Neutral Buffer Research Articles

L-Methionine Selenoxide as an Oxidizing and Deprotection Reagent for the Synthesis of Multiple Disulfide Bonds in Peptides.

The regioselective synthesis of multiple disulfide bonds in peptides has been a significant challenge in synthetic peptide chemistry. In this work, two disulfide bonds in peptides were regioselectively synthesized via an approach of MetSeO oxidation and deprotection reaction (SeODR), in which the first disulfide bond was constructed through oxidation of dithiol by MetSeO in a neutral buffer, and the second disulfide bond was then directly constructed through the deprotection of two Acm groups or one Acm group and one Thz group by MetSeO in acidic media. Synthesis of two disulfide bonds by the SeODR approach was achieved through a one-pot manner. Moreover, the SeODR approach is compatible with the synthesis of peptides containing methionine residues. Both H+ and Br- drastically promoted the reaction rate of SeODR. The mechanistic picture for the SeODR approach was delineated, in which the formation of a stable Se-X-S bridge as the transition state plays a critical role. The SeODR approach was also utilized to construct the three disulfide bonds in linaclotide, conferring a reasonable yield.

Cost-effective electrodeposited Ni-Co-TiO2 electrodes for boosting hydrogen evolution reaction in acidic and neutral electrolytes

Despite the importance of energy for our life, the rapid consumption of fossil fuels with human population growth worldwide is imperative to find other sustainable sources of energy. A promising strategy is to assess earth-abundant and inexpensive materials as electrocatalysts for efficient green hydrogen production. Herein, nanocrystalline Ni-Co-TiO2 electrodeposited films on copper that have different Co atomic ratios are exploited as target cathodes for hydrogen creation from acid and neutral electrolytes. Different characterization techniques were utilized to identify the chemical composition, morphological structure, crystal lattice system, and unit cell parameters of the obtained bimetals-metal oxide nanocomposites. Ultrasonication with mechanically stirred plating baths was used to ensure the homogenous inclusion of TiO2 nanoparticles inside the bimetallic texture. The electrocatalytic activity of hydrogen evolution reaction (HER) over the designed nanocrystalline cathodes was investigated using voltammetric polarization, chronoamperometry, and electrochemical impedance spectroscopy techniques. The results showed that Ni-Co-TiO2 nanocomposites with somewhat low Co contents (8–9 at.%) displayed superior electrocatalytic HER activity in acid and neutral phosphate buffer (PB) electrolytes, outperforming by many times the other benchmark electrocatalysts. In 0.5 M H2SO4 solution, the Ni-8.2Co-4.8TiO2 cathode demonstrated the highest catalytic activity for the HER, minimal overpotential of 223 mV to deliver a current density of 10 mA cm−2, and small Tafel slope of 74.3 mV dec-1. While the Ni-9.2Co-4TiO2 electrocatalyst required a slightly higher Tafel slope of 79.5 mV dec-1 and an overpotential of 279 mV to convey the same current density of 10 mA cm−2 in 1 M PB electrolyte. The impedance data further confirm these results as these two cathodes showed the lowest resistance for the charge transport process on their surfaces among the other tested ones. The study confirmed that abundant transition bimetals/metal oxide nanocomposites are among the most promising long time stability electrodes for large-scale economic hydrogen fuel production technologies.

Water-Soluble Gd(III)-Porphyrin Complexes Capable of Both Photosensitization and Relaxation Enhancement.

With the aim of developing more stable Gd(III)-porphyrin complexes, two types of ligands 1 and 2 with carboxylic acid anchors were synthesized. Due to the N-substituted pyridyl cation attached to the porphyrin core, these porphyrin ligands were highly water-soluble and formed the corresponding Gd(III) chelates, Gd-1 and Gd-2. Gd-1 was sufficiently stable in neutral buffer, presumably due to the preferred conformation of the carboxylate-terminated anchors connected to nitrogen in the meta position of the pyridyl group helping to stabilize Gd(III) complexation by the porphyrin center. 1H NMRD (nuclear magnetic relaxation dispersion) measurements on Gd-1 revealed high longitudinal water proton relaxivity (r1 = 21.2 mM-1 s-1 at 60 MHz and 25 °C), which originates from slow rotational motion resulting from aggregation in aqueous solution. Under visible light irradiation, Gd-1 showed extensive photoinduced DNA cleavage in line with efficient photoinduced singlet oxygen generation. Cell-based assays revealed no significant dark cytotoxicity of Gd-1, while it showed sufficient photocytotoxicity on cancer cell lines under visible light irradiation. These results indicate the potential of this Gd(III)-porphyrin complex (Gd-1) as a core for the development of bifunctional systems acting as an efficient photodynamic therapy photosensitizer (PDT-PS) with magnetic resonance imaging (MRI) detection capabilities.

