Substitution of histidine 30 by asparagine in manganese superoxide dismutase alters biophysical properties and supports proliferation in a K562 leukemia cell line.

We have generated a mutant of C. elegans manganese superoxide dismutase at histidine 30 by site-directed mutagenesis. The structure was solved at a resolution of 1.52 Å by X-ray crystallography (pdb: 6S0D). His30 was targeted, as it forms as a gateway residue at the top of the solvent access funnel to the active site, together with Tyr34. In the wild-type protein, these gateway residues are involved in the hydrogen-bonding network providing the protons necessary for the catalytic reaction at the metal center. However, biophysical characterization and cell viability experiments reveal that a mutation from histidine to asparagine in the H30N mutant modifies metal selectivity in the protein, favoring the uptake of iron over manganese in minimal media conditions, alters active-site coordination from the characteristic trigonal bipyramidal to octahedral geometry, and encourages cellular proliferation in K562 cells, when added exogenously to the cells.

In Situ Determination of Illegal Drugs in Oral Fluid by Portable Capillary Electrophoresis with Deep UV Excited Fluorescence Detection.

The present study demonstrates the potential of a portable capillary electrophoresis (CE) instrument, coupled to deep UV fluorescence detector (FD) with a 230-255 nm excitation wavelength range, for the determination of the abuse of illegal drugs in oral fluids in situ. CE was introduced in this study due its exceptional power of separation and resolution, short analysis time, and ability for miniaturization for on-site assessment of different substances. The deep UV fluorescence detector was equipped with five interchangeable emission filters, in the emission wavelength range from 278 to 600 nm, and was successfully employed for determination of natively fluorescing illegal drugs, such as cocaine, cocaethylene, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxeamphetamine (MDA), 3,4-methylenedioxy- N-ethylamphetamine (MDEA), para-methoxyamphetamine (PMA), para-methoxy- N-methylamphetamine (PMMA), amphetamine (AMP), methamphetamine (METH), tetrahydrocannabinol (THC) and cannabidiol (CBD). The developed FD showed impressive sensitivity. The instrumental detection limit was 0.5 µg/L for MDMA. It also showed broad linearity, up to 50 mg/L for MDMA. The noise CV% was 1.1% for an empty capillary and 0.6% for a capillary filled with acetonitrile. The portable CE-FD with developed electrophoretic methodologies was successfully utilized for the determination of illegal abuse of drugs during "Weekend" 2016 and 2017 Music Festivals (Estonia). Moreover, CE-FD can be employed for detection of other natively fluorescing compounds in the proposed range (e.g., for different phenolic compounds, BTEX, naphthalene derivatives, and others), significantly widening the applicability of developed CE-FD instrument.

Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

Determination of γ-hydroxybutyric acid in saliva by capillary electrophoresis coupled with contactless conductivity and indirect UV absorbance detectors.

The aim of the current study was to optimise and validate the methodology for determination of γ-hydroxybutyric acid (GHB) in saliva by CE combined with a contactless conductivity detector (C(4)D) and indirect UV absorbance detection (λ(ABS) = 210 nm). The optimized BGE, consisting of 8.5 mM maleic acid, 17 mM arginine, 255 µM cetyltrimethylammonium bromide (CTAB), and 15% acetonitrile, was evaluated for the separation of GHB in saliva within 6 min. The performance characteristics of the CE-C(4)D-indirect UV methodology was validated. The instrument detection and quantification limits were 0.49 and 1.6 mg/L for C(4)D, and 5.1 mg/L and 17.0 mg/L for indirect UV, respectively. The linearity was obtained over the range from 2.5 to 400 mg/L for C(4)D and from 12.5 to 400 mg/L for indirect UV. The interday precisions were within 2.3-5.7% and intraday precisions were within 1.6-9.0% for C(4)D as well as 2.1-9.3%, 5.6-10.1% for indirect UV in spiked saliva, respectively. The recoveries were within 87.2-104.4%. The matrix effects were +53.2% for small concentrations up to 25 mg/L for C(4)D and +23.6% for concentrations up to 75 for mg/L for indirect UV detection. No matrix effects were observed for higher concentration levels. In conclusion, CE-C4D-indirect UV can offer a rapid, accurate, sensitive, and definitive method for the determination of GHB abuse in saliva samples as a forensic screening tool.

Determination of metal content in superoxide dismutase enzymes by capillary electrophoresis†.

