Acid Gradient Research Articles

Effect of H2O2/ascorbic acid degradation and gradient ethanol precipitation on the physicochemical properties and biological activities of pectin polysaccharides from Satsuma Mandarin

In this work, three degraded polysaccharides (DMPP-40, DMPP-60, DMPP-80) were successfully obtained by H2O2/ascorbic acid degradation and gradient ethanol precipitation from Satsuma mandarin peel pectin (MPP), and their physicochemical properties, antioxidant and prebiotic activities were investigated. The molecular weight of MPP, DMPP-40, DMPP-60, DMPP-80 were determined to be 336.83 ± 10.57, 18.93 ± 0.54, 26.07 ± 0.83 and 8.71 ± 0.27 kDa, respectively. The ethanol concentration significantly affected the physicochemical properties of DMPPs. DMPP-60 showed the highest yield (69.07 %) and uronic acid content (64.85 %), DMPP-80 showed the lowest molecular weight (8.71 kDa), and the composition and proportion of monosaccharides of DMPPs were significantly different. Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (1H NMR) confirmed that DMPPs exhibited similar functional groups, while X-ray diffraction (XRD) indicated that DMPP-40 possessed some crystallographic sequences. Scanning electron microscopy (SEM) images directly verified the fragmented structure and reduced surface area of DMPPs. Besides, the H2O2/ascorbic acid treatment could obviously reduce the apparent viscosity and thermal stability of MPP. Meanwhile, the results of bioactivity assay showed that DMPPs possessed better antioxidant activity and probiotics pro-proliferative effects compared with MPP. DMPP-80 could significantly inhibit lipopolysaccharides (LPS)-stimulated production of inflammatory factors (including nitric oxide (NO), interleukin (IL)-6, tumor necrosis factor (TNF)-α and interleukin (IL)-1β) in RAW264.7 cells. Results suggest that the H2O2/ascorbic acid combined with gradient ethanol precipitation has potential applications in degradation and separation of MPP to improve its biological activities.

B-167 Quantification of Thyroglobulin in Serum for Clinical Research using SISCAPA Workflow Combined with LC-MS/MS

Abstract Background Accurate measurement of Thyroglobulin (Tg) using existing immunoassay-based techniques can be challenging in the presence of anti-Tg antibodies (TgAb), which can prevent the binding of Tg to assay antibodies thus leading to non-quantifiable Tg concentrations. The Stable Isotope Standards and Capture with Anti-Peptide Antibodies (SISCAPA™) Workflow combined with LC-MS/MS has been successfully employed to circumvent this problem by digesting the serum sample thus eliminating interfering TgAb and measuring a Tg-specific surrogate peptide instead. Methods Using a Andrew+™ Pipetting Robot, denaturation and reduction buffer was added to 250µL serum samples and incubated at 37°C. Internal standard and trypsin were added and samples incubated at 37°C. The samples were quenched with protease inhibitor and Anti-Tg FSP mAb SISCAPA was added to the serum samples and mixed for 60 minutes. Using a magnetic array plate, the beads were left to pull down, samples were discarded, and the beads were twice washed with PBS/CHAPS prior to elution with acetonitrile with formic acid. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters™ XSelect™ HSS T3 Column using a water/acetonitrile/formic acid gradient elution profile and the Tg FSP peptide was quantified using a Waters Xevo™ TQ Absolute Mass Spectrometer. Results The method demonstrated no significant carryover or matrix effects and was shown to be linear from 0.1 - 50 ng/mL. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (<20%) at 0.1 ng/mL with S/N (PtP) >10:1. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all < 9.5% (n = 25) for both automated and manual precision assessments. Comparison with samples from the UK NEQAS scheme demonstrated significant method bias of -40% for the developed LC-MS/MS method. Re-assignment of the calibrator concentrations using the EQA samples reduced the bias to -5.5%, indicating differences in the calibration materials used for these measurements. Conclusions A LC-MS/MS clinical research method for serum thyroglobulin was developed using SISCAPA, followed by analysis using ACQUITY UPLC I-Class PLUS FL System and the Xevo TQ Absolute Mass Spectrometer. The method provides analytical sensitivity down to 0.1 ng/mL from 250 µL serum, while providing sufficient sample for re-analysis. The method demonstrates excellent linearity across the calibration range, with no significant carryover, interferences, and matrix effects. Total reproducibility and repeatability of the method was ≤9.5% RSD for manual and automated sample preparation, using the Andrew+ Pipetting Robot. In addition, the Andrew+ Pipetting Robot can minimize user touch-time, allowing a full plate to be prepared within four hours without user intervention.

