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1.
Biotechnol Bioeng ; 114(1): 46-52, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27427236

RESUMO

Self-assembling proteins forming amyloid fibrils are promising candidates for the fabrication of biomaterials, due to the chemical and mechanical stability of their structures. Among potential applications, their use as platforms for enzyme immobilization is rapidly gathering attention. In this work, we demonstrate that the production of the enzyme glutathione-S-transferase (GST) fused to the class I hydrophobin Vmh2 from Pleurotus ostreatus represents an invaluable tool for the development of self-immobilizing enzymes useful for high throughput analyses. The proposed immobilization strategy is versatile since it can be applied, in principle, to every recombinant protein able to refold from Escherichia coli inclusion bodies. A GST based biosensor has been developed to quantify toxic compounds, such as the pesticides molinate and captan, in aqueous environmental samples. The main advantages of this sensor include simplicity and speed of preparation, high sensitivity, reusability, and accuracy. Biotechnol. Bioeng. 2017;114: 46-52. © 2016 Wiley Periodicals, Inc.


Assuntos
Amiloide/metabolismo , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Proteínas Recombinantes de Fusão/química , Amiloide/química , Animais , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Pleurotus/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Schistosoma japonicum/enzimologia , Schistosoma japonicum/genética
2.
Biomacromolecules ; 17(3): 954-64, 2016 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-26828412

RESUMO

Hydrophobins are fungal proteins whose functions are mainly based on their capability to self-assemble into amphiphilic films at hydrophobic-hydrophilic interfaces (HHI). It is widely accepted that class I hydrophobins form amyloid-like structures, named rodlets, which are hundreds of nanometers long, packed into ordered lateral assemblies and do not exhibit an overall helical structure. We studied the self-assembly of the Class I hydrophobin Vmh2 from Pleurotus ostreatus in aqueous solutions by dynamic light scattering (DLS), thioflavin T (ThT), fluorescence assay, circular dichroism (CD), cryogenic trasmission electron microscopy (cryo-TEM), and TEM. Vmh2 does not form fibrillar aggregates at HHI. It exhibits spherical and fibrillar assemblies whose ratio depends on the protein concentration when freshly solubilized at pH ≥ 7. Moreover, it spontaneously self-assembles into isolated, micrometer long, and twisted amyloid fibrils, observed for the first time in fungal hydrophobins. This process is promoted by acidic pH, temperature, and Ca(2+) ions. A model of self-assembly into amyloid-like structures has been proposed.


Assuntos
Amiloide/química , Proteínas Fúngicas/química , Amiloide/metabolismo , Proteínas Fúngicas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Pleurotus/química , Ligação Proteica
3.
Anal Bioanal Chem ; 407(2): 487-96, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25395204

RESUMO

The development of efficient and rapid methods for the identification with high sequence coverage of proteins is one of the most important goals of proteomic strategies today. The on-plate digestion of proteins is a very attractive approach, due to the possibility of coupling immobilized-enzymatic digestion with direct matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) analysis. The crucial step in the development of on-plate immobilization is however the functionalization of the solid surface. Fungal self-assembling proteins, the hydrophobins, are able to efficiently functionalize surfaces. We have recently shown that such modified plates are able to absorb either peptides or proteins and are amenable to MALDI-TOF-MS analysis. In this paper, the hydrophobin-coated MALDI sample plates were exploited as a lab-on-plate for noncovalent immobilization of enzymes commonly used in protein identification/characterization, such as trypsin, V8 protease, PNGaseF, and alkaline phosphatase. Rapid and efficient on-plate reactions were performed to achieve high sequence coverage of model proteins, particularly when performing multiple enzyme digestions. The possibility of exploiting this direct on-plate MALDI-TOF/TOF analysis has been investigated on model proteins and, as proof of concept, on entire whey milk proteome.


