Ion-exchange Chromatography Research Articles

How to isolate channel-forming membrane proteins using the E. coli expression system.

The recombinant expression, isolation and characterization of pore-forming proteins is one of the most commonly used strategies for understanding the permeability properties of the biological membrane into which they are embedded. This protocol describes how to quantify the expression of your protein of interest and use this information to optimize its production using the Escherichia coli strain BL21Gold(de3)ΔABCF. It explains with a step-by-step approach how to separate the bacterial compartments according to their solubility and how to extract your protein of interest in its native conformation using detergent solutions. Finally, it describes how to improve its purity via ion-exchange chromatography and insert the purified porins into outer membrane vesicles, from which they can be copurified. The protocol is simpler and less empirical than those described for most channel-forming membrane proteins and also provides a solid foundation for the isolation of soluble proteins. Several parameters can be optimized on a case-by-case basis: expression time and temperature, concentration of the inducer, nature and concentration of the detergent, incubation time and temperature, pH and ionic strength of the purification buffers. This protocol is effective with prokaryotic channel-forming membrane proteins and can be employed for the production of pore-forming proteins from chloroplasts, mitochondria or eukaryotes in general. With minor optimization, this protocol can be adapted for the isolation of receptors, carrier, pumps or any other membrane-active proteins.

Anti-Melanogenic Effects of a Polysaccharide Isolated from Undaria pinnatifida Sporophyll Extracts

Undaria pinnatifida is a temperate brown alga known to exert free radical-scavenging and anti-inflammatory effects. In this study, we investigated the skin-whitening effects of U. pinnatifida sporophyll extracts (UPEs) in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells. The crude polysaccharide fraction (UPF) was obtained via ethanol precipitation. Four polysaccharide fractions (UPF1–4) were isolated and purified using ion-exchange column chromatography, and their anti-melanogenic activity was evaluated. UPF3 exhibited the highest anti-melanogenic activity, showing the highest sulfate (39.79%), fucose (143 μg/mg), and galactose (208 μg/mg) contents. UPF3 significantly inhibited intracellular tyrosinase activity in B16F10 cells. We also evaluated the melanogenic signaling pathway to determine the mechanism of action of UPF3 in melanongenesis. UPF3 reduced the expression of tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and tyrosinase, which play important roles in melanin production. Therefore, UPF3 has high potential for use in skin-whitening functional pharmaceuticals and cosmetics.

Ion Exchange Chromatography: A Versatile Technique for Comprehensive Characterization of Therapeutic Proteins and Peptides

Ion Exchange Chromatography (IEX) is an efficient and widely employed technique for the comprehensive characterization of therapeutic proteins and peptides. IEX separates biomolecules based on differences in their surface charge arising from amino acid composition and Post- Translational Modifications (PTMs). As a non-denaturing method, IEX is particularly valuable for resolving and quantifying charge variants like deamidation, oxidation, glycosylation, and terminal truncations in monoclonal antibodies, antibody-drug conjugates, and other biotherapeutics. The review provides insights into the principles, advantages, applications, recent advances, and limitations of using IEX (cation and anion exchange modes) for detailed analysis of proteins and peptides.

Lily bulb polyphenol oxidase obtained via an optimized multi-stage separation strategy for structural analysis and browning mechanism elucidation

An optimized multi-stage separation strategy was developed to purify lily bulb polyphenol oxidase (PPO) for revealing its molecular structure. The PPO was purified 14.64-fold with high specific activity of 153,900 U/mg via optimized conditions of phosphate buffer pH (6.5), solid-liquid ratio (1:3), PVPP content (2 %), extraction time (4 h), followed by 30 %–50 % ammonium sulfate, diethylaminoethyl ion-exchange chromatography (0.1 M NaCl), and size exclusion chromatography. The PPO was identified as a dimeric protein with molecular weight of 135 kDa, containing 58.79 % random coil, 20.78 % α-helix, 17.41 % β-folding, and 3.02 % β-corner. The three-dimensional structure via homology modeling suggested that active center CuA bound to His151, His172, and His181, CuB bound to His307, His311, and His341. Furthermore, molecular docking indicated that its Phe337 and Tyr312 residues were catalytic cavity gates of catechol and 4-methylcatechol, respectively. Therefore, this study successfully analyzed purified PPO structure and further provided a theoretical foundation for its browning mechanism.

