L. angustifolia × L. latifolia, was tested on human leukaemia cells (HL60). Based on the MTT results, the reduced cell viability of HL60 cells was further investigated to determine whether cell death was related to the apoptotic process. HL60 cells treated for 24 h with LEO were processed by flow cytometry, and the presence of Annexin V was measured. The activation of caspases-3 was evaluated by western blot and immunofluorescence techniques. Treated cells were also examined by scanning and transmission electron microscopy to establish the possible occurrence of morphological alterations during the apoptotic process. LEO main compounds, such as linalool, linalyl acetate, 1,8-cineole, and terpinen-4-ol, were also investigated by MTT and flow cytometry analysis. The set of obtained results showed that LEO treatments induced apoptosis in a dose-dependent, but not time-dependent, manner on HL60 cells, while among LEO main compounds, both terpinen-4-ol and linalyl acetate were able to induce apoptosis.Recent scientific investigations have reported a number of essential oils to interfere with intracellular signalling pathways and to induce apoptosis in different cancer cell types. In this paper, Lavandin Essential Oil (LEO), a natural sterile hybrid obtained by cross-breeding  It has been well established that natural compounds are a source of new molecules of potential pharmaceutical interest . The chePlants containing EOs are only a small percentage of the wider plant molecule population; these species are defined as aromatic plants and are distributed all over the world . The genThe main constituents of EOs are monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, oxygenated monoterepenes, and esters .The numerous medicinal benefits of EOs have been widely demonstrated. They have therapeutic effects and pharmaceutical uses in cardiovascular disease, cancer, and diabetes, alongside applications in skin penetration enhancement, antimicrobial, massage therapy, neuroprotection, and aromatherapy, as well as anti-aging properties . In partCurrent studies show how EOs can affect intracellular signalling pathways, inducing apoptosis or autophagy in different cancer cells models, thus representing excellent candidates for the treatment and prevention of cancer development ,14.Apoptosis, a type of programmed cell death characterized by several morphological and biochemical features, such as plasma membrane blebbing, cell shrinkage, chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies , plays aL. angustifolia have been reported to possess cytotoxic and proapoptotic activities on two human cancer cell lines, Hela and MCF-7 cells [Three different extracts and the essential oil of -7 cells . Sobral -7 cells , in a re-7 cells ,14, and -7 cells  reportedAs reported in our previous work , LavandiThe aim of this study was to evaluate the possible apoptotic activity induced by pure LEO and its most abundant components on HL60 cells. Techniques and assays such as MTT, flow cytometry, Western blot, immunofluorescence, and scanning (SEM) and transmission (TEM) electron microscopy were used.50 values of the LEO treatments. The data are shown as the mean ± standard deviation (SD) of three individual experiments. After 24 h of LEO treatment, the EC50 was 117.66 ± 5.50 µg/mL. No meaningful variation concerning EC50 values after 48 and 72 h  occurred. The EC50 for the positive puromycin control was 0.57 ± 0.05 µg/mL.An MTT assay was performed to evaluate the cell cytotoxicity induced by LEO and treatment with LEO’s main compounds . Based on the results obtained by the MTT assays, the reduced cell viability of HL60 was further investigated to assess cell death and detect if it was related to the apoptotic process. HL60 samples treated for 24 h with LEO were processed for flow cytometry, and the presence of Annexin V-FITC/PI was investigated a–b. As sTo confirm the flow cytometry results, an apoptosis assay using fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst 33,342 was carried out by confocal microscopy analysis. In The use of electron microscopy technique is considered the “gold standard” method for the identification of apoptotic cells . The ultTo define the HL60 apoptotic process, we investigated cleaved caspase-3 expression. Data indicated that after 24 h of LEO treatment, the cleaved caspase-3 monoclonal antibody recognized two bands (17 kDa and 19 kDa), which correspond to the large and small fragments of activated caspase-3 resulting from the cleavage adjacent to Asp175 in the caspase-3 aminoacidic sequence. β-actin expression served as an internal control, while the puromycin was used as a positive control to confirm the activation of caspase-3 a.Confocal microscopy immunofluorescence analysis confirmed cleaved-caspase-3 expression in accordance with the Western blotting analysis b–d. In D50 values. Main cytotoxic compounds that resulted were terpinen-4-ol and linalyl acetate with an EC50 of 6.30 ± 0.7 µg/mL and 4.93 ± 0.22 µg/mL, respectively. Linalool and 1,8-cineole were revealed to be less cytotoxic on HL60 cells, thus demonstrating the mortality of half-cell populations with higher doses (>30 µg/mL for both of the compounds).In our previous work , we inveFor LEO treatments, the reduced cell viability of HL60 was further investigated with flow cytometry. The presence of Annexin V-FITC/PI staining was carried out to test the potential role of the main LEO compounds in the apoptotic process .In It has been well established that numerous natural compounds have beneficial effects on human health, and, in some cases, they are also able to induce apoptosis in different cancer cells; such compounds have possible applications in cancer therapy. Among plant compounds, some EOs and their constituents show antimutagenic, antiproliferative, antioxidant, and detoxifying activities ,25.Origano onites EO has been studied in vitro and in vivo by testing its antiproliferative activity on CT26 and HT-29 colon cancer cells, and an apoptosis-related mechanism was detected in these studies [Blowman and coworkers  reviewed50 values obtained at different times of incubation did not show significant differences, ranging from 117.66 ± 5.50 µg/mL after 24 h to 111.00 ± 1.73 µg/mL after 72 h of treatment.As we already reported , LEO maiL. angustifolia EO have reported cytotoxic activity on different cancer cell lines, with EC50 values of 80.62 ± 1.04 µg/mL on Hela cells and 88.90 ± 1.71 µg/mL on A549, confirming the high cytotoxic properties of the EOs from the Lamiaceae family on cancer cells, as observed in our results [L. angustifolia Mill.) EOs at a low concentration and with minimum exposure time.Previous investigations on  results  determinL angustifolia EO were investigate on human prostate cancer cells, showing potent cytotoxicity against both DU145 and PC-3 cell lines, with an EC50 0.199% ± 0.026% and 0.037% ± 0.011% (v/v), respectively [Furthermore, the therapeutic effects of ectively .In the present paper, the observed cell viability reduction is due to the activation of an apoptotic process as a consequence of LEO treatments.Apoptosis is a programmed and tightly controlled type of cell death characterized by distinct morphological features, such as nuclear condensation, cell shrinkage, membrane blebbing, DNA fragmentation, and apoptotic bodies as a consequence of cell breakdown . ApoptosL. angustifolia than that inducted by its EO, for which the apoptotic rate was 27.4% with 400 µg/mL of treatment after 48 h. In prostate cancer cells, treatment with 0.05% (v/v) for 48 h determined an apoptotic rate (early and late) of 74.76% for PC3 cells and 10.64% for DU145 cells [Tayarani-Najaran et al.  found hi45 cells .Electron microscopy investigations allowed us to define the morphological alterations after LEO treatments and clearly showed the occurrence of the apoptotic process. The morphological changes accompanying different apoptotic stages were evinced by cell shrinkage (nucleus and cytoplasm condensation), karyorrhexis , and the formation of apoptotic bodies, in full agreement with the previously reported data .Most of the morphological modifications that occur in the apoptotic process are regulated by caspase family members, cysteine proteases consisting of “initiator” caspases and “executioner” caspases responsible for activating the proteins directly involved in programmed cell death . Cleaved50 values obtained after 24 h of treatment with these compounds revealed that relevant activity was obtained by terpinen-4-ol (6.30 ± 0.7 µg/mL) and by lynalyl acetate (4.93 ± 0.22 µg/mL). Linalool and 1,8-cineole had an EC50 > 30 µg/mLThe antiproliferative and apoptosis induction effects of LEO may be due to its composition ,18,35. ETo define if the apoptotic effects observed in HL60 were related to LEO’s main compounds, investigations were carried out via flow cytometry techniques. After 24 h of treatments, terpinen-4-ol and linalyl acetate showed high apoptotic rates , while minor apoptotic induction effects were observed by linalool and 1,8-cineole .50 of 30 μM, by both autophagy and apoptosis processes, whereas no activity was found in U937 cells [Terpinen-4-ol exhibited cytotoxicity in human leukemic HL-60 cells, with an EC37 cells . As desc37 cells .In HCT-116, a combination of linalyl acetate, terpineol, and camphor determined the induction of the apoptotic process with greater efficacy than observed in normal human intestinal cells . Zhao etLikewise, 1,8-cineole treatment on Molt 4B and HL60 cells induced DNA fragmentation and specific inductions of apoptosis in a dose-dependent (from 5 to 10 µM) and time-dependent (from 12 h to 48 h) manner . In RKO Lavandula x intermedia “Grosso”). Linalyl acetate (CAS 115-95-7), terpinen-4-ol (CAS 562-74-3), linalool (CAS 78-70-6), and 1,8-cineole (CAS 470-82-6) were purchased from Merck KGaA .The LEO used in the present experiments  was a kind gift of Azienda Agricola Podere dell’Arco  as a steam distilled sample obtained from a cultivar of lavandin , were maintained in an RPMI-1640 medium supplemented with 10% foetal bovine serum, 2 mM l-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin and incubated at 37 °C in a humidified atmosphere with 5% CO2.Human promyelocytic leukemia cells HL60, obtained from ATCC4 cells/well in complete RPMI medium were seeded in a 96-well micro plate. LEO was previously dissolved at a concentration of 1 g/mL (w/v) in dimethyl sulfoxide (DMSO), and ten different concentrations  were used for calculating the EC50 value. Pure compounds were dissolved in DMSO, and ten different concentrations were used . Puromycin was used as a positive control , and DMSO at 0.01% was used as a solvent control. After 24, 48, and 72 h for pure compounds, the medium containing the treatments was removed, and 100 µL of MTT solution (0.5 mg/mL) was added to each well and incubated in the dark at 37 °C for 3 h. The formazan crystals were dissolved in 100 µl of DMSO, and the absorbance was measured at 570 nm. The concentration at which a substance exerts half of its maximal response values (EC50) was calculated by using AAT Bioquest, Inc. .The cytotoxicity effects of LEO and pure compounds were investigated by an MTT-assay . 2 × 1045 cells/mL density onto a six well plate. After 24 h of incubation, the cells were treated with LEO at 0.2 mg/mL and with pure compounds at 77.1 µg/mL for terpinen-4-ol, linalool, and 1,8-cienole, and at 98.1 µg/mL for linalyl acetate. Cells grown in media containing an equivalent amount of DMSO served as the solvent control. After 24 h, the cells were stained with an Annexin V-FITC conjugate and propidium iodide, and the percentage of apoptotic, necrotic, and living cells was determined according to the protocol provided by the Annexin V-FITC and PI apoptosis kit. The cells’ emitted fluorescence was analyzed by flow cytometry  through the NovoExpress 1.3.0 software , acquiring 1 × 104 events per sample using the population plot “dot plot”, where each point corresponds to a single event with a specific fluorescence signal in reference to the axes; Annexin V-FITC green fluorescence in abscissa vs. PE red fluorescence in ordinate. The experiments were repeated three times.Apoptosis detection was performed by using an Annexin V-FITC and PI apoptosis kit . HL60 cells were plated at a 3 × 1050 concentrations for 24 h, the samples were centrifuged at 2000 rpm to collect cells and apoptotic bodies, and staining was performed with Propidium iodide (20 µg/mL), Fluorescein diacetate (30 µg/mL), and Hoechst 33,342 (20 µg/mL) in an RPMI medium at 37 °C for 5 m. After incubation, samples were mounted on slides and observed by a Zeiss LSM 710 Confocal Microscope  to capture images.After LEO treatments, HL60 cells were stained to detect apoptosis and necrosis. After treatments with LEO and puromycin at EC5 cells/mL density onto a six well plate and incubated for 24 h in appropriate culture conditions.For the scanning and transmission electron microscopy analysis, HL60 cells were seeded at a 3 × 1050 concentrations for 24 h, the cells were collected in tubes, washed with PBS, and fixed with 4% paraformaldehyde and 5% glutaraldehyde, pH 7.2, in a 0.1 M cacodylate buffer for 1 h at 4 °C [After treatments with LEO and puromycin at EC at 4 °C . After r2 in a Balzers Union CPD 020, sputter-coated with gold in a Balzers MED 010 unit and observed by a JEOL JSM 5200 electron microscope .For Scanning Electron Microscopy (SEM), cells were dried by the critical point method using COFor Transmission Electron Microscopy (TEM), samples were fixed and dehydrated as described above and embedded in an Epon mixture resin. Thin sections (50–70 nm) were cut with Reichert Ultracut  and LKB Nova ultramicrotomes  using a diamond knife, collected on copper grids, stained with uranyl acetate and lead citrate, and observed by a JEOL 1200EX II electron microscope.6 cells/mL density onto a petri dish and incubated for 24 h. After treatments with LEO and puromycin at EC50 concentrations, the cells were lysed with a RIPA buffer on ice for 30 m. Protein concentrations were determined by the Bradford test [HL60 cells were seeded at a 1 × 10ord test . Samples50 concentrations for 24 h, cells were processed, as reported in the literature [® 488 Goat Anti-Mouse IgG Antibody  diluted to 1:100 in a blocking buffer. For evaluation of possible unspecific staining, control samples were prepared by omitting the primary antibody. After three washings in PBS, nucleic acid staining was obtained by incubation with Fluoroshield with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) , and then samples were placed. The images were captured by a Zeiss LSM 710 Confocal Microscope (Carl Zeiss, Oberkochen, Germany.Immunofluorescence observations were performed to investigate cleaved caspase-3 expression in the LEO treated cell. After treatments with LEO and puromycin at ECterature , and subIn the present paper, a multidisciplinary research approach was performed to study the effects of LEO on HL60 human leukemia cells. Taken together, the obtained results clearly show that LEO treatments induced apoptosis in a dose- and not time-dependent manner, and, among the main LEO compounds, both terpinen-4-ol and linalyl acetate were able to induce apoptosis. Our findings open interesting possibilities for further deeper investigations into such compounds. In our laboratory, experimental work is being done on other human cancer cell lines.


