Fusarioid community diversity associated with conifer seedlings in forest nurseries across the contiguous USA.

Introduction: Fusarioid fungi that cause damping-off and root diseases can result in significant losses to conifer crops produced in forest nurseries across the USA. These nurseries are vital to reforestation and forest restoration efforts. Understanding the diversity of Fusarioid fungi associated with damping-off and root diseases of conifer seedlings can provide an approach for targeted management techniques to limit seedling losses and pathogen spread to novel landscapes. Methods: This study identifies 26 Fusarium spp. (F. acuminatum, F. annulatum, F. avenaceum, F. brachygibbosum, F. clavus, F. commune, F. cugenangense, F. diversisporum, F. elaeagni, F. elaeidis, F. flocciferum, F. fredkrugeri, F. fujikuroi, F. grosmichelii, F. ipomoeae, F. lactis, F. languescens, F. luffae, F. odoratissimum, F. oxysporum, F. queenslandicum, F. redolens, F. torulosum, F. triseptatum, F. vanleeuwenii, & F. verticillioides), 15 potential species within Fusarium and Neocosmospora species complexes (two from F. fujikuroi species complex, nine from F. oxysporum species complex, three from F. tricinctum species complex, and one from Neocosmospora species complex), and four Neocosmospora spp. (N. falciforme, N. metavorans, N. pisi, & N. solani) and associated host information collected from conifer-producing nurseries across the contiguous USA. Results: Phylogenetic analyses identified Fusarioid fungi haplotypes that were associated with 1) host specificity, 2) localization to geographic regions, or 3) generalists found on multiple hosts across diverse geographic regions. Discussion: The haplotypes and novel species identified on conifer seedlings should be considered for further analysis to determine pathogenicity, pathogen spread, and assess management practices.

Dietary supplementation with phytochemicals improves diversity and abundance of honey bee gut microbiota.

AIM: Determine the impact of beneficial phytochemicals on diversity and abundance of the gut microbiome in the honey bee (Apis mellifera). METHODS AND RESULTS: Eight-day-old honey bee workers were fed 25 ppm of phytochemical (caffeine, gallic acid, p-coumaric acid or kaempferol) in 20% sucrose. Guts of bees collected at 3 and 6 days were excised and subjected to next-generation sequencing for bacterial 16S and fungal ITS regions. Although phytochemical supplementation fostered gut microbial diversity and abundance, the patterns differed between phytochemicals and there was a temporal stabilization of the bacterial community. While bacterial and fungal communities responded differently, all phytochemical treatments displayed increased abundance of the most represented bacterial genera, Snodgrassella sp. and Lactobacillus sp. CONCLUSIONS: Phytochemical supplementation improves gut microbial diversity and abundance, reiterating the need for diverse habitats that provide bees with access to pollen and nectar rich in these micronutrients. Diverse gut microbiota can provide a strong line of defense for bees against biotic stressors while improving worker bee lifespan. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the impact of phytochemical supplementation on gut microbiota in honey bees and these findings have implications for strategic hive management through standardization of effective phytochemical and probiotic feed supplements.

Preventive Fungicidal Control of Cytospora leucostoma in Peach Orchards in Colorado.

In western Colorado, Cytospora leucostoma is ubiquitous in peach orchards and has developed into a major limiting factor of peach production. The pathogen is unable to invade healthy intact phloem tissue of the tree, but instead, it requires a wound as a mode of entry. Bark injuries caused by cold and pruning in commercial orchard systems provide infection courts that, in suitable environment conditions, can lead to many successful fungal infections. Preventive fungicide control is an integral component of management in tree fruit production. Eighteen fungicides were tested at selected label dose rates for C. leucostoma control. All treatments were initially tested in vitro in fungicide-amended media dishes. Successful treatments were then tested under controlled conditions on detached peach branch segments. Effective fungicides identified in the laboratory assays (thiophanate-methyl, captan, lime sulfur, and copper hydroxide) were further tested as spray applications in the field and as wound sealant applications in combination with latex paint and kaolin clay. Of the treatments evaluated, thiophanate-methyl, captan, 50% latex paint, thiophanate-methyl amended in 50% latex paint, captan amended in 50% latex paint, and lime sulfur were most effective in reducing C. leucostoma necrotic area. Copper hydroxide was ineffective in all field trials and in some instances, yielded larger necrotic areas than the nontreated positive control shoots.

