Green mutant cells of sycamore (Acer pseudoplatanus L.), which had been selected by mutagenic treatment of the white wild type, grow photoheterotrophically in auxin-depleted culture medium. In contrast to the wild-type cells, mutant cells exhibit photosynthetic O(2)-evolution activity during their growth coincident with increases of (a) chlorophyll, (b) protein, and (c) ribulose-1,5-bisphosphate (RuBP) carboxylase activity. Functionally competent chloroplasts were isolated from the green cells. Mechanism(s) governing gene expression of amyloplast DNA in the heterotrophically grown white cells were compared with those of the chloroplast DNA isolated from the mutant cells. We have demonstrated in both amyloplast and chloroplast DNAs the presence of sequences homologous to the maize chloroplast genes for photosynthesis, including the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)(rbcL), the 32 kDa Q(B) protein (PG32) (psbA), the apoprotein of P700 (psaA) and subunits of CF(1) (atpA, atpB, and atpE). However, employing either enzyme assays or immunological techniques, RuBisCO and CF(1) cannot be detected in the white wild type cells. Northern blot hybridization of the RNA from the white cells showed high levels of transcripts for the 16S rRNA gene and low level of transcripts for psbA; based on comparison with results obtained using the green mutant cells, we propose that the amyloplast genome is mostly inactive except for the 16S rRNA gene and psbA which is presumably regulated at the transcriptional level.

We have studied the relationship between the cytokine production induced in vivo by prolonged isometric exercise and the symptom complex marked by fatigue in patients with chronic fatigue syndrome (CFS). Twelve male patients and 13 matched male control subjects undertook an isometric hand-grip exercise protocol utilizing dynamometers. Subjects undertook 30 minutes of exercise, for which the target force was set at 40% of the maximal voluntary contraction and the duty cycle was 50%. Prior to, during, and for 24 hours following the exercise, blood samples were collected and assayed for the presence of cytokines, including interferon-gamma and interferon-alpha, interleukin-1 beta, and tumor necrosis factor-alpha. At those times subjects also completed the Profile of Mood States (POMS) questionnaire, which served as a measure of changes in subjective fatigue. No significant alteration in the level of any of the cytokines in the plasma of patients or control subjects was detected before, during, or after exercise. Surprisingly, the patients' levels of fatigue, depression, and confusion, as measured by the POMS, decreased in response to the exercise. These data do not confirm the presence of an immunologic process correlating with the exacerbation of fatigue after exercise experienced by patients with CFS. Limitations in the study design and in the sensitivity of the cytokine assays may have affected our results.

OBJECTIVE

Vascular endothelial growth factor (VEGF) is the prime regulator of angiogenesis and vascular permeability and its serum levels increase in cystic fibrosis (CF). The mechanisms of VEGF overproduction and its impact on CF lung pathology and pulmonary vascular permeability during lung transplantation are not fully understood.

METHODS

The expression of VEGF, its receptors, hypoxia inducible factor (HIF)-1alpha, beta, angiopoietins, and endothelial cell marker CD31 were studied in lung biopsies of CF and COPD patients and controls, using real time reverse transcription (RT)-PCR and Western blotting. DNA binding activity of HIF-1 to VEGF-A promoter was assessed by electrophoretic mobility shift assay (EMSA) and wet-to-dry lung weight ratios as well as microvascular density (MVD) were determined. Serum VEGF-A concentrations in enzyme-linked immunosorbent assay (ELISA) and wet-to-dry weight ratios of donor lungs were monitored during transplantation in CF and COPD patients. Primary graft dysfunction (PGD) was diagnosed and graded according to the guidelines of the International Society for Heart and Lung Transplantation.

RESULTS

VEGF-A165 and Flt-1 mRNA expression (P<0.05), VEGF-A (P<0.05), and HIF-1alpha (P<0.05) protein levels, DNA binding activity of HIF-1 to VEGF promoter (P<0.001) and extravascular lung water content (P<0.05) were increased in CF lungs versus controls, whereas MVD was unchanged. Before and during lung transplantation, VEGF-A serum concentrations were higher in CF versus COPD patients (P<0.05) and 60 min following reperfusion donor lungs transplanted to CF patients had higher tissue water contents than in COPD patients (P<0.05). PGD grade 3 occurred more frequently in CF (22.7%) versus COPD patients (4%). PGD grade 3 patients had significantly higher VEGF serum concentrations versus PGD grade 0-2 patients (P<0.001).

CONCLUSIONS

These data indicate that upregulated VEGF-A levels are most likely induced by enhanced HIF-1 binding to VEGF-A promoter, possibly contributing to elevated serum VEGF-A levels in CF. Furthermore, CF patients undergoing lung transplantation are possibly more susceptible to PGD because of increased VEGF-A expression that mediates increased lung graft vascular permeability.

