Valorization of agro-industrial residues using Aspergillus heteromorphus URM0269 for protease production: Characterization and purification.

This study aimed to produce, characterize and purify a protease from Aspergillus heteromorphus URM0269. After production by solid fermentation of wheat bran performed according to a central composite design, protease was characterized in terms of biochemical, kinetic, and thermodynamic parameters for further purification by chromatography. Proteolytic activity achieved a maximum value of 57.43 U/mL using 7.8 g of wheat bran with 40 % moisture. Protease displayed high stability in the pH and temperature ranges of 5.0-10.0 and 20-30 °C, respectively, and acted optimally at pH 7.0 and 50 °C. The enzyme, characterized as a serine protease, followed Michaelis-Menten kinetics with a maximum reaction rate of 140.0 U/mL and Michaelis constant of 11.6 mg/mL. Thermodynamic activation parameters, namely activation Gibbs free energy (69.79 kJ/mol), enthalpy (5.86 kJ/mol), and entropy (-214.39 J/mol.K) of the hydrolysis reaction, corroborated with kinetic modeling showing high affinity for azocasein. However, thermodynamic parameters suggested a reversible mechanism of unfolding. Purification by chromatography yielded a protease purification factor of 7.2, and SDS-PAGE revealed one protein band with a molecular mass of 14.7 kDa. Circular dichroism demonstrated a secondary structure made up of 45.6 % α-helices. These results show the great potential of this protease for future use in the industrial area.

Immunoproteomics of cow's milk allergy in Mexican pediatric patients.

Immunological mechanisms of non-IgE-mediated cow's milk protein allergy (CMPA) are not well understood. Such a circumstance requires attention with the aim of discovering new biomarkers that could lead to better diagnostic assays for early treatment. Here, we sought both to investigate the mechanism that underlies non-IgE-mediated CMPA and to identify cow's milk immunoreactive proteins in a Mexican pediatric patient group (n = 34). Hence, we determined the IgE and IgG1-4 subclass antibody levels against cow's milk proteins (CMP) by ELISA. Then, we performed 2D-Immunoblots using as first antibody immunoglobulins in the patients'serum that bound specifically against CMP together with CMP enrichment by ion-exchange chromatography. Immunoreactive proteins were identified by mass spectrometry-based proteomics. The serological test confirmed absence of specific IgE in the CMPA patients but showed significant increase in antigen-specific IgG1. Additionally, we identified 11 proteins that specifically bound to IgG1. We conclude that the detection of specific IgG1 together with an immunoproteomics approach is highly relevant to the understanding of CMPA's physiopathology and as a possible aid in making a prognosis since current evidence indicates IgG1 occurrence as an early signal of potential risk toward development of IgE-mediated food allergy. SIGNIFICANCE: Allergies are one of the most studied topics in the field of public health and novel protein allergens are found each year. Discovery of new principal and regional allergens has remarkable repercussions in precise molecular diagnostics, prognostics, and more specific immunotherapies. In this context, specific IgE is widely known to mediate physiopathology; however, allergies whose mechanism does not involve this immunoglobulin are poorly understood although their incidence has increased. Therefore, accurate diagnosis and adequate treatment are delayed with significant consequences on the health of pediatric patients. The study of type and subtypes of immunoglobulins associated with the immunoreactivity of cow's milk proteins together with an immunoproteomics approach allows better comprehension of physiopathology, brings the opportunity to discover new potential cow's milk protein allergens and may help in prognosis prediction (IgG1 occurrence as an early signal of possible risk toward development of IgE-mediated food allergy).

Identification of carboxymethyl (CM)-binding proteins derived from Lolium multiflorum pollen extract and antibody reactivity in Brazilian allergic patients

Lolium multiflorum grass is the major pollen allergen source in the southern region of Brazil, but most of its allergens remain poorly characterized. The aim of this study was to investigate antibody reactivity to L. multiflorum crude and carboxymethyl-ligand extracts in allergic patients and healthy individuals. Ion exchange carboxymethyl (CM) chromatography (CM-Sepharose) was used to isolate proteins (S2) from L. multiflorum crude extract (S1), which were assessed by SDS-PAGE. S1- and S2-specific IgE and IgG4 levels were measured by ELISA using sera from 55 atopic and 16 non-atopic subjects. Reactive polypeptide bands in S1 and S2 were detected by immunoblotting, and the most prominent bands in S2 were analyzed by mass spectrometry (MS-MS). Similar IgE and IgG4 levels were observed to both S1 (IgE median absorbance: 1.22; IgG4 median absorbance: 0.68) and S2 (IgE median absorbance: 1.26; IgG4 median absorbance: 0.85) in atopic subjects. S1 and S2 had positive correlations for IgE and IgG4 (IgE: r=0.9567; IgG4: r=0.9229; P<0.0001) levels. Homology between S1 and S2 was confirmed by IgE (84%) and IgG4 (83%) inhibition. Immunoblotting revealed that the 29-32 kDa band was recognized by 100% of atopic subjects in both S1 and S2. MS-MS analysis identified similarity profile to groups 1 and 5 grass allergens. This study revealed that carboxymethyl-ligand fraction played an important role for pollen allergy diagnosis by containing clinically relevant allergens and constituted a promising candidate for allergen-specific immunotherapy.

