Analysis of complex multidimensional optical spectra by linear prediction.

We apply Linear Prediction from Singular Value Decomposition (LPSVD) to two-dimensional complex optical data in the time-domain to generate spectra with advantages over discrete Fourier transformation (DFT). LPSVD is a non-iterative procedure that fits time-domain complex data to the sum of damped sinusoids, or Lorentzian peaks in the spectral domain. Because the fitting is linear, it is not necessary to give initial guess parameters as in nonlinear fits. Although LPSVD is a one-dimensional algorithm, it can be performed column-wise on two-dimensional data. The method has been extensively used in 2D NMR spectroscopy, where spectral peaks are typically nearly ideal Lorentzians, but to our knowledge has not been applied in the analogous optical technique, where peaks can be far from Lorentzian. We apply LPSVD to the analysis of zero, one, and two quantum electronic two-dimensional spectra from a semiconductor microcavity. The spectra consist of non-ideal, often overlapping peaks. We find that LPSVD achieves a very good fit even on non-ideal data. It reduces noise and eliminates discrete distortions inherent in the DFT. We also use it to isolate and analyze weak features of interest.

Assessment of methods for determination of glycan composition of erythropoietin.

Erythropoietin (EPO) is a monomeric, highly glycosylated, protein hormone (molecular size around 30-35 kD), produced mainly in adult kidneys, which acts principally on red blood cell progenitors and precursors to promote red cell production. Therapeutic EPO products are widely used biotherapeutics. They are mainly produced by recombinant DNA technology in mammalian cells and their biological activity is closely linked to the degree of N-glycan sialylation. Determination of the sialic acids' content and complexity by glycan mapping therefore appears critical to ensure the quality and efficacy of the EPO therapeutic products. The European Directorate for the Quality of Medicines & HealthCare organised a study (BSP144) under the aegis of the Biological Standardisation Programme to assess N-glycan mapping tests with the aim of incorporating a standard method into the European Pharmacopoeia monograph 'Erythropoietin concentrated solution' (1316). The use of a 'reagent panel' consisting of six EPO preparations with a range of iso-electric properties facilitated comparison between laboratories and methodologies. Based on the study results, a robust and repeatable HPAEC-PAD chromatographic method was identified and work to introduce it in the monograph as an example method has been initiated.

Automated polarization-dependent multidimensional coherent spectroscopy phased using transient absorption.

An experimental apparatus is described for multidimensional optical spectroscopy with fully automated polarization control, based on liquid crystal variable retarders. Polarization dependence of rephasing two-dimensional coherent spectra are measured in a single scan, with absolute phasing performed for all polarization configurations through a single automated auxiliary measurement at the beginning of the scan. A factor of three improvement in acquisition time is demonstrated, compared to the apparatus without automated polarization control. Results are presented for a GaAs quantum well sample and an InGaAs quantum well embedded in a microcavity.

Application of wavelet analysis on transient reflectivity in ultra-thin films.

Applications of wavelet analysis in ultra-thin film transient reflectivity (TR) measurements have been investigated. Advantages of utilizing different localized wavelet bases, in position and time, have been addressed on the residual TR signals. Morse wavelets have been used to obtain information from the abrupt oscillatory modes in the signal, which are not distinguishable with conventional methods such as Fourier transforms. These abrupt oscillatory modes are caused by the surface, interface, or any short-lived oscillatory modes which are suppressed in the TR signal in ultra-thin films. It is demonstrated that by choosing different Morse wavelets, information regarding different oscillatory modes in the TR signal of a heterostructure thin film is achievable. Moreover, by performing wavelet analysis on multiferroic heterostructures, oscillatory modes with very close energy ranges are easily distinguishable. For illustration, residuals of the TR signals have been obtained by a pump-probe setup in reflectivity mode on La0.7Sr0.3MnO3/SrTiO3 and BaTiO3/La0.7Sr0.3MnO3/SrTiO3 samples, where sufficient signal to noise ratios have been achieved by taking multiple scans. The residual signals have been analyzed with Morse wavelets, and multiple oscillatory modes with close energy ranges have been observed and distinguished. This approach can isolate the location of various oscillatory modes at the surface, interface and in the bulk of the heterostructure sample.

