To investigate the structural basis of anion selectivity of Drosophila GABA-gated Cl(-) channels, the permeation properties of wild-type and mutant channels were studied in Xenopus oocytes. This work focused on asparagine 319, which by homology is one amino acid away from a putative extracellular ring of charge that regulates cation permeation in nicotinic receptors. Mutation of this residue to aspartate reduced channel conductance, and mutation to lysine or arginine increased channel conductance. These results are consistent with an electrostatic interaction between this site and permeating anions. The lysine mutant, but not the arginine mutant, formed a channel that is permeable to cations, and this cannot be explained in terms of electrostatics. The lysine mutant had a 25-mV reversal potential in solutions with symmetrical Cl(-) and asymmetrical cations. The permeability ratio of K(+) to Cl(-) was determined as 0. 33 from reversal potential measurements in KCl gradients. Experiments with large organic cations and anions showed that cation permeation can only be seen in the presence of Cl(-), but Cl(-) permeation can be seen in the absence of permeant cations. Measurements of permeability ratios of organic anions indicated that the lysine mutant has an increased pore size. The cation permeability of the lysine-containing mutant channel cannot be accounted for by a simple electrostatic interaction with permeating ions. It is likely that lysine substitution causes a structural change that extends beyond this one residue to influence the positions of other channel-forming residues. Thus protein conformation plays an important role in enabling ion channels to distinguish between anions and cations.

Photocatalytic nitrogen fixation has been considered as one of the most potential alternatives for the synthesis of ammonia; however, how to make full use of solar energy and how to cleave the strong NN bond remain great challenges. Herein, the attapulgite (ATP) mineral was modified with iron followed by the immobilization of doped CeF 3 nanoparticles to form a CeF 3 :Yb 3+ , Er 3+ /Fe-ATP heterostructure, which was utilized as a photocatalyst for nitrogen fixation. Results showed that doping of the activator Yb 3+ and the sensitizer Er 3+ generated fluorine vacancies (F v ) in the CeF 3 matrix, which formed dual active sites with Fe ions in ATP, benefiting the adsorption and activation of N 2 molecules. In addition, Fe-reconstructed ATP had a narrow band gap responsive in the visible region, while CeF 3 -doped Yb 3+ and Er 3+ facilitated transforming near-infrared light (NIR) into ultraviolet (UV) and visible light, both of which contributed to extending the harvest range in the full spectrum. The influence of the Er 3+ doping ratio and loading amount of fluoride on the generation of ammonia was explored. Notably, the separation of photogenerated charge carriers and the redox potentials was enhanced due to the rational indirect Z-scheme heterostructure mediated by F v . Under solar light irradiation, the NH 4 + production rate achieved the highest value of 253.6 μmol•h −1 •g −1 when the Er doping amount was optimized to be 3 mol % and the loading of CeF 3 :Yb 3+ , Er 3+ was 20 wt %, and it even reached 40.3 μmol•h −1 •g −1 under NIR irradiation. The current study may offer a sustainable strategy to use full solar energy and natural minerals for efficient ammonia synthesis.

The use of genetic distances to identify species within the framework of DNA barcoding has to some extent improved the development of biodiversity studies. However, using a fixed empirical threshold to delimit species may lead to overestimating species diversity. In this study, we use a new data set of COI sequences for 366 specimens within the genus of Cletus as well as conduct an analysis on the same genetic data for collected morphologically defined species from previous phylogeographical studies, to test whether high intraspecific genetic divergences are common with the premises of comprehensive sampling. The results indicate C. graminis Hsiao & Cheng , is the same species with C. punctiger (Dallas, 1852) and should be synonymized and that the distributional record of C. pugnator (Fabricius, 1787) in China is correct. High intraspecific genetic differentiations (0%-4.35%) were found in C. punctiger. Furthermore, as to the mined data, the maximum intraspecific K2P distances of 186 species (48.44% of 384) exceed 3%, and 101 species (26.30%) can be divided into two or more clusters with a threshold of 3% in cluster analysis. If genetic distance is used to delimit species boundaries, the minimum interspecific K2P distance of the congeneric species should be considered rather than only using the fixed empirical value; otherwise, the species richness may be overestimated in some cases.

No abstract

Despite significant progress in the fabrication of prevascularized networks over the past decade, a number of challenges remain. One of the most relevant issues is the lack of three-dimensional (3D) structures, which limits the clinical applications of the engineered scaffolds. Another problem is the complexity of prevascularized networks in engineered scaffolds, which is still less than that of human tissues, especially in the case of mature and bulk tissues. Thus, there is still the need to develop more flexible methods to better simulate the structure of natural tissues. In this work, we used a versatile sacrificial template method to fabricate bulk scaffolds with spatial prevascularized networks. Soft poly­(vinyl alcohol) (PVA) filaments were used to print the sacrificial template, and the receiving platform was a stepped shaft, allowing the sacrificial template to have a complex 3D structure. The obtained template was embedded into gelatin and microbial transglutaminase (mTG). The inner PVA template could be extracted from the enzymatic cross-linking system, and an engineered scaffold with spatial prevascularized networks was obtained. In vitro experiments demonstrated that the fabrication process is biocompatible with cells.

Recombinant baculoviruses containing two alternative splice forms of the Drosophila Rdl GABA receptor gene were constructed. Spodoptera frugiperda (Sf21) cells infected with either splice form expressed a transcript of expected size (2.5 kb). Western blotting of cell membrane extracts and immunoprecipitation experiments with an anti-Rdl antiserum recognized a protein of the expected size of -65 kDa. Whole cell patch clamp analysis of cells infected with either splice form revealed functional expression of GABA gated chloride ion channels which were blocked by application of 1 PM picrotoxinin. Following replacement of alanine 302 with a serine, a mutation associated with resistance to picrotoxinin and cyclodiene insecticides, mutant channels showed similar levels of insensitivity to picrotoxinin (-RIO-fold) as those observed in recordiugs from cultured ~~0~0~~~~~ neurons. The significance of the expression of an insect GABA receptor in an insect cell line and the s~il~ty of the results from these functional expression studies to recordings from cultured neurons is discussed.

We are interested in establishing stably transformed insect cell lines efficiently expressing the insect ?-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl.In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selectable marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chloride ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the genetic transformation of non-Drosophilid insects.

No abstract