Proteínas recombinantes de interesse na área da saúde

Recombinant proteins have revolutionized modern medicine and therapeutics. Currently recombinant proteins are used to treat diseases of great worldwide impact, as they allow maintenance and improvement of the life’s quality of patients with rare genetic diseases, metabolic syndromes, and immunological diseases. Since 1982, after the production of the first therapeutic recombinant protein (PRT), recombinant insulin, all major pharmaceutical companies have tried to develop PRT and supported its growth in the market. The right choice of expression systems and purification processes become essential steps to produce high-quality recombinant proteins with high yield. The recombinant protein Lsa63, the target of this project, is a leptospiral adhesin involved in the bacteria adhesion to the host. Lsa63 is expressed on the surface of patogenic Leptospiras and is absent in saprophytic ones. That said, the use of this protein in the development of new therapies and diagnosis for leptospirosis could be interesting. During this project, Lsa63 was purified from the frozen biomass of E. coli previously produced using liquid chromatography techniques). In all purification steps Lsa63 was quantified by BCA (bicinchoninic acid) and analyzed by SDS-PAGE electrophoresis and Western Blotting. The relative purity of Lsa63 was estimated by densitometry of SDS-PAGE bands. Immobilized-metal affinity chromatography (IMAC) was chosen as the first purification step and 24.4% of Lsa63 was yield with relative purity of 54.8%, followed by size-exclusion chromatography (SEC) with recovery of 95.2% and last step ion-exchange chromatography (IEC) with 7.9% and 80.3% of relative purity. Despite having obtained 80.3% of relative purity the global yield was low (7.9%).

As proteínas recombinantes revolucionaram a medicina moderna e a terapêutica. Por isso, atualmente, as proteínas recombinantes são utilizadas para tratamento de doenças de grande impacto mundial, pois permitem manutenção e melhoria da qualidade de vida de pacientes portadores de doenças genéticas raras, síndromes metabólicas e doenças imunológicas. Desde 1982, após a produção da primeira proteína recombinante terapêutica (PRT), insulina recombinante, todas as grandes farmacêuticas começaram a desenvolver PRT e impulsionaram o crescimento destas no mercado. A escolha correta dos sistemas de expressão e processos de purificação tornam-se etapas essenciais para produção de proteínas recombinantes de alta qualidade e em alto rendimento. A proteína recombinante Lsa63, alvo deste projeto, é uma adesina de leptospira envolvida na adesão da bactéria no organismo dos hospedeiros. A Lsa63 é expressa na superfície de Leptospiras patogênicas e apresenta-se ausente em cepas saprofíticas. Dito isto, a utilização desta proteína, no desenvolvimento de novas terapias e diagnóstico para leptospirose pode ser interessante. Durante o decorrer deste projeto, a Lsa63 foi purificada a partir da biomassa congelada de E. coli utilizando técnicas de cromatografia líquida. Em todas as etapas de purificação a Lsa63 foi quantificada por BCA (ácido bicinchonínico) e analisada por eletroforese SDS-PAGE e Western Blotting. A pureza relativa da Lsa63 foi estimada por densitometria das bandas do gel de eletroforese. A cromatografia de afinidade por íons metálicos imobilizados (IMAC) foi escolhida como primeira etapa de purificação sendo que a Lsa63 foi obtida com rendimento de 24,4% e pureza relativa de 54,8%, seguida pela cromatografia de exclusão molecular com recuperação de 95,2% e a última etapa a cromatografia de troca iônica com recuperação de 52,0% e pureza relativa de 80,3%. Apesar de ter obtido uma pureza relativa de 80,3% a recuperação global incluindo todas as etapas cromatográficas foi baixa sendo 7,9%.

Ascidian (Chordata-Tunicata) Glycosaminoglycans: Extraction, Purification, Biochemical, and Spectroscopic Analysis.

