Ion-exchange Chromatography Research Articles

Testing for the ability to modify antibiotics of Panus tigrinus 8/18 Lentinus strigosus 1566 laccase.

In advanced biotechnology, the utilization of enzymes to achieve new or modified compounds with antibacterial, fungicidal, and anti-cancer specifications is crucial. Mushroom lactases are a hopeful biocatalyst for the synthesis and modification of different compounds. They are an accessible and inexpensive enzyme for the preparation of reaction objects and have recently received attention. Laccase purification was performed from basidiomycete Lentinus strigosus (LS) in several stages: Stage 1. On ion-exchange chromatography on TEAE Servacell 23 (400 ml), two distinctly separated laccase activity peaks were observed, eluted from the carrier at 0.21 and 0.27 M NaCl. In order to reduce the loss of enzymes, all fractions with laccase activity were collected, concentrated, and desalted using an ultrafiltration cell (Amicon, United States) with a UM-10 membrane. Stage 2. The resulting preparation with laccase activity was applied to a Q-Sepharose column (60 ml). Two well-separated peaks with laccase activity were obtained during the elution: laccase I (0.12 M NaCl) and laccase II (0.2 M NaCl). Stage 3. In the course of further purification of both enzymes, carried out on anion-exchange carrier Resource Q (6 ml), a broken gradient was used: 0 - 10%, 10 - 20%, and 20 - 100% with 1M NaCl. Stage 4. Both laccase I and laccase II, obtained after Resource Q, were desalted, concentrated to 1 ml each, and applied to a Superdex 75 gel filtration column. As a result, two laccases were obtained in a homogeneous form.

Phenylboronic acid modification-based novel dumbbell-shaped Au-Ag nanorod SERS substrates for ultrasensitive detection of SO42.

Based on the coordination principle of Lewis acids, a 4-mercaptophenylboronic acid (4-MPBA)-modified novel dumbbell-shaped Au-Ag nanorod (4-MPBA@DS Au-AgNR) substrate was developed, which could be combined with the surface-enhanced Raman scattering (SERS) technique to detect SO42- with high sensitivity and specificity. DS Au-AgNRs synthesized in this study with a dumbbell-shaped structure were verified by finite-difference time domain (FDTD) simulation to be capable of stimulating strong localized electromagnetic enhancement (EM) at nano-edge and gap, generating a large number of "hot spots" exhibiting excellent SERS performance. The 4-MPBA modified on its surface could specifically recognize SO42-, producing a change in the spectral peak at 1382 cm-1, thus realizing highly sensitive and specific sensing of SO42-. Under optimized conditions, this SERS sensor responded rapidly to SO42- within 2 minutes and demonstrated outstanding specificity. Calculation of the ratio of the characteristic peaks at 1382 and 1070 cm-1 (I1382/I1070) enabled the quantitative detection of SO42- in the range of 1 × 10-8-1 × 10-3 M, and the detection threshold was as low as 1 nM, which was superior to those of similar detection methods. Importantly, the utility and reliability of this SERS substrate for the determination of SO42- in actual samples were evaluated using ion chromatography as the gold standard, and there was no significant difference between the two protocols (P > 0.05), and the RSD was less than 6% with a satisfactory recovery rate (97.6-102.3%). Therefore, the present protocol has the advantages of simplicity and rapidity, high sensitivity, specificity, stability, and practicability in the determination of SO42- in aqueous solution, providing a reliable solution for tracing SO42- in the fields of food safety and environmental testing.

A compact and high-performance setup of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D).

