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The taste receptor type 1 (T1r) family perceives 'palatable' tastes. These receptors function as T1r2-T1r3 and T1r1-T1r3 heterodimers to recognize a wide array of sweet and umami (savory) tastes in sugars and amino acids. Nonetheless, it is unclear how diverse tastes are recognized by so few receptors. Here we present crystal structures of the extracellular ligand-binding domains (LBDs), the taste recognition regions of the fish T1r2-T1r3 heterodimer, bound to different amino acids. The ligand-binding pocket in T1r2LBD is rich in aromatic residues, spacious and accommodates hydrated percepts. Biophysical studies show that this binding site is characterized by a broad yet discriminating chemical recognition, contributing for the particular trait of taste perception. In contrast, the analogous pocket in T1r3LBD is occupied by a rather loosely bound amino acid, suggesting that the T1r3 has an auxiliary role. Overall, we provide a structural basis for understanding the chemical perception of taste receptors.
Linseed (Linum usitatissimum L.) is becoming more and more important in the health food market as a functional food, since its seeds and oil represent a rich source of bioactive compounds. Its chemical composition is strongly correlated with, and dependent on, genetic characteristics. The aim of this study was to evaluate the variation in seed yield, oil content, fatty acid composition and secondary metabolite profiles between a low-linolenic linseed variety, belonging to the Solin-type group (Solal), and a high-linolenic traditional one (Bethune), cultivated, both as spring crops, in open field conditions of Central Italy. The achieved results pointed out the different behavior of the two varieties in terms of growth cycle, oil content, and some important yield components, such as capsule number per plant and thousand seed weight. There were also significant differences in seed composition regarding total phenols, total flavonoids, antioxidant activities as well as in carotenoid, tocopherol, and tocotrienol profiles between the two varieties. In particular, Solal was characterized by the greatest contents of oil, phenols, flavonoids, α- and δ- tocotrienol, together with the highest antioxidant activity. Bethune, on the contrary, showed the highest amounts of carotenoids (lutein and β-carotene). These results indicate a clear effect of the genetic characteristics on the biosynthesis of these secondary metabolites and, consequently, on the related antioxidant activity. Our findings suggest that the mutation process, responsible for the selection of the low-linolenic cultivar, is able to modify the biosynthetic pathways of carotenoids and phenolics.
Metal bioaccessibility is an alarming issue in croplands of mining sites due to overloading of toxic metals. Hence, the present study is aimed to determine the overloading of toxic metal in croplands across the Tawag village, Hutti, Raichur, India. Correspondingly, to identify the soil bacterial growth, physiological oxidative stress enzyme activity and surface macromolecular functional group evolution were analysed in and around the toxic metal contaminated sites through FT-IR and FT-Raman spectrometry. The evaluated results attribute that the study area is heavily polluted with the toxic metals such as arsenic, cadmium, chromium, lead and zinc. However, biochemical and 16S rRNA gene sequence homology tree confirmed that the arsenic and cadmium-resistant isolate belongs to Bacillus sp. MDPMK-02 and retrieved unique Gene Bank ID KT596811 (accession number) at National Centre for Biotechnology information (NCBI), India. Additionally, sodium arsenite-amended culture media possessing reduced biomass and enhanced the activity of oxidative stress defence enzymes such as superoxide dismutase (SOD) and catalase (CAT) than cadmium chloride-amended medium and control. Subsequently, the infrared (IR) and Raman spectral analytical assessment distinguish that arsenic-treated Gram-positive isolate membrane fetched high percentage of hydration, elevation of surface polysaccharides, proteins and polyhydroxybutyric acid (PHBA) molecular specific stretch intensity compared to cadmium exposures. From these results, the study concluded that the mining wastes significantly pollute the surrounding croplands, and also Bacillus sp. MDPMK-02 possesses good chemosensing for cross-protection and bio-adaptation of toxic metal ions. Hence, these isolates can be compiled and implemented in environmental hazardous management techniques such as bioremediation, bioleaching and biodegradation.
