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Modern (bio)pharmaceutical process development requires thorough investigation of all process parameters that are critical to product quality. The impact of a disturbance of such a parameter during processing needs to be known so that a rational decision can be made about the release of the product. In cultivation processes the dissolved oxygen (DO) concentration is generally accepted as being a critical parameter. In this article the impact of a 90 min period of oxygen limitation during the cultivation of the strictly aerobic Bordetella pertussis bacterium is investigated. The cultivation is the most important process step for the manufacturing of a vaccine against whooping cough disease. Samples were taken immediately before and after oxygen limitation and at the end of cultivation of four oxygen limited and three control cultivations. DNA microarray analysis of the full transcriptome of the B. pertussis bacterium revealed that a 90 min period of oxygen limitation has a substantial effect on overall gene expression patterns. In total 104 genes were identified as a significant hit at any of the sample points, of which 58 were directly related to oxygen limitation. The other genes were mainly affected towards the end of cultivation. Of all genes involved in oxygen limitation none were identified to show a significant difference between the oxygen limited and control cultivations at the end of the batch. This indicates a fully reversible effect of oxygen limitation on gene expression. This finding has implications for the risk assessment of dissolved oxygen concentration as a critical process parameter.

Growing evidence suggests that antiepileptic drugs (AEDs) may be useful in managing some eating disorders. In the present paper, we provide a brief overview of eating disorders, the rationale for using AEDs in the treatment of these disorders and review the data supporting the effectiveness of specific AEDs in the treatment of patients with eating disorders. In addition, the potential mechanisms of action of AEDs in these conditions are discussed. Of the available AEDs, topiramate appears to have the broadest spectrum of action as an anti-binge eating, anti-purging and weight loss agent, as demonstrated in two placebo-controlled studies in bulimia nervosa and three placebo-controlled studies in binge-eating disorder (BED) with obesity. Topiramate may also have beneficial effects in night-eating syndrome and sleep-related eating disorder, but controlled trials in these conditions are needed. The results of one small controlled study suggest that zonisamide may have efficacy in BED with obesity. However, both topiramate and zonisamide are associated with adverse effect profiles that may limit their use in patients with eating disorders. Phenytoin may be effective in some patients with compulsive binge eating, particularly if co-morbid EEG abnormalities are present, but available data are too varied to allow definitive conclusions to be made. Carbamazepine and valproate may be effective in treating patients with bulimia nervosa or anorexia nervosa when they are used to treat an associated psychiatric (e.g. mood) or neurological (e.g. seizure) disorder; otherwise, both agents, particularly valproate, are associated with weight gain. In conclusion, AEDs have an emerging role in the management of some eating disorders.

Background Oleaginous yeast species are an alternative for the production of lipids or triacylglycerides (TAGs). These yeasts are usually non-pathogenic and able to store TAGs ranging from 20 % to 70 % of their cell mass depending on culture conditions. TAGs originating from oleaginous yeasts can be used as the so-called second generation biofuels, which are based on non-food competing “waste carbon sources”. Results In this study the selection of potentially new interesting oleaginous yeast strains is described. Important selection criteria were: a broad maximum temperature and pH range for growth (robustness of the strain), a broad spectrum of carbon sources that can be metabolized (preferably including C-5 sugars), a high total fatty acid content in combination with a low glycogen content and genetic accessibility. Conclusions Based on these selection criteria, among 24 screened species, Schwanniomyces occidentalis (Debaromyces occidentalis) CBS2864 was selected as a promising strain for the production of high amounts of lipids. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0276-7) contains supplementary material, which is available to authorized users.

There is a need for novel protein sources. Insects are a possible interesting source of protein. They are nutritious in terms of protein (40-75 g/100g dry weight) and minerals. Insect protein is of high quality and has a high digestibility (77-98%) and concentration of essential amino acids (46-96% of the nutritional profile). Also insect cells may be a promising novel source of protein. Choice of cell line, growth conditions and use of the baculovirus expression system opens up possibilities to engineer the nutritional value of the biomass. The technological limits as well as consumer acceptance of insect cell based food remains to be investigated.

Symptoms arising from disease or treatment are subjective experiences. Insight into pediatric oncology treatment side effects or symptoms is ideally obtained from direct inquiry to the ill child. A concept-elicitation phase in a patient-reported outcome (PRO) instrument design provides an opportunity to elicit children's voices to shape cancer symptom selection and terminology.Through semistructured, one-on-one, voice-recorded interviews, symptom data were collected from 96 children with cancer between the ages of 7 and 20 years who were undergoing oncologic treatment at 7 pediatric oncology sites in the United States and Canada.The mean number of symptoms reported per child over the prior 7 days was 1.49 (range, 0-7; median, 1; standard deviation, 1.56). The most common symptoms across all age groups were tiredness or fatigue, nausea or vomiting, aches or pains, and weakness. There was not a statistically significant correlation between self-reported wellness and the number of reported symptoms (r = -0.156, n = 65, P = .215) or the number of symptoms reported by age group or diagnosis type. Forty participants reported experiencing a change in their body in the past week, with one-third of these changes unanticipated. Only through direct questions about feelings were emotional symptoms revealed because 90.6% of interviewees who discussed feelings (48 of 53) did so only in the context of direct questioning on feelings. Adolescents were more likely than younger children to discuss feelings as part of the interview.Concept elicitation from children and adolescents has the potential to enable researchers to develop age-appropriate, accurately representative PRO measures.

