The activation and growth of tumour-initiating cells with stem-like properties in distant organs characterize colorectal cancer (CRC) growth and metastasis. Thus, inhibition of colon cancer stem cell (CCSC) growth holds promise for CRC growth and metastasis prevention. We and others have shown that farnesyl dimethyl chromanol (FDMC) inhibits cancer cell growth and induces apoptosis in vitro and in vivo. We provide the first demonstration that FDMC inhibits CCSC viability, survival, self-renewal (spheroid formation), pluripotent transcription factors  expression, organoids formation, and Wnt/β-catenin signalling, as evidenced by comparisons with vehicle-treated controls. In addition, FDMC inhibits CCSC migration, invasion, inflammation (NF-kB), angiogenesis , and metastasis (MMP9), which are critical tumour metastasis processes. Moreover, FDMC induced apoptosis  in CCSCs and CCSC-derived spheroids and organoids. Finally, in an orthotopic (cecum-injected CCSCs) xenograft metastasis model, we show that FDMC significantly retards CCSC-derived tumour growth (Ki-67); inhibits inflammation (NF-kB), angiogenesis (VEGF and CD31), and β-catenin signalling; and induces apoptosis (cleaved PARP) in tumour tissues and inhibits liver metastasis. In summary, our results demonstrate that FDMC inhibits the CCSC metastatic phenotype and thereby supports investigating its ability to prevent CRC metastases. Enhanced screening and improved therapies are being used; however, the survival rate at 5 years of CRC patients with late-stage disease is only 12.5%5. The process of colorectal cancer biology, starting from the normal colon epithelial cell to the metastatic colorectal cancer cell, is associated with the development and acquisition of genetic mutations that lead to the activation or suppression of specific genes10. One of the key signalling pathways that is disrupted in colon cancer formation is the Wnt/β-catenin signalling pathway. This disruption is caused by specific genetic alterations in genes in this pathway15. The most common mutations are related to the adenomatous polyposis coli (APC) gene, which is inactivated, and the β-catenin gene, which is activated17. The standard of care for treating patients with colorectal cancer includes surgical resection for early-stage disease and chemotherapy with or without surgical resection for late-stage disease. Unfortunately, after undergoing potentially curative treatment, over half of all patients relapse in the form of distant metastasis19. In 85% of patients, relapse is diagnosed within the first 2.5 years after surgery and a third of patients are found to have new precursor legions of colorectal cancer at follow-up colonoscopy21. Metastasis of CRC is the most common reason for treatment failure because of CRC’s complicated mechanism of invasion, angiogenesis, and chemotaxis of the tumour microenvironment24. Our current understanding of the underlying process that leads to colorectal cancer relapse implicates specific cells that initiate cancer formation, referred to as cancer stem cells (CSCs). These cells are tumorigenic, metastatic, and resistant to the radiation and chemotherapies that are used to prevent CRC relapse28. Colorectal CSCs are histologically characterized by the expression of cell surface markers, such as CD24, CD44, CD133, LGR5, and ALDH1, which are key hallmarks of colorectal CSCs32. Given the critical role of the colorectal CSCs in CRC initiation, relapse, and metastasis, targeting these cells using novel agents may lead to both primary and secondary prevention of CRC.Colorectal cancer (CRC) is a significant healthcare problem and is estimated to be the second leading cause of cancer-related deaths in the United States in 201936. FDMCs are a unique species that naturally occur in cereal grains and oils40. We and others have shown that FDMC inhibits proliferation and survival in different types of cancers, including CRCs48. Recently, we showed that FDMC prevented the development of colorectal cancer in Azoxymethane rat models of colon carcinogenesis and inhibited human colon cancer cell lines SW-620 and HCT-11649. However, the activity of FDMC on CRC-initiating cells has not been reported. In this study, we investigated the activity of FDMC on viability, survival, self-renewal, migration, invasion, inflammation, angiogenesis, apoptosis, and Wnt/β-catenin signalling in colon CSCs (CCSCs) and CCSC-derived spheroids or organoids in vitro. We also studied the in vivo effects of FDMC on metastatic tumour growth, inflammation, angiogenesis, liver metastasis, apoptosis, and Wnt/β-catenin signalling.Recently, farnesyl dimethyl chromanol (FDMC) was shown to interact with key signalling pathways that are important in caner formation and metastasis48. However, our group is the first to have evaluated the effects of FDMC on CCSC and CCSC-derived spheroid and organoid growth. We treated CCSCs, spheroids, and organoids with FDMC in a concentration ranging from 10 to 100 μM for 72 h (for CCSCs), 5 days (for spheroids), and 6 days (for organoids). We measured cell viability using the CellTiter Glo assay and found that FDMC significantly (p < 0.001) decreased the viability of CCSCs, spheroids, and organoids in a fashion that was directly related to the concentration of FDMC  values were 50 ± 5 μM for CCSCs, spheroids, and organoids. We subsequently used IC50 (50 μM) of FDMC for other experiments. In addition, we evaluated the effects of FDMC (50 μM) on CCSC growth on soft agar by performing a colony formation assay; spheroids proliferation (Ki-67), using suspension 3D culture; and organoids proliferation (Ki-67), using Matrigel 3D culture. FDMC significantly inhibited colony formation (p < 0.01) and spheroid as well as organoid formation (p < 0.02), as evidenced by comparisons with vehicle-treated controls  suppressed cell mobility in CCSCs, which was evidenced by comparisons with vehicle-treated controls  prevented CCSCs’ invasion into the matrix compared to that observed in vehicle-treated controls . Inflammatory marker NF-kB expression was less in FDMC-treated CCSCs than in the vehicle-treated control, indicating that FDMC suppressed the inflammation , organoid formation, and pluripotency transcription factors. We found that FDMC-treated CCSCs became spherical in shape, unlike vehicle-treated CCSCs Fig. A. FDMC i52. Induction of Wnt/β-catenin signalling is crucial for colorectal tumour growth and metastasis26. Therefore, we first investigated the Wnt/β-catenin activity in 3T3 cells with β-catenin/TCF reporter activity (luciferase units) and CCSCs using Wnt receptor agonist Wnt3a and antagonist DDK1. β-catenin/TCF reporter activity (luciferase units) was induced by the agonist (Wnt3a) and decreased by the antagonist (DKK1) in 3T3 cells 56, in soft agar 57, and in Matrigel (organoid) formation58. The maintenance of stemness, pluripotency, and self-renewal capacity in CCSCs is actively regulated by putative transcription factors such as Nanog, OCT4, SOX2, and KLF48. Our data clearly show that FDMC down-regulated the expression of Nanog, OCT4, and SOX2 expression in CCSCs, targeting the CSCs’ growth, tumorigenesis, and metastatic spread. Moreover the expression of OCT4 and SOX2 has been shown to be elevated in colorectal tumours and expression was correlated with liver metastasis and predicted poor patient survival59. Knock-down of OCT4 and SOX2 has been shown to inhibit colorectal tumour growth, deplete CSC populations, and induce apoptosis61, supporting our proposal to target CCSC survival and self-renewal using FDMC.FDMC is a natural compound from the family of vitamin E molecules that has received significant interest for its potential as an agent to prevent and treat cancer. We and others have demonstrated that FDMC has significant anticancer activities in several models63. Our data further demonstrate that FDMC significantly inhibited the migration and invasion of CCSCs, which are the hallmarks of the cancer stem cell behaviour that precedes distant metastasis31. Therefore, it appears that FDMC prevents CCSC metastasis to the liver by mediating some of the necessary steps of tumour metastasis, such as migration, invasion, and angiogenesis. CSCs are known to contribute to tumour angiogenesis, and they generate angiogenic factors such as VEGF to induce angiogenesis64. Earlier studies showed that FDMC inhibited VEGF-induced angiogenesis in colorectal cancer cells in vitro66. However, to our knowledge, this is the first report showing that both in vitro and in vivo, FDMC significantly inhibited colorectal tumour inflammation and angiogenesis in CCSCs and metastatic colon tumours by inhibiting NF-kB, VEGF, and MMP9 levels. These data further reflect the anti-inflammatory and antiangiogenic activity of FDMC.One of our novel findings was that FDMC induced apoptosis in CCSCs, spheroids, organoids, and CRC tumours, which are important, as CCSCs are well known to be resistant to chemo and radiation therapies67. Our in vitro and in vivo experiments demonstrated that FDMC inhibited the metastasis marker MMP9. In our mouse model of metastasis (involving cecum-injected CCSCs), FDMC significantly inhibited tumour volume, tumour weight, and liver metastases compared with vehicle-treated controls. The analysis of the CCSC-derived tumours revealed that FDMC treatment not only restricted tumour growth by inhibiting pluripotency transcription factors (OCT4 and SOX2), inflammation (NF-kB), and angiogenesis (VEGF) but also induced apoptosis (C-PARP). The role of Wnt signalling in the homeostasis of CSCs in human CRC and tumorigenesis in mice has been demonstrated52. Our data also show that FDMC significantly inhibited Wnt/β-catenin receptor activity and β-catenin expression in vitro and in vivo.Sixty percent of CRC patients experience cancer recurrence following remission. Relapse from colorectal cancer remission is often associated with the development of metastasis. The process of metastasis involves the reactivation of dormant cancer cells from the primary tumour that have to undergo multiple steps to accomplish a clinically relevant metastatic tumour. These steps include proliferation, invasion, motility, and modulation of the tumour microenvironment, such as the induction of angiogenesis and suppression of immune clearanceIn summary, we found that FDMC inhibits CCSC growth, self-renewal, pluripotency, migration, invasion, inflammation, angiogenesis, and Wnt/β-catenin signalling and induction of apoptosis in vitro. It also inhibits metastatic tumour growth, inflammation, angiogenesis, Wnt/β-catenin signalling, and liver metastasis spread and induces apoptosis in vivo. These data indicate a novel antimetastatic potential of FDMC in targeting CCSCs, and hence, possible clinical use for the prevention and treatment of metastatic CRC.46, all chemicals and reagents were purchased from Sigma-Aldrich  unless otherwise specified. FDMC was obtained from Davos Life . l-Glutamine, penicillin, streptomycin, and HEPES (4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid) buffer were purchased from Mediatech . Foetal bovine serum (FBS) was purchased from HyClone  and ethanol (100%) from Aaper Alcohol and Chemical . Human colon cancer stem cells  and growth media were purchased from Celprogen . Phosphate-buffered saline (PBS), GlutaMAX, N2 supplement, B27 supplement, and advanced Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) were purchased from Invitrogen . Human epidermal growth factor (EGF), fibroblast growth factor 4, Wnt3a, R-Spondin, and Noggin were purchased from R&D Systems . Bovine serum albumin (BSA), N-acetyl cysteine, nicotinamide, gastrin, A83-01, SB202190, prostaglandin E2, and Y-27632 were purchased from Sigma . Growth factor-reduced Matrigel was purchased from Corning , pimocin from InvivoGen , basic fibroblast growth factor, and EGF from Peprotech , and basement membrane extract (BME) from Trevigen .As previously described in Husain et al.46, cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% O2.Human  colon cancer stem cells were grown in human colon cancer stem cell complete growth medium (M36112-39PS). As previously described in Husain et al.