In-silico modeling of the mitotic spindle assembly checkpoint

Background

The Mitotic Spindle Assembly Checkpoint (^MSAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer.

Principle Findings

We have constructed and validated for the human ^MSAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the ^MSAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the “Dissociation” and the “Convey” model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments.

Conclusion

Only in the controlled case, our models show ^MSAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for ^MSAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding.     

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology

The RecA protein ofEscherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.     

Prolonged Intragastric Drug Delivery Mediated by Eudragit<Superscript>®</Superscript><Emphasis Type="Bold">E-Carrageenan Polyelectrolyte Matrix Tablets</Emphasis>

Interpolyelectrolyte (IPE) complexation between carrageenan (CG) and Eudragit E (EE) was studied in 0.1 M HCl and was used to develop floating matrix tablets aimed to prolong gastric-residence time and sustain delivery of the loaded drug. The optimum EE/CG IPE complexation weight ratio (0.6) was determined in 0.1 M HCl using apparent viscosity measurements. The IPE complex was characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry. Metronidazole matrix tablets were prepared by direct compression using EE, CG, or hybrid EE/CG with ratio optimal for IPE complexation. Corresponding effervescent tablets were prepared by including Na bicarbonate as an effervescent agent. Tablets were evaluated for in vitro buoyancy and drug release in 0.1 M HCl. Both CG and EE–CG effervescent matrices (1:2 drug to polymer weight ratio, 60 mg Na bicarbonate) achieved fast and prolonged floating with floating lag times less than 30 s and floating duration of more than 10 h. The corresponding EE effervescent matrices showed delayed floating and rapid drug release, and completely dissolved after 3 h of dissolution. CG matrices showed an initial burst drug release (48.3 ± 5.0% at 1 h) followed by slow drug release over 8 h. EE–CG matrices exhibited sustained drug release in almost zero-order manner for 10 h (68.2 ± 6.6%). The dissolution data of these matrices were fitted to different dissolution models. It was found that drug release followed zero-order kinetics and was controlled by the superposition of the diffusion and erosion.     

Improved and Robust Detection of Cell Nuclei from Four Dimensional Fluorescence Images

Segmentation-free direct methods are quite efficient for automated nuclei extraction from high dimensional images. A few such methods do exist but most of them do not ensure algorithmic robustness to parameter and noise variations. In this research, we propose a method based on multiscale adaptive filtering for efficient and robust detection of nuclei centroids from four dimensional (4D) fluorescence images. A temporal feedback mechanism is employed between the enhancement and the initial detection steps of a typical direct method. We estimate the minimum and maximum nuclei diameters from the previous frame and feed back them as filter lengths for multiscale enhancement of the current frame. A radial intensity-gradient function is optimized at positions of initial centroids to estimate all nuclei diameters. This procedure continues for processing subsequent images in the sequence. Above mechanism thus ensures proper enhancement by automated estimation of major parameters. This brings robustness and safeguards the system against additive noises and effects from wrong parameters. Later, the method and its single-scale variant are simplified for further reduction of parameters. The proposed method is then extended for nuclei volume segmentation. The same optimization technique is applied to final centroid positions of the enhanced image and the estimated diameters are projected onto the binary candidate regions to segment nuclei volumes.Our method is finally integrated with a simple sequential tracking approach to establish nuclear trajectories in the 4D space. Experimental evaluations with five image-sequences (each having 271 3D sequential images) corresponding to five different mouse embryos show promising performances of our methods in terms of nuclear detection, segmentation, and tracking. A detail analysis with a sub-sequence of 101 3D images from an embryo reveals that the proposed method can improve the nuclei detection accuracy by 9 over the previous methods, which used inappropriate large valued parameters. Results also confirm that the proposed method and its variants achieve high detection accuracies ( 98 mean F-measure) irrespective of the large variations of filter parameters and noise levels.     

