Evaluation of Solanum sisymbriifolium as a Potential Inoculum Source of Verticillium dahliae and Colletotrichum coccodes.

Solanum sisymbriifolium, the litchi tomato, is a perennial herbaceous plant from South America that is used as a trap crop to reduce soilborne populations of the pale cyst nematode Globodera pallida, an important potato pathogen. Possible interactions of soilborne potato pathogens Verticillium dahliae and Colletotrichum coccodes with litchi tomato are unknown, yet important for potato production if litchi tomato is to be planted as a trap crop. The goal of this research was to quantitatively assess if litchi tomato is a potential inoculum source for C. coccodes and V. dahliae by comparing colony forming units (CFU) observed in litchi tomato to susceptible and resistant potato cultivars. The potato cvs. Alturas (P = 0.0003), Ranger Russet (P = 0.0193), and Russet Norkotah (P = 0.0022) produced more CFUs of the potato pathotype of V. dahliae than litchi tomato the first of two years of greenhouse trials. Significantly more CFUs of the potato pathotype of V. dahliae were quantified from stems and roots of only cv. Russet Norkotah compared with litchi tomato (P = 0.0001) in the second year. The CFUs for C. coccodes varied between litchi tomato and the potato cvs., perhaps due to varying levels of resistance since litchi tomato is from a selected intermated seed source. Based on these data, the effect of litchi tomato in rotation with potato is likely to have limited effect on the proliferation of V. dahliae or C. coccodes populations in the soil when compared with a susceptible potato cultivar.

Diversity within the genus Elymus (Poaceae: Triticeae) as investigated by the analysis of the nr5S rDNA variation in species with St and H haplomes.

The genus Elymus ("Ryegrass") is a repository for a range of species with a variety of haplome contents; hence the pejorative name "dustbin" genus. We have analyzed 1,059 sequences from 128 accessions representing 24 species to investigate the relationships among the StH haplomes-containing species described by Yen and Yang (Genus Elymus Beijing 5:58-362, 2013). Sequences were assigned to "unit classes" of orthologous sequences and subjected to a suite of analyses including BLAST (Basic Local Alignment Search Tool) searches, phylogenetic analysis and population genetic analysis to estimate species diversity. Our results support the genome analyses in Yen and Yang (Genus Elymus Beijing 5:58-362, 2013), i.e., genomic constitution StStHH including variants restricted to Elymus. Population genetic analysis of the 5S nrDNA sequence data revealed that the within-species variance component is roughly ±89 %; thus, we were unable to identify molecular markers capable to separate the 24 species analyzed. Separate phylogenetic analyses of the two unit classes and of all the data exhibit a trend only of the species to cluster on the phylograms. Finally, the analysis provides evidence for the multiple origins of American and Eurasian species.

Phylogenetic relationships among the polyploid and diploid Aegilops species inferred from the nuclear 5S rDNA sequences (Poaceae: Triticeae).

Phylogenetic inferences of the polyploid Aegilops taxa were drawn based upon the analysis of 909 nuclear 5S rDNA sequences obtained from 15 Aegilops polyploid taxa (531 sequences new to this paper) and 378 sequences from our previous study on the diploid taxa. The 531 sequences can be split into two orthologous groups (unit classes), the long AE1 and short AE1 previously identified in the diploid set. An examination of the relationships between unit classes and their associated haplomes suggests that U haplome sequences found in Ae. umbellulata are the closest to the T sequences found in Amblyopyrum muticum and that sequences of the polyploid species expected to be the M type found in Ae. comos are more similar to the T haplome sequences, except in the three hexaploids Ae. glumiaristata, Ae. juvenalis, and Ae. vavilovii and the tetraploid Ae. crassa where they are found to be similar to the M haplome sequences. These three hexaploid taxa likely originated from the tetraploid Ae. crassa (DM), while the closest taxon to the fourth hexaploid, Ae. recta, is the tetraploid Ae. neglecta (UM). Based upon the distribution of the unit classes, several reticulate phylogenies depicting evolutionary relationships among diploid, tetraploid, and hexaploid taxa were constructed; however, none of these widely used methods could depict the expected reticulate relationship as previously drawn from cytogenetic analyses in this group of allopolyploid species. These results suggest that evolutionary relationships derived from models based upon the assumption of bifurcating species require careful interpretation when these same models are applied to species with reticulate evolution.