Establishment of an erythropoietin CRS with stable measurable dimer content for SEC system suitability qualification

The European Pharmacopoeia (Ph. Eur.) monograph 1316 'Erythropoietin concentrated solution' prescribes that the dimer content of therapeutic erythropoietin (EPO) preparations must not exceed 2% as determined by Size-Exclusion Chromatography (SEC). This report describes the evaluation of a candidate Chemical Reference Substance (cCRS) to serve as system suitability reference material for the qualification of SEC systems used to assess dimer and oligomer content in EPO solutions. The study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) was performed with the participation of six European laboratories which tested the candidate material and the EPO for physicochemical tests CRS batch 1. The candidate material was shown to be a suitable reference material for the determination of the resolving capability of the SEC system for separation of dimer and higher oligomers from monomeric EPO. The cCRS was adopted by the Ph. Eur. Commission as Erythropoietin for SEC system suitability CRS batch 1 following consideration of the report. The importance of the resolving capability of the SEC system, as defined by the peak ratios or the peak-to-valley resolution, together with the asymmetry of the peaks eluted, and the linear response of the UV detector were all seen as critical parameters. Therefore, the monograph Erythropoietin concentrated solution (1316) was revised concomitantly to take account of the CRS and to set acceptance criteria for these critical parameters..

Stimulated two-photon emission in bulk CdSe.

While two-photon emission processes are firmly established in atomic physics, their observation and use in semiconductor physics remains elusive. Here, we experimentally investigate stimulated two-photon emission in photoexcited bulk CdSe and identify requirements for the observation of stimulated two-photon emission. In particular, this process requires population inversion as well as two-photon transition energies close to the bandgap energy. In any regime investigated in the present study, net optical gain is not achieved, as the free-carrier absorption intrinsically linked to the photoexcitation completely masks the two-photon gain. The results are well in line with a recent study on nondegenerate versions of two-photon emission in GaAs and place clear limits for the practical use of two-photon emission in optically excited semiconductors.

Quantification of low levels of methionine oxidation in erythropoietin drug substance.

A pharmacopoeial monograph under development for recombinant human erythropoietin (rhEPO) drug substance is likely to contain a specification limit for the proportion of the methionine-oxidised variant. Methionine oxidation has no effect on the folded structure and global thermodynamic stability of rhEPO but can decrease biological activity [1]. We describe here the development of a reference standard, a calibrated mixture of the native and oxidised tryptic peptides which contain methionine-54, and an optimised peptide mapping procedure to support this assay. The approach may be developed for analysis of drug product or generalised for other assays in which product-related impurities are quantified by peptide mapping.

Establishment of the Ph. Eur. erythropoietin chemical reference substance batch 1.

The Erythropoietin (EPO) European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 3 was calibrated in 2006 by in vivo bioassay and was used as a reference preparation for these assays as well as for the physicochemical methods in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). In order to avoid the frequent replacement of this standard and thus reduce the use of animals, a new EPO Chemical Reference Substance (CRS) was established to be used solely for the physicochemical methods. Here we report the outcome of a collaborative study aimed at demonstrating the suitability of the candidate CRS (cCRS) as a reference for the physicochemical methods in the Ph. Eur. monograph. Results from the study demonstrated that for the physicochemical methods currently required in the monograph (capillary zone electrophoresis (CZE), polyacrylamide gel electrophoresis (PAGE)/immunoblotting and peptide mapping), the cCRS is essentially identical to the existing BRP. However, data also indicated that, for the physicochemical methods under consideration for inclusion in a revised monograph (test for oxidised forms and glycan mapping), the suitability of the cCRS as a reference needs to be confirmed with additional work. Further to completion of the study, the Ph. Eur. Commission adopted the cCRS as "Erythropoietin for physicochemical tests CRS batch 1" to be used for CZE, PAGE/immunoblotting and peptide mapping.

