Soluble Fraction Research Articles

Genome-wide analysis of TFIIB's role in termination of transcription.

Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIB's role in termination, we performed GRO-seq analyses in sua7-1 mutant (TFIIB sua7-1 ) and the isogenic wild type (TFIIB WT ) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIB sua7-1 mutant compared to the TFIIB WT cells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIB sua7-1 and TFIIB WT cells. TFIIB purified from the chromatin fraction of TFIIB WT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIB sua7-1 mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered nearly 90% decline in the mutant over wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.

Differentiation of cellular responses to particulate and soluble constituents in sunscreen products

Despite the growing awareness of potential human and environmental risks associated with sunscreens, identifying the specific constituents responsible for their potential toxicity is challenging. In this study, we applied three different types of sunscreens with contrasting compositions and compared the effects of their particulate and soluble fractions based on 15 cellular biomarkers of HaCaT cells. Multilinear regression analysis revealed that the internalized soluble fractions played a primary role in the overall cytotoxicity of sunscreen mixtures, which was primarily attributed to their biotransformation, generating metabolites with higher toxicity. The presence of plastic microspheres in sunscreens either inhibited the internalization of soluble fractions or led to their redistribution toward lysosomes. Conversely, subcellular toxicity resulting from the sunscreen mixture was predominantly influenced by particulates. Bio-transformable particulates such as ZnO dissolved in the organelles and induced higher subcellular toxicity compared to bioinert particulates such as microplastics. Subcellular biomarkers including lysosomal count, lysosomal size, mitochondrial count and mitochondrial shape emerged as the potential predictors of sunscreen presence. Our study provides important understanding of sunscreen toxicity by elucidating the differential impacts of particulate and soluble fractions in mixture contaminants.

Valorization of lignite to arene-rich compounds via two-stage selective cleavage of C-O bridged bonds over Co-MoS2

Directly converting lignite into arenes as commodity chemicals and drop-in fuels is a highly desirable target for researchers. However, this is severely limited by the presence of stable inter unit C-O linkages in lignite. Herein, a highly active catalyst, Co-MoS2, has been designed and prepared to be used for the selective catalytic cleavage of inter unit C-O bridged bonds (BBs) in the conversion of Naomaohu (NMH), a lignite, to yield arenes. Both catalytic hydrogenation conversion (CHC) and thermal dissolution (a non-catalytic process, named NCHC) of NMH were conducted using n-hexane as the solvent. The soluble fractions from the first stage of NCHC (SFNCHC-1) and CHC (SFCHC-1), as well as the second stage of NCHC (SFNCHC-2) and CHC (SFCHC-2) were obtained using the same solvent. After the first-stage catalytic treatment, the relative contents of arenes and alkanes in SFCHC-1 increased by 16.7 % and 9.3 %, respectively, compared to those in SFNCHC-1, and the relative content of oxygen-containing compounds (OCCs), such as alcohols, phenols, esters, ketones, acids, and furans, decreased by 13.2 %. For the second-stage conversion, the relative contents of arenes and alkanes in SFCHC-2 were 53.5 % and 15.5 % higher than those in SFNCHC-2, and the relative contents of OCCs was only 6.6 % in SFCHC-2. The catalytic mechanisms of cleavage of C-O BBs and deoxygenation were proposed based on the CHC of model compounds and density functional theory (DFT) calculation. The adsorption of OCCs and the activation of released hydrogen radical (H·) by Co-MoS2, followed by H· addition to the carbon atom connected to oxygen in C-O BBs and oxygen-containing functional groups, are crucial for the cleavage of C-O BBs and formation of deoxygenated compounds, particularly arenes.