Techniques for chronic monitoring of brain activity in freely moving sheep using wireless EEG recording.

BACKGROUND: Large mammals with complex central nervous systems offer new possibilities for translational research into basic brain function. Techniques for monitoring brain activity in large mammals, however, are not as well developed as they are in rodents. NEW METHOD: We have developed a method for chronic monitoring of electroencephalographic (EEG) activity in unrestrained sheep. We describe the methods for behavioural training prior to implantation, surgical procedures for implantation, a protocol for reliable anaesthesia and recovery, methods for EEG data collection, as well as data pertaining to suitability and longevity of different types of electrodes. RESULTS: Sheep tolerated all procedures well, and surgical complications were minimal. Electrode types used included epidural and subdural screws, intracortical needles and subdural disk electrodes, with the latter producing the best and most reliable results. The implants yielded longitudinal EEG data of consistent quality for periods of at least a year, and in some cases up to 2 years. COMPARISON WITH EXISTING METHODS: This is the first detailed methodology to be described for chronic brain function monitoring in freely moving unrestrained sheep. CONCLUSIONS: The developed method will be particularly useful in chronic investigations of brain activity during normal behaviour that can include sleep, learning and memory. As well, within the context of disease, the method can be used to monitor brain pathology or the progress of therapeutic trials in transgenic or natural disease models in sheep.

Avascular necrosis of the canine radial carpal bone: a condition analogous to Preiser's disease?

An eight-year-old male neutered Staffordshire bull terrier was presented for investigation of right forelimb lameness of 14 months' duration. Radiography showed mottled osteolysis of the right radial carpal bone. Histopathology of the bone demonstrated replacement of healthy bone with granulation tissue suggestive of ischaemic necrosis. Lameness resolved following pancarpal arthrodesis. In humans, Preiser's disease is a condition in which idiopathic ischaemic necrosis of the scaphoid bone, the equivalent of the canine radial carpal bone, occurs. This disease may be analogous to the presentation seen in this case. To the authors' knowledge, this is the first report of such a condition in a dog.

Recent fat intake modulates fat taste sensitivity in lean and overweight subjects.

OBJECTIVE: To evaluate the effects of a high-fat and low-fat diet on taste sensitivity to oleic acid (C18:1) in lean and overweight/obese (OW/OB) subjects. DESIGN: Randomized cross-over dietary intervention involving the consumption of a high-fat (>45% fat) and low-fat (<20% fat) diet, both consumed over a 4-week period. SUBJECTS: A total of 19 lean, mean age 33±13 years, mean body mass index (BMI) 23.2±2.2 kg m(-2) and 12 OW/OB, mean age 39.5±3 years, mean BMI 28±2.6 kg m(-2), subjects participated in the study, which measured taste thresholds for C18:1, fat perception and hedonic ratings for regular (RF) and lowered-fat (LF) foods before, and following consumption of a high- and low-fat diet. RESULTS: Consumption of the low-fat diet increased taste sensitivity to C18:1 among lean and OW/OB subjects (P<0.05) and increased the subjects ability to perceive small differences in the fat content of custard (P=0.05). Consumption of the high-fat diet significantly decreased taste sensitivity to C18:1 among lean subjects (P<0.05), with no change in sensitivity among OW/OB persons (P=0.609). The hedonic ratings for several RF and LF foods differed following the diets. CONCLUSION: Alterations in the fat content of the diet modulated taste sensitivity to C18:1 among lean subjects, which was increased following a 4-week period of fat restriction and attenuated following the high-fat diet. The failure of the high-fat diet to alter fatty acid taste thresholds among OW/OB subjects suggests that these individuals were 'adapted' to high-fat exposure, perhaps because of differences in habitual fat consumption. Taken together, these data suggest that excessive dietary fat attenuates nutrient sensing epithelia response in the oral cavity, which could be associated with changes in diet and weight status.

Tuberculosis in socio-economically deprived neighborhoods: missed opportunities for prevention.

