Opioid Analgesics Do Not Improve Abdominal Pain or Quality of Life in Crohn's Disease.

BACKGROUND: Abdominal pain and opioid analgesic use are common in Crohn's disease (CD). AIMS: We sought to identify factors associated with abdominal pain in CD and evaluate the impact of opioid analgesics on pain and quality-of-life scores in this setting. METHODS: We performed a longitudinal cohort study using a prospective, consented IBD natural history registry from a single academic center between 2009 and 2013. Consecutive CD patients were followed for at least 1 year after an index visit. Data were abstracted regarding pain experience (from validated surveys), inflammatory activity (using endoscopic/histologic findings), laboratory studies, coexistent psychiatric disorders, medical therapy, opioid analgesic, and tobacco use. RESULTS: Of 542 CD patients (56.6% women), 232 (42.8%) described abdominal pain. Individuals with pain were more likely to undergo surgery and were more frequently prescribed analgesics and/or antidepressants/anxiolytics. Elevated ESR (OR 1.79; 95%CI 1.11-2.87), coexistent anxiety/depression (OR 1.87; 95%CI 1.13-3.09), smoking (OR 2.08; 95%CI 1.27-3.40), and opioid use (OR 2.46; 95%CI 1.33-4.57) were independently associated with abdominal pain. Eighty patients (14.8%) were prescribed opioids, while 31 began taking them at or after the index visit. Patients started on opioids demonstrated no improvement in abdominal pain or quality-of-life scores on follow-up compared to patients not taking opioids. CONCLUSIONS: Abdominal pain is common in CD and is associated with significant opioid analgesic utilization and increased incidence of anxiety/depression, smoking, and elevated inflammatory markers. Importantly, opioid use in CD was not associated with improvement in pain or quality-of-life scores. These findings reinforce the limitations of currently available analgesics in IBD and support exploration of alternative therapies.

Evaluation of Therapeutics for Advanced-Stage Heart Failure and Other Severely-Debilitating or Life-Threatening Diseases.

Severely-debilitating or life-threatening (SDLT) diseases include conditions in which life expectancy is short or quality of life is greatly diminished despite available therapies. As such, the medical context for SDLT diseases is comparable to advanced cancer and the benefit vs. risk assessment and development of SDLT disease therapeutics should be similar to that of advanced cancer therapeutics. A streamlined development approach would allow patients with SDLT conditions earlier access to therapeutics and increase the speed of progression through development. In addition, this will likely increase the SDLT disease therapeutic pipeline, directly benefiting patients and reducing the economic and societal burden of SDLT conditions. Using advanced-stage heart failure (HF) as an example that illustrates the concepts applicable to other SDLT indications, this article proposes a streamlined development paradigm for SDLT disease therapeutics and recommends development of aligned global regulatory guidance.

Isolation and identification of ochratoxin A-producing Aspergillus section Nigri strains from California raisins.

AIMS: To determine incidence and levels of ochratoxin A (OTA) in California raisins and to isolate and characterize OTA-producing fungi from California raisin vineyard populations. METHODS AND RESULTS: Forty raisin clusters sampled from four California vineyards in the San Joaquin Valley were analysed for OTA content using immunoaffinity and HPLC methods. OTA was detected in 93% of the samples, at levels from 0·06 to 11·4 ng g⁻¹. From these raisin samples, a total of 400 strains of Aspergillus were isolated and analysed for OTA production. Twelve isolates (3%), from five raisin samples, produced OTA. These isolates were identified as Aspergillus carbonarius, based on morphological characteristics and multilocus sequence analysis. Levels of OTA produced by these isolates on raisin agar ranged from 0·9 to 15 µg g⁻¹. CONCLUSIONS: OTA is a common contaminant of raisin vineyards, but average levels are much lower than EU regulatory limits for dried fruit. The primary species responsible for OTA contamination in California raisins is A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates that low-level OTA contamination of raisins occurs in California and that ecological studies of A. carbonarius within the Aspergillus section Nigri population on raisins are warranted to monitor ochratoxigenic potential of the crop.

Use of chemosensitization to overcome fludioxonil resistance in Penicillium expansum.

AIM: To overcome fludioxonil resistance of Penicillium expansum, a mycotoxigenic fungal pathogen causing postharvest decay in apple, by using natural phenolic chemosensitizing agents. METHODS AND RESULTS: Fludioxonil-resistant mutants of P. expansum were co-treated with different oxidising and natural phenolic agents. Resistance was overcome by natural phenolic chemosensitizing agents targeting the oxidative stress-response pathway. These agents also augmented effectiveness of the fungicide, kresoxim-methyl. Results indicated that alkyl gallates target mitochondrial respiration and/or its antioxidation system. Fungal mitochondrial superoxide dismutase (Mn-SOD) plays a protective role against alkyl gallates. CONCLUSIONS: Natural chemosensitizing agents targeting the oxidative stress-response system, such as Mn-SOD, can synergize commercial fungicides. SIGNIFICANCE AND IMPACT OF THE STUDY: Redox-active compounds can serve as potent chemosensitizing agents to overcome resistance and lower effective dosages of fungicides. This can reduce costs with coincidental lowering of environmental and health risks.