Sulfate polysaccharides with unique structures of the chondroitin/dermatan and heparin/heparan families of sulfated glycosaminoglycans have been described in several species of ascidians (Chordata-Tunicata). These unique sulfated glycans have been isolated from the ascidians and characterized by biochemical and spectroscopic methods. The ascidian glycans can be extracted by different tissues or cells by proteolytic digestion followed by cetylpyridinium chloride/ethanol precipitation. The total glycans are then fractionated by ion-exchange chromatography on DEAE-cellulose and/or Mono Q (HR 5/5) columns. Alternatively, precipitation with different ethanol concentrations can be employed. An initial analysis of the purified ascidian glycans is carried out by agarose gel electrophoresis on diaminopropane/acetate buffer, before or after digestion with specific glycosaminoglycan lyases or deaminative cleavage with nitrous acid. The disaccharides formed by exhaustive degradation of the glycans are purified by gel-filtration chromatography on a Superdex Peptide column and analyzed by HPLC on a strong ion-exchange Sax Spherisorb column. 1H- or 13C-nuclear magnetic resonance spectroscopy in one or two dimensions is used to confirm the structure of the intact glycans.

Novel Thermotolerant Amylase from Bacillus licheniformis Strain LB04: Purification, Characterization and Agar-Agarose.

This study analyzed the thermostability and effect of calcium ions on the enzymatic activity of α-amylase produced by Bacillus licheniformis strain LB04 isolated from Espinazo Hot springs in Nuevo Leon, Mexico. The enzyme was immobilized by entrapment on agar-agarose beads, with an entrapment yield of 19.9%. The identification of the bacteria was carried out using 16s rDNA sequencing. The enzyme was purified through ion exchange chromatography (IEX) in a DEAE-Sephadex column, revealing a protein with a molecular weight of ≈130 kDa. The enzyme was stable at pH 3.0 and heat stable up to 80 °C. However, the optimum conditions were reached at 65 °C and pH 3.0, with a specific activity of 1851.7 U mg-1 ± 1.3. The agar-agarose immobilized α-amylase had a hydrolytic activity nearly 25% higher when compared to the free enzyme. This study provides critical information for the understanding of the enzymatic profile of B. licheniformis strain LB04 and the potential application of the microorganisms at an industrial level, specifically in the food industry.

Determination of the Fluoride Content in Water of Aqueducts of Cauca (Colombia) by Ion Exchange Chromatography.

Water for human consumption is the main source of fluoride exposure. The concentration in water should not exceed 1 mg/L of fluoride since, at higher levels; it increases the risk of dental fluorosis, among other adverse effects. The fluoride content of 149 water samples from different aqueducts in Cauca (Colombia) has been determined by ion exchange chromatography with the aim of fluoride risk assessment. The rural area of the Municipality of Santander de Quilichao registered fluoride concentrations between 0.012 and 0.150 mg/L. The urban area of Santander de Quilichao recorded lower fluoride levels than the rural area (0.027-0.068 mg/L). The urban area of the Municipality of Cajibío registered fluoride levels of 0.082-0.186 mg/L. The highest levels of fluoride were found in Timbío (0.121-0.210 mg/L). The fluoride levels recorded in this study are not considered sufficient to trigger dental fluorosis. Likewise, optimal levels are not considered to protect the child population against dental caries. However, a monitoring plan of fluoride concentrations in water should be implemented to assure the quality and safe of the water.

Streamlined downstream process for efficient and sustainable (Fab')2 antivenom preparation.

BACKGROUND: Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. METHODS: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. RESULTS: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. CONCLUSION: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.

Streamlined downstream process for efficient and sustainable (Fab)2 antivenom preparation

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)

Streamlined downstream process for efficient and sustainable (Fab)2 antivenom preparation

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)

Proteomic analysis of soluble proteins retrieved from Duttaphrynus melanostictus skin secretion by IEx-batch sample preparation.

Amphibians display a toxic secretion that works as chemical defenses against predators and/or microorganisms that is stored in specialized glands located in the tegument. For some animals, such glands have accumulated in specific regions of the body and formed prominent structures known as macroglands. The Bufonidae family displays conspicuous macroglands in a post-orbital position, termed parotoids, which secretions are known to be extremely viscous and rich in alkaloids and steroids. Few proteins have been described in this material, though. Mainly, because of the difficulties to handle such biological matrix. In this context, we have performed a proteomic study on the parotoid macrogland secretion of the Asian bufonid Duttaphrynus melanostictus. By employing the Ion-Exchange (IEx)-batch chromatography (anionic, cationic and both) we obtained six fractions - bound and unbound - that were submitted to an in solution-trypsin digestion followed by LC-MS/MS. Proteins related to: antioxidant activity, binding processes (carbohydrate/lipid/protein), energy metabolism, hydrolases, lipid metabolism and membrane traffic were identified. Moreover, IEx was able to preserve the biological activity of the retrieved proteins (peptidasic). The current study increases the knowledge on the proteins present in the bufonids parotoid macrogland secretion, providing a better understanding of the physiological role played by such molecules. SIGNIFICANCE: The current approach allowed a detailed proteomic analysis of the several proteins synthesized in the D. melanostictus parotoid macrogland (Bufonidae) that are secreted into the skins, but embedded within a complex viscous biological matrix. Moreover, our results aim to increase the knowledge on the biological role played by such proteins at the skin.

