Overcoming insecticide resistance through computational inhibitor design.

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase αE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp αE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

An ex vivo murine model to study poliovirus-induced apoptosis in nerve cells.

Paralytic poliomyelitis results from destruction of motor neurons owing to poliovirus (PV) replication. Using a mouse model, we have previously shown that PV kills neurons of the central nervous system (CNS) as a result of apoptosis (Girard et al., Journal of Virology 73, 6066-6072, 1999). We report the development of mixed mouse primary nerve cell cultures from the cerebral cortex of neonatal mice transgenic for the human PV receptor. These cultures contained all three main cell types of the CNS, i.e. neurons, astrocytes and oligodendrocytes. All three cell types were susceptible to PV infection and virus replication in the cultures led to DNA fragmentation characteristic of apoptosis. PV-induced apoptosis was inhibited by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD.FMK), indicating that this process involved caspases. Thus, these mixed mouse primary nerve cell cultures are a new in vitro model for studying the molecular mechanisms of PV-induced apoptosis in nerve cells.

Cell coupling and Cx43 expression in embryonic mouse neural progenitor cells.

Embryonic neural progenitors isolated from the mouse striatal germinal zone grow in vitro as floating cell aggregates called neurospheres, which, upon adhesion, can be induced to differentiate into the three main cell types of the central nervous system (CNS), that is, astrocytes, neurons and oligodendrocytes. To study the possible role of connexins and junctional communication during differentiation of neural progenitors, we assessed cell-to-cell communication by microinjecting Lucifer Yellow into neurospheres at various times after adhesion. Cells located in neurospheres were strongly coupled, regardless of the differentiation time. Microinjections performed on the cell layers formed by differentiated cells migrating out of the neurosphere established that only astrocytes were coupled. These observations suggest the existence of at least three distinct communication compartments: coupled proliferating cells located in the sphere, uncoupled cells undergoing neuronal or oligodendrocytic differentiation and coupled differentiating astrocytes. A blockade of junctional communication by 18-beta-glycyrrhetinic acid (betaGA) reduced, in a concentration-dependent manner, the viability of undifferentiated neural progenitor cells. This effect appeared to be specific, inasmuch as it was reversible and that cell survival was not affected in the presence of the inactive analog glycyrrhyzic acid. Addition of betaGA to adherent neurospheres also decreased cell density and altered the morphology of differentiated cells. Cx43 was strongly expressed in either undifferentiated or differentiated neurospheres, where it was found both within the sphere and in astrocytes, the two cell populations that were dye coupled. Western blot analysis further showed that Cx43 phosphorylation was strongly increased in adherent neurospheres, suggesting a post-translational regulation during differentiation. These results point to a major role of cell-to-cell communication and Cx43 during the differentiation of neural progenitor cells in vitro.

Sonic hedgehog is a potent inducer of rat oligodendrocyte development from cortical precursors in vitro.

Sonic Hedgehog (Shh) induces oligodendrocyte development in the ventral neural tube and telencephalon but its role in oligodendrocyte generation in dorsal telencephalon is debated. Transcripts for Shh and its receptor complex were detected in subventricular zone and neocortex from E17 to birth. As Shh is not yet expressed in E15 neocortex, we grew E15 cortical precursors (CP) into neurospheres in the presence of recombinant Octyl-Shh (O-Shh). After sphere adhesion and removal of O-Shh, enhanced neurite outgrowth and cell migration were already observed at 3 h. Three days after O-Shh treatment, oligodendrocyte progenitors (OP) emerged and continued to increase in number for 7 days while the ratio of neuronal cells decreased compared to control. Shh selectively triggered mitosis of OP but not neuronal progenitors and enhanced growth of neonatal OP. Thus Shh in E15-17 embryonic neocortex can signal CP to adopt an oligodendrocyte fate and favors expansion of this lineage.