Some yet unidentified factors released by both oocyte and embryonic microenvironments demonstrated to be non-permissive for tumor development and display the remarkable ability to foster cell/tissue reprogramming, thus ultimately reversing the malignant phenotype. In the present study we observed how molecular factors extracted from Zebrafish embryos during specific developmental phases (20 somites) significantly antagonize proliferation of breast cancer cells, while reversing a number of prominent aspects of malignancy. Embryo extracts reduce cell proliferation, enhance apoptosis, and dramatically inhibit both invasiveness and migrating capabilities of cancer cells. Counteracting the invasive phenotype is a relevant issue in controlling tumor spreading and metastasis. Moreover, such effect is not limited to cancerous cells as embryo extracts were also effective in inhibiting migration and invasiveness displayed by normal breast cells undergoing epithelial–mesenchymal transition upon TGF-β1 stimulation. The reversion program involves the modulation of E-cadherin/β-catenin pathway, cytoskeleton remodeling with dramatic reduction in vinculin, as well as downregulation of TCTP and the concomitant increase in p53 levels. Our findings highlight that—contrary to the prevailing current “dogma”, which posits that neoplastic cells are irreversibly “committed”—the malignant phenotype can ultimately be “reversed”, at least partially, in response to environmental morphogenetic influences. Some yet unidentified factors released by both oocyte and embryonic microenvironments demonstrated to be non-permissive for tumor development and display the remarkable ability to foster cell/tissue reprogramming, thus ultimately reversing the malignant phenotype ,2.Danio rerio), chick, and amphibian embryo/eggs extracts were used to give an insight into the molecular reprogramming of cancer cells in response to the embryonic environment [Pioneer studies from the sixties showed that carcinoma cells are “reprogrammed” when injected into a mouse blastocyst, ultimately resulting in normal tissue originating from cancer cells . Since tironment .This process may entail proliferation and apoptosis rate , as wellThese studies documented that the reversion program is a complex attempt, running through discrete steps in which differentiation that lead a stem cell toward a complete phenotypic commitment is recapitulated according to an ”inverse process” . This reThe fact that the observed reversal of malignancy in cancer cells frequently involves only some features of the malignant phenotype, evidences how limited is our knowledge of the molecular and biophysical mechanisms that orchestrated the reversion.Namely, only few factors extracted from embryo kept during a well-defined period of development share the capability to trigger tumor reversion . For insIndeed, the release of morphogenetic factors significantly differs according to the different stages of embryonic development  and we cWe have already shown that stage developmental factors extracted from embryos of Zebrafish can efficiently arrest cancer proliferation and induce apoptosis, both in vitro  and in vMature oocytes and undifferentiated embryonic stem (ES) cells contain reprogramming factors  that enable these cells to reprogram a somatic nucleus to pluripotency . HoweverOver the last decades, Zebrafish has proven to be a powerful model in cancer research. Indeed, Zebrafish displays more than 80% of all human disease-related genes, indicating that many human diseases can, in fact, be modeled in Zebrafish. Namely, an impressive body of studies demonstrated that Zebrafish can serve as a useful model to investigate tumor driving as well as anti-tumor mechanisms . MoreoveHerewith we seek to determine the stages of Zebrafish embryo development that display the most significant activity in inducing the reversal of prominent malignant features, like proliferation, migrating capacity, and invasiveness.We performed a preliminary screening to identify the most effective embryo fraction (EF). Biological activity was ascertained by considering as prominent parameter the reduction in cell viability, assessed with the Sulforhodamine B (SRB) colorimetric assay. Two concentrations (0.3 and 3.0 μg/mL) of each EF were tested in breast cancer cell lines—MCF7 and MDA-MB-231—at different time points . This preliminary survey identified F6 (corresponding to the 20-somite stage) to be the most active fraction, and significant effects were also recorded in F4 and F5, for both cell lines. No significant differences were found between 0.3 and 3 μg/mL concentrations . HereaftBesides the reduction in cell viability as assessed with the SRB test, we investigated if EFs could foster the apoptosis rate. As expected, fractions from F1 to F5 did not induce any significant increase in apoptosis either in MCF7 or MDA-MB-231 cancer cells, at any of the time points we considered (data not shown). Instead, F6 at both 0.3 and 3 μg/mL significantly raise the apoptosis rate in MDA-MB-231 cells, while in MCF7 cells only a trend toward increased apoptosis was observed, albeit not significant . Given tCell proliferation was investigated in MDA-MB-231 and MCF-7 cells at 24 h by comparing data recorded in cells treated with 5FU or F6 alone and in association. As shown in To ascertain to what extent F6 can significantly reverse the malignant phenotype, we plan to investigate some remarkable parameters belonging to the macroscopic–mesoscopic level, where microscopic elements are “channeled” and organized in a coherent manner in producing macroscopic features, as recorded by macroscopic parameters . Indeed,Changes in the migratory/invasive phenotype are indeed mirrored by cytoskeleton rearrangement under the influence of F6. While both control and 5FU-treated MDA-MB-231 cells harbored a dense texture of stress fibers, with actin filaments distributed all along the cytosol, in F6-treated cells actin was predominantly concentrated along the membrane border a. MoreovMDA-MB-231 breast cancer cells showed prominent mesenchymal features, as down-regulation of E-cadherin, while β-catenin was redistributed in the cytosol and the nucleus . ControlIn control cancer cells, overall β-catenin is lowered and mostly dispersed around in the cytoplasm, with only isolated spots at the membrane site of adhesion d. InsteaTranslationally Controlled Tumor Protein (TCTP) has emerged as a critical regulator of cell fate determination, as it regulates many different biological processes, all of which may converge to a limited set of key events that control cell fate determination and namely, tumor reversion. Aberrant expression of TCTP is frequently observed in cancer cells while silencing TCTP showed to be instrumental in promoting cancer reversion in different types of cancer cells. Namely, silencing TCTP expression in breast cancer was demonstrated to restore growth and morphological patterns reminiscent of the outgrowths generated by normal mammary epithelial cells . Down-reIn the present study, we showed that unknown molecular factors extracted from Zebrafish embryos during specific developmental phases (20 somites) significantly antagonize proliferation of breast cancer cells, while reversing some prominent aspects of the malignant phenotype. Embryo extracts reduce cell proliferation, enhance apoptosis, and dramatically inhibit both invasiveness and migrating capabilities of cancer cells. Counteracting the invasive phenotype is a relevant issue in controlling tumor spreading and metastasis. Moreover, such effect is not limited to cancerous cells as embryo extracts were also effective in inhibiting migration and invasiveness displayed by normal breast cells undergoing epithelial–mesenchymal transition upon TGF-β1 stimulation. The reversion program, as previously reported by several studies, involves downregulation of TCTP and the concomitant increase in p53 levels.TCTP is a key player in the process of tumor reversion, the process in which a tumor cell is transformed into a revertant cells by losing its malignant traits—uncontrolled growth, invasiveness, and metastasis-forming capability—through the activation of a complex cascade of biochemical events, including cytoskeleton remodeling, pathways modulation, and even gene reprogramming .TCTP knockdown in primary mammary tumor cells from ErbB2 transgenic mice resulted in increased p53 expression and fewer stem cell-like cancer cells, while in breast cancer patients a high-TCTP status is associated with aggressive tumors and predicts a poor prognosis . DownstrThe tumor reversion we observed in our model, notwithstanding how incomplete the process can be, has the merit to highlight some essential steps that are mandatory for suppressing/achieving malignancy. Indeed, tumor reprogramming proceeds along paces that, in a reverse mode, “recapitulate” carcinogenic steps, thus allowing in ascertaining critical crossroads, which still must be investigated in depth.The reversion process leads, sometimes, to a complete reversal but, frequently, reversion is only partially obtained. Indeed, the multistep nature of tumorigenesis is paralleled by the series of “uphill” steps required in order to achieve full reprogramming to pluripotency, and the requirement for different factors allows overcoming several barriers that are biologically designed to protect cells from the transformation, that is, to prevent cells from changing their identity. Similarly, during the reprogramming, several steps can be attained before a fully reprogrammed state could be achieved . MoreoveTreatment of breast cancer cells with 5FU is followed by a dramatic increase in apoptosis at 72 h, when cancer cells were almost all killed. However, 5FU exert only minimal, if any, effect on tumor reversion, especially when invasiveness and migrating capability are considered. Instead, addition of embryo extracts to 5FU-treated cells amplify the chemotherapy-induced cytostatic effect at early times and enhances the reversion of the invasive phenotype. This additive activity could be exploited in improving clinical response to conventional drugs, as previously reported in colon cancer patients treated with chemotherapy and embryo fish extracts .A major drawback of the present study is constituted by the lack of information about the identity of the “reprogramming” factors present in the pool of molecules extracted from zebrafish embryos. However, indirect evidence suggest that they could be represented by low-molecular weight components, as they are easily absorbed by oral mucosae. Studies are currently ongoing in our laboratory to ascertain the true nature of such molecular factors.The recognition of cancer as a disease of reprogramming opens the door to therapeutic strategies directed