A new approach to ion exchange chromatography with conductivity detection for adulterants investigation in dietary supplements.

The extent of adulteration of dietary supplements has significantly increased in recent years. This situation worries health authorities and requires auxiliary analytical tools for the investigation of illegal substances purposely added. Ion exchange chromatography with conductivity detection is a consolidated analytical technique for the determination of inorganic compounds in various matrices. This technique has been applied to the pharmaceutical characterization of mainly impurities and degradation products. This work presents a new approach to ion exchange chromatography as a screening method to investigate the presence of amfepramone, femproporex, sibutramine, bisacodyl and amiloride in dietary supplements advertised for weight loss. The method was optimized and validated using a Metrosep C4 100/4.0 cation exchange column. The mobile phase consisted of 1.8 mm HNO3 containing 2% acetonitrile (v/v), with a flow rate of 0.9 ml min-1 , and nonsuppressed conductivity detection was applied. The limits of detection and quantification varied from 1.01 to 3.62 mg L-1 and from 1.48 to 8.72 mg L-1 , respectively. The proposed method was successful applied to 78 solid dietary supplement samples, in two of which adulterations were found. Moreover, ion exchange chromatography with conductivity detection could be easily used for quality control without prior complex sample pre-treatment.

Proteomic analysis of soluble proteins retrieved from Duttaphrynus melanostictus skin secretion by IEx-batch sample preparation

Amphibians display a toxic secretion that works as chemical defenses against predators and/or microorganismsthat is stored in specialized glands located in the tegument. For some animals, such glands have accumulated inspecific regions of the body and formed prominent structures known as macroglands. The Bufonidae familydisplays conspicuous macroglands in a post-orbital position, termed parotoids, which secretions are known to beextremely viscous and rich in alkaloids and steroids. Few proteins have been described in this material, though.Mainly, because of the difficulties to handle such biological matrix. In this context, we have performed a pro-teomic study on the parotoid macrogland secretion of the Asian bufonidDuttaphrynus melanostictus. By em-ploying the Ion-Exchange (IEx)-batch chromatography (anionic, cationic and both) we obtained six fractions -bound and unbound–that were submitted to an in solution-trypsin digestion followed by LC-MS/MS. Proteinsrelated to: antioxidant activity, binding processes (carbohydrate/lipid/protein), energy metabolism, hydrolases,lipid metabolism and membrane traffic were identified. Moreover, IEx was able to preserve the biological ac-tivity of the retrieved proteins (peptidasic). The current study increases the knowledge on the proteins present inthe bufonids parotoid macrogland secretion, providing a better understanding of the physiological role played bysuch molecules.Significance:The current approach allowed a detailed proteomic analysis of the several proteins synthesized intheD. melanostictusparotoid macrogland (Bufonidae) that are secreted into the skins, but embedded within acomplex viscous biological matrix. Moreover, our results aim to increase the knowledge on the biological roleplayed by such proteins at the skin

Biochemical Analyses of Proteins from Duttaphrynus melanostictus (Bufo melanostictus) Skin Secretion: Soluble Protein Retrieval from a Viscous Matrix by Ion-Exchange Batch Sample Preparation.

A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.

Sulfanilic acid-modified chitosan mini-spheres and their application for lysozyme purification from egg white.

A cation exchange matrix with zwitterionic and multimodal properties was synthesized by a simple reaction sequence coupling sulfanilic acid to a chitosan based support. The novel chromatographic matrix was physico-chemically characterized by ss-NMR and ζ potential, and its chromatographic performance was evaluated for lysozyme purification from diluted egg white. The maximum adsorption capacity, calculated according to Langmuir adsorption isotherm, was 50.07 ± 1.47 mg g-1 while the dissociation constant was 0.074 ± 0.012 mg mL-1 . The process for lysozyme purification from egg white was optimized, with 81.9% yield and a purity degree of 86.5%, according to RP-HPLC analysis. This work shows novel possible applications of chitosan based materials. The simple synthesis reactions combined with the simple mode of use of the chitosan matrix represents a novel method to purify proteins from raw starting materials. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:387-396, 2018.

