Transmission of oocyte DNA damage to preimplantation embryos after in vivo mouse exposure to daunorubicin and cytarabine.

International audienceStudy question: Does oocyte DNA damage induced by a previous in vivo mouse exposure to chemotherapy agents is transmissible to preimplantation embryos?Summary answer: DNA damage was observed in preimplantation embryos issued from mice previously exposed to daunorubicin and cytarabine.What is known already: In acute leukemia, the emergency to start a chemotherapydon’t allow a fertility preservation at the time of diagnosis. Some authors have proposed to cryopreserve mature oocytes or embryos after a controlled ovarian stimulation applied shortly after the induction chemotherapy,which is mainly composed by daunorubicin and cytarabine, and reputated to be less gonadotoxic than alkylant agents. We previously observed DNA damage on mouse oocytes issued from antral follicles exposed in vivo to daunorubicin and cytarabine.Little is known about the risk of transmission of oocyte DNA damage to preimplantation embryos after fecundation of oocytes recently exposed to chemotherapy.Study design, size, duration: By three time, two groups of mice (n = 11) were exposed for four days to cytarabine (10 mg/kg IP) or every two days to daunorubicin (1 mg/kg IV). Each group was compared with a negative control group (n = 11) and with a positive control group (n = 11) injected with cyclophosphamide(75 mg/kg IP). Females were mated one week after exposure and preimplantation embryos were collected by flushing the oviducts.Participants/materials, setting, methods: 4 weeks female CD1 mice were mated one week after exposure for studying embryos conceived from oocytes exposed to chemotherapy at late pre-antral stage of follicular development.Cytotoxicity has been assessed by ovulation and fertilization rates and by embryo morphology. DNA embryonic damage was assessed by: (i) alkaline comet assay to quantify the tail DNA (ii) fluorescent immunohistochemical staining in blastomeres to quantify accumulating γH2AX foci.Main results and the role of chance: In mouse, a recent exposure to daunorubicin and cytarabine did not alter the ovarian response to controlled ovarian stimulation with no adverse impact on the fertilization rate and the number of embryo conceived. Ovulation and fertilization rates in mice previouslyexposed to daunorubicin and cytarabine were similar to those in our negative control group. One week after exposure, we observed with the comet assay a significant increase of embryonic DNA damage after exposure to daunorubicin (16.57 ± 1.3, p = 0.0003) and cytarabine (16.46 ± 1.4, p =0.0003) Vs 26.16 ± 2.5 after cyclophosphamide exposure (p < 0.0001) and 7,01 ± 1,1 in negative control group exposed to an injection of sterile saline solution. The analysis γ-H2AX on embryos showed a significant increase of foci corresponding to DNA double-strand breaks, after exposure to daunorubicin (7.97 ± 1.1; p = 0.001), cytarabine (6.47 ± 0.7, p = 0.0039), cyclophosphamide (5.92 ± 0.9; p = 0.0148) compared with negative control group (2,8 ±0,7).Limitations, reasons for caution: Mouse oocyte DNA is not exactly similar to human oocyte DNA, and would be more sensitive to genotoxic effects of chemotherapy agents. After chemotherapy, the kinetic of DNA repair before and after fertilization has to be studied by further assays in exposed oocyte andin embryos.Wider implications of the findings: DNA damage in preimplantation embryos conceived from oocytes exposed to chemotherapy at late pre-antral stage of follicular development lead us to hypothese a transmission of oocyte DNA damage to preimplantation embryo. In acute leukemia, we strongly adviseto not cryopreserve mature oocytes or embryo early after induction chemotherapy.Trial registration number: Experimental protocols and animal handling procedures were reviewed by the French National Ethics Committee on Animal Experimentation (N° 2017033010523688)

