Characterization of salt interferences in second-harmonic generation detection of protein crystals.

Studies were undertaken to assess the merits and limitations of second-harmonic generation (SHG) for the selective detection of protein and polypeptide crystal formation, focusing on the potential for false positives from SHG-active salts present in crystallization media. The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples. Out of 19 salts investigated, six produced significant background SHG and 15 of the 96 wells of a sparse-matrix screen produced SHG upon solvent evaporation. SHG-active salts include phosphates, hydrated sulfates, formates and tartrates, while chlorides, acetates and anhydrous sulfates resulted in no detectable SHG activity. The identified SHG-active salts produced a range of signal intensities spanning nearly three orders of magnitude. However, even the weakest SHG-active salt produced signals that were several orders of magnitude greater than those produced by typical protein crystals. In general, SHG-active salts were identifiable through characteristically strong SHG and negligible two-photon-excited ultraviolet fluorescence (TPE-UVF). Exceptions included trials containing either potassium dihydrogen phosphate or ammonium formate, which produced particularly strong SHG, but with residual weak TPE-UVF signals that could potentially complicate discrimination in crystallization experiments using these precipitants.

Selective imaging of active pharmaceutical ingredients in powdered blends with common excipients utilizing two-photon excited ultraviolet-fluorescence and ultraviolet-second order nonlinear optical imaging of chiral crystals.

Second order nonlinear optical imaging of chiral crystals (SONICC) and two-photon excited fluorescence measurements [both autofluorescence and two-photon excited UV-fluorescence (TPE-UVF)] were assessed for the selective detection of APIs relative to common pharmaceutical excipients. Active pharmaceutical ingredients (APIs) compose only a small percentage of most tabulated formulations, yet the API distribution within the tablet can affect drug release and tablet stability. Complementary measurements using either UV-SONICC (266 nm detection) or TPE-UVF were shown to generate signals >50-fold more intense for a model API (griseofulvin) than those produced by common pharmaceutical excipients. The combined product of the measurements produced signals >10(4)-fold greater than the excipients studied. UV-SONICC or TPE-UVF produced greater selectivity than analogous measurements with visible-light detection, attributed to the presence of aromatic moieties within the API exhibiting strong one and two photon absorption at ~266 nm. Complementary SONICC and fluorescence measurements allowed for the sensitive detection of the three-dimensional distribution of tadalafil within a Cialis tablet to a depth of >140 µm.

Single particle nonlinear optical imaging of trace crystallinity in an organic powder.

Microscopic characterization of crystallinity in powders can reveal information lost in ensemble-averaged measurements. Nonlinear optical imaging based on second harmonic generation (SHG) provides rapid and highly selective detection of individual chiral microcrystals, enabling insights into the fundamental mechanism of action for the observed crystallinity loss of an organic powder induced by mechanical grinding. Using griseofulvin as the model compound, the results from second order nonlinear optical imaging of chiral crystals (SONICC) compared favorably with those of powder X-ray diffraction (PXRD) over the linear dynamic range of the PXRD measurements. However, the SHG measurements demonstrated three decade improvements in linear dynamic range. The detection limit of SHG was estimated to be 4 ppm crystallinity in the powder. The rate of crystallinity loss induced by milling followed a first order process with a half-life of 15 ± 1 min. Recrystallization of cryomilled powder is ~40 times faster than that prepared by melt-quenched powder, suggesting that the disordered state obtained by exhaustive cryomilling appears to contain ordered domains that are larger than the critical nucleation size, but below the detection limit of SONICC. The presence of such domains provides a barrier-less nucleation source resulting in rapid crystallization, the kinetics of which depends only on crystal growth.

Detection of membrane protein two-dimensional crystals in living cells.

It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation.

Nanoengineered structures for holding and manipulating liposomes and cells.