Alteration of gene expression and protein solubility of the PI 5-phosphatase SHIP2 are correlated with Alzheimer’s disease pathology progression

A recent large genome-wide association study has identified EGFR (encoding the epidermal growth factor EGFR) as a new genetic risk factor for late-onset AD. SHIP2, encoded by INPPL1, is taking part in the signalling and interactome of several growth factor receptors, such as the EGFR. While INPPL1 has been identified as one of the most significant genes whose RNA expression correlates with cognitive decline, the potential alteration of SHIP2 expression and localization during the progression of AD remains largely unknown. Here we report that gene expression of both EGFR and INPPL1 was upregulated in AD brains. SHIP2 immunoreactivity was predominantly detected in plaque-associated astrocytes and dystrophic neurites and its increase was correlated with amyloid load in the brain of human AD and of 5xFAD transgenic mouse model of AD. While mRNA of INPPL1 was increased in AD, SHIP2 protein undergoes a significant solubility change being depleted from the soluble fraction of AD brain homogenates and co-enriched with EGFR in the insoluble fraction. Using FRET-based flow cytometry biosensor assay for tau-tau interaction, overexpression of SHIP2 significantly increased the FRET signal while siRNA-mediated downexpression of SHIP2 significantly decreased FRET signal. Genetic association analyses suggest that some variants in INPPL1 locus are associated with the level of CSF pTau. Our data support the hypothesis that SHIP2 is an intermediate key player of EGFR and AD pathology linking amyloid and tau pathologies in human AD.

Antioxidant inactivated yeast: High potential of non-Saccharomyces specific metabolome

Understanding the contribution of new natural sources of antioxidant compounds to the stability of wines is of great interest in a context of reduction of sulfites. Here, we investigated the antioxidant potential of selected inactivated non-Saccharomyces yeast (INSY) along with related chemical fingerprints, using combined untargeted UHPLC-Q-ToF MS and DPPH analyses. 4 INSY species were compared to a reference inactivated Saccharomyces cerevisiae yeast (ISY) selected for its high antioxidant capacity. Our results show that, all the INSY can accumulate GSH during the specific production process with yields ranging from +170 % to +360 % compared to the corresponding classical production process. The principal component analysis of the 3511 ions detected by UHPLC-Q-ToF-MS clearly grouped INSY by species, independently of the production process. One INSY exhibited equivalent antioxidant capacity to the control ISY, but with a GSH concentration four times lower (4.73 ± 0.09 mg/g against 20.95 ± 0.34 mg/g, respectively). 73 specific ions presenting strong and significant spearman correlation (rho < −0.6, p-value < 0.05) with the DPPH scores, clustered the most antioxidant INSY and the control Saccharomyces in different groups, indicating that the antioxidant capacity of these two products should be driven by different pools of compounds. These results point out that, GSH alone is not relevant to explain the antioxidant capacity of INSY soluble fractions and other more reactive compounds must be considered, which opens an avenue for the selection new species with great enological potential.

Application of pressure homogenization on whole human milk pasteurized by high hydrostatic pressure: Effect on protein aggregates in milk fat globule membrane and skim milk phases

This study explored the impact of homogenization (at pressures of 16, 30, and 45 MPa) on both raw and high hydrostatic pressure (HHP)-treated human milk (HM). It focused on protein compositions and binding forces of soluble and insoluble fractions for both milk fat globule membrane (MFGM) and skim milk. Mild homogenization of HHP-treated milk increased lactoferrin (LF) levels in the insoluble fractions of both MFGM and skim milk, due to insoluble aggregation through hydrophobic interactions. Intense homogenization of HHP-treated milk decreased the LF level in the MFGM fractions due to the LF desorption from the MFGM, which increased LF level in the insoluble skim milk fraction. Homogenized-HHP treated milk showed noticeably higher casein (CN) level at the MFGM compared to homogenized-raw milk, attributed to HHP effect on CN micelles. Overall, the combined use of HHP and shear-homogenization should be avoided as it increased the biological proteins in insoluble fractions.

Elevated Temperature Effects on Protein Turnover Dynamics in Arabidopsis thaliana Seedlings Revealed by 15N-Stable Isotope Labeling and ProteinTurnover Algorithm.

