Cell cycle analysis is important for cancer research. We present herein a novel method for accurate cell cycle analysis. This method analyzes the cell cycle by multiparameter flow cytometry based on simultaneously labeling the cell nuclear DNA, RNA, and phosphorylated mitotic nuclei protein, using Hoechst 33342, pyronin Y, and MPM-2-Cy5, respectively, and our results demonstrated that this method could effectively divide the cell cycle into G0, G1, S, G2, and M phases. We further tested this method using the clinical anticancer agents crizotinib and taxol, and the results clearly illustrated that crizotinib and taxol arrested Jurkat cells in G0 and M phase, respectively. These results indicate that this method could be a very useful tool for cytokinetic and pharmacological research. The cell cycle consists of five distinct phases: G0 phase (quiescence), G1 phase, S phase (synthesis), G2 phase (interphase), and M phase (mitosis). Studies have shown that cancer is a cell cycle-related disease cause by the loss of cell cycle regulation ,4. The cet al. have developed a method based on pyronin Y/Hoechst 33342 double staining to separate the G0 from the G1 phase [Several multiparameter flow cytometry analysis methods have been developed based on different expression of cyclins ,7,8. OthG1 phase . During G1 phase . TherefoG1 phase , and theIn the present studies, on the base of Shapiro’s method , which sBy measuring the DNA content of Jurkat cells using PI staining, the cells cycle distribution was analyzed. As shown in To overcome the limitations of the DNA content-based cell cycle analysis, we set up a multiparameter flow cytometry analysis using Hoechst 33342, pyronin Y, and MPM-2. As shown in To further validate this method in other cell lines, an adherent cell line SMMC-7721 was employed. In This multiparameter flow cytometry method is based on the increased RNA level in G1 phase and the mitotic phosphoproteins level in M phase. The staining of DNA/RNA by Hoechst 33342/pyronin Y is mainly the same as Shapiro’s method . HoweverIt has been reported that the cell cycle arrest caused by crizotinib is at the G0/G1 phase , and thaAs shown in Hoechst 33342 and pyronin Y were from Sigma ; MPM-2-Cy5 was purchased from Millipore ; criziotinib (pf-02341066) and taxol were purchased from Selleck Chemicals . Compounds were dissolved in dimethyl sulfoxide to a final concentration of 10 mM and stored at −20 °C until use.2. SMMC-7721 cells are maintained in Dulbecco’s modified Eagle medium  and the other incubation conditions are the same as for Jurkat cells.Human T lymphocyte Jurkat cells and human liver cancer cells SMMC-7721 were purchased from Culture Collection of the Chinese Academy of Sciences . Jurkat cells are maintained in RPMI 1640 medium supplemented with 10% fetal calf serum , 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified incubator with 5% COThe Jurkat cells were harvested and ﬁxed in ice-cold 70% (v/v) ethanol for 24 h at 4 °C. The cell pellet was collected by centrifugation at 500 × g, resuspended in PBS, and stained with a mixture of RNase (10 µg/mL) and PI (50 µg/mL) in sodium citrate containing 0.5% Triton X-100 for 20 min in the dark. Cell cycle analysis was performed using a Quanta SC flow cytometer .2. The cell suspensions were then centrifuged at 400 g for 5 min and washed once with ice-cold PBS. Then, the cells were vortexed and permeabilized with absolute cold methanol (−20 °C) overnight. After permerabilization, the cells were centrifuged, collected and rinsed once with PBS. The cells were then resuspended in 1 µg/mL Cy5 conjugated MPM-2 antibody in PBS for 1 h at 37 °C. After labeled with the antibody, the cells were rinsed and resuspended in HBSS containing Ca2+ and Mg2+, at a cell density about 2 × 106/mL. This suspension was finally incubated with equal volume of Hoechst 33342 (2 µg/mL) and of pyronin Y (4 µg/mL) diluted in a solution of Hanks’ balanced salt solution (HBSS) for 20 min.Cells were fixed in suspension in 0.5% formaldehyde for 15 min at 37 °C in the humidified incubator with 5% COCellular fluorescence was measured using a Moflo XDP instrument  using the following setup parameters: Hoechst 33342 was excited by 355 nm laser, emitted at 450 nm, in FL11 channel, and the signal mode was linar amplifer; pyronin Y was excited by 488 nm laser, emitted at 560 nm, in FL2 channel, and the signal mode was linar amplifer; MPM-2-Cy5 was excited by 633 nm laser, emitted at 650 nm, in FL6 channel, and the signal mode was logarithmic amplifer. The fcs mode data files were analyzed using FlowJo software .The negative control for Jurkat T and SMMC-7721 cells are stained by Hoechst 33342 and IgG-Cy5. We used the bivariate dot plots showing Hoechst 33342/pyronin Y negative control. As there was no pyronin Y in negative control, so all the cells in pyronin Y were negative, and this cluster cell (Hoechst 33342(2C) pyronin Y −) was G0, and the region of the (Hoechst 33342(2C) pyronin Y +) was G1 gate. In addition, we used the bivariate dot plots showing Hoechst 33342/MPM-2-Cy5 negative control. As there was no MPM-2-Cy5 in negative control, so (Hoechst 33342(4C) Cy5–) cluster was set as G2, and (Hoechst 33342(4C) Cy5+) was set as M gate. The cluster in (Hoechst 33342(4C) Cy5+) was the nonsepecific combined cells. At last, S phase was gated (Hoechst 33342(2C–4C)) from the Hoechst 33342 histogram manually, according to the DNA distribution. Jurkat cells were seeded in 24-well plates. Cells in exponentially growing phase were treated with crizotinib (100 nM) or taxol (25 nM) for 24 h. After the treatment with crizotinib or taxol, the cells were collected by centrifugation at 500 × g, washed and resuspended in PBS. Then the cells were fixed, stained with MPM-2-Cy5, Hoechst 33342, pyronin Y and analysis with flow cytometry as we described above.t-test. p values less than 0.05 were considered statistically significant and p values less than 0.01 were considered extremely striking.Data was expressed as mean ± standard deviation of the mean (SD). Statistics was analyzed using Student’s We have established a novel and alternate multiparameter flow cytometry method for cell cycle analysis by simultaneously labeling the cells with Hoechst 33342, pyronin Y, and MPM-2-Cy5. This method could accurately divide the cells into five groups, corresponding to the G0, G1, S, G2, and M phases, and resolve the ambiguous cell cycle arrest obtained by the single parameter method. Using this newly established method, we showed that the anticancer agent crizotinib and taxol arrested the Jurkat cells at the G0 and M phase, respectively. Our results indicate that this new method could be used for accurate cell cycle analysis in cytokinetic and pharmacological research.


Allium sativum) has potent antimicrobial activity due to allicin  synthesized by enzyme catalysis in damaged garlic tissues. Allicin gives crushed garlic its characteristic odor and its volatility makes it potentially useful for combating lung infections. Allicin was synthesized (>98% pure) by oxidation of diallyl disulfide by H2O2 using formic acid as a catalyst and the growth inhibitory effect of allicin vapor and allicin in solution to clinical isolates of lung pathogenic bacteria from the genera Pseudomonas, Streptococcus, and Staphylococcus, including multi-drug resistant (MDR) strains, was demonstrated. Minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were determined and compared to clinical antibiotics using standard European Committee on Antimicrobial Susceptibility Testing (EUCAST) procedures. The cytotoxicity of allicin to human lung and colon epithelial and murine fibroblast cells was tested in vitro and shown to be ameliorated by glutathione (GSH). Similarly, the sensitivity of rat precision-cut lung slices (PCLS) to allicin was decreased by raising the [GSH] to the approximate blood plasma level of 1 mM. Because allicin inhibited bacterial growth as a vapor, it could be used to combat bacterial lung infections via direct inhalation. Since there are no volatile antibiotics available to treat pulmonary infections, allicin, particularly at sublethal doses in combination with oral antibiotics, could make a valuable addition to currently available treatments.Garlic ( Allium sativum L.) was identified by Cavallito in 1944 as being due to diallylthiosulfinate which was given the trivial name allicin [S-allyl-l-cysteine sulfoxide)  . Enzyme However, allicin is a reactive sulfur species and undergoes a thiol-disulfide type exchange reaction a with avImportantly, allicin is a volatile substance and can be effective against microorganisms via the gas phase. Since there are no volatile antibiotics currently available for clinical use, this makes allicin a very interesting candidate molecule. It therefore makes sense to develop new strategies using allicin-based drugs, perhaps combined with other conventional antibiotics, for direct treatment of lung infections via the pulmonary rather than the oral route. In this regard, the successful treatment of tuberculosis patients by inhalation of garlic vapor has been reported in the ‘pre-antibiotic’ era .The antimicrobial effect of garlic or allicin was already documented for several human pathogenic bacteria in vitro and in animal trials ,22,23,24Currently, the emergence of bacteria pan-resistant to current clinical antibiotics is a threat to effective treatment of infectious disease. Resistance to penicillin, the first commercially available antibiotic, was already documented in 1940 before its release into clinical practice in 1943. The appearance of resistance, rapidly following discovery or introduction into clinical practice, is also the trend for later antibiotics ,26. The Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Acinetobacter baumannii, and Mycobacterium tuberculosis have been reported and there is desperate need for new strategies and new classes of antibiotics [Klebsiella pneumoniae against which all 26 antibiotics allowed for clinical use in the USA were ineffective [Several MDR (multiple drug resistant) strains of human pathogenic bacteria e.g., ibiotics ,31,32,33ffective .In the work reported here, we tested the antimicrobial effect of allicin as a vapor on several clinical isolates of lung pathogenic bacteria and used standardized EUCAST  guidelines to determine minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). The toxicity of allicin towards human lung epithelial cells and rat precision-cut lung tissue slices (PCLS) was investigated and the protective effect of glutathione was shown. The feasibility of developing allicin as part of a treatment regime for lung infections is discussed.P. aeruginosa strains PAO1 and PAO25 showed inhibition but P. aeruginosa DSM2659, which was very resistant to allicin in the EUCAST MIC and MBC tests, showed no inhibition  compared to P. aeruginosa PAO1 SBUG8 and PAO25 (MIC = 64 µg/mL). MDR and non-MDR S. pneumoniae strains tested were equally susceptible to allicin and showed MICs from 32 to 64 µg/mL allicin and MBCs from 64 to 128 µg/mL allicin, respectively  and for these MDR strains, allicin, including in absolute µM terms, compared favorably with those antibiotics.In comparison to conventional antibiotics, the MICs and MBCs for allicin were generally higher, both in terms of µg/mL and absolute concentrations in µM . Thus, eThe effect of allicin on mammalian cell cultures was tested with two human lung epithelial cell lines (A549 and Beas-2B), a human colon cancer epithelial cell line (Caco-2), and murine embryonic fibroblasts NIH/3T3. Two-week-old cell cultures in 96-well plates were incubated with allicin for one hour. After challenge, remaining allicin was titrated out by reaction with cysteine and the plates were incubated for 23 