A modified Poisson-Boltzmann equation applied to protein adsorption.

Ion-exchange chromatography has been widely used as a standard process in purification and analysis of protein, based on the electrostatic interaction between the protein and the stationary phase. Through the years, several approaches are used to improve the thermodynamic description of colloidal particle-surface interaction systems, however there are still a lot of gaps specifically when describing the behavior of protein adsorption. Here, we present an improved methodology for predicting the adsorption equilibrium constant by solving the modified Poisson-Boltzmann (PB) equation in bispherical coordinates. By including dispersion interactions between ions and protein, and between ions and surface, the modified PB equation used can describe the Hofmeister effects. We solve the modified Poisson-Boltzmann equation to calculate the protein-surface potential of mean force, treated as spherical colloid-plate system, as a function of process variables. From the potential of mean force, the Henry constants of adsorption, for different proteins and surfaces, are calculated as a function of pH, salt concentration, salt type, and temperature. The obtained Henry constants are compared with experimental data for several isotherms showing excellent agreement. We have also performed a sensitivity analysis to verify the behavior of different kind of salts and the Hofmeister effects.

Biochemical analyses of proteins from Duttaphrynus melanostictus (Bufo melanostictus) skin secretion: soluble protein retrieval from a viscous matrix by ion-exchange batch sample preparation

A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.

HbA1c levels in individuals heterozygous for hemoglobin variants / Dosagem de hemoglobina glicada em heterozigotos para hemoglobinas variantes

Summary Objective: To evaluate the levels of glycated hemoglobin (HbA1c) in patients heterozygous for hemoglobin variants and compare the results of this test with those of a control group. Method: This was an experimental study based on the comparison of HbA1c tests in two different populations, with a test group represented by individuals heterozygous for hemoglobin variants (AS and AC) and a control group consisting of people with electrophoretic profile AA. The two populations were required to meet the following inclusion criteria: Normal levels of fasting glucose, hemoglobin, urea and triglycerides, bilirubin > 20 mg/dL and non-use of acetylsalicylic acid. 50 heterozygous subjects and 50 controls were evaluated between August 2013 and May 2014. The comparison of HbA1c levels between heterozygous individuals and control subjects was performed based on standard deviation, mean and G-Test. Results: The study assessed a test group and a control group, both with 39 adults and 11 children. The mean among heterozygous adults for HbA1c was 5.0%, while the control group showed a rate of 5.74%. Heterozygous children presented mean HbA1c at 5.11%, while the controls were at 5.78%. G-Test yielded p=0.93 for children and p=0.89 for adults. Conclusion: Our study evaluated HbA1c using ion exchange chromatography resins, and the patients heterozygous for hemoglobin variants showed no significant difference from the control group.

Resumo Objetivo: Avaliar os níveis de hemoglobina glicada em pacientes heterozigotos para hemoglobinas variantes e comparar os resultados deste exame com grupo controle. Método: Trata-se de um estudo experimental, baseado na comparação do exame de hemoglobina glicada de duas populações diferentes, sendo um grupo teste, representado por indivíduos heterozigóticos para hemoglobinas variantes (AS e AC) e um grupo controle, constituído por pessoas com perfil eletroforético AA. As duas populações verificadas devem obedecer ao critério de inclusão: glicemia de jejum, hemoglobina, ureia e triglicérides normais, bilirrubina > 20 mg/dL e não fazer uso de ácido acetilsalicílico. Foram avaliados 50 indivíduos heterozigotos e 50 controles no período de agosto de 2013 a maio de 2014. A comparação dos valores de hemoglobina glicada entre indivíduos heterozigóticos e controle foi realizada por meio do desvio padrão, média e teste G. Resultados: O estudo analisou um grupo teste e um grupo controle, ambos com 39 adultos e 11 crianças. A média dos adultos heterozigotos para HbA1c foi de 5,0%, o grupo controle apresentou índice de 5,7%. Já as crianças heterozigóticas obtiveram média de HbA1c de 5,11%, enquanto as normais apresentaram valores médios de 5,78%. O valor do teste G foi de p=0,9 para crianças e p=0,89 para adultos. Conclusão: Este estudo avaliou HbA1c pela metodologia de cromatografia de coluna com resinas de troca iônica, em que pacientes com heterozigoses para hemoglobinas variantes não apresentaram uma diferença significativa em relação ao grupo controle.

Production of Brazilian human norovirus VLPs and comparison of purification methods

Abstract Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.(AU)

Production of Brazilian human norovirus VLPs and comparison of purification methods

Abstract Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.