+, CD44+, LGR5+, and ALDH1+; Celprogen) were grown in human colon CSC complete growth medium (M36112-49PS). Two thousand cells (100 µL) were seeded in 96-well plates  for 24 h to attach overnight. As previously described in Husain et al.46, cells were then incubated for 72 h with various concentrations of FDMC (10–5 to 10–4 M) or ethanol (< 5%) vehicle as control. To test spheroid viability, 1000 spheroids (100 µL) were added to each well of the nonadherent plates . The cells were then incubated for 5 days with various concentrations of FDMC (10–5 to 10–4 M) or ethanol (< 5%) vehicle as a control medium. To test organoid viability, organoids were diluted to 50 organoids/µL in growth medium containing 5% BME. First, a 96-well plate was coated with 30 µL BME then 20 µL of organoid cell suspension were added to each well of the plate. The following day, different concentrations of FDMC (10–5 to 10–4 M) or ethanol (< 5%) vehicle as control (50 µL) were added to each well and then incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% O2 for 6 days, as previously described in Husain et al.46 CellTiter-Glo reagent (100 µL) was added to each well, mixed on an orbital shaker, and incubated at room temperature for 30 min, and luminescence was recorded in a Multimode Plate Reader .Human  colon CSCs  for 24 h. This dose is the IC50 after 72 h of exposure, but at 24 h there are minimal effects on the death of these specific cells. As previously described by Yuan et al.68, the spread of wound closure was observed and photographed under light microscope. For invasion assays, 1 × 105 cells (CCSCs) in serum-free stem cell media were added into the upper chamber of an insert that was precoated with Matrigel . The lower chamber was filled with stem cell media with 10% FBS. After 48-h incubation, the cells remaining on the upper surface of the membrane were removed, whereas the cells that had invaded through the membrane were stained with 20% methanol and 0.2% crystal violet, imaged, and counted under light microscope.The cell migration assay was performed as previously described in Husain et al.46 In brief, 100 000 human  colon cancer stem cells  were plated in 3-dimensional culture, in ultra-low nonadherent plates  that contained stem cell-specific DMEM/F12 medium supplemented with 2% B27 supplement, 1% N2 supplement, 10 ng/mL basic fibroblast growth factor, 20 ng/mL EGF, and 1% antibiotics. This was treated with vehicle (5% ethanol) and FDMC (50 μM) and incubated at 37 °C for 5 days. A number of spheroids were formed, counted under microscope, and photographed.The spheroid formation assay was performed as previously described in Husain et al.56. The spheroids formed in 3D culture were then suspended in growth factor-reduced Matrigel (Corning) and pipetted (25 µL) on the bottom of the 24-well plate and incubated for 10 min at 37 °C. Five hundred microliters of DMEM/F12 organoid culture medium containing penicillin/streptomycin, 10 mM HEPES, GlutaMAX, 50% Wnt, 20% R-Spondin, 10% Noggin, 1 × B27, 1.25 mM n-Acetyl Cysteine, 10 mM Nicotinamide, 50 ng/ml human EGF, 10 nM Gastrin, 500 nM A83-01, 3 µM SB202190, 10 nM Prostaglandin E2, 100 mg/mL Primocin, and 10 µM of Y-27632 added to the wells of the plate without touching the Matrigel dome and plates were incubated at 37 °C for 10 days58. The media were refreshed every 2 to 3 days. Organoids treated with vehicle and FDMC were photographed under microscope.The cells were grown in RPMI medium supplemented with GlutaMAX, penicillin/streptomycin (100 µg/mL), and varying concentrations of defined FBS . On the fourth day, the cells were grown in DMEM/F12 medium containing 2% FBS, fibroblast growth factor 4 (500 ng/mL), and Wnt3a (500 ng/mL)49. Human  colon CSCs were plated and treated concurrently with vehicle (5% ethanol) or FDMC (50 μM) for 24 h. Cells were harvested, and 105 cells were transferred to 5 mL tubes containing PBS (100 μL), and then 2 μL of PI and 5 μL of annexin V-FITC (BD Bioscience) were added and mixed. The tubes were incubated for 15 min at room temperature in the dark, 400 μL of binding buffer were added, and the tubes were analyzed for apoptosis by flow cytometry.The apoptosis assay was performed as previously described by Husain et al.4) were seeded in two chambered glass slides and allowed to adhere overnight. As described in Francois et al.69, cells were treated the following day with FDMC and vehicle, and after 24 h, they were washed with PBS then fixed in 4% paraformaldehyde in PBS solution overnight at 4 °C. Cells were made permeable the next day via incubation in 0.1% sodium citrate-0.1% Triton X-100 solution at 4 °C for 2 min. They were then labelled for apoptotic DNA strand breaks using the in situ cell death detection kit, AP , in accordance with the manufacturer’s instructions. Cells were stained in Vectashield mounting medium  containing DAPI to counterstain DNA. Fluorescein-labelled DNA strand breaks (TUNEL-positive cells) were then visualized using a fluorescence microscope , and 5 representative pictures of the slide field were taken with a digital camera . TUNEL-positive nuclei (green) were scored and compared with DAPI-stained nuclei (blue) to determine the induction of apoptosis percentage for each treatment group.TUNEL assay for apoptosis was also carried out in CCSCs. Cells .The 3T3 cell line expressing luciferase reporter gene under the control of Wnt-responsive promoters (T-cell factor/lymphoid enhancer factor) were grown and maintained in DMEM medium . The cells  were added to each well of the 96-well plate  in growth medium and grown to 60% confluency. One hundred µL of PBS, 100 ng/mL Wnt receptor agonist (WNT3a) alone, agonist plus antagonist (DKK1) at a concentration of 100 ng/mL, and agonist plus FDMC (50 µM) prepared in assay medium were added to the control wells, agonist wells, agonist plus antagonist wells, and agonist plus FDMC wells, respectively. The plates were incubated at 37 °C in a humidified atmosphere of 5% CO70. This technique required the use of needle under binocular guidance. Female Athymic nude mice , obtained from Charles River , were kept in our animal facility for 1 week of quarantine. After 1 week, mice were anesthetized with isofurane, and their ceca were exteriorized via laparotomy. Luciferase-expressing CCSCs (1 × 105) were suspended in 50 µL of PBS and placed into a sterile syringe for each animal. We slowly injected the cell suspension at a depth of 5 mm into the wall of each cecum under a binocular lens, with the syringe at an approximately 30° angle. Afterward, we applied slight pressure with a cotton stick at 2 mm from the injection point in the direction of the needle. We pulled the needle out and cleaned the area around the injection with 3% iodine to avoid the unlikely seeding of refluxed tumour cells into the abdominal cavity. After injection, the gut was returned to the abdominal cavity and closed with surgical sutures and staples.We tested whether direct orthotopic cell microinjection, between the mucosa and the muscularis externa layers of the wall of cecum in nude mice, induces tumour foci in sites corresponding to those most often observed to be the location of metastases in humans46, 1 week after CCSC microinjection, mice were randomly divided into 2 treatment groups as follows: (1) control group, which received oral gavage of 100 µL of vehicle (ethanol-extracted olive oil) twice daily for 4 weeks and (2) FDMC group, which received 200 mg/kg FDMC oral gavage twice daily for 4 weeks. Mouse tumour volume was measured once per week (luciferase bioluminescence) using an IVIS 200 (Xenogen) in vivo imaging system machine. After 4 weeks of treatment, animals were sacrificed by CO2 inhalation, and tumour weights and liver metastases were recorded. One half of the tumour was immediately frozen in liquid nitrogen and stored at − 80 °C for biochemical analyses; the other half was fixed in buffered formalin for histologic analyses. The care and use of the animals included in this study were approved by the Moffitt Cancer Centre Institutional Laboratory Animal Care and Use Committee and were conducted following National Institutes of Health guidelines.As described in Husain et al.46, cells and tumour tissues were washed 3 times in cold PBS (pH 7) and then lysed in protein extraction reagent radioimmunoprecipitation assay (RIPA) buffer  that contained phosphatase and protease inhibitor cocktail. Protein concentration was determined using bicinchoninic acid assay reagents , in accordance with the manufacturer’s instructions.As previously described in Husain et al.71. Proteins were then electrotransferred onto nitrocellulose membranes. After blocking in 5% nonfat powdered milk for 1 h, the membranes were washed and treated overnight at 4 °C with antibodies to VEGF, MMP9, Nanog, Oct4, Sox-2, NF-kB, C-PARP, β-catenin, ubiquitin and β-actin (1:1000) . As described in Husain et al.46, after blots were washed, they were incubated with horseradish peroxidase-conjugated secondary antibody IgG (1:5000) for 1 h at room temperature. Washed blots were then treated with SuperSignal West Pico chemiluminescent substrate (Pierce) for positive antibody reaction. Membranes were exposed to radiographic film  for visualization and densitometric quantization of protein bands using AlphaEaseFC software .Extracted proteins from cells and tumour tissues (40 µg) were resolved on 12.5% sodium dodecyl sulphate polyacrylamide gel  and a 5% stacking gel. As described in Pimiento et al.Treated cells and spheroids/organoids were cytospin on glass slides then fixed in 4% paraformaldehyde in PME buffer overnight at 4 °C. Cells/spheroids were made permeable the next day via incubation in 0.5% Triton X-100 solution at 4 °C for 2 min and blocked with 2% BSA for 30 min. Slides were immunostained with primary antibodies  and 4′,6-diamidino-2-phenylindole (DAPI). The images were recorded using confocal microscopy , and representative pictures of the slide field were taken with a digital camera .46. Formalin-fixed, paraffin-embedded tumour tissues were sectioned (4 μm) and stained with haematoxylin and eosin. Immunohistochemistry was performed using the Ventana Discovery XT automated system  with proprietary reagents as per manufacturer’s protocol. Briefly, slides were deparaffinised on the automated system with EZ Prep solution. Sections were heated for antigen retrieval. For immunohistochemistry, tissue sections were incubated with Ki-67 and CD31 at 1:4000 dilutions for 60 min. Detection was performed using the Ventana OmniMap kit. The expression percentage was recorded for each area and then averaged for each mouse. For CD31, sections were examined at low power to identify cancers and associated hot spots. The number of vessels per ×400 field was counted manually. Single cells and groups expressing CD31 were counted as vessels in addition to groups with lumens. Sections not showing a cancer were assessed for hot spots at low power.Histologic evaluation was performed as previously described in Husain et al.49, the data were analyzed statistically using unpaired t tests or 1-way ANOVA  where appropriate. ANOVA was followed by Duncan's multiple range tests using SAS statistical software for comparisons between different treatment groups. Statistical significance was set at p < 0.05.The data were expressed as means ± standard errors of the means. As described in Husain et al.


