Gel Electrophoretic Method Research Articles

Thiosemicarbazone-based Dioxomolybdenum (VI) complexes as inorganic nucleases

This study focuses on the design of alkyl-substituted thiosemicarbazones from 5-bromosalicylaldehyde and substituted thiosemicarbazides and the corresponding dioxomolybdenum(VI) complexes, hitherto unreported. In addition to elemental analyses, the synthesized ligands and the corresponding complexes are further analyzed by physico-chemical and spectroscopic techniques. The DNA interaction studies are based on UV absorption titration and gel electrophoretic methods. The binding constants (Kb) for the ligands (L1-L4) are found to be 1.20 ± 0.1 × 104 M−1; 1.32 ± 0.2 × 104 M−1; 1.27 ± 0.3 × 104 M−1 and 1.25 ± 0.3 × 104 M−1 respectively and that of the complexes (C1–C4) are 1.45 ± 0.2 × 104 M−1; 2.10 ± 0.4 × 104 M−1; 1.54 ± 0.2 × 104 M−1 and 2.13 ± 0.2 × 104 M−1, respectively. The complexes have shown relatively higher binding propensity as compared to that of the ligands. Further, as the outcome of gel electrophoresis studies, the ligands show a cleavage pattern of nicked circular (Form I). However, the complexes show cleavage patterns of both nicked circular and supercoiled, Forms I & II respectively. This implies that both the ligands and complexes possess varying binding efficacy with DNA and accordingly the cleavage patterns also differ.

Comprehensive Study of Total Nitrogen Content and Microfluidic Profiles in Additive-Enriched Plant-Based Drinks.

The growing consumption of plant-based milk substitutes raises important questions about their composition. The various additives used by manufacturers, including those employed as flavor enhancers, protein additives, and stabilizers, may contain both protein and non-protein nitrogen components. In our study, we examined not only popular milk alternatives but also other milk substitutes made from specific plants. We present a reproducible and rapid method for the simultaneous qualitative and quantitative determination of the total nitrogen content in milk alternatives, focusing on applicability. Using the microchip gel electrophoretic method, we determined that the total nitrogen content differed from the protein content indicated on the packaging. Our results, along with statistical evaluations, supported the hypothesis that different brands of products, derived from the same plant source, resulted in different microfluidic profiles, likely due to the presence of additives. As expected, the microfluidic profiles of additive-free products differed from those of fortified products made from the same plant-based milk replacer. Total nitrogen content provides crucial information for individuals with kidney disease, as is essential to reduce the burden on the kidneys to slow deterioration, alleviate symptoms and avoid complications.

Repurposing the antihistamine drug bilastine as an anti-cancer metallic drug entity: synthesis and single-crystal X-ray structure analysis of metal-based bilastine and phen [Co(II), Cu(II) and Zn(II)] tailored anticancer chemotherapeutic agents against resistant cancer cells.

Bilastine (BLA), 2-(4-(2-(4-(1-(2-ethoxyethyl)-1H-benzo[d]imidazole-2-yl)-piperidin-1-yl)-ethyl)-phenyl)-2-methylpropanoic acid, is an active antihistamine drug. With the idea of repurposing drugs from the existing pool of 'active' pharmaceutical ingredients, the therapeutic potency of bilastine as an anticancer agent was investigated via the tailored synthesis of a metal-based anticancer drug formulation of the type [BLA(phen)2M(II)]+·X-, where M = Co, Cu, and Zn and X- = NO3 and ClO4. The synthesized metal-based chemotherapeutics derived from the bilastine drug that acts as a ligand were thoroughly characterized using spectroscopic techniques, namely, UV-vis, FT-IR, and EPR (in the case of 1 and 2); 1H-NMR and 13C-NMR (in the case of 3); ESI-MS and single-crystal X-ray diffraction studies. Comprehensive biological studies (DNA binding, cleavage, and cytotoxic activity) using various biophysical and gel electrophoretic methods were carried out to validate their potential as anticancer agents. The cytotoxic activity of 'therapeutically promising' copper(II)-based drug candidate 2 was evaluated against MCF-7, MBA-MD-231, HeLa, HepG2, and Mia-PaCa-2 cancer cells via an SRB assay, and the results demonstrated 2 as a potent anticancer agent at low nanomolar concentrations against all tested cancer cells, preferably with a much superior anticancer efficacy against human pancreatic cancer cells.