An improved high-resolution method for quantitative separation of empty and filled AAV8 capsids by strong anion exchange HPLC

Cell and gene therapy (CGT) is a field of therapeutic medicine that aims to treat, prevent, and cure diseases using engineered cells (stem cells, immune cells, and differentiated adult or fetal cells), vectors [Adeno Associated Virus (AAV), Adeno Virus (AV), Herpes Simplex Virus (HSV), Baculo Virus (BV), Lenti Virus (LV), Retro Virus (RV), etc.], and other carriers [non-viral vectors, virus-like particles (VLP), Lipid Nano-Particles (LNP), etc.]. Among viral CGT vectors, adeno-associated viruses and lentiviruses (AAV and LV) are the most widely applied vector platforms. The presence of non-functional (empty or non-infectious) vectors that carry null or partial genes in the final drug product is classified as an impurity by the FDA. These impurities impair dosage accuracy and induce non-specific immunogenicity and variability in drug efficacy. These non-functional viral vectors in the drug product need to be elucidated following International Conference on Harmonization (ICH) guidelines for clinical manufacturing of the final drug product. This article showcases an ion-exchange chromatography (IEX) high-resolution method supporting ICH guidelines using commercially available AAV8 filled and empty capsids as reference standards. Our method successfully separated empty to full capsids with a resolution of 15 and sustained a linearity greater than 0.98 even under a wide range of empty or full viral particle concentrations (E+9 to E+13 vp/mL), which is an upgrade to other IEX capsid separation methods. The medium-throughput capacity and shorter sample processing time improve testing efficiency and save costs while delivering quality as value. The discussed method is a reliable and reproducible platform to precisely evaluate the presence of non-functional viral particles in AAV8 samples. Aligned with other orthogonal results, the method is a powerful tool to improve the quality of rAAV analytics.

A readily accessible quaternized cellulose filter paper with high permeability for IgG separation

Anion-exchange chromatography (AEC) is recognized as a highly effective approach for the purification of immunoglobulin G (IgG). This study introduces an innovative strategy that employs waste cellulose filter paper in the production of AEC media. A quaternized cellulose fiber membrane (CFM-QCS) was successfully fabricated that cellulose fibers were as a structural framework, glutaraldehyde (GA) as a crosslinking agent, and quaternary chitosan (QCS) as a modifying agent. Morphological and chemical characterization revealed that GA and QCS were uniformly crosslinked on the surface of the cellulose fibers, resulting in excellent mechanical properties in both dry and wet states. Benefiting from its 3D network scaffold structure, CFM-QCS demonstrated a high adsorption capacity for bovine serum albumin (BSA), with static and dynamic adsorption capacities of 605.15 mg/g and 88.63 mg/ml, respectively. After treated with extreme conditions and 10 cyclic adsorption and elution, the adsorption capacity of CFM-QCS remains almost unchanged, highlighting its excellent stability. Additionally, a CFM-QCS packed chromatography column exhibited high flux of 10.38 L/h at 0.1 MPa, which can efficiently separate IgG from a mixed solution in the presence of BSA and IgG by gravity-driven. This work presents a straightforward approach for preparing high-performance ion-exchange chromatography membranes for IgG separation.

Multifunctional polymeric stationary phases with enhanced hydrophilicity grafted with polyethyleneimine and polyglycidol

Two methods for increasing the degree of hydrophilization and screening of the sorbent matrix based on a copolymer of styrene and divinylbenzene grafted with polyethyleneimine quaternized with glycidol are proposed. The first method involves the polymerization of glycidol in the functional layer by varying the pH of the reaction medium, and the second method involves modifying the matrix by oxidizing the double bonds on its surface to obtain anchor epoxy groups. It was demonstrated that in the first method, the optimal approach is the twofold addition of glycidol before and after the addition of alkali, as in this case, the first addition of glycidol is consumed for the quaternization of polyamine, and the second for polymerization in ion-exchange centers. The new method of matrix modification, combined with the developed method of creating hydrophilic layers, significantly reduced the retention of oxyhalides, haloacetic acids, and polarizable anions in ion chromatography with suppressed background conductivity and amino acids in hydrophilic chromatography up to the point of changing the elution order. The obtained stationary phases are suitable for the simultaneous determination of standard inorganic anions, oxyhalides, and anions of haloacetic or alkylphosphonic acids in ion chromatography, as well as for the separation of amino acids, sugars, and vitamins in hydrophilic chromatography.