at correcting the wrong differentiation program in an attempt to eliminate the cancerous clone from the root. Differentiation therapies are already successfully in use for some very specific cases of cancer . Our finTumor cells can indeed be as amenable to reprogramming as the normal ones have shown to be. Hopefully, subsequent studies will disclose the possibility to change the natural fate of tumors and, either force them to differentiate and disappear, or convert them into cells susceptible to the newly developed targeted therapies.2 flasks . MCF-7 and MDA-MB-231 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics , MCF-10A were grown in Dulbecco’s modified Eagle’s medium/ nutrient mixture F12 Ham  supplemented with 10% horse serum  and EGF 500 µ/5 mL , Hydrocortisone (50 µM), cholera toxin (0.5 mg/mL), insulin (10 mg/mL)  and antibiotics . The cells were cultured at 37 °C in an atmosphere of 5% CO2 in air. The medium was changed every third day. At confluence, the cells were sub-cultured after removal with 0.05% trypsin–0.01% EDTA. In MCF-7 and MDA-MB-231 0.1 mg/mL 5-Fluorouracil , 0.1 mg/mL 5-FU + 0.3µg/mL F6 and 0.3µg/mL F6 were added in DMEM supplemented with 0.1% FBS. MCF-10A cells were firstly treated with 10ng/mL TGFβ1  for five days and on fifth day 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3µg/mL F6 and 0.3 µg/mL F6 were added in F12 Ham supplemented with 0.1% horse serum.The human hormone-sensitive breast adenocarcinoma cell line MCF-7 (ECACC Cat# 86012803), the human Caucasian breast adenocarcinoma MDA-MB-231 (ECACC Cat# 92020424) were obtained from Sigma-Aldrich . The non-tumorigenic epithelial cell line MCF-10A (ATCC CRL-10317) were obtained from LGC Standards S.r.l, MI, Italy. Cells were seeded into 25 cmThe embryos of Zebrafish (cultured under standard conditions as previously described ) were ke4 cells were seeded in a 96-multiwell and stimulated with F1, F2, F3, F4, F5, F6 at concentration of 0.1, 1, 10, 0.3, 3, 30 µg/mL, respectively. After 24, 48, and 72 h the cells were fixed for 1 h at 4 °C by gently layering 1/4 volume of cold 50% (w/v) Trichloroacetic Acid (TCA Solution) on top of the growth medium, and then rinsed with water several times to remove TCA solution, serum proteins, etc. Plates were air dried and stored until use. Blank background optical density was measured in wells incubated with growth medium without cells. The 0.4% Sulforhodamine B Solution  was added in a sufficient amount to cover the culture surface area (∼50% of the culture medium volume). Cells were stained for 20–30 min and at the end of the staining period, the stain was removed, and the cells quickly rinsed with Wash Solution (1% acetic acid) until unincorporated dye was removed. The incorporated dye was then solubilized in a volume of Sulforhodamine B Assay Solubilization Solution (10 mM Tris) equal to the original volume of culture medium. Absorbance at a wavelength of 565 nm was spectrophotometrically measured.4 × 104 cells non-stimulated (ctrl) and stimulated 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3µg/mL F6 and 0.3µg/mL F6 respectively, were placed in 500 μL DMEM + 0.1% FBS medium (DMEM F12 + 0.1% horse serum + 10ng/mL TGF-β1 in case of MCF-10A cells) in the upper side of 8-µm filters  and placed in wells of a 24-well plate  (lower chamber), containing 0.8 mL of DMEM + 10% FBS medium (DMEM F12 + 10% horse serum in case of MCF-10A cells). After 24 h of incubation, the migratory cells on the lower surface of membranes were fixed, stained with Hemacolor®  and examined microscopically cellular migration was determined by counting the number of cells on membranes in at least 4–5 randomly selected fields using a Zeiss Axiovert 10 optical microscope. For each data point, four independent experiments in duplicate were performed.The 2.5 × 104 cells non stimulated (ctrl) and 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6 and 0.3 µg/mL F6 as single agent respectively, were placed in 500 μL DMEM + 0.1% FBS medium (DMEM F12 + 0.1% horse serum + 10 ng/mL TGF-β1 in case of MCF-10A cells) in the upper side of 8-µm filters  (upper chamber) and placed in wells of a 24-well plate  (lower chamber), containing 0.8 mL of DMEM 10% FBS medium (DMEM F12 + 10% horse serum in case of MCF-10A cells). After 24 h of incubation, the invasive cells on the lower surface of membranes were fixed, stained with Hemacolor®  and examined microscopically. Cellular invasion was determined by counting the number of cells on membranes in at least 4–5 randomly selected fields using a Zeiss Axiovert 10 optical microscope. For each data point, four independent experiments in duplicate were performed.The 2.5 × 106 cells/well in a complete medium. The following day, the cells were refed with DMEM supplemented with 0.1% FBS containing 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6 and 0.3 µg/mL F6. The plates were incubated for 24 h at 37 °C in an atmosphere of 5% CO2. Then, the cells were trypsinized and centrifuged, and cell pellets were resuspended in phosphate-buffered saline (PBS). Cell count was performed by a particle count and size analyzer . Three replicate wells were used for each data point, and the experiment was performed six times.MCF-7 and MDA-MB-231 cells were seeded in 6-well culture plates  at a concentration of 1 × 102 flasks  in a complete medium. The apoptotic assay was performed using the Muse Annexin V and Dead cell kit . Briefly, MCF-7 and MDA-MB-231 cells were stimulated with the different embryo fish factors in DMEM 0.1% FBS. On the day of the experiment they were trypsinized, centrifuged, and resuspended in DMEM, 0.1% FBS medium to have a cell suspension between 1 × 105 and 1 × 107 mL−1. 100 μL of Muse Annexin and Dead Reagent was added. Cells were incubated for 20 min in the dark, and then analyzed with the Muse TM Cell Analyzer. Each assay was performed in triplicate.Cells were cultured at confluence into 25 cmg for 10 min. The Bradford assay was used to determine protein contents. For western blot analysis, cellular extracts were separated on SDS-polyacrylamide gels and proteins were blotted onto nitrocellulose membranes . The following antibodies were analyzed: anti-vinculin (7F9): sc-73614; anti-Rock1 (H-85): sc-5560 and anti-beta-catenin sc-7963 all from Santa Cruz Biotechnology; anti-p53 (acetyl k382) ab-75754 from Abcam; anti-TPT1 (E-AB-31729) from Elabscience; anti-E-cadherin (610181) from BD Bioscience. Antigens were detected with an enhanced chemiluminescence kit , according to the manufacturer’s instructions. Control and stimulated cells were washed twice with ice-cold PBS and scraped in RIPA lysis buffer (Sigma Aldrich). A mix of protease inhibitors  and phosphatase inhibitors  was added just before use. Cellular extracts were then centrifuged at 8000× All Western blot images were acquired and analyzed through Imaging Fluor S densitometer . Optical density (OD) of each condition was normalized versus the signal of internal control GAPDH .4 cells were placed into both wells of each insert with 70 μL of complete medium. When cells were confluent, the culture inserts were gently removed, and cells were fed with 10% FBS DMEM (CTRL), 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6 and 0.3 µg/mL F6 for 24 h. Then, the medium was removed and the cells were fixed with 4% paraformaldehyde for 10 min at 4 °C and washed twice for 10 min with PBS. The cells were permeabilized for 30 min using PBS, 3% BSA, 0.1% Triton X-100, followed by anti-vinculin (7F9): sc-73614, or anti anti-beta-catenin sc-7963  staining in PBS, 3% BSA at 4 °C overnight. The cells were washed with PBS and incubated for 1 h at room temperature with appropriate secondary antibody FITC conjugated . Negative controls were processed in the same conditions besides primary antibody staining. For F-actin visualization, Rhodamine Phalloidin  was used. Cells were then washed in PBS and mounted in buffered glycerol . Finally, analysis was conducted using a Leica confocal microscope TCS SP2  equipped with Ar/ArKr and He/Ne lasers. Laser line were at 543 nm and 488 nm for TRITC and FITC excitation, respectively. The images were scanned under a 40× oil objective. To analyze the colocalization of actin and vinculin, optical spatial z series composed of about 8/10 optical section with a step size of 1 μm were performed. Color channels were merged and colocalization were analyzed with the Leica confocal software.To evaluate the migratory phenotype of treated or non-treated cells, we perform the wound-healing assay using special double well culture inserts . Each insert was placed in 8-well μ-slides  and 3.5 × 10To test the enzymatic activity of urokinase plasminogen activator (uPA), aliquots of conditioned media of MDA-MB-231 human breast cancer untreated control cells, 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6, and 0.3 µg/mL F6 treated cells were separated by electrophoresis in 10% polyacrylamide slab gels in the presence of SDS (SDS–polyacrylamide gels (PAGE)) under non-reducing conditions. The uPA was then visualized by placing the Triton-X100-washed gel on a casein–agar–plasminogen underlay. The lytic zones were plasminogen dependent. Molecular weights were calculated from the position of pre-stained markers subjected to electrophoresis in parallel lines. Densitometric scanning of zymographies was performed to derive a semi-quantitative estimation of protease activities. PA gelatin zimography was performed three times.p < 0.05. Statistical analysis was performed by using GraphPad Instat software .Data were expressed as mean ± standard deviation (SD). Data were statistically analyzed with the analysis of variance (ANOVA) followed by the Bonferroni post-test. Differences were considered significant at the level of In the present experimental study we showed that molecular factor extracted from Zebrafish embryos isolated at the 20-somite developmental stage can reverse several malignant feature of the cancerous phenotype in a model of human breast cancer. Embryo extracts reduce cell proliferation, enhance apoptosis, and dramatically inhibit both invasiveness and migrating capabilities of cancer cells. Inhibition of migrating and invasive properties is not restricted to breast cancer cells, as embryo extracts were also effective in inhibiting the migrating phenotype adopted by normal breast cells undergoing epithelial–mesenchymal transition upon TGF-β1 stimulation. In cancerous cells embryo-induced reversion entails E-cadherin/β-catenin pathway, cytoskeleton remodeling, as well as downregulation of TCTP and the concomitant increase in p53 levels. Our findings suggest that neoplastic transformation cannot be viewed as an irreversible commitment and can be “reversed”—even partially - in response to proper morphogenetic influences.