B-155 Analysis of Monoclonal Antibodies Using a Quantification LC-MS/MS Kit - Spotlight on Infliximab

Abstract Background The use of immunoassays for measuring mAbs is well established but has some limitations, including poor performance, lack of standardization, a high cost when processing a limited number of samples, limited dynamic range, and the potential for cross-reactivity. Using LC-MS/MS associated with the ready-to-use commercial mAbXmise Kit is a simple way to implement mAbs measurement for infliximab, providing high analytical performance, ease of use, and flexibility. Methods The Promise Proteomics Inflammation mAbXmise Kit contains all the calibrators, QCs, internal standard and sample preparation consumables to run the LC-MS/MS method for infliximab. Briefly, 20µL samples are dispensed into the mAbXmise plates containing lyophilized stable labelled infliximab. Samples are transferred to the PuriXmise plate to perform the immunocapture of infliximab and allow washing of the samples. Samples are eluted into a collection plate, evaporated to dryness, resuspended, and the protease (CutXmise) is added to the sample to digest infliximab overnight. The digestion is quenched, with the samples immediately ready for analysis. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters™ XSelect™ Premier HSS T3 Column, eluted using a water/acetonitrile/formic acid gradient profile and quantified with both a Waters Xevo™ TQ-XS and Xevo TQ-S micro Mass Spectrometer. Results The method was shown to be linear from 2 - 100 µg/mL. Analytical sensitivity at lowest calibrator of 2µg/mL provided S/N (PtP) ≥15:1 for all tryptic peptides on both mass spectrometers. Coefficients of variation (CV) at 4 and 25µg/mL QC concentrations were all ≤ 10.0% (n = 5). The relative concentrations of the tryptic peptides were compared across 29 anonymized samples and good agreement was observed for GLE, SAV, DIL and ASA peptides. Conclusions We have demonstrated a clinical research method for quantifying Infliximab in plasma using a commercially available kit for sample preparation followed by LC-MS/MS analysis. The Promise Proteomics mAbXmise Kit aids in the day-to-day reproducibility of the immunocapture and tryptic digestion of IFX for targeted LC-MS/MS analysis with the correct peptide selection. Compared to a direct digest surrogate peptide workflow, the process used in the kit reduces matrix interference, improves analytical sensitivity, and enables the use of lower sample volumes. The method can be run on both the Xevo TQ-XS and Xevo TQ-S micro Mass Spectrometers which provide the dynamic range to quantify IFX across the expected range, and the selectivity and analytical sensitivity to obtain low-level quantification of the IFX surrogate peptide in plasma samples. The data presented combines the use of a kit dedicated to the preparation of samples and the use of liquid chromatography and mass spectrometry instrumentation to perform the quantitative analyses. The mAbXmise Kit described has not been cleared by any regulatory entity for diagnostic purposes outside of Europe. Analytical performance depends on the instrument characteristics and its settings. The end user is responsible for completion of the method development and validation. Proteomics mAbXmise Kits are not available for sale in all countries. For Research Use Only. Not for use in diagnostic procedures.

Pharmacological inhibition reveals participation of the endocytic compartment in positive feedback IL-6 secretion in human skeletal myotubes

