Calcium-dependent aggregation and fusion of phosphatidylcholine liposomes induced by complexes of flavonoids with divalent iron

It was found that complexes of the flavonoids quercetin, taxifolin, catechin and morin with divalent iron initiated an increase in light scattering in a suspension of unilamellar 100nm liposomes. The concentration of divalent iron in the suspension was 10μM. Liposomes were prepared from 1-palmitoyl-2-oleoylglycero-3-phoshpatidylcholine. The fluorescent resonance energy transfer (FRET) analysis of liposomes labeled with NBD-PE and lissamine rhodamine B dyes detected a slow lipid exchange in liposomes treated with flavonoid-iron complexes and calcium, while photon correlation spectroscopy and freeze-fracture electron microscopy revealed the aggregation and fusion of liposomes to yield gigantic vesicles. Such processes were not found in liposomes treated with phloretin because this flavonoid is unable to interact with iron. Rutin was also unable to initiate any marked changes because this water-soluble flavonoid cannot interact with the lipid bilayer. The experimental data and computer calculations of lipophilicity (cLogP) as well as the charge distribution on flavonoid-iron complexes indicate that the adhesion of liposomes is provided by an iron link between flavonoid molecules integrated in adjacent bilayers. It is supposed that calcium cations facilitate the aggregation and fusion of liposomes because they interact with the phosphate moieties of lipids.     

Transcriptional regulation of central carbon and energy metabolism in bacteria by redox-responsive repressor Rex

Redox-sensing repressor Rex was previously implicated in the control of anaerobic respiration in response to the cellular NADH/NAD(+) levels in gram-positive bacteria. We utilized the comparative genomics approach to infer candidate Rex-binding DNA motifs and assess the Rex regulon content in 119 genomes from 11 taxonomic groups. Both DNA-binding and NAD-sensing domains are broadly conserved in Rex orthologs identified in the phyla Firmicutes, Thermotogales, Actinobacteria, Chloroflexi, Deinococcus-Thermus, and Proteobacteria. The identified DNA-binding motifs showed significant conservation in these species, with the only exception detected in Clostridia, where the Rex motif deviates in two positions from the generalized consensus, TTGTGAANNNNTTCACAA. Comparative analysis of candidate Rex sites revealed remarkable variations in functional repertoires of candidate Rex-regulated genes in various microorganisms. Most of the reconstructed regulatory interactions are lineage specific, suggesting frequent events of gain and loss of regulator binding sites in the evolution of Rex regulons. We identified more than 50 novel Rex-regulated operons encoding functions that are essential for resumption of the NADH:NAD(+) balance. The novel functional role of Rex in the control of the central carbon metabolism and hydrogen production genes was validated by in vitro DNA binding assays using the TM0169 protein in the hydrogen-producing bacterium Thermotoga maritima.     

Intact Cytoskeleton Is Required for Small G Protein Dependent Activation of the Epithelial Na+ Channel

Background

The Epithelial Na⁺ Channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is regulated, in part, by cortical cytoskeleton. Besides, the cytoskeleton is an established target for small G proteins signaling. Here we studied whether ENaC activity is modulated by changes in the state of the cytoskeleton and whether cytoskeletal elements are involved in small G protein mediated increase of ENaC activity.

Methods and Findings

First, the functional importance of the cytoskeleton was established with whole-cell patch clamp experiments recording ENaC reconstituted in CHO cells. Pretreatment with Cytochalasin D (CytD; 10 µg/ml; 1–2 h) or colchicine (500 µM; 1–3 h) to disassembly F-actin and destroy microtubules, respectively, significantly decreased amiloride sensitive current. However, acute application of CytD induced rapid increase in macroscopic current. Single channel measurements under cell-attached conditions revealed similar observations. CytD rapidly increased ENaC activity in freshly isolated rat collecting duct, polarized epithelial mouse mpkCCD_c14 cells and HEK293 cells transiently transfected with ENaC subunits. In contrast, colchicine did not have an acute effect on ENaC activity. Small G proteins RhoA, Rac1 and Rab11a markedly increase ENaC activity. 1–2 h treatment with colchicine or CytD abolished effects of these GTPases. Interestingly, when cells were coexpressed with ENaC and RhoA, short-term treatment with CytD decreased ENaC activity.

