HPLC Detection Research Articles

In site preparation of core-shell magnetic triazine functionalized hyper-crosslinking polymers as adsorbent for efficient enrichment of benzoyl urea insecticides

A new magnetic triazine functionalized hyper-crosslinking polymer (M-TPS-HCP) was fabricated and applied as the adsorbent for the magnetic solid-phase extraction (MSPE) of trace benzoyl urea insecticides in food analysis. The main parameters that influence the extraction efficiency of the MSPE were optimized. The established analytical method based on the MSPE and HPLC detection had good linearities with a high correlation coefficient (r ≥ 0.9965). The method achieved a good sensitivity and accuracy, with the limits of detection of 0.03–0.1 ng mL−1 for water, 0.3–1.00 ng g−1 for honey, and 0.8–3.00 ng g−1 for tomato samples, the relative recoveries being in the range of 80.0%–116%, and the relative standard deviations ≤9.8% (n = 5). The results demonstrate that the method was reliable and effective for detecting BUs in real samples. The experimental study on the adsorption mechanism indicates that hydrogen bonding and hydrophobic interactions play a pivotal role for the extraction.

Hollow-Fiber Liquid-Phase Microextraction (HF-LPME) Coupled On-Line to Liquid Chromatography for the Determination of the Herbicides 2,4-Dichlorophenoxyacetic Acid and 2-Methyl-4-chlorophenoxyacetic Acid and Their Main Metabolites in Soil Samples

The use of hollow-fiber liquid-phase microextraction (HF-LPME) is very promising for the clean-up, enrichment, and analysis of chlorophenoxyacetic acid herbicides and their metabolites from environmental and biological samples. An on-line methodology coupling HF-LPME (using a hydrophobic polypropylene hollow fiber of 57 cm length, 0.3 mm i.d., 0.2 μm pore size, and 75% porosity) and HPLC-UV has been developed for the analysis of these compounds in soil samples taken from environments treated with these herbicides. Di-hexyl ether was found to be the best solvent for the enrichment of the target herbicides. The use of supported liquid membranes has shown a high and efficient clean-up of the dissolved organic matter present in soil extracts. The enrichment factors achieved with the on-line methodology have allowed us to reach the detection limits of 0.1–0.3 µg·kg−1 soil, by analyzing 20 mL of alkaline extract as a donor solution and using 1 mL of stripping solution, which has permitted us to detect and follow the presence of these compounds in soil samples until nine days after the application of the herbicide. The on-line method has been applied in a preliminary study to assess the mobility of the chlorophenoxyacetic acid herbicides through soils. It has also been found that degradation of these compounds in soil is relatively fast and some metabolites were detected in soils just one day after the application of the herbicide. The high enrichment factors obtained with the HF-LPME procedure allow for the obtainment of low detection limits, which permits the use of a simple HPLC detector, such as UV, which simplifies and reduces the cost of analyses.

The gut symbiont Sphingomonas mediates imidacloprid resistance in the important agricultural insect pest Aphis gossypii Glover