Selective Fluorimetric Detection of Pyrimidine Nucleotides in Neutral Aqueous Solution with a Styrylpyridine-Based Cyclophane

A styrylpyridine-containing cyclophane with diethylenetriamine linkers is presented as a host system whose association with representative nucleotides was examined with photometric and fluorimetric titrations. The spectrometric titrations revealed the formation of 1:1 complexes with log Kb values in the range of 2.3–3.2 for pyrimidine nucleotides TMP (thymidine monophosphate), TTP (thymidine triphosphate) and CMP (cytidine monophosphate) and 3.8–5.0 for purine nucleotides AMP (adenosine monophosphate), ATP (adenosine triphosphate), and dGMP (deoxyguanosine monophosphate). Notably, in a neutral buffer solution, the fluorimetric response to the complex formation depends on the type of nucleotide. Hence, quenching of the already weak fluorescence was observed with the purine bases, whereas the association of the cyclophane with pyrimidine bases TMP, TTP, and CMP resulted in a significant fluorescence light-up effect. Thus, it was demonstrated that the styrylpyridine unit is a useful and complementary fluorophore for the development of selective nucleotide-targeting fluorescent probes based on alkylamine-linked cyclophanes.

Effects of different dithiolate ligands on electrocatalytic hydrogen production of nickel complexes in acetic acid or water

In order to solve the problems of fossil energy depletion and environmental pollution, it is necessary to find new energy sources that are recyclable and environmentally friendly. Among them, hydrogen is a high calorific value, clean and sustainable energy. At present, hydrogen is mainly obtained by electrolysis of water. The use of electrocatalysts in this process can effectively reduce the consumption of electrical energy. In this study, three molecular electrocatalysts based on nickel complexes, [BzTPP]2[Ni(mnt)2](1), [BzTPP]2[Ni(i-mnt)2](2), and [BzTPP]2[Ni(tdas)2](3) ([BzTPP]+ = 1-benzyltriphenylphosphinium, mnt = 1,2-dicyanoethylene-1,2-dithiolate, i-mnt = 1,1-dicyanoethylene-2,2-dithiolate and, and tdas = 1,2,5-thiadiazole-3,4-dithiolate), were systemically investigated. Electrochemical study showed three complexes can electrocatalyze hydrogen generation from acetic acid or aqueous buffer solution, and afford a turnover frequency (TOF) of 504.76, 591.52 and 1022.93 mol of hydrogen per mole of catalyst per hour at an overpotential (OP) of 0.838 V from a neutral buffer. It is shown that [NiII(tdas)2]2−exhibits higher electro-catalytic hydrogen production activity than [NiII(mnt)2]2− and [NiII(i-mnt)2]2−. This provides a new chemical paradigm for the design and fabrication of efficient molecular catalysts for water reduction.

Hydrazone-Based Amphiphilic Brush Polymer for Fast Endocytosis and ROS-Active Drug Release.

Due to the high reactivity of reactive oxygen species (ROS), it is essential to sweep them away in time. In this study, ClO--responsible amphiphilic brush polymers were prepared by free radical polymerization using two monomers consisting of polyethylene glycol as the hydrophilic part, and an alkyl chain connected by hydrazone as the hydrophobic part. The macromolecules assemble into particles with nanoscaled dimensions in a neutral buffer, which ensures quick cellular internalization. The polymer has a low critical micellization concentration and can encapsulate hydrophobic drug molecules up to 19% wt. The micelles formed by the polymer disassemble in a ClO--rich environment and release 80% of their cargo within 2 h, which possesses a faster release rate compared to the previous systems. The relatively small size and the quick response of hydrazone toward ClO- ensure a quick uptake and elimination of ROS in vitro and in vivo.

