Satellite Remote Sensing of Wheat Infected by Wheat streak mosaic virus.

The prevalence of wheat streak mosaic, caused by Wheat streak mosaic virus, was assessed using Landsat 5 Thematic Mapper (TM) images in two counties of the Texas Panhandle during the 2005-2006 and 2007-2008 crop years. In both crop years, wheat streak mosaic was widely distributed in the counties studied. Healthy and diseased wheat were separated on the images using the maximum likelihood classifier. The overall classification accuracies were between 89.47 and 99.07% for disease detection when compared to "ground truth" field observations. Omission errors (i.e., pixels incorrectly excluded from a particular class and assigned to other classes) varied between 0 and 12.50%. Commission errors (i.e., pixels incorrectly assigned to a particular class that actually belong to other classes) ranged from 0 to 23.81%. There were substantial differences between planted wheat acreage reported by the United States Department of Agriculture-National Agricultural Statistics Service (USDA-NASS) and that detected by image analyses. However, harvested wheat acreage reported by USDA-NASS and that detected by image classifications were closely matched. These results indicate that the TM image can be used to accurately detect and quantify incidence of wheat streak mosaic over large areas. This method appears to be one of the best currently available for identification and mapping disease incidence over large and remote areas by offering a repeatable, inexpensive, and synoptic strategy during the course of a growing season.

Multiplex real-time RT-PCR for detection of Wheat streak mosaic virus and Triticum mosaic virus.

Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are widespread throughout the southwestern Great Plains states. When using conventional diagnostics such as enzyme-linked immunosorbent assays (ELISA), these two viruses are commonly found together in infected wheat samples. Methods for molecular detection have been developed for wheat viral pathogens, but until recently no multiplex method for detection of both WSMV and TriMV within a single sample was available. Therefore, the objective of this study was to develop a multiplex real-time PCR technique for detection of both pathogens within a single plant sample. Specific primers and probe combinations were developed for detection of WSMV and TriMV, single and multiple reactions were run simultaneously to detect any loss in sensitivity during the multiplex reaction, as well as any cross-reaction with other common wheat viruses. The multiplex reaction was successful in detection of both pathogens, with little difference between single and multiplex reactions, and no cross-reaction was found with other common wheat viruses. This multiplex technique not only will be useful for diagnostic evaluations, but also as a valuable tool for ecological and epidemiology studies, and investigations of host/pathogen interactions, especially when the host is infected with both pathogens.

Inter-institutional pathology consultations for breast cancer: impact on clinical oncology therapy recommendations.

BACKGROUND: Despite recommendations favouring review of cancer pathology specimens for patients being treated at an institution other than the one that produced the initial pathology report, data regarding discordance rates and their potential clinical impact remain limited, particularly for breast cancer. At the QEII Health Sciences Centre in Halifax, Nova Scotia, it was routine practice to review histopathology when patients referred for adjuvant therapy had undergone their breast cancer surgery and pathology reporting at another institution. The aim of the present study was to determine the rate and clinical impact of discordance in inter-institutional pathology consultations for breast cancer in Nova Scotia. METHODS: We conducted a retrospective review of 100 randomly selected inter-institutional pathology consultations for breast cancer patients referred to the QEII in 2004. Cases were categorized as having either no discordance, discordance with no clinical impact, or discordance with potential for clinical impact. Cases with potential clinical impact were independently reviewed by 2 medical oncologists and 2 radiation oncologists, and the discordances were rated as having high, medium, or no clinical impact. RESULTS: The study cohort consisted of 93 cases that met the inclusion criteria. Of these 93 cases, 6 had no discordance, 7 had discordance with no clinical impact, and 80 had discordance with potential for clinical impact. Overall, 10 cases (11%) were rated as having either high or medium clinical impact, with agreement on the clinical impact ratings by oncologist reviewers in the same specialty. The remaining cases had either no clinical impact or disagreement on the clinical impact rating. CONCLUSIONS: Inter-institutional pathology consultations for breast cancer in Nova Scotia identified discordant findings with potential clinical impact as determined by oncologist reviewers. Further evaluation of inter-institutional pathology consultations and the impact on clinical decision-making is warranted.

Effects of Wheat streak mosaic virus on Root Development and Water-Use Efficiency of Hard Red Winter Wheat.