Collaborative study for the establishment of erythropoietin BRP batch 4.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for erythropoietin (EPO) is used as a working standard for potency determination of EPO preparations by in vivo bioassay as prescribed in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). The BRP batch 3 was calibrated in 2006 and its stocks are depleted. The European Directorate for the Quality of Medicines & HealthCare (EDQM) thus initiated a project to calibrate a replacement batch in International Units against the WHO 3(rd) International Standard (IS) for Erythropoietin, recombinant, for bioassay (11/170). A Ph. Eur. Chemical Reference Substance (CRS) was established recently for use as reference in some of the physicochemical tests prescribed in the monograph. Therefore, the EPO BRP batch 4 was only calibrated for the normocythaemic and polycythaemic mouse in vivo bioassays described in the Assay section of the Ph. Eur. monograph (1316). The collaborative study involved seven laboratories from Europe, the USA and South America. The results confirmed that the candidate BRP (cBRP) is suitable for use as a reference preparation in the potency determination of EPO medicinal products by bioassay (using the normocythaemic or polycythaemic mouse methods). The outcome of the study enabled the Ph. Eur. Commission to establish the proposed standard as erythropoietin BRP batch 4 in November 2014 for use as a reference preparation solely for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 13 000 IU/vial. Furthermore, the potency of BRP3 was confirmed during the study, thus warranting a good continuity of the IU.

Broadband terahertz pulse emission from ZnGeP2.

Optical rectification is demonstrated in (110)-cut ZnGeP(2) (ZGP) providing broadband terahertz (THz) generation. The source is compared to both GaP and GaAs over a wavelength range of 1150 nm to 1600 nm and peak-intensity range of 0.5 GW/cm(2) to 40 GW/cm(2). ZGP peak-to-peak field amplitude is larger than in the other materials due to either lower nonlinear absorption or larger second-order nonlinearity. This material is well suited for broadband THz generation across a wide range of infrared excitation wavelengths.

Regulatory requirements for therapeutic proteins: the relationship between the conformation and biological activity of filgrastim.

Higher order structure, including conformation, is considered a critical quality parameter of therapeutic proteins, and is mandatory information in development of first use and bio-similar therapeutic protein drugs, the assumption being that the biological activity of a protein is directly dependent on its adoption of a 'correct' conformation. Studies on the relationship between conformation and activity depend on the ability to induce conformational changes in proteins, and conventional approaches such as thermal or chemical denaturation are incompatible with bioactivity measurements. To explore the relationship between bio-activity and conformational studies, we have studied variants of the therapeutic protein filgrastim (rec met huGCSF) which have been mutated by the replacement of helical alanine residues with glycine, to destabilise the conformation of the molecule. In the GCSF A-G mutant series studied, single conformation-destabilising amino-acid substitutions significantly reduced the biological activity. These effects were not, however correlated with changes in secondary structure measurable by far-UV Circular Dichroism (CD) spectroscopy. Only the more extensively mutated double and triple substitutions showed measurable reductions in alpha-helical structure by CD. We conclude that in this system, GCSF does not readily adopt a reduced-activity altered conformational state which can be detected by low resolution techniques such as CD. In contrast, reductions in biological activity do reflect reductions in conformational stability, possibly caused by time-dependent degradation of the protein in the cell-proliferation bioassay. Although not a formal model of biosimilarity, we suggest that our results could inform the regulatory process in determining appropriate experimental approaches to meeting regulatory requirements for higher order structural analysis of therapeutic proteins.

Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA.

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

A versatile ultrastable platform for optical multidimensional Fourier-transform spectroscopy.

The JILA multidimensional optical nonlinear spectrometer (JILA-MONSTR) is a robust, ultrastable platform consisting of nested and folded Michelson interferometers that can be actively phase stabilized. This platform generates a square of identical laser pulses that can be adjusted to have arbitrary time delay between them while maintaining phase stability. The JILA-MONSTR provides output pulses for nonlinear excitation of materials and phase-stabilized reference pulses for heterodyne detection of the induced signal. This arrangement is ideal for performing coherent optical experiments, such as multidimensional Fourier-transform spectroscopy, which records the phase of the nonlinear signal as a function of the time delay between several of the excitation pulses. The resulting multidimensional spectrum is obtained from a Fourier transform. This spectrum can resolve, separate, and isolate coherent contributions to the light-matter interactions associated with electronic excitation at optical frequencies. To show the versatility of the JILA-MONSTR, several demonstrations of two-dimensional Fourier-transform spectroscopy are presented, including an example of a phase-cycling scheme that reduces noise. Also shown is a spectrum that accesses two-quantum coherences, where all excitation pulses require phase locking for detection of the signal.