The development of point-of-care detection methodologies for biologically relevant analytes that can facilitate rapid and appropriate treatment is at the forefront of current research efforts and interests. Among the various approaches, those exploiting host-guest chemistries where the optoelectronic signals of the chemical sensor can be modulated upon interaction with the target analyte are of particular interest. In aiding their rational development, judicious selection of peripheral functional groups anchored to core motifs with desired properties is critical. Herein, we report an in-depth investigation of the binding of three psychoactive substances, MDAI, mexedrone, and phenibut, to receptors of the monoamine transporters for dopamine, norepinephrine, and serotonin, particularly focusing on the role of individual amino acid residues. We first evaluated the conformational flexibility of the ligands by comparing their experimentally determined crystal structure geometries to those optimized by means of quantum as well as molecular mechanics, observing significant changes in the case of phenibut. Molecular docking studies were employed to identify preferential binding sites by means of calculated docking scores. In all cases, irrespective of the monoamine transporter, psychoactive substances exhibited preferred interaction with the S1 or central site of the proteins, in line with previous studies. However, we observed that experimental trends for their relative potency on the three transporters were only reproduced in the case of mexedrone. Subsequently, to further understand these findings and to pave the way for the rational development of superior chemical sensors for these substances, we computed the individual contributions of each nearest neighbor amino acid residue to the binding to the target analytes. Interestingly, these results are now in agreement with those experimental potency trends. In addition, these observations were in all cases associated with key intermolecular interactions with neighboring residues, such as tyrosine and aspartic acid, in the binding of the ligands to the monoamine transporter for dopamine. As a result, we believe this work will be of interest to those engaged in the rational development of chemical sensors for small molecule analytes as well as to those interested in the use of computational approaches to further understand protein-ligand interactions.
Many cyanobacteria produce extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides with unique characteristics that make them suitable for biotechnological applications. However, manipulation/optimization of EPS biosynthesis/characteristics is hindered by a poor understanding of the production pathways and the differences between bacterial species. In this work, genes putatively related to different pathways of cyanobacterial EPS polymerization, assembly, and export were targeted for deletion or truncation in the unicellular Synechocystis sp. PCC 6803. No evident phenotypic changes were observed for some mutants in genes occurring in multiple copies in Synechocystis genome, namely ∆wzy (∆sll0737), ∆wzx (∆sll5049), ∆kpsM (∆slr2107), and ∆kpsM∆wzy (∆slr2107∆sll0737), strongly suggesting functional redundancy. In contrast, Δwzc (Δsll0923) and Δwzb (Δslr0328) influenced both the amount and composition of the EPS, establishing that Wzc participates in the production of capsular (CPS) and released (RPS) polysaccharides, and Wzb affects RPS production. The structure of Wzb was solved (2.28 Å), revealing structural differences relative to other phosphatases involved in EPS production and suggesting a different substrate recognition mechanism. In addition, Wzc showed the ATPase and autokinase activities typical of bacterial tyrosine kinases. Most importantly, Wzb was able to dephosphorylate Wzc in vitro, suggesting that tyrosine phosphorylation/dephosphorylation plays a role in cyanobacterial EPS production.
Radix Scutellaria-Licorice drug pair (RSLDP), a frequently used herbal pair with the effect of clearing heat and detoxifying, is the commonly employed drug pair in TCM prescriptions for the treatment of COVID-19. Until now, the metabolism feature and anti-COVID-19 mechanism of RSLDP have not been fully elucidated. In this study, a sensitive and rapid method was developed for the separation and identification of the absorbed constituents of RSLDP in the rat plasma by UHPLC-QTOF-MS. Additionally, we optimized the conventional methodologies of network pharmacology and proposed a new concept called target network pharmacology (T-NP). It used the absorbed constituents and the corresponding targets to generate a compound-target network, and compared to conventional network pharmacology, it could reduce false-positive results. A total of 85 absorbed constituents were identified or tentatively characterized in dosed plasma, including 32 components in the group of Radix Scutellaria, 27 components in the group of Licorice, and 65 components in the group of RSLDP. The results showed that the compatibility of Radix Scutellaria and Licorice increased the number of components in vivo. We found that 106 potential targets among the 61 active compounds in RSLDP were related to COVID-19. And 12 targets (STAT3, AKT1, EGFR, HSP9AA1, MAPK3, JUN, IL6, VEGFA, TNF, IL2, RELA, and STAT1) could be core targets for RSLDP in treating COVID-19. Results from these targets indicate that RSLDP treatment of COVID-19 mainly involves response to chemical stress, response to oxygenates, positive regulation of cytokines, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway for diabetic complications, virus-related pathways such as novel coronavirus and human cytomegalovirus infection, inflammatory immune-related pathways, and so on. The metabolism feature of RSLDP in vivo was systematically uncovered. The combined use of the T-NP method could discover potential drug targets and disclose the biological processes of RSLDP, which will clarify the potential mechanisms of RSLDP in the treatment of COVID-19.
NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD(+) degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H(2)O(2) produced during the oxidative process or added exogenously.
