Role of boost radiotherapy for local control of pure ductal carcinoma in situ after breast-conserving surgery: a multicenter, retrospective study of 622 patients.

PURPOSE: To evaluate the effect of boost radiotherapy on ipsilateral breast tumor recurrence (IBTR) for ductal carcinoma in situ (DCIS) after breast-conserving surgery and whole breast radiotherapy (WBRT) with or without boost. METHODS AND MATERIALS: Retrospective, multicentre study of 622 patients (624 tumors) diagnosed with pure DCIS from 1993-2011. RESULTS: Most tumors (377/624; 60.4%) received a boost. At a median follow-up of 8.8 years, IBTR occurred in 64 cases (10.3%). A higher percentage of patients with risk factors for IBTR received a boost (p < 0.05). Boost was not associated with lower rates of IBTR than WBRT alone (HR 0.75, 95% CI 0.42-1.35). On the univariate analyses, IBTR was significantly associated with tumor size (11-20 mm, HR 2.32, 95% CI 1.27-4.24; and > 20 mm, HR 2.10, 95% CI 1.14-3.88), re-excision (HR 1.76, 95% CI 1.04-2.96), and tamoxifen (HR 2.03, 95% CI 1.12-3.70). Boost dose > 16 Gy had a protective effect (HR 0.39, 95% CI 0.187-0.824). Multivariate analyses confirmed the independent associations between IBTR and 11-20 mm (p = 0.02) and > 20 mm (p = 0.009) tumours, and re-excision (p = 0.006). On the margin-stratified multivariate analysis, tamoxifen was a poor prognostic factor in the close/positive margin subgroup (HR 4.28 95% CI 1.23-14.88), while the highest boost dose ( > 16 Gy) had a significant positive effect (HR 0.34, 95% CI 0.13-0.86) in the negative margin subgroup. CONCLUSIONS: Radiotherapy boost did not improve the risk of IBTR. Boost radiotherapy was more common in patients with high-risk disease. Tumor size and re-excision were significant independent prognostic factors.

Management of breast ductal carcinoma in situ in Catalonia, Spain: Results from the Grup Oncologic Calalà-Occità-Catalonia survey with 9-year follow up.

INTRODUCTION: Reliable data on DCIS incidence and management are not available in many countries. The present study describes the management of DCIS in Catalonia, Spain in the year 2005 and compares these findings to data obtained in France. Local recurrence and late toxicity rates from 2005 through the end of 2014 are reported. MATERIALS AND METHODS: Observational survey of patients with pure DCIS (n = 270) diagnosed during 2005. A written questionnaire, the same as used in the French survey, was completed by 14 doctors at 12 cancer centres in Catalonia, Spain. RESULTS: Median patient age was 55 years (range, 29-89). Diagnosis was mammographic in 225 cases (83.3%). Treatment approaches included: mastectomy (10.4% of cases), breast-conserving surgery (BCS) alone (3.7%), and BCS plus radiotherapy (RT) (85.5%). Sentinel node biopsy and axillary dissection were performed in 27.4% and 5.6% of patients, respectively. Hormonotherapy was prescribed in 45.2% of cases. Tumour nuclear grade was as follows: low (16.7% of cases), intermediate (23%), and high (55.6%). Excision was complete (margins ≥1 mm) in 75% of patients treated with BCS alone vs. 95.7% for BCS+RT. The treatment approach varied widely: mastectomy rates ranged from 7.1% to 26.7% of centres, BCS+RT from 55.5% to 87.8%, and hormonotherapy from 3.3% to 83.3%. At a median follow-up of 102.6 months, 14 patients (5.6%) presented ipsilateral breast tumour recurrence. CONCLUSIONS: These findings on DCIS management in Catalonia are consistent with previous international reports. The inter-centre differences observed are similar to those reported in other international surveys during the same period.

Single-pump heart-cutting two-dimensional liquid chromatography applied to the determination of fatty alcohol ethoxylates.

