a p53 mutation, silences wild-type p53 and is capable of proliferating with CA or loss of centrioles51. Therefore, MCPH1 depletion may not uniformly cause CA in all cell lines or contexts.Proliferation in the presence of CA requires additional adaptations, such as loss/mutation of p5352, but according to our analyses, neither genomic amplification nor mRNA overexpression/underexpression are commonly seen in human cancer. Increased centrosome size can be caused by overexpression of NLP/NINL53 and other mediators of microtubule nucleation, but our analyses demonstrate that neither genomic amplification nor mRNA overexpression are common. Increased centrosome size can also be caused by increased centriole number or length6, and the relative contribution of all these mechanisms to centrosome size remain unclear. Another potential limitation is that our study only focused on genes with reported localization to the centrosome; however, we know that other extra-centrosomal genes, such as transcriptional regulators or post-translational modifiers, can alter the levels of centrosome proteins and could drive CA. Lastly, inhibition of cell cycle progression can lead to CA, and this would not have been detected by our study.Our study focused mostly on causes of numerical CA. However, structural centrosome abnormalities have also been reported, such as increased centriole length. Increased centriole length is caused by CPAP overexpression or CP110 depletion54. Another caveat to our study is that our overexpression of candidate genes was limited; however, we addressed this by only analyzing centrosomes in cells with observable GFP expression. Another caveat is that we did not directly determine whether the increased centrosomal STIL in MCPH1-depleted cells is a consequence or a cause of CA.Despite our in-depth analysis of centrosome gene alterations, there still remains a significant fraction of CA that is unaccounted for by genomic alterations. There are a number of possible explanations. First, there could be mutations affecting gene regulatory regions that influence chromatin structure or gene expression levels that might not have been discovered by exome sequencing alone. Secondly, there could be changes in non-protein coding RNAs that regulate the levels of centrosome genes. Thirdly, there could be post-transcriptional and/or post-translational regulation of mediators of centriole duplication that were undetermined by our screen. Finally, the cause of CA could be non-genetic and heritable, as supported by some previous work on centriole amplification or cell doublingThe goal of this project was to determine the causes of CA in human cancer. Our analysis of alterations in centrosome genes and in vitro validation of the most common alterations demonstrated that MCPH1 deletion occurs in up to 10% of human cancers, depending on the disease site, and causes CA. Furthermore, the mechanism of MCPH1 depletion-mediated CA depends on increased CDK2 activity and is associated with increased levels of STIL at the centrosome. This study provides additional insight into the clinically-relevant causes of CA in human cancer, revealing MCPH1 deletion as a common and penetrant driver of CA. It is conceivable that there are other drivers of CA that warrant future investigation.Supplementary Information 1. Supplementary Information 2.


Passive commercial gauzes were turned into interactive wound dressings by impregnating them with a chitosan suspension. To further improve healing, and cell adhesion and proliferation, chitosan/bioactive glass wound dressings were produced with the addition of (i) 45S5, (ii) a Sr- and Mg-containing bioactive glass, and (iii) a Zn-containing bioactive glass to the chitosan suspension. SEM and FTIR analyses evidenced positive results in terms of incorporation of bioactive glass particles. Bioactivity was investigated by soaking chitosan-based bioactive glass wound dressings in simulated body fluid (SBF). Cell viability, proliferation, and morphology were investigated using NIH 3T3 (mouse embryonic fibroblast) cells by neutral red (NR) uptake and MTT assays. Furthermore, the wound-healing rate was evaluated by means of the scratch test, using NIH 3T3. The results showed that bioactive glass particles enhance cell adhesion and proliferation, and wound healing compared to pure chitosan. Therefore, chitosan-based bioactive glass wound dressings combine the properties of the organic matrix with the specific biological characteristics of bioactive glasses to achieve chitosan composites suitable for healing devices. In recent years, with the increase in diabetic problems, scientists have been exploring different materials for the treatment of wounds from natural and synthetic sources . An ideaThe regeneration of wound tissue is a complicated process that involves blood clotting, epithelialization, wound contraction, and synthesis of collagen and blood vessels ,3,4,5. TDisparate wound types go through a sequence of mechanisms: 1) Hemostasis, in which a fibrin clot is formed to prevent blood loss through vasoconstriction, as well as prevent microbial contamination ; (2) infCHT is a linear polysaccharide composed of N-acetyl-d-glucosamine (acetylated unit) and