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.     

Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse

Background  

The chondroitin sulphate proteoglycan NG2 blocks neurite outgrowth in vitro and has been proposed as a major inhibitor of axonal regeneration in the CNS. Although a substantial body of evidence underpins this hypothesis, it is challenged by recent findings including strong expression of NG2 in regenerating peripheral nerve.     

Characterization of the particulate methane monooxygenase metal centers in multiple redox states by X-ray absorption spectroscopy

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.     

The conformational bias of aryl, arylsulfonyl geminally substituted tertiary carbon centers: applications in substrate-based stereocontrol

Intramolecular nitrile oxide-olefin cycloaddition to form hexahydrobenzisoxazole 14, which engenders a phenylsulfonyl, 2,5-difluorophenyl geminally substituted carbon substructure, proceeds with up to 99% ds. A rationalization of the high level of substrate-based stereo-induction observed in this and related ketone and acrylonitrile metallohydride reductions, supported by single crystal X-ray crystallography, is presented.     

X-ray crystallography and biological metal centers: is seeing believing?

Metalloenzyme crystal structures have a major impact on our understanding of biological metal centers. They are often the starting point for mechanistic and computational studies and inspire synthetic modeling chemistry. The strengths and limitations of X-ray crystallography in determining properties of biological metal centers and their corresponding ligand spheres are explored through examples, including ribonucleotide reductase R2 and particulate methane monooxygenase. Protein crystal structures locate metal ions within a protein fold and reveal the identities and coordination geometries of amino acid ligands. Data collection strategies that exploit the anomalous scattering effect of metal ions can establish metal ion identity. The quality of crystallographic data, particularly the resolution, determines the level of detail that can be extracted from a protein crystal structure. Complementary spectroscopic techniques can provide crucial information regarding the redox state of the metal center as well as the presence, type, and protonation state of exogenous ligands. The final result of the crystallographic characterization of a metalloenzyme is a model based on crystallographic data, supported by information from biophysical and modeling studies, influenced by sample handling, and interpreted carefully by the crystallographer.     

A novel crystallization-induced diastereomeric transformation based on a reversible carbon-sulfur bond formation. Application to the synthesis of a gamma-secretase inhibitor

This paper describes a remarkably efficient process for the preparation of gamma-secretase inhibitor 1. The target is synthesized in only five steps with an overall yield of 58%. The key operation is a highly selective and practical, crystallization-driven transformation for the conversion of a mixture of tertiary benzylic alcohols into the desired sulfide diastereomer with 94:6 dr. This unprecedented process is based upon a reversible carbon-sulfur bond formation under acidic conditions.     

Study of global solutions of parabolic equations via a priori estimates. Part I: Equations with principal elliptic part equal to the laplacian

We study global solutions of u_t=Δu + f(u, Du) with zero boundary data. Under suitable hypotheses on f, we show that an energy, which is roughly the H¹ norm of u, cannot change too rapidly. When f depends only on u, this estimate implies that all global solutions are bounded.     

Time scale to ergodicity in the Fermi-Pasta-Ulam system

We study the approach to near-equipartition in the N-dimensional Fermi-Pasta-Ulam Hamiltonian with quartic (hard spring) nonlinearity. We investigate numerically the time evolution of orbits with initial energy in some few low-frequency linear modes. Our results indicate a transition where, above a critical energy which is independent of N, one can reach equipartition if one waits for a time proportional to N(2). Below this critical energy the time to equipartition is exponentially long. We develop a theory to determine the time evolution and the excitation of the nonlinear modes based on a resonant normal form treatment of the resonances among the oscillators. Our theory predicts the critical energy for equipartition, the time scale to equipartition, and the form of the nonlinear modes below equipartition, in qualitative agreement with the numerical results. (c) 1995 American Institute of Physics.