Endoglin expression in breast tumor cells suppresses invasion and metastasis and correlates with improved clinical outcome.

Tumor growth factor-ß (TGF-ß) signaling in cancer has been implicated in growth suppression of early lesions and enhancing tumor cell invasion and metastasis. However, the cellular mechanisms that determine this signaling output in individual tumors are still largely unknown. In endothelial cells, TGF-ß signaling is modulated by the TGF-ß co-receptor endoglin (CD105). Here we demonstrate that endoglin is expressed in a subset of invasive breast cancers and cell lines and is subject to epigenetic silencing by gene methylation. Endoglin downregulation in non-tumorigenic MCF10A breast cells leads to the formation of abnormal acini in 3D culture, but does not promote cell migration or transformation. In contrast, in the presence of activated ErbB2, endoglin downregulation in MCF10A cells leads to enhanced invasion into a 3D matrix. Consistent with these data, ectopic expression of endoglin in MDA-MB-231 cells blocks TGF-ß-enhanced cell motility and invasion and reduces lung colonization in an in vivo metastasis model. Unlike endothelial cells, endoglin does not modulate Smad-mediated TGF-ß signaling in breast cells but attenuates the cytoskeletal remodeling to impair cell migration and invasion. Importantly, in a large cohort of invasive breast cancers, lack of endoglin expression in the tumor cell compartment correlates with ENG gene methylation and poor clinical outcome.

First Report of Tobacco etch virus Infection in Coleus in the United States.

Virus-like disease symptoms consisting of foliar and veinal necrosis similar to those caused by Coleus vein necrosis virus (CVNV) (2) were observed in plants of coleus (Coleus blume Benth.) 'Rustic Orange' obtained from retail greenhouse outlets in Missouri and Minnesota. Flexuous, filamentous, 750 to 770 nm virus-like particles (vlps) were observed by transmission electron microscopy in negatively stained partially purified leaf tissue extracts from symptomatic 'Rustic Orange' leaf tissue. No other virus-like particles were observed and none were detected in extracts from asymptomatic leaves. These vlps were longer than those of CVNV (640 nm) (2) and were not detected by immunosorbent electron microscopy (ISEM) using antibodies to CVNV (2). Degenerate potyvirus primers PNIbF1 (5'GGBAAYAATAGTGGNCAACC3') and PCPR1 (5'GGGGAGGTGCCGTTCTCDATRCACCA3') (1) and total RNA extracted from 'Rustic Orange' leaf tissue with a Qiagen RNeasy Kit were used for reverse transcription-PCR with Ready-To-Go RT-PCR Beads (GE Healthcare). A 950-bp amplicon was obtained from total RNA from diseased but not from healthy leaf tissue. The nucleotide sequence of the amplicon (GenBank Accession No. GQ268818) had levels of identity to published Tobacco etch virus (TEV) sequences comprising portions of the nuclear inclusion body (NIb) and coat protein (CP) gene regions ranging from 89% (L38714) to 93% (M15239, M11458). The identity of the virus occurring in 'Rustic Orange' was further confirmed by ISEM. Virions were trapped and decorated by antibodies to TEV (ATCC PVAS 32). Systemically infected leaf tissue from Datura stramonium in which the coleus TEV isolate was propagated was used to mechanically inoculate Carborundum-dusted leaves of virus-free test plants of 'Rustic Orange' (Park Seed, Greenwood, SC). Inoculated plants developed foliar necrosis symptoms similar to those observed originally, and the presence of TEV was confirmed by ISEM and RT-PCR and nucleotide sequence analysis as described above. To our knowledge, this is the first report of a disease of coleus caused by TEV. Many of approximately 30 'Rustic Orange' plants in one nursery in Minnesota showed similar necrotic foliar symptoms and randomly selected plants tested positive for TEV by ISEM. This suggests that TEV infection in this variety may be spread by vegetative propagation from infected stock plants. References: (1) Y.-C. Hsu et al. J. Virol. Methods 128:54. 2005. (2) D. S. Mollov et al. Plant Dis. 91:754. 2007.

