Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.     

Gallium-desferrioxamine protects the cat retina against injury after ischemia and reperfusion

This study sought to determine whether gallium-desferrioxamine (Ga/DFO) can curb free radical formation and mitigate biochemical and electrophysiological parameters of injury in the cat retina subjected to ischemia followed by reperfusion.For the biochemical studies, cat eyes were subjected to 90 min of retinal ischemia followed by 5 min of reperfusion, and enucleation of one eye of each cat was used to measure retinal reperfusion injury. Before enucleation of fellow eyes, 2.5 mg/kg Ga/DFO was injected intravenously 5 min before reperfusion. The flux of hydroxyl radicals, as measured directly by conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA), was significantly lower in Ga/DFO-treated eyes. The mean normalized level of 2,3-DHBA (considered a specific marker of hydroxyl radicals) was 3.5 times higher in untreated eyes. Ga/DFO caused a significant reduction, by 2.56-fold, in lipid peroxidation, as reflected by levels of malondialdehyde. Ascorbic acid, a natural antioxidant present in the retina, is severely depleted in untreated eyes. In contrast, in Ga/DFO-treated eyes, levels were 10 times higher than the control. Energy charge was 2.38 times higher in treated eyes. Levels of purine catabolites (hypoxanthine, xanthine, and uric acid) that reflect excessive metabolism of purine nucleotides were approximately twice higher in untreated retinas. Electroretionographic studies, performed on a different subset of animals, substantiated the biochemical results. In Ga/DFO-treated eyes the amplitude of the mixed cone-rod response b-wave (as compared with fellow nonischemic eyes) fully recovered within 24 h after ischemia (b-wave ratio 1.04 +/- 0.09, [mean +/- SEM]) whereas ischemic/reperfused and nontreated eyes recovered to only 0.33 +/- 0. 05. The results show that severe biochemical and functional retinal injury occurs in cat eyes subjected to ischemia and reperfusion. These severe changes were significantly reduced by a single administration of Ga/DFO just before reperfusion. We hypothesize that the protection afforded by Ga/DFO is due to a combined effect of "Push-Pull" mechanisms interfering with transition metal-dependent and free radical-mediated injurious processes.     

Mutations in C8orf37, encoding a ciliary protein, are associated with autosomal-recessive retinal dystrophies with early macular involvement

Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166^∗]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156−2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156−2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.     

Proline-Rich Peptide from the Coral Pathogen Vibrio shiloi That Inhibits Photosynthesis of Zooxanthellae

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29°C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH₄Cl, pure natural or synthetic toxin P (10 μM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH₄Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH₄Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH₃ into the cell. It is known that uptake of NH₃ into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.     

Topographic features of the organization of lymphatic capillaries and lipid resorption in the jejunal villi of the white rat

As demonstrate serial semithin sections and transmissive electron microscopy, there is not one but a group (2-7) of lymphatic capillaries with anastomoses between them in the villus of the white rat jejunum. In the superior parts of the villus the lumen in the lymphatic capillaries is maximal, and their distance to the epithelial basal membrane of the anterior and posterior surfaces is small. In the inferior part of the villus, when the size of the lumen in the lymphatic microvessels is minimal, the greatest distance between them and the basal membrane of epithelium covering the mentioned surfaces of the villus is noted. In the superior and middle parts of the villus paracellular transport of lipids from the interstitial space into the lumen of the lymphatic capillaries predominate, in the inferior part--transcellular is the main way of transport. The topographic peculiarities of the lymphatic microvessels in the superior and middle parts of the villus make, in combination with the active paracellular transport, the morphological basis of a more intensive absorbtion of lipids from the intestinal lumen.     

