Biomedical students' satisfaction with and engagement in laboratory e-learning support are related to their self-regulation.

Laboratory e-learning support tools can assist students' learning while preparing for laboratory classes. To successfully work in such virtual experimental environments (VEEs) outside class, students require self-regulated learning (SRL) skills. A deeper understanding of the continuous reciprocal interactions between SRL, satisfaction, and online engagement is needed to develop more effective online learning experiences. This study therefore aimed to explore the interconnection between students' satisfaction with, effort/importance and engagement in an exemplary VEE, and to relate this to their perceived SRL and learning outcomes. Based on surveys in 79 university students, SRL was related to VEE engagement, effort/importance, and satisfaction. VEE engagement and satisfaction were not related to learning outcomes, while SRL and effort were. Students with different SRL also tended to interact differently with the VEE and experienced differing degrees of procedural and feedback support by the e-environment. We conclude that, for optimal learning experience and outcomes, students' effort regulation and SRL need to be supported while interacting with the VEE, preferably by interventions that integrate personalized and adaptive features. This study has implications for designing and optimizing VEEs and indicates that future research should focus on VEEs taking students' SRL and effort regulation into account to support individual learners effectively.

Consistent individual differences in the social phenotypes of wild great tits, Parus major.

Despite growing interest in animal social networks, surprisingly little is known about whether individuals are consistent in their social network characteristics. Networks are rarely repeatedly sampled; yet an assumption of individual consistency in social behaviour is often made when drawing conclusions about the consequences of social processes and structure. A characterization of such social phenotypes is therefore vital to understanding the significance of social network structure for individual fitness outcomes, and for understanding the evolution and ecology of individual variation in social behaviour more broadly. Here, we measured foraging associations over three winters in a large PIT-tagged population of great tits, and used a range of social network metrics to quantify individual variation in social behaviour. We then examined repeatability in social behaviour over both short (week to week) and long (year to year) timescales, and investigated variation in repeatability across age and sex classes. Social behaviours were significantly repeatable across all timescales, with the highest repeatability observed in group size choice and unweighted degree, a measure of gregariousness. By conducting randomizations to control for the spatial and temporal distribution of individuals, we further show that differences in social phenotypes were not solely explained by within-population variation in local densities, but also reflected fine-scale variation in social decision making. Our results provide rare evidence of stable social phenotypes in a wild population of animals. Such stable social phenotypes can be targets of selection and may have important fitness consequences, both for individuals and for their social-foraging associates.

No evidence for MHC class I-based disassortative mating in a wild population of great tits.

Genes of the major histocompatibility complex (MHC) are regarded as a potentially important target of mate choice due to the fitness benefits that may be conferred to the offspring. According to the complementary genes hypothesis, females mate with MHC dissimilar males to enhance the immunocompetence of their offspring or to avoid inbreeding depression. Here, we investigate whether selection favours a preference for maximally dissimilar or optimally dissimilar MHC class I types, based on MHC genotypes, average amino acid distances and the functional properties of the antigen-binding sites (MHC supertypes); and whether MHC type dissimilarity predicts relatedness between mates in a wild great tit population. In particular, we explore the role that MHC class I plays in female mate choice decisions while controlling for relatedness and spatial population structure, and examine the reproductive fitness consequences of MHC compatibility between mates. We find no evidence for the hypotheses that females select mates on the basis of either maximal or optimal MHC class I dissimilarity. A weak correlation between MHC supertype sharing and relatedness suggests that MHC dissimilarity at functional variants may not provide an effective index of relatedness. Moreover, the reproductive success of pairs did not vary with MHC dissimilarity. Our results provide no support for the suggestion that selection favours, or that mate choice realizes, a preference for complimentary MHC types.

Fewer invited talks by women in evolutionary biology symposia.

