Prediction of Degradation Profiles for Various Sartans under Solvent-Free Mechanochemical Conditions.

For the approval of a drug, the stability data must be submitted to regulatory authorities. Such analyses are often time-consuming and cost-intensive. Forced degradation studies are mainly carried out under harsh conditions in the dissolved state, often leading to extraneous degradation profiles for a solid drug. Oxidative mechanochemical degradation offers the possibility of generating realistic degradation profiles. In this study, a sustainable mechanochemical procedure is presented for the degradation of five active pharmaceutical ingredients (APIs) from the sartan family: losartan potassium, irbesartan, valsartan, olmesartan medoxomil, and telmisartan. High-resolution mass spectrometry enabled the detection of impurities already present in untreated APIs and allowed the elucidation of degradation products. Significant degradation profiles could already be obtained after 15-60 min of ball milling time. Many of the identified degradation products are described in the literature and pharmacopoeias, emphasizing the significance of our results and the applicability of this approach to predict degradation profiles for drugs in the solid state.

Valine Treatment Enhances Antimicrobial Component Production in Mammary Epithelial Cells and the Milk of Lactating Goats Without Influencing the Tight Junction Barrier.

The production of antimicrobial components and the formation of less-permeable tight junctions (TJs) are important in the defense system of lactating mammary glands and for safe dairy production. Valine is a branched-chain amino acid that is actively consumed in the mammary glands and promotes the production of major milk components like ß-casein; additionally, branched-chain amino acids stimulate antimicrobial component production in the intestines. Therefore, we hypothesized that valine strengthens the mammary gland defense system without influencing milk production. We investigated the effects of valine in vitro using cultured mammary epithelial cells (MECs) and in vivo using the mammary glands of lactating Tokara goats. Valine treatment at 4 mM increased the secretion of S100A7 and lactoferrin as well as the intracellular concentration of ß-defensin 1 and cathelicidin 7 in cultured MECs. In addition, an intravenous injection of valine increased S100A7 levels in the milk of Tokara goats without influencing milk yield and milk components (i.e., fat, protein, lactose, and solids). In contrast, valine treatment did not affect TJ barrier function either in vitro or in vivo. These findings indicate that valine enhances antimicrobial component production without influencing milk production and TJ barrier function in lactating mammary glands; thus, valine contributes to safe dairy production.

Improvement of valine and isobutanol production in sake yeast by Ala31Thr substitution in the regulatory subunit of acetohydroxy acid synthase.

The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds.

Chemical Composition Assessment of Structural Parts (Seeds, Peel, Pulp) of Physalis alkekengi L. Fruits.

In recent years there has been an extensive search for nature-based products with functional potential. All structural parts of Physalis alkekengi (bladder cherry), including fruits, pulp, and less-explored parts, such as seeds and peel, can be considered sources of functional macro- and micronutrients, bioactive compounds, such as vitamins, minerals, polyphenols, and polyunsaturated fatty acids, and dietetic fiber. The chemical composition of all fruit structural parts (seeds, peel, and pulp) of two phenotypes of P. alkekengi were studied. The seeds were found to be a rich source of oil, yielding 14-17%, with abundant amounts of unsaturated fatty acids (over 88%) and tocopherols, or vitamin E (up to 5378 mg/kg dw; dry weight). The predominant fatty acid in the seed oils was linoleic acid, followed by oleic acid. The seeds contained most of the fruit's protein (16-19% dw) and fiber (6-8% dw). The peel oil differed significantly from the seed oil in fatty acid and tocopherol composition. Seed cakes, the waste after oil extraction, contained arginine and aspartic acid as the main amino acids; valine, phenylalanine, threonine, and isoleucine were present in slightly higher amounts than the other essential amino acids. They were also rich in key minerals, such as K, Mg, Fe, and Zn. From the peel and pulp fractions were extracted fruit concretes, aromatic products with specific fragrance profiles, of which volatile compositions (GC-MS) were identified. The major volatiles in peel and pulp concretes were ß-linalool, α-pinene, and γ-terpinene. The results from the investigation substantiated the potential of all the studied fruit structures as new sources of bioactive compounds that could be used as prospective sources in human and animal nutrition, while the aroma-active compounds in the concretes supported the plant's potential in perfumery and cosmetics.

