Controls remained untreated and/or non-irradiated. Cell growth, proliferation, apoptosis, adhesion, and chemotaxis were evaluated, as was cell cycle regulating protein expression , and integrins of the α- and β-family. Curcumin or light alone did not cause any significant effects on tumor growth, proliferation, or metastasis. However, curcumin combined with light irradiation significantly suppressed tumor growth, adhesion, and migration. Phosphorylation of CDK1 decreased and expression of the counter-receptors cyclin A and B was diminished. Integrin α and β subtypes were also reduced, compared to controls. Irradiation distinctly enhances the anti-tumor potential of curcumin in vitro and may hold promise in treating prostate cancer.Although anti-cancer properties of the natural compound curcumin have been reported, low absorption and rapid metabolisation limit clinical use. The present study investigated whether irradiation with visible light may enhance the inhibitory effects of low-dosed curcumin on prostate cancer cell growth, proliferation, and metastasis in vitro. DU145 and PC3 cells were incubated with low-dosed curcumin (0.1–0.4 µg/mL) and subsequently irradiated with 1.65 J/cm Prostate cancer is the most common cancer in men, with 1,414,259 new cases and 375,304 recorded deaths worldwide in 2020 [During the last decade, reports pointing to the natural compound curcumin as a potential anti-cancer remedy have been published. Curcumin, a yellow-orange pigment extracted from the rhizome of the Curcuma longa plant, serves as a traditional food additive and spice ingredient. Preclinical studies have demonstrated that curcumin exerts anti-inflammatory, anti-oxidant, and anti-tumor activities . In vitrRecently, Bernd presented evidence of increased antitumor properties of curcumin on human epidermal keratinocytes in vitro when irradiated with UVA or visible light . The fin2) resulted in a significantly decreased growth rate after 48 and 72 h  or curcumin (0.2 µg/mL). Combined treatment induced a distinct loss of cell–cell contact and destruction of clone formation. Similar clone behavior was seen with PC3 cells, although the clones were less densely packed. 2). No effects were observed when DU145 or PC3 were treated with curcumin or light alone.2) led to a significant decrease in proliferation of both DU145 and PC3 cells. No significant effects, compared to untreated controls, were apparent when cells were treated with light or curcumin alone (Treatment with curcumin (0.2 µg/mL) and subsequent irradiation with visible light  or curcumin (0.2 µg/mL) did not result in significant alterations of apoptosis, related to the controls. However, the percentage of apoptotic DU145 and PC3 cells considerably increased when treated with light and curcumin , excepting CDK2, which was diminished in DU145 and PC3 cells and Rictor, which was suppressed in PC3 cells with all treatment modalities  or visible light alone . Curcumin application in concert with irradiation triggered maximum effects (Attachment of DU145 and PC3 cells was significantly inhibited when cells were treated with curcumin (0.2 µg/mL) or with curcumin plus visible light  significantly altered the expression level of some integrin subtypes. Curcumin and light exposure alone did not lead to any changes of the receptor expression profiles. The subtypes α5, α6, and αV were downregulated in DU145 and PC3 cells. Integrin β3 was reduced on DU145 but not on PC3, whereas the opposite was seen with β4—reduced on PC3 but not on DU145 (Curcumin (0.2 µg/mL) combined with visible light irradiation  curcumin being necessary to suppress the growth of gastric cancer cell lines by 50% . Even hiLight plus curcumin application triggered a G2/M cell cycle arrest in both cell lines evaluated. A- and B-type cyclins, along with their corresponding partners, CDK2 and CDK1, are crucial for mitotic transit with reduced expression correlated to G2/M-phase arrest ,25. A siThe reduced expression of Akt and pAkt by low-dosed curcumin is notable. Akt serves as a major target to block prostate cancer growth, proliferation, adhesion, and invasion. A phase 2 clinical trial has shown that blocking Akt, compared to second-line chemotherapy , is assoApart from being a growth suppressor, low-dosed curcumin combined with light irradiation also acted on adhesion and chemotactic properties of the tumor cells. One-hundred-fold higher concentrations are required to evoke similar responses by curcumin alone ,31. SincAside from α6, α5 and αV, further integrin family members were removed from the cell surface by curcumin and light. However, this did not occur consistently. Different responses of DU145 and PC3 cells were observed with β3 expression (down-regulated on DU145) and the integrins α2, α3, β1, and β4 (down-regulated on PC3). Earlier studies support the hypothesis that differences in the molecular fine tuning of invasive processes may (at least in part) depend on the initial integrin expression pattern . The intA low concentration of curcumin has been shown to potently reduce the proliferative and invasive activity of androgen-independent prostate cancer cells by interfering with the CDK-cyclin, the Akt-mTOR pathway, and integrin-related signaling. These effects were not apparent unless the tumor cells had been exposed to visible light. Several techniques have been reported, whereby light has been successfully applied to tumor tissue in in vivo models and in clinical practice. Neuschmelting et al. introduced an optical fiber into renal cell tumors in mice to successfully activate the photosensitizer WST11 by laser illumination with a wavelength of 750 nm or multispectrally of 700–800 nm . Kroeze 2. Subcultures from passages 5–30 were selected for experimental use.Prostate carcinoma androgen-insensitive DU145  and PC3  cell lines were obtained from DSMZ . The human castration-resistant tumor cell lines were grown and subcultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% Glutamax , 2% Hepes buffer and 1% penicillin/streptomycin , at 37 °C in a humidified incubator with 5% CO2). During that time the culture medium was replaced by PBS (Sigma-Aldrich). The untreated control and the curcumin treated control group were stored in PBS and kept light-protected to ensure identical experimental conditions. Thereafter, the cells were cultured in cell culture medium until assays were performed.Curcumin was stored at −20 °C and diluted in cell culture medium to the final concentration of 0.1–1 µg/mL (0.271–2.71 µM). Control cell cultures were cultivated in cell culture medium without treatment. To exclude significant effects of low-dosed curcumin and light on its own, two additional control groups were assessed. Treatment was performed at least 24 h prior to the experiment in the desired concentration. Cells were treated for 1 h with curcumin and/or were irradiated with visible light for 5 min (1.65 J/cm4 cells/mL) were plated into 96-well tissue culture plates. After 24, 48, and 72 h, MTT (0.5 mg/mL) was added for an additional 4 h. The reaction was stopped by lysing the cells in a buffer containing 10% SDS in 0.01 M HCl. After incubating the plates at 37 °C, 5% CO2 overnight, the absorbance at 570 nm was measured for each well using a microplate proliferation enzyme-linked immunosorbent assay (ELISA) reader. Each experiment was performed in triplicate. After subtracting background absorbance, results were expressed as mean cell number.Cell growth was measured using the 3--2,5-diphenyltetrazolium bromide (MTT) dye reduction assay . Tumor cells , seeded onto 96-well plates, were incubated with 20 μL BrdU-labeling solution per well for 8 h, fixed and detected using anti-BrdU mAb according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate ELISA reader.Cell proliferation was measured using a BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit . Tumor cells  that quantifies binding of Annexin V/propidium iodide. Tumor cells, incubated with SFN (controls were without SFN) were washed twice with PBS, and incubated with 5 μL Annexin V-FITC and 5 μL propidium iodide in the dark for 15 min at room temperature. Cell numbers were determined by flow cytometry . Percentages of early and late apoptotic, necrotic, and vital cells in each quadrant were calculated using CellQuest software (BD Biosciences). Apoptosis was verified by the evaluation of the PARP protein expression level . β-actin  served as the internal control.Five-hundred single PC3 or DU145 cells (treated with 0.2 µg/mL curcumin and irradiated versus non-irradiated or non-treated) were transferred to 6-well plates. Following 5 to 10 days incubation without medium change, cell colonies were fixed and counted. Clones of at least 50 cells were counted as one colony.Cell cycle analysis was carried out with subconfluent tumor cells. Tumor cell populations were stained with propidium iodide, using a Cycle TEST PLUS DNA Reagent Kit (BD Pharmingen), and then subjected to flow cytometry with a FACScan flow cytometer (Becton Dickinson) after 24 h of cultivation. A total of 10,000 events were collected for each sample. Data acquisition was carried out using Cell-Quest software and cell cycle distribution was calculated using the ModFit software (BD Biosciences). The number of gated cells in the G1, G2/M, or S-phase is presented as a percentage of the total cell number.To investigate the level of the cell cycle and movement related proteins in the three cell lines, tumor cell lysates were applied to 7% to 12% polyacrylamide gel and electrophoresed for 90 min at 100 V. The protein was then transferred to nitrocellulose membranes . After blocking with nonfat dry milk for 1 h, the membranes were incubated overnight with monoclonal antibodies directed against the cell cycle proteins: CDK1/Cdc2 , pCDK1/Cdc2 ), CDK2 , Cyclin A , Cyclin B , pCDK2 . The mechanistic target of rapamycin (mTOR) pathway was investigated by using the following monoclonal antibodies: Raptor (24C12), phospho Raptor (Ser792), Rictor (D16H9), phospho Rictor , PKBα/Akt (IgG1 clone 55) and anti-phospho Akt .Integrins, adhesion or chemotaxis-related proteins were incubated as described before: FAK , pFAK  ILK , Integrin alpha 5 (clone 1), Integrin alpha V (clone 21), Integrin beta 1 (clone 18) and Integrin beta 4 , Integrin alpha 2 (clone P1E6) and Integrin alpha 3 , Integrin alpha 6 .HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG  served as the secondary antibody. The membranes were briefly incubated with ECL detection reagent  to visualize the proteins and then analyzed by the Fusion FX7 system . β-actin  served as the internal control.6 tumor cells were then added to each well and left for 60 min incubation. Subsequently, non-adherent tumor cells were washed off, and the remaining adherent cells were fixed with 1% glutaraldehyde and counted microscopically. The mean cellular adhesion rate was calculated from five different observation fields (5 × 0.25 mm2).Six-well plates were coated with collagen G  overnight. Plastic dishes served as the background control. Plates were washed with 1% BSA  in PBS to block nonspecific cell adhesion. 0.5 × 106 cells/mL added. One well each was filled with cells from one of the different treatments . Plates were incubated for 30 min, one hour and two hours at 37 °C and 5% CO2. Subsequently, the cells were fixed with 1% glutaraldehyde. Adherent tumor cells were counted in five different fields of a defined size (5 × 0.25 mm2) using a phase contrast microscope and the mean cellular adhesion rate was calculated.Twenty-four hours before the start of the experiment, HUVECs were seeded onto 6-well plates  and filled with 2 mL HUVEC medium (M199) so that a subconfluent cell lawn was present on the day of the experiment. Three plates of four wells each were prepared for each cell line. Before the experiment, half of the HUVEC medium was removed, and 0.5 × 106 DU145 or PC3 cells/mL of the respective treatment were placed in the upper chamber in serum-free medium. Serum free medium in the upper chamber and 10% serum in the lower chamber provided the serum gradient necessary for tumor cell migration in this model. After 20 h incubation, fixation  and staining (hematoxylin), the upper surface of the Transwell membrane was gently wiped with a cotton swab to remove cells that had not migrated. Cells that had moved towards the serum gradient were counted microscopically. The mean migration rate was calculated from five different observation fields (5 × 0.25 mm2). In addition, the metastasis marker CD44  was evaluated by Western blotting using β-actin as the internal control.Serum-induced chemotactic movement was examined using 6-well Transwell chambers  with 8 µm pores. 0.5 × 104 cells/scan) and expressed as mean fluorescence units. A mouse IgG1-PE (MOPC-21), IgG2a-PE (G155–178) or rat IgG2b-PE  was used as an isotype control.Tumor cells were washed in blocking solution  and then incubated for 60 min at 4 C with phycoerythrin (PE)-conjugated monoclonal antibodies directed against the following integrin subtypes: anti-α1 , anti-α2 , anti-α3 , anti-α4 , anti-α5 , anti-α6 , anti-β1 , anti-β3  and anti-β4  or anti-αV . Integrin expression of tumor cells was then measured using a FACscan  channel histogram analysis; 1 × 10t-test. Differences were considered statistically significant at a p value less than 0.05.All experiments were performed three to six times. Statistical significance was calculated with the Wilcoxon–Mann–Whitney U test or the Low dosed curcumin coupled with visible light application led to reduced proliferation and growth of PC3 and DU145 cells in vitro and was associated with a cell-cycle arrest in the G2/M-phase. Cellular adhesion and invasion capacity were also reduced, presumably by modifying cell specific integrin subtypes. The integrative application of combined curcumin and visible light may enhance the anti-tumor potential of conventional treatment regimens.