A setup for heart-cutting bi-dimensional liquid chromatography (LC-LC), constructed with a chromatograph provided with a single pump, an auxiliary 6-port 2-position valve (V6/2) and a column selector valve (VCS), is described. The possible ways of connecting the two valves for LC-LC, namely with V6/2 first followed by VCS and vice versa, are compared. The possibility of using the setups for preconcentration followed by the backwards transfer of the preconcentrated solutes to the detector or to a second column is also shown. The V6/2-first configuration for LC-LC was applied to the characterization of industrial fatty alcohol ethoxylates (FAEs) using UV-vis detection. For this purpose, the phthalates of the FAE oligomers were first obtained. The hydrocarbon series were separated along the 1st dimension by MeOH/water gradient elution on a C8 column at 60°C. Selected segments of the eluate were transferred to the 2nd dimension, where the EO oligomers of the isolated series were resolved by gradient elution with a complementary ACN/water mobile phase on a C8 column at 25°C. In addition, an average response factor of the hydrocarbon series of FAEs was proposed. To apply the factors, the average EO number of the series is first established by chromatographing one of the series along the 2nd dimension. Then, the factors are used to correct the peak areas of the isolated series which are obtained along the 1st dimension chromatogram, thus allowing the fast and accurate determination of the series in industrial FAEs. The method is particularly useful to characterize FAEs having large average EO numbers or constituted by mixtures of even and odd series.

Determination of non-ionic and anionic surfactants in industrial products by separation on a weak ion-exchanger, derivatization and liquid chromatography.

A method for the determination of priority surfactants, including fatty alcohol ethoxylates (FAE), alkylether sulfates (AES) and linear alkylbenzene sulfonates (LAS) is described. The samples were diluted with 50% methanol at pH 4 prior to solid-phase extraction on a weak anionic exchanger (WAX). The AES and LAS surfactant classes were retained, whereas the non-ionic components, including most FAE oligomers were eluted. After washing the WAX cartridge to remove cations, the remaining hydrophobic FAE oligomers were eluted using hot 80% methanol at pH 4 (at ca. 50°C). These two eluates were combined to constitute the non-ionic fraction. Then, AES and LAS were eluted using 80% MeOH with 3M NH3 followed by 95% methanol with 0.75M NH3. The two eluates obtained in basic media were combined to constitute the anionic fraction. The solvents were evaporated, the residues were dissolved in 1,4-dioxane, and esterification of the alcohols and transesterification of AES with phthalic anhydride was performed. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid and 0.1M NaClO4. The chromatogram of the non-ionic fraction showed the peaks of the resolved FAE oligomers. The chromatogram of the anionic fraction showed the peaks of the LAS homologues well resolved from those of the AES oligomers. The method was applied to laundry and industrial cleaners, shampoos and a shower gel.

Survival and clinicopathological characteristics of breast cancer patient according to different tumour subtypes as determined by hormone receptor and Her2 immunohistochemistry. a single institution survey spanning 1998 to 2010.

As far as recent breast cancer molecular subtype classification is concerned, much work has dealt with clinical outcomes for triple negative and Her2 patients. Less is known about the course of patients in the remaining subtypes. Molecular classification based on immunohistochemistry is widely available and correlates well with genetic microarray assessment, but at a lower cost. The aim of our investigation was to correlate immunohistochemical subtypes of breast cancer with clinical characteristics and patient outcomes. Since 1998, 1167 patients operated for 1191 invasive breast tumours were included in our database. Patients were regularly followed up until March 2010. Disease-free survival, overall mortality, and breast cancer-specific mortality at 5 years were calculated for the cohort. 72% of tumours were ER+PR±HER2- group, 13% triple negative (ER-PR-HER2-), 10% ER+PR±HER2+ group, and 5% Her2 (ER-PR-HER2+). Cancer-specific survival was 94.2% for the ER+PR+HER2- subtype, 84.8% for the Her2 subtype, 83.3% for the ER+PR-HER2- subtype, and 78.6% for triple negatives. Distant metastases prevalence ranged from 7% to 22% across subtypes, increasing stepwise from ER+PR+HER2-, ER+PR+HER2+, ER+PR-HER2-, ER+PR-HER2+, ER-PR-HER2+ through triple negative. Small, low-grade tumours with low axillary burden were more likely to belong to the ER+PR±HER2- group. Conversely, larger high-grade tumours with significant axillary burden were more likely to belong to Her2 or triple negative groups. ER+PR±HER2- group patients with negative PR receptors performed more like Her2 or triple negative than like the rest of ER+PR±HER2± groups patients. Molecular classification of breast tumours based only on immunohistochemistry is quite useful on practical clinical grounds, as expected. ER+PR±HER2- group patients with negative PR receptors seem to be at high risk and deserve further consideration.