OBJECTIVE: To evaluate tuberculosis (TB) risk in three different US locations--Chicago, Illinois; Fulton County, Georgia; and the state of South Carolina--using two census-based measures of neighborhood-level deprivation and a geographic information system. METHOD: Individual-level data, including race and ZIP code of residence, were obtained for the three sites. TB cases were geocoded at the ZIP code tabulation area (ZCTA) level. Socio-economic status (SES) was defined at the ZCTA level using two Census 2000-based measures of socio-economic disadvantage: 1) percentage of population below poverty and 2) Townsend Deprivation Index. Based separately on the distributions of poverty and Townsend social deprivation scores, ZCTAs in each site were grouped into quartiles reflecting relative socio-economic well-being. To evaluate TB incidence in low- vs. high-SES neighborhoods, average annual TB incidence rates were calculated for the highest and lowest ZCTA quartiles. RESULTS: In all sites, TB incidence rates were significantly higher in high poverty/high social deprivation ZCTAs (P < 0.0001). CONCLUSIONS: Both census-based indicators performed well in distinguishing areas with high TB incidence rates from areas with little or no TB. Due to simplicity, the single poverty measure rather than the multifactorial Townsend index might be especially useful in identifying high-risk neighborhoods for targeted TB prevention efforts.

Serum cytokine profiles in Behçet's disease: is there a role for IL-15 in pathogenesis?

OBJECTIVES: Behçet's disease (BD) is a multisystem inflammatory disease characterised by recurrent orogenital ulceration, ocular inflammation and skin lesions whose aetiology is currently unknown. We hypothesized that levels of cytokines in the serum might provide either diagnostic or activity markers for the disease. METHODS: Levels of 10 cytokines were analysed in a multiplex bead analysis system as well as IL-15 by ELISA, in 79 serum samples from 52 patients with BD. The same cytokines were also measured in serum samples from 20 patients with recurrent aphthous stomatitis (RAS), as disease controls, and 15 healthy volunteers. The results were correlated with disease activity and current drug therapy. RESULTS: CXCL8 and TNF were the most abundant cytokines and were significantly raised compared to both patients with RAS and healthy controls. IL-15 was present in all samples and was significantly raised in both patients with BD and RAS compared to healthy controls. By comparison, cytokines associated with an adaptive immune response such as IFNgamma and IL-2 were found in few samples, while IL-4 and IL-10 were not detected in any sample. Levels of cytokines correlated with each other suggesting a response to the same stimulus, however, there was no association with either disease activity or treatment. CONCLUSION: Cytokines related to activity of the innate immune response were most prominent in this study and showed good correlation with each other. In particular, it was shown that IL-15 was raised in BD. However, there was no pattern of cytokine expression relating to disease activity or treatment.

Fingerprinting genomic instability in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a high-risk pre-cancerous condition where 7-13% of these patients develop head and neck squamous cell carcinoma (HNSCC). To date there is no cancer predictive markers for OSF patients. Genomic instability hallmarks early genetic events during malignant transformation causing loss of heterozygosity (LOH) and chromosomal copy number abnormality. However, to date there is no study on genomic instability in OSF. Although this condition is known as a high-risk pre-cancerous condition, there is no data regarding the genomic status of this disease in terms of genetic susceptibility to malignant transformation. METHODS: In this study, we investigated the existence of genetic signatures for carcinogenesis in OSF. We employed the high-resolution genome-wide Affymetrix Mapping single nucleotide polymorphism microarray technique to 'fingerprint' global genomic instability in the form of LOH in 15 patient-matched OSF-blood genomic DNA samples. RESULTS: This rapid high-resolution mapping technique has revealed for the first time that a small number of discrete hot-spot LOH loci appeared in 47-53% of the OSF tissues studied. Many of these LOH loci were previously identified regions of genomic instability associated with carcinogenesis of the HNSCC. CONCLUSION: To our knowledge, this is the first evidence that genomic instability in the form of LOH is present in OSF. We hypothesize that the genomic instability detected in OSF may play an important role in malignant transformation. Further functional association studies on these putative genes may reveal potential predictive oral cancer markers for OSF patients.