A novel method for the degradation of human blood clot by immobilised bromelain using multi-walled carbon nanotube and polyphenol oxidase

Pathological blood clot in blood vessels, which often leads to cardiovascular diseases, are one of the most common causes of death in humans. Therefore, enzymatic therapy to degrade blood clots is vital. To achieve this goal, bromelain was immobilized and used for the biodegradation of blood clots. Bromelain was extracted from the pineapple fruit pulp (Ananas comosus) and purified by ion exchange chromatography after precipitation with ammonium sulphate (0-80 %), resulting in a yield of 70%, purification fold of 1.42, and a specific activity of 1175 U/mg. Bromelain was covalently immobilized on functionalized multi-walled carbon nanotubes (MWCNT), with an enzyme loading of 71.35%. The results of the characterization of free and immobilized bromelain demonstrated that the optimum pH for free and immobilized bromelain activity was 7.0, while the pH range of stability was from 5.0 to 8.5 and 4.0 to 9.0, respectively. The optimum temperature for free and immobilized bromelain activity was 45ºC, whereas the stability was 15 to 50°C and 15 to 55°C, respectively. The immobilized bromelain activity was decreased after the fifth reuse, and the storage period of the free and immobilized bromelain was decreased after 6 and 123 days, respectively. Casein was the best substrate-free bromelain, and fibrin was the best substrate for immobilized bromelain. The results of the purification of polyphenol oxidases (PPO) from potatoes by ion exchange chromatography gave a yield of about 54 %, a purification fold of 1.27, and a specific activity of 2804 U/mg. The current study showed that the immobilized bromelain can significantly biodegrade human blood clots in vitro, while the PPO enzyme has no significant effect on blood clots.

Incorporating an artificially synthesized fluoride complex into urethane-acrylate-based 3D printing resin: Effects on mechanical properties, cytotoxicity, antimicrobial actions, and its long-term fluoride-releasing properties

ObjectivesTo synthesize a 3D printing resin with antibacterial and long-term fluoride-releasing properties. Methods(4,4-Bis-4-[2‑hydroxy-3-(2-methacryloyloxy)propoxy]-phenyl-pentanol-amine)-N,N-diacetic acid zirconium (IV) fluoride complex was synthesized from 4,4-bis-(4-hydroxyphenyl)-pentanoic acid and monitored using proton nuclear magnetic resonance spectroscopy. The synthesized complex was incorporated into a urethane-acrylate-based (UA) resin at 5 wt% and 10 wt% (5F-UA and 10F-UA groups, respectively). The UA resin without the synthesized complex was considered as the control group. All groups were 3D printed using a DLP printer, followed by 10 min of washing and 20 min of curing. Surface characteristics were observed using scanning electron microscopy. The mechanical properties were assessed by measuring its flexural strength and Vickers hardness. The antibacterial property was investigated with direct and indirect contact tests and a WST-8 metabolic activity assay. The suspension was fully mixed and diluted for counting the number of colony-forming units. The cell viability test was performed using a cell proliferation assay. The amount of fluoride released was measured daily for 28 days using ion chromatography. One-way analysis of variance was performed for statistical analyses using SPSS software. ResultsThe amount of fluoride released increased with the concentration of fluoride complex in the resin. The fluoride ions were constantly released at a low concentration from the 3D printed specimens (5F-UA: around 0.13 ppm daily; 10F-UA: around 0.22 ppm daily). The antibacterial efficacy was acceptable in both the 5F-UA and 10F-UA groups, and higher in the latter. No cytotoxicity of the resin was detected. The mechanical properties were significantly influenced by the addition of the fluoride-releasing complex. ConclusionsThe present 3D printing UA resin incorporating a fluoride complex effectively inhibited the growth of S. mutans and demonstrated the ability to slowly release fluoride over an extended period of time. Clinical significanceThis study provided informative composition of a fluoride-releasing UA-based 3D printing resin, ideal for dental applications such as crowns, bridges, removable partial dentures, and orthodontic appliances, which can benefit from sustained fluoride release and antimicrobial properties. Further modifications to the resin composition can be easily achieved to enhance the resin qualities.