IL-6 is an important cytokine involved in metabolic, immunological, and cell-fate responses. It is released upon stimulation by skeletal muscle cells through partially characterized mechanisms. In some cell types, IL-6 has been reported to activate a positive feedback loop involving endocytic vesicles, but evidence is mostly based on transcription and signal transduction mechanisms and is very scarce in muscle cells. Our aim was to directly demonstrate the presence of positive feedback in the ATP-induced release of IL-6 into the supernatant of human skeletal muscle cultures. The total release (production) of IL-6 was reduced for higher volumes of supernatant, when the secreted IL-6 molecules are more diluted, and enhanced when the supernatant volume was lower. In addition, secretion was impaired both by tocilizumab, a blocker of human IL-6 receptors, and by the soluble form of the receptor. The secretion in response to ATP was also inhibited by treatment with the endocytosis inhibitor dynasore, and by disruption of the acidic gradient of the endocytic compartment using different methods (chloroquine, NH4Cl or monensin). IL-6 secretion was also impaired by NED-19, a specific inhibitor of the two pore channels receptor mediating Ca2+ release from the endolysosomal compartment. IL-6 and ATP increased IL-6 mRNA levels, an effect blocked by tocilizumab. Altogether, our results demonstrate that ATP-secreted IL-6 activates a positive loop based on IL-6 receptors, endocytosis, two pore channels and IL-6 transcription. Given the importance of muscle IL-6 as a systemic regulator and as an inflammatory mediator, our study can help to understand muscle pathophysiology.

The hemostatic performance and mechanism of palygorskite with structural regulate by oxalic acid gradient leaching

Palygorskite (Pal) is a naturally available one-dimensional clay mineral, featuring rod-shaped morphology, nanoporous structure, permanent negative charges as well as abundant surface hydroxyl groups, exhibiting promising potential as a natural hemostatic material. In this study, the hemostatic performance and mechanisms of Pal were systematically investigated based on the structural regulate induced by oxalic acid (OA) gradient leaching from perspectives of structure, surface attributes and ion release. In vitro and in vivo hemostasis evaluation showed that Pal with OA leaching for 1 h exhibited a superior blood procoagulant effect compared with the raw Pal as well as the others leached for prolonging time. This phenomenon might be ascribed to the synergistic effect of the intact nanorod-like morphology, the increase in the surface negative charge, the release of metal ions (Fe3+ and Mg2+), and the improved blood affinity, which promoted the intrinsic coagulation pathway, the fibrinogenesis and the adhesion of blood cells, thereby accelerating the formation of robust blood clots. This work is expected to provide experimental and theoretical basis for the construction of hemostatic biomaterials based on clay minerals.

Three-dimensional (3D) polylactic acid gradient scaffold to study the behavior of osteosarcoma cells under dynamic conditions.

This study adopts an in vitro method to recapitulate the behavior of Saos-2 cells, using a system composed of a perfusion bioreactor and poly-L-lactic acid (PLLA) scaffold fabricated using the low-cost thermally-induced phase separation (TIPS) technique. Four distinct scaffold morphologies with different pore sizes were fabricated, characterized by Scanning electron microscopy and micro-CT analysis and tested with osteosarcoma cells under static and dynamic environments to identify the best morphology for cellular growth. In order to accomplish this purpose, cell growth and matrix deposition of the Saos-2 osteosarcoma cell line were assessed using Picogreen and OsteoImage assays. The obtained data allowed us to identify the morphology that better promotes Saos-2 cellular activity in static and dynamic conditions. These findings provided valuable insights into scaffold design and fabrication strategies, emphasizing the importance of the dynamic culture to recreate an appropriate 3D osteosarcoma model. Remarkably, the gradient scaffold exhibits promise for osteosarcoma applications, offering the potential for targeted tissue engineering approaches.

Genetic vulnerability to Crohn's disease reveals a spatially resolved epithelial restitution program.

Effective tissue repair requires coordinated intercellular communication to sense damage, remodel the tissue, and restore function. Here, we dissected the healing response in the intestinal mucosa by mapping intercellular communication at single-cell resolution and integrating with spatial transcriptomics. We demonstrated that a risk variant for Crohn's disease, hepatocyte growth factor activator (HGFAC) Arg509His (R509H), disrupted a damage-sensing pathway connecting the coagulation cascade to growth factors that drive the differentiation of wound-associated epithelial (WAE) cells and production of a localized retinoic acid (RA) gradient to promote fibroblast-mediated tissue remodeling. Specifically, we showed that HGFAC R509H was activated by thrombin protease activity but exhibited impaired proteolytic activation of the growth factor macrophage-stimulating protein (MSP). In Hgfac R509H mice, reduced MSP activation in response to wounding of the colon resulted in impaired WAE cell induction and delayed healing. Through integration of single-cell transcriptomics and spatial transcriptomics, we demonstrated that WAE cells generated RA in a spatially restricted region of the wound site and that mucosal fibroblasts responded to this signal by producing extracellular matrix and growth factors. We further dissected this WAE cell-fibroblast signaling circuit in vitro using a genetically tractable organoid coculture model. Collectively, these studies exploited a genetic perturbation associated with human disease to disrupt a fundamental biological process and then reconstructed a spatially resolved mechanistic model of tissue healing.

