Lateralization of Neural Speech Discrimination at Birth Is a Predictor for Later Language Development.

Newborns are able to neurally discriminate between speech and nonspeech right after birth. To date it remains unknown whether this early speech discrimination and the underlying neural language network is associated with later language development. Preterm-born children are an interesting cohort to investigate this relationship, as previous studies have shown that preterm-born neonates exhibit alterations of speech processing and have a greater risk of later language deficits.

Metabolism of selective 20-epi-vitamin D analogs in rat osteosarcoma UMR-106 cells: Isolation and identification of four novel C-1 fatty acid esters of 1α,25-dihydroxy-16-ene-20-epi-vitamin D.

Analogs of 1α,25-dihydroxyvitamin D (S1) with 20-epi modification (20-epi analogs) possess unique biological properties. We previously reported that 1α,25-dihydroxy-20-epi-vitamin D (S2), the basic 20-epi analog is metabolized into less polar metabolites (LPMs) in rat osteosarcoma cells (UMR-106) but not in a perfused rat kidney. Furthermore, we also noted that only selective 20-epi analogs are metabolized into LPMs.

Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) has become an established method for analysis of protein higher order structure. Here, we use HDX-MS methodology based on manual solid-phase extraction (SPE) to allow fast and simplified conformational analysis of proteins under pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup.

Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry.

When highly concentrated, an antibody solution can exhibit unusual behaviors, which can lead to unwanted properties, such as increased levels of protein aggregation and unusually high viscosity. Molecular modeling, along with many indirect biophysical measurements, has suggested that the cause for these phenomena can be due to short range electrostatic and/or hydrophobic protein-protein interactions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for investigating protein conformation, dynamics, and interactions.

Subvisible (2-100 μm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis.

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes.

Technical Decision-Making with Higher Order Structure Data: Detecting Reversible Concentration-Dependent Self-Association in a Monoclonal Antibody and a Preliminary Investigation to Eliminate It.

Protein self-association or aggregation is a property of significant concern for biopharmaceutical products due to the potential ability of aggregates to cause adverse toxicological and immunological effects. Thus, during the development of a protein biopharmaceutical, it is important to detect and quantify the level and nature of aggregate species as early as possible in order to make well-informed decisions and to mitigate and control potential risks. Although a deeper understanding of the mechanism of aggregation (i.

Subvisible (2-100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy.

Measurement and characterization of subvisible particles (defined here as those ranging in size from 2 to 100 μm), including proteinaceous and nonproteinaceous particles, is an important part of every stage of protein therapeutic development. The tools used and the ways in which the information generated is applied depends on the particular product development stage, the amount of material, and the time available for the analysis. In order to compare results across laboratories and products, it is important to harmonize nomenclature, experimental protocols, data analysis, and interpretation.

Size-exclusion chromatography-mass spectrometry with m-nitrobenzyl alcohol as post-column additive for direct characterization of size variants of monoclonal antibodies.

Size-exclusion chromatography (SEC) is commonly used to monitor low molecular weight fragments and aggregates present in recombinant monoclonal antibody (mAb) biopharmaceuticals. It has been previously demonstrated that SEC could be coupled with mass spectrometry (MS) to directly measure the molecular weights of these protein species to aid in their identification. However, the use of certain mobile phase modifiers led to compromised sensitivity in MS detection.

Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases.

Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance.

Isolation and identification of 1alpha-hydroxy-3-epi-vitamin D3, a potent suppressor of parathyroid hormone secretion.

Since our original demonstration of the metabolism of 1alpha,25(OH)2D3 into 1alpha,25(OH)2-3-epi-D3 in human keratinocytes, there have been several reports indicating that epimerization of the 3 hydroxyl group of vitamin D compounds is a common metabolic process. Recent studies reported the metabolism of 25OHD3 and 24(R),25(OH)2D3 into their respective C-3 epimers, indicating that the presence of 1alpha hydroxyl group is not necessary for the 3-epimerization of vitamin D compounds. To determine whether the presence of a 25 hydroxyl group is required for 3-epimerization of vitamin D compounds, we investigated the metabolism of 1alphaOHD3, a non-25 hydroxylated vitamin D compound, in rat osteosarcoma cells (ROS 17/2.

A phthalide with in vitro growth inhibitory activity from an oidiodendron strain.

A screening campaign was implemented utilizing capillary electrophoresis as a primary assay to discover binders to the cancer target Akt1 from a crude natural extract library. Fungal extracts with binding activities were characterized for biochemical inhibition of Akt1 to phosphorylate the downstream substrate protein Bad. One of the crude extracts with bioactivity selected for isolation and structure elucidation from fermentation of the fungal culture Oidiodendron sp.

Tissue specific metabolism of 1alpha,25-dihydroxy-20-epi-vitamin D3 into new metabolites with significant biological activity: studies in rat osteosarcoma cells (UMR 106 and ROS 17/2.8).