Global warming poses a threat to plant survival, impacting growth and agricultural yield. Protein turnover, a critical regulatory mechanism balancing protein synthesis and degradation, is crucial for the cellular response to environmental changes. We investigated the effects of elevated temperature on proteome dynamics in Arabidopsis thaliana seedlings using 15N-stable isotope labeling and ultra-performance liquid chromatography-high resolution mass spectrometry, coupled with the ProteinTurnover algorithm. Analyzing different cellular fractions from plants grown under 22 °C and 30 °C growth conditions, we found significant changes in the turnover rates of 571 proteins, with a median 1.4-fold increase, indicating accelerated protein dynamics under thermal stress. Notably, soluble root fraction proteins exhibited smaller turnover changes, suggesting tissue-specific adaptations. Significant turnover alterations occurred with redox signaling, stress response, protein folding, secondary metabolism, and photorespiration, indicating complex responses enhancing plant thermal resilience. Conversely, proteins involved in carbohydrate metabolism and mitochondrial ATP synthesis showed minimal changes, highlighting their stability. This analysis highlights the intricate balance between proteome stability and adaptability, advancing our understanding of plant responses to heat stress and supporting the development of improved thermotolerant crops.

Interfacial and foaming properties of soluble lupin protein isolates: Effect of pH

Lupins are rich in proteins (∼40%) and fibers (∼40%), and have been widely used for animal feed, but limitedly for human consumption due to the presence of alkaloids. Due to their high protein content, lupins have a high potential to be used as protein sources in diverse food manufacturing processes, including emulsion and foam stabilization. However, the interfacial properties of lupin protein isolates (LPI), especially their performance at different pH values, are not well understood so far. Here, we systematically investigated the air-water interfacial and foaming properties of the soluble fraction of LPI at pH 7.0, pH 6.0, pH 4.0, and pH 3.5. We observed that LPI at pH 4.0 (LPI-4) and pH 3.5 (LPI-3.5) adsorbed faster at the air-water interface than at pH 7.0 (LPI-7) and pH 6.0 (LPI-6) due to a smaller particle size and higher surface hydrophobicity of LPI-4 and LPI-3.5. This resulted in a higher foam overrun of LPI-4 and LPI-3.5 (300% and 331%, respectively). The air-water interfaces formed by LPI at different pH values were dramatically different in terms of interfacial structure and mechanical properties. LPI-4 and LPI-3.5 formed stiff and solid-like interfaces, leading to higher foam stability. In contrast, for LPI-7 there were clearly more protein aggregates at the interface, and this structure was weaker and less stretchable in response to shear and dilatational deformation, causing lower foam stability. LPI-6 also showed weaker air-water interfaces and thus formed less stable foams. Overall, LPI at acid pH (3.5 and 4) has a better performance in foam stabilization than at a pH close to neutral (6 and 7). Our results suggest that LPI can be potentially used as a plant-based clean-label additive in the production of foam-based food products in acidic environments.

Comments Regarding “Solubility determination and thermodynamic model analysis of L-α-glyceryl phosphorylcholine in different organic solvents of 278.15–323.15 K”

A polemic is given regarding several of the calculated curve-fit parameters that Zhou and coworkers reported in their published paper. The calculated curve-fit parameters for the Combined Nearly Ideal Binary Solvent/Redlich-Kister, Jouyban-Acree-van’t Hoff, Sun and modified Apelblat models were found to give mole fraction solubilities that exceeded unity. Our analysis also found that the mean relative absolute percent deviations provided by the authors were significantly underestimated.

