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Summary The increasing interest of animal and plant research communities for biomedical 3D imaging devices results in the emergence of new topics. The anatomy, structure and function of tissues can be observed non-destructively in time-lapse multimodal imaging experiments by combining the outputs of imaging devices such as X-ray CT and MRI scans. However, living samples cannot remain in these devices for a long period. Manual positioning and natural growth of the living samples induce variations in the shape, position and orientation in the acquired images that require a preprocessing step of 3D registration prior to analyses. This registration step becomes more complex when combining observations from devices that highlight various tissue structures. Identifying image invariants over modalities is challenging and can result in intractable problems. Fijiyama, a Fiji plugin built upon biomedical registration algorithms, is aimed at non-specialists to facilitate automatic alignment of 3D images acquired either at successive times and/or with different imaging systems. Its versatility was assessed on four case studies combining multimodal and time series data, spanning from micro to macro scales. Availability and implementation Fijiyama is an open source software (GPL license) implemented in Java. The plugin is available through the official Fiji release. An extensive documentation is available at the official page: https://imagej.github.io/Fijiyama Supplementary information Supplementary data are available at Bioinformatics online.

Rabies is a lethal zoonotic encephalomyelitis that causes an estimated 59,000 human deaths yearly worldwide. Although developing countries of Asia and Africa bear the heaviest burden, surveillance and disease detection in these countries is often hampered by the absence of local laboratories able to diagnose rabies and/or the difficulties of sample shipment from low-access areas to national reference laboratories. Filter papers offer a convenient cost-effective alternative for the sampling, shipment, and storage of biological materials for the diagnosis of many pathogens including rabies virus, yet the properties of diagnostic tests using this support have not been evaluated thoroughly. Sensitivity and specificity of molecular diagnosis of rabies infection using a reverse transcription followed by a hemi-nested polymerase chain reaction (RT-hn-PCR) either directly on brain tissue or using brain tissue dried on filter paper were assessed on 113 suspected field animal samples in comparison to the direct fluorescent antibody test (FAT) recommended by the World Health Organization as one of the reference tests for rabies diagnosis. Impact of the duration of the storage was also evaluated. The sensitivity and the specificity of RT-hn-PCR i) on brain tissue were 96.6% (95% CI: [88.1–99.6]) and 92.7% (95% CI: [82.4–98.0]) respectively and ii) on brain tissue dried on filter paper 100% (95% CI: [93.8–100.0]) and 90.9% (95% CI: [80.0–97.0]) respectively. No loss of sensitivity of RT-hn-PCR on samples of brain tissue dried on filter paper left 7 days at ambient temperature was detected indicating that this method would enable analyzing impregnated filter papers sent to the national reference laboratory at ambient temperature within a 1-week shipment time. It could therefore be an effective alternative to facilitate storage and shipment of samples from low-access areas to enhance and expand rabies surveillance., Author summary Dogs are responsible for 99% of human rabies deaths. Facilitating diagnostic of rabid dogs can help further identify i) people who were exposed to rabies in order to encourage them to seek post exposure prophylaxis and ii) other exposed animals to break the transmission chain (i.e. prevent them from further transmitting the virus to other people and animals). Yet, the reference diagnostic method for animal rabies requires brain samples to be collected post-mortem and shipped under temperature-controlled conditions. The shipping of such samples is complex in low-access areas, especially in low income countries, which are often those that bear the heaviest rabies burden. Filter papers offer a convenient alternative for biological sample shipment and storage. Here the efficiency of a molecular diagnostic method applied to brain tissue dried on filter paper is compared to one of the reference methods, the direct fluorescent antibody test. Our results show it has an excellent sensitivity (it does not miss any positive samples), even when filter papers are left 7 days at ambient temperature. These results let us foresee a cost-effective alternative facilitating shipment, storage and testing of samples from rabies suspected animals from low-access areas. This could considerably enhance and expand rabies surveillance in low-income countries, allowing a more comprehensive evaluation of rabies burden, and thus reinforcing arguments for allocating funds to rabies control policies.