Donepezil treatment restores the ability of estradiol to enhance cognitive performance in aged rats: evidence for the cholinergic basis of the critical period hypothesis.

Recent studies suggest that the ability of estradiol to enhance cognitive performance diminishes with age and/or time following loss of ovarian function. We hypothesize that this is due, in part, to a decrease in basal forebrain cholinergic function. This study tested whether donepezil, a cholinesterase inhibitor, could restore estradiol effects on cognitive performance in aged rats that had been ovariectomized as young adults. Rats were ovariectomized at 3 months of age, and then trained on a delayed matching to position (DMP) T-maze task, followed by a configural association (CA) operant condition task, beginning at 12-17 or 22-27 months of age. Three weeks prior to testing, rats started to receive either donepezil or vehicle. After one week, half of each group also began receiving estradiol. Acclimation and testing began seven days later and treatment continued throughout testing. Estradiol alone significantly enhanced DMP acquisition in middle-aged rats, but not in aged rats. Donepezil alone had no effect on DMP acquisition in either age group; however, donepezil treatment restored the ability of estradiol to enhance DMP acquisition in aged rats. This effect was due largely to a reduction in the predisposition to adopt a persistent turn strategy during acquisition. These same treatments did not affect acquisition of the CA task in middle-aged rats, but did have small but significant effects on response time in aged rats. The data are consistent with the idea that estrogen effects on cognitive performance are task specific, and that deficits in basal forebrain cholinergic function are responsible for the loss of estradiol effect on DMP acquisition in aged ovariectomized rats. In addition, the data suggest that enhancing cholinergic function pharmacologically can restore the ability of estradiol to enhance acquisition of the DMP task in very old rats following long periods of hormone deprivation. Whether donepezil has similar restorative effects on other estrogen-sensitive tasks needs to be explored.

Quality in the technical performance of screening flexible sigmoidoscopy: recommendations of an international multi-society task group.

BACKGROUND: Flexible sigmoidoscopy (FS) is a complex technical procedure performed in a variety of settings, by examiners with diverse professional backgrounds, training, and experience. Potential variation in technical quality may have a profound impact on the effectiveness of FS on the early detection and prevention of colorectal cancer. AIM: We propose a set of consensus and evidence based recommendations to assist the development of continuous quality improvement programmes around the delivery of FS for colorectal cancer screening. RECOMMENDATIONS: These recommendations address the intervals between FS examinations, documentation of results, training of endoscopists, decision making around referral for colonoscopy, policies for antibiotic prophylaxis and management of anticoagulation, insertion of the FS endoscope, bowel preparation, complications, the use of non-physicians as FS endoscopists, and FS endoscope reprocessing. For each of these areas, continuous quality improvement targets are recommended, and research questions are proposed.

Endoscopic therapy for gastroesophageal reflux disease: a systematic review.

Endoscopic therapy has emerged as an alternative for patients with symptomatic gastroesophageal reflux disease (GERD) who are seeking a non-pharmacologic, non-surgical intervention. To date, there are 3 basic categories of these endoscopic options: radiofrequency ablation, endoscopic suturing and endoscopic injection. Although the pivotal clinical studies to date have consistently demonstrated efficacy for defined endpoints of symptom control, GERD health related quality of life and discontinuance of acid secretory medications, depending on the therapy, there has been less consistent improvement in objective parameters of pH control and lower esophageal sphincter pressure. Although the data for these procedures has been favorable, extrapolation to justify a more widespread clinical use has been limited by the relative lack of controlled sham studies-although these are currently underway for all of the available therapies. Appropriate patient selection for endoscopic GERD therapy is critical and at present these therapies have the best clinical outcome data in patients who are responsive to therapy with a proton pump inhibitor. The risk/benefit profile, long term durability and cost effectiveness should be considered in all patients being considered when evaluating each of the endoscopic therapies for GERD.