Administration of recombinant adenoviral (AdV) vectors to animals can lead to inflammatory and immune responses. For therapeutic indications in which repeated treatment is necessary, such as cystic fibrosis (CF), these responses can limit the therapeutic usefulness of the vector. In principle, the utility of the vector can be improved by increasing its therapeutic index, that is, by either increasing its efficacy or decreasing its toxicity. A strategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given level of transgene expression, and thereby also reduce unwanted effects such as immune responses. Following up on our observation that treating polarized normal human bronchial epithelial cells with calcium (Ca(2+))-free medium or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) significantly enhanced the subsequent transfection of these cells with cationic lipid:pDNA complexes, we have now asked whether such a treatment protocol might also improve the ability of AdV to infect these cells. Treating polarized airway epithelial cells with EGTA led to a dramatic increase in AdV-mediated transduction, as demonstrated by an approximately 50-fold increase in transgene expression. This strategy was also tested in vivo and resulted in substantial increases (up to 50-fold) in the ability of AdV vectors to infect mouse tracheal epithelium. Transfection of mouse trachea with an AdV aerosol was also significantly increased by pretreatment with EGTA. The enhancing effects of EGTA could not be duplicated with hypo- or hyperosmotic treatments. Light microscopy of mouse trachea that had been EGTA treated and then infected with AdV demonstrated an EGTA-mediated AdV infection of airway epithelial cells. The apparent enhanced potency of AdV for airway cells resulting from this strategy provides a significant increase in the therapeutic index of this gene delivery vector, and may increase the likelihood that it can be used for clinical indications requiring chronic administration of the vector.

BACKGROUND

High-dose ibuprofen (HDI) is a clinically beneficial anti-inflammatory regimen that may be a useful reagent to study induced sputum inflammatory marker changes over short study periods appropriate for early-phase CF clinical trials.

METHODS

We conducted a 28-day, open-label, randomized, controlled trial among 72 clinically stable CF subjects (FEV1≥40% predicted) randomized to HDI or routine care that assessed IL-6, IL-8, TNF-α, IL-1-β, free neutrophil elastase, and white cell counts with differentials change from baseline in induced sputum.

RESULTS

IL-6 was the only biomarker with significant within-group change: 0.13 log10 pg/mL mean reduction among ibuprofen-treated subjects (p=0.04); and no change in the control group. IL-6 change between groups was statistically significant (p=0.024). No other inflammatory biomarker differences were observed between groups after 28 days.

CONCLUSIONS

Although we studied only one agent, HDI, these results suggest that one month may be inadequate to assess anti-inflammatory candidates using markers from induced sputum.

We have used monoclonal antibodies to study the expression of calgranulins by keratinocytes in inflammatory dermatoses. Calgranulins are intracellular calcium binding proteins which have inflammatory cytokine activity and are composed of at least two different chains, calgranulin A and B. Antibody CF 145 and CF 557 identify calgranulin A and B, respectively. MAC 387 recognizes a molecule probably containing both calgranulins. Keratinocytes in normal skin did not contain these molecules. The keratinocytes in 52 cases of different inflammatory dermatoses showed expression of both calgranulin chains in lesional but not in non-lesional skin. Keratinocytes in inflammatory dermatoses therefore express an intracellular calcium binding protein which has cytokine activity.

Phylogenetic inference regarding the biogeography and evolution of the family Cobitidae depends in large part on the correct interpretation of transitions between the morphological states of secondary sexual characters (e.g., the scale of Canestrini or lamina circularis). Here, we use the complete mitochondrial ATP synthase 8 and 6 and cytochrome b genes to provide an independent assessment of systematics and biogeographic relationships of species in the genus Cobitis, including geographic and subgeneric sampling of species with Canestrini's scale present, duplicated, or absent. The mtDNA-based phylogeny for the genus Cobitis provides the first formal hypothesis for the group and permits a phylogenetic-based assessment of the morphological transitions demonstrated by Canestrini's scale. Our data confirm the monophyly of the genus Cobitis and indicate that European Cobitis comprise six evolutionarily independent lineages. These lineages were defined by nucleotide synapomorphies permitting bootstrapped confidence estimates of 95% or greater and mtDNA genetic distances greater than 4.5% and correspond with moderate fidelity to the Cobitis groups defined by Bacescu (1962, Rev. Roum. Biol. 4, 435-448). The Caucasian lineage, C. cf. sibirica, represents the basal sister species of the genus Cobitis, supporting an eastern Asiatic origin of the European Cobitis: Cobitis sensu stricto, Acanestrinia, Bicanestrinia, Iberocobitis, and Cobitis calderoni. Phylogenetic relationships among Cobitis subgenera and species indicate that the ancestral condition of one scale of Canestrini was duplicated once at the origin of the Bicanestrinia lineage and has been independently lost by C. calderoni and C. elongata. The absence of the scale of Canestrini is the synapomorphy defining the subgenus Acanestrinia, but the mtDNA phylogeny indicates that Acanestrinia is not a natural group and places C. calderoni as the sister lineage to the subgenus Iberocobitis, a finding that is also geographically parsimonius.

