Antimicrobial activities of YycG histidine kinase inhibitors against Staphylococcus epidermidis biofilms

Staphylococcus epidermidis has become a significant pathogen causing infections due to biofilm formation on surfaces of indwelling medical devices. Biofilm-associated bacteria exhibit enhanced resistance to many conventional antibiotics. It is therefore, important to design novel antimicrobial reagents targeting S. epidermidis biofilms. In a static chamber system, the bactericidal effect of two leading compounds active as YycG inhibitors was assessed on biofilm cells by confocal laser scanning microscopy combined with viability staining. In young biofilms (6-h-old), the two compounds killed the majority of the embedded cells at concentrations of 100 microM and 25 microM, respectively. In mature biofilms (24-h-old), one compound was still effectively killing biofilm cells, whereas the other compound mainly killed cells located at the bottom of the biofilm. In contrast, vancomycin was found to stimulate biofilm development at the MBC (8 microg mL(-1)). Even at a high concentration (128 microg mL(-1)), vancomycin exhibited poor killing on cells embedded in biofilms. The two compounds exhibited faster and more effective killing of S. epidermidis planktonic cells than vancomycin at the early stage of exposure (6 h). The data suggest that the new inhibitors can serve as potential agents against S. epidermidis biofilms when added alone or in concert with other antimicrobial agents.     

Stiffening of human skin fibroblasts with age

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.     

Apelin: a new plasma marker of cardiopulmonary disease

OBJECTIVES: Dyspnea is a major symptom of both parenchymal lung disease and chronic heart failure. Underlying cardiac dysfunction can be assessed by measurement of cardiac-derived B-type natriuretic peptide or its precursor in plasma. However, no specific endocrine marker of the lung parenchyma has so far been identified. We therefore examined whether plasma concentrations of apelin, a novel inotropic hormone, is affected in patients with chronic parenchymal lung disease without cardiac dysfunction. METHODS AND RESULTS: Patients with severe chronic parenchymal lung disease and normal cardiac function (n=53), idiopathic pulmonary hypertension with increased right ventricular pressure (n=10), and patients with severe left ventricular systolic dysfunction (n=22) were enrolled. Plasma apelin-36 and proBNP concentrations were measured with radioimmunoassays. While proBNP plasma concentrations were unaffected in chronic parenchymal lung disease patients compared to normal subjects, the apelin-36 concentration was reduced 3.3-fold (median 35 pmol/l (0-162 pmol/l) vs. 117 pmol/l (55-232 pmol/l), P<0.001). Moreover, the apelin-36 concentration was decreased in chronic heart failure patients (2.1-fold, P<0.01) and in patients with idiopathic pulmonary hypertension (4.0-fold, P<0.001). In contrast, the proBNP concentration was highly increased in both chronic heart failure and idiopathic pulmonary hypertension patients. CONCLUSION: Plasma concentrations of apelin-36, a novel inotropic peptide, are decreased in patients with chronic parenchymal lung disease and preserved cardiac function. Combined measurement of apelin-36 and proBNP may be a new diagnostic approach in distinguishing pulmonary from cardiovascular causes of dyspnea.     

Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges

Traditional DNA transduction routes used for the modification of cellular genomes are subject to unpredictable alterations, as the cell-intrinsic repair machinery may affect both the integrity of the transgene and the recipient locus. These problems are overcome by recombinase-mediated cassette exchange (RMCE) approaches enabling predictable expression patterns by the nondisruptive insertion of a gene cassette at a pre-characterized genomic locus. The destination is marked by a “tag” consisting of two heterospecific recombination target sites (RTs) at the flanks of a selection marker. Provided on a circular donor vector, an analogous cassette encoding the gene of interest can cleanly replace the resident cassette under the influence of a site-specific recombinase. RMCE was first based on the yeast integrase Flp but had to give way to the originally more active phage-derived Cre enzyme. To be effective, both Tyr-recombinases have to be applied at a considerable concentration, which, in the case of Cre, triggers endonucleolytic activities and therefore cellular toxicity. This review addresses the particularities of both recombination routes depending on the structure of the synaptic complex and on improved integrase and RT variants. While the performance of Flp-RMCE can now firmly rely on optimized Flp variants and multiple sets of functional target sites (FRTs), the Cre system suffers from the promiscuity of its RT mutants, which is explained in molecular terms. At present, RMCE enters applications in the stem cell field. Remarkable efforts are noted in the framework of various mouse mutagenesis programs, which, in their first phase, have targeted virtually all genes and now start to shift their emphasis from gene trapping to gene modification.     