Superoxide dismutases are antioxidant scavenger enzymes that contain a metal cofactor (copper, zinc, iron, and manganese) in their active site. Metal content measurement is one of the essential steps to characterize enzyme biological activity. We have developed a capillary electrophoretic protocol for the determination of the metal content in superoxide dismutase enzymes. The background electrolyte containing 10 mM pyridine-2,6-dicarboxylic acid and 1 mM 1-methyl-3-tetradecylimidazolium chloride at pH 3.8 was optimized for on-column complexation of the above-mentioned metals. The minimum detectable levels of metals ranged from 0.3 to 1.2 µg/mL. The reliability of the method was checked by parallel quantitative determination of the metal content in superoxide dismutase enzymes by graphite furnace or flame atomic absorption spectrophotometry methods.

Chloromethylation of Lignin as a Route to Functional Material with Catalytic Properties in Cross-Coupling and Click Reactions.

We present a novel, greener chloromethylation procedure for organosolv aspen lignin under mild reaction conditions without Lewis acid as a catalyst and in acetic acid as a solvent. This synthetic protocol provides a reliable approach to chloromethylated lignin (CML) and means to obtain valuable lignin derivatives. The resulted CML was subsequently transformed into 1-methylimidazolium lignin (ImL), which effectively serves as a stabilizing agent for Pd/CuO nanoparticles (Pd/CuO-NPs). To evaluate the versatility of developed lignin-based catalyst, we investigate its performance in a series of carbon-carbon bond formation reactions, including Suzuki-Miyaura, Sonogashira, Heck reactions, and azide-alkyne cycloaddition (click) reaction. Remarkably, this catalyst exhibited a high degree of catalytic efficiency, resulting in reactions with yields ranging from average to excellent. The heterogeneous catalyst demonstrated outstanding recyclability, enabling its reuse for at least 10 consecutive reaction cycles, with yields consistently falling within the range of 42 % to 84 %. A continuous flow reactor cartridge prototype employing Lignin@Pd/CuO-NPs was developed, yielding results comparable to those achieved in batch reactions. The utilization of Lignin@Pd/CuO-NPs as a catalyst showcases its potential to facilitate diverse carbon-carbon bond formation reactions and underscores its promising recyclability, aligning with the green chemistry metrics and principles of sustainability in chemical processes.

Chloromethylation of Lignin as a Route to Functional Material with Catalytic Properties in Cross-Coupling and Click Reactions.

Invited for this issue's cover are researchers from Tallinn University of Technology (TalTech). The image depicts the lignin chemical evolution route from raw biomass through a greener chloromethylation procedure developed by the research team. It showcases the transformation into lignin-supported metal nanoparticles, serving as a catalyst for various chemical reactions in both batch and continuous flow conditions. The Research Article itself is available at 10.1002/cssc.202301588.

Can selenium levels act as a marker of colorectal cancer risk?

BACKGROUND: Selenium has attracted attention because of its antioxidant properties. Antioxidants protects cells from damage. Certain breakdown products of selenium are believed to prevent tumor growth by enhancing the immune cell activity and suppressing the development of tumor blood vessels. In this observational study, selenium level was measured in a series of patients from Poland and Estonia to determine a correlation between levels of this microelement and colorectal cancer risk. METHODS: A total of 169 colorectal cancer patients and 169 healthy controls were enrolled in the study after obtaining their informed consent. Selenium level in the blood serum was measured using Graphite Furnace Atomic Absorption Spectrometry (GFAAS). The statistical analysis was performed by Fisher's exact test. RESULTS: The threshold point of selenium level was 55 µg/l and 65 µg/l for Poland and Estonia respectively, for an increase in cancer risk. The lower levels of selenium were associated with greater risk of colorectal cancer. CONCLUSIONS: The result reveals a significant strong association between low selenium level and the colorectal cancer risk in both Estonian and Polish populations.

Capillary electrophoretic study of the synergistic biological effects of alkaloids from Chelidonium majus L. in normal and cancer cells.

In this study, the synergistic biological action of five celandine alkaloids in normal and cancer cells was investigated by capillary electrophoresis with light-emitting diode-induced native fluorescence detection. The specific capacity of each alkaloid to penetrate into the cells was estimated by monitoring alkaloid concentration decreases in the cell medium during incubation with murine fibroblast NIH/3T3, mouse melanoma B16F10, and human breast cancer MCF7 cell lines. Mixtures of isoquinoline alkaloids containing protopine, chelidonine, sanguinarine, allocryptopine, and stylopine were applied to cell cultures for 20 and 40 min, and the content of alkaloids in the cell media was measured by capillary electrophoresis (CE). CE separation of isoquinoline alkaloids was performed in 30 mM phosphate buffer (pH 2.5). As these alkaloids have native fluorescence, they were directly detected using the commercially available UV light-emitting diode without troublesome fluorescent derivatization. The results showed a differential ability of celandine alkaloids to penetrate into the normal and cancer cell interior, which was inversely proportional to their cytotoxic activity. While the most effective transport of celandine alkaloids from the cell medium to the cell interior was observed for normal murine fibroblast NIH/3T3 cells (about 55% of total content), cytotoxicity tests demonstrated selective and profound apoptotic effects of a five-alkaloid combination in the mouse melanoma B16F10 cell line.

