Acid Gradient Research Articles

A selective comprehensive reversed-phase×reversed-phase 2D-liquid chromatography approach with multiple complementary detectors as advanced generic method for the quality control of synthetic and therapeutic peptides.

There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3mm ID column is used in the second dimension. Recording data with a UV detector at 215nm after 1D separation provides the common generic 1D chromatogram. However, after the 2D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of 2D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed.

Multimodal chemo-/magneto-/phototaxis of 3G CNT-bots to power fuel cells

We report the development of a 3G microswimmer, namely, CNT-bot, capable of undergoing acid-, alkali-, magneto- and phototaxis inside acidic or alkaline baths of peroxide fuel and/or water. The use of carboxyl-functionalised multi-walled carbon nanotubes (MWCNTs) facilitated the propulsion of CNT-bots in an alkaline-water solution by ejecting carbon-dioxide bubbles. Furthermore, doping of magnetite nanoparticles (FeONPs), ferrous ions (Fe2+) and titanium dioxide nanoparticles (TiONPs) induces magnetic, chemical and photonic modes of propulsion. While FeONPs stimulated magnetotaxis at a rate of up to ~10 body lengths per second under the influence of a bar magnet, chemotaxis of a similar speed in a peroxide fuel was achieved by bubble-propulsion of oxygen gas originating from the Fenton reaction. In addition, the light-stimulated photo-Fenton reaction led to phototaxis of CNT-bots. A thin coating of magnesium imparted a half-faced Janus appearance to the CNT-bots, which facilitated motion in normal or acidic water media through the ejection of hydrogen gas bubbles. This chemotaxis could be transformed into pH-stimulated directional motion by establishing an acid or alkali concentration gradient across the peroxide and/or water baths. The capacity of CNT-bots to produce oxygen (hydrogen) bubbles in peroxide (acidic water) fuel was exploited to power a PEM fuel cell to generate electricity. The pure oxygen and hydrogen gases generated by CNT-bots in separate chambers were fed directly into the fuel cell in which the incessant motions of the particle facilitated the creation and release of the pure gases to achieve on-demand electricity generation. The motor could also induce dye degradation through advanced oxidation owing to the production of intermediate hydroxyl radicals during the Fenton reaction.

Choroid plexus LAT2 and SNAT3 as partners in CSF amino acid homeostasis maintenance

BackgroundCerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2–20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient.MethodsExpression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 (Slc7a8) antiporter deletion on CSF amino acids was determined.ResultsThe purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 (Slc38a1), SNAT3 (Slc38a3), LAT2 (Slc7a8), ASC1 (Slc7a10) and SIT1 (Slc6a20b). Three of them were visualized by immunofluorescence: SNAT1 (Slc38a1), a neutral amino acid-Na+ symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 (Slc38a3), an amino acid-Na+ symporter and H+ antiporter, as well as LAT2 (Slc7a8), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2–20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro).ConclusionThese results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.

Changes in spatiotemporal protein and amino acid gradients in wheat caryopsis after N-topdressing

Wheat grain nitrogen content displays large variations within different pearling fractions of grains because of radial gradients in the protein content. We identified how spatiotemporal mechanisms regulate this. The protein gradients emerged clearly at 19 days after anthesis, with the highest N content in aleurone and seed coat, followed by outer endosperm, whereas the lowest was in middle and inner endosperm. Laser microdissection, qRT-PCR and LC–MS were used to dissect tissue from aleurone, outer endosperm, middle endosperm, inner endosperm and transfer cells, measure gene expression and levels of free and protein-bound amino acids, respectively. The results showed that different FAA transportation pathways worked in parallel during grain filling stage while the grain protein gradient did not follow spatial expression of storage proteins. Additionally, two nitrogen (N) topdressing timings were conducted, either at the emergence of top third leaf (standard timing) or top first leaf (delayed timing), finding that delayed N topdressing enhanced both amino acids supply and protein synthesis capacity. The results provide insight into protein synthesis and amino acid transport pathways in endosperm and suggest targets for the enhancement of specialty pearled wheat with higher quality.

N-WASP Control of LPAR1 Trafficking Establishes Response to Self-Generated LPA Gradients to Promote Pancreatic Cancer Cell Metastasis.

SummaryPancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. We show that N-WASP drives pancreatic cancer metastasis, with roles in both chemotaxis and matrix remodeling. lysophosphatidic acid, a signaling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for cancer cells. Pancreatic cancer cells break lysophosphatidic acid down as they respond to it, setting up a self-generated gradient driving tumor egress. N-WASP-depleted cells do not recognize lysophosphatidic acid gradients, leading to altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. We describe a signaling loop whereby N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid-induced RhoA-mediated contractility and force generation by controlling lysophosphatidic acid receptor recycling and preventing degradation. This chemotactic loop drives collagen remodeling, tumor invasion, and metastasis and could be an important target against pancreatic cancer spread.

Surface Wettability-Directed Propulsion of Glucose-Powered Nanoflask Motors.

Chemically driven colloidal motors capable of implementing different movements under a common environment are of great importance for various complex tasks. However, the key parameters underlying different motion behaviors are incompletely understood. Here, we demonstrate that carbonaceous nanoflask (CNF) motors move spontaneously in glucose powered by the cascade reaction of glucose oxidase and catalase, and their directional propulsion can be premeditated by controlling the surface wettability of nanomotors. The hydrophilic CNF motors move from the round-bottom to the opening neck (backward), whereas the hydrophobic CNF motors swim from the opening neck to the round-bottom (forward). We demonstrate that the backward motion of the hydrophilic CNF motors is driven by the local glucose gradient due to self-diffusiophoresis, and the forward movement of the hydrophobic CNF motors is caused by the locally produced glucose acid gradient. The fluid simulation reveals that the hydrophilic and hydrophobic CNF motors correspond to the puller and pusher models, respectively. Our study offers a minimal strategy to manipulate the direction of motion of motors for specific applications and to change the hydrodynamic behaviors of glucose-powered motors.

Cambrian Chordates and Vetulicolians

Deuterostomes make a sudden appearance in the fossil record during the early Cambrian. Two bilaterian groups, the chordates and the vetulicolians, are of particular interest for understanding early deuterostome evolution, and the main objective of this review is to examine the Cambrian diversity of these two deuterostome groups. The subject is of particular interest because of the link to vertebrates, and because of the enigmatic nature of vetulicolians. Lagerstätten in China and elsewhere have dramatically improved our understanding of the range of variation in these ancient animals. Cephalochordate and vertebrate body plans are well established at least by Cambrian Series 2. Taken together, roughly a dozen chordate genera and fifteen vetulicolian genera document part of the explosive radiation of deuterostomes at the base of the Cambrian. The advent of deuterostomes near the Cambrian boundary involved both a reversal of gut polarity and potentially a two-sided retinoic acid gradient, with a gradient discontinuity at the midpoint of the organism that is reflected in the sharp division of vetulicolians into anterior and posterior sections. A new vetulicolian (Shenzianyuloma yunnanense nov. gen. nov. sp.) with a laterally flattened, polygonal anterior section provides significant new data regarding vetulicolians. Its unsegmented posterior region (‘tail’) bears a notochord and a gut trace with diverticula, both surrounded by myotome cones.

Simultaneous determination of multiple components in formula and preparations of Xiaoyaosan

Xiaoyaosan is one of the famous formulas treating for liver, spleen and blood deficiency syndrome along Chinese history. Their five main components, paeoniflorin, ferulic acid, glycyrrhizic acid, liquiritin, and atractylenolide I are believed to partly representative of this formula. Ultra performance liquid chromatography method was newly established, UPLC BEH-C18 column (2.1 × 100 mm, 1.7 μm) with acetonitrile – 0.1% phosphate acid gradient elution system, in 0.4 mL/min and the temperature was 30 °C; Detection wavelengths were optimized separately. The five components were linear within their linear range (r ≥ 0.9991), average recovery 97.63%∼102.83%, RSD 1.88%∼4.38%. The newly established method is accurate, rapid, and convenient, with satisfied separation performance to quantify multi-components in formula and preparations of Xiaoyaosan. It will provide a reliable reference for the quality evaluation of both Xiaoyaosan formula and preparations during the process of manufacture, administration and clinical application in the future.