Production, Characterization, Kinetics, and Thermodynamics Analysis of Amyloglucosidase from Fungal Consortium.

The current study aimed to produce an amyloglucosidase enzyme from the fungal consortium. The best amylolytic fungal consortia were identified as Alternaria alternata and Aspergillus niger through the 18S rDNA technique. Fermentation kinetics and various nutritional and cultural parameters were analyzed. Maximum production was obtained in M4 media, pH 5.5, 30°C, and 4mL inoculum at 150rpm after 72h of incubation. Along with that, sodium nitrate at 2.5%, maltose, beef extract 1%, zinc sulfate (0.1%), and Tween 80 (0.1%) supported the maximum amyloglucosidase production. Amyloglucosidase was partially purified up to 1.6 purification fold with a specific activity of 1.84 Umg-1 in a stepwise manner by ammonium sulfate purification, dialysis, and ion exchange chromatography. The AMG enzyme also revealed maximum activity at 50°C with 5.0 pH. Upon the kinetic analysis, the specific yield coefficient Yp/x and volumetric rates Qp and Qx were also found to be significant in the above optimized conditions. The Km value 0.33mgmL-1 and Vmax 26.31 U mL-1 were obtained at 1% soluble starch substrate. Thermodynamic parameters for soluble starch hydrolysis were as follows: ΔH = 48.78kJmol-1, (Ea) = - 46.0kJmol-1, and ΔS = - 43.10Jmol-1K-1. This finding indicates the indigenously isolated fungal consortium can be the best candidate for industrial applications.

An unconventional strategy for purifying recombinant SARS-CoV-2 spike protein

The soluble domain of the trimeric SARS-CoV-2 spike protein is a promising candidate for a COVID-19 vaccine. Purification of this protein from mammalian cell culture supernatant using conventional resin-based chromatography is challenging as its large size (∼550 kDa) restricts its access and mobility within the pores of the resin particles. This reduces binding capacity and process robustness very significantly as extremely low flow rates need to be used during purification. Convection-based ion-exchange membrane chromatography has been found to be suitable in this respect. However, the high ionic strength of mammalian cell culture supernatant makes it difficult to bind this protein on charged membranes without dilution with a suitable buffer. An unconventional strategy involving size-exclusion chromatography as the first step, followed by cation exchange membrane chromatography as the second step is proposed in this paper. In the size exclusion chromatography step, the spike protein is excluded from the pores and can therefore be isolated in the void volume fraction. This step removes small molecule impurities and also serves as a desalting and buffer exchange step, making the partially purified material suitable for the cation exchange membrane chromatography step. The proposed process is variant-independent, fast and scalable and addresses some of the challenges associated with the currently used purification methods.

Ex Ovo Chorioallantoic Membrane Assay as a Model of Bone Formation by Biomaterials.

Biomaterials play an increasingly critical role in bone tissue engineering. However, achieving effective clinical translation requires a careful choice of biomimetic materials and thorough assessment of their efficacy and safety. Existing in vitro and in vivo models have drawbacks including time and cost constraints, invasive procedures, and discordance between animal models and clinical outcomes. Therefore, there is a demand for an alternative model. We hypothesized that the chick embryo chorioallantoic membrane can serve as a bioreactor to evaluate the initial sign of bone formation on scaffolds. In parallel, we investigated the osteogenic potential of a previously fabricated fibrin-alginate-calcium phosphate biomaterial (FACaP). Blood vessels were observed to infiltrate the scaffolds with early signs of bone formation, confirmed via RUNX-2 and alpha smooth muscle actin markers. The scaffolds' chemical composition was evaluated by Fourier-transform infrared spectroscopy, and ion chromatography was used to assess calcium ion release. Finally, the topography was examined by atomic force microscopy. In conclusion, this system offers simple refinement for in vivo models in bone tissue engineering and highlights the great potential of FACaP as an angiogenic and osteogenic biomaterial for non-load-bearing applications.

