Fluorescent α-Conotoxin [Q1G, ΔR14]LvIB Identifies the Distribution of α7 Nicotinic Acetylcholine Receptor in the Rat Brain.

α7 nicotinic acetylcholine receptors (nAChRs) are mainly distributed in the central nervous system (CNS), including the hippocampus, striatum, and cortex of the brain. The α7 nAChR has high Ca2+ permeability and can be quickly activated and desensitized, and is closely related to Alzheimer's disease (AD), epilepsy, schizophrenia, lung cancer, Parkinson's disease (PD), inflammation, and other diseases. α-conotoxins from marine cone snail venom are typically short, disulfide-rich neuropeptides targeting nAChRs and can distinguish various subtypes, providing vital pharmacological tools for the functional research of nAChRs. [Q1G, ΔR14]LvΙB is a rat α7 nAChRs selective antagonist, modified from α-conotoxin LvΙB. In this study, we utilized three types of fluorescein after N-Hydroxy succinimide (NHS) activation treatment: 6-TAMRA-SE, Cy3 NHS, and BODIPY-FL NHS, labeling the N-Terminal of [Q1G, ΔR14]LvΙB under weak alkaline conditions, obtaining three fluorescent analogs: LvIB-R, LvIB-C, and LvIB-B, respectively. The potency of [Q1G, ΔR14]LvΙB fluorescent analogs was evaluated at rat α7 nAChRs expressed in Xenopus laevis oocytes. Using a two-electrode voltage clamp (TEVC), the half-maximal inhibitory concentration (IC50) values of LvIB-R, LvIB-C, and LvIB-B were 643.3 nM, 298.0 nM, and 186.9 nM, respectively. The stability of cerebrospinal fluid analysis showed that after incubation for 12 h, the retention rates of the three fluorescent analogs were 52.2%, 22.1%, and 0%, respectively. [Q1G, ΔR14]LvΙB fluorescent analogs were applied to explore the distribution of α7 nAChRs in the hippocampus and striatum of rat brain tissue and it was found that Cy3- and BODIPY FL-labeled [Q1G, ΔR14]LvΙB exhibited better imaging characteristics than 6-TAMARA-. It was also found that α7 nAChRs are widely distributed in the cerebral cortex and cerebellar lobules. Taking into account potency, imaging, and stability, [Q1G, ΔR14]LvΙB -BODIPY FL is an ideal pharmacological tool to investigate the tissue distribution and function of α7 nAChRs. Our findings not only provide a foundation for the development of conotoxins as visual pharmacological probes, but also demonstrate the distribution of α7 nAChRs in the rat brain.

A novel electrolytic gas lift reactor for efficient microbial electrosynthesis of acetate from carbon dioxide.

The low current density impedes the practical application of microbial electrosynthesis for CO2 fixation. Engineering the reactor design is an effective way to increase the current density, especially for H2-mediated microbial electrosynthesis reactors. The electrolytic bubble column microbial electrosynthesis reactor has shown great potential for scaling up, but the mixing and gas mass transfer still need to be enhanced to further increase the current density. Here, we introduced an inner draft tube to the bubble column to tackle the problem. The addition of draft tube resulted in a 76.6% increase in the volumetric mass transfer coefficient (kLa) of H2, a 40% increase in the maximum current density (337 A/m2) and a 72% increase in average acetate production rate (3.1 g/L/d). The computational fluid dynamics simulations showed that the addition of draft tube enhanced mixing efficiency by enabling a more ordered cyclic flow pattern and a more uniform gas/liquid distribution. These results indicate that the electro-bubble column reactor with draft tube holds great potential for industrial implementation.

Synthesis, Activity, and Application of Fluorescent Analogs of [D1G, Δ14Q]LvIC Targeting α6ß4 Nicotinic Acetylcholine Receptor.