BackgroundNeonicotinoid insecticides are applied worldwide for the control of agricultural insect pests. The evolution of neonicotinoid resistance has led to the failure of pest control in the field. The enhanced detoxifying enzyme activity and target mutations play important roles in the resistance of insects to neonicotinoid resistance. Emerging evidence indicates a central role of the gut symbiont in insect pest resistance to pesticides. Existing reports suggest that symbiotic microorganisms could mediate pesticide resistance by degrading pesticides in insect pests.ResultsThe 16S rDNA sequencing results showed that the richness and diversity of the gut community between the imidacloprid-resistant (IMI-R) and imidacloprid-susceptible (IMI-S) strains of the cotton aphid Aphis gossypii showed no significant difference, while the abundance of the gut symbiont Sphingomonas was significantly higher in the IMI-R strain. Antibiotic treatment deprived Sphingomonas of the gut, followed by an increase in susceptibility to imidacloprid in the IMI-R strain. The susceptibility of the IMI-S strain to imidacloprid was significantly decreased as expected after supplementation with Sphingomonas. In addition, the imidacloprid susceptibility in nine field populations, which were all infected with Sphingomonas, increased to different degrees after treatment with antibiotics. Then, we demonstrated that Sphingomonas isolated from the gut of the IMI-R strain could subsist only with imidacloprid as a carbon source. The metabolic efficiency of imidacloprid by Sphingomonas reached 56% by HPLC detection. This further proved that Sphingomonas could mediate A. gossypii resistance to imidacloprid by hydroxylation and nitroreduction.ConclusionsOur findings suggest that the gut symbiont Sphingomonas, with detoxification properties, could offer an opportunity for insect pests to metabolize imidacloprid. These findings enriched our knowledge of mechanisms of insecticide resistance and provided new symbiont-based strategies for control of insecticide-resistant insect pests with high Sphingomonas abundance.

The Antioxidative Properties of Selected Herbs Estimated Using Various Assays

Antioxidants can be determined using a variety of analytical assays. The total of antioxidant properties, depending on the sum of the products of their concentrations and antioxidant powers in the sample, is usually called total antioxidant potential (TAP). The direct method developed by us, based on the voltammetric measurements, was used to determine the TAPs of selected herbs. However, it is expected that the detection limit of this assay will be much lower when the convection current is increased and the capacitive current is eliminated. Such conditions are found in electrochemical HPLC detection. The advantage of this method is the ability to perform measurements at different potentials, which is analogous to the measurements related to various radicals in indirect methods. Measurements performed at high potentials of the working electrode allow us to test weak antioxidants. It turned out that electrochemical methods provided additional information about the antioxidant properties of the herbs. Some of them have a large part of TAP caused by weak antioxidants in high concentrations. The obtained results were correlated with the TAP values related to DPPH (2, 2-diphenyl-1-picrylhydrazyl) radicals and the total content of polyphenols in the sample.

The Metabolite Profile of Radioisotope-Labeled Biotin in Rats Indicates that Rat Biotin Metabolism is Similar to that in Humans

The egg white-fed rat is the most commonly used animal model of human biotin deficiency. We sought to determine whether the urinary excretion rates of biotin and biotin metabolites in rats are similar to those reported in humans. D-(Carbonyl-14C)biotin was injected intraperitoneally at physiologic doses in 6- to 10-wk-old rats; HPLC and radiometric flow detection were used to specifically identify and quantify biotin and metabolites in urine. Substantial amounts of bisnorbiotin and biotin sulfoxide, the two principal human metabolites, were detected. The excretion rates of biotin and metabolites were strikingly dependent on the dose of biotin. When the dose of [14C]biotin was 30% of the daily dietary intake (a physiologic dose), 50% of the administered 14C was excreted within 24 h; more than half of the excretion was the unchanged vitamin. After d 1, [14C]bisnorbiotin was the major form excreted. For the cumulative 5-d excretion, [14C]biotin accounted for 46 ± 9%, [14C]bisnorbiotin accounted for 47 ± 11%, and [14C]biotin sulfoxide accounted for 8 ± 4% of the total of biotin, bisnorbiotin, and biotin sulfoxide recovered radioactivity (mean ± 1 SD, n = 6). These proportions are similar to those reported in humans: biotin = 52 ± 12%, bisnorbiotin = 35 ± 9%, and biotin sulfoxide = 13 ± 4% of total biotin plus metabolites (mean ± 1 SD, n = 10). Thus, these, studies confirm the earlier identification of bisnorbiotin and biotin sulfoxide as the two principal biotin metabolites and provide evidence that the rat is an appropriate model for human biotin metabolism.

