Generation of human embryonic stem cell-derived mesoderm and cardiac cells using size-specified aggregates in an oxygen-controlled bioreactor

The ability to generate human pluripotent stem cell-derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and biopharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called "embryoid bodies" (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro-printing strategy to generate large numbers of size-specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size-control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT-PCR data for markers such as Brachyury, LIM domain homeobox gene Isl-1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells.     

Design and synthesis of substituted nicotinamides as inhibitors of soluble epoxide hydrolase

A series of potent nicotinamide inhibitors of soluble epoxides hydrolase (sEH) is disclosed. This series was designed using structure-based deconstruction and a combination of two HTS hit series, resulting in hybrid analogs that retained the optimal potency from one series, and acceptable in vitro metabolic stability from the other. Structure-guided optimization of these analogs gave rise to nanomolar inhibitors of human sEH that had acceptable plasma exposure to qualify them as probes to determine the in vivo phenotypic consequences of sEH inhibition.     

The vastus lateralis neuromuscular activity during all-out cycling exercise

ObjectiveThe objective of this work was to study modifications in motor control through surface electromyographic (sEMG) activity during a very short all-out cycling exercise.MethodsTwelve male cyclists (age 23 ± 4 years) participated in this study. After a warm-up period, each subject performed three all-out cycling exercises of 6 s separated by 2 min of complete rest. This protocol was repeated three times with a minimum of 2 days between each session. The braking torque imposed on cycling motion was 19 N m. The sEMG of the vastus lateralis was recorded during the first seven contractions of the sprint. Time–frequency analysis of sEMG was performed using continuous wavelet transform. The mean power frequency (MPF, qualitative modifications in the recruitment of motor units) and signal energy (a quantitative indicator of modifications in the motor units recruitment) were computed for the frequency range 10–500 Hz.ResultssEMG energy increased (P ? 0.05) between contraction number 1 and 2, decreased (P ? 0.05) between contraction number 2 and 3 then stabilized between contraction number 3 and 7 during the all-out test. MPF increased (P ? 0.05) during the all-out test. This increase was more marked during the first two contractions.ConclusionsThe decrease in energy and the increase in the sEMG MPF suggest a large spatial recruitment of motor units (MUs) at the beginning of the sprint followed by a preferential recruitment of faster MUs at the end of the sprint, respectively.     

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment

Aims:  Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages.
Methods and Results:  Different points during the production of a traditional fermented sausage type ( fuet ) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source.
Conclusions:  The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin.
Significance and Impact of the Study:  This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.     

Design and synthesis of substituted imidazole and triazole N-phenylbenzo[d]oxazolamine inhibitors of retinoic acid metabolizing enzyme CYP26

The design of N-phenylbenzo[d]oxazolamines as CYP26A1 inhibitors involved ligand docking experiments using molecular modeling (FlexX) and analysis of ligand interactions at the binding domain. The synthesis of the benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines was achieved by cyclisation of the corresponding isothiocyanates with subsequent introduction of the haem-binding heterocycle. Triazole and tetrazole derivatives were also prepared for comparison with the lead imidazole derivative. The benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines with small substituents in the phenyl ring were moderately potent CYP26A1 inhibitors (IC(50) 8 and 12 microM) and comparable with liarozole (IC(50) 7 microM).     

Extracellular regulators of axonal growth in the adult central nervous system

Robust axonal growth is required during development to establish neuronal connectivity. However, stable fibre patterns are necessary to maintain adult mammalian central nervous system (CNS) function. After adult CNS injury, factors that maintain axonal stability limit the recovery of function. Extracellular molecules play an important role in preserving the stability of the adult CNS axons and in restricting recovery from pathological damage. Adult axonal growth inhibitors include a group of proteins on the oligodendrocyte, Nogo-A, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein and ephrin-B3, which interact with axonal receptors, such as NgR1 and EphA4. Extracellular proteoglycans containing chondroitin sulphates also inhibit axonal sprouting in the adult CNS, particularly at the sites of astroglial scar formation. Therapeutic perturbations of these extracellular axonal growth inhibitors and their receptors or signalling mechanisms provide a degree of axonal sprouting and regeneration in the adult CNS. After CNS injury, such interventions support a partial return of neurological function.     

Multiscale modeling of light absorption in tissues: limitations of classical homogenization approach

In biophotonics, the light absorption in a tissue is usually modeled by the Helmholtz equation with two constant parameters, the scattering coefficient and the absorption coefficient. This classic approximation of "haemoglobin diluted everywhere" (constant absorption coefficient) corresponds to the classical homogenization approach. The paper discusses the limitations of this approach. The scattering coefficient is supposed to be constant (equal to one) while the absorption coefficient is equal to zero everywhere except for a periodic set of thin parallel strips simulating the blood vessels, where it is a large parameter ω. The problem contains two other parameters which are small: ε, the ratio of the distance between the axes of vessels to the characteristic macroscopic size, and δ, the ratio of the thickness of thin vessels and the period. We construct asymptotic expansion in two cases: ε --> 0, ω --> ∞, δ --> 0, ωδ --> ∞, ε2ωδ --> 0 and ε --> 0, ω --> ∞, δ --> 0, ε2ωδ --> ∞, and and prove that in the first case the classical homogenization (averaging) of the differential equation is true while in the second case it is wrong. This result may be applied in the biomedical optics, for instance, in the modeling of the skin and cosmetics.