Assuntos
Enzimas Imobilizadas/química , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fosfatase Alcalina/química , Sequência de Aminoácidos , Caseínas/química , Proteínas Fúngicas/química , Proteínas do Leite/química , Dados de Sequência Molecular , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Proteômica/métodos , Técnicas de Microbalança de Cristal de Quartzo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Tripsina/química
4.
Anal Biochem ; 449: 9-16, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24287080

RESUMO

Fungal hydrophobins are amphipathic self-assembling proteins. Vmh2 hydrophobin, prepared from mycelial cultures of the basidiomycete fungus Pleurotus ostreatus, spontaneously forms a stable and homogeneous layer on solid surfaces and is able to strongly absorb proteins even in their active forms. In this work, we have exploited the Vmh2 self-assembled layer as a novel coating of a matrix-assisted laser desorption/ionization (MALDI) steel sample-loading plate. Mixtures of standard proteins, as well as tryptic peptides, in the nanomolar-femtomolar range were analyzed in the presence of salts and denaturants. As evidence on a real complex sample, crude human serum was also analyzed and spectra over a wide mass range were acquired. A comparison of this novel coating method with both standard desalting techniques and recently reported on-plate desalting methods was also performed. The results demonstrate that Vmh2 coating of MALDI plates allows for a very simple and effective desalting method suitable for development of lab-on-a-plate platforms focused on proteomic applications.


Assuntos
Proteínas Sanguíneas/análise , Proteínas Fúngicas/química , Proteínas Imobilizadas/química , Peptídeos/análise , Pleurotus/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Sais/química
5.
Biomacromolecules ; 15(10): 3811-6, 2014 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-25224565

RESUMO

The spontaneous oxidative polymerization of 0.01-1% w/w 5,6-dihydroxyindole (DHI) in chicken egg white (CEW) in the absence of added solvents leads to a black, water-soluble, and processable artificial biomelanin (ABM) with robust and 1 order of magnitude stronger broadband light absorption compared to natural and synthetic eumelanin suspensions. Small angle neutron scattering (SANS) and transmission electron microscopy (TEM) analysis indicated the presence in the ABM matrix of isolated eumelanin nanoparticles (≤100 nm) differing in shape from pure DHI melanin nanoparticles (SANS evidence). Electron paramagnetic resonance (EPR) spectra showed a slightly asymmetric signal (g ∼ 2.0035) similar to that of solid DHI melanin but with a smaller amplitude (ΔB), suggesting hindered spin delocalization in biomatrix. Enhanced light absorption, altered nanoparticle morphology and decreased free radical delocalization in ABM would reflect CEW-induced inhibition of eumelanin aggregation during polymerization accompanied in part by covalent binding of growing polymer to the proteins (SDS-PAGE evidence). The technological potential of eumelanin nanosizing by biomimetic synthesis within a CEW biomatrix is demonstrated by the preparation of an ABM-based black flexible film with characteristics comparable to those of commercially available polymers typically used in electronics and biomedical applications.


Assuntos
Galinhas/metabolismo , Melaninas/química , Melaninas/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Animais , Biomimética/métodos , Clara de Ovo , Indóis/química , Indóis/metabolismo , Luz , Oxirredução , Polimerização , Polímeros/química , Polímeros/metabolismo , Espalhamento a Baixo Ângulo , Água/química
6.
Biomacromolecules ; 13(3): 743-50, 2012 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-22292968

RESUMO

Fungal hydrophobins are amphipathic, highly surface-active, and self-assembling proteins. The class I hydrophobin Vmh2 from the basidiomycete fungus Pleurotus ostreatus seems to be the most hydrophobic hydrophobin characterized so far. Structural and functional properties of the protein as a function of the environmental conditions have been determined. At least three distinct phenomena can occur, being modulated by the environmental conditions: (1) when the pH increases or in the presence of Ca(2+) ions, an assembled state, ß-sheet rich, is formed; (2) when the solvent polarity increases, the protein shows an increased tendency to reach hydrophobic/hydrophilic interfaces, with no detectable conformational change; and (3) when a reversible conformational change and reversible aggregation occur at high temperature. Modulation of the Vmh2 conformational/aggregation features by changing the environmental conditions can be very useful in view of the potential protein applications.