β-(1-4)-linked d-glucosamine (deacetylated unit) randomly distributed. It is produced by the deacetylation of shrimps and other crustacean shells with the alkali NaOH to eliminate the acetamide group. The conversion to the amine group is never absolute; it varies from 30% to 95%. The deacetylation degree (DD) influences the biocompatibility, which decreases with increasing DD, solubility, and degradation rate. Furthermore, CHT presents antimicrobial, hemostatic, and analgesic properties, stimulating closure, neovascularization, and permanent regeneration of the dermis in the wound . The catIn this work, novel chitosan-based bioactive glass wound dressings were developed starting from commercial gauzes; such commercial gauzes are simply inactive devices that safeguard the wound from other lesions while wound healing occurs naturally. Impregnating commercial gauzes with the chitosan suspension has the aim to transform passive gauzes into interactive products, which can change the local environment of wounds . For thi®) [The bioactive glasses were produced by a classical melt-quenching route, according to the process reported for other bioactive glasses ,45,46,47®) .2, CaCO3, Ca3(PO4)2, Na2CO3, K2CO3, SrCO3, MgCO3, ZnO, all reagent grade, Carlo Erba Reagenti, Milano, Italy) were mixed for 3 h in a laboratory shaker  and then melted in a Pt crucible in air. The thermal cycle was: (i) From room temperature to 1100 °C at 10 °C/min; (ii) decarbonation step at 1100 °C for 2 h; (iii) from 1100 °C to 1450 °C at 10 °C/min; (iv) and 45 min at 1450 °C. The molten glasses were quenched in room-temperature water to obtain a frit, and the frit of each glass composition was then dried at 110 °C. After a night in the oven, the frit was milled in dry conditions in a porcelain jar to obtain powders with a final grain size lower than 38 μm. Finally, the bioactive glass powders were incorporated into chitosan solution to obtain chitosan-based bioactive glass wound dressings.Briefly, the commercial raw powders  for 1 h in a chitosan solution. The amounts of materials used to prepare chitosan-based bioactive glass wound dressings are summarized in Wound dressings (2 cm × 2 cm squared shape) were obtained by soaking commercial gauzes  was dissolved in an aqueous acetic acid solution 2 wt.%/Vtot under stirring for 5 min at 32 °C. Then, a commercial gauze was immersed for 1 h in such chitosan suspension at room temperature without stirring and then dried for 24 h at 36 °C. For chitosan-based bioactive glass wound dressings, the same chitosan suspension  was prepared. Subsequently, different amounts of bioactive glasses were added under stirring to different chitosan suspensions: 5 and 10 wt.% of 45S5, BGMS10 and BGMS_2Zn, respectively. Then, a commercial gauze was immersed for 1 h at room temperature without stirring and then dried for 24 h at 36 °C.−1 with a resolution of 4 cm−1.The surfaces of the chitosan wound dressing (CHT) and chitosan-based bioactive glass wound dressings  were studied by Environmental Scanning Electron Microscopy  before and after immersing samples in simulated body fluid (SBF) solution see . Further2O at room temperature and their weights (I) were determined at 30 min, 1 h, 2 h, 3 h, 4 h, 1 day, 2 days, 3 days, and 4 days. The same experiment was repeated for 10 samples for each wound dressings’ composition, and the mean value was taken as the swelling ratio. The swelling ratio (SR%) was calculated as follows:To measure the swelling behavior, wound dressings were cut into 1 cm × 1 cm pieces and their dry weights (D) were immediately measured; then, the dry wound dressings were soaked in distilled H’ weight ,50,51.Wound dressings  were soaked in simulated body fluid (SBF) solution, prepared following the protocol developed by Kokubo , to inve® . The cytotoxicity of wound dressings was investigated according to International Standards ISO 10993: (i) 10993-1 for the selection of tests; (ii) 10993-12 for samples preparation and reference materials; and (iii) 10993-5 for the in vitro methods [The in vitro biocompatibility of wound dressings was evaluated using NIH 3T3 (mouse embryonic fibroblast) cells. The NIH 3T3 cell line was provided by ATCC methods ,54,55. T2/air ratio of 5% ± 1%. For each type of wound dressing, at least 3 replicates were prepared and tested at 24 and 72 h.Dulbecco’s modified Eagle’s Medium (DMEM) containing 1 mM sodium pyruvate, L-glutamine 2 mM, 10% (v/V) FBS , and pen-streptomycin was used for growing NIH 3T3 cells. Cells were cultured in 12-well plates; the plates were maintained in an incubator at a temperature of 37 °C ± 1 °C, humidity of 90% ± 5%, and CO2/mL ratio between standard surface and liquid volumes according to [2/mL (surface of sample/liquid volume). DMEM only was used as the negative control (CTRL-) instead. The test tubes were incubated at a temperature of 37 °C for 72 h. Before testing, wound dressings’ eluates were filtered using a 0.22 μm filter .CHT, CHT45S5_5, CHT45S5_10, CHTSrMg5, CHTSrMg10, CHTZn5, and CHTZn10 wound dressings were immersed in test tubes containing DMEM with a 3 cmrding to . As a po2/air ratio of 5% ± 1%. Then, 24 and 72 h after seeding, the morphology of cells was observed by an optical