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.

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.

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.

Description of a novel fusion transcript between HMGI-C, a gene encoding for a member of the high mobility group proteins, and the mitochondrial aldehyde dehydrogenase gene.

Aberrations involving the chromosomal region 12q24 are a nonrandom cytogenetic abnormality in frequent benign tumors mainly of mesenchymal origin, e.g., uterine leiomyomas, pleomorphic adenomas of the salivary gland, lipomas, or hamartomas of the lung. Mostly, these 12q24 abnormalities occur as a result of inversions also affecting chromosomal region 12q14-15. In addition to the frequent tumors mentioned above, these abnormalities have also been found in rare mesenchymal tumors, e.g., hemangiopericytomas. Although recently the molecular basis of the aberrations of chromosomal region 12q14-15, i.e., a rearrangement of the HMGI-C gene has been identified, the molecular roots of the 12q24 changes still remain to be elucidated. Herein we report on 3' rapid amplification of cDNA ends PCR results on cDNA from a primary uterine leiomyoma. As an ectopic sequence fused to exon 3 of the HMGI-C gene, we have identified a cDNA sequence that revealed 100% homology to exon 13 of the human mitochondrial aldehyde dehydrogenase gene (ALDH 2). Because ALDH 2 maps to 12q24.1, this fusion transcript is a good candidate underlying the chromosomal rearrangements involving 12q24.

Molecular characterization of 12q14-15 rearrangements in three pulmonary chondroid hamartomas.

Chromosomal aberrations involving the chromosomal breakpoint region 12q14-15 are frequently seen in a variety of mesenchymal tumors as uterine leiomyomas, lipomas, myxoid liposarcomas, enchondromas, or hemangiopericytomas. Therefore, this breakpoint region seems to be one of the most frequent chromosomal abnormality associated with the initiation of human mesenchymal neoplasms. To narrow down the breakpoint region on a molecular level in cells of three pulmonary chondroid hamartomas with 12q14-15 aberrations, we performed fluorescence in situ hybridization analysis with different cosmid clones originating from a YAC and cosmid contig overspanning parts of the region 12q14-15. We were able to narrow down the breakpoint to a region of 175 kb belonging to an area designated multiple aberration region because it also includes the breakpoints of leiomyomas, lipomas, and pleomorphic adenomas with 12q14-15 abnormalities. Our molecular and cytogenetic data suggest that hamartomas of the lung molecularly belong to the benign group of mesenchymal tumors showing multiple aberration region involvement.

Genomic changes in endometrial polyps associated with tamoxifen show no evidence for its action as an external carcinogen.

Eighty-eight endometrial specimens from 36 postmenopausal breast cancer patients treated with tamoxifen were investigated cytogenetically and molecularly using fluorescence in situ hybridization with appropriate probes for the HMGIC and HMGIY genes. Twenty control specimens, 10 endometrial polyps, and 10 endometrial biopsy specimens were investigated in the same way. Of the 88 specimens, 44 were from endometrial polyps; 3 were from endocervical polyps; 7 were from cystic endometrium; 30 were from normal or atrophic endometrium, normal endocervix, or myometrium; and 4 were from endometrial carcinomas. Chromosome investigation of the endometrial polyps showed the nature of the chromosome changes in tamoxifen-induced polyps to be the same as that in the controls and in sporadic endometrial polyps described in the literature. HMGIC and HMGIY gene rearrangements in both groups were identical as shown by fluorescence in situ hybridization, which also allowed for the detection of seven hidden paracentric inversions involving 12q15, one of which occurred in a cystic endometrium. The carcinomas did not exhibit any of these changes. Because abnormal expression of HMGIC or HMGIY as a consequence of structural chromosome changes in 12q15 or 6p21, respectively, is invariably associated with benign neoplasia, tamoxifen-associated endometrial polyps are unlikely to undergo further malignant transformation, and a mode of action of tamoxifen as an external carcinogen is unlikely.

HMGI-C rearrangements as the molecular basis for the majority of pulmonary chondroid hamartomas: a survey of 30 tumors.