The production of acellular pertussis in comparison with whole cell pertussis vaccines demands 5-25 times the amount of Bordetella pertussis' virulence factors, such as Pertussis Toxin (PT), to produce the same number of vaccine doses. An increase in the volumetric productivity by employing fed-batch rather than the currently used batch cultivations of B. pertussis could reduce the cost price of acellular pertussis vaccines. This study defined the conditions that enable fed-batch cultivations at high specific PT production. A solution containing lactate and glutamate was fed to the cultures at various rates. The feed rate and whether or not the fed substrates were completely consumed, significantly influenced cellular metabolism. If lactate was detectable in the culture broth while glutamate was not, poly-hydroxy-butyrate (PHB) was formed. Any PHB present was metabolized when glutamate became detectable again in the culture liquid. At higher lactate and glutamate concentrations, free fatty acids were produced. Though toxic, free fatty acids were not the reason the cultures stopped growing. By choosing appropriate conditions, a cell density of 6.5 g/L dry weight was reached, i.e. a 7-fold increase compared to batch culture. The metabolic mechanisms behind the formation of PHB and fatty acids are discussed, as well as how to increase the cell density further. The PT production stopped at 12 mg/L, well before growth stopped, indicating that regulatory mechanisms of PT production may be involved.

Objective. To determine changes in peri- and neonatal care concerning neonatal mortality and morbidity by comparing 2 cohorts of very prematurely born infants (gestational age [GA] Methods. The Leiden Follow-Up Project on Prematurity (LFUPP-1996/97), a regional, prospective study, includes all infants who were born alive after a GA Results. The absolute number of preterm births in the study region increased by 30%: 102 in 1983 to on average of 133 in 1996–1997. Centralization of perinatal care improved: the percentage of extrauterinely transported infants decreased from 61% in 1983 to 35% in 1996–1997. A total of 182 (73%) of the LFUPP-1996/97 infants were treated antenatally with glucocorticosteroids compared with 6 (6%) of the POPS-1983 infants. A total of 112 (42%) of the LFUPP-1996/97 infants received surfactant. In-hospital mortality decreased from 30% in the 1980s to 11% in the 1990s. Mortality of the extremely preterm infants (Conclusions. An increase in the absolute number of very preterm births in this study region was found, leading to a greater burden on the regional NICUs. Improvements in peri- and neonatal care have led to an increased survival of especially extremely preterm infants. However, increased survival has resulted in more morbidity, mainly bronchopulmonary dysplasia, at the moment of discharge from the hospital.

Perfusion systems have the possibility to be operated continuously for several months. It is important that the performance of the cell retention device does not limit the operation time of a perfusion process used in the production of active pharmaceutical ingredients. Therefore, the aim of this study was to investigate the reliability and long-term stability of an acoustic perfusion process using the 200 L/d BioSep. As the BioSep is an external device, it is possible that dependent on the recirculation rate nutrient gradients occur in the external loop, which could affect the cell metabolism. Therefore, the effect of possible nutrient gradients on cell metabolism, viability and productivity was studied by varying the recirculation rate. In this study, it is shown that a perfusion process using a pilot-scale acoustic cell-retention device (200 L/d) is reliable and simple to operate, resulting in a stable 75-day cultivation of a hybridoma cell line producing a monoclonal antibody. The recirculation rate had a significant effect on the oxygen concentration in the external loop, with oxygen being depleted within the cell-retention device at recirculation rates below 6 m3/m3reactor · d (=600 L/d). The oxygen depletion at low circulation rates correlated with a slightly increased lactate production rate. For all other parameters no effect of the recirculation rate was observed, including cell death measured through the release of lactate dehydrogenase and specific productivity. A maximum specific productivity of 12 pg/cell · d was reached.

Objective : To determine the effect of dissolved oxygen tension (DO) on the redifferentiation of dedifferentiated adult human nasal septum chondrocytes cultured as pellets. Design : After isolation, human nasal chondrocytes were expanded in monolayer culture, which resulted in their dedifferentiation. Dedifferentiated cells were pelleted, transferred to a bioreactor and maintained for up to 21 days at 100% DO (21% oxygen), 25% DO (5.25% oxygen) or 5% DO (1% oxygen), which was controlled in the liquid phase. Redifferentiation was assessed by staining the extracellular matrix with safranin-O and by the immunolocalization of collagen types I, II, IX and of a fibroblast membrane marker (11-fibrau). In addition, glycosaminoglycans (GAG) and DNA content were determined spectrophotometrically. Results : In monolayer culture, cells dedifferentiated and multiplied 90- to 100-fold. Cell pellets cultured in a bioreactor under conditions of low oxygen tension (25% DO or 5% DO) stained intensely for GAGs and for collagen type II, but very weakly for collagen type I. After 14 days of culturing, cell pellets maintained at 5% DO stained more intensely for collagen IX and more weakly for 11-fibrau than did those incubated at 25% DO. After 21 days of culturing the GAG content of cell pellets maintained at 5% DO was significantly greater than that of those incubated at 25% DO. Under air-saturated conditions (100% DO), the DNA and GAG contents of cell pellets decreased with time in culture. After 21 days of culturing, both parameters were substantially lower in cell pellets maintained at 100% DO than in those incubated at low oxygen tensions. The staining signals for collagen types II and IX were much weaker, and those for the markers of dedifferentiation (collagen type I and 11-fibrau) much stronger under air-saturated conditions than at low oxygen tensions. Conclusion : These observations demonstrate that using the present set-up, low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes in pellet cultures.

Continuous protein production with baculovirus expression vectors in insect-cell bioreactors is characterized by a dramatic drop in heterologous protein production within a few weeks. This is mainly due to the spontaneous deletion of the heterologous gene(s) from the baculovirus genome and/or to the rapid accumulation of defective interfering baculoviruses (DIs). Cell culture experiments with bacmid-derived baculoviruses showed that spontaneous deletions in the foreign bacterial artificial chromosome (BAC) sequences readily occurred, These deletions correlated with a low density of baculovirus homologous (repeat) regions (hrs), which are located dispersed throughout the baculovirus genome and are believed to act as origins of viral DNA replication (oris). To test the hypothesis that deletions are more likely to occur in regions with a low ori density, the properties of bacmidderived baculoviruses with an additional hr in the unstable BAC sequences were compared to the standard bacmidderived baculovirus in a continuous cascaded insect-cell bioreactor configuration. All viruses were equipped with a green fluorescent protein (GFP) gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). The insertion of an extra hr in the BAC vector led to improved genetic stability of adjacent sequences, resulting in prolonged protein expression. The maintenance of the BAC sequences appeared to be dependent on the orientation of the inserted hr. The advantages of the utilization of hrs to improve the stability of baculovirus expression vectors for the large-scale protein production in insect-cell bioreactors are discussed. (C) 2004 Wiley Periodicals, Inc.