Chemical composition, mutagenicity, and cytotoxicity of urban submicron particulate matter (PM1) in Agra, India

This study, conducted in Agra, India, examined the mass concentrations, chemical compositions, and seasonal variations of submicron particles (PM1). The concentrations of metals, water-soluble inorganic ions (WSIs) including anions (F−, Cl−, NO₃−, SO₄2−) and cations (Ca2+, K+, Mg2+, NH₄+, Na+), organic carbon (OC) and elemental carbon (EC) and polycyclic aromatic hydrocarbons (PAHs) in PM1 extract were determined using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES), Ion Chromatography, Thermogravimetric Analysis and Gas Chromatography-Mass Spectrometry (GC–MS) respectively. For morphological observation of PM1 particles, Field Emission Scanning Electron Microscopy-Energy Dispersive X-ray spectrometry (FESEM-EDS) was used. The annual average concentration of PM1 was 82.9 ± 33.4 μg/m3, which exceeds the World Health Organisation's (WHO) safe limit for PM2.5 of 5 μg/m3 by a factor of 17. The PM1 mass composition included metals (31 %), WSIs (28 %), OC and EC (9.8 %), and PAHs (0.4 %). Winter recorded the highest PM1 concentration (96.1 ± 25.8 μg/m3), followed by post-monsoon, summer, and monsoon seasons. The average concentration of PAHs was 364.6 ± 226.6 ng/m3. Positive Matrix Factorization (PMF) identified traffic, emissions from biomass/coal and wood combustion, industrial/stationary sources, and secondary aerosols as potential contributors. The Ames test revealed the presence of frameshift mutations and base pair substitutions, especially in winter and post-monsoon. Additionally, PM1 exhibited cytotoxic effects on V-79 cells, with heightened toxicity during winter and prolonged exposure in other seasons. This study underscores the urgent need to address local emission sources and establish regulatory standards for PM1 in urban areas.

Peptide ligands for the affinity purification of adenovirus from HEK293 and vero cell lysates

Adenovirus (AdVs) is the viral vector of choice in vaccines and oncolytic applications owing to its high transduction activity and inherent immunogenicity. For decades, AdV isolation has relied on ultracentrifugation and ion-exchange chromatography, which are not suitable to large-scale production and struggle to deliver sufficient purity. Immunoaffinity chromatography resins of recent introduction feature high binding capacity and selectivity, but mandate harsh elution conditions (pH 3.0), afford low yield (< 20%), and provide limited reusability. Seeking a more efficient and affordable alternative, this study introduces the first peptide affinity ligands for AdV purification. The peptides were identified via combinatorial selection and in silico design to target hexons, the most abundant proteins in the adenoviral capsid. Selected peptide ligands AEFFIWNA and TNDGPDYSSPLTGSG were conjugated on chromatographic resins and utilized to purify AdV serotype 5 from HEK293 and Vero cell lysates. The peptide-functionalized resins feature high binding capacity (> 1010 active virions per mL at the residence time of 2 min), provide high yield (> 50%) and up to 100-fold reduction of host cell proteins and DNA. Notably, the peptide ligands enable gentle elution conditions (pH 8) that prevent the “shedding” of penton and fiber proteins, thus affording intact adenovirus particles with high cell-transduction activity. The study of the peptide ligands by surface plasmon resonance and molecular docking and dynamics simulations confirmed the selective targeting of hexon proteins and elucidated the molecular-level mechanisms underlying binding and release. Collectively, these results demonstrate the strong promise of peptide ligands presented herein for the affinity purification of AdVs from cell lysates.