Transforming growth factor β (TGF-β) serves critical functions in brain injury, especially in cerebral ischemia; however, apart from its neuroprotective effects, its role in regulating neurogenesis is unclear. TGF-β acts in different ways; the most important, canonical TGF-β activity involves TGF-β receptor I (TβRI) or the activin receptor-like kinase 5 (ALK5) signaling pathway. ALK5 signaling is a major determinant of adult neurogenesis. In our previous studies, growth arrest and DNA damage protein 45b (Gadd45b) mediated axonal plasticity after stroke. Here, we hypothesized that ALK5 signaling regulates neural plasticity and neurological function recovery after cerebral ischemia/reperfusion (I/R) via Gadd45b. First, ALK5 expression was significantly increased in middle cerebral artery occlusion/reperfusion (MCAO/R) rats. Then, we knocked down or overexpressed ALK5 with lentivirus (LV) in vivo. ALK5 knockdown reduced axonal and dendritic plasticity, with a concomitant decrease in neurological function recovery. Conversely, ALK5 overexpression significantly increased neurogenesis as well as functional recovery. Furthermore, ALK5 mediated Gadd45b protein levels by regulating Smad2/3 phosphorylation. Finally, ALK5 coimmunoprecipitated with Gadd45b. Our results suggested that the ALK5 signaling pathway plays a critical role in mediating neural plasticity and neurological function recovery via Gadd45b after cerebral ischemia, representing a new potential target for cerebral I/R injury. Approximately 80–90% of all stroke cases are ischemic stroke, while 10–15% are hemorrhagic stroke2. At present, the most effective treatment for ischemic stroke is rapid restoration of the blood supply. However, blood reperfusion may aggravate brain injury and functional impairment3. The process of restoring blood flow and causing reperfusion injury after cerebral ischemia is called cerebral ischemia/reperfusion (I/R) injury. The mechanisms of cerebral I/R injury are complex, with multiple signaling pathways and biological processes involved5. Improving functional recovery after cerebral I/R injury is an urgent key problem that must be solved. However, information about the mechanisms of injury and protection to brain tissues after cerebral I/R remains limited, and there are no satisfactory therapeutic strategies. According to an increasing number of studies, enhancing brain plasticity and promoting neurogenesis after stroke can significantly improve functional recovery8.Stroke is one of the leading causes of disability in human adults and accounts for 44 million disabilities worldwide each year9. According to many studies, TGF-β has extensive neuroprotective functions11. Activin receptor-like kinase 5 (ALK5), also known as TGF-β type I receptor (TβRI), plays a key role in the canonical TGF-β signaling pathway. ALK5 can catalyze the phosphorylation of receptor-regulated Smad proteins , which form a heteromeric complex with common-mediated Smad4 and translocate into the nucleus where they regulate transcription12. Based on recent studies, the ALK5-dependent TGF-β signaling pathway is involved in neurogenesis13. However, the mechanisms of the ALK5 signaling pathway that promote neurogenesis and whether ALK5 is involved in neurogenesis after cerebral ischemia remain unclear.Transforming growth factor-β (TGF-β) is a multifunctional secreted cytokine that plays a crucial role in cell proliferation, differentiation and apoptosis15. We previously demonstrated that Gadd45b can improve axonal plasticity and functional recovery after stroke7. In addition, our studies indicated that Gadd45b is a critical mediator of neuronal apoptosis and autophagy16. Another study demonstrated that Gadd45b can promote adult neurogenesis17. According to an increasing number of studies, ALK5 signaling can regulate the expression of Gadd45b19. Based on all these findings, we hypothesize that after cerebral I/R injury, ALK5 signaling can promote neurogenesis through GADD45b, thereby promoting functional recovery.Growth arrest and DNA damage protein 45b (Gadd45b), a member of the GADD45 family, plays a key role in anti-apoptosis and DNA repairTo verify our hypothesis, we first employed a MCAO/R rat model and used anterograde neuronal tracer biotinylated dextran amine (BDA), Golgi staining and immunohistochemical staining of neurofilament protein 200 (NF-200) to demonstrate that ALK5 promotes neurogenesis. Moreover, ALK5 improved functional recovery after cerebral I/R injury. Furthermore, the ALK5/Smad2/Smad3 signaling pathway served as an upstream regulator of Gadd45b expression, and ALK5 increased Gadd45b expression by promoting Smad2/3 phosphorylation. Finally, ALK5 coimmunoprecipitated with Gadd45b.Healthy adult male Sprague-Dawley rats (200–250 g) were purchased from the Experimental Animal Centre of Chongqing Medical University . All rats were housed at a 12 h/12 h light/dark cycle room maintained at 21–22 °C with a relative humidity of 60% and allowed free access to food and water. All animal experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Chongqing Medical University.7. Briefly, the common carotid artery, external carotid artery (ECA) and internal carotid artery (ICA) were exposed. A nylon filament suture rounded by paraffin wax at the tip was advanced from the ECA into the ICA. After 2 h of occlusion, the filament was withdrawn for blood reperfusion. Rats that failed to exhibit neurological deficits after reperfusion  as evaluated by the Longa-Z method20 were excluded from the study.Right MCAO/R was performed as previously described8 TU/ml) were supplied by GeneCopoeia Inc. . Lentiviral vectors coding for GFP were used as the control (LV-con-RNAi).To knock down the expression of ALK5, we used a small interfering RNA (siRNA) with the sequence GGACCAAGCCTATGATGATAA. Lentiviral vector: psi-LVUGP, Promoter: U6, Reporter: EGFP, Resistance marker: Puromycin resistance gene. Lentiviral vectors expressing ALK5-RNAi  were also supplied by GeneCopoeia Inc. Lentiviral vector: pEZ-Lv201, Promoter: CMV, Reporter: EGFP, Resistance marker: Puromycin resistance gene. Lentiviral vectors coding for GFP were used as the control (LV-con-ALK5).Lentiviral vectors for overexpressing ALK5  and adhesive-removal somatosensory test were performed 3 d before and 1, 7 and 14 d after MCAO/R.22.The mNSS is a motor, sensory, reflex, and balance composite test. Neurological function was graded from 0 to 18 . In the severity scores of injury, 1 point indicates the inability to perform the test or the lack of a tested reflex2 adhesive-backed papers were used as bilateral tactile stimuli that were adhered to the distal-radial region of each forelimb. The time to remove each stimulus was recorded during 3 trials per day (maximum limit: 120 s). Individual trials were separated by at least 5 min. Before MCAO/R, the animals should be trained for 3 d23.An adhesive-removal somatosensory test was used to evaluate sensorimotor deficits. Two pieces of 113.1 mmProtein was extracted from ischemic hemisphere brain tissues from different groups using a whole protein extraction kit . The protein concentration was measured by BCA protein assay reagent . After denaturation by boiling, all protein samples were stored at −80 °C until analysis. Equal amounts of protein were loaded into each well of SDS-PAGE gels, electrophoresed on 10% or 12% separating gels and transferred onto a PVDF membrane . The membranes were blocked with 5% skim milk and incubated overnight at 4 °C with the following primary antibodies: anti-ALK5 , anti-Smad2/3 , anti-phospho-Smad2/3 , anti-Gadd45b , anti-growth-associated protein 43 (GAP-43) , and anti-GAPDH  antibodies. GAPDH was used as a loading control. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody  or anti-mouse IgG antibody  for 1 h at room temperature. Then, the membranes were washed with TBST again and visualized using an enhanced chemiluminescence (ECL) reagent . Images were captured by a Fusion FX5 analysis system  and quantified using Quantity One software .2O2 for 15 min at 37 °C. Next, the sections were blocked with normal goat serum for 30 min at 37 °C. Then, the sections were incubated overnight at 4 °C with the following primary antibodies: anti-ALK5  and anti-neurofilament-200  antibodies. Next, the sections were incubated with goat anti-rabbit IgG or goat anti-mouse IgG  for 30 min at 37 °C. Positive activity was revealed by 3-3′diaminobenzidine (DAB) . Images were captured by a LEICA DM600B automatic microscope  and quantified using Image-Pro Plus 6.0 software . Protein expression levels were reflected by the mean optical density value  divided by the relevant area).Animals were anesthetized at 24 h and 14 d after MCAO/R and fixed through the left ventricle with PBS followed by 4% paraformaldehyde. Five slides were randomly selected from each sample for analysis. Sections were deparaffinized in xylene, rehydrated in graded ethanol solutions, placed in 10 mM sodium citrate buffer (pH 6.0) and heated in a microwave for antigen retrieval. Endogenous peroxidase activity was blocked with 3% HBrain tissues were cut into frozen sections. The frozen sections were air-dried at room temperature, washed with PBS, permeabilized with 0.4% Triton X-100, and submitted to antigen retrieval as for immunohistochemistry. After washing with PBS, sections were blocked in normal goat serum for 1 h. Then, the sections were incubated overnight at 4 °C with the following primary antibodies: anti-ALK5 , anti-neuron-specific beta-III tubulin , anti-GFAP , anti-nestin , and anti-DCX  antibodies. The next day, the sections were washed with PBS and incubated with a mixture of goat anti-rabbit IgG-CFL 488  and goat anti-mouse IgG-CFL 555  at 37 °C for 1 h in the dark. Then, the sections were counterstained with DAPI for nuclei. Images were captured by a confocal laser scanning microscope .21.Two weeks after BDA injection, animals were transcardially perfused with PBS followed by 4% paraformaldehyde. The entire brains were removed, post-fixed overnight in 4% paraformaldehyde and dehydrated in graded sucrose solutions. Brain tissues were cut into 50 μm-thick coronal sections by a cryostat microtome . All sections were collected in PBS, incubated with 0.5% H2O2 for 20 min, and submitted to 0.5% Triton X-100 for 30 min. Then, the sections were incubated with avidin–biotin–peroxidase complex  overnight at room temperature and DAB  for 5 min8.Four weeks after MCAO/R, 15 μl of a 10% BDA solution  was injected into the left sensorimotor cortex at the following five coordinates: 1.0 mm rostral to bregma, 1.0 mm lateral to the midline, and 1.5 mm ventral to the dura; 4.0 mm caudal to bregma, 1.0 mm lateral to the midline, and 1.5 mm ventral to the dura; 1.0 mm rostral to bregma, 5.0 mm lateral to the midline, and 1.5 mm ventral to the dura; 4.0 mm caudal to bregma, 5.0 mm lateral to the midline, and 1.5 mm ventral to the dura; and 1.5 mm caudal to bregma, 2.5 mm lateral to the midline, and 1.5 mm ventral to the dura21. The method of counting positive fibers crossing the midline of