Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. Using mouse genetics, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase subunit Tir1. Rapid degradation of condensin I and condensin II, two essential regulators of mitotic chromosome structure, revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

Effect of Chemical Treatment Sequence on Pineapple Peel Fiber: Chemical Composition, Thermal Stability and Thermal Degradation Kinetics

Millions of tons of fruit wastes are generated globally every year from residual agriculture, which makes essential to find alternative uses to increase their aggregate value and reduce the impact of environmental damage. The present study aimed to explore pineapple peel as an alternative source of cellulose by evaluating its composition and physical properties, which are essential to provide a clue to its application function diverse. Cellulose was extracted by a sequence of chlorine-free treatments to delignify the fresh pineapple peels, followed by characterization using chemical composition, XRD, FTIR, SEM and TGA to determine its crystallinity, structural properties, morphology thermal characteristics, and thermal degradation kinetic study. The result revealed that the pineapple peel amorphous segments containing hemicelluloses and lignin were extensively removed with increasing chemical treatments, leading to increased purity, crystallinity index and thermal stability of the extracted materials. The maximum degradation, and crystallinity index of the 2B isolated from the PPF are 150 °C and 80.91% respectively. The cellulose content increased from 24.05% (pineapple peel) to 80.91% (bleached cellulose). These results indicated that pretreatment via bleaching has suitable potential applications in nanocrystal production and suggests possible uses in the development of cellulose nanocrystal and application for packaging films.

Effect of the Progressive Increase of Organic Loading Rate in an Anaerobic Sequencing Batch Reactor for Biodiesel Wastewater Treatment

The wastewater from the biodiesel industry is an environmental problem, and from a sanitation resources perspective, the anaerobic sequencing batch reactor (ASBR) is an interesting alternative for wastewater treatment. A better understanding of ASBR operation behavior under the progressive increase of the organic loading rate (OLR) is crucial for upscaling. The objective of this study was to monitor an ASBR operating with an OLR ranging from 1.3 to 9.3 kgCOD m−3 d−1. The average chemical oxygen demand (COD) removal efficiencies of the ASBR were 52, 41, 47, and 11% for phases 1, 2, 3, and 4, respectively. The apparent kinetic coefficient, i.e., the rate of degradation of organic matter, was between 0.10 and 1.80 h−1, considering the kinetic model that considers the residual substrate concentration, which was the one that best fit the obtained data. The progressive increase in applied OLR modified the microbial biomass diversity, which in turn influenced the degradation kinetics of the organic matter. In addition, the values of the applied OLR of 5.1 kgCOD m−3 d−1 and a food to microorganism ratio (F/M) of 0.6 kgCOD kgVSS−1 d−1 were shown to be limiting values that promoted the overload of ASBR.

Thermal Stability and Inhibitory Action of Red Grape Skin Phytochemicals against Enzymes Associated with Metabolic Syndrome

The present study focuses on heat-induced structural changes and the degradation kinetics of phytochemicals and antioxidant activity of red grape skin extract. The thermal degradation of anthocyanins, flavonoids, polyphenols, and antioxidant activity followed a first-order kinetic model, increasing with temperature due to the intensification of the degradation process. The activation energy (Ea) highlighted this phenomenon. Likewise, the kinetic and thermodynamic parameters certified the irreversible degradation of the bioactive compounds from the skin of the Băbească neagră grape variety. Both temperature and duration of heating had a significant impact on the content of bioactive compounds. In addition, the red grape skin extract inhibited certain enzymes such as α-amylase, α-glucosidase, lipase, and lipoxygenase, which are associated with metabolic syndrome and inflammation. Further knowledge on the possible inhibition mechanisms exerted by the major anthocyanins found in red grape skin extract on the metabolic syndrome-associated enzymes was gathered upon running molecular docking tests. Detailed analysis of the resulting molecular models revealed that malvidin 3-O-glucoside binds in the vicinity of the catalytic site of α-amylase and lipase, whereas no direct contact with catalytic amino acids was identified in the case of α-glucosidase and lipoxygenase.