microscope . Then, DMEM was removed and 150 μL of NR solution was poured in each well. At the end of 3 h at 37 °C ± 1 °C of incubation, the NR solution was removed and 150 µL pre-warmed D-PBS was used to carefully wash cells, in order to remove the excess of NR solution. Subsequently, 1500 µL NR Desorb  solution was added to each well, before incubating the 12-well plates at room temperature for 20 min, in order to extract NR dye from cells. Finally, the final solution was poured into 96 multi-well plates and the amount of NR dye bound by cells was investigated by a spectrophotometer at 540 nm absorbance.NIH 3T3 were cultured in direct contact with wound dressings in 12-well plates at a temperature of 37 °C ± 1 °C, humidity of 90% ± 5%, and CO4/mL) were put in contact with 50 μL of eluates of wound dressings for 24 and 72 h. After each time point, 10 μL of tetrazolium salt (5 mg/mL in PBS) was poured into each well. Then, the media were carefully removed 3 h after incubation at 37 °C; 100 μL of DMSO was used to solubilize formazan. Finally, the absorbance of each well was measured at 540 nm to quantify the concentration of formazan synthesized inside cells.In 96 multi-well plates, NIH 3T3 cells  was used to statistically evaluate the results, which were expressed as the mean ± standard deviation. Student’s t-test was performed considering a two-populations comparison. In the data, To ascertain that bioactive glass particles were embedded into wound dressings, SEM analyses before immersion in SBF solution were performed. Furthermore, FTIR spectra of wound dressings containing bioactive glass particles corroborate the good incorporation of bioactive glass particles .−1, which is related to the stretching vibrations of the hydroxyl group [−1, symmetric and asymmetric C–H stretching was visible in the spectrum [−1  and chitosan ). The bafound in . Other bectively ,63,65. T−1 [−1 and between 724 and 465 cm−1, which are the asymmetric stretching Si–O–Si and bending Si–O–Si of 45S5 particles, respectively, were detected as well [−1 associated with Si–O–Si bending are shifted compared to the same bands in the CHT45S5_5 spectra .The swelling behavior of CHT, CHT45S5_5, CHT45S5_10, CHTSrMg5, CHTSrMg10, CHTZn5, and CHTZn10 in distilled Hironment ,51. The ironment . The betironment . Figure One, three, and seven days after immersion in SBF, SEM analyses were performed on the surface of CHT, CHT45S5_5, CHT45S5_10, CHTSrMg5, CHTSrMg10, CHTZn5, and CHTZn10. Seven days after immersion in SBF solution, some HCA deposits are visible on CHT45S5_5, CHT45S5_10, CHTSrMg5, and CHTSrMg10 wound dressings. Such deposits are absent on CHT, CHTZn5, and CHTZn10 wound dressings. CHT shows low bioactivity a, which 42−, which bond Ca ions for charge balancing. This removes Ca cations from the silica network, decreasing bioactivity and reducing glass dissolution [SEM images of CHT45S5_10 and CHTSrMg10 b,d confisolution ,72. Evensolution ,73. In fBiological tests were performed on CHT, CHT45S5_5, CHT45S5_10, CHTSrMg5, CHTSrMg10, CHTZn5, and CHTZn10 to ascertain the biocompatibility of such wound dressings. At 24 and 72 h after direct contact with wound dressings, no significant differences in cellular morphology were observed . The morTherefore, to avoid any eventual problem caused by particulates released from materials, an MTT assay was carried out b. Then, Furthermore, to assess the effect of bioactive glass particles and chitosan on mouse embryonic fibroblasts (NIH 3T3), the closure of the gap in monolayers of mouse embryonic fibroblasts was investigated. Gaps on the monolayer of cells were created by the tip of a 200 μL pipette, and at 24 and 48 h, the closure of gaps was measured. At 24 h, there was a gradual closing defect for all wound dressings .A significant closure in terms of cell migration rate (%)  was registered for CHT and CHTZn10, i.e., 71.06% and 73.31%, respectively .2+ signaling affects every aspect of a cell’s life and death, as reported in [2+ binds to numerous proteins to effect changes in function, localization, and association, and those ions are involved in the regulation of mitochondrial shuttling, Golgi, endosomal and vesicular fusion events, etc. [Thus, at 24 h, CHT and CHTZn10 further narrowed the scratch. At 48 h, the cell migration rate (%) of wound dressings containing bioactive glasses was higher compared to that measured for CHT, and the scratch completely disappeared in CHTZn5 and CHTZn10, and almost disappeared in CHTSrMg10 . In partorted in . Additiots, etc. . Besidests, etc. . Furtherts, etc. . The relts, etc. . Additiots, etc. . The frets, etc.  by alterts, etc. . The intts, etc. . Furtherts, etc. . Therefots, etc. . Bioactits, etc. ,89. The ts, etc. . Furtherts, etc. ,41,42, wHowever, although the addition of bioactive glass particles to chitosan improves the wound healing rate, the bioactive glass particle addition is still far from the challenge of healing chronic wounds.Wound dressings are complex systems employed to protect and heal different types of wound. The research is driven by the aim to find innovative wound dressing or healing systems to enhance healing and reduce patient pain. In this work, commercial passive gauzes were immersed