Characterization and molecular identification of bacteria from mackerel bekasam in Sorong City, Southwest Papua Province, Indonesia

Abstract. Sukmawati S, Ratna R, Sipriyadi, Yunita M. 2023. Characterization and molecular identification of bacteria from mackerel bekasam in Sorong City, Southwest Papua Province, Indonesia. Biodiversitas 24: 4967-4977. Bekasam is traditional food type produced by traditional fermented fish. Microbes that grow through fermentation play an important role in forming the product’s aroma, texture, and overall quality. The study aimed to determine the biochemical characteristic of bacteria from mackerel (Scomberomorus sp.) bekasam in Sorong City and identify bacteria at the molecular level. This research was a descriptive study, which described the results of the characterization of bacteria from fermented mackerel fish and the results of molecular identification to the species level through the PCR (Polymerase Chain Reaction) technique. Then, the DNA sequences were further analyzed using the agarose gel electrophoretic separation method to visualize the bacterial DNA profile. The biochemical characterization of bacterial isolates from mackerel showed that all isolates were negative indole, and eight isolates were positive in reducing nitrate. In comparison, four isolates were negative in reducing nitrate, then all isolates had proteolytic activity except the FST 3.1 and FST 3.2 isolates. Eleven isolates were positive in hydrolyzing fat, and one isolate could not hydrolyze fat. According to the DNA patterns seen in electrophoresis and alignment of the 16 sRNA gene sequences, several types of bacteria had been identified as Bacillus paramycoides strain 2883 FST 1.1, Bacillus paramycoides strain 3665 FST 2.1, Bacillus mobilis strain ICA-144 FST 3.1, Bacillus cereus strain ATCC 14579 FNT 1.1, Bacillus mobilis strain ICA-144 FNT 2.1, and Bacillus cereus strain ATCC 14579 FNT 3.1.

Microfluidic Analysis for the Determination of Protein Content in Different Types of Plant-Based Drinks.

The widespread consumption of plant-based drinks, driven by health and dietary reasons (including cow's milk allergy, lactose intolerance, milk protein intolerance, following a vegetarian or vegan diet) necessitates automated and accurate test methods. Our study demonstrates the simultaneous determination of protein components and total protein concentrations in plant-based milk alternatives using a rapid and reproducible microchip gel electrophoretic method. As expected, the electrophoretic profiles of each plant-based drink differed. Based on our analyses and statistical evaluation, it can be determined that the protein profiles of different plant-based beverages do not differ significantly between different manufacturers or different expiry dates. The measured total protein content was compared with the nominal values, i.e., the values stated on the beverage labels. As the number of consumers of functional and specialized plant-based milk alternatives continues to rise, it is important to prioritize methods that provide qualitative and quantitative information on protein composition and other nutrients.

Synthesis and structural investigation of o-Vanillin scaffold Schiff base metal complexes: Biomolecular interaction and molecular docking studies

Schiff base metal complexes such as VNPV (VO(IV)), VNPCo (Co(II)), VNPNi (Ni(II)), VNPCu (Cu(II)) and VNPZn (Zn(II)) have been synthesised by using the ligand (VNP) which was prepared by the condensation reaction of o-Vanillin and N-phenyl-o-phenylenediamine. Elemental analysis, UV–visible, FT-IR, ESI-MS, ESR, 1H NMR, XRD, SEM and Cyclic Voltammogram methods were used to evaluate the above metal complexes. The metal center in the complexes is four coordinated by using the tetradentate N2O2 donar Schiff base ligand to form square pyramidal geometry for VO(IV) metal complex and square planar geometry for other metal complexes. The disc well-diffusion method was used on agar plate to assess the in vitro antibacterial activities of the ligand and its metal complexes against bacteria such B. amyloliquefaciens, B. cereus, B. subtilis, S. epidermidis and S. aureus. From the results demonstrate that the metal complexes of the ligand exhibit better antibacterial behaviour on S. aureus bacteria than the other bacterial strains. The antioxidant studies were carried by DPPH free radical scavenging assay method and from the results it is found that the VNPCu complex has better antioxidant activity than the ligand and other metal complexes. Additionally, the spectral techniques like UV–visible absorption, fluorescence and molecular docking methods were used to examine the interactions between the ligand and metal complexes with biomolecules (DNA and BSA). The CT-DNA cleavage activities of metal complexes with CT DNA were examined by gelelectrophoretic method. From the results, the VNPCu complex has only cleave the DNA molecules than the other metal complexes. According to the results of the investigations, the metal complex VNPCu exhibits better interaction with the biomolecules than the free ligand and other metal complexes.