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) has the advantages of high throughput (simultaneous detection of multiple ions), high separation efficiency (higher than 105 theoretical plates) and rapid analysis capability (less than 5 min for common inorganic ions). A compact CE-C4D system is ideal for water quality control and on-site analysis. It is suitable not only for common cations (e.g. Na+, K+, Li+, NH4+, Ca2+, etc.) and anions (e.g. Cl-, SO42-, BrO3-, etc.) but also for some ions (e.g. lanthanide ions, Pb2+, Cd2+, etc.) that require complex derivatization procedures to be detected by ion chromatography (IC). However, an obvious limitation of the CE-C4D method is that its sensitivity (e.g. 0.3-1 μM for common inorganic ions) is often insufficient for trace analysis (e.g. 1 ppb or 20 nM level for common inorganic ions) without preconcentration. For this technology to become a powerful and routine analytical technique, the system should be made compact while maintaining trace analysis sensitivity. In this study, we developed an all-in-one version of the CE-C4D instrument with custom-made modular components to make it a convenient, compact and high-performance system. The system was designed using direct digital synthesis (DDS) technology to generate programmable sinusoidal waveforms with any frequency for excitation, a kilovolt high-voltage power supply for capillary electrophoresis separation, and an "effective" differential C4D cell with a low-noise circuitry for high-sensitivity detection. We characterized the system with different concentrations of Cs+, and even a low concentration of 20 nM was detectable without preconcentration. Moreover, the optimized CE-C4D setup was applied to analyse mixed ions at a trace concentration of 200 nM with excellent signal-to-noise ratios. In typical applications, the limits of detection based on the 3σ criterion (without baseline filtering) were 9, 10, 24, 5, and 12 nM for K+, Cs+, Li+, Ca2+, and Mg2+, respectively, and about 7, 6, 6 and 6 nM for Br-, ClO4-, BrO3- and SO42-, respectively. Finally, the setup was also applied for the analysis of all 14 lanthanide ions and rare-earth minerals, and it showed an improvement in sensitivity by more than 25 times.

Evaluation of time-dependent ion-release and antibacterial activity of three adhesive resin cements

Aim This in-vitro study aimed to evaluate time-dependent ion-release and antibacterial activity of three self-adhesive resin cements. Materials and methods Sixty cement discs of the three tested self-adhesive resin cements were fabricated according to the manufacturer’s instructions in a plastic transparent mold, their dimensions were 6 mm in diameter and 2 mm in height. They were divided into three main experimental groups according to the self-adhesive resin cement type: group A (Thera-Cem), group B (G-Cem), and group C (RelyX Unicem 2) (20 each). Then, each main group was further subdivided into subgroup 1 (n = 10) for antibacterial assessment against Streptococcus mutans and subgroup 2 (n = 10) for ion-release assessment of fluoride and calcium. Direct contact test was used to monitor the bacterial outgrowth. Fluoride and calcium ions release were measured by ion chromatography and atomic absorption spectrometer respectively. All results were analyzed by one-way analysis of variance then multiple comparison Tukey’s test. Results All the evaluated cements exhibited antibacterial effects against S. mutans; fluoride and calcium were released by all the three cements in varying concentrations after aging. Conclusion Based on the results of this in-vitro study and within its limitations, it can be concluded that self-adhesive resin cements seem to provide promising antibacterial and remineralizing effects as being bioactive materials.

A method for the quantitative analysis of polar anionic pesticides in milk/infant formula, cereals and fruit and vegetables using ion chromatography coupled to tandem mass spectrometry.

Polar pesticides such as anionic or ionisable compounds have always provided a challenge for analytical chemists. Methods of analysis have been developed using a range of techniques including normal phase chromatography, ion-pairing, derivatisation and HILIC or multi-mode chromatography. These work well with some of these compounds but, except for HILIC, all of them have their limitations and none of them cover the range required by legislation. Some of these compounds, glyphosate, chlorate and phosphonic acid, are found regularly in a range of food matrices, and therefore reliable methods of analysis are essential. This study describes an ion chromatography method with tandem mass spectrometry detection which not only covers the full range of compounds required by legislation but also can be expanded to include other anionic or ionisable pesticides and metabolites. These include glyphosate and its metabolites, glufosinate and its metabolites, ethephon and its metabolites as well as fosetyl aluminium, chlorate and perchlorate. The method is fully validated according to the performance criteria from the SANTE guidelines for the analysis of pesticides in food and feed over a wide range of matrices, including milk, infant formula, cereals and fruits and vegetables. Over 300 food samples have analysed as part of our routine monitoring program.