Pulmonary chondroid hamartomas (PCH) are benign tumors of the lung characterized by a more or less high degree of mesenchymal metaplasia. In our series we investigated 30 PCH by a combination of cytogenetic and molecular methods. 18 tumors (60%) had cytogenetically detectable aberrations involving either 12q14-15 or 6p21 with a clear predominance of chromosomal abnormalities involving 12q14-15 (15 tumors). As in subgroups of pleomorphic adenomas of the salivary glands, leiomyomas of the uterus, and lipomas with 12q14-15 abnormalities the HMGI-C gene is frequently rearranged we tested PCH with either 12q14-15 abnormalities or normal karyotype by FISH and 3' RACE experiments for rearrangements of HMGI-C. Rearrangements were found in all cases with chromosomal 12q14-15 abnormalities and further six cases with an apparently normal karyotype. By the combination of cytogenetics with molecular techniques the percentage of cases with intragenic rearrangements of HMGI-C or rearrangements of its immediate surrounding was thus increased to 70% (21/30 cases). Considering all types of aberrations within this series 80% (24/30) of all PCH were aberrant. This is the first report on a combined molecular and cytogenetic analysis of a large series of pulmonary chondroid hamartomas indicating that rearrangements of HMGI-C, a member of the high mobility group protein gene family, are the leading molecular events in the genesis of PCH.

pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer.

Filamentous phage pIV is an outer membrane protein required for phage assembly and secretion. Chemical cross-linking and sedimentation experiments have been used to demonstrate that pIV from f1-infected Escherichia coli exists as a homo-multimer, probably composed of 10 to 12 subunits. pIV secreted from spheroplasts remains soluble and does not form multimers. Synthesis of pIV from distantly related filamentous phages or from a bacterial homolog that participates in a specialized form of extra-cellular protein secretion in the same cell with pIVf1 resulted in the formation of mixed multimers. This suggests that the homologous proteins themselves form homo-multimers. These structures could form gated channels that conduct assembling phage or specific substrate proteins across the outer membrane to the extracellular milieu.

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.

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.

Transcriptional activation of HMGI-C in three pulmonary hamartomas each with a der(14)t(12;14) as the sole cytogenetic abnormality.

Aberrations involving the chromosomal region 12q14-15 are non-random cytogenetic abnormalities in many benign tumors, e.g. pulmonary chondroid hamartomas (PCH). Recently, we identified rearrangements of the HMGI-C gene within the third or fourth intron as the molecular mechanism underlying most of these chromosomal aberrations. Herein we report our FISH and RACE studies on three PCHs each showing a rare variant type of the translocation t(12;14)(q14-15;q24) with presence of two normal chromosomes 12 and a der(14) but missing the der(12). The results revealed that in all three cases the breakpoint is located 5' to HMGI-C, suggesting that besides intragenic rearrangements also transcriptional activation of the gene can initiate tumor growth.

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.

Two human breast cancer cell lines showing decreasing telomeric repeat length during early in vitro passaging.

Specific DNA repeats serve as a molecular protection shield at the telomeric ends of mammalian chromosomes. The absence of telomerase activity leads to a gradual decrease of telomeric repeat length in normal somatic cells. In contrast, immortalized cells from malignant tumors are usually thought to re-express telomerase to overcome a self-limited growth. Following this hypothesis, re-expression of telomerase is due to a rare mutational event. Herein we describe our results of telomeric length determination in two newly established breast cancer cell lines. During in vitro establishment from pleural effusions, both cell lines showed a marked decrease of the upper border range of telomeric repeat length distribution. The lower border remained within a constant range characteristic for each cell line. In no case was decrease of repeat length accompanied by an increased incidence of telomeric associations or fusions. The results show that a constant telomeric repeat length does not constitute a characteristic feature of immortalized cells. Furthermore, the kinetics of repeat length decrease and the constant range of the lower border reveal that the onset of telomerase activity is not necessarily due to a rare, i.e., mutational, event.

Deletion of HMG17 in uterine leiomyomas with ring chromosome 1.

Uterine leiomyomas are characterized by several subgroups with characteristic chromosomal aberrations, mainly 12q14-15, 6p21, or interstitial deletions of chromosomes 3 and 7. For the first two subgroups, aberrations of the HMGIC and HMGIY genes have been described and are held responsible for tumor initiation. For other subgroups no molecular findings have been described as of yet. We focus here on a smaller subgroup of uterine leiomyomas with a ring chromosome 1 either as the only karyotypic deviation or occurring along with other abnormalities. In the p-arm of chromosome 1 HMG17, another member of the high-mobility group of proteins has been localized to the short arm of chromosome 1 (1p35) with two PAC clones on metaphase spreads of a uterine leiomyoma ring(1). Hybridization signals for these probes were not detected within the ring chromosome consistent with loss or deletion of HMG17. These findings suggest that HMG17 does not play a mechanistic role in leiomyoma similar to that observed with other high-mobility proteins.

