Genetic linkage maps are an important tool in genetic and genomic research. In this study, two hawthorn cultivars, Qiujinxing and Damianqiu, and 107 progenies from a cross between them were used for constructing a high-density genetic linkage map using the 2b-restriction site-associated DNA (2b-RAD) sequencing method, as well as for mapping quantitative trait loci (QTL) for flavonoid content. In total, 206,411,693 single-end reads were obtained, with an average sequencing depth of 57× in the parents and 23× in the progeny. After quality trimming, 117,896 high-quality 2b-RAD tags were retained, of which 42,279 were polymorphic; of these, 12,951 markers were used for constructing the genetic linkage map. The map contained 17 linkage groups and 3,894 markers, with a total map length of 1,551.97 cM and an average marker interval of 0.40 cM. QTL mapping identified 21 QTLs associated with flavonoid content in 10 linkage groups, which explained 16.30–59.00% of the variance. This is the first high-density linkage map for hawthorn, which will serve as a basis for fine-scale QTL mapping and marker-assisted selection of important traits in hawthorn germplasm and will facilitate chromosome assignment for hawthorn whole-genome assemblies in the future. Crataegus pinnatifida Bunge) belongs to the family Rosaceae and is a widespread fruit tree in China. The fruits are used for food and medicinal purposes. The leaves, fruits, roots, and twigs of hawthorn contain many nutrients, including proteins, fats, dietary fibre, vitamins, flavones, and many minerals. Many studies have focused on hawthorn flavones6, which are the primary bioactive components7. Flavones are polyphenol secondary metabolites that have low molecular weight and are common in plants; they are produced in response to environmental stress and play a role in defence against predators and pathogens8. Flavones have antioxidant and anticancer properties and can scavenge free radicals9, so their properties have been widely researched for use in agriculture, the chemical industry and medicine. Flavone production is under genetic regulation, but the regulation of flavones in hawthorn has been little studied.Hawthorn 13, specific length amplified fragment (SLAF) sequencing14, and genotyping by sequencing 15 are powerful tools for constructing high-density genetic linkage maps. For example, Pfender et al.16, Chutimanitsakun et al.17, and Wang et al.18 used RAD-seq to construct high-density genetic linkage maps for ryegrass, barley, and grape, respectively. Poland et al.15 constructed high-density genetic linkage maps for barley and wheat, and Zhang et al.19 constructed a map for jujube based on GBS technology. Several other studies have used SLAF to construct high-density genetic linkage maps for soybean20, sesame21, and Salvia miltiorrhiza Bunge22.As high-throughput technology and next-generation sequencing (NGS) methods have been developed, many can now quickly genotype thousands of markers in a single stepBsaXI or AlfI (both insensitive to methylation) to produce uniform tags. This is a simple and flexible method for genome-wide genotyping23.Type IIB endonuclease RAD (2b-RAD) uses restriction enzymes such as In this study, our aim was to construct a high-density genetic linkage map for hawthorn using the 2b-RAD method and then conduct fine-scale QTL mapping for flavonoid content in hawthorn leaves. This linkage map will be a powerful tool for research involving both fine-scale QTL mapping and marker-assisted selection of important economic traits of hawthorn germplasm. It will facilitate chromosome assignment for a future whole-genome assembly.24, 117,896 unique tags were generated, including 13,937 dominant tags and 103,959 codominant tags. RAD typing v1.0 software25 was used to genotype the reads, with 47,923 SNPs that were polymorphic markers, including 9,237 dominant markers and 38,686 codominant markers. A Mendelian fit test and genotyping percentage were used to trim SNP markers for 12,951 markers that fit Mendelian ratios (P ≥ 0.05) and possessed a high genotyping rate (the available genotype was found in over 80% of progeny), and the types of all these markers are shown in Fig. The Hiseq2500 v2 platform was used to conduct single-end sequencing of 2b-RAD libraries for the parents and 107 progenies. A total of 526,949,839 reads were recovered, including 11,265,798 reads from the seed parent, 12,977,353 reads from the pollen parent, and 502,706,688 reads from the progeny. The sequencing depth was 62× for the seed parent, 52× for the pollen parent, and 23× for the 107 progenies. The sequencing depth detail of the 107 progenies is shown in Fig. The number of homozygous and heterozygous SNPs are listed in Additional File 26 (logarithm of odds (LOD) ≥5) to map 1,890 markers for the seed parent and 2,149 markers for the pollen parent. The parental maps contained 17 linkage groups, which were consistent with the haploid chromosome number of hawthorn10.The high-density parental linkage maps were first constructed using Joinmap4.1 softwareFor the seed parent linkage map, 1,890 markers were mapped to 1,780 distinct positions. The total linkage map length was 1,266.81 cM, the longest linkage group, LG7, was 107.93 cM, the shortest linkage group, LG17, was 34.43 cM, and the average linkage group length was 74.52 cM. LG6, which contained 117 markers, had the highest number of markers, while LG17 contained the lowest number of markers at 15; the average marker number for these 17 linkage groups was 111. The longest average marker interval of 2.46 cM was found in LG17, the shortest average marker interval of 0.38 cM was found in LG6, and the average marker interval of these 17 linkage groups was 0.68 cM. The percentage of ‘Gap ≤5′ was used to reflect the linkage level between adjacent markers in the same linkage group. In the seed parent map, LG15 had the lowest percentage (82.05), while LG14 had the highest percentage (100.00) , while LG3 had the highest percentage (99.54) , the shortest linkage group was LG17 (35.03 cM), and the average linkage group length was 91.29 cM. LG1 contained the highest number of markers at 327, LG17 contained the lowest number of markers at 16, and the average number of markers for these 17 linkage groups was 229. The longest average marker interval of 2.34 cM was found in LG17, the shortest average marker interval of 0.30 cM was found in LG1, and the average marker interval of these 17 linkage groups was 0.40 cM. In the consensus map, LG17 had the lowest percentage of ‘Gap ≤5′ (92.86), while LG5 had the highest percentage (100.00) , randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs) were once the mainstays of genotyping. However, because they are based on gel electrophoresis, these methods usually require a substantial amount of time, with high labour costs, to sample a large population. In addition, these methods are prone to error from artificial bands. Moreover, because of marker number limitations, these methods were not suitable for constructing high-density linkage maps, assembling chromosomes, constructing fine-scale QTL maps, and breeding with marker-assisted selection33.Genotyping with molecular markers is useful for studies on phylogeny, evolution, plant breeding, and diseases34, SLAF36, and 2b-RAD23 have been introduced. As a high-throughput sequencing technique, 2b-RAD can be used for large-scale genotyping, and compared to traditional molecular marker sequencing techniques, it can construct the linkage map with high marker density and good uniformity. The 2b-RAD method has many advantages. It can provide a streamlined alternative to existing RAD-seq library construction methods, because all reactions occur consecutively in a single well within 4 h. At the same time, 2b-RAD can detect almost every restriction site in the genome in parallel, whereas other RAD-seq methods can only detect a subset of sites. The third advantage that 2b-RAD provides is a choice of selective adaptors, which can adjust to the marker density in the genome. This choice can balance the level of genotyping detail against sequencing throughput capabilities, depending on the type of study37.The emergence and development of NGS technology has made RAD-seq a feasible route to genotype the entire genome in a short time. However, library construction for RAD-seq is labour-intensive and time-consuming, and thus modifications such as ddRADet al.38 found 1,385 SNP markers in rice, Tian et al.39 found 7,389 SNPs in sea cucumber, and Fu et al.40 found 3,121 SNPs in their high-density linkage map for bighead carp. Compared to linkage maps constructed using RAD and GBS methods44, whose marker numbers ranged from several hundreds to thousands, the linkage map in this study contained 3,894 SNP markers, comparable to and suitable for QTL mapping of flavonoid content. Moreover, these mapped markers can also be used for candidate gene discovery and de novo chromosome assembly for hawthorn.In this study, we constructed the refined high-density linkage map for hawthorn using the 2b-RAD method. Other studies have reported the construction of high-density linkage maps using the 2b-RAD method. Guo We first conducted fine-scale QTL mapping analysis of flavonoid content for hawthorn leaves based on this high-density linkage map. Flavonoid content was measured in 2014 and 2015, and 21 QTLs related to flavonoid content were discovered in 10 linkage groups. This preliminary QTL mapping analysis for flavonoid content in hawthorn leaves will continue, especially for the QTLs that were adjacent within a single linkage group. Both qV14c and qV15d were related to vitexin content, had high LOD values , and explained 59.00% and 52.60% of the variance, respectively; these QTLs will be the focus of future research.The diploid seed parent material came from cv. Shandongdamianqiu, the most common cultivar in Shandong Province and Beijing, China. The diploid pollen parent came from cv. Damianqiu, which is a cultivar native to Anshan city in northeast China. The parents were hybridized to produce 107 progenies . Genomic DNA was extracted using the CTAB method, and RNA digestion was conducted by adding the proper quantities of RNase and incubating at 37 °C for 30 min. DNA samples were checked for quality and concentration and used for further experiments.−1, and the injection volume was 20 μL.In 2014 and 2015, the content of five flavonoid monomers, vitexin-rhamnoside, vitexin, rutin, hyperoside, and quercetin, was detected using an Agilent 1100 HPLC with a DAD detector and a C18 column . Acetonitrile (A) and 0.5% phosphoric acid solution (B) were used as the mobile phase with the following gradient elution protocol: 0–9 min, 18–20%(A); 9–25 min, 20–50%(A); 25–33 min, 50–18%(A); 33–38 min, 18%(A). The maximum absorption spectrum was 345 nm, the column temperature was 25 °C, the flow rate was 1.0 mL·minet al.23. A total of 100 ng genomic DNA was digested by 4 U BsaXI (New England Biolabs) in a 15-ml reaction at 37 °C for 3 h. A 1% agarose gel was used to verify the digestion of genomic DNA (~30 ng). A total of 12 ml ligation master mix containing 0.2 mM each of two library-specific adaptors, 1 mM ATP (New England Biolabs), and 800 U T4 DNA ligase (New England Biolabs) was added to the digestion product at 4 °C for 16 h, and the mixture was inactivated by heat at 65 °C for 20 min. Ligation products were amplified in three 20 ml reactions per sample, each composed of 7 ml ligated DNA, 0.1 mM each primer, 0.3 mM dNTPs, 1× Phusion HF buffer, and 0.4 U Phusion high-fidelity DNA polymerase (New England Biolabs). PCR was conducted in a DNA Engine Tetrad 2 thermal cycler (Bio-Rad) with 20 cycles of 98 °C for 5 s, 60 °C for 20 s, and 72 °C for 10 s, with a final extension at 72 °C for 10 min. A 2% agarose gel was used for excising the target band, and the DNA will be diffused into nuclease-free water at 4 °C for 12 h. Sample-specific barcodes were introduced by PCR with platform-specific barcode-bearing primers. Each 20 ml PCR reaction contained 25 ng of gel-extracted PCR product, 0.1 mM of each primer, 0.3 mM dNTPs, 1× Phusion HF buffer, and 0.4 U Phusion high-fidelity DNA polymerase; four or five cycles of the PCR profile listed above were performed. PCR products were purified using a QIAquick PCR purification kit (Qiagen) and pooled for sequencing using the Illumina HiseqXTen platform.The 2b-RAD libraries were prepared for 109 samples by following the protocol developed by Wang et al.37 Raw reads were first trimmed to remove adaptor sequences. The 3′ terminal positions were excluded from each read to eliminate artefacts that might have arisen at ligation sites. Reads with no restriction sites or containing ambiguous base calls (Ns), long homopolymer regions (>10 bp), and regions with more than five consecutive low-quality (score <10) positions were removed. The remaining trimmed, high-quality reads formed the basis of subsequent analyses. De novo 2b-RAD genotyping was performed using the program RADtyping v1.0.Genotyping was performed using procedures described by Jiao 26. An LOD score cut-off of 5.0 was used to determine the genetic positions of the markers. Map distances (cM) were converted using recombination frequencies through the Kosambi mapping function. The consensus map was generated by integrating the parental maps based on the shared markers using MergeMap45 with a map weight a 1.0. The visualized linkage maps were subsequently drawn using MapChart 2.246.lm × ll (markers from the pollen parent) or nn × np (markers from the seed parent) were categorized as the dominant markers, which segregated in a 1:1 ratio in the map population. Next, hk × hk (markers in both parents) was categorized as co-dominant markers, which were present in both parents and segregated in a ratio of 1:2:1. Before grouping, the markers were tested for goodness-of-fit based on the expected Mendelian ratios through chi-square tests to eliminate markers that significantly deviated from the expected ratios . The qualified markers were then used to construct paternal and maternal linkage maps using the JoinMap 4.1 software47. The LOD scores were first analysed using the interval mapping model; LOD statistics were calculated at an interval of 1 cM. Genome-wide and chromosome-wide LOD significance thresholds at the 95% level were determined with a 1000 permutation test for the content of five flavonoids, and QTLs with LOD scores greater than the LOD threshold at 95% were declared significant. Once a QTL was detected, the confidence interval was calculated using the protocol of Li48.QTL mapping analysis was performed for the content of five flavonoids in hawthorn using MapQTLAdditional File A1Additional File A2Additional File A3Supp Info


