RESUMEN

Huanglongbing, also known as citrus greening, is currently the most devastating citrus disease with limited success in prevention and mitigation. A promising strategy for Huanglongbing control is the use of antimicrobials fused to a carrier protein (phloem protein of 16 kDa or PP16) that targets vascular tissues. This study investigated the effects of genetically modified citrus trees expressing Citrus sinensis PP16 (CsPP16) fused to human lysozyme and ß-defensin-2 on the soil microbiome diversity using 16S amplicon analysis. The results indicated that there were no significant alterations in alpha diversity, beta diversity, phylogenetic diversity, differential abundance, or functional prediction between the antimicrobial phloem-overexpressing plants and the control group, suggesting minimal impact on microbial community structure. However, microbiota diversity analysis revealed distinct bacterial assemblages between the rhizosphere soil and root environments. This study helps to understand the ecological implications of crops expressing phloem-targeted antimicrobials for vascular disease management, with minimal impact on soil microbiota.

Asunto(s)

Bacterias , Citrus , Microbiota , Floema , Enfermedades de las Plantas , Rizosfera , Microbiología del Suelo , Floema/microbiología , Floema/metabolismo , Bacterias/genética , Bacterias/clasificación , Bacterias/metabolismo , Bacterias/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Citrus/microbiología , Plantas Modificadas Genéticamente/microbiología , Plantas Modificadas Genéticamente/genética , Filogenia , Metagenómica , Muramidasa/metabolismo , Muramidasa/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , beta-Defensinas/genética , ARN Ribosómico 16S/genética , Antiinfecciosos/farmacología , Antiinfecciosos/metabolismo , Citrus sinensis/microbiología , Raíces de Plantas/microbiología

RESUMEN

The use of computer simulation for binding affinity prediction is growing in drug discovery. However, its wider use is constrained by the accuracy of the free energy calculations. The key sources of error are the force fields used to depict molecular interactions and insufficient sampling of the configurational space. To improve the quality of the force field, we developed a Python-based computational workflow. The workflow described here uses the minimal basis iterative stockholder (MBIS) method to determine atomic charges and Lennard-Jones parameters from the polarized molecular density. This is done by performing electronic structure calculations on various configurations of the ligand when it is both bound and unbound. In addition, we validated a simulation procedure that accounts for the protein and ligand degrees of freedom to precisely calculate binding free energies. This was achieved by comparing the self-adjusted mixture sampling and nonequilibrium thermodynamic integration methods using various protein and ligand conformations. The accuracy of predicting binding affinity is improved by using MBIS-derived force field parameters and a validated simulation procedure. This improvement surpasses the chemical precision for the eight aromatic ligands, reaching a root-mean-square error of 0.7 kcal/mol.

Asunto(s)

Muramidasa , Unión Proteica , Termodinámica , Muramidasa/química , Muramidasa/metabolismo , Ligandos , Electrones , Bacteriófago T4/enzimología , Mutación , Conformación Proteica , Simulación de Dinámica Molecular , Modelos Moleculares

RESUMEN

This study is an extension of our previous studies in which the lysozyme was isolated and purified from Bacillus subtilis BSN314 (Naveed et al., 2022; Naveed et al., 2023). In this study, the lysozyme genes were cloned into the E. coli BL21. For the expression of lysozyme in E. coli BL21, two target genes, Lyz-1 and Lyz-2, were ligated into the modified vector pET28a to generate pET28a-Lyz1 and pET28a-Lyz2, respectively. To increase the production rate of the enzyme, 0.5-mM concentration of IPTG was added to the culture media and incubated at 37 °C and 220 rpm for 24 h. Lyz1 was identified as N-acetylmuramoyl-L-alanine amidase and Lyz2 as D-alanyl-D-alanine carboxypeptidase. They were purified by multi-step methodology (ammonium sulfate, precipitation, dialysis, and ultrafiltration), and antimicrobial activity was determined. For Lyz1, the lowest MIC/MBC (0.25 µg/mL; with highest ZOI = 22 mm) were recorded against Micrococcus luteus, whereas the highest MIC/MBC with lowest ZOI were measured against Salmonella typhimurium (2.50 µg /mL; with ZOI = 10 mm). As compared with Aspergillus oryzae (MIC/MFC; 3.00 µg/mL), a higher concentration of lysozyme was required to control the growth of Saccharomyces cerevisiae (MIC/MFC; 50 µg/mL). Atomic force microscopy (AFM) was used to analyze the disintegrating effect of Lyz1 on the cells of selected Gram-positive bacteria, Gram-negative bacteria, and yeast. The AFM results showed that, as compared to Gram-negative bacteria, a lower concentration of lysozyme (Lyz1) was required to disintegrate the cell of Gram-positive bacteria.