Antifungal Activity of Medicinal Mushrooms and Optimization of Submerged Culture Conditions for Schizophyllum commune (Agaricomycetes).

The main goal of the present study was the exploration of the antifungal properties of Agaricomycetes mushrooms. Among twenty-three tested mushrooms against A. niger, B. cinerea, F. oxysporum, and G. bidwellii, Schizophyllum commune demonstrated highest inhibition rates and showed 35.7%, 6.5%, 50.4%, and 66.0% of growth inhibition, respectively. To reveal culture conditions enhancing the antifungal potential of Sch. commune, several carbon (lignocellulosic substrates among them) and nitrogen sources and their optimal concentrations were investigated. Presence of 6% mandarin juice production waste (MJPW) and 6% of peptone in nutrient medium promoted antifungal activity of selected mushroom. It was determined that, extracts obtained in the presence of MJPW effectively inhibited the grow of pathogenic fungi. Moreover, the content of phenolic compounds in the extracts obtained from Sch. commune grown on MJPW was several times higher (0.87 ± 0.05 GAE/g to 2.38 ± 0.08 GAE/g) than the extracts obtained from the mushroom grown on the synthetic (glycerol contained) nutrient medium (0.21 ± 0.03 GAE/g to 0.88 ± 0.05 GAE/g). Flavonoid contents in the extracts from Sch. commune varied from 0.58 ± 0.03 to 27.2 ± 0.8 mg QE/g. Identification of phenolic compounds composition in water and ethanol extracts were provided by mass spectrometry analysis. Extracts demonstrate considerable free radical scavenging activities and the IC50 values were generally low for the extracts, ranging from 1.9 mg/ml to 6.7 mg/ml. All the samples displayed a positive correlation between their concentration (0.05-15.0 mg/ml) and DPPH radical scavenging activity. This investigation revealed that Sch. commune mushroom has great potential to be used as a source of antifungal and antioxidant substances.

Extraction and Fractionation of Bioactives from Dipsacus fullonum L. Leaves and Evaluation of Their Anti-Borrelia Activity.

Lyme disease (LD) is a tick-borne bacterial disease that is caused by Borrelia burgdorferi. Although acute LD is treated with antibiotics, it can develop into relapsing chronic form caused by latent forms of B. burgdorferi. This leads to the search for phytochemicals against resistant LD. Therefore, this study aimed to evaluate the activity of Dipsacus fullonum L. leaves extract (DE) and its fractions against stationary phase B. burgdorferi in vitro. DE showed high activity against stationary phase B. burgdorferi (residual viability 19.8 ± 4.7%); however, it exhibited a noticeable cytotoxicity on NIH cells (viability 20.2 ± 5.2%). The iridoid-glycoside fraction showed a remarkable anti-Borrelia effect and reduced cytotoxicity. The iridoid-glycoside fraction was, therefore, further purified and showed to contain two main bioactives-sylvestrosides III and IV, that showed a considerable anti-Borrelia activity being the least toxic to murine fibroblast NIH/3T3 cells. Moreover, the concentration of sylvestrosides was about 15% of DE, endorsing the feasibility of purification of the compounds from D. fullonum L. leaves.

Characterization of Organosolv Lignins and Their Application in the Preparation of Aerogels.

The production of novel materials and value-added chemicals from lignin has received considerable attention in recent years. Due to its abundant occurrence in nature, there is a growing interest in utilizing lignin as a feedstock for functional materials production, for example aerogels. Much like in the synthesis of phenol-based resins, the vacant ortho positions of the aromatic rings in lignin can crosslink with formaldehyde and form polymeric gels. After drying the hydrogels with supercritical CO2, highly porous aerogels are obtained. Current study focuses on the preparation and thorough parametrization of organosolv lignins from different types of lignocellulosic biomass (aspen, pine, and barley straw) as well as their utilization for the preparation of lignin-5-methylresorcinol-formaldehyde aerogels. The thorough structural characterization of the obtained aerogels was carried out by gas adsorption, IR spectroscopy, and scanning electron microscopy. The obtained lignin-based monolithic mesoporous aerogels had specific surface areas and total pore volumes in the upward ranges of 450 m2/g and 1.4 cm3/g, respectively.