Rigosertib, via reactive oxygen species (ROS), stimulates cJun N-terminal kinases 1/2 (JNK1/2), which inactivate RAS/RAF signaling and thereby inhibit growth and survival of tumor cells. JNK1/2 are not only regulated by ROS—they in turn can also control ROS production. The prooxidant and cell death function of p66Shc requires phosphorylation by JNK1/2. Here, we provide evidence that establishes p66Shc, an oxidoreductase, as a JNK1/2 effector downstream of Rigosertib-induced ROS production, DNA damage, and cell death. This may provide a common pathway for suppression of tumor cell growth by Rigosertib. Mutational activation of the three small G proteins HRAS, KRAS and NRAS is common in tumors, enabling them to interact with various effectors—among which the members of the serine-threonine kinases of the RAF family , PI3K, and RalGDS feature prominently ,2,3. RAS® SGM Plus® PCR amplification kit  at the Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria. PC3 and DU-145 cells were kept in RPMI1640 medium. All media contained 10% fetal calf serum (FCS), 2 mmol/L l-glutamine, and 1× penicillin/streptomycin . Cells were maintained at 37 °C in a 5% CO2/95% air-humidified atmosphere. The cells were passaged every alternate day. For drug treatment, 350,000 cells were seeded per well of a 6-well plate and grown for 24 h to obtain 70–80% confluence. The medium was replaced with 0.1% FCS containing medium [The MCF7 cell line was obtained from LGC Standards  for these experiments, and cells were maintained in Minimal Essential Medium (MEM) . PC3 and DU-145 cells were provided by Prof. Helmut Klocker, University Hospital for Urology, Division of Experimental Urology, Department of Surgery, Medical University Innsbruck, Austria, and cells have been authenticated by short tandem repeat analysis using the AmpFlSTRProteins were isolated and detected as described previously ,14,15. PCell viability was assessed using AnnexinV/PI staining, as previously described . DNA damMCF7 cells were transfected with 1 µg pRec_p66Shc-HA-His  using Lipofectamine 2000 . Transiently transfected cells were used 48 h after transfection.t-test or ANOVA analyses. Statistical analyses were done using GraphPad Prism 5 . Significance values were designated as follows: * p  <  0.05, ** p  <  0.005, and *** p  <  0.0005.All data are presented as a mean  ±  SD following Three different cancer cell lines were included in our studies: the breast cancer line MCF7 and the prostate cancer lines PC3 and DU-145. JNK1/2 activity was monitored by analyzing S63 phosphorylation of cJun . PronounWe next addressed whether JNK1/2 activation results in the phosphorylation of S36 on p66Shc, an event that is essential for the activation of its prooxidant and pro-death activity . As showTo confirm the involvement of JNK1/2 in the activation of p66Shc, cells were pretreated with the JNK1/2 inhibitor SP600125 for one h at a concentration of 20 µM prior to Rigosertib treatment. As expected, SP600125 efficiently prevented cJun phosphorylation, which we monitored to assess JNK1/2 activity A. In MCFTreatment with the JNK inhibitor SP600125 prior to Rigosertib prevented the phosphorylation of γH2AX A as wellRigosertib was discovered in a phenotypic screen for molecules that induce mitotic cell-cycle arrest in tumor cells while sparing non-transformed cells . The dirIn the work presented here, we confirm earlier findings on Rigosertib-induced JNK1/2 activation and concomitant inhibition of ERK signaling in MCF7 cells [JNK1/2 are also required for the activation of p66Shc , which i


Bacillus spp. supplementation on mortality, viral load, and expression of immune-related genes in red hybrid tilapia (Oreochromis spp.) upon TiLV infection. Fish were divided into three groups, and fed with: control diet, 0.5% probiotics-supplemented diet, and 1% probiotics-supplemented diet. After 21 days of experimental feeding, the three groups were infected with TiLV and monitored for mortality and growth performances, while organs were sampled at different time points to measure viral load and the transcription modulation of immune response markers. No significant difference was found among the groups in terms of weight gain (WG), average daily gain (ADG), feed efficiency (FE), or feed conversion ratio (FCR). A lower cumulative mortality was retrieved from fish fed 0.5% and 1% probiotics , compared to the control group (32%). Moreover, fish fed with 1% probiotic diet had a significantly lower viral load, than those fed with 0.5% probiotic and control diet at 5, 6, 9, and 12 days post infection-challenge (dpc). The expression patterns of immune-related genes, including il-8 , ifn-γ, irf-3, mx, rsad-2  showed significant upregulation upon probiotic treatment during the peak of TiLV pathogenesis (between 9 and 12 dpc) and during most of the study period in fish fed with 1% probiotics-supplemented diet. Taken together, these findings indicate that dietary supplementation using Bacillus spp. probiotics may have beneficial effects to strengthen tilapia immunity and resistance against TiLV infections. Therefore, probiotic treatments may be preventively administered to reduce losses caused by this emerging viral infection in tilapia aquaculture.Tilapia lake virus (TiLV) causes an emerging viral disease associated with high mortality and economic damage in tilapia farming around the world. The use of probiotics in aquaculture has been suggested as an alternative to antibiotics and drugs to reduce the negative impact of bacterial and viral infections. In this study, we investigate the effect of probiotic  Tilapia tilapinevirus, under the genus Tilapinevirus, but in the family Amnoonviridae  ÷ initial body weight(g)Feed efficiency (FE) = [weight gain(g) ÷ amount of ingested feed (g)]FCR = feed intake ÷ weight gainADG (g) = weight gain ÷ number of days on feed® solution , following the manufacturer’s instructions. RNA quality and concentration were estimated using spectrophotometry . One µg of total RNA was reverse transcribed using ReverTraAce® kit , following the manufacturer’s instructions. The resulting cDNA was diluted with TE buffer pH 8.0 and stored at −80 °C.Total RNA was extracted from sampled tissues at the defined experimental time points using TrizolThe amount of TiLV genomic RNA in liver, spleen, and head kidney of control and probiotic supplementation fish was analyzed by an RT-qPCR assay as previously described ,76. The ™ universal SYBR green supermix (BioRad), 0.3 µL of each forward and reverse primer, 0.4 µL of molecular grade water, and 4 µL of cDNA template. All samples were run in triplicate. General cycling parameters were set as follows: denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. At the end of the qPCR cycle, samples were subjected to melting curve analysis at a temperature ranging from 65 to 95 °C with 0.5 °C per 5 s increments. RT-qPCR assays were performed using a CFX96 Touch™ machine (BioRad). Primer pairs used in this experiment are listed in RT-qPCR was performed using a MicroAmp Optical 96-well reaction plate (BioRad) in a 10 µL reaction: consisting of 5 µL iTaqp < 0.05, where the different amounts of experimental probiotic diets were used as an explanatory variable. Cumulative mortality significance was set at p < 0.05 during the TiLV infection.Data retrieved from growth performance, viral load, and the expression of immune-related genes were expressed as mean ± standard error of mean in control and treatment groups. An ordinary one-way ANOVA with Tukey’s multiple comparisons test, using GraphPad Prism software 5.0.1 , was applied to the statistically significant difference at


Based on the results, we performed experiments in vivo. Before 90 min ARE induction, CLR was administered orally by concentration. ARE was derived by linking the metastatic junction between pylorus and forestomach and corpus in Sprague-Dawley rats. And rats were sacrificed 5 h after surgery. We analyzed the expression of antioxidant and inflammatory-related markers by western blot and observed the production of alanine aminotransferase (ALT), aspartate aminotransferase (AST), reactive oxygen species (ROS), peroxynitrite (ONOO−), and thiobarbituric acid reactive substance (TBARS). The administration of CLR reduced esophagus tissue damage in rats with acute reflux esophagitis and decreased the elevated ALT, AST, ROS, ONOO−, and TBARS. In addition, CLR effectively increased antioxidant-related factors and reduced inflammatory protein. Overall, these results suggest that CLR would be used as a therapeutic material in protection and treatment for ARE. Overall, CLR treatment informed that markedly ameliorated inactivation of NF-κB led to the inhibition of the expressions of proinflammatory proteins. These results suggest that CLR would be used as a therapeutic material in protection and treatment for ARE.Gastroesophageal reflux disease (GERD) is induced by the reflux of stomach contents or gastric acid, pepsin into the esophagus for prolonged periods of time due to defection of the lower esophageal sphincter. Reflux esophagitis is a disease found in less than 50% of GERD patients. This study is aimed at evaluating the protective effect of Curcumae longae Rhizoma 30% EtOH extract (CLR) in acute reflux esophagitis (ARE) rats. CLR measured antioxidant activity through  After m−), an oxidative stress biomarker, was measured according to the method of Kooy et al. [Peroxynitrite  levels were measured according to the method of Mihara and Uchiyama . 1,1,3,32, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, and 1,250 μL protease inhibitor mixture solution. The tissue homogenates were incubated (4°C for 20 min), and then, 10% NP-40 was mixed well. After centrifugation  using Eppendorf 5415R , the supernatant (cytosol fractions) was separated with new Eppendorf tubes. The pellets were washed twice by the lysis buffer and discard the supernatant. After that, the pellets were suspended with 20 mL ice-cold lysis buffer C containing 300 mM NaCl, 50 mM HEPES (pH 7.8), 50 mM KCl, 1 mM DTT, 0.1 mM PMSF, 0.1 mM EDTA, 1% (v/v) glycerol, and 100 μL protease inhibitor mixture solution which were suspended and incubated (4°C for 30 min). And in centrifugation , the supernatant (nuclear fractions) was collected with new Eppendorf tubes. Both cytosol and nuclear fractions were stored at -80°C before the analysis.The extraction of protein was performed according to the method of Komatsu with modifications . For cytκBp65, and histone, 12 μg of protein from each nuclear fraction was electrophoresed through 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Separated proteins were transferred to a nitrocellulose membrane, blocked with 5% (w/v) skim milk solution for 1 h, and then incubated with primary antibodies  overnight at 4°C. After the blots were washed, they were incubated with anti-rabbit or anti-mouse IgG HRP-conjugated secondary antibody for 1 h at room temperature. In addition, 8 μg proteins of each cytosol fraction of NOX4, p22phox, SOD, catalase, GPx-1/2, p-p38, p-JNK, p-IκBα, IκBα, Inos, COX-2, TNF-α, IL-6, claudin-3, claudin-4, and β-actin were electrophoresed through 10-12% SDS-PAGE. Each antigen-antibody complex was visualized using ECL Western Blotting Detection Reagents and detected by chemiluminescence with Sensi-Q 2000 Chemidoc . Band densities were measured using ATTO Densitograph Software  and quantified as the ratio to histone or β-actin. The protein levels of the groups are expressed relative to those of the normal rat (represented as 1) [For the estimation of Nrf2, c-Jun, NF-ed as 1) .