Features of the Distribution of Mast Cell Populations in Lungs and Spleen During Simulated Hypoxic Hypoxia

The aim of research was to study the distribution of mast cell populations in the lungs and spleen depending on their expression of proteases in normal conditions and different periods of simulated hypoxic hypoxia.Material and methods. Hypoxic hypoxia was modeled on 87 outbred male rats using 200 liter chambers. The animals were under experimental conditions for four months. Rats were removed from the experiment after 30, 60, 90 and 120 days. After the animals were removed from the experiment, the lungs and the spleen were removed and fixed in 10% neutral buffer solution of Labiko formalin. Lung and spleen preparations were stained with hematoxylin and eosin and according to Van Gieson. For immunohistochemical studies, a panel of monoclonal antibodies was used: Anti-Mast Cell Tryptase antibody, Anti-Mast Cell Chymase antibody.Results. When modeling hypoxia in the structures of the lungs at the terms of 30 and 60 days, the content of predominantly tryptase-positive cells increased, and by the 90th and 120th days, the content of chymase-positive cells increased. By the end of the experimental exposure, the number of tryptase-positive cells increased by 3 times, and chymase-positive – by 7,7 times compared with the control. As the duration of the experiment increased, the presence of tryptase-positive cells in the spleen increased by 3,5 times, chymase-positive cells – by 7 times in the structures of the red and white pulp.Conclusion. During the formation of chronic hypoxic hypoxia in the body of laboratory animals, there is a redistribution of mast cells expressing tryptase and chymase. The most significant increase in the number of chymase-positive mast cells was noted both in the lungs and in the spleen.

Perylene derivative and persulfate as highly efficient electrochemical system for constructing sensitive amperometric aptasensor

To design highly efficient electrochemistry system was important for construct simple and sensitive biosensors, which was crucial in clinical diagnosis and therapy. In this work, a novel electrochemistry probe N,N′-di (1-hydroxyethyl dimethylaminoethyl) perylene diimide (HDPDI) with positive charges was reported to show two-electron redox behavior in neutral phosphate buffer solution between 0 and -1.0 V. And K2S2O8 in solution could significantly increase the reduction current of HDPDI at −0.29 V, which was interpreted with cyclic catalysis mechanism of K2S2O8. Moreover, HDPDI as electrochemical probe and K2S2O8 as signal enhancer was used to design aptasensors for protein detection. Thrombin was used as target model protein. Thiolate ssDNA with thrombin-binding sequence was immobilized on gold electrode to selectively capture thrombin and adsorb HDPDI. The thiolate ssDNA without binding with thrombin was with random coil structure and could adsorb HDPDI through electrostatic attraction interaction. However, the thiolate ssDNA binding with thrombin became G-quadruplex structure and hardly adsorbed HDPDI. Thus, with increasing the concentration of thrombin, the current signal stepwisely decreased and was taken as detection signal. Compared with other aptasensors based on electrochemistry molecules without signal enhancer, the proposed aptasensors exhibited wider linear response for thrombin between 1 pg mL−1 and 100 ng mL−1 with lower detection limit 0.13 pg mL−1. In addition, the proposed aptasensor showed good feasibility in human serum samples.

Iodine substitution compromises the aggregation-induced emission (AIE) property and enhances the biothiol sensitivity of quinoline–malononitrile-based fluorescent probes

The development of long-wavelength fluorescent probes with a pH working range from weakly acidic to neutral is important for cell-imaging studies. In this study, quinoline-malononitrile (QM)-based fluorescent probes without and with iodine substitution surrounding its phenolic ring are synthesized. The iodine substitution manner was found to compromise the aggregation-induced emission of QM whilst resulting in a significantly enhanced sensitivity for biothiols including l-cystine (L-Cys) detection in weakly acidic to neutral aqueous buffer solutions through reducing the pKa of the QM fluorogen. The probe also effectively achieved the fluorescence visualization of the fluctuation in L-Cys levels in cells.

Pd quantum dot induced changes in the photocatalytic, electrocatalytic, photoelectrochemical and thermoelectric performances of galvanically synthesized Sb2Se3 thin films