the corpus callosum was similar to that of the red nucleus. BDA-positive fibers were counted using Image-Pro Plus 6.0 software.The sections were examined using a ×10 objective. Then, we counted individual axons under a ×20 objective. The fibers projecting to the right red nucleus were examined by counting all midline-crossing BDA-positive fibers. The number of midline-crossing BDA-positive fibers was divided by the total number of corticospinal tract fibers24 as follows: 2D reconstructions of the entire dendritic tree were generated, a series of concentric circles with 10 μm intervals were centered on the soma, and the number of intersections was used to estimate the total length and distribution of dendrites27.Golgi–Cox staining was conducted according to the manufacturer’s instructions for a HITO GOLGI-COX OPTIMSTAIN™ KIT . Briefly, whole brain tissues were removed rapidly and immersed into a mixture of solution 1 and solution 2 for 14 d in the dark and transferred to solution 3 for 24–72 h at 4 °C in the dark. Sections (200 μm-thick) were cut by a vibratome . For staining, sections were mounted on 3% gelatin-coated glass slides, air-dried, stained with solutions 4 and 5, followed by graded ethanol solutions, dehydration and clearing with xylene. Layer III or V pyramidal neurons were identified with a ×20 objective and captured under a ×40 objective. A total of 20–25 neurons were measured for each group. The length and distribution of dendrites were evaluated by Sholl analysisCoimmunoprecipitation was conducted according to the manufacturer’s instructions for Protein A/G Magnetic Beads . Briefly, after washing the magnetic beads with binding/wash buffer, the beads were incubated with anti-ALK5 antibody , anti-Gadd45b  or anti-IgG antibody  on the rotator for 2 h at 4 °C. Then, the supernatant was removed and discarded, the beads were washed, and protein samples were added and incubated with rotation for 2 h at 4 °C. After washing, the supernatant was discarded, and elution buffer was added. The supernatants were collected and used as samples for Western blotting. The antibodies used for Western blotting were as follows: anti-ALK5 , anti-Gadd45b , HRP-Mouse Anti-Rabbit IgG Light Chain Specific  and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody  antibodies.P < 0.05 was considered statistically significant.The data are presented as the means ± standard deviation (SD). SPSS 23.0 was used for statistical analyses, and GraphPad Prism 7.0 was used for graphing. Student’s t test, one-way or two-way analysis of variance (ANOVA) with a Bonferroni post hoc test were used to compare the differences between groups. P < 0.05, Fig. P < 0.05, Fig. The expression and location of ALK5 were examined by Western blotting, immunohistochemistry and immunofluorescence. Western blotting showed that the expression of ALK5 was higher in the ischemic cortex of the MCAO/R group than in the sham group 24 h and 14 d after MCAO/R  and colocalized with DCX (neuroblast and immature neuron marker) Fig. . These rP < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. Then, axonal regeneration was assessed by upregulation of GAP-43 and NF-200. Western blot analysis showed that GAP-43 protein expression was lower in the LV-ALK5-RNAi group than in the LV-con-RNAi group at the same time point after MCAO/R 37. Axonal regeneration is reflected by upregulated expression of GAP-43 and NF-200. GAP-43 is a pivotal component of the regenerative response in the nervous system and is present at high levels in neuronal growth cones during axonal regeneration. NF-200 is a major component of neuronal cytoskeletons and can reflect neuronal function and axonal regeneration. For axonal reorganization, we focused on BDA-positive fibers crossing the midline of the corpus callosum and corticorubral fibers originating from the contralateral sensorimotor cortex. Dendritic plasticity can be identified by Golgi–Cox staining with Sholl analysis. An increase in dendritic branches and dendritic length occurs after ischemic stroke38. As demonstrated in the present study, the overexpression or knockdown of ALK5 enhanced or reduced axonal plasticity and dendritic plasticity, respectively, indicating that ALK5 signaling mediates neurogenesis after cerebral I/R injury. However, a study suggested that ALK5 is involved in glial scar formation, thereby inhibiting neurological function recovery39. We speculate that this opposing study conclusion may be related to the important roles of ALK5 in multiple signaling and functional pathways. Therefore, we need to further investigate the mechanism of ALK5 signaling that mediates neurogenesis and find a more effective therapeutic strategy for neurological function recovery.To investigate whether ALK5 promotes neurological function recovery by affecting neuroplasticity, we focused on changes in axonal plasticity and dendritic plasticity. Axonal plasticity including axonal reorganization and axonal regeneration40. Gadd45b has been implicated as a stress-response gene for physiological or environmental stressors; it influences apoptosis, cell-cycle arrest, cell growth and DNA repair42. In our previous study, Gadd45b mRNA and protein were significantly increased after I/R43. Gadd45b knockout mice display a decrease in the expression and DNA demethylation of BDNF17. Additionally, our previous studies suggested that Gadd45b knockdown reduces the expression and demethylation of BDNF, with a concomitant decrease in neurological function recovery; we also speculate that Gadd45b promotes axonal plasticity by promoting the expression and DNA demethylation of BDNF7. The corticospinal tract (CST) is the direct central nervous structure that controls limb movement. The direct injury of CST caused by cerebral infarction is the direct neurological basis of motor dysfunction. The ipsilateral or contralateral CST undamaged axons regenerate and sprout to form lateral branches and reconstruct the neural control of the anterior horn cells of the spinal cord, which is the material basis for limb motor function rehabilitation45. In the present study, changes in Gadd45b expression were consistent with ALK5 expression and corresponded to changes in neural plasticity and neurological function recovery. Based on these data, we believe that ALK5 acts upstream of Gadd45b, which affects the protein levels and demethylation of BDNF by regulating the expression of Gadd45b, thereby promoting functional recovery.The Gadd45 family includes three members, Gadd45a, Gadd45b, and Gadd45y; of these family members, Gadd45b is the only member that can be regulated by TGF-β46.Based on all our findings, we propose the following possible mechanisms of ALK5-regulated neurological recovery after cerebral I/R injury. The activation of ALK5 signaling by I/R promotes the phosphorylation of Smad2/3, which form a heteromeric complex with Smad4 and translocate into the nucleus where they interact with Gadd45b Fig. . NotablyIn conclusion, our findings suggest that ALK5 signaling may be an important mediator of neurogenesis and functional recovery after cerebral I/R injury. Although the mechanisms require more in-depth investigation, our present study could provide insight into the role of ALK5 signaling in cerebral I/R injury. Targeting ALK5 may be a potential therapeutic strategy for cerebral I/R injury.


Megalobrama amblycephala fed high-carbohydrate diets. Fish (39.44 ± 0.06 g) were randomly fed three diets: a control diet (30% carbohydrate), a high-carbohydrate diet , and the HC diet supplemented with 0.04% resveratrol (HCR) for 12 weeks. Fish fed the HC diet had significantly high values of nitrogen and energy retention efficiency, hepatosomatic index, intraperitoneal fat ratio, whole-body lipid content and intraperitoneal fat glycogen and lipid contents compared to the control group, but showed little difference with the HCR treatment. Liver and muscle lipid contents and plasma levels of glucose, glycated serum protein, advanced glycation end products and total cholesterol of fish fed the HC diet were significantly higher than those of the control group, whereas the opposite was found with resveratrol supplementation. Fish fed the HC diet obtained significantly low values of plasma insulin levels and hepatic adenosine monophosphate (AMP) contents and NAD+/NADH ratio compared to HCR treatment, but showed little difference with the control group. The opposite was found for hepatic adenosine triphosphate (ATP) contents and the ATP/AMP ratio. In addition, fish fed the HC diet showed significantly high transcriptions of glucose transporter 2 (GLUT2), glucose-6-phosphate dehydrogenase, glycogen synthase, fatty acid synthetase (FAS), acetyl-CoA carboxylase α (ACCα), peroxisome proliferator-activated receptor γ and PPARα compared to the control group, whereas the opposite was found for protein levels of AMP-activated protein kinase α (t-AMPKα), phosphorylated AMP-activated protein kinase α (p-AMPKα), sirtuin-1 (SIRT1), and p-AMPKα/t-AMPKα ratio as well as the transcriptions of AMPKα1, AMPKα2, SIRT1, PPARγ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase (FBPase), glucose-6-phosphatase, carnitine palmitoyl transferase I (CPT I) and acyl-CoA oxidase. Resveratrol supplementation significantly up-regulated the protein levels of t-AMPK, p-AMPK, and SIRT1, p-AMPK/t-AMPK ratio as well as the transcriptions of AMPKα1, AMPKα2, SIRT1, PGC-1α, GLUT2, FBPase, and CPT I compared to HC group, while the opposite was found for sterol regulatory element-binding protein-1, FAS and ACCα. Furthermore, resveratrol improved glucose and insulin tolerance of fish fed the HC diet after glucose and insulin load.This study investigated the effects of resveratrol on the growth performance, energy sensing, glycolipid metabolism and glucose and insulin load of blunt snout bream  Carbohydrates are generally deemed to the most economical energy source in fishery, because of their relatively low cost and protein-sparing effect  . The incThe AMP-activated protein kinase (AMPK) is an evolutionary conserved serine/threonine protein kinase. As a key energy metabolic sensor, it plays a vital important role in maintaining the cellular energy homeostasis . PreviouDanio rerio, blunt snout bream Megalobrama amblycephala, Atlantic salmon  fed high-fat diets  is a naturally occurring phytoalexin. This compound is found in different pharmaceutical dosage forms, and is recommended as a dietary supplement . Previouat diets . HoweverBlunt snout bream is one of the most important economic freshwater fish in China. Due to its fast growth and high larval survival rate, this species has been widely cultured worldwide . HoweverAll experimental animals in the present study were approved by the Animal Care and Use Committee of Nanjing Agricultural University . All experimental animal operations were approved and guided by the Care and Use of Laboratory Animals in China.Table 1. Protein was supplied by fish meal, soybean meal, rapeseed meal and cottonseed meal. Dietary lipids derived from fish oil and soybean oil. Corn