μm sections and stained using hematoxylin and eosin (H&E) for microscopic evaluation. The stained slices were observed under an optical microscope and then analyzed using the i-Solution Lite software program .Histological examination through microscopy was performed to evaluate the separated esophagus tissues. The separated esophagus was fixed through a 10% neutral-buffered formalin and embedded in paraffin, and cut into 2 In vitro values were expressed as the means ± SEM and in vivo values as the means ± SD. Statistical comparisons were analyzed by one-way ANOVA tests followed by the least significant difference (LSD) test using SPSS . Values of p < 0.05 were considered significant.50 (μg/mL). IC50 represents half the maximum concentration of compound tested to remove DPPH radicals. The IC50 value of L-ascorbic acid (standard sample) was 1.22 ± 0.04 μg/mL, and the IC50 value of DPPH radical scavenging activity of CLR was 36.44 ± 0.76 μg/mL. The antioxidant effect of CLR was measured by ABTS radical scavenging assay, which is expressed in IC50 (μg/mL). IC50 represents half the maximum concentration of compound tested to remove ABTS radicals. The IC50 value of ABTS radical scavenging activity of L-ascorbic acid (standard sample) was 3.48 ± 0.01 μg/mL, and the IC50 value of CLR was 44.08 ± 1.89 μg/mL  is a common gastrointestinal disorder caused by abnormal reflux of gastric contents as a reflux symptom . Reflux First, DPPH and ABTS free radical scavenging activities of CLR were confirmed. In addition, total polyphenol and total flavonoid contents were measured. As a result, it was shown that CLR exhibits excellent antioxidant effects Tables  and 2. B2O2), hydroperoxyl radical (HO·2), hydroxyl (OH−), and superoxide (O2·−) radicals. Among them, O2·− and H2O2 activate multiple signaling pathways that cause cell proliferation and apoptosis, elevated vascular tone, fibrosis, and inflammation, and excessive ROS cause mitochondrial damage or dysfunction that causes mitochondrial cell oxidative stress [−, biomarkers of oxidative stress in serum, it was significantly decreased in CM and CH, and TBARS levels, lipid peroxides in serum and tissue, were also significantly decreased in CM and CH  is a collective common term that includes highly oxidative radicals such as hydrogen peroxide  to modulate intracellular antioxidant responses. Thus, the Nrf2 pathway is considered a major factor regulatory mechanism for reducing oxidative stress and major regulator of the antioxidant defense system . HO-1, rκB in response to inflammatory agonists. Activated MAPKs are involved in directing cellular responses to various stimuli and in the regulation of cellular processes, such as proliferation and differentiation, cell survival, and apoptosis, and NF-κB is found in many types of cells and is involved in cellular responses to various stimuli, such as stress, cytokines, oxidized low-density lipoproteins, and bacterial or viral antigens. Also, it regulates inflammation by promoting the expression of TNF-α, IL-6, and other proinflammatory cytokines such as COX-2 and iNOS [κB pathways NF-κBp65, p-IκBα, proinflammatory (such as COX-2 and iNOS), and inflammatory cytokines are reduced  are responsible for the formation of functional epithelial and endothelial barriers that regulate the passage of cells and solutes through the intercellular space as the most important structural component for the formation of constitutive barrier function in endothelial and epithelial cells. TJs include the claudin family, occludin, junctional adhesion molecules, and tricellulin, and decreased expression of its contribution to the increased permeability of the gastrointestinal epithelium in GERD –50. We oκB pathway-related factors, thereby suppressing inflammation of the esophageal mucosa.In our findings, CLR increased the expression of antioxidant-related factors by suppressing factors related to oxidative stress. In addition, it was suggested that the reduction of oxidative stress-related factors suppresses the expression of MAPKs and NF-κB pathways. Therefore, the results of this study suggest that CLR is a new material for the treatment of acute reflux esophagitis.The aim of our study was to demonstrate the ameliorating effects of bioactive CLR of esophageal damage caused by ARE. Taken together our research results, CLR suppressed excessive oxidative stress generation, thereby activating the expression of the antioxidant factor by the Nrf2 pathway and inhibiting the expression of proinflammatory proteins through the MAPKs and NF-


Ochrobactrum anthropi SH14, utilizing azoxystrobin as a sole carbon source, was isolated from contaminated soils. Strain SH14 degraded 86.3% of azoxystrobin (50 μg·mL−1) in a mineral salt medium within five days. Maximum specific degradation rate (qmax), half-saturation constant (sK), and inhibition constant (iK) were noted as 0.6122 d−1, 6.8291 μg·mL−1, and 188.4680 μg·mL−1, respectively.Conditions for strain SH14 based azoxystrobin degradation were optimized by response surface methodology. Optimum degradation was determined to be 30.2 °C, pH 7.9, and 1.1 × 107 CFU·mL−1 of inoculum. Strain SH14 degraded azoxystrobin via a novel metabolic pathway with the formation of N--acetamide,2-amino-4-(4-chlorophenyl)-3-cyano-5,6-dimethyl-pyridine, and 3-quinolinecarboxylic acid,6,8-difluoro-4-hydroxy-ethyl ester as the main intermediate products, which were further transformed without any persistent accumulative product. This is the first report of azoxystrobin degradation pathway in a microorganism. Strain SH14 also degraded other strobilurin fungicides, including kresoxim-methyl (89.4%), pyraclostrobin (88.5%), trifloxystrobin (78.7%), picoxystrobin (76.6%), and fluoxastrobin (57.2%) by following first-order kinetic model. Bioaugmentation of azoxystrobin-contaminated soils with strain SH14 remarkably enhanced the degradation of azoxystrobin, and its half-life was substantially reduced by 95.7 and 65.6 days in sterile and non-sterile soils, respectively, in comparison with the controls without strain SH14. The study presents O. anthropi SH14 for enhanced biodegradation of azoxystrobin and elaborates on the metabolic pathways to eliminate its residual toxicity from the environment.Azoxystrobin is one of the most popular strobilurin fungicides, widely used in agricultural fields for decades.Extensive use of azoxystrobin poses a major threat to ecosystems. However, little is known about the kinetics and mechanism of azoxystrobin biodegradation. The present study reports a newly isolated bacterial strain,  Strobilurin fungicides represent an outstanding new class of pesticides with higher biological activity and specific modes of action ,2,3. Ove−1. According to the European Food Safety Authority, azoxystrobin is one of the most frequently found pesticides in cereal crops and is classified as highly toxic to aquatic organisms or agricultural beneficial organisms  and Luria–Bertani medium (LB)  were used for the isolation of degraders [Mineral salt medium (MSM) [2.0 g of  plates containing 50 μg·mL−1 azoxystrobin, and incubated at 30 °C for 2 days. Individual colonies were picked out and their azoxystrobin degrading abilities were monitored by high-performance liquid chromatography (HPLC) according to the previous method [Approximately 5 g of activated sludge collected from pesticide-manufacturing wastewater treatment system was suspended in 250-mL Erlenmeyer flasks containing 100 mL MSM and 50 μg·mLs method . A highlMorphology of strain SH14 was observed on LB agar (1.8%) plates after incubation at 30 °C for 2 days. Genetic analysis was carried out by PCR amplification of 16S rDNA gene with universal primers . PCR cycg for 4 min, and washed twice with 0.9% sterile saline to prepare a concentrated inoculum solution. Colony-forming units (CFU) of this suspension werequantified by the dilution plate count technique [7 CFU·mL−1) was used as inoculum for all the degradationassays. The suspension was inoculated into 50 mL MSM supplemented with 50 μg·mL−1 azoxystrobin as a carbon source and cultivated at 30 °C and 200 rpm for 5 days. Each treatment consisted of three replicates and control cultures were prepared in sterilized MSM with the same concentration of azoxystrobin only. The growth of strain SH14 was determined by counting CFU per milliliter of serial dilutions and concentration of azoxystrobin residues was monitored by HPLC at an interval of 12 h [To initiate degradation experiments, strain SH14 was thawed and cultivated on LB agar plates. After 24 h cultivation, the individual colonywas transferred intoLB medium, harvested by centrifugationat 4000× echnique ,37. Unle of 12 h .−1 azoxystrobin in MSM on day 5 was considered as a dependent variable. Polynomial regression analyses were used to fit the quadratic regression equation (Equation(1)) by response surface regression procedure (REREG) of the statistical analysis system (SAS) software.Yi refers to predicted response(azoxystrobin degradation %), Xi and Xj refer to variable parameters. b0, bi, bij, and bii refer to constant, linear coefficient, interaction coefficient, and quadratic coefficient, respectively.Biodegradation conditions of strain SH14 were optimized by response surface methodology (RSM) ,41,42. B−1). Experiments were conducted in triplicate for each treatment, and all the applications were incubated at 30 °C on a rotary shaker (200 rpm) for 5 days. Non-inoculated applications served as controls. Andrews equation (Equation(2)) was followed to determine kinetics parameters of biodegradation at different initial azoxystrobin concentrations [S is azoxystrobin concentration(μg·mL−1), q is azoxystrobin degradation rate (d−1), maxq is maximum azoxystrobin degradation rate (d−1), sK is half-saturation constant (μg·mL−1), and iK is azoxystrobin inhibition constant (μg·mL−1). The Andrews equation was applied in Matrix Laboratory software .To test the effect of initial azoxystrobin concentration on its degradation, strain SH14 was inoculated into 250-mL Erlenmeyer flasks containing 50 mL sterilized MSM and different concentrations of azoxystrobin q=qm−1 azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, picoxystrobin, and fluoxastrobin, respectively. Three replicates were prepared for each treatment and non-inoculated applications served as controls. All cultures were performed in 250-mL Erlenmeyer flasks and cultivated at 30 °C on a rotary shaker (200 rpm) for 5 days. Concentrations of fungicide residues were monitored by HPLC at an interval of 12 h.Degradation efficacy of strain SH14 against various strobilurins was studied. Strain SH14 was inoculated into 50 mL sterile MSM containing 50 μg·mLCt is the concentration (μg·mL−1) of strobilurin fungicides at time t, C0 is the concentration (μg·mL−1) of strobilurin fungicides at time zero, and k is the degradation rate constant (d−1).The first-order kinetic model (Equation(3)) was created to elucidate fungicides degradation efficiency of strain SH14 .