Strategies for protein synthetic biology

Proteins are the most versatile among the various biological building blocks and a mature field of protein engineering has lead to many industrial and biomedical applications. But the strength of proteins—their versatility, dynamics and interactions—also complicates and hinders systems engineering. Therefore, the design of more sophisticated, multi-component protein systems appears to lag behind, in particular, when compared to the engineering of gene regulatory networks. Yet, synthetic biologists have started to tinker with the information flow through natural signaling networks or integrated protein switches. A successful strategy common to most of these experiments is their focus on modular interactions between protein domains or domains and peptide motifs. Such modular interaction swapping has rewired signaling in yeast, put mammalian cell morphology under the control of light, or increased the flux through a synthetic metabolic pathway. Based on this experience, we outline an engineering framework for the connection of reusable protein interaction devices into self-sufficient circuits. Such a framework should help to ‘refacture’ protein complexity into well-defined exchangeable devices for predictive engineering. We review the foundations and initial success stories of protein synthetic biology and discuss the challenges and promises on the way from protein- to protein systems design

Transmission of oocyte DNA damage to preimplantation embryos after in vivo mouse exposure to daunorubicin and cytarabine.

International audienceStudy question: Does oocyte DNA damage induced by a previous in vivo mouse exposure to chemotherapy agents is transmissible to preimplantation embryos?Summary answer: DNA damage was observed in preimplantation embryos issued from mice previously exposed to daunorubicin and cytarabine.What is known already: In acute leukemia, the emergency to start a chemotherapydon’t allow a fertility preservation at the time of diagnosis. Some authors have proposed to cryopreserve mature oocytes or embryos after a controlled ovarian stimulation applied shortly after the induction chemotherapy,which is mainly composed by daunorubicin and cytarabine, and reputated to be less gonadotoxic than alkylant agents. We previously observed DNA damage on mouse oocytes issued from antral follicles exposed in vivo to daunorubicin and cytarabine.Little is known about the risk of transmission of oocyte DNA damage to preimplantation embryos after fecundation of oocytes recently exposed to chemotherapy.Study design, size, duration: By three time, two groups of mice (n = 11) were exposed for four days to cytarabine (10 mg/kg IP) or every two days to daunorubicin (1 mg/kg IV). Each group was compared with a negative control group (n = 11) and with a positive control group (n = 11) injected with cyclophosphamide(75 mg/kg IP). Females were mated one week after exposure and preimplantation embryos were collected by flushing the oviducts.Participants/materials, setting, methods: 4 weeks female CD1 mice were mated one week after exposure for studying embryos conceived from oocytes exposed to chemotherapy at late pre-antral stage of follicular development.Cytotoxicity has been assessed by ovulation and fertilization rates and by embryo morphology. DNA embryonic damage was assessed by: (i) alkaline comet assay to quantify the tail DNA (ii) fluorescent immunohistochemical staining in blastomeres to quantify accumulating γH2AX foci.Main results and the role of chance: In mouse, a recent exposure to daunorubicin and cytarabine did not alter the ovarian response to controlled ovarian stimulation with no adverse impact on the fertilization rate and the number of embryo conceived. Ovulation and fertilization rates in mice previouslyexposed to daunorubicin and cytarabine were similar to those in our negative control group. One week after exposure, we observed with the comet assay a significant increase of embryonic DNA damage after exposure to daunorubicin (16.57 ± 1.3, p = 0.0003) and cytarabine (16.46 ± 1.4, p =0.0003) Vs 26.16 ± 2.5 after cyclophosphamide exposure (p < 0.0001) and 7,01 ± 1,1 in negative control group exposed to an injection of sterile saline solution. The analysis γ-H2AX on embryos showed a significant increase of foci corresponding to DNA double-strand breaks, after exposure to daunorubicin (7.97 ± 1.1; p = 0.001), cytarabine (6.47 ± 0.7, p = 0.0039), cyclophosphamide (5.92 ± 0.9; p = 0.0148) compared with negative control group (2,8 ±0,7).Limitations, reasons for caution: Mouse oocyte DNA is not exactly similar to human oocyte DNA, and would be more sensitive to genotoxic effects of chemotherapy agents. After chemotherapy, the kinetic of DNA repair before and after fertilization has to be studied by further assays in exposed oocyte andin embryos.Wider implications of the findings: DNA damage in preimplantation embryos conceived from oocytes exposed to chemotherapy at late pre-antral stage of follicular development lead us to hypothese a transmission of oocyte DNA damage to preimplantation embryo. In acute leukemia, we strongly adviseto not cryopreserve mature oocytes or embryo early after induction chemotherapy.Trial registration number: Experimental protocols and animal handling procedures were reviewed by the French National Ethics Committee on Animal Experimentation (N° 2017033010523688)

