Tris-HCl Buffer Research Articles

A comparative application of spectrophotometric and spectrofluorimetric methods to estimate levofloxacin-DNA and ofloxacin-DNA interactions.

Ofloxacin (OFL) and its (S)-enantiomer, levofloxacin (LEV), are among members of the fluoroquinolone antibiotic class, renowned for their broad-spectrum efficacy against both gram-negative and gram-positive bacteria. These potent drugs have been widely used in both human and veterinary medicine, working as bactericidal agents by binding to DNA gyrase, an essential enzyme for bacterial DNA replication. Understanding the binding constants of these drugs to DNA is vital for elucidating their interaction mechanisms and enhancing our grasp of gene expression regulation. The interactions of LEV and OFL with calf thymus DNA under a physiological medium (0.02M tris-HCl buffer, pH 7.4) using UV spectrophotometry and spectrofluorimetry were investigated. The assay results obtained by applying two spectroscopic approaches confirmed the presence of the interaction of LEV and OFL antibiotics with DNA. In the LEV-DNA and OFL-DNA interactions, hyperchromic effect and fluorescence quenching were observed for UV spectrophotometric and spectrofluorometric measurements, respectively. In the spectrophotometric analysis, the binding constants for the LEV-DNA and OFL-DNA complexes at 298K were determined as (1.24 ± 0.047) x 103 and (1.39 ± 0.040) x 103 M- 1, respectively. In the spectrofluorimetric analysis of the interaction of LEV and OFL with DNA, the thermodynamic properties were examined at three distinct temperatures. Based on the fluorescence signal changes the binding constants at 293, 298, and 310K were calculated as (8.91 ± 0.161) x 103, (7.62 ± 0.098) x 103, and (6.08 ± 0.041) x 103 M- 1 for LEV-DNA and, (3.14 ± 0.053) x 103, (3.04 ± 0.031) x 103, and (2.78 ± 0.023) x 103 M- 1 for OFL-DNA, respectively. In these assays, the Gibbs free energy (ΔG0), entropy (ΔS0), and enthalpy (ΔH0) were determined using the Van't Hoff equation. The negative ΔG⁰ values indicate that both LEV-DNA and OFL-DNA interactions are spontaneous. Furthermore, the positive ΔS⁰ and negative ΔH⁰ values revealed that electrostatic forces played a significant role in the binding LEV and OFL to DNA.

Optimization of extraction and purification of the M protein fused GCN4pII motif and elastin like polypeptide of porcine epidemic diarrhea virus from <i>Nicotiana benthamiana</i>

Porcine Epidemic Diarrhea (PED) has significantly impacted the swine farming industry in several countries worldwide, including Vietnam. Porcine Epidemic Diarrhea Virus (PEDV) has been proven to be the cause of PED. The COE and M proteins are potential candidates for subunit vaccine research against PEDV. The M protein, a largest envelope protein of PEDV, is highly conserved and plays a crucial role in virus assembly as well as in inducing the production of virus-neutralizing antibodies in the presence of complement. In a previous study, we constructed and expressed the M protein fused with the GCN4pII motif and Elastin-Like Polypeptide (M-pII-ELP) separately from the COE/G2a-pII protein in Nicotiana benthamiana. In this study, we assessed the co-expression of M-pII-ELP and COE/G2a-pII proteins in N. benthamiana using SDS-PAGE and Western blot. Next, we verified the assembly of virus-like particles (VLPs) by the M-pII-ELP protein alone through Transmission Electron Microscopy (TEM) analysis after ultracentrifugation with a sucrose gradient. Suitable buffers for the extraction and purification of M-pII-ELP protein using immobilized affinity chromatography (IMAC) were also selected. Results from SDS-PAGE and Western blot confirmed the co-expression of M-pII-ELP and COE/G2a-pII proteins in the plant; however, the expression of COE/G2a-pII protein was suppressed when co-expressed with M-pII-ELP. TEM analysis confirmed the formation of virus-like particles based on the assembly of the M-pII-ELP protein. Among the buffers tested for M-pII-ELP protein extraction, Tris-HCl buffer yielded the highest amount of M-pII-ELP protein. It was determined that the optimal imidazole concentrations for extraction and washing buffers in M-pII-ELP protein purification are 0 mM and 10 mM, respectively. These results lay the groundwork for further studies on developing plant-based subunit vaccines against PEDV.