Multidrug resistance (MDR) is the main obstacle in anticancer therapy. The use of drug combinations to circumvent tumor resistance is a well-established principle in the clinic. Among the therapeutic targets, glycoprotein-P (P-gp), an energy-dependent transmembrane efflux pump responsible for modulating MDR, is highlighted. Many pharmacological studies report the ability of calcium channel blockers to reverse tumor resistance to chemotherapy drugs. Isolated for the first time from parsley, the phenylpropanoid apiole is described as a potent calcium channel inhibitor. Taking this into account, herein, the ability of apiole to potentiate the action of well-established chemotherapeutics in the clinic, as well as the compound’s relationship with the reversal of the resistance phenomenon by blocking P-gp, is reported. The association of apiole with both chemotherapeutic drugs doxorubicin and vincristine resulted in synergistic effect, in a concentration-dependent manner, as evaluated by the concentration reduction index. Molecular docking analysis demonstrated the affinity between apiole and the active site of P-gp, corroborating the inhibitory effect. Moreover, apiole demonstrated druglikeness, according to ADME analysis. In conclusion, apiole possibly blocks the active P-gp site, with strong binding energy, which, in turn, inhibits doxorubicin and vincristine efflux, increasing the antiproliferative response of these chemotherapeutic agents. Including more than 100 different diseases, cancer is characterized by disordered cells growth with the ability to invade tissues and organs; some types of tumor cells can also spread to other body parts in a process called metastasis  at 37 °C with 5% CO2.Human tumor cell lines  were kindly donated by the National Cancer Institute at Frederick MA-USA. Stock cultures were grown in complete medium [RPMI 1640 medium supplemented with 5% fetal bovine serum and 1% penicillin:streptomycin mixture (1000 U∙mL®—Europharma) and vincristine (Tecnocris Sulfato de Vincristina®—Zodiac) were prepared aseptically in DMSO (100 mg/mL) and diluted in in complete medium to afford the final concentration. The final DMSO concentration (≤0.25%) in the experiments did not affect cell viability [, incubated for 24 h, treated with apiole , doxorubicin  and vincristine , in triplicate, and then incubated for 48 h at 37 °C in 5% CO2. A second plate, denominated T0, was prepared to infer the absorbance value of untreated cells at the moment of sample addition. Untreated (T0 and T1 plates) and treated (T1 plates) cells were fixed with 50% trichloroacetic acid and stained with sulforhodamine dye (0.4% in acetic acid 1%). Absorbance was recorded at 540 nm using a microplate reader . Using the absorbance values, the cell growth (%) for each cell line, at each sample concentration, was calculated considering at 100% of cell growth the difference between the absorbances of untreated cells after 48 h incubation (T1) and at the sample addition moment (T0). The curve cell growth vs. sample concentration was plotted and GI50 (concentration required for 50% growth inhibition) was calculated by sigmoidal regression using the Origin 8.0 software  [Briefly, the tumor cells were seeded in 96-well plates  .4 cells/mL, respectively, 100 µL/well). After 24 h of incubation, NCI/ADR-RES cell was treated doxorubicin  associated with apiole at 10, 100 µM and 1 mM assay or vincristine  associated with apiole at 25, 50 and 100 µM. For treatment of OVCAR-3 cells, both doxorubicin  and vincristine  were associated with apiole at 100 µM. A T0 plate was prepared for each experiment. After 48h-exposure, cell proliferation was determined as described for the antiproliferative activity evaluation.Following the protocol already described for the antiproliferative activity evaluation, NCI/ADR-RES and OVCAR-3 cells were seeded in 96-well plates  was calculated as CRI = GIbination .® 2018; small-molecule drug discovery suite) using Protein Preparation Wizard (Epik pH: 7.4 ± 0.5 and OPLS3e force field). The grid was defined by 20 Å × 20 Å × 20 Å box centred in the central ligand QZ59 position . The ligand docking was carried out by replacing the QZ59 with Apiole, using all atoms for docking and hydrogen atoms added, considering pH 7.The target (P-gp) for molecular docking was selected in the RCSB PDB (Protein Data Bank)—accession code 3G60, chain A, with 4.40 Å resolution. OpenBabel Cheminformatics tools of ChemInfo  was usedThe apiole structure was also submitted to ADME analysis, in order to verify the compound’s physico-chemical properties, solubility, lipophilicity, pharmacokinetics parameters  and druglikeness, following the medicinal chemistry alert .®. Comparisons among groups (three or more), considering only one independent variable/factor, were performed using One-way ANOVA followed by Tukey post-hoc tests. The results were expressed as the mean ± SD and the level of significance was 5 %.Statistical analysis was performed using GraphPad Prism


Crassostrea gigas, and their possible immune functions are studied. Oyster α/β integrins exhibit a higher degree of sequence and structural variability than the members from Homo sapiens and Drosophila melanogaster. The analysis reveals that oyster RGD- and laminin-binding receptor homologs are present in the phylogenetic tree of α integrins, but the other six oyster α integrins mainly form a species-specific branch; meanwhile, oyster β integrins are clustered with insect β integrins but distinct from a member from the mollusk Biomphalaria glabrata. Although phylogenetically lacking the important α integrin branches of LDV-binding, PS3-type, and αI-containing integrins, oyster integrins can bind to most ECM ligands, including RGDCP, LDVCP, GFOGERCP, and laminin protein in a distinct binding pattern. Besides, oyster integrins are distributed in different hemocyte subpopulations, while only specific integrins are selectively involved in hemocyte phagocytosis, migration, and encapsulation, and some of them participate in more than one immune response in a sophisticated pattern. Especially, oyster β integrins are arranged in the core to mediate complex immune responses, unlike the counterparts in humans that mainly depend on αI-containing integrins to incite immune reactions. This study represents the first comprehensive attempt to reveal the structural and evolutionary features of the integrin family and their involvement in cellular immune responses in the non-model invertebrate C. gigas and sheds light on the characteristics specific to the innate immune system in the integrin family.Studies on invertebrate immune receptors can provide insights into characteristics specific to innate immune system. Here, eight α and three β integrins are identified from an invertebrate, the Pacific oyster  Integrins are α-β heterodimeric cell surface receptors that exert various cell adhesion-related functions in multiple biological processes –5. They Drosophila melanogaster , a sessile filter feeder exposed to a wide range of biotic and abiotic stresses, is an attractive model to study the diversity of immune receptor families and the mediation of associated immune responses in invertebrates  to investigate their functional characteristics during the ligand binding process; and (3) to determine their mediation mechanism in different cellular immune responses, and to clarify their possible functional patterns. The results will provide systematic data to improve our understanding of the classification, structure, and evolutionary characteristics of the integrin family in a marine invertebrate animal and thus add to the evidence essential for the diversity and specificity of immune responses in invertebrates.Although information regarding the evolutionary and functional relationships among invertebrate integrins is not clear, and limited by the few genome-sequenced species, more and more integrin family members are identified in non-model invertebrates, such as in the Mollusca, which is the biggest phylum in the marine animal kingdom with the most species. The molecular aspects of some integrins in mollusks such as tagnalis , Mytilusrossulus , 41, B. glabrata , 42, Mytincialis , and C. C. gigas , 44–46 htebrates , 48. TheC. gigas genome  using HMMER3.1 software (http://smart.embl-heidelberg.de/). The presence/absence of the transmembrane region was evaluated by TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/).Because the reported integrins in human and drosophila commonly contain conserved domains with two to five extracellular Intα domains (beta-propeller repeats) and an integrin_alpha2 (INA) in α integrins, or an extracellular integrin_beta (INB)  in β integrins, in addition to a transmembrane domain in both α and β integrins , oyster software  with a mD. melanogaster, and eighteen α and eight β integrins from H. sapiens were retrieved from NCBI (http://www.ncbi.nlm.nih.gov). The information regarding integrins and GenBank accession numbers had been shown in http://www.bio-soft.net/sms/) (http://itol.embl.de/)  . The MEGmbl.de/) .2, 3.9 g/L MgCl2, 1 g/L MgSO4)  (1 mg/mL) after 12 h were included in the LPS stimulation group, and the other untreated oysters were used in the blank control group. Twenty individuals were randomly sampled from each group, and the hemolymph samples (1 mL per oyster) were collected from oyster hematococoel using an injection syringe. Afterwards, the hemocytes were pelleted from the hemolymph by centrifugation at 800 g for 10 min at 4°C. The hemocytes were transferred and resuspended in modified L15 medium  , after wTotal RNA was extracted from oysters using the Trizol reagent (Invitrogen), and cDNA strands were synthesized for use as templates by M-MLV reverse transcriptase (Promega), according to the manufacturer's instructions. The partial cDNA sequences of oyster integrin genes  were ampEscherichia coli Rosetta (DE3) system (TransGen Biotech). Nickel affinity column chromatography was employed to purify recombinant His-tagged integrin protein fragments (The Polymerase Chain Reaction (PCR) products of partial cDNA sequences of oyster integrin genes, mainly the extracellular part of integrin protein, with 5′ BamHI and 3′ EcoRI restriction site, were integrated into the pET-30a expression vector (Novagen) and expressed in the ragments . The purPolyclonal antibodies were prepared as described previously . Briefly2 conversion, after which heatmaps were drawn to display the expression patterns of integrin family genes. The α-β integrin pairings were predicted according to heatmap clusters of gene co-expression, based on Pearson correlation coefficients (The available reads per kilobase million (RPKM) values of integrin genes for the stages of tissue expression and development were obtained from the previous transcriptome data, released by the oyster genome project . Fragmenficients .Peptide ligands, including RGD-containing peptide (RGDCP), LDV-containing peptide (LDVCP), GFOGER-containing peptide (GFOGERCP), FITC labeled-RGDCP, LDVCP, and GFOGERCP with a purity of >95% were synthesized by Sangon Biotech . The protein ligand of laminin was purchased from Abcam . All were stored at −20°C before use.The binding activities of the recombinant integrins toward four ligands, including RGDCP, LDVCP, GFOGERCP, and laminin proteins were detected on a BIAcore T200 Surface Plasmon Resonance (SPR) instrument . The detailed setups of