Optimization of extraction solvents, solid phase extraction and decoupling for quantitation of free isoprenoid diphosphates in Haematococcus pluvialis by liquid chromatography with tandem mass spectrometry.

Isoprenoid diphosphates are important precursors actively participating in many downstream metabolisms; they are often in modified forms, e.g., protein-coupled or esterified form. Therefore, in vivo level of free isoprenoid diphosphates is quite low, ˜0.07 nmol/g fresh weight in plants. In order to directly measure the isoprenoid diphosphate pool during stress-induced accumulation of astaxanthin in Haematococcus pluvialis, the present study optimized several pretreatment procedures to enrich free isoprenoid diphosphates for high-pressure liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) detection. Specifically, different extraction solvents, e.g., water, methanol, chloroform, and mixture of water, methanol, and chloroform (1:1:1, V/V/V), and solid phase extraction (SPE) columns (OASIS@ WAX and HLB Cartridges) were compared; and gentle decoupling by NaOH or trifluoroacetic acid (TFA) was introduced to release free isoprenoid diphosphates. Results found that solvent mixture of water, methanol and chloroform (1:1:1, V/V/V) showed the highest extraction efficiency (RE) for five isoprenoid diphosphates, ranging from 76.83% to 92.43%; HLB column showed the balanced recoveries ranging from 75.29% to 87.54%; and incubation with low NaOH (˜4.7 mmol/L) at 4 °C significantly increased detectable isoprenoid diphosphates in algal cells, some of which were undetectable or in trace level before NaOH decoupling. The method was applied to H. pluvialis cells under various stresses. Low levels of isoprenoid diphosphates were determined in most of the stresses used, e.g., 0.19 ± 0.09 to 0.98 ± 0.06 mg/g fresh weight (FW) for IPP/DMAPP, 0.35 ± 0.07 mg/g FW for GGPP and undetectable for FPP and GPP; while isoprenoid diphosphates were significantly accumulated in the dark to 3.27 ± 0.05, 0.17 ± 0.09, 1.81 ± 0.16 and 0.58 ± 0.07 mg/g FW for IPP/DMAPP, GPP, FPP and GGPP, respectively. These results implied that isoprenoid diphosphates were exhausted by downstream carotenogenesis under stress. Our work emphasizes NaOH decoupling for exact quantitation of in vivo isoprenoid diphosphates.

A rapid UHPLC-HILIC method for algal guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and the potential separation mechanism.

A fast and facile hydrophilic interaction liquid chromatography (HILIC) method was developed and applied to quantify physiologically important ppGpp and its analogues in a tough sample, the astaxanthin-accumulating alga Hameatococcus pluvialis. The method is able to analyze simultaneously seven nucleotides, including ppGpp at the order of pmol g-1 cells within 12 min. Mechanism on the elution order was investigated. It was found that 1) phosphate salt competed for the amide groups on the HILIC column with the phosphate groups of the nucleotides; 2) intramolecular hydrogen bonds might contribute to the elution order by offsetting and reducing the number of free hydrogen acceptor/donor of the nucleotide molecules interacting with the amide groups. This is the first HILIC method for ppGpp, which is feasible and applicable to a wide range of samples, especially tough samples, e.g., algae and plants.

A Novel Hydrolytic Activity of Tri-Functional Geranylgeranyl Pyrophosphate Synthase in Haematococcus pluvialis.

Under environmental stresses, Haematococcus pluvialis accumulates large amounts of carotenoids. Scale of carotenoid biosynthesis depends on availability of geranylgeranyl pyrophosphate (GGPP) precursor, which is supplied by GGPP synthase (GGPPS) through sequential 1'-4 condensation of three isopentenyl pyrophosphates (IPPs) into dimethylallyl pyrophosphate (DMAPP). Using IPP and DMAPP as substrates, a tri-functional HpGGPPS was identified in this study to promiscuously synthesize allylic prenyl pyrophosphates (PPPs), e.g. C10 geranyl pyrophosphate (GPP), C15 farnesyl pyrophosphate (FPP), and C20 GGPP. Intriguingly, HpGGPPS can utilize GPP or FPP as a single substrate to synthesize GGPP by hydrolyzing the allylic PPP substrate into C5 IPP. Transcription of HpGGPPS and key carotenogenesis genes, morphological transformation, and carotenoid biosynthesis were differentially induced by environmental stresses, while HpGGPPS's products were low in vivo, implying that most of PPP flux had been shunted into carotenoid biosynthesis. Hydrolyzing allylic PPP intermediates into C5 building blocks by promiscuous HpGGPPS may be a fail safe for carotenoid accumulation against environmental stress.

Simultaneous determination of 13 carotenoids by a simple C18 column-based ultra-high-pressure liquid chromatography method for carotenoid profiling in the astaxanthin-accumulating Haematococcus pluvialis.