Calculation of diagnostic parameters of advanced serological and molecular tissue-print methods for detection of Citrus tristeza virus: a model for other plant pathogens.

Citrus tristeza virus (CTV) is one of the most important virus diseases that affect citrus. Control of CTV is achieved by grafting selected virus-free citrus scions onto CTV-tolerant or -resistant rootstocks. Quarantine and certification programs are essential for avoiding the entry and propagation of severe strains of CTV. Citrus nurseries in Spain and central California (United States) maintain zero-tolerance policies for CTV that require sensitive, specific, and reliable pathogen-detection methods. Tissue-print (TP) real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was compared with the validated TP enzyme-linked immunosorbent assay (ELISA), using the CTV-specific monoclonal antibodies 3DF1 and 3CA5, for CTV detection. In total, 1,395 samples from healthy and CTV-infected nursery and mature tree plants were analyzed with both methods. The total agreement between both detection methods was substantial (Cohen's kappa index of 0.77 ± 0.03). The diagnostic parameters of each technique (i.e., the sensitivity, specificity, and likelihood ratios) were evaluated in a second test involving 658 Citrus macrophylla nursery plants. Mexican lime indexing was used to evaluate samples with discrepant results in the analysis. For TP-ELISA, a sensitivity of 0.8015, a specificity of 0.9963, and a positive and negative likelihood ratio of 216.42 and 0.199, respectively, were estimated. For TP real-time RT-PCR, a sensitivity of 0.9820, a specificity of 0.8519, and a positive and negative likelihood ratio of 6.63 and 0.021, respectively, were estimated. These diagnostic parameters show that TP real-time RT-PCR was the most sensitive technique, whereas TP-ELISA showed the highest specificity, validating the use of the molecular technique for routine CTV-detection purposes. In addition, our results show that the combination of both techniques can accurately substitute for the conventional biological Mexican lime index for the detection of CTV. The calculation of diagnostic parameters is discussed, as a necessary tool, to validate detection or diagnostic methods in plant pathology. Furthermore, assessment of the post-test probability of disease after a diagnostic result and CTV prevalence allows selection of the best method for accurate and reliable diagnosis.

Determination of fatty alcohol ethoxylates and alkylether sulfates by anionic exchange separation, derivatization with a cyclic anhydride and liquid chromatography.

A method for the separation, characterization and determination of fatty alcohol ethoxylates (FAE) and alkylether sulfates (AES) in industrial and environmental samples is described. Separation of the two surfactant classes was achieved in a 50:50 methanol-water medium by retaining AES on a strong anionic exchanger (SAX) whereas most FAE were eluted. After washing the SAX cartridges to remove cations, the residual hydrophobic FAE were eluted by increasing methanol to 80%. Finally, AES were eluted using 80:20 and 95:5 methanol-concentrated aqueous HCl mixtures. Methanol and water were removed from the FAE and AES fractions, and the residues were dissolved in 1,4-dioxane. In this medium, esterification of FAE and transesterification of AES with a cyclic anhydride was performed. Phthalic and diphenic anhydrides were used to derivatizate the surfactants in industrial samples and seawater extracts, respectively. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid. Good resolution between both the hydrocarbon series and the successive oligomers within the series was achieved. Cross-contamination of FAE with AES and vice versa was not observed. Using dodecyl alcohol as calibration standard, and correction of the peak areas of the derivatized oligomers by their respective UV-vis response factors, both FAE and AES were evaluated. After solid-phase extraction on C18, the proposed method was successfully applied to the characterization and determination of the two surfactant classes in industrial samples and in seawater.

Comparison of monolithic and microparticulate columns for reversed-phase liquid chromatography of tryptic digests of industrial enzymes in cleaning products.