Measurement and correlation of solubility data for sodium picosulphate in selected pure solvents and mixed-solvent systems from 298.15 K to 328.15 K

An investigation was carried out to determine the solubility of the stimulant laxative drug Sodium Picosulphate (SP). A wide range of pure solvents were studied, including methanol, ethanol, 1-propanol, 1-butanol, dimethyl formamide, dimethyl sulfoxide, acetonitrile, 1,4-dioxane, acetic acid, and water. More to the point, the solubility of the compound was determined using mixed solvent systems; methanol + acetonitrile and water + acetonitrile. Experimental mole fraction solubility of SP was determined by the gravimetric method over a range of temperatures (298.15 K to 328.15 K) under atmospheric pressure (p=0.1MPa). According to experimental results, a temperature-dependent relationship exists between solubility and temperature. Further, we correlated the experimental mole fraction solubility data with theoretical models including Apelblat, Yaws, Van't Hoff, Modified Jouyban-Acree, CNIBS/R-K models. It appears that the results are well in agreement with the experimental results due to the small values of relative average deviation (RAD) and root mean square deviation (RMSD).The solubility data might be useful in the various areas such as modification, production, crystallization, and separation of SP.

Parascedosporium putredinis NO1 tailors its secretome for different lignocellulosic substrates.

Parascedosporium putredinis NO1 is a plant biomass-degrading ascomycete with a propensity to target the most recalcitrant components of lignocellulose. Here we applied proteomics and activity-based protein profiling (ABPP) to investigate the ability of P. putredinis NO1 to tailor its secretome for growth on different lignocellulosic substrates. Proteomic analysis of soluble and insoluble culture fractions following the growth of P. putredinis NO1 on six lignocellulosic substrates highlights the adaptability of the response of the P. putredinis NO1 secretome to different substrates. Differences in protein abundance profiles were maintained and observed across substrates after bioinformatic filtering of the data to remove intracellular protein contamination to identify the components of the secretome more accurately. These differences across substrates extended to carbohydrate-active enzymes (CAZymes) at both class and family levels. Investigation of abundant activities in the secretomes for each substrate revealed similar variation but also a high abundance of "unknown" proteins in all conditions investigated. Fluorescence-based and chemical proteomic ABPP of secreted cellulases, xylanases, and β-glucosidases applied to secretomes from multiple growth substrates for the first time confirmed highly adaptive time- and substrate-dependent glycoside hydrolase production by this fungus. P. putredinis NO1 is a promising new candidate for the identification of enzymes suited to the degradation of recalcitrant lignocellulosic feedstocks. The investigation of proteomes from the biomass bound and culture supernatant fractions provides a more complete picture of a fungal lignocellulose-degrading response. An in-depth understanding of this varied response will enhance efforts toward the development of tailored enzyme systems for use in biorefining.IMPORTANCEThe ability of the lignocellulose-degrading fungus Parascedosporium putredinis NO1 to tailor its secreted enzymes to different sources of plant biomass was revealed here. Through a combination of proteomic, bioinformatic, and fluorescent labeling techniques, remarkable variation was demonstrated in the secreted enzyme response for this ascomycete when grown on multiple lignocellulosic substrates. The maintenance of this variation over time when exploring hydrolytic polysaccharide-active enzymes through fluorescent labeling, suggests that this variation results from an actively tailored secretome response based on substrate. Understanding the tailored secretomes of wood-degrading fungi, especially from underexplored and poorly represented families, will be important for the development of effective substrate-tailored treatments for the conversion and valorization of lignocellulose.

Pharmacological Profile Analysis of a Vegetable Crop Pumpkin (Cucurbita maxima Linn.) Seeds