(3)Ct=Ct1/2) of different strobilurin fungicides.k is degradation rate constant (d−1).Equation (4) was followed to calculate the theoretical half-life  and a PAD detector at a column temperature of 28 ± 1 °C. Then, 10 μL of each sample was injected and azoxystrobin concentration was determined at 230 nm wavelengths. A mobile phase of 70:30 acetonitrile/water (v/v) was used at a flow rate of 1.0 mL·min−1 [Azoxystrobin concentration was monitored by HPLC equipped with a CmL·min−1 .−1. Temperatures corresponding to the transfer line and ion trap were 280 °C and 230 °C, respectively, at ionization energy of 70 eV. The column was initially held at 200 °C for 3 min and then raised at 25 °C·min−1 to 280 °C for 20 min.To identify the azoxystrobin degradation metabolites, extracts were analyzed by GC-MS on a DB-5MS capillary column (30.0 m × 250 μm × 0.25 μm) with an Agilent 6890N/5975 GC-MS system equipped with an auto-sampler and on-column, split/splitless capillary injection system, and an array detection from 40–430 nm . Operating conditions were as follows: injection volume was 1.0 μL with splitless sampling at 260 °C and helium (>99.999% purity) was used as a carrier gas at a flow rate of 1.0 mL∙minAzoxystrobin-degrading bacterial strain SH14 was isolated from the activated sludge that utilized azoxystrobin as a sole source of carbon for growth in MSM. Strain SH14 is an obligate aerobic Gram-negative bacterium that exhibited significantly high azoxystrobin degrading efficiency of 86.3% after 5 days of inoculation. Colonies grown on LB agar plates appeared white, round, convex and circular with a smooth margin. It was positive to glucose, arabinose, maltose, citric acid, and oxidase tests whereas negative in gelatin liquefaction, urea and esculin. Ochrobactrum anthropi and closely clustered with O. anthropi CCUG 34,735 (GenBank accession number AM114407) showing over 99% similarity (O. anthropi with 99.9% identification. This isolated strain was deposited in the China Center for Type Culture Collection (collection number: CCTCC M 2013681).Based on the phylogenetic analysis of 16S rDNA gene sequences, strain SH14 was tentatively identified as milarity . API 20 O. anthropi strain SH14 was isolated from azoxystrobin-contaminated soils by enrichment culture technique and it exhibited superb degradation efficiency against azoxystrobin and other strobilurin fungicides.Previous research demonstrated that the bacterial strains from genus Ochrobactrum are metabolically active microbes, and they are able to degrade and metabolize various xenobiotics [Ochrobactrum genus of bacteria.Large-scale applications of azoxystrobin cause several adverse effects on the ecosystem and human health ,49,50. Mobiotics ,26,27,28The dynamic relationship between the growth of strain SH14 in MSM with azoxystrobin as a sole carbon source and its degradation efficiency is shown in X1), media pH (X2), and inoculum (X3) were designed as three critical independent variables for the enhancement of azoxystrobin biodegradation conditions. Data about the residual amount of azoxystrobin (Y1) represent the combined effect of these three factors at various levels. The CCRD matrix and response of the dependent variable for azoxystrobin degradation by strain SH14 are presented in X1 × 3 and X2 × 3 combinations).Y1 is the predicted response, X1 is the temperature,X2 is the media pH and X3 is inoculum.Box-Behnken design based response surface methodology (RSM) was used to optimize biodegradation conditions for strain SH14. Temperature (R2) value 0.9806 indicates that there is a perfect agreement between predicted and experimental values. Azoxystrobin degradation model reliably represented the relationship between response and independent variables at high significance (p < 0.01).Analysis of variance (ANOVA) of the fitted quadratic polynomial model for azoxystrobin degradation by strain SH14 is listed in X1), pH (X2), and inocula(X3). As shown in X1), media pH (X2), and inocula(X3) were significant (p < 0.05). Three-dimensional response surface plot  were determined as temperature 30.2 °C, pH 7.9, and 1.1 × 107 CFU·mL−1 of inoculum, respectively.Single-factor experiments were conducted to determine the effects of important variables such as temperature  to explore azoxystrobin degradation kinetics. As shown in −1 at degradation of 62.4%. However, some previous studies had reported complete repression of pesticide-degraders at high concentrations [−1, degradation reached up to 89.1%, 86.3%, 78.5%, and 71.7%, respectively within 5 days. q) at different initial azoxystrobin concentrations.Strain SH14 was inoculated in the media containing different initial azoxystrobin concentrations ) were determined as 0.6122 d−1, 6.8291 μg·mL−1, and 188.4680 μg·mL−1, respectively. The determination coefficient (R2) of 0.9657 indicated a clear correlation among experimental values and the Andrews equation. Furthermore, a critical inhibitor concentration of azoxystrobin was found as 35.8757 μg·mL−1. At azoxystrobin’s initial concentration of lower than 35.8757 μg·mL−1, the q value was increased rapidly (−1), inhibition of degradation became prominent. Our results indicated that strain SH14 possesses high azoxystrobin degrading efficiency within the initial concentration of 400 μg·mL−1 (q > 0.2 d−1), highlighting its promising potentials as an ideal microorganism to be employed for bioremediation of variable environments.Kinetics parameters  rapidly b. At a hm/z). In the sample of 12 h, a significant peak of compound A was detected at an RT of 22.869 min. The protonated molecular ion of this compound at m/z 403 exactly matched with the authentic standard of azoxystrobin in the NIST library database -acetamide, 2-amino-4-(4-chlorophenyl)-3-cyano-5,6-dimethyl-pyridine and 3-quinolinecarboxylic acid,6,8-difluoro-4-hydroxy-ethyl ester  library database. database . Furtheryl ester . At the yl ester . HydrolyO. anthropi SH14 further degraded azoxystrobin by cleavage of the aromatic ring, leading to complete metabolism and detoxification of azoxystrobin.The complex structure of azoxystrobin provides several sites for metabolic reactions by microorganisms . It was The degradation rate of various strobilurins by strain SH14 was detected and explained in R2 varied from 0.9419 to 0.9902 indicating that the degradation data reliably fitted with the first-order kinetic model. As shown in k) varied from 0.1857 to 0.5161 d−1 that characterized the degradation process of various strobilurin fungicides by strain SH14. Theoretical half-life (t1/2) of azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, picoxystrobin, and fluoxastrobin was noted as 1.52, 1.34, 1.35, 1.80, 2.06, and 3.73 days, respectively. The t1/2 for strobilurin fungicides in field conditions often ranges from3.31 to 279 days [t1/2 of various strobilurin fungicides as compared to under field conditions.The first-order kinetic model (Equation(3)) was created to elucidate the degradation ability of strain SH14 against various strobilurinfungicides. 279 days ,72,73. SR2 varied from 0.9447 to 0.9892 indicating that the experimental data were well fitted with the first-order model. k characterized the degradation process and ranged between 0.1857 to 0.5161 d−1. t1/2 of azoxystrobin was calculated as 108.3 and 75.3 d in sterile and non-sterile soils, which drastically reduced to 12.6 and 9.7 days, respectively with the application of strain SH14. These results suggest that strain SH14 is quite potent for the bioremediation of azoxystrobin-contaminated environments.To investigate the bioremediation potential of strain SH14 in azoxystrobin-contaminated soils, tests were conducted under controlled conditions. The degradation process was further studied with first-order kinetic model and kinetic parameters are tabulated in O. anthropi is a widespread bacterium in natural habitats and has broad catabolic abilities, and it is capable of degrading various xenobiotic compounds including chlorate, cyhalothrin, cypermethrin, etc. [O. anthropi has been described as an opportunistic pathogen that may cause infections in immunocompromised persions [Previously, low activity of bacterial isolates under field conditions restricted its field applications ,76,77,78in, etc. ,84,85. Hpersions . Thus, fO. anthropi SH14 having superior azoxystrobin degradation activity was characterized. Strain SH14 was capable of rapidly degrading azoxystrobin without a lag phase over a broad range of temperature (22 to 38 °C) and pH (5.0 to 10.0). This feature gives the pesticide degrader a competitive advantage in variable environments. It is noteworthy that strain SH14 tolerated and degraded azoxystrobin up to a concentration of 400 mg·L−1, thus giving it an exceptional ability to colonize ecological niches where pesticide concentration is high. Strain SH14 transformed azoxystrobin by hydrolysis of the ester linkage and cleavage of the aromatic ring and to yield three intermediates, without any persistent accumulative product, suggesting that this particular strain may harbor a complete metabolic pathway for degradation and detoxification of azoxystrobin. This is the first report of a pathway of degradation of azoxystrobinin a microorganism, which is of vital importance in azoxystrobin biogeocycle. Another important feature which is worth mentioning is that strain SH14 significantly enhanced the removal rate of azoxystrobin in various soils, suggesting a high potentialof applying O. anthropi SH14 for bioremediation of an azoxystrobin-contaminated environment. However, further in-depth studies based on genetics and molecular biology of strain SH14 are needed to develop a more safe and efficient strategy to clean up azoxystrobin contamination.In this study, a newly isolated bacterial strain


