Rho GTPase-activating bacterial toxins: from bacterial virulence regulation to eukaryotic cell biology

Studies on the interactions of bacterial pathogens with their host have provided an invaluable source of information on the major functions of eukaryotic and prokaryotic cell biology. In addition, this expanding field of research, known as cellular microbiology, has revealed fascinating examples of trans-kingdom functional interplay. Bacterial factors actually exploit eukaryotic cell machineries using refined molecular strategies to promote invasion and proliferation within their host. Here, we review a family of bacterial toxins that modulate their activity in eukaryotic cells by activating Rho GTPases and exploiting the ubiquitin/proteasome machineries. This family, found in human and animal pathogenic Gram-negative bacteria, encompasses the cytotoxic necrotizing factors (CNFs) from Escherichia coli and Yersinia species as well as dermonecrotic toxins from Bordetella species. We survey the genetics, biochemistry, molecular and cellular biology of these bacterial factors from the standpoint of the CNF1 toxin, the paradigm of Rho GTPase-activating toxins produced by urinary tract infections causing pathogenic Escherichia coli. Because it reveals important connections between bacterial invasion and the host inflammatory response, the mode of action of CNF1 and its related Rho GTPase-targetting toxins addresses major issues of basic and medical research and constitutes a privileged experimental model for host-pathogen interaction.     

Development and pilot evaluation of novel genetic educational materials designed for an underserved patient population

Genetic counseling for BRCA1 and BRCA2 mutations involves teaching about hereditary cancer, genetics and risk, subjects that are difficult to grasp and are routinely misunderstood. Supported by a grant from the Avon Foundation, the UCSF Cancer Risk Program started the first genetic testing and counseling service for a population of traditionally underserved women of varied ethnic and social backgrounds at the San Francisco General Hospital (SFGH). Informed by educational theory and clinical experience, we devised and piloted two simplified explanations of heredity and genetic risk, with the aim of uncovering how to best communicate genetics and risk to this underserved population. A "conventional" version comprised pictures of genes, pedigrees, and quantitative representations of risk. A "colloquial" pictorial version used an analogy of the "information book" of genes, family stories and vignettes, and visual representations of risk, without using scientific words such as genes or chromosomes. A verbal narrative accompanied each picture. We presented these modules to four focus groups of five to eight women recruited from the SFGH Family Practice Clinic. Overall, women preferred a picture-based approach and commented that additional text would have been distracting. The majority of women preferred the colloquial version because it was easier to understand and better conveyed a sense of comfort and hope. We conclude that simplicity, analogies, and familiarity support comprehension while vignettes, family stories, and photos of real people provide comfort and hope. These elements may promote understanding of complex scientific topics in healthcare, particularly when communicating with patients who come from disadvantaged backgrounds.     

Cardiac resynchronization therapy: a meta-analysis of randomized controlled trials

Background

Studies of cardiac resynchronization therapy in addition to an implantable cardioverter defibrillator in patients with mild to moderate congestive heart failure had not been shown to reduce mortality until the recent RAFT trial (Resynchronization/Defibrillation for Ambulatory Heart Failure Trial). We performed a meta-analysis including the RAFT trial to determine the effect of cardiac resynchronization therapy with or without an implantable defibrillator on mortality.

Methods

We searched electronic databases and other sources for reports of randomized trials using a parallel or crossover design. We included studies involving patients with heart failure receiving optimal medical therapy that compared cardiac resynchronization therapy with optimal medical therapy alone, or cardiac resynchronization therapy plus an implantable defibrillator with a standard implantable defibrillator. The primary outcome was mortality. The optimum information size was considered to assess the minimum amount of information required in the literature to reach reliable conclusions about cardiac resynchronization therapy.