Development of a thermal stabilizer formulation optimized by response surface methodology for Senecavirus A antigen

Numerous members of the family Picornaviridae, such as the Senecavirus A (SVA) and foot-and-mouth disease virus (FMDV), exhibit thermal instability, resulting in the dissociation of viral particles, which affects the insufficient potency of the vaccine. Based on this characteristic, this study aimed to maintain the thermal stability of SVA by supplementing it with a stabilizer. Excipients, such as sucrose, mannitol, sorbitol, polyethylene glycol (PEG), L-arginine (L-Arg), glutamic acid (Glu), polyvinyl pyrrolidone (PVP), bovine serum albumin (BSA), and potassium chloride (KCl) dissolved in Tris-HCl buffer solution, retained the infectivity of SVA in the thermostability assay. Thermal stability formulations were developed by combining different excipients in disaccharide polyol systems and optimizing formulations using the Box-Behnken experimental design (BBD) combined with response surface methodology (RSM). Three significant factors were studied: sucrose 9.9%, sorbitol 9.9%, and L-Arg 0.06 mol/L against virus titer of thermal-resistance of SVA as a response. The formulation improved the stability of SVA, whose viral infectivity titer decreased by 1.0 TCID50/mL at 4°C, 25°C, and 37°C, respectively, until it decreased by 1.21 TCID50/mL at 7 d of incubation at 42°C. The combinational thermal stabilizer generated in this study enabled the stabilization of the SVA, which might contribute to storage and transportation when the cold chain is unavailable, especially in rural areas. Therefore, the thermal stabilizer is an efficient candidate stabilizer for picornavirus formulations, which keep picornavirus infectivity at various temperatures. Further optimization of this approach will provide new opportunities for the generation of stabilizer formulation from different stabilizers.

Marine chitinase AfChi: green defense management against Colletotrichum gloeosporioides and anthracnose.

Anthracnose disease, caused by the Colletotrichum gloeosporioides species, affects vegetables, fruits, pulses, and cereals, leading to significant economic losses worldwide. Although many synthetic fungicides are used to control this pathogen, eco-friendly biological alternatives are gaining popularity. This study focuses on isolating and purifying chitinase ( Af Chi)from a marine bacterium and testing its antifungal efficacy against C. gloeosporioides spore germination by targeting the chitin in the fungal cell wall. The chitinase was purified from a marine bacterium A. faecalis from the Arabian Sea and had a molecular mass of 45kDa and a specific activity of 84.6 U/mg. Af Chi worked best at 50°C and pH 7.0 in Tris HCl buffer. Na+ ion was the highest cofactor, highlighting the halophilic nature of this chitinase. K+, Ca2+, Cu2+, Mg2+, Mn2+, and EDTA also increased activity, while Fe3+, Zn2+, Co2+, and Pb2+ decreased it. The Km and Vmax values were 1.87µg/mL and 17.45 U/mL, respectively. Purified Af Chi at 10mg/mL completely inhibited spore germination within 8h and reduced the size of the spores.

Harnessing co-expressed L-arabinose and L-ribose isomerases to enhance the biosynthesis of L-ribose

L-Ribose is an essential rare sugar used in several industries, including pharmaceutical, food, cosmetics, and agriculture. Recently, the enzymatic production of L-ribose has garnered significant attention due to its considerable advantages over synthetic methods. This study introduces a novel approach for producing L-ribose from L-arabinose. Initially, the genes encoding L-arabinose isomerase (L-AI) from Bacillus subtilis str. 168 (BsL-AI) and L-ribose isomerase (L-RI) from Actinotalea fermentans ATCC 43279 (AfL-RI) were cloned to construct the recombinant plasmid containing the pANY1-BsL-AI/AfL-RI vector and co-expressed in Escherichia coli BL21(DE3). Subsequently, the of co-expression exhibited optimal activity at pH 8.5 and 40 °C in 50 mM Tris-HCl buffer, with 1 mM Mn2+ ion. The activity was increased by 33 % and 12 % with Mn2+ and Co2+ ions, respectively, compared to the control having no metal ions. The scheme comprising 50 g L−1 of co-expressed cells remained comparatively stable up to 60 °C for 2 h. Finally, the co-expression scheme produced 23.52 g L−1 (24 %), 25.87 g L−1 (26 %) and 27.97 g L−1 (28 %) of L-ribose when utilizing L-arabinose concentration of 30, 50 and 100 g L−1, respectively, with 50 g L−1 of co-expressed cells. This study presents a viable methodology for the utilization of L-arabinose to produce L-ribose in slightly alkaline conditions utilizing a co-expression scheme concealing BsL-AI and AfL-RI genes.

