A two-galectin network establishes mesenchymal condensation phenotype in limb development.

Previous work showed that Gal-1A and Gal-8, two proteins belonging to the galactoside-binding galectin family, are the earliest determinants of the patterning of the skeletal elements of embryonic chicken limbs, and further, that their experimentally determined interactions in the embryonic limb bud can be interpreted via a reaction-diffusion-adhesion (2GL: two galectin plus ligands) model. Here, we use an ordinary differential equation-based approach to analyze the intrinsic switching modality of the 2GL network and characterize the network behavior independent of the diffusive and adhesive arms of the patterning mechanism. We identify two states: where the concentrations of both the galectins are respectively, negligible, and very high. This bistable switch-like system arises via a saddle-node bifurcation from a monostable state. For the case of mass-action production terms, we provide an explicit Lyapunov function for the system, which shows that it has no periodic solutions. Our model therefore predicts that the galectin network may exist in low expression and high expression states separated in space or time, without any intermediate states. We test these predictions in experiments performed with high density cultures of chick limb mesenchymal cells and observe that cells inside precartilage protocondensations express Gal-1A at a much higher rate than those outside, for which it was negligible. The Gal-1A and -8-based patterning network is therefore sufficient to partition the mesenchymal cell population into two discrete cell states with different developmental (chondrogenic vs. non-chondrogenic) fates. When incorporated into an adhesion and diffusion-enabled framework this system can generate a spatially patterned limb skeleton.

A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa.

The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa's outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic.IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

Cancer spectrum and frequency among children with Noonan, Costello, and cardio-facio-cutaneous syndromes.

BACKGROUND: Somatic mutations affecting components of the Ras-MAPK pathway are a common feature of cancer, whereas germline Ras pathway mutations cause developmental disorders including Noonan, Costello, and cardio-facio-cutaneous syndromes. These 'RASopathies' also represent cancer-prone syndromes, but the quantitative cancer risks remain unknown. METHODS: We investigated the occurrence of childhood cancer including benign and malignant tumours of the central nervous system in a group of 735 individuals with germline mutations in Ras signalling pathway genes by matching their information with the German Childhood Cancer Registry. RESULTS: We observed 12 cases of cancer in the entire RASopathy cohort vs 1.12 expected (based on German population-based incidence rates). This corresponds to a 10.5-fold increased risk of all childhood cancers combined (standardised incidence ratio (SIR)=10.5, 95% confidence interval=5.4-18.3). The specific cancers included juvenile myelomonocytic leukaemia=4; brain tumour=3; acute lymphoblastic leukaemia=2; rhabdomyosarcoma=2; and neuroblastoma=1. The childhood cancer SIR in Noonan syndrome patients was 8.1, whereas that for Costello syndrome patients was 42.4. CONCLUSIONS: These data comprise the first quantitative evidence documenting that the germline mutations in Ras signalling pathway genes are associated with increased risks of both childhood leukaemia and solid tumours.

Reaction-diffusion model of atherosclerosis development.

Atherosclerosis begins as an inflammation in blood vessel walls (intima). The inflammatory response of the organism leads to the recruitment of monocytes. Trapped in the intima, they differentiate into macrophages and foam cells leading to the production of inflammatory cytokines and further recruitment of white blood cells. This self-accelerating process, strongly influenced by low-density lipoproteins (cholesterol), results in a dramatic increase of the width of blood vessel walls, formation of an atherosclerotic plaque and, possibly, of its rupture. We suggest a 2D mathematical model of the initiation and development of atherosclerosis which takes into account the concentration of blood cells inside the intima and of pro- and anti-inflammatory cytokines. The model represents a reaction-diffusion system in a strip with nonlinear boundary conditions which describe the recruitment of monocytes as a function of the concentration of inflammatory cytokines. We prove the existence of travelling waves described by this system and confirm our previous results which suggest that atherosclerosis develops as a reaction-diffusion wave. The theoretical results are confirmed by the results of numerical simulations.

De novo partial trisomy 18p and partial monosomy 18q in a patient with anorectal malformation.