experimental instrument were performed as described with modifications . HBS-EP 6 cells/mL) in modified L15 medium was seeded onto positively charged glass slides for 30-min cell adhesion and fixed with 4% paraformaldehyde prepared in PBS for 15 min at room temperature, followed by blocking with 3% BSA solution (in PBS) for 1 h. After extensively washing the slides, the hemocytes were incubated with a mouse-anti integrin antibody solution at a dilution of 1:250 (in 3% BSA solution) for 1 h. The slides were washed with PBS, after which the hemocytes were incubated with the Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (Abcam) solution at a dilution of 1:250 (in 3% BSA solution), for another 1 h. Finally, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and observed under a laser confocal scanning microscope (Carl Zeiss LSM 710).To detect the subcellular localization of integrin proteins, 20 μL of hemocytes (106 cells/mL) and separately incubated with FITC-labeled RGDCP, LDVCP, and GFOGERCP (0.01 mg/mL) for 30 min. After washing the hemocytes extensively with modified L15 medium, they were fixed with 4% paraformaldehyde on glass slides and stained with DAPI, followed by observation under a laser confocal scanning microscope (Carl Zeiss LSM 710). The percentage of hemocytes stained with FITC was detected by flow cytometry . To block the binding of hemocytes to FITC-labeled ligands, samples in the blocking group were incubated with four different mouse-anti integrin antibodies, including three antibodies against the α integrins (Cgα1~3) and one β integrin (Cgβ1) (diluted by 1:100 in modified L15 medium). There were three replicates for each sample.An immunofluorescence assay was performed to detect the binding activity of hemocytes to three FITC-labeled integrin ligands. First, the collected hemocytes were resuspended in modified L15 medium , and then incubated with 1 mg/mL FITC (Sigma-Aldrich) overnight. After the washing of unbound FITC with PBS buffer for three times, the FITC-labeled bacteria were prepared for phagocytosis assay. Hemocytes  were incubated with FITC-labeled E. coli  at room temperature for 1 h to incite phagocytosis, and the unphagocytosed bacteria were washed by modified L15 medium for three times. The phagocytosis rate was finally detected by flow cytometry  (Cgα1~3) and one β integrin protein (Cgβ1) for 1 h prior to the incubation with FITC-labeled E. coli for cell phagocytosis. There were three replicates for each sample.The phagocytosis assay was conducted according to the method described previously . FirstlyCSArial) . For the6 cells/mL). The mixture was added into a well in a 1% agarose-coated cell culture plate (Costar) and incubated at 18°C for 6 h. The encapsulation of the agarose beads was observed under a microscope after incubation, and encapsulation rates were calculated as described previously  was added into the suspension to label the hemocytes for 30 min at room temperature. After washing the unbound Calcein AM, 50 μL of labeled hemocytes were mixed with 150 μL of modified L15 medium containing 10% fetal bovine serum (TransGen Biotech). The fluorescence of each well was measured to indicate the total number of hemocytes . After 60 min, the undersides of inserts and the receiver were carefully washed and swabbed with modified L15 medium to remove all migrated hemocytes. Afterward, the fluorescence measurement of each well was repeated to indicate the total number of non-migrant cells . The migration rate was calculated after all trials were conducted with three replicates . Negative controls employed the hemocytes that were seeded into the wells in which FBS was omitted from the modified L15 medium. For the blocking group, the migration rates of hemocytes were determined after hemocytes were incubated with the four integrin ligands (0.01 mg/mL) or the four mouse-anti integrin antibodies (diluted by 1:100 in modified L15 medium) for 1 h.Cell migration was surveyed using EMD Millipore MultiScreen™ 96-well assay plates , based on the method described previously . Brieflyt-test was performed for the comparisons conducted between groups; *stands for statistical significance (p < 0.05) and **stands for extremely statistical significance (p < 0.01).Results were shown as mean ± S.D values. The two-sample Student's C. gigas  in the extracellular and intracellular regions, respectively  and the other type containing both α helices and β sheets (Cgα1~4). The tridimensional structure of INB domains showed that the conserved amino acid sequences appeared to be mainly located in the β sheet regions and revealed a remarkable conservation of the central structure with four to six β sheets that were surrounded by six to eight α helices. The modeled tridimensional structure of Cgβ1_INB2 formed the only conserved central structure of the INB domain, while Cgβ1_INB1 and Cgβ3_INB contained six β sheets in their N-terminus, and Cgβ2_INB contained five β sheets and one α helix in the C-terminus, in addition to the conserved central structure of the INB domain (As the lengths of INA ~146–499 aa) and INB (~225–354 aa) domains from oyster integrins were much more varied and shorter than human integrins  were clustered with the insecta βV and were distantly related to the β integrin from the mollusk species B. glabrata  and two β integrin genes (Cgβ1 and Cgβ2) as one subgroup of genes were highly expressed before gastrulation (Cgβ3) and three α integrin genes (Cgα4~6) were expressed after the D7 developmental stage  were highly expressed at the early and late developmental stages, which formed another two distinct subgroups  had relatively low expression levels in all tissues  had expression levels of >100 RPKM in hemocytes, whereas these genes showed expression levels of <100 RPKM in other tissues  and Cgβ1 were up-regulated 6 and 24 h after LPS stimulation , and Cgβ2 might co-express with Cgα1 and Cgα8 (the blue continuous lines), while Cgβ3 might co-express with Cgα4, Cgα5, and Cgα6 (the green continuous lines). The pairing pattern indicated a complex coordination relationship between oyster α and β integrins in different tissues or development stages during their mediation to various responses , 7.06% in Cgα2 group (p < 0.05), 4.04% in Cgα3 group (p < 0.01), and 3.83% in Cgβ1 group (p < 0.01). Meanwhile, the antibodies to Cgα3 and Cgβ1 reduced significantly the percentage of hemocytes that bound the FITC-labeled GFOGERCP from 9.73% in blocking control to 4.52% in Cgα3 group (p < 0.01) and 6.17% in Cgβ1 group (p < 0.01). In addition, the percentage of hemocytes that bound to FITC-labeled LDVCP were also significantly reduced after the blocking of Cgβ1 antibodies, compared to the percentage bound to the blocking control . Collectively, the integrin ligand-binding activities of hemocytes were significantly inhibited by specific integrin antibodies, which confirmed the binding abilities of oyster integrins to multiple ECM ligands at the cellular level.The binding activities of oyster integrins to different ECM ligands were also investigated at the cellular level. FITC-labeled RGDCP, LDVCP, and GFOGERCP were used as specific probes, and the results from confocal scanning microscopy showed that some of the hemocytes selectively bound to the three FITC-labeled peptides . Furtherng assay . After tCgα2 and Cgα3 bound to the hemocyte subpopulation with a smaller cell size (5–10 μm) and a smaller cytoplasm to nucleus ratio relative to the other hemocyte subpopulations  were detected, and the results were summarized as following.In order to investigate the immune responses mediated by different oyster integrins, the cellular immune reactions including hemocyte phagocytosis, migration, and encapsulation by preincubating oyster hemocytes that were LPS-activated or not with four integrin ligands  and four integrin antibodies (E. coli by hemocytes were significantly blocked by the four integrin ligands (p < 0.05), 14.6% in LDVCP group (p < 0.05), 16.9% in GFOGERCP group (p < 0.05), and 14.7% in laminin protein group (p < 0.05). The greatest inhibitory effect (31.9%) on phagocytosis was observed after the preincubation with RGDCP , 18.3% (p < 0.05), and 17.6% (p < 0.05), respectively, compared to that for the blocking control (20.4%). However, the inhibition was weaker than that observed in blocking assays using the four integrin ligands. Besides, the antibody used for blocking Cgα1 on hemocytes did not significantly inhibit the phagocytosis rate for FITC-labeled E. coli, compared to that of the blocking control . This suggested that Cgα1 may have been unnecessary for phagocytosis of E. coli. Furthermore, the antibodies to Cgβ1 inhibited hemocyte phagocytosis with the highest inhibition rate of 15.2%, compared to the other three α integrin antibodies  after LPS stimulation compared to 20.4% in the blank control group , 3.8% (p < 0.01), 8.0% (p < 0.01), and 18.3% (p < 0.01), respectively, compared to that observed with the blocking control  and 20.3% (p < 0.01), respectively  and Cgα3  did not alter hemocyte migration rates, compared to that for the blocking control (p < 0.05), compared to that for the blank group  was significantly hindered after the blocking by integrin ligands, including the RGDCP, LDVCP, GFOGERCP, and laminin proteins on hemocytes, with migration rates of 23.3% , 10.1% (p < 0.05), and 13.3% (p < 0.05), respectively, compared to the rate of 17.1% for the blocking control (p > 0.05). The antibodies to Cgβ1 and Cgα3 also inhibited significantly the hemocyte encapsulation of beads at encapsulation rates of 13.4% (p < 0.05) and 14.7% (p < 0.05), respectively  and Cgα1  did not significantly inhibit hemocyte encapsulation (p < 0.01), compared to that for the blank group. GFOGERCP inhibited hemocyte encapsulation at a higher inhibition rate of 36.8%  domain in β integrin proteins, they all contain conserved domains, including the extracellular INA domain, Intα domain in the α integrin proteins, or extracellular INB domain in the β integrin proteins, and a short transmembrane domain and cytoplasmic domain in both α and β integrin proteins, as most integrins in other species  family from the sea urchin , including four distinct branches of chordate β, protostome β, insect βV, and early metazoan β, which underlines the phyla lineage-specific features of invertebrate β integrins  that can pair with both Cgβ1 and Cgβ2. Besides, the number of α-β integrin pairings in oysters is obviously more than in N. vectensis (D. melanogaster (C. elegans (Now only a few reports have shown that some single invertebrate integrins are involved in cell immune responses , 82, 83,ectensis , 20, D. nogaster , and C.  elegans , indicatCgα1 appears to occur on both the smaller and larger cytoplasmic-nucleo ratio hemocytes with uniform expression levels, which may be related to its co-expression with both Cgβ1 and Cgβ2 that may make it expressed on at least two different hemocyte subpopulations. Similarly, the localization of Cgβ1 on different hemocyte subpopulations is probably related to its broad ECM ligand-binding ability and cooperation with three α integrins, especially that the co-localization of Cgβ1 and Cgα3 on the hemocyte subpopulation with a smaller cytoplasmic-nucleo ratio is accordant with the results that these two integrins may function as a pair in some hemocytes. Hemocytes with a different cytoplasmic-nucleo ratio, cell size, or cytoplasmic granules play