Catabolism of volatile sulfur compounds precursors by Brevibacterium linens and Geotrichum candidum, two microorganisms of the cheese ecosystem

Two Brevibacterium linens strains and the cheese-ripening yeast Geotrichum candidum were compared with regard to their ability to produce volatile sulfur compounds (VSCs) from three different precursors namely L-methionine, 4-methylthio-2-oxobutyric acid (KMBA) and 4-methylthio-2-hydroxybutyric acid (HMBA). All microorganisms were able to convert these precursors to VSCs. However, although all were able to produce VSCs from L-methionine, only G. candidum accumulated KMBA when cultivated on this amino acid, contrary to B. linens suggesting that the transamination pathway is not active in this microorganism. Conversely, a L-methionine gamma-lyase activity--which catalyses the one step L-methionine to methanethiol (MTL) degradation route--was only found in B. linens strains. Several other enzymatic activities involved in the catabolism of the precursors tested were investigated. KMBA transiently accumulated in G. candidum cultures, and was then reduced to HMBA by a KMBA dehydrogenase (KDH) activity. This activity was not detected in B. linens. Despite no HMBA dehydrogenase (HDH) was found in G. candidum, a strong HMBA oxidase (HOX) activity was measured in this microorganism. This latter activity was weakly active in B. linens. KMBA and HMBA demethiolating activities were found in all the microorganisms. Our results illustrate the metabolic diversity between cheese-ripening microorganisms of the cheese ecosystem.     

Diel variations in Acartia tonsa feeding, respiration and egg production in a Mediterranean coastal lagoon

Short-term changes in abundance, feeding, respiration and spawningby the copepod Acartia tonsa were studied during four 24 h periodsat two stations in a Mediterranean lagoon in May and June of1989 and 1990. Nightly increases of abundance in the upper layer,attributed to nocturnal upward migration, were observed in threeout of the four time series. Nevertheless, occasional dailyincreases were observed too, so that no clear diel patternsin abundance could be defined. Contrarily, diel patterns seemedto appear: (i) for feeding (higher at the beginning of the night),based on gut fluorescence and clearance and ingestion rate measurements;(ii) for respiration (lower at night and based on 8 h incubationperiods); and (iii) for spawning (higher at the end of the night,based on experimental data). The origin of these patterns, aswell as their relationships to environmental data and theirpossible association, are discussed with regard to the availableliterature.     

Evidence for distinct L-methionine catabolic pathways in the yeast Geotrichum candidum and the bacterium Brevibacterium linens

Tracing experiments were carried out to identify volatile and nonvolatile L-methionine degradation intermediates and end products in the yeast Geotrichum candidum and in the bacterium Brevibacterium linens, both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium B. linens is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast G. candidum produces, we examined whether the L-methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and alpha-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of L-methionine catabolism. However, the L-methionine degradation pathways differ in the first steps of L-methionine degradation. L-Methionine degradation is initiated by a one-step degradation process in the bacterium B. linens, whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast G. candidum. Since G. candidum develops earlier than B. linens during the ripening process, MOBA and MHBA generated by G.candidum could also be used as precursors for VSC production by B. linens.     

Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate

Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.     

Chlorophyll budget in a productive tropical pond: algal production,sedimentation, and grazing by microzooplankton and rotifers

Chlorophyll a and pheopigment standing stocks and fluxes were used during a two weeks colonization experiment in a productive tropical pond (Layo, Côte d'Ivoire) in order to establish a chlorophyll budget. The experiment started from an azoïc state (the pond was dried, limed and progressively filled with ground water). Algal production was the only input to the phytoplanktonic system, while grazing and algal sedimentation were the main outputs. Chlorophyll a reflected the algal biomass, and degradation pigments were considered as an index of grazing by zooplankton (here, protozoans and rotifers). An estimation of the input through the algal growth rate was performed for the two main biological events observed during the study. The first algal bloom, with a large picoplankton participation, was mainly regulated by microzooplankton (increase of the peak) and rotifers (decrease of the peak). The second bloom (exclusively nanoplankton) was regulated by rotifers (increase) and by sedimentation of living cells (decrease). This last process was related to a sudden exhaustion of ammonia in the water column. Because of the time-lag between algal proliferation and zooplanktonic bloom, the phytoplanktonic biomass was able to be adjusted according to the availability of nutrients. This self-regulation took the form of sinking of active algal cells, resulting in a transient reduction of the food available for rotifers. This process had drastic consequences in these shallow waters, since a major part of the phytoplankton produced was removed from the pelagic system. For an optimal exploitation of the natural resources of an aquaculture pond, a study of the equilibrium nutrients-phytoplankton-zooplankton would provide a basis for artificial intervention, with a view to limit the impact of this mode of natural regulation.     

RhoB Promotes γH2AX Dephosphorylation and DNA Double-Strand Break Repair

Unlike other Rho GTPases, RhoB is rapidly induced by DNA damage, and its expression level decreases during cancer progression. Because inefficient repair of DNA double-strand breaks (DSBs) can lead to cancer, we investigated whether camptothecin, an anticancer drug that produces DSBs, induces RhoB expression and examined its role in the camptothecin-induced DNA damage response. We show that in camptothecin-treated cells, DSBs induce RhoB expression by a mechanism that depends notably on Chk2 and its substrate HuR, which binds to RhoB mRNA and protects it against degradation. RhoB-deficient cells fail to dephosphorylate γH2AX following camptothecin removal and show reduced efficiency of DSB repair by homologous recombination. These cells also show decreased activity of protein phosphatase 2A (PP2A), a phosphatase for γH2AX and other DNA damage and repair proteins. Thus, we propose that DSBs activate a Chk2-HuR-RhoB pathway that promotes PP2A-mediated dephosphorylation of γH2AX and DSB repair. Finally, we show that RhoB-deficient cells accumulate endogenous γH2AX and chromosomal abnormalities, suggesting that RhoB loss increases DSB-mediated genomic instability and tumor progression.     

Lâcher de ballons chez une femme de 35 ans révélant une granulomatose sarcoïdosique nécrosante diagnostiquée sur biopsie guidée par la TEP/TDM au FDG (18F)

This case report exemplifies the role of FDG (18F) PET/CT in case of scattered multiple pulmonary nodules to assist the aetiologic diagnosis and guide the surgical biopsy evidencing a necrotising sarcoid granulomatosis while CT guided biopsy is non-contributive.