Asunto(s)

Antiinfecciosos , Muramidasa , Muramidasa/genética , Muramidasa/farmacología , Muramidasa/metabolismo , Escherichia coli , Antiinfecciosos/farmacología , Bacillus subtilis/genética

RESUMEN

The amyloid fibres have been related to many diseases. The molten globule intermediate has been proposed to form part of the folding pathway of many proteins. In the present study, we investigated the mechanism of amyloid-fibres formation of hen egg-white lysozyme (HEWL) incubated in a potassium phosphate buffer, pH 11.8, 100 mM, at 37 °C for 30 h, and evaluated the influence of Cu(II) present in two salts (CuSO4 and CuCl2) during fibrillogenesis. Co-incubation and post-incubation of lysozyme with copper salts reduced the fluorescence signal of thioflavin T with an increment in the intrinsic fluorescence of the protein. The ANS fluorescence test showed that incubation of HEWL for 6 h generated a molten globule intermediate state that formed amyloid fibres when incubation was carried out for a 30-h timespan. Dynamic light scattering showed a heterogeneous population of states in samples incubated in the absence or the presence of salts during the fibrillation process. The existence of a reducing potential was verified during the formation of HEWL amyloid fibres with the bathocuproine disulphonate test. Transmission electron microscopy confirmed the presence and absence of fibres in solutions incubated with and without Cu(II). This work demonstrated that lysozyme formed amyloid fibres at 37 °C and copper inhibited its formation.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Muramidasa , Sales (Química) , Sales (Química)/farmacología , Muramidasa/metabolismo , Cobre , Dispersión Dinámica de Luz , Amiloide

RESUMEN

Several applications arise from the confinement of proteins on surfaces because their stability and biological activity are enhanced. It is also known that the way in which a protein adsorbs on the surface is important for its biological function since its active sites should not be obstructed. In this study, the adsorption properties of hen egg-white lysozyme, HEWL, into a negatively charged silica pore is examined by employing a coarse-grained model and constant-pH Monte Carlo simulations. The role of electrostatic interactions is taken into account via including the Debye-Hückel potentials into the Cα structure-based model. We evaluate the effects of pH, salt concentration, and pore radius on the protein preferential orientation and spatial distribution of its residues regarding the pore surface. By mapping the residues that stay closer to the pore surface, we find that the increase of pH leads to orientational changes of the adsorbed protein when the solution pH gets closer to the HEWL isoelectric point. Under these conditions, the pKa shift of these important residues caused by the adsorption into the charged confining surface results in a HEWL charge distribution that stabilizes the adsorption in the observed protein orientation. We compare our observations to the results of the pKa shift for HEWL available in the literature and to some experimental data.

Asunto(s)

Muramidasa/química , Adsorción , Animales , Pollos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Método de Montecarlo , Muramidasa/metabolismo , Protones

RESUMEN

Different evidences suggest that pericardial cells play an important role during the immune response against pathogens that invade the mosquito hemocoel. Previously, we identified two lysozyme genes in Anopheles albimanus heart transcriptome. The present study showed that one of these genes (IDVB: AALB004517) has high percentage of identity to mosquito lysozyme genes related to immunity, suggesting its possible participation during the mosquito immune response. This An. albimanus gen, constitutively expressed lysozyme c-1 mRNA (albLys c-1) in mosquito heart; however, it was overexpressed in bacteria-injected mosquitoes. In heart extract samples, we identified a protein of approximately 14 kDa (likely lysozyme c-1), which lysed M. luteus. In addition, mRNA-FISH assay in heart samples, showed specific fluorescent hybridization signal in pericardial cells from M. luteus-injected mosquitos. We conclude that for the first time an inducible immune factor (lysozyme c-1) is identified in Anopheles albimanus mosquito pericardial cells, which could be a key component in the response against pathogens that interact with the mosquito heart.