A highly efficient liquid-liquid microextraction pretreatment method for determination of ginkgolic acids based on hydrophobic deep eutectic solvent

For the quantification of ginkgolic acids (GAs) within ginkgo products, a very effective liquid–liquid microextraction pretreatment method based on hydrophobic deep eutectic solvent (HDES) was created. The HPLC detection conditions for GAs were optimized before establishing the pretreatment method. Then, the DES composition, DES volume, sample amount, extraction temperature and time were investigated to obtain the optimal pretreatment conditions. This developed method had satisfactory analytical performance, the linearity was r2 = 0.9997, and its low limit of detection (LOD) was 0.81 mg/kg. The recovery value ranged from 95.10 % to 98.75 %. When compared with the Ch.P.2010 and EP10.0 recommended methods, this developed method gave a higher efficiency and showed obvious advantages. Furthermore, the developed method was effectively used to identify GAs in a variety of Ginkgo biloba medications, Ginkgo biloba leaves from various cultivars and collection times. These results highlighted the superiorities of the HDES-based liquid–liquid microextraction pretreatment technique for the measurement of GAs, providing insights for future development of efficient methods in active ingredients analysis.

Varietal Influence on the Formation of Bioactive Amines during the Processing of Fermented Cocoa with Different Pulp Contents.

During cocoa processing, there can be the formation of bioactive amines, which are compounds that play relevant roles not only in plant development but also in human health. Thus, we aimed to investigate the presence and levels of bioactive amines during the processing of two important varieties of cocoa (PS 1319 and Parazinho). The seeds were fermented using five different pulp proportions: 100% (E1), 80% (E2), 60% (E3), and 0% (total pulp removal) (E4). The beans were fermented and dried on a farm following traditional procedures. Soon after, they were roasted and processed into chocolates with 60% cocoa in the laboratory. Bioactive amine contents were determined by ion-pair reversed-phase HPLC and fluorometric detection in the samples before, during, and after fermentation, after drying and roasting (nibs), and in the liquor and chocolate. The only amines found before processing in PS 1319 and Parazinho, respectively, in dry weight basis (dwb), were putrescine (pulp, 13.77 and 12.31; seed, 5.88 and 4.58) and serotonin (seed, 2.70 and 2.54). Fermentation was shorter for Parazinho (156 h) compared to PS 1319 (180 h). The changes in amines were affected by the cocoa variety. During drying, the presence of cadaverine stood out, appearing in all treatments of the PS 1319 variety, reaching 17.96 mg/kg dwb, and in two treatments of the Parazinho variety (100 and 60% pulp). During roasting, most of the amines decreased, except for phenylethylamine, which increased up to 2.47 mg/kg dwb for Parazinho and 1.73 mg/kg dwb for PS 1319. Most of the amines formed and built up (e.g., tyramine, putrescine, and cadaverine) during fermentation were not available or were at low levels in the nibs. Most of the amines found during processing did not reach the final product (chocolate), except for cadaverine in PS 1319 without pulp (7.54 mg/kg dwb). Finally, we confirmed how pulp content, processing, and variety influence the content of bioactive amines in cocoa and chocolate. These changes can be better demonstrated through a heatmap and principal component analysis.

Isolation, bioassay and 3D-QSAR analysis of 8-isopentenyl flavonoids from Epimedium sagittatum maxim. as PDE5A inhibitors