Assuntos
Amiloide/metabolismo , Cálcio/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solventes/química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Meio Ambiente , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Pleurotus/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
7.
World J Psychiatry ; 11(11): 1095-1105, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34888176

RESUMO

BACKGROUND: The subsequent waves of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have represented a dramatic health emergency characterized by significant consequences on mental health. Diachronic variations in the incidence rates of acute relapse of psychiatric disorders may represent significant "sentinel events" for assessing the mental health response to an unprecedented stressful event. AIM: To investigate the variation in psychiatric hospitalization rates and differences in sociodemographic and clinical-psychopathological peculiarities at Bologna "Maggiore" General Hospital Psychiatric Ward (GHPW) between the first two waves SARS-CoV-2 pandemic and the same periods of the previous 3 years. The secondary purpose of the study was to suggest a diachronic response pathway to stress by reporting additional literature data on coping strategies. METHODS: This observational and retrospective study collected information on admission to the GHPW at the "Maggiore" Hospital in Bologna in the index periods defined as follows: the first period between February 24, 2020 and April 30, 2020 (first epidemic wave) and the second period between October 8, 2020, and January 7, 2021 (second pandemic wave). Absolute numbers and proportion of admitted patients, their sociodemographic and clinical-psychopathological characteristics were compared with the same parameters recorded in the two same periods of the previous 3 years. No strict inclusion or exclusion criteria were provided in the data collection to collect information on all patients requiring acute psychiatric hospitalization. RESULTS: During the first wave, there was a significant reduction in hospitalization rates, although there was a simultaneous increase in compulsory hospitalizations and the acute relapse of schizophrenia spectrum and other psychotic disorders. During the second wave, hospitalization rates reached those recorded during the same period of the previous 3 years, mainly due to the rise of bipolar and related disorders, depressive disorders, anxiety disorders, trauma- and stressor-related disorders and suicidal behaviors. CONCLUSION: The coping strategies adopted during the first wave of the SARS-CoV-2 pandemic protected the vulnerable population from the general risk of clinical-psychopathological acute relapse, even if they increased the susceptibility to run into schizophrenia spectrum and other psychotic disorder relapses. In the medium-long term (as in the second pandemic wave), the same strategies do not play protective roles against the stress associated with the pandemic and social restriction measures. Indeed, during the second wave of the SARS-CoV-2 pandemic, an increase in total hospitalization rate, suicidal behaviors and the incidence rate of bipolar and related disorders, depressive disorders, anxiety disorders, trauma- and stressor-related disorders was observed.

8.
Glycobiology ; 20(5): 594-602, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20100692

RESUMO

Hydrophobins are small self-assembling proteins produced by fungi. A class I hydrophobin secreted by the basidiomycete fungus Pleurotus ostreatus was purified and identified. The pure protein is not water soluble, whereas complexes formed between the protein and glycans, produced in culture broth containing amylose, are soluble in water. Glycan structure matched to cyclic structures of alpha-(1-4) linked glucose containing from six to 16 monomers (cyclodextrins). Moreover, it was verified that not only pure cyclodextrins but also a linear oligosaccharide and even the simple glucose monomer are able to solubilize the hydrophobin in water. The aqueous solution of the protein-in the presence of the cyclic glucans-showed propensity to self-assembly, and conformational changes towards beta structure were observed on vortexing the solution. On the other hand, the pure protein dissolved in less polar solvent (60% ethanol) is not prone to self assembly, and no conformational change was observed. When the pure protein was deposited on a hydrophobic surface, it formed a very stable biofilm whose thickness was about 3 nm, whereas the biofilm was not detected on a hydrophilic surface. When the water-soluble protein-in the presence of the cyclic glucans-was used, thicker (up to 10-fold) biofilms were obtained on either hydrophilic or hydrophobic surfaces.