Multifunctional Proximity Labeling Strategy for Lipid Raft-Specific Sialic Acid Tracking and Engineering.

Lipid raft-specific glycosylation has been implicated in many biological processes, including intracellular trafficking, cell adhesion, signal transduction, and host-pathogen interactions. The major predicament in lipid raft-specific glycosylation research is the unavailability of tools for tracking and manipulating glycans on lipid rafts at the microstructural level. To overcome this challenge, we developed a multifunctional proximity labeling (MPL) platform that relies on cholera toxin B subunit to localize horseradish peroxidase on lipid rafts. In addition to the prevailing electron-rich amino acids, modified sialic acid was included in the horseradish peroxidase-mediated proximity labeling substrate via purposefully designed chemical transformation reactions. In combination with sialic acid editing, the self-renewal of lipid raft-specific sialic acid was visualized. The MPL method enabled tracking of lipid raft dynamics under methyl-β-cyclodextrin and mevinolin treatments; in particular, the alteration of lipid rafts markedly affected cell migration. Furthermore, we embedded functional molecules into the method and implemented raft-specific sialic acid gradient engineering. Our novel strategy presents opportunities for tailoring lipid raft-specific sialic acids, thereby regulating interactions associated with lipid raft regions (such as cell-virus and cell-microenvironment interactions), and can aid in the development of lipid raft-based therapeutic regimens for tumors.

B-305 A Novel Dilute-n-shoot Method for Quantitating Isotonitazene and Desmetonitazene in Human Urine By LC/MS

Abstract Background Isonitazene is a benzimidazole derived opioid analgesic drug related to etonitazene, which has been sold as a designer drug. According to doctors isotonitazene has the potency of 20–200 times of fentanyl. The side effects of isotonitazene are like fentanyl and has been linked to many fatalities in Europe and the US. It has led to a number of overdoses through secondhand contact, by smelling or coming in contact with skin. It is slowly replacing fentanyl as the deadliest street drug in the US. We developed a dilute-n-shoot method that would test for isotonitazene and its metabolite metodesnitazene in human urine. This method used filtration to remove unwanted particulates and protein. It reduces the number of steps needed to prepare samples for liquid chromatography/mass spectrometry analysis compared to methods that require centrifugation. This assay allows for easier training of staff. In addition, it is easily adaptable to increase sample load. Method Isotonitazene and metodesnitazene urine samples were prepared for LC/MS analysis by diluting them 1:20 in 0.1% formic acid in water. These samples were spiked with internal standard and filtered through a 0.2 μm Captiva 96-well filter plate. Isotonitazene and metodesnitazene concentrations were measured using LC/MS in the positive ion mode. The separation was obtained using a phenylhexyl UPLC column, 50 × 2.1 mm, 1.8 μm, along with a 50:50 acetonitrile/methanol/water formic acid gradient mobile phase. Results The separation was completed in a proximately 4.5 min with good resolution between isotonitazene and metodesnitazene. Validation studies demonstrated that this method was linear from 2 to 100 ng/mL, with r2 ≥ 0.990 for both isotonitazene and metodesnitazene. Interday and intraday precision for both analytes were acceptable, with CV values < 5.0%. Accuracy measurements at 5, 25 and 80 ng/mL gave results where the percent difference was ≤20% for the 4 ng/mL concentration and ≤15% for the 25 and 80 ng/mL. A study of interfering compounds showed that there were no interferences that would affect the accuracy of isotonitazene and metodesnitazene. Recovery studies showed that > 90% of each analyte was recovered from the filtration step. The LOQ was determined to be 2 ng/mL and the LOD was determined to be 0.5 ng/mL. The reportable range was determined to be 2 to 200 ng/mL. The results of this validation will be discussed in detail in this poster. Conclusion The method was linear and precise for both analytes over a range of 2 to 100 ng/mL. The method was reproducible and easy to use. Isotonitazene and metodesnitazene could easily be quantitated in human urine using this method.