Enzymes of several classes used in the formulations of cleaning products were characterized by trypsin digestion followed by HPLC with UV detection. A polymeric monolithic column (ProSwift) was used to optimize the separation of both the intact enzymes and their tryptic digests. This column was adequate for the quality control of raw industrial enzyme concentrates. Then, monolithic and microparticulate columns were compared for peptide analysis. Under optimized conditions, the analysis of tryptic digests of enzymes of different classes commonly used in the formulation of cleaning products was carried out. Number of peaks, peak capacity and global resolution were obtained in order to evaluate the chromatographic performance of each column. Particulate shell-core C18 columns (Kinetex, 2.6 µm) showed the best performance, followed by a silica monolithic column (Chromolith RP-18e) and the conventional C18 packings (Gemini, 5 µm or 3 µm). A polymeric monolithic column (ProSwift) gave the worst performances. The proposed method was satisfactorily applied to the characterization of the enzymes present in spiked detergent bases and commercial cleaners.

First Report on Almond in Europe of Bacterial Spot Disease of Stone Fruits Caused by Xanthomonas arboricola pv. pruni.

Symptoms characteristic of bacterial spot disease of Prunus spp. (4) were observed on almond trees (Prunus dulcis (Mill.) Webb) in 14 localities of Comunidad Valenciana (eastern Spain) and Aragón (northeastern Spain) between 2006 and 2009. Symptoms were first noted in the spring and were observed until leaf fall. Initial infections began on leaves as small, angular, water-soaked spots, which mainly developed toward the tip and along the leaf margins. These water-soaking lesions were surrounded by chlorotic tissue, although chlorosis did not extend more than a few millimeters. Subsequently, the lesions turned light brown, necrotic, and sometimes the necrotic spots fell out. When the lesions coalesced, they produced large necrotic areas. Sometimes premature leaf drop of infected leaves was observed in severely affected trees. Infected fruits initially displayed sunken, corky lesions that oozed gum, which later became raised when the mesocarp dehydrated. Infected fruits either dropped prematurely or remained on trees after harvest. Cankers typical of bacterial spot disease of stone fruit trees were observed on branches and shoots. Isolations from diseased leaves and fruits yielded Xanthomonas-like colonies on YPGA medium (yeast extract, peptone, and glucose agar), which were subsequently purified and characterized. All strains were gram-negative rods, oxidase negative, and strictly aerobic and showed typical biochemical characteristics of the Xanthomonas genus (3). A collection of 70 strains were further identified by PCR with primers Y17CoF/Y17CoR (1) as Xanthomonas arboricola pv. pruni by comparison with reference strains ISPaVe B4 and ISPaVe B6 isolated from Prunus salicina in Italy. A selection of 46 strains were also analyzed by immunofluorescence (IF) and ELISA using commercial polyclonal antibodies from NEOGEN Europe Ltd. (Ayrshire, Scotland, UK) and SEDIAG S.A.S. (Longvic, France), respectively), although ELISA antibodies proved to be not specific for X. arboricola pv. pruni. Pathogenicity was confirmed by inoculation of 70 almond strains and the reference strains on leaves of potted almond trees and/or on detached leaves (2) with bacterial suspensions (107 CFU per ml). One leaf was inoculated at 8 to 10 sites per strain. Characteristic bacterial spot disease symptoms (4) appeared on all inoculated leaves after 1 week of incubation at 25°C and high humidity, but not on the negative controls infiltrated with sterile distilled water. The original pathogen was reisolated from lesions of inoculated leaves and confirmed by biochemical tests, IF and PCR. As observed in Spain, the disease produces serious damage on the most susceptible almond cultivars like Antoñeta, Guara, Marta, Mas Bovera, and Vayro and can be very harmful, with severity of infection depending upon the relative cultivar susceptibility and environmental conditions. Appropriate eradication measures were taken after the causal agent was confirmed as X. arboricola pv. pruni. This pathogen was previously reported on almond in Japan and New Zealand (4). To our knowledge, this is not only the first report on almond in Spain but also in Europe. References: (1) M. C. Pagani. Ph. D. thesis, North Carolina State University, Raleigh, 2004. (2) P. S. Randhawa and E. L. Civerolo. Phytopathology 75:1060, 1985. (3) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995. (4) J. M. Young. N. Z. J. Agric. Res. 20:105, 1977.