different immune functions in oysters (C. gigas (Circulating hemocytes are thought functionally analogous to vertebrate leukocytes and play crucial roles during host defense in oyster , 85; the oysters , 84. ForE. coli. LDVCP and GFOGERCP exert relatively higher level of inhibitory effects on hemocyte migration, and RGDCP and GFOGERCP show relatively higher levels of inhibitory effects on hemocyte encapsulation for large foreign agents. The results from the inhibitory effects of different integrin antibodies on various immune responses also reveal that Cgβ1, Cgα2, and Cgα3 may be responsible for hemocyte phagocytosis, but the presence of Cgα1 seems to be unnecessary for this process. Cgβ1 and Cgα2 induce hemocyte migration, but Cgα1 and Cgα3 seem to be inactive. Cgβ1 and Cgα3 participate in hemocyte encapsulation, while Cgα1 and Cgα2 are not required. Meantime, it is also found that various integrin members exhibit synergistic cooperation, in order to mediate complex cellular immune responses as in vertebrates (Cgα2, Cgα3, and Cgβ1 are involved in hemocyte phagocytosis, Cgα2 and Cgβ1 are involved in hemocyte migration, and Cgα3 and Cgβ1 participate in hemocyte encapsulation. These findings suggest that the entire oyster integrin family members probably have tightly integrated with the innate immune system, by selecting the specific members to mediate different immune responses in a sophisticated cooperation manner.Our experiments further confirm that oyster integrins indeed participate in multiple cellular immune responses of hemocyte phagocytosis, migration, and encapsulation with a clear function division and sophisticated cooperation. In ligand-blocking experiments, RGDCP and laminin protein show relatively higher levels of inhibitory effects on hemocyte phagocytosis for tebrates , 67. Fortebrates . In the L. stagnalis hemocytes can modulate the phosphorylation of focal adhesion kinase, phagocytosis, and cell spreading (D. melanogaster βV (A. gambiae BINT2 (C. gigas β integrin (Cgβ1 is the only member of the tested integrins appears to be involved in the all detected cellular immune responses, and the antibody to Cgβ1 exhibits a relatively higher level of inhibitory effects on these immune responses than the other three α integrin antibodies. Hence, it is reasonable to infer that the involvement of oyster integrin in multiple cellular immune responses is largely dependent on the presence of β integrins.In mammals, integrins exert essential functions in cellular immunity, which greatly relies on several αI-domain-containing integrins, mainly leukocyte-specific receptors . In invepreading . The D. aster βV , A. gambae BINT2 , and a Cintegrin  have beeintegrin . In our C. gigas genome. Oyster integrins contain conserved domains but also exhibit a high degree of sequence and structural variability compared to those in H. sapiens and D. melanogaster. Eight α integrins are evolutionarily present in RGD-binding receptors, laminin-receptors, and mainly grouped as oyster-specific receptors. Three β integrins are phylogenetically similar to insect βV branch and are distantly related to one β integrin from the mollusk B. glabrata, indicating that oyster α and β integrins may have different evolutionary histories, and both have developed into distinct evolutionary branches. The study on the functional characteristics of oyster integrins involved in ligand-binding activities and cellular immune responses demonstrates that oyster integrins can bind to multiple ECM ligands, including RGDCP, LDVCP, GFOGERCP, and the laminin protein and are involved in hemocyte phagocytosis, migration, and encapsulation, in a sophisticated cooperation pattern and β-dependence (In summary, eight α and three β integrins are identified from the oyster pendence . The prehttp://ensemblgenomes.org. Other raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.Publicly available datasets were analyzed in this study. This data can be found here: oyster_v9, All experiments involving animals reported in this study were approved by the Ethics Committee of the Institute of Oceanology, Chinese Academy of Sciences.ZLv, LQ, LS, and LW conceived and designed the experiments and wrote the manuscript. ZLv performed the experiments and analyzed the data. LQ and QL contributed reagents, materials, and analysis tools. ZLv, WW, and ZLi contributed to the discussion. All authors read and approved the final 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.


Oxidative stress and inflammation are known to be linked to the development of chronic inflammatory conditions, such as type 2 diabetes and cardiovascular disease. Dietary polyphenols have been demonstrated to contain potent bioactivity against specific inflammatory pathways. Rice bran (RB), a by-product generated during the rice milling process, is normally used in animal feed or discarded due to its rancidity. However, RB is known to be abundant in bioactive polyphenols including phenolic acids. This study investigates the antioxidant and anti-inflammatory effects of RB phenolic extracts  on RAW264.7 mouse macrophage cells stimulated with hydrogen peroxide and lipopolysaccharide. Biomarkers of oxidative stress and inflammation such as malondialdehyde (MDA), intracellular reactive oxygen species, nitric oxide and pro-inflammatory cytokines such as interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), interleukin-12, p70 (IL-12p70), and interferon-γ (IFN-γ) were measured in vitro. Treatment with RB extracts significantly decreased the production of MDA, intracellular reactive oxygen species, nitric oxide and pro-inflammatory cytokines  when compared to the control. It is proposed that RB phenolic extracts, via their metal chelating properties and free radical scavenging activity, target pathways of oxidative stress and inflammation resulting in the alleviation of vascular inflammatory mediators. Oxidative stress is generated due to an imbalance between the endogenous antioxidant systems in our body and free radicals such as reactive oxygen species . This coMDA is generated as a result of the peroxidation between polyunsaturated fatty acids , as lipiSeveral synthetic drugs are currently available in the treatment of acute inflammatory states, however, their long-term use has been associated with gastrointestinal, cardiac and renal complications . MoreoveOryza sativa, and has been cultivated around the globe for centuries and is considered a staple food. During rice milling, the rice bran (RB) layer is removed due to its rancidity associated with storage and is primarily used as animal feed. However, RB is known to be abundant in macronutrients and bioactive compounds such as p-coumaric acid, ferulic acid, and caffeic acid [2O2) and lipopolysaccharide (LPS). Biomarkers of oxidative stress such as MDA, intracellular reactive oxygen species, nitric oxide and pro-inflammatory cytokines such as IL-6, MCP-1, IL-10, TNF-α, IL-12p70, and IFN-γ were measured in vitro.Rice is the seed of the grass species eic acid . Althougeic acid , their iUnless otherwise stated, all the chemicals and reagents used in this study were acquired from Sigma-Aldrich  and BD Biosciences .v/v) mixture, and characterized using ultra-high-performance liquid chromatography coupled to an 2,2’-Azino-bis 3-ethylbenzothiazoline-6-sulfonic acid-online system and mass spectrometry (Agilent Technologies) [Stabilization of RB was previously conducted by SunRice, Leeton, Australia using a drum-drying method. Extraction of the RB phenolic compounds was performed using an acetone/water/acetic acid  . Prior t2. Cultured RAW264.7 cells were used before reaching the 8th passage.Experiments were conducted on RAW264.7 cells purchased from Sigma-Aldrich . RAW264.7 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich: D6429) supplemented with 10% fetal bovine serum and 1% 10,000 U/mL penicillin–10 mg/mL streptomycin at 37 °C in 5% CO® Cell Analyzer from Luminex Corporation . RAW264.7 cells were then treated with 200 µL of DMEM complete media containing RB phenolic extract  for 6 h. For the positive control, 5 mM concentration of H2O2 was used, and 0.5% DMSO served as the negative control. Subsequently, all the treatment wells were emptied before incubation with 200 µL of resazurin red solution for an additional 4 h. The absorbance at 570 and 600 nm was measured on a microplate reader  against a resazurin red blank. The percentage of cell viability was calculated. Each treatment was measured in quintuplicate.A resazurin red cytotoxicity assay, as described by Saji, Francis , was uti2O2. For MDA determination, a Lipid Peroxidation (MDA) Assay Kit (Sigma-Aldrich: MAK085) was used according to the manufacturer’s instructions. Briefly, the cell lysate was centrifuged for 10 min at 13,000× g to remove any insoluble material. Subsequently, 600 µL of freshly prepared thiobarbituric acid (TBA) solution was combined with 200 µL of the sample supernatant and the previously prepared MDA standards  to form the MDA–TBA adduct. All the samples were incubated at 95 °C for 1 h and subsequently placed in an ice bath for 10 min. The sensitivity of each sample was enhanced by adding 300 µL of 1-butanol, followed by centrifugation for 3 min at 16,000× g to separate the layers. The 1-butanol layer (the top layer) was then transferred to another tube and evaporated at 55 °C. The remaining residue was dissolved in 200 µL of water and transferred to a 96-well plate for analysis. Absorbance was measured at 532 nm using a microplate reader . Each treatment was measured in quintuplicate.RAW264.7 cells  were seeded into 6-well plates and incubated for 24 h. RB phenolic extracts at varying concentrations  were then incubated with the cells for 6 h. To induce oxidative stress, RAW264.7 cells were subjected to 12 h incubation with a 500 µM concentration of H2O2 for 12 h. Formation of reactive oxygen species was determined by removing the old media and replenishing with 50 µM 2,7-dichlorofluorescein diacetate (Sigma-Aldrich: 35845) and incubating for an additional 30 min in the dark at 37 °C. The assessment of intracellular reactive oxygen species levels was conducted by measuring the fluorescence using a fluorescence microplate reader  at 480 nm excitation and 530 nm emission at 37 °C. Each treatment was measured in octuplicate.RAW264.7 cells were seeded at a density of 50,000 cells/well into a black, clear-bottom 96-well plate and incubated for 24 h. The cells were then incubated with RB extracts  for 6 h. The DMSO-treated cells served as the control. Oxidative stress was induced by treatment with 500 µM concentration of Hg, 10 min, 4 °C). To assess the nitric oxide levels, the cell supernatants and standards (400 µL) were combined with 400 µL of Griess reagent (Sigma-Aldrich: G4410) and incubated for 15 min in the dark at room temperature. Sodium nitrite was used to generate a standard curve . Absorbance was measured at 540 nm using a microplate reader . Each treatment was measured in quintuplicate.RAW264.7 cells at a density of 500,000 cells/well were seeded into a 6-well plate and incubated for 24 h, followed by treatment with 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL RB phenolic extracts for 6 h. DMSO-treated cells served as the control. To induce inflammation, RAW264.7 cells were further subjected to 24 h incubation with 1 µg/mL of LPS. The supernatant was collected by centrifugation  and stored at −20 °C