The seeds of pumpkin (Cucurbita maxima Linn.) can be utilized as both a conventional and multifunctional dietary constituent. In this study the antioxidant, antibacterial, and cytotoxic effects of various fractions such as petroleum ether soluble fraction (PESF), chloroform soluble fraction (CFSF), dichloromethane soluble fraction (DCMSF), and aqueous soluble fraction (AQF) were obtained from crude extract (CE) of the C. maxima seeds were assessed by using 1,1-Diphenyl-2- Picrylhydrazyl (DPPH) method, disc diffusion technique, and brine shrimp lethality bioassay respectively. The results showed that all the fractions have remarkable antioxidant, antibacterial, and cytotoxic activities. The study revealed that both the AQF and PESF and the CF confirmed significant antioxidant properties. The antibacterial activity of different fractions against Gram-positive and Gram-negative bacteria was examined to determine their significant effect. PESF and CFSF fractions exhibited substantial inhibitory activity against Gram-positive bacteria, particularly Bacillus cereus. Bacillus subtilis exhibited significant susceptibility to both CFSF and AQF. Salmonella typhi and Shigella dysenteriae exhibited significant susceptibility to PESF and CFSF, while Escherichia coli was the Gram-negative bacterium most effectively inhibited by CFSF. The IC50 values revealed that the DCMSF exhibited the highest inhibitory activity, followed by other fractions. The new study emphasizes the significance of pumpkin seeds in terms of their pharmacological and biological properties, which may encourage more research on the potential benefits of this plant for human health. Jagannath University Journal of Life and Earth Sciences, 9(1): 7-18, 2023 (June)

L-fuculokinase-catalyzed phosphorylation and its use for the straightforward synthesis of L-fuculose-1-phosphate

L-Fuculokinases play a key role in both natural and synthetic metabolic pathways, not only for the utilization of L-fucose, but also for related monosaccharides, and catalyze selective phosphorylation of L-fuculose in the 1-position or of D-ribulose in the 5-position. Therefore, L-fuculokinases have been selected, the fucK gene from Escherichia coli K12 has been synthesized and cloned into an appropriate expression vector for the transformation of E. coli BL21(DE3). The cultivation of the transformed strain and the overexpression of the fucK gene has led to highly active L-fuculokinase after straightforward downstream processing. The soluble fraction from the crude cell extract could be directly applied in the enzymatic phosphorylation of L-fuculose. Recycling of the ATP cofactor was done with pyruvate kinase (PK) and the high energy phosphoenolpyruvate (PEP) as donor. The time course of the phosphorylation reactions has been monitored by 31P-NMR and 1H-NMR spectroscopy and L-fuculose could be phosphorylated with nearly 100% conversion. After standard workup, the L-fuculose-1-phosphate has been obtained in high yield and purity.

Supplementation with soluble or insoluble rice-bran fibers increases short-chain fatty acid producing bacteria in the gut microbiota in vitro.

Studies have shown that a diet high in fiber and prebiotics has a positive impact on human health due largely to the fermentation of these compounds by the gut microbiota. One underutilized source of fiber may be rice bran, a waste product of rice processing that is used most frequently as an additive to livestock feed but may be a good source of fibers and other phenolic compounds as a human diet supplement. Previous studies focused on specific compounds extracted from rice bran showed that soluble fibers extracted from rice bran can improve glucose response and reduce weight gain in mouse models. However, less is known about changes in the human gut microbiota in response to regular rice bran consumption. In this study, we used a Simulator of the Human Intestinal Microbial Ecology (SHIME®) to cultivate the human gut microbiota of 3 different donors in conditions containing either soluble or insoluble fiber fractions from rice bran. Using 16S rRNA amplicon sequencing and targeted metabolomics via Gas Chromatography-Mass Spectrometry, we explored how gut microbial communities developed provided different supplemental fiber sources. We found that insoluble and soluble fiber fractions increased short-chain fatty acid production, indicating that both fractions were fermented. However, there were differences in response between donors, for example the gut microbiota from donor 1 increased acetic acid production with both fiber types compared with control; whereas for donors 2 and 3, butanoic acid production increased with ISF and SF supplementation. Both soluble and insoluble rice bran fractions increased the abundance of Bifidobacterium and Lachnospiraceae taxa. Overall, analysis of the effect of soluble and insoluble rice bran fractions on the human in vitro gut microbiota and the metabolites produced revealed individually variant responses to these prebiotics.