Comparison on photo-initiators for the preparation of methacrylate monolithic columns for capillary electrochromatography.

The synthesis of lauryl methacrylate monoliths for capillary electrochromatography by UV polymerization using several free-radical initiators (alpha,alpha'-azobisisobutyronitrile, 2,2-dimethoxy-2-phenylacetophenone, dibenzoyl peroxide (BPO) and lauroyl peroxide (LPO)) has been investigated. Using a 1,4-butanediol/1-propanol mixture as porogenic solvent, the influence of each initiator and its content on the morphological and electrochromatographical properties of beds was evaluated. Under their respective optimum content, satisfactory separations of a test mixture of PAHs with similar efficiencies (minimum plate heights of 8.0-12.7 microm obtained from Van Deemter plots) were achieved for the four investigated photo-initiators. The columns photo-polymerized with LPO provided the best compromise between chromatographic performance and analysis time. Moreover, this initiator showed a fine control in the column retention properties. The resulting monolithic columns exhibited a good run-to-run repeatability in the tested chromatographic parameters (RSD<2.4%) for all initiators investigated; and satisfactory column-to-column repeatability (RSD<6.0%), except for beds photo-polymerized with BPO (RSD<10.8%).

Characterization and determination of poly(vinylpyrrolidone) by complexation with an anionic azo-dye and nonequilibrium capillary electrophoresis.

Using capillary zone electrophoresis in nonequilibrium conditions, the complexes of poly(vinylpyrrolidone) (PVP) with anionic azo-dyes dissociate following a first-order kinetics. Two peaks due to the remaining PVP-dye complexes and the equilibrium concentration of the free dye, plus an exponential region due to the dye liberated by the complexes during the electrophoretic run, are obtained. This behaviour was closely similar to that described in the literature for protein-probe and DNA-protein mixtures, upon application of the technique known as nonequilibrium capillary electrophoresis of equilibrium mixtures or NECEEM. Using Congo Red and Acid Blue 113, information about the maximal stoichiometry and average stability of the PVP-dye complexes was obtained. The procedure was also useful to predict the average molecular mass of PVP and to determine PVP in cleaning products and pharmaceutical preparations. By using an appropriate probe, the procedure should be also useful to characterize and determine many other synthetic or natural nonionic polymers, and to study polymer-probe interactions.

Enzyme class identification in cleaning products by hydrolysis followed by derivatization with o-phthaldialdehyde, HPLC and linear discriminant analysis.

The enzymes present in raw materials of the cleaning industry (enzyme industrial concentrates) and in household cleaners were isolated by precipitation with acetone and hydrolyzed with HCl. The resulting amino acids were derivatized with o-phthaldialdehyde, and the derivatives were separated by HPLC. The peaks of 14 amino acids were observed using a C18 column and a multi-segmented gradient of acetonitrile-water in the presence of a 5 mM citric/citrate buffer of pH 6.5. Using either normalized peak areas (divided by the sum of the peak areas of the chromatogram) or ratios of pairs of peak areas as predictor variables, linear discriminant analysis models, capable of predicting the enzyme class, including proteases, lipases, amylases and cellulases, were constructed. For this purpose, both enzyme industrial concentrates and detergent bases spiked with them were included in the training set. In all cases, the enzymes of the evaluation set, including industrial concentrates, spiked detergent bases and commercial cleaners were correctly classified with assignment probabilities higher than 99%.

Graft transmission efficiencies and multiplication of 'Candidatus Liberibacter americanus' and 'ca. Liberibacter asiaticus' in citrus plants.