starch was adopted to obtain the dietary carbohydrate levels required. Microcrystalline cellulose was included as the filler.Four isonitrogenous (32% crude protein) and isolipidic (5% crude lipid) diets were formulated, including a control diet (containing 30% nitrogen-free extract), a high-carbohydrate diet  and a HC diet supplemented with 0.04% resveratrol (HCR). Dietary carbohydrate levels were adopted on the basis of our previous studies , 2014. RThe diets were produced in our laboratory. Ingredients were finely ground, carefully weighed, well mixed, and pelletized using a laboratory pellet machine . All diets were dried at 30°C for 24 h. After drying, the diets were stored at −20°C in plastic bags until use.Blunt snout bream were purchased from the National Fish Hatchery Station in Yangzhou . Before the experiment, fish were acclimated to the experimental conditions by feeding a commercial diet  containing 32% protein and 5% lipid for 2 weeks. After that, a total of 216 fish (average weight: 39.44 ± 0.06 g) were randomly distributed into 12 tanks (300 L each) in a flow-through recirculating aquaculture system  at a stocking density of 18 fish per tank. Then, fish were fed to visual satiation thrice daily  for 12 weeks with one of three experimental diets. Each diet was tested in four tanks. Throughout the feeding period, a 12:12 h light: dark regime (07:00 to 19:00 h light period) was maintained by timed fluorescent lighting. Water temperature was maintained at 27 ± 1°C, and dissolved oxygen was maintained above 5.0 mg/L. Total ammonia nitrogen and nitrite were maintained below 0.2 and 0.01 mg/L, respectively.Before the feeding trial, six fish were randomly collected from the acclimated fish for the analysis of initial body composition. Then, the rest of fish were assigned to the feeding trial. After the last meal, fish were fasted for 24 h to empty gut contents prior to sampling. Then, all fish in each tank were counted and weighed. Four fish from each replicate with a total of 16 fish from each treatment were randomly selected, and were slightly anesthetized by MS-222  at a concentration of 100 mg/L. Thereafter, blood sample was rapidly taken from the caudal vein using heparinized syringes, and was centrifuged at 3000 rpm at 4°C for 10 min. The supernatant was collected and stored at −80°C for subsequent analysis. It should be mentioned here that fish among different treatments were sampled at equally timed intervals (about 20 min for each replicate) to average diurnal fluctuations in hormone titres across the groups. Then, individual liver, muscle and intraperitoneal fat were also sampled. These samples were stored in liquid nitrogen for further analysis. In addition, two fish were randomly collected from each tank, and were stored at −20°C for the determination of whole-body composition.The growth performance parameters adopted in this study were calculated as follows:W is body weight, W0 is initial weight, Wt is final weight, T is the culture period in days, N0/E0 and Nt/Et are the initial and final nitrogen/energy contents in whole body, respectively, and Ndiet/Ediet are the nitrogen/energy contents in the diets.The After initial sampling, the remaining fish in each group were assembled in one tank, and were starved for 24 h before the GTT and ITT.For GTT, according to the method detailed by The ITT was carried out as described in our previous study . BrieflyDiets and fish were analyzed for proximate composition. Moisture was determined by oven drying at 105°C until constant weight. Crude protein (nitrogen × 6.25) was determined by the micro-Kjeldahl method using an Auto Kjeldahl System . Crude lipid was determined via ether extraction using a Soxtec System . Ash content was analyzed by burning at 550°C for 4 h. Gross energy was measured using an adiabatic bomb calorimeter . Crude fiber was analyzed by fritted glass crucible method using an automatic analyzer . In addition, the crude protein, crude lipid, ash and gross energy in whole body were measured on the air-dry basis samples, which were converted to wet-weight basis samples. Tissue GS activities were determined following the procedures detailed by Cyprinus carpio L.) (+) and nicotinamide adenine dinucleotide phosphate (NADH) were determined by The levels of plasma glucose was measured using the glucose oxidase method . Plasma rpio L.) , which srpio L.) . Hepaticg at 4°C for 15 min. The supernatant was collected and stored at −80°C for subsequent analysis. Then, the protein concentration was determined using a Bio-Rad Protein Assay Kit . Subsequently, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Mini-Protean system  for 1–2 h at 100 V, then they were transferred to polyvinylidene fluoride (PVDF) membranes . The specific primary antibodies used were anti-β-actin , anti-AMPKα , anti-phospho-AMPKα  and anti-SIRT1 . Then PVDF membranes were washed and incubated with anti-rabbit  secondary antibody for 1–2 h at room temperature. Immune complexes were detected by a chemiluminescent substrate  based on the manufacturer’s instructions, and were visualized with a luminescent image analyzer . The protein levels were normalized by β-actin, and the intensities of each lane were quantified using the densitometry band analysis tool in Image J 1.44p .Total protein was extracted from the liver using RIPA lysis buffer  containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, 1 mM PMSF and 1× protease inhibitor cocktail . Then, it was centrifuged at 12,000 × Total RNA in hepatopancreas of blunt snout bream was extracted using Trizol  according to the manufacturer’s instructions, and was treated with RQ1 RNase-free DNase  to eliminate genomic DNA amplification. The quantity and purity of extracted RNA were determined by absorbance measures at 260 and 280 nm, respectively. The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA. Accordingly, a ratio near 2.0 is generally accepted as “pure” for RNA. Its integrity was further measured by electrophoresis in 1.0% formaldehyde denaturing agarose gels.−1), 0.5 μL dT-AP primer (50 mM), 0.25 μL ExScriptTM RTase (200 U μL−1) and 6.5 μL DEPC water. Cycling conditions were 42°C for 40 min, 90°C for 2 min, and 4°C thereafter.cDNA was generated using 500 ng DNase-treated RNA by a RT-PCR kit  following the manufacturer’s instructions. The reaction volume was 10 μL, containing 2 μL buffer (5×), 0.5 μL dNTP mixture (10 mM each), 0.25 μL RNase inhibitor (40 U μLTable 2). RT-PCRs were carried out on the Mini Option real-time detector . The assays were performed with a reaction mixture of 20 μL per sample, each of which contained 2 μL cDNA template , 0.4 μL of each primer (10 μmol L−1), 10 μL SYBR® premix Ex TaqTM (TaKaRa), 6.8 μL dH2O and 0.4 μL ROX Reference DyeII (TaKaRa). The PCR reaction was piloted under the following conditions: initial denaturation at 95°C for 5 s followed by 40 cycles, annealing at 60°C for 34 s and a final extension at 95°C for 5 s, followed by a melt curve analysis of 15 s from 95 to 60°C, 1 m for 60°C and then up to 95°C for 15 s. To analyze the relative transcriptional levels, the transcriptions of target genes were normalized by a reference gene-elongation factor 1 alpha (EF1α) (−ΔΔCT method (R2 > 0.983), and all primer pairs had equivalent PCR efficiencies (from 0.89 to 1.14).After reverse transcription, real-time PCR was employed to determine the mRNA levels based on the SYBR Green II Fluorescence Kit . The PCR primers sets were designed using the Primer 5.0 software according to the available sequences of blunt snout bream (a (EF1α)  using thT method . Four saP < 0.05) differences were found in the interaction, each factor was further analyzed separately by one-way ANOVA. All data were reported as mean ± SEM (standard error of the mean).Data on growth performance, body composition, tissue GS activities and glycogen and lipid contents, plasma parameters as well as protein and gene expressions were subjected to one-way ANOVA using the SPSS 20.0 software package  for Windows, after testing the homogeneity of variances with the Levene test. Unlikely, the data regarding plasma glucose levels in the GTT and ITT were analyzed by two-way ANOVA for significant differences among treatment means based on sampling time, dietary treatments and their interaction. If significant  among dietary treatments. The NRE, ERE, HSI, IPF and whole-body lipid contents of fish fed the HC diet were significantly (P < 0.05) higher than those of the control group. But they showed no statistical difference (P > 0.05) with those of the HCR treatment.Growth performance, feed utilization and whole-body composition of blunt snout bream were presented in Table 4, intraperitoneal fat GS activities and muscle glycogen contents showed no significant difference (P > 0.05) among dietary treatments. Intraperitoneal fat lipid and glycogen contents of fish fed the HC diet were significantly (P < 0.05) higher than those of the control group. But, they showed no significant difference (P > 0.05) with those of the HCR group. In addition, liver and muscle lipid contents of fish fed the HC diet were significantly (P < 0.05) higher than those of the other groups. However, the HCR group obtained a significantly (P < 0.05) high value of liver glycogen than the other treatments.As was shown in Figure 1, hepatic ATP, AMP and NADH contents as well as the ATP/AMP and NAD+/NADH ratios of fish fed the HC diet showed no statistical difference (P > 0.05) with those of the control group. The NAD+ content in fish fed the HC diet was significantly (P < 0.05) lower than that of the control group. In addition, the ATP content and the ATP/AMP ratio of the HC group were significantly (P < 0.05) higher than those of the HCR treatment, whereas the opposite was found for AMP content and NAD+/NADH ratio.As was shown in Table 5. No statistical difference (P > 0.05) was found in triglyceride levels among dietary treatments. Plasma levels of glucose, GSP and total cholesterol in fish fed the HC diet were significantly (P < 0.05) higher than those of the other groups. Plasma insulin levels of the HCR group was significantly (P < 0.05) higher than that of the other treatments. In addition, fish fed the HC diet obtained significantly (P < 0.05) high plasma AGES levels compared to the control group, but HC and HCR showed similar results (no significant difference).Plasma metabolites of blunt snout bream were shown in Figure 2 and Supplementary Figure S1, fish fed the HC diet obtained significantly (P < 0.05) lower t-AMPKα, p-AMPKα, the p-AMPKα/t-AMPKα, and SIRT1 protein contents compared with other groups.As can be seen from Figure 3, the transcriptions of AMPKα1, AMPKα2, SIRT1, and PGC-1α of fish fed the HC diet were significantly (P < 0.05) lower than those of the other groups.As can be seen from Figure 4. Hepatic GK and PK expressions showed no significant difference (P > 0.05) among dietary treatments. The GLUT2 and G6PDH expressions of the control group were significantly (P < 0.05) lower than those of the other groups, whereas the opposite was found