Stress-Induced Changes in Optical Properties,Pigment and Fatty Acid Content of <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp.: Implications for Non-destructive Assay of Total Fatty Acids

In order to develop a practical approach for fast and non-destructive assay of total fatty acid (TFA) and pigments in the biomass of the marine microalga Nannochloropsis sp. changes in TFA, chlorophyll, and carotenoid contents were monitored in parallel with the cell suspension absorbance. The experiments were conducted with the cultures grown under normal (complete nutrient f/2 medium at 75 μmol PAR photons/(m² s)) or stressful (nitrogen-lacking media at 350 μmol PAR photons/(m² s)) conditions. The reliable measurement of the cell suspension absorbance using a spectrophotometer without integrating sphere was achieved by deposition of cells on glass–fiber filters in the chlorophyll content range of 3–13 mg/L. Under stressful conditions, a 30–50% decline in biomass and chlorophyll, retention of carotenoids and a build-up of TFA (15–45 % of dry weight) were recorded. Spectral regions sensitive to widely ranging changes in carotenoid-to-chlorophyll ratio and correlated changes of TFA content were revealed. Employing the tight inter-correlation of stress-induced changes in lipid metabolism and rearrangement of the pigment apparatus, the spectral indices were constructed for non-destructive assessment of carotenoid-to-chlorophyll ratio (range 0.3–0.6; root mean square error (RMSE) = 0.03; r ² = 0.93) as well as TFA content of Nannochloropsis sp. biomass (range 5.0–45%; RMSE = 3.23 %; r ² = 0.89) in the broad band 400–550 nm normalized to that in chlorophyll absorption band (centered at 678 nm). The findings are discussed in the context of real-time monitoring of the TFA accumulation by Nannochloropsis cultures under stressful conditions.     

The contribution of hydrophobic residues in the pore-forming region of the ryanodine receptor channel to block by large tetraalkylammonium cations and Shaker B inactivation peptides

Although no high-resolution structural information is available for the ryanodine receptor (RyR) channel pore-forming region (PFR), molecular modeling has revealed broad structural similarities between this region and the equivalent region of K(+) channels. This study predicts that, as is the case in K(+) channels, RyR has a cytosolic vestibule lined with predominantly hydrophobic residues of transmembrane helices (TM10). In K(+) channels, this vestibule is the binding site for blocking tetraalkylammonium (TAA) cations and Shaker B inactivation peptides (ShBPs), which are stabilized by hydrophobic interactions involving specific residues of the lining helices. We have tested the hypothesis that the cytosolic vestibule of RyR fulfils a similar role and that TAAs and ShBPs are stabilized by hydrophobic interactions with residues of TM10. Both TAAs and ShBPs block RyR from the cytosolic side of the channel. By varying the composition of TAAs and ShBPs, we demonstrate that the affinity of both species is determined by their hydrophobicity, with variations reflecting alterations in the dissociation rate of the bound blockers. We investigated the role of TM10 residues of RyR by monitoring block by TAAs and ShBPs in channels in which the hydrophobicity of individual TM10 residues was lowered by alanine substitution. Although substitutions changed the kinetics of TAA interaction, they produced no significant changes in ShBP kinetics, indicating the absence of specific hydrophobic sites of interactions between RyR and these peptides. Our investigations (a) provide significant new information on both the mechanisms and structural components of the RyR PFR involved in block by TAAs and ShBPs, (b) highlight important differences in the mechanisms and structures determining TAA and ShBP block in RyR and K(+) channels, and (c) demonstrate that although the PFRs of these channels contain analogous structural components, significant differences in structure determine the distinct ion-handling properties of the two species of channel.     

