Transiently responsive protein-polymer conjugates via a 'grafting-from' RAFT approach for intracellular co-delivery of proteins and immune-modulators.

We report on transiently responsive protein-polymer conjugates that temporarily change their protein conformation from the soluble to the particle-like state. 'Grafting-from' RAFT polymerization of a dioxolane-containing acrylamide with a protein macroCTA is used to design polymer-protein conjugates that self-assemble into nanoparticles at physiological temperature and pH. Acid triggered hydrolysis of the dioxolane units into diol moeities rendered the conjugates fully water soluble irrespective of temperature.

First Report of Pineapple Black Rot Caused by Ceratocystis paradoxa on Ananas comosus in French Guiana.

Ceratocystis paradoxa (Dade) C. Moreau is a polyphagous wound parasite causing black rot post-harvest disease in pineapple. This fungus is responsible for high losses of fruit destined for the fresh market and is a common problem in many countries (2). C. paradoxa is officially listed as a quarantine pathogen for French Guiana. In November 2013, the Plant Protection Service of French Guiana observed damage in crops of Ananas comosus var. perolera located in Corossony (4°19'10.8″ N, 52°10'17.1″ W) and Wayabo (5°01'02.3″ N, 52°36'18.7″ W) (eastern and middle part of the country). The three plants collected at each location showed a soft base rot of the stem and of young leaves, and developed a foul smell. Plant tissues were collected from the edge of the lesions, chopped in small pieces, and plated on malt extract agar supplemented with 100 ppm chloramphenicol. The plates were incubated at 25°C with a 12-h photoperiod. After 5 days, a fungal colony, first white and downy, then becoming black and velvety after 10 days, was transferred on potato dextrose agar (PDA) and incubated in the same conditions. After 7 days, the colonies produced phialides releasing cylindrical or doliform conidia that were unicellular, colorless to pale brown, in long chains (3.09 to 20.17 × 3.1 to 5.57 µm, n = 20) and oval, pyriform, brown chlamydospores (8.02 to 21.32 × 4.20 to 9.76 µm, n = 20), occurring in long chains or singly with a vertical slit, usually not very visible. Furthermore, the colonies emitted a fruity odor. On the basis of these morphological characteristics, the fungus was identified as the anamorph of C. paradoxa (Thielaviopsis paradoxa (De Seynes) Höhn.) (1). The species designation was confirmed by sequencing the ITS region of the rDNA followed by comparison with reference sequences available in GenBank. Fungal material was collected from PDA culture by scraping the mycelium with a sterile needle and transferring into 2-ml microtubes. Fungal total DNA was then extracted and the ITS region was amplified by PCR using the ITS1-ITS4 primer pair. Nucleotide sequence was determined and deposited in GenBank (KJ667047). BLAST analysis showed 100% identity with C. paradoxa. The pathogenicity of the fungus was confirmed by inoculating two pineapples with mycelium from the C. paradoxa isolate grown on PDA. The peel of fruits and the base of the crowns were wounded with a sterile scalpel blade, each at five locations. Mycelial plugs (avg. 4 mm diameter) were placed on the wounds. Inoculation sites were wrapped with Parafilm to prevent dehydration and to hold the mycelial plugs in position. Negative controls received five sterile PDA plugs. The samples were incubated at 25°C in a moist chamber with a 12-h photoperiod. Eight days after inoculation, negative controls remained symptomless, whereas characteristic soft, watery, and black rot lesions developed on the base of all the crowns that were inoculated with C. paradoxa. The pathogen was successfully re-isolated from symptomatic tissues, fulfilling Koch's postulate. To our knowledge, this is the first report of C. paradoxa on A. comosus in French Guiana, and quarantine measures have been enforced to prevent the spread of this pathogen that might also cause severe losses on other susceptible plant species that are important for the local market (e.g., banana, coconut, sugar cane). Pineapple has become a major crop in French Guiana, and is now subjected to a more intensive monitoring, which may explains why this disease was discovered recently. References: (1) T. R. Nag Raj and W. B. Kendrick. A Monograph of Chalara and Allied Genera. Wilfrid Laurier University Press, Waterloo, Ontario, 1975. (2) R. C. Ploetz et al., eds. Compendium of Tropical Fruit Diseases. American Phytopathological Society, St. Paul, MN, 1994.