Degradation kinetics and in vitro digestive stability of selected bioactive compounds from a beverage formulated with butterfly pea flowers

ABSTRACT: This study evaluated the effects of temperature on the pH of extracts of ascorbic acid and anthocyanins from petals of butterfly pea, as well as their in vitro digestive stability in model systems at 60, 70, and 80 °C. The pH values significantly decreased with an increase in the temperature (P < 0.05). The findings were similar for the degradation of anthocyanins and ascorbic acid, which followed first-order kinetics in all the systems. The samples heated at 80 °C presented the highest degradation rate (kobs), as well as higher percentages of degradation at the end of digestive stability in vitro.

Ethoxy acetalated dextran-based nanocarriers accomplish efficient inhibition of leukotriene formation by a novel FLAP antagonist in human leukocytes and blood

Leukotrienes are pro-inflammatory lipid mediators generated by 5-lipoxygenase aided by the 5-lipoxygenase-activating protein (FLAP). BRP-201, a novel benzimidazole-based FLAP antagonist, inhibits leukotriene biosynthesis in isolated leukocytes. However, like other FLAP antagonists, BRP-201 fails to effectively suppress leukotriene formation in blood, which limits its therapeutic value. Here, we describe the encapsulation of BRP-201 into poly(lactide-co-glycolide) (PLGA) and ethoxy acetalated dextran (Ace-DEX) nanoparticles (NPs), aiming to overcome these detrimental pharmacokinetic limitations and to enhance the bioactivity of BRP-201. NPs loaded with BRP-201 were produced via nanoprecipitation and the physicochemical properties of the NPs were analyzed in-depth using dynamic light scattering (size, dispersity, degradation), electrophoretic light scattering (effective charge), NP tracking analysis (size, dispersity), scanning electron microscopy (size and morphology), UV–VIS spectroscopy (drug loading), an analytical ultracentrifuge (drug release, degradation kinetics), and Raman spectroscopy (chemical attributes). Biological assays were performed to study cytotoxicity, cellular uptake, and efficiency of BRP-201-loaded NPs versus free BRP-201 to suppress leukotriene formation in primary human leukocytes and whole blood. Both PLGA- and Ace-DEX-based NPs were significantly more efficient to inhibit leukotriene formation in neutrophils versus free drug. Whole blood experiments revealed that encapsulation of BRP-201 into Ace-DEX NPs strongly increases its potency, especially upon pro-longed (≥ 5 h) incubations and upon lipopolysaccharide-challenge of blood. Finally, intravenous injection of BRP-201-loaded NPs significantly suppressed leukotriene levels in blood of mice in vivo. These results reveal the feasibility of our pharmacological approach using a novel FLAP antagonist encapsulated into Ace-DEX-based NPs with improved efficiency in blood to suppress leukotriene biosynthesis.

Stability-Indicating UHPLC Method for the Determination of Desvenlafaxine: Application to Degradation Kinetics

This study was aimed to investigate the degradation behavior and physicochemical stability of desvenlafaxine using reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) system. The chromatogram was developed on Eclipse XDB-C8 column (150 x 4.6 mm, 5μm). The eluents were monitored through a photo diode array plus (PDA+) detector at 210 nm using an isocratic method with a flow rate of 1.5 ml/min. Mobile phase composition was 30:70 v/v mixture of 0.1 % trifluoroacetic acid (TFA) in water and methanol. Forced degradation studies were performed on drug substance of desvenlafaxine as per International Conference on Harmonization (ICH) prescribed stressed conditions (Q1A(R2) and Q1B) using hydrolytic (acidic, basic, and neutral), oxidative and photolytic methods. The drug substance was found highly labile to acidic (0.5 N hydrochloric acid, 18.65 % degradation in 2 hours at 70°C), basic (1.0 N sodium hydroxide, 11.01 % degradation in 12 hours at 70°C) and oxidative (3 % hydrogen peroxide, 17.05 % degradation in 2 hours at 50°C) stressed conditions, but a great resistance was observed towards dry heat (maximum degradation 0.27 % in 10 days from ambient to higher temperature, 80°C), moist heat (maximum degradation 0.25 % in 2 hours at 80°C and 75 % relative humidity) as well as in photolytic degradation (maximum degradation 0.23 % in 10 days at UV light of 315 - 400 nm). A pseudo-first order kinetic was followed in acidic, basic and peroxide degradation methods which paved a way to calculate the half-life of the drug substance desvenlafaxine under ICH mentioned stressed conditions. The results were also statistically analyzed and the % RSD values were compared with recommended guidelines. Dhaka Univ. J. Pharm. Sci. 20(2): 167-176, 2021 (December)