for PEPCK and G6Pase expressions. The GS transcription of the control group was significantly (P < 0.05) lower than that of the HCR treatment, but showed no statistical difference (P > 0.05) with that of the HC group. In addition, the FBPase expression of fish fed the HC diet was significantly (P < 0.05) lower than that of the other groups.The transcriptions of the enzymes involved in glucose metabolism were presented in Figure 5, the hepatic expressions of FAS and ACCα of the HC treatment were significantly (P < 0.05) higher than those of the other groups, whereas the opposite was found for CPT I expression. The mRNA expressions of PPARα and PPARγ in the HC group were significantly (P < 0.05) higher than those of the control group, but showed no statistical difference (P > 0.05) with that of the HCR group; whereas, the opposite was found for ACO expression. In addition, the SREBP1 expression of fish fed the HC diet was significantly (P < 0.05) higher than that of the HCR treatment, but HC and control group showed similar results (no significantly difference).In the Figure 6, plasma glucose levels were significantly (P < 0.001) affected by sampling time, dietary treatments and their interaction. The glucose injecting resulted in a significantly (P < 0.05) elevated plasma glucose concentration with the highest value being obtained at 1 h after injection. Thereafter, plasma glucose levels decreased significantly (P < 0.05) to the basal value, which was attained within 8 h after the glucose load. Then gradually reduced to the minimum at 12 h. In terms of dietary treatments, plasma glucose level of the HCR treatment was significantly (P < 0.001) lower than that of the other groups. In addition, plasma glucose levels were significantly affected by the interaction between sampling time and dietary treatments with significant (P < 0.001) differences observed during the period of 0–4 h among different groups.As was shown in Figure 7. Plasma glucose levels were significantly (P < 0.001) affected by sampling time, dietary treatments and their interaction. The insulin administration resulted in a significantly (P < 0.05) decreased plasma glucose concentration with lowest levels being obtained at 1 h after injection. Thereafter, plasma glucose levels decreased significantly (P < 0.05) to the basal value, which was obtained within 12 h after the insulin load. In terms of dietary treatments, plasma glucose level of the HCR treatment was significantly (P < 0.001) lower than that of the other groups. In addition, plasma glucose levels were significantly affected by the interaction between sampling time and dietary treatments with significant (P < 0.001) differences observed during the period of 0–8 h among different groups.Plasma glucose levels in blunt snout bream after ITT were presented in In this study, microcrystalline cellulose was incorporated at 13% in diets as a filler to compensate for the carbohydrate levels required. This could not have negative effects on the growth performance of fish, since as a herbivorous fish blunt snout bream has a high tolerance for cellulose than most of the carnivorous and omnivorous species . In fact+ contents in liver. This result may be due to the fact that high-carbohydrate intake usually induced a hyperglycemia state of fish, inhibiting the pyruvate dehydrogenase complex, thereby leading to a decreased NAD+ content  SREBP1, FAS, and ACCα played an important role in hepatic adipogenesis ; (2) PPACyprinus carpio and tilapia Oreochromis niloticus × O. aureus  reslin dose , thus reIn conclusion, the present study suggested that dietary supplementation of resveratrol could improve the energy sensing and glycolipid metabolism of blunt snout bream fed high-carbohydrate diets by activating the AMPK–SIRT1–PGC-1α network, the up-regulation of the genes related to glucose transportation, glycogenesis, and fatty acid β-oxidation coupled with the depression of lipogenesis. In addition, resveratrol supplementation slightly compromised the growth performance of blunt snout bream.W-BL and X-FL designed the experiments. H-JS, CX, B-KW, D-HC, and M-YL carried out the experiments. H-JS, X-FL, CX, B-KW, LZ, and C-YX contributed reagents, materials, and analysis tools. H-JS and X-FL wrote the paper. All authors read and approved the final version of 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.


Dinophysis (D. acuminata-complex and D. caudata) and in scallops (Argopecten purpuratus), the most important commercial bivalve species in Perú. LC-MS analyses revealed two different toxin profiles associated with species of the D. acuminata-complex: (a) one with OA (0.3–8.0 pg cell−1) and PTX2 (1.5–11.1 pg cell−1) and (b) another with only PTX2 which included populations with different toxin cell quota (9.3–9.6 pg cell−1 and 5.8–9.2 pg cell−1). Toxin results suggest the likely presence of two morphotypes of the D. acuminata-complex in the north, and only one of them in the south. Likewise, shellfish toxin analyses revealed the presence of PTX2 in all samples (10.3–34.8 µg kg−1), but OA (7.7–15.2 µg kg−1) only in the northern samples. Toxin levels were below the regulatory limits established for diarrhetic shellfish poisoning (DSP) and PTXs (160 µg OA kg−1) in Perú, in all samples analyzed. This is the first report confirming the presence of OA and PTX in Dinophysis cells and in shellfish from Peruvian coastal waters.Causative species of Harmful Algal Bloom (HAB) and toxins in commercially exploited molluscan shellfish species are monitored weekly from four classified shellfish production areas in Perú (three in the north and one in the south). Okadaic acid (OA) and pectenotoxins (PTXs) were detected in hand-picked cells of  O-acyl-derivatives (“DTX3”) are liposoluble polyethers that have been designated as diarrhetic shellfish toxins Dinophysio, Italy , where oD. caudata from the same bay had 5.8 pg PTX2 cell−1. Therefore, there was a co-occurrence of two toxic Dinophysis species in this area. Scallop samples (“concha de abanico”) during the occurrence of these species reached toxin levels ranging from 7.7 to 15.2 µg OA kg−1 and from 10.7 to 34.8 µg PTX2 kg−1  of toxin monitoring of scallops, A. purpuratus, by LC-MS/MS. During this period, toxin levels never reached regulatory limits, although Dinophysis densities above 104·cells L−1 were recorded. Dinophysis densities of around 103 cells L−1 are considered a bloom, and have often been related to toxic outbreaks in other parts of the world [The low toxin content found in Dinophysis and of scallops (Argopecten purpuratus) from shellfish production areas in Perú were characterized. Different species included in the Dinophysis acuminata-complex and D. caudata, producers of toxins regulated by the EU, co-occur in northern Perú. The toxic species of Dinophysis from Sechura Bay, Samanco Bay, Salinas, and Independencia Bay showed low cell-toxin content (pg cell−1) in comparison with those reported for the same species in other parts of the world, although more studies, including physiological and genetic factors affecting toxin profiles and content, are needed. Dinophysis acuminata-complex and D. caudate are most likely the main species concerning molluscan shellfish safety in Perú.The toxin profiles, including OA and PTX2, of several species of Argopecten purpuratus “concha de abanico”) samples were collected at the fixed monitoring stations in Sechura Bay, Samanco Bay, Salinas, and Independencia Bay  of the National Fisheries Health Organization of Perú (SANIPES), which is the national competent authority for the control of seafood safety. Samples from classified shellfish production areas were analyzed at the SANIPES official laboratory. During 2017 and the first half of 2018, seawater and scallops  (Cells of f. ovum)  were iso3CN) were used for the extraction and analyses of toxins by liquid chromatography coupled to mass spectrometry (LC-MS). Analytical-grade ammonium hydroxide, sodium hydroxide (NaOH), and hydrochloric acid (HCl) were used for the mobile phase and hydrolysis. Ultrapure water was obtained with a Sartorius (Arium Pro) purification system. Certified reference solutions for okadaic acid CRM-OA-d (batch #20141119), dinophysistoxin-2 CRM-DTX2-b (batch #20150819), dinophysistoxin-1 CRM-DTX1-b (batch #20151209), and pectenotoxin-2 CRM-PTX2-b (batch #20120516) were obtained from NRC-CNRC.LC-MS grade methanol (MeOH) and acetonitrile  as described in .Argopecten purpuratus, were homogenized and a 2 ± 0.05 g subsample, weighed on an analytical scale , was placed in a 50 mL centrifuge tube and extracted twice with 9 mL methanol, stirred with a vortex  for 3 min, and centrifuged at 2000× g  for 10 min at a temperature of 20 °C. The supernatants were transferred and mixed in a volumetric flask and made up to 20 mL with methanol. To explore the presence of esterified derivatives of OA and DTXs, an aliquot of the methanolic extract was taken for alkaline hydrolysis. Whole flesh samples of 12–15 scallops, The alkaline hydrolysis was carried out by adding 125 µL of 2.5 N NaOH to 1 mL of the methanolic extract, vortexing the mixture for 0.5 min, heating it for 40 min at 76 °C, and finally neutralizing the added NaOH with an equivalent amount of 2.5 N HCl. ®Xtra, 25 mm) following the recommended protocols from the European Union Reference Laboratory for Marine Biotoxins [Finally, all extracts (raw and hydrolyzed) were filtered through a 0.2 µm pore size nylon filter  column kept at 40 °C. A binary gradient elution was used, with phase A consisting of H2O and phase B of 90% CH3CN, both containing ammonium hydroxide 6.7 mM (approximate pH of 11). The gradient started with 30% B, that proportion was kept for 1 min and then linearly increased to 90% B in 4 min. It was maintained at that proportion for 1 min, returned to the initial proportion in 0.1 min, and maintained for equilibration during 1.5 min before the next injection. The flow rate was 0.4 mL min−1 and the injection volume was 2 µL. The mass spectrometer was operated in both ESI positive and negative modes, the cone voltage was 3.0 kV, desolvation gas temperature was 500 °C with an N2 flow of 1000 L h−1 and a source temperature of 150 °C. Voltage parameters of the cone and collision energy were optimized during the tuning phase by direct infusion in alkaline medium. Product ions used for the quantification of each toxin in microalgae and shellfish and the MS/MS conditions for the multiple reaction monitoring (MRM) for each molecule are shown in For LC-MS/MS analysis of the lipophilic toxins, a Waters Acquity I Class chromatograph coupled to a triple quadrupole mass spectrometer Waters XEVO-TQS by means of an electrospray interface (ESI) was used. Analytical separation was performed following a modification of the method developed by Gerssen et al. , with an