Polysorbate 80 for injection (TW80) is a common excipient used for injection whose macromolecular impurities, including those that cause anaphylactoid reactions, are frequently ignored. The main aim of this study was to prove that the macromolecular impurities in the excipient are an important cause of anaphylactoid reactions. Component A (containing macromolecules > 100 kDa), Component B (containing macromolecules from 10 to 100 kDa), and Component C (containing substances < 10 kDa) were prepaired from the original TW80 using ultrafilters. The original TW80 contained numerous substances with molecular weights > 10kD. The original TW80 and Components A and B caused strong anaphylactoid reactions in both guinea pigs and rabbits by intravenous administration. Moreover, the original TW80 and Components A and B even caused strong passive cutaneous anaphylactoid (PCA) reactions and pulmonary capillary permeability. The PCA reaction and increased permeability were partly prevented by cromolyn sodium. Additionally, the original TW80 and Components A and B caused vasodilation and severe hemolysis in vitro. The anaphylactoid reactions were associated with histamine release but not with mast cell degranulation. Nevertheless, Component C almost caused no anaphylactoid reactions or hemolysis and was weaker in the few reactions that ocurred. Taken together, these results suggest that macromolecular substances are one of the main risk factors responsible for anaphylactoid reactions and hemolysis caused by TW80.
Here we present a new biomaterial based on cellulose, collagen and polyurethane, obtained by dissolving in butyl imidazole chloride. This material served as a matrix for the incorporation of tannin and lipoic acid, as well as bioactive substances with antioxidant properties. The introduction of these bioactive principles into the base matrix led to an increase of the compressive strength in the range 105-139 kPa. An increase of 29.85% of the mucoadhesiveness of the film containing tannin, as compared to the reference, prolongs the bioavailability of the active substance; a fact also demonstrated by the controlled release studies. The presence of bioactive principles, as well as tannins and lipoic acid, gives biomaterials an antioxidant capacity on average 40%-50% higher compared to the base matrix. The results of the tests of the mechanical resistance, mucoadhesiveness, bioadhesiveness, water absorption and antioxidant capacity of active principles recommend these biomaterials for the manufacture of cosmetic masks or patches.
Cell-penetrating peptides (CPPs) are commonly used substances enhancing the cellular uptake of various cargoes that do not easily cross the cellular membrane. CPPs can be either covalently bound directly to the cargo or they can be attached to a transporting system such as a polymer carrier together with the cargo. In this work, several CPP-polymer conjugates based on copolymers of N-(2-hydroxypropyl)methacrylamide (pHPMA) with HIV-1 Tat peptide (TAT), a minimal sequence of penetratin (PEN), IRS-tag (RYIRS), and PTD4 peptide, and the two short hydrophobic peptides VPMLK and PFVYLI were prepared and characterized. Moreover, the biological efficacy of fluorescently labeled polymer carriers decorated with various CPPs was compared. The experiments revealed that the TAT-polymer conjugate and the PEN-polymer conjugate were internalized about 40 times and 15 times more efficiently than the control polymer, respectively. Incorporation of dodeca(ethylene glycol) spacer improved the cell penetration of both studied polymer-peptide conjugates compared to the corresponding spacer-free polymer conjugates, while the shorter tetra(ethylene glycol) spacer improved only the penetration of the TAT conjugate but it did not improve the penetration of the PEN conjugate. Finally, a significantly improved cytotoxic effect of the polymer conjugate containing anticancer drug pirarubicin and TAT attached via a dodeca(ethylene glycol) was observed when compared with the analogous polymer-pirarubicin conjugate without TAT.
Today, the application of polyaniline in biomedicine is widely discussed. However, information about impurities released from polyaniline and about the cytotoxicity of its precursors aniline, aniline hydrochloride, and ammonium persulfate are scarce. Therefore, cytotoxicity thresholds for the individual precursors and their combinations were determined (MTT assay) and the type of cell death caused by exposition to the precursors was identified using flow-cytometry. Tests on fibroblasts revealed higher cytotoxicity of ammonium persulfate than aniline hydrochloride. Thanks to the synergic effect, both monomers in combination enhanced their cytotoxicities compared with individual substances. Thereafter, cytotoxicity of polyaniline doped with different acids (sulfuric, nitric, phosphoric, hydrochloric, and methanesulfonic) was determined and correlated with impurities present in respective sample (HPLC). The lowest cytotoxicity showed polyaniline doped with phosphoric acid (followed by sulfuric, methanesulfonic, and nitric acid). Cytotoxicity of polyaniline was mainly attributed to the presence of residual ammonium persulfate and low-molecular-weight polar substances. This is crucial information with respect to the purification of polyaniline and production of its cytocompatible form.