Results

Of 3071 reports identified, 12 studies (n = 7538) were included in our meta-analysis. Compared with optimal medical therapy alone, cardiac resynchronization therapy plus optimal medical therapy significantly reduced mortality (relative risk [RR] 0.73, 95% confidence interval [CI] 0.62–0.85). Compared with an implantable defibrillator alone, cardiac resynchronization therapy plus an implantable defibrillator significantly reduced mortality (RR 0.83, 95% CI 0.72–0.96). This last finding remained significant among patients with New York Heart Association (NYHA) class I or II disease (RR 0.80, 95% CI 0.67–0.96) but not among those with class III or IV disease (RR 0.84, 95% CI 0.69–1.07). Analysis of the optimum information size showed that the sequential monitoring boundary was crossed, which suggests no need for further clinical trials.

Interpretation

The cumulative evidence is now conclusive that the addition of cardiac resynchronization to optimal medical therapy or defibrillator therapy significantly reduces mortality among patients with heart failure.Congestive heart failure is currently reaching epidemic proportions in Canada, with 500 000 Canadians affected and 50 000 new patients identified each year.¹ It accounts for more than 100 000 hospital admissions per year and has a one-year mortality ranging from 15% to 50%, depending on the severity of heart failure.² By 2050, the number of patients with heart failure is projected to increase threefold.²Advances in medical therapies have resulted in substantial reductions in mortality associated with congestive heart failure.^3–7 The use of devices has recently become an important adjuvant therapy.⁸ Cardiac resynchronization therapy involves pacing from both the right and left ventricles simultaneously to improve myocardial efficiency (see radiographs in Appendix 1, at www.cmaj.ca/cgi/content/full/cmaj.101685/DC1). Cardiac resynchronization therapy has been shown to reduce morbidity and, when compared with medical therapy alone, to reduce mortality.^9–13 Until recently, it was not shown to reduce mortality among patients who also received an implantable cardioverter defibrillator. Among patients receiving optimal medical therapy, the Resynchronization/Defibrillation for Ambulatory Heart Failure Trial (RAFT) showed the superiority of cardiac resynchronization therapy in addition to an implantable defibrillator over a standard implantable defibrillator in reducing mortality and the combined outcome of death from any cause or hospital admission related to heart failure.¹⁴We performed a meta-analysis to further assess the effect on mortality of cardiac resynchronization therapy with and without an implantable defibrillator among patients with mildly symptomatic and advanced heart failure.     

Assessment of micronucleus frequency in normal oral mucosa of patients exposed to carcinogens

OBJECTIVE: To assess the presence of micronuclei in exfoliated oral mucosal cells collected from 3 anatomic sites in patients exposed to tobacco and alcohol. STUDY DESIGN: Smears were prepared with normal oral mucosal cells obtained from the lower lip, tongue border and floor of the mouth of 21 controls, 28 tobacco users and 19 tobacco/alcohol users. Slides were stained with Feulgen stain for quantification of micronucleated cells, karyorrhexis and "broken eggs." RESULTS: The groups were similar in terms of the mean number of micronucleated cells and cells undergoing karyorrhexis. In the comparison of anatomic sites, the mean number of cells undergoing karyorrhexis was higher on the lower lip than on the tongue border or floor of the mouth (all groups). A significantly higher number of broken eggs was observed in the control group when compared to the tobacco and tobacco/alcohol groups at all anatomic sites. CONCLUSION: The higher number of broken eggs in patients not exposed to tobacco and/or alcohol suggests that this nuclear alteration may be associated with DNA repair or a healthy mucosa. A trend toward an increased number of micronucleated cells was observed for tobacco and/or alcohol users at all anatomic sites.     

Distinct action of the retinoblastoma pathway on the DNA replication machinery defines specific roles for cyclin-dependent kinase complexes in prereplication complex assembly and S-phase progression