in chitosan-based bioactive glass suspension to produce active wound dressings with enhanced cell proliferation and healing. Bioactive glasses of different compositions  were added to the chitosan solution in different amounts . Bioactive glass could elicit a specific cellular response and could introduce antibacterial properties, enhancing the biological properties of chitosan. Among other features, an ideal wound dressing shows the capacity to preserve high humidity while removing excess exudates; for this reason, the swelling test was performed. Wound dressings containing bioactive glass particles showed a lower swelling capacity compared to chitosan wound dressings because bioactive glass particles inhibit the capacity of chitosan to absorb exudates. On the other hand, bioactive glass particles enhance chitosan bioactivity because HCA precipitate was observed 7 days after immersion in SBF solution on wound dressings with bioactive glass particles only. Therefore, wound dressings with bioactive glass particles showed a higher bioactivity compared to wound dressings with chitosan. Furthermore, to exclude cytotoxicity, NR uptake and MTT assays using NIH 3T3 cells were carried out. Furthermore, once again, 24 and 72 h after seeding, wound dressings with bioactive glass particles showed higher cellular viability values compared to the cellular viability of chitosan wound dressings; this could be ascribed to the ions released by bioactive glass particles. Finally, the in vitro scratch test showed better migration of cells for wound dressings containing BGMS10 and BGMS_2Zn compared to wound dressings containing 45S5 and to chitosan wound dressings. In the case of CHTZn5 and CHTZn10, the cellular gap was almost already closed 24 h after the scratch, and 48 h after the scratch, the gap was completely closed. Then, 48 h after the scratch, for CHTSrMg5 and CHTSrMg10, the gap was closed, while for other wound dressings, the gap was still visible. The release of Sr, Mg, and Zn ions improved cell proliferation and wound healing rate. Therefore, wound dressings containing BGMS10 and BGMS_2Zn showed favorable properties for healing, and this makes them suitable materials for in vivo studies. Thus, wound dressings with bioactive glass particles could represent a starting composite for biomedical application, even if a better comprehension of molecular and biological response is still needed.


Tisochrysis lutea is a marine haptophyte rich in omega-3 polyunsaturated fatty acids ) and carotenoids . Because of the nutraceutical applications of these compounds, this microalga is being used in aquaculture to feed oyster and shrimp larvae. In our earlier report, T. lutea organic crude extracts exhibited in vitro cytotoxic activity against human hepatocarcinoma (HepG2) cells. However, so far, the compound(s) accountable for the observed bioactivity have not been identified. Therefore, the aim of this study was to isolate and identify the chemical component(s) responsible for the bioactivity observed. Bioassay-guided fractionation through a combination of silica-gel column chromatography, followed by preparative thin layer chromatography (PTLC), led to the isolation of two diastereomers of a monoterpenoid lactone, namely, loliolide (1) and epi-loliolide (2), isolated for the first time in this species. The structural elucidation of both compounds was carried out by GC-MS and 1D (1H and 13C APT) and 2D  NMR analysis. Both compounds significantly reduced the viability of HepG2 cells and were considerably less toxic towards a non-tumoral murine stromal (S17) cell line, although epi-loliolide was found to be more active than loliolide.  Tisochrysis lutea (Haptophyta), formerly known as Isochrysis galbana T-ISO [T. lutea is able to accumulate significant levels of n-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), and of xanthophylls such as fucoxanthin, which play an important role in the prevention and treatment of human diseases [In recent years, natural products derived from microalgal biomass produced at pilot scale have attracted great attention in drug discovery . Becausena T-ISO ,4, is a na T-ISO . More rediseases . T. lutea have been evaluated concerning their immunomodulatory properties by the induction of IL-1 in murine macrophages [T. lutea has exhibited promising therapeutic effects by inducing weight loss and decreasing glucose, triacylglycerol (TAG), and cholesterol levels in diabetic rats [Mycobacterium tuberculosis [T. lutea displayed promising antioxidant properties, inhibition of acetylcholinesterase (AChE), and cytotoxicity against tumor cell lines (HepG2) [In addition, several isolated compounds have been reported to display a wide spectrum of medicinal properties. For example, crude polysaccharides of rophages . Moreoverophages . T. lutetic rats . In additic rats , compounrculosis , as wellrculosis  and inhirculosis . In a pr (HepG2) .T. lutea with cytotoxic activity against hepatocarcinoma HepG2 cells. Hepatocarcinoma is a prevalent cancer of the liver and occurs particularly in patients with underlying chronic liver disease and cirrhosis. It is the fourth leading cause of cancer mortality and third most common cause of cancer-related death