Alginate is one of the main extracellular polymeric substances (EPS) in biofilms of Cystic Fibrosis (CF) patients suffering from pulmonary infections. Gentamicin sulfate (GS) can strongly bind to alginate resulting in loss of pharmacological activity; however neither the mechanism nor its repercussion is fully understood. In this study, we investigated how GS modifies the alginate macromolecular network and its microenvironment.
Several ruminant animals rely almost exclusively on the complex polysaccharide matrix from the plant cell wall (CW) as their primary energy source via volatile fatty acids produced through ruminal and some hindgut fermentation processes. The CW contains different types and proportions of polysaccharides, proteins, phenolic compounds, and minerals in their macromolecular structure that influence the rate and extent of fiber digestion and selective retention of particulate matter due to its physical characteristics (buoyancy and comminuting) in the reticulorumen. The biosynthetic formation of the CW dictates possible manipulation mechanisms (targeted plant and microbes selection) and processing methods (physical, chemical, microbial, and enzymatic treatments and the use of genetically engineered bacteria) to increase its digestibility, leading to better utilization of the CW by the ruminant animal and hopefully lower the contribution of ruminants' greenhouse gas emissions. Early studies on lignin biosynthesis have led to more advanced studies focusing on replacing traditional monolignols with homopolymers that are easier to deconstruct or degrade. Concurrently, laboratory methods must be developed, evaluated, and modified to accurately reflect the digestibility and nutritive value of CW brought about by modern manipulation mechanisms or processing methods. However, the laboratory methods must also be reliable, precise, feasible, trivial, easy to implement, and cost-effective, but at the same time environmentally friendly and aware. For instance, although the acid detergent lignin has been demonstrated to behave uniformly as a nutritional entity, its chemical determination and association with carbohydrates still lack consensus. Spectroscopy (near-infrared and Raman) and in vitro gas production techniques have been adopted to assess plant chemical composition and nutritive value, but an incomplete understanding of the impacts caused by disrupting the CW for sample processing still exists. Different variations of multicompartmental and time- and age-dependent mathematical models have been proposed to determine the ruminal rates of degradation and passage of fiber. However, low-quality and incomplete data due to inconsistent marker results used to determine passage rates and transit time of fiber in the gastrointestinal tract have hindered advancements and adoptions of the next generation of computer models to understand ruminal fiber degradation.
New Delhi metallo-β-lactamase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to all common β-lactam antibiotics. Understanding the molecular basis of β-lactam hydrolysis by NDM is crucial for designing NDM inhibitors or β-lactams resistant to their hydrolysis. In this study, for the first time, NMR was used to study the influence of Zn(II) ions on the dynamic behavior of NDM-1. Our results highlighted that the binding of Zn(II) in the NDM-1 active site induced several structural and dynamic changes on active site loop 2 (ASL2) and L9 loops and on helix α2. We subsequently studied the interaction of several flavonols: morin, quercetin, and myricetin were identified as natural and specific inhibitors of NDM-1. Quercetin conjugates were also synthesized in an attempt to increase the solubility and bioavailability. Our NMR investigations on NDM-1/flavonol interactions highlighted that both Zn(II) ions and the residues of the NDM-1 ASL1, ASL2, and ASL4 loops are involved in the binding of flavonols. This is the first NMR interaction study of NDM-1/inhibitors, and the models generated using HADDOCK will be useful for the rational design of more active inhibitors, directed against NDM-1.
In order to address the potential toxicity of metal-based magnetic resonance imaging (MRI) contrast agents (CAs), a concept of non-metallic MRI CAs has emerged. Currently, paramagnetic nitroxides (such as (2,2,5,5-tetramethylpyrrolidine-1-oxyl, PROXYL), (2,2,6,6-tetramethylpiperidine-1-oxide, TEMPO), etc.) are being extensively studied because their good stability and imaging mechanism are similar to metal-based contrast agents (such as Gd3+ chelate-based clinical CAs). However, a lower relaxivity and rapid in vivo metabolism of nitroxides remain to be addressed. Previous studies have demonstrated that the construction of macromolecular nitroxides contrast agents (mORCAs) is a promising solution through macromolecularization of nitroxides (i.e., use of large molecules to carry nitroxides). Macromolecular effects not only increase the stability of nitroxides by limiting their exposure to reductive substances in the body, but also improve the overall 1H water relaxation by increasing the concentration of nitroxides and slowing the molecular rotation speed.