Characterization of nuclease-resistant ribozymes directed against hepatitis B virus RNA.

Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide and 1.2 million deaths each year. To explore the use of ribozymes as a novel therapy for HBV infection, nuclease-resistant ribozymes that target highly conserved regions of HBV RNA were screened in cell culture. These synthetic ribozymes have the potential to cleave all four major HBV RNA transcripts and to block the HBV lifecycle by cleavage of the pregenomic RNA. A number of the screened ribozymes demonstrate activity in cell culture systems, as measured by decreased levels of HBV surface antigen, HBV e antigen and HBV DNA. In addition, a lead anti-HBV ribozyme maintains activity against a lamivudine-resistant HBV variant in cell culture. Treatment of HBV transgenic mice with lead anti-HBV ribozymes significantly reduced viraemia compared with saline-treated animals and was as effective as treatment with lamivudine. In conclusion, the therapeutic use of a ribozyme alone or in combination with current therapies (lamivudine or interferons) may lead to improved HBV therapy.

Activation of the antioxidant response element in primary cortical neuronal cultures derived from transgenic reporter mice.

Many phase II protective genes contain a cis -acting enhancer region known as the antioxidant response element (ARE). Increased expression of these genes contributes to the protection of cells from oxidative stress. Transgenic reporter mice were created that carry in their genome the core ARE coupled to the human placental alkaline phosphatase (hPAP) reporter gene. Primary cortical cultures derived from these mice were treated with tBHQ resulting in a dose-dependent increase in hPAP activity. Histochemical staining for hPAP activity was observed in both glia and neurons from tBHQ-treated cultures. The tBHQ-mediated increase in hPAP was not affected by the antioxidant glutathione monoethyl ester (GSHEE), whereas the increase in hPAP following DEM treatment was completely blocked by GSHEE. Pre-treatment of cultures with the PI3-kinase inhibitor LY 294002 demonstrated a dose-dependent decrease in tBHQ-induced hPAP activity. In addition, the tBHQ-mediated expression of ARE-driven genes in primary cortical cultures was blocked by LY 294002. Interestingly, basal expression of Nrf2 was also inhibited by LY 294002. We theorize that increased levels of genes controlled by the ARE are important for cellular protection against oxidative stress. These ARE-hPAP transgenic mice will be an important in vivo model for testing our hypothesis.

Persistent vertical binocular diplopia after cataract surgery.

PURPOSE: To report the incidence of, and factors associated with, persistent vertical diplopia after cataract surgery. DESIGN: Consecutive interventional case series. METHODS: Retrospectively, all adult patients examined during a five year, five month period because of new onset persistent (>3 months) vertical binocular diplopia after cataract surgery were analyzed. All patients had their cataract surgery at the same outpatient ophthalmic surgery center, and were referred to the author, enabling calculation of incidence. Trends in anesthesia type and strabismus complications therefrom were also assessed. Comparison was made between ophthalmologist-administered retrobulbar anesthesia versus anesthesia staff-administered retrobulbar anesthesia. Incidence during a period in which hyaluronidase was not incorporated in the retrobulbar anesthetic was calculated. RESULTS: Persistent vertical diplopia occurred after cataract surgery in 32 (0.18%) of 17,531 eyes that had cataract surgery. No patient whose cataract surgery was conducted with topical anesthesia (3817 eyes) had persistent vertical diplopia, whereas 32 (0.23%) of the 13714 eyes whose cataract surgery was done after retrobulbar anesthesia were affected. No cases of persistent postoperative diplopia were found among 7410 cataract surgery eyes after retrobulbar injection given by one cataract surgeon. There was a threefold greater number of left eyes involved than right eyes (P <.005). No significant (P >.20) increase in cases of persistent vertical diplopia was noted during a period of hyaluronidase shortage. CONCLUSIONS: In this study, persistent binocular vertical diplopia after cataract surgery occurred in 0.23% of cases in which retrobulbar anesthesia was performed. No cases were found after topical anesthesia. Occurrence may be technique-related.