Repair of articular cartilage defects using tissue engineered constructs composed of a scaffold and cultured autologous cells holds promise for future treatments. However, nutrient limitation (e.g. oxygen) has been suggested as a cause of the onset of chondrogenesis solely within the peripheral boundaries of larger constructs. In the present study, oxygen gradients were evaluated by microelectrode measurements in two porous polyethylene glycol terephthalate/polybutylene terephthalate (PEGT/PBT) scaffold architectures, a compression-molded and particle-leached sponge (CM) and a 3D-deposited fiber (3DF) scaffold. During the first 14 days in vitro, gradients intensified, after which a gradual decrease of the gradients was observed in vitro. In vivo, however, gradients changed instantly and became less pronounced. Although similar gradients were observed regardless of scaffold type, significantly more cells were present in the center of 3DF constructs after 2 weeks of in vivo culture. Our results stress the importance of a rationally designed scaffold for tissue-engineering applications. Organized structures, such as the 3DF PEGT/PBT polymer scaffolds, offer possibilities for regulation of nutrient supply and, therefore, hold promise for clinical approaches for cartilage repair. (C) 2004 Elsevier Ltd. All rights reserved.

Articular cartilage lacks the ability to repair itself and consequently defects in this tissue do not heal. Tissue engineering approaches, employing a scaffold material and cartilage producing cells (chondrocytes), hold promise for the treatment of such defects. In these strategies the limitation of nutrients, such as oxygen, during in vitro culture are of major concern and will have implications for proper bioreactor design. We recently demonstrated that oxygen gradients are indeed present within tissue engineered cartilaginous constructs. Interestingly, oxygen, besides being an essential nutrient, is also a controlling agent of developmental processes including cartilage formation. However, the specific role of oxygen in these processes is still obscure despite the recent advances in the field. In particular, the outcome of published investigations is inconsistent regarding the effect of oxygen tension on chondrocytes. Therefore, this article describes the possible roles of oxygen gradients during embryonic cartilage development and reviews the data reported on the effect of oxygen tension on in vitro chondrocyte proliferation and differentiation from a tissue engineering perspective. Furthermore, possible causes for the variance in the data are discussed. Finally, recommendations are included that may reduce the variation, resulting in more reliable and comparable data.

To get a better understanding of sponge feeding biology and efficiencies, the fatty acid (FA) composition and (13)C natural abundance of sponges and of suspended particulate matter (SPM) from surrounding seawater was studied in different seasons at three locations. Haliclona oculata and Haliclona xena from the Oosterschelde, the Netherlands, Halichondria panicea and H. xena from Lake Veere, the Netherlands, and Aplysina aerophoba and Dysidea avara from the Mediterranean, Spain, were studied. Several FA biomarkers for different algal groups, bacteria and sponge biomass were identified in all sponges. The FA concentration variation in sponges was related to changes in fatty acid concentration in SPM. Stable carbon isotopic ratios (δ(13)C) in sponge specific FAs showed very limited seasonal variation at all sites. Algal FAs in sponges were mainly acquired from the SPM through active filtration in all seasons. At the two sites in the Netherlands only in May (spring), the sponge specific FAs had similar δ(13)C ratios as algal FAs, suggesting that sponges were mainly growing during spring and probably summer. During autumn and winter, they were still actively filtering, but the food collected during this period had little effect on sponge δ(13)C values suggesting limited incorporation of filtered material into the sponge body. The sponge A. aerophoba relied mostly on the symbiotic bacteria. In conclusion, fatty acid composition in combination with stable carbon isotope analysis can be used to analyze the food source of sponges.

OBJECTIVE: To examine the association between weight loss in obese pregnant women and relevant maternal and neonatal outcomes. METHODS: All liveborn singleton term (37 weeks of gestation or greater) births in obese women between 2009 and 2011 in Flanders (the northern part of Belgium) were included (N=18,053). Outcomes assessed included gestational hypertension, low (2,500 g or less) birth weight, small-for-gestational-age (less than the 10th percentile) neonates, macrosomia (birth weight 4,000 g or greater), large-for-gestational-age (greater than 90th percentile) neonates, emergency caesarean delivery, and admission to a neonatal intensive care unit. Risk for adverse outcomes was calculated by multiple logistic regression analysis for weight change categories (greater weight loss [5 kg or greater], lesser weight loss [between 0 and 5 kg], low gestational weight gain [0 or greater and less than 5 kg], adequate gestational weight gain [5 or greater to 9 kg or less, reference], and excessive gestational weight gain [greater than 9 kg]) in each obesity class (I 30-34.9, II 35-39.9, III 40 or greater) adjusted for parity and maternal and gestational age. RESULTS: In the total population, 854 (4.7%) obese pregnant women reported weight loss. Weight loss and low weight gain were associated with a decreased incidence of gestational hypertension for women with class I obesity (greater weight loss adjusted odds ratio [OR] 0.31, 95% confidence interval [CI] 0.11-0.84; lesser weight loss adjusted OR 0.46 95% CI 0.21-0.99; low gain adjusted OR 0.71 95% CI 0.54-0.93), a reduction in the rate of emergency cesarean delivery, but only in those with class II obesity (greater weight loss adjusted OR 0.24, 95% CI 0.07-0.78; lesser weight loss adjusted OR 0.50, 95% CI 0.26-0.97; low gain adjusted OR 0.55, 95% CI 0.38-0.79), and decreased macrosomia and large-for-gestational-age neonates in women in all classes of obesity, with the highest decrease for women with class III obesity (greater weight loss adjusted OR 0.15, 95% CI 0.05-0.49; lesser weight loss adjusted OR 0.37, 95% CI 0.15-0.90 for macrosomia). No association between weight loss and low birth weight, small-for-gestational-age neonates, or admission to the neonatal intensive care unit was shown in the different obesity classes. CONCLUSION: Weight loss in obese pregnant women was associated with reduced perinatal risks but not with the rate of low birth weight or small-for-gestational-age neonates in obese women from class III in this affluent region. Stratification of recommended gestational weight gain ranges in obese women should be considered.

