Isolation of highly polymorphic microsatellite loci for a species with a large genome size: sharp-ribbed salamander (Pleurodeles waltl)

Fourteen highly polymorphic microsatellite markers were developed and characterized for the sharp-ribbed salamander, Pleurodeles waltl. Isolating microsatellites with more than 12 single repeat type units was only successful for a tetranucleotide repeat (ATAG). Compared to microsatellite libraries constructed simultaneously for two anuran amphibian species, a greater number of primer pairs designed for P. waltl had to be discarded, due to consistent amplification problems. Low amplification success rate for P. waltl may be due to its larger genome size. Consequently, to avoid nonspecific binding and to increase amplification success, polymerase chain reaction programmes with touchdown cycles were used. For 14 microsatellite markers, amplification was successful and consistent with number of alleles and expected heterozygosity ranging from seven to 22 and from 0.79 to 0.94, respectively. All 14 microsatellite markers will be extremely useful for metapopulation studies of this unique amphibian species.     

Three‐dimensional correlative single‐cell imaging utilizing fluorescence and refractive index tomography

Cells alter the path of light, a fact that leads to well‐known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, facilitating the segmentation of the 3D‐correlated RI and fluorescence data for a quantitative interpretation of the nuclear RI. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.      

Clinical Manifestation of Depression after Stroke: Is It Different from Depression in Other Patient Populations?

Background

Despite ample research on depression after stroke, the debate continues regarding whether symptoms such as sleep disturbances, loss of energy, changes in appetite and diminished concentration should be considered to be consequences of stroke or general symptoms of depression. By comparing symptoms in depressed and non-depressed stroke patients with patients in general practice and patients with symptomatic atherosclerotic diseases, we aim to further clarify similarities and distinctions of depression after stroke and depression in other patient populations. Based on this, it is possible to determine if somatic symptoms should be evaluated in stroke patients in diagnosing depression after stroke.

Methods

An observational multicenter study is conducted in three hospitals and seven general practices including 382 stroke patients admitted to hospital with a clinical diagnosis of intracerebral hemorrhage or ischemic infarction, 1160 patients in general practice (PREDICT-NL), and 530 patients with symptomatic atherosclerotic diseases (SMART-Medea).

Results

The prevalence of major depressive disorder according to DSM-IV criteria was 14.1% (95% CI 11.0%-18.0%) in the stroke cohort, 5.4% (95% CI 3.8%-7.9%) in the symptomatic atherosclerotic diseases cohort and 12.9% (95% CI 11.1%-15.0%) in the general practice cohorts. Comparing depressed patients of the three cohorts demonstrated broadly similar symptom profiles, as well as comparable levels of individual symptom prevalence. However, the stroke patients suffered more severely from these symptoms than patients in the other populations.

Conclusions

The findings suggest that depression after stroke is not a different type of depression. This finding indicates that all depressive symptoms should be evaluated in stroke patients, including somatic symptoms.     

Cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome

Adjuvants are vaccine additives that stimulate the immune system without having any specific antigenic effect of itself. In this study we show that alum adjuvant induces the release of IL-1beta from macrophages and dendritic cells and that this is abrogated in cells lacking various NALP3 inflammasome components. The NALP3 inflammasome is also required in vivo for the innate immune response to OVA in alum. The early production of IL-1beta and the influx of inflammatory cells into the peritoneal cavity is strongly reduced in NALP3-deficient mice. The activation of adaptive cellular immunity to OVA-alum is initiated by monocytic dendritic cell precursors that induce the expansion of Ag-specific T cells in a NALP3-dependent way. We propose that, in addition to TLR stimulators, agonists of the NALP3 inflammasome should also be considered as vaccine adjuvants.     