Preferential expression of antioxidant response element mediated gene expression in astrocytes.

Transcriptional control of target genes by antioxidant/electrophile response elements has been well described in peripheral tissues. Genes that are regulated by this mechanism include the antioxidant enzymes NAD(P)H:quinone oxidoreductase, gamma-glutamyl cystine synthetase and glutathione-S-transferase. Antioxidant/electrophile response elements within a gene's promoter confer induction by low-molecular-weight electrophilic compounds such as tert-butylhydroquinone and dimethyl fumarate. We have now examined the ability of antioxidant/electrophile response elements to elicit gene expression in neurons and astrocytes in both brain slices and primary cultures using transient transfection of promoter reporter constructs. Our results using a heat-stable human placental alkaline phosphatase reporter indicate that antioxidant/electrophile response element mediated gene expression is largely restricted to astrocyte cell populations. Placental alkaline phosphatase expression was significantly elevated in astrocytes treated with the antioxidant/electrophile response element inducer dimethyl fumarate. Mutant constructs lacking a functional antioxidant/electrophile response element abolished all placental alkaline phosphatase expression in astrocytes. We suggest that astrocytic metabolic processes that normally aid and/or protect neurons may be controlled via this inducible system.

Esomeprazole once daily for 6 months is effective therapy for maintaining healed erosive esophagitis and for controlling gastroesophageal reflux disease symptoms: a randomized, double-blind, placebo-controlled study of efficacy and safety.

OBJECTIVE: Esomeprazole, the S-isomer of omeprazole, achieves a significantly greater healing rate and symptom resolution of erosive esophagitis than that achieved by omeprazole. The objective of this study is to assess the efficacy of the new proton pump inhibitor esomeprazole in preventing relapse over a prolonged period in patients with healed erosive esophagitis. METHODS: A total of 318 gastroesophageal reflux patients whose erosive esophagitis was healed in a comparative study of esomeprazole 40 mg, 20 mg, or omeprazole 20 mg, were randomized to maintenance therapy with once daily esomeprazole 40 mg, 20 mg, or 10 mg, or placebo in a U.S., double-blind multicenter trial. RESULTS: After 6 months, healing was maintained (cumulative life table rates) in 93.6% (95% CI 87.4-99.7) of patients treated with esomeprazole 40 mg, 93.2% (95% CI 87.4-99.0) treated with esomeprazole 20 mg, and 57.1% (95% CI 45.2-69) treated with esomeprazole 10 mg; p < 0.001 vs placebo (29.1%; 95% CI 17.7-40.3). Of patients relapsing, mean time to first recurrence of esophagitis increased with dose, from 34 days (placebo) to 78 days (10 mg), 115 days (20 mg), and 163 days (40 mg). Patients treated with esomeprazole had less frequent and less severe heartburn than those treated with placebo. At month 6, more than 70% of patients being treated with esomeprazole remained symptom-free. CONCLUSIONS: Esomeprazole is effective and well tolerated in the maintenance of a healing erosive esophagitis. Esomeprazole 40 mg and 20 mg maintain healing in over 90% of patients while providing effective control of heartburn symptoms.

Consequences of cAMP and catalytic-subunit binding on the flexibility of the A-kinase regulatory subunit.