Glehnia littoralis has been reported to have several pharmacological properties but no reports describing the antiadipogenic effect of this plant have been published. This study was conducted to investigate the effects of Glehnia littoralis root hot water extract (GLE) and its underlying mechanism on 3T3-L1 cell adipogenesis and in high-fat diet- (HFD-) induced obese mice. We measured intracellular lipid accumulation using oil red O staining in vitro. For in vivo study, twenty-eight C57BL/6J male mice were randomly divided into four groups, Control, HFD, HFD + 1% GLE, and HFD + 5% GLE, which was performed for eight weeks. We determined the expression levels of the adipogenesis-related proteins by RT-PCR and western blotting in HFD-induced obese mice. The GLE dose-dependently inhibited 3T3-L1 adipocyte differentiation and intracellular lipid accumulation in differentiated adipocytes. Further, body weight gain and fat accumulation were significantly lower in the GLE-treated HFD mice than in the untreated HFD mice. GLE treatment suppressed the expression of adipogenic genes such as peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer-binding protein (C/EBP) α, fatty acid synthase (aP2), and fatty acid synthase (FAS). These results suggest that the GLE inhibits adipocyte differentiation and intracellular lipid accumulation by downregulating the adipogenic gene expression both in vitro and in vivo. The prevalence of obesity has increased dramatically worldwide owing to lifestyle and diet changes and is rapidly becoming a threat to human health. Obesity has recently attracted increasing attention owing to its association with several metabolic diseases including type II diabetes, cardiovascular disease, and hypertension .Obesity is caused by excess adipose tissue mass, which is the major energy reserve in the body . As the Glehnia littoralis Fr. Schmidt ex Miq. (Umbelliferae) is a perennial herb that grows on the sandy beaches of eastern China, Korea, Japan, and North-west America [ G. littoralis has been reported to have several pharmacological properties including antioxidant [ G. littoralis have been identified as quercetin, isoquercetin, rutin, chlorogenic acid, and caffeic acid [ America . Its roo America , have trioxidant , anticanioxidant , 13, antioxidant , and somioxidant , 15. Theeic acid . Glehnia littoralis root extract (GLE) on the adipogenic differentiation of 3T3-L1 cells by measuring intracellular lipid accumulation. We also investigated the mechanism underlying the inhibitory effects of GLE on adipocyte differentiation in HFD-induced obese mice to determine the potential medicinal benefits of G. littoralis as an antiobesity agent.To date, no reports describing the antiadipogenic effect of this plant have been published. High-fat diet- (HFD-) induced animal models of obesity and 3T3-L1 cells have been widely used for studying the antiobesity properties of various compounds . TherefoG. littoralis roots obtained from Fine Food Tech Co., Ltd. , were air-dried at 50°C at an air velocity of 1.5 m/s for 4 days, blended, and further ground, to obtain a fine powder. The powder (300 g) was soaked in 3 L of distilled water and then heated at 100°C for 4 h. The crude extract was collected, filtered with a sterilized cloth, freeze-dried at −60°C, and stored in a deep freezer (−70°C) until use.µm, Shiseido Capcell Pak MG). The mobile phase was solvent A  and solvent B . The analysis was performed under the following gradient conditions: 100% A to 0% B (0–30 min), 100% B (30–40 min), 100% B to 0% A (40–42 min), and 100% A (42–60 min), with a flow rate of 1 mL/min and a detection wavelength of 280 nm with 1 nm bandwidth. All standards were purchased from Sigma-Aldrich .The HPLC analysis was performed on a Dionex Summit™ system  equipped with an UVD 340U-photodiode array detector  using a reverse-phase C18 analytical column , cell differentiation was induced with the MDI differentiation medium containing 1 µM dexamethasone , 0.5 mM 3-isobutyl-1-methylxanthine , 10 µg/mL insulin , and DMEM supplemented with 10% FBS. After 48 h (day 2), the culture medium was replaced with DMEM supplemented with 10% FBS, and this was repeated every 48 h until day 8. The cells were treated with different concentrations of the GLE  from day 0 to 8, and untreated cells were used as a control.Murine 3T3-L1 preadipocytes were obtained from the Korean Cell Bank  and cultured to confluence in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  in a humidified 5% CO4 cells/well and treated with the GLE (0–400 µg/mL) for 24 h. 10 µL of CCK-8 reagent was then added to each well and the absorbance was measured at 450 nm using an Infinite® F50 microplate reader . The viability of the GLE-treated cells was expressed as a percentage of the control cell viability.The effect of different concentrations of the GLE on 3T3-L1 preadipocyte viability was determined by the cell counting kit-8 (CCK-8) assay . Briefly, the cells were seeded in a 96-well plate at a density of 1 × 10The lipid accumulation in adipocytes, which indicates the extent of differentiation, was measured using oil red O staining. Briefly, differentiated 3T3-L1 cells were fixed in 10% formaldehyde in PBS for 1 h, washed with distilled water, and dried completely. The cells were then stained with 0.5% oil red O solution in 60 : 40 (v/v) isopropanol : triple distilled water for 15 min at room temperature, washed four times with triple distilled water, and dried. The treated cells were observed under an Olympus microscope , and representative images were captured using an Olympus DP70 camera. The cell differentiation was quantified by elution of the stain with isopropanol and measurement of the absorbance at 520 nm.C57BL/6J male mice (6- to 8-week-old) were purchased from Samtako Bio Korea Co., Ltd.,  and initially acclimated to laboratory conditions for 1 week, prior to experimental use. After acclimatization, 28 mice were randomly divided into four groups, namely, the American Institute of Nutrition- (AIN-) 93G diet , high-fat diet (HFD), HFD with 1% GLE (HFD + 1% GLE), and HFD with 5% GLE (HFD + 5% GLE) groups. The HFD contained 45.5% fat (as soybean oil and lard), 20% protein, and 34.5 % carbohydrate .The mice were housed under a 12 : 12 h light-dark cycle at 22 ± 2°C and 55 ± 5% relative humidity with ad libitum access to the specified diets and sterile drinking water for 8 weeks. The food intake and body weight were measured every week, and the feed efficiency ratio (FER) was calculated as the total weight gain/total food intake. All experiments on animals were carried out in accordance with the institutional guidelines of the Hankyong National University, Anseong, Korea. This study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , and was approved by the Hankyong National University Animal Welfare Committee (Hankyong. 2015-2).At the end of the experimental period, the animals were fasted overnight and administered mild ether anesthesia, and blood was collected via puncture of the retroorbital sinus in ethylenediaminetetraacetic acid- (EDTA-) coated vials. The animals were then euthanized by cervical dislocation under mild ether anesthesia and the abdominal, perirenal, and epididymal fat pads were excised. The fat samples were rinsed with saline and stored at −70°C until further analysis.µg of the total RNA in a 20 µL reaction volume using a Maxime RT PreMix kit  containing the OptiScript™ reverse transcriptase and i-StarTaq™ DNA polymerase, following the manufacturer's recommended protocol. The oligonucleotide primers are shown in Total RNA was isolated from the epididymal fat samples of the experimental mice using the RNAiso Plus reagent  according to the manufacturer's instructions. cDNA was synthesized from 1 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis  and transferred to nitrocellulose membranes. The membranes were blocked overnight with 5% skim milk in Tris-buffered saline-Tween 20  and incubated with the following primary antibodies (1 : 1000 dilution): PPARγ, C/EBPα, SREBP-1c, aP2, leptin, FAS, and β-actin . The membranes were then washed four times with TBST buffer and incubated with the corresponding horseradish-peroxidase- (HRP-) conjugated secondary antibody (1 : 2000 dilution). The immunoreactive protein bands were visualized using an enhanced chemiluminescence plus kit , and their relative intensities were quantified using the ImageJ 1.41 software.Proteins were extracted from the epididymal fat samples using a protein extraction kit . The lysates were centrifuged at 15,000 rpm for 15 min at 4°C, and the protein content of the supernatant was determined by Bio-Rad™ assay kit . Diluted protein samples  software package version 9.2  was used for the analysis.The results are expressed as the mean ± standard deviation (SD) of at least three independent experiments. Statistical differences between the groups were evaluated by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test. Values of When the composition of the GLE was investigated by comparing its HPLC profile with that of nine standard compounds including cnidilide, ligustilide, neocnidilide, butylphthalide, senkyunolide, tetramethylpyrazine, caffeic acid, ferulic acid, and perlolyrine eluted under the same conditions, two compounds, namely, caffeic acid and ferulic acid, were identified as the active constituents of the GLE .µg/mL) of the GLE for 24 h stimulated the proliferation of 3T3-L1 cells with no cytotoxicity observed following the treatment with 400 µg/mL of the GLE for 24 h . At the end of the experiment, mice in the HFD + 5% GLE group exhibited a drastic reduction in body weight gain compared to that reported for the HFD group mice (8.2 ± 3.4 versus 17.3 ± 2.6 g). However, the antiadipogenic effect in the HFD + 1% GLE group was not as pronounced as that in the HFD + 5% GLE group. The feed efficiency ratio (FER) of the HFD + 5% GLE group was significantly lower than that of the HFD and HFD + 1% GLE groups (p < 0.05). The fat weight, which comprises the abdominal, perirenal, and epididymal fat pad weights, of mice in the HFD + 5% GLE group (8.2 ± 0.3 g) was approximately 50% lower than that of mice in the HFD (16.3 ± 0.3 g) and HFD + 1% GLE (15.8 ± 0.2 g) groups. The fat weight per 100 g body weight of mice in the HFD + 5% GLE group (27.7 ± 1.0 g) was significantly lower than that of mice in the HFD (42.7 ± 0.8 g) and HFD + 1% GLE (40.3 ± 0.5 g) groups (p < 0.05).We further elucidated the antiadipogenic effects of the GLE by performing anE groups  (p < 0.0) groups  , with the levels in the HFD + 5% GLE group being reduced by 59.5, 118.3, and 41.3%, respectively, compared to those in the HFD group  . In particular, the leptin mRNA expression in the HFD + 5% GLE group decreased by 44.6 ± 2.7%. The GLE treatment also suppressed the expression of SREBP-1c and FAS. SREBP-1c accelerates adipogenesis by inducing the expression of FAS. Leptin, which is one of the best-known hormone markers for obesity, was also downregulated following the ingestion of an HFD with 5% GLE. These findings also indicate that GLE might contain FAS or leptin inhibitors and present its efficiency against fat accumulation through this pathway in addition to adipogenesis inhibition. It has been reported that caffeic acid phenethyl ester suppresses the production of leptin during differentiation of 3T3-L1 preadipocytes [Preadipocyte differentiation is regulated by transcriptional activators including members of the C/EBP and PPARfamilies –21. Currntiation . The comSREBP-1c . C/EBPα  and aP2 , while b Figures . The mRN Figures . Interesipocytes . Therefoγ and C/EBPα are major regulators of the preadipocyte differentiation process, and C/EBPα mediates the transactivation of leptin transcription [α, which is expressed rather late in the adipogenesis process, has been widely reported to be both necessary and sufficient for the differentiation of 3T3-L1 preadipocytes to adipocytes [γ by cross-regulation [γ ligands [γ expression. SREBP-1c also reportedly binds to the promoter region of FAS to activate its transcription [γ, C/EBPα, and SREPB-1c [γ, C/EBPα, SREPB-1c, aP2, leptin, and FAS, in the epididymal fat of the GLE-treated HFD mice followed the same trend as their respective mRNA levels (Figures γ and C/EBPα decreased following the treatment with 1 and 5% GLE (Figures γ expression [PPARcription . C/EBPα,ipocytes , 27, 28 gulation . SREBP-1 ligands . The rescription . The expcription . AdditioSREPB-1c . Further Figures . Thus, t Figures . In connpression .Obesity is related to adipocyte differentiation and excess fat accumulation . In our γ, C/EBPα, and SREBP-1c. The consequent downregulation of lipid metabolizing mediators such as aP2, leptin, and FAS, which are involved in the transport, uptake, and synthesis of lipids, resulted in the reduced fat accumulation in adipocytes.Our results showed that the abdominal, perirenal, and epididymal fat weight of 5% GLE-treated mice was less than half of that of the untreated HFD-induced obese mice, which may have been due to the GLE-mediated inhibition of the mRNA and protein expression of SREBP-1c and FAS. We also demonstrated that the antiobesity effects of the GLE on various genes involved in adipogenesis, which is a differentiation pathway, are mediated via the downregulation of major transcription factors including PPARIn conclusion, the GLE strongly inhibited adipogenesis by reducing the expression of adipogenesis-related transcription factors. Therefore, the GLE may act as an effective nutraceutical for the treatment of obesity by suppressing either adipocyte differentiation or lipid accumulation.