h. The experiment was repeated with 1 mM GSH in the medium since this partially simulates the situation for cells in the body, which are continually surrounded by body fluids containing GSH. Cell viability was determined with the MTT -2,5-diphenyltetrazolium bromide) test .50 values of 460 and 250 µM allicin  which increased to 800 and 500 µM  in the presence of 1 mM GSH  which increased to 450 µM (73 µg/mL) in both cases in the presence of 1 mM GSH . The EC50 value increased in the presence of 1 mM GSH to 656 µM (106 µg/mL) in the LDH-test and 280 µM (45 µg/mL) in the WST-test, respectively  and WST (water soluble tetrazolium) tests. In the absence of GSH cell in the lung slices were extremely sensitive to allicin with ECectively .E. coli [Pseudomonas aeruginosa and led to the disappearance of the bacteria in the infected lungs [We have previously shown in agar diffusion assays that allicin compared well on a mol-for-mol basis with ampicillin and kanamycin against E. coli , and theE. coli ,22,36. HE. coli . Subcutaed lungs . Howevered lungs . Becauseed lungs .P. aeruginosa DSM2659 strain .MIC for the bacteria tested ranged from 32 to 128 µg/mL allicin with the majority of strains being completely inhibited by 64 µg/mL allicin, except for  strains . The majS. pneumoniae CSR14-10, S. Africa19A-13 and Poland23F-16 and erythromycin and clindamycin MIC > 256 µg/mL and for allicin 64 µg/mL. On this basis, allicin might be considered as a treatment for MDR strains. Where there is allergy to a particular antibiotic, allicin might also be considered.Despite the MDR strains being as susceptible to allicin as the non-MDR strains, the overall performance of allicin compared to clinical antibiotics was poor . Neverth50 values for the lung epithelial cell lines near the 64 µg/mL value taken as the general MIC for most bacteria  and three-fold 45.5 µg/mL allicin (WST test) when the rat slices were supplied with 1 mM GSH  with Heviously .Pseudomonas aeruginosa strains PAO1 SBUG8, PAO25, and DMS2659 were from the culture collection of the Institute for Applied Microbiology . The Streptococcus pneumoniae strains (MDR strains: Spain23F-1 (PMEN-1), CSR14-10 (PMEN-10), S. Africa19A-13 (PMEN-13), Poland23F-16 (PMEN-16) (http://www.pneumogen.net/pmen), clinical isolates: SNo 67715, SNo 68668, SNo 68665), Streptococcus pyogenes , Streptococcus dysgalactiae equisilimlis , Streptococcus agalactiae , Staphylococcus aureus , Klebsiella pneumoniae strains , and Acinetobacter baumannii  were from the culture collection of the German National Reference Center for Streptococci (GNRCS), University Hospital, RWTH Aachen.The 600) = 0.2. Defibrinated sheep blood was added to 25 mL molten CASO-agar at 50 °C (5% v/v), mixed with 300 µL of bacterial suspension and poured immediately into a Petri dish to make bacteria-seeded agar plates. Bacteria were either spread onto the agar surface. Different concentrated allicin solutions (20 µL) were pipetted onto the Petri dish lid and the solidified agar plate with bacteria was placed inverted over the lid. The amount of allicin in the droplet is indicated in Bacteria were grown over night at 37 °C on blood-CASO-agar plates. Bacterial colonies were resuspended in 5 mL MHB2 medium to an optical density at 600 nm  up to 1 McFarland. Double concentrated MHB2 (2.2 mL), 141 µL lysed horse blood  and 10 µL bacterial suspension were mixed and kept on ice until pipetting into microtitre plates.Bacteria were grown over night at 37 °C on blood-CASO-agar plates , A two-power dilution series of allicin in water (2–2048 µg/mL = 12 µM–12.6 mM) was prepared and 50 µL was mixed with 50 µL of bacteria suspended in blood-MHB2 medium (see above) in 96-well plates so that the final bacterial suspension in the test wells was 0.5 McFarland. Plates were covered with air-permeable, self-adhesive cling film  and incubated at 37 °C for 20 h without shaking. The lowest allicin concentration without growth gave the MIC. For the determination of MBC 10 µL from each well were pipetted onto agar plates (see above) and incubated at 37 °C overnight. The lowest concentration without bacterial growth was the MBC.v/v)  and fetal bovine serum (FBS) 10% (v/v) . NIH/3T3 cells were cultivated in Roti®-CELL RPMI-1640  media with penicillin/streptomycin 1% (v/v) and FBS 10% (v/v). Cells were cultivated in TC (tissue culture) dishes  at 37 °C and 5% atmospheric CO2 and sub-cultured weekly. For experiments, the cell culture was grown directly in 96-well plates (100 µL per well).Lung epithelial cell lines Beas-2B  and A549 , Caco-2  and fibroblast NIH/3T3 (murine embryonic fibroblast) cells were tested for their sensitivity to allicin. Beas-2B, A549 and Caco-2 were cultivated in DMEM medium with penicillin/streptomycin 1% -2,5-diphenyltetrazolium bromide, Carl Roth GmbH, Karlsruhe, Germany). 50 µL of MTT 0.5% (w/v) dissolved in phosphate-buffered saline (PBS) was added to each well and the plate incubated for 3 h at 37 °C and 5% CO2. Cells were lysed by adding 100 µL isopropanol and the A570 subtracted from A630 automatically in the plate reader . The results are presented as half maximal effective concentration (EC50) showing a 50% reduction in MTT response.Cells were exposed to allicin (diluted in medium) for 1 h (controls with medium only). Allicin concentrations between 78 and 2500 µM (12.7–406 µg/mL) (two-power dilution series) were tested. In a further experiment, cells were incubated with GSH 1 mM . After incubation, unreacted allicin was titrated out by the addition of 100 µL cysteine solution 5 mM . Cells were post-incubated for 23 h at 37 °C and 5% CORattus norvegicus) (270 ± 10 g) were purchased from Charles River . Animal studies were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen  and performed following the Directive 2010/63/EU of the European Parliament. Animals were euthanized with a lethal dose of pentobarbital (60 mg/kg). After confirming that the animals did not show any reflexes, the abdomen was opened and the lung was removed. The lung was filled via the trachea with 37 °C 1.5% (w/v) low-melting agarose and the lung was transferred to ice-cold PBS. After the agarose solidified, the lung was sliced first into 10 mm thick cylinders and afterwards into 250 µm thin slices with a Krumdieck tissue slicer . The slices were placed in a 24-well plate (two slices per well). One well per treatment and per animal was used. Each experiment was performed with three animals.Female Wistar rats  supplemented with CaCleriments .The slices were incubated for 1 h with allicin  . Allicin was removed after incubation by washing the slices 3 times with PBS and post-incubating the tissue slices for 23 h.Cell viability was determined using the lactate dehydrogenase activity test  and via measurement of the mitochondrial metabolic activity with WST-1 (water soluble tetrazolium) kit .In conclusion, allicin in the gas phase is antimicrobial towards the majority of pathogenic isolates tested, including antibiotic resistant strains. However, the relatively low differential sensitivity to allicin in vitro between animal and bacterial cells in liquid culture suggests that allicin alone may not be a suitable alternative to conventional antibiotics to treat lung infections, although the ameliorating effect of continual GSH supply to lung cells in vivo is an unknown variable. Nevertheless, in light of the historical precedents and the increasingly urgent need for new alternatives , the use


Synthetic biology approach has been frequently applied to produce plant rare bioactive compounds in microbial cell factories by fermentation. However, to reach an ideal manufactural efficiency, it is necessary to optimize the microbial cell factories systemically by boosting sufficient carbon flux to the precursor synthesis and tuning the expression level and efficiency of key bioparts related to the synthetic pathway. We previously developed a yeast cell factory to produce ginsenoside Rh2 from glucose. However, the ginsenoside Rh2 yield was too low for commercialization due to the low supply of the ginsenoside aglycone protopanaxadiol (PPD) and poor performance of the key UDP-glycosyltransferase (UGT) (biopart UGTPg45) in the final step of the biosynthetic pathway. In the present study, we constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of P450 expression levels. The new yeast chassis could produce 529.0 mg/L of PPD in shake flasks and 11.02 g/L in 10 L fed-batch fermentation. Based on this high PPD-producing chassis, we established a series of cell factories to produce ginsenoside Rh2, which we optimized by improving the C3–OH glycosylation efficiency. We increased the copy number of UGTPg45, and engineered its promoter to increase expression levels. In addition, we screened for more efficient and compatible UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Combining all engineered strategies, we built a yeast cell factory with the greatest ginsenoside Rh2 production reported to date, 179.3 mg/L in shake flasks and 2.25 g/L in 10 L fed-batch fermentation. The results set up a successful example for improving yeast cell factories to produce plant rare natural products, especially the glycosylated ones. Since then, a series of plant natural products have been successfully heterologously produced in microbial cell factories4, highlighting the potential for metabolic engineering of microbial chassis to provide reliable approaches for obtaining natural plant products on a large scale, especially rare natural products.High-efficiency production of artemisinic acid via yeast fermentation has become a milestone for the application of synthetic biology to the production of natural plant productsPanax species, is a promising candidate for cancer prevention and therapy7. However, the ginsenoside Rh2 accounts for less than 0.01% of dried Panax ginseng roots8. Commercially available ginsenoside Rh2 is currently produced mainly via the deglycosylation of major protopanaxadiol (PPD)-type ginsenosides isolated from Panax spp. using chemical or biotransformation approaches11. However, such approaches rely heavily on the cultivation of Panax plants and the successive cultivation of Panax plants is unsustainable, resulting in low productivity and vulnerability to disease outbreaks12. A synthetic biology strategy to artificially produce ginsenoside Rh2 from glucose in a metabolically engineered microbial cell factory is expected.Ginsenoside Rh2, derived from 13. Nearly concurrently, we also characterized two UGTs (UGTPg45 and UGTPg29) taking parts in the biosynthesis of ginsenosides Rh2 and Rg3 independently14. In this previous work, we constructed the strain D20RH18 for de novo production of ginsenoside Rh2 from glucose based on the PPD-producing chassis strain ZW-PPD-B 14. However, the ginsenoside Rh2 production of D20RH18 was relatively low (16.95 mg/L in shake flasks), and the yield of total dammarane-type triterpenoids  was also low. Kinetic analysis of the biopart UGTPg45 to produce ginsenoside Rh2 revealed a low catalytic efficiency, only 1/2500 of that of UGTPg29, which further catalyzes ginsenoside Rh2 into Rg3. Therefore, we reasoned that the limited supply of the precursor for dammarane-type triterpenoid synthesis and poor performance of the key UGT biopart were the two key factors hindering the high-efficiency production of ginsenoside Rh2.PPD is the common precursor of all dammarane-type ginsenosides, including ginsenoside Rh2. A key enzyme to glycosylate PPD to produce ginsenoside Rh2 is required. A Korean research group reported the isolation of two glycosyltransferase (UGT) genes involved in the biosynthesis of the ginsenosides Rh2 and Rg3Bacillus subtilis could catalyze the C3–OH and C12–OH glycosylation of DM, PPD, and protopanaxatriol (PPT)15. Another