Assuntos
Proteínas Fúngicas/química , Glucanos/química , Pleurotus/química , Solubilidade , Água/química
9.
Front Neurol ; 11: 302, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32477234

RESUMO

Background: Parkinson's disease (PD) starts asymmetrically and it maintains a certain degree of asymmetry throughout its course. Once functional disability proceeds, people with PD can change their dominant hand due to the increased disease severity. This is particularly true for hand dominance, while no studies have been performed so far exploring the behavioral changes of lower limb utilization in PD according to the lateralized symptom dominance. In the current study, we aim to track the foot preference of participants with PD to respond to the Pull Test. Methods: Forty-one subjects suffering from PD, with a H&Y scale ≤ 2, were recruited. A motor evaluation was performed, including the motor part of the MDS-UPDRS, its axial and lateralized scores (for more and less affected side), two Timed Tests, namely Time to Walk a standard distance (TW, in seconds) and Time Up and Go Test (TUG, in seconds), and the Pull Test. The preferred foot (right or left) involved in the step backward was recorded. Thirty-seven healthy controls underwent a motor assessment which included the Pull Test and the Timed Tests. Both participants with PD and controls were right-handed. To evaluate the relationship between the response to Pull-Test and PD-symptoms, subjects with PD were further divided into two groups: (1) Right more affected side (Right-MAS), and (2) Left more affected side (Left-MAS). Results: Both groups of subjects with PD (Right-MAS and Left-MAS) during the Pull Test shifted significantly their leg use preference toward the opposite side than the more affected side: Right-MAS used preferentially their left leg (71%) and vice versa (p < 0.001). The limb preference shift was especially true for Left-MAS group that almost invariably used their right, dominant leg to respond to the Pull Test (95%). Similar results were obtained comparing people with PD and Controls. Conclusions: This study shows that the limb used to respond to the Pull Test generally predicts the contralateral side of worse PD involvement. As the disease takes place, it prevails over hemispheric dominance: right-handed subjects with left side PD-onset and worse lateralization tend to be hyper-right-dominant, while right-handed subjects with right side PD-onset and worse impairment tend to behave as left-handers. Lateralization of symptoms in PD is still a mysterious phenomenon; more studies are needed to better understand this association and to optimize tailored rehabilitation programs for people with PD.

10.
Colloids Surf B Biointerfaces ; 136: 214-21, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26402419

RESUMO

We report a simple and original method to synthesize gold nanoparticles in which a fungal protein, the hydrophobin Vmh2 from Pleurotus ostreatus and dicarboxylic acid-terminated polyethylene-glycol (PEG) has been used as additional components in a one step process, leading to hybrid protein-metal nanoparticles (NPs). The nanoparticles have been characterized by ultra-violet/visible, infrared and X-ray photoelectron spectroscopies, dynamic light scattering and also by electron microscopy imaging. The results of these analytical techniques highlight nanometric sized, stable, hybrid complexes of about 12 nm, with outer surface rich in functional chemical groups. Interaction with protein and antibodies has also been exploited.


Assuntos
Proteínas Fúngicas/química , Ouro/química , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão , Polietilenoglicóis/química , Análise Espectral
11.
J R Soc Interface ; 9(75): 2450-6, 2012 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-22572031

RESUMO

Hydrophobins are small proteins secreted by fungi, which self-assemble into amphipathic membranes at air-liquid or liquid-solid interfaces. The physical and chemical properties of some hydrophobins, both in solution and as a biofilm, are affected by poly or oligosaccharides. We have studied the interaction between glucose and the hydrophobin Vmh2 from Pleurotus ostreatus by spectroscopic ellipsometry (SE), atomic force microscopy (AFM) and water contact angle (WCA). We have found that Vmh2-glucose complexes forms a chemically stable biofilm, obtained by drop deposition on silicon, 1.6 nm thick and containing 35 per cent of glucose, quantified by SE. AFM highlighted the presence of nanometric rodlet-like aggregates (average height, width and length being equal to 3.6, 23.8 and 40 nm, respectively) on the biofilm surface, slightly different from those obtained in the absence of glucose (4.11, 23.9 and 64 nm). The wettability of a silicon surface, covered by the organic layer of Vmh2-glucose, strongly changed: WCA decreased from 90° down to 17°.


Assuntos
Materiais Biocompatíveis/química , Biofilmes/crescimento & desenvolvimento , Proteínas Fúngicas/química , Glucose/química , Pleurotus/química , Pleurotus/fisiologia , Microscopia de Força Atômica , Nanoestruturas/química , Nanotecnologia , Espectrofotometria Ultravioleta
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