Isatis indigotica has a long history in treating virus infection and related symptoms in China. Nevertheless, its antivirus evidence in animal studies is not satisfactory, which might be due to the lack of appropriate animal model. Previously, we had utilized restraint stress to establish mouse H1N1 susceptibility model which was helpful in evaluating the anti-virus effect of medicines targeting host factors, such as type I interferon production. In this study, this model was employed to investigate the effect and mechanism of indirubin, a natural bisindole alkaloid from Isatis indigotica, on influenza A virus susceptibility. In the in vitro study, the stress hormone corticosterone was used to simulate restraint stress. Our results demonstrated that indirubin decreased the susceptibility to influenza virus with lowered mortality and alleviated lung damage in restraint-stressed mice model. Moreover, indirubin promoted the expression of interferon-β and interferon inducible transmembrane 3. In addition, indirubin maintained the morphology and function of mitochondria following influenza A virus infection. Further study revealed that indirubin promoted interferon-β production through promoting mitochondrial antiviral signaling pathway. Our study indicated that indirubin could be a candidate for the therapy of influenza. Sarcandra glabra extract, and ReDuNing injection [Influenza, as a highly contagious disease, poses a health threat to humans. According to an online report by Centers for Disease Control and Prevention, 310 thousand people were hospitalized because of influenza-related illness during the 2015-2016 influenza season . In factnjection –11.Isatis indigotica, a traditional medicine, is widely applied in clinic in China to treat viral diseases such as hepatitis, influenza and encephalitis [Isatis indigotica. Mak and his colleagues proved the antiviral activity of indirubin against influenza by inhibiting the production of the chemokine regulated on activation, normal T cell expressed and secreted in human bronchial epithelial cells infected with influenza virus [in vitro studies, further in vivo studies are required. Indirubin also shows the anti-inflammatory effect as it has been reported to inhibit the inflammatory reactions of delayed-type hypersensitivity [phalitis , 13. Recphalitis , 14–18. za virus . Chang'ssitivity . These p−1. As shown in Figure P < 0.01), 50% (P < 0.05) and 79%, respectively. The data above indicated a role of indirubin on alleviating the morbidity of stressed mice infected with influenza virus, which was also supported by the improvement of behavioral changes, such as the altered respiration and ruffed fur. Then, we calculated the survivals and estimated the mortality of mice in each group. As shown in Figure P < 0.05). In comparison, oseltamivir, indirubin-H and indirubin-L treatment significantly improved the survival rate to 92% (P < 0.01), 64% and 57% (P < 0.05), respectively.After H1N1 infection by intranasal inoculation, body weight changes and survivals of mice in each group were monitored daily for 21 days. As illustrated in Figure th day after influenza virus challenge to detect the pathological changes. As shown in Figure −1. It increased to 11.0 ± 1.3 mg·g−1 (P < 0.01) in Virus group and further increased to 15.8 ± 4.4 mg·g−1 (P < 0.05) in “Stress+Virus” group. Nevertheless, oseltamivir, indirubin-H and indirubin-L notably decreased it to 9.7 ± 1.4 mg·g−1 (P < 0.01), 9.3 ± 4.1 mg·g−1 (P < 0.05) and 12.0 ± 1.8 mg·g−1, respectively.Severe pneumonia primarily accounts for the morbidity and mortality after influenza infection. The lung tissues were removed on the 5P < 0.01). In contrast, indirubin treatment induced an elevated production of IL-10, a pleiotropic cytokine limiting inflammatory responses, in the lung tissues of stressed mice after virus infection (P < 0.01). These results showed that indirubin navigated a balance of pro-inflammatory responses and protected against pneumonia associated with influenza infection in restraint-stressed mice.Pro-inflammatory responses are essential for the early control of influenza viral replication. However, excessive inflammation results in tissue damaging cytotoxic in the lungs . WesternP < 0.01), comparing with Virus group. However, indirubin treatment obviously reduced NP protein level (P < 0.01). Influenza virus is recognized by host sensors, which results in type I IFNs secretion [P < 0.01), comparing with Virus group. Indirubin treatment elevated MAVS expression and promoted the phosphorylation of IRF3 in the lung tissues  level indicates the replication state of influenza virus. As shown in Figure ecretion . Type I ecretion . In the ecretion . In our s Figure . On the s Figure , indicatin vitro, CORT  treatment was used to simulate stress in A549 cells and NP, IFN-β and IFITM3 protein levels were measured. The concentrations of 5 μM, 7.5 μM and 10 μM of indirubin were selected based on its influence on cell viability in normal cells and CORT-loaded cells (data not shown). Western blotting results showed that indirubin at 7.5 μM and 10 μM inhibited influenza virus replication and enhanced the expression of antiviral protein IFN-β and IFITM3   Figure . Neverth) Figure . We also) Figure .STING, a protein located in endoplasmic reticulum reticular, is well defined in the control of DNA viruses , 24. RecP < 0.01)  treatment Figure . On the ) Figure .MAVS locates the outer membrane of mitochondria and forms aggregates when it is activated. The morphology and function of mitochondria play an important role in MAVS signal transduction . Mitofusin vitro and in vivo observations demonstrated an improving effect of indirubin on reducing H1N1 susceptibility caused by stress.In clinic, elder adults are susceptible to influenza virus and 90% of deaths occur in the elder population . A recenEvidence has shown excessive inflammatory infiltrates and lung tissue injury are main factors accounting for high mortality following influenza virus infection . Our resin vivo studies, IFITM3 is found to be critical for protecting the host from influenza A virus, resulting in restricting the morbidity and mortality [κB level was reduced by indirubin treatment, pointing out that other pathways may be involved the depressing effect on NF-κB, such as Toll-like receptors or inflammasomes. Although this speculation remains to be proved, it coincides with the efficacy of indirubin on lung damage.Upon influenza virus infection, PAMPs recognize virus by PRRs and results in type I IFNs secretion . Type I ortality . In the ortality . In our Upon defining the role of MAVS-mediated IFN-β generation in the anti-virus effect of indirubin, we took deeper insights into its mechanism. For such purpose, we employed Surflex-Dock program to do molecular docking and STING was found a candidate target of indirubin , 52. STIAs the morphology and function of mitochondria are essential for the aggregation of MAVS and the interaction between MAVS and STING , 26, we Overall, results have demonstrated that indirubin decreased the susceptibility to H1N1 virus and reduced the production of pro-inflammatory cytokines to alleviate pneumonia in restraint-stressed mice. Based on both restraint-stressed mice model and CORT-loaded A549 cell model, indirubin was found to maintain the morphology and function of mitochondria to ensure IFN-β production mediated by MAVS signaling following H1N1 infection Figure Indirubin (Batch No. 110717-200204) was obtained from National Institutes for Food and Drug Control . Oseltamivir (Batch No. 0221301004) was purchased from Yichang Changjiang Pharmaceutical Co., Ltd. . CORT was purchased from Sigma .50) was measured in mice and a 2×LD50 amount was applied for the animal experiments related to influenza virus. The median tissue culture infective dose (TCID50) was measured in A549 cells and 10 TCID50 amount was applied to the in vitro experiments.Influenza A/FM/1/47 (H1N1) virus was donated by Prof. Jianxin Chen in South China Agricultural University . New viral stocks were passaged in the allantoic cavities of specific pathogen-free fertilized eggs. Then, virus fluid was kept at −80°C before use. The median lethal dose .Specific-pathogen-free male Kunming mice  were obtained from Guangdong Medical Laboratory Animal Center . The animals were raised in plastic cages with bedding material of corn straw and lived at 23 ± 2°C with a cycle of equal light and dark. The relative humidity was 50 ± 10%. Animal experiments conformed to the Animal Care and Use Committee of Jinan University , as well as the National Institute of Health's Guide for the Care and Use of Laboratory Animals (7−1·d−1 oseltamivir), and two indirubin (5 or 2.5 mg·kg−1·d−1) groups which are described as “Indirubin-H+Stress+Virus” and “Indirubin-L+Stress+Virus” respectively. Oseltamivir and indirubin were administered orally to mice for seven days, while mice in groups were orally treated with equal volume of water. On the first day of administration, mice except those in Normal and Virus groups were restricted in ventilated plastic centrifuge tubes of 50 mL for 22 h. On the 3rd day, mice were anesthetized using diethyl ether vapor and immediately infected with influenza A virus suspension (2×LD50) in PBS of 35 μL. Subsequently, the daily changes of mice in survival, body weight, typical influenza symptoms, such as hunched back, ruffled fur, altered respiration and unresponsiveness, were observed and recorded for 21 days or until death. Consequently, the data of morbidity rate and survival rate were calculated.In the first animal experiment, mice were distributed at random to six groups: Normal, Virus, “Stress+Virus”, “Oseltamivir+Stress+Virus” (30 mg·kg−1) = lung weight/body weight.The second animal experiment was also conducted in restraint-stressed mice. The effect of indirubin on pneumonia caused by influenza virus was evaluated. The animal grouping is the same as the first experiment. However, five days post virus infection, the weights of mice were recorded. Subsequently, mice were anesthetized using ether vapor and sacrificed to harvest lungs for histopathologic examination and western blotting analysis of inflammation mediators. The lungs were weighted and lung index was counted in accordance with the formula: Lung index (mg·g−1 indirubin). The following treatment was the same as the second experiment. The lung tissues were collected to determine the protein expressions related with IFN-β and MAVS signaling.The third animal experiment was conducted to investigate the effect and mechanism of indirubin on type I IFN secretion in stressed mice. Mice were distributed at random to four groups: Normal, Virus, “Stress+Virus” and “Indirubin+Stress+Virus”   containing 10% heat-inactivated fetal bovine serum (FBS) . The CO4 cells·ml−1 for experiments. After one day, the cells were cultured with CORT (100 μM) or CORT (100 μM) plus indirubin (10 μM) for 48 h and were subsequently overlaid with H1N1 influenza virus of 10 TCID50 for 45 min at 4°C, then PBS was used to clean cells for two times and serum-free DMEM was added. Eight hours post influenza virus infection, the cells were collected for the determination of protein expression.Cells were randomly divided into four groups: Normal, Virus, “CORT+Virus”, “Indirubin+CORT+Virus”. Cells were seeded onto cell culture dish in 8×10Lungs were removed and fixed in 4% paraformaldehyde with a hand-held 5 mL syringe to pump out air. Then the lung tissue was embedded in paraffin wax and sliced in 4 μm thickness. The slices were stained with H&E. Histopathologic changes were observed using ×10 objectives .For western blotting analysis, the proteins of lung samples and cell lysates lysed by RIPA buffer  were separated using SDS-polyacrylamide gel electrophoresis. Subsequently, the proteins resolved in the gel were transferred to the polyvinylidene fluoride membrane . Western blots were visualized using the ECL system  and Quantity One software  was used to analyze the intensity of individual bands. Data were normalized with β-actin as a loading control in each individual sample. The following antibodies were used in western blotting analysis: influenza virus NP rabbit pAb (1:2000), IFITM3 rabbit pAb (1:2000), IL-1β rabbit pAb (1:1200) and IL-6 rabbit pAb (1:1200) were provided by Abcam . Mfn2 mouse mAb  and IL-10 mouse mAb (1:2000) were obtained from Santa Cruz Biotechnology . β-actin rabbit pAb (1:2000) was from Bioworld . IFN-β rabbit pAb (1:500) was from OriGene Technology . p-IRF3 rabbit mAb (1:1000), NF-κB p65 rabbit mAb (1:2000) and TNF-α rabbit pAb (1:2000) were provided by Cell Signaling Technology . MAVS rabbit pAb (1:500) and STING rabbit pAb (1:500) antibodies were obtained from Proteintech Group .