worldwide after lung and gastric cancer [1) and epi-loliolide (2), from T. lutea. The evaluation of their cytotoxic effects against human HepG2 cells is also discussed in comparison to those against a non-tumoral cell line (S17 cells).Nevertheless, to the best of our knowledge, there are no reports on the isolation of compounds from c cancer ,15. Despc cancer . Consequc cancer . Interesc cancer . Hence, T. lutea were tested for their in vitro cytotoxic activity against HepG2 cells. In our study, dimethyl sulfoxide (DMSO) and the chemotherapeutic drug etoposide were used as negative and positive controls, respectively. Etoposide is a semisynthetic derivative of podophyllotoxin, which is well-known and widely used as an anti-cancer drug [T. lutea on mitochondrial metabolic activity . It was found that the DCM extract was the most active compared to the hexane and acetone extracts, decreasing HepG2 cell viability down to 24.7% at a concentration of 125 µg/mL (50 of 85.1 µg/mL (CI95% 73.1 to 99.1 µg/mL).The extraction was first carried out sequentially using organic solvents with increasing polarity, such as hexane (Hex), dichloromethane (DCM), and acetone (Ace), in order to obtain an enriched bioactive extract. Later, the organic extracts of cer drug ,20. It i25 µg/mL , resultiTo identify the bioactive molecule(s) responsible for the cytotoxic properties, the active DCM extract was subjected to bioguided fractionation using silica-gel chromatography, which yielded five fractions named as F1 through F5 A. Each f1H and 13C NMR. Firstly, subfractions F5–3 and F5–4 were analyzed by GC-MS  as a mobile solvent system. This experiment showed two different spots at 0.43 and 0.34, respectively. To further elucidate the structures of both compounds, 1D (1H and 13C Attached Proton Test (APT) and 2D  NMR spectral analyses were carried out. The chemical shifts and structural assignments (1) and epi-loliolide (2)   upon comTisochrysis lutea [T. lutea showed cytotoxicity towards HepG2 and S17 cell lines, this effect was lower than that of epi-loliolide (2); this result might be explained by a different configuration around C–6 and/or C–7a  as a dark green powder material produced by lyophilization. Human hepatocellular carcinoma (HepG2) and murine bone marrow stromal (S17 cell) cell lines were kindly provided by Dr. Vera Marques and Dr. Nuno Santos, Center for Molecular and Structural Biomedicine (CBME), University of Algarve , respectively. Hexane (Hex), methanol, ethyl acetate (EA), dichloromethane (DCM), acetone (Ace), and dimethyl sulfoxide (DMSO) from Fisher Scientific  and 3--2,5-diphenyltetrazolium bromide  were purchased from VWR International . TLC-plates ALUGRAM® Xtra Sil G/UV254, pre-coated with silica gel 60 (1 mm) and silica gel (70–130 mesh), were purchased from M/s Merck . All the other chemicals used were of reagent grade.Tisochrysis lutea was dispersed in the chosen solvent and homogenized using a disperser IKA T10B Ultra-Turrax at room temperature. The extractions were made sequentially using 200 mL of hexane, dichloromethane, and acetone at room temperature to obtain crude extracts. The extractions were done in triplicate, filtered through Whatman filter paper n° 4, and the supernatants combined and concentrated under reduced pressure using a rotary evaporator, at 40 °C. All extracts were dissolved in DMSO at a concentration of 50 mg/mL, aliquoted, and stored at 4 °C until use.Lyophilized biomass (50 g) of 2 in humidified atmosphere at 37 °C overnight, and later treated with the extracts  at concentrations ranging from 3.9 to 125 µg/mL for 72 h. Negative controls for cell lines were performed with DMSO at a maximum concentration of 0.5% (v/v). The MTT colorimetric assay was carried out to assess their effect on mitochondrial metabolic activity, as an indicator of cell viability [TM 4, Winooski, VT, USA), and results were calculated as percent of cell viability and as IC50 values (µg/mL). Furthermore, the selective index (SI) of the subfractions , and positive control etoposide was evaluated at a concentration of 125 µg/mL. The SI was calculated using the following equation: SI = VNT/VT, where VNT and VT represent the cell viability of non-tumoral cells (S17) and tumoral cells (HepG2), respectively, after exposure to the same concentration of extract or fraction [Cell lines were maintained in culture media RPMI-1640 supplemented with glucose (1000 mg/mL), 10% fetal bovine serum (FBS), L-glutamine (2 mM), streptomycin (50 µg/mL), and penicillin (50 µg/mL). Both HepG2 and S17 cell lines were seeded on 96-well plates, incubated under 5.0% COiability . Brieflyfraction .v/v), F3 , F4 , and F5 . After identification of the active fraction  this was again subjected to silica-gel column chromatographic fractionation to afford fractions F5–1, F5–2, F5–3, and F5–4, which were eluted using mixtures ranging from pure hexane to pure ethyl acetate, particularly fractions F5–3 and F5–4, which were obtained in a solvent system containing 40–50% hexane in ethyl acetate. The resulting active fractions (F5–3 and F5–4) were further purified by preparative TLC which yielded 29 mg of F5–3 (1) and 13 mg of F5–4 (2). These pure compounds were stored at 4 °C until further