Natural carotenoids such as astaxanthin, β,β-carotene and lutein are pigments with a high market value. We studied the effects of nitrogen depletion on the carotenoid metabolism of Chlorella zofingiensis (Chlorophyta) and the subsequent treatment with diphenylamine (DPA), an inhibitor of the biosynthesis of secondary ketocarotenoids. Pigments were identified and quantified based on reversed phase ultra-high performance liquid chromatography photodiode array tandem mass spectrometry (RP-UHPLC-PDA-MSn). Nitrogen depletion (without DPA) resulted in a degradation of chlorophylls and primary carotenoids and an accumulation of astaxanthin, ketolutein, canthaxanthin, adonixanthin and β,β-carotene. The DPA treatment decreased the overall production of β,β-carotene derivatives (sum of astaxanthin, canthaxanthin, echinenone and adonixanthin); however, the production of ketolutein and degradation of primary carotenoids were not modified. This suggests that the regulatory mechanisms controlling the flux towards ketolutein and primary carotenoids were not affected by the decreased levels of β,β-carotene derivatives. In addition, DPA increased production of the individual carotenoids, adonixanthin and echinenone. Insight into the regulation of microalgal carotenoid biosynthesis as demonstrated in this paper is essential when a large-scale carotenoid production process is to be optimised or a recombinant C. zofingiensis strain is to be designed with the intention of excessively producing primary or secondary carotenoids.

Insect cells have been cultured for over 30 years, but their application is still hampered by low cell densities in batch fermentations and expensive culture media. With respect to the culture method, the fed-batch culture mode is often found to give the best yields. However, optimization of the feed composition is usually a laborious task. In this report, the successful use of genetic algorithms (GAs) to optimize the growth of insect cells is described. A feed was developed from 11 different medium components, each used at a wide range of concentrations. The feed was optimized within four sets of 20 experiments. The optimized feed was tested in bioreactors and the addition scheme was further improved. The viable-cell density of HzAm1 (Helicoverpa zea) insect cells improved 550% to 19.5 × 106 cells/mL compared to a control fermentation in an optimized commercial medium. No accumulation of waste products was found, and none of the amino acids was depleted. Glucose was depleted, which suggests that even further improvement is possible. We show that GAs are a successful method to optimize a complex fermentation in a relatively short time frame and without the need of detailed information concerning the cellular physiology or metabolism. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 269–278, 2003.

The growth process of Lactobacillus curvatus colonies was quantified by a coupled growth and diffusion equation incorporating a volumetric rate of lactic acid production. Analytical solutions were compared to numerical ones, and both were able to predict the onset of interaction well. The derived analytical solution modeled the lactic acid concentration profile as a function of the diffusion coefficient, colony radius, and volumetric production rate. Interaction was assumed to occur when the volume-averaged specific growth rate of the cells in a colony was 90% of the initial maximum rate. Growth of L. curvatus in solid medium is dependent on the number of cells in a colony. In colonies with populations of fewer than 10 5 cells, mass transfer limitation is not significant for the growth process. When the initial inoculation density is relatively high, colonies are not able to grow to these sizes and growth approaches that of broth cultures (negligible mass transfer limitation). In foods, which resemble the model solid system and in which the initial inoculation density is high, it will be appropriate to use predictive models of broth cultures to estimate growth. For a very low initial inoculation density, large colonies can develop that will start to deviate from growth in broth cultures, but only after large outgrowth.

Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches ’bottle-necks’ are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected ’rate-limiting’ steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.

Mixed cultures of Lactobacillus curvatus and Enterobacter cloacae were chosen as a model system to quantitatively study microbial interactions involved in food spoilage and food preservation. In this paper models were developed to predict the individual behaviour of L. curvatus and E. cloacae in pure suspension cultures as a function of the glucose and lactate concentration and the pH. In a second paper these models will be used to predict the moment of interaction in mixed suspension cultures and the cell concentrations at this moment. The effect of pH on the maximum specific growth rate could be described by a parabolic equation for L. curvatus and E. cloacae. E. cloacae is clearly more sensitive to a pH decrease than L. curvatus, as may be concluded from the theoretical minimum pH for growth of 5.6 for E. cloacae and 4.3 for L. curvatus. For both organisms no effect of glucose on the maximum specific growth rate could be detected and Monod kinetics with a low Monod constant was assumed. For L. curvatus the effect of lactate on the maximum specific growth rate at different initial pH values could be described by a linear relationship. For E. cloacae a slight effect of lactate on the maximum specific growth rate could only be detected at pH 7. However, this effect was negligible compared to the effect of the pH. For both organisms the lag time was modelled by the inverse of the specific growth rate. It can be concluded that, with regard to interactions between L. curvatus and E. cloacae, the pH is likely to be the most important factor in the case where L. curvatus is the dominant organism. Substrate limitation is likely to become important in the case where E. cloacae is the dominant organism or for media with a high buffer capacity.

