A Strategy Utilizing Protein-Protein Interaction Hubs for the Treatment of Cancer Diseases.

We describe a strategy for the development of a rational approach of neoplastic disease therapy based on the demonstration that scale-free networks are susceptible to specific attacks directed against its connective hubs. This strategy involves the (i) selection of up-regulated hubs of connectivity in the tumors interactome, (ii) drug repurposing of these hubs, (iii) RNA silencing of non-druggable hubs, (iv) in vitro hub validation, (v) tumor-on-a-chip, (vi) in vivo validation, and (vii) clinical trial. Hubs are protein targets that are assessed as targets for rational therapy of cancer in the context of personalized oncology. We confirmed the existence of a negative correlation between malignant cell aggressivity and the target number needed for specific drugs or RNA interference (RNAi) to maximize the benefit to the patient's overall survival. Interestingly, we found that some additional proteins not generally targeted by drug treatments might justify the addition of inhibitors designed against them in order to improve therapeutic outcomes. However, many proteins are not druggable, or the available pharmacopeia for these targets is limited, which justifies a therapy based on encapsulated RNAi.

Environmentally friendly rhamnolipid production for petroleum remediation.

Biosurfactants have potential applications in the remediation of petroleum-contaminated sites. Several strategies can be used to reduce the production costs of these surfactants and make the process more environmentally friendly. In this study, we combined some of these strategies to produce the rhamnolipid-type biosurfactant, including the use of the genetically modified strain Pseudomonas aeruginosa-estA, an industrial coproduct as a carbon source, a simple and low-cost medium, and a simple downstream process. The process resulted in a high yield (17.6 g L-1), even using crude glycerin as the carbon source, with substrate in product conversion factor (YRML/s) of 0.444. The cell-free supernatant (CFS) was not toxic to Artemia salina and selected mammalian cell lineages, suggesting that it can be used directly in the environment without further purification steps. Qualitative analysis showed that CFS has excellent dispersion in the oil-displacement test, emulsifying (IE24 = 65.5%), and tensoactive properties. When salinity, temperature and pressure were set to seawater conditions, the values for interfacial tension between crude oil and water were below 1.0 mN m-1. Taken together, these results demonstrate that it is possible to obtain a nontoxic crude rhamnolipid product, with high productivity, to replace petroleum-based surfactants in oil spill cleanups and other environmental applications.

A Brazilian cohort of individuals with Phelan-McDermid syndrome: genotype-phenotype correlation and identification of an atypical case.

BACKGROUND: Phelan-McDermid syndrome (PMS) is a rare genetic disorder characterized by global developmental delay, intellectual disability (ID), autism spectrum disorder (ASD), and mild dysmorphisms associated with several comorbidities caused by SHANK3 loss-of-function mutations. Although SHANK3 haploinsufficiency has been associated with the major neurological symptoms of PMS, it cannot explain the clinical variability seen among individuals. Our goals were to characterize a Brazilian cohort of PMS individuals, explore the genotype-phenotype correlation underlying this syndrome, and describe an atypical individual with mild phenotype. METHODOLOGY: A total of 34 PMS individuals were clinically and genetically evaluated. Data were obtained by a questionnaire answered by parents, and dysmorphic features were assessed via photographic evaluation. We analyzed 22q13.3 deletions and other potentially pathogenic copy number variants (CNVs) and also performed genotype-phenotype correlation analysis to determine whether comorbidities, speech status, and ASD correlate to deletion size. Finally, a Brazilian cohort of 829 ASD individuals and another independent cohort of 2297 ID individuals was used to determine the frequency of PMS in these disorders. RESULTS: Our data showed that 21% (6/29) of the PMS individuals presented an additional rare CNV, which may contribute to clinical variability in PMS. Increased pain tolerance (80%), hypotonia (85%), and sparse eyebrows (80%) were prominent clinical features. An atypical case diagnosed with PMS at 18 years old and IQ within the normal range is here described. Among Brazilian ASD or ID individuals referred to CNV analyses, the frequency of 22q13.3 deletion was 0.6% (5/829) and 0.61% (15/2297), respectively. Finally, renal abnormalities, lymphedema, and language impairment were found to be positively associated with deletion sizes, and the minimum deletion to cause these abnormalities is here suggested. CONCLUSIONS: This is the first work describing a cohort of Brazilian individuals with PMS. Our results confirm the impact of 22q13 deletions on ASD and several comorbidities, such as hypotonia. The estimation of a minimal deletion size for developing lymphedema and renal problem can assist prediction of prognosis in PMS individuals, particularly those diagnosed in early infancy. We also identified one atypical individual carrying SHANK3 deletion, suggesting that resilience to such mutations occurs. This case expands the clinical spectrum of variability in PMS and opens perspectives to identify protective mechanisms that can minimize the severity of this condition.

Intermediate purification of CHO-derived recombinant human Factor IX using hydrophobic interaction membrane-based chromatography and its comparison to a sulfated resin.