Fluorine and boron co doped porous carbon materials as an electrochemical platform in trace-level sodium nitrite sensing

Sodium nitrite (NaNO2) is a common chemical oilfield tracer in the field of reservoir geological analysis. A simple, sensitive, and accurate electrochemical detection method for detection of NaNO2 has been studied. The electrode carbon porous material was based on coal tar pitch n-pentane insoluble extractants (CTPNI) and doped with the elements of fluorine (F) and boron (B) and labeled as FB-CTPNI-PC and an electrochemical sensor modified with FB-CTPNI-PC material was constructed for rapid detection of NaNO2. The characterization results of SEM and TEM indicate that this material has certain number of pore structures, also the BET analysis showed that has main pore size distribution is around the range of 2–20 nm, with abundant mesoporous structure. Moreover, the modified electrode has a good electrocatalytic effect on NaNO2. The electrochemical reaction mechanism of NaNO2 on the surface of the modified electrode was a diffusion-controlled process involving protons. The detection range of NaNO2 can reach 0.1 mM–9.5 mM. When the S/N is 3, the sensitivity is 219.8592 A·L·cm2·mol−1 and the detection limit (LOD) is 0.011 μM. The constructed electrochemical sensor has been applied to the rapid detection of NaNO2 in real oilfield water, with recovery rates ranging from 99.7 % to 103.4 %. It is also roughly consistent with the results of ion chromatography (IC) in detecting NaNO2 in oilfield water, indicating that the sensor has high accuracy. This has provided a new prospect for oil filed tracer and detection technologies.

Cold atmospheric plasma activated media selectively affects human head and neck cancer cell lines.

Cold atmospheric plasma (CAP) is a novel approach for cancer treatment. It can be used to treat liquids-plasma-activated media (PAM)-which are then transferred to the target as an exogenous source of reactive oxygen and nitrogen species (RONS). The present study aimed at chemically characterizing different PAM and assessing their invitro selectivity against head and neck cancer cells (HNC). PAM were obtained by exposing 2 and 5 mL of cell culture medium to CAP for 5, 10 and 20 min at a 6 mm working distance. Anions kinetics was evaluated by ion chromatography. Cell proliferation inhibition, apoptosis occurrence, and cell cycle modifications were assessed by MTS and flow cytometry, on human epidermal keratinocyte (HaCaT) and HNC cell lines HSC3, HSC4 and A253. The 2 mL conditions showed a significant reduction in cell proliferation whereas for the 5 mL the effect was milder, but the time-dependence was more evident. HaCaT were unaffected by the 5 mL PAM, indicating a selectivity for cancer cells. The media chemical composition modified by CAP exposure influenced cell proliferation by modulating cell cycle and inducing apoptosis in cancer cells, without affecting normal cells.

Analytical Spectral Semi-quantitative Dating Method of Printed Text Toner Using Raman Spectroscopy and the Fluorescence signal of Paper Sheet Cellulose