In Brazil 'Candidatus Liberibacter asiaticus' and 'Ca. L. americanus' cause huanglongbing (also known as greening), the most destructive citrus disease. A shift in pathogen prevalence was observed over time, with a disproportional increase in 'Ca. L. asiaticus' occurrence. Graft transmission experiments were used for a comparative study of both species using budsticks from symptomatic branches of field-affected trees as inoculum. The plants were inoculated with 'Ca. L. asiaticus' or 'Ca. L. americanus' alone, or simultaneously with both species. Symptom manifestation and conventional and quantitative real-time polymerase chain reaction were used for plant evaluations. 'Ca. L. americanus' was detected mainly in symptomatic plants and 'Ca. L. asiaticus' was detected in symptomatic plants as well as in infected plants prior to symptom manifestation. Transmission percentages varied from 54.7 to 88.0% for 'Ca. L. asiaticus' and 10.0 to 45.2% for 'Ca. L. americanus' in two experiments. In co-inoculated plants, 12.9% contained 'Ca. L. americanus' only, 40.3% contained 'Ca. L. asiaticus' only, and 19.3% contained both species. Average bacterial titers for 'Ca. L. asiaticus' and 'Ca. L. americanus', in log cells per gram of leaf midrib, were 6.42 and 4.87 for the experimental plants and 6.67 and 5.74 for the field trees used as the source of inoculum. The higher bacterial populations of the 'Ca. L. asiaticus'-infected plants provided an explanation for the disproportional increase in field prevalence of this species over time, based on the greater likelihood for pathogen transmission by the insect vector.

First Report of Pepper as a Natural Host for Pelargonium zonate spot virus in Spain.

Pelargonium zonate spot virus (PZSV) was first reported on Pelargonium zonale (L.) L'Hér. ex Aiton and later on tomato in Italy, Spain, France (1), and the United States (2). In Spain, PZSV was first detected in 1996 in tomato plants of cv. Royesta from greenhouses in Zaragoza Province (3) and subsequently in tomato in the Catalonia and Navarra areas. In April 2006, symptoms of PZSV were found at high incidence on tomato in a greenhouse in Huesca, Aragón (northeastern Spain). Randomly distributed pepper plants (Capsicum annuum L.) of cv. Estilo F1 growing in the same greenhouse showed severe foliar chlorotic ringspots and line patterns similar to those observed in tomato. Samples from symptomatic peppers and tomatoes and one asymptomatic weed of Rubia tinctorum L. tested positive by double-antibody sandwich (DAS)-ELISA using polyclonal antibodies against PZSV (Agdia Inc., Elkhart, IN and DSMZ, Braunschweig, Germany) as did a Spanish PZSV isolate used as a positive control (3). Sap extracts from two tomatoes, three peppers, and the single R. tinctorum plant were mechanically inoculated to 22 indicator species, including pepper and tomato. On 17 of 22 species inoculated, sap from symptomatic tomatoes and peppers elicited local or systemic symptoms similar to those reported earlier for PZSV isolates (3). Systemic symptoms were mainly mosaic, chlorotic, and necrotic line patterns and ringspots on leaves of most indicator species, closely resembling those observed on the greenhouse pepper and tomato plants. Symptoms on inoculated tomatoes also included stem necrosis and death. Reactions of indicator species did not indicate the presence of any other pepper- or tomato-infecting viruses. Both field infected and mechanically inoculated plants of pepper cvs. Yolo Wonder and Doux des Landes were maintained in the greenhouse until the development of fruit symptoms. Only fruits of cv. Yolo Wonder showed dark green and slightly depressed circles on their surface. Local and systemic infection by PZSV was confirmed by DAS-ELISA in most inoculated plants. Total RNA from leaves of field or inoculated plants was used as template for amplification by reverse transcription (RT)-PCR with primers R3-F and R3-R that are specific for the PZSV 3a gene (2), and amplicons were sequenced directly. The sequences of 697 nt from pepper and tomato isolates from the same greenhouse were identical (GenBank Accession Nos. CQ178217 and CQ178216, respectively) and had 96.1% identity to nucleotides 384 to 1,080 in PZSV RNA-3 (NC_003651). Our results confirm the natural infection of pepper plants in Huesca by PZSV. To our knowledge, this is the first report of pepper as a natural host for PZSV, a significant finding considering the potential risks of PZSV dispersion whenever tomato and pepper coexist, particularly in greenhouses and nurseries. References: (1) M. Finetti-Sialer and D. Gallitelli. J. Gen. Virol. 84:3143, 2003. (2) H. Y. Liu and J. L. Sears. Plant Dis. 91:633, 2007. (3) M. Luis-Arteaga and M. A. Cambra. Plant Dis. 84:807, 2000.