Paintable, Fast Gelation, Highly Adhesive Hydrogels for High-fidelity Electrophysiological Monitoring Wirelessly.

High-fidelity wireless electrophysiological monitoring is essential for ambulatory healthcare applications. Soft solid-like hydrogels have received significant attention as epidermal electrodes because of their tissue-like mechanical properties and high biocompatibility. However, it is challenging to develop a hydrogel electrode that provides robust contact and high adhesiveness with glabrous skin and hairy scalp for high-fidelity, continuous electrophysiological signal detection. Here, a paintable, fast gelation, highly adhesive, and conductive hydrogel is engineered for high-fidelity wireless electrophysiological monitoring. The hydrogel, consisting of gelatin, gallic acid, sodium citrate, lithium chloride, glycerol, and Tris-HCl buffer solution exhibits a reversible thermal phase transition capability, which endows it with the attributes of on-skin applicability and fast in situ gelation with 15 s, thereby addressing the aforementioned limitations. The introduction of gallic acid enhances the adhesive properties of the hydrogel, facilitating secure electrode attachment to the skin or hairy scalp. To accentuate the potential applications in at-home electrophysiological health monitoring, the hydrogel electrodes are demonstrated for high-fidelity electrocardiogram recording for one hour during various daily activities, as well as in simultaneous electroencephalogram and electrocardiogram recording during a 30 min nap.

Aminothiophenol and 7-diethylamino-4-hydroxycoumarin derived probe for reversible turn off–on–off detection of Cu2+ ions and cysteine

Here, we present a simple disulfide linked probe HTP for rapid detection of Cu2+ ions, which was prepared by a condensation reaction between 7-diethylamino-4-hydroxycoumarin aldehyde and 2-aminothiophenol. The disulfide linked probe HTP was characterized using 1H NMR, 13C NMR, and HRMS spectroscopic analysis and confirmed by single crystal X-ray diffraction analysis. The photophysical behavior of HTP in various solvents (non-polar to polar) was studied and HTP displayed aggregation induced emission (AIE) characteristics in CH3CN-water mixtures (0–99 %). Upon binding with Cu2+ ions, emission enhancement occurs along with color changes from weak green to intense yellow emission in CH3CN/Tris-HCl buffer (20 μM, 9:1, 10 mM Tris HCl Buffer, pH = 7.4). Detection limit for Cu2+ ions was found to be 0.97 nM which is lower than the recommended tolerance limit by the WHO and the association constant 0.42 × 108 M−1 was obtained using B–H plot. Furthermore, the stoichiometric ratio 1:1 was confirmed by Job’s plot, 1H NMR, mass spectral analysis and DFT calculations were supported the formation of HTP-Cu2+ complex. The reversibility of HTP with Cu2+ ions was achieved by cysteine with detection limit and association constant value of 1.64 µM and 0.15 × 107 M−1 respectively. The reversible sensing nature of HTP with Cu2+/cysteine was further applied for constructing a molecular logic gate (INHIBIT) and practical applications such as paper strips, cotton swabs and real water analysis.

Optimizing PES microfiltration membranes for sterile filtration: A comparative study of polydopamine coating in acidic and basic conditions to minimize protein adsorption

The increasing need for effective sterile filtration in the pharmaceutical industry calls for advancements in microfiltration (MF) membrane technology that can effectively reduce fouling and preserve essential protein products. Our study highlights the crucial role of improving the hydrophilicity throughout the entire membrane, not just on the surface, to decrease both protein adsorption and fouling. We have developed a facile but effective method for producing hydrophilic porous polyethersulfone (PES) MF membranes using vapor-induced phase separation (VIPS) followed by a hydrophilic coating. By meticulously adjusting the vapor exposure time, we fabricated two specific types of symmetric membranes: a standard one with a mean pore size of 0.22 μm and another designed with slightly larger pores for the subsequent polydopamine (PDA) coating. All membranes developed in this study showed an outstanding sterilization performance (over 99.99999 % bacteria rejection) due to the absence of defects. Our comparative studies of PDA coating in both acidic and basic environments revealed that the PDA-modified membranes in a Tris-HCl buffer (pH 8.0) (PDA-b-PES2M) outperform others in performance of protein microfiltration. While the membrane modified under acidic conditions underwent a gradual degradation of PDA leading to a poor filtration performance, the PDA-b-PES2M membranes showcased remarkable anti-fouling properties, greatly minimizing the adsorption of bovine serum albumin (BSA) and simplifying the removal of adsorbed materials. Our optimized membrane has a great potential for sterile filtration applications where minimizing protein loss is crucial.