Upregulation of HIF-1alpha in malignant transformation of oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous condition showing extensive fibrosis of the submucosa and affects most parts of the oral cavity, including pharynx and upper third of the oesophagus. The molecules involved in the biological pathways of the fibrotic process appeared to be either down- or upregulated at different stages of the disease. Despite the precancerous nature, malignant transformation of the epithelium in the background of fibrosis has not been studied in detail. HIF-1alpha is a known transcription factor that is induced by hypoxia. AIMS: To test the hypothesis that hypoxia plays a role in malignant transformation and progression of OSF. MATERIALS AND METHODS: We used both formalin-fixed and frozen samples of OSF and normal mucosa to investigate the relationship between HIF-1alpha and epithelial dysplasia using immunohistochemistry and RT-PCR. CONCLUSIONS: Our data indicate that HIF-1alpha is upregulated at both protein and mRNA levels in OSF and the correlation with epithelial dysplasia is statistically significant (P < 0.001). We propose that HIF-1alpha may play a role in malignant transformation of OSF. Further, over-expression of HIF-1alpha may contribute to the progression of fibrosis. It may be possible to use HIF-1alpha as a marker for malignant transformation of OSF.

HEF1 is a necessary and specific downstream effector of FAK that promotes the migration of glioblastoma cells.

The highly invasive behavior of glioblastoma cells contributes to the morbidity and mortality associated with these tumors. The integrin-mediated adhesion and migration of glioblastoma cells on brain matrix proteins is enhanced by stimulation with growth factors, including platelet-derived growth factor (PDGF). As focal adhesion kinase (FAK), a nonreceptor cytoplasmic tyrosine kinase, has been shown to promote cell migration in various other cell types, we analysed its role in glioblastoma cell migration. Forced overexpression of FAK in serum-starved glioblastoma cells plated on recombinant (rec)-osteopontin resulted in a twofold enhancement of basal migration and a ninefold enhancement of PDGF-BB-stimulated migration. Both expression of mutant FAK(397F) and the downregulation of FAK with small interfering (si) RNA inhibited basal and PDGF-stimulated migration. FAK overexpression and PDGF stimulation was found to increase the phosphorylation of the Crk-associated substrate (CAS) family member human enhancer of filamentation 1 (HEF1), but not p130CAS or Src-interacting protein (Sin)/Efs, although the levels of expression of these proteins was similar. Moreover downregulation of HEF1 with siRNA, but not p130CAS, inhibited basal and PDGF-stimulated migration. The phosphorylated HEF1 colocalized with vinculin and was associated almost exclusively with 0.1% Triton X-100 insoluble material, consistent with its signaling at focal adhesions. FAK overexpression promoted invasion through normal brain homogenate and siHEF1 inhibited this invasion. Results presented here suggest that HEF1 acts as a necessary and specific downstream effector of FAK in the invasive behavior of glioblastoma cells and may be an effective target for treatment of these tumors.

Extracellular matrix metabolites as potential biomarkers of disease activity in wound fluid: lessons learned from other inflammatory diseases?

The new era of pharmacogenetics has identified a potential for individuals to receive customized treatments for a variety of disease states. For such individualized treatments to fulfil their potential, it will be essential for clinicians to be able to monitor disease activity, ideally in a rapid, noninvasive fashion. The accessibility of the skin offers much potential to develop noninvasive tests of metabolic and disease activity for clinical use. Impaired human wound healing in the skin is a chronic inflammatory disorder in which the development of such tests has considerable potential, aiding clinical decision making and monitoring responses to treatment. This review article discusses how studies in other human diseases have highlighted potential biochemical markers (biomarkers) of disease activity in secreted biofluids, as aids to determining disease and metabolic activity within tissues. Using, as examples, lessons learned in the study of disease activity and prognosis of other chronic inflammatory conditions, such as osteoarthritis and periodontal disease, this review highlights the potential of dermal extracellular matrix (ECM) components (collagens, proteoglycans, hyaluronan and glycoproteins) for such uses. The limitations of currently utilized techniques and the concept that analysis of ECM components in wound fluid may represent useful biomarkers of disease activity are also discussed.

Viability of Sirococcus clavigignenti-juglandacearum Conidia on Exoskeletons of Three Coleopteran Species.