Composition of feathers and pulp of two broiler genotypes.

1. Cobb and Ross broilers (200 of each sex and breed) were fed four phases of diets ad libitum formulated with balanced protein to match their amino acid requirements throughout growth. Ten birds per genotype were sampled and euthanised at two-weekly intervals from 14 to 112 d of age. All feathers were dry-plucked from each of the seven tracts (specific skin areas) and pulp (the centre of the feather filament) was removed from primary and secondary remiges.2. Daily losses of feathers were collected from an additional 20 individually-caged broilers of each breed. These feathers were separated into natal down, contour feathers, remiges and rectrices and then pooled by type, sex and genotype to quantify water and protein contents. Only those feathers collected from male Cobb 500 MX broilers were analysed for amino acid content.3. Amino acid contents of feathers from the seven tracts were measured only in Cobb males on days 1, 28 and 70; for pulp on days 28 and 70; and for the four types of moulted feathers.4. Protein content on a dry matter basis remained relatively constant over all ages and tracts during growth. Water content decreased with age in both sexes and genotype. Lysine and methionine content in feathers decreased with age while cystine, valine, leucine and serine increased. Lysine, methionine and histidine levels were higher in pulp than in mature feathers whereas cystine and valine were higher in mature feathers than in pulp.5. These results, together with information about moulting patterns in broilers, enabled the effects of age of the bird and of the type of feather, to be taken into account when determining the rate of deposition of amino acids in feathers.

Ethylene oxide review: characterization of total exposure via endogenous and exogenous pathways and their implications to risk assessment and risk management.

This review is intended to provide risk assessors and risk managers with a better understanding of issues associated with total exposures of human populations to ethylene oxide from endogenous and exogenous pathways. Biomonitoring of human populations and lab animals exposed to ethylene oxide has relied upon the detection of hemoglobin adducts such as 2-hydroxyethylvaline (HEV), which provides a useful measure of total exposure to ethylene oxide from all pathways. Recent biomonitoring data from CDC provide an excellent characterization of total exposure to ethylene oxide to the general U.S. population by demographic factors such as age, gender, and race as well as smoking habit, which might be comparable to previous measurements reported for humans and lab animals. The biochemical pathways including gastrointestinal (production by bacteria) and systemic (enzymatic production) pathways by which endogenous ethylene is generated and converted to ethylene oxide are described. The relative importance of endogenous pathways and exogenous pathways via ambient air or tobacco smoke was quantified based upon available data to characterize their relative importance to total exposure. Considerable variation was noted for HEV measurements in human populations, and important sources of variation for all pathways are discussed. Issues related to risk assessment and risk management of human populations exposed to ethylene oxide are provided within the context of characterizing total exposure, and data needs for supporting future risk assessment identified.

Determination and structural characterization of ravidasvir metabolites by LC coupled to triple quadrupole linear ion trap MS: Application to pharmacokinetics and phase I metabolism in rats.

Hepatitis C virus (HCV) is an infectious disease that has become a global clinical issue because of its significant morbidity and mortality. Novel anti-hepatitis C drugs are continuously developed to decrease the pervasiveness of the infection globally. A synthetic ravidasvir, benzimidazole-naphthylene-imidazole derivatives, has been used as an anti-HCV drug. This study determined the metabolites of ravidasvir and its pharmacokinetics in rats using information-dependent acquisition and multiple reaction monitoring scanning modes in linear ion trap LC-MS/MS instrument, respectively. Two time-programming linear-gradient chromatographic methods were employed using a Kinetex C18 column (50 × 3 mm, 2.6 µm) and a Luna HILIC column (100 × 4.6 mm, 3 µm) for the qualitative and quantitative determination of ravidasvir and its metabolites, respectively. In silico prediction where sites in a molecule are susceptible to metabolism by cytochrome P450 was implemented, which helped in proposing the metabolic pathway of ravidasvir. The most dominant metabolite in rat liver microsomal samples was oxidative ravidasvir, where one O-demethylated metabolite and eight isomers of the oxidative ravidasvir metabolites were identified. The study provides essential data for proposing the metabolic pathway and successfully applied it to determine the pharmacokinetics of ravidasvir in rat plasma.