before analysis. The anti-inflammatory effect of RB phenolic extracts on inflammatory cytokines  was determined using a BD™ Cytometric Bead Array Mouse Inflammation Kit (BD Biosciences: 552364). Briefly, 50 μL of the diluted samples (1:40) and standards were combined with 50 μL of the mixed capture beads and 50 μL of the mouse inflammation PE detection reagent. This was then incubated for 2 h in the dark at room temperature. After which, 1 mL of wash buffer was added to each assay tube and centrifuged . Following centrifugation, the supernatant was discarded, and the bead pellet was suspended in 300 μL of fresh wash buffer. Each sample was vortexed for 3–5 s and immediately inspected using a GalliosTM Flow Cytometer . Kaluza Flow Cytometry Analysis Software  was used for conducting the data analysis. Each treatment was measured in quintuplicate.Seeding of RAW264.7 cells  was conducted in a 6-well plate and incubated for 24 h. After confluency was reached, the RAW264.7 cells were treated with 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL RB phenolic extracts and DMSO (control) for 6 h. Inflammation was induced with the addition of LPS (1 µg/mL) and the cells were incubated for an additional 24 h, after which, the supernatant was collected by centrifugation .GraphPad Prism 7 software  was utilized for statistical analysis by one-way analysis of variance (ANOVA), followed by post-hoc Tukey’s multiple comparisons test. Data were considered statistically significant when A time-course study was conducted on a range of incubation periods varying from 2–24 h (data not shown) from which a 6 h incubation period was selected as the optimal time point associated with cell viability. It was observed that RAW264.7 cells, post-exposure to RB phenolic extracts for 6 h, did not display any toxic effects at any of the doses tested (25–1000 µg/mL) when compared to the DMSO control. Therefore, the optimal and physiologically attainable concentrations of RB phenolic extract selected for further examinations were between 25 and 250 µg/mL .p < 0.05), 50 µg/mL (p < 0.05) and 100 µg/mL (p < 0.01) of RB phenolic extract compared to the H2O2 control (MDA concentrations (nmol/mL) were significantly reduced in oxidative stress-induced RAW264.7 cells after treatment with 25 µg/mL ( control . However2O2 showed a significant reduction in the generation of intracellular reactive oxygen species after treatment with 25–250 µg/mL (p < 0.0001) of RB phenolic extract when compared to the control  reduced the expression of nitric oxide levels in LPS-stimulated RAW264.7 cells .p < 0.01) of RB phenolic extract reduced the expression of IL-12p70 and IL-6 under a pro-inflammatory environment. Furthermore, the RB phenolic extract at 100 µg/mL (p < 0.05) and 250 µg/mL (p < 0.01) concentrations reduced IFN-γ compared to the LPS control  after treatment with 25, 50 and 100 µg/mL of RB phenolic extract  and mitogen-activated protein kinase (MAPK) pathways ,26. MacrIn the present study, the stimulation of RAW264.7 cells with LPS was observed to up-regulate several inflammatory markers which were subsequently suppressed by pre-treatment with the RB phenolic extract. This included a significant decrease in the nitric oxide levels and pro-inflammatory cytokine  production in RAW264.7 cells stimulated with LPS . HoweverThe results observed in this study demonstrate that RB phenolic compounds effectively down-regulated the expression of IL-6, IL-12p70, and IFN-γ production in LPS-stimulated RAW264.7 cells. IL-12 is a heterodimeric interleukin that plays an important role in defence against intracellular pathogens . It is cIn the body, phytochemicals are processed as xenobiotics. This is because the human body is only capable of distinguishing between nutrients and constituents that are not nutrients, and is unable to differentiate between beneficial, neutral and toxic compounds. However, many of these phytochemicals are known to activate the adaptive cellular response pathways to oxidative stress by acting as low-dose stressors or pro-oxidants . In the This study highlights that RB phenolic extracts play a significant role in modulating the biomarkers of oxidative stress and inflammation in an oxidative stress/inflammatory environment in vitro. It is believed that the RB phenolic compounds, via their synergistic action, target pathways of inflammation and oxidative stress through their free radical scavenging potential, and modulate pro-inflammatory cytokine expression. The outcomes of this study suggest that RB phenolic extracts may potentially be used as a functional food alternative and could have preventive or therapeutic implications in chronic oxidative stress and inflammatory conditions. Further in vivo investigations that evaluate the bioactivity of RB phenolic extracts in pro-inflammatory populations are warranted.


G12D; Ptf1a-CreER™ (iKC) mouse model, in which Kras is activated postnatally at week 16 upon tamoxifen (TAM) administration. Upon TAM treatment, iKC mice develop pancreatic intraepithelial neoplasia (PanIN) lesions and PC with metastasis at the fourth and fortieth weeks, respectively, and exhibited acinar-to-ductal metaplasia (ADM) and transdifferentiation. Kras activation upregulated the transcription factors Ncoa3, p-cJun and FoxM1, which in turn upregulated expression of transmembrane mucins  and secretory mucin (Muc5Ac). Interestingly, knockdown of KrasG12D in multiple PC cell lines resulted in downregulation of MUC1, MUC4, MUC5AC and MUC16. In addition, iKC mice exhibited ADM and transdifferentiation. Our results show that the iKC mouse more closely mimics human PC development and can be used to investigate pancreatic ductal adenocarcinoma (PDAC) biomarkers, early onset of PDAC, and ADM. The iKC model can also be used for preclinical strategies such as targeting mucin axis alone or in combination with neo-adjuvant, immunotherapeutic approaches and to monitor chemotherapy response.Pancreatic cancer (PC) is acquired postnatally; to mimic this scenario, we developed an inducible Kras Summary: In this study, we developed a tamoxifen-inducible transgenic mouse model that closely mimics the age-related onset of pancreatic cancer (PC). We showed that activation of Kras (iKC) specifically regulates transcription factors that promote mucin expression and acinar to ductal metaplasia, whereas, loss of G12DKras allele in PC cells resulted in the reduction of mucin proteins such as MUC1, MUC4, MUC5AC and MUC16. KRAS, INK4A (p16), Trp53, DPC4 and BRCA2 as well as other risk factors like cigarette smoking, obesity and diabetes mellitus  is a major driver in PDAC development and the most frequently mutated gene, with constitutive activation in 70–90% of PDAC patients  is a lethal malignancy with an extremely poor prognosis, having a 5-year survival rate of less than 9% . The letmellitus . Kirstenpatients . Furtherpatients .G12D;Pdx1-CreKras (KC) and G12D;Trp53R172H;Pdx1-CreKras (KPC) mouse models  are diagnosed with advanced disease. Since pre-neoplastic human samples are rarely available, genetically engineered mouse models (GEMM) can be used to identify and analyze molecules specifically involved in the initiation, progression and advancement of the disease. Since 2003, the e models  have beee models . HoweverKras or inactivation of Trp53 , and the earliest reported cases at approximately age 30 . The risof Trp53 . PanIN lof Trp53 . This eaof Trp53 , thus seKRAS expression in postnatal life using CreER  in PC patients  lead to a significant reduction in proliferation, migration, anchorage-dependent growth and ability to form metastasis in mice   cassette] and activation of G12DKras in pancreatic acinar cells. Thus, tamoxifen administration in the compound strain, G12D;Ptf1a-CreERKras™ (iKC), postnatally, will specifically activate G12DKras only in the acinar compartment, i.e. after acinar cell terminal differentiation and not in ductal and endocrine cells. We examined the pancreas-specific expression of mucin proteins  and the transcriptions factors  involved in their expression. We also investigated if activation of G12DKras in the acinar compartment could drive reprogramming of acinar cells to express the ductal cell marker cytokeratin-19 (CK19).In our study design, we generated an inducible KC mouse model, by intercrossing rasG12D)  with PtfrasG12D)  that havG12DKras in the adult acinar cell lineage, we crossed LSL G12DKras mice with Ptf1a-CreER™ mice and generated the iKC inducible mouse model. In this model, tamoxifen is used to activate Cre, which induces mutant G12DKras expression in acinar cells of 16-week-old animals, while littermate controls are injected with corn oil. Mice were euthanized at 4, 10, 20, 30, 40, and 50 weeks (n=4 for each time point) post-treatment. Time to onset of tumor initiation, progression (degree of disease), and the impact of G12DKras activation on mucin expression were determined. During necropsy, tumors were excised, and distant organs were examined for metastatic lesions. In tamoxifen-treated animals, we observed both micro- and gross metastases to regional lymph nodes, liver, spleen, diaphragm, and mesenteric lymph nodes (data not shown). Pancreata from tamoxifen- and corn-oil-treated mice at each time point were stained with Hematoxylin and Eosin (H&E) to assess differences in histopathology between the groups. Mice treated with tamoxifen displayed pre-neoplastic PanIN I lesions by 4 weeks post-tamoxifen treatment , 40 (P<0.01) and 50 (P<0.01) weeks post-treatment  to 7.33 at 50 weeks (P<0.05) post-treatment . The Muc5Ac average IHC composite score increased from 6.5 at 40 weeks (P<0.05) to 7.0 at 50 weeks (P<0.001) post-treatment . No reactivity was observed in the corn-oil-treated mouse pancreas from 4 to 50 weeks of age , which may indicate weak immunoreactivity of the antibody in normal pancreas  mucin expression in human PC cells .In a previous study, we showed that mosaic  of PDAC . HoweverKras regulates mucin expression, we investigated the expression profile of three transcription factors that have been previously shown by us . We also observed upregulation of p-cJun expression in the nucleus of pancreatic ductal cells of iKC mice from 10 to 50 weeks after tamoxifen injection, with no expression of p-cJun detected in the pancreas of control mice . A similar upregulation of FoxM1 was observed in mice 30 (P<0.01) to 50 (P<0.01) weeks of age, treated with tamoxifen as compared to control mice  , which has allowed us to induce Kras activation postnatally at 16 weeks of life, specifically in acinar cells. This design enabled our study of PanIN progression from the acinar cell lineage and confirmed findings previously made in the KC model under the control of the Pdx1 promoter. The iKC mice developed low-grade PanIN lesions by 4 weeks of treatment, with PDAC and metastasis by 40 weeks post-tamoxifen induction.For the last two decades, PC researchers have debated the pancreatic cell type from which PDAC originates. Several studies were performed to address this mystery but were inconclusive because human pre-neoplastic tissues were not available  and studregions) . Furtherregions) . Similarregions) . Yet, thregions) . To miniKras activation. Thus, like the KC model, the iKC model can be appropriately used to explore genes related to the ADM transdifferentiation process  and was expressed at a basal level in the corn-oil-treated control littermates. The