Rhizobiales declined under CF. Furthermore, the results of partial least square path modeling revealed that soil available phosphorus (AP) negatively affected bacterial diversity while it positively affected bacterial community structure in fluvo-aquic soils. In contrast, soil pH was positively linked with both bacterial diversity and community structure in black soil. Overall, our study demonstrated that RF is an environmentally friendly approach which not only maintained above ground plant productivity, but also preserved belowground microbial populations and important soil variables regulating bacterial communities varied in different soil types.Shifts in soil bacterial diversity and community composition are suggested to be induced by elevated input of nitrogen (N) fertilization with implications for soil quality, and consequently production. In this study, we evaluated the impacts of recommended fertilization (RF) and conventional fertilization (CF) on soil chemical properties, crop yield, bacterial diversity, and community composition from two long-term experiments conducted in fluvo-aquic soil and black soil of China. Each site comprised of four treatments, i.e., RF N−, RF N+, CF N−, CF N+. No N fertilization was indicated by N− and N fertilization was indicated by N+. Across both sites, N fertilization significantly increased crop yield compared with no N fertilization and RF successfully enhanced crop yield over CF. Interestingly, the RF maintained bacterial diversity, while CF depressed bacterial diversity in the two soils. Microbial taxa performing important ecological roles such as order Rhodospirillales and Bacillales were significantly enhanced in the RF approach, while  Mineral fertilizers, especially nitrogen (N), constitute a large contributor to global reactive N input, which was 86 Tg N in 1995 and was predicted to hit 135 Tg N by 2050 , and chrIt is well established that changes in soil properties followed by N fertilization such as, soil acidification and nutrient availability can cause ambient shifts in microbial diversity and community composition . HoweverWith a goal of higher yields, Chinese farmers tend to apply high quantities of fertilizers, which not only degrade soil quality but also induced shifts in soil microbial communities . To presThe long-term field trial was established in 2009 at the Dahe Experimental Station located in Shijiazhuang City, Hebei Province (38°07ʹ N and 114°29ʹ E) north central China. That region has fluvo-aquic soil . The study area has typical warm temperate and sub-humid continental monsoon climate. The average annual temperature is 14.3 °C and precipitation is 400 mm. Another field trial was established in 2009 in Liufangzi County, Gongzhuling City, Jilin Province (43°34.86ʹ N and 124°53.92ʹ E) northeastern China. This study area has black soils and is called as  and as a Mollisol in the USDA classification. This site has a temperate and semi-humid continental monsoon climate. The average annual temperature is 4–5 °C. The mean annual precipitation is 500–900 mm. This study area has black soils and is called as .2 (5 × 9 m) and 40 m2 (10 × 4 m) in fluvo-aquic soil and black soil site, respectively. There were four treatments in fluvo-aquic soil: RF N0, RF N182, CF N0, CF N225 kg ha−1. Similarly, four treatments were conducted in black soils, namely RF N0, RF N180, CF N0, and CF N251 kg ha−1  and ammonium-nitrogen (NH4+ N) were extracted from a 12 g fresh soil sample using a 1:10 ratio of soil to 0.01 mol L−1 CaCl2 solution and then analyzed using continuous flow analysis (Foss FlAstar 5000). The determination of soil water content was conducted by oven drying soil at 105 °C. Soil total nitrogen (TN) and soil organic carbon (SOC) was analyzed by Kjeldahl digestion [2SO4 and determined by a total organic C/N analyzer  [Soil samples were collected in September 2016 from both locations at maize harvest. Generally, five soil cores (2 cm in diameter) were collected from each experimental plot to a depth of 0–20 cm and pooled to create a composite sample. One part of the subsample was held at −80 °C for further molecular analysis. The nitrate-nitrogen (NOigestion  and dichigestion . The Olsigestion . The soiGermany) . In late® Bacterial DNA Kit  according to the manufacturer’s instructions. DNA quality was tested by 1% agarose gel electrophoresis and spectrophotometry . 16S rRNA gene V3-V4 hypervariable regions were sequenced with Illumina MiSeq PE300 sequencing . The pair of universal primers 338F (5′ ACTCCTACGGGAGGCAGCAG-3) and 806R (5′ GACTACHVGGGTWTCTAAT-3′) was used to amplify V3-V4 regions of 16S rRNA. Conditions for the PCR amplification were as follows: 95 °C for 5 min, and 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and at 72 °C for 30 s with a final extension of 72 °C for 10 min. PCR was conducted in triplicate in a 25 μL mixture containing 2.5 μL of 10 × Pyrobest buffer, 2 μL of 2.5 mM dNTPs, 1 μL of each primer (10 μM), 0.4 U of Pyrobest DNA polymerase (TaKaRa), and 15 ng of template DNA. Amplicon mixtures were transfered to the MiSeq Genome Sequencer .Genomic DNA was extracted from 0.5 g soil samples using the E.Z.N.A. Amplicons were extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions and quantified using QuantiFluor™ -ST . We pooled purified amplicons in equimolar and paired-end sequences (2 × 300) on an Illumina MiSeq platform according to the standard protocols. High-quality sequence extraction was conducted with the Quantitative Insights into Microbial Ecology (QIIME) (v1.2.1) package. The raw sequences were selected, and the sequences with low-quality were eliminated (i) if raw reads were shorter than 110 nucleotides; (ii) if 300 bp reads were truncated at any site receiving an average quality score <20 over a 50 bp sliding window, discarding the truncated reads that were shorter than 50 bp; or (iii) if exact barcode matching or a two nucleotide mismatch in primer matching were obtained and reads contained ambiguous characters; only sequences that overlap more than 10 bp were assembled according to their overlap sequence. Reads that could not be assembled were discarded. The unique sequence set was classified into operational taxonomic units (OTUs) under the threshold of 97% identity using UCLUST. Chimeric sequences were identified and removed using USEARCH (version 8.0.1623). The taxonomy of each 16S rRNA gene sequence was analyzed by UCLUST against the Silva119 16S rRNA database using a confidence threshold of 90%.p ≤ 0.05) to compare the treatment means. Two-way ANOVA was used to evaluate the effect of treatments and soil types on soil biochemical traits, maize yield, and alpha diversity index. To identify taxa which were significantly affected by RF and CF, we carried out the linear discriminant analysis (LDA) effect size (LEfSe) algorithm [2) and the estimate of path coefficient in our model were validated using “plspm” package (1000 bootstraps) in R software version 3.4.4. Nonmetric multidimensional scaling (NMDS) based on Bray–Curtis dissimilarities was employed to identify shifts in soil bacterial community structures. For each variable measured  the data were analyzed by one-way analysis of variance (ANOVA) using Fisher’s least significant difference (LSD) at  significantly reduced the soil pH and lowest pH was found in CF N225 treatment (p ≤ 0.05), and highest TN was recorded in RF N182 treatment. Nitrate (NO3− N) levels significantly increased as a result of the N addition (p ≤ 0.01), with the highest values observed in CF N225. By contrast, N fertilization (p ≤ 0.05) significantly decreased AP, and the lowest AP was found in RF N182. N fertilization did not significantly influence NH4+ N, SOC, C:N ratio, MBC, and MBN stocks. In black soil, soil pH significantly declined as a result of the N addition  and highest value was found in the CF N251. Furthermore, NO3− N levels increased as a result of the N fertilization (p ≤ 0.01), and highest level was found in the RF N180 treatment. Ammonium levels increased as a result of N fertilization (p ≤ 0.01). MBC was declined as a result of N fertilization (p ≤ 0.05) and lowest MBC was recorded in the CF N251 treatment. Both the MBN and C:N ratio declined as a result of N fertilization , with the lowest values observed in CF N251 treatment. N fertilization did not significantly impacted SOC stocks. The maize yield was evaluated . In black soil, the N Fertilization altered (p ≤ 0.01) bacterial richness and taxonomic diversity. Interestingly, at both locations, in the RF treatments, bacterial diversity was maintained, but in CF treatments, there was a gradual decline in bacterial diversity and richness that paralleled N addition, and lowest values were found in the CF N225 and CF N251 treatments respectively. The effect of N fertilization, soil type, and their interaction with the bacterial alpha diversity index is shown in the In the present study, the Shannon index  and ChaoTo determine whether the management system and N fertilization led to shifts in soil bacterial communities, we profiled the structural changes in bacterial communities using NMDS based on Bray-Curtis dissimilarities. It was evident from the NMDS ordinations that there was clear separation among bacterial communities associated with recommended and conventional N fertilizer rates in the two soils . BacteriWe further identified high-dimensional biomarkers at the order level using LEfSe, a robust algorithm that recognizes features among biological groups that are significantly different , and explored which taxa were significantly altered under RF and CF treatments. In fluvo-aquic soil, Rhodospirillales (LDA score = 3.83) and Bacillales (LDA score = 3.80) were significantly altered taxa in RF N182 treatment A. Conver3− N, had significant positive effects on maize yield (0.63)  and community structure (46%) , whereasd (0.63) A. Microbd (0.63) . AP had d (0.63) B. Soil p+ to the soil. Additionally, H+ is liberated during the oxidation of NH4+ N to NO3− N [The recommended and conventional fertilization significantly altered the physiochemical properties of fluvo-aquic and black soils . Earliero NO3− N . Soil aco NO3− N . Differeo NO3− N . For exao NO3− N . The AP o NO3− N .3− N, TN, MBC, and MBN levels were the key drivers regulating maize grain yield either interactively or individually, through mediating soil bacteria . These processes are extremely important for proper ecosystem functioning because N availability has a profound role in plant productivity . Furthersubgroup_6 and Acidobacteriales were affiliated to plots with higher soil pH  B. Previominished . Taken tTo safeguard the planet earth from adverse effects of over-fertilization at the same time as producing a higher yield to feed the mouths of the ever-increasing population, it is recommended fertilization could be a potential alternative farming system. Our results demonstrated that conventional fertilization, which is characterized by elevated N input, not only deteriorated soil quality by soil acidification, but also depressed bacterial diversity. It is well recognized that bacterial diversity is imperative for ecosystem functions and stability. Therefore, if N enrichment consistently increased in the future, then the reduction in bacterial diversity would worsen, which will eventually hamper ecosystem functions. Conversely, recommended fertilization successfully maintained bacterial diversity. Recommended fertilization also improved beneficial bacterial taxa which perform a remarkable role in ecosystem functions. Meanwhile, our results revealed that AP and pH are determining soil factors that couple with bacterial diversity and bacterial community structure in fluvo-aquic and black soil, respectively. Our study provides novel information pertaining to how belowground microbial communities respond to recommended and conventional fertilization regimes. Furthermore, in comparison with community-level changes in soil bacteria, the important high-magnitude biomarkers differentiating bacterial communities under recommended and conventional fertilization regimes require further detailed examination in future studies for sustainable crop production.


