Optical mapping of Langendorff-perfused human hearts: establishing a model for the study of ventricular fibrillation in humans

Our objective was to establish a novel model for the study of ventricular fibrillation (VF) in humans. We adopted the established techniques of optical mapping to human ventricles for the first time to determine whether human VF is the result of wave breaks and singularity point formation and is maintained by high-frequency rotors and fibrillatory conduction. We describe the technique of acquiring optical signals in human hearts during VF, their characteristics, and the feasibility of possible analyses that could be performed to elucidate mechanisms of human VF. We used explanted hearts from five cardiomyopathic patients who underwent transplantation. The hearts were Langendorff perfused with Tyrode solution (95% O(2)-5% CO(2)), and the potentiometric dye di-4-ANEPPS was injected as a bolus into the coronary circulation. Fluorescence was excited at 531 +/- 20 nm with a 150-W halogen light source; the emission signal was long-pass filtered at 610 nm and recorded with a mapping camera. Fractional change of fluorescence varied between 2% and 12%. Average signal-to-noise ratio was 40 dB. The mean velocity of VF wave fronts was 0.25 +/- 0.04 m/s. Submillimetric spatial resolution (0.65-0.85 mm), activation mapping, and transformation of the data to phase-based analysis revealed reentrant, colliding, and fractionating wave fronts in human VF. On many occasions the VF wave fronts were as large as the entire vertical length (8 cm) of the mapping field, suggesting that there are a limited number of wave fronts on the human heart during VF. Phase transformation of the optical signals allowed the first demonstration ever of phase singularity point, wave breaks, and rotor formation in human VF. This method provides opportunities for potential analyses toward elucidation of the mechanisms of VF and defibrillation in humans.     

Cysteine-cysteine contact preference leads to target-focusing in protein folding

We perform a statistical analysis of amino-acid contacts to investigate possible preferences of amino-acid interactions. We include in the analysis only tertiary contacts, because they are less constrained--compared to secondary contacts--by proteins' backbone rigidity. Using proteins from the protein data bank, our analysis reveals an unusually high frequency of cysteine pairings relative to that expected from random. To elucidate the possible effects of cysteine interactions in folding, we perform molecular simulations on three cysteine-rich proteins. In particular, we investigate the difference in folding dynamics between a Gō-like model (where attraction only occurs between amino acids forming a native contact) and a variant model (where attraction between any two cysteines is introduced to mimic the formation/dissociation of native/nonnative disulfide bonds). We find that when attraction among cysteines is nonspecific and comparable to a solvent-averaged interaction, they produce a target-focusing effect that expedites folding of cysteine-rich proteins as a result of a reduction of conformational search space. In addition, the target-focusing effect also helps reduce glassiness by lowering activation energy barriers and kinetic frustration in the system. The concept of target-focusing also provides a qualitative understanding of a correlation between the rates of protein folding and parameters such as contact order and total contact distance.     

Influence of microbial growth kinetics on steady state multiplicity and stability of a two-step nitrification (SHARON) model

In this paper, the influence of microbial growth kinetics on the number and the stability of steady states for a nitrogen removal process is addressed. A two-step nitrification model is studied, in which the maximum growth rate of ammonium oxidizers is larger than the one of nitrite oxidizers. This model describes the behavior of a SHARON reactor for the treatment of wastewater streams with high ammonium concentrations. Steady states are identified through direct calculation using a canonical state space model representation, for several types of microbial kinetics. The stability of the steady states is assessed and the corresponding phase portraits are analyzed. Practical operation of a SHARON reactor aims at reaching ammonium conversion to nitrite while suppressing further conversion to nitrate. Regions in the input space are identified that result in this desired behavior, with only nitrite formation. It is demonstrated that not only the dilution rate plays a role, as is commonly known, but also the influent ammonium concentration. Besides, the type of microbial (inhibition) kinetics has a nonnegligible influence. While the results indicate that product inhibition does not affect the number of steady states of a (bio)reactor model, it is shown that substrate inhibition clearly yields additional steady states. Particular attention is devoted to the physical interpretation of these phenomena.     

Designing lipid nanostructures for local delivery of biologically active macromolecules

