Endosomal Phosphatidylinositol-3-Phosphate-Associated Functions Are Dispensable for Establishment of the Cytomegalovirus Pre-Assembly Compartment but Essential for the Virus Growth

Murine cytomegalovirus (MCMV) initiates the stepwise establishment of the pre-assembly compartment (pre-AC) in the early phase of infection by the expansion of the early endosome (EE)/endosomal recycling compartment (ERC) interface and relocation of the Golgi complex. We depleted Vps34-derived phosphatidylinositol-3-phosphate (PI(3)P) at EEs by VPS34-IN1 and inhibited PI(3)P-associated functions by overexpression of 2xFYVE- and p40PX PI(3)P-binding modules to assess the role of PI(3)P-dependent EE domains in the pre-AC biogenesis. We monitored the accumulation of Rab10 and Evectin-2 in the inner pre-AC and the relocation of GM130-positive cis-Golgi organelles to the outer pre-AC by confocal microscopy. Although PI(3)P- and Vps34-positive endosomes build a substantial part of pre-AC, the PI(3)P depletion and the inhibition of PI(3)P-associated functions did not prevent the establishment of infection and progression through the early phase. The PI(3)P depletion in uninfected and MCMV-infected cells rapidly dispersed PI(3)P-bond proteins and reorganized EEs, including ablation of EE-to-ERC transport and relocation of Rab11 endosomes. The PI(3)P depletion one hour before pre-AC initiation and overexpression of 2xFYVE and p40PX domains neither prevented Rab10- and Evectin-2 accumulation, nor Golgi unlinking and relocation. These data demonstrate that PI(3)P-dependent functions, including the Rab11-dependent EE-to-ERC route, are dispensable for pre-AC initiation. Nevertheless, the virus growth was drastically reduced in PI(3)P-depleted cells, indicating that PI(3)P-associated functions are essential for the late phase of infection.

RNase P inhibitors identified as aggregators

RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a small protein cofactor termed RnpA. Several studies reported small molecule inhibitors directed against bacterial RNase P that were identified by high-throughput screenings. Using the bacterial RNase P enzymes from Thermotoga maritima, Bacillus subtilis and Staphylococcus aureus as model systems, we found that such compounds, including RNPA2000 and derivatives, iriginol hexaacetate and purpurin, induce the formation of insoluble aggregates of RnpA rather than acting as specific inhibitors. In the case of RNPA2000, aggregation was induced by Mg2+ ions. These findings were deduced from solubility analyses by microscopy and HPLC, RnpA-inhibitor co-pulldown experiments, detergent addition and RnpA titrations in enzyme activity assays. Finally, we used a B. subtilis RNase P depletion strain, whose lethal phenotype could be rescued by a protein-only RNase P of plant origin, for inhibition zone analyses on agar plates. These cell-based experiments argued against RNase P-specific inhibition of bacterial growth by RNPA2000. We were also unable to confirm the previously reported non-specific RNase activity of S. aureus RnpA itself. Our results indicate that high-throughput screenings searching for bacterial RNase P inhibitors are prone to the identification of “false positives” that are also termed Pan-assay interference compound s (PAINS).

Effect of P depletion on the functional pools of diatom carbohydrates, and their utilization by bacterial communities

Phosphorus (P) limitation of phytoplankton growth is known to affect the accumulation and release of carbohydrates (CHO) by micro-algae. However, relatively little is known about the fate of algal exudates, notably their bacterial degradation. The CHO chemical characterization is also not exhaustive, especially in ‘functional’ pools relevant for phytoplankton physiology (particulate reserve [R] or structural [S] CHO) and for bacterial degradation (dissolved mono- [MDCHO] and polysaccharides [P-DCHO]). In this study, we investigated how P depletion and repletion affect the CHO composition in diatom Thalassiosira weissflogii cultures, and the shortterm response of free and diatom-attached bacteria in terms of abundance and potential βglucosidase activity (βGlc). The bacterial inoculum was composed of the bacterial consortiums of diatom precultures and a natural bacterial community from the Bay of Brest. P depletion favored CHO accumulation in diatom cells, mainly as R i.e. soluble CHO accumulated in cytoplasm, but also as S, polysaccharides linked to the cell wall. The R:S ratio was high in the present diatom cultures. The high M-DCHO observed in P-deplete cultures (twice that of P-replete cultures) when P-DCHO remained quite similar is explained both by active polysaccharide hydrolysis (very high potential βGlc of attached bacteria) and reduced uptake of M-DCHO by Pdepleted bacteria. P depletion of heterotrophic bacteria favors labile CHO accumulation, which may affect particle potential aggregation. However, the remarkably constant M-DCHO concentration over time for both conditions suggests tight coupling between phytoplankton accumulation, release, polymer hydrolysis and monomer uptake by bacteria.