Zobellia galactanivorans (ZgHGPRT/AMPK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMPK. Biochemical characterization of the recombinant protein indicates that the enzyme is a homodimer, with high activity in the pH range 6-7 and in a temperature interval from 30 to 80°C. Thermal denaturation experiments revealed that ZgHGPRT/AMPK exhibits an apparent unfolding temperature (Tm) of 45°C and a retained activity of around 80% when incubated at 40°C for 240 min. This bifunctional enzyme shows a dependence on divalent cations, with a remarkable preference for Mg2+ and Co2+ as cofactors. More interestingly, substrate specificity studies revealed ZgHGPRT/AMPK as a bifunctional enzyme, which acts as phosphoribosyltransferase or adenylate kinase depending upon the nature of the substrate. Finally, to assess the potential of ZgHGPRT/AMPK as biocatalyst for the synthesis of nucleoside-5′-mono, di- and triphosphates, the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase) and the effect of water-miscible solvents on enzyme activity were studied.In our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from  In the de novo pathway, purine nucleotides are synthesized from simple precursors like glycine, glutamine, or aspartate. In contrast, salvage pathway employs purine nucleobases to generate the corresponding nucleoside-5′-monophosphates (NMPs). This requirement for purines is satisfied by means of different endogenous and/or exogenous sources of preformed nitrogen bases  synthesis. IMP is converted to guanosine-5′-monophosphate (GMP) and adenosine-5′-monophosphate (AMP), which are subsequently phosphorylated to get guanosine-5′-triphosphate (GTP) and adenosine-5′-triphosphate (ATP), respectively.Purine nucleotides are involved in multitude of biochemical processes, but they are also particularly important as building blocks for RNA and DNA synthesis. Biosynthesis of purine nucleotides is performed through two different metabolic routes, en bases . Both meBiocatalysis aims to reproduce, implement and expand nature’s synthetic strategies to perform the synthesis of different organic compounds using whole cells or enzymes.3, or phosphorus pentoxide, P2O5), acidic conditions, and organic solvents, which are expensive and environmentally harmful  are essential enzymes in the purine salvage pathway . 6-oxo P2+ .2+  belongs to nucleoside-5′-monophosphate kinase (NMPK) family. AMPK catalyzes the reversible transfer of the terminal phosphate group between ATP and AMP to give 2 molecules of ADP in presence of Mg2+ . AMPK is and ATP .Zobellia galactanivorans which contains both HGPRT  and AMPK  domains (ZgHGPRT/AMPK) was shown as a homodimer, active in the pH interval 6-7 and in a broad temperature range (30–80°C). Moreover, ZgHGPRT/AMPK displays an apparent unfolding temperature (Tm) of 45°C. Finally, to assess the potential of ZgHGPRT/AMPK the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase) and the effect of divalent cations and water-miscible solvents on enzyme activity were assayed.Herein we report, for the first time, a bifunctional protein from  domains . In the Cell culture medium reagents were purchased from Difco . Triethyl ammonium acetate buffer was provided by Sigma-Aldrich . All other reagents and organic solvents were purchased from Symta . Nucleosides, nucleotides and nucleobases were provided by Carbosynth Ltd. .in silico mining of phosphoribosyltransferase (PRT) candidates, we found a gene which encodes a putative bifunctional protein HGPRT/AMPK in Zobellia galactanivorans genome . The corresponding hgprt/ampk gene was provided by GenScript (United States) as a NheI-BamHI fragment subcloned into the expression vector pET28b (+). The recombinant vector pET28bZgHGPRT/AMPK encodes an N-terminal His6-tagged fusion with a thrombin cleavage site between the tag and the enzyme. ZgHGPRT/AMPK was expressed in Escherichia coli BL21(DE3) grown at 37°C in Luria Bertani medium supplemented with kanamycin (50 μg/mL). Protein overexpression was induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to exponential cultures and the cells were further grown for 4 h. Then, cells were harvested by centrifugation at 3,800 × g and the resulting pellet was resuspended in 10 mM sodium phosphate buffer pH 7. Crude extracts were prepared by cellular disruption of cell suspensions using a digital sonicator. The lysates were centrifuged at 16,500 × g for 30 min at 4°C. The cleared lysates were loaded onto a 5-mL HisTrap FF column , pre-equilibrated in a binding buffer . Bound proteins were eluted using a linear gradient of imidazole (from 20 to 500 mM). Fractions containing ZgHGPRT/AMPK were identified by SDS-PAGE, pooled, concentrated and loaded onto a HiLoad 16/60 Superdex 200 prep grade column  pre-equilibrated in 20 mM sodium phosphate, pH 7. Fractions with the protein of interest were identified by SDS-PAGE. Protein concentration was determined spectrophotometrically by UV absorption 280 nm using a ε280 = 22,350 M–1cm–1.After an ZgHGPRT/AMPK were carried out in 20 mM Tris–HCl  using an Optima XL-I analytical ultracentrifuge (Beckman-Coulter Inc.) equipped with UV-VIS absorbance and Raleigh interference detection systems, an An-60Ti rotor and standard  double-sector center pieces of Epon-charcoal. Sedimentation profiles were recorded at 292 nm. Sedimentation coefficient distributions were calculated by least-squares boundary modeling of sedimentation velocity using the continuous distribution c(s) Lamm equation model as implemented by SEDFIT 14.7 g.Sedimentation velocity experiments for g. The apparent sedimentation coefficient distribution, c(s), and sedimentation coefficients were calculated from the sedimentation velocity data using SEDFIT  (Mw) were determined from the buoyant masses, considering the partial specific volumes of the protein (0.4 mg/ml) obtained from the amino acid composition using the program SEDNTERP  . The corSEDNTERP .2 in 50 mM Tris–HCl pH 8 at 50°C and 300 r.p.m. for 5–10 min After this, the enzyme was inactivated as previously described , sodium phosphate (pH 6–8.5), MES (pH 5.5–7), Tris–HCl (pH 7–9) and sodium borate (pH 8–11) as reaction buffers (50 mM). The optimum temperature was determined using the standard assay over a 20–80°C range.ZgHGPRT/AMPK activity, different divalent salts  were added to the reaction mixture at different concentrations (2–20 mM). The reaction was performed using the standard phosphoribosyltransferase activity assay under the optimal pH and temperature conditions previously determined. In this respect, 6.5 μg of purified enzyme were incubated in a 40 μL solution containing with 2 mM PRPP, 2 mM Hyp and 2–20 mM divalent salts, in 50 mM sodium phosphate buffer pH 7 at 50°C and 300 r.p.m., 5–10 min.To determine the effect of divalent cations on 2 in 50 mM sodium phosphate buffer pH 7, in the presence of 20% (v/v) water-miscible organic solvents. The reaction mixture was incubated at 50°C for 5–10 min (300 r.p.m.).To determine the effect of organic solvents on enzymatic activity, the phosphoribosyltransferase activity was assayed in the presence of different protic and aprotic organic solvents. To this end, 6.5 μg of purified enzyme were added to a 40 μL solution containing 2 mM PRPP, 2 mM Hyp, 2.4 mM MgClZgHGPRT/AMPK was stored at 4 and −80°C in 20 mM sodium phosphate buffer, pH 7 for 365 days. Periodically, samples were taken and the enzymatic activity was evaluated. Storage stability was defined as the relative activity between the first and the successive reactions. Moreover, thermal stability was studied by incubating 6.5 μg of purified enzyme in 20 mM sodium phosphate buffer, pH 7, for 240 min at different temperatures (40–60°C).Tm) was measured using differential scanning fluorimetry in a Rotor GeneTM 6000 (Corbett Life Sciences) essentially as previously described . The samples were heated over 35–95°C range at 1°C/min. The fluorescent signal was measured with excitation and emission filters of 460 and 510 nm, respectively. The Tm was approximated to the melt peak obtained from a graph plotting the negative first derivative of the melting curve with respect to the temperature (dRFU/dT vs. T), where RFU stands for relative fluorescence units.The melting temperature  was assayed to evaluate adenylate kinase activity. To this end, 13 μg of purified enzyme were added to a 40 μL solution containing 3.2 mM ATP and 3.2 mM AMP (or 6.4 mM ADP), 12 mM MgClKM, kcat, and cat/KMk, for both HGPRTase and AMPKase activities were determined. The kinetic analysis of HGPRT was performed at varying concentrations of one substrate , fixing the concentration of the other substrate at constant saturating level (5 mM). An identical approach was followed for the kinetic analysis of AMPKase activity  using 3.2 mM as constant saturation concentration. Apparent KM, kcat, and cat/KMk, were determined by non-linear regression assuming Michaelis–Menten kinetics. Calculations were