Molecular adhesion development in a neural cell monolayer forming in an ultrasound trap

A 2-dimensional aggregate of C6 neural cells was formed rapidly (within 30 s) in suspension in a recently developed 1.5 MHz ultrasound standing wave trap. A typical 1 mm diameter aggregate contained about 3,500 cells. Spreading of membrane occurred between the aggregated cells. The rate of spreading of the tangentially developing intercellular contact area was 0.19 microm/min. The form of the suspended aggregate changed from one of a hexagonal arrangement of cells to one of a cell-monolayer-like continuous sheet of mostly quadrilateral and pentagonal cells as in a cell monolayer on a solid substratum. A range of fluorescent indicators showed that the >99% viability of the cells did not change during 1 h exposures; therefore cell viability was not compromised during the monolayer development. The average integral intensities from stained actin filaments at the spreading cell-cell interfaces after 1, 8 and 30 min were 14, 25 and 46 microm(2) respectively. The cells in this work progressed from physical aggregation, through molecular adhesion, to displaying the intracellular consequences of receptor interactions. The ability to form mechanically strong confluent monolayer structures that can be monitored in situ or harvested from the trap provides a technique with general potential for monitoring the synchronous development of cell responses to receptor-triggered adhesion.     

The predictive role of serum and bronchoalveolar lavage cytokines and adhesion molecules for acute respiratory distress syndrome development and outcome

Chorioamnionitis is frequently associated with preterm deliveries before 30 weeks gestation. Chorioamnionitis correlates both with an increased risk of bronchopulmonary dysplasia and with a decreased risk of respiratory distress syndrome. Both interleukin-1α and endotoxin can induce inflammation in the fetal lungs and lung maturation after preterm birth when given by intra-amniotic injection. Inflammation can also result in an arrest of alveolarization, and this lung developmental abnormality is prominent in the lungs of preterm infants that die of bronchopulmonary dysplasia. The mechanisms by which infection/inflammation can have both beneficial and injurious effects on the preterm lung remain to be characterized.     

Elicitation of Expert Prior Opinion: Application to the MYPAN Trial in Childhood Polyarteritis Nodosa

Objectives

Definitive sample sizes for clinical trials in rare diseases are usually infeasible. Bayesian methodology can be used to maximise what is learnt from clinical trials in these circumstances. We elicited expert prior opinion for a future Bayesian randomised controlled trial for a rare inflammatory paediatric disease, polyarteritis nodosa (MYPAN, Mycophenolate mofetil for polyarteritis nodosa).

Methods

A Bayesian prior elicitation meeting was convened. Opinion was sought on the probability that a patient in the MYPAN trial treated with cyclophosphamide would achieve disease remission within 6-months, and on the relative efficacies of mycophenolate mofetil and cyclophosphamide. Expert opinion was combined with previously unseen data from a recently completed randomised controlled trial in ANCA associated vasculitis.

Results

A pan-European group of fifteen experts participated in the elicitation meeting. Consensus expert prior opinion was that the most likely rates of disease remission within 6 months on cyclophosphamide or mycophenolate mofetil were 74% and 71%, respectively. This prior opinion will now be taken forward and will be modified to formulate a Bayesian posterior opinion once the MYPAN trial data from 40 patients randomised 1:1 to either CYC or MMF become available.

Conclusions

We suggest that the methodological template we propose could be applied to trial design for other rare diseases.     

The anti-sigma factor TcdC modulates hypervirulence in an epidemic BI/NAP1/027 clinical isolate of Clostridium difficile

Nosocomial infections are increasingly being recognised as a major patient safety issue. The modern hospital environment and associated health care practices have provided a niche for the rapid evolution of microbial pathogens that are well adapted to surviving and proliferating in this setting, after which they can infect susceptible patients. This is clearly the case for bacterial pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococcus (VRE) species, both of which have acquired resistance to antimicrobial agents as well as enhanced survival and virulence properties that present serious therapeutic dilemmas for treating physicians. It has recently become apparent that the spore-forming bacterium Clostridium difficile also falls within this category. Since 2000, there has been a striking increase in C. difficile nosocomial infections worldwide, predominantly due to the emergence of epidemic or hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Various hypotheses have been proposed for the emergence of these strains, and for their persistence and increased virulence, but supportive experimental data are lacking. Here we describe a genetic approach using isogenic strains to identify a factor linked to the development of hypervirulence in C. difficile. This study provides evidence that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor in the development of hypervirulence in epidemic C. difficile isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for C. difficile pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype.     