A total of 19 QTL for stripe rust resistance were mapped on 12 chromosomes using phenotypic data from multiple field tests over the course of 6 years. These chromosomes included 1B (2), 1D (2), 2A (2), 2B (2), 2D (1), 4B (2), 4D (1), 5A (3), 5B (1), 6A (1), 6B (1), and 7B (1). Two stable QTL on chromosomes 2AS (Qyr.gaas.2A) and 6AL (Qyr.gaas.6A) were detected in six and five different environments, respectively; in both QTL, positive allele was contributed by GX3 variety. Qyr.gaas.2A was found to be crucial for increasing adult plant resistance, which may explain the large phenotypic variation of 45.52%. Our results provide theoretical and molecular insight for wheat breeding and suggest the cloning of genes associated with the GX3 variety may be beneficial in future studies.Development of cultivars with multiple resistances has proven to be an effective way to prevent diseases in wheat breeding. The Guixie 3 variety (GX3) has shown excellent performance in resistance to stripe rust in field for many years. The purpose of this study was to detect quantitative trait loci (QTL) associated with resistance to stripe rust in the adult plant stage and determine closely linked molecular markers. A population of recombinant inbred lines (The online version contains supplementary material available at 10.1007/s13353-022-00686-z. Puccinia striiformis f. sp. tritici (Pst), is one of the most damaging diseases associated with global wheat production  officially designated resistance genes and 47 proposed resistance genes  and adult plant disease resistance genes (APR)  still maintain effective resistance to wheat stripe rust  arrays have been widely used in wheat. An example includes the construction of a high-density genetic map with stripe rust resistance gene/quantitative trait locus (QTL) mapping , a wild tetraploid ancestor of common wheat, has good resistance for wheat stripe rust and importantly shows great potential for wheat breeding. A number of resistant genes have been previously identified in wild emmer wheat, such as YrH52, Yr15, Yr35, and Yr36  and abiotic  stress  with wild oat (Avena fatua L. var. glabrata pat) and then backcrossed with common wheat (Guinong 22). It has shown resistance to the current wheat stripe rust epidemic for many years. In this study, we use a wheat 55 K SNP array to map QTL for APR to stripe rust employing recombinant inbred line (RIL) population of “M96-5/GX3,” to identify tightly linked molecular markers for their use in future marker-assisted breeding.Wheat wild relatives can be used as a resource bank of disease resistance genes. Some genes have been officially classified for stripe rust resistance . Avocet S (AVS) and SY95-71 were used as susceptible controls throughout the study.The susceptible winter landrace line M96-5 and the resistant line GX3 were used as the parental lines for this study. The mapping population comprised of 228 F2O5 + K2O ≥ 45%) per hectare in the field before planting wheat. The ratio of nitrogen, phosphorus, and potassium is 19–15-11 in the compound fertilizer.In these field trials, two parents and a RIL population were used to test resistance in mixed races of stripe rust within a natural setting. These were planted in October 2016 in Mianyang, Sichuan Province ; in October 2017, 2018, and 2019 in Guiyang, Guizhou Province ; and in November 2019 in Anshun, Guizhou Province  and Shuangliu, Sichuan Province . During planting season (October of the sowing year — May of harvest year) in different environments, the total rainfall of MY17 was 265.08 mm, and the average temperature was 13.53 °C, GY18 was 611.35 mm/12.17 °C, GY19 was 736.97 mm/10.94 °C, GY20 was 730.40 mm/12.24 °C, AS20 was 785.64 mm/13.01 °C, and SL20 was 333.52 mm/13.97 °C  was planted every five lines as the control line. The first severe degree was recorded when the disease severity of AVS (or SY95-71) in the control group and the susceptible parent M96-5 reached 50% or more . In accordance with the percentage of the total leaf area occupied by rust fungus, the severity of stripe rust was also recorded visually for each wheat family. More than 5 individual plants were observed in each row, and data of each row was the overall average. Recordings were taken every other week until the susceptible control reached 100%, known as the maximum disease severity (MDS), and got the MDS data at each environment (two replications × one MDS data × six environments). The modified Cobb scale was referred to for the phenotypic data required for QTL analysis  (Li and Zeng f − MSfe)/re, fe − MSe)/r, and e, f = mean square of genotypes, MSfe = mean square of genotype × environment interaction, MSe = mean square of error, r = number of replications, and e = number of environments. The correlation between multiple field conditions was analyzed by the Pearson method in SPSS v20 software.To estimate the genetic and environmental effects in each line, we compared the environments and line × environment interactions using the AOV function in IciMapping 4.1 software . Basic quality control (QC) tests were performed on samples by measuring markers based on genotype data detection rate, minimum allele frequency (MAF), and heterozygosity. The criteria used for sample quality control were as follows: DQC > 0.82, detection rate ≥ 85%, and heterozygosity rate ≤ 10%; the criteria for marking quality control were as follows: detection rate greater than or equal to 95%, and MAF of 5% or more, heterozygosity rate of 50% or less, and the number of alleles was 2.Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method   followed by digesting of restriction enzyme DpnII. Detecting procedure for URIC/LN2 was processed according to previously report  markers located on the 2AS chromosome were selected for genetic map construction (see Table http://www.isbreeding.net/)  was used to draw images (Voorrips The BIN function in QTL IciMapping v4.1 software (Voorrips . The R pVoorrips .http://www.wheatgenome.org/). The specific parameters of blastn are default as follows: expect threshold was 0.001, word size was 15, max target sequence was 50, match/mismatch scores were 2 or − 3, gap costs were existence: 5 and extension: 2, at the same time, excluding low complexity region.In order to get the physical location of the polymorphic SNP marker, the SNP probes of these markers were aligned with respect to the released Chinese Spring sequence through a BLAST search , http://202.194.139.32); the Triticeae Multi-omics Center provides genome, transcriptome, proteome, and epigenome dataset resources for common wheat and relatives as well as useful tools such as the Basic Local Alignment Search Tool (BLAST), sequence extraction, and the design of molecular markers and primers. It also aligned the flanking sequences of the Wheat 55 K SNP array to the Chinese Spring reference genome V1.0. Based on the alignment results in the Triticeae Multi-omics Center, unique probes with reliable physical positions in the 55 K SNP array were extracted   Table . ANOVA a1) Table .Fig. 1FrWhole genome analysis of the two parental lines and 228 RILs was performed using the wheat 55 K SNP array. Within the 55,000 SNPs, 7570 were identified as polymorphic markers to distinguish between M96-5 and GX3. A total of 589 were excluded because they had missing data (> 10%) or showed segregation distortion. The remaining 6981 SNPs fell into 1543 bins, and 5438 SNPs were excluded.R), and a susceptible pool (BS). Four markers  showed successful amplification consistent between the resistant parent and the resistant pool, and then, there are polymorphisms in the resistant pool and the sensitive pool. The four SSR markers were then used in combination with the 1543 SNPs obtained from genotyping data to analyze the QTL for resistance to wheat stripe rust at the adult plant stage. The final genetic map included 21 linkage groups corresponding to the 21 chromosomes , a disease-resistant pool , whereas the D genome had the fewest 382, 24.69%). Among the seven homologous groups of wheat, the fifth homology group had the highest number of SNPs , while the sixth homology group had the fewest ; for the 21 chromosomes in wheat, there was an average of 75 markers for each chromosome. Chromosome 5A harbored the highest number of markers , while chromosome 6A had the fewest  (Table .69%. AmoQyr.gaas.2A) and 6AL (Qyr.gaas.6A) were detected in six and five different environments, respectively (Table Qyr.gaas.2A) was located at the position of 5 cM on chromosome 2AS. The 6-year LOD values ranged from 8.01 to 44.61 with confidence intervals of cfd36 ~ AX-110576889 (3.5–5.5 cM), which explains the high phenotypic variation of 45.52%. Qyr.gaas.6A was located at 4 cM of the distal chromosome 6AL with a confidence interval of AX-109558600 ~ AX-109542604 (3.5–4.5 cM), accounting for a phenotypic variation of 3.27–21.73%. In addition, a QTL (Qyr.gaas.6BL) was identified in three different environments (Mianyang in 2017 and Guiyang in 2018 and 2019), with a confidence interval of AX-109408478 ~ AX-110409180 (54.5–60.5 cM) and a phenotypic variation of 2.01–6.66%. The additive effect of the above three QTL was negative, indicating that their disease resistance originated from GX3. Moreover, another QTL (Qyr.gaas.2D) was identified in three environments , with a phenotypic variation of 2.56–3.55%. The additive effects were positive, indicating that its disease resistance originated from the other parental line, M96-5.A total of 19 QTL for resistance to wheat stripe rust were located on 12 chromosomes, including 1B (2), 1D (2), 2A (2), 2B (2), 2D (1), 4B (2), 4D (1), 5A (3), 5B (1), 6A (1), 6B (1), and 7B (1), of which two stable QTL on chromosome 2AS  and Qyr.gaas.6A , the sequences located within the interval of two QTL were searched in Triticeae Multi-omics Center to identify the wheat gene ID, annotation, and corresponding sequence. The results identified 620 and 61 segments at the confidence intervals of Qyr.gaas.2A and Qyr.gaas.6A, respectively  and 3 WRKY transcription factors , while 11 fragments related to disease resistance were clustered on a region of the 6AL chromosome spanning from 609,635,388 bp to 609,796,663 bp, including 8 LRR , 2 NBS-LLR , and 1 TIR-NBS-LRR (TraesCS6A01G401100.1).According to the physical locations of ly Table . The twoUntil 2016, China had officially classified 34 stripe rust races (CYR1–CYR34) and more than 40 pathogenic types  of Guinong 22 was first identified in Sichuan in 2009, it has gradually increased to become the dominant pathogenic group  was detected at the interval of cfd36 ~ AX-110576889 (16.63 ~ 31.85 Mb) in all six environments assessed in this study. Five genes for the resistance of stripe rust were identified on chromosome 2AS, Yr17 , Yr56 , YrR61 , Yr69 (X2AS33–Xmag3807), and YrZM175 . Through pedigree analysis, we found that YrR61 was derived from the American soft red wheat Pioneer 26R61  present medium resistance to the pathogenic group of Guinong 22 in Guiyang. Moreover, Yr17 had the closest position to Qyr.gaas.2A. The allelism tests will perform to cross between GX3 and the carrier line of Yr17, VPMI in the future. Furthermore, 13 QTL have been previously reported on chromosome 2AS  is located in the interval spanning 3.5 ~ 4.5 cM (609.11–609.89 Mb). YrLM168, derived from Chuannong 16, was