B-151 Development and Validation of Plasma Catecholamines by Mass Spectrometry

Abstract Background Our established method for measuring plasma catecholamines utilized a Shimadzu HPLC coupled to an ESA Coulochem III electrochemical detector. Chromatographic run times were long, resulting in slow turnaround times. Instrument maintenance was labor intensive, and the assay was prone to interferences. To overcome these limitations, we changed the method to liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements. Methods After the addition of a labeled internal standard for each analyte, samples are loaded onto 50 mg of activated alumina at pH 8.6, utilizing a Tris-HCl, sodium carbonate and EDTA buffer. After incubation the alumina is then washed with 50% acetonitrile followed by water. Elution off the alumina is performed with 2% acetic acid. Derivatization is then performed on the eluate utilizing acetaldehyde and a cyanoborohydride buffer. Following derivatization, the samples are analyzed for epinephrine, norepinephrine, and dopamine by LC-MS/MS using a Sciex 7500 coupled to a ThermoFisher Scientific TLX-4 equipped with Vanquish pumps. Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 um BiPhenyl 50x3 mm analytical column. Results Runtimes were significantly shorter than with the previous method, and no interferences were observed. Assay validation consisted of: intra/inter assay precision, accuracy, recovery, linearity, carryover, limit of detection (LOD), and interferences. Three levels were assessed for intra assay precision and each of the analytes monitored yielded %CVs of less than 6%. Four levels were assessed for inter assay precision and each of the analytes monitored yielded %CVs of less than 10%. When using linear regression analysis to compare results with the previous clinical assay, the slope, y-intercept and R2 were the following; dopamine: 1.06, 2.74, 0.978; epinephrine: 0.905, 2.20, 0.989; norepinephrine: 0.919, 9.75, 0.953. Spike and recovery from five samples yielded a mean average recovery of 93.4% for dopamine, 100% for epinephrine and 98.1% for norepinephrine. For linearity 4 samples were diluted and the mean % difference from expected was -1.66% for dopamine, -0.25% for epinephrine and 1.78% for norepinephrine. Blanks were injected following the high standard and carryover was negligible. LOD was determined by running a low sample pool along with a blank over 20 assays. The LOD was determined to be less than the LLOQ. Sixty-two common drugs/vitamins along with varying levels of hemolysis, lipemia, and bilirubin were also tested to ensure no interferences were seen. Conclusions The LC-MS/MS method exceeded the performance of the historical Shimdazu HPLC system with ESA Coluochem III electrochemical detector method. We anticipate that the better analytical performance will improve the testing quality of our patients.

Understanding the Effect of Ionic Liquid-Mediated Solvent Engineering on the Kinetics and Thermodynamic Stability of Phenylalanine Ammonia-Lyase.

Phenylalanine ammonia-lyase (PAL) plays a central role in the phenylpropanoid pathway and in the treatment of phenylketonuria. However, the integration of PAL into sustainable industrial biocatalysis is hampered by its instability under harsh conditions. This study demonstrates that ionic liquid (IL)-assisted solvent (Tris-HCl buffer) engineering enables improvement of the reaction kinetics and thermodynamic stability of Rhodotorula glutinisPAL (RgPAL) under various stresses. Under optimized conditions, a 66.2% higher Kcat value, >60% remaining activity after 5 weeks of storage at room temperature, and >80% activity of RgPAL after incubation at 60 °C for 1 h were obtained in the [Ch][Ac]-blended Tris-HCl solvent compared to pristine Tris-HCl. The spectroscopic and molecular docking results suggest that the higher extent of hydration and the soft interactions complemented by the ILs with the D-chain residues of RgPAL jointly contributed to achieving more stable and active conformations of RgPAL. The enzyme showed a higher melting temperature (Tm) in ILs+Tris-HCl compared to that in pristine Tris-HCl, with less change in enthalpy (ΔHfu) and entropy (ΔSfu) of unfolding. Overall, IL-mediated solvent engineering alters the microenvironment of RgPAL and allows the development of a robust PAL-based biocatalytic system.