CXCL12 chemokine and CXCR4 receptor: association with susceptibility and prognostic markers in triple negative breast cancer

CXCL12/CXCR4 signaling has been implicated in breast carcinogenesis, and genetic polymorphisms in these molecules have been associated with different types of cancer. The present study analyzed genetic polymorphisms in CXCL12 (rs1801157, G?>?A) and CXCR4 (rs2228014, C?>?T) and CXCR4 immunostaining in tumor tissues from patients with triple negative breast cancer (TNBC) aiming to evaluate their possible role in its’ susceptibility and prognosis. Genetic polymorphisms were analyzed in 59 TNBC patients and 150 control women; age-adjusted logistic regression showed no association when variants were considered in isolation; however, a statistically significant interaction was noted for heterozygosis for both allelic variants increasing the odds for TNBC (CXCL12-GA by CXCR4-CT: OR 7.23; 95% CI 1.15–45.41; p?=?0.035). CXCL12 polymorphism was correlated negatively with proliferation index (Ki67) (Tau-b?=???0.406; p?=?0.006). CXCR4 immunostaining was evaluated in 37 TNBC patients (22 with paired tumor-normal adjacent tissue). CXCR4 was detected more intensely in cell cytoplasm than in membrane, and was more expressed in tumor than in normal adjacent tissues, although not statistically significant. CXCR4 expression on the membrane of tumor cells was correlated positively with histopathological grade (Tau-b?=?0.271; p?=?0.036) and negatively with lymph node metastasis (Tau-b?=???0.478; p?=?0.036). The present study indicates that CXCL12 and CXCR4 polymorphisms and CXCR4 immunostaining might have susceptibility and prognostic roles in TNBC pathogenesis.     

New Family of Ulvan Lyases Identified in Three Isolates from the Alteromonadales Order

Ulvan is the main polysaccharide component of the Ulvales (green seaweed) cell wall. It is composed of disaccharide building blocks comprising 3-sulfated rhamnose linked to d-glucuronic acid (GlcUA), l-iduronic acid (IdoUA), or d-xylose (Xyl). The degradation of ulvan requires ulvan lyase, which catalyzes the endolytic cleavage of the glycoside bond between 3-sulfated rhamnose and uronic acid according to a β-elimination mechanism. The first characterized ulvan lyase was identified in Nonlabens ulvanivorans, an ulvanolytic bacterial isolate. In the current study, we have identified and biochemically characterized novel ulvan lyases from three Alteromonadales isolated bacteria. Two homologous ulvan lyases (long and short) were found in each of the bacterial genomes. The protein sequences have no homology to the previously reported ulvan lyases and therefore are the first representatives of a new family of polysaccharide lyases. The enzymes were heterologously expressed in Escherichia coli to determine their mode of action. The heterologous expressed enzymes were secreted into the milieu subsequent to their signal sequence cleavage. An endolytic mode of action was observed and studied using gel permeation chromatography and ¹H NMR. In contrast to N. ulvanivorans ulvan lyase, cleavage occurred specifically at the GlcUA residues. In light of the genomic context and modular structure of the ulvan lyase families identified to date, we propose that two ulvan degradation pathways evolved independently.     

Soil and water and its relationship to the origin of life

Soils of the terrestrial planets form at the boundaries between lithosphere, atmosphere and hydrosphere. Biogenesis occurred in these zones; thus, it is axiomatic that some, perhaps many, stages of biogenesis occurred in intimate association with the mineral constituents of soils. Because of a high surface to mass ratio and, consequently, a high surface reactivity, the layer lattice clay minerals are the most important of these. according to the geological record, clay minerals appeared very early on the primordial Earth. Recent investigations have confirmed their presence in carbonaceous meteorites and have indicated their occurrence on Mars. In this paper we collect pertinent physico-chemical data and summarize the organic reactions and interactions that are induced or catalyzed by clays. Many clay-organic reactions that do not occur readily at high water contents proceed rapidly at adsorbed water contents corresponding to surface coverages of one or two molecular layers. One or two monolayers of adsorbed water correspond to extremely dry on cold planetary environments. Some consequences of these factsvis à vis biogenesis on Mars are considered.