UGT from B. subtilis, Bs-YjiC, which shares 94% identity with UGT109A1, could also catalyze the C3–OH and C12–OH glycosylation of PPD to yield ginsenoside Rh2 and an unnatural ginsenoside F12 (3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxadiol)16. Coupling this enzyme with a UDP-glucose regeneration system powered by sucrose synthase, an in vitro one-pot ginsenoside synthesis platform was established to produce 0.2 g/L of ginsenoside Rh2 and 3.98 g/L of F12 using PPD as the substrate16. Another group built an efficient ginsenoside Rh2 biosynthetic cell factory by repurposing an inherently promiscuous UDP-glycosyltransferase UGT51 from Saccharomyces cerevisiae17. Based on a structure-guided semi-rational engineering approach, the catalytic efficiency of UGT51 to convert PPD into ginsenoside Rh2 increased by more than 1800-fold. The yeast cell factory with this engineered glycosyltransferase could produce ~300 mg/L of ginsenoside Rh2 in a 5L fed-batch fermentation system. However, there are still many disadvantages of the use of UGT from bacteria or yeast, such as its poor substrate specificity. For example, the engineered UGT51 can also catalyze the C3–OH glycosylation of many native yeast sterols , and thus produce diverse by-products and waste UGT enzyme activity17. Meanwhile, Bs-YjiC exhibits poor site specificity to C3–OH, resulting in lower ginsenoside Rh2 yield (0.2 g/L) compared with the high F12 yield (3.98 g/L)16.Since the first two reports of the ginsenoside Rh2 biosynthetic pathway, several studies have attempted to optimize this glycosylation step by identifying UGT bioparts from microbial sources or by engineering UGTs to catalyze ginsenoside Rh2 formation. UGTs from bacteria often show substrate flexibility and are potential candidates for ginsenoside Rh2 synthesis. UGT109A1 from tHMG1 (truncated HMG-CoA reductase gene) and three key precursor genes, the resulting PPD-producing strain could produce 148.1 mg/L PPD in shake flasks, and the titer reached more than 1 g/L in fed-batch fermentation18. Recently, to overcome the poor coupling between P. ginseng cytochrome P450 and the Arabidopsis thaliana cytochrome P450 reductase ATR1, as well as the high DM accumulation in PPD-producing yeast recombinants, the fusion of PPDS and ATR1 encoding genes was introduced and the PPD production increased significantly, reaching 265.7 mg/L PPD in shake flasks and 4.25 g/L in 5 L fed-batch fermentation, which is the highest PPD production ever reported20.Besides, optimization of the PPD-producing chassis may also improve ginsenoside Rh2 yield. In the past several years, much progress has been made in engineering microbes for high-level production of PPD. For example, by integrating the PPD biosynthesis pathway into a yeast chromosome and overexpressing the rate-limiting gene In this study, we constructed a new PPD-producing chassis strain to improve the conversion of glucose into PPD by overexpressing all mevalonic acid (MVA) pathway genes and optimizing the expression levels of cytochrome P450 enzymes in yeast. In addition, we employed several strategies to optimize the cell factories to produce ginsenoside Rh2 by improving UGTPg45 expression level and activity, including: (1) increasing UGTPg45 expression by increasing its copy number and engineering its promoter and (2) increasing the in vivo activity of UGTPg45 in yeast via protein engineering based on in vivo directed evolution and searching for novel UGTs with higher C3–OH glycosylation efficiencies from other plant species.P. ginseng, which encode three enzymes that convert 2, 3-oxidosqualene into PPD, namely PgDDS, CYP716A47, and PgCPR1, are also required for PPD biosynthesis. Therefore, we aimed to overexpress these 13 genes  that converted acetyl-CoA into the isoprenoid building blocks isopentenyl phosphate (IPP) and dimethylallyl phosphate (DMAPP). The second group, designated as the downstream-module, contained the remaining six downstream genes , which converted IPP and DMAPP into the target product PPD. The three heterologous genes PgDDS, CYP716A47, and PgCPR1 were codon-optimized for expression in yeast and renamed synPgDDS, synPgPPDS, and synPgCPR1, respectively. Because HMG-CoA reductase catalyzes the rate-limiting step in the MVA pathway, multi-copy overexpression of tHMG1 benefits the production of different terpenoids21. Therefore, we added an additional copy of tHMG1 to the second group  in the MVA pathway. Then three heterologous genes from nes Fig. . The 13 delta DNA site of the yeast strain BY4742, the resulting strain, ZW03BY, produced 2.7 mg/L DM and 237.9 mg/L PPD from glucose in shake flasks  production  constructed by Blazeck et al. to replace the TDH3 promoter24, yielding the strain ZWDRH2-3. As a result, the ginsenoside Rh2 yield further increased to 74.7 mg/L , and this poor catalytic activity has limited the biosynthesis of ginsenosides Rh2 and Rg3 in previously constructed strains14. As such, UGTs with higher efficiencies are necessary to improve the yield of ginsenoside Rh2. Many studies have demonstrated that some UGTs do not show strict substrate specificity, only regio-specificity26. The substrate flexibility of these UGTs has been applied in combinatorial biosynthesis and glycol diversification of natural products28. Because of the poor performance of UGTPg45 from P. ginseng, we searched for novel alternative UGT bioparts from other plants to more efficiently convert PPD into ginsenoside Rh2. Three UGT genes, UGT73F3 from Medicago truncatula29, UGT73C10 from Barbarea vulgaris30, and UGTPn50 from Panax notoginseng, were characterized able to produce ginsenoside Rh2 in in vitro reactions by incubating the raw enzyme solution with PPD and UDP-glucose  in vitro, as shown in Supplementary Fig. To compare their ginsenoside Rh2 synthesis efficiencies, the encoding genes ely Fig. ; howeverely Fig. . UGT73C1kcat/Km of UGTPn50 (0.127 mM−1 s−1) was significantly higher than that of UGTPg45 (0.074 mM−1 s−1) , we acquired the transformant P8-E7 (renamed strain ZWDRH2-8 hereafter), with a ginsenoside Rh2 yield of 60.5 mg/L, 1.7-fold that of strain ZWDRH2-1 with wild-type UGTPg45 Fig. . We perfIn order to explore the molecular mechanism for the catalytic efficiency promotion of the mutant UGTPg45-HV, the wild-type UGTPg45 was analyzed under homology modeling and ligand docking. The analyzing data demonstrated that the two mutated sites on UGTPg45-HV were not included in the binding pocket, in which the enzyme directly contact with the substrates PPD or UDP-glucose . The final DM and PPD titers reached 7.1 g/L and 11.02 g/L, respectively  extremely limited the final ginsenoside Rh2 yield. Besides, the introducing of UGTPg45 into the PPD-producing yeast chassis dramatically reduced its total triterpenoids production14. To screen alternative bioparts with higher compatibility from other source has usually been applied in the optimization of cell factory36. In fact, some UGTs from bacteria  and yeast (UGT51) have been adopted to produce ginsenoside Rh217. The original substrates of those UGTs might be quite different from PPD, however, owing to their lower substrate specificity and even lower regio-specificity, those UGTs turn to be potential bioparts for ginsenoside Rh2 synthesis. Nevertheless, the substrate flexibility or low regio-specificity of those UGTs from microbes often resulted in the production of various by-products17. Some plant UGTs exhibit lower substrate specificity but high regio-specificity, i.e., these UGTs might catalyze the glycosylation of different aglycons with similar structure at the same position. For example, four UGTs, UGT73C10, UGT73C11, UGT73C12 and UGT73C13, from B. vulgaris could glucosylate the C3–OH of different triterpenoids including oleanolic acid, hederagenin, and betulinic acid30. Those plant UGTs catalyzing the C3–OH glycosylation of triterpenoids with high regio-specificity provide potential alternative bioparts for ginsenoside Rh2 synthesis. Besides, P. notoginseng, another ginsenoside producing plant, may also provide potential UGT bioparts for ginsenoside Rh2 synthesis37. In this work, three UGTs, UGT73F3 from M. truncatula29, UGT73C10 from B. vulgaris30, and UGTPn50 from P. notoginseng were characterized to catalyze the C3–OH glycosylation of PPD yielding ginsenoside Rh2 for the first time , which gave 70% increase of ginsenoside Rh2 yield compared with UGTPg45, was acquired via in vivo directed evolution  of UGTPg45-HV increased by more than 40%, which may explain the increase in ginsenoside Rh2 production  was used for UGTs heterologous expression. S. cerevisiae strain BY4742  obtained from EUROSCARF was used as the parent strain for all engineering. Codon-optimized genes synPgDDS, synPgPPDS, and synPgCPR1  were synthesized by Genscript Corporation . All the strains used or constructed in this study are listed in Supplementary Table Authentic ginsenoside samples compound K, ginsenoside Rh2, F2, Rg3, Rd, Rb1, Rb2, and protopanaxadiol (PPD) were purchased from Nanjing Zelang Medical Technology Co., Ltd. . Dammarenediol II (DM) was purchased from BioBioPha Co., Ltd. . Plant materials M. truncatula using primer 73F3-F and 73F3-R and cloned into the pMD18T vector . The coding sequence of UGTPn50 was a PCR-amplified form of P. notoginseng using primer Pn50-F and Pn50-R and cloned into the pMD18T vector. UGT73C10 (Genbank accession no. AFN26666.1) was synthesized by Genscript Corporation.The coding sequence of UDP-glycosyltransferase UGT73F3 (Genbank accession no. ACT34898.1) was a PCR-amplified form of E. coli was carried out as described previously14. Briefly, UGT genes with a C-terminally 6×His-tag was ligated into the pET-28a vector via ClonExpress II One Step Cloning Kit  and transformed into E. coli BL21 (DE3). Protein expression was conducted by 0.2 mM IPTG induction for 18 h at 18 °C. The cells were collected by centrifugation and suspended in 100 mM phosphate buffer (pH 7.5) supplemented with 1 mM PMSF and then disrupted with a French Press (25 kpsi). The cell debris was removed by centrifugation , and the supernatant was used for enzymatic assays.Heterologous expression of the UGT genes in 40. Generally, the reaction was carried out in a 100 μL system containing 100 mM phosphate buffer (pH 7.5), 1% Tween-20, 5 mM UDP-glucose, 0.5 mM substrate, and 50 μL of UGT crude enzyme (~ 400 ng/mL) for 2 h in a 35 °C water bath. Reaction products was extracted by adding 100 μL of n-butanol, the organic phase was then evaporated and dissolved in methanol for TLC and HPLC analysis.Enzymatic assays for glycosyltransferases were performed as described previously41. The purified C-terminal 6×His-tagged UDP-glycosyltransferase OleD (Genbank accession no. ABA42119.2) was diluted serially  to make a standard curve for protein quantitative. The reaction mixtures contained 100 mM phosphate buffer (pH 7.5), 1% Tween-20, 0.5 mM PPD, 40-300 μM UDP-glucose, and 60 µl crude enzyme of UGTs (~ 400 ng/mL) in a final volume of 300 μL. The reactions were incubated at 30 °C for 30 min. HPLC analysis was used to quantify the target product in each reaction. The Michaelis–Menten parameters were calculated by Lineweaver–Burk plot. All data are presented as means ± SD of two or three independent repeats.For the kinetic study of UDP-glycosyltransferases, the His-tagged UGTs in the crude enzyme were firstly quantified by dot bolt as described previously42.The general procedure for each yeast strain construction was descript as following. First, each promoter, gene, terminator, selection marker, and homologous arm was PCR amplified to give the basic fragments, the adjacent basic fragments all sharing 40–75 bp homologous sequences for recombination or fusion PCR; Second, using the adjacent 2–4 basic fragments as