Forty Klebsormidium strains isolated from soil crusts of mountain regions (Alps, 600–3,000 m elevation) were analyzed. The molecular phylogeny (internal transcribed spacer rDNA sequences) showed that these strains belong to clades B/C, D, E, and F. Seven main (K. flaccidum, K. elegans, K. crenulatum, K. dissectum, K. nitens, K. subtile, and K. fluitans) and four transitional morphotypes (K. cf. flaccidum, K. cf. nitens, K. cf. subtile, and K. cf. fluitans) were identified. Most strains belong to clade E, which includes isolates that prefer humid conditions. One representative of the xerophytic lineage (clade F) as well as few isolates characteristic of temperate conditions (clades B/C, D) were found. Most strains of clade E were isolated from low/middle elevations (<1,800 m above sea level; a.s.l.) in the pine-forest zone. Strains of clades B/C, D, and F occurred sporadically at higher elevations (1,548–2,843 m a.s.l.), mostly under xerophytic conditions of alpine meadows. Comparison of the alpine Klebsormidium assemblage with data from other biogeographic regions indicated similarity with soil crusts/biofilms from terrestrial habitats in mixed forest in Western Europe, North America, and Asia, as well as walls of buildings in Western European cities. The alpine assemblage differed substantially from crusts from granite outcrops and sand dunes in Eastern Europe (Ukraine), and fundamentally from soil crusts in South African drylands. Epitypification of the known species K. flaccidum, K. crenulatum, K. subtile, K. nitens, K. dissectum, K. fluitans, K. mucosum, and K. elegans is proposed to establish taxonomic names and type material as an aid for practical studies on these algae, as well as for unambiguous identification of alpine strains. New combination Klebsormidium subtile (Kützing) Mikhailyuk, Glaser, Holzinger et Karsten comb. nov. is made.

We evaluated the cardioprotection against myocardial ischemia-reperfusion injury induced by sevoflurane postconditioning (SpostC) in chronically-infarcted rat hearts, and investigated the roles of phosphoinositide 3-kinase (PI3K)-protein kinase B/Akt (PKB/Akt), mitogen-activated extracellular regulated kinase 1/2 (MEK 1/2)-extracellular regulated kinase 1/2 (ERK 1/2), and mitochondrial permeability transition pore (mPTP). Left anterior descending (LAD) coronary artery was ligated to induce myocardial infarction in rats. Six weeks later, chronically-infarcted hearts were isolated and subjected to 30 min of global ischemia, followed by 1 h of reperfusion with Krebs-Henseleit (K-H) buffer. SpostC was administered by perfusing the hearts with K-H buffer saturated with 3% sevoflurane during the first 15 min of reperfusion. To evaluate the role of PI3K-PKB/Akt and MEK 1/2-ERK 1/2 in SpostC, PI3K inhibitor LY294002 (15 microM) and MEK 1/2 inhibitor PD98059 (20 microM) were administered alone or together with sevoflurane during the first 15 min of reperfusion. We found that exposure of 3% sevoflurane during early reperfusion significantly improved functional recovery (improved left ventricular developed pressure (LVDP), +/-dp/dt, CF, HR and reduced left ventricular end-diastolic pressure (LVEDP)), decreased myocardial infarct size and reduced LDH and CK-MB release, when compared with unprotected hearts. However, these protective effects were abolished in the presence of either LY294002 or PD98059, which was accompanied by the prevention of PKB/Akt and ERK 1/2 phosphorylation, and reduction of myocardial nicotinamide adenine dinucleotide (NAD+) content. These findings suggest that sevoflurane postconditioning protects chronically-infarcted rat hearts against ischemia-reperfusion injury by inhibiting mPTP opening via recruitment of PKB/Akt and ERK 1/2.