E. coli global transcription regulators, affects T4 infection. Both ppGpp0 and ΔdksA increase T4 wild type (wt) plaque size. However, ppGpp0 does not significantly alter burst size or latent period, and only modestly affects T4 transcript abundance, while ΔdksA increases burst size (2-fold) without affecting latent period and increases the levels of several Pe transcripts at 5 min post-infection. In a T4ammotA infection, ΔdksA increases plaque size and shortens latent period, and the levels of specific middle RNAs increase due to more transcription from Pe’s that extend into these middle genes. We conclude that DksA lowers T4 early gene expression. Consequently, ΔdksA results in a more productive wt infection and ameliorates the poor expression of middle genes in a T4ammotA infection. As DksA does not inhibit Pe transcription in vitro, regulation may be indirect or perhaps requires additional factors.Bacteriophage T4 relies on host RNA polymerase to transcribe three promoter classes: early , middle , and late . Using primer extension, RNA-seq, RT-qPCR, single bursts, and a semi-automated method to document plaque size, we investigated how deletion of DksA or ppGpp, two  The integrity and regulation of gene expression is essential for proper cellular function and adaptation. Bacteria have evolved myriad mechanisms to tightly regulate gene expression to ensure that genes are expressed under the correct environmental conditions . BacteriEscherichia coli (E. coli), the primary σ factor, σ70, recognizes specific −10 and −35 elements within the promoter sequence and is essential for the transcription of genes needed for exponential growth under nutrient-rich conditions. Efficient transcription requires the interaction of RNAP with promoter DNA to create an “open complex” with a single-stranded region at the transcription start site, initiation of RNA synthesis, promoter clearance, processive transcription elongation, and accurate termination , which consists of five core factors , along with a σ subunit that specifies the transcription start site . In EE. coli promoters use specific activators, repressors, and/or other transcriptional regulators to control initiation, elongation, and termination. While activators and repressors typically interact with specific DNA sequence motifs, DksA is a member of a growing class of global transcription regulators, called secondary (2°) channel proteins, that interact with just RNAP. These proteins, which also include GreA and GreB, all share two superimposable structural domains: a coiled-coil domain that penetrates the 2° channel of RNAP and a globular domain that binds the rim helices of RNAP atop the 2° channel [ channel . The aci channel . These f channel .E. coli gene expression are complicated. DksA/ppGpp inhibition of RNAP initiation from the ribosomal promoter PrrnB1 is the most widely studied example, where DksA and ppGpp act synergistically to further destabilize already unstable open complexes [E. coli cells [The interaction of DksA with RNAP generates one of two binding sites for the small molecule ppGpp on polymerase ,12, and omplexes ,15,16. Iomplexes . In otheli cells . In addili cells . Furtherli cells . It is nli cells ,22.70-dependent consensus −10 and −35 elements, do not require any viral factors for activity and are active immediately after infection. Thus, Pe’s successfully compete with host DNA for the same pool of RNAP. Pm’s, which become active ~1 min after infection at 37 °C, require two T4-encoded proteins, MotA and AsiA, to direct RNAP to recognize a specific element, a MotA Box, centered at −30. Importantly, middle genes are also expressed through the extension of early transcription from Pe’s into downstream middle genes. Finally, Pl’s become active ~5–7 min after infection at 37 °C. Pl’s harbor a novel −10 sequence known as the late TATA box and require the T4 proteins gp55 (late sigma factor) and gp33 (co-activator) for activity. In addition, late transcription requires active T4 DNA replication through T4 protein gp45, which is the replication sliding clamp that also serves as an enhancer for Pl’s [In a bacteriophage T4 infection, there are three types of T4 promoters (early (Pe), middle (Pm), and late (Pl)) based on the stage of development in which they are active (reviewed in ). Pe’s, for Pl’s . As a fumotA knockdown (T4ammotA) infections of a wild-type laboratory strain and a dksA mutant host strain, and we investigate the effects of a DksA or ppGpp deletion on T4 growth and transcription. We demonstrate that a deletion of either DksA or ppGpp results in significantly larger plaques for either T4 wt or T4ammotA. Infections in a ppGpp0 host have a marginal effect on burst size, latent period, or phage transcript abundance. However, a deletion of DksA results in a 2-fold increase in T4 wt burst size and increased levels of several Pe RNAs. As the T4 activator MotA is required for Pm activation, a T4 motA knockdown results in poor phage growth. Our transcriptome analyses reveal that middle genes are affected differentially, ranging from slight to severe inhibition of gene expression. We show that the absence of DksA in the T4 motA knockdown background also increases transcription from specific Pe’s. This increase ameliorates the poor growth of T4ammotA by increasing expression of downstream middle genes needed for replication, recombination, and late transcription. As we do not observe an effect of DksA on Pe transcription in a purified in vitro transcription system, we speculate that DksA decreases T4 early transcription through other changes present in the infected ΔdksA host or by using additional host/phage factors.In this study, we report the first global transcriptome analyses of T4 wild type (wt) and a T4 + wt and T4amG1 (T4ammotA) [–BompT gal dcm lon hsdS(B− mB−r) λ(DE3 (lacI lacUV5-T7p07ind1 sam7 nin5)) K-12(λS)) [r, r–m−, T6r, ara–) [dksA, relA, and spoT loci are referred to as wild type. B606 ∆dksA::kan (B606 ∆dksA) was constructed by P1 transduction using a K-12 lysate containing the ∆dksA::kan insertion [0, an isogenic mutant strain that is unable to generate ppGpp, was constructed by introducing a ∆relA::kan insertion followed by a ∆spoT::cat insertion as previously described [ksA, relA and spoT mutants were constructed by P1 transduction with E. coli K-12 donor strains and the recipient B606 strain. The ∆dksA::kan insertion recombinant in B606 grows on LB but not on M9 glucose minimal, like the MG1655 K-12 sister strain. Transduction of the ΔrelA::kan recombinant allele into B606 was phenotypically verified by loss of resistance to 3-aminotriazole as well as the absence of ppGpp accumulation after adding 1 mg/mL serine hydroxamate. The B606 ΔrelA::kan ΔspoT::cat double deletion construct was made by introducing the ΔrelA mutant first, then transduction of the ΔspoT mutant since the ΔspoT relA+ combination is not viable owing to excessive ppGpp levels. Features of the double mutant again mimic those of the K-12 sister strain and are unable to grow on minimal medium without amino acid supplementation.T4D4motAam)  were use-12(λS))  and B606r, ara–)  were thensertion . B606 ppescribed . BrieflydksA, and B606 ppGpp0 were freshly streaked out on Lennox L Broth : 5 g/L sodium chloride, 10 g/L tryptone, and 5 g/L yeast extract) plates and grown at 37 °C overnight. For B606 ∆dksA, a final concentration of 3 μg/mL adenine was added to plates to improve growth for single colony selection. From a single colony, cultures were grown overnight in LB at 32 °C. For plating, cultures were diluted in fresh LB to an OD600 ~ 0.1 and grown to early-log phase (OD600 ~ 0.3) at 32 °C. Cells were then transferred to ice until ready to titer. For each titer, ~50 to 100 phages in 200 μL LB were incubated with an equal volume of culture for 5 min, followed by 3 mL of 0.65% (w/v) Drake Top agar at 48 °C [w/v) LB agar. Plates were incubated at 37 °C overnight.BL21(DE3), B606, B606 ∆at 48 °C , and theDigital images of each plate were documented using a Protein Simple Molecular Imager Gel Doc . TIFF image files were converted to JPEG files with 256 gray levels (0–255) and 650 × 515 pixels resolution in ImageJ . Plaque dksA, and B606 ppGpp0 were grown overnight at 32 °C from a single colony, diluted to an OD600 ~ 0.1 in LB, and then cultured to mid-log phase (OD600 ~ 0.5) at 37 °C. For all conditions, B606 wt was kept on ice and used as the plating strain. For each condition, 900 μL of culture was transferred to a 1.5 mL microcentrifuge tube (pre-warmed to 37 °C) containing either T4 wt or T4ammotA at a multiplicity of infection (MOI) of <0.04. After 10 min, the phage infected sample was diluted 10,000-fold for T4 wt or 1000-fold for T4ammotA. Dilutions were gently vortexed to mix and immediately returned to 37 °C. For pre-burst (≤30 min) and post-burst (35 to 80 min) time points, 100 μL or 10 μL of the diluted phage culture, respectively, was added to 100 μL of B606 wt. This was immediately followed by the addition of 3 mL of 0.65% (w/v) Drake Top agar (at 50 °C) and plating on 1.5% (w/v) LB agar. Plates were incubated overnight at 37 °C. Burst size was calculated by dividing the average number of plaques for the post-burst time points (50 to 70 min for T4 wt infection) by the average number of plaques for pre-burst time points (15 to 30 min for T4 wt infections). The latent period is defined as the last time point post-infection for which the relative titer remains less than or equal to 2.Burst size experiments were performed as previously described  for singdksA, and B606 ppGpp0 were grown at 37 °C to early/mid log phase (OD600 ~ 0.4), then infected at an MOI of 10 with either T4 wt or T4ammotA. RNA was isolated at 1, 5, or 12 min post-infection using method II [B606, B606 ∆ethod II . Brieflyw/v) agarose gel stained with ethidium bromide.Total RNA used for RNA-seq was assessed on a Bioanalyzer using the Agilent RNA 6000 Nano Kit  to evaluate the quality of samples; all samples used for library preparation had an RNA integrity number (RIN) of 9.3 or better . The quaTotal RNA (2.5 µg) from each sample was treated with a Ribo-Zero rRNA Removal Kit  to deplete ribosomal RNAs. A TruSeq Stranded mRNA Library Prep Kit  was then used for the cDNA library preparation, and the library was sequenced using Illumina MiSeq in order to obtain 100 bp paired end reads.E. coli B str. DE3 (NC_012971.2) as a reference genome using STAR v2.5.2 retaining unmapped reads [p value ≤ 0.05 were considered significant. RNA-seq data is available in the National Center for Biotechnology Information (NCBI) database (GEO number GSE111808) and in After trimming, FASTQ files were aligned to ed reads . Unmappeed reads . Differe32P-labeled primer. Unless otherwise indicated, oligonucleotides annealed ~100 nucleotides (nt) downstream of the predicted start site of the promoter. Primer sequences are available upon request. Primer extension products were separated on 5% (w/v) denaturing polyacrylamide  gels and visualized by autoradiography. Product levels were quantified using a GS-800 calibrated densitometer  and Quantity One Software Primer extension was performed using Avian Myeoloblastosis Virus (AMV) reverse transcriptase  as previously described ,41,42, uT method, also known as 2Ct-ΔΔ [nrdA (B606 ∆dksA vs. B606 wt) and 