template, fusion PCR was performed to give each fusion fragments. Third, the fusion fragments were purified, quantified, and co-transformed into yeast strain via standard LiAc/ssDNA method, because each adjacent fragment sharing 40–75 bp homologous sequences they will be join together via yeast homologous recombination and integrated into chromosomeYeast strains were grown in YPD medium composed of 10 g/L Bacto Yeast Extract , 20 g/L Bacto peptone , and 20 g/L dextrose . For auxotrophic marker selection yeast clones were grown in SD medium composed of 6.7 g/L Difco Yeast Nitrogen Base without amino acids , 20 g/L dextrose and 2 g/L Drop-out Mix Synthetic minus appropriate amino acids . For KanMx marker selection yeast clones were grown in YPD medium containing 200 mg/L G418 sulfate .n-butanol and used for the analysis of DM, PPD, and ginsenoside Rh2.Shake flask fermentation of engineered yeast strains. Individual clones were inoculated into the YPD medium and cultivated at 30 °C, 250 rpm for 16 h. Aliquots were diluted to an initial OD600 of 0.05 in 10 mL of YPD medium in 50 mL shake flasks and grown at 30 °C, 250 rpm for 96 h. The fermentation broth was extracted with 18. About 0.3 L seed of strains ZW04BY-RS or ZWDRH2-10 was inoculated into 2.7 L of batch medium in the 10-L bioreactor and then started the fermentation. Temperature of the fermentation was set at 30 °C. pH was controlled at 5.0 by addition of ammonia hydroxide. Dissolved O2 was controlled above 30% by varying speed of agitation or air flow rate. Feeding rate was controlled by maintaining ethanol < 0.5 g/L.Fed-batch fermentation of yeast strain ZW04BY-RS and ZWDRH2-10 were conducted in a 10 L bioreactor . The medium for seed, batch fermentation, and the control of feed rate was conducted as previously describedn-butanol was added into each well, and PPD and Rh2 were extracted. The organic phase was transformed into a new 96-well plate for quantification of PPD and Rh2 by HPLC.Random mutagenesis of UGTPg45 (Genbank accession no. AKA44586.1) by error-prone PCR was performed using the GeneMorph II random mutagenesis kit . The PCR reaction condition was set as suggested in the user manual, error rate was controlled to be 1–2 mutations per gene. The UGTPg45 mutants was transformed into PPD-producing yeast ZW04BY-RS to generate a mutant library  followed by heating at 110 °C for 5 min. For identification, a mixture of ginsenoside authentic samples containing compound K, ginsenoside Rh2, F2, Rg3, Rb1, Rb2, and Rd, and PPD of concentration 0.2 mg/mL (dissolved in methanol) was also spotted on each plate.TLC analysis. The TLC analysis was performed using silica gel 60 F254 plates  with chloroform/methanol/water  as the developing solvent. The spots on the TLC plates were visualized by spraying with 1% (w/v) vanillin in HHPLC analysis. The HPLC analysis was performed on a Shimadzu LC20A system  equipped with a LC20ADXR pumper, an auto-sampler and a diode array detector. Chromatographic separation of ginsenoside Rh2, PPD, and DM was carried out at 35 °C on a Shim-pack XR-ODS column . The gradient elution system consisted of water (A) and acetonitrile (B). Separation was achieved using the following gradient: 0–2.5 min (60% B), 2.5–10 min (60%–90% B), 10–11 min (90% B), and 11–13 min (60% B), and the flow rate was kept at 0.45 mL/min. For rapid detection products of mutants of UGTPg45, Accucore C18 columns  were used and separation was achieved using the following gradient: 0–1 min (55% B), 1–3.5 min (90% B), and 3.5–5 min (55% B), and the flow rate was kept at 0.45 mL/min. Therefore, each sample can be finished within 5 min, and a 96-well plate samples can be finished within 8 h.45. PyMOL v1.746 was used to generate the mutant UGTPg45-HV. UDP-glucose was placed in the binding pocket in the reference of a previous released crystal structure of the complex of a UGT enzyme and UDP-glucose (PDB code: 2ACW47). PPD was docked and refined into the binding pocket with AutoDock Vina v1.1.248. MD simulations and most analysis procedures were conducted using the Amber18 software package49. Hydrogen atoms were added using the LEaP module. Counter-ions were used to maintain system neutrality. All systems were solvated in a truncated octahedron box of TIP3P waters with a buffer of 10 Å. The ff14SB force field50 was used for proteins. The parameters of ligands were created using Antechamber with GAFF as basic force field. All the MD simulations were accelerated with the CUDA version of PMEMD and run on NVIDIA® Tesla® P100. Up to 20000-step, steepest descent minimization was performed to relieve any further structural clash in the solvated systems. This was followed by a 400-ps heating up and a 200-ps equilibration in the NVT ensemble at 298K. The 60 ns’ production runs were simulated in the NPT ensemble at 298K with a time step of 2 fs in the Berendsen’s thermostat and barostat with default settings. Detailed simulation information was listed in Supplementary Table SInitial structure of WT UGTPg45 was modeled with I-Tasser web serverSupplementary Information


Increased cancer cell motility constitutes a root cause of end organ destruction and mortality, but its complex regulation represents a barrier to precision targeting. We use the unique characteristics of small molecules to probe and selectively modulate cell motility. By coupling efficient chemical synthesis routes to multiple upfront in parallel phenotypic screens, we identify that KBU2046 inhibits cell motility and cell invasion in vitro. Across three different murine models of human prostate and breast cancer, KBU2046 inhibits metastasis, decreases bone destruction, and prolongs survival at nanomolar blood concentrations after oral administration. Comprehensive molecular, cellular and systemic-level assays all support a high level of selectivity. KBU2046 binds chaperone heterocomplexes, selectively alters binding of client proteins that regulate motility, and lacks all the hallmarks of classical chaperone inhibitors, including toxicity. We identify a unique cell motility regulatory mechanism and synthesize a targeted therapeutic, providing a platform to pursue studies in humans. In this study, the authors identify and validate a halogen-substituted isoflavanone able to inhibit prostate cancer cell motility, invasion and metastasis in vitro and in vivo. They demonstrate its ability to selectively inhibit activation of client proteins that stimulate cell motility. It is required in order for cells to invade through the basement membrane, represents an initial step in the metastatic cascade, and is necessary for cells to move from their primary organ of origin to distant metastatic sites. The movement of cancer cells out of their primary organ of origin greatly reduces the chances of survival2. Movement of cells to distant organs, and their resultant destruction, constitutes a primary cause of cancer-associated morbidity and mortality3. Processes that drive the development of increased cell motility represent high-value therapeutic targets. However, comprehensive endeavors aimed at selectively inhibiting cancer cell motility and resultant metastasis have met with failure5. While many pathways have been shown to regulate cell motility, they constitute pathways whose regulatory effects are pleiotropic5. It has therefore not been possible to identify regulators of cell motility possessing the selective capacity to support targeted manipulation.Increased cell motility is a fundamental characteristic of cancer cellsRecognizing the critical importance and intractable nature of this problem, we reasoned that it needed to be approached in a unique manner. We did so by considering that small chemicals have potent biological properties, that single atom changes in their structure can affect those properties, that chemical structure can be modulated, and that as such they constitute highly refined biological probes. We hypothesized that we could use them to identify novel and selective sites that regulate cancer cell motility and that such sites would constitute high-value therapeutic targets.Herein, we delineate a novel and selective regulatory mechanism for these processes using efficient synthesis routes and resultant small chemicals as biological probes. We go on to demonstrate the therapeutic potential of the resultant probe, KBU2046. We do so by demonstrating selectivity across comprehensive molecular, cellular, and systemic assays. Efficacy of KBU2046 is demonstrated across several different in vitro models and across multiple murine models of human cancer metastasis, which includes decreased metastasis, decreased bone destruction, and prolonged survival. Also, comprehensive pharmacokinetic and toxicity studies further support therapeutic potential. Finally, we go on to characterize the molecular mechanism and its ability to perturb the novel regulatory process.6. We began with 4′,5,7-trihydroxyisoflavone (genistein) as our starting point because of its known anti-motility properties. We previously demonstrated that nanomolar concentrations of genistein inhibit human prostate cancer (PCa) cell invasion in vitro7, metastasis in a murine orthotopic model8, and in the context of a prospective human trial that it downregulates matrix metalloproteinase 2 (MMP-2) expression in prostate tissue9. While its diverse spectrum of biological effects render it unusable as a selective and potent biological probe, these same properties maximize its potential to selectively probe a wide spectrum of bioactive sites upon chemical diversification.We selected flavonoids as a chemical scaffold to advance probe synthesis because they exert a wide range of biological effects10, we also de-selected for ER-binding. Through this strategy, (±)-3(4-fluorophenyl)chroman-4-one (KBU2046), a halogen-substituted isoflavanone, was discovered . These compounds were advanced by iterative selection for inhibition of human PCa cell invasion Fig. . A majorred Fig. .Fig. 1KB11, and KBU2046 inhibited the migration of human prostate, breast, colon, and lung cancer cells  cells were delivered by ultrasound-guided intracardiac (IC) injection and metastatic outgrowth monitored weekly via IVIS imaging 14. This translated into inhibition of MMP-2 expression and cell invasion in vitro, inhibition of human PCa metastasis in mice8 and decreased MMP-2 expression in human prostate tissue9. In contrast to genistein, KBU2046 did not bind to MKK4 nor inhibit its kinase activity in vitro, and it did not inhibit downstream phosphorylation of p38 MAPK or of HSP27 in cells ® panel of antibodies  detect established protein phosphorylation motifs, and were used to probe for KBU2046-induced changes in protein phosphorylation , we pursued alternative methods. The KinomeViewion Fig. . We prioion Fig. , that co® antibody used in Fig. 226 on HSP90β by 6.6-fold . As expected, transfection of cells with (S226A)-HSP90β inhibited cell invasion, compared to vector control (VC) transfected cells -HSP90β transfected cells, KBU2046 does not further inhibit cell invasion, while it significantly inhibits invasion in VC cells -HSP90β were more invasive than VC cells -HSP90β transfected cells, KBU2046 did not significantly inhibit cell invasion, while it significantly inhibited invasion in both VC and WT-HSP90β cells -HSP90β construct lacks a Serlls Fig. . Furtherlls Fig. . The sellls Fig. . HSP90β-lls Fig. . Converslls Fig. . Recognills Fig. . These f15. In contrast, KBU2046 was not cytotoxic and its effects on protein phosphorylation were highly specific, demonstrating a lack of effects on kinase function. HSP90β is part of a large multiprotein chaperone complex whose function involves binding a large but specific set of regulatory proteins. We reasoned that KBU2046 was changing the signature