Abstract: The chemical processes that occur during the surface degradation of document paper under the influence of external factors were studied by Raman spectroscopy. The possibility of comparing the same processes occurring on clean free paper and under printed text toner letters is of interest. It is assumed that the transformation of cellulose can occur at different rates in the areas of paper exposed to direct atmospheric and light exposure and in the areas containing an intermediate layer of the toner. The most stable temporal changes occur in the region of C–C–C and C– C–O vibrations of glycosidic rings in the short-wavelength region of the Raman spectrum. Background: The main areas of research in the field of document dating, mainly focused on chromatographic methods. These methods are based on studying the dynamics of evaporation of volatile ink components. There is also a variety of chromatography techniques, including gas chromatography, ion chromatography, high performance liquid chromatography, thin layer chromatography and gas chromatography-mass spectrometry. This indicates the wide range of approaches and techniques used to determine the age of written materials. However, all these methods are used mainly during the first two years after writing the document details, which does not allow examining documents that are more than two years old. Methods: Raman spectroscopy was used as the main research method. The object of the study was sheets of white A4 paper with printed text printed on them, produced on a laser printer. The subject of the study was areas of paper free of text, as well as areas of paper under toner. The integrated fluorescence of a paper optical brightener based on stilbene was studied in the wavelength range of 400–500 nm. Also, all objects were studied using optical microscopy. Results: The comparison of peak intensity values averaged over 10 brands over the years made it possible to reveal the following regularities: – the ratio of peaks in the paper spectra from which the toner was removed is greater than in the blank paper spectra, and the difference becomes less noticeable as the age of the paper decreases. The ratio of the intensity of the 330 cm-1 peak to the intensity of the 1603 cm-1 peak: the older the document becomes, the lower the average ratios of these peaks in both the blank paper spectra and in the spectra of paper from which the toner has been removed. –the intensity of the peak in the region of 280 cm-1 and 1 086 cm-1 of the Raman shift increases with decreasing the age of the toner-free paper.– a gradual increase in the intensity of the 380 cm-1 peak is observed when comparing the paper samples from which the toner was removed for 2016, 2018, 2020, and 2022. Regularity was also found in the change in the ratio of the average statistical values of the peak at 280 cm-1 in the spectra of the paper under the toner to a similar peak in the spectra of the paper free from text: the value of this ratio decreases with decreasing the paper age. At the same time, the paper fluorescence signal is an informative parameter that makes it possible to establish some facts from the document’s history. With artificial document aging, a significant broadening of the dispersion region and the appearance of its “splitting” into two or three strata are observed in the distribution of the fluorescence signal level of a paper sheet relative to the average value. The average fluorescence signal of a naturally aged sheet decreases with the age of the document. Conclusion: As a result of research, it has been established that Raman spectroscopy can be successfully used as a method for dating documents. When comparing the degree of degradation of the surface of open areas of paper and areas protected by toner of printed text, different effects of external factors on these areas were established, leading to different rates of degradation processes. The use of Raman peak intensity ratios for paper components in exposed areas compared to peaks under printed text further expands the methodology. The emphasis on spectral methods, in particular fluorescence signal distribution and Raman spectroscopy, to detect surface changes is well justified. It demonstrates a holistic approach to document analysis, taking into account both the chemical changes observed using Raman spectroscopy and the fluorescence signal distribution that may indicate signs of artificial aging through light exposure. This combination of methods appears to be reliable for assessing the age of documents and can make significant contributions to the field of forensic document examination.

Determination of tetramethylammonium hydroxide in air using ion chromatography or ultra-performance liquid chromatography - high-resolution mass spectrometry

Tetramethylammonium hydroxide (TMAH) is extensively utilized in the semiconductor and optoelectronic industries, serving as either a developer or an etchant. TMAH possesses acute toxicity and alkaline corrosiveness, having caused several fatal and injurious accidents in Taiwan. However, there is currently a lack of methods available for assessing TMAH exposure. This study developed a method involving sampling with quartz fiber filters followed by ultrasonic agitation in 10 mL mobile phase, filtering, and analysis using either ion chromatography (IC) or ultra-performance liquid chromatography - high-resolution mass spectrometry (UPLC-HRMS). Utilizing the characteristics of HRMS, the compound analyzed was identified as tetramethylammonium ion (TMA+) through mass spectrometry analysis. The method exhibits excellent trapping capacity and recovery rate. The linear range of TMA+ for IC is 0.3 to 100 μg/mL, with a correlation coefficient of R = 0.9998. The linear range of TMA+ for UPLC-HRMS is 0.002 to 1 μg/mL, with a correlation coefficient of R = 0.996. The recovery rates for IC and HRMS were 116.6% and 90.6%, with corresponding coefficient of variation (CV) of 2.25% and 3.53%, respectively. Samples can be stored at room temperature for 28 days. Verification of this method was conducted at the TMAH factory, and the air concentrations measured in the TMAH filling area were 3.29 and 4.75 μg/m3 from IC and UPLC-HRMS, respectively. This method will be applied in the on-site analysis of occupational hazards for operators in chemical plants, technology factories, and recycling plants, aiming to evaluate risk values to protect the safety and health of workers and prevent occupational diseases.

Biosynthesis of Piceatannol from Resveratrol in Grapevine Can Be Mediated by Cresolase-Dependent Ortho-Hydroxylation Activity of Polyphenol Oxidase.