Increasing P-stress and viral infection impact lipid remodeling of the picophytoplankter <i>Micromonas pusilla</i>

The intact polar lipid (IPL) composition of phytoplankton is plastic and dependent on environmental factors. Previous studies have shown that phytoplankton under phosphorus (P)-stress substitute phosphatidylglycerols (PGs) with sulphoquinovosyldiacylglycerols (SQDGs) and digalactosyldiacylglycerols (DGDGs). However, these studies focused merely on P-depletion, while phytoplankton in the natural environment often experience P-limitation whereby the degree of limitation depends on the supply rate of the limiting nutrient. Here we demonstrate a linear increase in SQDG : PG and DGDG : PG ratios with increasing cellular P-stress in the picophotoeukaryote Micromonas pusilla, obtained by P-replete, P-limited (chemostat) and P-starved (no supply of P) culturing conditions. These ratios were not affected by the degree of the P-limiting conditions itself (i.e. 0.97 and 0.32 μmax chemostats), suggesting there is a minimum requirement of PGs for the maintenance of cell growth. Viral infection reduced the increase in SQDG : PG and DGDG : PG ratios in P-starved cells, but the extent did depend on the growth rate of the cultures before infection. The membrane of M. pusilla virus MpV itself was lacking some IPLs compared to the host as, e.g. no monogalactosyldiacylglycerols could be detected. Growth of the phytoplankton cultures under enhanced CO2 concentration did not affect the lipid remodeling results. The present study provides new insights into how the P-related trophic state of an ecosystem as well as viral infection can affect phytoplankton IPL composition, and therefore influence food web dynamics and biogeochemical cycling.

Efficient microfluidic negative enrichment of circulating tumor cells in blood using roughened PDMS

Depletion of >99.7% WBCs enabling tumor cell recovery from blood with nano-rough PDMS microfluidic negative enrichment devices functionalised with anti-CD45.

Trains, tails and loops of partially adsorbed semi-flexible filaments

Depletion induced adsorption of semi-flexible polymers onto a hard-wall is investigated by a combination of microscopy and computer simulations. In contrast to long flexible filaments which primarily desorb by the formation of loops, the desorption of stiff, finite-sized filaments is largely driven by fluctuating filament tails.

Direct measurement of interaction forces between charged multilamellar vesicles†

Depletion-attraction induced adhesion of two giant (∼40 μm), charged multilamellar vesicles is studied using a new Cantilevered-Capillary Force Apparatus, developed in this laboratory.

Nutrient Uptake among Subspecies of Cucurbita pepo L. Is Related to Exudation of Citric Acid

Exudation of organic acids by roots has been implicated in uptake of minerals from soil. Three cultivars within each of two subspecies of summer squash (Cucurbita pepo ssp. ovifera D. S. Decker var. ovifera and C. pepo ssp. pepo var. pepo) were grown in the field. Plants of ssp. pepo had higher concentrations of K, P, and Zn than those of ssp. ovifera. These same cultivars were grown under P sufficient and depleted conditions in hydroponics, to measure exudation of organic acids from roots. When grown in hydroponics, tissues of ssp. ovifera had similar or higher concentrations of nutrients than ssp. pepo. Therefore, differences in tissue composition of field-grown plants are likely due to differences in nutrient uptake ability, not inherent differences in tissue composition between subspecies. Phosphorus nutrition played a significant role in exudation of organic acids into the hydroponics solution. For both subspecies, P depletion resulted in exudation of more citric and succinic acid, and less oxalic and tartaric acid. Under P depletion, ssp. pepo exuded more citric acid than ssp. ovifera. When soil was eluted with solution containing root exudates, the exudates from ssp. pepo eluted more K, Mg, Fe, and Zn than did those from ssp. ovifera. Among subspecies of C. pepo, exudation of organic acids, particularly exudation of citric acid in response to P depletion, is associated with the plant's ability to accumulate more inorganic nutrients when grown in the field.