Liberibacters Associated with Citrus Huanglongbing in Brazil: 'Candidatus Liberibacter asiaticus' Is Heat Tolerant, 'Ca. L. americanus' Is Heat Sensitive.

In São Paulo State, Brazil, 'Candidatus Liberibacter americanus' and 'Candidatus Liberibacter asiaticus' are associated with huanglongbing (HLB). Affected municipalities occur mainly in the central and southern regions, where the annual number of hours above 30°C is two to five times lower than that in the extreme northern and western regions. The influence of temperature on sweet orange trees infected with 'Ca. L. asiaticus' or 'Ca. L. americanus' was studied in temperature-controlled growth chambers. Symptom progression on new shoots of naturally infected and experimentally graft-inoculated symptomatic sweet orange trees was assessed. Mottled leaves developed on all infected trees at 22 to 24°C, but not on any 'Ca. L. americanus'-infected trees at 27 to 32°C. Quantitative, real time-PCR was used to determine the liberibacter titers in the trees. After 90 days, 'Ca. L. asiaticus'-infected trees had high titers at 32 and 35°C, but not at 38°C, while 'Ca. L. americanus'-infected trees had high titers at 24°C, but at 32°C the titers were very low or the liberibacters could not be detected. Thus, the multiplication of 'Ca. L. asiaticus' is not yet affected at 35°C, while a temperature of 32°C is detrimental to 'Ca. L. americanus'. Thus, 'Ca. L. americanus' is less heat tolerant than 'Ca. L. asiaticus'. The uneven distribution of these two liberibacters in São Paulo State might be in relation with these results.

La relación sueño-epilepsia / The sleep epilepsy relation

En la presente revisión se agrupan los distintos aspectos de la relación entre el sueño y la epilepsia. Se analiza el impacto del sueño en la epilepsia concretado en fenómenos como la presentación circadiana de las crisis epilépticas y distinguiendo entre epilepsias relacionadas con el sueño, con el despertar y difusas. Asimismo, se describe la influencia de los distintos elementos del sueño en la actividad epileptiforme, tanto crítica como intercrítica, y se enfatiza en el efecto facilitador del sueño NREM, del despertar y de la privación de sueño. En sentido inverso, se describe el efecto de la epilepsia en la arquitectura del sueño, tanto de las crisis y del propio síndrome epiléptico, como de los fármacos anticomiciales. Por último, se reseña la importancia de valorar la posible coexistencia de trastornos del sueño en los pacientes epilépticos y la necesidad de tratar esos trastornos para un mejor manejo y control de las crisis y del funcionamiento cognitivo de los pacientes (AU)

In this paper sleep/epilepsy interaction is analized. We review the sleep impact on epilepsy including the circadian presentation of seizures and the effects of NREM, REM and sleep deprivation on inctal and interictal epileptiform discharges. Inversely we describe the impact of individual seizures and anticonvulsant drugs on sleep structure. Finally recognition and treatment of sleep disorders coexisting in epileptic patients is important for optimizing their management and may improve seizure control and daytime alertness (AU)

Field Trials of Plum Clones Transformed with the Plum pox virus Coat Protein (PPV-CP) Gene.

Transgenic clones C2, C3, C4, C5, C6, and PT-6, of plum (Prunus domestica L.) transformed with the coat protein (CP) gene of Plum pox virus (PPV), PT-23 transformed with marker genes only, and nontransgenic B70146 were evaluated for sharka resistance under high infection pressure in field trials in Poland and Spain. These sites differed in climatic conditions and virus isolates. Transgenic clone C5 showed high resistance to PPV at both sites. None of the C5 trees became naturally infected by aphids during seven (Spain) or eight (Poland) years of the test, although up to 100% of other plum trees (transgenic clones and nontransgenic control plants) grown in the same conditions showed disease symptoms and tested positively for PPV. Although highly resistant, C5 trees could be infected artificially by chip budding or via susceptible rootstock. Infected C5 trees showed only a few mild symptoms on single, isolated shoots, even up to 8 years post inoculation. These results clearly indicate the long-term nature and high level of resistance to PPV obtained through genetically engineered resistance.

QTL analysis of citrus tristeza virus-citradia interaction.

