Heritability of fumonisin B1 production in Gibberella fujikuroi mating population A.

Fumonisins are mycotoxins produced by strains belonging to several different mating populations of Gibberella fujikuroi (anamorphs, Fusarium section Liseola), a major pathogen of maize and sorghum worldwide. We studied the heritability of fumonisin production in mating population A by crossing fumonisin-producing strains collected from maize and sorghum in the United States with fumonisin-nonproducing strains collected from maize in Nepal. Random ascospore and tetrad progeny from three of these crosses were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography for their ability to produce fumonisins on autoclaved cracked maize. In all three crosses, the ability to produce fumonisins, predominately fumonisin B1, segregated as a single gene or group of closely linked genes. Intercrosses between appropriate progeny and parents were poorly fertile, so we could not determine if the apparent single genes that were segregating in each of these crosses were allelic with one another. Mating type and spore-killer traits were scored in some crosses, and each segregated, as expected, as a single gene that was unlinked to the ability to produce fumonisins. We conclude that G. fujikuroi mating population A provides a powerful genetic system for the study of this important fungal toxin.     

A tripartite culture system for endomycorrhizal inoculation of micropropagated strawberry plantlets in vitro

The objective of the current investigation was to develop a reliable method to obtain vesicular arbuscular mycorrhizae (VAM) in micropropagated plantlets and to determine their influence on growth. An in vitro system for culturing the VA mycorrhizal fungus Glomus intraradices with Ri T-DNA-transformed carrot roots or nontransformed tomato roots was used in this study as a potential active source of inoculum for the colonization of micropropagated plantlets. After root induction, micropropagated plantlets grown on cellulose plugs (sorbarod) were placed in contact with the primary mycorrhizae in growth chambers enriched with 5000 ppm CO₂ and fed with a minimal medium. After 20 days of tripartite culture, all plantlets placed in contact with the primary symbiosis were colonized by the VAM fungus. As inoculum source, 30-day-old VA mycorrhizal transformed carrot roots had a substantially higher infection potential than 5-, 10-or 20-day-old VAM. Colonized plantlets had more extensive root systems and better shoot growth than control plants. The VAM symbiosis reduced the plantlet osmotic potential. This response may be a useful pre-adaptation for plantlets during transfer to the acclimatization stage.     

Photoacoustic imaging of the human placental vasculature

Minimally invasive fetal interventions require accurate imaging from inside the uterine cavity. Twin‐to‐twin transfusion syndrome (TTTS), a condition considered in this study, occurs from abnormal vascular anastomoses in the placenta that allow blood to flow unevenly between the fetuses. Currently, TTTS is treated fetoscopically by identifying the anastomosing vessels, and then performing laser photocoagulation. However, white light fetoscopy provides limited visibility of placental vasculature, which can lead to missed anastomoses or incomplete photocoagulation. Photoacoustic (PA) imaging is an alternative imaging method that provides contrast for hemoglobin, and in this study, two PA systems were used to visualize chorionic (fetal) superficial and subsurface vasculature in human placentas. The first system comprised an optical parametric oscillator for PA excitation and a 2D Fabry‐Pérot cavity ultrasound sensor; the second, light emitting diode arrays and a 1D clinical linear‐array ultrasound imaging probe. Volumetric photoacoustic images were acquired from ex vivo normal term and TTTS‐treated placentas. It was shown that superficial and subsurface branching blood vessels could be visualized to depths of approximately 7 mm, and that ablated tissue yielded negative image contrast. This study demonstrated the strong potential of PA imaging to guide minimally invasive fetal therapies.      

Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study

Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages.Innate immunity is the first line of immune defense and is common to all metazoans (1, 2). In this immune system, macrophages play a crucial role in the maintenance of tissue homeostasis. These cells are involved in almost every disease through their immunological and wound-healing functions (1, 2). During a pathogenic infection, trauma or neurodegeneration, macrophages are recruited and activated contributing to the phagocytosis of pathogens and the secretion of cytokines and chemokines activating other immune cells. Macrophages can develop into classically pro-inflammatory (M1) or alternatively (M2) activated macrophages. M1 macrophages are characterized by the secretion of pro-inflammatory cytokines whereas M2 macrophages secrete anti-inflammatory cytokines (3). Stimulation of macrophages with LPS activates TLR4 signaling leading to the nucleus translocation of NF-κB or IRF3 which activate genes encoding proteins involved in innate immune response (4). Many of these proteins are secreted (cytokines, chemokines…) to attract and activate other immune cells like T lymphocytes. In tumors, macrophages are oriented toward the M2 phenotype and promote cancer growth by suppressing immune cells function (5). Current research in the therapeutic field focus on ways to reactivate macrophages.Surprisingly, we have shown that during immune responses, macrophages secrete typical neuroendocrine molecules (6–8), such as neuropeptides (9) or the proprotein convertases (PC)¹ PC2 and PC1/3 and that PC1/3 is an important regulator of innate immune responses (10–12). Proprotein convertases cleave precursor proteins which can lead to the activation, inactivation or functional changes. PC2 and PC1/3 operate within the regulated secretory pathway. Their expression is not restricted to neuroendocrine tissues, they are also expressed in macrophages and lymphocytes (12). In a previous study from our group, PC1/3 knockout (KO) in mice challenged with LPS caused innate immune defects and uncontrolled cytokine secretion (10). Th1 pathway is enhanced in PC1/3 KO mice. Following LPS treatment, PC1/3 colocalized with TLR4 in the endosomal compartment (11). We concluded that PC1/3 contributes to the regulation of TLR4 signaling and the resulting cytokine secretion.The NR8383 rat pulmonary macrophage cell line was previously shown as a good model to study the role of PC1/3 in the macrophage innate immune response (13). In the present study, we developed a PC1/3-knockdown (K_D) NR8383 cell line using lentiviral-delivered shRNAs. Our aim is to understand the cellular impact of PC1/3 inhibition in macrophages and the consequences on their activation. Proteomic analysis of secreted proteins allowed us to identify pro-inflammatory cytokines and alarmins already at 24h of LPS stimulation in PC1/3-K_D secretomes which was confirmed by cytokines array. Proteomic studies of PC1/3-K_D NR8383 cellular extracts revealed an important perturbation in the intracellular trafficking machinery through the disorganization of cytoskeletal protein expression. These results were confirmed on macrophages from PC1/3 KO mice. Cytokines secretion and cytoskeleton reorganization can be linked to intracellular calcium increase in PC1/3-K_D cells. Moreover, we showed that MyD88-dependant TLR4 signaling was sustained when PC1/3 is down-regulated. We describe here that inhibition of PC1/3 induced classically activated phenotype (M1) in macrophages. The chemotactic and anti-tumor properties of the PC1/3-K_D macrophage secretome promoted the cytotoxic immune response and inhibited cancer cell viability. The down-regulation of PC1/3 could be used in cancer immunotherapy to reactivate macrophages.