In vivo assessment of TiO2 based wear nanoparticles in periprosthetic tissues

A multimodal approach combining inductively coupled plasma mass spectrometry (ICP-MS), single-particle ICP-MS (spICP-MS), scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS) and Raman spectroscopy enabled a deeper insight into the balance between total titanium (Ti), the soluble titanium fraction and titanium dioxide based particle fraction levels in periprosthetic tissues collected from patients undergoing revision surgery. Hydrofluoric acid usage in the sample digestion allowed for complete digestion of TiO2 particles, thus enabling accurate estimation of total Ti levels. The TiO2 fraction represents 38–94% of the titanium load in the six samples where particles were detected, and the fraction is present mainly in samples from patients with aseptically loosened total hip arthroplasty. Further attention was given to this fraction determining the elemental composition, particle count, particle size and modification of TiO2. The spICP-MS analysis confirmed the presence of the TiO2-derived (nano)particles (NPs) with a 39- to 187-nm median size and particle count up to 2.3 × 1011 particles per gram of tissue. On top of that, the SEM-EDS confirmed the presence of the TiO2 nanoparticles with 230-nm median size and an anatase crystal phase was determined by Raman spectroscopy. This study presents a novel multimodal approach for TiO2 particle determination and characterization in tissue samples and is the first in vivo study of this character.

Efficient Removal of Water Soluble Fraction of Diesel Oil by Biochar Sorption Supported by Microbiological Degradation

The contamination of the water bodies by diesel oil (DO) and its water-soluble fraction (WSF) represents one of the most challenging tasks in the management of polluted water streams. This paper contains data related to the synthesis and characteristics of the plum stone biochar material (PmS-B), which was made from waste plum stones (PmS), along with its possible application in the sorption of the WSF of DO from contaminated water. Techniques applied in sample characterisation and comparisons were: Elemental Organic Analysis (EOA), Scanning Electron Microscopy−Energy Dispersive X-ray Spectroscopy (SEM-EDX), Fourier Transform Infrared Spectroscopy (FTIR), pH (pHsus) and point of zero charge (pHpzc). In order to increase the overall efficiency of the removal process, sorption and bioremediation were subsequently combined. Firstly, PmS-B was used as a sorbent of WSF, and then the remaining solution was additionally treated with a specific consortium of microorganisms. After the first treatment phase, the initial concentration of diesel WSF was reduced by more than 90%, where most of the aromatic components of DO were removed by sorption. The sorption equilibrium results were best fitted by the Sips isotherm model, where the maximum sorption capacity was found to be 40.72 mg/g. The rest of the hydrocarbon components that remained in the solution were further subjected to the biodegradation process by a consortium of microorganisms. Microbial degradation lasted 19 days and reduced the total diesel WSF concentration to 0.46 mg/L. In order to confirm the non-toxicity of the water sample after this two-stage treatment, eco-toxicity tests based on a microbial biosensor (Aliivibrio fischeri) were applied, confirming the high efficiency of the proposed method.

Expression of soluble moloney murine leukemia virus-reverse transcriptase in Escherichia coli BL21 star (DE3) using autoinduction system.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

New 19(10 → 9)abeo-euphane-type triterpenoids from Trichilia dregeana leaves and NO production inhibitory activities

Phytochemical investigation of the EtOAc soluble fraction from leaves of Trichilia dregeana Sond. (Meliaceae) afforded naturally rare four new pentacyclic triterpenoids (1–4), together with five known pentacyclic analogs (5–8, and 11) and two steroids (9 and 10). Their structures were elucidated by extensive spectroscopic techniques such as 1D and 2D NMR and HRESIMS data analyses. The absolute configuration of 1 was determined by using the single-crystal X-ray diffraction analysis. The nitric oxide (NO) production inhibitory assay indicated that the EtOAc fraction as well as 4 and 7 inhibited the NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with the IC50 values of 83.53 μg/mL and 81.31 and 85.71 μM, respectively. Compounds 1–4 are rare 19(10 → 9)abeo-euphane-type triterpenoids bearing a 3,10-ether bridge. To the best of our knowledge, this study is the first isolation of triterpenoids with the 3,10-ether bridge in their skeleton from the genus Trichilia, providing new insights into the chemodiversity of the terpenoids in T. dregeana.