Asunto(s)

Anopheles/inmunología , Escherichia coli/fisiología , Infecciones por Bacterias Grampositivas/inmunología , Proteínas de Insectos/metabolismo , Micrococcus luteus/fisiología , Muramidasa/metabolismo , Pericardio/metabolismo , Animales , Clonación Molecular , Biología Computacional , Proteínas de Escherichia coli/inmunología , Inmunidad Innata , Proteínas de Insectos/genética , Muramidasa/genética , Pericardio/patología , Filogenia , Transcriptoma , Regulación hacia Arriba

RESUMEN

Wound repair is a complex process that calls for strategies to allow a rapid and effective regeneration of injured skin, which has stimulated the research of advanced wound dressings. Herein, highly porous membranes of N,O-carboxymethylchitosan (CMCh), and poly (vinyl alcohol) (PVA) were successfully prepared via a green and facile freeze-drying method of blend solutions containing CMCh/PVA at weight ratio 25/75. Membranes composed only by CMCh were also prepared and genipin was used for crosslinking. Different contents of TiO2 nanoparticles were incorporated to both type of membranes, which were characterized in terms of morphology, porosity (Φ), swelling capacity (S.C.), mechanical properties, susceptibility to lysozyme degradation and in vitro cytotoxicity toward human fibroblast (HDFn) and keratinocytes (HaCaT) cells. Larger apparent pores were observed in the surface of the genipin-crosslinked CMCh membrane, which resulted in higher porosity (Φ ≈ 76%) and swelling capacity (S.C. ≈ 1720%) as compared to CMCh/PVA membrane (Φ ≈ 68%; S.C. ≈ 1660%). The porosity of both types of membranes decreased upon the addition of TiO2 nanoparticles while swelling capacity increased. Due to their high porosity and swelling capacity, adequate mechanical properties, controlled degradability, and cytocompatibility, such carboxymethylchitosan-based membranes are potentially useful as wound dressings.

Asunto(s)

Vendajes , Quitosano/análogos & derivados , Membranas Artificiales , Cicatrización de Heridas/efectos de los fármacos , Muerte Celular , Supervivencia Celular/efectos de los fármacos , Quitosano/farmacología , Reactivos de Enlaces Cruzados/química , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células HaCaT , Humanos , Iridoides/química , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Muramidasa/metabolismo , Alcohol Polivinílico/química , Porosidad , Espectrometría por Rayos X , Estrés Mecánico , Titanio/química

RESUMEN

In this work, the influence of Sodium Acetate Trihydrate (SAT) on the gelling stage of a chitin hydrogel was studied. Characterization techniques, such as FTIR, Raman, solid-state NMR, Dielectric Spectroscopy, Small-angle X-ray scattering (SAXS), Wide-angle X-ray scattering (WAXS), and X-ray diffraction (XRD) were used to study the effect of SAT on the micro and nanostructure of the material in the wet, dry and freeze-dried states. It was demonstrated that the amount of SAT in the gelling solution can induce a variation in the supramolecular interaction among the polysaccharide chains, which leads to a change in the structural characteristics. In addition, it was observed that the polymer-water interactions are also altered by this structural ordering. Also, the affinity interaction with lysozyme was evaluated and an influence on the adsorption capacity was evidenced with the use of SAT. This could be an advance for biotechnological, biomedical, and food applications.

