3D reconstruction of the hemocyanin subunit dimer from the chiton Acanthochiton fascicularis

Procedures are presented for the purification of the subunit dimer from Acanthochiton fasicularis hemocyanin. Electron microscopy of negatively stained specimens revealed a uniform population of macromolecules possessing the characteristic "boat shape". A 3D reconstruction from this EM data generated a approximately 3 nm resolution model that correlates well with earlier data of the purported subunit dimer, extracted from the 3D reconstruction of the didecamer of Haliotis tuberculata hemocyanin type 1.     

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.     

Quasiperiodic Raman technique for ultrashort pulse generation

We report the experimental demonstration of a new Raman technique that produces 200 sidebands, ranging in wavelength from 3 microm to 195 nm. By studying multiphoton ionization of nitric oxide (NO) molecules, we show mutual phase coherence among 15 visible sidebands covering 0.63 octaves of bandwidth.     

Photocurrent, rectification, and magnetic field symmetry of induced current through quantum dots

We report mesoscopic dc current generation in an open chaotic quantum dot with ac excitation applied to one of the shape-defining gates. For excitation frequencies large compared to the inverse dwell time of electrons in the dot (i.e., GHz), we find mesoscopic fluctuations of induced current that are fully asymmetric in the applied perpendicular magnetic field, as predicted by recent theory. Conductance, measured simultaneously, is found to be symmetric in field. In the adiabatic (i.e., MHz) regime, in contrast, the induced current is always symmetric in field, suggesting its origin is mesoscopic rectification.     

Accurate Jones matrix expansion for all orders of polarization mode dispersion

I present a simple method for extracting the dispersive elements from a general unitary Jones matrix in such a way that the parts comprising the phase delays and higher-order dispersion in the two principal states of polarization are separated from those describing the frequency-dependent coupling between them. I then rigorously expand these two parts into power series in relative optical frequency, wherein the coefficients are explicit functions of the polarization mode dispersion vectors of various orders. This accurate expansion of the Jones matrix reveals in particular the amplitudes and phases of the cross-coupled components of the principal states of polarization, which are incorrectly described in most polarization mode dispersion models.     

Reconstitution and biochemical characterization of a new pyridoxal-5'-phosphate biosynthetic pathway

The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified.     

Generation of a single-cycle optical pulse

We make use of coherent control of four-wave mixing to the ultraviolet as a diagnostic and describe the generation of a periodic optical waveform where the spectrum is sufficiently broad that the envelope is approximately a single-cycle in length, and where the temporal shape of this envelope may be synthesized by varying the coefficients of a Fourier series. Specifically, using seven sidebands, we report the generation of a train of single-cycle optical pulses with a pulse width of 1.6 fs, a pulse separation of 11 fs, and a peak power of 1 MW.     

Enantioselective syntheses of isoaltholactone, 3-epi-altholactone, and 5-hydroxygoniothalamin

A flexible enantioselective route to highly functionalized alpha, beta-unsaturated delta-lactones has allowed for the syntheses of the styryllactones: isoaltholactone, 3-epi-altholactone, and 5-hydroxygoniothalamin in 10%, 5%, and 13% overall yields from furfural, respectively. This approach derives its asymmetry by applying the Sharpless catalytic asymmetric dihydroxylation to vinylfuran. The resulting diols are produced in high enantioexcess and can be stereoselectively transformed into alpha,beta-unsaturated delta-lactones via a short highly diastereoselective oxidation and reduction sequence.     

Visualization of Taylor-Couette and spiral Poiseuille flows using a snapshot FLASH spatial tagging sequence

A new magnetic resonance imaging technique was applied to the Taylor-Couette and spiral Poiseuille (Taylor-Couette with superposed mean axial flux) flows for the first time. The experimental technique is a combination of spatial tagging methods and a snapshot FLASH imaging sequence, which allows the full-field visualization of 2-D slices of the flow field, with image acquisition times approximately half a second. By acquiring images every few seconds, direct visualization of flow patterns can be obtained in the form of cinematography. Tagged images of the Taylor-Couette flow were acquired in both the axial and transverse planes and confirmed previously reported numerical predictions of Taylor cell size. Tagged images of the spiral Poiseuille flows verified that the cells in this flow propagate at a higher velocity than the mean axial flow. In addition, intermittent cell formation was observed as the axial flow was increased.