Comparative Quality Assessment of Five Bread Wheat and Five Barley Cultivars Grown in Romania.

Cereals whole grains contain vitamins, phytochemicals, antioxidants, resistant starch, and minerals with potential benefits to human health. The consumption of whole grains is correlated with a lowered risk of the most important chronic diseases, including type II diabetes, cardiovascular diseases, and some cancers. This study aimed to characterize and evaluate the content of five cultivars of wheat (Triticum aestivum L.) and five cultivars of barley (Hordeum vulgare L.) obtained by conventional plant breeding using crossing and selection methods. The novelty and the purpose of this research was to quantitatively and qualitatively analyze these ten cultivars from Romania and to show the importance of, and the changes produced by, crossing and selection methods when these are aimed at the physiological or morphological development of the cultivars. Studies based on gluten dosing; spectrophotometry using Bradford, fructan and protein dosing; Kjeldahl protein dosing; GC-MS/MS-protein and amino acid dosing; and identification of protein fractions using polyacrylamide gel electrophoretic method were conducted. This study demonstrates the possibility of developing future cultivars using conventional methods of improvement to modify the content and composition of nutrients to increase their health benefits.

Structure elucidation, DNA interaction, potential anticancer, molecular docking activities, and γ‐ray irradiation studies on novel mono‐ and binuclear Fe (II), Ni (II), Co (II), and Hg (II) Schiff base complexes

Bis Schiff baseN,N–((1E,2E)‐ethane 1,2diylidene)bis(azanylylidene)di(isonicotinohydrazide) ligand L was reacted with Fe (III), Ni (II), Co (II), and Hg (II) chlorides to yield complexes (1–6). The structure of the synthesized ligand and metal complexes was well established by various spectroscopic techniques (Fourier‐transform infrared [FT‐IR], UV–Vis., and1H NMR), mass spectrometry, elemental analysis (CHN) and physical studies (thermogravimetric analysis [TGA]/first derivative thermogravimetric analysis [DTG], powder X‐ray diffraction, molar conductance, and magnetic moment). Solid powder samples of the binuclear complexes (3and6) were irradiated at two different irradiation doses (150 and 200 kGy) to yield complexes (3Fand3F*) and (6Fand6F*), respectively. For the γ‐irradiated samples, FT‐IR, UV–Vis., and physical studies (TGA/DTG, X‐ray diffraction, molar conductance, and magnetic moment) were performed using the same methods as for the as‐prepared complexes. In addition, calf thymus ct‐DNA binding results monitored by agarose gel electrophoretic method indicated that the as‐prepared binuclear [Hg2LCl4(OH)2] complex (6) and its γ‐irradiated [Hg2LCl4(OH)2] complex (6F) have the ability to cleave ct‐DNA at a concentration of 30 and 70 μg/ml, respectively. Inhibitory activity against hepatocellular carcinoma cells (HepG‐2) and breast carcinoma cells (MCF‐7) of ligand and as‐prepared complexes (3,6, and6F) was evaluated. The results revealed that metal complexes exhibited enhanced activity in comparison to the free ligand, and the as‐prepared complex (6) shows an IC50value of 5.7 ± 0.2 and 8.72 ± 0.6 μg/ml for the two cell lines, respectively. Docking process for the ligand and complexes (3) and (6) with liver cancer protein (ID: 4FM9) and breast cancer protein (ID: 1H7K) has been evaluated.

Histopathology of Duchenne muscular dystrophy in correlation with changes in proteomic biomarkers.