Equilibrium and Kinetic Study of l- and d-Valine Adsorption in Supramolecular-Templated Chiral Mesoporous Materials.

Adsorption kinetic studies are conducted to investigate the potential to use chiral mesoporous materials nanoporous guanosine monophosphate material-1 (NGM-1) and nanoporous folic acid material-1 (NFM-1) for the enantiomeric separation of l- and d-valine. A pseudo-second-order (PSO) kinetic model is applied to test the experimental adsorption equilibrium isotherms, according to both the Langmuir and Freundlich models and the characteristic parameters for each model are determined. The calcined versions of both NGM-1 and NFM-1 fit the Langmuir model with maximum sorption capacities of 0.36 and 0.26 g/g for the preferred adsorption enantiomers, d-valine and l-valine, respectively. Experimental results and the analysis of adsorption models suggest a strong adsorbate-adsorbent interaction, and the formation of a monolayer of tightly packed amino acid on the internal mesopore surface for the preferred enantiomers.

RP-HPLC method development and validation for quantification of daclatasvir dihydrochloride and its application to pharmaceutical dosage form.

Daclatasvir dihydrochloride is an antiviral drug used in the treatment of Hepatitis C and for its estimation in drug product, no Pharmacopeial method is available. Therefore, a simple, rapid, precise and accurate isocratic RP-HPLC method was developed and validated for quantification of daclatasvir dihydrochloride in pharmaceutical dosage form. The quantification was carried out using Hypersil ODS - C18 Column (250mm, 4.6mm, 5µm), Shimadzu LC-2030 Prominence-I Series. The mobile phase composed of phosphate buffer (pH 3.5, adjusted with ortho phosphoric acid) and acetonitrile (60:40 v/v). The flow rate was 1.0ml/min with UV detection at 308 nm. The validation of developed method was conducted for specificity, linearity, accuracy, precision, LOD and LOQ. A linearity was established in the concentration range of 0.5-150% with coefficient of correlation 0.9993. The limit of detection (LOD) was 0.005µg/ml and the limit of quantification (LOQ) was 0.01µg/ml. The method was successfully applied to the assay and in-vitro dissolution studies of daclatasvir dihydrochloride in tablet dosage form. It can be concluded that this method can be very helpful in the quality control estimation of daclatasvir dihydrochloride in different pharmaceutical products intended for hepatitis C infections.

Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis.

Bacillus licheniformis has been regarded as an outstanding microbial cell factory for the production of biochemicals and enzymes. Due to lack of genetic tools to repress gene expression, metabolic engineering and gene function elucidation are limited in this microbe. In this study, an integrated CRISPR interference (CRISPRi) system was constructed in B. licheniformis. Several endogenous genes, including yvmC, cypX, alsD, pta, ldh, and essential gene rpsC, were severed as the targets to test this CRISPRi system, and the repression efficiencies were ranged from 45.02 to 94.00%. Moreover, the multiple genes were simultaneously repressed with high efficiency using this CRISPRi system. As a case study, the genes involved in by-product synthetic and L-valine degradation pathways were selected as the silence targets to redivert metabolic flux toward L-valine synthesis. Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) led to 90.48% and 80.09 % increases in L-valine titer, respectively. Compared with the control strain DW9i△leuA (1.47 g/L and 1.79 g/L), the L-valine titers of combinatorial strain DW9i△leuA/pHYi-alsD-bcd were increased by 1.27-fold and 2.89-fold, respectively, in flask and bioreactor. Collectively, this work provides a feasible approach for multiplex metabolic engineering and functional genome studies of B. licheniformis.