Molecular insights into the impacts of acid mine drainage on dissolved organic matter dynamics in pit lakes

Pit lakes are artificial hydrological features created by mining operations that typically suffer from acid mine drainage (AMD), which not only endangers water quality but also exacerbates carbon loss. However, the impacts of AMD on the fate and role of dissolved organic matter (DOM) in pit lakes remain unclear. This study employed negative electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) combined with biogeochemical analysis to examine DOM molecular variations and environmental controls across the AMD-induced acidic and metalliferous gradients in five pit lakes. The results demonstrated distinct DOM pools in pit lakes characterized by the prevalence of smaller aliphatic compounds compared to other waterbodies. AMD-induced geochemical gradients promoted DOM heterogeneity among pit lakes, with acidic pit lakes containing more lipid-like compounds. Acidity and metals enhanced DOM photodegradation, reducing the content, chemo-diversity and aromaticity. Organic sulfur was detected in high abundance, potentially from sulfate photo-esterification and mineral flotation agent. Furthermore, microbial involvements in carbon cycling were revealed by DOM-microbe correlation network, but microbial contributions to the DOM pools decreased under acidic and metal stresses. These findings highlight abnormal carbon dynamics caused by AMD pollution and integrate DOM fate into pit lake biogeochemistry, thereby contributing to management and remediation.

PH-Regulated lignin as a green catalyst for Highly-Efficient CO2 cycloaddition

Lignin, one of the most abundant sources of renewable biomass on earth, has the potential to be used as an alternative and value-added green catalyst via the up-grading of lignin waste. However, the application of lignin-based catalysts is still a significant challenge due to the complexity of the lignin structure. Herein, lignin enriched with COOH and Ar-OH groups were generated after subjecting to the acid gradient grading method, and lignin with sharply raised COOH/Ar-OH ratio (0.48) was obtained when it was precipitated at pH 2. This up-graded lignin exhibited outstanding catalytic activity (Yield: 93 %, TON: 131) for the coupling reaction between styrene oxide (SO) and CO2 under mild condition (T: 120 ℃, P: 0.5 MPa). The 31P NMR and 2D HSQC NMR analysis showed that enriching of Ar-OH and COOH groups was due to the cracking of the β-O-4′ interunits and ester units such as p-coumarates (PCA) and ferulates (FA) under acidic conditions. Particularly, the Ar-OH groups showed significant increase only under stronger acid condition (pH 1), while the COOH groups were constantly unleashed with the pH decreasing (pH from 3 to 1). Furthermore, the combined experiments and DFT study for a series of lignin model monomers catalysts revealed the outstanding synergistic promoting effect of COOH and Ar-OH in lignin on the conversion of CO2 and epoxides due to the declined energy barrier of the ring-opening step, while the synergistic effect could be weakened by –OCH3 in lignin. Thus, lignin waste can be converted into excellent catalyst for transforming CO2 into high-value chemicals by regulating their catalytic centres through the acid gradient grading method. The fundamental results in this work can also provide theoretical guidance for the design and development of green lignin-based catalysts in the future.

Speciation of inorganic arsenic with mixed mode HPLC-ESI-MS and Arsenite Oxidation

It has been challenging to analyze inorganic arsenic (iAs) with anion exchange HPLC-Electrospray Ionization-Mass spectrometry (HPLC-ESI-MS), because arsenite (As(III)) is difficult to retain on column and the salts in mobile phase causes ionization suppression of iAs. To address these issues, a method has been developed involving the determination of arsenate (As(V)) with mixed mode HPLC-ESI-MS and the conversion of As(III) to As(V) for total iAs. As(V) was separated from other chemicals on Newcrom B, a bi-modal HPLC column involving anion exchange and reverse phase interaction. The elution employed a two-dimensional gradient, including a formic acid gradient to elute As(V) and a concurrent alcohol gradient to elute organic anions used in sample preparations. As(V) was detected by Selected Ion Recording (SIR) in negative mode at m/z = 141 with a QDa (single quad) detector. As(III) was quantitatively converted to As(V) by mCPBA oxidation and measured for total iAs. By replacing salt with formic acid in elution, the ionization efficiency for As(V) was greatly enhanced in ESI interface. The limit of detection (LOD) for As(V) and As(III) were 0.0263 μM (1.97 ppb) and 0.0398 μM (2.99 ppb), respectively. The linear range was 0.05–1 μM.The method has been used to characterize iAs speciation change in the solution and precipitation in a simulated iron-rich groundwater caused by air exposure.