Duchenne muscular dystrophy is an inherited disorder of early childhood that affects multiple systems in the body. Besides late-onset cardio-respiratory syndrome and various body-wide pathophysiological changes, X-linked muscular dystrophy is primarily classified as a disorder of the skeletal musculature. This is reflected by severe histopathological alterations in voluntary contractile tissues, including progressive fibre degeneration, fat substitution, reactive myofibrosis and chronic inflammation. The underlying cause for dystrophinopathy are genetic abnormalities in the DMD gene, which can result in the almost complete loss of the membrane cytoskeletal protein dystrophin, which triggers the collapse of the dystrophin-associated glycoprotein complex and disintegration of sarcolemmal integrity. This in turn results in an increased frequency of membrane micro-rupturing and abnormal calcium ion fluxes through the impaired plasmalemma, which renders muscle fibres more susceptible to enhanced proteolytic degradation and necrosis. This review focuses on the complexity of skeletal muscle changes in X-linked muscular dystrophy and outlines cell biological and histological alterations in correlation to proteome-wide variations as judged by mass spectrometric analyses. This includes a general outline of sample handling, subcellular fraction protocols and modern proteomic approaches using gel electrophoretic and liquid chromatographic methods for efficient protein separation prior to mass spectrometry. The proteomic profiling of the dystrophic and highly fibrotic diaphragm muscle is described as an example to swiftly identify novel proteomic markers of complex histopathological changes during skeletal muscle degeneration. The potential usefulness of new protein markers is examined in relation to key histopathological hallmarks for establishing improved diagnostic, prognostic and therapy-monitoring approaches in the field of dystrophinopathy.

Synthesis, structural, spectral, antidiabetic, DNA interactions and molecular docking investigations of a piperidine derivative

A new organic piperidine derivative named as 4‐methyl‐1‐({2,3,5,6‐tetramethyl‐4‐[(4‐methylpiperidinyl)methyl]phenyl}methyl)piperidine (D24MP) was synthesized and characterized by single crystal X-ray diffraction (SCXRD) and diverse spectroscopic (1H &13C NMR and FT-IR) techniques. The monoclinic structure of D24MP crystallises with the space group P-1. Density Function theory (DFT) using the B3LYP/6–31++G(d,p) level basis set was used to get the optimised geometry and non-linear optical properties. MEP surface analysis was used to investigate the crystal's nucleophilic and electrophilic reactive sites. The molecular shape and intermolecular interactions in the crystal structure were determined using Hirshfeld surface analysis and fingerprint plots. The physicochemical, pharmacokinetic and toxicological properties of D24MP have been evaluated by using drug-likeness and insilico ADMET studies. The molecule's first hyperpolarizability (β) estimation is 3.65 times higher than urea. Antidiabetic, antioxidant and DNA binding ability of D24MP were investigated by experimental and theoretical techniques. The obtained results suggest that D24MP possesses better antidiabetic, antioxidant and DNA binding abilities. Gel-electrophoretic method results suggest that D24MP has better cleaving capability against CT-DNA at higher concentrations (100 µg).

α-Tomatin`nin Antiproliferatif Aktivitesi ve Moleküler Hedef Tanımlaması

α-tomatine is a glycoalkaloid derived from tomato varieties that has been reported to possess various anticancer properties. However, its inhibitory effects on epidermal growth factor receptor is still poorly understood. The aim of this study is to investigate the anticancer effect of α-tomatine and its related mechanisms in lung cancer cells. Cytotoxicity and apoptosis induction of α-tomatine were determined by MTT assay and annexin V-FITC staining methods, respectively. For tyrosine kinase activity, TK-1 kinase selectivity profiling assay and molecular modelling study were performed. The DNA cleavage activity of α-tomatine was investigated using agarose gel electrophoretic method. α-tomatine proved to possess an outstanding antiproliferative activity against A549 and Jurkat cells without noticeable toxicity on PBMC. The results indicated that α-tomatine has a significant inhibition effect on both EGFR and HER2. α-tomatine formed some key interaction into ATP binding sites of EGFR and HER2. Furthermore, α-tomatine strongly disintegrated DNA at low concentrations in the presence of iron(II) complexes. The current findings suggest that α-tomatine has a distinguished receptor tyrosine kinase inhibition profile from erlotinib and might be a potential drug candidate for treatment of NSCLC.

Viroid research and its significance for RNA technology and basic biochemistry.