pattern of Muc1 expression in iKC and KC models, and in human PDAC tissues  expression during PDAC pathogenesis arising from the acinar cell lineage. The findings in the iKC model support our previous findings in the KC model, and further implicate aberrant mucin expression as a mechanism associated with PDAC initiation, progression and aggressiveness. We plan to develop mouse syngeneic cell lines mimicking the biological features of PDAC progression, such as PanIN and PDAC, from the iKC model, as demonstrated previously  using specific primer sets. All positive animals (G12DKras and Ptf1a-CreER™) carried the mutant G12DKras allele and Cre recombinase in the pancreas. To activate the Kras (transgenic floxed KrasG12D) alleles in the mouse pancreata postnatally , we intraperitoneally injected iKC mice (three or four mice/group/time point) with tamoxifen  or corn oil on three consecutive days, which led to excision of the LSL cassette in the tamoxifen group. Corn-oil-injected mice acted as control with an intact LSL cassette in the pancreas. Animals were housed five mice in a group and were kept under a 12 h light/dark cycle at 22±1°C and 50±10% relative humidity with ad libitum access to food and water. Animals were euthanized by CO2 asphyxiation followed by cervical dislocation at 4, 10, 20, 30, 40 and 50 weeks after tamoxifen/corn-oil injections, and the pancreata were resected and observed for gross metastatic lesions on distant organs. A separate schema demonstrating the design of the study is shown in Fig. S1. A portion of the pancreas tissue was stored in buffered formalin (10%) for IHC analysis. The rest of the pancreas was flash frozen in liquid nitrogen and stored at -80°C for analysis of mucin gene expression . The animals involved in this proposed study were treated humanely and in all situations in which the mice are used, pain and discomfort were minimized. B6.129-Krastm4Tyj (01XJ6) mice were obtained from the National Cancer Institute Mouse Models of Human Cancers Consortium . The Ptf1a-CreER™ mice were a kind gift from Dr Christopher V. Wright, Vanderbilt University. To generate iKC mice, we crossed pression .® 16 mouse tail DNA purification kit  according to the manufacturer's protocol. Genotyping PCR was performed using 100 ng of genomic DNA in 2x PCR master mix  and 5 pmol of each forward and reverse primers of Kras and Ptf1a-CreER genes: forward (Kras), 5′-CCT TTA CAA GCG CAC GCA GAC TGT AGA-3′; reverse (Kras), 5′-AGC TAG CCA CCA TGG CTT GAG TAA GTC TGC A-3′; forward (Ptf1a-CreER), 5′-GTG ATC ATG CAA GCT GGT GGC T-3′; reverse (Ptf1a-CreER), 5′-CCG ACT TGA CGT AGC AAG CAA C-3′ in a final volume of 20 μl. PCR conditions consisted of 5 min at 95°C, followed by 40 cycles at 94°C for 1 min, annealing at 59°C for 2 min, followed by extension at 72°C for 45 s. A final extension at 72°C was performed for 10 min. PCR products were analyzed using 1.5% agarose gel electrophoresis  at 37°C with 5% COeviously . The humG12D using the mirVana™ miRNA isolation kit  according to the manufacturer's instructions. RNA purity and integrity was determined by spectrophotometry  at λ 260/280 and bioanalyzer microfluid chip assays , respectively , DTT (0.1 M) and 1 µl RNase inhibitor were added to the above mixture, and reactions were incubated at 42°C for 2 min. To extend the reaction, each reaction tube received SuperScript II RT (50 units) and was incubated at 42°C for 50 min. RT-PCR was carried out on 2 μl cDNA in 1× SYBRGreen PCR Master Mix  and primers (MUC5AC/β-actin) in a total volume of 10 μl. PCR conditions consisted of 10 min at 95°C, followed by 40 cycles of annealing and extension at 95°C for 15 s and 60°C for 1 min. Transcript copy numbers were normalized to β-actin, which served as an internal control. PCR products were analyzed using 1.5% agarose gel electrophoresis .G12D) and corn-oil- (unfloxed KrasG12D) administered animals (n=4/group) was fixed in 10% (v/v) buffered formalin  and embedded in paraffin. Sections of 4 μm thickness were mounted on glass slides, deparaffinized in xylenes, hydrated in a decreasing gradient of ethanol, and stained with H&E as described previously , a portion of the pancreas from the F1 progeny of tamoxifen-  solution containing 0.05% Tween® 20 (PBST). Anti-mouse universal secondary antibody conjugated with horseradish peroxidase was added for 30 min. Following three PBST washes, mucin antibodies were detected using, 3,3′-diaminobenzidine  prior to counterstaining with Hemotoxylin. The slides were dehydrated with increasing concentrations of ethanol followed by two xylene washes, and mounted with Permount mounting medium . Slides were observed and scored by a certified pathologist. The differential expression of mucins was scored from 0–3, where 0 was considered to be negative; 1, weak; 2, moderate and 3, strong. The percentage of positive cells for each respective antibody was given a score on a scale of 1–4 . A composite score (0–12) was calculated by multiplying the value of staining intensity by the value representing percentage of positive cells , Muc5Ac , Muc16 , Ncoa3, pcJun and FoxM1, amylase, CK19  and Muc4 , the latter was designed in our lab and developed by GenScript . IHC analysis was performed on pancreatic tissue from different age groups as mentioned above. Briefly, after formalin fixation of tissues for 48 h, sections of 4 μm thickness were placed on slides. The slides were incubated overnight at 56°C and then were washed with xylene to remove paraffin. Tissue sections were rehydrated with decreasing concentrations of ethanol, and endogenous peroxidase activity was quenched with 5% Hve cells .Immunofluoresence was performed as described previously .Immunoblot analysis was performed as described in our previous publications  using tht-test to assess staining patterns for each mucin at each interval (different stage) of PC progression.Results are expressed as mean±standard error of mean (s.e.m.). The statistical significance of differences was analyzed by Student's


Calycera, Orites, and Chusquea plant genera were reported. More than 50% of the 130 isolates showed ≥33% bacterial cell survival at temperatures below zero. Seventy strains of Pseudomonas, Curtobacterium, Janthinobacterium, Stenotrophomonas, Serratia, Brevundimonas, Xanthomonas, Frondihabitans, Arthrobacter, Pseudarthrobacter, Paenarthrobacter, Brachybacterium, Clavibacter, Sporosarcina, Bacillus, Solibacillus, Flavobacterium, and Pedobacter genera were identified by 16S rRNA gene sequence analyses. Ten strains were selected based on psychrotolerance, auxin production, phosphate solubilization, presence of nifH (nitrogenase reductase) and acdS (1-aminocyclopropane-1-carboxylate (ACC) deaminase) genes, and anti-phytopathogenic activities. Two of the three bacterial consortia formulated promoted tomato plant growth under normal and cold stress conditions. The bacterial consortium composed of Pseudomonas sp. TmR5a & Curtobacterium sp. BmP22c that possesses ACC deaminase and ice recrystallization inhibition activities is a promising candidate for future cold stress studies.Cold stress decreases the growth and productivity of agricultural crops. Psychrotolerant plant growth-promoting bacteria (PGPB) may protect and promote plant growth at low temperatures. The aims of this study were to isolate and characterize psychrotolerant PGPB from wild flora of Andes Mountains and Patagonia of Chile and to formulate PGPB consortia. Psychrotolerant strains were isolated from 11 wild plants (rhizosphere and phyllosphere) during winter of 2015. For the first time, bacteria associated with  The production and quality of agricultural crops are decreased by a wide range of abiotic stresses, including cold and high temperatures, drought, and salinity. It is estimated that more than 50% of global yield loss for major agricultural crops is due to abiotic stress . Every yPseudomonas, Bacillus, Serratia, Pantoea, and Paraburkholderia strains on canola, wheat, grapevine, and tomato under cold stress [Bacillus and Brevibacillus strains protects and promotes the growth of rice seedlings under cold stress [Bacillus and Serratia strains promotes the survival of tomato plants subjected to cold stress [In this context, the plant growth-promoting microorganisms play an essential role for plants during their growth and adaptation to the changing environment through some mechanisms, such as nitrogen fixation, phosphate solubilization, modifying plant hormone levels, and improving plant defense responses to biotic and abiotic stresses ,9,10,11.d stress ,25,26,27d stress . Anotherd stress .The application of PGPB consortia is an eco-friendly alternative to agriculture that has the potential to increase crop yield under normal and stressful conditions. In addition, the use of PGPB reduces the application of chemical products, decreasing the environmental impact in agriculture , and increasing the microbial diversity and functionality of agricultural soils with the concomitant production of healthy and safe foods . To searnifH (nitrogenase reductase) and acdS (1-aminocyclopropane-1-carboxylate (ACC) deaminase) genes, and antimicrobial activities to phytopathogens. Three bacterial consortia were selected for tomato growth-promoting assays; two consortia promoted growth of tomato plants. Finally, the ACC deaminase and ice recrystallization inhibition activities allowed the selection of one bacterial consortium composed of Pseudomonas sp. TmR5a & Curtobacterium sp. BmP22c (BC3), which possess diverse plant growth-promoting activities. The BC3 is an attractive candidate for future plant frost protection assays.The aims of this study were to isolate and characterize psychrotolerant PGPB from the wild flora of Chile, and to formulate PGPB consortia. The bioprospecting of psychrotolerant PGPB with novel characteristics was carried out from wild plants that inhabit three areas of the Andes Mountains and one area of the Patagonia of Chile during the winter of 2015. These areas have low temperatures and frosts throughout the year ,34. The L-tryptophan (>99% purity), α-ketobutyrate (97% purity), and 2,4-dinitrophenylhydrazine (97% purity) were purchased from Sigma-Aldrich . Cycloheximide was obtained from United States Biological . Sulfuric acid, toluene, sodium chloride, sodium hydroxide, glucose, sucrose, glycerol, gluconic acid, citric acid, KH2PO4, Na2HPO4, MgSO4 × 7H2O, FeSO4 × 7 H2O, H3BO3, MnSO4 × H2O, ZnSO4 × 7H2O, CuSO4 × 5H2O, MoO3, FeCl3 × 6H2O, and (NH4)2SO4 were obtained from Merck . Yeast extract, Tryptone, Bacto Proteose Peptone No. 3, Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), and Müller-Hinton (MH) were purchased from Difco Laboratories ; 1-aminocyclopropane-1-carboxylate (ACC) was obtained from Calbiochem . Type III AFP was purchased from A/F Protein, Inc. . GoTaq Green Master Mix was purchased from Promega . Bacterial Protein Extraction Reagent (B-PER) and Halt protease inhibitor cocktail were obtained from Thermo Fisher Scientific . Quick Start Bradford Dye was purchased from Bio-Rad Laboratories . Primers ,  Primers  were obtParaburkholderia xenovorans LB400 (positive control for IAA biosynthesis) [Pseudomonas protegens CHA0 (plant growth-promoting bacterium with biocontrol activity) [Escherichia coli JM109  [Achromobacter sp. 188 (positive control for ACC deaminase activity) [Pseudomonas syringae pv. syringae Cc1  bacterial strain and a causal agent of bacterial