We present a novel solution-based galvanic (electrochemical) synthesis of antimony selenide light absorber thin films on FTO (Fluorine doped tin oxide) coated glass substrates. The structural, surface morphological, compositional, optical and electrical properties of the samples were characterized using X-Ray Diffraction (XRD), Field Emission Scanning Electron Microscopy (FESEM), Transmission Electron Microscopy (TEM), Fast Fourier Transform (FFT), Energy Dispersive X-ray spectroscopy (EDS), UV–Vis and Photoluminescence (PL) spectroscopy and Electrochemical Impedance spectroscopy (EIS). X-ray Photoelectron Spectroscopy (XPS) was employed to determine the elemental state composition of as-deposited and annealed Sb2Se3 films containing Pd. The annealed Sb2Se3:Pd films exhibited hydrogen evolution in neutral buffer medium with small potential of 164 mV and a Tafel slope of 39 mV/decade. Pd incorporation enhances the efficiency towards HER performance, which shows the 10 mAcm−2 current density at a low overpotential of 244 mV with reasonable durability. Photoelectrochemical (PEC) water splitting was observed at a potential of 0.41 V versus a reversible hydrogen electrode (RHE) with 3.2 mA cm−2 at 0 V versus RHE, under air mass 1.5 global illumination in a neutral electrolyte. To check the performance of this material for waste water treatment photodegradation of hazardous dyes and metal ion reduction experiments were performed. The photodegradation percentages of MG, MB and RhB dyes with annealed Sb2Se3:Pd thin film catalysts and H2O2 were 99.32% in 10 min, 98.95% in 20 min and 99.16% in 30 min, respectively. Cu2+ ions were photocatalytically reduced with the same catalyst to an extent of 98.9% in 20 min. Thermoelectric measurements on annealed Sb2Se3:Pd yielded the best Figure of merit and power factor values of 39.8 μW/cmK2 and 10.81 × 10−3, respectively at 373 K.

In Situ Electro-organic Synthesis and Functionalization of Catechol Derivative on Carbon Black and Its Interference-free Voltammetric pH Sensor Application ∗

We report an easy electrochemical route for preparing a catechol derivative (di-ferulyl sesamol derivative, designated as CA-Fer) functionalized low-cost carbon black (CB) modified electrode in a neutral pH buffer solution. As synthesized precursor organic compound, CA-Fer has been electrochemically oxidized over the CB by potential cycling treatment in pH 7 PBS that leads to a high redox active CA-Fer-catechol derivative (CB@CA-Fer-Redox), which exhibited a well-defined and stable surface-confined redox response at Eo’ = 0.150 V vs Ag/AgCl with a surface excess value = 50.46 nmol cm−2. Unlike the conventional redox active mediators, GCE/CB@CA-Fer-Redox modified electrode has failed to show any mediated oxidation/reduction response to various electro-active biochemicals and chemicals, viz., ascorbic acid, glucose, cysteine, caffeic acid, hydrazine, hydrogen peroxide, uric acid, dopamine, creatinine, urea, nitrite, sulfide and sulfate ions, depicting a clear advantage of using it as a voltammetric pH sensor for real-time applications. The modified electrode showed a linear voltammetric potential signal against pH in a window, 3–11, with a slope value of (∂Ep/∂pH) = −59 ± 3 mV pH−1. As an independent study, a CA-Fer-Redox modified three-in-one screen printed electrode was developed, and a sensitive voltammetric pH analysis of some chemical biochemical real sample systems were demonstrated.

Growth, pectoralis muscle performance, and testis of pelung cockerels (Gallus gallus gallus [Linnaeus, 1758]) supplemented with blood clam shell powder (Anadara granosa [Linnaeus, 1758]).