Viroids were described 47 years ago as the smallest RNA molecules capable of infecting plants and autonomously self-replicating without an encoded protein. Work on viroids initiated the development of a number of innovative methods. Novel chromatographic and gelelectrophoretic methods were developed for the purification and characterization of viroids; these methods were later used in molecular biology, gene technology and in prion research. Theoretical and experimental studies of RNA folding demonstrated the general biological importance of metastable structures, and nuclear magnetic resonance spectroscopy of viroid RNA showed the partially covalent nature of hydrogen bonds in biological macromolecules. RNA biochemistry and molecular biology profited from viroid research, such as in the detection of RNA as template of DNA-dependent polymerases and in mechanisms of gene silencing. Viroids, the first circular RNA detected in nature, are important for studies on the much wider spectrum of circular RNAs and other non-coding RNAs.

Determination of baclofen and vigabatrin by microchip electrophoresis with fluorescence detection: application of field-enhanced sample stacking and dynamic pH junction

A novel microchip gel electrophoretic method for the assay of baclofen and vigabatrin for the first time.

Synthesis, spectral characterization, DNA interaction, antioxidant, and antimicrobial studies of new water soluble metal(II) complexes of morpholine based ligand

A new series of metal(II) complexes 1–5 of the type [M(L)(AcO)2H2O], where L is a bidentate ligand, 2-(3-morpholinopropylimino)methylphenol and M is Mn(II) (1), Co(II) (2), Ni(II) (3), Cu(II) (4), and Zn(II) (5), have been isolated and characterized by physico-chemical and spectral methods. Spectroscopic data supported the square pyramidal geometry around the central metal ion in the complexes 1–5. Absorption spectra and viscometric data indicated that the complexes 1–5 interacted with calf thymus (CT) DNA via intercalative strategy. Cleavage activities of complexes 1–5 with CT DNA were analyzed by gelelectrophoretic method. The in vitro antioxidant activity of complexes 1–5 was tested using the DPPH assay. The complexes 1–5 have been tested for antimicrobial activity against some pathogenic bacterial and fungal species by the agar well diffusion method.

Scaffold-free approach produces neocartilage tissue of similar quality as the use of HyStem\u2122 and Hydromatrix\u2122 scaffolds

Numerous biomaterials are being considered for cartilage tissue engineering, while scaffold-free systems have also been introduced. Thus, it is important to know do the scaffolds improve the formation of manufactured neocartilages. This study compares scaffold-free cultures to two scaffold-containing ones. Six million bovine primary chondrocytes were embedded in HyStem™ or HydroMatrix™ scaffolds, or suspended in scaffold-free chondrocyte culture medium, and then loaded into agarose gel supported culture well pockets. Neocartilages were grown in the presence of hypertonic high glucose DMEM medium for up to 6 weeks. By the end of culture periods, the formed tissues were analyzed by histological staining for proteoglycans (PGs) and type II collagen, gene expression measurements of aggrecan, Sox9, procollagen α1(II), and procollagen α2(I) were performed using quantitative RT-PCR, and analyses of PG contents and structure were conducted by spectrophotometric and agarose gel electrophoretic methods. Histological stainings showed that the PGs and type II collagen were abundantly present in both the scaffold-free and the scaffold-containing tissues. The PG content gradually increased following the culture period. However, the mRNA expression levels of the cartilage-specific genes of aggrecan, procollagen α1(II) and Sox9 gradually decreased following culture period, while procollagen α2(I) levels increased. After 6-week-cultivations, the PG concentrations in neocartilage tissues manufactured with HyStem™ or HydroMatrix™ scaffolds, and in scaffold-free agarose gel-supported cell cultures, were similar to native cartilage. No obvious benefits could be seen on the extracellular matrix assembly in HyStem™ or HydroMatrix™ scaffolds cultures.Graphical abstract

Gel electrophoretic and liquid chromatographic methods for the identification and authentication of cave and house edible bird's nests from common adulterants

The increasing demand for edible bird's nest (EBN) have remarkably increased its price.