Microwell fluoride assay screening for enzymatic defluorination.

There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating enzymes are readily monitored by fluoride determination. Fluorine is the most electronegative element. Consequently, all mechanisms of enzymatic C-F bond cleavage produce fluoride anion, F-. Therefore, methods for the determination of fluoride are critical for C-F enzymology and apply to any fluorinated organic compounds, including PFAS, or per- and polyfluorinated alkyl substances. The biodegradation of most PFAS chemicals is rare or unknown. Accordingly, identifying new enzymes, or re-engineering the known defluorinases, will require rapid and sensitive methods for measuring fluoride in aqueous media. Most studies currently use ion chromatography or fluoride specific electrodes which are relatively sensitive but low throughput. The methods here describe refashioning a drinking water test to efficiently determine fluoride in enzyme and cell culture reaction mixtures. The method is based on lanthanum alizarin complexone binding of fluoride. Reworking the method to a microtiter well plate format allows detection of as little as 4nmol of fluoride in 200μL of assay buffer. The method is amenable to color imaging, spectrophotometric plate reading and automated liquid handling to expedite assays with thousands of enzymes and/or substrates for discovering and improving enzymatic defluorination.

A new pepper allergen Zan b 1.01 of 2S albumins: Identification, cloning, characterization, and cross-reactivity.

Zanthoxylum bungeanum (Sichuan pepper; in Chinese) is used as a spice worldwide and is a potentially life-threatening allergenic food source, as first reported by our team in 2005. However, its allergen components are unknown. We aim to identify and characterize its major allergen and determine its cross-reactivities with citrus seeds, pistachios, and cashew seeds. Ionic exchange and molecular exclusion chromatography were used to isolate the protein components from Sichuan pepper seed. A protein fraction was characterized by SDS-PAGE, analytical ultracentrifugation, mass spectrometry, and circular dichroism spectroscopy. The coding region of it was amplified from the genome. ELISA and competitive ELISA assays were used to investigate the allergenicity and cross-reactivity of allergens. This protein allergen was around 14 kDa. It was a 2S albumin similar to an α-Amylase inhibitor (AAI) domain-containing protein of Citrus sinensis. Circular dichroism spectroscopy showed its thermal stability was high. A 303 bps DNA sequence of the AAI domain was cloned from the genome of the Sichuan pepper. Competitive ELISA assays showed positive cross-reactivities between this allergen and citrus seeds, pistachios, and cashew seeds. A major allergen of around 14 kDa from Sichuan pepper seed was confirmed, which belongs to the 2S albumin of plant seed storage proteins. Based on the nomenclature of the IUIS Subcommittee for Allergen Nomenclature, this allergen is designated as Zan b 1.01. The cross-reactivities were demonstrated between Zan b 1.01 and citrus seeds, pistachios, and cashew seeds.

Expression, purification, characterization and crystallization of Panax quinquefolius ginsenoside glycosyltransferase Pq3-O-UGT2

Pq3-O-UGT2, derived from Panax quinquefolius, functions as a ginsenoside glucosyltransferase, utilizing UDP-glucose (UDPG) as the sugar donor to catalyze the glycosylation of Rh2 and F2. An essential step in comprehending its catalytic mechanism involves structural analysis. In preparation for structural analysis, we expressed Pq3-O-UGT2 in the Escherichia coli (E. coli) strain Rosetta (DE3). The recombinant Pq3-O-UGT2 was purified through Ni-NTA affinity purification, a two-step ion exchange chromatography, and subsequently size-exclusion chromatography (SEC). Notably, the purified Pq3-O-UGT2 showed substantial activity toward Rh2 and F2, catalyzing the formation of Rg3 and Rd, respectively. This activity was discernible within a pH range of 4.0–9.0 and temperature range of 30–55 °C, with optimal conditions observed at pH 7.0–8.0 and 37 °C. The catalytic efficiency of Pq3-O-UGT2 toward Rh2 and F2 was 31.43 s−1 mΜ−1 and 169.31 s−1 mΜ−1, respectively. We further crystalized Pq3-O-UGT2 in both its apo form and co-crystalized forms with UDPG, Rh2 and F2, respectively. High-quality crystals were obtained and X-ray diffraction data was collected for all co-crystalized samples. Analysis of the diffraction data revealed that the crystal of Pq3-O-UGT2 co-crystalized with UDP-Glc belonged to space group P1, while the other two crystals belonged to space group P212121. Together, this study has laid a robust foundation for subsequent structural analysis of Pq3-O-UGT2.