This work investigated the use of hydrophobic interaction membrane chromatography for intermediate purification of recombinant human Factor IX (rFIX) produced by CHO cells. The first purification step was based on a strong anion exchange monolith, thus forming a purification process fully based on convective media, which allow operation at high flow rates and low pressure drops, as well as modular scale-up. Although the starting material was challenging (CHO cell culture supernatant harvested at 70% cell viability), the two-step purification process showed promising results, with a global purification factor of 298, a global recovery of 69%, and DNA and endotoxin levels close to regulatory limits. Final host cell DNA (68.8 ng per dose of 500 IU), endotoxins (60 EU per dose of 500 IU) and activated FIX (FIXa/FIX = 2.33%) were in levels close to those recommended by regulatory authorities. HCP removal was of 99.98%, decreasing from 9 424 358 ppm in the supernatant to a final HCP value of 2071 ppm. The use of a supernatant harvested at higher viability and/or the addition of a third polishing step focusing on HCP removal could allow meeting the desired HCP range of 50-100 ppm, as well as the regulatory requirements for the other critical contaminants.

Development of functionalized polyetherimide/polyvinylpyrrolidone membranes for application in hemodialysis.

The present study aimed to synthesize membranes for hemodialysis based on polyetherimide (PEI) and polyvinylpyrrolidone (PVP), with chemical immobilization of heparin on its surface to increase blood compatibility. The synthesized PEI/PVP membranes were characterized by morphological analysis and transport properties, as well by infrared spectroscopy (FT-IR), protein adsorption, contact angle, activated partial thromboplastin time (aPTT), and platelet adhesion. Hydraulic permeability of the synthesized PEI membranes were comparable to those of current high flux clinical membranes; values of diffusive permeability and rejection for typical solutes were similar to those reported in literature. The immobilization of heparin, in turn, resulted in more hydrophilic membranes, with insignificant protein adsorption and platelet adhesion (as opposed to actual clinical membranes), indicating anti-thrombogenic characteristics as confirmed by increased aPTT.

Nanostructured screen-printed electrodes based on titanate nanowires for biosensing applications.

This work demonstrates the successful modification of screen-printed electrodes using functionalized titanate nanowires for producing a peroxide biosensor. Titanate nanowires were synthesized by the hydrothermal method and characterized using physico-chemical techniques. The surface of the nanowires was modified with (3-aminopropyl)trimethoxysilane and glutaraldehyde to immobilize horseradish peroxidase through covalent bound, obtaining a surface coverage of 1.62mg of enzyme/m2. The surface of screen-printed carbon electrodes was modified with peroxidase-containing nanowires. Cyclic voltammetry and chronoamperometry were employed to study the electrochemical properties of the nanostructured electrode. A low hydrogen peroxide reduction potential around -0.98V (vs Ag, pH7.0) was observed, with linear response in the range of 40 to 560µmolL-1, detection limit of 10.7µmolL-1 and good stability. Reproducibility relative standard deviation was as low as 4.7%. For repeatability, deviation was 3.3%.

Membrane adsorber for endotoxin removal

ABSTRACT The surface of flat-sheet nylon membranes was modified using bisoxirane as the spacer and polyvinyl alcohol as the coating polymer. The amino acid histidine was explored as a ligand for endotoxins, aiming at its application for endotoxin removal from aqueous solutions. Characterization of the membrane adsorber, analysis of the depyrogenation procedures and the evaluation of endotoxin removal efficiency in static mode are discussed. Ligand density of the membranes was around 7 mg/g dry membrane, allowing removal of up to 65% of the endotoxins. The performance of the membrane adsorber prepared using nylon coated with polyvinyl alcohol and containing histidine as the ligand proved superior to other membrane adsorbers reported in the literature. The lack of endotoxin adsorption on nylon membranes without histidine confirmed that endotoxin removal was due to the presence of the ligand at the membrane surface. Modified membranes were highly stable, exhibiting a lifespan of approximately thirty months.

RESUMO A superfície de membranas planas de nylon foi modificada utilizando-se bisoxirano como espaçador e poli(álcool vinílico) para recobrimento das membranas. O aminoácido histidina foi utilizado como ligante para endotoxinas, visando à sua aplicação na remoção de endotoxinas a partir de soluções aquosas. São discutidas as etapas de caracterização do adsorvedor com membranas, análise do procedimento de despirogenização e avaliação da eficiência de remoção em modo estático. A densidade de ligantes nas membranas foi em torno de 7 mg/g membrana (massa seca), permitindo uma remoção de endotoxinas de até 65%. O desempenho das membranas preparadas com nylon e recobertas com poli(álcool vinílico) contendo histidina como ligante foi superior ao de outros adsorvedores com membranas descritos na literatura. A ausência de adsorção de endotoxinas em membranas sem histidina confirma que a remoção das endotoxinas deve-se exclusivamente à presença do ligante na superfície da membrana. As membranas modificadas mostraram-se bastante estáveis, exibindo um tempo de vida superior a 30 dias.

Evaluation of the stability of concentrated emulsions for lemon beverages using sequential experimental designs.