The European Network of Trainees in Obstetrics and Gynaecology (ENTOG) is a newly founded organisation which is closely associated with the European Board and College of Obstetrics and Gynaecology. The main objectives of ENTOG are: (i) to provide a forum for trainees to discuss and express their opinions on issues in Obstetric/Gynaecology (Ob/Gyn) training; (ii) to establish a continuous means of communication between European trainees; (iii) to promote high standards of training and thus improve the quality of medical care. To reach these objectives, ENTOG organizes a yearly exchange programme and educational meeting for trainees. Recently, a meeting was held in Greece with harmonization of training programmes in Ob/Gyn serving as a main topic.

Background: Proton pump inhibitors are effective for the healing of oesophagitis. Standard doses of omeprazole, lansoprazole or pantoprazole are sufficient for healing in mild to moderate cases of oesophagitis. Aim: To compare the efficacy of double the standard doses of omeprazole, lansoprazole or pantoprazole for maintenance treatment of severe oesophagitis complicated by a stricture. Methods: Thirty-six patients with reflux oesophagitis and stricture confirmed by endoscopy were included in a prospective study comparing three maintenance therapies. In all cases weekly dilatation of the stenosis was performed and patients were treated with omeprazole 20 mg b.d. until healing of oesophagitis and dysphagia relief were achieved. Thirty participants responded to therapy and were then randomly assigned to 4 weeks of maintenance treatment with omeprazole (20 mg b.d.; n=10), lansoprazole (30 mg b.d.; n=10) or pantoprazole (40 mg b.d.; n=10). Subsequently, endoscopies were performed—the endoscopists were blinded to the therapy assignment. The endpoints were defined as the absence of oesophagitis, oesophageal stricture and complaints. Results: After 4 weeks of treatment, the number of patients remaining in remission (no oesophagitis or stricture and no symptoms) was nine out of 10 (90%) in the omeprazole group, two out of 10 (20%) in the lansoprazole group (P

Microalgae are a promising future source for sustainable edible oils. To make microalgal oil a cost-effective alternative for common vegetable oils, increasing TAG productivity and TAG content are of high importance. Fulfilling these targets requires proper understanding of lipid metabolism in microalgae. Here, we provide an overview of our current knowledge on the biology of TAG accumulation as well as the latest developments and future directions for increasing oil production in microalgae, considering both metabolic engineering techniques and cultivation strategies.

Reproducing the native collagen structure and glycosaminoglycan (GAG) distribution in tissue-engineered cartilage constructs is still a challenge. Articular cartilage has a specific nutrient supply and mechanical environment due to its location and function in the body. Efforts to simulate this native environment have been reported through the use of bioreactor systems. However, few of these devices take into account the existence of gradients over cartilage as a consequence of the nutrient supply by diffusion. We hypothesized that culturing chondrocytes in an environment, in which gradients of nutrients can be mimicked, would induce zonal differentiation. Indeed, we show that glucose gradients facilitating a concentration distribution as low as physiological glucose levels enhanced a zonal chondrogenic capacity similar to the one found in native cartilage. Furthermore, we found that the glucose consumption rates of cultured chondrocytes were higher under physiological glucose concentrations and that GAG production rates were highest in 5 mM glucose. From these findings, we concluded that this condition is better suited for matrix deposition compared to 20 mM glucose standard used in a chondrocyte culture system. Reconsidering the culture conditions in cartilage tissue engineering strategies can lead to cartilaginous constructs that have better mechanical and structural properties, thus holding the potential of further enhancing integration with the host tissue.

There is increasing interest in naturally produced colorants, and microalgae represent a bio-technologically interesting source due to their wide range of colored pigments, including chlorophylls (green), carotenoids (red, orange and yellow), and phycobiliproteins (red and blue). However, the concentration of these pigments, under optimal growth conditions, is often too low to make microalgal-based pigment production economically feasible. In some Chlorophyta (green algae), specific process conditions such as oversaturating light intensities or a high salt concentration induce the overproduction of secondary carotenoids (β-carotene in Dunaliella salina (Dunal) Teodoresco and astaxanthin in Haematococcus pluvialis (Flotow)). Overproduction of all other pigments (including lutein, fucoxanthin, and phycocyanin) requires modification in gene expression or enzyme activity, most likely combined with the creation of storage space outside of the photosystems. The success of such modification strategies depends on an adequate understanding of the metabolic pathways and the functional roles of all the pigments involved. In this review, the distribution of commercially interesting pigments across the most common microalgal groups, the roles of these pigments in vivo and their biosynthesis routes are reviewed, and constraints and opportunities for overproduction of both primary and secondary pigments are presented.

To describe the obstetric outcome in women at ages >= 45. A retrospective cohort study on a large existing database covering all deliveries in the Flanders region, Belgium, was performed, comparing obstetric outcomes at age 25, 35, 40 and 45 or older, for the period 2005-2010. In the period studied, 421 women gave birth at maternal age >= 45 vs 3,405, 15,206, 22,586 at ages 40, 35 and 25, respectively. With advancing maternal age, a significant linear increase for low birth weight (< 2,500 g) and preterm delivery (< 37, < 35 weeks and < 29 weeks), maternal hypertension and diabetes, both primary and secondary caesarean section, was noted. Between ages 40 and >= 45, both fetal and early neonatal death demonstrated a significant rise from 4.9/1,000 and 1.8/1,000 to 26/1,000 and 9.5/1,000, respectively. With advancing maternal age, a gradual but not statistically significant decline in the proportion of male fetuses was noted, from 51.6% at 25, to 47.2% in the oldest group. Multivariate analysis confirmed advanced maternal age to be a significant factor in low birth weight, preterm delivery, hypertension, diabetes, caesarean section and perinatal mortality. In Flanders, mothers at age 45 and older have a significantly increased risk for low birth weight, preterm delivery, hypertension, diabetes, caesarean section and perinatal mortality.