−1, Beyotime, Shanghai, China) was used to stain cell nuclei. Cells were observed and analyzed using a confocal laser scanning microscope .Cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton-X100 for 5 min, then either stained with a rabbit polyclonal antibody against NP  or a rabbit polyclonal antibody against MAVS  in 1:50 dilution at 4°C for 12 h. Cells were then incubated with Alexa Fluor 488 conjugated goat anti-rabbit IgG  in 1:500 dilution. 4′, 6-diamidino-2-phenylindole . Docking poses with a polar score of smaller than 0.8 and shape similarity Tanimoto coefficient of smaller than 0.7 were discarded.The Surflex-Dock program implemented in SYBYL-8.1 (http://bioinfo-pharma.u-strasbg.fr/scPDB/) was used to dock the indirubin into protein structures from the scPDB database (http://cheminfo.u-strasbg.fr/scPDB). This database contains 9283 protein structures, including 3678 unique targets . Docking50 H1N1 virus as before. Eight hours later, the cells were harvested and lysed for the determination of protein expressions.The siRNA transfection was performed using targeting sequences 5′-GAAGAGGUAUUGAAUGCUA-3′ and 5′-CUUCUCCAUAACUUACGAU-3′ against STING . A non-targeting siRNA  was used as negative control. The transfection, using lipofectamine 2000 , was carried out as described in the instructions of the manufacturer. Six hours after transfection, cells were incubated with vehicle, CORT (100 μM), or indirubin  plus CORT (100 μM) for 48 h. Cells were then overlaid with 10 TCID4 cells·ml−1 and subsequently prepared for pAcGFP1-Mito  transfection to reach 50-60% confluence. A non-targeting vector  was used as negative control. The transfection, using lipofectamine 2000 , was carried out as described in the instructions of the manufacturer. Six hours after transfection, cells were incubated with CORT or CORT plus indirubin for 48 h. Subsequently, cells were overlaid with 10 TCID50 H1N1 virus as before. Eight hours later, cells were stained with MAVS rabbit pAb in 1:50 dilution at 4°C for 12 h and then stained with Alexa Fluor 555 conjugated goat anti-rabbit IgG  in 1:500 dilution. Cell nuclei were stained using DAPI (5 μg·ml−1) . The fluorescence was analyzed using a confocal laser scanning microscope .A549 cells were cultured on glass bottom cell culture dish of 15 mm  in 8×10−1) at 37°C for 20 min. Subsequently, cells were cleaned with PBS, resuspended in 1 mL of sheath fluid and filtered through a 40 μm nylon mesh. Cells were then harvested and analyzed by detecting the JC-1 fluorescence emission using a flow cytometry .JC-1 probe  was used to detect MMP of A549 cells. After harvested, cells were incubated with the JC-1 solution (10 μg·mlThese studies complied with the recommendations on experimental design and analysis in pharmacology . All dat


Rb−/− murine retina. Deleting adenovirus E2 promoter binding factor 3 (E2f3), which rescues starburst amacrine cell differentiation, or E2f2, had no effect, but deleting E2f1, which promotes neuronal cell cycle exit and survival, restored retinal vasculature. We specifically linked cell loss to the defect because removing Bax rescued rod and bipolar neurons and the vasculature, but not cell cycle exit. Despite rescuing Rb−/− neurons, Bax deletion exacerbated a delay in outer retina lamination, and exposed a requirement for Rb in inner retina lamination. The latter resembled Sem5 or FAT atypical cadherin 3 (Fat3) mutants, but expression of Sem5/Fat3 pathway components, or that of Neogenin, which perturbs migration in the Rb−/− cortex, was unchanged. Instead, lamination defects correlated with ectopic division, and were E2f1-dependent, implicating the cell cycle machinery. These in vivo studies expose new developmental roles for Rb, pinpoint aberrant E2f1 and Bax activity in neuronal death and vascular loss, and further implicate E2f1 in defective lamination. Links between Rb, angiogenesis and lamination have implications for the treatment of neovascularization, neurodegeneration and cancer.Retinoblastoma tumor suppressor (Rb) promotes cell cycle exit, survival, differentiation, and tumor suppression in the retina. Here, we show it is also essential for vascularization and lamination. Despite minimal effects on Hif1a target expression, intraretinal vascular plexi did not form in the  In mice, the retinal vasculature consists of three interconnected parallel vascular plexi. A superficial vascular plexus (SVP) in the nerve fiber layer (NFL) develops from the optic nerve head and progresses radially to the peripheral retina between postnatal day 0 (P0) and P8. Subsequently, vessels sprout vertically into the retina. Around P7, sprouting vessels descend and advance into the outer plexiform layer (OPL) to establish the deep vascular plexus (DVP). Around P11, the DVP vessels ascend into the inner plexiform layer (IPL) and form the intermediate vascular plexus (IVP)4. Previous studies revealed that Frizzled-4 (Fzd4), Lrp5, Norrin, and Tetraspanin 12 (Tspan12) are required for intraretinal vascular development6, and retinal Hif1a is required for IVP development7. Retinal neurons form the neurovascular unit to interact with endothelial cells8. Retinal ganglion cells (RGCs) are essential for the SVP development10, photoreceptors are important for the development of the intraretinal vascular plexus12, and amacrine and horizontal cells are critical for developing and maintaining the intraretinal vasculature4.Angiogenesis is a critical step in development and disease and is regulated by pro-angiogenic and anti-angiogenic factors13. The Rb/E2f pathway plays critical roles in angiogenesis1. For example, Rb binds Hif-1α and enhances its transcriptional activity14. Atypical E2fs (E2F7/8) bind Hif-1α to stimulate Vegfa induction15. As such, deletion of Rb1, or E2f7/8, or Hif1 in mice all result in vascular defects in the placenta and early embryonic lethality18.The retinoblastoma tumor suppressor (Rb) plays a major role in regulating cell cycle and other cellular processes by interacting with adenovirus E2 promoter binding factors (E2fs)19, such as Dscam21, FAT atypical cadherin 3 (Fat3)22, semaphorins (Sema), and plexins23. Proper development of retinal lamination is important for physiological retinal responses and function. Recent studies have revealed that Rb can regulate neuronal migration and cortical lamination25.The retina comprises three distinct nuclear layers , separated by two synaptic layers (OPL and IPL). Retinal lamination is guided by many different cuesα-Cre)-mediated Rb gene knockout (KO) in mouse retina cause ectopic cell division, cell death, and differentiation defects27. α-Cre is active from embryonic day 10 (E10) in the peripheral progenitors of the temporal and nasal retina29. Employing this model we now implicate Rb in formation of the two intraretinal vascular plexi, as well as lamination of the outer and inner retina. We show that the angiogenesis defect is related to E2f1 and Bax-induced retinal cell death, whereas the lamination defects are cell death-independent and instead correlate with ectopic cell division.Previously we reported that Pax6 alpha enhancer Cre  control), and the WT areas of Rbf/f;α-Cre retinas  cells were found in the NFL, IPL, and OPL, corresponding to the SVP, IVP, and DVP, respectively. However in RbKO retinal sections, IB4+ cells were only found in the NFL, but not in the IPL and OPL  and GCL in WT and Rb−/− retinas  from the microarray data of P8 RbKO retinas (Gene Expression Omnibus (GEO) accession: GSE86372)32. Gene enrichment analysis by Enrichr34 indicated that the most enriched pathways of RbKO-related DEGs included the cell cycle and DNA replication, consistent with Rb function, and phototransduction, explained by rod death, but not the Hif1 or Vegf pathways  differentiation, and while E2f2 does not cause defects in the RbKO retina, it mediates the ectopic division and death of cones of Rb/p107 double KO (DKO) retina39. To define whether any E2f contributes to the vascular defects, we crossed Rbf/f;α-Cre mice with E2f1−/−, or E2f2−/−, or E2f3f/f mice  than outer retina (photoreceptors).Next, we defined which death-prone bly Fig. . Rhodopsbly Fig. . Thus, BBaxKO affects the disrupted vasculature of the Rb−/− retina. Strikingly, we observed both IVP and DVP in the P18 Rb/Bax DKO retina 45 45 Fig. .Fig. 6LaWT or BaxKO retina, SV2A+ synapses were disorganized in the RbKO or DKO retinas  horizontal cells were almost superimposed, but it remained disorganized in the DKO tissue, even though positioning of the cone and horizontal cells was restored  induction causes migration and lamination defects40. However Neo1 was not induced in the Rb−/− retina , which plays important roles in neuronal migration and lamination by phosphorylating doublecortin (Dcx)ed Figs.  and 6b. lls Fig. . In P8 Rlls Fig. . These dRbKO or BaxKO retinas  in P8 DKO retinas, which split the INL Figs. . It was nas Fig. . Excess pes Fig. , retinallls Fig. . Many amlls Fig. . A similMPL Fig. , arrows.Rb/Bax DKO and Fat3 or Sem5 KO retinas, we assessed the mRNA levels of Fat3 and Sem5A & B, as well as those of Dachsous (Dchs), a Fat3 ligand, and Four jointed box 1 (Fjx1), which modifies Fat activity. The levels of Dchs, Fat3, and Sema5A were not different between WT, RbKO, BaxKO, and DKO retina at three different time points, and while Fjx1 and Sema5B were slightly increased in DKO retinas, this was also the case in the BaxKO control which has no OMPL , Rbf/f mice (A. Berns), E2f1–/– mice (M. Greenberg), E2f2–/– mice (G. Leone), E2f3f/f mice (G. Leone), Z/Red , and Bax−/−  were maintained on a mixed (NMRI × C57/BL6 × FVB/N × 129sv) background.Mice were treated according to institutional and national guidelines. All procedures were performed in compliance with the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and visual research. Bax-null males are infertile, to generate Rbf/f;Bax−/−;α-Cre mice we first generated Rbf/f;Bax+/−;α-Cre males and Rbf/f;Bax−/− females, and inter-bred them. Of 96 pups we obtained 50 with α-Cre and 48 with Bax−/− alleles as predicted, but only 5 pups (5.2%) with Bax−/−;α-Cre, far less than the