use, such as structural elucidation and biological activities evaluation.The DCM crude extract  was subjected to a bioguided fractionation using silica-gel (70–130 mesh) column chromatography eluting with mixtures of hexane, ethyl acetate, and methanol of increasing polarity to obtain five fractions as follows: F1 (pure hexane), F2  were dissolved in deuterated chloroform  and analyzed by NMR. 1H (400.1 MHz) and 13C (100.6 MHz) NMR were recorded on a Bruker Avance spectrometer ; chemical shifts were expressed in δ values and referenced to the residual CDCl3 peak (δH = 7.26 ppm and δC = 77.00 ppm); coupling constants were reported in hertz (Hz). Unequivocal assignments of all proton and carbon signals were achieved by 1D  and 2D  NMR experiments. The NMR data obtained for both compounds were in accordance with previously reported data [m/z 33–1000; source at 230 °C and quadruple at 150 °C) in EI mode with a ZB-5 ms capillary column . The column temperature was initially maintained at 60 °C for 1 min, and gradually increased as follows: 60 °C to 120 °C at 30 °C/min, 120 °C to 250 °C at 4 °C/min, then 250 °C to 270 °C at 20 °C/min, and finally 270 °C to 300 °C at 2.5 °C/min, where it remained for 5 min. The carrier gas was helium at a flow rate of 1.0 mL/min, the inlet temperature was maintained at 300 °C and split-less mode was used.Pure samples of loliolide (1) and ted data ,26,27,28T. lutea can be a source of compounds for the prevention and treatment of human hepatocarcinoma. Two cytotoxic and selective compounds were isolated, loliolide (1) and epi-loliolide (2), which are likely to be degradation products of fucoxanthin and/or zeaxanthin. Therefore, comparative biological studies of pure carotenoids  and their respective catabolites are warranted in order to find a structure–activity relationship (SAR) of loliolide and its stereoisomers. A possible research avenue would include the modification of the hydroxyl group occurring in loliolides in order to generate various analogues and determine their bioactivities, which could further improve the usefulness of these lactones.Our results indicate that crude extracts and/or biomass of


Due to their capacity to proliferate, migrate, and differentiate, mesenchymal stem cells (MSCs) are considered to be good candidates for regenerative medicine applications. The mechanisms underlying proliferation and differentiation of MSCs have been studied. However, much less is known about the mechanisms regulating the migration of MSCs. Platelet lysate (PL), a supplement used to promote cell expansion, has been shown to promote MSCs migration; however, the underlying mechanism are unknown. Here, by using adipose-derived rat MSCs (rMSCs) and the scratch assay in the absence and presence of various BK channels modulators, we evaluated the role of BK channels in mediating the PL-stimulated migration of rMSCs. We found that 5% PL increased rMSCs migration, and this effect was blocked by the addition of the BK channel selective antagonist Iberiotoxin (IBTX). In the absence of PL, the BK channel agonist NS1619, stimulated rMSCs migration to similar level as 5% PL. Addition of both NS1619 and 5% PL resulted in an increase in rMSCs migration, that was higher than when either one was added individually. From whole-cell recordings, it was found that the addition of 5% PL increased the magnitude of BK current density. By using Western blot and flow cytometry, it was found that PL did not affect the expression of BK channels. Together, our results indicate that as shown in other cell types, activation of BK channels by themselves also promote rMSC migration, and show that activation of BK channels contribute to the observed PL-induced increase in migration of rMSC. The efficient migration of stem cells and their subsequent differentiation and long-term survival are essential characteristics for a successful use of stem cell-mediated treatments . Mesench2+ concentration levels (2 + -activated K+ channels (BK) channels, through intracellular signaling pathways, have been identified as an essential step in the basic fibroblast growth factor (bFGF)-induced proliferation of endothelial cells , which modify their biophysical and pharmacological properties , directed against β1 and α subunits, respectively, were from Alomone Labs and Novus biological. Anti-rabbit Alexa Fluor 488 was purchased from Invitrogen, Carlsbad, CA, United States.The BK channel antagonist, Iberiotoxin (IBTX), and agonist, NS1619 , were solubilized in water and dimethyl sulfoxide, respectively. CD45-allophycocyanin (APC), CD90-phycoerythrin (PE), and CD29-Alexa Fluor 488 conjugated antibodies were purchased from Biolegend, San Diego, CA, United States. Anti-sloβ1 (NB300-535) and anti-Kg for 5 min. The sample was then resuspended in DMEM supplemented with 10% FBS and maintained at 37°C and 5% CO2. After 72–96 h, the cells were washed twice with PBS to remove any non-MSCs. For all the assays, MSCs from passages 2–7 were used assays  supplemented with penicillin/streptomycin (P/S) 5% and treated with 0.075% collagenase type I in PBS plus 2% P/S at 37°C. After 1 h, collagenase was neutralized by adding Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and P/S 2%. The