Greenhouse and field studies were conducted to determine the effects of Wheat streak mosaic virus (WSMV), a member of the family Potyviridae, on root development and water-use efficiency (WUE) of two hard red winter wheat (Triticum aestivum) cultivars, one susceptible and one resistant to WSMV. In the greenhouse studies, wheat cultivars were grown under three water regimes of 30, 60, and 80% soil saturation capacity. After inoculation with WSMV, plants were grown for approximately 4 weeks and then harvested. Root and shoot weights were measured to determine the effect of the disease on biomass. In all water treatments, root biomass and WUE of inoculated susceptible plants were significantly less (P < 0.05) than those of the noninoculated control plants. However, in the resistant cultivar, significance was only found in the 30 and 60% treatments for root weight and WUE, respectively. Field studies were also conducted under three water regimes based on reference evapotranspiration rates. Significant reductions in forage, grain yield, and crop WUE were observed in the inoculated susceptible plots compared with the noninoculated plots. Both studies demonstrated that wheat streak mosaic reduces WUE, which is a major concern in the Texas Panhandle because of limited availability of water.

Characterization and Epidemiological Significance of Potato Plants Grown from Seed Tubers Affected by Zebra Chip Disease.

An emerging disease of potato in the United States, known as "Zebra Chip" or "Zebra Complex" (ZC), is increasing in scope and threatens to spread further. Here, we report on studies performed to understand the role of tuberborne ZC in the epidemiology of this disease. Depending on variety, up to 44% of ZC-affected seed tubers (ZCST) were viable, producing hair sprouts and weak plants. Chip discoloration in progeny tubers of ZCST was more severe than those from ZC-asymptomatic seed tubers but varied depending on whether progeny tubers or foliage were positive or negative for 'Candidatus Liberibacter solanacearum'. A low percentage of greenhouse-grown plants produced by ZCST tested positive for 'Ca. Liberibacter'. No adult potato psyllids became infective after feeding upon these plants but they did acquire 'Ca. Liberibacter' from field-grown plants produced by ZCST. Plants with new ZC infections near plants produced by ZCST were not significantly different from healthy plants, whereas plants affected with ZC from infectious potato psyllids had significantly more ZC infections near either plants produced by ZCST or healthy plants. We conclude that, in areas where ZC is currently established, plants produced by ZCST do not significantly contribute to ZC incidence and spread within potato fields.

[Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors.

An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala12]MCD was 120-fold less potent in histamine-releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala12]MCD showed a 50% inhibition of IgE binding to the Fc epsilon RI alpha mast cell receptor by using rat basophilic leukemia (RBL-2H3) mast cells and fluorescence polarization. Furthermore, in a beta-hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala12]MCD analog and the parent MCD peptide. The present results show that [Ala12]MCD may provide a base for designing agents to prevent IgE/Fc epsilon RI alpha interactions and, consequently, allergic conditions.

Histamine-releasing activity and binding to the FcepsilonRI alpha human mast cell receptor subunit of mast cell degranulating peptide analogues with alanine substitutions.

We have investigated the effects on mast cell binding and the histamine-releasing activity of l-alanine substitutions for the five lysine residues and the proline residue in the MCD peptide (1) sequence. All synthesized analogues Ala(2) (2), Ala(6) (3), Ala(11) (4), Ala(12) (5), Ala(17) (6), and Ala(21) (7) showed a loss of histamine release compared to the parent MCD peptide 1. The order of decreased potency was 1 > 6 > 7 > 4 > 2 > 3 > 5. The alanine-substituted analogues showed a 5- to 6-fold decrease in histamine release for analogues 6, 7, and 4 and a 10-fold decrease for analogue 2. A more significant loss was observed in analogue 3 with a 75-fold loss of activity. The greatest loss of activity was observed with alanine substituting for proline in position 12. This analogue 5 showed a 130-fold loss of histamine release compared to the parent peptide 1. The ability of each analogue to interact with the FcepsilonRIalpha subunit of the human mast cell receptor was analyzed by competitive binding of the fluorescent peptide 1 and the alanine analogues using fluorescence polarization. The binding affinities of analogues 4, 6, and 7 for the mast cell receptor were less than the affinity of the native peptide 1. Analogues 2, 3, and 5 showed an increase in binding affinity, with analogue 5 showing the highest increase compared to the native peptide 1. The order of increased affinity was 5 > 3 > 2 > 1 > 4, 6, 7. On the basis of these results, the possibility that analogue 5 inhibits peptide 1-stimulated histamine release was examined. We found that peptide 5 did not inhibit histamine release by peptide 1. The analogues 2, 3, and especially analogue 5 may be useful leads toward study of agents that prevent binding of IgE to mast cell receptors.