α6ß4* nicotinic acetylcholine receptor (nAChR) (* represents the possible presence of additional subunits) is mainly distributed in the central and peripheral nervous system and is associated with neurological diseases, such as neuropathic pain; however, the ability to explore its function and distribution is limited due to the lack of pharmacological tools. As one of the analogs of α-conotoxin (α-CTx) LvIC from Conus lividus, [D1G, Δ14Q]LvIC (Lv) selectively and potently blocks α6/α3ß4 nAChR (α6/α3 represents a chimera). Here, we synthesized three fluorescent analogs of Lv by connecting fluorescent molecules 6-carboxytetramethylrhodamine succinimidyl ester (6-TAMRA-SE, R), Cy3 NHS ester (Cy3, C) and BODIPY-FL NHS ester (BDP, B) to the N-terminus of the peptide and obtained Lv-R, Lv-C, and Lv-B, respectively. The potency and selectivity of three fluorescent peptides were evaluated using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes, and the potency and selectivity of Lv-B were almost maintained with the half-maximal inhibition (IC50) of 64 nM. Then, we explored the stability of Lv-B in artificial cerebrospinal fluid and stained rat brain slices with Lv-B. The results indicated that the stability of Lv-B was slightly improved compared to that of native Lv. Additionally, we detected the distribution of the α6ß4* nAChR subtype in the cerebral cortex using green fluorescently labeled peptide and fluorescence microscopy. Our findings not only provide a visualized pharmacological tool for exploring the distribution of the α6ß4* nAChR subtype in various situ tissues and organs but also extend the application of α-CTx [D1G, Δ14Q]LvIC to demonstrate the involvement of α6ß4 nAChR function in pathophysiology and pharmacology.

Treatment of cadmium and zinc-contaminated water systems using modified biochar: Contaminant uptake, adsorption ability, and mechanism.

Cd and Zn contamination in water occurs frequently that threatens water supply, human health, and food production. MnFeB, a novel absorbent biochar modified using KMnO4 and hematite, was prepared and used for the treatment of Cd2+ and Zn2+solutions. MnFeB exhibits a rough surface structure, large specific surface area, higher total pore volume, massive functional groups, and abundant iron oxide, all of which contribute to higher Cd2+ and Zn2+ adsorption capacity. In single metal systems, maximum Cd2+ and Zn2+ adsorption capacities of MnFeB were 1.88 and 1.79 times higher than those of unmodified biochar (CSB). The maximum Cd2+ and Zn2+ adsorption capacities of MnFeB were 2.73 and 2.65 times higher than CSB in the binary metal system. Key adsorption mechanisms of Cd2+ and Zn2+ by MnFeB included electrostatic interaction, co-precipitation, π-π interaction, complexation, and ion exchange. Thus, MnFeB can be used as a novel absorbent to treat Cd and Zn-polluted water.

A novel phytoremediation technology for polluted cadmium soil: Salix integra treated with spermidine and activated carbon.

A variety of plants have been used as phytoremediation materials to remove Cd from polluted soil. However, the disadvantages of using plants for decontamination include low biomass, low uptake, and inefficiency. We conducted experiments to determine the effects of spermidine and activated carbon treatments of Salix integra on Cd removal. The results showed that exogenous spermidine and activated carbon increased plant growth and root development compared with the CK. The increased dry mass (39.65-92.95%) with the combined spermidine and activated carbon treatments was higher than that with either single treatment (14.79-62.80%). The root length, surface area, root volume, and root diameter with the combined spermidine and activated carbon treatments (53.51-189.35%, 113.08-207.62%, 111.71-499.27%, and 32.51-106.62%, respectively) were higher than those of the lone application treatments (19.35-132.23%, 52.33-111.57%, 35.08-297.07%, and 24.22-81.38%, respectively). In addition, spermidine and activated carbon application reduced the toxicity of Cd to S. integra by improving the antioxidant capacity, thereby increasing the accumulation of Cd. The application of spermidine and activated carbon also changed the distribution of Cd in each part of S. integra. There was increased accumulation of Cd in the shoots and better absorption by the S. integra shoots, thereby improving their Cd remediation efficiency. The combined 0.8 mM spermidine and 0.5 g kg-1 activated carbon were most effective on removing Cd from the soil. The Cd removal efficiency was 78.11-120.86% higher than that of the CK. Our results may provide foundational information for understanding the mechanisms for the sustainable remediation of Cd-contaminated soil using a combination of spermidine and activated carbon.

Peptides from the Sea Anemone Metridium senile with Modified Inhibitor Cystine Knot (ICK) Fold Inhibit Nicotinic Acetylcholine Receptors.

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1-Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1-Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1-Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors.

α-Conotoxin TxID and [S9K]TxID, α3ß4 nAChR Antagonists, Attenuate Expression and Reinstatement of Nicotine-Induced Conditioned Place Preference in Mice.