sample was pipetted up and down several times to fragment the tissue aggregates and then filtered and centrifuged at 360 × d assays .g for 5 min and suspended in DMEM at a density of 2 × 105 cells/ml. Cells were fixed with 4% formaldehyde and centrifuged at 360 × g for 5 min. The pellet was suspended in methanol; left overnight at −20°C, washed in PBS plus 2% FBS, and incubated with antibodies CD45-APC, CD90-PE, and CD29-Alexa Fluor 488 for 1 h. Samples were evaluated using a Guava cytometer  and analyzed with the Guava Incyte Software.Rat MSCs were characterized by evaluating the expression of the surface markers CD45, CD29, and CD90 by flow cytometry . Cells were trypsinized after reaching 90% confluence, centrifuged at 360 × g for 8 min. 50% of the plasma volume was removed to produce a platelet-rich plasma, which was then centrifuged, the plasma was removed, and the platelet pellet was resuspended in PBS. Finally, the platelets were frozen to −80°C and thawed to 37°C (this cycle was repeat for three times), and then used in subsequent experiments.Platelets were isolated from healthy volunteers after obtaining the written informed consent and with the approval of the Institutional Ethics Committee at the Pontificia Universidad Javeriana. Blood was collected and centrifuged at 800 × g for 5 min, suspended in DMEM at a density of 2 × 105 cells/ml, fixed with 4% formaldehyde, and centrifuged again at 360 × g for 5 min. Then, cell pellet was resuspended in methanol and left overnight at −20°C. Cells were washed in PBS–2% FBS, centrifuged at 360 × g for 5 min, and incubated with primary antibody for 1 h , followed by secondary antibody  for 30 min, and then washed in PBS buffer. Cells incubated only with secondary antibody were used as a control. Samples were evaluated using a Guava cytometer  and analyzed with Guava Incyte software. All experiments were performed in triplicate, and the number of samples (n) was 5.Total expression of BK subunits was evaluated by flow cytometry and Western blot, using specific antibodies in permeabilized cells. For flow cytometry, cells at 80–90% confluence were detached, centrifuged at 360 × Ca1.1; anti-sloβ1 antibody prepared in blocking buffer with 5% Bovine serum albumin (1:500). The membrane was then washed three times with TBS-T 0.1%, incubated for 1 h with horseradish peroxidase-conjugated secondary antibody  and washed with TBS-T 0.1% for 5 min. Blots were visualized by using a chemiluminescence substrate .For Western blot assays, MSCs were trypsinized, washed twice with PBS and lysed at 4°C with RIPA buffer containing protease/phosphatase inhibitor cocktail. Lysates were centrifugated (16000 rpm for 1 min at 4°C), and protein concentration was measured by using the Bicinchoninic acid kit (ThermoFisher Scientific). 20 μg of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a PVDF membrane (BioRad) using standard techniques. The membranes were blocked with Tris-buffered saline with Tween (TBS-T) 0.1% + 5% milk, and stained with anti-Kg for 5 min and suspended in DMEM at a density of 2 × 105 cells/ml. Cells were fixed with 4% formaldehyde, washed in PBS–2% FBS, centrifuged at 360 × g for 5 min, incubated with primary antibody for 1 h , followed by secondary antibody  for 30 min, and then washed in PBS buffer. Cells incubated only with secondary antibody were used as a control. Samples were evaluated using a Guava cytometer  and analyzed with Guava Incyte software. All experiments were performed in triplicate, and the number of samples (n) was 5.In order to evaluate BK channel surface expression, we used flow cytometry in non-permeabilized cells. Cells at 80%–90% confluence were detached, centrifuged at 360 × 5 cells/well. The cells were then subjected to the following treatments after 16 h: IBTX 10 nM; NS1619 10 μM; 5% PL plus heparin (1% v/v); IBTX 10 nM + 5% PL plus heparin (1% v/v); and NS1619 10 μM + 5% PL plus heparin (1% v/v). Cells treated with doxorubicin 30 μM were used as a positive control and cells without treatment were used as a negative control. After 24 h, the cells were detached, stained with propidium iodide, and analyzed by flow cytometry. All experiments were performed in triplicate and n was 5.Rat MSCs were detached and plated in 12-well plates at a density of 2 × 10Rat MSCs were plated in 24-well plates in DMEM. When the cells reached confluence, a linear wound was made by scratching the cell monolayer with a sterile 1000 μl plastic pipette tip. Cells were incubated with fresh medium for 24 h in the presence of BK channel modulators (IBTX 10 nM and NS1619 3 and 10 μM), with or without 5% PL (v/v) plus heparin (1% v/v). Cells without treatment were used as controls. The area of the wound gap was then observed and photographed under an inverted microscope (20X)  and cells on the wound area were counted at 0 and 24 h after scratching to assess cell mobility under different treatment conditions. Number of cells in wound area after each treatment was dividing by number of cells in control experiments to analyze changes in migration induced by the treatments. Five experiments were performed in triplicate.2, 0.5 mM MgCl2, and 5 mM [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] HEPES, pH 