Butternut canker, caused by Sirococcus clavigignenti-juglandacearum, is the primary cause of decline of butternut (Juglans cinerea). Conidia of the fungus have been isolated from several insect species. The vector potential of three species of Coleoptera, Astylopsis macula, Eubulus parochus, and Glischrochilus sanguinolentus, was studied during 2001 and 2002. Beetles were collected, rinsed, and artificially inoculated with conidia. All three species carried viable conidia up to 16 days. The mean number of conidia carried per beetle in 2001 was as follows: 3.21 million at 0 h to 11,371 at 384 h for A. macula; 3.91 million at 0 h to 57 at 384 h for E. parochus; and 355,742 at 0 h to 314 at 384 h for G. sanguinolentus. In 2002, the numbers were: 1.42 million at 0 h to 2,814 at 384 h for A. macula; 1.29 million at 0 h to 85 at 384 h for E. parochus; and 72,342 at 0 h to 0 at 192 h for G. sanguinolentus. Using scanning electron microscopy, conidia were observed on the abdomen, thorax, and legs of artificially inoculated individuals of each species and on the thorax and abdomen of field-collected A. macula and E. parochus. These data suggest that all three species are potential vectors of S. clavigignenti-juglandacearum; however, A. macula and E. parochus may be more effective vectors.

TGF-beta1 up-regulates paxillin protein expression in malignant astrocytoma cells: requirement for a fibronectin substrate.

Cytokines can influence the interactions between members of the integrin cell adhesion receptor family and the extracellular matrix thereby potentially affecting cell function and promoting cell adhesion, growth and migration of malignant astrocytoma tumor cells. As malignant astrocytoma cells synthesize TGF-beta1 in vivo, we analysed the effects of TGF-beta1 on signaling events associated with integrin receptor ligation, focusing on the effects on paxillin, a phosphorylated adaptor protein, that acts as a scaffold for signaling molecules recruited to focal adhesions. TGF-beta1-stimulation of primary astrocytes and serum-starved U-251MG malignant astrocytoma cells attached to fibronectin induced a substantial increase in the levels of paxillin protein (fivefold increase at 2.0 ng/ml) in a dose- and time-dependent manner compared to the levels observed on plating onto fibronectin in the absence of stimulation. In the astrocytoma cells, this resulted in an increase in the pool of tyrosine-phosphorylated paxillin, although it did not appear to alter the extent of phosphorylation of the paxillin molecules. In contrast, in primary astrocytes the protein levels were upregulated in the absence of a parallel increase in phosphorylation. The TGF-beta1-stimulated increase in paxillin levels required ligation of the fibronectin receptor, as it was not induced when the cells were plated onto vitronectin, collagen or laminin. The increase in the pool of paxillin on TGF-beta1 stimulation of the fibronectin-plated astrocytoma cells was associated with an increase in translation, but was not associated with an increase in the steady-state levels of paxillin mRNA. Stimulation with TGF-beta1 on a fibronectin substrate increased subsequent attachment and spreading of U-251MG cells onto fibronectin and, to a lesser extent, vitronectin, but not collagen. Our results indicate that physiologic levels of TGF-beta1 stimulate the expression of paxillin protein at the level of translation through a process that requires engagement of the fibronectin receptor, and promotes attachment and spreading of malignant astrocytoma cells on fibronectin.

Purification and identification of a human dermal extract component inhibitory to fibroblast proliferation.

It was previously shown that a citric acid buffer extract of human dermis (extract D) inhibited growth of human diploid fibroblasts in monolayer culture (Muir et al., 1997). Further fractionation has shown that the active principle is probably a proteoglycan, and that retention of its inhibitory activity is dependent on the use protease inhibitors throughout the extraction procedure. Elution of extract D from a DEAE-cellulose column produced four major peaks, each of which was subjected to SDS-PAGE as well as being tested for inhibitory activity on the growth of fibroblasts in culture. Peaks III and IV had no inhibitory effect, but peak I contained highly active material. Gels of this peak showed prominent bands of 120 kDa (corresponding to dermatan sulphate proteoglycan II, DS-PG II) and at 45 kDa (corresponding to the core protein). The latter band became more prominent when extract D which had been treated with chrondroitinase ABC was electrophoresed. Their identities were verified by Western blotting. Peak II also contained some slower-acting inhibitory material which has as yet to be identified, but contains little or no protein corresponding to the decorin core-protein. The data indicate that the intact decorin molecule, DS-PG II, is the main inhibitory principle in human skin.