Integration of animal linkage and BAC contig maps using overgo hybridization.

The alignment of genome linkage maps, defined primarily by segregation of sequence-tagged site (STS) markers, with BAC contig physical maps and full genome sequences requires high throughput mechanisms to identify BAC clones that contain specific STS. A powerful technique for this purpose is multi-dimensional hybridization of "overgo" probes. The probes are chosen from available STS sequence data by selecting unique probe sequences that have a common melting temperature. We have hybridized sets of 216 overgo probes in subset pools of 36 overgos at a time to filter-spotted chicken BAC clone arrays. A four-dimensional pooling strategy, including one degree of redundancy, has been employed. This requires 24 hybridizations to completely assign BACs for all 216 probes. Results to date are consistent with about a 10% failure rate in overgo probe design and a 15-20% false negative detection rate within a group of 216 markers. Three complete rounds of overgo hybridization, each to sets of about 39,000 BACs (either BAMHI or ECORI partial digest inserts) generated a total of 1853 BAC alignments for 517 mapped chicken genome STS markers. These data are publicly available, and they have been used in the assembly of a first generation BAC contig map of the chicken genome.

Analysis of antibody-antigen interactions in mixtures containing reactive and nonreactive components using size-exclusion high-performance (pressure) liquid chromatography.

Analysis of antibody-antigen interactions using size-exclusion high-performance (pressure) liquid chromatography was applied to a polyvalent system composed of both reactive and nonreactive components. Mixtures containing varying concentrations of antivenin (Crotalidae) polyvalent (equine origin) and either Crotalus atrox (Western diamondback rattlesnake) venom (CV) or isolated C. atrox phospholipase A2 (PLA2) were separated using a Bio-Sil SEC 250-5 size-exclusion column (300 x 7.8 mm). Major regions containing high-molecular-weight aggregates, antivenin IgG, and CV components or PLA2 could be identified from the elution profiles. Changes in elution profile areas could be modeled by equations derived from the law of mass action that included values for the maximum fraction of reactivity, antigen valence, apparent binding constants, profile area proportionality constants, and nonreactive profile area. The analysis was simple, fast, and readily interpretable and may be applicable to a variety of situations in which stable antigen-antibody complexes are formed in the presence of nonreactive components.

Further studies on the structural requirements for mast cell degranulating (MCD) peptide-mediated histamine release.

Mast cell degranulating (MCD) peptide was modified in its two disulfide bridges and in the two arginine residues in order to measure the ability of these analogs to induce histamine release from mast cells in vitro. Analogs prepared were [Ala(3,15)]MCD, [Ala(5,19)]MCD, [Orn(16)]MCD, and [Orn(7,16)]MCD. Their histamine-releasing activity was determined spectrofluorometrically with peritoneal mast cells. The monocyclic analogs in which the cysteine residues were replaced pairwise with alanine residues showed three-to ten-fold diminished histamine-releasing activity respectively, compared with the parent MCD peptide. Substantial increases in activity were observed where arginine residues were replaced by ornithines. The ornithine-mono substituted analog showed an almost six-fold increase and the ornithine-doubly substituted analog three-fold increase in histamine-releasing activity compared with the parent MCD peptide. The structural changes associated with these activities were followed by circular dichroism (CD) spectroscopy. Changes in the shape and ellipticity of the CD spectra reflected a role for the disulfide bonds and the two arginine residues in the overall conformation and biological activity of the molecule.

Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding.

Fluorescent and biotinylated analogs of mast cell degranulating (MCD) peptide were synthesized and the labels fluoresceinisothiocyanate and N-hydroxysuccinimidobiotin were conjugated at position 1 in the MCD peptide sequence. The analogs with these moieties retained histamine-releasing activity as high as that of the parent MCD peptide in rat peritoneal mast cell assays. These labeled analogs were used in rat basophilic leukemia cells (RBL-2H3) to demonstrate by confocal microscopy and flow cytometry the specific binding of MCD peptide to mast cell receptors. Consequently MCD peptide was found to compete with and inhibit the binding of fluorescent IgE on RBL cells as monitored by flow cytometry. Thus MCD peptide may prove to be useful in the study of IgE receptor-bearing cells.

Identification and characterization of PRKCBP1, a candidate RACK-like protein.