Background Many microalgae accumulate carbohydrates simultaneously with triacylglycerol (TAG) upon nitrogen starvation, and these products compete for photosynthetic products and metabolites from the central carbon metabolism. As shown for starchless mutants of the non-oleaginous model alga Chlamydomonas reinhardtii, reduced carbohydrate synthesis can enhance TAG production. However, these mutants still have a lower TAG productivity than wild-type oleaginous microalgae. Recently, several starchless mutants of the oleaginous microalga Scenedesmus obliquus were obtained which showed improved TAG content and productivity. Results The most promising mutant, slm1, is compared in detail to wild-type S. obliquus in controlled photobioreactors. In the slm1 mutant, the maximum TAG content increased to 57¿±¿0.2% of dry weight versus 45¿±¿1% in the wild type. In the wild type, TAG and starch were accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The starchless mutant did not produce starch and the liberated photosynthetic capacity was directed towards TAG synthesis. This increased the maximum yield of TAG on light by 51%, from 0.144¿±¿0.004 in the wild type to 0.217¿±¿0.011 g TAG/mol photon in the slm1 mutant. No differences in photosynthetic efficiency between the slm1 mutant and the wild type were observed, indicating that the mutation specifically altered carbon partitioning while leaving the photosynthetic capacity unaffected. Conclusions The yield of TAG on light can be improved by 51% by using the slm1 starchless mutant of S. obliquus, and a similar improvement seems realistic for the areal productivity in outdoor cultivation. The photosynthetic performance is not negatively affected in the slm1 and the main difference with the wild type is an improved carbon partitioning towards TAG.

Background: Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results: Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions: The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under photoautotrophic conditions. These results can pave the way towards a more feasible microalgae-driven TAG production platform. Keywords: Starchle

The death rate of Vero cells grown on Cytodex-3 microcarriers was studied as a function of the gas flow rate in a small air-lift loop reactor. The death rate may be described by first-order death-rate kinetics. The first-order death-rate constant as calculated from the decrease in viable cells, the increase in dead cells and the increase in LDH activity is linear proportional to the gas flow rate, with a specific hypothetical killing volume in which all cells are killed of about 2·10(-3) m(3) liquid per m(3) of air bubbles. In addition, an experiment was conducted in the same air-lift reactor with Vero cells grown inside porous Asahi microcarriers. The specific hypothetical killing volume calculated from this experiment has a value of 3·10(-4) m(3) liquid per m(3) of air bubbles, which shows that the porous microcarriers were at least in part able to protect the cells against the detrimental hydrodynamic forces generated by the bubbles.

Despite its demonstrated effectiveness, colorectal cancer (CRC) testing is suboptimal, particularly in vulnerable populations such as those who are publicly insured. Prior studies provide an incomplete picture of the importance of the intersection of multilevel factors affecting CRC testing across heterogeneous geographic regions where vulnerable populations live. We examined CRC testing across regions of North Carolina by using population-based Medicare and Medicaid claims data from disabled individuals who turned 50 years of age during 2003–2008. We estimated multilevel models to examine predictors of CRC testing, including distance to the nearest endoscopy facility, county-level endoscopy procedural rates, and demographic and community contextual factors. Less than 50% of eligible individuals had evidence of CRC testing; men, African-Americans, Medicaid beneficiaries, and those living furthest away from endoscopy facilities had significantly lower odds of CRC testing, with significant regional variation. These results can help prioritize intervention strategies to improve CRC testing among publicly insured, disabled populations.

Lab-scale stirred-tank bioreactors (0.2-20 l) are used for fundamental research on animal cells and in process development and troubleshooting for large-scale production. In this chapter, different configurations of bioreactor systems are shortly discussed and setting up these different configurations is described. In addition, online measurement and control of bioreactor parameters is described, with special attention to controller settings (PID) and online measurement of oxygen consumption and carbon dioxide production. Finally, methods for determining the oxygen transfer coefficient are described.

Lab-scale stirred-tank bioreactors (0.2–20 l) are used for fundamental research on animal cells and in process development and troubleshooting for large-scale production. In this chapter, different configurations of bioreactor systems are shortly discussed and setting up these different configurations is described. In addition, online measurement and control of bioreactor parameters is described, with special attention to controller settings (PID) and online measurement of oxygen consumption and carbon dioxide production. Finally, methods for determining the oxygen transfer coefficient are described.

Objective: To examine the association between interpregnancy weight change and the risk for adverse maternal and neonatal outcomes.Methods:All live-born singleton births delivered at 21–42 weeks of gestation in women who had their first two consecutive births between 2009 and 2011 in Flanders (the northern part of Belgium) and who were included in the Study Center for Perinatal Epidemiology database (N=7,897) were included. Interpregnancy weight change was calculated as the difference between the prepregnancy body mass index (BMI) of the first pregnancy and the prepregnancy BMI of the second pregnancy. Multivariate logistic regression analysis to predict gestational diabetes mellitus, pregnancy-induced hypertension, cesarean delivery, macrosomia (4,000 g or greater), low birth weight (less than 2,500 g), and congenital malformations were performed.Results: The adjusted odds ratio (OR) for gestational diabetes mellitus was 2.25 (95% confidence interval [CI] 1.33–3.78; P=.002) for interpregnancy weight retention of 2 or more BMI units, and the adjusted OR for pregnancy-induced hypertension was 3.76 (95% CI 2.16–6.57; PConclusion: We show that weight retention between the first and second pregnancy is associated with an increased risk for perinatal complications, even in underweight and normal-weight women. Stabilizing interpregnancy weight appears an important target for reducing adverse perinatal outcomes in a second pregnancy.