Assessment of synthetic cannabinoid FUB-AMB and its ester hydrolysis metabolite in human liver microsomes and human blood samples using UHPLC-MS/MS.

FUB-AMB, an indazole carboxamide synthetic cannabinoid recreational drug, was one of the compounds most frequently reported to governmental agencies worldwide between 2016 and 2019. It has been implicated in intoxications and fatalities, posing a risk to public health. In the current study, FUB-AMB was incubated with human liver microsomes (HLM) to assess its metabolic fate and stability and to determine if its major ester hydrolysis metabolite (M1) was present in 12 authentic forensic human blood samples from driving under the influence of drug cases and postmortem investigations using UHPLC-MS/MS. FUB-AMB was rapidly metabolized in HLM, generating M1 that was stable through a 120-min incubation period, a finding that indicates a potential long detection window in human biological samples. M1 was identified in all blood samples, and no parent drug was detected. The authors propose that M1 is a reliable marker for inclusion in laboratory blood screens for FUB-AMB; this metabolite may be pharmacologically active like its precursor FUB-AMB. M1 frequently appears in samples in which the parent drug is undetectable and can point to the causative agent. The results suggest that it is imperative that synthetic cannabinoid laboratory assay panels include metabolites, especially known or potential pharmacologically active metabolites, particularly for compounds with short half-lives.

NMR-Based Serum Metabolomics Revealed Distinctive Metabolic Patterns in Reactive Arthritis Compared with Rheumatoid Arthritis.

Reactive arthritis (ReA) is a member of seronegative spondyloarthropathy (SSA), which involves an acute/subacute onset of asymmetrical lower limb joint inflammation weeks after a genitourinary/gastrointestinal infection. The diagnosis is clinical because it is difficult to culture the microbes from synovial fluid. Arthritis patients with a similar clinical picture but lapsed history of an immediate preceding infection that do not fulfill the diagnostic criteria of other members of SSA, such as ankylosing spondylitis, psoriatic arthritis, and arthritis associated with inflammatory bowel disease, are labeled as peripheral undifferentiated spondyloarthropathy (uSpA). Both ReA and uSpA patients show a strong association with class I major histocompatibility complex allele, HLA-B27, and a clear association with an infectious trigger; however, the disease mechanism is far from clear. Because the clinical picture is largely dominated by rheumatoid-arthritis (RA)-like features including elevated levels of inflammatory markers (such as ESR, CRP, etc.), these overlapping symptoms often confound the clinical diagnosis and represent a clinical dilemma, making treatment choice more generalized. Therefore, there is a compelling need to identify biomarkers that can support the diagnosis of ReA/uSpA. In the present study, we performed NMR-based serum metabolomics analysis and demonstrated that ReA/uSpA patients are clearly distinguishable from controls and further that these patients can also be distinguished from the RA patients based on the metabolic profiles, with high sensitivity and specificity. The discriminatory metabolites were further subjected to area under receiver operating characteristic curve analysis, which led to the identification of four metabolic entities (i.e., valine, leucine, arginine/lysine, and phenylalanine) that could differentiate ReA/uSpA from RA.

Discovery of Novel N-(4-Hydroxybenzyl)valine Hemoglobin Adducts in Human Blood.