mapped in the interval of wmc59-wmc145 on chromosome 6AL, which is adjacent to Qyr.gaas.6A  was derived from the CIMMYT variety Saar and has two important gene loci (Lr34/Yr18/Pm38 and Lr46/Yr29/Pm39)  came from the susceptible parent “Avocet S”  was from the hard red winter wheat cultivar “Einstein” from Limagrain, UK , Tdurum_contig 29087_757 , TA002369-0369 , and wsnp_Ex_c965_1846161 . We considered that the four SNPs were likely to be associated with new QTLs/genes that confer resistance to race CYR34. In this study, we detected a QTL  that was located closely to Tdurum_contig29087_628 and Tdurum_contig29087_757, which may not be associated with YrExp1 (637.39 Mb) (Lin and Chen Qyr.gaas.6A (609.11–609.89 Mb) was inconsistent with the SNP locus wsnp_Ex_c965_1846161  on chromosome 6A.In our last study about genome-wide association analysis of resistance to strip rust  released by the pathogen into the cell and trigger a disease resistance response. This response is referred to as effector triggered immunity  CC-NBS-LRR, such as stripe rust resistance gene Qyr.gaas.2A (cfd36 ~ AX-110576889) and found a structural region (chr2A:17411781_17601016) containing 10 NBS-LRR and 3 WRKY transcription factor near cfd36 marker (16.63 Mb). We therefore speculated that the structural region in combination with a relatively complete NBS-LRR may represent a novel candidate gene, and these 13 identified segments will be further investigated. We also found the similar candidate gene structure on chromosome 6AL; this area  contained eight LRR, two NBS-LLR, and one TIR-NBS-LRR. We hence considered that one of the 11 candidate segments in this region is most likely to be novel Yr gene.In the present study, we identify T. dicoccoides) and wild oat (Avena fatua L. var. glabrata pat) varieties and was subsequently obtained by backcrossing with the common wheat Guinong 22 variety. Field observations spanning many years have shown that wild emmer wheat and Guinong 22 are susceptible to CYR34, while the wild oat is immune. Therefore, we speculate that the major QTL on chromosome 2AS originated from Avena fatua L. var. glabrata. However, in situ hybridization analysis showed no signal detection in the wild oat variety samples (unpublished data). These findings may be due to the fact that the introgression fragment is too small. These experiments therefore require further validation.GX3 was derived from wild crossing between wild emmer wheat (Qyr.gaas.2A and Qyr.gaas.6A) were identified from biparental populations, and four closely linked markers could be transformed into KASP or SSR markers for marker-assisted selection breeding. Several resistance genes/QTL has been previously mapped on chromosomes 2AS and 6AL; we hence will perform further research to determine whether they are new disease resistance genes/QTLs and simultaneously increase the density of genetic maps around the two QTLs to clone the underlying gene.High-yield and disease-resistant new lines could be bred by crossing GX3 with susceptible varieties possessing excellent agronomic traits. Two stable and reliable QTLs (Supplementary file1 (XLSX 63 kb)Below is the link to the electronic supplementary material.


Botrytis cinerea, a fungal pathogen that causes gray mold, is damaging more than 200 plant species, and especially tomato. Photosystem II (PSII) responses in tomato (Solanum lycopersicum L.) leaves to Botrytis cinerea spore suspension application were evaluated by chlorophyll fluorescence imaging analysis. Hydrogen peroxide (H2O2) that was detected 30 min after Botrytis application with an increasing trend up to 240 min, is possibly convening tolerance against B. cinerea at short-time exposure, but when increasing at relative longer exposure, is becoming a damaging molecule. In accordance, an enhanced photosystem II (PSII) functionality was observed 30 min after application of B. cinerea, with a higher fraction of absorbed light energy to be directed to photochemistry (ΦPSΙΙ). The concomitant increase in the photoprotective mechanism of non-photochemical quenching of photosynthesis (NPQ) resulted in a significant decrease in the dissipated non-regulated energy (ΦNO), indicating a possible decreased singlet oxygen (1O2) formation, thus specifying a modified reactive oxygen species (ROS) homeostasis. Therefore, 30 min after application of Botrytis spore suspension, before any visual symptoms appeared, defense response mechanisms were triggered, with PSII photochemistry to be adjusted by NPQ in a such way that PSII functionality to be enhanced, but being fully inhibited at the application spot and the adjacent area, after longer exposure (240 min). Hence, the response of tomato PSII to B. cinerea, indicates a hormetic temporal response in terms of “stress defense response” and “toxicity”, expanding the features of hormesis to biotic factors also. The enhanced PSII functionality 30 min after Botrytis application can possible be related with the need of an increased sugar production that is associated with a stronger plant defense potential through the induction of defense genes. Botrytis cinerea, a fungal pathogen that causes gray mold, is damaging more than 200 plant species, especially tomato, cucumber, strawberry, potato, and ornamental plants [B. cinerea releases ethylene to accelerate leaf wither, decreasing photosynthetic efficiency and affecting plant production resulting in huge economic losses [B. cinerea influences growth performance of tomato plants [B. cinerea induces increased respiration on the infected tissues, leading to a severe drop in the O2 concentration in an otherwise fully aerobic leaf [l plants ,2. B. cic losses . This pac losses ,5. Sympto plants . Sugars o plants . Infectibic leaf .B. cinerea [In plant cellular metabolism, and especially in chloroplasts, reactive oxygen species (ROS) are continuously produced at basal levels that are incapable to cause damage, as they are being scavenged by different antioxidant mechanisms ,10,11,12 cinerea .2•−), hydrogen peroxide (H2O2), and singlet oxygen (1O2), produced in chloroplasts play dual roles as they generate oxidative stress and also confer essential biological function as redox signaling in response to biotic and abiotic stress conditions [ROS, such as superoxide anion radical  in tomato leaves, combined with ROS detection at the inoculated leaf area. We monitored the time-lapsed PSII functionality and try to gain an insight into the mechanisms that play a role in plant defense response to the fungal pathogen.In the present work we used CF-IA to quantify the impact of 5–107 spores/mL)  a, with tores/mL) b. Leafleores/mL) a.B. cinerea spore suspension application, H2O2 production was detected at the drop’s application area only , is lost by regulated heat dissipation (ΦNPQ), and that of non-regulated energy loss (ΦNO), that add up to unity [PSII) 30 min after spore suspension application increased significantly compared to control, while 120 min after spore suspension application it did not differ compared to control, at both LL and HL treatments. ΦPSII decreased significantly compared to control, 240 min after spore suspension application, at both LL and HL (NPQ) increased significantly up to 120 min after spore suspension application, but later on decreased to control level at LL  decreased compared to control, but increased significantly 240 min after spore suspension application at LL  increased significantly up to 120 min after spore application, but later on decreased to control level at LL a. At HL B. cinerea suspension, ΦPSII decreased in the direct vicinity of the spore application area, compared to control values  at the application spot and the adjacent area, and on most of the leaflet, with a high photosynthetic heterogeneity to be observed , 30 min after spore suspension application increased at the whole leaflet (54% open), compared to control (44% open) , and also on most of the leaflet, with only a 5% of reaction centers to remain open at the whole leaflet (Following the pattern of Φ4% open) . In acco leaflet .Botrytis cinerea is a necrotrophic plant pathogen causing gray mold disease on several crop plant species, producing enormous damage in crop production [Plant pathogens are divided into biotrophics, that attack the host cells conserving host viability and gaining nutrients from living cells, and necrotrophics, that get their nutrients by killing the host plant . Botrytioduction . Thus, toduction .B. cinerea is able to induce ROS generation, especially H2O2, in a plethora of plant species [2O2 was detected 30 min after spore suspension application and gradually increased  oxidized, thus, to have a higher fraction of open PSII reaction centers (qp) compared to controls , the key photoprotective process in plants that dissipates excess light energy as heat and protects photosynthesis ,54,55,56 cinerea . After 2 control b. This id demand ,58,59,60oduction  as we oboduction ,56. Chlooduction ,63. Tomacontrols , indicatPSII in the direct vicinity of the application spot compared to control values was detected  and increases in NPQ values before any visual symptoms appeared were observed in cashew seedlings inoculated with Lasiodiplodia theobromae [At 30 min after spore suspensions application, a decreased Φdetected , while an plants ,14,16,23recorded , but, wirecorded . Decays eobromae .PSII 30 min after B. cinerea spore application (NPQ) (NO)  [B. cinerea on tomato leaflets, the decreased ΦNO indicates decreased 1O2, that is considered as a highly damaging ROS produced by PSII [B. cinerea application, the increased ΦNO suggests high levels of 1O2 (2O2 level (Botrytis cinerea is a necrotrophic fungus that produces constantly toxic compounds which ultimately cause cell death [The enhancement of Φlication  and the on (NPQ) , resultePQ) (NO) . ΦNO com (3chl*) ,66,67,68 by PSII ,70,71,72s of 1O2  that couO2 level d, in chlll death . Then, tll death .1O2 creation via 3chl* decreased 30 min after spore application compared to control (2O2 was detected to appear 30 min after spore application (B. cinerea in strawberry leaves [B. cinerea –induced leaf damage [1O2), and electron transport (H2O2) is simultaneous, it seems likely that their signaling pathways occasionally antagonize each other [While  control a, H2O2 wlication c, and grlication d. Hydrogy leaves , while, f damage , that evf damage . As ROS ch other . A low ich other ,50,75,76ch other ,13, whicBotrytis cinerea that has been placed second in rank order of the top 10 fungal plant pathogens [athogens  can deveathogens .Botrytis application can possible be related with the need of an increased sugar production that is associated with a stronger plant defense potential through the induction of defense genes [B. cinerea growth in tomato leaves [Rosa chinensis leaves, exogenous jasmonate (JA) spraying was shown to be essential for inducing defense response against B. cinerea [B. cinerea.The enhanced PSII functionality 30 min after se genes . Howevero leaves . In Rosa cinerea . Plant d cinerea , or by e cinerea , and γ-a cinerea , that arB. cinerea inoculation can be described as a time-dependent hormetic response (Photosystem II responses of tomato leaflets to response  in termsSolanum lycopersicum L.) plants (20 cm in height) were obtained from the market and transported to a growth chamber