We describe the fabrication of nanoengineered holding pipets with concave seating surfaces and fine pressure control. These pipets were shown to exhibit exceptional stability in capturing, transporting, and releasing single cells and liposomes 1-12 microm in diameter, which opens previously inaccessible avenues of research. Three specific examples demonstrated the utility and versatility of this manipulation system. In the first, carboxyrhodamine was selectively incorporated into individual cells by electroporation, after which nearly all the medium (hundreds of microliters) surrounding the docked and tagged cells was rapidly exchanged (in seconds) and the cells were subsequently probed by laser-induced fluorescence (LIF). In the second study, a single liposome containing carboxyrhodamine was transported to a dye-free solution using a transfer pipet, docked to a holding pipet, and held firmly during physical agitation and interrogation by LIF. In the third study, pairs of liposomes were positioned between two microelectrodes, held in contact, and selectively electrofused and the resulting liposomes undocked intact.

Influence of threonine intake on whole-body protein deposition and threonine utilization in growing pigs fed purified diets.

The relationship between available threonine (Thr) intake and whole-body protein deposition (PD) was established using the serial slaughter method in 36 individually housed growing gilts between 39 and 77 kg live BW. Pigs were prescreened for their maximum PD (PDmax), based on a N balance starting at 25 kg BW while they consumed semi-ad libitum a nonlimiting diet. Pigs were fed combinations of a casein and cornstarch-based diet that was confirmed to be first-limiting in Thr and a protein-free diet starting at approximately 30 kg BW. Casein-bound Thr was provided at 60, 70, 80, 90, 100, or 120% of estimated Thr requirements for PDmax. Energy intake was kept constant across treatments and exceeded requirements for PDmax. Pigs were fed three equal meals per day; feeding levels were adjusted weekly based on BW. Pigs were killed at either 39 kg BW (n = 2 per treatment) or 77 kg BW (n = 4 per treatment) for determining chemical body composition. Composition of 39-kg BW pigs was not different across treatments (P > 0.10); therefore, an overall mean initial body composition was used to estimate body protein content at the initial BW. Across treatments, mean daily ME intake was 25.3 (SE 0.08) MJ/d and did not differ (P > 0.10) among treatments. Average daily true ileal digestible Thr intake varied between 5.33 and 9.66 g/d, representing means for pigs on the lowest and the highest Thr intakes, respectively. Mean PD was 93, 102, 118, 124, 139, and 133 (SE 4.2) g/d for pigs on the six respective treatments. Dietary Thr intake did not influence (P > 0.10) Thr content of body protein at the final BW or the partitioning of body protein between carcass, viscera, and blood. The efficiency of Thr utilization for PD was lowest (P < 0.05) at the highest Thr intake level and highest (P < 0.05) at the lowest Thr intake level. It was similar (P > 0.10) at the four intermediate Thr intake levels, in which the relationship between true ileal digestible Thr intake and PD was linear. Based on these four treatments, calculated Thr disappearance, which is closely associated with inevitable Thr catabolism, was 23.5 (SE 0.55)% of available Thr intake. This value is consistent with an efficiency of using available Thr intake above maintenance Thr requirements (54 mg/kg BW0.75) for Thr retention with PD of 73.4 (SE 1.11)%. Based on N balances conducted at approximately 40 and 75 kg BW, the marginal efficiency of Thr utilization was not influenced by BW.

Measurement of orientation in organic thin films.

As the potential for applications utilizing oriented thin films grows, so grows the need for accurate, reliable measurements of molecular orientation and surface coverage. Recent work in our laboratory has been directed toward this goal. In this paper the theoretical and experimental effects of surface roughness and the width of the molecular orientation distribution on spectroscopically measured orientation angles are reviewed, the combination of linear and nonlinear spectroscopic techniques for accurate determination of both the mean and width of an orientation distribution is described, and the theory and methodology necessary to obtain orientation-insensitive surface coverage measurements by second harmonic generation for adsorption isotherm and kinetics investigations are reviewed.