Benign recurrent sixth (abducens) nerve palsies in children.

Sixth nerve palsy can occur as a result of elevated intracranial pressure, neoplasm or trauma. Reports from tertiary centres indicate that between 5% and 16% of referred cases have no ascribed aetiology and are classified as benign. Rarely, these benign palsies can recur. A retrospective chart review of a cohort of 253 paediatric patients with sixth nerve palsies was analysed and uncovered 30 cases of benign sixth nerve palsy, nine of which recurred. Our data and review of other studies on the subject imply that a new onset sixth nerve palsy presenting in children can be benign in approximately 13% of cases, so a thorough history and physical examination to evaluate for any other neurological symptoms or signs followed by MRI of the brain with and without contrast is recommended.

Pathogenesis of Eutypa lata in grapevine: identification of virulence factors and biochemical characterization of cordon dieback.

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism.

Enhanced activity of strobilurin and fludioxonil by using berberine and phenolic compounds to target fungal antioxidative stress response.

AIMS: Identify natural products that effectively target antioxidative signal transduction/stress response systems [i.e., mitogen-activated protein kinase (MAPK) pathway, mitochondrial superoxide dismutase (Mn-SOD)] of fungi. Enhance activity of strobilurin or fludioxonil with discovered compounds. METHODS AND RESULTS: Enhancement of antifungal activity of strobilurins, inhibitors of complex III of the mitochondrial respiratory chain, was tested using berberine hemisulfate and different phenolic compounds. The Saccharomyces cerevisiae sod2Delta, a deletion mutant lacking Mn-SOD gene, was highly sensitive to berberine and veratraldehyde. Functional complementation analysis verified these compounds target Mn-SOD. Activity of strobilurin (25-50 micromol l(-1)) was elevated on most aspergilli and Penicillium expansum by co-application with berberine or veratraldehyde (2-4 mmol l(-1)). These compounds also prevented Aspergillus fumigatus MAPK mutants (sakADelta and mpkCDelta) from escaping toxicity of fludioxonil (a phenylpyrrole fungicide potentiated by the MAPK pathway), a typical phenotype of fungal MAPK mutants. CONCLUSIONS: Strobilurin activity or prevention of fungal escape from fludioxonil toxicity can be enhanced by co-application of certain alkaloids or phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural products can be used to target cellular stress response systems in fungal pathogens and serve as alternatives/additives to commercial antifungal agents.

Targeting antioxidative signal transduction and stress response system: control of pathogenic Aspergillus with phenolics that inhibit mitochondrial function.

AIMS: The aim of this study was to show whether antioxidative response systems are potentially useful molecular targets for control of Aspergillus fumigatus and Aspergillus flavus. Selected phenolic agents are used in target-gene-based bioassays to determine their impact on mitochondrial respiration. METHODS AND RESULTS: Vanillyl acetone, vanillic acid, vanillin, cinnamic acid, veratraldehyde, m-coumaric acid (phenolic agents to which Saccharomyces cerevisiae sod2delta mutant showed sensitivity), carboxin (inhibits complex II of the mitochondrial respiratory chain), strobilurins/antimycin A (inhibits complex III of the mitochondrial respiratory chain) and fludioxonil/fenpiclonil [antifungals potentiated by mitogen-activated protein kinase (MAPK)] were examined in A. fumigatus, A. flavus and S. cerevisiae. Individual or combined application of phenolics with inhibitors of mitochondrial respiration showed some of the phenolics effectively inhibited fungal growth. Target-gene bioassays were performed using a sakAdelta (MAPK deletion) strain of A. fumigatus and a complementation analysis using the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) of A. flavus in the ortholog mutant, sod2delta, of S. cerevisiae. The results demonstrated that mitochondrial antioxidative stress system plays important roles in fungal response to antifungal agents tested. CONCLUSIONS: Antioxidative response systems of fungi can be an efficient molecular target of phenolics for pathogen control. Combined application of phenolics with inhibitors of mitochondrial respiration can effectively suppress the growth of fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural compounds that do not pose any significant medical or environmental risks could serve as useful alternatives or additives to conventional antifungals. Identifying the antioxidative response systems in other pathogens could improve methods for fungal control.

A Reassessment of the Species Concept in Eutypa lata, the Causal Agent of Eutypa Dieback of Grapevine.