Burkholderia cepacia, a major pathogen amongst individuals with cystic fibrosis (CF), is intrinsically resistant to most clinically available antibiotics. We report the identification of an immunodominant antigen in CF patients infected with B. cepacia, a multidrug-resistance efflux pump called BcrA. The bcrA gene encodes a 46 kDa peptide with 14 potential alpha-helices that belongs to the major facilitator superfamily of drug transporters. A recombinant Escherichia coli strain was constructed containing the bcrA gene, which resulted in a four-fold increase in resistance to tetracycline and an eight-fold increase in resistance to nalidixic acid. These results demonstrate that the bcrA gene is part of a drug efflux system that is potentially a major contributor to the high-level antibiotic resistance observed in B. cepacia and thus a potential target for novel therapeutics.

1. We have studied the physiology of sensory neurons innervating skin of the rat hindlimb, in three groups of animals: 1) normal animals; 2) animals in which the sural nerve (Sn) had regenerated to its original cutaneous target; and 3) animals in which the gastrocnemius muscle nerve (Gn) had previously been cut and cross anastomosed with the distal stump of the cut Sn so that its axons regenerated to a foreign target, skin. 2. Single-unit recordings were made from 222 afferents in normal, intact animals. They had conduction velocities of 0.5-53.1 m/s. The conduction velocity distribution had distinct peaks at approximately 37.5, 2.5, and 1.25 m/s, presumably corresponding to A alpha beta-, A delta-, and C-fiber populations. Eighty-two percent of the characterized myelinated fibers had low-threshold mechanosensitive receptive fields, whereas 16% were high threshold, and only 2% appeared to have no receptive field. The very large majority of low-threshold mechanosensitive receptive fields (87%) were rapidly adapting hair follicle afferents. 3. In animals with regenerated Sn, 308 afferents were recorded with conduction velocities of 0.4-58.8 m/s. However, the mean conduction velocity was lower than in control animals (P less than 0.05), and only one peak, at 27.5 m/s, was apparent for myelinated fibers. Eighty-six percent of myelinated fibers were low-threshold mechanosensitive afferents, 8.5% were high-threshold mechanoreceptors (HTMRs), and 5.5% appeared to have no receptive fields. Fewer low-threshold mechanoreceptors (LTMRs; compared with controls) were activated by hair movement (63 vs. 87%). Most of the remainder appeared to be field receptors (which were therefore more commonly observed here than in normal animals). 4. In animals in which the Gn had regenerated to skin, 430 afferents were recorded. These had conduction velocities ranging from 0.6 to 71.4 m/s, and again only one peak was apparent in the myelinated conduction velocity histogram, at approximately 17.5 m/s. Of the myelinated fibers, 79% had low-threshold mechanosensitive receptive fields in skin and 10% high-threshold mechanosensitive receptive fields. The remaining 11% apparently had no receptive field (cf. 5.5% in regenerated Sn). In contrast to normal or regrown sural afferents, only 58% of low-threshold gastrocnemius afferents in skin were rapidly adapting. Of the 42% slowly adapting afferents, many surprisingly responded to hair movement. Thus some gastrocnemius afferents seemed to have retained the adaptation properties characteristic of muscle afferents. Also surprisingly, given that the Gn contains fewer fibers than the Sn, receptive-field areas were not significantly different from regrown or normal sural fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

<A<em>b</em>stractText>Progressive lung injury in Cystic Fi<em>b</em>rosis (<em>CF</em>) patients can lead to chronic colonization with <em>b</em>acteria and fungi. Fungal colonization is o<em>b</em>tained from the environment which necessitates locally performed epidemiology studies. We prospectively analyzed respiratory samples of <em>CF</em> patients during a 3-year period, using a uniform fungal culture protocol, focusing on filamentous fungi and azole resistance in Aspergillus fumigatus.</A<em>b</em>stractText><A<em>b</em>stractText>Over a 3-year period, all respiratory specimens collected from <em>CF</em> patients in 5 Dutch <em>CF</em> centers, were analyzed. Samples were inoculated onto the fungal culture media Sa<em>b</em>ouraud dextrose agar (SDA) and Medium <em>B</em>+. All fungal isolates were collected and identified in one centre, using Amplified Fragment Length Polymorphism (AFLP) fingerprinting, rDNA PCR and ITS, calmodulin and <em>β</em>-tu<em>b</em>ulin sequencing. Azole resistance was assessed for all A. fumigatus using a qPCR assay followed <em>b</em>y phenotypic confirmation.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Filamentous fungi were recovered from 699 patients from at least one respiratory sample, corresponding with 3787 cultured fungal species. A. fumigatus was cultured most often with a mean prevalence of 31.7%, followed <em>b</em>y Penicillium species (12.6%), non-fumigatus Aspergillus species (5.6%), Scedosporium species (4.5%) and Exophiala dermatitidis and Cladosporium species (1.1% each). In total 107 different fungal species were identified, with 39 Penicillium species and 15 Aspergillus species. Azole resistance frequency in A. fumigatus was 7.1%, with TR<su<em>b</em>)34</su<em>b</em>)/L98H <em>b</em>eing the dominant resistance mechanism.</p><A<em>b</em>stractText>A vast diversity of filamentous fungi was demonstrated, dominated <em>b</em>y Aspergillus and Penicillium species. We o<em>b</em>served a mean azole resistance prevalence of 7.1% of A. fumigatus culture positive patients.</A<em>b</em>stractText>