Recently, suitably sized polymer-based nanogels containing functional groups for the binding of biologically active substances and ultimately degradable to products that can be removed by glomerular filtration have become extensively studied systems in the field of drug delivery. Herein, we designed and tailored the synthesis of hydrophilic and biodegradable poly[N-(2-hydroxypropyl) methacrylamide-co-N,N'-bis(acryloyl) cystamine-co-6-methacrylamidohexanoyl hydrazine] (PHPMA-BAC-BMH) nanogels. The facile and versatile dispersion polymerization enabled the preparation of nanogels with a diameter below 50 nm, which is the key parameter for efficient and selective passive tumor targeting. The effects of the N,N'-bis(acryloyl) cystamine crosslinker, polymerization composition, and medium including H2O/MetCel and H2O/EtCel on the particle size, particle size distribution, morphology, and polymerization kinetics and copolymer composition were investigated in detail. We demonstrated the formation of a 38 nm colloidally stable PHPMA-BAC-BMH nanogel with a core-shell structure that can be rapidly degraded in the presence of 10 mM glutathione solution under physiologic conditions. The nanogels were stable in an aqueous solution modeling the bloodstream; thus, these nanogels have the potential to become highly important carriers in the drug delivery of various molecules.
Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules' diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a βM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been characterized as a pandemic around the world. Cardiac complications can occur in patients with COVID-19 and can be fatal in severe cases. Recently, it was reported that SARS-CoV-2 used the angiotensin-converting enzyme 2 (ACE2) as a cellular receptor to gain entry into the host cell. However, whether SARS-CoV-2 can directly infect heart tissues and the potential mechanism of cardiac injury in COVID-19 have not been determined.
The present study was directed to investigate the effect of precotreatment with (E)-N'-(1-(7-hydroxy-2-oxo-2H-chromen-3-yl) ethylidene) benzohydrazide (7-hyd.HC), a novel potent synthesized coumarin, on isoproterenol- (ISO-) induced myocardial infarction (MI) in rats. The hydrazone compound was characterized by IR, 1D, and 2D NMR analyses. Experimental induction of MI in rats was established by ISO (85 mg/kg/day, s.c) for two consecutive days (6th and 7th days). 7-hyd.HC or sintrom was given for 7 days prior and simultaneous to ISO injection. 7-hyd.HC offered a cardiopreventive effect by preventing heart injury marker leakage (LDH, ALT, AST, CK-MB, and cTn-I) from cardiomyocytes and normalizing cardiac function and ECG pattern, as well as improving lipid profile (TC, TG, LDL-C, and HDL-C), which were altered by ISO administration. Moreover, 7-hyd.HC precotreatment significantly mitigated the oxidative stress biomarkers, as evidenced by the decrease of lipid peroxidation and the increased level of the myocardial GSH level together with the SOD, GSH-Px, and catalase activities. 7-hyd.HC inhibited the cardiac apoptosis by upregulating the expression of Bcl-2 and downregulating the expression of Bax and caspase-3 genes. In addition, 7-hyd.HC reduced the elevated fibrinogen rate and better prevented the myocardial necrosis and improved the interstitial edema and neutrophil infiltration than sintrom. Overall, 7-hyd.HC ameliorated the severity of ISO-induced myocardial infarction through improving the oxidative status, attenuating apoptosis, and reducing fibrinogen production. The 7-hyd.HC actions could be mediated by its antioxidant, antiapoptotic, and anti-inflammatory capacities.
Crystal structure of photosystem II (PSII) has been reported from prokaryotic cyanobacteria but not from any eukaryotes. In the present study, we improved the purification procedure of PSII dimers from an acidophilic, thermophilic red alga Cyanidium caldarium, and crystallized them in two forms under different crystallization conditions. One had a space group of P222(1) with unit cell constants of a=146.8 A, b=176.9 A, and c=353.7 A, and the other one had a space group of P2(1)2(1)2(1) with unit cell constants of a=209.2 A, b=237.5 A, and c=299.8 A. The unit cell constants of both crystals and the space group of the first-type crystals are different from those of cyanobacterial crystals, which may reflect the structural differences between the red algal and cyanobacterial PSII, as the former contains a fourth extrinsic protein of 20 kDa. X-ray diffraction data were collected and processed to a 3.8 A resolution with the first type crystal. For the second type crystal, a post-crystallization treatment of dehydration was employed to improve the resolution, resulting in a diffraction data of 3.5 A resolution. Analysis of this type of crystal revealed that there are 2 PSII dimers in each asymmetric unit, giving rise to 16 PSII monomers in each unit cell, which contrasts to 4 dimers per unit cell in cyanobacterial crystals. The molecular packing of PSII within the unit cell was constructed with the molecular replacement method and compared with that of the cyanobacterial crystals.
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