Two-Dimensional Nonclose-Packed Colloidal Crystals by the Electrostatic Adsorption of Three-Dimensional Charged Colloidal Crystals.

We demonstrate that nonclose-packed two-dimensional (2D) colloidal crystals fixed on flat solid surfaces can be obtained by the electrostatic adsorption of three-dimensional (3D) charged colloidal crystals onto oppositely charged substrates. 3D colloidal crystals of negatively charged polystyrene (diameter d = 500 nm) and silica (d = 510 and 550 nm) particles were formed in their aqueous dispersions. Then, a single layer of the 3D crystals (the particle volume fraction = ∼0.07-0.3) was adsorbed onto a glass surface, which was earlier modified with 3-aminopropyltriethoxysilane (APTES), a cationic silane coupling reagent. Under salt-free conditions, the lowermost layer of the 3D crystals, which was oriented parallel to the substrate, was adsorbed onto the substrate surface, forming 2D crystals. Centimeter-sized, large 2D silica crystals were produced by combining a unidirectional 3D crystallization of the silica colloid under a base concentration gradient and a unidirectional adsorption under an acidic concentration gradient, which allowed tuning of the charge number on the APTES-modified substrate. The interparticle separations of the resulted 2D crystals did not vary greatly (within 5%) over a large area (length: 2 cm); however, the separations were smaller than the initial value because of gravitational sedimentation. We also produced 2D crystals of gold particles (d = 250 nm), which we expect to be applicable as plasmonic materials. The present study will provide a facile strategy to produce nonclose-packed 2D colloidal crystals of various types of particles, including large and high-density particles.

Multi-dimensional life cycle assessment of decentralised energy storage systems

The intermittent nature of renewable energy sources like solar and wind energy stimulates the use of centralised and decentralised energy storage systems. The sustainability of lead acid, lithium-ion and concentration gradient flow batteries, compressed air and pumped hydro energy storage (PHES) systems is investigated by conducting a multi-dimensional life cycle assessment. The environmental, economic and exergetic sustainability are assessed by calculating ReCiPe 2016 indicators, the present worth ratio and the Total Cumulative Exergy Loss, respectively. The multi-dimensional sustainability assessment did not lead to one preferred system. The PHES causes the lowest damage to human health, ecosystem diversity and resource availability and results in the lowest global warming potential. The concentration gradient flow battery system named BBS is preferred from an economic viewpoint, while the PHES is second-best. The lithium-ion battery system causes the lowest exergy losses, followed by the PHES. It is recommended to pay attention to the exergetic sustainability of technological systems as exergy losses are independent of environmental models, weighting factors, market prices, subsidies etc. More research into the specifications of the energy storage systems is needed to be able to draw firm conclusions with regard to which system is preferred.

Foliar application of uniconazole improves yield through enhancement of photosynthate partitioning and translocation to tuberous roots in sweetpotato

ABSTRACTTo examine the effects of uniconazole (UCZ) on the distribution and transport of photosynthate in sweetpotatoes, three spraying levels of UCZ, 0 (control), 50 and 100 mg L−1, were exposed to an experimental field cultivated with Jishu26 (‘J26ʹ) and Xushu32 (‘X32ʹ) varieties. Compared to control, the distribution of carbon-13 (13C) was significantly higher in the tuberous roots of both varieties treated with UCZ. In addition, UCZ treatments promoted a desirable top-to-base sucrose gradient and significant greater declines in the top-to-bottom amino acid and potassium ion (K+) gradients along the stem. Higher tuberous root yields were found in the UCZ-treated ‘J26ʹ and ‘X32ʹ. In comparison to 50 mg L−1 UCZ, ‘J26ʹ treated with 100 mg L−1 UCZ exhibited significantly higher distribution ratio of 13C in tuberous root, and reductions in the top-to-bottom amino acid and K+ gradients along the stems at 105 and 150 days after planting. Furthermore, 100 mg L−1 of UCZ application exhibited a significantly higher tuberous root yield. For ‘X32ʹ, the two concentrations of UCZ demonstrated no significant differences in these parameters. A reasonable distribution structure of photosynthates that responsible for high yields is associated with the desirable sucrose, ammonia nitrogen (N) or K+ gradient along the stems caused by UCZ application.