BACKGROUND

New designs and alloys have been developed to increase cyclic fatigue (CF) resistance of rotary files. The aim of this study was to compare CF resistance of ProTaper Universal (PTU; Dentsply Tulsa Dental, Tulsa, OK) and ProTaper Next (PTN, Dentsply Tulsa Dental) instruments at different points of curvature.

METHODS

A total of 420 files (240 PTU, S1, F1, F2, and F3 and 180 PTN, X1, X2, and X3) were divided in 14 groups of 30 instruments each. Instruments in groups S1-5, F1-5, X1-5, F2-5, X2-5, F3-5, and X3-5 were tested at 5 mm from the tip. Groups S1-12, X1-12, and F1-12 were tested at 12 mm from the tip because S1, X1, and F1 instruments have the same diameter at that level. Groups F2-8, X2-8, F3-8, and X3-8 were tested at 8 mm (F2/X2 and F3/X3, respectively, had the same diameter at 8 mm). All files were rotated at 300 rpm until fracture. CF resistance was tested in stainless steel curved canals (60°, r = 3 mm). Time to fracture was recorded. The mean half-life and beta and eta were calculated for each group and were compared with Weibull analysis.

RESULTS

PTN instruments will last significantly longer than PTU files with a probability higher than 98% at all tested levels except for S1, which was the significantly the most resistant instrument to CF at 5 mm from the tip.

CONCLUSIONS

PTU S1 was significantly the most resistant instrument at 5 mm from the tip. PTN files were significantly more resistant to CF than PTU instruments at all the other tested levels.

BACKGROUND

New designs and alloys and different motions have been introduced to increase the cyclic fatigue (CF) resistance of nickel-titanium (NiTi) files. The aim of this study was to compare the CF resistance of K3 (SybronEndo, Orange, CA), K3XF (SybronEndo), and TF (SybronEndo) files under continuous rotation and reciprocating motion.

METHODS

A total of 210 files (30-tip diameter, 0.06 fixed taper), 60 K3, 60 K3XF, and 90 TF files, were divided into 7 groups (30 files each): K3-C, K3XF-C, and TF1-C were rotated at 300 rpm; TF2-C was rotated at 500 rpm; and K3-R, K3XF-R, and TF1-R were used in a reciprocating motion. CF resistance was tested in stainless steel, curved canals (60°, r = 3 mm) until fracture, and the time to fracture was recorded. The mean half-life, beta, and eta were calculated for each group and were compared with Weibull analysis.

RESULTS

The probability of a longer mean life was greater under reciprocating motion for all of the files (100% for K3, 87% for K3XF, and 99% for TF). Under continuous rotation, K3XF was more resistant than K3 and TF. TF lasted significantly longer than K3. TF was more resistant to CF when rotated at 300 rpm instead of 500 rpm. Under reciprocating motion, there were no significant differences between K3XF and TF mean lives, but both were significantly longer than the K3 mean life (78% for TF and 86% for K3XF).

CONCLUSIONS

Reciprocating motion and R-phase increase CF resistance.

Infection-triggered disease onset, chronic immune activation and autonomic dysregulation in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) point to an autoimmune disease directed against neurotransmitter receptors. We had observed elevated autoantibodies against ß2 adrenergic receptors, and muscarinic 3 and 4 acetylcholine receptors in a subset of patients. Immunoadsorption (IA) was shown to be effective in removing autoantibodies and improve outcome in various autoimmune diseases.

10 patients with post-infectious CFS/ME and elevated ß2 autoantibodies were treated with IA with an IgG-binding column for 5 days. We assessed severity of symptoms as outcome parameter by disease specific scores. Antibodies were determined by ELISA and B cell phenotype by flow cytometry.

IgG levels dropped to median 0.73 g/l (normal 7-16 g/l) after the 4th cycle of IA, while IgA and IgM levels remained unchanged. Similarly, elevated ß2 IgG antibodies rapidly decreased during IA in 9 of 10 patients. Also 6 months later ß2 autoantibodies were significantly lower compared to pretreatment. Frequency of memory B cells significantly decreased and frequency of plasma cells increased after the 4th IA cycle. A rapid improvement of symptoms was reported by 7 patients during the IA. 3 of these patients had long lasting moderate to marked improvement for 6-12+ months, 2 patients had short improvement only and 2 patients improved for several months following initial worsening.