Le protocole vise a diagnostiquer, en laboratoire, les principales especes de Mycosphaerella pathogenes des bananiers et plantains. Les trois agents pathogenes Mycosphaerella fijiensis (anamorphe de Paracercospora fijiensis), M. musicola (anamorphe de Pseudocercospora musae) et M. eumusae (anamorphe de Pseudocercospora eumusae) sont respectivement responsables de la maladie des raies noires, de la maladie de Sigatoka et de la cercosporiose due a eumusae. Le principe, les principaux avantages, le materiel vegetal et le temps necessaires a la methode sont presentes. Materiel et methodes. Le materiel de laboratoire necessaire et le detail des treize etapes de realisation du protocole (prelevement des tissus et observations microscopiques in situ, isolement sur milieu artificiel et clonage d'isolats monospores, sporulation in vitro et observations microscopiques des conidies, conservation a long terme des isolats) sont decrits. Resultats. Le diagnostic est base sur les observations des anamorphes (conidiophores et conidies) qui peuvent etre observes directement a partir des feuilles de bananes ou apres sporulation des isolats mis en culture s'il n'y a pas de lesions sporulantes sur les echantillons de bananiers. (Resume d'auteur)

The nut yield and fruit composition of 'nature (30-40 years old) West African Tall coconut palms were evaluated in seven trials during the first three years (from June 2000 to May 2003) of implementation of a five-year fertilization schedule recommended to the Coconut Sector Development Project (CSDP) for the rehabilitation of old farms in the coastal belt of the Western Region of Ghana. The seven trials are located at Jeway Wharf, Takinta, Ellenda, Nawule, AB Bokazo, Anwia and Edwakpole. Three fertilizer treatments were applied between June 2000 and May 2003. The first two fertilizers were applied in June 2000 and May 2001 at the rate of 0. 75kg urea and 2.5kg muriate of potash (MOP) per palm. For the third fertilization, 1 kg MOP only was applied per palm in May 2002. Fertilizer treatment did not affect nut yield in year one of fertilization. But in year two, the fertilized plots gained remarkably 27.7, 20.2, 19.4 and 14.9 nuts per palm at Jeway Wharf, Nawule, Ellenda and Takinta respectively. The remarkable nut gain was further boosted or sustained in year three except at Jeway wharf which lost 9.8 nuts per palm. In economic terms, Ellenda trial broke even on MOP fertilizer investment in year three while Jeway Wharf was close to breaking even. Takinta and Nawule trials made profits of 925 and 1, 750 cedis (3.7 and 7 nuts) per palm respectively in year three. For Anwia, AB Bokazo and Edwakpole trials there was no yield response to MOP fertilizer due to P deficiency. Fertilizer treatment in the first two years of fertilization did nor influence fruit composition. (Resume d'auteur)

Introduction. Chlordecone (CLD), an organochlorine formerly used to control the banana black weevil, is strongly adsorbed on soils, particularly on andosols. A simplified analytical procedure for the quantitative determination of chlordecone residues in water and micro-samples of soil and plants was compared with a standard method. Materials and methods. The procedure combines a simplified sampling protocol and a 10-min solid phase microextraction (SPME), followed by gas chromatographic separation (GC) and mass spectrometric (MS and MS/MS) identification. Quantitation of CLD used a standard addition method with zero extrapolation. First, seventy samples were analysed using the proposed method and the standard method based on hot solvent extraction. Second, fifteen soil samples were analysed with two SPME methods followed by GC-MS but using CLD labelled with C13 as an internal standard or the proposed method. Results and discussion. The detection (LOD) and quantitation (LOQ) limits of our SPME extraction procedure were determined for GC-MS and GC-MS/MS with water, plant (pineapple roots) and soil samples: in water for MS/MS, LOD MS/MS-water = 0.5 ng⋅L -1 , LOQ MS/MS-water = 2.0 ng⋅L -1 ; in andosol for MS/MS, LOD MS/MS-soil = 15.0 ng⋅kg -1 dw, LOQ MS/MS-soil = 80.0 ng⋅kg -1 dw. Data from the seventy contaminated soils obtained with the proposed method and the standard method showed a correlation coefficient of r = 0.86. Data obtained by the two SPME/GC-MS quantitation procedures showed a correlation of r = 0.8073. Conclusion. The method proposes a simplified sample preparation and extraction of CLD in water, plant and soil samples, with no solvent manipulation and which is not time-consuming. The LOD and LOQ were similar to those obtained with other currently used methods. The method is reli- able and accurate and may be considered as a good tool for research purposes. France (Martinique) / chlordecone / extraction / analytical methods / quantitative analysis / water analysis / soil analysis / tissue analysis