Anorectal malformations (ARM) encompass a broad clinical spectrum which ranges from mild anal stenosis to severe anorectal anomalies such as complex cloacal malformations. The overall incidence of ARM is around 1 in every 2,500 live births. Although causative genes for a few syndromic forms have been identified, the molecular genetic background of most ARM remains unknown. The present report describes a patient with a de novo 13.2-Mb deletion of chromosome 18q22.3-qter and a 2.2-Mb de novo duplication of chromosomal region 18pter-p11.32 located at the telomeric end of chromosome 18q. The patient presented with ARM and the typical features of 18q- syndrome (De-Grouchy syndrome). The combination of a partial duplication of the short arm and a partial deletion of the long arm of chromosome 18 has been described in 16 previous cases. However, this is the first report of an association between this complex chromosomal rearrangement and ARM.

Spatial gradients in kinase cascade regulation.

The spatiotemporal kinetics of proteins and other substrates regulate cell fate and signaling. In this study, we consider a reaction-diffusion model of interaction of membrane receptors with a two-step kinase cascade. The receptors activate the 'up-stream' kinase, which may diffuse over cell volume and activate the 'down-stream' kinase, which is also diffusing. Both kinase species and receptors are inactivated by uniformly distributed phosphatases. The positive feedback, key to the considered dynamics, arises since the up-stream kinase activates the receptors. Such a mutual interaction is characteristic for immune cell receptors. Based on the proposed model, we demonstrated that cell sensitivity (measured as a critical value of phosphatase activity at which cell maybe activated) increases with decreasing motility of receptor-interacting kinases and with increasing polarity of receptors distribution. These two effects are cooperating, the effect of receptors localisation close to one pole of the cell grows with the decreasing kinase diffusion and vanishes in the infinite diffusion limit. As the cell sensitivity increases with decreasing diffusion of receptor-interacting kinase, the overall activity of the down-stream kinase increases with its diffusion. In conclusion, the analysis of the proposed model shows that, for the fixed substrate interaction rates, spatial distribution of the surface receptors together with the motility of intracellular kinases control cell signalling and sensitivity to extracellular signals. The increase of the cell sensitivity can be achieved by (i) localisation of receptors in a small subdomain of the cell membrane, (ii) lowering the motility of receptor-interacting kinase, (iii) increasing the motility of down-stream kinases which distribute the signal over the whole cell.

Mathematical modelling of atherosclerosis as an inflammatory disease.

Atherosclerosis is an inflammatory disease. The atherosclerosis process starts when low-density lipoproteins (LDLs) enter the intima of the blood vessel, where they are oxidized (ox-LDLs). The anti-inflammatory response triggers the recruitment of monocytes. Once in the intima, the monocytes are transformed into macrophages and foam cells, leading to the production of inflammatory cytokines and further recruitment of monocytes. This auto-amplified process leads to the formation of an atherosclerotic plaque and, possibly, to its rupture. In this paper we develop two mathematical models based on reaction-diffusion equations in order to explain the inflammatory process. The first model is one-dimensional: it does not consider the intima's thickness and shows that low ox-LDL concentrations in the intima do not lead to a chronic inflammatory reaction. Intermediate ox-LDL concentrations correspond to a bistable system, which can lead to a travelling wave that can be initiated by certain conditions, such as infection or injury. High ox-LDL concentrations correspond to a monostable system, and even a small perturbation of the non-inflammatory case leads to travelling-wave propagation, which corresponds to a chronic inflammatory response. The second model we suggest is two-dimensional: it represents a reaction-diffusion system in a strip with nonlinear boundary conditions to describe the recruitment of monocytes as a function of the cytokines' concentration. We prove the existence of travelling waves and confirm our previous results, which show that atherosclerosis develops as a reaction-diffusion wave. The results of the two models are confirmed by numerical simulations. The latter show that the two-dimensional model converges to the one-dimensional one if the thickness of the intima tends to zero.

Phenotype and genotype associations of lung carcinoma with atypical adenomatoid hyperplasia, squamous cell dysplasia, and chromosome alterations in non-neoplastic bronchial mucosa.