Lactococcus lactis subsp. cremoris MG1363 is a paradigm strain for lactococci used in industrial dairy fermentations. However, despite of its importance for process development, no genome-scale metabolic model has been reported thus far. Moreover, current models for other lactococci only focus on growth and sugar degradation. A metabolic model that includes nitrogen metabolism and flavor-forming pathways is instrumental for the understanding and designing new industrial applications of these lactic acid bacteria. A genome-scale, constraint-based model of the metabolism and transport in L. lactis MG1363, accounting for 518 genes, 754 reactions, and 650 metabolites, was developed and experimentally validated. Fifty-nine reactions are directly or indirectly involved in flavor formation. Flux Balance Analysis and Flux Variability Analysis were used to investigate flux distributions within the whole metabolic network. Anaerobic carbon-limited continuous cultures were used for estimating the energetic parameters. A thorough model-driven analysis showing a highly flexible nitrogen metabolism, e.g., branched-chain amino acid catabolism which coupled with the redox balance, is pivotal for the prediction of the formation of different flavor compounds. Furthermore, the model predicted the formation of volatile sulfur compounds as a result of the fermentation. These products were subsequently identified in the experimental fermentations carried out. Thus, the genome-scale metabolic model couples the carbon and nitrogen metabolism in L. lactis MG1363 with complete known catabolic pathways leading to flavor formation. The model provided valuable insights into the metabolic networks underlying flavor formation and has the potential to contribute to new developments in dairy industries and cheese-flavor research.

A kinetic model is presented that describes functional biomass, starch and storage lipid (TAG) synthesis in the microalga Neochloris oleoabundans as a function of nitrogen and light supply rates to a nitrogen-limited turbidostat cultivation system. The model is based on the measured electron distribution in N. oleoabundans, which showed that starch is the primary storage component, whereas TAG was only produced after an excess of electrons was generated, when growth was limited by nitrogen supply. A fixed 8.6% of the excess electrons ended up in TAG, suggesting close metabolic interactions between nitrogen assimilation and TAG accumulation, such as a shared electron pool. The proposed model shows that by manipulating the cultivation conditions in a light or nitrogen limited turbidostat, algal biomass composition can be customised and the volumetric productivities and yields of the major biomass constituents can be changed on demand.

To assess preliminarily the effectiveness of zonisamide in bulimia nervosa.This was an open-label, prospective, 12-week, flexible dose study of zonisamide in bulimia nervosa. The primary outcome was binge-purge episode frequency.Twelve individuals received zonisamide, 10 completed at least one post-baseline evaluation, and six completed the study. Mean dose at endpoint was 420 (SD = 215) mg/day. Zonisamide was associated with significant reductions in frequency of binge-purge episodes and binge-purge days as well as measures of binge eating behavior, purging behavior, clinical severity, obsessive-compulsive features, and depressive symptoms. Weight was unchanged.In this open-label trial, zonisamide was effective in bulimia nervosa, but associated with a high discontinuation rate.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes. With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of. This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other. The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

Summary In a survey of the metazoan ecto- and endoparasites of Siganus sutor (Cuvier & Valenciennes, 1835), a commercially important herbivorous fish from the Kenyan coast, sixteen species of parasites are found. The gill parasites include Monogenea (Tetrancistrum sigani Goto & Kikuchi, 1917; Microcotyle mouwoi Ishii & Sawada, 1938; Pseudohaliotrema sp. 1 and sp. 2, and an unidentified Microcotylidae species); Copepoda (Caligus sp. and Hatschekia sp.); and Isopoda (one Gnathiidae species). The intestinal parasites found are Digenea (Opisthogonoporoides cf. hanumanthai Madhavi, 1912; Gyliauchen papillatus Goto & Matsudaira, 1918; Hexangium sigani Goto & Ozaki, 1929; and three other unidentified digeneans); Acanthocephala (Sclerocollum rubrimaris Schmidt & Paperna, 1978); and Nematoda (Procamallanus sigani Yamaguti, 1935). The species listed are first records for the Kenyan coast. Infection prevalence, mean intensity and site specificity are determined for the different parasite species. The parasites have an aggregated frequency distribution in the host population: some individuals of the siganid population are more heavily infected than expected in a random distribution, while others are very little or not at all infected. Resume On a decouvert seize especes de parasites lors d'une etude des metazoaires ecto-et endoparasites de Siganus sutor (Cuvier & Valenciennes, 1985), un Poisson herbivore commercialement important de la cote keyane. Les parasites des branchies comprennent des Monogeneae (Tetrancistrum sigani Goto & Kikuchi, 1917; Microcotyle mouwoi Ishii & Sawada, 1938; Pseudohaliotrema sp.1 & sp.2, et une espece non identifiee de Microcotylideae); des Copepodes (Caligus sp. et Hatschekia sp.) et un Icopode (une espece de Gnathiideae). Les parasites intestinaux trouves appartenaient aux Digeneae (Opisthogonoporoides cf. hanumanthai Madhavi, 1972; Gyliauchen papillatus Goto & Matsudaira, 1918; Hexangium sigani Goto & Ozaki, 1929; et trois autres Digenaeae non identifies); aux Acanthocephaleae (Sclerocollum rubrimaris Schmidt & Paperna, 1978), et aux Nematodes (Procamallanus sigani Yamaguti 1935). Ces especes sont rapportees pour la premiere fois sur la cote kenyane. Le degre, l'intensite

The Model presented in this work demonstrates the combination of cell-cycle model with a model describing the growth and conversion kinetics of hybridoma cells in a steady-state continuous culture. The cell-cycle model is based upon a population balance model as described by Cazzador et al. and assumes the existence of a cycling-and apoptotic-cell population, which together form the viable-cell population. In this part the fraction of apoptotic cells, the age distribution of the cycling and apoptotic-cell population, the mean volume and biomass content per cell of the cycling, apoptotic, and viable cells, and the specific growth and death rates of the cells are calculated. The metabolic part consists of a Monod-type growth equation, four elemental balances, an equation assuming a constant yield of ammonia on glutamine, an equation for product formation, and the relation of Glacken for energy production. Furthermore, a maintenance-energy model for the consumption of glucose and glutamine is introduced, which combines the approaches of Herbert and Pirt into one model in a way similar to Beeftink et al. For energy consumption a Pirt model is assumed. The model is capable of predicting trends in steady-state values of a large number of variables of interest like specific growth rate, specific death rate, viability, cell numbers, mean viable-cell volume, and concentrations and conversion rates of product, glucose, glutamine, lactate, and ammonia. Also the concentrations and conversion rates of oxygen and carbon dioxide are qualitatively predicted. The values of the model predictions are generally close to experimental data obtained from literature.