of bound client proteins, that the change was highly selective in terms of number of affected proteins, and that it was highly specific for proteins that regulate cell motility.KBU2046’s effect upon HSP90β function is completely different from that of classical HSP90 inhibitors. The latter induce cytotoxicity and work by binding directly to HSP90, thereby inhibiting its enzyme activity, in turn affecting the function of large numbers of cellular kinases and other client proteins16. CDC37 is a flexible arm-like structure (protein data bank (PDB) ID: 2WOG), is highly dynamic17, enables binding of large numbers of kinases, defines their positioning and thereby their potential to affect HSP90β phosphorylation status. We reasoned that KBU2046 was binding to either CDC37 or HSP90β, that this altered the function of the CDC37/HSP90β heterocomplex resulting in a change in the spectrum of bound client kinase proteins, that changes were highly selective and that this altered binding spectrum would in turn be responsible for KBU2046’s effects upon cell motility.CDC37 is a co-chaperone that mediates the binding of over 350 client proteins to HSP90β, including over 190 kinases18. Although KBU2046 did not bind CDC37 or HSP90β individually, because CDC37 and HSP90β associate to form a heterocomplex17, we went on to combine CDC37 and HSP90β in a DARTS assay, demonstrating that KBU2046 protected both proteins from digestion  assay  and chemical cross-linker physical mapping analysisore Fig. . In this16 to detect KBU2046-induced changes in client protein binding to CDC37/HSP90β heterocomplexes in intact cells. Of 420 kinase proteins screened, KBU2046 had highly selective effects, significantly changing the binding of only 17 (4%): binding was increased in 10 and decreased in 7 , from structural information reported in the literature, from our findings implicating KBU2046 interaction with HSP90/CDC37 heterocomplexes, and only took into consideration heterocomplex structure . In contrast, one recent model used cyroEM to evaluate HSP90/CDC37 heterocomplex structure in the context of its binding to the CDK4 client protein21. There are many other potential explanations. It is likely that each model describes a particular state, and different states are possible. Our model should therefore be considered a speculative framework from which future studies can be launched. Future investigations will need to be performed in order to determine the effects of bound vs. unbound client protein, different client proteins, binding of small molecules to the HSP90/CDC37 heterocomplex structure, and the ultimate validity of each of these models.We go on to present a structural model in which KBU2046 binds within a cleft that is created through the binding of HSP90 and CDC37, and exists at the interface between these two proteins. While our model integrates information from physical mapping, biochemical analysis, and crystallographic structure, its final construct was in silico, and it should therefore be considered speculative. Several other models also describe the structure of HSP90/CDC37 interactionsFrom our findings, we propose an integrated structural and functional model. KBU2046 binds to a cleft that is formed when HSP90β and CDC37 bind to form a heterocomplex. This in turn affects the ability of the hetercomplex to bind client kinase proteins in a very precise manner, selectively affecting those that regulate cell motility. Of these, RAF1 is of particular importance. KBU2046 decreases RAF1 binding to the heterocomplex, resulting in decreased activation, and inhibition of cell motility. KBU2046’s precision type of effect on chaperone function differentiates it from classical HSP90 inhibitors, which broadly disrupt client protein function, and underlie KBU2046’s lack of toxicity and selective modulation of cell motility.Together, the findings of this study provide a rational platform to move investigations into humans. That platform includes mechanistic strategy and the physical tools with which to affect it. Also, this study provides proof of principle findings that through pharmacologic means it is possible to induce precision modulation of the signature of client protein binding to chaperone scaffold proteins, in turn resulting in highly selective functional effects at the cellular and systemic level. In parallel, this approach serves to inform us about novel pathways for regulating critical biological processes. Finally, our approach that coupled efficient chemical synthesis routes with a well-designed phenotypic screening strategy has the potential to be broadly applied as a tool to interrogate other critical biological processes.Procedure for Large-Scale Production of 4′-fluoroisoflavanone (KBU2046): 3-(dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one. The starting materials 2′-hydroxyacetophenone  and N,N-dimethylformamide dimethyl acetal  were added to a 10–20 mL microwave vial. The vial was capped and heated in a Biotage Initiator microwave synthesizer at 150 °C and 11 bar for 10 min. The resulting dark orange liquid was allowed to cool to 23 °C, at which time yellow–orange crystals crashed out of solution. The crystals were collected and washed with hexanes (50 mL), then dried and weighed to give 3-(dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one  as orange–yellow needles. Analytical data for 3-(dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one: 1H nuclear magnetic resonance (NMR)  δ 13.97 , 7.92 , 7.72 , 7.38 , 6.96 , 6.85 , 5.81 , 3.23 , 3.01 ; 13C NMR : δ 191.5, 163.0, 154.8, 134.0, 128.2, 120.3, 118.3, 118.0, 90.1, 45.5, 37.5; ultra performance liquid chromatography/mass spectrometry (UPLCMS): mass calculated for C11H13NO2, [M + H]+, 192. Found 192. 3-bromochromone.23. To a flame-dried 250 mL round bottom flask, was added 3-(dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one , which was dissolved in CHCl3 (70 mL). The reaction flask was cooled to 0 °C in an ice bath, then Br2  was added dropwise through an addition funnel. After all of the Br2 was added, water (70 mL) was added slowly to the reaction and it was stirred at 23 °C for 10 min. The dark orange–yellow organic layer was then separated from the aqueous layer, which was back-extracted with 3 × 50 mL CHCl3. The combined organic layers were then dried over Na2SO4 and concentrated to give a dark orange oil. This was purified by flash column chromatography  to afford 3-bromochromone  as an off-white solid. Analytical data for 3-bromochromone: 1H NMR  δ 8.31 , 8.27 , 7.75 , 7.57–7.44 ; 13C NMR : δ 172.3, 156.1, 153.8, 134.2, 126.5, 125.9, 123.2, 118.2, 110.7; UPLCMS: mass calculated for C9H5BrO2, [M + H]+, 226. Found 226.3-bromochromone was prepared by a procedure taken from Gammill3)4). The catalyst for the Suzuki-Miyaura cross-coupling reaction to synthesize 4′-fluoroisoflavone was made using a procedure by Coulson24. To a flame-dried 100 mL Schlenk flask was added PdCl2  and triphenylphosphine . The solids were dissolved in DMSO (60 mL), then the mixture was purged with N2 and heated to 145 °C, at which time it turned a bright yellow–orange color. The reaction was removed from heat and allowed to stir at room temperature for 15 min, then hydrazine hydrate  was added via syringe, with a vent needle in place to account for the formation of N2 gas. After the hydrazine hydrate had been added, the reaction was cooled to 23 °C, during which time a yellow solid crashed out of solution. The solid was washed under Schlenk filtration conditions with 2 × 50 mL EtOH, then 2 × 50 mL ether to yield Pd(PPh3)4  as a canary yellow solid that was stored under N2 in the glovebox.Palladium tetrakis (triphenylphosphine) , 4-fluorophenylboronic acid , and Na2CO3 . The solids were dissolved in a mixture of benzene (100 mL) and water (50 mL), and the system was purged with N2 for 10-15 min. The Pd(PPh3)4 catalyst  was then added, at which time the reaction turned a bright orange. The flask was equipped with a reflux condenser and the reaction was heated to reflux (80 °C) overnight. After ~16 h, the reaction was cooled to 23 °C and was diluted with EtOAc (250 mL), then the crude material was passed through a plug of silica with EtOAc as the eluent. The organic material was dried over Na2SO4 and concentrated to give a dark brown solid that was adsorbed onto silica gel using DCM. Material purified by flash column chromatography  to afford 4′-fluoroisoflavone  as a yellow–orange solid that showed minor impurities by 1H NMR spectroscopy. Slightly impure material was taken onto the next step of the synthesis without further purification. 1H NMR  δ 8.35 , 8.05 , 7.73 , 7.61–7.54 , 7.53 , 7.48 , 7.17 ; 13C NMR : δ 176.2, 163.8, 161.8, 156.2, 152.9, 133.8, 130.7, 127.8, 126.4, 125.4, 124.5, 118.1, 115.5; UPLCMS: mass calculated for C15H9FO2, [M + H]+, 241. Found 241.4′-fluoroisoflavone was prepared on large scale according to a procedure from Suzuki and Miyaura 4′-fluoroisoflavanone (KBU2046).26. To a flame-dried 500 mL round bottom flask was added 4′-fluoroisoflavone , and the solid was dissolved in dry THF (100 mL). The solution was cooled to −78 °C (dry ice/acetone bath), monitored by a thermocouple. Once the solution had cooled to the desired temperature, l-selectride  was added dropwise over a period of 30–45 min. The reaction was then allowed to stir at −78 °C for 2 h, after which time it was quenched with MeOH (55 mL) at −78 °C. The mixture was then poured into 300 mL of water, and the aqueous layer was adjusted to pH 7 with 2 M HCl. The aqueous layer was extracted 2 × 200 mL with EtOAc, then the combined organic layers were dried over Na2SO4 and concentrated to give a dark brown oily solid. This was purified by flash column chromatography  to give 4.5 g of crude material that was recrystallized in hexanes to afford 4′-fluoroisoflavanone  as a fluffy white solid. It was checked for purity by both 1H NMR and HPLC analysis, with material that was >98% pure taken onto animal studies. Analytical data for isoflavanone 4′-fluoroisoflavanone: 1H NMR  δ 7.98 , 7.55 , 7.33–7.24 , 7.14–6.99 , 4.77–4.54 , 4.02 ; 13C NMR  δ 192.0, 163.3, 161.5, 136.2, 130.7, 130.2, 127.8, 121.8, 120.9, 117.9, 115.8, 71.4, 51.5; UPLCMS: mass calculated for C15H11FO2, [M + H]+, 243. Found 243.The reaction conditions to synthesize 4′-fluoroisoflavanone on large scale were adapted from a procedure reported by Wähälä1H and 13 C) as well as by UPLCMS . All compounds were isolated and stored in powdered form (in the absence of light) and were formulated into DSMO stock solutions just prior to use.Synthesis of additional compounds. A series of related analog compounds was synthesized in addition to the parent 4′-fluoroisoflavanone (KBU2046). These compounds were prepared in the same general manner of KBU2046 and the structures of each such compound are depicted in supplementary figures. The structure and purity of the additional analogs were confirmed by NMR spectroscopy  and bioluminescent (Luc2) genes as described29. All cells were cultured as described28, were maintained at 37 °C in a humidified atmosphere of 5% carbon dioxide with biweekly media changes, were drawn from stored stock cells, and replenished on a standardized periodic basis and were routinely monitored for Mycoplasma , at least every 3 months. Cells were authenticated by the following: they were acquired from the originator of that line, grown under quarantine conditions, expanded and stored as primary stocks and not used until following conditions were met: mycoplasma negative; through morphologic examination; growth characteristics; hormone responsiveness or lack thereof, when applicable; replenished from primary stocks at least every 3 months; working with a single primary stock cell line at a time with hood sterilization in