with 20 ± 1/18 ± 1 °C day/night temperature, 10-h photoperiod with photosynthetic photon flux density (PPFD) 220 ± 20 μmol quanta m−2 s−1 and relative humidity 50 ± 5/60 ± 5% day/night.Tomato  containing 0.5 mL L−1 lactic acid 1N at 23 °C until sporulation. Sporulated cultures were transferred in 100 mL sterilized distilled water and gently vortexed for spore release.B. cinerea aqueous spore suspension (105–107 spores/mL) was applied on tomato leaflets with a micropipette [B. cinera spores) were considered as controls.Tomato 5th leaflets were detached and laid on Petri dishes containing two sterilized blotting filter papers , wetted with sterile water. A drop (20 μL) of opipette  as shown2O2 imaging detection, in control, and at 30, 120, and 240 min after spore suspension application, was performed as described earlier [2O2 production.manyHInoculated and control tomato leaflets were observed under a Zeiss Axioplan epifluoresence microscope equipped with an AxioCam MRc 5 digital camera. Photographs were obtained with the ZEN 2 version software according to the manufacturer’s instructions.Imaging PAM M-Series system  was used for the chlorophyll fluorescence measurements of the dark adapted (15 min) control and infected tomato leaflets as described in detail previously [B. cinera spores, control, 0 min) and after 30, 120 and 240 min of B. cinerea aquatic spore suspension application. The light intensities that were used for the photosynthetic efficiency measurements were 230 μmol photons m−2 s−1, a low light (LL) intensity similar to the growth light, a moderate light (ML) intensity at 640 μmol photons m−2 s−1, and a high light (HL) one, at 900 μmol photons m−2 s−1. Representative areas of interest (AOIs) were selected in each leaflet so as to cover the whole leaflet area. The chlorophyll fluorescence parameters that were measured together with their definitions are shown in PSΙΙ) and the redox state of the plastoquinone pool (qp), obtained at ML intensity at 640 μmol photons m−2 s−1, are also presented.An eviously . Measurep < 0.05.Statistically significant differences among the means were determined, from three independent treatments with each treatment consisting of three leaflets from three different tomato plants, using two-way ANOVA. Means (± SD) were considered statistically different at a level of Botrytis spore suspension in tomato leaflets, before any visual symptoms appeared, defense response mechanisms were triggered, with light energy use to be adjusted by NPQ in a such way that PSII functionality to be enhanced. The underlying mechanism involved was possible activated by H2O2, that was detected 30 min after Botrytis application with an increasing trend up to 240 min. This, is possibly convening tolerance against B. cinerea at short-time exposure, but at relatively longer exposure time when H2O2 is increasing, it is becoming a damaging molecule. Hence, the response of tomato PSII to B. cinerea, indicates a hormetic temporal response, in terms of “stress defense response” and “toxicity”, expanding the features of hormesis to biotic factors also.In the present study, 30 min after application of


Clinical interpretation of thyroid labs is usually straightforward. However, there are rare instances when the atypical profile of thyroid labs warrants systematic investigation to determine the underlying cause. We report the case of a 90-year-old Caucasian male with a chronic history of atrial fibrillation with chronic pacemaker dependence who presented with significantly elevated free thyroxine level (>7.77 ng/dL) but normal thyroid-stimulating hormone level (2.15 µIU/mL). After ruling out pituitary tumors and artifactual errors due to lab interference, the diagnosis of thyroid hormone resistance was made. Thyroid labs are one of the most commonly ordered lab tests by primary care physicians. On most occasions, interpretation of thyroid labs is straightforward. However, on rare occasions, primary physicians encounter thyroid lab panels that do not make sense. Normally thyrotropin-releasing hormone (TRH) from the hypothalamus stimulates the production of thyroid-stimulating home (TSH) which, in turn, acts on the thyroid gland to regulate the synthesis and release of tetraiodothyronine (T4) and triiodothyronine (T3). Three carrier proteins, namely, albumin, thyroxine-binding prealbumin, and thyroxine-binding globulin (TBG) bind most of the circulating thyroid hormones in the blood and only a small fraction are available as free hormones (about 0.3% T3 and 0.02% T4) . The secA 90-year-old Caucasian male with a history of atrial fibrillation, hypertension, and primary hypogonadism was referred to endocrinology for evaluation of abnormal thyroid labs and suspected hyperthyroidism.The patient had a thyroid lab panel ordered routinely which was found to be significantly abnormal. Labs showed TSH of 2.15 µIU/mL (0.46-4.68 µIU/mL), free T4 of >7.77 ng/dL, total T4 of 19.5 µg/dL (reference range: 4.5-12 µg/dL), and free T3 of 5 pg/mL (reference range: 2.0-4.4 pg/mL). He was not taking any biotin supplement and did not receive any iodinated contrast before the labs. The patient had a history of atrial fibrillation and had undergone radiofrequency ablation in 1991. He had been on apixaban and testosterone replacement but not on amiodarone, vitamins, or biotin supplements before the lab draw. The patient was asymptomatic. He had been completely dependent on a pacemaker for his heart rate. His heart rate varied from 70-130 beats per minute, and it was programmed for dynamic changes during physical activity and exercise. The patient denied tremors, palpitations, and heat intolerance.The patient had a history of hypogonadism. He got hot sweats if he missed his testosterone replacement dose. None of his family members had abnormal thyroid blood tests, including his two sons. Physical examination showed a heart rate of 96 beats per minute, blood pressure 136/86 mmHg, respiratory rate 16 breaths per minute, oxygen saturation 97% on room air, height 180.3 cm, weight 91.5 kg, and body mass index of 28. The thyroid gland was not palpable. The rest of the physical examination was unremarkable except for bilateral atrophic testicles.The patient had a mumps infection in the mid-20s during which one of the testicles was inflamed. Subsequently, he had a minor hernia repair, following which the other testicle also atrophied. Thyroid-stimulating immunoglobulin and thyrotropin receptor antibody levels were undetectable. The patient could not get an MRI because of the pacemaker. CT of the pituitary gland was normal. A summary of pertinent lab workup is provided in Table thyroid hormone receptor beta (THRβ) gene. Although the majority of TRβ-mediated thyroid hormone resistance is inherited as autosomal dominant, up to 20% can occur sporadically due to de novo mutations. In addition, negative THRβ gene sequencing does not exclude THRβ defects.Our patient had an elevated free T4 level in the presence of normal TSH, which, in the absence of a pituitary tumor or evidence of artifactual errors in lab assay, is suspicious for thyroid hormone resistance. However, our patient did not have any family history suggestive of thyroid hormone resistance. Many medications can also affect thyroid labs -4. He waTHRβ mutations have been described in the majority of the cases, up to 20% of cases can be due to de novo mutations [The thyroid hormones are responsible for the regulation of various physiological functions by binding to four identified thyroid hormone receptors isoforms, namely, thyroid hormone receptor α1 (THRα1), thyroid hormone receptor α2 (THRα2), thyroid hormone receptor β1 (THRβ1), and thyroid hormone receptor β2 (THRβ2). THRβ1 is expressed in the liver, kidney, and thyroid and THRβ2 is expressed in the pituitary, hypothalamus, and ear . THRβ2 hutations ,8. Theseutations -11.THRβ gene [The incidence of RTH has not been clearly defined; however, a study conducted in Oregon reported an incidence of 1 in 40,000 live births and approximately 1,000 cases have been reported in the literature between 1967 and 2006 . Where gHRβ gene . ApproxiHRβ gene . RTHβ caHRβ gene . The treHRβ gene . Our patTHRβ gene mutation.In the setting of normal TSH and elevated free T4, after ruling out the pituitary tumor and artifactual lab interference, a high index of clinical suspicion is needed to diagnose thyroid hormone resistance, in the absence of a family history of thyroid hormone resistance and negative


Combining genetic and cell-type-specific proteomic datasets can generate biological insights and therapeutic hypotheses, but a technical and statistical framework for such analyses is lacking. Here, we present an open-source computational tool called Genoppi (lagelab.org/genoppi) that enables robust, standardized, and intuitive integration of quantitative proteomic results with genetic data. We use Genoppi to analyze 16 cell-type-specific protein interaction datasets of four proteins  involved in cancer and neurological disease. Through systematic quality control of the data and integration with published protein interactions, we show a general pattern of both cell-type-independent and cell-type-specific interactions across three cancer cell types and one human iPSC-derived neuronal cell type. Furthermore, through the integration of proteomic and genetic datasets in Genoppi, our results suggest that the neuron-specific interactions of these proteins are mediating their genetic involvement in neurodegenerative diseases. Importantly, our analyses suggest that human iPSC-derived neurons are a relevant model system for studying the involvement of BCL2 and TDP-43 in amyotrophic lateral sclerosis. Genetic variation can impact protein complexes and interaction networks, but reconciling genetic and proteomic information remains challenging. To address this need, the authors develop Genoppi —a computational tool for integrating genetics and cell-type-specific proteomics data. Integration of genomic and proteomic datasets has revealed that genetic variation implicated in rare and common diseases often manifests at the proteome level, for example, by impacting protein complexes or cellular networks4. Data from genetics and cell-type-specific quantitative proteomics, therefore, have the potential to inform each other and lead to key molecular and biological insights. However, even for experts in these fields, the relevant data types are often not interoperable. This creates a bottleneck for functionally interpreting genetic data and dissecting the molecular biology of human diseases. In the longer term, difficulties in reconciling these data types will hamper efforts towards gaining mechanistic insights from genetic data and designing therapeutic interventions. To enable the robust, standardized, and intuitive integration of data from genetics and cell-type-specific quantitative proteomics, we have developed an open-source computational tool named Genoppi5 , which we apply to 16 cell-type-specific protein–protein interaction datasets to illustrate the functionalities of the tool.Large-scale genetic datasets, such as those obtained from genome-wide association studies (GWAS) or exome sequencing are becoming increasingly available. Simultaneously, advanced proteomic technologies can generate high-quality cell- and tissue-specific