BackgroundAs known, inhibition of phosphodiesterase 5 (PDE5) has the therapeutic effect on male erectile dysfunction (ED), and the processed folium of Epimedium sagittatum Maxim. (PFES) characterized by 8-isopentenyl flavonoids is a famous herb for treating ED. However, the main flavonoids inhibitory activities, structure–activity relationship (SAR) and signaling pathway have been not systematically studied so that its pharmacodynamic mechanism is unclear.MethodsWe aimed to initially reveal the PFES efficacy mechanism for treating ED. For the first time, 6 main 8-isopentenyl flavonoids (1–6) from PFES were isolated and identified. Then based on HPLC detection, we proposed a novel method to screen inhibitors among them. We further analyze the three-dimensional quantitative structure–activity relationship (3D-QSAR) for those inhibitors.ResultsThe results were verified by cellular effects of the screened flavonoids. Among 6 compounds, Icariin: (1), 2-Oʹʹrhamnosylicaridide II (2) and Baohuoside I (3) were identified with significant activities (IC50 = 8.275, 3.233, 5.473 μM). Then 3D-QSAR studies showed that the replacement of C8 with bulky steric groups as isopentenyl, C3 with positive charge groups and C4' with a hydrogen bond acceptor substituent could increase inhibitory effects. In contrast, the substitution of C7 with bulky steric groups or hydrophilic groups tended to decrease the efficacies. And compounds 1, 2, 3 could increase cGMP level and decrease cytoplasmic Ca2+ of rat corpus cavernosum smooth muscle cells (CCSMCs)by activating PKG.Conclusion8-isopentenyl flavonoids could be the main pharmacodynamic substances of PFES in the treatment for ED, and some had significant PDE5A1 inhibitory activities so as to activate cGMP/PKG/Ca2+ signaling pathway in CCSMCs, that was related to the substituents at the key sites such as C8, C3, C4ʹ and C7 in the characteristic compounds.

Selective and ratiometric fluorescence sensing of bisphenol A in canned food based on portable fluorescent test strips

In this study, a conversion method and molecular imprinting technology were used to design molecularly imprinted polymers (MIP)-based ratiometric fluorescence test papers. The ZnO quantum dots (ZnO QDs) acted as the background quantum dots and ZIF-8 raw material. Carbon dots (CDs) were used as the identification signals. The imprinting layer achieved a selective function. Therefore, a ZnO@ZIF-8/CDs@MIPs sensor was designed for the detection of Bisphenol A (BPA). The sensor exhibited a fast response time for BPA detection. In addition, the sensor demonstrated that effective detection of BPA can still be achieved in complex environments. The detection limit of this sensor was 0.778 nM with a linear range of 0–60 nM. The corresponding test solutions exhibited clear changes from blue to yellow. The selectivity experiments results demonstrated that ZnO@ZIF-8/CDs@MIPs only exhibit excellent selective recognition effect for BPA. ZnO@ZIF-8/CDs@MIPs-2 was used for the detection of BPA in canned food and compared with the results of HPLC detection of BPA. The two spiked recovery ranges were 96.58–102.04% and 97.43–103.82%, respectively. In addition, the prepared ZnO@ZIF-8/CDs@MIPs-2 test paper visually recognized BPA under ultraviolet light. This study provides guidelines for the design and application of fluorescent test papers for quick detection in practical applications.

Optimization of biosurfactant production by Pseudomonas aeruginosa using rice water and its competence in controlling Fusarium wilt of Abelmoschus esculentus