Toxicity assessment and bioaccumulation in zebrafish embryos exposed to carbon nanotubes suspended in Pluronic® F-108

Carbon nanotubes (CNTs) are often suspended in Pluronic® surfactants by sonication, which may confound toxicity studies because sonication of surfactants can create degradation products that are toxic to mammalian cells. Here, we present a toxicity assessment of Pluronic® F-108 with and without suspended CNTs using embryonic zebrafish as an in vivo model. Pluronic® sonolytic degradation products were toxic to zebrafish embryos just as they were to mammalian cells. When the toxic Pluronic® fragments were removed, there was little effect of pristine multi-walled CNTs (pMWNTs), carboxylated MWNTs (cMWNTs) or pristine single-walled carbon nanotubes (pSWNTs) on embryo viability and development, even at high concentrations. A gel electrophoretic method coupled with Raman imaging was developed to measure the bioaccumulation of CNTs by zebrafish embryos, and dose-dependent uptake of CNTs was observed. These data indicate that embryos accumulate pMWNTs, cMWNTs and pSWNTs yet there is very little embryo toxicity.

Biomass production from fermented starch wastewater in photo-bioreactor with internal overflow recirculation

A photo-bioreactor with internal overflow recirculation was applied to treat real fermented starch wastewater and convert it to photosynthetic biomass for further utilization. The photo-bioreactor was operated at a hydraulic retention time of 10days by circulating mixed liquor through overflow pipes and penetrating light through infrared transmitting filter. During the operation of 154days, the average BOD and COD removals were 95% and 88%, respectively. Majority of photosynthetic bacteria was found attached on pipes as biofilm contributed to 82% of total biomass production. Photosynthetic biomass yield was 0.51g dried solid/g BOD removed and crude protein content of 0.58g/g dried solid. Rhodopseudomonas palustris was found in the photosynthetic system as the predominant bacterial group by denaturing gradient gel electrophoretic analysis (DGGE) and 16S rDNA sequencing method.

Identification of the long-acting erythropoiesis-stimulating agent darbepoetin alfa in human urine by liquid chromatography–tandem mass spectrometry

The misuse of recombinant human erythropoietin (rhEPO) increases the proliferation/production of erythrocytes, which enhance oxygen transport capacities, and has grave consequences with respect to human health and fairness in sports. For sports drug testing, the current analytical methods for rhEPOs are mainly gel electrophoretic methods, such as isoelectric focusing-polyacrylamide gel electrophoresis. Mass spectrometry is fundamentally necessary for the reliable identification of rhEPOs in doping control. In this study, a high-sensitivity and high-throughput mass spectrometric qualitative detection method for darbepoetin alfa in human urine was established by a bottom-up approach. The novel method involves the immunopurification of human urine (10 mL), protease digestion with endoproteinase Glu-C (V8-protease) in an ammonium bicarbonate buffer (pH 7.8) and ultra-performance liquid chromatography using a charged surface hybrid C18 column coupled with electrospray-ionisation high-sensitivity tandem mass spectrometry for improved selectivity of the target molecules. The specific fragment digested from darbepoetin alfa was (90)TLQLHVDKAVSGLRSLTTLLRALGAQKE(117) (V11). The lower limit of detection of urinary darbepoetin alfa was 1.2 pg/mL. The limit of detection for the confirmation analysis was estimated to be 5 pg/mL. The developed method allows high-throughput confirmation analysis, namely 6 h for sample preparation and an analytical run time of only 10 min per sample; this high-throughput method dramatically decreases the workload in the laboratory. Darbepoetin alfa could be identified in human urine collected after the intravenous administration of 15 μg darbepoetin alfa (n = 3). This mass spectrometric method is an innovative and powerful tool for detecting darbepoetin alfa in human urine for doping control testing.

Gel Electrophoretic Methods for The Analysis of Biosimilar Pharmaceuticals Using The Example of Recombinant Erythropoietin

Due to their versatility and cost-effectiveness, gel electrophoretic methods provide an important set of tools for the analysis of therapeutic proteins. As an increasing number of biosimilar pharmaceuticals are entering the market, techniques are required that allow reliable demonstration of comparability of these products with the reference products. Isoelectric focusing, SDS-PAGE, native PAGE and 2D electrophoresis (2D-PAGE) have been frequently applied for this purpose. Supplementary techniques are fluorophore-assisted carbohydrate electrophoresis and sarcosyl-PAGE. Of additional importance is the comparison of recombinant with endogenously synthesized glycoproteins. Reagent array analysis combined with SDS-PAGE and western blotting proved especially useful for this purpose. As an example for the application of these methods, the analysis of recombinant originator erythropoietins and some of their biosimilar counterparts is described.