Lower visibility of female scientists, compared to male scientists, is a potential reason for the under-representation of women among senior academic ranks. Visibility in the scientific community stems partly from presenting research as an invited speaker at organized meetings. We analysed the sex ratio of presenters at the European Society for Evolutionary Biology (ESEB) Congress 2011, where all abstract submissions were accepted for presentation. Women were under-represented among invited speakers at symposia (15% women) compared to all presenters (46%), regular oral presenters (41%) and plenary speakers (25%). At the ESEB congresses in 2001-2011, 9-23% of invited speakers were women. This under-representation of women is partly attributable to a larger proportion of women, than men, declining invitations: in 2011, 50% of women declined an invitation to speak compared to 26% of men. We expect invited speakers to be scientists from top ranked institutions or authors of recent papers in high-impact journals. Considering all invited speakers (including declined invitations), 23% were women. This was lower than the baseline sex ratios of early-mid career stage scientists, but was similar to senior scientists and authors that have published in high-impact journals. High-quality science by women therefore has low exposure at international meetings, which will constrain Evolutionary Biology from reaching its full potential. We wish to highlight the wider implications of turning down invitations to speak, and encourage conference organizers to implement steps to increase acceptance rates of invited talks.

Human tissue-engineered bone produced in clinically relevant amounts using a semi-automated perfusion bioreactor system: a preliminary study.

The aim of this study was to evaluate a semi-automated perfusion bioreactor system for the production of clinically relevant amounts of human tissue-engineered bone. Human bone marrow stromal cells (hBMSCs) of eight donors were dynamically seeded and proliferated in a perfusion bioreactor system in clinically relevant volumes (10 cm(3)) of macroporous biphasic calcium phosphate scaffolds (BCP particles, 2-6 mm). Cell load and distribution were shown using methylene blue staining. MTT staining was used to demonstrate viability of the present cells. After 20 days of cultivation, the particles were covered with a homogeneous layer of viable cells. Online oxygen measurements confirmed the proliferation of hBMSCs in the bioreactor. After 20 days of cultivation, the hybrid constructs became interconnected and a dense layer of extracellular matrix was present, as visualized by scanning electron microscopy (SEM). Furthermore, the hBMSCs showed differentiation towards the osteogenic lineage as was indicated by collagen type I production and alkaline phosphatase (ALP) expression. We observed no significant differences in osteogenic gene expression profiles between static and dynamic conditions like ALP, BMP2, Id1, Id2, Smad6, collagen type I, osteocalcin, osteonectin and S100A4. For the donors that showed bone formation, dynamically cultured hybrid constructs showed the same amount of bone as the statically cultured hybrid constructs. Based on these results, we conclude that a semi-automated perfusion bioreactor system is capable of producing clinically relevant and viable amounts of human tissue-engineered bone that exhibit bone-forming potential after implantation in nude mice.

Expansion of human mesenchymal stromal cells on microcarriers: growth and metabolism.

Adult stem cells, or mesenchymal stromal cells (MSCs), are of great potential for cell therapy and tissue-engineering applications. However, for therapeutic use, these cells need to be isolated from tissue or a biopsy and efficiently expanded, as they cannot be harvested in sufficient quantities from the body. In our opinion, efficient expansion of MSCs can be achieved in a microcarrier-based cultivation system. This study selected a suitable microcarrier for human bone marrow-derived stromal cells (HBMSCs), optimized cell-seeding strategies by varying serum concentrations, and optimized dynamic expansion of the HBMSCs in a microcarrier-based spinner flask cultivation system by applying various feeding regimes. Cytodex 1 microcarriers in combination with a low-serum concentration (0-5%) in the medium resulted in the highest seeding efficiency for the HBMSCs. Subsequently, significant expansion of the HBMSCs on these carriers has been observed. The highest number of HBMSCs population doublings (4.8 doublings) was obtained by a combination of 50% medium refreshment combined with addition of 30% medium containing microcarriers every 3 days. Exponential cell growth was observed for at least 9 days after seeding, provided that sufficient nutrients (such as glucose) were present, metabolite concentrations (such as ammonia) were kept below growth-inhibitory concentrations and adequate surface area was present for the cells. After dynamic expansion of the HBMSCs, the cells retained their differentiation potential and their cell surface markers, indicating that HBMSCs expansion on Cytodex 1 microcarriers did not alter the phenotypic properties of the cells.