Five types of cellular aggregates have been characterised in embryogenic cell suspensions of banana (#Musa# 'AAA' 'Grande naine' cv.). Type I corresponded to isolated cells or to small cell aggregates. Type II were composed of embryogenic cells. Type III can be distinguished from type II due to the presence of peripheral proliferation zones with embryonic cells. Type IV were composed of protodermic masses histologically comparable to proembryos. Type V were nodules composed of a central zone of meristematic cells and of an external zone of starchy cells. Each culture flask of a cell line contained a majority of one of the above-mentioned aggregate types. Histological studies of somatic embryo developement on semi-solid regeneration medium showed that there were close similarities between the initial steps of ontogenesis of the embryos and the different cell aggregates in liquid multiplication medium. It appeared that aggregates II-IV of the suspension belong to the same development continuum which reproduces the initial phases of somatic embryo ontogenesis on semi-solid medium. Type V resulted from the development of type IV, for which ontogenesis is hindered by direct contact with 2,4-dichlorophenoxyacetic acid and the shaken liquid multiplication medium. Type I aggregates probably do not belong to the development continuum but rather correspond to the degeneration of the other types of aggregates in the suspension. The presence of intermediate types in the liquid medium reinforces the hypothesis of a relationship between the aggregates. The aggregates tended to develop through time from a majority of type II or III at the beginning of their culture to types IV-V for older suspensions.

Foliar mineral concentration may provide a basis for monitoring the consequences of long-term environmental changes, such as eutrophication and acidification of soils, or increase in atmospheric CO(2) concentration. However, analytical drifts and inter-tree and year-to-year variations may confound environmental effects on long-term changes in foliar mineral concentration. We have characterized the relative effects of these potentially confounding factors on foliar carbon, nitrogen, phosphorus, calcium, potassium, magnesium and manganese concentrations in 118 pure beech (Fagus sylvatica L.) stands, sampled in 1969-71 and 1996-97. Interannual fluctuations of these elements were quantified in a subset of six beech stands monitored for 5 years. Intercalibration between the methods used at each sampling period for nitrogen and phosphorus analyses showed significant, but low, relative differences (0.8 and 3.3% for N and P, respectively). Based on inter-tree variability, elements could be arranged in four groups: C (constant), N and P (low variability), K and Ca (medium variability), Mn and Mg (high variability). Inter-tree coefficients of variation were 2, 6, 8, 15, 18, 22 and 27%, respectively. Year-to-year fluctuations increased in the order N, P, Mg, K, Ca, and Mn coefficients of variation of 4, 4, 7, 9, 11, 15 and 29%, respectively). Between the two sampling periods, foliar N concentration increased 12%, whereas decreases were observed for P (-23%), Mg (-38%) and Ca (-16%). Ratios of N/P, N/K and N/Mg increased by 42, 19 and 77%, respectively. These changes were larger than the interannual variations for P, Mg, N/P, N/Mg and Mg/Ca. Decreasing concentrations of P and cations were particularly marked for trees growing on acidic soils, whereas the positive N trend did not depend on soil type. Both increasing atmospheric CO(2) concentrations and acidification of forest soils could contribute to decreasing P and cation concentrations in foliage. The increase in foliar N concentration with time suggests a nitrogen deposition effect. Whatever the causes of these changes, the large shift in element ratios indicates an accelerating imbalance between nitrogen and cation status.