To evaluate cardiotocography (CTG) alone versus CTG and ST-analysis (STAN) in daily obstetric practice in a complete region.Prospective registration in the region of Flanders in combination with standard registration of perinatal outcome.Of 62,606 term deliveries registered, 57,141 (91.3%) were available for complete analysis. In 50,748 (88.8%) CTG alone and in 6,393 (11.6%) CTG+STAN was used. STAN was used significantly more in case of hypertension, diabetes and induction of labour and was associated both in univariate and multivariate analysis with significantly more secondary caesarean section for suspected foetal distress, instrumental vaginal delivery, low Apgar score and need for neonatal intensive care. There was no difference in perinatal death or asphyxia.ST-analysis versus CTG results in more caesarean sections, instrumental vaginal deliveries and neonatal intensive care. This can not be explained solely by its use in more complicated cases as in multivariate analysis including hypertension, diabetes and induction of labour ST analysis persists as a significant factor. We hypothesise that this could be explained by less well trained users not adhering to STAN-guidelines.

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

Marine sponges are a rich source of bioactive compounds with pharmaceutical potential. Since biological production is one option to supply materials for early drug development, the main challenge is to establish generic techniques for small-scale production of marine organisms. We analysed the state of the art for cultivation of whole sponges, sponge cells and sponge symbionts. To date, cultivation of whole sponges has been most successful in situ; however, optimal conditions are species specific. The establishment of sponge cell lines has been limited by the inability to obtain an axenic inoculum as well as the lack of knowledge on nutritional requirements in vitro. Approaches to overcome these bottlenecks, including transformation of sponge cells and using media based on yolk, are elaborated. Although a number of bioactive metabolite-producing microorganisms have been isolated from sponges, and it has been suggested that the source of most sponge-derived bioactive compounds is microbial symbionts, cultivation of sponge-specific microorganisms has had limited success. The current genomics revolution provides novel approaches to cultivate these microorganisms.

Both pre-pregnancy body mass index (BMI) and gestational weight gain (GWG) are important determinants of a healthy pregnancy outcome and may show important variation. To study the influence of socio-demographic and obstetrical correlates on pre-pregnancy BMI and GWG, data of 54 022 singleton term pregnancies were analysed using adjusted regression models. In 2009, in the Northern region of Belgium, one-third of women were overweight (21.6%) or obese (10.1%) and GWG as recommended by the Institute of Medicine occurred in only 28% of obese women. A high pre-pregnancy BMI was significantly associated with low maternal education, high maternal age and multiparity, belonging to ethnic minority groups and a lower professional state. Compared to adequate GWG, excessive GWG was more common in younger (20 years) women, with higher pre-pregnancy BMI and pregnancy-induced hypertension. Moreover, younger (20-24 years), single women, belonging to ethnic minority groups showed higher odds for excessive as well as insufficient GWG, while those with high/highest educational level had lower odds for excessive (odds ratio [OR] 0.76; confidence interval [CI] 0.72-0.80) and insufficient (OR 0.93; CI 0.89-0.98) GWG. The results of this study highlight the scale of the problem of maternal obesity and excessive GWG for this region and offer opportunities to target educational campaigns and intervention programmes in the clinical setting.

Microalgae-derived lipids are an alternative to vegetable and fossil oils, but lipid content and quality vary among microalgae strains. Selection of a suitable strain for lipid production is therefore of paramount importance. Based on published results for 96 species, nine strains were selected to study their biomass, total fatty acid, and triacylglycerol (TAG) production under nitrogen-sufficient and deficient cultivation conditions. Under nitrogen-deficient conditions, Chlorella vulgaris, Chlorella zofingiensis, Neochloris oleoabundans , and Scenedesmus obliquus , accumulated more than 35% of their dry weight as TAGs. Palmitic and oleic acid were the major fatty acids produced. The main difference between these strains was the amount of biomass that was produced (3.0–7.8-fold increase in dry weight) and the duration that the biomass productivity was retained (2–7 days) after nitrogen depletion. S. obliquus (UTEX 393) and C. zofingiensis (UTEX B32) showed the highest average TAG productivity (322 and 243 mg l −1 day −1 ).

Marine sponges are a rich source of bioactive compounds with pharmaceutical potential. Since biological production is one option to supply materials for early drug development, the main challenge is to establish generic techniques for small-scale production of marine organisms. We analysed the state of the art for cultivation of whole sponges, sponge cells and sponge symbionts. To date, cultivation of whole sponges has been most successful in situ; however, optimal conditions are species specific. The establishment of sponge cell lines has been limited by the inability to obtain an axenic inoculum as well as the lack of knowledge on nutritional requirements in vitro. Approaches to overcome these bottlenecks, including transformation of sponge cells and using media based on yolk, are elaborated. Although a number of bioactive metabolite-producing microorganisms have been isolated from sponges, and it has been suggested that the source of most sponge-derived bioactive compounds is microbial symbionts, cultivation of sponge-specific microorganisms has had limited success. The current genomics revolution provides novel approaches to cultivate these microorganisms.