A Biosensor-Based Quantitative Analysis System of Major Active Ingredients in Lonicera japonica Thunb. Using UPLC-QDa and Chemometric Analysis.

In the study, a surface plasmon resonance-based (SPR-based) competitive assay was performed to analyze different compounds’ inhibitory activity to TNF-α, an important pro-inflammatory cytokine in the pathogenesis of chronic inflammatory diseases. Moreover, the single mass spectrometry (MS) detection method was coupled with an ultra-high-performance liquid chromatography (UPLC) system for the routine quality control (QC) of a traditional Chinese medicine (TCM). The above quality control strategy was evaluated with Lonicera japonica Thunb. Analytes were firstly separated on a Waters ACQUITYTM UPLC HSS T3 column (2.1 × 50 mm; particle size = 1.8 μm) using a 0.1% formic acid gradient elution, then detected by negative ESI mass spectrometry. The limits of quantification (LOQ) for analytes reached 0.005–0.56 μg/mL. The LOD of the QDa detector was lower than that of the PDA detector, indicating its wider detection range. The QDa detector was also more suitable for the analysis of the complex matrix of TCM. The method showed excellent linearity, with regression coefficients higher than 0.9991. The average recoveries of the investigated analytes were in the range of 98.78–105.13%, with an RSD below 3.91%. The inter-day precision range (n = 3 days) was 2.51–4.54%. Compared to other detectors, this strategy could be widely applied in the quantitative analysis of TCM. In addition, the chemically latent data could be revealed using chemometric analysis. Importantly, this study provides an efficient screening method for small-molecule inhibitors targeting the TNF-α pathway.

Spatial patterns of benthic silica flux in the North Pacific reflect upper ocean production

Diatoms are the dominant algal group that cycles dissolved silicic acid in the ocean; they also play an important role in the oceanic carbon cycle. It is therefore important to quantify the spatial distribution of silica cycling for defining global ocean biogeochemical cycles. On the research cruise CDisK-IV, water samples and sediment cores were collected at 5 stations along a North Pacific transect near 150°W from 22°N to 50°N to evaluate benthic remineralization rates of biogenic silica (bSi). Two independent methods, core incubation and diffusive transport based on porewater profiles, were utilized to estimate benthic silicic acid fluxes, and these independent methods yield fluxes that agree within uncertainties. The benthic fluxes are reported as 0.04 ± 0.01, 0.04 ± 0.01, 0.05 ± 0.01, 0.67 ± 0.14, 0.40 ± 0.08 mmol Si m−2 day−1 for Stations 1 to 5, south to north, respectively. Burial fluxes were estimated using measurements of solid phase bSi in sediments and literature values of sediment accumulation rate. Burial efficiencies of bSi at all stations were <5% and show reasonable agreement with previous estimates. When burial rates were added to benthic fluxes to calculate rain rates, the rain observed under the subarctic gyre (Stations 4–5), was far larger than in the lower latitudes of the subtropics (Stations 1–3), corresponding to higher surface diatom productivity at higher latitudes. At the two northern stations, the bottom 500 m of the water column shows a near-bottom increase in silicic acid that is consistent with the measured benthic flux and the estimated vertical eddy diffusivity. Above this horizon, water column density stratification increases and vertical diffusivity decreases, but the silicic acid gradient decreases. This reduction in gradient indicates that above this horizon, horizontal transport by deep waters, rather than vertical diffusion, becomes the dominant process removing the silicic acid released by benthic remineralization.

Theoretical minimal RNA rings recapitulate the order of the genetic code's codon-amino acid assignments