• Rice water was selected as the cheapest carbon source for formulating rice water medium in order to optimize biosurfactant synthesis by Pseudomonas aeruginosa . • The in vitro and in vivo antifungal efficacy and biocontrol strategy of the biosurfactant against Fusarium wilt of Abelmoschus esculentus were demonstrated. • The identification of the rhl gene, TLC, FT-IR, HPLC, and GC–MS analyses suggested the biosurfactant was rhamnolipid. • The biosurfactant that has been produced will aid in large-scale commercialization and will replace the usage of chemical surfactants in agricultural wastes. Biosurfactants, in comparison to chemical surfactants, have a greater impact on various applications due to their stability, degradability, and other physicochemical characteristics. Increasing yield and lowering production costs are critical to enhancing biosurfactant efficiency. Because biosurfactant production costs are determined by the least expensive substrate, agro-based industrial waste is one of the most promising economic strategies. Therefore, in this study we aimed to achieve the biosurfactant production in Pseudomonas aeruginosa by low cost rice medium using Response surface methodology (RSM) and assessed their competence in controlling the Fusarium wilt of Abelmoschus esculentus . Initially, the selected strain P. aeruginosa PBS29 was confirmed using 16S rRNA sequencing. Then, the 20% (v/v) of rice water was chosen as the cheapest carbon source for formulating rice water medium to optimize biosurfactant production using Central Composite Design. Enhanced biosurfactant yield of 9.35 g/l was attained through RSM by 0.59 fold higher than preliminary analysis. The model was significant with a regression coefficient of 0.98. The optimal condition was identified as 1.18% (w/v) nitrogen source [glutamic acid], pH 6.8, temperature at 37.4 °C, 2.5% (v/v) inoculum size, and 167.9 rpm agitation. In-vitro antifungal activity and biocontrol strategy of the biosurfactant demonstrated against Fusarium wilt of Abelmoschus esculentus at 100 μg/ml concentration by both soil drenching and foliar spray by pot trial. Furthermore, the detection of rhl gene, TLC, FT-IR, HPLC and GC–MS analyses suggested that the biosurfactant is rhamnolipid. Overall, in this study, the biosurfactant was synthesized using inexpensive rice water and substantiated its safe use as a biocontrol agent, which will help large-scale commercialization and interchange chemical surfactant used in agricultural wastes.

HPLC Study of Product Formed in the Reaction of NBD-Derived Fluorescent Probe with Hydrogen Sulfide, Cysteine, N-acetylcysteine, and Glutathione

Hydrogen sulfide (H2S) and its bioderivatives analogs, such as L-cysteine (L-Cys) and glutathione (GSH), are ubiquitous biological thiols in the physiological and pathological processes of living systems. Their aberrant concentration levels are associated with many diseases. Although several NBD-based fluorescence probes have been developed to detect biological thiols, the HPLC-detection of H2S, GSH, L-Cys, and N-acetylcysteine-specific products has not been described. Herein, a novel NBD-derived pro-coumarin probe has been synthesized and used to develop a new strategy for the triple mode detection of H2S and such thiols as GSH, L-Cys, and NAC. Hydrogen sulfide and those biothiols at physiological pH release fluorescent coumarin from the probe and cause a significant fluorescence enhancement at 473 nm. The appropriate NBD-derived product for H2S, L-Cys, GSH, and NAC has a different color and retention time that allows distinguishing these biological thiols meaning the probe has a great possibility in the biological application. Fluorescent imaging combined with colorimetric and HPLC detection of H2S/biothiol-specific product(s) brings a potential tool for confirming the presence of biological thiols and determining concentrations in various aqueous biological samples.

Selective capture and determination of doxycycline in marine sediments by using magnetic imprinting dispersive solid-phase extraction coupled with high performance liquid chromatography.

Antibiotics are frequently used in aquaculture as feed additives and finally enter the marine environment that can pose potential threat to humans. In this study, magnetic molecularly imprinted nanocomposites were prepared by surface imprinting and applied as selective sorbents for specific capture of doxycycline. A multivariate approach based on response surface methodology with Box-Behnken design was adopted to optimize the dispersive solid-phase extraction of doxycycline from marine sediment. Three key parameters, including adsorbent amount and type of washing/eluting solvent, were screened. Under optimum conditions, the limit of detection was 0.03μgg-1 with good linearity from 0.5 to 20μgg-1 followed by HPLC detection. Finally, two sediment samples were analysed and satisfactory recoveries between 90.60% and 93.76% were obtained with acceptable relative standard deviations (≤4.12%), suggesting a promising applicability of the developed method for efficient extraction and sensitive quantification of antibiotics in complex marine environmental matrix.