Development and optimization of a LC-MS/MS compatible method for quantification of losartan, hydrochlorothiazide and their impurities using AQbD approach and measurement uncertainty evaluation

The concept of Analytical Quality by Design (AQbD) comes as a more robust, economical, and scientifically based alternative for analytical development, to the detriment of OFAT (one factor at a time). This new understanding applicable to analytical development is recommended since regulatory flexibility can be achieved and ensure more reliable results throughout the life of the product. This new approach was applied to develop an analytical procedure indicative of stability for a pharmaceutical product of association of Losartan Potassium and Hydrochlorothiazide, considered a potential first line for the treatment of hypertension. The first stage of the analytical development consisted of defining analytical target profile (ATP), follow by a bibliographic survey of the physicochemical properties of the molecules in question to define an initial method. After defining the initial analytical conditions, statistical tools for design of experiments (DoE) were used for the screening and optimization steps. In the screening stage, the Plackett-Burman design was chosen, using 11 factors and 2 levels, through which it was possible to evaluate numerous variables and determine their significance in relation to the responses. Next, optimization was carried out with the experimental design of a central composite with 4 factors and 5 levels, which allowed modeling a complex response surface and evaluating the phenomena of interactions between the factors. Thus, the optimized analytical conditions were defined, considering a 0.3% formic acid gradient as eluent A and a mixture of acetonitrile and methanol (80:20) as eluent B, X-Bridge C18 chromatographic column (150 mm × 4 .6 mm × 3.5 μm), column temperature of 40°C, flow rate of 1.3 mL/min, injection volume of 10 μL. Through this methodology, it was possible to identify an unknown degradation product of Hydrochlorothiazide, formed by the reaction with lactose (excipient present in the drug formulation), proving that the method can be applicable both to DAD detectors and to spectrometry and mass detectors. It was also proven through the forced degradation study that the method is indicative of stability, in addition to being validated and robust for its purpose.

Exploring Rhenium Arene Piano-Stool Chemistry with [Re(η6-C6H6)(NCCH3)3]+: A Powerful Semi-Solvated Precursor.

Thermal treatment of the ReIII hydride complex [ReH(η5-C6H7)(η6-C6H6)]+ in CH3CN results in the formation of [Re(η6-C6H6)(NCCH3)3]+. This semi-solvated complex is remarkably stable under an ambient atmosphere and exhibits a fast CH3CN self-exchange, which facilitates substitution reactions. The CH3CN ligands are replaced by σ-donating phosphines such as trimethyl phosphine (PMe3), triphenyl phosphine (PPh3), or the bidentate 1,2-bis(diphenylphosphino)ethane (dppe) to afford [Re(η6-C6H6)(NCCH3)3-x(PR3)x]+ (if R = Me, then x = 2; if R = Ph, then x = 1 or 2) or [Re(η6-C6H6)(dppe)(NCCH3)]+, respectively. [Re(η6-C6H6)(NCCH3)3]+ also reacts with π-acceptors such as 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen), or CO (1 atm) to give [Re(η6-C6H6)(L)(NCCH3)]+ (L = bipy or phen) and [Re(η6-C6H6)(CO)(NCCH3)2]+, respectively. The latter does not show any signs of decomposition after being exposed to an ambient atmosphere for multiple days. Additionally, [Re(η6-C6H6)(NCCH3)3]+ reacts with π-donors such as the dienes 2,3-dimethyl-1,3-butadiene (DMBD), norbornadiene (NBD), or 1,5-cyclooctadiene (COD) to give [Re(η6-C6H6)(η4-diene)(NCCH3)]+ (diene = DMBD, NBD, and COD). All three complexes are extremely stable and do not decompose during purification by preparative high-performance liquid chromatography (aqueous acidic gradient). In the presence of 18-crown-6, [Re(η6-C6H6)(NCCH3)3]+ reacts with lithium cyclopentadienyl to give the sandwich complex [Re(η5-C5H5)(η6-C6H6)]. Loss of the coordinated benzene was observed when treating [Re(η6-C6H6)(NCCH3)3]+ with diphenylacetylene (PhC≡CPh), yielding the tetra-coordinated [Re(NCCH3)(η2-PhC≡CPh)3]+.