BackgroundTheoretical minimal RNA rings form stem-loop hairpins coding for each of the 20 amino acids and a stop, presumably mimicking life's first minimal coding and self-replicating RNAs. They resemble consensual tRNAs. Mean amino acid positions in proteins follow the genetic code's consensual amino acid inclusion order, a 5′-late-to-3′-early amino acid gradient. HypothesisWe translated minimal RNA rings to test whether translated peptides share that gradient with modern proteins, using a) ribosomal translation, non-overlapping consecutive codons; and b) frameless translation advancing nucleotide by nucleotide, producing partially overlapping codons. ResultsFor frameless translation, most RNA rings code for a 5′-late-to-3′early amino acid gradient. Gradients indicate decreasing amino acid metabolic costs, from large to small amino acids. For ribosomal translation, the 5′-late-to-3′early amino acid gradient evolves from early to late RNA rings when ranked according to yields in Miller's experiment of their predicted anticodon's cognate amino acid. ConclusionsSimulations that produced in silico minimal RNA rings didn't account for coded amino acid properties. Yet, produced peptides remind actual proteins, and suggest ancestral frameless translation of partially overlapping trinucleotides advancing by single nucleotide steps, constrained by resource scarcity. Minimal RNA rings reflect the transition from frameless to ribosomal translation and are realistic candidates for ancestral tRNAs.

Quantitative AOP based teratogenicity prediction for mixtures of azole fungicides

Exposure of embryos to mixtures of environmental chemicals can result in congenital malformations. Mixture experiments can provide an indication of the joint effects of substances, but it is practically infeasible to test all possible combinations. The development of mechanistic approaches and integrated models able to predict the effects of mixtures from the concentrations of their individual components, are crucial to assess mixtures associated risks. Azole fungicides can induce craniofacial defects, both after in utero and in vitro exposure. Results obtained in vitro have shown a significant enhancement of teratogenic effects after co-exposure to azoles in comparison to the single exposures. In this project, we evaluated the hypothesis that those molecules concur to imbalance the retinoic acid pathway in specific responsive embryonic tissues. We developed a quantitative adverse outcome pathway for craniofacial malformations, able to simulate the formation of the physiological retinoic acid gradient in the rat embryo hindbrain and its perturbation after exposure to cyproconazole, flusilazole, triadimefon and to their binary mixtures. The underlying system biology model was calibrated using in vitro data and is reasonably predictive of mixtures’ effects for those azoles, thereby confirming the plausibility of the hypothesized pathogenic pathway. This quantitative AOP could have mechanistic or predictive applications in pesticides risk assessment.

The Effect of Buffers on Weak Acid Uptake by Vesicles.

The assessment of weak acid membrane permeability (Pm) frequently involves large unilamellar vesicles. It relies on measurements of the intravesicular pH drop, ΔpHin, in response to a sudden augmentation of external acid concentration. However, ΔpHin may be primarily governed by non-instantaneous protonation and deprotonation reactions of (i) the acid itself, (ii) the buffer molecules, and (iii) the fluorescent pH reporter dye. Moreover, buffer concentration and acid gradient also serve as determinants of ΔpHin, as we show here. The uniexponential time constant (τ) of ΔpHin(t) is an invalid measure of Pm as Arrhenius plots of Pm and reveal different activation energies for acid influx. We calculate Pm by fitting a mathematical model to experimental stopped-flow traces. The model takes into account not only the time course of total internal buffer capacity but also (i) water self-dissociation, (ii) volume changes due to acid induced osmotic water flow, and (iii) the spontaneous membrane proton leak. It allows extracting a Pm of 30.8 ± 3.5 μm/s for formic acid for 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles.

A Novel UPLC-MS/MS Method for the Determination of Salvianolic Acid A in Rat Urine, Feces, and Bile and its Application to Excretion Study

Background: Salvianolic acid A (SAA) is a polyphenolic acid extracted from Salvia miltiorrhiza Bunge. It showed protective effect against diabetic complications after oral administration with a low bioavailability of 1.42%. Attempts have been made to develop it into a new medication. Intracorporal process of SAA is indistinct and no report regarding the excretion is available. Our preliminary experiment revealed that previous reported methods were unsuitable for the excretion study due to the serious matrix effect. Methods: To better clarify its pharmacokinetics and avoid the interference of complex endogenous substances, a sensitive UPLC-MS/MS method with a better resolution was developed for the excretion study of SAA for the first time. The analytes were separated by reversed-phase chromatography with acetonitrile-water (containing 0.1% formic acid) gradient elution. The mass spectrometer was operated in the negative ESI mode and multiple reaction monitoring mode. Results: This method was linear over the concentration range of 2.5-100, 5-100 and 5-100 ng/mL in urine, feces and bile, respectively. The accuracy, precision, stability, recovery and matrix effect were satisfactory in all matrices examined. The validated method was successfully applied to an excretion study in rats. After oral administration of 20 mg/kg, the average accumulated excretion amount of SAA in urine, feces and bile were 99.80, 32046.30 and 161.03 ng, respectively. Conclusion: A quick but low elimination was observed. The date is useful for the clinical trial design of SAA.