Key motif to gain selectivity at the neuropeptide Y5-receptor: structure and dynamics of micelle-bound [Ala31, Pro32]-NPY

The structure of [Ala(31), Pro(32)]-NPY, a neuropeptide Y mutant with selectivity for the NPY Y(5)-receptor (Cabrele, C., Wieland, H. A., Stidsen, C., Beck-Sickinger, A. G., (2002) Biochemistry XX, XXXX-XXXX (companion paper)), has been characterized in the presence of the membrane mimetic dodecylphosphocholine (DPC) micelles using high-resolution NMR techniques. The overall topology closely resembles the fold of the previously described Y(5)-receptor-selective agonist [Ala(31), Aib(32)]-NPY (Cabrele, C., Langer, M., Bader, R., Wieland, H. A., Doods, H. N., Zerbe, O., and Beck-Sickinger, A. G. (2000) J. Biol. Chem 275, 36043-36048). Similar to wild-type neuropeptide Y (NPY) and [Ala(31), Aib(32)]-NPY, the N-terminal residues Tyr(1)-Asp(16) are disordered in solution. Starting from residue Leu(17), an alpha helix extends toward the C-terminus. The decreased density of medium-range NOEs for the C-terminal residues resulting in larger RMSD values for the backbone atoms of Ala(31)-Tyr(36) indicates that the alpha helix has become interrupted through the [Ala(31), Pro(32)] mutation. This finding is further supported by (15)N-relaxation data through which we can demonstrate that the well-defined alpha helix is restricted to residues 17-31, with the C-terminal tetrapeptide displaying increased flexibility as compared to NPY. Surprisingly, increased generalized order parameter as well as decreased (3)J(HN)(alpha) scalar coupling constants reveal that the central helix is stabilized in comparison to wild-type NPY. Micelle-integrating spin labels were used to probe the mode of association of the helix with the membrane mimetic. The Y(5)-receptor-selective mutant and NPY share a similar orientation, which is parallel to the lipid surface. However, signal reductions due to efficient electron, nuclear spin relaxation were much less pronounced for the surface-averted residues in [Ala(31), Pro(32)]-NPY when compared to wild-type DPC-bound NPY. Only the signals of residues Asn(29) and Leu(30) were significantly more reduced in the mutant. The postulation of a different membrane binding mode of [Ala(31), Pro(32)]-NPY is further supported by the faster H/D exchange at the C-terminal amide protons. We conclude that arginine residues 33 and 35, which are believed to be directly involved in forming contacts to acidic receptor residues at the membrane-water interface, are no longer fixed in a well-defined conformation close to the membrane surface in [Ala(31), Pro(32)]-NPY.     

Membrane-facilitated bioproduction of 3-methylcatechol in an octanol/water two-phase system

Bioproduction of 3-methylcatechol from toluene by Pseudomonas putida MC2 was studied in the presence of an additional 1-octanol phase. This solvent was used to supply the substrate and extract the product, in order to keep the aqueous concentrations low. A hollow-fibre membrane kept the octanol and aqueous phase separated to prevent phase toxicity towards the bacterium. Volumetric production rates increased approximately 40% as compared to two-phase 3-methylcatechol production with direct phase contact. Preliminary investigations on downstream processing of 3-methylcatechol showed that 1 M of sodium hydroxide selectively extracted the disodium salt of 3-methylcatechol into an aqueous phase.     

Opinion: Building epithelial architecture: insights from three-dimensional culture models

How do individual cells organize into multicellular tissues? Here, we propose that the morphogenetic behaviour of epithelial cells is guided by two distinct elements: an intrinsic differentiation programme that drives formation of a lumen-enclosing monolayer, and a growth factor-induced, transient de-differentiation that allows this monolayer to be remodelled.     

Large-scale identification of disease genes involved in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of diseases in which chromosomal aberrations, small insertions or deletions, or point mutations in certain genes have profound consequences for prognosis. However, the majority of AML patients present without currently known genetic defects. Retroviral insertion mutagenesis in mice has become a powerful tool for identifying new disease genes involved in the pathogenesis of leukemia and lymphoma. Here we have used the Graffi-1.4 strain of murine leukemia virus, which causes predominantly AML, in a screen to identify novel genes involved in the pathogenesis of this disease. We report 79 candidate disease genes in common integration sites (CISs) and 15 genes whose family members previously were found to be affected in other studies. The majority of the identified sequences (60%) were not found in lymphomas and monocytic leukemias in previous screens, suggesting a specific involvement in AML. Although most of the virus integrations occurred in or near the 5' or 3' ends of the genes, suggesting deregulation of gene expression as a consequence of virus integration, 18 CISs were located exclusively within the genes, conceivably causing gene disruption.