carried out using the GraphPad Prism 8  and AMPK structure from Geobacillus stearothermophilus (PDB id 1ZIN) were selected as templates. Then, a complete ZgHGPRT/AMPK 3D model was built through assembling the best homology models for both, HGPRT and AMPK domains, using replica-exchange Monte Carlo simulations integrated in Domain Enhanced Modeling server  implemented in Domain linker pRediction using OPtimal features server (DROP) . Flexibi AMBER16 . Finallyj module  in AMBERThe production of nucleotides was measured quantitatively using an ACE EXCEL (5 μm CN-ES 250 × 4.6 mm) equilibrated with 100% triethyl ammonium acetate at a flow rate of 0.8 mL/min. Retention times for the reference natural compounds  were as follows: adenine (Ade), 10.4 min; adenosine-5′-monophosphate (AMP), 5.5 min; adenosine-5′-diphosphate (ADP), 4.4 min; adenosine-5′-triphosphate (ATP), 3.8 min; guanine (Gua), 4.8 min; guanosine-5′-monophosphate (GMP), 3.2 min; hypoxanthine (Hyp), 4.5 min; inosine-5′- monophosphate (IMP), 3.0 min; xanthine (Xan), 4.4 min; cytosine (Cyt), 3.9 min; uracil (Ura), 4.1 min; thymine (Thy), 7.2 min; thymidine-5′-monophosphate (TMP), 4.4 min.Zobellia galactanivorans has been analyzed and published online (NC_015844.1). After an in silico mining, we discovered an ORF that potentially encodes a putative HGPRT/AMPK bifunctional protein annotated throughout the genome. To confirm this, a bioinformatic analysis was performed. The pairwise amino acid sequence alignment using BLASTP1 against Protein Data Bank revealed the presence of both canonical, PRT and adenylate kinase domains, encoded in the amino acid sequence  in their amino acid sequence , as well as in the homology model. Finally, the well-known purine pocket of purine PRTs was also found in ZgHGPRT/AMPK amino acid sequence.According to the multiple sequence alignment of amino acid sequences of closest PRT homologs, sequence . Type I sequence . Some ofsequence . As showZgHGPRT/AMPK amino acid sequence  thesequence . Moreovesequence . Like otlization . As infelization .To form the complete monomer of the protein, both HGPRT and AMPK domains are connected by an interdomain linker consisting of 10 amino acids (REVYQLNQKH). This sequence is characterized by charged and polar uncharged amino acids, similar to other linkers from multidomain proteins . MoreoveZobellia galactanivorans genome (NCBI reference sequence NC_015844.1) displayed the presence of a putative ZgHGPRT/AMPK. The putative hgprt/ampk gene from Zobellia galactanivorans was cloned and overexpressed in E. coli BL21(DE3) as described above. The recombinant N-terminal His6-tagged ZgHGPRT/AMPK was purified by an affinity and an exclusion size chromatographic procedure. SDS-PAGE analysis of the purified enzyme shows only one protein band with an apparent molecular mass around 42 kDa  (97%) and 7.10 S  (3%). The major species found in solution corresponds to a dimer state (Mw = 83.33 kDa) and is compatible with a monomer subunit of 41.65 kDa, a molecular mass similar to that calculated from the amino acid sequence of the His6-tagged protein (43.77 kDa). Since ZgHGPRT/AMPK is the first dual domain PRT/AMPK protein, there are not previous examples to compare it with. However, different oligomeric states have been described for 6-oxopurine PRTs from several sources, such as dimeric 6-oxo PRTs from Sulfolobus solfataricus (SsHGXPRT) and GPRT from Giardia lamblia (GlGPRT), tetrameric 6-oxo PRTs from Thermus thermophilus (TtHGXPRT and TtXPRT), Toxoplasma gondii (TgHGPRT) and E. coli (EcXGPRT and EcHPRT), or the hexameric HGPRT from Pyrococcus horikoshii (PfHGXPRT)  . In thisArchaea, and monomeric class more common in Eubacteria  across a broad temperature range (from 30 to 80°C), with a maximum at 50–60°C, which is higher than those reported for other mesophilic HGPRTs, such as HGXPRT from Plasmodium falciparum (T = 22°C) (T = 22°C) (Trypanosoma cruzi (T = 37°C) (The effect of temperature and pH on  = 22°C) , human H = 22°C)  or HPRT  = 37°C) .ZgHGPRT/AMPK displays high activity in a narrow pH range 6–7, with a maximum peak when it was incubated in 50 mM sodium phosphate pH 7  was observed when stored at −80°C. Furthermore, the effect of temperature on enzyme stability was evaluated by incubating ZgHGPRT/AMPK for 4 h in 20 mM sodium phosphate pH 7, in the temperature range 40–60°C (ZgHGPRT/AMPK suffered a high loss of activity when incubated at 50 and 60°C in a brief period of time (relative activity <70% for incubation periods longer than 10 min). In contrast, ZgHGPRT/AMPK displayed a high relative activity (around 80%) when stored at 40°C for 240 min. These results agree with thermal denaturation curves, which allowed us to estimate a moderate melting temperature (Tm) of 45.25°C  and temperature (50°C) were stablished, the effect of divalent cations on enzyme activity was assayed . As desc2+ 2 mM. Since high activity values were also observed when using Mn2+, it might act as an effective substitute for Mg2+, as also described for other HGPRTs such as those from E. coli, Salmonella typhimurium  polar protic  and aprotic co-solvents  . As showlog P > 0), such as ethyl acetate  or chloroform , whereas a significant activity decrease is observed for hydrophilic solvents , such as acetone , acetonitrile , DMF , DMSO  (ε) decreases, displaying the highest activity values for ethyl acetate (ε = 6.0) and chloroform (ε = 4.8), and the lowest values for DMF (ε = 38) and acetone (ε = 21) (A correlation between hydrophobicity and activity  was obse = −1.3) . Moreove(ε = 21) .ε), in the following order: glycerol (ε = 47) > ethylene glycol (ε = 37) > propylene glycol (ε = 32) ≈ MeOH (ε = 32.7) > EtOH (ε = 25) > isopropanol (ε = 20)  seems to be linked to the increase of dielectric constant ((ε = 20) . HoweverZgHGPRT/AMPK for both, phosphoribosyltransferase and adenylate kinase activities. As expected for previous in silico analysis, ZgHGPRT/AMPK can perform the phosphoribosyltransferase reaction on 6-oxopurines (Hyp and Gua), while neither 6-aminopurines nor pyrimidine bases are substrates for ZgHGPRT/AMPK. In addition, no transferase reaction was observed when xanthine was used as acceptor. These results agree with previous reports for purine PRTs since, for the purine salvage, organisms usually display different PRTs, one specific for adenine and one or more responsible for the salvage of 6-oxopurines (aprt gene encoding a putative APRT (GenBank: CAZ96936.1) in Zobellia galactanivorans genome would support our findings.opurines . In thisZgHGPRT/AMPK was built   . As showZgHGPRT/AMPK also catalyzed the phosphotransfer reaction between adenine nucleotides. As shown in ZgHGPRT/AMPK is more active (up to 3 times) when transferring γ-phosphate moiety from ATP to α-phosphate of AMP, than it is for the reverse reaction. In this sense, the reaction equilibrium seems to be shifted toward ADP synthesis. Moreover, the PRTase/kinase activity ratio is around 5-6/1, which indicates a strong preference for Hyp and PRPP instead of adenine nucleotides as substrates. These results might suggest ZgHGPRT/AMPK plays a key role in salvage pathway, whereas it is not essential for maintaining the cellular homeostasis of adenine nucleotides.Furthermore, Zobellia galactanivorans genome coding other putative enzymes which could also be involved in the metabolism of adenine nucleotides, such as an APRT (GenBank: CAZ96936.1), a ribonucleotide reductase (GenBank: CAZ95963.1), a nucleoside diphosphate kinase (GenBank: CAZ97836.1) and adenylate cyclase (GenBank: CAZ95571.1), would reinforce this hypothesis.The presence of the genes in M, kcatK, and cat/KMk). The results are summarized in ZgHGPRT/AMPK displays a lower KM value for Hyp than for PRPP, which agrees with kinetic data described for other type I PRTs . ZgHGPRT/AMPK is the only two in one HGPRT/AMPK described up to date. Experimental results revealed ZgHGPRT/AMPK as a homodimer, active in the pH range 6–7 and in a temperature interval from 30 to 80°C, and also displaying a moderate thermal stability. More interestingly, substrate specificity studies revealed ZgHGPRT/AMPK as a bifunctional enzyme, which acts as phosphoribosyltransferase or adenylate kinase depending upon the nature of substrates. Finally, the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase), as well as the effect of divalent cations and water-miscible solvents on enzyme activity, were described for a suitable application as biocatalyst for the synthesis of nucleoside-5′-mono, -di and triphosphates.Herein we report a novel bifunctional hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from All datasets presented in this study are included in the article/supplementary material.JF-L and JA are mainly responsible for the experiment design and for coordinating the research. JA, AH, JB, BH-T, VC-S, and MD contributed to the development and analysis of experimental data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.