Asunto(s)

Quitina/química , Geles/química , Acetato de Sodio/química , Acetatos/química , Adsorción , Coloides , Liofilización/métodos , Espectroscopía de Resonancia Magnética/métodos , Muramidasa/metabolismo , Nanoestructuras/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodos

RESUMEN

We describe the synthesis of polymer monoliths inside polypropylene tubes from ink pens. These tubes are cheap, chemically stable, and resistant to pressure. UV-initiated grafting with 5 wt% benzophenone in methanol for 20 min activated the internal surface, thus enabling the covalent binding of ethylene glycol dimethacrylate, also via photografting. The pendant vinyl groups attached a poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith prepared via photopolymerization. These tubes measured 100-110 mm long, with 2 mm of internal diameter. The parent monoliths were functionalized with Na2 SO3 or iminodiacetate to produce strong and weak cation exchangers, respectively. The columns exhibited permeabilities varying from 2.7 to 3.3 × 10-13  m2 , which enabled the separation of proteins at 500 µL/min and back pressures <2.8 MPa. Neither structure collapse nor monolith detachment occurred at flow rates as high as 2.0 mL/min, which produced back pressures between 6.9 and 9.0 MPa. The retention times of ovalbumin, ribonuclease A, cytochrome C, and lysozyme in salt gradient at pH 7.0 followed the order of increasing isoelectric points, confirming the cation exchange mechanism. Separation and determination of lysozyme in egg white proved the applicability of the columns to the analysis of complex samples.

Asunto(s)

Citocromos c/aislamiento & purificación , Tinta , Muramidasa/aislamiento & purificación , Ovalbúmina/aislamiento & purificación , Polipropilenos/química , Ribonucleasa Pancreática/aislamiento & purificación , Resinas de Intercambio de Catión/química , Cromatografía por Intercambio Iónico , Citocromos c/química , Muramidasa/química , Muramidasa/metabolismo , Ovalbúmina/química , Ribonucleasa Pancreática/química

RESUMEN

Lysozymes play a key role in innate immune response to bacterial pathogens, catalyzing the hydrolysis of the peptidoglycan layer of bacterial cell walls. In this study, the genes encoding the c-type (TmLyzc) and g-type (TmLyzg) lysozymes from Totoaba macdonaldi were cloned and characterized. The cDNA sequences of TmLyzg and TmLyzc were 582 and 432 bp, encoding polypeptides of 193 and 143 amino acids, respectively. Amino acid sequences of these lysozymes shared high identity (60-90%) with their counterparts of other teleosts and showed conserved functional-structural signatures of the lysozyme superfamily. Phylogenetic analysis indicated a close relationship with their vertebrate homologues but distinct evolutionary paths for each lysozyme. Expression analysis by qRT-PCR revealed that TmLyzc was expressed in stomach and pyloric caeca, while TmLyzg was highly expressed in stomach and heart. These results suggest that both lysozymes play important roles in defense of totoaba against bacterial infections or as digestive enzyme.

Asunto(s)

Antibacterianos/metabolismo , Proteínas de Peces/genética , Peces/inmunología , Mucosa Gástrica/metabolismo , Muramidasa/genética , Miocardio/metabolismo , Animales , Pollos/genética , Clonación Molecular , Digestión , Evolución Molecular , Proteínas de Peces/metabolismo , Gansos/genética , Perfilación de la Expresión Génica , Inmunidad Innata , Muramidasa/metabolismo , Especificidad de Órganos , Filogenia , Alineación de Secuencia

RESUMEN

BACKGROUND: Protein engineering has many applications for industry, such as the development of new drugs, vaccines, treatment therapies, food, and biofuel production. A common way to engineer a protein is to perform mutations in functionally essential residues to optimize their function. However, the discovery of beneficial mutations for proteins is a complex task, with a time-consuming and high cost for experimental validation. Hence, computational approaches have been used to propose new insights for experiments narrowing the search space and reducing the costs. RESULTS: In this study, we developed Proteus (an acronym for Protein Engineering Supporter), a new algorithm for proposing mutation pairs in a target 3D structure. These suggestions are based on contacts observed in other known structures from Protein Data Bank (PDB). Proteus' basic assumption is that if a non-interacting pair of amino acid residues in the target structure is exchanged to an interacting pair, this could enhance protein stability. This trade is only allowed if the main-chain conformation of the residues involved in the contact is conserved. Furthermore, no steric impediment is expected between the proposed mutations and the surrounding protein atoms. To evaluate Proteus, we performed two case studies with proteins of industrial interests. In the first case study, we evaluated if the mutations suggested by Proteus for four protein structures enhance the number of inter-residue contacts. Our results suggest that most mutations proposed by Proteus increase the number of interactions into the protein. In the second case study, we used Proteus to suggest mutations for a lysozyme protein. Then, we compared Proteus' outcomes to mutations with available experimental evidence reported in the ProTherm database. Four mutations, in which our results agree with the experimental data, were found. This could be initial evidence that changes in the side-chain of some residues do not cause disturbances that harm protein structure stability. CONCLUSION: We believe that Proteus could be used combined with other methods to give new insights into the rational development of engineered proteins. Proteus user-friendly web-based tool is available at < http://proteus.dcc.ufmg.br >.