The frequency of preneoplastic lesions of the lung and bronchial mucosa as well as potential genotype alterations in spatial relationship to pulmonary malignancies still need intensive investigations in order to understand the occurrence and manifestation of lung cancer in detail. To investigate the contemporary manifestation of lung cancer precursor lesions, peripheral (non-neoplastic) lung parenchyma and bronchial mucosa of operated lung carcinomas were analyzed at distinct distances (1, 2, 3, and 4 cm) from the tumor boundary for pre-neoplastic lesions--atypical adenomatoid hyperplasia (AAH) and squamous cell dysplasia (SCD), in 150 surgical specimens. Short-term tissue cultures of additional 55 primary and secondary lung tumors and their surrounding non-neoplastic bronchial mucosa were performed at the same distances in order to search for chromosome alterations, i.e. genotype aberrations. In phenotype observations, atypical adenomatoid hyperplasia was noted in 19/150 (13%) cases, and squamous cell dysplasia in 46/150 (31%) cases. The degree of cellular atypia decreased with increasing distance from the tumor boundary in both AAH and SCM. AAH was observed more frequently in adenocarcinomas, SCQ more frequently in squamous cell carcinomas. In genotype observations, the average number of abnormal metaphases measured 4.5/10 high power fields (HPF) in primary lung carcinomas, and only 2/10 in metastases. Data indicate that the so-called preneoplastic lesions in the lung are not completely tumor-precursor lesions, but, in addition, induced by the tumor itself.

On multiscale approaches to three-dimensional modelling of morphogenesis.

In this paper we present the foundation of a unified, object-oriented, three-dimensional biomodelling environment, which allows us to integrate multiple submodels at scales from subcellular to those of tissues and organs. Our current implementation combines a modified discrete model from statistical mechanics, the Cellular Potts Model, with a continuum reaction-diffusion model and a state automaton with well-defined conditions for cell differentiation transitions to model genetic regulation. This environment allows us to rapidly and compactly create computational models of a class of complex-developmental phenomena. To illustrate model development, we simulate a simplified version of the formation of the skeletal pattern in a growing embryonic vertebrate limb.

Homoclinic solutions in mechanical systems with small dissipation. Application to DNA dynamics.

This paper analyzes the problem of persistence of homoclinic solutions to perturbed systems of second order ODE's. These systems arise from PDE's, when considering solutions in the form of travelling waves. It is shown that homoclinic solutions persist in the presence of dissipation. Dissipation can be balanced by nonautonomous terms of compact support, which are controlled by a single parameter. This result is applied to prove the existence of torsional pulse-like travelling waves propagating along a nonelastic DNA molecule. In this case the energy is added to system by advancing the RNA polymerase.

Pseudomonas aeruginosa ExoT acts in vivo as a GTPase-activating protein for RhoA, Rac1, and Cdc42.

The Pseudomonas aeruginosa protein ExoT is a bacterial GTPase-activating protein (GAP) that has in vitro activity toward Rho, Rac, and Cdc42 GTPases. Expression of ExoT both inhibits the internalization of strain PA103 by macrophages and epithelial cells and is associated with morphological changes (cell rounding and detachment) of infected cells. We find that expression of ExoT leads to the loss of GTP-bound RhoA, Rac1, and Cdc42 in transfected HeLa cells, demonstrating that ExoT has GAP activity in vivo toward all three GTPases. GAP activity is absolutely dependent on the presence of arginine at position 149 but is not affected by whether ExoT is expressed in the absence or presence of other P. aeruginosa type III secreted proteins. We also demonstrate that expression of ExoT in epithelial cells is sufficient to cause stress fiber disassembly by means of ExoT's GAP activity toward RhoA.

Interaction of bacterial pathogens with polarized epithelium.

Many pathogens must surmount an epithelial cell barrier in order to establish an infection. While much has been learned about the interaction of bacterial pathogens with cultured epithelial cells, the influence of cell polarity on these events has only recently been appreciated. This review outlines bacterial-host epithelial cell interactions in the context of the distinct apical and basolateral surfaces of the polarized epithelium that lines the lumens of our organs.

Pseudomonas aeruginosa ExoT inhibits in vitro lung epithelial wound repair.

The nosocomial pathogen Pseudomonas aeruginosa causes clinical infection in the setting of pre-existing epithelial tissue damage, an association that is mirrored by the increased ability of P. aeruginosa to bind, invade and damage injured epithelial cells in vitro. In this study, we report that P. aeruginosa inhibits the process of epithelial wound repair in vitro through the type III-secreted bacterial protein ExoT, a GTPase-activating protein (GAP) for Rho family GTPases. This inhibition primarily targets cells at the edge of the wound, and causes actin cytoskeleton collapse, cell rounding and cell detachment. ExoT-dependent inhibition of wound repair is mediated through the GAP activity of this bacterial protein, as mutations in ExoT that alter the conserved arginine (R149) within the GAP domain abolish the ability of P. aeruginosa to inhibit wound closure. Because ExoT can also inhibit P. aeruginosa internalization by phagocytes and epithelial cells, this protein may contribute to the in vivo virulence of P. aeruginosa by allowing organisms both to overcome local host defences, such as an intact epithelial barrier, and to evade phagocytosis by immune effector cells.