Conclusions

We conclude that cytoskeleton is involved in regulation of ENaC and is necessary for small G protein mediated increase of ENaC activity.     

Antigen-specific B cells reactivate an effective cytotoxic T cell response against phagocytosed Salmonella through cross-presentation

Background

The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8⁺ T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8⁺ T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons.

Methodology/Principal Findings

Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8⁺ T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8⁺ T cells is dependent on CD4⁺ T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis.

Conclusions/Significance

B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.     

Illumina-microarray analysis of mycophenolic acid-induced cell death in an insulin-producing cell line and primary rat islet cells: New insights into apoptotic pathways involved

Mycophenolic acid (MPA), widely used to prevent organ transplant rejection, may induce toxicity and impair function in β-cells. Mechanisms of MPA-induced cell death have not been fully explored. In this study, we examined gene expression patterns in INS-1E cells and isolated primary rat islets following MPA treatment using the Illumina-cDNA microarray. The MPA treatment decreases RhoGDI-α gene expression, which points to apoptosis by JNK activation through a MAPKs-dependent pathway. A strong association between RhoGDI-α and Rac1 activation during MPA-induced apoptosis is also consistent with apoptosis through JNK. Suppression of RhoGDI-α using siRNA and gene over-expression both affected the cell death rate, consistent with Rac1 activation and downstream activation of MAPKs signaling. We confirmed that Rac1 protein mediates the interaction between RhoGDI-α and JNK signaling. We conclude that MPA-induced cell death in primary β-cells and an insulin-secreting cell line proceeds through RhoGDI-α down-regulation linked to Rac1 activation, with subsequent activation of JNK. The RhoGDI-α/Rac1/JNK pathway may present a key to intervention in MPA-induced islet apoptosis.     

Small GTPases of the Rab family in the brain of Bombyx mori

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm.     

Multiple Euryhaline Lineages of Centrohelids (Haptista: Centroplasthelida) in Inland Saline Waters Revealed with Metabarcoding

The diversity of centrohelids in inland saline waters was studied with metabarcoding for the first time. The fragment of V6–V7 regions of 18S rDNA was sequenced with newly designed primers. Obtained OTUs were identified with molecular phylogenetic analysis and comparison of the signatures in 39es9 hairpin of V7. The obtained data included some OTUs, which could be attributed to four described species, but the majority belonged to previously established or novel environmental clades. Along with some presumably marine/brackish clades and freshwater/low salinity (0–2 ppt) clades, seven presumable species demonstrating broad (from 1–2 up to 78 ppt) salinity tolerance were detected. A number of OTUs belonged to Raphidocystis contractilis, which is known from three independent findings in brackish habitats only. Thus, it was assumed that this species is stenohaline and specifically adapted to salinity 5–15 ppt. The high level of salinity tolerance was suggested for centrohelids before based on morphology, which was used to justify their cosmopolitan distribution. Later these views were criticized based on environmental sequencing, but the results of the current survey indicate, that at least some species are present at salinities from almost freshwater (1–2 ppt) to twice oceanic (78 ppt) and are presumably capable of overcoming oceanic salinity barriers for their distribution.     

Techno-economic analysis of a plant-based platform for manufacturing antimicrobial proteins for food safety

Continuous reports of foodborne illnesses worldwide and the prevalence of antibiotic-resistant bacteria mandate novel interventions to assure the safety of our food. Treatment of a variety of foods with bacteriophage-derived lysins and bacteriocin-class antimicrobial proteins has been shown to protect against high-risk pathogens at multiple intervention points along the food supply chain. The most significant barrier to the adoption of antimicrobial proteins as a food safety intervention by the food industry is the high production cost using current fermentation-based approaches. Recently, plants have been shown to produce antimicrobial proteins with accumulation as high as 3 g/kg fresh weight and with demonstrated activity against major foodborne pathogens. To investigate potential economic advantages and scalability of this novel platform, we evaluated a highly efficient transgenic plant-based production process. A detailed process simulation model was developed to help identify economic “hot spots” for research and development focus including process operating parameters, unit operations, consumables, and/or raw materials that have the most significant impact on production costs. Our analyses indicate that the unit production cost of antimicrobial proteins in plants at commercial scale for three scenarios is $3.00–6.88/g, which can support a competitive selling price to traditional food safety treatments.