Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A.

Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.

Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics.

Both conventional and innovative biomedical approaches require cost-effective protein drugs with high therapeutic potency, improved bioavailability, biocompatibility, stability and pharmacokinetics. The growing longevity of the human population, the increasing incidence and prevalence of age-related diseases and the better comprehension of genetic-linked disorders prompt to develop natural and engineered drugs addressed to fulfill emerging therapeutic demands. Conventional microbial systems have been for long time exploited to produce biotherapeutics, competing with animal cells due to easier operation and lower process costs. However, both biological platforms exhibit important drawbacks (mainly associated to intracellular retention of the product, lack of post-translational modifications and conformational stresses), that cannot be overcome through further strain optimization merely due to physiological constraints. The metabolic diversity among microorganisms offers a spectrum of unconventional hosts, that, being able to bypass some of these weaknesses, are under progressive incorporation into production pipelines. In this review we describe the main biological traits and potentials of emerging bacterial, yeast, fungal and microalgae systems, by comparing selected leading species with well established conventional organisms with a long run in protein drug production.

Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis.

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.

Long term operation of a thermophilic anaerobic reactor: process stability and efficiency at decreasing sludge retention time.

The aim of this study was to evaluate the performance of thermophilic sludge digestion at decreasing sludge retention time (SRT) and increasing organic loading rate (OLR), in terms of methane production, effluent stabilisation, hygienisation and dewaterability. Focus was put on determining indicators to help prevent process failure. To this end, a lab-scale reactor was operated for nearly 2 years at 55 degrees Celsius. Methane production rate was increased (from 0.2 to 0.4-0.6 m(3)((ch)(4)) m(-3)(reactor) d(-1)) by decreasing the SRT from 30 to 15-10 days, while increasing the OLR from 0.5 to 2.5-3.5 kg VS m(-3)(reactor) d(-1). Sludge dewaterability was worsened at SRT below 15 days; while pathogen destruction was always successful. The following concentrations might be used to prevent process failure: VFA C2-C5 (3.7 g CODL(-1)), acetate (0.6 g L(-1)), acetate/propionate (0.5), intermediate alkalinity (1.8 g CaCO(3) L(-1)), intermediate/partial alkalinity (0.9), intermediate/total alkalinity (0.5), CH(4) in biogas (55%).

The use of composting for the treatment of animal by-products: Experiments at lab scale.

Animal by-products (ABP), containing mainly rabbit and chicken carcasses were composted at laboratory scale. Results indicate that if proper conditions are used, wastes can be successfully composted and stabilised meeting current European hygienisation standards regarding the disposal of this type of wastes. During the process, temperatures above 60 degrees C were easily reached and maintained for 2 days at least, due to the high energy potential of these materials. However, care must be taken to ensure that these temperatures are reached in the entire reactor to guarantee proper hygienisation of the material. These high temperatures may bring about operational problems such as moisture losses due to very high airflows required for their control. Biological activity indices, such as respiration index (RI) and oxygen uptake rate (OUR) used for the monitoring of the process, were able to indicate potential and actual conditions within the composting reactor, respectively.

In search of a reliable technique for the determination of the biological stability of the organic matter in the mechanical-biological treated waste.

The biological stability determines the extent to which readily biodegradable organic matter has decomposed. In this work, a massive estimation of indices suitable for the measurement of biological stability of the organic matter content in solid waste samples has been carried out. Samples from different stages in a mechanical-biological treatment (MBT) plant treating municipal solid wastes (MSW) were selected as examples of different stages of organic matter stability in waste biological treatment. Aerobic indices based on respiration techniques properly reflected the process of organic matter biodegradation. Static and dynamic respirometry showed similar values in terms of aerobic biological activity (expressed as oxygen uptake rate, OUR), whereas cumulative oxygen consumption was a reliable method to express the biological stability of organic matter in solid samples. Methods based on OUR and cumulative oxygen consumption were positively correlated. Anaerobic methods based on biogas production (BP) tests also reflected well the degree of biological stability, although significant differences were found in solid and liquid BP assays. A significant correlation was found between cumulative oxygen consumption and ultimate biogas production. The results obtained in this study can be a basis for the quantitative measurement of the efficiency in the stabilization of organic matter in waste treatment plants, including MBT plants, anaerobic digestion of MSW and composting plants.