The study of the stability of concentrated oil-in-water emulsions is imperative to provide a scientific approach for an important problem in the beverage industry, contributing to abolish the empiricism still present nowadays. The use of these emulsions would directly imply a reduction of transportation costs between production and the sales points, where dilution takes place. The goal of this research was to evaluate the influence of the main components of a lemon emulsion on its stability, aiming to maximize the concentration of oil in the beverage and to correlate its physicochemical characteristics to product stability, allowing an increase of shelf life of the final product. For this purpose, analyses of surface and interface tension, electrokinetic potential, particle size and rheological properties of the emulsions were conducted. A 2(4-1) fractional factorial design was performed with the following variables: lemon oil/water ratio (30% to 50%), starch and Arabic gum concentrations (0% to 30%) and dioctyl sodium sulfosuccinate (0 mg/L to 100 mg/L), including an evaluation of the responses at the central conditions of each variable. Sequentially, a full design was prepared to evaluate the two most influential variables obtained in the first plan, in which concentration of starch and gum ranged from 0% to 20%, while concentration of lemon oil/water ratio was fixed at 50%, without dioctyl sodium sulfosuccinate. Concentrated emulsions with stability superior to 15 days were obtained with either starch or Arabic gum and 50% lemon oil. The most stable formulations presented viscosity over 100 cP and ratio between the surface tension of the emulsion and the mucilage of over 1. These two answers were selected, since they better represent the behavior of emulsions in terms of stability and could be used as tools for an initial selection of the most promising formulations.

Anion-exchange purification of recombinant factor IX from cell culture supernatant using different chromatography supports.

Both recombinant and plasma-derived factor IX concentrates are used in replacement therapies for the treatment of haemophilia B. In the present work, the capture step for a recombinant FIX (rFIX) purification process was investigated. Different strong anion-exchange chromatography media (the resins Q Sepharose(®) FF and Fractogel(®) TMAE, the monolith CIM(®) QA and the membrane adsorber Sartobind(®) Q) were tested for their rFIX binding capacity under dynamic conditions. In these experiments, crude supernatant from CHO cells was used, thus in the presence of supernatant contaminants and mimicking process conditions. The highest dynamic binding capacity was obtained for the monolith, which was then further investigated. To study pseudoaffinity elution of functional rFIX with Ca(2+) ions, a design of experiments to evaluate the effects of pH, NaCl and CaCl2 on yield and purification factor was carried out. The effect of pH was not statistically significant, and a combination of no NaCl and 45mM CaCl2 yielded a good purification factor combined with a high yield of active rFIX. Under these conditions, activity yield of rFIX was higher than the mass yield, confirming selective elution of functional, γ-carboxylated rFIX. Scaling-up of this process 8 fold resulted in very similar process performance. Monitoring of the undesired activated FIX (FIXa) revealed that the FIXa/FIX ratio (1.94%) was higher in the eluate than in the loaded sample, but was still within an acceptable range. HCP and DNA clearances were high (1256 and 7182 fold, respectively), indicating that the proposed process is adequate for the intended rFIX capture step.

Analysis of experimental errors in bioprocesses. 1. Production of lactobionic acid and sorbitol using the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of Zymomonas mobilis.

The proper determination of experimental errors in bioprocesses can be very important because experimental errors can exert a major impact on the analysis of experimental results. Despite this, the effect of experimental errors on the analysis of bioprocess data has been largely overlooked in the literature. For this reason, we performed detailed statistical analyses of experimental errors obtained during the production of lactobionic acid and sorbitol in a system utilizing as catalyst the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of the bacteria Zymomonas mobilis. The magnitude of the experimental errors thus obtained were then correlated with the process operation conditions and with the composition of the culture media used for bacterial growth. It is shown that experimental errors can depend very significantly on the operation conditions and affect the interpretation of available experimental data. More specifically, in this study, experimental errors depended on the nutritional supplements added to the cultivation medium, the inoculation process, and the reaction time, which may be of fundamental importance for actual process development. The results obtained also indicate, for the first time, that GFOR activity can be affected by the composition of the medium in which cells are cultivated.

Structural stability of myoglobin in organic media.

The structural stability of metmyoglobin in organic solvents and cosolvents was investigated aiming the choice of a suitable medium to perform its dissolution with maintenance of the native folding. The spectroscopic behavior of metmyoglobin solution in UV-Visible and circular dichroism was used to evaluate the solubility and the secondary structure. The results were dependable of the chemical structure of the organic compounds, their polarity and content, in the case of cosolvents. Protic solvents showed better ability than the aprotic ones for the biomolecule dissolution, since they are able to establish hydrogen bonds. Solvents with high polarity usually damage the secondary structure of the protein. Myoglobin was dissolved in pure methanol, ethylene glycol and glycerol. The secondary structure was retained in some extent. The controlled addition of sodium dodecyl sulfate to myoglobin aqueous solution changed the surface moiety of the protein. The complex was extracted to hexane with efficiency of 77%.