Multifunctional-Separation-Mode Ion Chromatography Method for Determining Major Metabolites during Multiple Parallel Fermentation of Rice Wine

Multifunctional-Separation-Mode Ion Chromatography Method for Determining Major Metabolites during Multiple Parallel Fermentation of Rice Wine

Identification and quantification of fluorinated polymers in consumer products by combustion ion chromatography and pyrolysis-gas chromatography-mass spectrometry.

Total fluorine was determined in 45 consumer product samples from the Swedish market which were either suspected or known to contain fluorinated polymers. Product categories included cookware (70-550 000 ppm F), textiles (10-1600 ppm F), electronics (20-2100 ppm F), and personal care products (10-630 000 ppm F). To confirm that the fluorine was organic in nature, and deduce structure, a qualitative pyrolysis-gas chromatography-mass spectrometry (pyr-GC/MS) method was validated using a suite of reference materials. When applied to samples with unknown PFAS content, the method was successful at identifying polytetrafluoroethylene (PTFE) in cookware, dental products, and electronics at concentrations as low as 0.1-0.2 wt%. It was also possible to distinguish between 3 different side-chain fluorinated polymers in textiles. Several products appeared to contain high levels of inorganic fluorine. This is one of the few studies to quantify fluorine in a wide range of consumer plastics and provides important data on the concentration of fluorine in materials which may be intended for recycling, along with insights into the application of pyr-GC/MS for structural elucidation of fluorinated polymers in consumer products.

A wearable AuNP enhanced metal-organic gel (Au@MOG) sensor for sweat glucose detection with ultrahigh sensitivity.

The demand for sensitive and non-invasive sensors for monitoring glucose levels in sweat has grown considerably in recent years. This study presents the development of a wearable sensor for sweat glucose detection with ultrahigh sensitivity. The sensor was fabricated by embedding Au nanoparticles (AuNPs) and metal-organic gels (MOGs) on nickel foam (NF). A non-enzymatic electrocatalytic glucose sensor has been developed to combine the three-dimensional network of MOGs with more active sites favourable for glucose diffusion and the transfer of electrons from glucose to the electrode. These results show that the sensor has an ultrahigh sensitivity of 13.94 mA mM-1 cm-2, a linear detection range between 2 and 600 μM, and a lower detection limit as low as 1 μM (signal/noise = 3) with comparable accuracy and reliability under non-alkaline conditions to those of high-pressure ion chromatography (HPIC). Furthermore, a wearable sweat glucose sensor has been constructed by sputtering an Au conductive layer on a flexible polydimethylsiloxane (PDMS) substrate and coating it with Au@MOGs. Our work demonstrates that the combination of Au NPs and MOGs can enhance the sensitivity and activity of these materials, making them useful for electrocatalytic glucose monitoring with ultrahigh sensitivity.

PARTIAL PURIFICATION AND CHARACTERIZATION OF PROTEASE ENZYMES FROM NATIVE Bacillus Cereus STRAINS

Proteases are the main enzymes responsible for the breakdown of protein molecules into amino acids. These enzymes can easily be obtained from plants, animals and microorganisms. However, the easiest, fastest and cheapest source is microorganisms. Bacillus cereus strains B16, B17, B18, B19 and B20 were selected for use in this study. The optimum conditions and other parameters of the strains in the production process of proteases were investigated. The strains were characterized using biochemical methods. Different culture conditions were analyzed for the production efficiency of protease activity. Based on the proteolytic activity analysis, Bacillus cereus B17 strain was selected for detailed studies. Protease purification was done by dialysis, ion exchange chromatography and gel filtration chromatography. It was determined that the growth and the protease activity of Bacillus cereus B17 was greatly affected by parameters such as pH and temperature of the medium, presence of metal ions and inhibitors, and incubation time.