Molecular Codes Through Complex Formation in a Model of the Human Inner Kinetochore

We apply molecular code theory to a rule-based model of the human inner kinetochore and study how complex formation in general can give rise to molecular codes. We analyze 105 reaction networks generated from the rule-based inner kinetochore model in two variants: with and without dissociation of complexes. Interestingly, we found codes only when some but not all complexes are allowed to dissociate. We show that this is due to the fact that in the kinetochore model proteins can only bind at kinetochores by attaching to already attached proteins and cannot form complexes in free solution. Using a generalized linear mixed model we study which centromere protein (CENP) can take which role in a molecular code (sign, meaning, context). By this, associations between CENPs (CenpA, CenpQ, CenpU and CenpI) and code roles are found. We observed that CenpA is a major risk factor (increases probability for code role) while CenpQ is a major protection factor (decreases probability for code role). Finally we show, using an abstract model of copolymer formation, that molecular codes can also be realized solely by the formation of stable complexes, which do not dissociate. For example, with particular dimers as context a molecular code mapping from two different monomers to two particular trimers can be realized just by non-selective complex formation. We conclude that the formation of protein complexes can be utilized by the cell to implement molecular codes. Living cells thus facilitate a subsystem allowing for an enormous flexibility in the realization of mappings, which can be used for specific regulatory processes, e.g. via the context of a mapping.     

Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis

Introduction  

Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum.     

Effects of cardiotonic steroids on dermal collagen synthesis and wound healing.

We previously reported that cardiotonic steroids stimulate collagen synthesis by cardiac fibroblasts in a process that involves signaling through the Na-K-ATPase pathway (Elkareh et al. Hypertension 49: 215-224, 2007). In this study, we examined the effect of cardiotonic steroids on dermal fibroblasts collagen synthesis and on wound healing. Increased collagen expression by human dermal fibroblasts was noted in response to the cardiotonic steroid marinobufagenin in a dose- and time-dependent fashion. An eightfold increase in collagen synthesis was noted when cells were exposed to 10 nM marinobufagenin for 24 h (P < 0.01). Similar increases in proline incorporation were seen following treatment with digoxin, ouabain, and marinobufagenin (10 nM x 24 h, all results P < 0.01 vs. control). The coadministration of the Src inhibitor PP2 or N-acetylcysteine completely prevented collagen stimulation by marinobufagenin. Next, we examined the effect of digoxin, ouabain, and marinobufagenin on the rate of wound closure in an in vitro model where human dermal fibroblasts cultures were wounded with a pipette tip and monitored by digital microscopy. Finally, we administered digoxin in an in vivo wound healing model. Olive oil was chosen as the digoxin carrier because of a favorable partition coefficient observed for labeled digoxin with saline. This application significantly accelerated in vivo wound healing in rats wounded with an 8-mm biopsy cut. Increased collagen accumulation was noted 9 days after wounding (both P < 0.01). The data suggest that cardiotonic steroids induce increases in collagen synthesis by dermal fibroblasts, as could potentially be exploited to accelerate wound healing.     

Antioxidant therapy for management of oxidative stress induced hypertension

Hypertension is considered as the most common risk factor for cardiovascular diseases, also is regarded as a leading cause of the mortality and morbidity worldwide. The mechanisms underlying the pathological process of hypertension are not completely explained. However, there is growing evidence that increased oxidative stress plays an important role in the pathophysiology of hypertension. Several preclinical studies and clinical trials have indicated that antioxidant therapy is important for management of hypertension, using antioxidants compounds such as alpha tocopherol (Vit E) and ascorbic acid (Vit C), polyphenols with others and some antihypertensive drugs that are now in clinical use (e.g. ACEIs, ARBs, novel B-blockers, dihydropyridine CCBs) which have antioxidative pleiotropic effects. The purpose of this review is to highlight the importance of antioxidant therapy for management of oxidative stress induced hypertension. Furthermore, we review the current knowledge in the oxidative stress and its significance in hypertension.