An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi

RNA interference (RNAi) mediates gene silencing in many eukaryotes and has been widely used to investigate gene functions. A common method to induce sustained RNAi is introducing plasmids that synthesize short hairpin RNAs (shRNAs) using Pol III promoters. While these promoters synthesize shRNAs and elicit RNAi efficiently, they lack cell specificity. Monitoring shRNA expression levels in individual cells by Pol III promoters is also difficult. An alternative way to deliver RNAi is to use Pol II-directed synthesis of shRNA. Previous efforts in developing a Pol II system have been sparse and the results were conflicting, and the usefulness of those Pol II vectors has been limited due to low efficacy. Here we demonstrate a new Pol II system that directs efficient shRNA synthesis and mediates strong RNAi at levels that are comparable with the commonly used Pol III systems. In addition, this system synthesizes a marker protein under control of the same promoter as the shRNA, thus providing an unequivocal indicator, not only to the cells that express the shRNA, but also to the levels of the shRNA expression. This system may be adapted for in vivo shRNA expression and gene silencing

Inhibition of activity of GABA transporter GAT1 by δ-opioid receptor

Analgesia is a well-documented effect of acupuncture. A critical role in pain sensation plays the nervous system, including the GABAergic system and opioid receptor (OR) activation. Here we investigated regulation of GABA transporter GAT1 by δOR in rats and in Xenopus oocytes. Synaptosomes of brain from rats chronically exposed to opiates exhibited reduced GABA uptake, indicating that GABA transport might be regulated by opioid receptors. For further investigation we have expressed GAT1 of mouse brain together with mouse δOR and μOR in Xenopus oocytes. The function of GAT1 was analyzed in terms of Na(+)-dependent [(3)H]GABA uptake as well as GAT1-mediated currents. Coexpression of δOR led to reduced number of fully functional GAT1 transporters, reduced substrate translocation, and GAT1-mediated current. Activation of δOR further reduced the rate of GABA uptake as well as GAT1-mediated current. Coexpression of μOR, as well as μOR activation, affected neither the number of transporters, nor rate of GABA uptake, nor GAT1-mediated current. Inhibition of GAT1-mediated current by activation of δOR was confirmed in whole-cell patch-clamp experiments on rat brain slices of periaqueductal gray. We conclude that inhibition of GAT1 function will strengthen the inhibitory action of the GABAergic system and hence may contribute to acupuncture-induced analgesia

Early Exposure to Paraquat Sensitizes Dopaminergic Neurons to Subsequent Silencing of PINK1 Gene Expression in Mice

Environmental exposure, genetic modification, and aging are considered risky for Parkinson's disease (PD). How these risk factors cooperate to induce progressive neurodegeneration in PD remains largely unknown. Paraquat is an herbicide commonly used for weed and grass control. Exposure to paraquat is associated with the increased incidence of PD. In contrast to familial PD, most sporadic PD cases do not have genetic mutation, but may suffer from partial dysfunction of neuron-protective genes as aging. Using conditional transgenic RNAi, we showed that temporal silencing of PINK1 expression in adult mice increased striatal dopamine, the phenotype that could not be induced by constitutive gene silencing. Moreover, early exposure to paraquat sensitized dopaminergic neurons to subsequent silencing of PINK1 gene expression, leading to a significant loss of dopaminergic neurons. Our findings suggest a novel pathogenesis of PD: exposure to environmental toxicants early in the life reduces the threshold of developing PD and partial dysfunction of neuron-protective genes later in the life initiates a process of progressive neurodegeneration to cross the reduced threshold of disease onset

Pol II–Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species

{4-Bromo-2-[3-(diethyl­ammonio)propyl­imino­meth­yl]phenolato}diiodidozinc(II) methanol solvate

In the title complex, [ZnI2(C14H21BrN2O)]·CH3OH, the asymmetric unit consists of a mononuclear zinc(II) complex mol­ecule and a methanol solvent mol­ecule. The compound was derived from the zwitterionic form of the Schiff base 4-bromo-2-[3-(diethyl­amino)propyl­imino­meth­yl]phenol. The ZnII atom is four-coordinated by the imine N and phenolate O atoms of the Schiff base ligand and by two iodide ions in a distorted tetra­hedral coordination. In the crystal structure, the methanol mol­ecules are linked to the Schiff base mol­ecules through N—H⋯O and O—H⋯O hydrogen bonds. One I atom is disordered over two positions in a 0.702 (19):0.298 (19) ratio

{4-Bromo-2-[2-(piperidin-1-ium-1yl)ethyl­iminometh­yl]phenolato}diiodido­zinc(II)

In the title complex, [ZnI2(C14H19BrN2O)], the ZnII atom is four-coordinated by the imine N and phenolate O atoms of the Schiff base ligand and by two iodide ions in a distorted tetra­hedral coordination. In the crystal structure, mol­ecules are linked through inter­molecular N—H⋯O hydrogen bonds, forming chains running along the b axis

Diiodido[N′-(2-methoxy­benzyl­idene)-N,N-dimethyl­ethane-1,2-diamine]zinc(II)

In the title complex, [Zn(C12H18N2O)I2], the ZnII ion is four-coordinated by the imine N and amine N atoms of the Schiff base ligand and by two iodide ions in a distorted tetra­hedral coordination

Perspective on Epitaxial NiCo2O4 Film as an Emergent Spintronic Material: Magnetism and Transport Properties

The ferrimagnetic inverse spinel NiCo2O4 has attracted extensive research interests for its versatile electrochemical properties, robust magnetic order, high conductivity, and fast spin dynamics, as well as its highly tunable nature due to the closely coupled charge, spin, orbital, lattice, and defect effects. Single-crystalline epitaxial thin films of NiCo2O4 present a model system for elucidating the intrinsic physical properties and strong tunability, which are not viable in bulk single crystals. In this perspective, we discuss the recent advances in epitaxial NiCo2O4 thin films, focusing on understanding its unusual magnetic and transport properties in light of crystal structure and electronic structure. The perpendicular magnetic anisotropy in compressively strained NiCo2O4 films is explained by considering the strong spin-lattice coupling, particularly on Co ions. The prominent effect of growth conditions reveals the complex interplay between the crystal structure, cation stoichiometry, valence state, and site occupancy. NiCo2O4 thin films also exhibit various magnetotransport anomalies, including linear magnetoresistance and sign change in anomalous Hall effect, which illustrate the competing effects of band intrinsic Berry phase and impurity scattering. The fundamental understanding of these phenomena will facilitate the functional design of NiCo2O4 thin films for nanoscale spintronic applications