Partial Purification of 5'-Nucleotidase Enzyme in Patients with Renal Failure and Kinetic Study of the Purified Enzyme in Salah al-Din

This study involved separating and partially purifying the 5'-Nucleotidase enzyme isoforms from the sera of patients with renal failure. The purification process consisted of several steps, beginning with precipitation using 30% ammonium sulfate, resulting in a purification fold of 1.2625 and an enzyme yield of 65.63%. This was followed by membrane filtration using a buffer solution, achieving a purification fold of 1.651 and an enzyme yield of 49%. The protein fraction separated from the dialysis bag was then passed through a gel filtration column, reaching a purification fold of 51.88 and an enzyme yield of 43.1%. The enzyme isoforms from the separated protein fraction were further separated using gel filtration, employing ion-exchange chromatography techniques. This resulted in three isoforms of the 5'-Nucleotidase enzyme with varying degrees of purity, the highest for isoform. at 2.297-fold. The study also included kinetic studies of the enzyme purified by gel filtration, which involved determining the Km value under the influence of varying concentrations of the enzyme's substrate, 5'-AMP, for the purified enzyme from the sera of patients with renal failure, which was found to be 12.4. The effects of temperature, pH, and reaction time on enzyme activity were also studied.

Production and purification of Clostridium perfringens type C beta-toxin and IgG and IgY antitoxins

ObjectivesThis study aimed to produce and purify Clostridium perfringens type C beta-toxin, sheep anti-beta toxin immunoglobulin G (IgG) and chicken immunoglobulin Y (IgY). MethodsTwo methods were used for beta-toxin purification: single-step metal affinity chromatography (MAC) using zinc as a chelator and ion exchange chromatography (IEX). The purified and inactivated beta-toxoids were then administered to sheep and chickens in order to produce IgG and IgY. ResultsAll assays using the IEX failed. In contrast, MAC purified more than 21 mg of toxin per run in a single-step protocol. The purified and inactivated beta-toxoids were then administered to sheep and chickens, and IgG and IgY were purified with a high yield, medium antibody titer of 50 IU/mL, and high avidity (73.2 %). ConclusionsC. perfringens type C beta-toxin and sheep or chicken anti-beta toxin IgG and IgY antibodies were successfully produced and purified using a simple protocol. This protocol can be used for the production of components used in the diagnosis and research of necrotic enteritis caused by C. perfringens type C, as well as for the evaluation of existing vaccines and the development of new preventive methods against this disease.

New l-Rhamnose-Binding Lectin from the Bivalve Glycymeris yessoensis: Purification, Partial Structural Characterization and Antibacterial Activity.

In this study, a new l-rhamnose-binding lectin (GYL-R) from the hemolymph of bivalve Glycymeris yessoensis was purified using affinity and ion-exchange chromatography and functionally characterized. Lectin antimicrobial activity was examined in different ways. The lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as l-rhamnose, d-galactose, lactose, l-arabinose and raffinose. Using the glycan microarray approach, natural carbohydrate ligands were established for GYL-R as l-Rha and glycans containing the α-Gal residue in the terminal position. The GYL-R molecular mass determined by MALDI-TOF mass spectrometry was 30,415 Da. The hemagglutination activity of the lectin was not affected by metal ions. The lectin was stable up to 75 °C and between pH 4.0 and 12.0. The amino acid sequence of the five GYL-R segments was obtained with nano-ESI MS/MS and contained both YGR and DPC-peptide motifs which are conserved in most of the l-rhamnose-binding lectin carbohydrate recognition domains. Circular dichroism confirmed that GYL is a α/β-protein with a predominance of the random coil. Furthermore, GYL-R was able to bind and suppress the growth of the Gram-negative bacteria E. coli by recognizing lipopolysaccharides. Together, these results suggest that GYL-R is a new member of the RBL family which participates in the self-defense mechanism against bacteria and pathogens with a distinct carbohydrate-binding specificity.