quantitative proteomic data  values between studied conditions  for multiple experimental replicates, Genoppi identifies proteins with statistically significant log2 FC by applying user-defined thresholds, displays the data in scatter and volcano plots, and provides options for integrating the results with other forms of data  derived from GWAS of a disease of interest. Genoppi automatically uses linkage disequilibrium (LD) information from the 1000 Genomes Project 15 Fig. . Genoppions Fig. . When th17, GO19, and MSigDB21, to visually identify groups of proteins that may be overrepresented in a particular dataset 24 . For example, it is possible to dynamically explore a dataset by changing various technical thresholds and visualizing the results in real-time; furthermore, a search function enables the quick identification of proteins of interest in various plots  in four distinct cell lines (“Methods” and Supplementary Data 25) and three cancer cell lines -derived neuronal cell line 30. However, little is known about the specific role of BCL2 in different human cell types, and insights into its overlapping and differential interaction partners in neurons compared to cancer cell lines could generate actionable biological hypotheses of therapeutic relevance.We first explored BCL2, which is a well-studied oncoprotein functioning as an apoptosis suppressor in a variety of cell types;2 FC > 0 and false discovery rate (FDR) ≤ 0.1 in the experiment compared to the cell-type-matched IgG control (“Methods”), while non-interactors are proteins that do not fulfill either criteria. Among BCL2 interactors, only five were previously reported in InWeb . This indicates that, despite little overlap with InWeb interactors and the identification of many cell-type-specific interactors (10 in GPiNs and 85 in G401 cells), a significant subset of new BCL2 interaction partners (n = 23) were common to two different cellular backgrounds, and thus are likely to be true interaction partners that have not been reported before 33, or schizophrenia (SCZ)34, as well as a curated list of genes involved in ALS36 . TDP-43 aggregation is also a common pathological hallmark of both neurodegenerative disorders37. To gain insights into neuronal functions of TDP-43, we analyzed the IP-MS/MS data of TDP-43 in GPiNs  are involved in RNA metabolism, suggesting that the role of TDP-43 in human neurons is mostly related to transcriptional and post-transcriptional regulation, as highlighted using Genoppi’s gene set annotation feature Fig. . Importa38, or in HEK cells39 that are less relevant to its role in ALS or FTD. Our Genoppi analyses illustrate that GPiNs recapitulate the known biology of this protein and its physical interactions to a number of known ALS-related proteins. To confirm the interactors identified in GPiNs, we made biological replicates of the TDP-43 IP, quality-controlled 23 interactor-specific antibodies  and newly identified interactors  with the same degree of success. Among the newly identified interactors, we validated eight out of nine (or 88.9%) interactors found in multiple cell lines and three out of three (or 100%) GPiN-specific interactors. Our validation experiments also span 19 interactors with ≥ two values imputed prior to log2 FC calculation (see “Methods”); 17 out of 19 (or 89.5%) could be confirmed, strongly supporting the procedure we used to impute missing values in the mass spectrometry data.TDP-43 has previously been studied in human brain homogenates, which are an aggregate of many different cell typesWe further executed reciprocal IPs of five of the TDP-43 interactors validated by western blots, including three ALS-relevant proteins  and two non-ALS proteins  that are also hypothesized to have divergent functions in cancer and neurodevelopment. Similar to the observations for BCL2 and TDP-43, we observed a large set of new interaction partners that can be replicated across multiple cell types, as well as a set of cell-type-specific interaction partners that can inform targeted hypotheses and follow-up experiments . This means that high-quality interaction experiments executed in specific human cell types can lead to the discovery of many novel interactions. Indeed, when we combined the IP-MS/MS results of BCL2, TDP-43, MDM2, and PTEN across four different cell lines, we found that only 16.6% (144/870) of the interaction partners were reported in InWeb, meaning that up to 83.4% (726/870) of these interactions were new, offering potentially exciting insights into the biology of these proteins. Stratified by protein, 97.2%, 65.8%, 72.0%, and 93.0% of the interaction partners were new for BCL2, TDP-43, MDM2, and PTEN, respectively. Across cell lines, 73.4%, 80.6%, 79.7%, and 78.8% of the interaction partners identified for the four proteins were new in GPiN, G401, T47D, and A375 cells, respectively. We note that, while the statistics here were calculated based on known interactions curated in InWeb, most of the interaction partners were also new according to the iRefIndex or BioPlex database .Finally, we clustered the four cell lines based on the overlap of interaction partners for each bait between pairs of cell lines, and observed a clear clustering of cancer cells  versus GPiNs for three out of four bait proteins . In the examples we show in this paper, the cell-type-specific protein–protein interactions in a model of human neurons link the tested proteins more strongly to neurodevelopmental diseases than the interactions identified in cancer cell lines.41, while other tools, such as ProHits-viz42, provide visualization capabilities to summarize protein interaction data as well as communicate quantitative differences between a protein of interest and its potential interaction partners. However, none of these tools focuses specifically on creating a systematic and unified workflow for integrating cell-type-specific quantitative proteomic datasets and genetic information. Genoppi is designed so it can be easily incorporated into any functional genomics pipeline by allowing users to integrate datasets, download the results, and modify the code as needed to extend the software and meet different usability requirements.Several programs are available to the community to analyze raw MS/MS dataBeyond providing Genoppi as an accessible tool, we also apply it to analyze a large set of cell-type-specific protein interaction experiments. Together, these datasets provide insights into the interactome landscape of BCL2, TDP-43, MDM2, and PTEN, and open potential avenues to explore their links to cancers and neurological disorders based on the newly found cell-type-specific interactions. We use the generated data to showcase Genoppi as a resource that can be employed to combine original and published datasets in a simple and clear format, allowing systematic analysis, visualization, and exploration of otherwise heterogeneous proteomic and genetic datasets. Genoppi is available as both an R package and as a Shiny application with documentation and test datasets to get the users started (Supplementary Note https://github.com/lagelab/Genoppi). This section provides additional technical details for analyses performed by Genoppi.A user-friendly documentation of analytical and visualization features implemented in the Genoppi application v1.0.0) is provided in Supplementary Note .0.0 is p2 FC values from ≥ 2 replicates, Genoppi performs a one-sample moderated t test from the limma43 R package to calculate a two-tailed P value and Benjamini–Hochberg FDR for each protein. Limma was originally developed to robustly identify differentially expressed genes in microarray experiments and has since been used on a variety of data types, including proteomic results45. The empirical Bayes moderated t test is used in Genoppi, as it is less sensitive to underestimated sample variances and performs best on small sample sizes compared to the classical t test44. Throughout the paper, we define significant interaction partners of a bait protein as proteins with log2 FC > 0 and FDR ≤ 0.1 in the bait versus IgG IP-MS/MS data, but we note that Genoppi allows the user to adjust these thresholds according to their needs.Given protein log12 (phase 3) dataset to obtain genotype data for unrelated individuals residing in Utah with Northern and Western European ancestry and SNPs with minor allele frequency ≥ 0.05 and missing rate ≤ 0.1. Pairwise LD between SNPs was calculated using a sliding window of 200 kb, which is the default haplotype block estimation distance used in PLINK46 (v1.07). Next, for each SNP, the LD genomic locus was defined as the region covered by other SNPs that have r2 > 0.6 with the SNP, ± 50 kb on either end. These parameters were chosen to comply with established community standards49. Using the precalculated LD locus boundaries, Genoppi can then identify all Ensembl50 protein-coding genes whose coordinates overlap with LD loci given a SNP list of interest. If multiple genes are present in the locus defined by a SNP of interest, all genes are mapped to that SNP.To generate the precalculated data Genoppi uses for SNP-to-gene mapping, we filtered the 1000 Genomes Project51 , which is a standard tool for SNP-to-gene mapping. DAPPLE uses the following definition for LD locus of a SNP: “the region containing SNPs with r2 > 0.5… extended to the nearest recombination hotspot.” Nonetheless, in our comparison test, 100% of genes mapped from SNPs using DAPPLE were analogously mapped using the Genoppi algorithm, illustrating the robustness of our approach.To verify that SNPs are robustly mapped to genes using the mapping method in Genoppi, we mapped 20 random SNPs to genes using both Genoppi and Disease Association Protein-Protein Link EvaluatorP values are calculated using a hypergeometric distribution to assess the enrichment of overlap between experimental proteomic results and other gene lists, known protein interactors from InWeb8, iRefIndex9, or BioPlex11, genes intolerant of loss-of-function (LoF) mutations derived from gnomAD16, and tissue-specific genes derived from GTEx23 or HPA24. To test overlap with a gene list , the “population” (N) is defined as all genes encoding proteins identified in the experimental data and “success in population” (k) is defined as the subset of N that pass the user-defined significance threshold . The “sample” (n) contains genes from the gene list that are found in N and “success in sample” (x) is the overlap between k and n. Similar definitions apply to test overlap with InWeb, iRefIndex, BioPlex, gnomAD, GTEx, or HPA data, except in this case the “population” (N) is the intersection of all genes in the experimental data and in the respective database, while the “sample” (n) is the subset of N consisting of known interactors for a chosen bait in InWeb, iRefIndex, or BioPlex, LoF-intolerant genes defined using a gnomAD pLI score cutoff, or tissue-specific genes in GTEx or HPA  are identified in the proteomic data, meaning that the statistical test is conditional on the proteome of the cell type being tested.One-tailed P values. Confounders include whether the mapped gene is from a single gene or multigenic locus, the gene length, and its tissue-specific expression pattern, to name a few. To accurately perform this analysis requires a workflow that is dataset-specific