expected 24 pups , and oIMR7338 and oIMR7339 for internal positive control (324 bp). The primers for genotyping Bax−/− mice are primers oIMR0661 and oIMR0662 for mutant allele (507 bp), and oIMR0661 and oIMR0663 for WT allele (304 bp). The sequences of the above primers are listed in Jackson Laboratory genotyping protocols (www2.jax.org).Mice of different genotypes were compared within the same litter and across a minimum of three litters. We have not noted any phenotypic differences in separate litters. Genotyping was performed as before27. Primary antibodies or labeled cells were visualized using donkey anti-mouse, donkey anti-rabbit, and donkey anti-goat antibodies conjugated with Alexa-488, Alexa-568, or Alexa-647 . Nuclei were counter-stained with 4′, 6-diamidino-2-phenyindole  and mounted with Mowiol mounting medium.Eyeballs were fixed in 4% paraformaldehyde for 1 h at 4 °C, embedded in OCT (TissueTek 4583), frozen on dry ice, and cut into 12–14 µm sections on Superfrost slides. For immunohistochemistry, the retinal sections were dried at room temperature and incubated in blocking solution ) for 1 h, then were incubated with primary antibodies such as active caspase-3 , Ap2α , Brn3 , Calbindin , Calretinin , Cdk5 , Cone arrestin , cyclin E , Dcx , glutamine synthetase , Ki67 , M opsin , Onecut2 , phospho-histone H3 , protein kinase Cα , rhodopsin , SV2a , and Syt1 . Vascular endothelial cells were labeled by fluorescein isothiocyanate (FITC)-conjugated isolectin B4 . Antigen retrieval was performed as described by boiling sections in citric acid For whole-mount staining, eyeballs were enucleated and incubated for 30 min in 4% paraformaldehyde in PBS. With a dissection microscope, a circumferential incision was made around the limbus, followed by removal of the anterior segment, lens, and vitreous body. The retinas were incubated at 4 ℃ with FITC-conjugated isolectin B4  and DAPI in PBS for 1–2 days. After briefly washing with PBS, radial cuts were made to divide the retina into four quadrants to flatten the retina, and flat retinas were mounted with Mowiol.https://imagej.nih.gov/ij/) was used for cell counting following the online guide. The positive cells of active caspase-3, Ki67, and cell-type markers  were counted manually. The thickness of ONLs was measured by the microscope program. For vascular blood vessel analysis, representative images were analyzed using the AngioTool software (https://ccrod.cancer.gov/confluence/display/ROB2/Home) to quantify the vessel coverage , average vessel length, and lacunarity (distribution of the gap sizes surrounding the object).Stained sections and slides were analyzed using a Zeiss Axio Imager Z2 fluorescence microscope and Nikon C1si confocal microscope. Image J 1.50b with cell counter plugin (http://www.ncbi.nlm.nih.gov/geo/) was used to compare WT vs. RbKO mouse P8 retinas, which include three WT and three RbKO mouse P8 retinas. The data were analyzed by GEO2R from the GEO website. The genes, of which expression fold changes are >2 or <0.5, and adjusted p < 0.05 were selected as the RbKO-related DEGs. Totally 677 DEGs were identified. The heatmap was generated using Heatmapper55. The function enrichment of DEG was performed using Enrichr34, the pathways with adjusted p < 0.05 were chosen to report.The dataset GSE86372 at NCBI GEO database  to remove DNA contamination. First-strand cDNA was synthesized from 0.2–0.5 µg of total RNA using the RT reagent kit with gDNA Eraser  or SuperScript II first-strand synthesis system (Invitrogen). PCR primers are listed in Supplementary Table Total RNA was isolated from dissected peripheral retina using the TriPure isolation reagent  or RNeasy mini kit (Qiagen) followed by digestion with RNase-Free DNase . The results were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni correction for multiple comparisons. The threshold for significance was set at Supplementary table 1


Chrysanthemum morifolium. However, the mechanism underlying the response to waterlogging and post-waterlogging reoxygenation in C. morifolium remains unknown. In this study, we compared the differences between the transcriptomes of two chrysanthemum cultivars, i.e., the waterlogging-tolerant cultivar “Nannongxuefeng” and the waterlogging-sensitive cultivar “Qinglu”, by performing RNA-seq to elucidate the possible mechanism of waterlogging and reoxygenation in C. morifolium. “Nannongxuefeng” had a higher ethylene production under the waterlogging and reoxygenation conditions. Furthermore, the expression of transcription factors and genes that are involved in the hormone response, N-end rule pathway and ROS signaling significantly differed between the two cultivars. “Nannongxuefeng” and “Qinglu” significantly differed in their response to waterlogging and reoxygenation, providing a deeper understanding of the mechanism underlying the response to waterlogging and guidance for the breeding of C. morifolium.Waterlogging stress is among the most severe abiotic stressors in the breeding and the production of  Water is crucial for plant growth and development. However, many plants are often subjected to flooding stress due to excess water remaining in soils. Flooding stress is among the most widespread abiotic stressors during the lifespan of plants . FloodinPDC), alcohol dehydrogenase (ADH), and sucrose synthase (SUS), are involved in sugar metabolism and are significantly up-regulated during the waterlogging process [SOD), catalase (CAT), and ascorbate peroxidase (APX), are impaired under waterlogging stress due to the disturbed balance of ROS generation and removal [When oxygen is limited, aerobic respiration is suppressed in the roots . Anaerob process ,6,7,8,9. process ,12,13,14 removal ,16,17,18Arabidopsis thaliana, waterlogging induces the expression of AtACS5, AtACS7, and AtACS8 [RpACS1 in Rumex palustris, and enzyme activity assays have shown that the activity of ACS is significantly higher in flooding plants than that in non-flooded plants [Rumex palustris can be inhibited by Aminoethoxyvinyl glycine (AVG), which is an inhibitor of ethylene biosynthesis [Ethylene is a pivotal hormone in the response to waterlogging in plants. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is produced in the plant roots and is transported to the xylem, where it is oxidized to ethylene to mediate waterlogging tolerance in plants . Ethylend plants . Howeverynthesis .SNORKEL1 (SK1)/SNORKEL2 (SK2) and SUB1A and have been extensively investigated [SK1/SK2 expression and it then activates the gibberellin (GA) pathway to promote the elongation of the rice internode, thus enabling the rice to escape the flooding environment [Sub1A-1 is induced by ethylene [Arabidopsis VII ERFs, including the five members RAP2.12 (AtERF75), RAP2.2 (AtERF74), RAP2.3 (AtERF72), HRE1 (AtERF73), and HRE2 (AtERF71) [Arabidopsis plants with 35S::RAP2.12 are more tolerant to submergence conditions [Arabidopsis mutants, i.e., rap2.12, rap 2.2, and rap 2.3, all show a more sensitive phenotype than wild type Arabidopsis following submergence stress [PDC1 and ADH1, in order to enhance hypoxia tolerance in Arabidopsis [ERF transcription factors play an important role in ethylene signal transduction . ERF trastigated ,28,29,30ironment . In the ethylene ,33 and iethylene . The AraAtERF71) , are regAtERF71) . Transgenditions . Three Ae stress . Stabilie stress . The conbidopsis ,40.Arabidopsis, hypoxia stress results in a 50% reduction in polysome content, and this change can be reversed by reoxygenation for 1 h [Arabidopsis [Reoxygenation refers to the recovery period following the removal of hypoxia stress, and it is closely interrelated with waterlogging stress . However for 1 h . In rice for 1 h . Reoxygebidopsis . Moreovebidopsis . Based oChrysanthemum is a leading ornamental species that is widely popular worldwide . HoweverHere, we compared the morphological changes in two different cultivars, i.e., “Nannongxuefeng” and “Qinglu”, following 6 day of waterlogging stress. No morphological changes were observed in “Nannongxuefeng”, which displayed green leaves and an upright stem C,D. HoweThe ethylene production was measured 0 h, 3 h, 6 h, 12 h, and 24 h after waterlogging and after 2 h of reoxygenation after 12 h of waterlogging of the roots. The following four groups were assessed: control, waterlogging, waterlogging + ACC and waterlogging + AVG. The ethylene production in “Nannongxuefeng” was higher than that in “Qinglu” at each time point in all groups regardless of the waterlogging stage or reoxygenation stage . During To investigate the differences in the mechanisms of the response to waterlogging and reoxygenation between the sensitive and tolerant cultivars, ten libraries from the root tissues of the cultivars, i.e., Q (“Qinglu”) 0 h, QCK (control) 12 h, QW (waterlogging) 12 h, QCK 12 h + 2 h, and QW 12 h + 2 h (waterlogging 12 h followed by 2 h reoxygenation recovery), X (“Nannongxuefeng”) 0 h, XCK 12 h, XW 12 h, XCK 12 h + 2 h, and XW 12 h + 2 h, were generated using Illumina HiSeq. After filtering the raw reads, we obtained 67.01 Mb, 65.06 Mb, 65.43 Mb, 65.43 Mb, 65.97 Mb, 65.48 Mb, 65.48 Mb, 65.02 Mb, 65.52 Mb, and 67.03 Mb of clean reads, containing 6.70 Gb, 6.51 Gb, 6.54 Gb, 6.54 Gb, 6.60 Gb, 6.55 Gb, 6.55 Gb, 6.50 Gb, 6.55 Gb, and 6.70 Gb clean bases, respectively. Of the ten libraries of clean reads, Q20 were above 97.31% and Q30 were above 93.36% . The cleThe numbers of transcripts in the libraries from Q 0 h, QCK 12 h, QW 12 h, QCK 12 h + 2 h, QW 12 h + 2 h, X 0 h, XCK 12 h, XW 12 h, XCK 12 h + 2 h, and XW 12 h + 2 h were 154,416, 76,075, 134,241, 149,040, 70,777, 149,070, 151,868, 148,994, 147,947 and 50,049, respectively . The totTo compare the differentially expressed genes (DEGs) between the two cultivars in response to waterlogging and reoxygenation, we separated the up-regulated and down-regulated genes in the four groups  . The numVenn diagrams of the abovementioned DEGs during the waterlogging and reoxygenation stages were also performed B,C. In cTo deeply understand the DEGs between the two cultivars in response to waterlogging stress and reoxygenation, the DEGs were further investigated by performing a GO functional analysis. In the Q 0 h vs. X 0 h comparison, 6921 DEGs were assigned a GO term. Nine GO classes significantly fell into the category ‘cellular component’, 8 GO classes significantly fell into the category “molecular function”, and 8 GO classes significantly fell into the category ‘biological process’ . In the A KEGG pathway analysis of the abovementioned DEGs was also performed. In the Q 0 h vs. X 0 h comparison, 7623 DEGs were assigned to 135 pathways in the KEGG database. “DNA replication”, “Isoquinoline alkaloid biosynthesis”, “Tyrosine metabolism”, and “Mismatch repair” were the most significantly enriched terms . In the AP2/ERF, bHLH and MYB families. A heatmap analysis of 11 AP2/ERFs, six bHLHs, and five MYBs was performed  and ERF (Unigene18230) were induced more strongly in “Nannongxuefeng” than in “Qinglu” at 12 h of waterlogging (DREB2A (CL1489.Contig6), AIL5 (Unigene31729), ERF017 (Unigene42092), DREB1D (CL16775.Contig2), and ERF109 (Unigene30139 and Unigene22576) were more suppressed in “Nannongxuefeng” than in “Qinglu” during the reoxygenation stage , bHLH130 (Unigene35505 and CL2224.Contig1), and SPATULA (CL19413.Contig6) were all more up-regulated in “Nannongxuefeng” than in “Qinglu” during the waterlogging