Asunto(s)

Proteínas/química , Interfaz Usuario-Computador , Algoritmos , Bases de Datos de Proteínas , Muramidasa/química , Muramidasa/genética , Muramidasa/metabolismo , Mutagénesis , Ingeniería de Proteínas/métodos , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismo

RESUMEN

Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards. Although the lipids and volatile substances secreted by the femoral glands have been investigated in several biochemical studies, the protein composition and functions of secretions remain completely unknown. Applying a proteomic approach, we provide the first attempt to comprehensively characterize the protein composition of femoral gland secretions from the Galápagos marine iguana. Using samples from several organs, the marine iguana proteome was assembled by next-generation sequencing and MS, resulting in 7513 proteins. Of these, 4305 proteins were present in the femoral gland, including keratins, small serum proteins, and fatty acid-binding proteins. Surprisingly, no proteins with discernible roles in partner recognition or inter-species communication could be identified. However, we did find several proteins with direct associations to the innate immune system, including lysozyme C, antileukoproteinase (ALP), pulmonary surfactant protein (SFTPD), and galectin (LGALS1) suggesting that the femoral glands function as an important barrier to infection. Furthermore, we report several novel anti-microbial peptides from the femoral glands that show similar action against Escherichia coli and Bacillus subtilis such as oncocin, a peptide known for its effectiveness against Gram-negative pathogens. This proteomics data set is a valuable resource for future functional protein analysis and demonstrates that femoral gland secretions also perform functions of the innate immune system.

Asunto(s)

Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Iguanas/metabolismo , Sistema Inmunológico/metabolismo , Inmunidad Innata , Proteoma/metabolismo , Transcriptoma , Animales , Apoproteínas/genética , Apoproteínas/metabolismo , Bacillus subtilis/efectos de los fármacos , Encéfalo/metabolismo , Factores Quimiotácticos/genética , Factores Quimiotácticos/metabolismo , Ecuador , Endopeptidasas/genética , Endopeptidasas/metabolismo , Escherichia coli/efectos de los fármacos , Galectinas/genética , Galectinas/metabolismo , Corazón/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Iguanas/genética , Iguanas/inmunología , Inmunidad Innata/genética , Pulmón/metabolismo , Muramidasa/genética , Muramidasa/metabolismo , Músculos/metabolismo , Miocardio/metabolismo , Especificidad de Órganos , Proteoma/genética , Proteoma/inmunología , Proteómica , Proteínas Asociadas a Surfactante Pulmonar/genética , Proteínas Asociadas a Surfactante Pulmonar/metabolismo , Piel/metabolismo , Espectrometría de Masas en Tándem , Transcriptoma/genética

RESUMEN

This paper describes the preparation of polymer monolithic columns in the confines of fluorinated ethylene propylene (FEP) tubes. These tubes are cheap, chemically stable, and widely used in flow analysis laboratories. UV-initiated grafting with 5 wt% benzophenone in methanol for 1 h activated the internal surface walls, thus enabling the further covalent binding of ethylene glycol dimethacrylate (EDMA) from a 15 wt% solution in methanol, also via photografting. Both steps used 254 nm radiation under a potency of 120 mJ cm2. ATR-FTIR measurements revealed the presence of carbonyl, alkyl and vinyl groups in the functionalized FEP. The density of vinyl groups was high enough to firmly attach a poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) monolith in 120 × 1.57 mm i.d. tubes, prepared via photopolymerization. The total preparation lasts less than 2-h. The columns were permeable, (1.58 ± 0.06) × 10-13 m2, providing reproducible chromatographic parameters of retention times, retention factor, selectivity, and resolution. The monoliths were stable at flow rates of 500 µL min-1, collapsing only at flow rates >700 µL min-1, a condition that increased the backpressure over 1000 psi (experiments at the room temperature). The separation of proteins by reversed-phase liquid chromatography demonstrated the efficiency of the columns. Determination of egg white proteins (ovalbumin and lysozyme) and myoglobin in spiked urine proved the applicability to the analysis of real samples.