Improving the Efficiency of Luminescent Zn(II)-Modified N-Doped GOQD Nanomaterials in Parkinson's Disease Treatment: A Theoretical Mechanistic Framework Exploring Doping Effect.

Levodopa, a widely prescribed drug in Parkinson's disease treatment, stands as the foremost prodrug of dopamine. An affordable self-testing kit is utilized to monitor levodopa content in anti-parkinson drugs in human serum. A photoluminescent trinuclear Zn(II) complex [Zn3(L)2(κ1-OAc)2(κ2-OAc)2] has been synthesized, which cleaves into mononuclear ZC in aqueous solution. ZC was found to detect L-Dopa in Tris-HCl buffer, exhibiting a moderate decrease in PL-emission. The real-life utility of the ZC probe is limited, for its lower sensitivity (LOD 35.3 μM) and separation challenges. Therefore, an interface between homogeneous and heterogeneous supports has been explored, leading to the strategic development of NGOZC, where ZC was grafted onto NGOQD (Graphene oxide quantum dots). This material enables naked- eye detection under both ambient and UV light with color change from bright cyan to green, followed by dark. The nitrogen doping effect was investigated by several comparative investigations involving the synthesis of ZC-grafted GOQD, leading to enhanced quenching performance. Steady-state and time-resolved fluorescence titration study, morphological analysis, and computational calculations have been performed to get insights into the sensing mechanism. To the best of our knowledge, this as-synthesized NGOZC (LOD 1.78 nM) represents a promising strategy and platform for applications in biosensors, especially for Parkinson's and Alzheimer's diseases.

Mg2+ - Ca2+ chelating citric acid crosslinked gelatin, a bone adhesive composed of hydroxyapatite and calcined dolomite: Physicochemical characteristics and in-vitro biological activity

Hydroxyapatite-based bone adhesives have strong bond strength and good adsorbability, however, they also have some disadvantages such as poor mechanical strength, fast curing reaction rate and high exothermic strength, which limit their wide application in bone tissue engineering. In this study, hydroxyapatite-based bone adhesive was prepared by solid-liquid blended crosslinking followed by heating. The prepared bone adhesive has two components: (i) is a mineral composed of calcined dolomite and montmorillonite and (ii) is organic composite composed of citric acid and gelatine. The optimum solid-liquid ratio (0.50 g/mL) of the bone adhesive was determined based on the bond strength of nano-hydroxyapatite/calcined dolomite/citric acid/gelatin system. The best physicochemical properties of the bond adhesive were obtained when the composition of the bone adhesive was as follows: 20 wt% citric acid, 17 wt% gelatin and 12 wt% calcined dolomite. The acquired curing time of bone adhesive was 10.5 min, comparable to the necessity for surgery (5–30 min); moreover, the adhesive strength and compressive strength of the bone adhesive were 12.18 MPa and 9.36 MPa respectively, obtained after being placed in the air for 168 h; besides, the porosity (16.8 %) was found quite suitable. Upon this, a certain amount of montmorillonite was added with calcined dolomite to prepare bone adhesive, resulting reduction in the adhesive strength (10.19 MPa) and compressive strength (7.109 MPa), but improved the biocompatibility of the system. The absorbency of the hydroxyapatite-based bone adhesives in deionized water and Tris-HCl buffer solution was found to be 27.19 % and 18.68 %, respectively, ascertaining that such bone adhesives are efficient in absorbing certain blood and tissue fluid in the biological body and promoting the absorption of required nutrients in the process of bone repair. In addition, the formation of osteoid apatite in the In-vitro study, proved by FTIR and EDAX analysis, rationalized for improving the osteogenic activity. Furthermore, after 56 days of immersion in the Tris-HCl buffer solution, the weight loss rate of the bone adhesive reached up to 42.73 %, indicating that the bone adhesive had satisfactory degradation performance. Finally, in the cytotoxicity test, the survival rate of mouse precranial bone cells was found to be greater than 80 % after adding the prepared bone adhesive, demonstrating its sufficient biocompatibility.