canker in cherry trees), Pectobacterium carotovorum NCPPB 312  [Clavibacter michiganensis subsp. michiganensis OP3  [Agrobacterium tumefaciens C58C1  [The following model strains were used in this study: nthesis) , Pseudomctivity) ,40, Eschctivity) , and Achctivity) . The phyl crops) , Clavibaathogen) , and Agr plants) . These sHaplopappus sp., Thlaspi sp., and Calycera sp. From the vicinity of El Teniente Mine (34°11′37.9″ S 70°34′29.4″ W), Machalí, Libertador General Bernardo (L.G.B.) O’Higgins Region at 1508 m.a.s.l, two plants were sampled: Baccharis sp. and Gnaphalium sp. From Shangri-La EcoPark (36°53′29″ S 71°28′23″ W), Las Trancas Valley, Ñuble Region at 1550 m.a.s.l, four plants were sampled: Orites sp., Gaultheria sp., Chusquea sp., and Nothofagus sp. in association with the lichen Usnea sp. From Chabunco Park (52°59′19.4″ S 70°48′50.0″ W), Punta Arenas, Magallanes, and Chilean Antarctica (C.A.) The region at 12 m.a.s.l, one plant was sampled: Berberis sp. and from the vicinity of Club Andino Ski Center (53°09′44.1″ S 71°01′27.6″ W) Punta Arenas, Magallanes and C.A. Region at 342 m.a.s.l., one plant was sampled: Nothofagus sp. samples (only phyllosphere). A sampling of rhizosphere (near the root) and phyllosphere  were performed in triplicate (n = 3) for each plant. The samples were kept in a close sterile plastic bag on ice and transported to the laboratory, where the samples were stored at 4 °C until analysis.The wild flora samplings were carried out in four regions of Chile during the winter of 2015 . From thw v−1) and stirred for 1 h in a Multi Reax vibrating shaker . Then, the suspensions were serially diluted  and 100 µL of each dilution were spread on TSA (pH 7) plates, supplemented with cycloheximide (100 µg mL−1) to inhibit the growth of fungi. The plates were incubated at 4 °C for 2 weeks. Morphologically distinct individual colonies were individually seeded on fresh TSA plates at least twice to isolate pure strains. The colonies of pure cultures were grown in TSA medium at room temperature (22 ± 3 °C) for 24–48 h, and stored in 15% glycerol (v v−1) at −20 and −80 °C.For bacterial isolation, the protocols from Kucheryava et al.  and BarrPseudomonas protegens CHA0 (30.7% BCS).The psychrotolerant potential was evaluated with a bacterial cell survival test at subzero temperatures described by Sun et al. , with moFor the identification of the 70 selected psychrotolerant bacteria (≥33% BCS), the 16S rRNA gene was amplified, using the universal primers 27F and 1492R . The extnifH and acdS genes, and finally (iii) antimicrobial activity against phytopathogenic bacteria.For the preliminary selection of bacterial consortia, the following activities were determined. First, (i) auxin production; followed by (ii) phosphate solubilization, and detection of L-tryptophan (500 µg mL−1) for 48 h [g in a Rotina 380R centrifuge . The supernatant (0.5 mL) was mixed with 2 mL of Salkowski’s reagent  and was incubated for 20 min at room temperature before measuring the absorbance at 535 nm, in a Jenway 6320D spectrophotometer . The auxin concentration in each sample was calculated based on a standard curve that ranged from 1 to 20 µg mL−1 of IAA. Paraburkholderia xenovorans LB400 was used as a positive control for IAA production [−1)/Turbidity600nm ≥ 0.5 µg mL−1 and/or BCS ≥50%.Auxin production was determined by Salkowski colorimetric method ,52 for tfor 48 h . SubsequPseudomonas protegens CHA0 was used as a positive control for phosphate solubilization [For the 41 selected bacterial strains, the phosphate solubilization was analyzed by visualizing solubilization halos in Pikovskaya agar medium . Bacterilization . The resnifH (nitrogenase reductase) and acdS (ACC deaminase) genes were carried out using the primers nifH-F-Rösch and nifH-R-Rösch [nifH gene detection was performed with an initial denaturation of DNA at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, alignment at 55 °C for 30 s, and elongation at 72 °C for 30 s, with a final extension at 72 °C for 5 min. The reaction for acdS gene detection started with an initial denaturation of DNA at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 1 min, alignment at 50 °C for 1 min, and elongation at 72 °C for 40 s, with a final extension at 72 °C for 7 min. For detection of both genes, amplification of the 16S rRNA gene was additionally carried out as a positive control. The genomic DNA of P. xenovorans LB400 was used as a positive control for the detection of both genes.The amplification of -R-Rösch , and Deg-R-Rösch , respectTwenty-one bacterial isolates that showed multiple activities/determinations were selected.Pseudomonas syringae pv. syringae Cc1, Pectobacterium carotovorum NCPPB 312, Clavibacter michiganensis subsp. michiganensis OP3, and Agrobacterium tumefaciens C58C1 was evaluated twice per duplicate. Selected bacteria were grown on Yeast Malt  and MH (Difco Laboratories) media for ~12 h at 25 °C. Then the inoculum was adjusted to turbidity at 600 nm of 1, and 100 µL of each isolate were deposited in the center of the plate, air drying and incubated for 72 h at room temperature. The phytopathogenic bacteria were previously grown in YM and MH media for ~12 h and were adjusted to turbidity at 600 nm of 1. Then, 10 µL of each phytopathogenic bacteria were arranged in radial lines surrounding growth of selected bacteria and incubated at room temperature. Pseudomonas protegens CHA0 was used as a positive control for antimicrobial activity.Antimicrobial activities of the 21 selected bacterial strains were carried out by the radial streak method according to Coman et al. , with moTen bacterial isolates that exhibited the highest microbial growth inhibition were selected for the formation of the bacterial consortia.The compatibility tests through a direct method among the 10 selected bacterial strains were carried out twice per duplicate. The selected bacteria were grown in YM and MH media for ~12 h at 25 °C. Then the inocula of the strains were adjusted to turbidity at 600 nm of 0.3 and 0.6. A volume of 100 µL (0.3) were spread throughout the plate and 10 µL (0.6) drops were placed in plates of each strains grown previously and incubated at room temperature. Inhibitions were observed at 24, 48, and 72 h.Three bacterial consortia formulated with two bacterial strains  were selected according to their compatibility  and in accordance with those that presented the highest values of all the activities/determinations.Solanum lycopersicum L. cv. San Pedro) was evaluated. Tomato seeds were germinated for 2 weeks in a pot with peat as substrate. Then, the tomato seedlings were placed individually in pots (0.52 L) with 100 g peat and treated with bacterial consortia every 2 weeks for 2 months in a growth chamber at 25 °C with a photoperiod of 16 h light and 8 h dark, with seven replicates each treatment. The bacterial isolates of each bacterial consortia  were mixed in the same concentration ratio in distilled water, obtaining a final bacterial concentration of 2 × 108 CFU mL−1. The consortia were applied in a foliar and radicular treatment, soaking all the plant with a concentration of 2 × 108 CFU mL−1 [6 CFU g−1 [To determine the plant growth-promoting effect of the three selected bacterial consortia, a pot assay with tomato plants (CFU mL−1  and inoc CFU g−1  in each Solanum lycopersicum L. cv. San Pedro) was evaluated at 14 and 25 °C. A biopriming assay was performed as described by Subramanian et al. [8 CFU mL−1. Sterile distilled water was used as a negative control. The positive control (Nutrisac) was used and diluted according to the manufacturer’s instructions. The disinfected tomato seeds were immersed in 10 mL of the different treatments  for 4 h at 20 °C with shaking at 200 rpm, and then 10 seeds were placed in sterile plates with filter paper soaked with 1 mL of sterile distilled water (3 replicates for each treatment). This test was carried out at two different temperatures, 14 and 25 °C for 12 days in the dark. At the end of the test, the germination percentage and root length were measured in tomato seeds at 14 °C, and complete germination % (root + hypocotyl), root length, and hypocotyl length were evaluated in tomato seeds at 25 °C.To determine the plant growth-promoting effect of the three selected bacterial consortia, an in vitro germination test of tomato seeds  activities were determined.w v−1) and centrifuged at 8000× g for 10 min at 4 °C. A volume of 500 µL of the culture was resuspended in saline solution and incorporated in 4.5 mL of DF (Dworkin and Foster) medium 2SO4 (2 g L−1) as sole N source and glucose (2 g L−1) as the carbon source) for 1–2 days at 30 °C. The bacterial cells were washed three times with saline solution and centrifuged at 8000× g for 10 min at 4 °C. The bacterial pellets were resuspended in 1 mL of saline solution and 100 µL of each inoculum were incorporated in 3 mL of DF medium with 3 mM ACC as sole N source and glucose as carbon source, in triplicate. Growth was evaluated for 5 days at 30 °C.The ACC deaminase enzyme converts ACC into α-ketobutyrate (α-KB) and ammonium. The bacterial strains of selected consortia were analyzed to determine their ability to grow in culture medium with ACC as the sole nitrogen (N) source, according to the procedures described by Penrose and Glick  and Barr−1 h−1 produced in the assays. The procedure described was carried out again for bacteria with positive growth in ACC, to evaluate ACC deaminase activity. A volume of 250 µL of inoculum was incorporated into 7.5 mL of DF medium with 3 mM ACC as sole nitrogen source and glucose as carbon source. On the fourth day, the cells were washed two times with saline solution and centrifuged at 8000× g for 10 min at 4 °C. Bacteria pellets were resuspended in 1 mL of 0.1 M Tris-HCl pH 7.6 and centrifuged at 16,000× g for 5 min, the supernatant was removed, and the pellet was resuspended in 600 µL of 0.1 M Tris-HCl pH 8.5. A volume of 30 µL of toluene was added and stirred for 30 s, and a volume of 100 µL was used to measure the protein concentration by Bradford method [g at room temperature. A volume of 1 mL of supernatant was taken, 800 µL of 0.56 M HCl was added, stirred, and placed in glass tubes to later incorporate 300 µL of 2,4-dinitrophenylhydrazine , stirred and incubated at 30 °C for 30 min. A volume of 2 mL of 2 N NaOH was added and it was stirred. Then, the absorbance at 540 nm was measured on a Multiskan GO spectrophotometer . A standard curve of ɑ-KB from 0 to 2 mM was performed. The µmoles α-KB mg protein−1 h−1 were calculated according to the values of α-KB and the protein concentration (mg mL−1) after an incubation of 15 min.The ACC deaminase activity was determined by measuring µmoles α-KB mg proteind method . A volumg for 10 min at 4 °C and gently resuspended  with 200 µL of B-PER in 2 mL Eppendorf tubes for protein extraction. A volume of 200 µL of sterile distilled water was added and gently homogenized. It was centrifuged at 24,000× g for 20 min at 4 °C. The supernatant was transferred to a fresh Eppendorf tube and 4 µL of a Halt protease inhibitor cocktail (10 µL mL−1) was added. A volume of 100 µL was used to measure the protein concentration by Bradford method [−1 with a solution of B-PER: sterile distilled water (1:1). A volume of 50 µL of protein from each bacterium was mixed with 50 µL of a sucrose solution (60% w v−1) and this volume (100 µL) was placed