Expansion of mesenchymal stem cells using a microcarrier-based cultivation system: growth and metabolism.

For the continuous and fast expansion of mesenchymal stem cells (MSCs), microcarriers have gained increasing interest. The aim of this study was to evaluate the growth and metabolism profiles of MSCs, expanded in a microcarrier-based cultivation system. We investigated various cultivation conditions to expand goat mesenchymal stem cells on Cytodex 1 microcarriers. These conditions differed in feeding regime, i.e. the addition of fresh proliferation medium, with or without new microcarriers. For all conditions, cell attachment, cell proliferation, energy source consumption, metabolite production, and cell distribution on the microcarriers were studied. Attachment efficiencies of 40% were obtained followed by successful expansion up to 15 cultivation days. Depending on the feeding regime, an exponential growth, stationary growth, and decline growth phase could be distinguished. Addition of 30% fresh medium containing microcarriers every three days showed the longest continuous proliferation of goat MSCs on microcarriers. This feeding regime has the advantage that metabolites, such as ammonia, are diluted and that new energy sources, such as glucose and glutamine, and additional surface area are provided to the cells. In addition, by adding extra microcarriers a more homogenous cell distribution on the microcarriers is obtained as a result of bead-to-bead transfer. A correlation between nutrient consumption, metabolite production and cell growth was observed. The decreasing yield of lactate from glucose over time indicated a possible shift in cellular metabolism.

An optimized DHPLC protocol for molecular testing of the EXT1 and EXT2 genes in hereditary multiple osteochondromas.

Hereditary multiple osteochondromas (MO) is an autosomal dominant bone disorder characterized by the presence of bony outgrowths (osteochondromas or exostoses) on the long bones. MO is caused by mutations in the EXT1 or EXT2 genes, which encode glycosyltransferases implicated in heparan sulfate biosynthesis. Standard mutation analysis performed by sequencing analysis of all coding exons of the EXT1 and EXT2 genes reveals a mutation in approximately 80% of the MO patients. We have now optimized and validated a denaturing high-performance liquid chromatography (DHPLC)-based protocol for screening of all EXT1- and EXT2-coding exons in a set of 49 MO patients with an EXT1 or EXT2 mutation. Under the optimized DHPLC conditions, all mutations were detected. These include 20 previously described mutations and 29 new mutations - 20 new EXT1 and nine new EXT2 mutations. The protocol described here, therefore, provides a sensitive and cost-sparing alternative for direct sequencing analysis of the MO-causing genes.

Controlled release of proteins from degradable poly(ether-ester) multiblock copolymers.

A new series of multiblock poly(ether-ester)s based on poly(ethylene glycol) (PEG), butylene terephthalate (BT) and butylene succinate (BS) segments were introduced as matrices for controlled release applications. The release of two model proteins, lysozyme and bovine serum albumin (BSA), from poly(ether-ester) films were evaluated and correlated to the swelling and degradation characteristics of the polymer matrices. First- and zero-order profiles were found for the release of lysozyme, depending on the composition of the polymer matrix. The initial diffusion coefficient was correlated to the swelling of the matrix, which increased with longer PEG segments and lower BT/BS ratios of the polymer. High swelling matrices released the lysozyme according to diffusion-controlled first-order release profiles. Zero-order release profiles were obtained from less swollen matrices due to a combination of diffusion and degradation of the matrix. In contrast to the release of lysozyme, BSA was released from the poly(ether-ester) matrices via delayed release profiles. Both the delay time and the release rate could be tailored by varying the matrix composition. The BSA release rate was mainly determined by the degradation, whereas the delay time was determined by a combination of the swelling and the degradation rate of the polymer matrix.

Biodegradable poly(ether-ester) multiblock copolymers for controlled release applications: An in vivo evaluation.