Simple, rapid and enzyme-free assay for potentiometric determination of L-cysteine based on meso-2,3-dimercaptosuccinic acid self-assembled gold electrode

As a sulfur containing α-amino acid in nature, L-cysteine plays a crucial role in a variety of metabolic pathways with many important physiological functions. However, it is still a great challenge to construct a simple and rapid approach for L-cysteine up to now. In this paper, a simple, rapid and enzyme-free method was developed for potentiometric determination of L-cysteine by using meso-2,3-dimercaptosuccinic acid self-assembled monolayer on gold electrode. The surface morphology and property of self-assembled membrane with and without L-cysteine combined were characterized by means of scanning electron microscopy, X-ray photoelectron spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry, respectively. The recognition mechanism may be attributed to formation of hydrogen bonds between meso-2,3-dimercaptosuccinic acid and the target molecule of L-cysteine, producing a double electric layer at the sensing interface, that the membrane potential decreases with increasing concentration of L-cysteine. The assay results showed that good linear potential response to L-cysteine in Tris-HCl buffer solution (pH=7.0) was obtained in a range of 1.0 × 10-8 to 1.0 × 10-3 mol/L, with a slope of −57.00 ± 0.63 mV/-pc (25 °C) and a detection limit of 7.9 × 10-9 mol/L. The electrode possessed fast response time (45 s), good reproducibility, and strong anti-interference performance. Moreover, the electrode can be well applied to the detection of L-cysteine in pig serums with a recovery rate of 94.3 ∼ 106.3%, indicating a promising application prospect.

Preparation of micron-sized benzamidine-modified magnetic agarose beads for trypsin purification from fish viscera

The effective method for trypsin purification should be established because trypsin has important economic value. In this work, a novel and simple strategy was proposed for fabricating micron-sized magnetic Fe3O4@agarose-benzamidine beads (MABB) with benzamidine as a ligand, which can efficiently and selectively capture trypsin. The micro-sized MABB, with clear spherical core-shell structure and average particle size of 6.6 μm, showed excellent suspension ability and magnetic responsiveness in aqueous solution. The adsorption capacity and selectivity of MABB towards target trypsin were significantly better than those of non-target lysozyme. According to the Langmuir equation, the maximum adsorption capacity of MABB for trypsin was 1946 mg g−1 at 25 °C, and the adsorption should be a physical sorption process. Furthermore, the initial adsorption rate and half equilibrium time of MABB toward trypsin were 787.4 mg g−1 min−1 and 0.71 min, respectively. To prove the practicability, MABB-based magnetic solid-phase extraction (MSPE) was proposed, and the related parameters were optimized in detail to improve the purification efficiency. With Tris-HCl buffer (50 mM, 10 mM CaCl2, pH 8.0) as extraction buffer, Tris-HCl buffer (50 mM, 100 mM CaCl2, pH 8.0) as rinsing buffer, acidic eluent (0.01 M HCl, 0.5 M NaCl, pH 2.0) as eluent buffer and alkaline buffer (1 M Tris-HCl buffer, pH 10.0) as neutralization solution, the MABB-based MSPE was successfully used for trypsin purification from the viscera of grass carp (Ctenopharyngodon idella). The molecular weight of purified trypsin was determined as approximate 23 kDa through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified trypsin was highly active from 30 °C to 60 °C, with an optimum temperature of 50 °C, and was tolerant to pH variation, exhibiting 85 % of maximum enzyme activity from pH 7.0 to 10.0. The results demonstrated that the proposed MABB-based MSPE could effectively purify trypsin and ensure the biological activity of purified trypsin. Therefore, we believe that the novel MABB could be applicable for efficient purification of trypsin from complex biological systems.

Automated Standardization of Melanin Bleaching Procedures of Heavily Pigmented Melanocytic Lesions With Low-Concentration Hydrogen Peroxide on an Automated Platform.