IA can remove autoantibodies against ß2 adrenergic receptor and lead to clinical improvement. B cell phenotyping provides evidence for an effect of IA on memory B cell development. Data from our pilot trial warrants further studies in CFS/ME.

BACKGROUND

The aim of this study is to evaluate the efficacy and safety of medium-dose formoterol-budesonide combined inhaled treatment in a single inhaler compared with high-dose budesonide treatment in patients with non-cystic fibrosis (non-CF) bronchiectasis.

METHODS

This is a 12-month randomized, double-blind, parallel-groups clinical trial, to run in 40 patients with non-CF bronchiectasis diagnosed by high-resolution CT scan of the chest, receiving formoterol-budesonide combined treatment (18/640 μg daily) or budesonide treatment (1,600 μg daily). Variables concerning clinical condition, health-related quality of life (HRQL), lung function, β(2)-adrenergic agonist use, potentially pathogenic microorganism (PPM) isolates, and medication side effects were analyzed by intention-to-treat analysis.

RESULTS

The study group receiving a formoterol-budesonide combined treatment showed a significant improvement, both clinically and statistically, of symptoms (dyspnea, number of coughs, and rescue β(2)-adrenergic agonist-free days). Furthermore, we observed an HRQL improvement, with no changes in functional parameters or in PPM isolates, together with an important reduction in overall side effects, especially for those related to inhaled steroids, compared with the high-dose budesonide treatment group.

CONCLUSIONS

Inhaled medium-dose formoterol-budesonide combined treatment in a single inhaler is more effective and safe compared with high-dose budesonide treatment in patients with non-CF bronchiectasis.

BACKGROUND

ClinicalTrials.gov; No.: NCT00728715; URL: www.clinicaltrials.gov.

Cytotoxicity-directed purification of a Symploca cf. hydnoides sample from Cetti Bay, Guam, afforded seven new cyclic depsipeptides, veraguamides A-G (1-7), together with the known compound dolastatin 16. The planar structures of 1-7 were elucidated using NMR and MS experiments, while enantioselective HPLC and Mosher's analysis of acid and base hydrolysates, respectively, were utilized to assign the absolute configurations of the stereocenters. Veraguamides A-G (1-7) are characterized by the presence of an invariant proline residue, multiple N-methylated amino acids, an α-hydroxy acid, and a C8-polyketide-derived β-hydroxy acid moiety with a characteristic terminus as either an alkynyl bromide, alkyne, or vinyl group. These compounds and a semisynthetic analogue (8) showed moderate to weak cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cell lines. Preliminary structure-activity relationship analysis identified several sensitive positions in the veraguamide scaffold that affect the cytotoxic activity of this compound class. Dolastatin 16 showed only weak cytotoxic activity on both cell lines tested. The complete stereostructure of dolastatin 16 was proposed for the first time through degradation followed by a combination of advanced Marfey's analysis and modified Mosher's analysis using phenylglycine methyl ester as a chiral anisotropic reagent.

Bacteria of the Burkholderia cepacia complex (Bcc) are pathogens of humans, plants, and animals. Burkholderia cenocepacia is one of the most common Bcc species infecting cystic fibrosis (CF) patients and its carriage is associated with poor prognosis. In this study, we characterized a general O-linked protein glycosylation system in B. cenocepacia K56-2. The PglLBc O-oligosaccharyltransferase (O-OTase), encoded by the cloned gene bcal0960, was shown to be capable of transferring a heptasaccharide from the Campylobacter jejuni N-glycosylation system to a Neisseria meningitides-derived acceptor protein in an Escherichia coli background, indicating that the enzyme has relaxed specificities for both the sugar donor and protein acceptor. In B cenocepacia K56-2, PglLBc is responsible for the glycosylation of 23 proteins involved in diverse cellular processes. Mass spectrometry analysis revealed that these proteins are modified with a trisaccharide HexNAc-HexNAc-Hex, which is unrelated to the O-antigen biosynthetic process. The glycosylation sites that were identified existed within regions of low complexity, rich in serine, alanine, and proline. Disruption of bcal0960 abolished glycosylation and resulted in reduced swimming motility and attenuated virulence towards both plant and insect model organisms. This study demonstrates the first example of post-translational modification in Bcc with implications for pathogenesis.