61.4 (T4ammotA vs. T4 wt) that appeared unaffected in the RNA-seq data were used as internal controls. Primers used in this study were designed using Primer3web [RT-qPCR was used to validate RNA-seq data. Briefly, RNA samples were collected under the same conditions used for RNA-seq, and complementary DNA (cDNA) was generated using a High Capacity cDNA Reverse Transcription Kit . Target genes were amplified with SYBR Green PCR Master Mix (Applied Biosystems) in a LightCycler 480 instrument . Relative levels of gene expression were determined with the comparative Cs 2-ΔΔCt . Genes nimer3web ,45; prim70 [6 tag was isolated and purified from CF9413:MG1655 (λDE3)/pHM1501 [600 of ~0.35, protein synthesis was induced by the addition of 0.2 mM IPTG  for 2 h with shaking. Cells were harvested by centrifugation at 13,000× g for 10 min. The following procedures were performed on ice or at 4 °C. Pellets were resuspended in 12 mL of lysis buffer (40 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM imidazole, 2 mM 2-mercaptoethanol, 0.1 mM benzamidine; 2–5 mL per gram wet weight) and lysed by sonication. Clarified supernatant was obtained by centrifugation at 15,000× g for 30 min and added to 2 mL of a 50% slurry of equilibrated Ni-NTA agarose  in lysis buffer and rocked gently overnight. The lysate/Ni-NTA mixture was loaded onto a Bio-Rad 10 mL disposable column, washed twice with five bed volumes of wash buffer (40 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 2 mM 2-mercaptoethanol), and then with 4 mL each of wash buffer containing increasing imidazole concentrations: 60 mM, 150 mM, and 250 mM. Although all imidazole fractions contained DksA, the 250 mM fraction was highly purified  [S17 (gift of J. Nguyen) that contains a σ70-dependent promoter that is not affected by DksA as a negative control. Transcription reactions (10 µL) were assembled with 5 nM DNA, 30 nM RNAP  that had been reconstituted with or without 250 µM ppGpp , the indicated concentration of DksA, and transcription buffer (50 mM Tris-acetate (pH 8.0), 10 mM magnesium acetate, 10 mM 2-mercaptoethanol, 90 mM potassium glutamate, 10 µg/mL bovine serum albumin). Solutions were incubated at 37 °C for 10 min before single round transcription was initiated by the addition of heparin  and ribonucleoside triphosphates  UTP at 1.7 × 104 dpm/pmol). After incubation for 10 min at 37 °C, reactions were collected on dry ice. A solution (10 μL) containing 10 mM EDTA (pH 7.0), 0.1% bromophenol blue, and 0.1% xylene cyanol in deionized formamide was added. Samples were then heated for 2 min at 95 °C and 10 μL aliquots were electrophoresed on 4% polyacrylamide, 7 M urea denaturing gels run in 1× TBE (Tris-borate-EDTA). Gels were imaged by autoradiography as described above.In order to check the activity of purified DksA, in vitro transcription assays were performed using a supercoiled template DNA that contains the ribosomal promoter, Pto +109)  as a postac promoter. Before RNA isolation, a 10 μL aliquot of the Ptac reaction was added as an internal control into each sample, and then the RNA was extracted and purified by phenol extraction/ethanol precipitation as described above. 5′-ends of the RNA were detected by primer extension analyses as described above.For in vitro transcriptions with T4 DNA, T4 genomic DNA was purified from concentrated T4 wt by extraction with phenol, phenol:chloroform:isoamyl alcohol (25:25:1), and then chloroform:isoamyl alcohol (24:1); each extraction step was repeated twice. The purified DNA was subsequently dialyzed against TE buffer, and then treated with T4 DNA ligase  in the presence of ATP to seal any intramolecular nicks. Transcription reactions (100 μL) were assembled with 0.06 pmol of T4 DNA, 7.5 pmol pre-reconstituted RNAP (sigma:core ratio of 2.5:1), transcription buffer, and as indicated, 112.5 pmol AsiA, 1.88 pmol MotA and/or 60 pmol DksA. After incubation at 37 °C for 10 min, a single round of transcription was initiated by adding heparin  and rNTPs . Samples were incubated for 10 min at 37 °C before collection on dry ice. An independent in vitro transcription reaction was performed as described  using pGE. coli wt strains typically yield plaques with similar sizes. For example, using two different E. coli B strains, B606 wt or BL21(DE3) wt, we observed similar T4 wt plaques with minimal heterogeneity  . An amber mutation in the T4 gene motA (T4ammotA) is known to significantly impair T4 infection due to a dramatic decrease in middle gene expression and delayed replication  compared to B606 wt (average size of ~7 pixels2).Plaque size can be used as an indirect measure of phage fitness since larger plaques can arise from better phage growth. T4 wt infections of different ctively) . In conty) , diffusivity, absorption rate, and length of the latent period ,51. To a ~2-fold a,b, whil ~2-fold . In addi ~2-fold . We concammotA. In this case, we did not observe a significant difference in burst size for T4ammotA in host wt vs. ∆dksA strain . GendsbA and Pl15).As shown schematically in ammotA infection have indicated that when MotA-dependent promoters are not activated, expression of some early genes remains high, while late transcription, which is dependent on the middle products gp55, gp33, and gp45, is significantly diminished . Howevespecific ,42,55. Imination ,57. EvenDifferential expressions determined by RNA-seq analysis were compared to RT-qPCR assays of 68 genes using RNA isolated 5 min after infection and by primer extension analyses using RNA isolated at 1 and 5 min after infection. One min after infection, early promoters are active, but middle transcription has just initiated ,54. A coammotA infections. Expression of 44% of early genes increased ≥2-fold, while expression of 79% of late genes decreased ≥2-fold , gene 45 (DNA polymerase clamp and late transcription enhancer), and genes 44/62 (clamp loader) and (2) the region downstream of Pe148.6  that includes gene 33 (co-activator required for late transcription), rnh (RNase H), gene 59 (DNA helicase loader for replication), and gene 32 (single-stranded DNA binding protein). However, other sections with Pe’s and Pm’s, whose genes also encode important replication and late transcription factors, were not significantly MotA-dependent. These included: (1) the region downstream of Pe26.4, Pe20.3, and Pe19.8  containing genes 41/61 (DNA helicase/primase) and uvsX , and (2) the region downstream of Pe40.4  containing gene 55 (sigma factor for late transcription). Because the Pm’s for both these regions (Pm55 and PmuvsX) are highly dependent on MotA , the recombination protein UvsX, EndoVII (gp49) that resolves recombination structures, nucleases gp46 and gp47, and DNA ligase (gp30). Such a combination of proteins suggests a minimal system for recombination in which nucleases 46/47 might generate ssDNA substrates suitable for homologous recombination by UvsX/gp41. The nicked DNA would then be sealed by DNA ligase. However, whether such a system is used is not known.Thus, our analyses indicate that at 5 min post-infection in the t  and 38 , increased in the T4ammotA infection  or degradation of host nucleic acid  [45, Pm45.2) by the absence of DksA  . RNA-seq of DksA . The inc of DksA  and Pe14 of DksA , two Pe’ of DksA .Somewhat surprisingly, the expression level of several late genes  and the Taken together, our results are consistent with a role of DksA in lowering the burst of T4 wt phage in a wt infection by decreasing the level of transcription from many Pe’s. As mentioned above, the function of many early genes remains unknown. However, these early genes are predicted to be involved in host takeover and transition to middle gene expression . It is pammotA by significantly decreasing the latent period  or absence (B606 ∆dksA) of DksA.The absence of DksA alleviated the poor growth of T4t period . To deteammotA/wt infection  and the region containing genes 33, rnh, 59, and 32 ), the level of RNA from the T4ammotA infection in the absence of DksA increases from 1.5 to 7-fold over that seen in the T4ammotA infection in the presence of DksA. These are the middle genes that are most severely affected by the motA knock-down.The overall result of these analyses is that the DksA deletion partially ameliorates the poor expression of many T4 middle genes through the increase in transcription from certain Pe’s. This result is most easily seen in motA transcription in the ammotA background by ~2-fold , we observed that the overall increase in transcription resulted from a significant increase in transcription from Pe148.6 rather than an increase in PmdsbA  was not significantly reduced by the motA knockdown, yet gene 55 expression increased even more in a T4ammotA infection in the absence of DksA . As expected, the presence of ppGpp improved this inhibition. Next, we performed in vitro transcription reactions using T4 DNA under the same buffer conditions. We assayed for transcription from specific Pe’s using primer extension  that are needed for DksA action ,62, a coxtension . DksA dixtension a, Pe35.3xtension b, or Pe1xtension c. The pron  that are immediately recognized by the host transcriptional machinery [70 holoenzyme, allowing it to recognize a specific −30 sequence (the MotA box) rather than the host −35 element. Late transcription is initiated approximately 5–7 min after infection by the activation of Pl’s. These promoters do not contain either the bacterial −35 element or the MotA box sequence, but instead contain a novel −10 sequence, known as the late TATA box, that requires a new sigma factor encoded by T4 gene 55, as well as the activator encoded by gene 33 [To our knowledge, no study has reported whether DksA contributes defense systems by modulating gene expression of phages during infection. Bacteriophage T4 is a good model system for investigating this since it primarily regulates gene expression at the level of transcription. Temporal gene expression by T4 is controlled by the recognition of three distinct promoter architectures . Pe’s contain very strong σachinery ,72. Pm’sachinery ). Activa gene 33 . In addi gene 33 .dksA strain also explains the increased growth of T4ammotA . In facadequacy ,77.The enigmatic absence of regulatory effects associated with ppGpp might indicate the need for different conditions to detect effects of ppGpp on T4 burst size and latent period. For example, slow growth in minimal medium with a poor carbon source, which elevates the level of ppGpp, might give different results than a rich nutrient broth where the cellular ribosomal content is high and the ppGpp concentration is very low at the onset of infection. It also might be revealing to measure ppGpp levels during phage T4 lytic development to evaluate if ppGpp levels change. Alternatively, ppGpp levels could be artificially elevated to high levels at the onset of T4 infection to ask if development is altered.E. coli transcription terminator Rho and lambdoid phage antiterminators, N and Q . In addiewed in ). Intereiewed in , the T4 iewed in , and theiewed in  have eac