Combination of HPLC and SERS detection applied to the analysis of the trace content of amoxicillin in milk

The systematic consumption of food contaminated with antibiotics leads to emergence of resistant forms of microorganisms and also causes dysbiosis. Therefore, it is very important to ensure the necessary control of the residues of these contaminants in food by using fast and reliable methods. A simple and low-cost method for determining the trace content of amoxicillin in milk based on surface enhanced Raman spectroscopy (SERS) has been developed. To remove interfering components, a simple and fast "one-tube" method is proposed. Subsequent chromatographic purification makes it possible to almost completely eliminate the matrix effect of the sample, which ensures high accuracy and specificity of the SERS analysis. The conditions for chromatographic separation were selected to avoid the sorption of amoxicillin by free silanol groups of the stationary phase of the chromatographic column (C18), as well as to immediately carry out SERS analysis without intermediate steps. A calibration model was developed using the PLS chemometric algorithm, which further improved the accuracy and reproducibility of the analysis (R2 =0.9957 for the range 0.1–1.0 µM, RMSEC = 0.031 µM, RMSEP = 0.017 µM). This method allows to determine the content of amoxicillin in milk in the range of 1.1–11.0 µM and is characterized by good repeatability (RSD) ≤ 5.5% and a low limit of quantitation = 0.19 µM, which is considerably lower compared to the UV-HPLC method. Verification of the proposed method on samples of UHT (Ultra-High Temperature processed) milk of various fat content (1.8%, 2.5% and 3.2%) showed the recovery value = 100.6–105.7%.

High-Sensitive FAM Labeled Aptasensor Based on Fe3O4/Au/g-C3N4 for the Detection of Sulfamethazine in Food Matrix.

In this study, we developed a fluorescent aptasensor based on Fe3O4/Au/g-C3N4 and a FAM-labeled aptamer (FAM-SMZ1S) against sulfamethazine (SMZ) for the specific and sensitive detection of SMZ in food matrix. The FAM-SMZ1S was adsorbed by the Fe3O4/Au/g-C3N4 via π–π stacking and electrostatic adsorption, serving as a basis for the ultrasensitive detection of SMZ. Molecular dynamics was used to explain the reasons why SMZ1S and SMZ were combined. This aptasensor presented sensitive recognition performance, with a limit of detection of 0.16 ng/mL and a linear range of 1–100 ng/mL. The recovery rate ranged from 91.6% to 106.8%, and the coefficient of variation (CV) ranged from 2.8% to 13.4%. In addition, we tested the aptasensor for the monitoring of SMZ in various matrix samples, and the results were well-correlated (R2 ≥ 0.9153) with those obtained for HPLC detection. According to these results, the aptasensor was sensitive and accurate, representing a potentially useful tool for the detection of SMZ in food matrix.

HPLC fluorescence assay for measuring the activity of diacylglycerol lipases and the action of inhibitors thereof

1,2-Diacylglycerol lipases (DAGLs) are the most important enzymes for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG), and their role in various pathophysiological conditions is currently under investigation. We synthesized a new 1,2-diacylglycerol substrate for these enzymes with a fluorogenic 4-(pyren-1-yl)butanoyl residue in sn-2 position. Using the fluorescent substrate, we measured DAGL activity in rat liver S9 fraction and brain microsomes. To this end, 2-acylglycerol release was directly determined via HPLC and fluorescence detection without further sample clean-up. The method was used to evaluate the action of several known DAGL inhibitors. These showed partly significant differences in their inhibitory effect on DAGLs in liver versus brain preparations. The method was verified by measuring the IC50 values for a subset of inhibitors by HPLC and single-quad MS detection using the deuterated natural DAGL substrate 1-stearoyl-2-arachidonoyl-sn-glycerol-d8. DAGL activity could also be measured with the new pyrene-labeled substrate by HPLC and UV instead of fluorescence detection, if larger quantities of the samples were injected into the HPLC system. Furthermore, using intact human sperm, we show that the substrate is also converted by DAGL enzymes in human cells.