Pelung cockerels (Gallus gallus gallus [Linnaeus, 1758]) are different from other native cockerels in that they have a long and unique voice, in addition to their tall, large, and sturdy body with a relatively heavy body weight (BW). The sound quality of pelung cockerels is affected by the structure of the syrinx and their large and strong chest muscles. The performance of the chest muscles, and subsequently its voice, is influenced by the hormone testosterone. The shell of blood clams (Anadara granosa Linnaeus, 1758), a saltwater bivalve is known to contain a natural aromatase blocker (NAB) capable of blocking the aromatase enzyme from converting testosterone to estradiol. This generates consistently high levels of testosterone. This study aimed to determine the effect of blood clam shell powder (BCSP) as an NAB on the growth, pectoralis muscle performance, and testes of pelung cockerels. The study design was a completely randomized design, with 16 pelung cockerels aged 40-56 weeks divided into four treatment groups: T0 (control); T1 (BCSP [A. granosa] 0.9 mg/kg BW); T2 (zinc sulfate [ZnSO4] 0.9 mg/kg BW); and T3 (testosterone 3 mg/day). The animals were acclimatized for 7 days and then given dietary treatments for 56 days. The measurement of the comb, wattle, and chest circumference (CC) of pelung cockerels was performed on days 0, 14, 28, 42, and 56. At the end of the treatment, the pelung cockerels were sacrificed and the data of the pectoralis muscle weight (PMW), testis weight (TW), and area of the pectoralis muscle (APM) were measured. Samples of pectoralis muscle and testes were taken and fixed in 10% neutral buffer formalin for histology. The proliferating cell nuclear antigen (PCNA) was identified by immunohistochemical staining. To measure fascicle area (FA), myofiber area (MA), and enumerate, the fascicle myofibers (NM) histology preparations were stained with hematoxylin and eosin (H and E). Testicular preparations were stained with H and E to measure the diameter of the seminiferous tubules (DST) using ImageJ software. The growth performance on day 56 showed significantly (p < 0.05) higher differences of CC in T1 compared to T2 and T0, in T1 and T3 compared to T0, and in T3 and T2 compared to T0. Pectoralis muscle results, that is, FA, NM, MA, and PCNA-positive cells, showed that cockerels on treatment T3 had significantly higher results than other treatments, T1 was significantly different from T2 and T0, and T2 was significantly different from T0. In addition, the TW and DST measurement of cockerels on treatment T3 were significantly reduced (p < 0.05) than the other treatment groups. The oral administration of BCSP in the role of a NAB at a dose of 0.9 mg/kg BW for 56 days improved the growth performance and pectoralis muscle, especially the CC, FA, NM, MA, and PCNA-positive cells parameters, but did not affect the PMW, APM, and testis of pelung cockerels. The administration of testosterone at 3 mg/day for 56 days contributed to the decrease in TW and DST, as well as atrophy of the seminiferous tubules of pelung cockerels.

Influence of Biological and Environmental Factors in the Extraction and Concentration of Foodborne Pathogens using Glycan-Coated Magnetic Nanoparticles

Rapid detection of foodborne pathogens is essential to preventing foodborne illness outbreaks. Before detection can occur, however, it is often necessary to extract and concentrate bacteria. Conventional methods such as centrifugation, filtration, and immunomagnetic separation can often be time-consuming, ineffective, or costly when working with complex food matrices. This work used cost-effective glycan-coated magnetic nanoparticles (MNPs) for rapid concentration of Escherichia coli O157, Listeria monocytogenes, and Staphylococcus aureus. Glycan-coated MNPs were used to concentrate bacteria from both buffer solution and food matrices while examining the effect of factors including solution pH, bacterial concentration, and target bacterial species. In both pH 7 and reduced pH experiments, successful extraction of bacterial cells occurred in all food matrices and bacteria tested. In neutral pH buffer solution, bacteria were concentrated to 4.55 ± 1.17, 31.68 ± 6.10 and 64.27 ± 16.78 times their initial concentration (mean ± standard deviation) for E. coli, L. monocytogenes and S. aureus, respectively. Successful bacterial concentration occurred in several food matrices, including S. aureus in milk (pH 6), L. monocytogenes in sausage (pH 7), and E. coli O157 in flour (pH 7). The insights gained may facilitate future applications of glycan-coated MNPs to extract foodborne pathogens.

Fusion between bare liposomes and exosomes is enhanced through induced depletion attraction or in acidic conditions.

Exosomes are cell-secreted, nanometer scale extracellular vesicles (EVs) involved in functional miRNA and mRNA transfer from donor to acceptor cells and alteration of gene expression in acceptor cells. In line with their biological function, existing evidence points to reduced immunogenicity, cell-specific binding in vivo, and endosomal escape upon entry into target cells, properties which strongly support the use of exosomes as drug delivery vehicles. Previously, we isolated EVs, defined as a mixture of exosomes and microvesicles, from human prostate (PC3) and melanoma (M21) cancer cells using serial velocity ultracentrifugation. We confirmed these EVs were within the expected 30 - 200 nm diameter range for exosomes and contained the EV marker protein CD63 with nanoparticle tracking analysis and western blot assays [W.S. Fisher, C. Tchounwou, S. Wei, L. Roberts, K.K. Ewert, C.R. Safinya, Exosomes are secreted at similar densities by M21 and PC3 human cancer cells and show paclitaxel solubility, Biochimica et Biophysica Acta (BBA) - Biomembranes, 1864 (2022) 183841]. To enable EVs to serve as drug delivery vehicles, fusion between synthetic liposomes and EVs must be optimized to allow for transfer of desired hydrophobic drugs or nucleic acids from the liposomes to the EVs. We explored two different pathways leading to enhanced fusion. We induced depletion attraction, in reaction mixtures containing polyethylene oxide (PEO) as the depletant, to crowd labeled EVs and liposomes into micron-scale clusters observable in DIC and fluorescence microscopy. The crowding of EVs and synthetic liposomes enhances fusion verified via a Förster resonance energy transfer (FRET) based lipid-mixing assay. With this FRET assay, we also identified a statistically significant enhancement of EV-liposome fusion in acidic (pH 5) compared to neutral (pH 7.4) buffer conditions in mixtures containing no crowding depletants.

