Many roads to resistance: how invertebrates adapt to Bt toxins

The Cry family of Bacillus thuringiensis insecticidal and nematicidal proteins constitutes a valuable source of environmentally benign compounds for the control of insect pests and disease agents. An understanding of Cry toxin resistance at a molecular level will be critical to the long-term utility of this technology; it may also shed light on basic mechanisms used by other bacterial toxins that target specific organisms or cell types. Selection and cross-resistance studies have confirmed that genetic adaptation can elicit varying patterns of Cry toxin resistance, which has been associated with deficient protoxin activation by host proteases, and defective Cry toxin-binding cell surface molecules, such as cadherins, aminopeptidases and glycolipids. Recent work also suggests Cry toxin resistance may be induced in invertebrates as an active immune response. The use of model invertebrates, such as Caenorhabditis elegans and Drosophila melanogaster, as well as advances in insect genomics, are likely to accelerate efforts to clone Cry toxin resistance genes and come to a detailed and broad understanding of Cry toxin resistance.     

Large-scale phenotyping of an accurate genetic mouse model of JNCL identifies novel early pathology outside the central nervous system

Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development.     

Neuronal Goα and CAPS Regulate Behavioral and Immune Responses to Bacterial Pore-Forming Toxins

Pore-forming toxins (PFTs) are abundant bacterial virulence factors that attack host cell plasma membranes. Host defense mechanisms against PFTs described to date all function in the host tissue that is directly attacked by the PFT. Here we characterize a rapid and fully penetrant cessation of feeding of Caenorhabditis elegans in response to PFT attack. We demonstrate via analyses of C. elegans mutants that inhibition of feeding by PFT requires the neuronal G protein Goα subunit goa-1, and that maintenance of this response requires neuronally expressed calcium activator for protein secretion (CAPS) homolog unc-31. Independently from their role in feeding cessation, we find that goa-1 and unc-31 are additionally required for immune protection against PFTs. We thus demonstrate that the behavioral and immune responses to bacterial PFT attack involve the cross-talk between the nervous system and the cells directly under attack.     

The heart in Duchenne muscular dystrophy: early detection of contractile performance alteration

Progressive cardiomyopathy is a major cause of death in Duchenne muscular dystrophy (DMD) patients. Coupling between Ca²⁺ handling and contractile properties in dystrophic hearts is poorly understood. It is also not clear whether developing cardiac failure is dominated by alterations in Ca²⁺ pathways or more related to the contractile apparatus. We simultaneously recorded force and Ca²⁺ transients in field‐stimulated papillary muscles from young (10–14 weeks) wild‐type (wt) and dystrophic mdx mice. Force amplitudes were fivefold reduced in mdx muscles despite only 30 % reduction in fura‐2 ratio amplitudes. This indicated mechanisms other than systolic Ca²⁺ to additionally account for force decrements in mdx muscles. pCa‐force relations revealed decreased mdx myofibrillar Ca²⁺ sensitivity. ‘In vitro’ motility assays, studied in mdx hearts here for the first time, showed significantly slower sliding velocities. mdx MLC/MHC isoforms were not grossly altered. Dystrophic hearts showed echocardiography signs of early ventricular wall hypertrophy with a significantly enlarged end‐diastolic diameter ‘in vivo’. However, fractional shortening was still comparable to wt mice. Changes in the contractile apparatus satisfactorily explained force drop in mdx hearts. We give first evidence of early hypertrophy in mdx mice and possible mechanisms for already functional impairment of cardiac muscle in DMD.     

Information that should be given to HIV cohort participants during ongoing research: the viewpoints of patient representatives and research professionals

While investigators have a duty to provide research participants with summary findings at the end of a study, providing general information during the course of research is rarely considered. However, this raises an important ethical issue in the context of long-term studies such as cohorts or biobanks. We investigated this issue in the context of two ANRS cohorts of HIV-infected patients, AQUITAINE and COPILOTE. Face-to-face interviews were conducted with HIV patient representatives and research professionals concerning the delivery of information in the course of the research. Respondents stated that participants wish to be informed of research results (both aggregate and individual) but also expect general information about the cohort itself, research progression, and what their participation may provide. It was concluded that information provided during the course of the research may help participants to distinguish between care and research. The essential role of clinicians-investigators in providing information was emphasized.     

The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways

Bacterial toxins and effector proteins hijack eukaryotic enzymes that are spatially localized and display rapid signaling kinetics. However, the molecular mechanisms by which virulence factors engage highly dynamic substrates in the host cell environment are poorly understood. Here, we demonstrate that the enteropathogenic Escherichia coli (EPEC) type III effector protein EspF nucleates a multiprotein signaling complex composed of eukaryotic sorting nexin 9 (SNX9) and neuronal Wiskott-Aldrich syndrome protein (N-WASP). We demonstrate that a specific and high affinity association between EspF and SNX9 induces membrane remodeling in host cells. These membrane-remodeling events are directly coupled to N-WASP/Arp2/3-mediated actin nucleation. In addition to providing a biochemical mechanism of EspF function, we find that EspF dynamically localizes to membrane-trafficking organelles in a spatiotemporal pattern that correlates with SNX9 and N-WASP activity in living cells. Thus, our findings suggest that the EspF-dependent assembly of SNX9 and N-WASP represents a novel form of signaling mimicry used to promote EPEC pathogenesis and gastrointestinal disease.     

Modified free paraumbilical perforator flap: the next logical step in breast reconstruction

The free paraumbilical perforator flap that was developed with only the perforator penetrating the rectus abdominis muscle seems nearly ideal from the donor-site perspective, because it reduces morbidity virtually to that of an abdominoplasty. However, it requires "supermicrosurgery" skills for suturing of vessels with diameters of 0.7 mm or less, making the procedure technically demanding and risky. Another significant disadvantage of that technique is that the flap has very short vascular pedicles. To overcome the disadvantages of the free paraumbilical perforator flap, a minor modification in flap harvesting that improves the vessel size and length is described. Results for a total of 15 patients who underwent breast reconstruction with modified free paraumbilical perforator flaps are presented, and the advantages and disadvantages of this flap are discussed.     

E.S.R. Spin Trapping Analysis of Gamma Induced Radicals in Sucrose

Radicals induced by gamma-irradiation in sucrose, in the solid state at different temperatures and in aqueous solution, have been studied by the spin-trapping method. Electron spin resonance (ESR) combined with high performance liquid chromatography (HPLC) then spectra analysis with a simulation program (VOYONS), revealed seven main radical species. Their nitrogen and hydrogen splitting constants were compared with those obtained from fructose and glucose units. Assignments of chemical structures are discussed for three radical species.