In the liver, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) regulates bile secretion and other functions at the apical membrane of biliary epithelial cells (i.e., cholangiocytes). CF-related liver disease is a major cause of death in patients with CF. CFTR dysfunction affects innate immune pathways, generating a para-inflammatory status in the liver and other epithelia. This study investigates the mechanisms linking CFTR to toll-like receptor 4 activity. We found that CFTR is associated with a multiprotein complex at the apical membrane of normal mouse cholangiocytes, with proteins that negatively control Rous sarcoma oncogene cellular homolog (Src) activity. In CFTR-defective cholangiocytes, Src tyrosine kinase self-activates and phosphorylates toll-like receptor 4, resulting in activation of nuclear factor kappa-light-chain-enhancer of activated B cells and increased proinflammatory cytokine production in response to endotoxins. This Src/nuclear factor kappa-light-chain-enhancer of activated B cells-dependent inflammatory process attracts inflammatory cells but also generates changes in the apical junctional complex and loss of epithelial barrier function. Inhibition of Src decreased the inflammatory response of CF cholangiocytes to lipopolysaccharide, rescued the junctional defect in vitro, and significantly attenuated endotoxin-induced biliary damage and inflammation in vivo (Cftr knockout mice).

These findings reveal a novel function of CFTR as a regulator of toll-like receptor 4 responses and cell polarity in biliary epithelial cells; this mechanism is pathogenetic, as shown by the protective effects of Src inhibition in vivo, and may be a novel therapeutic target in CF-related liver disease and other inflammatory cholangiopathies. (Hepatology 2016;64:2118-2134).

BACKGROUND

Large artery stiffening is increased in advanced chronic kidney disease (CKD) but likely develops progressively in earlier stages of CKD. Active matrix Gla-protein (MGP) is a potent vitamin K-dependent inhibitor of vascular calcification. A recent animal model demonstrated intrinsic abnormalities in vitamin K metabolism even in early CKD, but whether early human CKD is associated with vascular vitamin K deficiency is unknown.

METHODS

We enrolled 137 adults without HF with varying degrees of renal function: normal estimated glomerular filtration rate (eGFR; >90 ml/min; n = 59), mildly reduced eGFR (stage 2 CKD: eGFR = 60-89 ml/min; n = 53) or at least moderately reduced eGFR (stage 3-5 CKD; eGFR < 60 ml/min; n = 25). Carotid-femoral pulse wave velocity (CF-PWV) was measured with carotid and femoral tonometry. Dephospho-uncarboxylated matrix gla-protein (dp-ucMGP) was measured with enzyme-linked immunosorbent assay (ELISA) (VitaK; Maastricht University; The Netherlands).

RESULTS

Dp-ucMGP levels were progressively increased with decreasing renal function (eGFR ≥ 90: 247 pmol/l; eGFR 60-89: 488 pmol/l; eGFR < 60: 953 pmol/l; P < 0.0001). These differences persisted after adjustment for multiple potential confounders (eGFR ≥ 90: 314 pmol/l; eGFR 60-89: 414 pmol/l; eGFR < 60: 770 pmol/l; P < 0.0001). In a multivariable model adjusted for various confounders, dp-ucMGP was a significant independent predictor of CF-PWV (β = 0.21; P = 0.019). In formal mediation analyses, dp-ucMGP mediated a significant relationship between eGFR and higher CF-PWV (β = -0.16; P = 0.005), whereas no significant dp-ucMGP-independent relationship was present (β = -0.02; P = 0.80).

CONCLUSIONS

CKD is associated with increased (inactive) dp-ucMGP, a vitamin K-dependent inhibitor of vascular calcification, which correlates with large artery stiffness. Further studies are needed to assess whether vitamin K2 supplementation represents a suitable therapeutic strategy to prevent or reduce arterial stiffening in CKD.

Burkholderia cepacia complex strains are genetically related but phenotypically diverse organisms that are important opportunistic pathogens in patients with cystic fibrosis (CF,) as well as pathogens of onion and banana, colonizers of the rhizospheres of many plant species, and common inhabitants of bulk soil. Genotypic identification and pathogenicity characterization were performed on B. cepacia complex isolates from the rhizosphere of onion and organic soils in Michigan. A total of 3,798 putative B. cepacia complex isolates were recovered on Pseudomonas cepacia azelaic acid tryptamine and trypan blue tetracycline semiselective media during the 2004 growing season from six commercial onion fields located in two counties in Michigan. Putative B. cepacia complex isolates were identified by hybridization to a 16S rRNA gene probe, followed by duplex PCR using primers targeted to the 16S rRNA gene and recA sequences and restriction fragment length polymorphism analysis of the recA sequence. A total of 1,290 isolates, 980 rhizosphere and 310 soil isolates, were assigned to the species B. cepacia (160), B. cenocepacia (480), B. ambifaria (623), and B. pyrrocinia (27). The majority of isolates identified as B. cepacia (85%), B. cenocepacia (90%), and B. ambifaria (76%) were pathogenic in a detached onion bulb scale assay and caused symptoms of water soaking, maceration, and/or necrosis. A phylogenetic analysis of recA sequences from representative B. cepacia complex type and panel strains, along with isolates collected in this study, revealed that the B. cenocepacia isolates associated with onion grouped within the III-B lineage and that some strains were closely related to strain AU1054, which was isolated from a CF patient. This study revealed that multiple B. cepacia complex species colonize the onion rhizosphere and have the potential to cause sour skin rot disease of onion. In addition, the onion rhizosphere is a natural habitat and a potential environmental source of B. cenocepacia.