This study focuses on the possible therapeutic utility of liposomes in the local treatment of inflammatory disorders, specifically rheumatoid arthritis (RA). Our purpose was to design a depot delivery system of an anti-inflammatory glycoprotein, lactoferrin (Lf), using positive multivesicular liposomes and to investigate its in vivo efficiency. Lactoferrin (Lf) has previously been shown to have therapeutic potential in mice with collagen-induced arthritis (CIA) after intra-articular (i.a.) injection. In order to protect Lf from enzymatic degradation and to maintain an adequate concentration in the joint, liposomes have been used as carriers for controlled drug delivery. Based on our previous findings we compared the ability of free Lf and Lf encapsulated in liposomes to suppress established joint inflammation and to modulate the cytokine response of lymph node (LN) T lymphocytes in DBA/1 mice with CIA. The anti-inflammatory effect of Lf formulated in positive liposomes was more pronounced compared with the free protein. After a single i.a. injection of liposomal Lf the arthritic score significantly decreased continuously for 2 weeks while in the case of free Lf for only 3-4 days. The cytokine levels produced by LN T cells showed decreased pro-inflammatory cytokines (TNF-alpha and IFN-gamma) accompanied by increased anti-inflammatory cytokines (IL-5 and especcialy IL-10) in encapsulated compared with free Lf. When compared with free Lf, liposomal Lf decreased the expression of costimulatory molecules on DCs, reduced pro-inflammatory (TNF) and increased anti-inflammatory (IL-10) cytokine production. Using CIA model we have studied the liposome trafficking following i.a. administration and we have identified DCs as a target for liposomes in the draining LN. Our results suggest that the entrapment of Lf in liposomes may modify its pharmacodynamic profile and could have great potential as controlled delivery system in the treatment of RA and other local inflammatory conditions.     

Molecular dynamics simulations of the interaction of phospholipid bilayers with polycaprolactone

The molecular interaction between common polymer chains and the cell membrane is unknown. Molecular dynamics simulations offer an emerging tool to characterise the nature of the interaction between common degradable polymer chains used in biomedical applications, such as polycaprolactone, and model cell membranes. Herein we characterise with all-atomistic and coarse-grained molecular dynamics simulations the interaction between single polycaprolactone chains of varying chain lengths with a phospholipid membrane. We find that the length of the polymer chain greatly affects the nature of interaction with the membrane, as well as the membrane properties. Furthermore, we next utilise advanced sampling techniques in molecular dynamics to characterise the two-dimensional free energy surface for the interaction of varying polymer chain lengths (short, intermediate, and long) with model cell membranes. We find that the free energy minimum shifts from the membrane-water interface to the hydrophobic core of the phospholipid membrane as a function of chain length. Finally, we perform coarse-grained molecular dynamics simulations of slightly larger membranes with polymers of the same length and characterise the results as compared with all-atomistic molecular dynamics simulations. These results can be used to design polymer chain lengths and chemistries to optimise their interaction with cell membranes at the molecular level.     

Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments

EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.     

Stem/progenitor cells and obstructive sleep apnea syndrome-new insights for clinical applications

Obstructive sleep apnea syndrome(OSAS) is a widespread disorder, characterized by recurrent upper airway obstruction during sleep, mostly as a result of complete or partial pharyngeal obstruction. Due to the occurrence of frequent and regular hypoxic events, patients with OSAS are at increased risk of cardiovascular disease, stroke, metabolic disorders, occupational errors, motor vehicle accidents and even death. Thus, OSAS has severe consequences and represents a significant economic burden. However, some of the consequences, as well as their costs can be reduced with appropriate detection and treatment. In this context, the recent advances that were made in stem cell biology knowledge and stem cell- based technologies hold a great promise for various medical conditions, including respiratory diseases. However, the investigation of the role of stem cells in OSAS is still recent and rather limited, requiring further studies, both in animal models and humans. The goal of this review is to summarize the current state of knowledge regarding both lung resident as well as circulating stem/progenitor cells and discuss existing controversies in the field in order to identify future research directions for clinical applications in OSAS. Also, the paper highlights the requisite for inter-institutional, multi-disciplinary research collaborations in order to achieve breakthrough results in the field.     

Lymphangiogenesis and tumor metastasis

The lymphatic system transports interstitial fluid and macromolecules from tissues back to the blood circulation, and plays an important role in the immune response by directing the traffic of lymphocytes and antigen-presenting cells. The lymphatic system also constitutes one of the most important pathways of tumor dissemination. In many human cancers, increased expression of vascular endothelial growth factor-C (VEGF-C) is correlated with regional lymph node metastases. Experimental studies using transgenic mice overexpressing VEGF-C or xenotransplantation of VEGF-C-expressing tumor cells into immunodeficient mice have demonstrated a role for VEGF-C in tumor lymphangiogenesis and the subsequent formation of lymph node metastases. However, there is at present little evidence for lymphangiogenesis in human tumors and the relative importance of preexisting vs. newly formed lymphatics for metastasis in humans remains to be determined. Nonetheless, the striking correlation between the levels of VEGF-C in primary human tumors and lymph node metastases predicts its importance in cancer spread. Aside from promoting lymphangiogenesis, VEGF-C may also activate lymphatics to promote tumor cell chemotaxis, lymphatic intravasation and hence tumor cell dissemination.Work in the authors' laboratories was supported by grants from the Swiss National Science Foundation (no. 3100–064037.00) (to M.S.P), the Speaker's Fund for Biomedical Research (to M.S.) and the Peter Sharp Foundation (to M.S.). Parts of this review will be published in abbreviated form in Thrombosis and Haemostasis