Asunto(s)

Muramidasa/aislamiento & purificación , Mioglobina/aislamiento & purificación , Ovalbúmina/aislamiento & purificación , Polímeros/química , Politetrafluoroetileno/análogos & derivados , Ribonucleasa Pancreática/aislamiento & purificación , Animales , Bovinos , Pollos , Cromatografía de Fase Inversa , Caballos , Muramidasa/química , Muramidasa/metabolismo , Mioglobina/química , Ovalbúmina/química , Politetrafluoroetileno/química , Ribonucleasa Pancreática/química

RESUMEN

We present a new model to describe DNA interactions with large ligands such as proteins, based on a quenched-disorder equation for ligand binding along the double helix and on Manning's description for the local changes of the persistence length at the binding sites. Such a model allows one to extract the physical chemistry of the interactions from pure mechanical measurements, such as those typically performed with DNA-protein complexes in force spectroscopy assays. We have tested the proposed methodology here to investigate the DNA interaction with the protein lysozyme, determining binding parameters such as the equilibrium association constant, the cooperativity degree of the binding reaction, and the fraction of neutralized charges on the phosphate backbone. The model also allows one to get information on the size and positional conformation of the bound proteins.

Asunto(s)

ADN Viral/química , Muramidasa/química , Química Física , Ligandos , Fenómenos Mecánicos , Modelos Moleculares , Muramidasa/metabolismo

RESUMEN

Chromium (Cr) is a micromineral that is involved in the metabolism of carbohydrates, lipids, ammonia, and nucleic acids; thus, its supplementation can influence the nutritional status of ruminants, and consequently, colostrum profile, since this secretion depends on products secreted by the mammary gland and elements of the maternal bloodstream. The present study investigated the influence of supplementation with Cr bound to organic molecule on the nutritional, immune, and antioxidant quality of ewe colostrum. Thirty-two multiparous Santa Ines ewes (55.3 ± 8.00 kg body weight) were randomly assigned into four groups: T1 (0.0 mg of chromium picolinate (CrPic) supplementation per ewe, n = 8), T2 (0.15 mg of CrPic per ewe, n = 9), T3 (0.30 mg of CrPic per ewe, n = 7), and T4 (0.45 mg of CrPic per ewe, n = 8). Supplementation was supplied during the breeding season, pregnancy, and lactation. Shortly after calving, the first milking colostrum was collected to determine its chemical composition, activity of lysozyme, lactoperoxidase, ceruloplasmin, catalase, glutathione peroxidase, and oxygen radical absorbance capacity. The results show that lactoperoxidase activity decreased with CrPic supplementation (P < 0.01), revealing that this micromineral reduces an important component of defense mechanism in the body. Therefore, the results of this work show that supplementation with chromium picolinate influences colostrum quality.

Asunto(s)

Cromo/farmacología , Calostro/efectos de los fármacos , Lactoperoxidasa/metabolismo , Ácidos Picolínicos/farmacología , Animales , Animales Recién Nacidos , Catalasa/metabolismo , Ceruloplasmina/metabolismo , Cromo/administración & dosificación , Cromo/análisis , Calostro/química , Calostro/metabolismo , Suplementos Dietéticos , Femenino , Glutatión Peroxidasa/metabolismo , Muramidasa/metabolismo , Ácidos Picolínicos/administración & dosificación , Embarazo , Ovinos