Rho GTPase activity modulates Pseudomonas aeruginosa internalization by epithelial cells.

The Gram-negative pathogen Pseudomonas aeruginosa invades epithelial cells in vivo and in vitro. We have examined the pathway(s) by which epithelial cells internalize P. aeruginosa strain PA103 using Madin-Darby canine kidney (MDCK) cells. We have recently demonstrated that P. aeruginosa internalization occurs by an actin-dependent Toxin B-inhibited pathway which becomes downregulated as epithelial cells become polarized, suggesting that one or more of the Rho family GTPases is involved in bacterial internalization. Here, we demonstrate that activation of the Rho family GTPases by cytotoxic necrotizing factor 1 (CNF-1) stimulates P. aeruginosa internalization. Examination of the roles of the individual Rho family GTPases in internalization shows that expression of a constitutively active allele of RhoA (RhoAV14), but not of constitutively active Rac1 (Rac1V12) or Cdc42 (Cdc42V12), is sufficient to increase uptake of PA103pscJ. This relative increase persists when bacterial infection is established at the basolateral surface of polarized cells, suggesting that the effect of RhoAV14 is not simply due to its known ability to disrupt tight junction integrity in polarized cells. RhoAV14-mediated stimulation of bacterial uptake is actin dependent as it is abrogated by exposure to latrunculin A. We also find that endogenous Rho GTP levels in epithelial cells are increased by infection with an internalized strain of P. aeruginosa; conversely, a poorly internalized isogenic strain expressing the bacterial anti-internalization protein ExoT causes decreased Rho GTP levels. Experimental inhibition of Rho, either by expressing dominant negative RhoAN19 or by inhibiting native Rho using a membrane permeable fusion construct of a Rho-specific inhibitor, C3 ADP-ribosyltransferase, does not inhibit PA103pscJ internalization in MDCK or HeLa cells. Models consistent with these data are presented.

An identical HMGIC-LPP fusion transcript is consistently expressed in pulmonary chondroid hamartomas with t(3;12)(q27-28;q14-15).

The high frequency of the t(3;12)(q27-28;q14-15) in lipomas and pulmonary chondroid hamartomas (PCHs) makes the HMGIC-LPP fusion gene the most common fusion gene in a human tumor known so far. Nevertheless, there is no in-depth molecular analysis of the HMGIC-LPP fusion transcripts in PCHs. Certainly, a possible molecular variability of the HMGIC-LPP fusion may contribute to a better understanding of the histologic differences between lipomas and PCHs and the intratumoral histologic heterogeneity of PCHs. By RT-PCR and restriction analysis, we have investigated the HMGIC-LPP fusion transcripts in a series of 13 PCHs with t(3;12)(q27-28;q14-q15). HMGIC-LPP fusion transcripts of identical size were found in all PCHs tested. In all tumors investigated, the fusion transcripts had the same structure, i.e., exons 1 to 3 of HMGIC and exons 9 to 11 of LPP encoding a protein composed of three AT-hooks and two LIM-domains. Our results clearly show that neither the histologic differences between lipomas and PCHs nor the histologic heterogeneity of PCHs can be explained by a molecular diversity of the HMGIC-LPP fusion transcript.

The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages.

Pseudomonas aeruginosa, an important nosocomial pathogen of humans, expresses a type III secretion system that is required for virulence. Previous studies demonstrated that the lung-virulent strain PA103 has the capacity to be either cytotoxic or invasive. Analyses of mutants suggest that PA103 delivers a negative regulator of invasion, or anti-internalization factor, to host cells via a type III secretion system. In this work we show that the type III secreted protein ExoT inhibits the internalization of PA103 by polarized epithelial cells (Madin-Darby canine kidney cells) and J774.1 macrophage-like cells. ExoS, which is closely related to ExoT but has additional ADP-ribosylating activity, can substitute for ExoT as an anti-internalization factor. ExoT contains a signature arginine finger domain found in GTPase-activating proteins. Mutation of the conserved arginine in ExoT diminished its anti-internalization activity and altered its ability to disrupt the actin cytoskeleton. Cell fractionation experiments showed that ExoT is translocated into host cells and that mutation of the arginine finger did not disrupt translocation. In a mouse model of acute pneumonia, PA103DeltaUDeltaT reached the lungs as efficiently as PA103DeltaU but showed reduced colonization of the liver. This finding suggests that the ability to resist internalization may be important for virulence in vivo.