Diversity of Ralstonia solanacearum in French Guiana expands knowledge of the "emerging ecotype".

Although bacterial wilt remains a major plant disease throughout South America and the Caribbean, the diversity of prevalent Ralstonia solanacearum populations is largely unknown. The genetic and phenotypic diversity of R. solanacearum strains in French Guiana was assessed using diagnostic polymerase chain reactions and sequence-based (egl and mutS) genotyping on a 239-strain collection sampled on the families Solanaceae and Cucurbitaceae, revealing an unexpectedly high diversity. Strains were distributed within phylotypes I (46.9%), IIA (26.8%), and IIB (26.3%), with one new endoglucanase sequence type (egl ST) found within each group. Phylotype IIB strains consisted mostly (97%) of strains with the emerging ecotype (IIB/sequevar 4NPB). Host range of IIB/4NPB strains from French Guiana matched the original emerging reference strain from Martinique. They were virulent on cucumber; virulent and highly aggressive on tomato, including the resistant reference Hawaii 7996; and only controlled by eggplant SM6 and Surya accessions. The emerging ecotype IIB/4NPB is fully established in French Guiana in both cultivated fields and uncultivated forest, rendering the hypothesis of introduction via ornamental or banana cuttings unlikely. Thus, this ecotype may have originated from the Amazonian region and spread throughout the Caribbean region.

Removal of the organic surface layer in combined sewer sediment using a flushing gate.

Recent research identified the different sources of pollution of wet weather Combined Sewers Overflows (CSOs): it appeared that the deposits in sewers, and especially an organic layer situated at the water-sediment interface, may contribute 40-70% to the total pollution load of CSOs. Using the cyclic flush Hydrass gate, we generated increased water flows during dry weather. The effects of flushing the deposits have been analysed: the eroded particles sampled during the first flush wave show pollutant characteristics similar to characteristics measured in the organic layer. The organic layer that has formed on the surface of deposits can thus be washed off before rainstorms occur using the cyclic flushing technique.

Hydraulics of a sewer flushing gate.

This paper presents field experiments carried out in a 1.8 m height egg-shaped sewer in Lyon, France in order to contribute to the knowledge and the simulation of Hydrass flushing gates. The main results are: i) definition of an empirical relationship giving the flow discharged by the gate as a function of the upstream water level and its use in modelling the gate behaviour, and ii) observation of the flush propagation along the sewer and evaluation of the potential cleansing efficiency.

A cell culture system for study of the development of Toxoplasma gondii bradyzoites.

Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of gamma-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.

Diagnosis of Encephalitozoon cuniculi infection by western blot and the use of cross-reactive antigens for the possible detection of microsporidiosis in humans.

Microsporidia are very primitive, eukaryotic, obligate, intracellular, protozoan parasites. Encephalitozoon cuniculi, a microsporidian originally described from a rabbit infection, has been described in humans as well as in many species of laboratory animals. We report the detection of E. cuniculi by Western blotting in a rabbit with torticollis that was obtained from an Encephalitozoon-free colony. Cross-reactivity of this serum was observed with antigens prepared from several genera of microsporidia. Identical Western blotting patterns were obtained with sera obtained from a rabbit immunized with E. cuniculi that was purified from tissue culture cells. In addition, we were able to demonstrate cross-reactivity between E. cuniculi rabbit antisera and Enterocytozoon bieneusi antigens by indirect immunofluorescent assay techniques in human intestinal biopsy samples. These cross-reactions between microsporidia may be useful in developing diagnostic tests for non-cultivatable microsporidia such as Enterocytozoon bieneusi.