Humans are exposed to a wide range of electrophilic compounds present in our diet and environment or formed endogenously as part of normal physiological processes. These electrophiles can modify nucleophilic sites of proteins and DNA to form covalent adducts. Recently, powerful untargeted adductomic approaches have been developed for systematic screening of these adducts in human blood. Our earlier untargeted adductomics study detected 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. We now describe a full characterization of one of these adducts, which corresponds to the addition of a 4-hydroxybenzyl (4-OHBn) group to N-terminal valine in Hb to form N(4-hydroxybenzyl)valine (4-OHBn-Val). The adduct structure was determined by comparison of its accurate mass, HPLC retention time, and MS/MS fragmentation to that of authentic standards prepared by chemical synthesis. Average 4-OHBn-Val adduct concentrations in 12 human blood samples were estimated to 380 ± 160 pmol/g Hb. Two possible routes of 4-OHBnVal adduct formation are proposed using two different precursor electrophiles: 4-quinone methide (4-QM) and 4-hydroxybenzaldehyde (4-OHBA). We found that 4-QM reacts rapidly with valine to form the 4-OHBn-Val adduct; however, the quinone methide is unstable under physiological conditions due to hydrolysis. It was shown that 4-OHBA forms reversible Schiff base adducts with valine, which can be stabilized via reduction in blood generating the 4-OHBn-Val adduct. In addition, trace amounts of isomeric 2-hydroxybenzyl-valine (2-OHBn-Val) adducts were detected in 12 human blood samples (estimated mean adduct level, 5.0 ± 1.4 pmol/g Hb). Further studies are needed to quantify the contributions from identified possible precursor electrophiles to the observed hydroxybenzyl adducts in humans.

Varying the ratio of Lys:Met while maintaining the ratios of Thr:Phe, Lys:Thr, Lys:His, and Lys:Val alters mammary cellular metabolites, mammalian target of rapamycin signaling, and gene transcription.

Amino acids are not only precursors for but also signaling molecules regulating protein synthesis. Regulation of protein synthesis via AA occurs at least in part by alterations in the phosphorylation status of mammalian target of rapamycin (mTOR) pathway proteins. Although the ideal profile of Lys:Met to promote milk protein synthesis during established lactation in dairy cows has been proposed to be 3:1, aside from being the most-limiting AA for milk protein synthesis, the role of Met in other key biologic pathways such as methylation is not well characterized in the bovine. The objective of this study was to determine the influence of increasing supplemental Met, based on the ideal 3:1 ratio of Lys to Met, on intracellular metabolism related to protein synthesis and mTOR pathway phosphorylation status. MAC-T cells, an immortalized bovine mammary epithelial cell line, were incubated (n = 5 replicates/treatment) for 12 h with 3 incremental doses of Met while holding Lys concentration constant to achieve the following: Lys:Met 2.9:1 (ideal AA ratio; IPAA), Lys:Met 2.5:1 (LM2.5), and Lys:Met 2.0:1 (LM2.0). The ratios of Thr:Phe (1.05:1), Lys:Thr (1.8:1), Lys:His (2.38:1), and Lys:Val (1.23:1) were the same across the 3 treatments. Applying gas chromatography-mass spectrometry metabolomics revealed distinct clusters of differentially concentrated metabolites in response to Lys:Met. Lower Phe, branched-chain AA, and putrescine concentrations were observed with LM2.5 compared with IPAA. Apart from greater intracellular Met concentrations, further elevations in Met level (LM2.0) led to greater intracellular concentrations of nonessential AA (Pro, Glu, Gln, and Gly) compared with IPAA and greater essential AA (EAA; Met, Ile, and Leu) and nonessential AA (Pro, Gly, Ala, Gln, and Glu) compared with LM2.5. However, compared with IPAA, mRNA expression of ß-casein and AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, and SLC43A1) and mTOR phosphorylation were lower in response to LM2.5 and LM2.0. Overall, the results of this study provide evidence that increasing Met while Lys and the ratios of Phe, Thr, His, and Val relative to Lys were held constant could increase the concentration and utilization of intracellular EAA, in particular branched-chain AA, potentially through improving the activity of AA transporters partly controlled by mTOR signaling. Because EAA likely are metabolized by other tissues upon absorption, a question for future in vivo studies is whether formulating diets for optimal ratios of EAA in the metabolizable protein is sufficient to provide the desired levels of these AA to the mammary cells.

Utility of Novel Plasma Metabolic Markers in the Diagnosis of Pediatric Tuberculosis: A Classification and Regression Tree Analysis Approach.