A simple ultra-high-pressure liquid chromatography (UHPLC) method for rapidly and simultaneously identifying thirteen carotenoids in Haematococcus pluvialis was developed in this study. The method is capable of effectively separating two astaxanthin isomers, two ζ-carotene isomers, and three phytoene isomers on two simple C18 columns within 9 and 12min only by using methanol and acetonitrile, respectively. To our best knowledge, this is the rapidest method for these carotenoid isomers, currently. Using this method, carotenoid profiling in the astaxanthin-accumulating H. pluvialis under environmental stresses was successfully carried out. Results indicated that carotenoid biosynthesis was differentially perturbed by environmental stresses, indicating that this simple and rapid method is suitable to not only bacterial but also algal samples, with potential applications for a wide range of samples from plant to animal. Finally, possible reasons for the elution order of carotenoids were studied.

Q-switched 1329 nm Nd:CNGG laser.

We demonstrate a laser-diode-pumped Q-switched 1329 nm neodymium-doped calcium-niobium-gallium-garnet (Nd:CNGG) laser using a V:YAG crystal as a saturable absorber. An average output power of 353 mW and a repetition rate of 13.43 kHz for Q-switched pulses were obtained. The pulse width was from 124 to 151.4 ns under different pump powers. Output power of 685 mW was obtained without the V:YAG crystal inserted.

Ion-implanted optical-stripe waveguides in neodymium-doped calcium barium niobate crystals.

We report on the fabrication and characterization of optical-stripe waveguides in neodymium-doped calcium barium niobate (Nd:CBN) crystals by using He ion implantation assisted with a mask. The guided-mode profiles are successfully modeled through numerical simulations. After annealing at 200 degrees C for 30 min, the waveguide propagation loss could be reduced down to ~3 dB/cm. Room-temperature microluminescence and micro-Raman investigations reveal that the crystal bulk features are well preserved in the active volume of the waveguide, suggesting possible applications as integrated photonic devices.

High average power 1.5 microm eye-safe Raman shifting in BaWO4 crystals.

A nanosecond 1.3 microm Nd:YAG laser-pumped external cavity eye-safe Raman laser based on BaWO(4) crystal is efficiently demonstrated. The average power of 1.5 microm was 0.60 W operating at a pulse repetition rate of 1.7 kHz and an incident pump power of 4.14 W. Conversion efficiency of 14.5% and a high slope efficiency of 54.5% have been achieved. Pulse shortening was obviously observed. The pulse duration of the first Stokes was about 40 ns, much shorter than the fundamental pulse with a duration of approximately 220 ns.

Characterization of optical waveguides in YbVO4 crystals formed by 3.0 MeV oxygen ion implantation.

We report optical planar waveguide formation in vanadate laser crystals YbVO(4) by oxygen ion implantation at an energy of 3.0 MeV and doses from 5 x 10(13) to 2 x 10(15) ions/cm(2) at room temperature. Positive changes of the ordinary refractive indices occur in the implanted regions, which define a waveguide structure in the near surface. The relationship between refractive index changes and doses is presented. Waveguide modes were measured at wavelengths of 633 and 1539 nm, respectively. The magnified field pattern of the propagation light in the annealed waveguide was captured and studied by an end-coupling method. The refractive index profiles were reconstructed by the reflectivity calculation method.

Optical ridge waveguides in SBN crystal produced by low-dose carbon ion implantation followed by a sputter etching technique.

This paper demonstrates, for the first time, a method to fabricate optical ridge waveguides in SBN photorefractive crystal, i.e. by first using high-energy carbon ion implantation (forming planar waveguide substrate) followed by Ar+ ion sputter etching (constructing ridged stripes). A two-dimensional (2D) cross-sectional refractive index profile of ridge waveguide is reconstructed by carefully considering the ridged topography as well as the index distributions of the planar waveguide. Based on this profile, the waveguide modes are calculated, in which shows a reasonable agreement with the experimentally observed modal near-field intensity distributions.

Rubidium gadolinium bis(tungstate).

The structure of rubidium gadolinium bis(tungstate), RbGd(WO4)2, has been determined. The crystal is built up from corner- and edge-sharing WO6 octahedral and GdO8 polyhedral groups, giving rise to a Gd-WO4 polyhedral backbone surrounding structural cavities filled with Rb+ cations. The Gd and Rb atoms lie on twofold axes.

Optical planar waveguide fabricated in Nd:LuVO4 crystal by MeV oxygen implantation.

We report the first time optical planar waveguide fabricated in vanadate laser crystal Nd:LuVO4 by 3.0 MeV oxygen ion implantation with the dose of 6x1014 ions/cm2 at room temperature. After the implantation, an enhanced ordinary refractive index region was formed with a width of about 2.1 microm beneath the sample surface to act as a waveguide structure. The modes were observed at 633 nm, while only one mode was observed at 1539 nm after annealing at 300 degrees C for 60 min in air. The changes of ordinary refractive index in the guiding region were about 4.42% and 4.07% before and after annealing.