Phenolic compounds, which are present in the epidermis zone, parenchyma zone, core zone, and seeds of fruit (Malus domestica var. Kermerrien), were extracted from freeze-dried material by three successive solvent extractions. The dry methanol extract and the dry aqueous acetone extracts were analyzed using reversed-phase HPLC coupled with diode array detection following thiolysis to quantify phenolic compounds according to their classes (hydroxycinnamic acid derivatives, flavan-3-ols, flavonols, and dihydrochalcones). The method is suitable for the determination of the relative proportions of the different classes of polyphenols and provided information on the constitutive units and the average degree of polymerization of oligomeric and polymeric procyanidin structures. Results showed that procyanidins are the predominant phenolic constituents in cider apple fruits, much of them corresponding to highly polymerized structures.

SUMMARYOver several decades agronomists have developed a mineral nutrition management method for perennial oil crops based on leaf analysis combined with reference field trials. This method has been used in a coconut plantation on the peats of the east coast of Sumatra to optimize mineral nutrition in a very short time, at minimum cost and with maximum reliability, through successive adjustments of the fertilizer schedules. Two reference trials were set up to study a wide range of mineral nutrients assumed to be deficient on this area. Further experiments were set up as new nutritional problems occurred at the plantation, and as soon as the first results were obtained from these two trials. In this way, the iron and copper deficiency problems at the plantation were dealt with satisfactorily taking an experimental approach. New experiments were set up recently to test the hypothesis of a silicon deficiency. The fertilization schedules for the commercial plantation were developed by successive adjustments based on the trial results. Leaf analysis data showed that the coconut palms in the commercial plots always complied with the experimental models. In addition to being rapid and reliable, this method was also cost-effective, especially when larger areas were involved.

Les sélectionneurs et agronomes ont donc besoin de moyens pour suivre la profondeur d'enracinement du riz. Une technique manuelle simple, basée sur l'injection d'herbicide dans le sol à une profondeur choisie, a été expérimentée. La période d'émergence des symptômes de phytotoxicité sur les feuilles de 4 génotypes de riz, ayant des systèmes racinaires différents, a été observée au champ et en serre. Trois herbicides ont été testés. Après injection de metribuzine à différentes profondeurs, des variations significatives dans le nombre de plants affectés sont apparues entre les génotypes au champ. Le classement des génotypes en fonction des résultats obtenus lors des expérimentations confirme les connaissances existantes sur leur système racinaire respectif. Bien que les risques de propagation de l'herbicide aient été maîtrisés, la fiabilité de la technique nécessite de prendre des précautions accroissant ainsi le temps nécessaire au traitement et limitant l'application de cette technique. De plus, les effets résiduels de metribuzine, observés 7 mois après l'application, posent des problèmes sur l'environnement

International audience

This paper illustrates the advantages of the hyperspectral data for sugar cane crop management. In 2002, a casi airborne campaign and a ground measurement experiment were set up over sugar cane sites in Réunion Island. Early results indicate that LAI and biomass are significantly correlated with some narrow spectral bands in the NIR and that chemical leaf contents are well correlated with the PRI index. A multi-regression procedure was used to calculate transfer functions in order to produce thematic maps of LAI, biomass and nitrogen. [Author's abstract]

We developed a hydroponic cultivation system for growing Arabidopsis plantlets under sterile, controlled environmental conditions. The system consists of a piece of stainless-steel wire cloth (125 [mu]m mesh size) that is fixed between 2 flat rings and held in place by 3 legs, placed in a commercially-available glass jar, and covered by the original glass lid or a sheet of sterilized cellophane. Sterilized seeds were distributed evenly across the mesh piece, the size of which allowed root growth and kept the seeds in place. After 3 weeks of cultivation, shoot and root tissues were easily harvested without mechanical damage. Proteome and metabolite analyses were performed on root and shoot tissues and demonstrated excellent reproducibility, indicating that the system is advantageous when biological variation is minimized. Induction experiments can be performed by transferring the apparatus (with plants) to a new jar containing a different nutrient solution. The apparatus is reusable and can easily be sterilized by autoclaving or dry heat. The system can be adapted to other small-seed plants by varying the mesh size.

Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi‐solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro‐regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0·86 cm2) had the highest rates of plant conversion ex vitro (63 %), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi‐solid media, those produced in soil were branched, fine (30–50 % had a diameter of less than 0·5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm–2). Moreover, they were more turgid, as indicated by higher pressure potential (ψP) (0·91 vs. 0·30 MPa) and relative water content values (97 vs. 93 %). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.

With the Trade Union of the French Poultry Selectors and Aquacoles (SYSAAF) and French National Institute of Agronomical Research (INRA) we studied the farmed brown sea-trout development according to genetic improvement and feeding composition by color image analysis. They want to value the effects of feeding which contains various levels of lipid. In this research project a vision system was devised to research and measure brown trout visual features. Various segmentation methods were applied to locate various tissues of brown trout cutlets and to measure their area and color. The results obtained on fish flesh by the image analysis were compared with chemical measurements of fat and with NMR measurements. The color image features give data on the development of various tissues and the effect of feeding on morphological development can be tested on efficient aspect variables., Nous avons étudié par analyse d'image couleur, la distribution des différents tissus et de la couleur de la chair dans la truite d'élevage en fonction de l'amélioration génétique et la composition de l'alimentation. Nous avons développé un système de vision couleur pour caractériser les tissus de la truite. Des méthodes de segmentation sont appliquées pour localiser les différents tissus, mesurer leur surface et leur couleur. Ces caractéristiques sont comparées au taux de lipides mesuré dans la chair par méthode chimique et par RMN.

La production de bananes pour l'exportation est soumise à des contraintes de taille et de qualité des fruits. Pour être exportable des Antilles vers la métropole, un fruit doit présenter des caractéristiques minimales en terme de diamètre (30 mm), de longueur (17 cm) et de maturité à la récolte (estimée par la durée de vie verte ou DVV qui doit excéder 20 jours). En conséquence, la forte variabilité de poids et de qualité des fruits observée au champ entraîne des pertes de rendement importantes pour les planteurs. Cette variabilité pondérale dépend des conditions de croissance mais également de la position des fruits sur le régime: les fruits de la partie distale du régime (mains du bas) sont 30 à 40% plus petits que ceux de la partie basale (mains du haut). L'étude conjointe du déterminisme du poids et de la qualité (maturité) du fruit à la récolte, réalisée dans ce travail, est donc nécessaire pour optimiser le rendement quantitatif et qualitatif des bananiers. Dans un premier temps, nous avons déterminé les causes de la variabilité pondérale des fruits au sein du régime (inflorescence). Pour cela, une étude histologique du développement a été réalisée afin de déterminer la chronologie des phases de division cellulaire et de remplissage des cellules à l'échelle du fruit et du régime. A l'échelle du fruit, la phase de forte accumulation de matière sèche (remplissage) a lieu après la fin des divisions cellulaires dans le fruit. Au sein du régime, il y a un décalage de développement entre les mains basales (mains du haut initiées en premier) et les mains distales (mains du bas initiées en dernier). Ce décalage a pour conséquence un nombre de cellules plus important dans les fruits des mains basales. A partir des données histologiques, un schéma conceptuel du développement de l'inflorescence a été proposé. Dans un deuxième temps, des modifications du rapport source/puits ont été réalisées au moyen d'ombrage des feuilles (diminution des sources) ou par adaptation de techniques culturales couramment usitées telles que le gainage des régimes (augmentation de l'activité du puits) et l'ablation de mains (diminution de la taille du puits). Il est apparu que le nombre de cellules des fruits est un facteur déterminant de la variabilité pondérale au sein du régime. En revanche, la vitesse de remplissage des cellules est identique pour tous les fruits du régime et déterminée par le rapport source/puits. Une relation de type Michaelis-Menten permet de relier la vitesse de remplissage moyenne des cellules d'un régime au rapport source/puits moyen pendant la période de remplissage. Dans un troisième temps, ces connaissances ont été intégrées dans un modèle de répartition des assimilats au sein du régime qui simule le poids des fruits et des régimes à la récolte. Une relation permettant de calculer la DVV des fruits en fonction de leur âge exprimé en somme de température a été adjointe au modèle, lui conférant ainsi une valeur prédictive de la qualité de la récolte. Le modèle construit présente un double intérêt cognitif et prédictif. Il perrnet d'abord de comprendre et de simuler le fonctionnement de la plante, selon des connaissances histologiques nouvelles et des règles validées de répartition des assimilats. Il constitue ensuite un outil pertinent de diagnostic du rendement et d'aide à la décision dans le choix de certaines interventions culturales telles que le gainage des régimes et l'ablation de mains ainsi que pour la détermination de dates de récolte optimales.