Melanin is a natural pigment in the human body that is primarily found in the skin and hair. It protects the skin from damage by ultraviolet radiation. Although this pigment plays a crucial role in protecting the human body, it represents a challenge for pathologists to evaluate highly pigmented tissue samples from melanoma or pigmented skin lesions. Abundant melanin may obscure tissue morphology, which makes it very difficult for pathologists to make a differential diagnosis. Melanin pigment is brown-to-black and granular, and its distribution is often uneven in tissues. The presence of these pigments can complicate the analysis of immunohistochemistry (IHC) for 2 reasons. First, they have a direct physical masking effect on antigen-antibody interactions. Second, 3,3-diaminobenzidine, the most commonly used chromogen, has a brown color that is difficult to distinguish from melanin pigment. Therefore, melanin bleaching has become a crucial step in handling pigmented melanocytic lesions. Bleaching techniques aid pathologists in histopathologic examination of melanin-rich tissue. In this study, we integrated melanin bleaching and IHC on an automated IHC platform to set up a rapid and fully automated procedure. Bleaching steps were performed before antigen retrieval. Samples were treated with 1% hydrogen peroxide solution in Tris-HCl buffer (pH 10) at 80°C for 8 minutes, achieving optimal conditions for melanin bleaching while preserving tissue morphology and antigenicity. This rapid, effective, fully automated, and standardized workflow can be applied to routine staining procedures in clinical laboratories, thereby improving the quality of pathological diagnosis.

Novel cold-active amylase-producing bacterium from Chukchi Sea and its enzyme properties

A cold-active amylase was purified from Alteromonas sp. KS7913 isolated from the Chukchi Sea in the Arctic Ocean. After purification with use of ammonium sulfate precipitation, phenyl column chromatography, and size exclusion chromatography, 200.34 U mg−1 of purified amylase was obtained. The final yield was 3.4%, and the activity was 5.7-fold higher than that of the initial culture broth. KS7913 origin amylase showed a molecular weight of 70 kDa and optimal activity at 25℃, pH 7.0 in Tris-HCl buffer. The amylase was highly active, especially at 5℃, and maintained stability at basic conditions below 25℃. Copper and zinc ions inhibited enzyme activity, whereas manganese, barium, and calcium ions exhibited positive effects. This activity was maintained even in the presence of alcohol. The findings of this study supplement our understanding of cold-active amylases, and may have practical applications in low-temperature industries.

Rational Design of a Flavoenzyme for Aerobic Nicotine Catabolism.

Enzymatic therapy with nicotine-degrading enzyme is a new strategy in treating nicotine addiction, which can reduce nicotine concentrations and weaken withdrawal in the rat model. However, when O2 is used as the electron acceptor, no satisfactory performance has been achieved with one of the most commonly studied and efficient nicotine-catabolizing enzymes, NicA2. To obtain more efficient nicotine-degrading enzyme, we rationally designed and engineered a flavoenzyme Pnao, which shares high structural similarity with NicA2 (RMSD = 1.143 Å) and efficiently catalyze pseudooxynicotine into 3-succinoyl-semialdehyde pyridine using O2. Through amino acid alterations with NicA2, five Pnao mutants were generated, which can degrade nicotine in Tris-HCl buffer and retained catabolic activity on its natural substrate. Nicotine-1'-N-oxide was identified as one of the reaction products. Four of the derivative mutants showed activity in rat serum and Trp220 and Asn224 were found critical for enzyme specificity. Our findings offer a novel avenue for research into aerobic nicotine catabolism and provides a promising method of generating additional nicotine-catalytic enzymes.

Two electrochemical sensors based on pencil graphite electrodes modified with azo dye and a novel Schiff base for lead and cadmium ion quantification

This research introduces a cost-effective and straightforward approach to fabricating two novel electrochemical sensors. The initial novel sensor, termed AZOD/PLE, involves coating the surface of a pencil lead electrode (PLE) with the azo dye molecule (AZOD). The second novel sensor, named HDBE/PLE, is created by coating the pencil lead electrode (PLE) with a newly synthesized novel Schiff base molecule (HDBE), characterized through techniques such as IR, 1H NMR, and 13C NMR. The reason for the choice of the modifier materials in this research is their complexing ability with metal ions since they contain electron donor atoms such as O and N, which can form coordination bonds with the toxic heavy metal’s ions, cadmium and lead. This is the first time using the azo dye molecule (AZOD) and the novel Schiff base molecule (HDBE) as modifiers on PLE. The characterization of the electrode surfaces was made by energy-dispersive X-ray spectroscopy (EDS), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). The surface morphology of the electrode was investigated by scanning electron microscopy (SEM). CV results indicate that the modified electrodes possess larger electroactive surface areas compared to the bare PLE. Square wave voltammetry (SWV) results demonstrate that both sensors exhibit high sensitivity in detecting low concentrations of Cd (II) and Pb (II) in tris-HCl buffer (0.1 M) for both single-metal and multi-metal solutions. These sensors can be easily produced in a single step and exhibit excellent selectivity, stability, reproducibility, and repeatability, making them well-suited for detecting Pb (II) and Cd (II) in aqueous environments.