Optimization of the hydrolytic-acidogenic anaerobic digestion stage (55 degrees C) of sewage sludge: influence of pH and solid content.

In conventional single-stage anaerobic digestion processes, hydrolysis is regarded as the rate-limiting step in the degradation of complex organic compounds, such as sewage sludge. Two-stage systems have been proposed to enhance this process. However, so far it is not clear which are the best conditions for a two-stage anaerobic digestion process of sewage sludge, in terms of temperature and hydraulic retention time of each stage. The aim of this work was to determine the optimal conditions for the hydrolytic-acidogenic stage treating real sludge with a high concentration of total solids (40-50gL(-1)) and volatile solids (25-30gL(-1)), named high concentration sludge. The variables considered for this first stage were: hydraulic retention time (1-4 days) and temperature (55 and 65 degrees C). Maximum volatile fatty acids generation was obtained at 4 days and 3 days hydraulic retention time for 55 degrees C and 65 degrees C, respectively. Consequently, 4 days hydraulic retention time and temperature of 55 degrees C were set as the working conditions for the hydrolytic-acidogenic stage treating high concentration sludge. The results obtained when operating with high concentration sludge were compared with a low concentration sludge consisting of 17-28gL(-1) total solids and 13-21gL(-1) volatile solids. The effect of decreasing the influent sludge pH, when working at the optimal conditions established, was also evaluated.

Dehydrogenase activity as a method for monitoring the composting process.

Dehydrogenase enzymatic activity was determined to monitor the biological activity in a composting process of organic fraction of municipal solid waste. Dehydrogenase activity is proposed as a method to describe the biological activity of the thermophilic and mesophilic stages of composting. The maximum dehydrogenase activity was detected at the end of the thermophilic stage of composting, with values within 0.5-0.7mgg dry matter(-1)h(-1). Also, dehydrogenase activity can be correlated to static respiration index during the maturation mesophilic stage.

Co-composting of hair waste from the tanning industry with de-inking and municipal wastewater sludges.

Production of waste hair in the leather manufacturing industry is increasing every year due to the adoption of hair-save unhairing techniques, leaving the tanners with the problem of coping with yet another solid by-product. Numerous potential strategies for hair utilisation have been proposed. However, the use of hair waste as agricultural fertiliser is one of its most promising applications due to the high nitrogen content of hair. Agricultural value of hair can be increased by composting. This paper deals with the composting of hair from the unhairing of bovine hide. Results indicated that hair cannot be either composted on its own or co-composted with de-inking sludge, a chemical complementary co-substrate. However, good results were obtained when co-composted with raw sludge from a municipal wastewater treatment plant at hair:raw sludge weight ratios 1:1, 1:2 and, 1:4 in lab scale and pilot plant scale composters. In all cases, a more stable product was achieved at the end of the process. Composting in the pilot plant composter was effectively monitored using Static Respiration Indices determined at process temperature at sampling (SRI(T)) and at 37 degrees C (SRI(37)). Notably, SRI(T) values were more sensitive to changes in the biological activity. In contrast, Respiratory Quotient (RQ) values were not adequate to follow the development of the process.

Terapia grupal en síndrome ansioso depresivo y situaciones de estrés / Group therapy in anxietydepressive disorder and stress situations

En el centro de Asistencia Primaria Turó deBarcelona se ha iniciado la utilización de la terapiagrupal como intervención terapéutica en mujeresque presentan síndromes ansioso depresivos y/osituaciones de estrés.La intervención ha sido llevada a cabo por unprofesional de Enfermería con formación en saludmental, dinámica de grupos y meditación seitai.Dicha intervención tiene continuidad, una vezfinalizadas las sesiones iniciales, en un centrocívico de la zona.Como resultado de esta experiencia se hacomprobado una disminución tanto en las visitas alcentro de asistencia como en el consumo demedicamentos, así como una significativa mejoraen la calidad de vida de las participantes del grupo

The Turó Primary Care Centre in Barcelona hasstarted a programme of group therapy as atherapeutic intervention in women presentinganxiety-depressive disorder and/or stress situations.A nursing professional trained in mental health,group dynamics and seitai meditation hasconducted the intervention. Once the initialsessions are completed, the intervention will becontinued in a civic centre of the area. As a resultof this experience, a decreased consumption ofdrugs as well as a decreased number inconsultations, have been observed. A noticeableimprovement in the quality of life of the groupparticipants has also been noted