stage and down-regulated during the reoxygenation stage (bHLH25 (CL1188.Contig3) and bHLH63 (CL17646.Contig1) were induced more strongly in “Qinglu” than in “Nannongxuefeng” at 12 h of waterlogging , MYB86 (Unigene1394), and MYB5 (Unigene26544) were more up-regulated in “Nannongxuefeng” than in “Qinglu” at 12 h (MYB-related genes (Unigene4726 and Unigene26622) were more down-regulated in “Nannongxuefeng” than in “Qinglu” during the reoxygenation stage , ACS1 (CL4253.Contig1), ERF1 (Unigene42398), ACS6 (CL15412.Contig4), and ACS7 (Unigene24656) were significantly up-regulated at 12 h of waterlogging and down-regulated after 2 h of recovery  was suppressed by waterlogging for 12 h and was up-regulated by reoxygenation for 2 h in “Nannongxuefeng”, while ERF2 remained stable in the three samples of “Qinglu”  and TGA1 (Unigene18225), the other six DEGs were more up-regulated in “Qinglu” than in “Nannongxuefeng” at 12 h of waterlogging stress (ARF19-like (Unigene41775), ARF7-like (Unigene38340), and ARF5 (Unigene31182) were more down-regulated in “Nannongxuefeng” than in “Qinglu” during the reoxygenation stage (GRAS4 (Unigene35562) and GID1C-like (Unigene16747), were more up-regulated in “Nannongxuefeng” than in “Qinglu” during the waterlogging stage at 12 h of waterlogging  were also identified , IAA (Auxin), JA (Jasmonic acid), and GA (Gibberellin), were also identified. Eight DEGs were involved in SA signaling, and except for g stress . Among ton stage . Auxin ton stage . Two GA-rlogging . Three Jentified .ERFs and other N-end rule pathway genes , RAP2.3 (CL4854.Contig1), RAP2.12 (CL13652.Contig1), ATE1-like (CL11416.Contig1), PCO1 (CL4000.Contig4), and PCO2 (CL16190.Contig1) were up-regulated at 12 h of waterlogging stress and down-regulated after 2 h of reoxygenation in “Qinglu” and “Nannongxuefeng” , were continuously down-regulated after waterlogging and recovery  and AOX1a (CL9414.Contig3) were more up-regulated by waterlogging of 12 h in “Nannongxuefeng” than in “Qinglu” (CAT (CL3646.Contig2), CSD1 (CL8215.Contig1), and APX1 (CL5448.Contig2), and were more up-regulated during reoxygenation stage in “Nannongxuefeng” than in “Qinglu” (RBohC (Unigene18044) was more up-regulated in “Qinglu” than in “Nannongxuefeng” during waterlogging stage , three ADHs , and SUS1 (CL20525.Contig1) were significantly up-regulated at 12 h of waterlogging and were down-regulated after 2 h of recovery in the two cultivars , autophagy-related protein 18a , and multiprotein-bridging factor 1c  were more up-regulated in “Nannongxuefeng” than in “Qinglu” at 12 h of waterlogging , two-on-two hemoglobin (CL9173.Contig2), ADH (Unigene15445), SUS1 (CL20525.Contig1), POD (CL15514.Contig3), v-type proton ATPase (CL15644.Contig2), ubiquitin-conjugating enzymes (Unigene35809), and a zinc finger protein (Unigene56291). The final qRT-PCR results of the other DEGs were highly consistent with the RNA-seq data , were down-regulated under the waterlogging condition  is strongly induced in the roots under the hypoxia condition in wheat [HYPOXIA RESPONSE ATTENUATOR1 (HRA1) is a trihelix family TF that is highly up-regulated under hypoxic conditions and it acts as a negative regulator of the key hypoxia transcription factor RAP2.12 [HRA1 negatively regulates the activation of its own promoter [DREB2A, ERF017, ERF109 (Unigene30139 and Unigene22576), DREB1D, AIL5, and two MYB-related proteins were more strongly suppressed during the reoxygenation stage in “Nannongxuefeng” than “Qinglu” (ERF109 (Unigene27829), ERF (Unigene18230), bHLH93 (Unigene49514) , bHLH130 (Unigene35505 and CL2224.Contig1), SPATULA, MYB59, MYB86, and MYB5 were more strongly induced by waterlogging in “Nannongxuefeng” than in “Qinglu” , ARF19-like (Unigene41775) were more induced in “Qinglu” than in “Nannongxuefeng” during the waterlogging stage (ARF7-like (Unigene38340) and ARF5 (Unigene31182) were more suppressed by waterlogging in “Nannongxuefeng”  in waterlogged Rumex plants . Higher on of AR . The trepersicum . In thisng stage . Auxin tng stage . Howeverxuefeng” , suggestArabidopsis, AtERF1 and AtERF2 are induced by various abiotic stresses, such as drought, salt, and cold [AtERF1 and AtERF2 function as transcription activators and bind the GCC-box, thereby meditating down-stream gene expression [CmERF1 was induced by the waterlogging stress and was suppressed by reoxygenation , TGA1 (Unigene18225), GRAS4 (Unigene35562), GID1C-like (Unigene16747), and LPPD (Unigene29271) were more strongly induced in “Nannongxuefeng” than in “Qinglu” under the waterlogging condition (TGA1 (Unigene23794 and Unigene26068), TGA1-like (Unigene26946 and CL18253.Contig3), TGA4-like (Unigene25344 and Unigene4634), and BAK1-like were more highly induced in “Qinglu” than in “Nannongxuefeng” under the waterlogging condition  [TGAs likely play a different role via SA signaling in waterlogging in chrysanthemum. Waterlogging and reoxygenation exert great impacts on the expression of hormone signaling and biosynthesis.The crosstalk among ethylene, JA, SA, and other hormones in response to multiple abiotic stresses has been demonstrated ,66,67,68ondition . Howeverondition . A recenPODs and AOX1a were more significantly induced by waterlogging in “Nannongxuefeng” than in “Qinglu”  mixture of perlite, vermiculite and leaf mold. The rooted seedlings were grown in a greenhouse under constant conditions of 22 °C during the day and a minimum of 15 °C during the night. The relative humidity varied from 70 to 75% (m/m), and no artificial light was provided. The healthy, uniform cuttings were transplanted into a small pot  containing a 3:1:1 mixture of garden soil, perlite and vermiculite until the plants developed 10–12 leaves. Then, all of the plants were transferred to the culture room under the 16 h photoperiod (80–100 μmol/m2/s illumination) at 22  ±  1 °C conditions for waterlogging treatment.The “Qinglu” and “Nannongxuefeng” Chrysanthemum cultivars were obtained from the Chrysanthemum Germplasm Resource Preserving Center of Nanjing Agricultural University, China. All of the plants were propagated with cuttings. The medium contained a 2:2:1 , and the ethylene content was measured, as previously described . At leasThe abluent “Qinglu” and “Nannongxuefeng” root samples were immediately collected at 0 h, 12 h, and 12 h + 2 h, snap-frozen in liquid nitrogen and stored at −80°C. Each sample was 0.5 g and three biological replicates were performed. The total RNA was isolated from each sample using the RNAiso Plus reagent  following the manufacturer’s protocol. The quality and concentration of each RNA sample were verified using an ND-1000 Spectrophotometer , and only RNA samples delivering the OD260/280 ratio of 1.8–2.1 and the OD260/230 ratio > 1.8 were retained. A 3 μg pool of RNA was formed by combining 1 μg from each biological replicate that was used for the construction of libraries.The total RNA was treated with DNase I , and Oligos (dT) were used to isolate the mRNA. The mRNA was fragmented using a fragmentation buffer. Then, the first cDNA strand was synthesized using the mRNA fragments as templates, which was achieved by random hexamer priming. The second cDNA strand was synthesized by a reaction that was driven by DNA polymerase I . The short double-stranded DNA (dsDNA) fragments were purified using a QiaQuick PCR Extraction Kit  and resolved with EB buffer for the end repair and single nucleotide A (adenine) addition. Sequencing adapters were ligated onto dsDNAs and suitable fragments were selected for the PCR amplification.During the quality control steps, an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System were used for the quantification and qualification of the sample library. The libraries were paired-end sequenced using an Illumina HiSeq 2000 system. In total, ten sets of raw reads corresponding to Q (“Qinglu”) 0 h, QCK (control) 12 h, QW (waterlogging) 12 h, QCK 12 h + 2 h and QW 12 h + 2 h (waterlogging 12 h followed by 2 h reoxygenation recovery), X (“Nannongxuefeng”) 0 h, XCK 12 h, XW 12 h, XCK 12 h + 2 h, and XW 12 h + 2 h were obtained.http://blast.ncbi.nlm.nih.gov/Blast.cgi) by default parameters, the unigenes were assigned functions based on the homologs present in the NR (www.ncbi.nlm.nih.gov/refseq), NT (www.ncbi.nlm.nih.gov/nuccore), Swiss-Prot (www.uniprot.org), KEGG (www.genome.jp/kegg/kegg1.html), COG (www.ncbi.nlm.nih.gov/COG/), and GO (geneontology.org) databases [https://www.blast2go.com) by default parameters with NR annotation to get the GO annotation [2 fold change| ≥ 1, based on the PossionDis method [Clean reads were obtained from the raw reads after filtering the low-quality reads, adaptor-polluted reads, and reads with a high content of unknown bases (N). After the filtering, the remaining clean reads were stored in the FASTQ format. The de novo assembly using the clean reads was performed using Trinity to obtain the unigenes. Using BLAST+ (v2.2.23) software (atabases . Using Bnotation . The expnotation . The DEGs method . The datEF1α gene (GenBank accession number KF305681) was used as a reference, and the gene primers are listed in Eight DEGs were selected from all DEGs to validate the reliability of the libraries. The primers that were used for these validations were designed using Primer 5.0 software, and qRT-PCR was performed, as previously described by Ren et al. . The chr


TGFβ1 expression is associated with survival and proliferation markers in patients with early-stage PDAC. We acquired clinicopathologic, treatment, and mRNA expression data from The Cancer Genome Atlas data set for 106 patients identified with stage I/II PDAC who underwent pancreaticoduodenectomy. Patients were categorized as high expression when mRNA expression was ≥75th percentile for each gene. Average log2 mRNA expression of TGFβ1 in patients with high expression was 11.6 ± 0.2 and 10.5 ± 0.6 in patients with low expression (P<0.001). Low TGFβ1 expression is associated with shorter median survival compared with high TGFβ1 expression . Patients with tumors demonstrating high MKI67 (the gene encoding Ki-67) expression have shorter median survival versus those with lowerMKI67 expression . TGFβ1 and MKI67 are inversely associated (P=0.009). On multivariate analysis, improved survival is associated with TGFβ1 overexpression (P=0.017), adjuvant chemotherapy (P=0.001), and adjuvant radiotherapy (P=0.017), whereas positive surgical margins are associated with worse survival (P=0.002). In patients who undergo pancreaticoduodenectomy for PDAC, high TGFβ1 expression may counteract the worse survival associated with high proliferation.The role of transforming growth factor beta-type-1 (TGFβ1) in pancreatic ductal adenocarcinoma (PDAC) progression is stage-dependent. We hypothesized that  TGFβ expression in early tumors (AJCC stage I and II) is a favorable characteristic but a detrimental characteristic in advanced-stage III or IV disease [The precise role of transforming growth factor beta type 1 (TGFβ1) in the progression of pancreatic ductal adenocarcinoma (PDAC) is unknown, and its relation to survival is context dependent . This is disease .TGFβ is a complex signaling molecule that exists in 3 isoforms and as part of large family of polypeptides . AlthougTGFB1 and RNA expression of the proliferation marker Ki-67 in PDAC.Although the role of TGFβ has been better delineated in late-stage PDAC, its role in early-stage PDAC is unclear. In late-stage PDAC, targeted therapeutics against the TGFβ pathway are undergoing clinical trials as data are more mature in this area  and suggTGFβ is both a proto-oncogene and tumor suppressor gene, depending on the context [Because of its complex signaling cascade, it has been suggested that  context , 13. Wor context , 10.