In a recent study, we investigated the metabolism of 1alpha,25-dihydroxy-20-epi-vitamin D3 (1alpha,25(OH)2-20-epi-D3), a potent synthetic vitamin D3 analog in the isolated perfused rat kidney and proposed that the enhanced biological activity of 1alpha,25(OH)2-20-epi-D3 is in part due to its metabolism into stable bioactive intermediary metabolites derived via the C-24 oxidation pathway (Siu-Caldera et al. [1999] J. Steroid.

1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 and 1alpha,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3: analogs of 1alpha,25-dihydroxyvitamin D3 that resist metabolism through the C-24 oxidation pathway are metabolized through the C-3 epimerization pathway.

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the previously well established main side chain modification pathway, is initiated by hydroxylation at C-24 of the side chain. The C-3 epimerization pathway, the newly discovered A-ring modification pathway, is initiated by epimerization of the hydroxyl group at C-3 of the A-ring.

Examination of structurally selective derivatization of vitamin D(3) analogues by electrospray mass spectrometry.

The structural specificity of vitamin D derivatization by PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) was probed using synthetic analogues and ion trap mass spectrometry. EB 1089, a vitamin D(3) analogue which contains a second site for Diels--Alder cycloaddition on its side-chain, allowed the examination of derivatization modes and comparisons of ion fragment structures. The origins of a PTAD-vitamin D(3) ion fragment, commonly used in metabolite characterization and quantitation of vitamin D(3) analogues (m/z 314), were established; ion trap mass spectrometry revealed that the PTAD comprises a portion of this diagnostic fragment, and is not lost by a retro-Diels--Alder step.

Enhanced biological activity of 1alpha,25-dihydroxy-20-epi-vitamin D3, the C-20 epimer of 1alpha,25-dihydroxyvitamin D3, is in part due to its metabolism into stable intermediary metabolites with significant biological activity.

1alpha,25-dihydroxy-20-epi-vitamin D3 (1alpha,25(OH)2-20-epi-D3), the C-20 epimer of the natural hormone 1alpha,25(OH)2D3, is several fold more potent than the natural hormone in inhibiting cell growth and inducing cell differentiation. At present, the various mechanisms responsible for the enhanced biological activities of this unique vitamin D3 analog are not fully understood. In our present study we compared the target tissue metabolism of 1alpha,25(OH)2D3 with that of 1alpha,25(OH)2-20-epi-D3 using the technique of isolated perfused rat kidney.

Production of 1alpha,25-dihydroxy-3-epi-vitamin D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed.

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized into calcitroic acid through the carbon 24 (C-24) oxidation pathway. It is now well established that the C-24 oxidation pathway plays an important role in the target tissue inactivation of 1alpha,25(OH)2D3. Recently, we reported that 1alpha,25(OH)2D3 is also metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)2-3-epi-D3] through the carbon 3 (C-3) epimerization pathway in human keratinocytes, human colon carcinoma cells (Caco-2), and bovine parathyroid cells.

1alpha,25-dihydroxy-3-epi-vitamin D3: in vivo metabolite of 1alpha,25-dihydroxyvitamin D3 in rats.

We recently identified 1alpha,25-dihydroxy-3-epi-vitamin D3 as a major in vitro metabolite of 1alpha,25-dihydroxyvitamin D3, produced in primary cultures of neonatal human keratinocytes. We now report the isolation of 1alpha,25-dihydroxy-3-epi-vitamin D3 from the serum of rats treated with pharmacological doses of 1alpha,25-dihydroxyvitamin D3. 1alpha,25-dihydroxy-3-epi-vitamin D3 was identified through its co-migration with synthetic 1alpha,25-dihydroxy-3-epi-vitamin D3 on both straight and reverse phase high performance liquid chromatography systems and by mass spectrometry.

Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides.

Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4.

Differentiation-related pathways of 1 alpha,25-dihydroxycholecalciferol metabolism in human colon adenocarcinoma-derived Caco-2 cells: production of 1 alpha,25-dihydroxy-3epi-cholecalciferol.

We used the human colon adenocarcinoma-derived cell line Caco-2, which spontaneously differentiates in vitro, as a model system to investigate the metabolism of 1 alpha,25-dihydroxycholecalciferol in colon cancer cells. Subconfluent proliferating and confluent differentiating cells were incubated with 1 microM 1 alpha,25-dihydroxycholecalciferol for a period of 24 to 48 h. HPLC analysis of the lipid extract of both cells and media was performed to isolate and identify the various metabolites of 1 alpha,25-dihydroxycholecalciferol.

Characterization of oligosaccharide composition and structure by quadrupole ion trap mass spectrometry.

The use of electrospray ionization-quadrupole ion trap mass spectrometry for the characterization of linear oligosaccharides and N-linked protein oligosaccharide mixtures is described. Tandem mass spectrometry (MS/MS) experiments with orders higher than two offer a number of ways to enhance MS/MS spectra and to derive information not present in MS and MS2 spectra. Three such methods are presented in this paper.