ABSTRACT Eutypa dieback is a vascular disease of several cultivated crops and trees worldwide. The attribution of the name to the agent responsible for branch dieback is ambiguous. Pathogenicity of Eutypa sp. first was reported on apricot and the causal agent was named E. armeniacae. However, no morphological differences were reported with the previously described E. lata, and some authors considered both species synonymous. Others regarded them as distinct species on the basis of pathogenesis and molecular analysis. We further investigated the relatedness of both species by phylogenetic analyses of the internal transcribed spacer region and beta-tubulin gene. These analyses included several other taxa placed in the same family (Diatrypaceae), and yielded three groups. The isolates referred to as E. lata in previous work clustered with Diatrype stigma in one group. Isolates of E. armeniacae and E. lata clustered in a second group, supporting the synonymy of these species. The third group included other Eutypa spp. supporting the polyphyletic origin of this genus. Measurements of conidia length and secondary metabolite production of isolates supported the phylogenetic analyses. Secondary metabolites appeared to be a synapomorphic character shared by several taxa including E. lata, E. armeniacae, E. laevata, and E. petrakii var. petrakii.

Gene targets for fungal and mycotoxin control.

It was initially shown that gallic acid, from hydrolysable tannins in the pelliele of walnut kernels, dramatically inhibits biosynthesis of aflatoxin byAspergillus flavus. The mechanism of this inhibition was found to take place upstream from the gene cluster, including the regulatory gene,aflR, involved in aflatoxin biosynthesis. Additional research using other antioxidant phenolics showed similar antiaflatoxigenic activity to gallic acid. Treatment ofA. flavus withtert-butyl hydroperoxide resulted in an almost doubling of aflatoxin biosynthesis compared to untreated samples. Thus, antioxidative response systems are potentially useful molecular targets for control ofA. flavus. A high throughput screening system was developed using yeast,Saccharomyces cerevisiae, as a model fungus. This screening provided an avenue to quickly identify fungal genes that were vulnerable to treatment by phenolic compounds. The assay also provided a means to quickly assess effects of combinations of phenolics and certain fungicides affecting mitochondrial respiration. For example, theS. cerevisiae sod2† mutant was highly sensitive to treatment by certain phenolics and strobilurins/antimycin A, fungicides which inhibit complex III of the mitochondrial respiratory chain. Verification of stress to this system in the target fungus,A. flavus, was shown through complementation analysis, wherein the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) ofA. flavus in the ortholog mutant,sod2†, ofS. cerevisiae, relieved phenolic-induced stress. Mitochondrial antioxidative stress systems play an important role in fungal response to antifungals. Combined treatment of fungi with phenolics and inhibitors of mitochondrial respiration can effectively suppress growth ofA. flavus in a synergistic fashion.

Germline BRCA1-2 mutations in non-Ashkenazi families with double primary breast and ovarian cancer.

OBJECTIVE: Ashkenazi women with double primary breast and ovarian cancer have a high prevalence (57%) of germline Jewish founder mutations in the BRCA1 (185delAG, 5382insC) and BRCA2 (6174delT) genes. The purpose of this study was to determine the frequency and type of BRCA1-2 mutations in non-Ashkenazi families with at least one member having double primary breast and ovarian cancer. METHODS: Women at increased risk for cancer based upon their family history were enrolled at the University of Texas Southwestern Familial Cancer Registry between 1992 and 2000. Blood samples from patients desiring genetic testing were sent for complete DNA sequencing of the BRCA1 and BRCA2 genes. Families with a member having both breast and ovarian cancer were identified and clinical data were obtained. RESULTS: Sixty-two (7%) of 900 enrolled families were non-Ashkenazi and had at least one member with double primary breast and ovarian cancer. Twenty-one families had members who underwent genetic testing; 41 did not. Thirteen (62%) families had a germline BRCA1 (n = 11) or BRCA2 (n = 2) mutation; only one Jewish founder mutation (185delAG) was detected. Eight (38%) families tested negative. Six (86%) of seven women undergoing genetic testing who themselves had double primary breast and ovarian cancer were BRCA1-2 mutation carriers. CONCLUSIONS: Germline BRCA1-2 mutations are common in non-Ashkenazi families with a member having double primary breast and ovarian cancer. These mutations occurred throughout both genes, emphasizing the need for comprehensive sequencing. One family had the BRCA2 6985delCT mutation, which lies beyond the "ovarian cancer cluster" region.

Identification of benzethonium chloride in commercial grapefruit seed extracts.

Commercial grapefruit seed extracts (GSE) were extracted with chloroform. The solvent was evaporated, and the resulting solid was subsequently analyzed by high-performance liquid chromatography, electrospray ionization mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and elemental analysis (by proton-induced X-ray emission [PIXE] analysis). The main constituent was identified as benzethonium chloride, a synthetic antimicrobial agent commonly used in cosmetics and other topical applications. This compound comprised 8.03% (n = 2) of the liquid GSE sample. Higher amounts of benzethonium chloride were found in powder GSE samples.

