Dual-Targeting Approach Degrades Biofilm Matrix and Enhances Bacterial Killing.

Biofilm formation is a key virulence factor responsible for a wide range of infectious diseases, including dental caries. Cariogenic biofilms are structured microbial communities embedded in an extracellular matrix that affords bacterial adhesion-cohesion and drug tolerance, making them difficult to treat using conventional antimicrobial monotherapy. Here, we investigated a multitargeted approach combining exopolysaccharide (EPS) matrix-degrading glucanohydrolases with a clinically used essential oils-based antimicrobial to potentiate antibiofilm efficacy. Our data showed that dextranase and mutanase can synergistically break down the EPS glucan matrix in preformed cariogenic biofilms, markedly enhancing bacterial killing by the antimicrobial agent (3-log increase versus antimicrobial alone). Further analyses revealed that an EPS-degrading/antimicrobial (EDA) approach disassembles the matrix scaffold, exposing the bacterial cells for efficient killing while concurrently causing cellular dispersion and "physical collapse" of the bacterial clusters. Unexpectedly, we found that the EDA approach can also selectively target the EPS-producing cariogenic bacteria Streptococcus mutans with higher killing specificity (versus other species) within mixed biofilms, disrupting their accumulation and promoting dominance of commensal bacteria. Together, these results demonstrate a dual-targeting approach that can enhance antibiofilm efficacy and precision by dismantling the EPS matrix and its protective microenvironment, amplifying the killing of pathogenic bacteria within.

Nanosized Building Blocks for Customizing Novel Antibiofilm Approaches.

Recent advances in nanotechnology provide unparalleled flexibility to control the composition, size, shape, surface chemistry, and functionality of materials. Currently available engineering approaches allow precise synthesis of nanocompounds (e.g., nanoparticles, nanostructures, nanocrystals) with both top-down and bottom-up design principles at the submicron level. In this context, these "nanoelements" (NEs) or "nanosized building blocks" can 1) generate new nanocomposites with antibiofilm properties or 2) be used to coat existing surfaces (e.g., teeth) and exogenously introduced surfaces (e.g., restorative or implant materials) for prevention of bacterial adhesion and biofilm formation. Furthermore, functionalized NEs 3) can be conceived as nanoparticles to carry and selectively release antimicrobial agents after attachment or within oral biofilms, resulting in their disruption. The latter mechanism includes "smart release" of agents when triggered by pathogenic microenvironments (e.g., acidic pH or low oxygen levels) for localized and controlled drug delivery to simultaneously kill bacteria and dismantle the biofilm matrix. Here we discuss inorganic, metallic, polymeric, and carbon-based NEs for their outstanding chemical flexibility, stability, and antibiofilm properties manifested when converted into bioactive materials, assembled on-site or delivered at biofilm-surface interfaces. Details are provided on the emerging concept of the rational design of NEs and recent technological breakthroughs for the development of a new generation of nanocoatings or functional nanoparticles for biofilm control in the oral cavity.

Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: from concept to process.

An aqueous two-phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back-extraction, and washing) was set up and validated in a pump mixer-settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155-fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22-fold reduction in the host cell protein/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.

Backpacks and school children's obesity: challenges for public health and ergonomics.

This descriptive study of cross sectional has focused on analyzing the school material weight transported by students associated with children and adolescents overweight in primary and secondary schools. Participants 339 students of both genders, aged 10-19 years. 243 students carrying and average load of 12.65% of his body weight. 53 students were overweight with a BMI of 20,00 to 35,6. 20 overweight students carrying backpacks more than 10% of his body weight. 21% of the students rated complained of back and shoulder pain. This data is very important in the preventive aspects for the individuals studied, as well as others with the same anthropometric characteristics and the same demand.

Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing.

The therapeutic use of proteins has created an increasing demand for feasible and economical methods for both up- and downstream processes. However, whereas upstream processes have attracted substantial investment and commercial attention, downstream processing has been overlooked, causing a production bottleneck that is shifting the costs of production. This review focuses on the use of aqueous two-phase extraction as an option for the downstream processing of therapeutic proteins. It is a potential and promising liquid-liquid extraction technique for the purification of biomolecules, such as monoclonal antibodies, growth factors and hormones, that combines a high selectivity and biocompatibility with an easy scale-up and continuous operation mode.

Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography.

In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.

Purification of human immunoglobulin G by thermoseparating aqueous two-phase systems.

The purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant was studied using an aqueous two-phase system (ATPS) composed of ethylene oxide/propylene oxide (UCON) and dextran. In UCON/dextran systems IgG partitions preferentially to the less hydrophobic dextran-rich phase (Kp<1). The addition of triethylene glycol-diglutaric acid (TEG-COOH) shifted the IgG partition into the upper phase showing significant improvements in both the recovery yields and purity. The purification of IgG from a CHO cell supernatant with UCON 2000/dextran/TEG-COOH system was optimised using a central composite design. Using an ATPS composed of 8% UCON, 6% dextran and 20% TEG-COOH, IgG was purified, in two steps, with a global yield of 85% and 88% purity. Statistical valid models were obtained to predict the effect of the experimental conditions on the IgG yield and purity, for both extraction and back-extraction steps. A system composed of 10% UCON, 5.5% dextran and 20% TEG-COOH was identified as the best compromise between final purity and yield.

Optimisation of aqueous two-phase extraction of human antibodies.