between.PCa , breast cancer (MDA-MB-231 and MCF-7), colon cancer (HCT110 and HT29), and lung cancer cells (H226 and A549) were obtained from American Type Culture Collection and not further authenticated. The origin, characteristics, for PC3-M, as well as for human papilloma virus (HPV) transformed primary 1532NPTX , 1532CPTX (cancer), 1542NPTX , and 1542CP3TX (cancer) cell lines, have previously been described by usPhospho-HSP27 , phospho-p38 MAPK (#4631), p38 MAPK (#9212), phospho-CK2 substrate (#8738), CDC37 (#3618), HSP90β (#5087), GST (#2622), phsopho-c-RAF (ser338) (#9427), SGK3 (#8573), GAPDH (#2118), anti-mouse IgG-HRP linked secondary (#7076), and anti-rabbit IgG-HRP linked secondary (#7074) antibodies were purchased from Cell Signaling Technology. MAP3K6 (#SAB1300114) antibody, estradiol, and 4′,5,7-trihydroxyisoflavone, genistein, were purchased from Sigma-Aldrich. Pierce ECL western blotting substrate (#32106) and SuperSignal West Femto maximum sensitivity substrate (#34096) were purchased from Thermo Scientific. All primary antibodies were used at a dilution of 1:1000 and corresponding secondary antibodies used at a dilution of 1:5000. The following recombinant proteins were purchased: RAF1 , MAP3K6 , and SGK3 .30, using either denatured collagen (BD Biosciences) or type IV human collagen (BD Biosciences), with all experiments repeated, each in N = 4 replicates. In some experiments, as indicated, cells were transfected with an expression plasmid, using Lipofectamine 2000™ (Invitrogen), or with siRNA, using DharmaFect Duo (Thermo Scientific) and co-transfected with β-Galactosidase .Boyden chamber cell invasion assays were performed as previously described by us31. Assays were in replicates of N = 3, and were repeated.Three-day MTT cell growth inhibition assays were performed as described by us32. All western blots were repeated at least once. Uncropped scans of the most important blots are depicted in Supplementary Fig. Western blots were performed as described by us4 cells to 35 mm tissue culture dishes  coated with collagen I (BD Biosciences), incubating at 37 °C in 5% CO2, performing time-lapse imaging using a Biostation (Nikon Instruments), tracking the path of N ≥ 35 cells using ImageJ software and the Manual Tracker plug-in, and using Chemotaxis and Migration Tool plug-in for data analysis.Single-cell motility assays were conducted by adding 1033. All experiments were conducted in N = 4 replicates, and repeated.Cells were transfected with the indicated siRNA constructs per manufacture protocol (GE-Dharmacon), cultured 48 h with 10 µM KBU2046 or vehicle, and scratch wound assay performed as described by us34, estrogen-responsive promoter-luciferase reporter construct, pERE-Luc, was from Craig Jordan (Georgetown University)35, human CDC37 in pET15b plasmid was from Avrom Caplan (City College of New York)36. siRNA used Dharmacon ON-Targetplus SMARTpool™ siRNA directed against HSP90β (cat # L-005187-00-0010) non-targeting siRNA (cat # D-001810-10-05) used TransIT-LT1 Transfection Reagent (Mirus Bio LLC), or with Dharmafect Duo  for co-delivery of plasmid. Luciferase assays were performed as described by us30.Constructs were purchased or gifts: constitutive active MEK4EE , pRL-TK-Renilla luciferase (Promega), pCMV-β-galactosidase (Agilent Technologies), and pcDNA-GFP (Invitrogen), HSP90β was from Pawel Bieganowski α = 0.05, and a pre-specified effect size of 30%.All animal studies adhered to the NIH Guide for the Care and Use of Laboratory Animals, were treated under institutional IACUC-approved protocols by Oregon Health and Sciences University and Northwestern University, complied with all federal, state, and local ethical regulations, housed in barrier (for immunocompromised mice) or conventional facilities, with a 12-h light/dark cycle and given soy-free food and water ad libitum. Animal study sample size determination: sample sizes were determined using the samples size estimation formula for differences in means with power set 80%, two-sided 37. Treatment with KBU2046, incorporated into Harlan Teklad 2016S® chow, began 1 week prior to implantation. Animals were excluded from the analysis if they died and/or met the criteria for killing in the 7 day post-operative period. Experimental groups were randomly assigned to cages prior to the initiation of the study. Metastasis were scored in a blinded fashion. Specifically, animals were assigned an ID number, resultant histologic slides contained a separate pathological ID number, slides were scored in a random fashion, after which the two numbers were linked up.Prostate cancer: orthotopic implantation: Orthotopic implantation of human GFP-PC3-M PCa cells into 6–8 week male Balb/c athymic mice (Charles River Laboratories) and quantification of distant metastasis was performed as described by us6 dTomato-LM2-4H2N cells in matrigel:PBS human breast cancer cells into 5–6 week old female SCID-Beige mice (Taconic), followed by resection of resultant primary tumor, provides a model wherein survival is dictated by metastatic burden, and was performed as described12. KBU2046 treatment by oral gavage 5 days per week began 4 weeks after resection, with weekly IVIS imaging. Animals were excluded from the analysis if they died and/or met the criteria for euthanasia in the 7 day post-operative period. Experimental groups were randomly assigned based upon size of primary tumor before treatment, in and manner that ensured equal distribution of tumor sizes across treatment groups.Breast cancer: orthotopic implantation: Orthotopic implantation of 2 × 105 PC3-Luc cells into male 6–8 week old athymic mice with IVIS imaging was performed as described13, and was done so under ultrasound guidance  injection: IC injection of 4.0 × 10Systemic effects: Off-target effects were sought by performing histologic examination of tissue and by measuring organ function.38  in a blinded fashion, and toxicity scored using an established semi-quantitative histological scoring system39, using human cord blood CD34+ stem cells (AllCells Inc.), using MethoCultExpress™ colony growth media (StemCell Technologies Inc.), performed in replicates of N = 2.Fourteen-day colony formation assays were conducted as described by usN = 3 CD1(ICR) mice at 0 , 25, or 100 mg/kg. Blood was collected into EDTA by terminal cardiac puncture before drug administration  and at 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 960, 1440 min after administration. In separate experiments, as described above, Balb/c athymic mice were dosed with KBU2046 incorporated into chow for 35 days, after which blood was collected by terminal cardiac puncture. Resultant plasma (~250 μl/mouse) was stored at −80 °C.For determination of pharmacokinetic (PK) parameters, KBU2046 was administered by either intravenous injection or oral gavage to groups of 2, reconstituted with 100 μL of mobile phase, and 20 µL was analyzed on an API 3000 liquid chromatography-tandem mass spectrometry system (Applied Biosystems) with an Agilent 1100 series HPLC system (Agilent Technologies). Samples were eluted isocratically from a Synergi 4-μm MAX-RP 100 Å column  by a mobile phase consisting of 10 mM ammonium formate in water and methanol (30:70 [vol/vol]) at a flow rate of 0.20 mL/min. The tandem mass spectrometer was operated with its electrospray source in the positive ionization mode. The mass-to-charge ratios of the precursor-to-product ion reactions monitored were 243.5 → 125.1 for KBU2046 and 257.0 → 165.0 for the internal standard. The retention time of KBU2046 was ~2.7 min while that of the internal standard was ~2.3 min. The linear range for plasma KBU2046 standard curves was 0.1–25.0 ng/mL, with coefficients of variation of 10% or less throughout the entire concentration range. Fresh plasma standard curves were prepared in blank plasma and run on the day of analysis of plasma samples.Plasma KBU2046 concentrations were measured in duplicate by liquid chromatography-tandem mass spectrometry after sample preparation by solid-phase extraction. Specifically, 0.1 mL of a sample, 10 μL of 0.1 μg/mL internal standard solution (3-(2-chlorophenyl)-, 3800 μL of water, and 10 μl of 85% phosphoric acid were added, vortexed, and stored at 4 °C for 2 h. After washing a 96-well Strata-X 33 µm polymeric reversed phase 30 mg/well solid-phase extraction plate (Phenomenex) with methanol and water, sample was applied, washed with 20% methanol in water, eluted with 70% acetonitrile/30% methanol, dried at 50 °C under N40. Oral drug absorption was characterized by a tanks-in-series delay element, to account for the non-instantaneous appearance of drug in the body. Simultaneous estimation of PK model parameters for both routes of administration permitted estimation of the bioavailability of the orally administered drug (F)41. The SAAM II objective function used was the extended least-squares maximum likelihood function using data weighted with the inverse of the model-based variance of the data at the observation times42. Model misspecification was sought by visual inspection of the measured and predicted marker concentrations vs. time relationships , implemented on a WindowsTM-based PC (see PK modeling schema). Plasma concentrations were modeled with a three-compartment PK model using a naive pooled data approachips Fig. 42.Fig. 743, analyzed by the 2−ΔΔCt method44, using primer/probes sets (ABI), HSP90α (Hs00743767_sH), HSP90β (Hs00427665_g1), trefoil factor 1 , cathepsin D , progesterone receptor , and GAPDH (Hs99999905_m1). Assays were repeated, each in replicates of N = 2.RNA was isolated and qRT-PCR was performed as described by us45, isothermal titration calorimetry19, and biolayer interferometry assays46 were all performed as described by us using MEK4EE  cloned into pMCSG7 and KRX Competent E. coli cells (Promega Inc.) and above denoted full-length HSP90B and CDC37 vectors and Rosetta BL-21 and BL-21 DE-3 competent E. coli cells, respectively. DARTS assays were conducted as described18, using equimolar amounts of CDC37 and HSP90β, thermolysin digestion, and silver stain visualization .Fluorescent thermal shift45. HSP90β/CDC37 heterocomplex kinase assays with RAF1, SGK3, and MAP3K6 assays incubated indicated proteins in 20 mM MOPS, pH 7.2, 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 0.25 mM ATP, and 37.5 mM MgCl2 at 30 °C for the indicated times.MEK4/MKK4/MAP2K4 in vitro kinase assay was performed as described by usThe Kinoview™ and PhosphoScan™ assays were performed by Cell Signaling Technology Inc.® assay, using the Human Protein Microarray platform, was performed by Invitrogen.The ProtoArray47, using a multi-stage pipeline that considers protein–protein/ligand interactions through evolutionary protein surface analysis50, robust homology modeling51, massively parallel docking simulations using mixed strategies56, and advanced, physics-based rescoring methodologies58, all as described by us.Modeling and docking used the APPLIED Pipeline  at the Argonne Leadership Computing Facility, Argonne National Laboratory, tuned for the 786,432 core BlueGene/Q Mira16.The LUMIER assay was performed as described, using reagents generously provided by that groupt-test using a threshold of P < 0.05. All experiments, unless otherwise stated, were conducted in replicates of at least N = 2–6  and were repeated at a separate time, also in replicates of N = 2–6. The relationship between dose and metastasis, and between drug concentration and effect on protein degradation, was evaluated by two-sided analysis of variance. The survival of mice was evaluated by the log-rank (Mantel-Cox) test.All results were analyzed by a dedicated statistician (B.J.). Unless otherwise stated, statistical significance was evaluated with the two-sided Student’s The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