-mediated carcinogenesis is an overwhelming, proliferative peritumoral desmoplastic response [in vivo data have suggested that collagen deposition by pancreatic stellate cells and pancreatic carcinoma cells both lead to a pro-carcinogenic desmoplastic response [COL1A2, has been associated with proliferative events and markers of aggressive tumor biology, including epithelial to mesenchymal transition [COL1A2) may be a serum biomarker for pancreatic carcinoma [A potential mechanism for TGFβ1response . In a noresponse , 15, 16.response –19. In aresponse –22. Inteansition . AnotherTGFβ1 and MKI67 expression (the gene encoding the proliferation marker Ki-67). To control for unknown features of cancer biology, we utilized a strict inclusion/exclusion criteria to identify a relatively homogenous patient population from The Cancer Genome Atlas (TCGA) with stage I and II PDAC who underwent pancreaticoduodenectomy.We hypothesized that survival in patients with resected early-stage PDAC is associated with Our TCGA data set consisted of 175 tumor samples with pancreatic malignancies. Five of these patients demonstrated histologies not consistent with “typical” PDAC to include neuroendocrine and mucinous colloid carcinomas. Likewise, a minority of patients underwent distal pancreatectomy, total pancreatectomy, or other pancreatic resection (n = 38) and 10 patients had stage III or IV disease.After applying the inclusion and exclusion criteria, 106 patients were identified for analyses. Of these patients, the average age was 65 ± 11 years, 58% of patients were male, and 91% were white. The median follow-up time was 15 months . Seven patients (7%) had a documented family history of PDAC. Only 1 patient received neoadjuvant therapy. In our patient group, 13%, 58%, and 28% of tumors were histologically grade I, II, and III/IV, respectively. The mean number of lymph nodes examined was 18 ± 8 per patient with an average of 3 ± 3 positive lymph nodes per patient. Overall, 60 patients (57%) received a margin-negative (R0) resection, while 56 patients received margin-positive resections (R1 or R2).TGFβ1 in the top quartile (high expression group) was 11.6 ± 0.2, whereas it was 10.5 ± 0.6 in patients with low expression . All long-term survivors (> 40 months) were in the high TGFβ1 expression group. The univaraiate Cox proportion hazard ratio (HR) for high TGFβ1 group was 0.35 .The clinical and treatment characteristics for patients with tumors expressing high or low levels of TGFβ1 (each P > 0.1).Interestingly, high mRNA expression of SMAD3, SMAD4, and TGFβR1 were not associated with survival . Likewise, there was no association between the mRNA expressions of these genes with expression of MKI67 expression ≥75th percentile have shorter median survival versus patients with tumors demonstrating lower MKI67 expression . Furthermore, 32% of patients with low tumor TGFβ1 expression have high MKI67 expression, whereas only 7% of patients in the high TGFβ1 expression group have high MKI67 expression (P = 0.009).Because Ki-67 is an established marker for cell proliferation in cancer, we investigated its mRNA expression in PDAC. Patients with tumors demonstrating TGFβ1 and MKI67 expression, we investigated survival in patients by gene expression status . All long-term survivors were in the high/low group.Because there was correlation between us Table . The medTGFβ1 expression remained associated with longer survival . Neither MKI67 expression nor COL1A2 expression was independently associated with survival on multivariate Cox regression .On multivariate Cox regression analyses, high COL1A2, a component of collagen production, is associated with high TGFβ1 expression. We found that 51% of tumors in the high TGFβ1 expression group had high COL1A2 expression, whereas only 24% of tumors in the low TGFβ1 expression group had high COL1A2 expression (P < 0.001). Furthermore, median survival was significantly lower in the low COL1A2 expression group . In the low TGFβ1 expression group, high COL1A2 expression was associated with improved survival . There was no association between MKI67 and COL1A2 expression .Overexpression of TGFβ1 expression, high MKI67 expression, high COL1A2 expression, and positive lymph node spread were all statistically associated with survival (Table P = 0.01), whereas radiotherapy was not significantly associated with longer survival in margin-negative (R0) resection .Of the 106 patients, the overall Kaplan-Meier median survival was 19 months. On univariate analyses, age, positive surgical margins, chemotherapy, radiotherapy, high The TGFβ1 signaling pathway activates transcription factors intimately involved in many normal and benign functions that, when commandeered by PDAC, permit the cancer to be refractory to standard therapy. For example, fibrosis, a critical response to wound healing, also provides PDAC with a mechanical barrier to chemotherapeutic delivery , 24. AddTGFβ1 expression may be able to ameliorate the decreased survival associated with highly proliferative tumors that have high levels of Ki-67 mRNA. Alternatively, high levels of Ki-67 may overwhelm tumor suppressor effects of TGFβ1. Importantly, in the multivariate analysis, MKI67 expression was no longer significant when analyzed in conjunction with the other variables in the model, specifically TGFβ1 and radiotherapy, a treatment that targets proliferating cells. High TGFβ1 expression remained statistically significant and has the greatest association with survival except for family history of PDAC and positive lymph node disease  to aberrant TGFβ1 signaling pathways.We investigated the role of for PDAC , 19, 26.for PDAC , 24, we MKI67 expression and TGFβ1 levels. However, these data can serve to outline future research targets, specifically the interaction between cellular proliferation and TGFβ1 pathways. Furthermore, we attempted to limit the effects of the TGFβ1 paradox by using strict inclusion/exclusion criteria to identify patients with “early” disease. However, there is no method to objectively define this patient population other than the self-fulfilling prophecy of investigating patients with only stage I and II PDAC who went to resection. This is selection bias is difficult to quantify.While analyzing a large data set such as the TCGA is quite useful to investigate the interaction of gene expression and clinical data, there are some significant limitations. The major limitation of this analysis is the inability to interrogate tissue specimens or use patient-derived samples to test hypotheses. Specifically, correlating TGFβ1 serum levels, TGFβ1 tissue levels, and clinical outcomes is a future research target to help exact the relationship between TGFβ1 expression is high, which may be due to limited cellular proliferation. Further work is needed to delineate the mechanism of this interaction, investigate it in patients with advanced PDAC, and identify opportunities to modulate the intra- and extracellular TGFβ1 signaling cascade to improve survival and decrease PDAC proliferation.In summary, our study demonstrates the potential role of proliferation to help understand the TGFβ1 paradox. Patients with early-stage PDAC have longer median survival when Data from TCGA were downloaded on February 5, 2016. Clinical data, tumor DNA sequence, and mRNA expression data were acquired from the TCGA data set from the data access committee. Except for a single patient, this represents an untreated patient population with PDAC who went directly to surgery for resection. After resection, tumor specimens were dissected from surrounding stroma. mRNA was sequenced with next generation sequencing, usually on the Illuminia platform, as part of the TCGA network with quality controls in place at multiple steps. The TCGA project has been well-described elsewhere , but briInclusion criteria were complete data available, pancreatic adenocarcinoma or ductal carcinoma histology, American Joint Committee on Cancer (AJCC) stage I or II, and underwent pancreaticoduodenectomy. Patients with stage III or IV disease based on the 7th edition of the AJCC staging system were excluded.https://tcga-data.nci.nih.gov/) open access HTTP directory. Gene expression data were further normalized against the median sample using IRON v2.1.6 (iron_generic --rnaseq --unlog) to correct for the remaining minor differences in dynamic range between samples [2 mRNA expression value for each gene. Low gene expression was based on values < 75th percentile log2 mRNA expression value for each gene. We specifically investigated the relationship among TGFβ1, MKI67, and COL1A2 and survival in patients with resected PDAC in the head of the pancreas and staged as I or II.Patient clinical data (Level 2.0.46.0) and RNASeqV2 normalized gene expression data (Level 3.1.8.0) were downloaded from the TCGA data portal . The multivariate survival model was based on clinical and gene expression data as described below utilizing a cut-off of univariate P = 0.1 to be included in the model.Data analyses were performed with STATA 13 . Significance was set to α = 0.05. Differences in variables between the two groups were investigated with Student


Coronary heart disease (CHD) is an important complication of atherosclerosis. Biomarkers, which associate with CHD development, are potential to predict CHD risk. To determine whether genes showing altered expression in hyperlipidaemia (H) and coronary heart disease (CHD) patients compared with controls could be CHD risk biomarkers.Control, H, and CHD groups represented atherosclerosis to CHD development. Gene profiling was investigated in peripheral blood mononuclear cells using DNA microarrays. Eight selected genes expressed only in H and CHD groups were validated by real-time quantitative reverse transcription PCR and plasma protein determination.DEFA1/DEFA3), pro-platelet basic protein (PPBP), and beta and alpha2 hemoglobin mRNA expression was significantly increased in H and CHD groups compared with controls, but only plasma PPBP and α-defensin proteins were correspondingly increased.