Biochemical properties of encapsulated high-density 3-D HepG2 aggregates formed in an ultrasound trap for application in hepatotoxicity studies

This paper describes the alginate encapsulation of preformed high-density 3-D HepG2 cell aggregates that guarantees good maintenance of liver-specific biomarker expression. The process involves forming a high-density (≥7 × 10⁴ cells/aggregate) discoid 3-D cell aggregate in an ultrasound trap, which is subsequently recovered and encapsulated in alginate/CaCl₂ hydrogel. Glucose secretion/consumption, lactate release, detoxifying enzyme capacity, cytokeratin-18 expression as well as hypoxia were characterized in encapsulated 3-D HepG2 aggregates over 10 days in culture. Encapsulated 3-D HepG2 aggregates released glucose into the media, although this ability was exhibited only after 1 day in culture and was subsequently lost over the ensuing 9 days. In contrast, lactate was constantly released into the media. Significantly more lactate was secreted after 3 days in culture indicating a more hypoxic environment and hence a higher rate of anaerobic glycolysis. Aggregates consistently expressed cytokeratin-18. Cytochrome P450-1A1 activity reached a maximum on day 1 of culture followed by a progressive reduction to basal levels, while P450-3A4 activity was up-regulated in a time-dependent manner reaching a peak on day 7 in culture. Glutathione-S-transferase activity, on the other hand, was at more physiological levels and remained constant over the 10-day culture period. The ultrasound trap allowed the rapid (within 5 min) generation of uniformly shaped and sized aggregates. The results reported here suggest that ultrasound-formed 3-D HepG2 aggregates can serve as alternative in vitro models providing a quick outlook on toxicity, in a tissue-mimetic manner, thus offering the future option of a cost-effective screening platform for pharmaceutical development.     

Chloroperoxidase-catalyzed cyclodimerization of methyl (2E)-2,4-pentadienoate: a [4+2] cycloaddition product

The chloroperoxidase (CPO)-catalyzed oxidation of the methyl (2E)-2,4-pentadienoate gives the terminal double bond epoxide (25%) and a cyclodimerization compound (63%) as the major products.     

Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif

tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, we show that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. We propose a model in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold.     

Folic acid modified gelatine coated quantum dots as potential reagents for in vitro cancer diagnostics

Background

Highly hydrophobic surfaces can have very low surface energy and such low surface energy biological interfaces can be obtained using fluorinated coatings on surfaces. Deposition of biocompatible organic films on solid-state surfaces is attained with techniques like plasma polymerization, biomineralization and chemical vapor deposition. All these require special equipment or harsh chemicals. This paper presents a simple vapor-phase approach to directly coat solid-state surfaces with biocompatible films without any harsh chemical or plasma treatment. Hydrophilic and hydrophobic monomers were used for reaction and deposition of nanolayer films. The monomers were characterized and showed a very consistent coating of 3D micropore structures.

Results

The coating showed nano-textured surface morphology which can aid cell growth and provide rich molecular functionalization. The surface properties of the obtained film were regulated by varying monomer concentrations, reaction time and the vacuum pressure in a simple reaction chamber. Films were characterized by contact angle analysis for surface energy and with profilometer to measure the thickness. Fourier Transform Infrared Spectroscopy (FTIR) analysis revealed the chemical composition of the coated films. Variations in the FTIR results with respect to different concentrations of monomers showed the chemical composition of the resulting films.

Conclusion

The presented approach of vapor-phase coating of solid-state structures is important and applicable in many areas of bio-nano interface development. The exposure of coatings to the solutions of different pH showed the stability of the coatings in chemical surroundings. The organic nanocoating of films can be used in bio-implants and many medical devices.