C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism Dictyostelium. C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. c2gapB- cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, c2gaB- cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

Harvesting Electricity from Atmospheric Moisture by Engineering an Organic Acid Gradient in Paper.

Moisture-activated electric generators (MEGs) that harvest clean energy from atmospheric humidity offer exciting opportunities for upgraded energy conversions. However, it is challenging to obtain MEGs that are both easy to fabricate and of high output power, due to the requirement for particular functional materials and the cumbersome manufacturing process. Herein, a simple and general method is adopted to prepare MEGs with chemically gradient structures. As a specific example, a gradient distribution of citric acid was successfully constructed inside an A4 printer paper by asymmetric drying, which can generate a continuous voltage of tens of millivolts by ambient humidity, and even to volts (275 mV and 7.6 μA cm-2) under asymmetric humidity stimulation, and the maximum power density output was 2.1 μW cm-2. The driving force behind this energy conversion is a self-maintained ionic gradient created within the paper by the asymmetric ionization of gradient organic acids when exposed to gradient or nongradient humid air. This work broadens the class of materials and possibilities for the rapid development of MEGs, shedding new light on the revolution of generators that harvest green and sustainable energy for power generation.

A new HPLC methodology for the analysis of metronidazole and dexibuprofen: Application to pharmacokinetic study and comparative greenness assessment

For the simultaneous analysis of metronidazole and dexibuprofen in real human plasma samples, a new HPLC method with a 6-minute analysis time was developed. Furthermore, the method was used to investigate their pharmacokinetics and bioavailability. The internal standard was chosen to be guaifenesin (IS). Following acetonitrile precipitation of plasma proteins, the drugs were chromatographically separated using a Zorbax Eclipse® C 18 column with acetonitrile: water (pH 4 with aqueous phosphoric acid) gradient mixture. Metronidazole and dexibuprofen calibration curves were found to be linear over the ranges of 0.5–20 and 1–30 µg/mL, respectively, with good correlation coefficients. The analysis of quality control samples revealed outstanding accuracy and precision. Drug stability was also examined throughout storage and processing, and all coefficients of variation were under 8.23 %. After this optimized method was successfully applied to plasma samples and the pharmacokinetic parameters of each of the targeted medicines were examined separately and compared to their parameters when they were administered concurrently. The outcomes supported each drug's significant impact on the pharmacokinetic characteristics of the other. Using five metrics, including the Analytical Eco-scale, Analytical GREEnness Calculator (AGREE), RGB-12, Hexagon, and AGREE-prep, the environmental impact of the suggested approach and some reported methods utilized for the determination of either medicines in biological matrix were evaluated. The promising results demonstrated lower environmental and health risks associated with sample preparation and analysis.

PH Gradient Liposomes Extract Protein Bound Amitriptyline in Peritoneal Dialysis-Exploratory Work.

Poisoning is a significant cause of injury-related death worldwide. Dialysis is usually ineffective in removing the toxin once it has been absorbed because of drug protein binding and high volumes of distribution. In this work, we explore whether the addition of liposomes to peritoneal dialysate could extract protein bound amitriptyline. Liposomes were prepared using the thin film hydration method. In the in vitro experiment, 3 mL of 20% albumin with a concentration of 6000 nmol/L amitriptyline in a proprietary dialysis cartridge was dialysed against 125 mL of phosphate-buffered saline with and without 80 mg 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) liposomes. In the in vivo arm, peritoneal dialysis was undertaken in 6 rats with pH gradient liposome augmented dialysate after intravenous amitriptyline injection. Peritoneal blood flow was estimated by CO2 extraction. Total amitriptyline extracted was compared to freely dissolved (non-protein bound) and total amitriptyline perfusing the membrane during the peritoneal dwell. Mean liposome size for DOPG and acidic centre pH gradient liposomes was 119 nm and 430 nm, respectively. In the in vitro experiment, more amitriptyline was extracted into the liposome containing dialysate than the control dialysate (40 +/− 2 nmol/L vs. 27 +/− 1 nmol/L). In the in vivo experiment, the total amitriptyline in dialysate was 5240 +/− 2750 nmol. Mean total free amitriptyline perfusing the peritoneal membrane was 93 +/− 46 nmol. Mean total blood amitriptyline perfusing the peritoneal membrane was 23,920 +/− 6920 nmol. Two of the six animals were excluded due to overestimation of peritoneal blood flow. This exploratory work suggests the addition of liposome nanoparticles to peritoneal dialysate extracted protein bound amitriptyline from blood.