RESUMEN

Silica nanoparticles present an enormous potential as controlled drug delivery systems with high selectivity towards diseased cells. This application is directly related to the phenomenon of protein corona, characterized by the spontaneous adsorption of proteins on the nanoparticle surface, which is not fully understood. Here, we report an investigation on the influence of pH, ionic strength and temperature on the thermodynamics of interaction of bovine serum albumin protein (BSA) with non-functionalized silica nanoparticles (SiO2NPs). Complementary, we also investigated the ability of polyethylene glycol (PEG) and zwitterionic sulfobetaine (SBS) surface-modified nanoparticles to prevent the adsorption of BSA (protein negatively charged at physiological pH) and lysozyme (protein positively charged at physiological pH). We showed that BSA interaction with SiO2NPs is enthalpically governed. On the other hand, functionalization of silica nanoparticles with PEG and SBS completely prevented BSA adsorption. However, these functionalized nanoparticles presented a negative zeta potential and were not able to suppress lysozyme anchoring due to strong nanoparticle-protein electrostatic attraction. Due to the similarity of BSA with Human Serum Albumin, this investigation bears a resemblance to processes involved in the phenomenon of protein corona in human blood, producing information that is relevant for the future biomedical use of functionalized nanoparticles.

Asunto(s)

Muramidasa/química , Nanopartículas/química , Albúmina Sérica Bovina/química , Dióxido de Silicio/química , Adsorción , Animales , Bovinos , Muramidasa/metabolismo , Tamaño de la Partícula , Soluciones , Propiedades de Superficie

RESUMEN

Colloidal Liquid Aphrons (CLAs) are micron sized discrete spherical solvent droplets formed by the dispersion of polyaphrons into a bulk aqueous phase at a low phase volume ratio where they can be kept homogenously suspended with only minimal agitation. CLAs have high stability due to the presence of a surfactant 'shell' surrounding the solvent core, and possess large surface areas per unit volume for mass transfer due to their small size. Therefore, CLAs are well suited for applications in pre-dispersed solvent extraction (PSE), enzyme immobilization, and have the potential to be used as a drug delivery system. Using PSE, CLAs have been used to remove metals such as Ni2+, Cu2+, Fe3+, Cr3+ and Mg2+ from dilute streams, separate organic dyes such as Yellow 1 from wastewater, extract succinic and lactic acid, reactively extract phenylalanine, and separate suspensions. CLAs have also been used to immobilize enzymes such as lipase, lysozyme and albumins with cases of superactivity being reported due to the influence of surfactant and solvent interactions with the enzyme. Furthermore, due to their similarity to current drug delivery systems such as microemulsions and hydrogels, and other advantages, CLA systems have the potential to be adapted for drug delivery systems also. This article provides a complete list of the current applications of Colloidal Liquid Aphrons (CLAs) in PSE and enzyme immobilization, and also presents insight into how CLAs can be utilized as a drug delivery method in the future. Finally, this review ends by summarizing potentially interesting research areas to pursue in this field.

Asunto(s)

Albúminas/química , Sistemas de Liberación de Medicamentos , Enzimas Inmovilizadas/química , Lipasa/química , Muramidasa/química , Coloides/química , Coloides/aislamiento & purificación , Coloides/metabolismo , Enzimas Inmovilizadas/metabolismo , Lipasa/metabolismo , Muramidasa/metabolismo , Tamaño de la Partícula , Solventes/química , Propiedades de Superficie

RESUMEN

The periosteum is a membrane that surrounds bones, providing essential cellular and biological components for fracture healing and bone repair. Tissue engineered scaffolds able to function as periosteum substitutes can significantly improve bone regeneration in severely injured tissues. Efforts to develop more bioactive and tunable periosteal substitutes are required to improve the success of this tissue engineering approach. In this work, a chemical modification was performed in chitosan, a polysaccharide with osteoconductive properties, by introducing phosphate groups to its structure. The phosphorylated polymer (Chp) was used to produce chitosan-xanthan-based scaffolds for periosteal tissue engineering. Porous and mechanically reinforced matrices were obtained with addition of the surfactant Kolliphor® P188 and the silicone rubber Silpuran® 2130A/B. Scaffolds properties, such as large pore sizes (850-1097 µm), micro-roughness and thickness (0.7-3.5 mm in culture medium), as well as low thrombogenicity compared to standard implantable materials, extended degradation time and negligible cytotoxicity, enable their application as periosteum substitutes. Moreover, the higher adsorption of bone morphogenetic protein mimic (cytochrome C) by Chp-based formulations suggests improved osteoinductivity of these materials, indicating that, when used in vivo, the material would be able to concentrate native BMPs and induce osteogenesis. The scaffolds produced were not toxic to adipose tissue-derived stem cells, however, cell adhesion and proliferation on the scaffolds surfaces can be still further improved. The mineralization observed on the surface of all formulations indicates that the materials studied have promising characteristics for the application in bone regeneration.