Antimicrobial and cytotoxic effects of marine sponge extracts <i>Agelas clathrodes</i>, <i>Desmapsamma anchorata</i> and <i>Verongula rigida</i> from a Caribbean Island

International audienceAlthough marine sponges are known for their antimicrobial, antifungal and cytotoxic activity, very few studies have been carried out on endemic species of Martinique. Martinique is part of the Agoa Sanctuary, a marine protected area that includes the exclusive economic zones (EEZ) of the French Caribbean islands, making it an abundant source of marine species. To highlight the potential of this area for the discovery of marine biomolecules with antipathogenic and antitumor activities, we tested the aqueous and ethanolic extracts of sponge species Agelas clathrodes, Desmapsamma anchorata and Verongula rigida. Five bacterial strains: Bacillus cereus (CIP 78.3), Escherichia coli (CIP 54.127), Pseudomonas aeruginosa (CIP A22), Staphylococcus aureus (CIP 67.8) and Staphylococcus saprophyticus (CIP 76125) were evaluated, as well as four tumor cell lines: breast cancer (MDA-MB231), glioblastoma (RES259) and leukemia (MOLM14 and HL-60). Antimicrobial activity was evaluated using the disc diffusion technique by determining the minimum inhibitory and minimum bactericidal concentrations. Tumor cytotoxic activity was determined in vitro by defining the minimum concentration of extracts that would inhibit cell growth. Ethanolic extracts of Agelas clathrodes were bactericidal for Staphylococcus aureus and Staphylococcus saprophyticus strains, as well as strongly cytotoxic (IC 50 < 20 µg/mL) on all cancer cell lines. Verongula rigida also showed strong cytotoxic activity on cell lines but no antimicrobial activity. These results are innovative for this species on these bacterial lines, highlighting the potential of sponge extracts from this area as bioactive compounds sources

Oral Delivery of miR-320-3p with Lipidic Aminoglycoside Derivatives at Mid-Lactation Alters miR-320-3p Endogenous Levels in the Gut and Brain of Adult Rats According to Early or Regular Weaning

International audienceTo investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning. Animals from a new transgenic rat line expressing the green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received a single oral bolus of miR-320-3p or miR-375-3p embedded in DiOleyl-Succinyl-Paromomycin (DOSP) on D-12. The pups were weaned early (D-15), or regularly (D-30). The expression of relevant miRNA, mRNAs, chromatin complexes, and duodenal cell density were assessed at 8 h post-inoculation and on D-45. The miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (p < 0.05). On regular weaning, on D-45, miR-320-3p and 375-3p were found to be downregulated in the stomach and upregulated in the hypothalamus (p < 0.001), whereas miR-320-3p was upregulated in the duodenum. After early weaning, miR-320-3p and miR-375-3p were downregulated in the stomach and the duodenum, but upregulated in the hypothalamus and the hippocampus. Combination of miR-320-3p/DOSP with early weaning enhanced miR-320-3p and chromogranin A expression in the duodenum. In the female brain stem, miR-320-3p, miR-504, and miR-16-5p levels were all upregulated. Investigating the oral miRNA-320-3p loads in the duodenal cell lineage paved the way for designing new therapeutics to avoid unexpected long-term impacts on the brain

Epigenetic alterations in gut and brain of adult rats after oral administration of miR-320-3p and miR-375-3p at mid-lactation, and preventive potential of miR-320-3p on early weaning stress