Multiblock poly(ether-ester)s based on poly(ethylene glycol), butylene terephthalate, and butylene succinate segments were evaluated for their in vivo degradation and biocompatibility in order to establish a correlation with previously reported in vitro results. Porous polymer sheets were implanted subcutaneously for 32 weeks in rats. The degradation was monitored visually (histology), by molecular weight (GPC), and by copolymer composition (NMR). Substitution of the aromatic terephthalate units by aliphatic succinate units was shown to accelerate the degradation rate of the copolymers. Direct correlation of the in vivo and in vitro degradation of the porous implants showed a slightly faster initial molecular weight decrease in vivo. Besides hydrolysis, oxidation occurs in vivo due to the presence of radicals produced by inflammatory cells. In addition, the higher molecular weight plateau of the residue found in vivo indicated a higher solubility of the oligomers in the extracellular fluid compared to a phosphate buffer. Minor changes in the poly(ether-ester) compositions were noted due to degradation. Microscopically, fragmentation of the porous implants was observed in time. At later stages of degradation, macrophages were observed phagocytozing small polymer particles. Both in vitro cytotoxicity studies and histology on in vivo samples proved the biocompatibility of the poly(ether-ester)s.

In vitro/in vivo correlation for 14C-methylated lysozyme release from poly(ether-ester) microspheres.

PURPOSE: The purpose of this study was to obtain an in vitro/in vivo correlation for the sustained release of a protein from poly(ethylene glycol) terephthalate (PEGT)/poly(butylene terephthalate) (PBT) microspheres. METHODS: Radiolabeled lysozyme was encapsulated in PEGT/PBT microspheres via a water-in-oil-in-water emulsion. Three microsphere formulations varying in copolymer composition were administered subcutaneously to rats. The blood plasma was analyzed for radioactivity content representing released lysozyme at various time points post-dose. The in vitro release was studied in phosphate-buffered saline. RESULTS: The encapsulation efficiency, calculated from the radioactivity in the outer water phase of the emulsion, varied from 60-87%. Depending on the PEG segment length and wt% PEGT, the lysozyme was released completely in vitro within 14 to 28 days without initial burst. 14C-methylated lysozyme could be detected in the plasma over the same time courses. The in vitro/in vivo correlation coefficients obtained from point-to-point analysis were greater than 0.96 for all microsphere formulations. In addition, less then 10% of administered radioactivity remained at dose site at 28 days for the microsphere formulations, indicating no notable retention of the protein at the injection site. CONCLUSION: The in vitro release in phosphate-buffered saline and the in vivo release in rats showed an excellent congruence independent of the release rate of 14C-methylated lysozyme from PEGT/PBT microspheres.

Biodegradable poly(ether-ester) multiblock copolymers for controlled release applications.

Multiblock poly(ether-ester)s based on poly(ethylene glycol), butylene terephthalate, and butylene succinate units were synthesized by a two-step melt polycondensation reaction, with the aim of developing a new series of degradable polymers for controlled release applications. The copolymers were characterized with respect to their composition (NMR), thermal properties (DSC), and swelling. The main focus was on the degradation kinetics and release properties of the copolymers. The crystallinity and swelling could be tailored by the PEG segment length and the ratio of the building units. With increasing mol fraction succinate in the hard segment, the swelling increased. The in vitro degradation was found to occur by molecular weight decrease and mass loss. Substitution of the aromatic terephthalate units by aliphatic succinate units increased the degradation rate of the copolymers. Polymers with PEG segments of 1000 kg/mol showed a more pronounced degradation than copolymers containing shorter and longer PEG segments. Model proteins were successfully incorporated and released from the poly(ether-ester) films. Depending on the size of the protein, the release mechanism was based on diffusion of the protein and degradation of the matrix.

Release of small water-soluble drugs from multiblock copolymer microspheres: a feasibility study.

Poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) multiblock copolymer was investigated as a possible matrix for controlled delivery of small water-soluble drugs. Two molecules were selected as sustained release candidates from microspheres: leuprorelin acetate (peptide of Mw = 1270 D) and vitamin B(12) (Mw = 1355 D). First, vitamin B(12)-loaded microspheres were prepared using a double emulsion method and preparation parameters were varied (surfactant in the first emulsion and copolymer composition). The resulting microsphere structure, entrapment efficiency and release rate were evaluated. Vitamin B(12)-loaded microsphere parameters could easily be tailored to achieve specific requirements. The addition of surfactant in the first preparation process led to a significant increase of the microsphere entrapment efficiency, whereas the decrease of the PEGT copolymer content allowed the release rates from microspheres to be precisely decreased. However, leuprorelin acetate-loaded microspheres did not show the same characteristics when prepared with the same parameters, possibly because of a high water solubility discrepancy between the vitamin B(12) and the peptide. This study shows the suitability of PEGT/PBT microspheres as a controlled release system for vitamin B(12), but not for leuprorelin acetate. It also underlines the necessity of tailored development for each individual drug and emphasizes the risk of using model molecules.

Stability aspects of salmon calcitonin entrapped in poly(ether-ester) sustained release systems.

Poly(ether-ester)s composed of hydrophilic poly(ethylene glycol)-terephthalate (PEGT) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks were studied as matrix for the controlled release of calcitonin. Salmon calcitonin loaded PEGT/PBT films were prepared from water-in-oil emulsions. The initial calcitonin release rate could be tailored by the copolymer composition, but incomplete release of calcitonin was observed. FTIR measurements indicated aggregation of calcitonin in the matrix, which was not due to the preparation method of the matrices, but due to the instability of calcitonin in an aqueous environment. Release experiments showed the susceptibility of calcitonin towards the composition of the release medium, in particular to the presence of metal ions. With increasing amount of sodium ions, a decrease in the total amount of released calcitonin was observed due to enhanced aggregation. The calcitonin had to be stabilized in the matrix to prevent aggregation. Incorporation of sodium dodecyl sulphate (SDS) as a stabilizer in PEGT/PBT matrices increased the percentage of calcitonin released, but could not avoid aggregation on a longer term.

Biocompatibility and degradation of poly(ether-ester) microspheres: in vitro and in vivo evaluation.

Microspheres of a hydrophobic and a hydrophilic poly(ether-ester) copolymer were evaluated for their in vitro and in vivo biocompatibility and degradation. The microspheres prior to and after sterilization were tested for in vitro cytotoxicity. The in vivo biocompatibility of the poly(ethylene glycol) terephthalate and poly(butylene terephthalate) (PEGT/PBT) microspheres was evaluated subcutaneously and intramuscularly for 24 weeks in rabbits. The in vivo degradation of the microspheres was studied microscopically and compared to the in vitro degradation. The in vitro and in vivo studies showed the biocompatibility of the microspheres of both the hydrophobic and the hydrophilic PEGT/PBT copolymer. Extracts of these microspheres showed no cytotoxic reactivity in the in vitro cytotoxicity test. Sterilization of the microspheres by gamma irradiation did not affect the cytotoxicity. PEGT/PBT microspheres injected subcutaneously and intramuscularly in rabbits showed a mild tissue response in vivo, in terms of the inflammatory response, the foreign body reaction and the granulation tissue response. Although an in vitro degradation experiment showed a decrease in molecular weight due to hydrolysis, the in vivo degradation of the microspheres was slower than previously published.

Control of vitamin B12 release from poly(ethylene glycol)/poly(butylene terephthalate) multiblock copolymers.

The release of vitamin B12 (1355 Da) from matrices based on multiblock copolymers was studied. The copolymers were composed of hydrophilic poly(ethylene glycol)-terephthalate (PEGT) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks. Vitamin B12 loaded films were prepared by using a water-in-oil emulsion method. The copolymer properties, like permeability, could be varied by increasing the PEG-segment length from 300 up to 4,000 g/mol and by changing the wt% of PEGT. From permeation and release experiments. the diffusion coefficient of vitamin B12 through PEGT/PBT films of different compositions was determined. The diffusion coefficient of Vitamin B12 was strongly dependent on the composition of the copolymers. Although an increased wt% of PEGT (at a constant PEG-segment length) resulted in a higher diffusion coefficient, a major effect was observed at increasing PEG-segment length. By varying the copolymer composition, a complete release of vitamin B12 in 1 day up to a constant release for over 12 weeks was obtained. The release rate could be effectively tailored by blending copolymers with different PEG-segment lengths. The swelling and the crystallinity of the matrix could explain the effect of the matrix composition on the release behavior.