BACKGROUND

A nationwide multicenter study was performed in Spain to evaluate the clonal diversity and antimicrobial susceptibility of Acinetobacter baumannii clinical isolates.

METHODS

A total of 221 consecutive A. baumannii isolates recovered from clinical samples from 25 Spanish hospitals during November 2000 were studied. Isolate identification was performed by phenotyping methods and by amplified rDNA restriction analysis. Clonal relationships among A. baumannii isolates were determined by pulsed field gel electrophoresis. MICs of amikacin (AK), ampicillin (AP), cephalothin (CF), cefoxitin (FX), ceftazidime (CZ), ciprofloxacin (CP), cotrimoxazole (T/S), doxycycline (DX), gemifloxacin (GX), gentamicin (GN), imipenem (IP), meropenem (MP), minocycline (MI), piperacillin (PP), polymyxin B (PB), rifampicin (RI), tetracycline (TT), sulbactam (SB) and tobramycin (TO) were determined by microdilution (NCCLS guidelines).

RESULTS

Seventy-nine A. baumannii clones were differentiated. MIC50/MIC90 (mg/L) values for the 221 A. baumannii isolates were PP:>> 512>> 512; AP, CF, FX:>> 256>> 256; TT, GN:>> 128>> 128; CZ: 128>> 256; CP:>> 64>> 64; FP: 64/256; AK: 32/256; DX: 32/64; GX:>> 16>> 16; TO: 16/128; SB, T/S: 16/64; MP: 8>> 128; IP: 4/128; RF: 4/8; MI: 2/16 and PB: 1/2. Percentages of susceptible isolates were PB: 100%; MI: 65.8%; IP: 52.5%, RF: 49.3%; SB: 46.7%; MP: 43.1%; AK: 34.7%; DX: 32.0%; TO: 21.3% and CZ, FP, GN, T/S, TT, GX, CP, AP, PP, CF and FX: < 20%.

CONCLUSIONS

A. baumannii isolates show high clonal variability in Spain. The most active antimicrobial agents against this organism were polymyxin B, minocycline, rifampicin, imipenem, sulbactam, meropenem, amikacin and doxycycline.

Four unsaturated polyketide lactone derivatives, coibacins A-D, were isolated from a Panamanian marine cyanobacterium, cf. Oscillatoria sp. The two different types of termini observed in these co-occurring metabolites, either a methyl cyclopropyl ring as seen in curacin A or a methyl vinyl chloride similar to that observed in the jamaicamides, suggest an intriguing flexibility in the "beta branch" forming biosynthetic process. The coibacins possess selective antileishmanial activity as well as potent anti-inflammatory activity.

Cystic fibrosis (CF) is one of the most common life-shortening genetic disorders, and the CF transmembrane conductance regulator (CFTR) is the major causal gene. However, a substantial clinical variability among patients with identical CFTR genotypes suggests the presence of modifier genes. We tested the effect of four genes involved in Pseudomonas aeruginosa infection. Analysis of a primary cohort detected eight candidate polymorphisms that were genotyped in the secondary cohort of 1579 patients; lung function and age at first infection with P. aeruginosa were considered as the phenotypes. Both additive and codominant models were considered, adjusting for confounding variables but not for multiple comparisons. In the secondary cohort, heme oxygenase-1 (HMOX1) rs2071749 had the most significant effect on lung function in the pediatric group (P=0.01; P(corrected)=0.03), and complement factor 3 (C3) rs11569393 and HMOX1 rs2071746 in the adult groups (P=0.03 for both variants; P(corrected)=0.16, 0.09). No polymorphism of complement factor B (CFB) or toll-like receptor 4 (TLR4) had a significant modifying effect on lung function in either group. We have identified two genes that showed nominal association with disease severity among CF patients. However, because of the multiple comparisons made, further studies are required to confirm the interaction between these modifying genes and CFTR.