The retinoblastoma (RB) and p16ink4a tumor suppressors are believed to function in a linear pathway that is functionally inactivated in a large fraction of human cancers. Recent studies have shown that RB plays a critical role in regulating S phase as a means for suppressing aberrant proliferation and controlling genome stability. Here, we demonstrate a novel role for p16ink4a in replication control that is distinct from that of RB. Specifically, p16ink4a disrupts prereplication complex assembly by inhibiting mini-chromosome maintenance (MCM) protein loading in G1, while RB was found to disrupt replication in S phase through attenuation of PCNA function. This influence of p16ink4a on the prereplication complex was dependent on the presence of RB and the downregulation of cyclin-dependent kinase (CDK) activity. Strikingly, the inhibition of CDK2 activity was not sufficient to prevent the loading of MCM proteins onto chromatin, which supports a model wherein the composite action of multiple G1 CDK complexes regulates prereplication complex assembly. Additionally, p16ink4a attenuated the levels of the assembly factors Cdt1 and Cdc6. The enforced expression of these two licensing factors was sufficient to restore the assembly of the prereplication complex yet failed to promote S-phase progression due to the continued absence of PCNA function. Combined, these data reveal that RB and p16ink4a function through distinct pathways to inhibit the replication machinery and provide evidence that stepwise regulation of CDK activity interfaces with the replication machinery at two discrete execution points.     

Selection and inertia in the evolution of holocentric chromosomes in sedges (Carex, Cyperaceae)

? Changes in chromosome number as a result of fission and fusion in holocentrics have direct and immediate effects on the recombination rate. We investigate the support for the classic hypothesis that environmental stability selects for increased recombination rates. ? We employed a phylogenetic and cytogenetic data set from one of the most diverse angiosperm genera in the world, which has the largest nonpolyploid chromosome radiation (Carex, Cyperaceae; 2n = 12-124; 2100 spp.). We evaluated alternative Ornstein-Uhlenbeck models of chromosome number adaptation to the environment in an information-theoretic framework. ? We found moderate support for a positive influence of lateral inflorescence unit size on chromosome number, which may be selected in a stable environment in which resources for reproductive investment are larger. We found weak support for a positive influence on chromosome number of water-saturated soils and among-month temperature constancy, which would be expected to be negatively select for pioneering species. Chromosome number showed a strong phylogenetic signal. ? We argue that our finding of small but significant effects of life history and ecology is compatible with our original hypothesis regarding selection of optima in recombination rates: low recombination rate is optimal when inmediate fitness is required. By contrast, high recombination rate is optimal when stable environments allow for evolutionary innovation.     

Beyond working hours: part II. incentive to improve

Efforts to limit resident work hours are conceptually flawed. A fundamental solution to resident service requires funding, which currently is not feasible. An equally fundamental structuring of the system of surgical resident education is feasible and should be attained.     

Relaxivities of paramagnetic liposomes: on the importance of the chain type and the length of the amphiphilic complex

Nuclear magnetic relaxation dispersion (NMRD) profiles of unilamellar DPPC liposomes incorporating Gd-DTPA-bisamides with alkyl chains of 12 to 18 C atoms in their external and internal layers were recorded in order to study the influence that the chain length and structure of Gd-bisamides incorporated in the liposomal membrane have on their proton relaxivity. The NMRD profiles recorded at 310 K show that the relaxivity reaches a minimum value when the carbon chain lengths of the phospholipid and of the Gd complex match and is at a maximum in the presence of a carbon-carbon double bond. For these DPPC paramagnetic liposomes, the longer the aliphatic chains of the complex, the larger will be its immobilization in the membrane. In addition, the presence of an unsaturated carbon-carbon bond in the alkyl chain of the Gd complex induces an increase of its mobility and of its water exchange rate with, as a result, a much greater efficiency as an MRI contrast agent.     

Stereospecific binding of MRI contrast agents to human serum albumin: the case of Gd-(S)-EOB-DTPA (Eovist) and its (R) isomer

The water proton relaxation rate enhancement of the hepatospecific Gd-(S)-EOB-DTPA (Eovist) and of its (R) isomer in aqueous solutions free of protein, in serum and in 4% human serum albumin solution, are compared. In the absence of proteins, both compounds exhibit, as expected, the same proton relaxivity, as measured by the nuclear magnetic relaxation dispersion (NMRD) profiles. In serum and albumin solution, non-covalent binding of the paramagnetic complexes to macromolecules is observed. Both isomers are likely to bind to the same site of human serum albumin, but the affinity of the (S) isomer is larger than for the (R) isomer.