α-defensin (PPBP and DEFA1/DEFA3 could be potential CHD biomarkers in Thai hyperlipidaemia patients. Atherosclerosis is a complicated, progressive disease characterized by the accumulation of lipids and fibrous elements in large and medium-sized arteries. It is the major underlying cause of cardiovascular disease (CVD), which in turn is the leading cause of death in the developed world, and an important cause of morbidity worldwide. Abundant previous studies have linked dyslipidaemia to atherogenesis, and roles have been identified for inflammatory mechanisms coupled with dyslipidaemia in atheroma formation both in humans and animal models –3. EarlyAtherogenesis and CHD involve a long preclinical process. Multiple risk factors have been identified for CHD without familial hypercholesterolaemia including behavioral, dietary, and lifestyle factors such as smoking, dietary fat intake, level of physical activity, infections (exogenous exposure), alteration of endogenous blood constituents such as lipid and lipoprotein particles, inflammation and coagulation proteins, intermediary metabolites, and oxidant markers of stress, adiposity, blood pressure, and diabetes mellitus . SeveralBlood is an accessible source for diagnosing various disease processes , and is ® reagent were purchased from Invitrogen , the IsoPrep RNeasy total RNA kit was from Qiagen , and the Affymetrix GeneChip® Human Gene 1.0 ST Array was from Affymetrix . Quantitative reverse transcription (qRT)-PCR primers were designed using Primer3 (v.0.4.0) software, GenBank sequences and based on previous studies, and were synthesized by Pacific Science Co., Ltd. . The specificity of the primers for the target genes was also determined using the BLAST program [® Biotech . All other reagents were from Sigma-Aldrich .Dulbecco’s phosphate-buffered saline (D-PBS) and TRIzol program . Human HThe patient flow and experimental design are summarized in Fig. All volunteers were unrelated males born to Thai parents. Twenty healthy controls were recruited who carried no infections, and had no underlying disease or CVD risk factors (N group). Forty-five patients were diagnosed, classified, and treated by a specialist (KP) at Pramongkutklao Hospital. They were classified into two groups based on their clinical manifestations according to the American College of Cardiology/American Heart Association criteria (2013) , includiHeparinized blood samples (5 ml) were collected once from healthy controls and from all patients before hyperlipidaemia treatment or coronary bypass grafting. Plasma (2 ml) was immediately collected by centrifugation of whole blood. Plasma aliquots were prepared for lipid measurement, and kept at −70 °C for the detection of plasma proteins encoded by the selected genes.6 PBMCs in TRIzol (Invitrogen) were kept at −70 °C for gene expression profiling by DNA microarray analysis using Affymetrix GeneChip® Human Gene 1.0 ST (Affymetrix).Packed blood cells were resuspended in D-PBS  and used to isolate mononuclear cells. Approximately 2 × 10Lipid markers including TC, triglycerides (TG), LDL cholesterol (LDL-c), and HDL cholesterol (HDL-c) were analyzed enzymatically using kits  and a biochemistry analyzer .PBMCs were separated by Isoprep gradient centrifugation according to the manufacturer’s recommendations .6 PBMCs of all patients and controls (n = 7) using RNA isolation kits (Qiagen). Total RNA was measured using a NanoDrop ND-1000 spectrophotometer with ND-1000 3.3 software, and RNA integrity (RIN) was determined using an Agilent Bioanalyzer . The Affymetrix GeneChip® Human Gene 1.0 ST array was performed using 5 μg of total RNA with RIN ≥ 8.0, according to the manufacturer’s protocol (Affymetrix Inc). The data were analyzed by Agilent GeneSpring GX Software version 12.0. Differentially expressed genes correlating with inflammation were identified using the criteria of a > 2.0-fold increase/decrease in expression in H and CHD patients compared with the N group [Total RNA was extracted from 2 × 10 N group . Figure  N group .Fig. 2GeDEFA1/DEFA3), pro-platelet basic protein (PPBP), beta haemoglobin (HBB), alpha 2 haemoglobin (HBA2), superoxide dismutase 2, (SOD2), chemokine ligand 3(CCL3), and 4 (CCL4), and interleukin-1β (IL-1β). Table - (ΔΔCt) was used to calculate the relative expression level.Eight intersected genes, showing increased expression in H and CHD patients, were selected for further validation. These were: α-defensin DEFA/DEFA3, p®MAP human cytokine/chemokine magnetic bead Panel III kit  according to the manufacturer’s instructions and based on a previous study [® (BioRad) and interpreted by xPONENT® software (Merck Millipore).Plasma levels of IL-1β, CCL3, CCL4, and PPBP were assayed using the MILLIPLEXus study . BrieflyPlasma α-defensin 1–3 concentrations were measured by ELISA (Hycult Biotechnology) according to the manufacturer’s instructions and as described in our previous study .2CO3 solution (10 mg/100 ml) as a reagent as described in a previous study [Free haemoglobin (Hb) in plasma samples was measured spectrophotometrically  with Naus study . AbsorbaACTB) mRNA in PBMCs. Plasma levels of PPBP, α-defensin 1–3, Hb, CCL3, CCL4, and IL -1β are non-parametric data and are also expressed as medians (upper and lower range limits). The significance of the difference between two groups was determined by the Mann–Whitney U test, and differences among the N, H, and CHD groups were determined by the Kruskal–Wallis test. mRNA expression and plasma PPBP and α-defensin 1–3 levels are represented as whisker plots, with boxes denoting the interquartile range and whiskers the minimum/maximum values. Correlations between CHD development, mRNA expression, plasma protein levels, and characteristics/clinical manifestations were determined by Spearman’s rho correlation analysis. The α level was set at <0.05 with a 95% confidence interval. All statistical analyzes were performed using SPSS version 18 software .Clinical data are reported as medians (upper and lower range limits). mRNA expression is represented as fold changes relative to β-actin (p = 0.000), but there was no significant difference in age between the N and H groups. Levels of TC in the H group were significantly higher than those in N (p = 0.004) and CHD groups (p = 0.008). The LDL level in the H group tended to be higher than in the N group (p = 0.072) and was significantly higher than in the CHD group (p = 0.049). All groups showed similar TG levels (p > 0.05).General descriptions and clinical manifestations of patients and controls are compared in Table PPBP, DEFA1/DEFA3, HBB, HBA2, SOD2, CCL3, CCL4, and IL-1β in N, H, and CHD groups are shown in Fig. PPBP, DEFA1/DEFA3, HBB, and HBA2 showed significant differences in mRNA expression in both H and CHD groups compared with the N group  of oup Fig. . The corr = 0.655, p = 0.034) and α-defensin 1–3  levels among the three groups, which supports the PPBP and DEFA1/DEFA3 expression results.Plasma levels of proteins encoded by the selected genes were compared among groups using ELISA and Milliplex bead techniques (data not shown). As shown in Fig. PPBP and DEFA1/DEFA3  and plasma PPBP and α-defensin 1–3 levels  were detected  PPBP and DEFA1/DEFA3 expression was significantly correlation with CHD development. Based on these data, we suggest that significantly increased expression of both PPBP and DEFA1/DEFA3 and their encoded proteins has the potential to be established as a synergistic predictive biomarker for CHD risk in hyperlipidaemia patients.In this cross-sectional study, N, H, and CHD groups were representative of the long-term development of CHD, which is a common complication of atherosclerosis. This study focused on verifying markers for potential CHD prediction in non-familial Thai hyperlipidaemia patients. Our findings revealed that: 1) there was significant association between increased genotypic and phenotypic Previous histological studies of atherogenesis revealed that early-stage (fatty streak) atherosclerotic lesions to more complicated lesions demonstrate chronic inflammation. This develops from an interaction between plasma lipoproteins, cellular components such as monocytes, macrophages, T lymphocytes, B lymphocytes, endothelial cells (ECs), and smooth muscle cells, and the extracellular matrix of the arterial wall . PolymorDEFA1/DEFA3 encode HNP-1, 2, and 3 [During inflammation, large amounts of intracellular proteins are released from activated PMNs into the extracellular milieu as an outcome of PMN degranulation, leakage during phagosome formation, and cell death. The amount of highly homologous human neutrophil peptides (HNPs)-1, −2, and −3, or α-defensin is more than half of the total protein content within PMN azurophilic granules . α-defen2, and 3 , 32. Pre2, and 3 . Moreove2, and 3 .A role for platelets and platelet-derived factors in atherosclerosis has long been suggested beyond their function in the hemostatic system. Platelets are also involved in thrombus formation in response to vascular injury, and affect coronary, cerebral, and peripheral circulation . FollowiPro-platelet basic protein (PPBP) or chemokine (C-X-C motif) ligand 7 (CXCL7) is an encoded protein, which is a small cytokine of the CXC chemokine family. PPBP is released in large amounts from activated platelets and is involved in the response to vascular injury . It stimPPBP, DEFA1/DEFA3, HBA2, and HBB was significantly increased in H and CHD groups of patients compared with controls, although plasma protein validation only revealed significant increases in PPBP and α-defensin 1–3. This indicates that the transcriptional and post-transcriptional processes of PPBP and DEFA1/DEFA3 were successful. In agreement with a previous study [In the present study, we found that the mRNA expression of us study , our obsus study  and α-deus study  are relers = 0.363, p = 0.041) and phenotype correlations  between PPBP and DEFA1/DEFA3 shown in Fig. Hypercholesterolaemia is typically an asymptomatic condition that is often detected during routine screening. Our findings suggest the possibility of applying the plasma proteins PPBP and α-defensin 1–3 as CHD risk markers in hyperlipidaemia patients. The significant genotype . We therefore suggest that HBA2 and HBB should be investigated to determine whether they could be alternative prognostic biomarkers for CHD.Several studies have demonstrated correlations between Hb and chronic stress in humans , as well in mice . Prior s in mice –47. In tPPBP, DEFA1/DEFA3, HBA2, and HBB demonstrate interplay within the network. Further investigation and confirmation of mRNA and protein expression may provide more effective predictions of CHD in hyperlipidemia patients. We observed no significant increase in the expression of mRNAs of the other selected genes in H and CHD groups Fig. . Additiop = 0.000). Our result agrees with long-term atherogenesis and the development of CHD complications [Table ications , 7. Thisications , 57.DEFA1/DEFA3 expression using a qRT-PCR with our designed primers, the amplicon size of the DEFA1/DEFA3 gene (204 bp) was slightly larger than ideal (70–200 bp) for maximum efficiency [DEFA1/DEFA3 showed a single peak. Similarly, agarose gel electrophoresis of the amplicon was a single band. Therefore, our DEFA1/DEFA3 gene primer was sufficiently specific and suitable for qRT-PCR, 3) the lack of Milliplex kit optimization, which may have accounted for the observed variation in its performance; future work should determine if heparin in plasma interferes with the binding capacity, whether the 1:100 dilution of plasma samples is appropriate, the optimal incubation time, and if artifacts interfere with plasma protein levels, and 4) the lack of plasma SOD2 level determination which reflects the fact that SOD2 enzyme and cytokine/chemokine analysis could not be conducted using the same Milliplex set. An appropriate SOD2 assay should therefore be performed to verify its importance as a CHD biomarker in future studies.The present study had some limitations, including 1) its small sample size and single-center analysis, 2) In determining ficiency . Our PCRPPBB and DEFA1/DEFA3 were the most strongly correlated with CHD development, and show promise for further application as inflammatory markers to synergistically predict the risk of CHD development in Thai hyperlipidaemia patients.We conducted a cross-sectional study of the evaluation of CHD predictive biomarkers in non-familial hyperlipidaemia. Among eight potential markers,