Asunto(s)

Quitosano/farmacología , Oseointegración/efectos de los fármacos , Periostio/fisiología , Polisacáridos Bacterianos/farmacología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Tejido Adiposo/citología , Adsorción , Fosfatasa Alcalina/metabolismo , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Citocromos c/metabolismo , Módulo de Elasticidad , Humanos , L-Lactato Deshidrogenasa/metabolismo , Muramidasa/metabolismo , Osteogénesis/efectos de los fármacos , Periostio/efectos de los fármacos , Fosforilación , Porosidad , Espectroscopía Infrarroja por Transformada de Fourier , Células Madre/citología , Células Madre/efectos de los fármacos , Estrés Mecánico , Trombosis/patología

RESUMEN

This work examined the hypothesis that interactions of Rose Bengal (RB2-) with lysozyme (Lyso) might mediate type 1 photoreactions resulting in protein cross-linking even under conditions favoring 1O2 formation. UV-visible spectrophotometry, isothermal titration calorimetry (ITC), and docking analysis were employed to characterize RB2--Lyso interactions, while oxidation of Lyso was studied by SDS-PAGE gels, extent of amino acid consumption, and liquid chromatography (LC) with mass detection (employing tryptic peptides digested in H218O and H2O). Docking studies showed five interaction sites including the active site. Hydrophobic interactions induced a red shift of the visible spectrum of RB2- giving a Kd of 4.8 µM, while data from ITC studies, yielded a Kd of 0.68 µM as an average of the interactions with stoichiometry of 3.3 RB2- per Lyso. LC analysis showed a high consumption of readily-oxidized amino acids (His, Trp, Met and Tyr) located at different and diverse locations within the protein. This appears to reflect extensive damage on the protein probably mediated by a type 2 (1O2) mechanism. In contrast, docking and mass spectrometry analysis provided evidence for the generation of specific intra- (Tyr23-Tyr20) and inter-molecular (Tyr23-Trp62) Lyso cross-links, and Lyso dimer formation via radical-radical, type 1 mechanisms.

Asunto(s)

Reactivos de Enlaces Cruzados/metabolismo , Colorantes Fluorescentes/metabolismo , Muramidasa/metabolismo , Fármacos Fotosensibilizantes/metabolismo , Rosa Bengala/metabolismo , Triptófano/química , Tirosina/química , Animales , Pollos , Reactivos de Enlaces Cruzados/química , Colorantes Fluorescentes/química , Muramidasa/química , Oxidación-Reducción , Fotoquímica , Fármacos Fotosensibilizantes/química , Conformación Proteica , Rosa Bengala/química

RESUMEN

X-ray crystallography provides structural information of molecules at the atomic level, being a central technique at the forefront of science and technology. However, crystallography teaching is not usually implemented in biochemistry lab classes due to its complex execution by nonexpert users. Here, we report the basic step-by-step workflow performed by crystallographers in order to solve the three-dimensional structure of a protein. All these activities were executed in a course for Latin-American graduate students with no previous knowledge on X-ray crystallography entitled "Crystallography in Structural Biology: why do we need a protein crystal, and how do we get it?." We would like to share our experience with the educational research community, with the main purpose being to enrich teaching in biochemistry and structural molecular biology by performing a series of interesting laboratory and computer experiments. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):700-707, 2019.

Asunto(s)

Laboratorios , Muramidasa/química , Animales , Bioquímica/educación , Pollos , Cristalografía por Rayos X , Curriculum , Humanos , Modelos Moleculares , Biología Molecular/educación , Muramidasa/metabolismo , Estudiantes