The purification of human antibodies in an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) 6000 and phosphate was optimised by surface response methodology. A central composite design was used to evaluate the influence of phosphate, PEG and NaCl concentration and of the pH on the purity and extraction yield of IgG from a simulated serum medium. The conditions that maximise the partition of IgG into the upper phase were determined to be high concentrations of NaCl and PEG, low concentrations of phosphate and low pH values. An ATPS composed of 12% PEG, 10% phosphate, 15% NaCl at pH 6 was further used to purify human monoclonal antibodies from a Chinese Hamster Ovary (CHO) concentrated cell culture supernatant with a recovery yield of 88% in the upper PEG-rich phase and a purification factor of 4.3. This ATPS was also successfully used to purify antibodies from a hybridoma cell culture supernatant with a recovery yield of 90% and a purification factor of 4.1.

Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies.

The partition of human antibodies in aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and phosphate was systematically studied using first pure proteins systems and then an artificial mixture of proteins containing 1mg/ml human immunoglobulin G (IgG), 10mg/ml serum albumin and 2mg/ml myoglobin. Preliminary results obtained using pure proteins systems indicated that the PEG molecular weight and concentration, the pH value and the salts concentration had a pronounced effect on the partitioning behaviour of all proteins. For high ionic strengths and pH values higher than the isoelectric point (pI) of the contaminant proteins, IgG could be selectively recovered on the top phase. According to these results, a face centred composite design was performed in order to optimise the purification of IgG from the mixture of proteins. The optimal conditions for the isolation of IgG were observed for high concentrations of NaCl and low concentrations of both phase forming components. The best purification was achieved using an ATPS containing 8% (w/w) PEG 3350, 10% (w/w) phosphate pH 6 and 15% (w/w) NaCl. A recovery yield of 101+/-7%, a purity of 99+/-0% and a yield of native IgG of 97+/-4% were obtained. Back extraction studies of IgG to a new phosphate phase were performed and higher yields were obtained using 10% phosphate buffer at pH 6. The total extraction yield was 76% and the purity 100%.

Comparative study of lormetazepam and flurazepam in the treatment of insomnia.

Lormetazepam (1 and 2 mg) was tested against flurazepam (30 mg) and placebo in 60 patients (15 per treatment group) with initial, intermediate, or late insomnia. Patients were randomly assigned to treatment groups, and a double-blind protocol was followed. The four-week study included one week of use of the placebo, followed by two weeks of treatment with the active drug (in three groups) or continued use of the placebo (one group), and one week of use of the placebo (all four groups). Results showed that the active drugs were efficient in controlling insomnia. In some subjective parameters, only 2 mg of lormetazepam was significantly better than placebo. Tolerability of the drugs was considered good, with no interruption of treatment required because of adverse reactions. There was a rebound effect in one patient receiving 1 mg of lormetazepam and in three patients receiving flurazepam.

Comparative study of zopiclone and pentobarbitone as hypnotics.

The safety and efficacy of zopiclone (7.5 mg) and pentobarbitone (100 mg) were compared in 60 adult outpatients suffering from insomnia. The patients were randomly assigned to one of the two treatment groups, and the medication was taken at bedtime for 16 days. Zopiclone and pentobarbitone, compared with placebo, improved sleep onset, duration of sleep, number of night time awakenings, and quality of sleep. Zopiclone was superior to pentobarbitone with regard to sleep quality, judgement of therapy, and condition in the morning. Side effects were reported in each treatment group, but were less frequent in the zopiclone group (p less than 0.005). Zopiclone is a useful drug for the treatment of sleep disturbances, not only because of its efficacy, but also because of its tolerability.

Comparative study of zopiclone and pentobarbitone as hypnotics.

The safety and efficacy of zopiclone (7.5 mg) and pentobarbitone (100 mg) were compared in 60 adult outpatients suffering from insomnia. The patients were randomly assigned to one of the two treatment groups, and the medication was taken at bedtime for 16 days. Zopiclone and pentobarbitone, compared with placebo, improved sleep onset, duration of sleep, number of night time awakenings, and quality of sleep. Zopiclone was superior to pentobarbitone with regard to sleep quality, judgement of therapy, and condition in the morning. Side effects were reported in each treatment group, but were less frequent in the zopiclone group (p less than 0.005). Zopiclone is a useful drug for the treatment of sleep disturbances, not only because of its efficacy, but also because of its tolerability.

Clorimipramina de liberacao lenta comparada a formulacao convencional no tratamento de disturbios fobicos e obsessivos. / Slow-release chlorimipramine versus conventional formulation in the treatment of phobic and obsessional disorders

Sao estudadas as propriedades da posologia unica diaria de uma formulacao de liberacao lenta de clorimipramina, conhecido antidepressivo triciclico, versus a formulacao convencional (tres doses diarias) do mesmo farmaco, no tratamento de neuroses fobicas e obsessivas.Fazem-se algumas consideracoes sobre as razoes que levaram a tendencia a prescrever dose unica diaria de antidepressivos triciclicos,e especialmente de clorimipramina. Analisam-se os resultados do estudo, encontrando-se 88,6% de resultados excelentes e bons no grupo que usou a formulacao de liberacao lenta, e 79,4% no grupo que usou a formulacao convencional,sendo 84,1% no total dos dois grupos. Observam-se efeitos colaterais de intensidade variavel em 45,7% no grupo que usou a formulacao de liberacao lenta e 58,8% no grupo que usou a formulacao convencional, sendo 52,2% no total dos dois grupos. Embora tais resultados demonstrem superioridade da formulacao de liberacao lenta, essa nao e estatisticamente significativa. No entanto, considerando-se as vantagens de ordem pratica dessa formulacao, conclui-se que a mesma representa excelente opcao potencial para o tratamento de disturbios fobicos e obsessivos