We observed the following: (i) Diminished beta adrenergic signaling mainly reflects in shifts in the Firmicutes phyla, with a significant increase in abundance of largely beneficial Bacilli Lactobacillales in the KO mice; (ii) This was associated with increased colonic production of beneficial short chain fatty acids (SCFAs) butyrate, acetate and propionate, confirming functional microbiota shifts in the KO mice; (iii) Dampened systemic immune responses in the KO mice reflected in reduction on circulating CD4+.IL17+ T cells and increase in young neutrophils, both previously associated with shifts in the gut microbiota. Taken together, these observations demonstrate that reduced expression of beta adrenergic receptors may lead to beneficial shifts in the gut microbiota and dampened systemic immune responses. Considering the role of both in hypertension, this suggests that dietary intervention may be a viable option for manipulation of blood pressure via correcting gut dysbiosis.Emerging evidence suggests an associative link between gut dysbiosis, the autonomic nervous system (ANS) and the immune system in pathophysiology of neurogenic hypertension (HTN). However, the close interplay between these three systems presents us with difficulties in deciphering the cause-effect relationship in disease. The present study utilized beta 1 and 2 adrenergic receptor knock out (AdrB1 Hypertension (HTN), or high blood pressure (BP), is one of the most modifiable risk factors of cardiovascular disease (CVD), which is among the leading causes of death in the United States. HTN is regarded as the “silent killer” as there are no obvious symptoms until development of overt clinical signs. Untreated or uncontrolled HTN typically results in end-organ damage such as hemorrhagic stroke, heart disease, peripheral arterial disease, retinopathy, and renal failure  tract provides one of the best examples of symbiosis in nature. The gut represents a complex ecosystem of host-bacteria interaction comprised of approximately 100 trillion bacteria  knockout (b1.b2 Adr KO) mice, characterized by reduced BP and heart rate responses  to Bacteroidetes (B), the F/B ratio as a biomarker for pathological conditions  knockout (KO) mice  and their wild type (WT) C57BL/6J controls  were used in all experiments. Genoytyping was performed prior to all experiments to confirm the strain differences, as per the Jacksonville Laboratories protocols. Briefly, mouse tails were snipped and lysed in 200 μl of Direct PCR Lysis Reagent (Viagen Biotech cat#101-T) supplemented with 4 μl of 200 mg/ml Proteinase K solution (Fisher cat#BP1700-100). Following 6 h of incubation at 55°C, and additional 45 min incubation at 85°C, the samples were briefly centrifuged and the DNA lysate was used for PCR. The following primers were used in regular PCR reaction, as per The Jackson Laboratory protocol for Adrb1tm1Bkk-Probe (see https://www2.jax.org/protocolsdb/f?p=116:1:0::NO:RP::) (i) to confirm the KO strain: F: TTG GGA AGA CAA TAG CAG GC, and R: CGC TGT CCA CAG TGG TTG T; (ii) to confirm the wild type strain: F: ATC TGG TCA TGG GAT TGC TG, and R: ACA CAG CAC ATC TAC CGA AG. The PCR reaction was performed using Amplitaq Gold 360 Master Mix (Invitrogen cat#4398876), and products were run on a 1% agarose gels. Using this protocol, the KO strain showed one band at 113 bps, while the wild type strain showed two bands, one at 113 bps, and one and 225 bps, as per the Jacksonville Laboratories instructions.All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee and complied with the standards stated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were housed in a temperature-controlled room (22°C to 23°C) with a 12:12-h light-dark cycle, in specific-pathogen free cages, and had access to standard mice chow and water n = 7 per strain) was performed using 1mL TRIzol® Reagent , as per manufacturer's protocol. Final RNA pellets were resuspended in 30μL of RNAse-DNAse free water to avoid potential genomic contamination. RNA concentration was determined using the NanoDrop-2000 spectrophotometer (Thermo Scientific), then subsequently diluted to 100 ng/μL. After dilution, 1 μg of RNA was used for cDNA synthesis using the High Capacity cDNA Reverse Transcriptase Kit . PCR was performed to evaluate the expression of beta 1 and 2 adrenergic receptors  using the CFX96 Touch™ Real-Time PCR Detection System . Two additional transcripts were analyzed in order to evaluate potential presence of overall inflammatory markers in the colon: TNFalpha  and IL-6 . The conditions of the PCR were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, primer annealing at 60°C for 30 s, and 72°C for 30 s. Dissociation curves were generated, starting at 75°C and ending at 95°C with increments of 0.5°C every 5 s, to confirm the specificity of reaction. Results were analyzed using the 2-ΔCt method as previously described , as an endogenous control. Additional no reverse transcriptase control was used.Strains were confirmed by genotyping as per The Jackson Laboratory protocols prior to commencement of experiments. RNA extraction was performed as previously described . Bacterial DNA was extracted using ZR Fecal DNA MiniPrep  as per manufacturer's protocols. DNA concentrations were determined using the NanoDrop-2000 spectrophotometer (Thermo Scientific).All mice were euthanized with 4% isoflurane in a mixture of 95%/5% of OThe analyses of microbial composition were performed, as previously described  as previously described  to quantify the levels of circulating inflammatory cells (ICs) as previously described , (ii) CD8a , (iii) CD3e , (iv) CD11b , (v) Ly6+CD184+CD62L+ . In the case of IL17, which is an intracellular marker, following incubation with anti-CD4, cells were washed in normal PBS and fixed in 1% PFA. Following this, cells were incubated with 0.5% Tween20 in normal PBS for 1 h at RT, in order to permeabilize the cell membrane and allow penetration of anti-IL17 inside the cell. Following this, cells were washed in normal PBS, and incubated with anti-IL17 at 1:250 ratio for 30 min at 4°C. SYTOX blue cell stain  was added to all samples per manufacturer protocol for exclusion of dead cells during flow cell analysis. Following incubation with antibodies, the cells were washed in normal PBS and fixed with 1% PFA, apart from IL17-stained samples, which were processed as described above. All samples were read on an LSR-II (BD Biosystems) in University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) and the data were analyzed with FACS Diva software, version 6.1.2. Fractions of young (Ly6+CD184+CD62Lhi) and old (Ly6+CD184+CD62Llo) neutrophils was determined as previously described  along with effect size measurements (LEfSe) in Galaxy and ANOSIM in R were used to distinguish differentially significant features at each taxonomic level.Statistical analysis was performed using GraphPad Prizm and two-tailed unpaired p < 0.05; Table p < 0.05; Table To confirm the relative expression levels of beta 1 adrenergic receptors in colon of WT and KO mice, we performed quantitative RT PCR. We observed a significant downregulation in beta 1 adrenergic receptor relative expression levels (Mean ± SEM: 1 ± 0.237 (WT) and 0.289 ± 0.018 (KO); n = 5 (WT) vs. 0.0009475 ± 0.0003198 (KO) (Table n = 7 (WT) and 0.0001558 ± 0.00004057 (KO) (Table We performed quantitative RT PCR to assess the relative gene expression levels of select inflammatory genes in the colon of WT and KO mice Table . Both cyO) Table .P < 0.05). Analysis of taxonomy indicated a significant increase in the abundance of Bacilli class of Firmicutes, and more specifically in the order of Lactobacillales, with significantly increased abundance of families of Lactobacillaeae and Carnobacteriaceae in the KO compared to WT mice  vs. 5.5 ± 1 μmol/g (KO), +: Mean ± SEM: 0.13637% ± 0.0318% (WT) and 0.1067% ± 0.02028% (KO); (ii) CD8+: Mean ± SEM: 4.593% ± 0.6136% (WT) and 5.863% ± 0.4781% (KO); and (iii) CD4+: Mean ± SEM: 5.817% ± 0.9342% (WT) and 4.92% ± 0.1943% (KO) (Table + monocytes/macrophages between the two strains (Mean ± SEM: 10.86% ± 1.468% (WT) and 9.48% ± 0.5786% (KO) (Table +.IL17+ T cells in the KO mice (Mean ± SEM: 0.34% ± 0.1% (WT) and 0.11% ± 0.06% (KO); p < 0.05; Figure +.CD184+.CD62Lhi) in the KO mice (Mean ± SEM: 29.28% ± 2.349% (WT) and 37.33% ± 0.4447% (KO); p < 0.05, Table Flow cytometry analysis was performed to assess the levels of circulating ICs  we observed no major differences in the diversity, richness or F/B ratio in b1.b2 Adr KO mice compared to C57 WT controls, suggesting no presence of gut dysbiosis in these mice. Moreover, there were no differences in the abundance of the major phyla between the two strains. (ii) However, unifrac unweighted PCoA revealed a significant separation between microbiota of C57 WT and b1.b2 Adr KO mice. This was reflected in significantly increased abundance of the Bacilli class of Firmicutes in the b1.b2 Adr KO mice, and specifically the family of Lactobacillaceae. Conversely, we observed an increased abundance of family of Peptococcaceae and Eubacteriaceae of the Firmicutes phyla, among several others, in the b1.b2 Adr KO mice. (iii) The increase in levels of butyrate, acetate and propionate in the feces of b1.b2 Adr KO mice, linked to anti-inflammatory and anti-hypertensive effects   Wells, . Strains) Wells, . However) Wells, , a prope) Wells, . Similar) Wells, . FurtherA significant increase in circulating young neutrophils in our b1.b2 Adr KO mice may also be driven by changes in the microbiota in these mice. Neutrophils represent a large population of leucocytes in the mouse, and they have a unique property in that they follow daily cycles of release from the bone marrow into circulation as “young” neutrophils, where their “aging” is positively correlated with pro-inflammatory properties (Casanova-Acebes et al., In summary, our data illustrates an association between reduced beta-adrenergic receptor signaling, associated with dampened effects of the SNS on the whole body including the gut and the immune system, and a significant shift in Lactobacillaceae family in the gut microbiota and reduced immune responses in mice with reduced BP and heart rate responses (Rohrer et al., This study was carried out in accordance with the recommendations and approval by the IACUC protocol number 201708217.AB and TY designed and performed experiments and analyzed the data. They also contributed to writing of the manuscript. RA, WM, RL, and KM contributed to collection and analysis of data. TV contributed to writing and editing of the manuscript. JZ designed the experiments, oversaw analysis of the data and interpretation of results, and contributed to writing and final editing of the manuscript. Authors would like to thank Niousha Ahmari for technical assistance with the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brassica napus L.), one of the main oil crops in China, shows higher sensitivity to boron (B) deficiency than other species. Here, we demonstrated AS changes that largely increased the diversity of the mRNA expressed in response to B deficiency in B. napus. Each gene had two or more transcripts on average. A total of 33.3% genes in both Qingyou10  and Westar10  showed AS in both B conditions. The types of AS events were mainly intron retention, 3′ alternative splice site, 5′ alternative splice site and exon skipping. The tolerance ability of QY10 was higher than that of W10, possibly because there were far more differential alternative splicing (DAS) genes identified in QY10 at low B conditions than in W10. The number of genes with both DAS and differentially expressed (DE) was far lower than that of the genes that were either with DAS or DE in QY10 