Bioremediation of lindane contaminated soil: Exploring the potential of actinobacterial strains.

Lindane, an organochlorine pesticide, causes detrimental impacts on the environment and human health owing to its high toxicity, low degradation, and bioaccumulation. Its toxic nature can be overcome by biological and eco-friendly approaches involving its degradation and detoxification. The biodegradation of lindane was assessed using actinobacterial species Thermobifida cellulosilytica TB100 (T. cellulosilytica), Thermobifida halotolerans DSM 44931 (T. halotolerans) and Streptomyces coelicolor A3 (S. coelicolor). The degradation conditions of Lindane such as pH, temperature, inoculum volume, glucose concentration and number of days were optimized under broth conditions. Lindane degradation at different concentrations was studied in soil using reverse phase-high performance liquid chromatography over a 30 day period. A bioassay test was performed on seeds of Lactuca sativa (Lettuce) to assess the success of bioremediated soil. Maximum lindane degradation in soil was observed using T. cellulosilytica sp. The degradation trend for different concentrations of lindane using T. halotolerans in sterilized soil was 55 mg kg-1 (82%) ˃ 155 mg kg-1 (75%) ˃ 255 mg kg-1 (70%) after an incubation period of 30 days. Lindane degradation in soil followed the first order reaction kinetics. Phytotoxicity test on seeds of Lactuca sativa showed considerably good vigor index values for the bioremediated sterilized and non-sterilized soil by T. cellulosilytica, T. halotolerans and S. coelicolor in comparison to the contaminated soil without bacteria. This confirms that these actinobacterial species can be implemented in bioaugmentation of contaminated sites to efficiently remediate high lindane concentrations.

Analysis of Total Phenols, Sugars, and Mineral Elements in Colored Tubers of Solanum tuberosum L.

The use of colored tubers of Solanum tuberosum L. is growing worldwide due to their health benefits and attractive color. The positive health effects of purple-fleshed tubers are a result of anthocyanins and various phenolic compounds. The aim of this study was to evaluate and compare variety Blue Congo and its cross-breeds of Desiree and Granola to yellow-fleshed tubers. The concentration of total phenols, anthocyanins, sugars, and mineral elements were evaluated in all tubers. The results showed differences between all tested materials, with largest differences in sugar content. Moreover, the results confirmed the preservation of health improving compounds of Blue Congo when cross-bred with yellow-fleshed tubers. The total phenolic content and anthocyanin concentrations of all analyzed tubers were above the comparison yellow ones.

Use of a newly-developed portable capillary electrophoresis analyser to detect drugs of abuse in oral fluid: A case study.

The aim of the current study was to develop and validate an analytical method to determine whether drugs of abuse (DOA) were present in oral fluid (OF) using a newly-developed, portable capillary electrophoresis (CE) instrument coupled to a deep ultra-violet fluorescence detector (FD). The performance of this portable CE-FD DOA analyser was tested at the Weekend Festival Baltic (Pärnu, Estonia) between 2016 and 2018 as well as on the roadside OF samples collected by the police. The study reported 128 analysed cases in which persons were allegedly found to have been under the influence of DOA. The samples were analysed for amphetamine (AMP), methamphetamine (METH), 3,4-methylenedioxy-methamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), cocaine (COC) and cocaethylene (COET). Subsequent toxicological reports revealed that 26% cases were negative, and 74% were positive. The most frequently detected and quantified DOA was MDMA (68 cases, 62%). A comparative study was conducted to validate the accuracy of using the CE-FD DOA analyser versus classic high-performance liquid chromatography coupled to mass spectrometry (HPLC-ESI-MS). Diagnostic statistics for CE-FD DOA were also evaluated and were higher than 99.5%. In addition, all zeta-scores were lower than 2 when both methods were compared, showing that the CE-FD analyser can be implemented as a reliable, sensitive and convenient tool for roadside and workplace testing for DOA.

CE separation of various analytes of biological origin using polyether ether ketone capillaries and contactless conductivity detection.