in a 96-well plate. This step was performed six times for each protein extract of each strain. The plate was placed at −50 °C for 20 min, removed, and observed that all wells were frozen, otherwise, a small blow was provided to accelerate freezing. The plate was then transferred to −6 °C for 48 h, and at 24 h it was verified that the wells were still frozen. Escherichia coli JM109 was used as a negative control for IRI activity. The strain JM109 was grown for 1 week at room temperature plus 3 days at 4 °C. The antifreeze protein AFP type III was used as a positive control, and 30% w v−1 sucrose solution was used as blank. After 48 h, the absorbance at 500 nm was measured in a Multiskan GO spectrophotometer. The bacterial strains that presented a significantly higher absorbance than that obtained with the strain JM109 protein extract were considered IRI positive as reported by Cid et al. [For bacterial strains of selected consortia, IRI activity was evaluated according to the protocol of Cid et al.  and Gilbd method . Proteind et al. .p < 0.05) among the treatments. Data were analyzed using the non-parametric Kruskal–Wallis test if transformational data did not satisfy the assumption of homogeneity of variance. The Kruskal–Wallis test was followed by all pairwise multiple comparisons.One-way ANOVA were used to analyze the main effects. After carrying out one-way ANOVA, the Fisher’s LSD test was used to detect significant differences  and 21 were isolated from the phyllosphere (30%) . Thirty-Pseudarthrobacter, Arthrobacter, Brachybacterium, Curtobacterium, Paenarthrobacter, Frondihabitans, and Clavibacter), followed by Proteobacteria phylum with 43 identified strains from six genera , Firmicutes phylum with six identified strains from three genera , and Bacteroidetes phylum, with two identified strains from two genera (Flavobacterium and Pedobacter). The most abundant isolates corresponded to Pseudomonas (18), Stenotrophomonas (17), Pseudarthrobacter (6), Serratia (5), Arthrobacter (4), and Sporosarcina (4) genera. Strains from Pseudomonas, Stenotrophomonas, Sporosarcina, Curtobacterium, Arthrobacter, and Pseudarthrobacter genera were isolated from rhizosphere and phyllosphere. On the other hand, Serratia, Xanthomonas, Paenarthrobacter, and Janthinobacterium genera were only isolated from rhizosphere, while Solibacillus, Bacillus, Brachybacterium, Frondihabitans, and Clavibacter genera were only isolated from the phyllosphere.The 70 selected isolates were identified through the analysis of the partial 16S rRNA gene sequence . The isoHaplopappus sp. is closely related to Xanthomonas translucens [Berberis sp. is closely related to Clavibacter michiganensis [Of the 70 isolates, only two were closely related to phytopathogenic bacteria. The strain HsR18 that was isolated from the rhizosphere of nslucens ,61. The ganensis ,62,63. B600nm ≥0.5. Forty-one strains were selected based on auxin synthesis or BCS ≥50%. Strains of the same genus with similar activities and origins were removed for the following assays.The capability to synthesize auxins in the presence of the precursor tryptophan was evaluated in the 68 selected strains . It was nifH and the acdS genes were analyzed in 41 selected strains. Fifteen strains showed phosphate solubilization activity at room temperature and 12 of these strains showed phosphate solubilization activity at 4 °C , Pectobacterium carotovorum NCPPB 312 (Pc), Clavibacter michiganensis subsp. michiganensis OP3 (Cmm), and Agrobacterium tumefaciens C58C1 (At) were evaluated for the 21 selected strains. Ten isolates showed activity against Pss, eight against Pc, five against At, and 14 against Cmm (Pss and Pc) were selected for the following studies. Six selected strains belong to Pseudomonas genus , whereas four strains are member of the Actinobacteria phylum   .Pseudomonas sp. TmR7 and Frondihabitans sp. GpP26d, BC2: Pseudomonas sp. CcR1d and Pseudomonas sp. NUR4a, and BC3: Pseudomonas sp. TmR5a and Curtobacterium sp. BmP22c  with 0.59 cm . After tThe plant growth–promotion on tomato seeds by the three selected consortia were analyzed at 25 and 14 °C. For treatments at 25 °C, BC2 and BC3 treatments presented a significant increase in hypocotyl length, with values of 4.26 and 4.24 cm, respectively, compared to the NC (3.15 cm) a,b. The For treatments at 14 °C, the germination % and the root length were analyzed c,d. The Pseudomonas sp. TmR7 & Frondihabitans sp. GpP26d) did not show any plant growth–promoting effect in these assays and, therefore, was not used for the following studies.The BC1  (E. coli JM109).The ice recrystallization inhibition (IRI) activity and the ACC deaminase activity were evaluated in strains that composed the bacterial consortia, to select one bacterial consortium for future cold stress analyses. To determine the antifreeze potential of the bacteria, the IRI activity was evaluated, subjecting protein extracts to temperatures below zero degrees Celsius . PseudomPseudomonas sp. TmR5a (BC3) showed growth under these conditions. Strain TmR5a presented an ACC deaminase activity of 1.516 µmol α-KB mg protein−1 h−1  was evaluated in DF medium with ACC as sole carbon and nitrogen source a. Only Pin−1 h−1 b.Pseudomonas sp. TmR5a & Curtobacterium sp. BmP22c) was selected because it presented all the evaluated activities together.Therefore, BC3 , obtaining a number <103 CFU mL−1 (no isolates on the plate) for Baccharis sp., Nothofagus sp., Orites sp., and Chusquea sp., while 2 × 103 CFU mL−1 (2 isolates) were obtained for Nothofagus sp. in association with the lichen Usnea sp., Haplopappus sp., and Gaultheria sp. The low bacterial number can be partly explained due to the antimicrobial activities of some of these wild plants and the lichen [In the present study, for the isolation of psychrotolerant bacteria, rhizosphere and phyllosphere of 11 wild plants were collected from three areas of the Andes Mountains and one area of Patagonia during winter of the year 2015. In this study, 130 psychrotolerant bacterial isolates were characterized, 79% from the rhizosphere and 21% from the phyllosphere . A highef plants ,64,65,66ultheria ,71,72,73Berberis . In our e lichen ,81,82, ae lichen ,84 underhttp://www.gobernacionlosandes.gov.cl/inicio/ (accessed on 5 February 2021)). The identification by the 16S rRNA gene sequence analysis indicated that these 70 strains belong to 18 different bacterial genera of four bacterial phyla . These bacteria were isolated from Paso Internacional Los Libertadores , which was the highest altitude wild flora sampling site (3149 m.a.s.l.) and presents a winter (from April to October) with temperatures below 0 °C, reaching −20 °C  and the phyllosphere (2). Stenotrophomonas strains genus were also isolated from the rhizosphere (12) and the phyllosphere (5). Pseudomonas is the genus of Gram-negative bacteria with the largest number of recognized species, where more than 220 species have been characterized [Pseudomonas strains have been isolated from each of the ecological niches within plants including roots (rhizosphere), leaves (phyllosphere), and tissues (endosphere) [Pseudomonas strains isolated from Chilean agricultural soils with nematicidal and herbicide-degrading activities have been characterized [Stenotrophomonas genus has been associated with soil and plants [Stenotrophomonas strains isolated from the four regions were closely related to Stenotrophomonas rhizophila species. Stenotrophomonas rhizophila is a rhizosphere-associated species that display antifungal properties [In the present study, ectively . Strainscterized . The ubiosphere) ,109. In cterized ,111. On d plants ,113,114,d plants . In our operties .Pseudomonas syringae and Pectobacterium carotovorum that magnify the stress caused by cold in plants [Agrobacterium tumefaciens that infects a wide range of plants and causes plant tumors called crown galls [Clavibacter michiganensis subsp. michiganensis, which is an important tomato phytopathogen [The search for psychrotolerant PGPB that can exert their role of plant growth-promoting at low temperatures is of high importance for agriculture ,105,106.n plants ,116, Agrwn galls , and Clapathogen ,63,101, pathogen ,121,122.Pseudomonas sp. CcR1d & Pseudomonas sp. NUR4a (BC2) and Pseudomonas sp. TmR5a & Curtobacterium sp. BmP22c (BC3), after 12 days at 14 °C, promoted the germination of tomato seeds by ≥90% compared to 33% control germination, and significantly increased the root lengths  was selected as the most promising consortium due to its plant protective and growth-promoting activities. The BC3 is composed of strains from the rhizosphere of Thlaspi and the phyllosphere of Berberis  for the germination of cotton seeds [Pseudomonas syringae pv. tomato [Pseudomonas and Curtobacterium genera are the most abundant in ecosystems related to poplar, willow, grapevine, rice, and nectarine plants [Bacillus amyloliquefaciens Bk7 and Brevibacillus laterosporus B4, which solubilizes phosphates and produces IAA and siderophores, protects and improves the growth of rice under cold stress [Bacillus cereus AR156, Bacillus subtilis SM21, and Serratia sp. XY21, which produces IAA and has antagonistic activity against phytopathogenic INA bacterium P. syringae, fungi, and oomycetes, improves the survival rate of tomato seedlings subjected to chilling stress (4 °C for 7 days) [In our study, the psychrotolerant bacterial mixture formulated with Berberis . This isnditions . Pseudomd stress ,126. On nditions ,128,129,syringae , and allsyringae ,132,133.on seeds , the pro. tomato , and the. tomato . In addie plants ,139,140,Pseudomonas sp. TmR5a and Curtobacterium sp. BmP22c, which exhibits several plant growth-promoting and protective activities including plant growth-promoting under chilling stress (<15 °C), is an attractive candidate for future applications in agriculture. Additional assays will be carried out to study its protective effect on plants under freezing stress, which are required prior to its evaluation on the field against frost.The selected bacterial consortium formulated with Calycera, Orites, and Chusquea plants; one of the Chusquea strains is a member of BC2. Two bacterial consortia: Pseudomonas sp. CcR1d & Pseudomonas sp. NUR4a (BC2) and Pseudomonas sp. TmR5a & Curtobacterium sp. BmP22c (BC3) presented auxin production, phosphate solubilization activity, nifH and acdS genes, antimicrobial activities against important phytopathogens, and growth promotion of tomato plants and seeds under normal and cold stress conditions. Due to its additional anti-stress ACC deaminase and IRI activities, the bacterial consortium composed of Pseudomonas sp. TmR5a and Curtobacterium sp. BmP22c is an attractive candidate for genome analyses, evaluation of its epiphytic and rhizosphere colonization potential, and characterization of its plant protection against frost. A bioproduct based on this psychrotolerant bacterial consortium is an eco-friendly alternative for the protection of agricultural crops to cold stress and, in addition, to phytopathogens.In this study, seventy highly psychrotolerant bacteria with plant growth-promoting activities of one hundred thirty isolates from wild flora from the Andes Mountains and Patagonia of Chile were characterized. These bacteria belong to Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes phyla, and 18 genera. Notably, this is the first report of bacteria associated with