Label-free detection of enhanced saccharide binding at pH 7.4 to nanoparticulate benzoboroxole based receptor units

Nanoparticles modified with either 6-amino-1-hydroxy-2,1-benzoxaborolane (3-aminobenzoboroxole) or 3-aminophenylboronic acid were prepared by nucleophilic substitution of a styrene-co-DVB-co-vinylbenzylchloride latex (25 nm). Isothermal titration calorimetry (ITC) was used as a label-free detection method for the analysis of the binding between monosaccharides and these two differently derivatized nanoparticle systems at pH 7.4. Because ITC reveals, thermodynamical parameters such as the changes in enthalpy ΔH, free energy ΔG, and entropy ΔS, possible explanations for the higher binding constants can be derived in terms of entropy and enthalpy changes. In case of the modified nanoparticles, the free energy of binding is dominated by the entropy term. This shows that interfacial effects, besides the intrinsic affinity, lead to a higher binding constant compared with the free ligand. The highest binding constant was found for fructose binding to the benzoboroxole modified nanoparticles: Its value of 1150 M(-1) is twice as high as for the free benzoboroxole and five times as high as with phenylboronic acid or 3-aminophenylboronic acid. In contrast to the binding of fructose to free boronic acids, which is an enthalpically driven process, the binding of fructose to the modified nanoparticles is dominated by the positive entropy term.     

A general framework for quantifying the effects of DNA repair inhibitors on radiation sensitivity as a function of dose

In this paper, we consider the ocular lens in the context of contemporary developments in biological ideas. We attempt to reconcile lens biology with stem cell concepts and a dearth of lens tumors. Historically, the lens has been viewed as a closed system, in which cells at the periphery of the lens epithelium differentiate into fiber cells. Theoretical considerations led us to question whether the intracapsular lens is indeed self-contained. Since stem cells generate tumors and the lens does not naturally develop tumors, we reasoned that lens stem cells may not be present within the capsule. We hypothesize that lens stem cells reside outside the lens capsule, in the nearby ciliary body. Our ideas challenge the existing lens biology paradigm. We begin our discussion with lens background information, in order to describe our lens stem cell hypothesis in the context of published data. Then we present the ciliary body as a possible source for lens stem cells, and conclude by comparing the ocular lens with the corneal epithelium.     

Genetic dissociation of ethanol sensitivity and memory formation in Drosophila melanogaster

The ad hoc genetic correlation between ethanol sensitivity and learning mechanisms in Drosophila could overemphasize a common process supporting both behaviors. To challenge directly the hypothesis that these mechanisms are singular, we examined the learning phenotypes of 10 new strains. Five of these have increased ethanol sensitivity, and the other 5 do not. We tested place and olfactory memory in each of these lines and found two new learning mutations. In one case, altering the tribbles gene, flies have a significantly reduced place memory, elevated olfactory memory, and normal ethanol response. In the second case, mutation of a gene we name ethanol sensitive with low memory (elm), place memory was not altered, olfactory memory was sharply reduced, and sensitivity to ethanol was increased. In sum, however, we found no overall correlation between ethanol sensitivity and place memory in the 10 lines tested. Furthermore, there was a weak but nonsignificant correlation between ethanol sensitivity and olfactory learning. Thus, mutations that alter learning and sensitivity to ethanol can occur independently of each other and this implies that the set of genes important for both ethanol sensitivity and learning is likely a subset of the genes important for either process.     

Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.