Computational model for fetal skeletal defects potentially linked to disruption of retinoic acid signaling.

All-trans retinoic acid (ATRA) gradients determine skeletal patterning morphogenesis and can be disrupted by diverse genetic or environmental factors during pregnancy, leading to fetal skeleton defects. Adverse Outcome Pathway (AOP) frameworks for ATRA metabolism, signaling, and homeostasis allow for the development of new approach methods (NAMs) for predictive toxicology with less reliance on animal testing. Here, a data-driven model was constructed to identify chemicals associated with both ATRA pathway bioactivity and prenatal skeletal defects. The phenotype data was culled from ToxRefDB prenatal developmental toxicity studies and produced a list of 363 ToxRefDB chemicals with altered skeletal observations. Defects were classified regionally as cranial, post-cranial axial, appendicular, and other (unspecified) features based on ToxRefDB descriptors. To build a multivariate statistical model, high-throughput screening bioactivity data from >8,070 chemicals in ToxCast/Tox21 across 10 in vitro assays relevant to the retinoid signaling system were evaluated and compared to literature-based candidate reference chemicals in the dataset. There were 48 chemicals identified for effects on both in vivo skeletal defects and in vitro ATRA pathway targets for computational modeling. The list included 28 chemicals with prior evidence of skeletal defects linked to retinoid toxicity and 20 chemicals without prior evidence. The combination of thoracic cage defects and DR5 (direct repeats of 5 nucleotides for RAR/RXR transactivation) disruption was the most frequently occurring phenotypic and target disturbance, respectively. This data model provides valuable AOP elucidation and validates current mechanistic understanding. These findings also shed light on potential avenues for new mechanistic discoveries related to ATRA pathway disruption and associated skeletal dysmorphogenesis due to environmental exposures.

Structural evolution of palygorskite-rich clay as the nanocarriers of silver nanoparticles for efficient improving antibacterial activity

Clay minerals with rod-like or layered structures have been widely used as the carrier to fabricate metal nanoparticles-based antibacterial composites. Palygorskite-rich clay (PALC) contains not only one-dimensional nanorods (palygorskite) but also two-dimensional nanolamella (mainly illite), where its structure can be regulated by oxalic acid (OA) gradient leaching to obtain multi-dimensional nanoparticles (MDP). To further understand the activity of different rod-lamellar nanostructures of PALC and MDP induced by the OA leaching process, and also to prepare high-performance antibacterial materials, a series of nanocomposites based on silver nanoparticles (AgNPs) were prepared in this study, using PALC and MDP as the nanocarrier. TEM, FTIR, XRD, Zeta potential and other characterizations together proved that OA leaching of PALC in the range of 8–48 h (MDP-8 h, MDP-24 h, MDP-48 h) was the most beneficial for in-situ synthesis of monodispersed AgNPs. The antibacterial evaluation results showed that the growth of 99.30 %, 84.94 %, 60.72 %, 79.48 %, 87.78 % of E. coli, S.aureus, ESBL-E. coli, P. aeruginosa and MRSA (1.5 × 105 CFU/mL) were inhibited after treatment with 0.5 µg/mL of MDP-24 h/AgNPs, confirming its excellent and broad-spectrum antibacterial performance, which is mainly due to the most ideal natural multi-dimensional nanostructure of MDP after 24 h of OA leaching. In brief, a simple and efficient method was used in this study to realize the high-value application of natural multi-dimensional nanoclay and to prepare antibacterial composites with promising applications.