Chemical, Crystallinity and Morphological Changes of Rubberwood (Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg.) Hydrothermally Treated in Different Buffered Media

Hydrothermal treatment is an efficient and environmentally friendly method of enhancing the dimensional stability and durability of wood. The destructive effects of formic and acetic acids generated during thermal treatment, on the other hand, are known to have a negative impact on wood strength. As a result, hydrothermal treatment in different buffered media (acidic, neutral, and alkaline buffer) was performed in this study to mitigate the effects of the acids. As heating media, acidic buffer (pH 4 and 6), alkaline buffer (pH 8 and 10), and tap water (pH 7.43) were used to treat rubberwood at three different temperatures (160 °C, 180 °C, and 200 °C). The effects of different buffered media and treatment temperatures on the chemical components, crystallinity, and morphological changes in treated and untreated rubberwood were studied. The results of the experiments revealed that the chemical constituents of rubberwood changed along with the pH of the buffered media and treatment temperature. Rubberwood treated in alkaline media showed the least degradation, whereas water medium caused the most severe degradation. The crystallinity of wood increased initially (from 160 to 180 °C) and then decreased further at 200 °C. In addition, minimal damage to the wood cell was observed in an alkaline medium.

Towards molecularly imprinted polymers that respond to and capture phosphorylated tyrosine epitopes using fluorescent bis-urea and bis-imidazolium receptors.

Early detection of cancer is essential for successful treatment and improvement in patient prognosis. Deregulation of post-translational modifications (PTMs) of proteins, especially phosphorylation, is present in many types of cancer. Therefore, the development of materials for the rapid sensing of low abundant phosphorylated peptides in biological samples can be of great therapeutic value. In this work, we have synthesised fluorescent molecularly imprinted polymers (fMIPs) for the detection of the phosphorylated tyrosine epitope of ZAP70, a cancer biomarker. The polymers were grafted as nanometer-thin shells from functionalised submicron-sized silica particles using a reversible addition-fragmentation chain-transfer (RAFT) polymerisation. Employing the combination of fluorescent urea and intrinsically cationic bis-imidazolium receptor cross-linkers, we have developed fluorescent sensory particles, showing an imprinting factor (IF) of 5.0. The imprinted polymer can successfully distinguish between phosphorylated and non-phosphorylated tripeptides, reaching lower micromolar sensitivity in organic solvents and specifically capture unprotected peptide complements in a neutral buffer. Additionally, we have shown the importance of assessing the influence of counterions present in the MIP system on the imprinting process and final material performance. The potential drawbacks of using epitopes with protective groups, which can co-imprint with targeted functionality, are also discussed.

Cholesterol Assay Based on Recombinant Cholesterol Oxidase, ABTS, and Horseradish Peroxidase.

Cholesterol determination by cholesterol oxidase reaction is a fast, convenient, and highly specific approach with widespread use in clinical diagnostics. Routinely, endpoint measurements with 4-aminophenazone or 4-aminoantipyrine as chromogens and sodium cholate, surfactants, or alcohols as solubilizing agents are used. Here we describe a novel kinetic method to determine cholesterol in 0.05-0.75mM range in neutral or acidic buffers by use of recombinant cholesterol oxidase from Nocardioides simplex in a coupled reaction with horseradish peroxidase, ABTS as a chromogen, and methyl-β-cyclodextrin as a solubilizing agent.

Protocol for the High-quality Plasmid Isolation from Different Recalcitrant Bacterial Species: Agrobacterium spp., Rhizobium sp., and Bacillus thuringiensis.