7.4. The pipette solution consisted of 150 mM KCl, 1 mM MgCl2, 5 mM ethyleneglycol tetraacetic acid, 2 mM Mg-ATP, and 10 mM HEPES, pH 7.4. Pipettes were made from borosilicate glass capillaries , pulled in a horizontal pipette puller , and heat polished . The pipette resistance was 3–5 MΩ. Currents were recorded using an EPC-7 amplifier  and a Nikon TS100 inverted microscope, filtered at 1/5 of the acquisition rate and sampled with an A/D converter . The acquisition software was developed by Dr. Patricio Orio, CINV, Valparaiso, Chile, using the LabView programming environment . Data analysis was performed using Clampfit 9 software . Currents were activated by a voltage step protocol with a holding potential of −80 mV, voltage pulses between −100 and +250 mV in 10 mV increments with durations of 70 ms, followed by a step to −80 mV. Assays were carried out after 24 h of treatment with 5% PL and currents were measured in the presence or absence of IBTX 100 nM. Series resistance was compensated, and changes were no greater than 10% during the experiments. To confirm the presence of BK channel current in the recordings, we subtract the current measured in the presence of IBTX from that obtained before the addition of the antagonist.Whole-cell currents were recorded using the patch-clamp technique. Cells were detached and seeded onto glass coverslips 30 min before assay. Recordings were carried out at room temperature (20–22°C) in a bath solution containing 143 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, and 5 mM HEPES, pH 7.4, for 1 h at 37°C in darkness. Fluorescence measurements were made by using a Zeiss inverted microscope Axio Observer and a AxioCam ICc1 camera. After record basal fluorescence, 5% PL was added by perfusion to the recording chamber. Ionomycin 5 μM was added at the end of the experiment to elicit a maximum Ca2+ influx for signal normalization. Changes in fluorescence signal induced by PL were analyzed using ImageJ.Rat MSCs were loaded with 5 μM Fluo-4AM (Invitrogen) in a solution with 143 mM NaCl, 5.4 mM KCl, 1.8 mM CaClp < 0.05 considered significant. T test was used to analyze channel expression. ANOVA analysis was used with Kruskal–Wallis post hoc to analyze cell viability and migration. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.The results are shown as the mean ± standard error of the mean (SEM). The significance of differences in electrophysiological analysis was determined by analysis of variance with a value of n = 5, p < 0.0001) . Cells treated with 5% PL plus IBTX migrated at a similar level to control cells without PL  showed that 97–98% of cells were positive for CD90 and CD29, and less than 0.1% were positive for CD45 ruling out a hematopoietic cell contamination and confirming a mesenchymal phenotype of the isolated cells . MSCs ex 0.0001)  suggesti 0.0001) . Previou 0.0001)  that can 0.0001) . To evaln = 5, p < 0.01)  agonist produced a 36% increase in cell migration  . The effn = 3, p < 0.001, n = 3, p < 0.01, To examine the mechanism by which BK channel regulates rMSCs migration, we used whole-cell patch-clamp recordings to determine whether 5% PL changes BK channel activity. Our results showed that the treatment of rMSCs with 5% PL for 24 h produced an ionic current increase  includinTo determine if the PL-dependent increase in BK currents was mediated by changes in BK channel protein expression, the levels of α and β1 subunits in permeabilized cells in the presence or absence of 5% PL were evaluated. The results showed that rMSCs express BK channel α and β1subunits; however, 5% PL (24 h) did not change the total level expression of these subunits . Similar2+ in cell migration mechanisms  or G (PKG) (BK channels are activated by membrane depolarization and increases in intracellular Cachannels . Neverth G (PKG) . Some re G (PKG)  making i+ efflux and changes in cell volume  . IK chanl volume . Moreovel volume . A role ignaling . Consideignaling , it is p2+ concentration and increases in BK channel activity. The later event could allow K+ efflux, changes in cell volume, and cell movement by retraction of the rear as reported previously in other migrating cells (In summary, the mechanism underlying PL-induced migration involves changes in intracellular Cang cells . Thus, tAll experiments were approved by the ethics committee at the Pontificia Universidad Javeriana, Bogotá, Colombia, and conducted following the institutional guidelines for the use of experimental animals. All procedures performed in studies involving human participants were under the ethical standards of the ethics committee at the Pontificia Universidad Javeriana, Bogotá, Colombia, and with the 1964 Helsinki declaration and its later amendments.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Comité de Investigación y Ética, Pontificia Universidad Javeriana; Acta No. 19 de septiembre 30 de 2010.SE, AG, and SG performed the research and analyzed the data. CSá, CSi and JH performed the research. J-JS analyzed the data and wrote the manuscript. YT designed the experiments, directed the research activities, performed the research, analyzed the data, and wrote the manuscript.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.