The receptors for activated C-kinase (RACK) family of proteins function as anchors for activated protein kinase C (PKC) isoenzymes. Using a monoclonal antibody to RACK1 in the screening of a human hippocampus cDNA library, we identified a novel member of the RACK family, designated PRKCBP1. Immunoprecipitation assays performed with a GST-fused PRKCBP1 protein suggest the carboxy terminus of PRKCBP1 interacts specifically with PKCbetaI. Northern analysis detected a 3.1-kb PRKCBP1 transcript in all tissues examined including brain, heart, liver, lung, pancreas, skeletal muscle, kidney, and spleen. The PRKCBP1 gene has been localized to human Chromosome (Chr) 20q12-13.1. Several groups have reported evidence for genetic linkage of Type II diabetes to this region in Caucasian families. This localization, combined with the observation that abnormalities in the activation, translocation, and inhibition of PKC occur in the development of Type II diabetes, suggested that PRKCBP1 was a candidate for contributing to Type II diabetes. We determined the PRKCBP1 coding sequence is 1845 bp in length, dispersed over 9 exons, spanning approximately 34 kb of genomic DNA. SSCP analysis was used to screen PRKCBP1 coding regions for mutations or polymorphisms in 100 Caucasian Type II diabetics and 100 Caucasian unaffected individuals. A silent C/T transition (bp1413, Thr137) was present in 23% of both diabetic and control individuals. A C/T transition (bp198) was also identified in a single diabetic individual, which resulted in no coding change (Ser66). The results of this analysis suggest that PRKCBP1 coding variations are uncommon and do not contribute to Type II diabetes in the general population.

The effects of conjugated deferoxamine in porcine skin flaps.

BACKGROUND: The free radical scavenger, deferoxamine (DFO) has been shown to reduce skin flap necrosis; however, its shortcomings are its toxicity and short plasma half-life. METHODS: This study investigates the effects of the less toxic, longer acting conjugated form, DFO-Hespan (DFO-H), to ischemic porcine skin flaps. During the study, DFO-H plasma concentrations and flap viability were evaluated over 10 days. RESULTS: Steady DFO serum levels were maintained with no evidence of systemic side effects. However, DFO-H was not effective in increasing porcine skin flap viability. Mean treated flap viability (n = 18) was 36.2% +/- 1.7% (mean +/- SE ) vs control (n = 16) 35.8% +/- 2.6%, p =.9. CONCLUSION: DFO-H conjugation increases its half-life and its systemic tolerance for DFO. However, this conjugation may also reduce DFO's effectiveness to preserve flap survival probably by decreasing its ability to reach the intracellular oxygen free radicals. In addition, further studies are needed to investigate whether longer DFO administration given postoperatively can be more effective in reducing ischemic injury.

Analysis of the HNF4 alpha gene in Caucasian type II diabetic nephropathic patients.

AIMS/HYPOTHESIS: Linkage and association studies in Caucasian patients with Type II (non-insulin-dependent) diabetes mellitus suggest that one or more diabetes susceptibility gene(s) reside within human chromosome 20q12-13.1. This region of chromosome 20 contains the maturity-onset diabetes of the young type 1 gene, HNF4 alpha. The purpose of this study was to assess the possible involvement of HNF4 alpha in Type II diabetes. METHODS: Mutation analysis was done on the 12 exons and promoter regions of the HNF4 alpha gene in 182 Caucasian diabetic nephropathic patients and 100 Caucasian control subjects. The functional consequences of a novel promoter mutation were examined using a reporter system in the HepG2 liver cell line and electrophoretic mobility shift assays. RESULTS: We identified two novel mutations in the HNF4 alpha, an R323H missense mutation in exon 8, and a 7 bp deletion (delta 7) in the proximal promoter region resulting in deletion of a single putative Sp1 binding site. Using a reporter assay system, the delta 7 sequence was found to exhibit a 51.2% (standard error +/- 4.2%) reduction in promoter activity relative to the normal sequence. In electrophoretic mobility shift assays using specific and non-specific competitors, the delta 7 sequence had a 45.5% (range 40.4-46.6) reduction in binding compared with the normal sequence. The delta 7 allele occurs in a family with multiple cases of Type II diabetes in a pattern consistent with coinheritance of the delta 7 allele and diabetes. CONCLUSION/INTERPRETATION: Analysis of the HNF4 alpha gene revealed two possible mutations in 182 diabetic patients which suggests that the HNF4 alpha gene does not make a large contribution to diabetes susceptibility in the general population of Caucasian diabetic nephropathic patients. Functional analysis of the delta 7 promoter deletion suggests, however, that promoter mutations in otherwise normal genes could contribute to diabetes susceptibility.

A physical map of the 20q12-q13.1 region associated with type 2 diabetes.