The development of efficient and sensitive analytical methods for the separation, identification and quantification of complex biological samples is continuously a topic of high interest in biological science. In the present study, the possibility of using a polyether ether ketone (PEEK) capillary for the CE separation of peptides, proteins and other biological samples was examined. The performance of the tubing was compared with that of traditional silica capillaries. The CE analysis was performed using contactless conductivity detection (C(4)D), which eliminated any need for the detection window and was suitable for the detection of optically inactive compounds. In the PEEK capillary the cathodic EOF was low and of excellent stability even at extremes pH. In view of this fast biological anions were analyzed using an opposite end injection technique without compromising separation. A comparison of the performances of fused-silica and polymer capillaries during the separation of model sample mixtures demonstrated the efficiency and separation resolution of the latter to be higher and the reproducibility of the migration times and peak areas is better. Furthermore, PEEK capillaries allowed using simple experimental conditions without any complicated modification of the capillary surface or use of an intricate buffer composition. The PEEK capillaries are considered as an attractive alternative to the traditional fused-silica capillaries and may be used for the analysis of complex biological mixtures as well as for developing portable devices.

Rapid and sensitive capillary electrophoresis method for the analysis of Ecstasy in an oral fluid.

In the present study, a capillary electrophoresis method, with a native fluorescence detection for the quantification of three amphetamine derivatives, methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-methamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) in an oral fluid is described. The reported CE method has made it possible to assess Ecstasy abuse in approximately 15 min, including a saliva sample collection, pretreatment procedures and capillary electrophoresis (CE) analysis. The proof of the principle that was demonstrated on a home-made lab scale instrument has had the potential to be easily translated onto a truly portable instrument for on-site measurements. The baseline CE separation of the three illegal drugs was achieved in 10 min, by applying an aqueous background electrolyte (BGE) that was composed of 40 mM phosphoric acid and 10 mM triethanolamine. The amphetamine derivatives were detected at their λex/λem maximum (280/326 nm) with LOD values of about 3 ng/mL for each amphetamine. The recovery of the compounds from the collection pad was about 40% of the LOQ concentrations and the inter-day precision was less than 6% for all of the analytes. The procedure was applied to a quantitation of oral fluid (OF) samples that were collected during the Baltic Weekend Music Festival that was held in Estonia.

On-line monitoring of enzymatic conversion of adenosine triphosphate to adenosine diphosphate by micellar electrokinetic chromatography.

Capillary electrophoresis can be a valuable tool for the on-line monitoring of bioprocesses. The enzymatic conversion of nucleotide adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by hexokinase (HK) was monitored in the bioreactor interfaced by a laboratory-built microsampler to a capillary electrophoresis unit. The use of this specially designed sampling device enabled rapid consecutive injections to be performed without high-voltage (HV) interruptions. No additional sample preparation was required. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow (EOF) by cationic surfactant and reversed polarity mode provided a good resolution and short analysis time of less than 5 min. The samples were injected electrokinetically, using -25 kV voltage for 3 s and detected by their UV absorbance at 254 nm. The analytes were detected at a microg/ml level with a reproducibility of about 7%. To demonstrate the potential of CE in understanding the processes of biological interest, such as nucleotide degradation and metabolism, the investigation of the efficiency and the time course of the enzymatic transformation was carried out.

Capillary electrophoresis with LED-induced native fluorescence detection for determination of isoquinoline alkaloids and their cytotoxicity in extracts of Chelidonium majus L.

In this study, we introduced a simple and sensitive method of capillary electrophoresis with ultraviolet light-emitting diode-induced native fluorescence (UV-LEDIF) detection for the determination of isoquinoline alkaloids in extracts of Chelidonium majus L. Samples were extracted with acidic methanol and the extracts were directly analysed by CE. Simultaneous determination of protopine, chelidonine, coptisine, sanguinarine, allocryptopine, chelerythrine and stylopine was performed in 20mM phosphate buffer (pH 3.1). The baseline separation of these alkaloids was finished within 20 min. As these alkaloids have native fluorescence, they were directly detected using the commercially available UV light emitting diode without troublesome fluorescent derivatisation. Satisfactory LOD values were obtained for the studied compounds considering their appearance in natural extracts. Lower limits of detection were 0.05 µg/mL for protopine, 0.06 µg/mL for stylopine and allocryptopine, 0.07 µg/mL for chelidonine, 0.22 µg/mL for sanguinarine, 1.7 µg/mL for chelerythrine and 5.5 µg/mL for coptisine. The developed method was successfully applied to determine the contents of seven alkaloids in the aerial parts of Chelidonium majus L, which varied from 0.025 to 0.763% (w/w). Also, to demonstrate the potential of the proposed CE method, an estimation of the cytotoxic properties of selected Celandine alkaloids in a natural extract was carried out.

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography.

A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.