Glycan mapping of glycotherapeutics.

Glycosylation of proteins may, or may not, affect biological activity, either directly or indirectly. In addition, variability in glycosylation arises within the manufacturing procedure. Therefore, glycosylation would be an important parameter when assessing product quality. Regulatory authorities require biotechnological and biological products to be characterised, including determination of their biological activity, physicochemical and immunochemical properties, their purity and their impurity profiles. Here we outline how we can decide if and which types of glycan analysis and methodologies to use for glycoprotein therapeutics to answer the scientific questions as well as meeting regulatory requirements that already exist or are going to be developed/implemented.

Can computerised tomography replace bone scintigraphy in detecting bone metastases from breast cancer? A prospective study.

BACKGROUND: The aim of this study was to determine whether bone scans (BS) can be avoided if pelvis was included in CT thorax and abdomen to detect bony metastases from breast cancer. MATERIALS AND METHODS: Results of 77 pairs of CT (thorax, abdomen, and pelvis) and BS in newly diagnosed patients with metastatic breast cancer (MBC) were compared prospectively for 12 months. Both scans were blindly assessed by experienced radiologists and discussed at multidisciplinary team meetings regarding the diagnosis of bone metastases. RESULTS: CT detected metastatic bone lesions in 43 (98%) of 44 patients with bone metastases. The remaining patient had a solitary, asymptomatic bony metastasis in shaft of femur. BS was positive in all patients with bone metastases. There were 11 cases of false positive findings on BS. CONCLUSION: Our findings suggest routine BS of patients presenting with MBC is not required if CT (thorax, abdomen, and pelvis) is performed.

The role of the vagus nerve in mediating the long-term anorectic effects of leptin.

Leptin, the product of the obese (ob) gene, is mainly known for its regulatory role of energy balance by direct activation of hypothalamic receptors. Recently, its function in the acute control of food intake was additionally attributed to activation of the vagus nerve to regulate meal termination. Whether vagal afferent neurones are involved in longer term effects of leptin on food intake, however, remains undetermined. Using vagotomised (VGX) rats, we sought to clarify the contributions of vagal afferents in mediating the long-lasting effect of leptin on appetite suppression. Intraperitoneal (i.p.) injection of leptin (3.5 mg/kg) attenuated food intake at 4, 6, 8 and 24 h and body weight at 24 h postinjection in SHAM-operated rats; however, this response was not abrogated by vagotomy. In a separate study using immunohistochemistry, we observed leptin-induced Fos expression in the nucleus tractus solitarii, a brain structure where vagal afferent fibres terminate. This signal was not attenuated in VGX animals compared to the SHAM group. Moreover, leptin treatment led to a similar level of nuclear STAT3 translocation, a marker of leptin signalling, in the hypothalami of SHAM and VGX animals. In addition to the effects of leptin, vagotomy surgery itself resulted in a decrease of 24 h food intake. Analyses of brains from saline-treated VGX animals revealed a significant induction of Fos in the nucleus tractus solitarii and changes in agouti-related peptide and pro-opiomelanocortin mRNA expression in the hypothalamus compared to their SHAM counterparts, indicating that the vagotomy surgery itself induced a modification of brain activity in areas involved in regulating appetite. Collectively, our data suggest that vagal afferents do not constitute a major route of mediating the regulatory effect of leptin on food intake over a period of several hours.

Collaborative study for the establishment of erythropoietin BRP batch 3.