Comparing the wound healing potential of natural rubber latex serum and F1-protein: An in vivo approach

Chronic wounds pose significant health concerns. Current treatment options include natural compounds like natural rubber latex (NRL) from Hevea brasiliensis. NRL, particularly the F1 protein fraction, has demonstrated bioactivity, biocompatibility, and angiogenic effects. So far, there is no study comparing F1 protein with total NRL serum, and the necessity of downstream processing remains unknown. Here, we evaluated the angiogenic potential of F1 protein compared to total NRL serum and the need for downstream processing. For that, ion exchange chromatography (DEAE-Sepharose), antioxidant activity, physicochemical characterization, cell culture in McCoy fibroblasts, and wound healing in Balb-C mice were performed. Also, the evaluation of histology and collagen content and the levels of inflammatory mediators were quantified. McCoy fibroblast cell assay showed that F1 protein (0.01 %) and total NRL serum (0.01 %) significantly increased cell proliferation by 47.1 ± 11.3 % and 25.5 ± 2.5 %, respectively. However, the AA of F1 protein (78.9 ± 0.8 %) did not show a significant difference compared to NRL serum (77.0 ± 1.1 %). F1 protein and NRL serum were more effective in wound management in rodents. Histopathological analysis confirmed accelerated healing and advanced tissue repair. Similarly, the F1 protein (0.01 %) increased collagen, showing that this fraction can stimulate the synthesis of collagen by fibroblastic cells. Regarding cytokines production (IL-10, TNF-α, IFN-γ), F1 protein and NRL serum did not exert an impact on the synthesis of these cytokines. Furthermore, we did not observe statistically significant changes in dosages of enzymes (MPO and EPO) among the groups. Nevertheless, Nitric Oxide dosage was reduced drastically when the F1 protein (0.01 %) protein was applied topically. These findings contribute to the understanding of F1 protein and NRL serum properties and provide insights into cost-effectiveness and practical applications in medicine and biotechnology. Therefore, further research is needed to assess the economic feasibility of downstream processing for NRL-based herbal medicine derived from Hevea brasiliensis.

Importance of lighting color and period for CO2 fixation and C-phycocyanin production during Thermosynechococcus sp. CL-1 growth

Lighting arrangement is a critical factor in cultivation of photosynthetic microorganisms such as a cyanobacterium, Thermosynechococcus sp. CL-1 (TCL-1). This study observed the impact of various light intensities, light colors, and lighting periods on TCL-1 CO2 fixation rate and C-phycocyanin production. TCL-1 growth and CO2 fixation rate were not affected by the light color during continuous illumination. The best CO2 fixation rate of 2.29 ± 0.54 and 2.26 ± 0.14 g-CO2/L/d were achieved under white and red lighting, respectively. However, application of 12/12 h light-dark illumination diminished TCL-1 performance especially under red light and it might be caused by the reduction of accumulated chlorophyll-a. Furthermore, extended studies were also conducted for the efficient C-phycocyanin extraction and purification strategies, along with accumulated cyanotoxins analysis. Purification using ion-exchange chromatography elevated C-phycocyanin purity index to 3.34. Besides, treatment using distilled water and freeze dryer could remove the accumulated cylindrospermopsin and saxitoxin from TCL-1.