Citrus tristeza virus (CTV) has caused the death of millions of trees grafted on sour orange ( Citrus aurantium). However, this rootstock is very well adapted to the Mediterranean, semi-arid conditions. The aim of the present research is to genetically analyze the accumulation of CTV in a progeny derived from the cross between C. aurantium and Poncirus trifoliata, both resistant to CTV isolate T-346. Graft propagation of 104 hybrids was done on healthy sweet orange as a rootstock. Three months later, each rootstock was graft inoculated with two patches of infected tissue (isolate T-346). One, 2, and sometimes, 3 and 4 years after inoculation, hybrids and infected patches were tested for CTV by tissue-blot immuno-assay. Additionally, CTV multiplication was evaluated every year as the optical density of double-antibody sandwich enzyme-linked immuno-sorbent assay reactions. Linkage maps for P. trifoliata based on 63 markers, and for C. aurantium based on 157 markers, were used. Most molecular markers were microsatellites and IRAP (inter-retrotransposon amplified polymorphisms). Some analogues of resistance and expressed sequences were also included for candidate gene analysis. Resistance against CTV was analyzed as a quantitative trait (CTV accumulation) by QTL (quantitative trait loci) analysis to avoid the assumption of monogenic control. Three major resistance QTLs were detected where the P. trifoliata resistance gene, Ctv-R, had been previously located in other progenies. Up to five minor QTLs were detected ( Ctv-A(1) to Ctv-A(5)). A significant epistatic interaction involving Ctv-R(1) and Ctv-A(1) was also found. An analogue of a resistance gene is a candidate for Ctv-A(3), and two expressed sequences are candidates for Ctv-A(1) and Ctv-A(5). Single-strand conformational polymorphism analysis of CTV genes QTL P20 and P25 (coat protein) in susceptible hybrids, was carried out to test whether or not any QTL accumulation was a defeated resistance gene. Since the same haplotype of the virus was visualized independently on the CTV titer, differences in the amount of virions are not explained through the selection of CTV genotypes by the host, but through differences among citradias in CTV replication and/or movement.

Borage (Borago officinalis) is a New Host of Pseudomonas cichorii in the Ebro Valley of Spain.

Borage (Borago officinalis L.) is an important vegetable crop for consumption in the Ebro Valley of northeastern Spain. During the autumn and spring of the last 10 years, black necrotic lesions were observed in leaf petioles of white-flowered borage plants in greenhouses and seedbeds in Zaragoza, Spain. These lesions developed from the soil line and spread upward to the central vein of leaves. Severely infected leaves of mature and occasionally young plants become rotted. Longitudinal sections through the crown revealed severely necrotic cortical, vascular, and pith tissues. Isolations from infected roots, petioles, and leaves onto King's B medium yielded a gram-negative, rod-shaped bacterium with colonies that were fluorescent under UV light. Bacterial colonies were purified and characterized. The isolates were strictly aerobic, negative for levan production, soft rot of potato, and arginine dihydrolase activity, oxidase positive, and induced hypersensitive reaction in tobacco leaves (2). The bacteria were further identified as Pseudomonas cichorii by comparison of the 49 carbohydrate utilization profiles, API 50 CH (bioMérieux, Marcy l'Etoile, France), with the reference strain ICPPB 2827. Ten lettuce plants used as indicators and borage plants were inoculated by root and petiole injections of bacterial suspensions (108 CFU/ml) of the borage strains and the P. cichorii reference strain ICPPB 2827. Inoculated plants and controls were maintained in a growth chamber at 20 to 25°C with nearly 100% relative humidity. Symptoms of varnish spot, described in lettuce (1), and the black lesions initially observed in borage roots, petioles, and leaves were evident on all inoculated plants at 7 and 5 days after inoculation, respectively. No symptoms developed on control plants. A bacterium with identical characteristics to those described above was reisolated from the black lesions on inoculated plants. To our knowledge, this is the first report of P. cichorii as a pathogen of B. officinalis. Successful infection of borage plants was dependent on high humidity conditions, which is present because of the greater density of mature crops. References: (1) R. G. Grogan et al. Phytopathology 67:957, 1977. (2) R. A. Lelliot et al. J. Appl. Bacteriol. 29:470, 1966.