Tobacco smoking has become a prominent health problem faced around the world. The α3ß4 nicotinic acetylcholine receptor (nAChR) is strongly associated with nicotine reward and withdrawal symptom. α-Conotoxin TxID, cloned from Conus textile, is a strong α3ß4 nAChR antagonist, which has weak inhibition activity of α6/α3ß4 nAChR. Meanwhile, its analogue [S9K]TxID only inhibits α3ß4 nAChR (IC50 = 6.9 nM), and has no inhibitory activity to other nAChRs. The present experiment investigates the effect of α3ß4 nAChR antagonists (TxID and [S9K]TxID) on the expression and reinstatement of nicotine-induced conditioned place preference (CPP) and explores the behaviors of acute nicotine in mice. The animal experimental results showed that TxID and [S9K] TxID could inhibit the expression and reinstatement of CPP, respectively. Moreover, both had no effect in acute nicotine experiment and the locomotor activity in mice. Therefore, these findings reveal that the α3ß4 nAChR may be a potential target for anti-nicotine addiction treatment. [S9K]TxID, α3ß4 nAChR antagonist, exhibit a superior effect for anti-nicotine addiction, which is promising to develop a novel smoking cessation drug.

Influence of disulfide connectivity on structure and bioactivity of α-conotoxin TxIA.

Cone snails express a sophisticated arsenal of small bioactive peptides known as conopeptides or conotoxins (CTxs). Through evolutionary selection, these peptides have gained the ability to interact with a range of ion channels and receptors, such as nicotinic acetylcholine receptors (nAChRs). Here, we used reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization-mass spectrometry (ESI-MS) to explore the venom peptide diversity of Conus textile, a species of cone snail native to Hainan, China. One fraction of C. textile crude venom potently blocked α3ß2 nAChRs. Subsequent purification, synthesis, and tandem mass spectrometric analysis demonstrated that the most active compound in this fraction was identical to α-CTx TxIA, an antagonist of α3ß2 nAChRs. Then three disulfide isoforms of α-CTx TxIA were synthesized and their activities were investigated systematically for the first time. As we observed, disulfide isomerisation was particularly important for α-CTx TxIA potency. Although both globular and ribbon isomers showed similar retention times in RP-HPLC, globular TxIA potently inhibited α3ß2 nAChRs with an IC50 of 5.4 nM, while ribbon TxIA had an IC50 of 430 nM. In contrast, beads isomer had little activity towards α3ß2 nAChRs. Two-step oxidation synthesis produced the highest yield of α-CTx TxIA native globular isomer, while a one-step production process based on random oxidation folding was not suitable. In summary, this study demonstrated the relationship between conotoxin activity and disulfide connectivity on α-CTx TxIA.

Azathioprine hepatotoxicity and the protective effect of liquorice and glycyrrhizic acid.

This study aimed to evaluate the responses of human hepatocytes to azathioprine hepatotoxicity in comparison with the well-studied azathioprine hepatotoxicity in rat hepatocytes and the effects of protective agents to suppress azathioprine hepatotoxicity. Azathioprine presented its hepatotoxicity at clinically relevant concentrations (lower than 10 microm) in primary rat hepatocytes after 48 h of treatment as shown by a severe decrease in cell viability as well as intracellular GSH depletion. However, primary human hepatocytes exhibited only significant intracellular GSH depletion after treatment with azathioprine at these clinically relevant concentrations, while a reduction in cell viability by 29% was only evidenced after 48 h of treatment with azathioprine at the high concentration of 50 microm. In addition, a monolayer culture of primary rat hepatocytes was used as an in vitro model to examine the protective effects of antihepatotoxic drugs including glutathione (GSH), N-acetylcysteine (NAC, a GSH precursor), liquorice and glycyrrhizic acid (GA), a major bioactive component of liquorice, against hepatotoxicity of 1 microm azathioprine. It was found that both liquorice and GA showed substantial protection according to assays of cell viability and intracellular GSH, while neither GSH nor NAC had such a protective function. Similarly, GA protected human hepatocytes from intracellular GSH depletion on exposure to 1 microm azathioprine. These results implied that GA or liquorice could be considered as potent protection agents against azathioprine hepatotoxicity.