This visit is the third and last one planned in the consultancy contract for the modernization of the PTAL. The objectives of this visit are i) to check the state of the project about the building and the analytical equipment, ii) to give some suggestions about the organisation of the lab and the data management, iii) to propose some complementary services to the leaves analysis, i.e. soil analysis, fertilizer analysis, methodology.

Récaitulatif des essais de fertilisation et nutrition des palmiers à huile pour la campagne 1997/1998 : AK CP 01, AL CP 06 A1, AL CP 06 A2, MP CP 02, MP CP 03, SY CP 01, SY CP 02, TG CP 03, TG CP 04

chap. 11; National audience

Lors de la régénération des premiers vitroplants obtenus par embryogenèse somatique chez le palmier à huile, on a observé l'apparition d'une anomalie florale qui pourrait être induite par un stress lors de la culture #in vitro#. Des travaux effectués sur d'autres espèces végétales ont mis en évidence que l'acide 2,4-D apporté dans les milieux de culture à certaines étapes du processus d'embryogenèse somatique pourrait être à l'origine de l'apparition de variants somaclonaux. D'autres études laissent à supposer que le 2,4-D aurait une action sur le degré de méthylation de façon indirecte via la teneur en cytokinines endogènes. Au cours de ce travail, on a étudié l'effet de différentes concentrations de 2,4-D apportées au stade suspensions cellulaires, sur les teneurs en cytokinines endogènes à 2 stades de culture. Une technique de quantification hormonale a été adaptée pour permettre le dosage des différentes formes hormonales (2,4-D et cytokinines de formes zéatine et isopentényladénine) au sein d'un même extrait végétal et appliquée sur des échantillons de palmier à huile pour doser leurs teneurs en 2,4-D et en cytokinines. Les résultats obtenus montrent que les cytokinines sont absentes au stade de prolifération cellulaire (qui contient du 2,4-D dans les milieux de culture), alors que des traces allant de 0,2 à 0,6 pmol/mg MF sont détectées au stade pro-embryons (qui ne contient pas de 2,4-D dans les milieux de culture). Par ailleurs, il a été constaté que le 2,4-D est accumulé dans les tissus végétaux aux deux stades étudiés (avec des teneurs allant de 1,6 à 10,9 pmol/ mg MF). Enfin, les dosages obtenus indiquent une plus faible accumulation du 2,4-D au stade pro-embryons par rapport au stade suspensions cellulaires, ce qui permet de faire l'hypothèse d'une action du 2,4-D sur la concentration en cytokinines endogènes dans les tissus végétaux

The building of the laboratory is conforming the layouts and facilities described in the report mission of October 1995. However, later a third floor extension has been decided. The works have to be done without any disturbance for the PTAL laboratory. The delivery of the equipment is now completed, with some minor defects without consequence for the operation. The tests of the equipment are satisfactory, except for the elemental analyser which needs some more adjustments to be operational. For the ICP spectrometer, the typical relative standard deviation (%RSD) is between 1 and 2%. This spectrometer is steady and easy to use. The test of the atomic absorption spectrometer was made by analysing 20 mineralization solutions of the same sample. The %RSD was 1.72%. This result, obtained without the sampler, is good. For the colorimeter, the typical %RSD is lower than 1%. Detailed results and error computation are in the annexe of the report. The training and mission program was revised because of the end of the project in December 1999. Two trainings will take place in June (Perkin-Elmer in Malaysia) and probably in August (Bran & Luebbe in Germany). some other trainings (LIMS and computer, quality management) will be organised out of the project. The laboratory is expected to be fully operational by October, but it is already able to process the leaves analyses. The third consultancy mission is scheduled in October 1999 (two weeks).