Regional fine mapping of HMG17 to chromosomal band 1p35.

HMG17, a member of the high-mobility group of proteins, is ubiquitously expressed in higher eukaryotes. This protein has been shown to enhance transcriptional activity of many other genes. Recently, the HMG17 gene has been mapped to chromosomal band 1p36.1. Because this region is frequently involved in chromosomal aberrations of various human neoplasms, two PAC clones containing an HMG17 sequence were isolated. By fluorescence in situ hybridization (FISH) on prometaphase spreads, HMG17 was mapped to chromosomal band 1p35.

Inflammatory myofibroblastic tumor with HMGIC rearrangement.

Inflammatory pseudotumors or inflammatory myofibroblastic tumors (IMT) are lesions of extreme heterogeneity showing a highly variable mixture of bland-looking spindle cells, inflammatory cells, and collagen fibers. We describe our results of molecular cytogenetic and rapid amplification of cDNA ends (RACE-PCR) studies on an IMT characterized by a translocation involving 12q15. Chromosomal aberrations involving this region are very frequent among other benign tumors, such as lipomas, uterine leiomyomas, or pulmonary chondroid hamartomas. Recently, we have shown that, by these structural chromosomal aberrations, the HMGIC gene is affected. Fluorescence in situ hybridization (FISH) analysis and 3' RACE-PCR on cells of the present case of an inflammatory myofibroblastic tumor indicated an intragenic rearrangement of HMGIC, resulting in an aberrant transcript of that gene. Clonal cytogenetic aberrations have been described in very few cases of IMT. The results presented herein indicate that this case of IMT represents a true benign mesenchymal neoplasm associated with, or due to, a rearrangement of HMGIC.

Leiomyoma cells with 12q15 aberrations can be transformed in vitro and show a relatively stable karyotype during precrisis period.

Tumor cells from three uterine leiomyomas showing translocations involving 12q14-15 were transformed by transfection using the "early regions" of the SV40 genome. The cells had a higher proliferative capacity, were able to form colonies in soft agar, and showed an increased growth potential. Karyotype analyses of these transformed leiomyoma cells showed that the cells had retained the initial t(12;14) and t(12;15).

Identification of the chromosome 12 translocation breakpoint region of a pleomorphic salivary gland adenoma with t(1;12)(p22;q15) as the sole cytogenetic abnormality.

Cell line Ad-312/SV40, which was derived from a primary pleomorphic salivary gland adenoma with t(1;12)(p22;q15), was used in fluorescence in situ hybridization (FISH) analysis to characterize its translocation breakpoint region on chromosome 12. Results of previous studies have indicated that the chromosome 12 breakpoint in Ad-312/SV40 is located proximally to locus D12S8 and distally to the CHOP gene. We here describe two partially overlapping yeast artificial chromosome (YAC) clones, Y4854 (500 kbp) and Y9091 (460 kbp), which we isolated in the context of a chromosome walking project with D12S8 and CHOP as starting points. We present a composite long-range restriction map encompassing the inserts of these two YAC clones and show by FISH analysis that both YACs span the chromosome 12 breakpoint as present in Ad-312/SV40 cells. Subsequently, we have isolated cosmid clones corresponding to various sequence-tagged sites (STSs) mapping within the inserts of these YAC clones. These included cRM51, cRM69, cRM85, cRM90, cRM91, cRM110, and cRM111. In FISH studies, cosmid clones cRM85, cRM90, and cRM111 appeared to map distally to the chromosome 12 breakpoint, whereas cosmid clones cRM51, cRM69, cRM91, and cRM110 were found to map proximally to it. These results assign the chromosome 12 breakpoint in Ad-312/SV40 to a DNA region of less than 165 kbp. FISH evaluation of the chromosome 12 breakpoints in five other pleomorphic salivary gland adenoma cell lines indicated that these are located proximally to the one in Ad-312/SV40, at a distance of more than 0.9 Mbp from STS RM91. These results, while pinpointing a potentially critical region on chromosome 12, also provide evidence for the possible involvement of 12q13-q15 sequences located elsewhere.