Although tuberculosis (TB) has been the greatest killer due to a single infectious disease, pediatric TB is still hard to diagnose because of the lack of sensitive biomarkers. Metabolomics is increasingly being applied in infectious diseases. But little is known regarding metabolic biomarkers in children with TB. A combination of a NMR-based plasma metabolic method and classification and regression tree (CART) analysis was used to provide a broader range of applications in TB diagnosis in our study. Plasma samples obtained from 28 active TB children and 37 non-TB controls (including 21 RTIs and 16 healthy children) were analyzed by an orthogonal partial least-squares discriminant analysis (OPLS-DA) model, and 17 metabolites were identified that can separate children with TB from non-TB controls. CART analysis was then used to choose 3 of the markers, l-valine, pyruvic acid, and betaine, with the least error. The sensitivity, specificity, and area under the curve (AUC) of the 3 metabolites is 85.7% (24/28, 95% CI, 66.4%, 95.3%), 94.6% (35/37, 95% CI, 80.5%, 99.1%), and 0.984(95% CI, 0.917, 1.000), respectively. The 3 metabolites demonstrated sensitivity of 82.4% (14/17, 95% CI, 55.8%, 95.3%) and specificity of 83.9% (26/31, 95% CI, 65.5%, 93.9%), respectively, in 48 blinded subjects in an independent cohort. Taken together, the novel plasma metabolites are potentially useful for diagnosis of pediatric TB and would provide insights into the disease mechanism.

Differentiation of Norvaline and Valine in Peptides by Hot Electron Capture Dissociation.

During the production of recombinant proteins, misincorporation of Nva (norvaline) is common and causes heterogeneity or even toxicity. To characterize Nva and differentiate it from Val (Valine), a systematic study was conducted using hot electron capture dissociation (HECD) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The thorough investigation of the fragmentation behaviors of a set of model peptides led us to reveal the characteristic/diagnostic fragment ions, w ions, which can be used to differentiate Val and Nva. However, when both Nva and Val were present in one peptide, the observation of interfering ions may mislead the interpretation. Interestingly, HECD also produced v ions, which have the same nominal mass as the M+1 isotope of the w ion and can only be determined by MS with ultrahigh mass resolution and high mass accuracy. The energy-dependent study of the v ion provided an unambiguous identification of Nva and Val since the v ion was generated only when Val was present, not Nva within the electron energy range we studied. In addition, an electron energy-dependent curve provided an overall picture on how w ions and v ions, as well as interfering ions, behaved as the electron energy increased from 1.5 to 14 eV. The results suggest that careful selection of electron energy during a HECD experiment is crucial for the unambiguous differentiation of Val and Nva.

Direct UV determination of Amadori compounds using ligand-exchange and sweeping capillary electrophoresis.

The detection of Amadori compounds poorly absorbing in the UV region is challenging. To cope with this problem, a simple and effective method using ligand-exchange and sweeping capillary electrophoresis was first developed. With this method, Amadori compounds can be detected by UV at 236 nm without derivatization steps and separated based on the stable levels of Amadori compound-Cu(2+) complexes. Under optimal conditions with background electrolyte of 50 mmol/L copper sulfate, pH 3.94, and separation voltage 25 kV, baseline separations of the following five Amadori compounds, N-(1-deoxy-D-fructos-1-yl) valine (Fru-Val), N-(1-deoxy-D-fructos-1-yl) leucine (Fru-Leu), N-(1-deoxy-D-fructos-1-yl) alanine (Fru-Ala), N-(1-deoxy-D-fructos-1-yl) methionine (Fru-Met), N-(1-deoxy-D-fructos-1-yl) glutamic acid (Fru-Glu), were achieved within a 20 min run. The method was applied to analyze these Amadori compounds in two tomato products. Fru-Glu could be quantitated sensitively down to 0.706 mg/L in MS powder (a kind of tomato powder), 0.726 mg/L in MS liquid (a kind of concentrated tomato juice), and the limit of quantification for the others ranged from 0.792 to 1.084 mg/L. The most abundant Amadori compound was Fru-Glu (1057.5 mg/100 g) in MS powder; however, Fru-Met was rarely detected in MS liquid. The accuracy of the method was tested by recovery measurement of the spiked samples, which yielded good results (94.2-106.0%) with relative standard deviation lower than 5%. Simultaneously, the method was also applied in the analysis of Fru-Glu content during a tomato puree evaporating concentration process. The first significant increase in Fru-Glu content coincided with the increment rate of soluble solids, which indicated that fast evaporation of water could be a critical step for the formation of Amadori compounds. These results show that this method is convenient and reliable for rapid analysis of Amadori compounds in tomato products.

Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2.

Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly (13)C/(15)N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap. The challenges involved in studies of larger proteins led to efficient methods for (13)C labeling of side chain methyl groups, which have favorable relaxation properties and high signal-to-noise. These methyls are often still assigned by linking them to the previously assigned backbone, thus limiting the applications for larger proteins. Here, a structure-based procedure is described for assignment of (13)C(1)H3-labeled methyls by comparing distance information obtained from three-dimensional methyl-methyl nuclear Overhauser effect (NOE) spectroscopy with the X-ray structure. The Ile, Leu, or Val (ILV) methyl type is determined by through-bond experiments, and the methyl-methyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed "NOE-methyl cluster" onto the structure. The combination of identification of ILV methyl type with mapping of the NOE-methyl clusters greatly simplifies the assignment process. This method was applied to the inactive and active forms of the 42-kDa ILV (13)C(1)H3-methyl labeled extracellular signal-regulated kinase 2 (ERK2), leading to assignment of 60% of the methyls, including 90% of Ile residues. A series of ILV to Ala mutants were analyzed, which helped confirm the assignments. These assignments were used to probe the local and long-range effects of ligand binding to inactive and active ERK2.

Bisoprolol and bisoprolol-valsartan compatibility studied by differential scanning calorimetry, nuclear magnetic resonance and X-ray powder diffractometry.

PURPOSE: The objective of this study was to evaluate the thermal behavior of crystalline and amorphous bisoprolol fumarate and its compatibility with amorphous valsartan. This pharmacologically relevant drug combination is a potential candidate for fixed-dose combination formulation. METHODS: DSC and TMDSC were used to examine thermal behavior of bisoprolol fumarate. SSNMR and XRPD were applied to probe the solid state forms. The thermal behavior of physical mixtures with different concentrations of bisoprolol and valsartan were examined by DSC and TMDSC, and the observed interactions were investigated by XRPD, solution- and solid-state NMR. RESULTS: The phase transitions from thermal methods and solid-state NMR spectra of crystalline and amorphous bisoprolol fumarate are reported. Strong interactions between bisoprolol fumarate and valsartan were observed above 60 C, resulting in the formation of a new amorphous material. Solution- and solid-state NMR provided insight into the molecular nature of the incompatibility. CONCLUSIONS: A combined analysis of thermal methods, solution- and solid-state NMR and XRPD experiments allowed the investigation of the conformational and dynamic properties of bisoprolol fumarate. Since bisoprolol fumarate and valsartan react to form a new amorphous product, formulation of a fixed-dose combination would require separate reservoirs for bisoprolol and valsartan to prevent interactions. Similar problems might be expected with other excipients or APIs containing carboxylic groups.

Quantification of plasma microRNAs in a group of healthy smokers, ex-smokers and non-smokers and correlation to biomarkers of tobacco exposure.

The stability of circulating miRNAs, their non-invasive sampling techniques and deregulation in diseases make them potential candidate biomarkers of biological effect. Here, we profiled the level of 84 plasma miRNAs in 30 smokers, 20 non-smokers and 20 ex-smokers. A robust statistical strategy was applied with replicate samples to account for reproducibility of the results. We identified differential expression of miR-124 and let-7a between the smoking and control groups. We further explored the dose-response relationship of miR-124 and let-7a with two biomarkers of tobacco exposure and found that this relationship was affected by adjustments based on age, pack-year and gender.