Several recent genetic studies have suggested linkage of Type 2 diabetes (non-insulin-dependent diabetes mellitus) susceptibility to a region of chromosome 20q12-q13.1. To facilitate the identification and cloning of a diabetes susceptibility gene(s) in this region, we have constructed correlated radiation hybrid and YAC/BAC contig physical maps of the region. A high-resolution radiation hybrid map encompassing 9.5 Mb between the PLC and the CEBPB genes was constructed using 68 markers: 25 polymorphic markers, 15 known genes, 21 ESTs, and 7 random genomic sequences. The physical order of the polymorphic markers within this radiation hybrid map is consistent with published genetic maps. A YAC/BAC contig that gives continuous coverage between PLC and CEBPB was also constructed. This contig was constructed from 24 YACs, 34 BACs, and 1 P1 phage clone onto which 71 markers were mapped: 23 polymorphic markers, 12 genes, 24 ESTs, and 12 random genomic sequences. The radiation hybrid map and YAC/BAC physical map enable precise mapping of newly identified transcribed sequences and polymorphic markers that will aid in linkage and linkage disequilibrium studies and facilitate identification and cloning of candidate Type 2 diabetes susceptibility genes residing in 20q12-q13.1.

Tricho-dento-osseous syndrome and amelogenesis imperfecta with taurodontism are genetically distinct conditions.

Amelogenesis imperfecta of the hypomaturation-hypoplasia type with taurodontism (AIHHT) is inherited as a highly penetrant autosomal dominant trait. These dental findings are similar to those of another autosomal dominant condition, the tricho-dento-osseous syndrome (TDO), from which AIHHT differs primarily by lack of changes in the hair and bones. TDO is characterized by a highly variable clinical phenotype. While enamel hypoplasia and taurodontism appear to be present in all TDO cases, non-dental features may be absent, with approximately half of TDO cases losing the kinky/curly hair phenotype seen in infancy by adolescence, and in almost 20% of cases, osseous changes are not evident. The genetic basis for AIHHT is unknown and it has been questioned whether AIHHT and TDO are separate conditions or a spectrum of disease. The genetic basis for TDO has recently been identified as a deletion mutation in the distal-less 3 (DLX3) transcription factor gene. To determine if AIHHT and TDO represent variable expression of a common DLX3 gene mutation, allelic mutations of the DLX3 gene, or mutations in DLX7 (the linked paralogue to DLX3 on chromosome 17), we have performed mutational analysis and sequencing studies of the DLX3 and DLX7 genes in three individuals (two affected and one unaffected) from a family with AIHHT. Results of the analysis demonstrate that AIHHT and TDO are not due to a common DLX3 gene mutation. Sequence analyses of the DLX3 and DLX7 genes suggest AIHHT is not due to genetic mutations or polymorphisms in the exons of these genes. These results suggest that AI-HHT and TDO are two genetically distinct conditions.

Melanoma vaccines as a therapeutic option.

BACKGROUND: In 1998, 41,600 new cases of melanoma with 7,300 deaths were expected. Worldwide, the incidence has risen 5% a year against a backdrop of generally decreasing cancer trends. Later stages of melanoma carry a severe prognosis. The need for newer, more effective therapeutic strategies for cancer is obvious. For melanoma, early diagnosis and surgical treatment are the only options that are currently curative. Chemotherapy and radiation therapy are of limited efficacy. METHODS: We reviewed the various forms of immunotherapy, concentrating on vaccine therapy. We then reviewed the history of our own vaccine in the context of the field of immunotherapy, and compared efficacy, immune response, production methods, and survival. RESULTS: Survival is improved among recipients of melanoma vaccine when compared with patients receiving conventional therapy. CONCLUSIONS: Imnmunotherapy in the form of melanoma vaccines is better than conventional therapy and is trending toward purer antigenic preparations.

Postoperative ventilatory management.

Immediate postoperative evaluation of the patient remains a crucial role of the intensivist. Postoperative patients can be divided into the otherwise healthy, chronically ill, and acutely ill for strategizing about care. For chronically ill and acutely ill patients who require ongoing ventilation, ventilator management continues to evolve toward modes that are more interactive with patient needs. Newer modes of ventilation are also being explored to protect the lung against damage attributable to mechanical ventilation. Weaning indexes and associated protocols have become more sophisticated and now allow physicians greater certainty in evaluating patients' readiness for extubation. This article will discuss factors to be considered prior to extubation as well as the latest ventilatory and weaning strategies.