Experimental design of size variation in albumin nanoparticles synthesized by electron beam

Protein-based nanoparticles have garnered significant interest for their potential in theranostic applications, including cancer treatment and nuclear medicine. Plasma proteins are particularly appealing due to their ability to bypass the rapid clearance typically associated with synthetic particles. Human serum albumin (HSA), for example, is already utilized in diagnostic procedures such as lymphoscintigraphy and sentinel lymph node detection and in cancer treatments where it delivers therapeutic agents in nanoparticulate form. Among the various techniques for synthesizing protein nanoparticles, ionizing radiation stands out for its ability to maintain good size control and preserve the protein's three-dimensional structure. Factors such as the reaction precursor, pH, protein concentration, presence of a stabilizer, and irradiation dose can all influence the size and shape of the resulting nanoparticles. This study sought to identify a correlation between the size of albumin nanoparticles and reaction parameters, including protein concentrations, the ionic content of the buffer solution, and the e-beam irradiation dose. We conducted different synthesis methods with varying BSA concentrations, using two different buffers and varying radiation doses. The resulting nanoparticles were evaluated using Dynamic Light Scattering. The size data were statistically analyzed using SAS Studio, SAS JMP, and WEKA software to identify correlations among the synthesis variables. Our observations primarily revealed that lower protein concentrations consistently resulted in smaller particles. Additionally, using Tris-HCl buffer as a medium led to a more proportional growth of particles with increases in concentration and irradiation dose. These findings suggest that Tris buffer is more suitable for BSA nanoparticle synthesis, as increased albumin concentration and radiation dose resulted in more consistent size control.

Adaptation of Ochrobactrum rhizosphaerae IMV B-7956 Bacteria to the Influence of Copper (II) Chloride

In technologically altered habitats, an increased content of organic compounds, nitrogen, phosphorus, sulfur compounds, antibiotic substances, etc. is found. Therefore, microorganisms that are systematically exposed to various stressors have developed adaptation mechanisms. The strain Ochrobactrum rhizosphaerae IMV B-7956, isolated from the infiltrate lakes of the Lviv solid waste landfill, is resistant to copper, chromium, manganese, and iron in concentrations exceeding the maximum permissible concentrations. The work aimed to study the response of O. rhizosphaerae IMV B-7956 cells to CuCl2 exposure by detecting changes in the content of lipid peroxidation products, products of the oxidative modification of proteins, activity of antioxidant defense system enzymes, and synthesis of extracellular polymers. Methods. To study the effect of CuCl2 on prooxidant indicators and the activity of enzymes of the antioxidant defense system, bacteria were pre-incubated in Tris-HCl buffer containing 2–10 mM CuCl2. After one hour of incubation, the bacterial cells were washed and cultured for 1, 12, 24, and 48 h in metal-free media. The copper content in the bacterial cells was determined by atomic absorption spectrometry. The content of lipid peroxidation indicators, carbonyl groups in proteins, total low-molecular-weight thiols, enzymatic activity, and the content of exopolysaccharides and extracellular proteins were determined photometrically. Results. Within an hour, O. rhizosphaerae IMV B-7956 bacteria accumulated 2.3–7.8 mg Cu/g of biomass. Under these conditions, an increased content of lipid peroxidation products was detected. During the first hour of growth in bacterial cells, enzymes with catalase and peroxidase activity were activated. During further cultivation, an increase in the activity of other antioxidant defense enzymes was detected. Carbonyl groups in proteins are probably formed due to an increase in the content of lipid peroxidation products, as they are formed later. Within 12–48 h of growth, the copper content in the bacterial cells decreases. This leads to the restoration of growth. Conclusions. The main damaging effect of CuCl2 on bacterial cells was detected during the first 24 h of growth. Activation of the enzymes of the antioxidant system and synthesis of exopolysaccharides are among the adaptations that ensure the survival of bacteria under these conditions.