and is beyond the scope of Genoppi. To not mislead users, and to ensure that other statistical tests in Genoppi can be considered reliable, we do not test the statistical significance of the overlap between proteomic data and genes mapped from SNPs.The hypergeometric test is not performed for genes derived from the SNP-to-gene mapping feature in Genoppi. Correctly testing the overlap between these genes and the proteomic results is a complicated statistical problem that can easily lead to confounded results and inflated 25. Plates were coated with Geltrex  adhesion matrix (1:100 in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F:12); Gibco) and seeded at a density of 40,000 cells cm−2 in Stemflex media  containing 1:400 Genetecin  as selective antibiotic and rock inhibitor Y27632 . After expansion, day 0 cells were passaged onto plates coated with Geltrex and adhesion matrix (1:100 in DMEM/F:12) in DMEM/F:12 media containing 1% N2 supplement (Gibco), 1% Glutamax, 0.3% glucose, 0.2% normocin (Invitrogen), 1:10,000 doxycycline hyclate , 1:10,000 LDN-193189 , 1:5000 XAV939 , 1:1000 SB431542 , and 1:1000 RI. Day 1 cells were differentiated in DMEM/F:12 media supplemented with 1% N2 supplement, 1% Glutamax, 0.3% glucose, 0.2% normocin, 1:10,000 DOX, 1:20,000 LDN, 1:10,000 XAV, and 1:2000 SB. Day 2 media were DMEM/F:12 supplemented with 1% N2 supplement, 1% Glutamax, 0.3% glucose, 0.2% normocin, and 1:10,000 DOX. On day 3, cells were passaged onto plates coated with Geltrex and adhesion matrix (1:100 in DMEM/F:12) in neurobasal media (Gibco) supplemented with 2% B27 , 1% Glutamax, 0.3% glucose, 0.2% normocin, 0.5% Minimum Essential Medium-Eagle with non-essential amino acid (MEM NEAA) (Gibco), 1:10,000 DOX, 1:10,000 brain-derived neurotrophic factor (BDNF), 1:10,000 ciliary neurotrophic factor (CTNF), 1:10,000 glial cell-derived neurotrophic factor (GDNF) . On day 6, cells were fed by replacing 50% of the media with neurobasal media supplemented with 2% B27, 1% Glutamax, 0.3% glucose, 0.2% normocin, 0.5% MEM NEAA, 1:10,000 DOX, 1:10,000 BDNF, 1:10,000 CTNF, 1:10,000 GDNF, 2.5 µg mL−1 laminin, and 1:10,000 floxuridine (Sigma-Aldrich). On days 9 and 13, cells were fed by replacing 50% of the media with neurobasal media supplemented with 2% B27, 1% Glutamax, 0.3% glucose, 0.2% normocin, 0.5% MEM NEAA, 1:10,000 DOX, 1:10,000 BDNF, 1:10,000 CTNF, 1:10,000 GDNF, and 2.5 µg mL−1 laminin. From day 13 onwards, cells were fed every 3–4 days by replacing 50% of the media with neurobasal media supplemented with 2% B27, 1% Glutamax, 0.3% glucose, 0.2% normocin, 0.5% MEM NEAA, and 1:10,000 DOX.GPiNs were differentiated to day 31 from a clonally selected induced PSC line (iPS hDFn 83/22 iNgn2#9 [iPS3]) by conditional expression of the neuralizing transcription factor NGN2−2 (uncoated plates). Cell maintenance media contained 10% fetal bovine serum and PenStrep (1:1000). A375 cells were cultured in DMEM (Thermo Scientific) and split every 2 days (at a 1:12 ratio), G401 were cultured using McCoy’s 5A (Thermo Scientific) media and split every 2 days (at a 1:10 ratio), and T47D cells were cultured in RPMI no phenol red (Thermo Scientific) media and split every 3 days (at a ratio of 1:4).We used the following cancer cell lines: A375 (ATCC CRL-1619), a human malignant melanoma cell line exhibiting a wild-type p53 genotype; G401 (ATCC CRL-1441), a kidney rhabdoid tumor cell line with a wild-type p53 genotype; and T47D (ATCC HTB-133), a human breast tumor cell line with a mutated p53 genotype. All cell lines were plated at a density of 40,000 cells cm2. To achieve detachment during passaging, all cell lines were exposed to TrypLE (Thermo Scientific).All cell lines were incubated at 37 °C, 5% CO53. In both cases, cell pellets were washed with phosphate-buffered saline (PBS) and resuspended in 10× packed cell volume (PCV) IP lysis buffer (Pierce), with freshly added Halt protease and phosphatase inhibitors (Thermo Scientific). After a 20 min incubation at 4 °C, cells were collected by centrifugation  and resuspended in 3× PCV lysis buffer. The concentration of the samples was quantified using the Thermo BCA protein assay, and when not used immediately, samples were stored at −80 °C. Samples for immunoblotting were diluted in 6×SMASH buffer , 0.02% bromophenol blue, 1% β-mercaptoethanol), boiled for 10 min at 95 °C, separated on a NuPAGE 4–12% Bis-Tris Protein Gel (Thermo Scientific), and transferred onto a PVDF membrane (Thermo Scientific) by wet transfer (100 V for 2 h). Membranes were blocked by incubation for 1 h at room temperature in 10 mL Tris-buffered saline (TBS) and 0.1% Tween (TBST) with 5% (w/v) Bio-Rad Blotting-grade Blocker. Blots were incubated overnight at 4 °C with the primary antibody (1:1000), washed three times for 10 min with TBST, and incubated for 45 min with the secondary horseradish peroxidase-conjugated antibody. After washing three times for 5 min with TBST, bands were visualized using ECL . All antibodies used in this study are listed in Supplementary Data Total protein extract was obtained by harvesting cells and either processing them immediately or snap-freezing them on dry ice for storage at −80 °CFor each individual experiment, 1–2 mg of protein extract was incubated at 4 °C overnight in the presence of 1–2 μg of the relevant antibody. The next day, 50 μL of Protein G beads (Pierce) were added to each sample and incubated at 4 °C for 4 h. Flow-through was collected and beads were washed once with 1 mL lysis buffer (Pierce) supplemented with Halt protease and phosphatase inhibitors (Thermo Scientific), and twice with PBS. Beads were resuspended in 60 μL of PBS and 10% of the volume was employed for immunoblotting, after being boiled in 6×SMASH buffer  for 10 min at 95 °C. The remaining volume was stored at −80 °C and subsequently used for MS analysis.n = 48) and IgG controls (n = 12) were in PBS buffer on beads. PBS was removed and samples were dissolved in 50 µL TEAB  buffer, followed by trypsin (Promega) digestion for 3 h at 38 °C. Digested samples were dried to 20 and 10 µL of each sample was injected in the mass spectrometer.All immunoprecipitated samples . MS survey scan was performed in the Orbitrap, in a range 400–1800 m/z at a resolution of 6 × 104, followed by the selection of the 20 most intense ions (TOP20) for CID-MS2 fragmentation in the ion trap using a precursor isolation width window of 2 m/z, automatic gain control setting of 10,000, and a maximum ion accumulation of 100 ms. Singly charged ion species were not subjected to collision-induced dissociation fragmentation. Normalized collision energy was set to 35 V and an activation time of 10 ms. Ions within a 10 p.p.m. m/z window around ions selected for MS2 were excluded from further selection for fragmentation for 60 s.LC-MS/MS was performed on a Lumos Tribrid Orbitrap Mass Spectrometer (Thermo Scientific) equipped with Ultimate 3000 (Thermo Scientific) nano-high-performance liquid chromatography. Peptides were separated onto a 150-µm inner diameter microcapillary trapping column, packed with ~2 cm of C18 Reprosil resin , followed by separation on a 50-cm analytical column . Separation was achieved by applying a gradient from 5 to 27% acetonitrile in 0.1% formic acid for > 90 min at 200 nL min54 as well as other known contaminants such as human keratins and common laboratory contaminants. Quantitative analysis between samples was performed by label-free quantitation (LFQ) between different sets of samples. Sequest HT searches were performed using a 10 p.p.m. precursor ion tolerance and requiring each peptide’s N/C termini to adhere with trypsin protease specificity, while allowing up to two missed cleavages. CID-MS2 spectra were searched with 0.5 Da ion tolerance for fragmentation. Methionine oxidation (+15.99492 Da) was set as variable modification. An MS2 spectra assignment FDR of 1% was applied to both proteins and peptides using the Percolator target-decoy database search.Raw data were analyzed with Proteome Discoverer . Assignment of MS/MS spectra was performed using the Sequest HT algorithm by searching the data against a protein sequence database including all entries from the Uniprot_Human2018_SPonly database2 transformation and median normalization of protein intensity values in each experimental sample; (2) filtered out contaminants and protein entries supported by < 2 unique peptides; (3) imputed missing protein intensity values in each sample (see details below); (4) calculated log2 FC for each pair of replicate samples . All preprocessed data and subsequent analysis results  can be found in Supplementary Data Starting with protein level LFQ reports, we performed the following preprocessing steps before inputting the data into Genoppi: (1) performed log2 FC statistics for each protein detected in the proteomic data, we imputed missing protein intensity values in each sample using a well-established approach55 prior to calculating log2 FC for a pair of samples. Specifically, to replace each missing value in a sample, we randomly sampled from a normal distribution with a mean of μ − 1.8σ and standard deviation of 0.3σ, where μ and σ are the mean and standard deviation of the observed intensity values in the sample. This procedure works under the assumption that proteins with missing values likely have low intensities below MS detection threshold, and therefore place these proteins in the lower tail of the observed intensity distribution. As positive control examples for this imputation strategy: some of our bait proteins and their known InWeb interaction partners needed to be imputed as they were not detected in the IgG controls; after imputation, they generally showed statistically significant log2 FC values that overlaid nicely with the non-imputed log2 FC distribution. As further support, we also successfully validated 17 out of 19 (89.5%) TDP-43 interactors with ≥ 2 imputed values prior to log2 FC calculation in GPiNs (Supplementary Note In order to derive logt test from the limma R package) implemented in Genoppi when applied to our experimental data, we also used an alternative statistical method, SAINTexpress56, to identify significant interaction partners of the four bait proteins in the four cell lines (Supplementary Data 2 FC > 0 and FDR ≤ 0.1) and by SAINTexpress (BFDR ≤ 0.1; Supplementary Data To assess the robustness of the statistical method (moderated 31. For neuropsychiatric disease, we aggregated a total of 571 unique genes that have been implicated in ASD or SCZ. This list includes ASD genes identified by exome sequencing32 and mapped from genome-wide significant GWAS index SNPs33 using Genoppi’s SNP-to-gene mapping framework. For SCZ, we mapped genome-wide significant GWAS regions34 to genes overlapping these regions. For ALS, we curated a list of 53 genes based on literature review36.In order to investigate the overlap between interactors identified in our proteomic data and disease-associated genes, we compiled several gene lists from published genetic studies (Supplementary Data Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Reporting Summary