High yield of good quality plasmid DNA from gram -ve bacteria (Agrobacterium tumefaciens, A. rhizogenes, and Rhizobium sp.) and gram +ve bacterium (Bacillus thuringiensis) is difficult. The widely used plasmid extraction kits for Escherichia coli yield a low quantity of poor-quality plasmid DNA from these species. We have optimized an in-house modification of the QIAprep Spin Miniprep kit protocol of Qiagen, consisting of two extraction steps. In the first, the centrifugation after adding neutralization buffer is followed by ethanol (absolute) precipitation of plasmid DNA. In the second extraction step, the precipitated DNA is dissolved in Tris-EDTA (TE) buffer, followed by an addition of 0.5 volumes of 5 M sodium chloride and 0.1 volumes of 20% (w/v) sodium dodecyl sulfate. After incubation at 65 °C for 15 min, the plasmid DNA is extracted with an equal volume of chloroform:isoamyl alcohol (CIA). RNase (20 mg/mL) is added to the upper phase retrieved after centrifugation and is incubated at 37 °C for 15 min. The extraction of the plasmid DNA with an equal volume of CIA is followed by centrifugation and is precipitated from the retrieved upper phase by adding an equal volume of absolute ethanol. The pellet obtained after centrifugation is washed twice with 70% (v/v) ethanol, air dried, dissolved in TE buffer, and quantified. This easy-to-perform protocol is free from phenol extraction, density gradient steps, and DNA binding columns, and yields high-quality plasmid DNA. The protocol opens an easy scale up to yield a large amount of high-quality plasmid DNA, useful for high-throughput downstream applications. Key features The protocol is free from density gradient steps and use of phenol. The protocol is an extension of the QIAprep Spin Miniprep kit (Qiagen) and is applicable for plasmid DNA isolation from difficult-to-extract bacterial species. The protocol facilitates the direct transformation of the ligation product into Agrobacterium by skipping the step of E. coli transformation. The plasmids isolated are of sequencing grade and the method is useful for extracting plasmids for metagenomic studies. Graphical overview Overview of the plasmid isolation protocol (modified QIAprep Spin Miniprep kit) of the present study.

Semisynthesis of Aminomethyl-Insulin: An Atom-Economic Strategy to Increase the Affinity and Selectivity of a Protein for Recognition by a Synthetic Receptor.

Advancements in the molecular recognition of insulin by nonantibody-based means would facilitate the development of methodology for the continuous detection of insulin for the management of diabetes mellitus. Herein, we report a novel insulin derivative that binds to the synthetic receptor cucurbit[7]uril (Q7) at a single site and with high nanomolar affinity. The insulin derivative was prepared by a four-step protein semisynthetic method to present a 4-aminomethyl group on the side chain of the PheB1 position. The resulting aminomethyl insulin binds to Q7 with an equilibrium dissociation constant value of 99 nM in neutral phosphate buffer, as determined by isothermal titration calorimetry. This 6.8-fold enhancement in affinity versus native insulin was gained by an atom-economical modification (-CH2NH2). To the best of our knowledge, this is the highest reported binding affinity for an insulin derivative by a synthetic receptor. This strategy for engineering protein affinity tags induces minimal change to the protein structure while increasing affinity and selectivity for a synthetic receptor.

Liver Tissue Examination of Mice Using 10% BNF Fixation For 6 Hours And 16 Hours

The fixation process is the first stage in the manufacture of histopathological preparations which aims to preserve the tissue and harden the tissue, so that the tissue to be observed does not change in shape or size. BNF fixation solution (Neutral Buffer Formalin) 10% has been used as a routine fixative and It has been be the gold standard in histology laboratories for decades. The material used in this study was a 10% BNF solution because it is easier to use and it can be used to preserve tissue for a long period of time. This study aims to determine the differences in the microscopic results of the liver tissue of mice (Mus musculus) fixed with 10% BNF for 6 hours and 16 hours. This type of research is descriptive analytic. The research was carried out at the Barokah Laboratory/ dr.Mezfi Unita on 04-05 March 2022 with a total sample of 20 samples of mice liver tissue (Mus musculus). The results of the study on 10% BNF fixation for 6 hours and 16 hours showed that the average results were not good. The conclusion from the results of the study that there was no difference in the microscopic results of liver tissue that were fixed with 10% BNF for 6 hours and 16 hours using Hematorylin-Eosin staining. 10% BNF fixation with a time of 6 hours and 16 hours can be used as an alternative in histopathological examination in the short term and long term.