A combination of site-directed labeling and time-resolved fluorescence anisotropy was used to further elucidate the structure and underlying dynamic features of the type I regulatory (R(I)(alpha)) subunit of the cAMP-dependent protein kinase. Specifically, the consequences of cAMP and the catalytic (C)-subunit binding on the backbone flexibility around seven sites of cysteine substitution and fluorescein maleimide labeling (Thr(6)Cys, Leu(66)Cys, Ser(75)Cys, Ser(81)Cys, Ser(99)Cys, Ser(145)Cys, and Ser(373)Cys) in the R(I)(alpha) subunit were assessed. Focusing on the fast rotational correlation time, the results indicate that most of the interdomain segment connecting the dimerization/docking (D/D) and tandem cAMP-binding domains is probably weakly associated with the latter domain. Also, this segment becomes more tightly bound to the C subunit upon holoenzyme formation. The results also suggest that there is a short 'hinge' segment (around Leu(66)Cys) that could allow the structured interdomain/cAMP-binding and D/D domains to pivot about each other. Finally, cAMP binding dramatically reduces the backbone flexibility around only the two sites of cysteine substitution in the cAMP-binding domains, suggesting a selective structural stabilization caused by cAMP and a "tight" coupling of low-nanosecond fluctuations selectively within the tandem cAMP-binding domains.

Cloning and molecular characterisation of the trout (Oncorhynchus mykiss) vacuolar H(+)-ATPase B subunit.

The current model of transepithelial ion movements in the gill of freshwater fish incorporates an apically oriented vacuolar H(+)-ATPase (H(+)V-ATPase; proton pump) that is believed to facilitate both acid excretion and Na(+) uptake. To substantiate this model, we have cloned and sequenced a cDNA encoding the B subunit of the rainbow trout (Oncorhynchus mykiss) H(+)V-ATPase. The cloning of the B subunit enabled an examination by northern analysis of its tissue distribution and expression during external hypercapnia. Degenerate oligonucleotide primers to the B subunit of the H(+)V-ATPase were designed and used in a semi-nested polymerase chain reaction (PCR) to amplify an 810 base pair (bp) product from a trout gill/kidney cDNA library. This PCR product was cloned and sequenced and then used to screen the same cDNA library. The assembled 2262 bp cDNA included an open reading frame coding for a deduced protein of 502 amino acid residues. A BLAST search of the GenBank nucleotide database revealed numerous matches to other vertebrate and invertebrate H(+)V-ATPase B subunits. Protein alignment demonstrated that the trout H(+)V-ATPase B subunit is more than 85 % identical and more than 90 % similar to those in other vertebrate species. An initial analysis of H(+)V-ATPase mRNA tissue distribution revealed significant expression in blood. Although a comparison of perfused tissues (blood removed) with non-perfused tissues demonstrated no obvious contribution of the blood to total tissue H(+)-ATPase mRNA levels, all subsequent experiments were performed using perfused tissues. Levels of H(+)V-ATPase mRNA expression were high in the gill, kidney (anterior or posterior), intestine, heart and spleen, but lower in liver and white muscle. Exposure of the fish to 12 h of external hypercapnia (water P(CO2)=7. 5 mmHg; 1 kPa) was associated with a transient increase (at 2 h) in the levels of H(+)V-ATPase B subunit mRNA in gill and kidney; liver mRNA levels were unaffected. These results are consistent with the hypothesis of an apically localised plasma membrane H(+)V-ATPase in the freshwater trout gill and that the expression of this proton pump is increased during periods of acidosis, at least in part because of an increased steady-state level of H(+)V-ATPase mRNA.

3-O-Desacyl monophosphoryl lipid A derivatives: synthesis and immunostimulant activities.

The synthesis of a series of novel analogues of lipid A, the active principle of lipopolysaccharide, is reported. In these compounds, the 1-O-phosphono and (R)-3-hydroxytetradecanoyl moieties of native Salmonella minnesota R595 lipid A have been replaced with hydrogen and the length of the normal fatty acyl residues has been systematically varied. Normal fatty acid chain length in the 3-O-desacyl monophosphoryl lipid A (MLA) series is shown to be a critical determinant of iNOS gene expression in activated mouse macrophages and the induction of proinflammatory cytokines in human peripheral monocytes. Examination of pyrogenicity in rabbits and lethal toxicity in D-galactosamine-treated mice shows that toxic effects in the MLA series can be ameliorated by modifying fatty acid chain length. When used as an adjuvant for tetanus toxoid vaccines, certain MLA derivatives enhance the production of tetanus toxoid-specific antibodies in mice.