SIMULTANEOUS METHOD DEVELOPMENT AND VALIDATION OF EPROSARTAN AND HYDROCHLORTHIAZIDE IN HUMAN PLASMA BY RPHPLC METHOD

A new simple and precise stability indicating bioanalytical reverse-phase High Performance Liquid Chromatography (RP-HPLC) method was developedand validated for the simultaneous estimation of Eprosartan and Hydrochlorothiazidein their formulation and in human plasma.The developed method was successfully used for assaying drug contents in plasma. Isocratic elution mode was carried at Symmetry C 18 (150mm x 4.6 mm, 5) column using 0.1% Orthophosphoric acidpH (2.2): Acetonitrile (65:35v/v) as mobile phase at flow rate 1.0mlmin at detection wavelength 240 nm. Valsartan was taken as an internal standandard. The method was validated as per ICH guidelines. It is concluded that the present validated method can be successfully applied for the estimation of Eprosartan and Hydrochlorothiazidein human plasma over the concentration range of 80 to 3200 ng /ml of Eprosartan, 8.5 to 340 ng /ml of Hydrochlorothiazide. The method for determination of Eprosartan and Hydrochlorothiazidein human plasma using HPLC detection met the acceptance criteria with respect to selectivity, precision, accuracy, linearity, recovery. Key words: ICH guidelines, Eprosartan, Hydrochlorothiazide, K2EDTA Plasma,Valsartan, Internal standard, Validation

P1521: A SEVERE MOUSE MODEL OF ALPHA-THALASSEMIA SHOWS ABNORMAL IRON METABOLISM, ERYTHROPOIESIS AND COAGULATION, AND CAN BE RESCUED BY A NOVEL GENE THERAPY APPROACH

Background: Clinical presentation of deletional a-thal varies from an asymptomatic condition (one inactivated a-globin gene) to a complete knockout (Hb Bart’s Hydrops Fetalis). In patients with severe a-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation. Aims: The aims of this study are to develop a novel adult mouse model of a-thal and a gene therapy approach for this disease. Methods: We generated adult animals that do not produce a-globin chains (a-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. To generate a gene therapy tool, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human a-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human a-globin genes. Results: The a-KO animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only b-globin chains (HbH). RBC show aberrant morphology and aggregation of b-globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events, associated with neutrophil infiltration, fibrinogen staining, von Willebrand factor (vWF) released, platelet recruitment and activation. These animals also show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney. We identified ALS20a, a vector where a-globin is under control of the b-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous a-globin at a level comparable to that produced by one endogenous a-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Use of ALS20a resulted in correction of the disease phenotype in a dose-dependent manner in a-KO mice. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels. We tested ALS20a in CD34 cells isolated from four patients with both deletional and non-deletional HbH disease. We measured the change of b/a-globin mRNA ratio (b/aR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial b/aR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a b/aR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced a-thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2). Image:Summary/Conclusion: We generated an adult mouse model of lethal a-thal and, in preliminary experiments, we rescue it with ALS20a. Furthermore, ALS20a successfully improves a-globin levels in patient cells.

Determination of sequence and absolute configuration of peptide amino acids by HPLC–MS/CD-based detection of liberated N-terminus phenylthiohydantoin amino acids

We report a method for the simultaneous determination of the sequence and absolute configuration of peptide amino acids using a combination of Edman degradation and HPLC–MS/CD. Phenylthiohydantoin (PTH) derivatives of 20 pairs of standard d- and l-amino acids were synthesized by the Edman reaction. The CD spectra of the derivatives revealed that each pair of the PTH derivatives exhibited the absorption with opposite signs at around 270 nm. These standard PTH derivatives showed well-resolved resolution without interference from byproducts in the ion chromatogram and clear positive/negative CD absorptions when subjected on a reversed phase HPLC–MS system coupled with a CD-2095 HPLC detector. This method was applied for the detection of a synthetic pentapeptide and a natural depsipeptide (halicylindramide C). The sequence and configuration of the pentapeptide and up to eight residues of halicylindramide C were successfully analyzed by this method. The amino acid configuration of the pentapeptide was also determined successfully by subjecting its acid hydrolysates to the Edman reaction followed by HPLC–MS/CD.