Length variation in HV2 of the human mitochondrial DNA control region.

Hair samples were typed from three individuals who exhibited length heteroplasmy in the homopolymeric cytosine stretches (C-stretch) in hypervariable region 2 (HV2). The study demonstrated that for different hairs within an individual, the HV2 C-stretch region can vary with respect to the number of cytosines and/or proportion of C-stretch length variants. Length heteroplasmy may occur regardless of the prominent length variant present in this region. Differences in the number of cytosines at the C-stretch region, or a variation in the relative amounts of heteroplasmic length variants, cannot be used to support an interpretation of exclusion.

Quantitative three-dimensional analysis and diffusion modeling of oligonucleotide concentrations after direct intraparenchymal brain infusion.

We compared quantitative experimental results on the diffusion of 35S-labeled phosphorothioate oligonucleotide (PS-ODN) after intraparenchymal infusion in rat brain, with the distributions predicted by Fick's second law of diffusion. Fischer 344 rats underwent identical intracerebral infusions of 36S-PS-ODN. After 0, 5, 11, 23, and 47 h, groups of animals were sacrificed and sequential brain cryosections subjected to autoradiography. The resulting experimental data were compared to the predicted distributions, for estimation of the apparent free diffusion coefficient, D*. Volumes of distribution and total content of 36 S-PS-ODN in the parenchyma were also computed, to monitor loss of total material. The values for D* were within the expected range for the 21-mer PS-ODN used, but a progressive decrease in D* over time was noted. The model requires D* to remain constant and, thus, does not adequately explain the spread of 35S-PS-ODN following infusion. The progressive slowing of spread over time suggests that at later time points, 35S-PS-ODN may be fixed by tissue binding or cellular uptake in the brain. Loss of material via vascular and CSF clearance may also contribute to the lack of fit. Our results highlight issues to be addressed in the modeling and experimental design of the intraparenchymal infusion process.

The effect of periodontal treatment on glycemic control in patients with type 2 diabetes mellitus.

BACKGROUND, AIMS: This study was designed to explore the effect of periodontal therapy on glycemic control in persons with type 2 diabetes mellitus (DM). METHODS: 36 patients with type 2 DM (treatment group) received therapy for adult periodontitis during an 18-month period. A 36-person control group was randomly selected from the same population of persons with type 2 DM who did not receive periodontal treatment. RESULTS: These groups were well matched for most of the parameters investigated. During the nine-month observation period, there was a 6.7% improvement in glycemic control in the control group when compared to a 17.1% improvement in the treatment group, a statistically significant difference. Several parameters that could confound or moderate this glycemic control were explored. These included the treatment of non-dental infections, weight and medication changes. No moderating effect was associated with any of these variables. However, there were too few subjects in the study to have the statistical power necessary to assess these possible moderators of glycemic control. CONCLUSIONS: We interpret the data in the study to suggest that periodontal therapy was associated with improved glycemic control in persons with type 2 DM.

Hypocarbia during the first 24 postnatal hours and white matter echolucencies in newborns < or = 28 weeks gestation.

The purpose of the present study was to test the hypothesis that newborns < or = 28 wk gestation who have a PCO(2) measurement in the lowest gestational age-specific quartile (hypocarbia) on the first day of life are not at increased risk for ultrasonographic white matter echolucency (EL) after adjustment for confounders. The sample consisted of 799 infants < or = 28 wk gestation born during 1991-1993. Forty-eight infants with EL were classified as cases and compared with 751 controls, i.e. those without EL. We performed univariable comparisons, stratified analyses, and multivariable logistic regression. In the univariable analyses, hypocarbia on the first day of life was associated with an increased EL risk. The odds ratios for the hypocarbia-EL relationship were prominently elevated in the strata of infants who did not have other major risk factors for EL (e.g. gestational age 26-28 wk, normothyroxinemia, no characteristics of antenatal infection). In the multivariable analyses, the association diminished after adjustment with a hypocarbia propensity score (odds ratio = 1.7; 95 % confidence interval, 0.8-3.2) or with potential confounders.