Myrciaria jaboticaba) is a Brazilian berry rich in phenolic compounds, much appreciated for its sweet and slightly acid taste, and highly perishable. Thus, we aimed at producing jaboticaba juice by steam extraction and at investigating its microbiological, sensorial and chemical qualities during storage for up to 168 days. Juice was microbiologically safe and even though unsweetened juice was well accepted, sucrose addition further improved flavor (21%), overall impression (11%) and purchase intent (21%) scores. Cyanidin-3-O-glucoside (C3G) was the major phenolic (40%), followed by gallic (28%) and ellagic acids (21%). Total phenolics contents decreased from 27% (50 °C) to 50% (25 °C), mainly driven by C3G degradation. At 60 °C, total phenolics contents did not change after 42 days since C3G degradation was counterbalanced by gallic acid formation (129%), which followed zero-order reaction kinetics. Anthocyanins degradation followed first-order reaction kinetics  and was associated with color changes during storage. In conclusion, steam extraction followed by hot-filling technique ensured a juice with at least six months of shelf life.Jaboticaba ( Myrtaceae family and Plinia genus, also referred as Myrciaria  against time and ln ([C]t/[C]0) against time, respectively.Zero and first-order rate constants (t1/2), which is the time required for anthocyanins to degrade to 50% of their initial contents at given temperatures, was calculated from the rate constant k, as t1/2 = ln 2/k.The half-life time  of the reactions were calculated from rate constants (k) values observed at each experimental temperature, using the Arrhenius equation (Equation (4))A is the frequency factor (day−1), R the universal gas constant (8.3145 J mol−1 K−1) and T the absolute temperature (K) [The activation energy (ture (K) .Salmonella spp., heterotrophic bacteria, lactic bacteria, yeasts and molds, every 56 days up to 168 days.Microbiological analyses of UJJ were performed according to Downes and Ito  investigSensory acceptance and purchase intent of both UJJ and SJJ were performed at the beginning and at the end of the storage (168 days) at 25 °C. All the consumers reported at least once a week fruit juice consumption.Juices were presented at ~10 °C in plastic cups coded with three-digits numbers and offered monadically in balanced order. Overall impression, aroma, color, flavor and viscosity were evaluated by consumers using a 9-points structured hedonic scale. Consumers were also asked to report the presence of any residual taste. The purchase intent was evaluated using a 5-points structured scale.The study was approved by the ethics committee of the Federal University of Rio de Janeiro .t-test. Differences in chemical composition between storage times for UJJ were evaluated by one-way ANOVA followed by Dunnett’s post hoc test. Pearson correlation analysis was performed between instrumental color and anthocyanins content. The effects of sucrose addition and of storage on the sensory acceptance and purchase intent scores were investigated using Wilcoxon matched-pairs signed rank test and Mann–Whitney test, respectively. Statistical analyses were performed using GraphPad Prism software version 8.0  and results were considered significant when p < 0.05.Data are expressed as mean ± standard deviation. Differences in phenolic compounds profile between UJJ and SJJ at each storage time were evaluated by unpaired Student’s UJJ contained glucose, fructose and sucrose, and oxalic, tartaric, malic and citric acids . The twoThe main fruit phenolic compounds were investigated. No relevant differences were observed between UJJ and SJJ phenolic compounds profile . CyanidiIn addition to yielding a juice rich in phenolics, the lixiviation of anthocyanins to the juice had a positive impact on its sensory acceptance, as color was one of the sensorial attributes with the highest scores in UJJ . In factExcept for sucrose, sugar contents of juice remained stable during the whole storage time at 25 °C . Among oConsidering that phenolic compounds profile during storage were similar in UJJ and SJJ, henceforth only data from UJJ will be presented and discussed. An expressive decrease in anthocyanins contents was observed A,B, withp < 0.0001). At 25 °C, ΔE was distinguishable (1.5 < ΔE < 5) to the human eye [E = 4.0), became evident (ΔE > 5) after 42 days (ΔE = 5.7) and progressively increased thereafter (up to ΔE = 8.0) (p < 0.0001) and b* values  values. Nevertheless, the juice still presented its purplish color even after 112 days of storage, which may be explained by different rates between anthocyanins degradation and color fading [E changed more rapidly. At 40 °C, evident color changes were observed after 21 days after storage (ΔE = 6.2) whereas at 50 and 60 °C, as soon as 7 days of storage   . Color cr fading . At highctively) .Storage of jaboticaba juices for 168 days at 25 °C did not affect their sensory acceptance and purchase intent, with the exception of color, which scores slightly decreased (~6%) . TherefoSalmonella sp. and with low numbers of possibly spoilage microorganisms (up to 90 colony-forming units per mL of juice) as heterotrophic bacteria, lactic acid bacteria, yeasts and molds. The extraction process was also effective to ensure adequate juice microbiological quality during storage. No growth of thermotolerant coliforms and Salmonella spp. was observed during 168 days of storage and presence of heterotrophic bacteria, lactic acid bacteria, yeasts and molds was limited to less than 10 colony-forming units per mL of juice for each of them  or formed  during storage, we performed kinetic studies, in order to investigate their transformation pathways.2 values (>0.96) confirmed that the Arrhenius model fitted the experimental data at temperatures ranging from 25 to 50 °C. Degradation rates constants (k) increased with temperature and results are in accordance with previously published data [k value was higher than that of D3G, indicating that the former was degraded faster than the latter. Consequently, the half-life times for D3G and C3G degradation at 25 °C were 23.9 days and 21.7 days, respectively. Together, these results indicate that D3G is more stable than C3G in jaboticaba juice, which is the opposite behavior observed for these anthocyanins isolated from grapes [a) obtained from the Arrhenius plot (a = 70.55 kJ mol−1) and C3G (Ea = 72.25 kJ mol−1) suggest that the reaction rate of the latter is more sensitive to an increase in temperature than the former. Ea values are similar to that reported for total anthocyanins (Ea = 72.2 kJ mol−1) in black carrot juice [2 > 0.89). Gallic acid formation followed a zero-order reaction model and showed increasing formation rates (k) with temperature. The 7-fold increase between formation rate constants at 40 °C (k = 2.43 × 10−6 mol L−1 day−1) and 60 °C (k = 1.73 × 10−5 mol L−1 day−1)  C suggesty−1) (−1 C and, toThe scheme in Jaboticaba juice produced by steam extraction incorporated the phenolic compounds from the fruit and showed adequate microbiological quality and good sensorial acceptance requiring neither the employment of any additional preservation technology nor the use of chemical preservatives. While sugars and organic acids contents were not modified during juice storage at 25 °C for 6 months, phenolics profile and contents changed, mainly because anthocyanins were almost completely degraded. Nevertheless, sensorial acceptance was not affected. Finally, steam extraction seems to be a viable method to produce jaboticaba juice with commercial potential, even though scale-up and marketing studies are still needed for this product to reach the market.


Background: Similarities in the biology of pulmonary hypertension and cancer suggest that anticancer therapies, such as sanguinarine, may also be effective in treating pulmonary hypertension. This, along with underlying biochemical pathways, is investigated in this study.Methods: Rats were subjected to 4-week hypoxia (or control) with or without sanguinarine treatment. In addition, pulmonary artery smooth muscle cells (PASMCs) were examined after 24–48 h hypoxia (with normoxic controls) and with or without sanguinirine. Pulmonary artery pressures and plasma survivin levels were measured in vivo. Ex vivo tissues were examined histologically with appropriate staining. mRNA and protein levels of survivin, HIF-1α, TGFb1, BMPR2, Smad3, P53, and Kv 1.2, 1.5, 2.1 were determined by real-time PCR and Western blot in PASMCs and distal PAs tissue. PASMC proliferation and changes of TGFb1 and pSmad3 induced by sanguinarine were studied using MTT and Western blot. Electrophysiology for Kv functions was measured by patch-clamp experiments.Results: Four-week hypoxia resulted in an increase in serum survivin and HIF-1α, pulmonary artery pressures, and pulmonary vascular remodeling with hypertrophy. These changes were all decreased by treatment with sanguinarine. Hypoxia induced a rise of proliferation in PASMCs which was prevented by sanguinarine treatment. Hypoxic PASMCs had elevated TGFb1, pSmad3, BMPR2, and HIF1α. These increases were all ameliorated by sanguinarine treatment. Hypoxia treatment resulted in reduced expression and function of Kv 1.2, 1.5, 2.1 channels, and these changes were also modulated by sanguinarine.Conclusion: Sanguinarine is effective in modulating hypoxic pulmonary vascular hypertrophy via the survivin pathway and Kv channels. Pulmonary hypertension (PH) refers to a variety of conditions characterized by elevations in pulmonary arterial pressure. It is a life-threatening disease with about 1% incidence in adults . The vasThere are several underlying causes and five categories are recognized. These are pulmonary arterial hypertension , pulmonary hypertension due to left heart or lung/hypoxia diseases , PH due to pulmonary arterial stenosis (Type 4), and PH due to miscellaneous cause (Type 5). The various types of PH differ widely with respect to their prevalence and treatment. Two most frequent causes of pulmonary hypertension are left heart diseases and hypoxic lung diseases. Global studies of chronic obstructive lung disease (COPD) stage IV showed that up to 90% have mPAP >20 mmHg, with most ranging between 20 and 35 mmHg. Approximately 1–5% of COPD patients have mPAP >35–40 mmHg at rest . WhereasSanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, has antioxidant, anti-inflammatory, proapoptotic, and growth inhibitory effects on different tumor cells . Survivin = 28, respectively). Hypoxic rats were housed in a hypoxic (10% O2) Plexiglas cabin for 8 h/day for 4 weeks  were purchased from the Laboratory Animal Center at Xi’an Jiaotong University . The rats were randomized into control and hypoxia groups  intraperitoneally and placed on an operating table. Subsequently, a percutaneous jugular vein-catheter, through the right atrium and ventricular, was installed into the main pulmonary artery and then connected to a pre-corrected zero value pressure transducer of a multiple electroconductive physiological recorder  to record the pulmonary artery pressure  with the heart rate.After the rats were sacrificed, the heart, lung, and liver were immediately isolated and weighed. The right ventricle (RV) was dissected from the left ventricle and septum (LV + S), and the Fulton index [RV wet weight/(LV + S)] was calculated as an index of right ventricular hypertrophy (RVHI). Then, the right lower lobes of the lung tissues were collected and fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded, cut into 10 μm sections, stained with hematoxylin and eosin (HE), and observed for pulmonary arteriole morphology under a light microscope . 