Abstract Aim To investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning. Methods Animals from a new transgenic rat line expressing green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received at Day-12, near neural diversification, a single oral bolus of mir-320-3p or miR-375-3p, embedded in DiOleyl-Succinyl-Paromomycin (DOSP), and were further early (Day-15) or regularly (Day-30) weaned. Relevant miRNA (miR-320-3p, miR-375-3p, miR-375-5p, miR-16-5p, miR-132-3p, miR-504), polr3d , hspb6 , inflammation, enteroendocrine, and circadian clock-related mRNAs, chromatin complexes, and duodenal cell density were assayed at 8h post-inoculation and at Day-45. Results The miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (p<0.05) but no long-term effects. On regular weaning, at Day-45, both miR-320-3p and 375-3p were down-regulated in the stomach, up-regulated in the hypothalamus (p<0.001) but only miR-320-3p was up-regulated in the duodenum. After early weaning, the miR-320-3p and miR-375-3p levels were down-regulated in the stomach and the duodenum, but up-regulated in the hypothalamus and the hippocampus. Combining miR-320-3p/DOSP with early weaning enhanced miR-320-3p and chromogranin A expression in the duodenum. In the hippocampus, the miR-504 was down-regulated for both sexes, but in the brain stem, up regulated only for females, along with miR-320-3p and miR-16-5p levels. In the hypothalamus, clock levels were up regulated for both sexes. In the miR-375-3p/DOSP group, the density of enteroendocrine duodenal cells increased. The long-term effect of miR-375-3p/DOSP was more limited, according to the fourfold lower number of predicted targets than with miR-320-3p. Conclusion Addressing oral miRNA-320-3p loads to duodenal cell lineage is paving the way for the design of new therapeutics, manipulating long term consequences of early life stress

A Novel Covalent mTOR Inhibitor, DHM25, Shows in Vivo Antitumor Activity against Triple-Negative Breast Cancer Cells

International audienceConstitutive activation of the PI3K/mTOR signaling pathway contributes to carcinogenesis and metastasis in most, if not all, breast cancers. From a chromene backbone reported to inhibit class I PI3K catalytic subunits, several rounds of chemical syntheses led to the generation of a new collection of chromologues that showed enhanced ability to kill PI3K-addicted cancer cells and to inhibit Akt phosphorylation at serine 473, a hallmark of PI3K/mTOR activation. This initial screen uncovered a chromene designated DHM25 that exerted potent antitumor activity against breast tumor cell lines. Strikingly, DHM25 was shown to be a selective and covalent inhibitor of mTOR using biochemical and cellular analyses, modeling, and a large panel of kinase activity assays spanning the human kinome (243 kinases). Finally, in vivo, this novel drug was an efficient inhibitor of growth and metastasis of triple-negative breast cancer cells, paving the way for its clinical application in oncolog

Human V 9V 2 T Cells Specifically Recognize and Kill Acute Myeloid Leukemic Blasts

International audienceVγ9Vδ2 T cells are attractive candidates for antileukemic activity. The analysis of Vγ9Vδ2 T cells in newly diagnosed acute myeloid leukemia (AML) patients revealed that their absolute cell numbers were normal in the blood as well as in the bone marrow but showed a striking imbalance in the differentiation subsets, with preponderance of the effector memory population. This unusual phenotype was restored after removal of leukemic cells in patients, which reached complete remission after chemotherapy, suggesting that leukemic cells might be involved in the alteration of γδ T cell development in AML. Accordingly, coculture between AML cells and Vγ9Vδ2 T cells induced selection of effector cells. In accordance with their effector memory status, in vitro proliferation of Vγ9Vδ2 T cells was reduced compared with normal controls. Nevertheless, Vγ9Vδ2 T cells efficiently killed autologous AML blasts via the perforin/granzyme pathway. The ligands for DNAM-1 were expressed by AML cells. We showed that killing of AML blasts was TCR and DNAM-1 dependent. Using a xenotransplantation murine model, we showed that Vγ9Vδ2 T cells homed to the bone marrow in close proximity of engrafted leukemic cells and enhanced survival. These data demonstrate that Vγ9Vδ2 T cells are endowed with the ability to interact with and eradicate AML blasts both in vitro and in a mouse model. Collectively, our data revealed that Vγ9Vδ2 T cells have a potent antileukemic activity provided that optimal activation is achieved, such as with synthetic TCR agonists. This study enhances the interest of these cells for therapeutic purposes such as AML treatment