Pb(II)-inducible proviolacein biosynthesis enables a dual-color biosensor toward environmental lead.

With the rapid development of synthetic biology, various whole-cell biosensors have been designed as valuable biological devices for the selective and sensitive detection of toxic heavy metals in environmental water. However, most proposed biosensors are based on fluorescent and bioluminescent signals invisible to the naked eye. The development of visible pigment-based biosensors can address this issue. The pbr operon from Klebsiella pneumoniae is selectively induced by bioavailable Pb(II). In the present study, the proviolacein biosynthetic gene cluster was transcriptionally fused to the pbr Pb(II) responsive element and introduced into Escherichia coli. The resultant biosensor responded to Pb(II) in a time- and dose-dependent manner. After a 5-h incubation with Pb(II), the brown pigment was produced, which could be extracted into n-butanol. Extra hydrogen peroxide treatment during n-butanol extract resulted in the generation of a stable green pigment. An increased brown signal was observed upon exposure to lead concentrations above 2.93 nM, and a linear regression was fitted from 2.93 to 3,000 nM. Extra oxidation significantly decreased the difference between parallel groups. The green signal responded to as low as 0.183 nM Pb(II), and a non-linear regression was fitted in a wide concentration range from 0.183 to 3,000 nM. The specific response toward Pb(II) was not interfered with by various metals except for Cd(II) and Hg(II). The PV-based biosensor was validated in monitoring bioaccessible Pb(II) spiked into environmental water. The complex matrices did not influence the regression relationship between spiked Pb(II) and the dual-color signals. Direct reading with the naked eye and colorimetric quantification enable the PV-based biosensor to be a dual-color and low-cost bioindicator for pollutant heavy metal.

Mu-Opioid Receptors Expressed in Glutamatergic Neurons are Essential for Morphine Withdrawal.

Although opioids still remain the most powerful pain-killers, the chronic use of opioid analgesics is largely limited by their numerous side-effects, including opioid dependence. However, the mechanism underlying this dependence is largely unknown. In this study, we used the withdrawal symptoms precipitated by naloxone to characterize opioid dependence in mice. We determined the functional role of mu-opioid receptors (MORs) expressed in different subpopulations of neurons in the development of morphine withdrawal. We found that conditional deletion of MORs from glutamatergic neurons expressing vesicular glutamate transporter 2 (Vglut2+) largely eliminated the naloxone-precipitated withdrawal symptoms. In contrast, conditional deletion of MORs expressed in GABAergic neurons had a limited effect on morphine withdrawal. Consistently, mice with MORs deleted from Vglut2+ glutamatergic neurons also showed no morphine-induced locomotor hyperactivity. Furthermore, morphine withdrawal and morphine-induced hyperactivity were not significantly affected by conditional knockout of MORs from dorsal spinal neurons. Taken together, our data indicate that the development of morphine withdrawal is largely mediated by MORs expressed in Vglut2+ glutamatergic neurons.