and W10, suggesting that the DAS and DE genes were independent. Four Serine/Arginine-rich (SR) splicing factors, BnaC06g14780D, BnaA01g14750D, BnaA06g15930D and BnaC01g41640D, underwent differentially alternative splicing in both cultivars. There existed gene–gene interactions between BnaC06g14780D and the genes associated with the function of B in oilseed rape at low B supply. This suggests that oilseed rape could regulate the alterative pre-mRNA splicing of SR protein related genes to increase the plant tolerance to B deficiency.Alternative splicing (AS) can increase transcriptome diversity, protein diversity and protein yield, and is an important mechanism to regulate plant responses to stress. Oilseed rape ( Alternative splicing (AS) is a widespread phenomenon in the majority of eukaryotic organisms that can generate one or multiple mRNAs isoforms from the same precursor mRNA (pre-mRNA) by using different splice sites ,2. The bArabidopsis, 42–61% of the intron-containing genes are alternatively spliced [Arabidopsis, such as CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) that is a MYB-related transcription factor [With the development of sequencing technique, more and more AS genes have been detected in plants. Four major types of AS events have been identified in plants: intron retention, alternative 3′ splice site, alternative 5′ splice site and exon skipping. Exon skipping is seen in the vast majority of the AS events . However spliced ,12. Most spliced ,14. It iPlant stress-related genes are particularly prone to AS events, which often modulate the ratio between active and inactive isoforms in response to abiotic stress, thus fine-tuning the expression of key stress regulators . Many alBrassica napus L.) requires much more B than other species for growth and development and is sensitive to B deficiency [B. napus cultivar Qingyou10 (QY10) and a B-inefficient B. napus cultivar Westar10 (W10) were screened by two-pace method (biomass screens at seedling and seed yield assessment at maturity) under contrasting B supply [NIP), which encodes a likely boric acid channel. A co-expression network analysis of the putative B transporters also highlighted its central role in the B uptake efficiency of B. napus [B. napus [Boron (B) deficiency is a major problem in China and worldwide, which causes the low yield and poor quality of many crops . In vascficiency . A B-effB supply . ProteomB supply ,23. qBECB supply ,25. ExplB. napus .B. napus under B deficiency have not been reported. In this study, RNA-Seq was used to investigate the AS events and DAS events in response to B deficiency in B. napus cultivars QY10 and W10. This should contribute to our understanding of the B deficiency response mechanism in oilseed rape, and therefore assist in efforts to improve B-efficiency in B. napus.Alternative splicing is an important mechanism to regulate plants responses to abiotic stress ,27,28,29B. napus cultivar Qingyou 10 (QY10) and B-inefficient B. napus cultivar Westar 10 (W10) were used in this study. The seedlings were cultured using Hoagland solution [3BO3 (10 µmol/L) and 0.25 µL H3BO3 (10 µmol/L) were applied in the solution to create a treatment with sufficient B and a treatment with deficient B with three replicates, respectively. The seedlings were grown in an illuminated culture room at 20 °C under a 16 h light/8 h dark cycle. The plants were arranged on a plant tray that had 9 × 6 holes with 5.6 cm between plants. The nutrient solution was oxygenated by an air pump and was replaced weekly. The pH of the nutrient solution was maintained at 5.7–6.0. When the seedlings showed B deficient symptoms (30 days), the root (R), old leaves (OL) and juvenile leaves (JL) of three plants of QY10 and W10 were sampled for RNA extraction, respectively. All samples were immediately frozen in liquid nitrogen and then stored at −80 °C.B-efficient solution  in a groTM 2000 platform . All raw data were deposited in the NCBI Sequence Read Archive  [Total RNA was extracted using an RNeasy Plant Kit (Bioteke RP1202) according to the manufacturer’s instructions. First, 1.5 µg total RNA from each sample were used for preparing RNA-Seq libraries (cDNA libraries). The manufacturer’s protocol  was used to generate sequencing libraries. The PCR amplification products were checked, excised and purified by agarose gel electrophoresis and MinElute PCR Purification Kit . The final products were loaded onto flow cell channels with a concentration of 2 pM by using 2 × 100 bp pair-end sequencing strategy. The sequencing analysis were conducted in the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University using Illumina HiseqA393069) .bzh  was also used to predict the splice junctions, and to identify the known and novel splice junctions according to the gene information with default parameters. In principal component analysis (PCA) of all genes, the replicate samples showed a high similarity with respect to the first two principal components, little variation within group and a good separation of groups  was used to calculate the AS events numbers. This study focused on four main types of AS: intron retention, exon skipping (cassette exons), alternative 3′ splice site  and alternative 5′ splicing site .All RNA CEL files were normalized using the RPKM (Reads Per kb per Million reads) method . Alternap value of splicing events with Δ ψ. A stringent threshold, p ≤ 0.05 and |Δ ψ| ≥ 0.05, was used to define the DAS events of AS events between different cultivars or different tissues of oilseed rape. The events with p values less than 0.05 were identified as significantly different events. Differentially expressed genes (DEGs) were calculated using a t-test  multiple testing). Probe-sets with a FDR corrected p-value ≤ 0.05 and fold change of >2 were considered to be differentially expressed.Differential alternative splicing (DAS) events were analyzed using the program Replicate Multivariate Analysis of Transcript Splicing  . A T-tesTotal RNA was extracted using an RNeasy Plant Kit (Bioteke RP1202) according to the manufacturer’s instructions. The DNAase treatment is mentioned above. Synthesizing the cDNA used a reaction solution containing 1 µg RNA with HiFiScript, dNTP Mix, Primer Mix, 5 × RT Buffer, DTT and HiFiScript. The solution was incubated at 42 °C for 50 min, followed by 85 °C for 5 min. The cDNA was used as a PCR template in a 20 µL reaction system of semi-quantitative RT-PCR. To validate the DAS events, 12 pairs of primers were designed based on different DAS events including intron retention, alternative 3′ splice site and alternative 5′ splice site. The primers used are listed in http://purl.obolibrary.org/obo). Biochemical pathway was analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology based on annotation system version 2.0 (KOBAS) [p < 0.05).Blast2GO was used to determine the gene functional category with a cut-off of 1E-5 . An inte (KOBAS) . The difAssembly of QY10 and W10 under B sufficient and deficient conditions identified 135,036 expressed transcripts and 56,203 expressed genes, and each gene had two or more transcripts on average . Many neIn total, 11,040,252 splice junctions were identified in all 36 RNA-Seq libraries . Four maThe AS genes with the types of splicing of “intron retentions (RI)” and “alternative 3′ splice site (A3SS)” were the largest fraction, whereas the AS genes with “exon skipping (ES)” were the least fraction among all the AS genes in both cultivars whether under B sufficient or B deficient conditions . The numThe categories of “cell wall modification”, “glucose-6-phosphate transport”, “ion transport”, and “response to stress” were mostly enriched for AS genes, while the categories of “DNA packaging”, “ribosomal export” and “protein location” were enriched for no AS genes . These dIn total, 159, 190 and 163 DAS genes were identified in root, juvenile leaves and old leaves of QY10, respectively, under B deficient conditions . Among tThe percentages of the four major types of DAS genes in root, juvenile leaves and old leaves of QY10 under B deficiency, and juvenile leaves and old leaves of W10 under B deficiency, all showed that RI > A3SS, A5SS > ES. However, in the root of W10, the percentage of “ES” DAS genes was the highest, and that of “A5SS” DAS genes was the least among the four major types of DAS genes . If we uB. napus, such as “Glycolysis/Gluconeogenesis”, “Calcium signaling pathway”, “MAPK signaling pathway” and “Peroxisome”  are degraded by the “nonsense-mediated mRNA decay (NMD)” surveillance pathway or are targeted by the microRNAs ,14. SequDAS and DE was lower than that of the genes that were either with DAS or DE in QY10 and W10, which demonstrated that the DAS and DE genes were independent.Combined analysis of DE genes and DAS genes in root, juvenile leaves and old leaves of QY10 and W10 indicated that about 0.06% of DE genes showed AS . Under BFunctional categorization of the DAS and DE genes in the root, juvenile leaves and old leaves of QY10 and W10 under B deficient conditions revealed that most of DAS and DE genes enriched in different functional pathways in biological process. For example, the categories of “mRNA processing”, “Succinyl–CoA metabolic process”, “signal transduction” and “cellular response to stress” were only enriched for DAS genes, whereas the categories of “cell wall formation”, “cell wall pectin metabolic process” and “cell wall organization” were only enriched for DE genes . Only a BnaA07g33860D (Sulphate anion transporter); and alternative 3′ splice site (DA3SS), such as BnaA01g30340D (SANT/Myb domain) and BnaA06g17710D  , such as 2, PHS2) . These g2, PHS2) .BnaC06g14780D, BnaA01g14750D, BnaA06g15930D and BnaC01g41640D, underwent DAS. Under B deplete conditions, A3SS occurred in the genes of BnaC06g14780D in the root and juvenile leaves; A5SS occurred in the genes of BnaA01g14750D in old leaves of QY10; and ES occurred in the gene of BnaC01g41640D in the root and old leaves of QY10 and BnaA06g15930D in the juvenile leaves of W10 .nditions . In addificiency . BnaA01gwith PTC . The exoBnaC06g14780D was a seed gene, and its downstream target genes were associated with the function of B [BnaA09g52970D) and bZIP transcription factor (BnaC04g52770D)  .B. napus cultivar “Darmor-bzh” [B. napus under B deficient conditions , whereas exon skipping is rare (3%) [Physcomitrella patens [Physcomitrella patens, the AS genes with the types of “RI” constituted the largest fraction of alternatively spliced genes [BnaC05g18490D, BnaC07g18010D and BnaA02g29830D, which were associated with cell wall synthesis and integrity of cell membrane, showed differential RI during B deficiency  . High prare (3%)  and Physa patens . Althouged genes . Moreoveficiency . AS evenficiency , which iB. napus is highly susceptible to B deficiency [B. napus cultivars [ficiency . There aultivars . Under Bultivars . FurtherB. napus adaption to B deficiency.The number of genes with both DAS and DE were far lower than that of the genes that were either DAS or DE in QY10 and W10 , which dBnaC06g14780D, BnaA01g14750D, BnaA06g15930D and BnaC01g41640D, showed differential alternative spliced genes ” and “alternative 3′ splice site (A3SS)” in both cultivars showed the largest fraction whether under B deficient or sufficient conditions. Further experiments should be conducted to confirm whether the tolerance ability of QY10 was higher than that of W10 was attributed to far more DAS genes were identified in QY10 under low B conditions than in W10. To find the functional transcript of the genes, such as SR splicing factor BnaC06g14780D, which responds to low boron stress, would provide a new way to increase the ability of B. napus to cope with B deplete stress.A total of ~33.3% genes showed AS in