Piceatannol is a naturally occurring hydroxylated analogue of the stilbene phytoalexin resveratrol that can be found in grape fruit and derived products. Piceatannol has aroused great interest as it has been shown to surpass some human health-beneficial properties of resveratrol including antioxidant activity, several pharmacological activities and also bioavailability. The plant biosynthetic pathway of piceatannol is still poorly understood, which is a bottleneck for the development of both plant defence and bioproduction strategies. Cell cultures of Vitis vinifera cv. Gamay, when elicited with dimethyl-β-cyclodextrin (MBCD) and methyl jasmonate (MeJA), lead to large increases in the accumulation of resveratrol, and after 120 h of elicitation, piceatannol is also detected due to the regiospecific hydroxylation of resveratrol. Therefore, an ortho-hydroxylase must participate in the biosynthesis of piceatannol. Herein, three possible types of resveratrol hydroxylation enzymatic reactions have been tested, specifically, a reaction catalyzed by an NADPH-dependent cytochrome, P450 hydroxylase, a 2-oxoglutarate-dependent dioxygenase and ortho-hydroxylation, similar to polyphenol oxidase (PPO) cresolase activity. Compared with P450 hydoxylase and the dioxygenase activities, PPO displayed the highest specific activity detected either in the crude extract, the particulate or the soluble fraction obtained from cell cultures elicited with MBCD and MeJA for 120 h. The overall yield of PPO activity present in the crude extract (107.42 EU) was distributed mostly in the soluble fraction (66.15 EU) rather than in the particulate fraction (3.71 EU). Thus, partial purification of the soluble fraction by precipitation with ammonium sulphate, dialysis and ion exchange chromatography was carried out. The soluble fraction precipitated with 80% ammonium sulphate and the chromatographic fractions also showed high levels of PPO activity, and the presence of the PPO protein was confirmed by Western blot and LC-MS/MS. In addition, a kinetic characterization of the cresolase activity of partially purified PPO was carried out for the resveratrol substrate, including Vmax and Km parameters. The Km value was 118.35 ± 49.84 µM, and the Vmax value was 2.18 ± 0.46 µmol min-1 mg-1.

An evolutionarily conserved function of C-reactive protein is to prevent the formation of amyloid fibrils.

C-reactive protein (CRP) binds to phosphocholine (PCh)-containing substances and subsequently activates the complement system to eliminate the ligand. The PCh-binding function of CRP has been conserved throughout evolution from arthropods to humans. Human CRP, in its structurally altered conformation at acidic pH, also binds to amyloid-β (Aβ) and prevents the formation of Aβ fibrils. It is unknown whether the Aβ-binding function of CRP has also been evolutionarily conserved. The aim of this study was to determine whether CRP isolated from American horseshoe crab Limulus polyphemus was also anti-amyloidogenic and whether this function required structural alteration of Limulus CRP (Li-CRP). Two CRP species Li-CRP-I and Li-CRP-II were purified from hemolymph by employing PCh-affinity chromatography and phosphoethanolamine-affinity chromatography, respectively. Both Li-CRP-I and Li-CRP-II bound to immobilized Aβ at physiological pH. Unlike human CRP, Li-CRP did not require any changes in its overall structure to bind to Aβ. Both Li-CRP-I and Li-CRP-II bound to Aβ in the fluid phase also and prevented the fibrillation of Aβ. Additionally, ion-exchange chromatography of purified Li-CRP indicated that a variety of Li-CRP molecules of different subunit compositions were present in Limulus hemolymph, raising the possibility that the presence of various Li-CRP species in hemolymph facilitates the recognition of a range of proteins with differing amyloidogenicity. We conclude that the binding of CRP to Aβ is an ancient function of CRP. In invertebrates, the Aβ-binding function of CRP can protect the host from toxicity caused by amyloidogenic and pathogenic proteins. In humans, the Aβ-binding function of CRP can protect against inflammatory diseases in which the host proteins are ectopically deposited on either host cells or foreign cells in an inflammatory milieu since immobilized proteins may expose Aβ-like structures after deposition at places where they are not supposed to be.