3 HE–stained lung tissue sections, each with at least three pulmonary arterioles, were randomly selected from every group for pulmonary arteriole measurement. Three views for each arteriole was observed, and the outer and inner circumference and wall thickness were calculated using Image-Pro Plus 6.0 software. The average values were used to assess vascular proliferation.The content of survivin and HIF-1α in rat serum was determined by the ELISA method, using the Rat Survivin  and HIF1α  ELISA kits following the manufacturer’s instructions. In brief, 20 μL of each standard, control, and samples (serum) in duplicate was dispensed with new disposable tips into appropriate wells on a microplate coated with survivin and HIF1α antibodies. After that, each microplate well was filled with 200 μL enzyme conjugate, thoroughly mixed, and then incubated for 60 min at room temperature. After incubation, the solution in the wells was briskly shaken out and rinsed out four times with 350 μL diluted washing solution per well. Furthermore, 100 μL substrate solution was added to each well of the microplate, which was then re-incubated for 15–20 min at room temperature. After that, the enzymatic reaction was stopped by adding 100 μL stop solution to each well. Finally, absorbance was determined by using an ELISA reader  at 450 nm for quantification of protein abundances.Second- to third-degree branches of the pulmonary artery were isolated from the lobes of the lungs, cleaned off blood in cold phosphate-buffered saline, dissected and removed the endothelium, frozen in liquid nitrogen, and stored at −80°C for subsequent mRNA or protein extraction.2+ Krebs solution, small pieces of muscle layers were stood for 20 min at room temperature prior to digestion with 4 mg/ml papain, 1.25 mg/ml BSA, and 2 mg/ml DTT in 2 ml hypoxic low-Ca2+ dissolution solution. Following threefold washing in low-Ca2+ solution, separated PASMCs were stored in hypoxic low-Ca2+ solution with 0.5% BSA at 4°C for use within 3–4 h.To functionally characterize Kv channels by whole-cell patch-clamp, PASMCs were freshly dissociated from intralobar pulmonary arteries of rats from control, Control + Sang, Hypoxia, and Hypoxia + Sang groups, as described previously . In brie2, 5% CO2, and rest N2) in the 1% (v/v) FCS and 1% (m/v) penicillin and streptomycin M199 medium for the indicated period of time. All other measurements were performed under normoxic conditions, starting 30 min after termination of hypoxia.PASMCs for the remaining cell experiments were isolated from normal adult SD rats and identified by immunohistochemical staining with α-SMC. Rat PASMCs were exposed to hypoxia  was separately added either in the hypoxia or normoxia medium for the indicated period. 1-10-100 nmol/L survivin selective inhibitor YM155  was used as a positive control versus DMSO as a negative control. Cells were harvested at 24 h for 24-h MTT experiments, and the rest were collected at 48 h for 48-h MTT and RNA and protein extraction.Isolated cells grown on glass slides were fixed with 4% formaldehyde in PBS for 30 min and incubated in blocking solution for 30 min (PBS containing 2% BSA-0.1% Triton X-100). Cells were incubated with primary antibodies against α-SMA, F-actin, collagen I, and collagen III for 1 h and incubated for 30 min at 37°C with secondary antibodies (Jackson ImmunoResearch). The cell nuclei were stained with 4′,6′-diamidino-2-phenylindole . Cells were observed with fluorescence microscopy (Olympus).PASMC proliferation was determined by a MTT method. In brief, at the end of treatments, 20 mg of 5 mg/ml MTT solution was added to each well, and PASMCs were incubated for 24 and 48 h at 37°C. Then, the culture medium was removed, and 150 l DMSO was added to each well and incubated for 10 min to solubilize the formazan salt crystals. The formazan within cells was quantified at 490 nm using an enzyme-linked immunosorbent assay reader . Each group had six wells, and the average optical density value of those six wells was used as the result of each experiment.-ΔΔCt (Livak) method.Total RNA was extracted using a TRIzol reagent , according to the manufacturer’s instructions. The concentration and quality of isolated RNAs were determined using an ultraviolet spectrophotometer and by electrophoresis on agarose gel, respectively. The cDNA was synthesized using a Reverse Transcriptase kit . The specific primer sequences used for the real-time PCR (Rt-PCR) are presented in Total protein was extracted using RIPA buffer, and the concentration was quantified using a BCA Protein Assay Kit . Equal amounts of protein were loaded onto 10% SDS-PAGE gels for electrophoresis, transferred onto the PVDF membrane, blocked by 5% skim milk, and incubated with the primary antibody overnight at 4°C, including anti-survivin , anti-TGFb1, anti-pSmad3 , anti-BMPR2 , anti-HIF1α, anti-P53 , anti-Kv1.2 , anti-Kv1.5 , anti-Kv2.1 , and anti-GAPDH . The membrane was washed and incubated with HRP-conjugated goat anti-rabbit IgG  at room temperature for 1 h. The blots were visualized using the chemiluminescence system and quantized by Quantity One.Cells for patch-clamp experiments were performed under hypoxic or normoxic conditions as indicated. Drug interventions were as indicated in the “Results” section. The record of K+ current (IK) and EM in the PASMCs was performed using an Axopatch 200B patch-clamp amplifier and 3- to 5-M micropipettes. PASMCs were voltage-clamped at −70 mV, and currents were evoked by steps of 200-ms duration from −70 to +70 mV. Membrane currents were filtered at 5 kHz, digitized using a Digidata 1320A interface , and analyzed using pCLAMP software.2 test was used to evaluate the differences among groups with ordinal data. Dose–response was analyzed by a four-parameter logistic regression model. A p-value of less than 0.05 was considered significant.Values are given as mean ± SEM (standard error of the mean) or SD (standard deviation) or percentage (N). Calculations were performed using the GraphPad Prism software package . Statistical tests were two-sided, and data were tested for normality. Comparisons between two groups were assessed with a student “t” test. The “t” test was used with a Bonferroni correction for multiple comparisons too. The χThere was no significant difference among the groups at baseline in body weight. After 4 weeks, weight gain was less in hypoxic that control rates. However, sanguinarine treatment significantly improved the nutritional status . MeanwhiHistological examination showed that the right ventricles were enlarged, the lungs and livers looked edematous and congested, and pleural effusion and ascites were visible in rats subjected to hypoxia when rats were sacrificed after 4 weeks. All the aforementioned signs were significantly less after sanguinarine treatment (data not shown here). Furthermore, rats exposed to hypoxia showed hypertrophy of pulmonary arteries with a thicker medial smooth muscle layer and smaller inner lumen area. All these indices indicative of remodeling of the pulmonary arteries and right heart were reduced dramatically in the Sang + Hypoxia group . SanguinIn the cell experiments, the PASMCs and the proliferation were identified by immunofluorescence staining with α-SMC  and thenHIF1α/TGFβ/Smad pathway or/and by its feed-forward loop with survivin. The results showed that both serums HIF1α and survivin were significantly increased in rats after 4-week hypoxic exposure , but these effects were reduced by sanguinarine treatment  (Bone morphogenetic protein type 2 receptor (BMPR2)/BMP signaling is a typical molecular mechanism for not only primary but also hypoxic PH. It was proposed TGFβ, working partially via its downstream Smad, is critical in lung vascular remodeling . In this < 0.05) . These r2 sensor and O2-sensitive voltage-dependent potassium (Kv) channels in resistance pulmonary arteries. A reduction in Kv channels, in particular Kv1.2, Kv1.5, and Kv2.1, have been proven in the pathogenesis of hypoxia-induced pulmonary hypertension, leading to pulmonary vasoconstriction and vascular remodeling, while the upregulation of Kv channels is of therapeutic significance for pulmonary hypertension (p < 0.05). However, the above reduction of IK was almost recovered completely by treating with sanguinarine (p < 0.05), whereas when PASMCs were pre-treated by the Kv channel blocker 4-AP, all above interventions had much less effect on whole-cell voltage K+ current . These results give another possibility that sanguinarine positively changed PA remolding by directly or indirectly  restoring Kv channels.Gene expressions of Kv1.2, Kv1.5, and Kv2.1 were also examined in PAs of experimental animals. The three Kv channels were down-expressed in the hypoxic group  is a fatal disease, classified into five types according to clinical characteristics (as enumerated in the Introduction). However, irrespective of the clinical type, a consistent feature is pulmonary artery remodeling with features similar to those seen in cancer and resulting in increased pulmonary vascular resistance, pulmonary hypertension, right heart failure, and ultimately death. Regarding the mechanism of this cancer-like vascular remodeling, TGF-β/Smad3, BMPR2 pathways, and O and p53 . It has  and p53 . The rol and p53 . We ther and p53 , 7, cons and p53 . In the  and p53 , 6. ThesMackraj et al. (Cardiovasc Ther. 2008). These effects of sanguinarine were also involved in the development of pulmonary artery remodeling. So, it suggests that sanguinarine would be a potential new therapeutic approach for pulmonary artery remodeling from PH. Importantly, it is enhanced by the previous demonstration in animal and cell studies of a low level of toxicity as well  is a plant alkaloid with the chemical structure of 13-methyl benzodioxole-1,3-dioxolo phenanthradinium. An important intermediate in its synthesis is protopine. Dihydrosanguinarine (DHSA) is formed by hydroxylation of protopine with NADPH, as a reduction cofactor, and molecular oxygen. It has diverse biological activities, including modulation of TGFb and HIF1α. It is also known to induce apoptosis, inflammation, and immunity as reviewed by  as well . In acco as well , 4. Ther as well , 6. The  as well .Taken together, our findings presented that sanguinarine has a multi-function in suppressing the pathogenesis of PH. Sanguinarine alleviates pulmonary vascular remodeling and fibrosis during hypoxia-induced PH, suppresses the excessive proliferation, and induces apoptosis of PASMCs via inactivation of survivin/HIF1α and their interactions with TGF-β/Smad3/p53. Moreover, hypoxia-induced decrease in function and expression of Kv channels is repressed by sanguinarine, which could be directly or indirectly via survivin/HIF1α, while further study is needed on this (Table 8).via survivin/HIF1α and by inhibiting their interactions with BMPR-TGFb/SMAD/p53 signals. It indicated that it is possible that sanguinarine could be a breakthrough anti-vascular remodeling agent for PH, possibly an ideal agent to use in combination with vasodilating agents.The study demonstrated that sanguinarine improving the hypoxia-induced characters and pulmonary vascular remodeling