Heterologous expression of nattokinase in E. coli: Biochemical characterization and functional analysis of fibrin binding residues

Heterologous expression of nattokinase, a potent fibrinolytic enzyme, has been successfully carried out in various microorganisms. However, the successful expression of this enzyme as a soluble protein was not achieved in E. coli. This study delves into the expression of nattokinase in E. coli as a soluble protein followed by its biochemical characterization and functional analysis for fibrinolytic activity. E. coli BL21C41 and pET32a vector host strain with pGro7 protein chaperone induced with IPTG at 16 °C 180 rpm for 16 h enabled the production of recombinant nattokinase in soluble fraction. Enzymatic assays demonstrated its protease activity, while characterization revealed optimal catalytic conditions at 37 °C and pH 8.0, with remarkable stability over a broad pH range (6.0–10.0) and up to 50 °C. The kinetic constants were determined as follows: Km = 25.83 ± 3.43 μM, Vmax = 62.91 ± 1.68 μM/s, kcat = 38.45 ± 1.06 s−1, and kcat/Km = 1.49 × 106 M−1 s−1. In addition, the fibrinolytic activity of NK, quantified by the fibrin plate hydrolysis assay was 1038 ± 156 U/ml, with a corresponding specific activity of 1730 ± 260 U/mg and the assessment of clot lysis time on an artificial clot (1 mg) was found to be 51.5 ± 2.5 min unveiling nattokinase's fibrinolytic potential. Through molecular docking, a substantial binding energy of −6.46 kcal/mol was observed between nattokinase and fibrin, indicative of a high binding affinity. Key fibrin binding residues, including Ser300, Leu302, and Asp303, were identified and confirmed. These mutants affected specifically the fibrin binding and not the proteolytic activity of NK. This comprehensive study provides crucial conditions for the expression of protein in soluble form in E. coli and biochemical properties paving the way for future research and potential applications in medicine and biotechnology.

In-vitro and molecular docking studies of plant secondary metabolites isolated from Hypericum oblongifolium as antibacterial agents and lipoxygenase (5-LOX) inhibitors

Natural Products are bases for modern pharmaceuticals as they are precursors for many synthetic drugs. People also use natural products directly in the form of herbal medicines due to their less harmful effects. This research project aimed to investigate the new pharmacological potential of natural products found in Hypericum oblongifolium. Five compounds were identified from chloroform and ethyl acetate soluble fractions of H. oblongifolium. These compounds were identified as 21-β-hydroxyursolic acid (1), β-sitosterol (2), 7,4′-dihydroxy-5,3′-dimethoxyisoflavone (3), α-D-glucopyranosyl-6′-O-hexadecanoate (4), Quercetin-3′-O-ß-D-glucopyranoside (5). When these compounds were tested for antibacterial potential compound 5 showed maximum inhibition against gram-positive Bacillus subtilis and was highly active against Escherichia coli gram-negative bacterium. Minimum inhibition was shown by compounds 2 and 3 against Shigella flexneri among gram-negative bacteria. Compound 5 also exhibited the least binding energy in the molecular modeling of these compounds against Escherichia coli proteins GlcN-6-P synthase (PDB ID: 1MOQ). Compound 5 also showed good inhibition of lipoxygenase (5-LOX) enzyme with an IC50 value of 30.1 ± 0.20 as it exhibited maximum hydrogen bondings and minimum energy when docked with LOX-5 (PDB ID 3V99) using a patch dock.