Metabolites, ions, and the mechanisms behind seasonal cold hardening of Pyrochroa coccinea (Pyrochroidae) larvae

The larvae of the black headed cardinal beetle Pyrochroa coccinea, overwinters under the bark of dead logs in northern European dioecious forests, and are thus exposed to temperatures below the melting point of their bodily fluids. Here we explore the mechanisms behind their seasonal cold hardening by characterising field samples collected monthly throughout the year. Both the lower lethal temperature and supercooling point dropped as much as 10℃ in the second half of November, reaching values around –15℃ by the beginning of December. This change was accompanied by a 320 mosmol/kg increase in hemolymph osmolality, which is a doubling compared to the summer levels. We used NMR metabolomics to identify and measure the absolute concentrations of the responsible cryoprotective C-H containing metabolites in the hemolymph. The largest increase was found to be in either glucose or trehalose, with an average total increase of 120 mM. Proline, alanine, and choline concentrations were found to increase by around 10 mM each. Contrarily, phosphocholine and phosphoethanolamine were halved, resulting in a total decrease of around 50 mM. These measurements were complemented with ion exchange chromatography measurements. This allowed us to account for all the osmotic pressure in the summer hemolymph, and the measured concentration changes explained as much as 40 % of the observed osmolality increase upon cold hardening. Preliminary results indicate that the remainder may be explained by non-colligative protein contributions.

Chelation chemistry of manganese-52 for PET imaging applications

IntroductionDue to its decay and chemical properties, interest in manganese-52 has increased for development of long-lived PET radiopharmaceuticals. Its long half-life of 5.6 days, low average positron energy (242 keV), and sufficient positron decay branching ratio make it suitable for radiolabeling macromolecules for investigating slow biological processes. This work aims to establish suitable chelators for manganese-52 that can be radiolabeled at mild conditions through the evaluation of commercially available chelators. MethodsManganese-52 was produced through the nuclear reaction NatCr(p,n)52Mn by irradiation of natural chromium targets on a TR24 cyclotron followed by purification through ion exchange chromatography. The radiolabeling efficiencies of chelators: DOTA, DiAmsar, TETA, DO3A, NOTA, 4′-Formylbenzo-15-crown-5, Oxo-DO3A, and DFO, were assessed by investigating the impact of pH, buffer type, and temperature. In vitro stability of [52Mn]Mn(DO3A)−, [52Mn]Mn(Oxo-DO3A)−, and [52Mn]Mn(DOTA)2− were evaluated in mouse serum. The radiocomplexes were also evaluated in vivo in mice. Crystals of [Mn(Oxo-DO3A)]− were synthesized by reacting Oxo-DO3A with MnCl2 and characterized by single crystal X-ray diffraction. ResultsYields of 185 ± 19 MBq (5.0 ± 0.5 mCi) (n = 4) of manganese-52 were produced at the end of a 4 h, 15 μA, bombardment with 12.5 MeV protons. NOTA, DO3A, DOTA, and Oxo-DO3A chelators were readily radiolabeled with >96 % radiochemical purity at all conditions. Manganese radiocomplexes of Oxo-DO3A, DOTA, and DO3A remained stable in vitro up to 5 days and exhibited different biodistribution profiles compared to [52Mn]MnCl2. The solid-state structure of Mn-Oxo-DO3A complex was determined by single-crystal X-ray diffraction. ConclusionsDO3A and Oxo-DO3A are suitable chelators for manganese-52 which are readily radiolabeled at mild conditions with high molar activity, and demonstrate both in vitro and in vivo stability.

Complexity of the effects of pre-fermentation oxygenation, skin contact and use of pectolytic enzymes in white winemaking as revealed by comprehensive proteomics and volatilomics analysis

Ion exchange chromatography and SDS-PAGE followed by identification by MALDI-TOF/TOF, and two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-TOF-MS) were used for comprehensive proteomics and volatilomics evaluation of the effects of pre-fermentative oxygenation, skin contact and use of pectolytic enzymes in production of Malvazija istarska white wine, respectively. Many protein species and an unprecedented number of volatiles have been identified and (semi)quantified, revealing high complexity of the observed effects. Compared to a standard control wine, oxygenation treatment modulated the protein composition and resulted with a volatilome characterized by decreased levels of several important volatiles. Skin contact treatments, especially in combination with pectolytic enzymes, significantly increased the levels of a large number of proteins, but were also deprived of particular protein species found in other wines. Wines obtained by skin contact with exogenous enzymes exhibited the most complex volatile composition with increased levels of many key monoterpenoids, alcohols and esters.