Investigating Substrate Specificity For 6‐Hydroxynicotinate 3‐Monooxygenase (NicC) With Coumalic Acid: Consequences To Catalysis Of Replacing or Removing The Ring Nitrogen

6‐Hydroxynicotinate 3‐monooxygenase (NicC) is a group A flavin dependent monooxygenase (FDM) that catalyzes the regioselective decarboxylative hydroxylation of 6‐HNA into 2,5‐dihydroxypyridine (DHP) with the concomitant oxidation of NADH to NAD+. This enzyme‐catalyzed reaction involves three segments: (1) substrate binding, (2) FAD reduction by NADH, and (3) hydroxylation‐decarboxylation of 6‐HNA through a hydroperoxy‐flavin intermediate. Past studies have shown that NicC can catalyze this reaction with alternate aromatic substrates including 4‐hydroxybenzoic acid (4‐HBA), a substrate analogue with a homocyclic ring (i.e. lacking the ring nitrogen). However, 4‐HBA is bound weakly (355 ± 66 μM) and only 50% of the reaction progresses to substrate hydroxylation, versus hydroperoxy‐flavin degradation to H2O2, suggesting the importance of the ring nitrogen in the coupling efficiency of NicC. To further explore the role of the ring nitrogen, the efficiency of the reaction with coumalic acid (CA) was determined. The NicC‐CA dissociation constant measured by monitoring the change in absorbance at 450nm (flavin) shows that NicC binds CA (Kd = 5 ± 1 μM) 10x more tightly than 6‐HNA (Kd = 58 ± 12 μM). Binding of CA by NicC is an isothermal process (∆H = 0), indicating that it is driven by a favorable change in entropy. Analysis of the reaction products by Q‐TOF MS indicates that NicC catalyzes the decarboxylative hydroxylation of CA into 5‐hydroxy‐2H‐pyrane‐2‐one. However, coupling experiments by HPLC analysis and detection of H2O2 by a fluorescence assay both indicate that the reaction with CA is highly uncoupled with only minor 5‐hydroxy‐2H‐pyrane‐2‐one formation. Steady‐state kinetic analyses show that CA bound to NicC enhances its rate of NADH oxidation 16‐fold (by comparison, 6‐HNA enhances the rate of NicC NADH oxidation 30‐fold). Transient state kinetic analyses by stopped flow under anaerobic conditions to permit single‐turnover kinetics shows that, unlike in the binding of 6‐HNA, the binding of CA does not enable the formation of a charge‐transfer complex prior to FAD reduction. These results indicate that an H‐bond accepting group in the aromatic ring at position 1 is important for establishing the correct conformation of the ES complex to enable effective binding, flavin reduction and, ultimately, substrate hydroxylation.

Integration of In Silico and In Vitro Analysis of Gliotoxin Production Reveals a Narrow Range of Producing Fungal Species.

Gliotoxin is a fungal secondary metabolite with impact on health and agriculture since it might act as virulence factor and contaminate human and animal food. Homologous gliotoxin (GT) gene clusters are spread across a number of fungal species although if they produce GT or other related epipolythiodioxopiperazines (ETPs) remains obscure. Using bioinformatic tools, we have identified homologous gli gene clusters similar to the A. fumigatus GT gene cluster in several fungal species. In silico study led to in vitro confirmation of GT and Bisdethiobis(methylthio)gliotoxin (bmGT) production in fungal strain cultures by HPLC detection. Despite we selected most similar homologous gli gene cluster in 20 different species, GT and bmGT were only detected in section Fumigati species and in a Trichoderma virens Q strain. Our results suggest that in silico gli homology analyses in different fungal strains to predict GT production might be only informative when accompanied by analysis about mycotoxin production in cell cultures.