The European Pharmacopoeia (Ph. Eur.) monograph on Erythropoietin concentrated solution (1316) specifies that identification and assay are performed using pharmacopoeial methods requiring the use of a reference preparation. To replace the current erythropoietin Biological Reference Preparation (BRP) of Ph. Eur., in 2006, the European Directorate for the Quality of Medicines undertook a collaborative study designed to establish a replacement batch. In order to guarantee continuity, the formulation of the candidate batch was similar to that of previous batches (1 and 2). The methods chosen to qualify the new standard were those included in the current monograph. The study was defined to allow calibration of the candidate by in vivo bioassay in terms of the current World Health Organization (WHO) International Standard (IS) and to assign a unitage. The suitability of the candidate preparation to serve as a reference standard for the other pharmacopoeial analytical procedures was also investigated. The collaborative study involved 16 laboratories from Europe, Australia, Canada, China, Japan, South Korea and the United States of America. Participants carried out biological and physicochemical assays on the candidate erythropoietin BRP batch 3 (cBRP3), using batch 2 (BRP2) and where necessary the 2nd World Health Organization International Standard (WHO 2nd IS) for recombinant erythropoietin as the reference standards. It was demonstrated that the replacement batch is appropriate for use as erythropoietin BRP in the context of the control of erythropoietin concentrated solutions according to the Ph. Eur. monograph (1316). However as regards the potency of BRP2 and cBRP3 in the mouse bioassay unexpected observations were made. Direct calibration of BRP2 against the WHO 2nd IS yielded, in all laboratories, results that were systematically higher than the potency of 32,500 IU/vial assigned by direct calibration against the WHO 2nd IS in the former study. It was therefore recommended to assign the potency of cBRP3 against BRP2, using the average of all results that were not considered as outlying obtained in the collaborative study, in order to guarantee continuity of unitage between the successive BRP batches. The outcome of the study enabled the Ph. Eur. Commission to establish the proposed standard as 'erythropoeitin BRP batch 3' (BRP3). BRP3 was established in June 2007 for use as a reference preparation for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 35,280 IU/vial, the identification by capillary zone electrophoresis, by polyacrylamide gel electrophoresis, immunoblotting and peptide mapping and as a reference for checking the system suitability of size-exclusion chromatographic procedures used in the test for dimers and related substances of higher molecular mass in the Ph. Eur. monograph (1316).

Enhanced all-optical tuning of leaky eigenmodes in photonic crystal waveguides.

We demonstrate all-optical switching in an active two-dimensional photonic crystal waveguide, observing as large as 16 nm blueshifts of a leaky eigenmode at 839 nm and switching ratios of almost 70%. These results are larger than those previously observed in similar experiments performed on passive photonic crystal waveguides; the enhancement is due to resonant photogeneration of carriers by In(0.12)Al(0.2)Ga(0.68)As quantum wells in the core of the waveguide. The effective change in the refractive index of the structure is approximately10(-2), with a rise time of approximately1 ps and a decay time of approximately10 ps, potentially allowing high-speed switching and fast modulation rates.

Collaborative study for the establishment of erythropoietin BRP batch 2.

The preparation and establishment of the 2nd European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for erythropoietin was the goal of a project run within the framework of the European Biological Standardisation Programme. The project, coded BSP062, was carried out between October 2002 and July 2003. The candidate preparation (cBRP2) was prepared in a similar manner to the first BRP batch (BRP1), as follows: -50:50 (weight/weight) blending of the two erythropoietin preparations currently available on the European market (epoietin-alpha and epoietin-beta), -lyophilisation using a protein-free carrier formulation to allow use of the standard for both biological and physico-chemical assay methods, -each vial contains approximately 250 microg erythropoietin. The cBRP2 was analysed in a collaborative study, carried out with the following aims: -to calibrate cBRP2 by in vivo bioassay in terms of the International Standard for erythropoietin, and assign a unitage, -to demonstrate continuity of unitage with BRP1, -to evaluate the suitability of cBRP2 to serve as a reference material for physico-chemical tests of erythropoietin. The collaborative study involved 14 laboratories both from Europe, and from Australia, Canada, South-Korea and the United States of America. Participants carried out biological and physicochemical assays on the candidate BRP batch 2, using BRP 1 and the 2nd World Health Organization (WHO) International Standard (IS) for recombinant erythropoietin as the reference standards. It was demonstrated that: -an assigned potency of 32,500 U per vial would maintain continuity between BRP1 and BRP2 in terms of the IS for erythropoietin, -the replacement batch was appropriate for use as erythropoietin BRP in the context of the control of erythropoietin concentrated solutions according to the Ph. Eur. monograph 1316. In July 2003, the Ph. Eur. Commission established the proposed standard as 'Erythropoeitin BRP batch 2' for use as a reference preparation for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 32,500 U/vial, the identification by capillary zone electrophoresis (CZE), by polyacrylamide gel electrophoresis, immunoblotting and peptide mapping and as a reference for checking the system suitability of size exclusion chromatographic procedures used in the test for 'Dimers and related substances of higher molecular mass'.