Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

Background

As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped.

Results

Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (~300 μm by ~150 μm) to the rootlets of cranial nerve XII (~20 μm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is ~30 μm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated.

Conclusion

In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of ~20 μm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development.     

Positive integer solutions of the diophantine equation x 2 − L n xy + (−1) n y 2 = ±5 r

In this paper, we consider the equation x ²?L _n x y+(?1) ⁿ y ² = ±5 ^r and determine the values of n for which the equation has positive integer solutions x and y. Moreover, we give all positive integer solutions of the equation x ²?L _n x y+(?1) ⁿ y ² = ±5 ^r when the equation has positive integer solutions.     

Aromatic hydrogenation over nickel-silica prepared by deposition-precipitation

Three different nickel/silica catalysts prepared by decomposition-precipitation are characterized by adsorption technique and evaluated for aromatic hydrogenation. Depending on the metal content and the available surface nickel, activity and selectivity are affected for the reactions studied.     

Stereoselective total synthesis of (+)-aspicilin

A stereoselective synthesis of (+)-aspicilin is described. Regio- and stereoselective functionalization by intramolecular participation of the sulfinyl group, ene reaction, and macrolactonization by Wadsworth-Emmons reaction employing Masamune-Roush protocol are the key steps of the route.     

Microwave Assisted Synthesis and Antimicrobial Activity of Novel Pyrrolidine Derivatives

Microwave assisted synthesis of 1-acetyl-2-benzylpyrrolidine-2-carboxylic acid and its derivatives has been developed with highly encouraging yields. 2-Benzyl-tert-butylpyrrolidine-1,2-dicarboxylate is used as an initial compound in the four steps synthesis of the target compounds. Structures of the products 5a–5h have been confirmed by spectroscopic methods. According to antimicrobial tests 1-acetyl-2-benzylpyrrolidine-2-carboxamide 5c is determined to be the most potent product.     

Retaining in‐gel zymographic activity of cysteine proteases via a cysteine‐supplemented running buffer

Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α‐benzoyl‐l ‐arginine p‐nitroanilide, without at the same time altering the mobilities of the gel proteins.     

Oxidation of toluidine blue by chlorite in acid and mechanisms of the uncatalyzed and Ru(III)-catalyzed reactions: a kinetic approach

The complex mechanism of the uncatalyzed and Ru(III)-catalyzed oxidation of toluidine blue [(7-amino-8-methylphenothiazin-3-ylidene)dimethyl ammonium chloride, TB(+)Cl(-)] (λ(max) = 626 nm) by acidic chlorite is elucidated by a kinetic approach. Both the uncatalyzed and catalyzed reactions had a first-order dependence on the initial ClO(2)(-) and H(+) concentrations ([ClO(2)(-)](0) and [H(+)](0), respectively). The catalyzed reaction had a first-order dependence on the initial Ru(III) concentration ([Ru(III)](0)). The overall reaction of toluidine blue and chlorite ion was as follows: TB(+) + 5ClO(2)(-) + H(+) = P + 2ClO(2) + 2HCOOH + 3Cl(-) + H(2)O, where P is (7-amino-8-methyl-5-sulfoxophenothiazin-3-ylidene)amine. Consistent with the experimental results, the pertinent reaction mechanisms are proposed.     

Synthesis and Insecticidal Activity of Tetrazole‐Linked Triazole Derivatives

A new series of 4‐(4‐substitutedbenzylideneamino)‐5‐((1‐methyl‐1H‐tetrazol‐5‐ylthio)methyl)‐4H‐1,2,4‐triazole‐3‐thiol derivatives ( 5a , 5b , 5c , 5d , 5e , 5f , 5g , 5h , 5i , 5j , 5k ) are prepared using 4‐amino‐5‐((1‐methyl‐1H‐tetrazol‐5‐ylthio)methyl‐4H‐1,2,4‐triazole‐3‐thiol ( 4 ), as an intermediate compound. The structures of all the newly synthesized products are established supported by their spectral ¹H NMR, ¹³C NMR, FTIR, electrospray ionization mass spectrometry (mass), and analytical data. All the compounds are screened for their insecticidal activity against Plodia interpunctella, and six compounds exhibited significant activity.     

Sorption of Cadmium from Aqueous Solutions at Different Temperatures by Mexican HEU-type Zeolite Rich Tuff

Many factors may affect the heavy metals sorption on natural zeolites among them the temperature, for this reason in this paper the cadmium retention behavior on Mexican zeolitic rich tuff as a function of temperature is considered. The kinetic and the isotherms were determined at 303, 318, and 333 K, the remaining cadmium in the solution samples was analyzed by atomic absorption spectrometry. The pseudo-second order rate constant, k, as well as the apparent diffusion coefficients were calculated from the cadmium uptake by the zeolitic rock as a function of the contact time and temperature, the highest amounts were found for the experiments done at 333 K. The maximum cadmium adsorption capacity by the zeolitic material was 12.2 mg Cd²⁺/g at 318 K corresponding to 20% of the effective ion exchange capacity of the Chihuahua zeolitic rock. In order to explain the cadmium sorption behavior different kinetics and isotherm models were considered.