Regulation of aflatoxin production by naphthoquinones of walnut (Juglans regia).

Walnuts are a valuable crop the sale and export potential of which may be severely limited by contamination with aflatoxins, metabolites produced on infection with Aspergillus flavus. The effect of a series of four naphthoquinones [1,4-naphthoquinone (1); juglone (5-hydroxy-1,4-naphthoquinone) (2); 2-methyl-1, 4-naphthoquinone (3); and, plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) (4)] (Figure 1), which occur in walnut husks, on fungal viability and aflatoxigenesis was studied in vitro. The quinones delayed germination of the fungus and were capable of completely inhibiting growth at higher concentrations. Their effect on aflatoxin levels was highly dependent on the concentration of individual naphthoquinones in the media. At higher concentrations, aflatoxin production was decreased or completely inhibited, but at lower concentrations there was a stimulatory effect on aflatoxin biosynthesis, with a >3-fold increase at 20 ppm of 3. Structural features associated with decreased fungal viability and greatest effect on aflatoxigenesis are the presence of a 5-hydroxyl or 2-methyl substituent, but there is no significant additive effect when both of these substituents are present.

In vivo importance of actin nucleotide exchange catalyzed by profilin.

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly.

Profilin is required for optimal actin-dependent transcription of respiratory syncytial virus genome RNA.

Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-L-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-L-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-L-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome.

Profilin binds proline-rich ligands in two distinct amide backbone orientations.

The actin regulatory protein profilin is targeted to specific cellular regions through interactions with highly proline-rich motifs embedded within its binding partners. New X-ray crystallographic results demonstrate that profilin, like SH3 domains, can bind proline-rich ligands in two distinct amide backbone orientations. By further analogy with SH3 domains, these data suggest that non-proline residues in profilin ligands may dictate the polarity and register of binding, and the detailed organization of the assemblies involving profilin. This degeneracy may be a general feature of modules that bind proline-rich ligands, including WW and EVH1 domains, and has implications for the assembly and activity of macromolecular complexes involved in signaling and the regulation of the actin cytoskeleton.

Crystallization and preliminary X-ray analysis of human platelet profilin complexed with an oligo proline peptide.

Profilin is an actin-monomer binding protein that regulates the distribution and dynamics of the actin cytoskeleton. Profilin binds poly-L-proline and proline-rich peptides in vitro and co-localizes with proline-rich proteins in focal adhesions and at the site of actin tail assembly on the surface of intracellular parasites such as Listeria monocytogenes. The crystallization of the complex between human platelet profilin (HPP) and an L-proline decamer [(Pro)10] is reported here. Diffraction from these crystals is consistent with the space group P21212 with unit-cell constants a = 68.25, b = 97.64, c = 39.10 A. The crystals contain two HPP molecules per asymmetric unit and diffract to 2.2 A.

Anger management style and the prediction of treatment outcome among male and female chronic pain patients.

Anger is a prominent emotion experienced by chronic pain patients. Anecdotes suggest that anger predicts poor outcome following multidisciplinary pain programs, but no empirical evidence documents this link. We expected that patient anger expression or suppression would predict poor outcome following a pain program and that gender differences would emerge. Pre- to posttreatment measures of lifting capacity, walking endurance, depression, pain severity and activity level were collected from 101 chronic pain patients. An 'anger expression x gender' interaction was found such that anger expression among males was correlated negatively with lifting capacity improvements. 'Anger suppression x gender' interactions emerged such that anger suppression among males was correlated negatively with improvements in depression and general activities. These effects remained significant after controlling for trait anger. Thus, how anger is managed may exert unique influence on outcomes apart from the effects of mere anger proneness, at least among male pain patients.

Structure determination and characterization of Saccharomyces cerevisiae profilin.

The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.

Cognitive and physical capacity process variables predict long-term outcome after treatment of chronic pain.

Cognitive-behavioral and physical therapies are incorporated into multidisciplinary chronic pain programs because changes in pain cognitions and physical capacity may represent therapeutic processes that facilitate favorable outcome. Decreases in depression, however, may explain treatment responses more parsimoniously. Measures of pain helplessness, lifting capacity, walking endurance, depression, pain severity, and activity level were collected from 94 chronic pain patients at pre- and posttreatment and at 3- to 6-month follow-up evaluations. Decreases in pain helplessness were linked to pain severity reduction, whereas walking endurance increases were related to improvements in activity levels and downtime even after controlling for effects of depression decreases. Thus, cognitive and physical capacity changes that occur through pain treatment may make unique contributions to long-term outcome.