Microspheres for protein delivery prepared from amphiphilic multiblock copolymers. 1. Influence of preparation techniques on particle characteristics and protein delivery.

The entrapment of lysozyme in amphiphilic multiblock copolymer microspheres by emulsification and subsequent solvent removal processes was studied. The copolymers are composed of hydrophilic poly(ethylene glycol) (PEG) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks. Direct solvent extraction from a water-in-oil (w/o) emulsion in ethanol or methanol did not result in the formation of microspheres, due to massive polymer precipitation caused by rapid solvent extraction in these non-solvents. In a second process, microspheres were first prepared by a water-in-oil-in-water (w/o/w) emulsion system with 4% poly(vinyl alcohol) (PVA) as stabilizer in the external phase, followed by extraction of the remaining solvent. As non-solvents ethanol, methanol and mixtures of methanol and water were employed. However, the use of alcohols in the extraction medium resulted in microspheres which gave an incomplete lysozyme release at a non-constant rate. Complete lysozyme release was obtained from microspheres prepared by an emulsification-solvent evaporation method in PBS containing poly(vinyl pyrrolidone) (PVP) or PVA as stabilizer. PVA was most effective in stabilizing the w/o/w emulsion. Perfectly spherical microspheres were produced, with high protein entrapment efficiencies. These microspheres released lysozyme at an almost constant rate for approximately 28 days. The reproducibility of the w/o/w emulsion process was demonstrated by comparing particle characteristics and release profiles of three batches, prepared under similar conditions.

Microspheres for protein delivery prepared from amphiphilic multiblock copolymers. 2. Modulation of release rate.

Amphiphilic multiblock copolymers, based on hydrophilic poly(ethylene glycol) (PEG) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks were used as matrix material for protein-loaded microspheres. The efficiency of lysozyme entrapment by a double emulsion method was found to depend on the swelling behavior of the polymers in water and decreased from 100% for polymers with a degree of swelling of less than 1.8 to 11% for PEG-PBT copolymers with a degree of swelling of 3.6. The particle size could be controlled by varying the concentration of the polymer solution used in the microsphere preparation. An increase in the polymer concentration resulted in a proportional increase in the particle size. The in vitro release profiles of the encapsulated model protein lysozyme could be precisely tailored by variation of the copolymer composition and the size of the microspheres. Both a slow continuous release of lysozyme, and a fast release which was completed within a few days could be obtained. The release behavior, attributed to a combination of diffusion and polymer degradation, could be described by a previously developed model.

Zero-order release of lysozyme from poly(ethylene glycol)/poly(butylene terephthalate) matrices.

Protein release from a series of biodegradable poly(ether ester) multiblock copolymers, based on poly(ethylene glycol) (PEG) and poly(butylene terephthalate) (PBT) was investigated. Lysozyme-containing PEG/PBT films and microspheres were prepared using an emulsion technique. Proteins were effectively encapsulated and dense polymer matrices were formed. The swelling in water of PEG/PBT films reached equilibrium within 3 days. The degree of swelling increased with increasing PEG content and with increasing molecular weight of the PEG segment. The release rate of lysozyme from PEG/PBT films could be tailored very precisely by controlling the copolymer composition. Release rates increased with increasing PEG/PBT weight ratio and increasing molecular weight of the PEG segment. For films prepared from block copolymers with PEG blocks of 4000 g/mol, first-order lysozyme release was observed. For matrices prepared from polymers with PEG segments of 1000 and 600 g/mol, the lysozyme release profile followed near zero-order kinetics. A mathematical description of the release mechanism was developed which takes into account the effect of polymer hydrolytic degradation on solute diffusion. The model was found to be consistent with the experimental observations. Finally, determination of the activity of released protein showed that lysozyme was not damaged during the formulation, storage and release periods.