A 12q13 translocation involving the HMGI-C gene in richter transformation of a chronic lymphocytic leukemia.

We report a case of Richter transformation of a chronic lymphocytic leukemia with a 12q13 translocation involving the HMGI-C gene. Fluorescence in situ hybridization analysis with the use of two different cosmid pools spanning the entire HMGI-C region showed that the breakpoint on chromosome 12 was located in the HMGI-C gene, presumably within intron 3. In fact, the 3' region of HMGI-C had been translocated to a derivative chromosome 6. This translocation was not visible at the cytogenetic level. Immunohistochemical analysis performed on the bone marrow smear demonstrated the expression of the HMGI-C protein specifically in the blasts, suggesting that the aberrant expression of the HMGI-C gene might have an important role in the process of leukemogenesis.

Back to the roots of a new exon--the molecular archaeology of a SP100 splice variant.

Retropseudogenes are intronless DNA sequences sharing a high degree of homology with the cDNA of their corresponding active genes. They are thought to have originated by reverse transcription of messenger RNA and reintegration of the cDNA into the genome. Usually considered a type of evolutionary waste, they melt into the background of their surrounding DNA by the loss of similarity to the active gene or disappear from the genome by the accumulation of deletions. On the other hand, in this paper we describe the evolutionary recycling of this genomic waste. Recently, a splice variant of the gene encoding the nuclear protein SP100 was identified in which the 3' part of the cDNA is replaced by an alternative exon apparently encoding an HMG1-DNA-binding domain. We were able to show that this HMG box is contributed by a new exon arising from an HMG1 retropseudogene that we have molecularly characterized in detail. In addition to being found in human cells, corresponding fusion transcripts were shown in Pan troglodytes, Gorilla gorilla, and Hylobates lar, but not in Macaca mulatta. Genomic DNA from M. mulatta enabled us to amplify by PCR the 5' part but not the 3' part of the HMG1 retropseudogene. From our data we thus can date the underlying retrotransposition to more than 35 million years ago. Our findings offer a model as to how new exons may evolve during evolution. To our knowledge this is the first example of a retropseudogene becoming part of an active gene in which both parental parts are well characterized and remain in-frame with their cDNA.

HMGI-C and HMGI(Y) immunoreactivity correlates with cytogenetic abnormalities in lipomas, pulmonary chondroid hamartomas, endometrial polyps, and uterine leiomyomas and is compatible with rearrangement of the HMGI-C and HMGI(Y) genes.

High-mobility group (HMG) proteins are nonhistone nuclear proteins that play an important role in the regulation of chromatin structure and function. HMGI-C and HMGI(Y) are members of the HMGI family of HMG proteins, and their expression in adult tissues generally correlates with malignant tumor phenotypes. However, HMGI-C and HMGI(Y) dysregulation as a result of specific rearrangements involving 12q15 and 6p21, the respective chromosomal sites in which the HMGI-C and HMGI(Y) genes are located, is also identified in a variety of common benign mesenchymal tumors, such as lipomas and uterine leiomyomata. The general prevalence of HMGI-C and HMGI(Y) protein expression and its correlation with chromosomal alterations in these benign tumors are unknown. We analyzed 95 human tumors (20 lipomas, 21 pulmonary chondroid hamartomas, 26 uterine leiomyomata, and 28 endometrial polyps) representing a selection of the benign lesions in which karyotypic alterations involving the chromosomal regions 12q15 and 6p21 are frequently detected. All cases were successfully karyotyped and some of them analyzed by fluorescent in situ hybridization with probes spanning the HMGI-C and HMGI(Y) genes. The results of this study demonstrate that expression of HMGI-C or HMGI(Y) is a common occurrence in lipomas, pulmonary chondroid hamartomas, leiomyomata, and endometrial polyps; that it correlates with 12q15 and 6p21 chromosomal alterations (p < 0.001); and that it is compatible with rearrangement of the HMGI-C and HMGI(Y) genes. The expression pattern and cellular localization of the immunoreactivity support the view that in biphasic lesions composed of a mixture of both stromal and epithelial cells, such as pulmonary chondroid hamartoma and endometrial polyps, the mesenchymal component is the site of the HMGI genetic alterations.