A microfluidic patterned model of non-alcoholic fatty liver disease: applications to disease progression and zonation.

Non-alcoholic fatty liver disease (NAFLD) and its progressive form non-alcoholic steatohepatitis (NASH) affect 25% of the world population. NAFLD is predicted to soon become the main cause of liver morbidity and transplantation. The disease is characterized by a progressive increase of lipid accumulation in hepatocytes, which eventually induce fibrosis and inflammation, and can ultimately cause cirrhosis and hepatic carcinoma. Here, we created a patterned model of NAFLD on a chip using free fatty acid gradients to recapitulate a spectrum of disease conditions in a single continuous liver tissue. We established the NAFLD progression via quantification of intracellular lipid accumulation and transcriptional levels of fatty acid transporters and NAFLD pathogenesis markers. We then used this platform to create oxygen driven steatosis zonation mimicking the sinusoidal lipid distribution on a single continuous tissue and showed that this fat zonation disappears under progressed steatosis, in agreement with in vivo observations and recent computational studies. While we focus on free fatty acids and oxygen as the drivers of NAFLD, the microfluidic platform here is extensible to simultaneous use of other drivers.

Quantification of Phenolic Compounds from Fadogia agrestis and Dietary Supplements using UHPLC-PDA-MS.

Fadogia agrestis is used in traditional African medicine as an analgesic and for anti-inflammatory and aphrodisiac activities. An ultra-high-performance liquid chromatography method was developed for the determination of 11 chemical constituents from roots and aerial parts of F. agrestis. The separation was achieved within 7 min by using C-18 column material and a water/acetonitrile mobile phase, both containing 0.1% formic acid gradient system with a temperature of 45 °C. The method was validated for linearity, repeatability, limits of detection, and limits of quantification. The limits of detection of phenolic compounds were found to be in the range from 0.025 to 0.1 µg/mL. The wavelengths used for quantification with the photodiode array detector were 238, 254, 291, and 325 nm. Twelve of 17 dietary supplements contained phenolic compounds in the range from 0.3 to 2.7 mg/d. The phenolic compounds were not detected in five dietary supplements. Liquid chromatography-mass spectrometry coupled with electrospray ionization interface method is described for the identification and confirmation of compounds from plant samples and dietary supplements claiming to contain F. agrestis. This method involved the use of [M + H]+ and [M + Na]+ ions in the positive mode and [M - H]- ions in the negative mode with extractive ion monitoring. The developed method is simple, economic, rapid, and especially suitable for quality control and chemical fingerprint analysis of F.agrestis.

Development of simultaneous quantification method of loteprednol etabonate (LE) and its acidic metabolites, and analysis of LE metabolism in rat

Loteprednol etabonate (LE) is a soft corticosteroid with two labile ester bonds at 17α- and 17β-positions. Its corticosteroidal activity disappears upon hydrolysis of either ester bond. Hydrolysis of both ester bonds produces the inactive metabolite, Δ1-cortienic acid (Δ1-CA).The simple high-performance liquid chromatography method using acetic acid gradient was developed for the simultaneous determination of LE and its acidic metabolites.LE was hydrolyzed in rat plasma with a half-life of 9 min. However, LE hydrolysis was undetectable in rat liver and intestine. LE hydrolysis in rat plasma was completely inhibited by paraoxon and bis(p-nitrophenyl) phosphate, thus identifying carboxylesterase as the LE hydrolase. Rat plasma carboxylesterase had a Km of 6.7 μM for LE.In contrast to the disappearance rate of LE in rat plasma, the formation rate of 17α-monoester and Δ1-CA was markedly low, and a main hydrolysate of LE was not detected in rat plasma.The metabolism of LE proceeded via different pathways in human and rat plasma. LE was slowly hydrolyzed by paraoxonase in human plasma to 17α-monoester with a half-life of 12 h, but by carboxylesterase in rat plasma to yield undetectable products, presumed to include the unstable 17β-monoester.