Impact of short-term acidification on nitrification and nitrifying bacterial community dynamics in soilless cultivation media

Soilless medium-based horticulture systems are highly prevalent due to their capacity to optimize growth of high-cash crops. However, these systems are highly dynamic and more sensitive to physiochemical and pH perturbations than traditional soil-based systems, especially during nitrification associated with ammonia-based fertilization. The objective of this study was to assess the impact of nitrification-generated acidification on ammonia oxidation rates and nitrifying bacterial community dynamics in soilless growth media. To achieve this goal, perlite soilless growth medium from a commercial bell pepper greenhouse was incubated with ammonium in bench-scale microcosm experiments. Initial quantitative real-time PCR analysis indicated that betaproteobacterial ammonia oxidizers were significantly more abundant than ammonia-oxidizing archaea, and therefore, research focused on this group. Ammonia oxidation rates were highest between 0 and 9 days, when pH values dropped from 7.4 to 4.9. Pyrosequencing of betaproteobacterial ammonia-oxidizing amoA gene fragments indicated that r-strategist-like Nitrosomonas was the dominant ammonia-oxidizing bacterial genus during this period, seemingly due to the high ammonium concentration and optimal growth conditions in the soilless media. Reduction of pH to levels below 4.8 resulted in a significant decrease in both ammonia oxidation rates and the diversity of ammonia-oxidizing bacteria, with increased relative abundance of the r-strategist-like Nitrosospira. Nitrite oxidizers (Nitrospira and Nitrobacter) were on the whole more abundant and less sensitive to acidification than ammonia oxidizers. This study demonstrates that nitrification and nitrifying bacterial community dynamics in high-N-load intensive soilless growth media may be significantly different from those in in-terra agricultural systems.     

Synthetic lethality of PARP inhibition in BRCA-network disrupted tumor cells is associated with interferon pathway activation and enhanced by interferon-γ

Tumor suppressor genes BRCA1 and BRCA2 function in a complex gene network that regulates homologous recombination and DNA double-strand break repair. Disruption of the BRCA-network through gene mutation, deletion, or RNAi-mediated silencing can sensitize cells to small molecule inhibitors of poly (ADP-ribose) polymerase (PARPi). Here, we demonstrate that BRCA-network disruption in the presence of PARPi leads to the selective induction and enhancement of interferon pathway and apoptotic gene expression in cultured tumor cells. In addition, we report PARPi cytotoxicity in BRCA1-deficient tumor cells is enhanced >10-fold when combined with interferon-γ. These findings establish a link between synthetic lethality of PARPi in BRCA-network disrupted cells and interferon pathway activation triggered by genetic instability.     

Modulation of immune responses by Plasmodium falciparum infection in asymptomatic children living in the endemic region of Mbita,western Kenya

Individuals living in malaria endemic areas become clinically immune after multiple re-infections over time and remain infected without apparent symptoms. However, it is unclear why a long period is required to gain clinical immunity to malaria, and how such immunity is maintained. Although malaria infection is reported to induce inhibition of immune responses, studies on asymptomatic individuals living in endemic regions of malaria are relatively scarce. We conducted a cross-sectional study of immune responses in asymptomatic school children aged 4–16 years living in an area where Plasmodium falciparum and Schistosoma mansoni infections are co-endemic in Kenya. Peripheral blood mononuclear cells were subjected to flow cytometric analysis and cultured to determine proliferative responses and cytokine production. The proportions of cellular subsets in children positive for P. falciparum infection at the level of microscopy were comparable to the negative children, except for a reduction in central memory-phenotype CD8⁺ T cells and natural killer cells. In functional studies, the production of cytokines by peripheral blood mononuclear cells in response to P. falciparum crude antigens exhibited strong heterogeneity among children. In addition, production of IL-2 in response to anti-CD3 and anti-CD28 monoclonal antibodies was significantly reduced in P. falciparum-positive children as compared to -negative children, suggesting a state of unresponsiveness. These data suggest that the quality of T cell immune responses is heterogeneous among asymptomatic children living in the endemic region of P. falciparum, and that the responses are generally suppressed by active infection with Plasmodium parasites.