Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles

In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.     

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition—Control; after 60 h—PES Day 2.5 of embryo culture; and after 96 h—PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (Control: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (Control: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification.     

Surfactant protein D increases phagocytosis and aggregation of pollen-allergen starch granules

Recent studies have shown that surfactant components, in particular the collectins surfactant protein (SP)-A and -D, modulate the phagocytosis of various pathogens by alveolar macrophages. This interaction might be important not only for the elimination of pathogens but also for the elimination of inhaled allergens and might explain anti-inflammatory effects of SP-A and SP-D in allergic airway inflammation. We investigated the effect of surfactant components on the phagocytosis of allergen-containing pollen starch granules (PSG) by alveolar macrophages. PSG were isolated from Dactylis glomerata or Phleum pratense, two common grass pollen allergens, and incubated with either rat or human alveolar macrophages in the presence of recombinant human SP-A, SP-A purified from patients suffering from alveolar proteinosis, a recombinant fragment of human SP-D, dodecameric recombinant rat SP-D, or the commercially available surfactant preparations Curosurf and Alveofact. Dodecameric rat recombinant SP-D enhanced binding and phagocytosis of the PSG by alveolar macrophages, whereas the recombinant fragment of human SP-D, SP-A, or the surfactant lipid preparations had no effect. In addition, recombinant rat SP-D bound to the surface of the PSG and induced aggregation. Binding, aggregation, and enhancement of phagocytosis by recombinant rat SP-D was completely blocked by EDTA and inhibited by d-maltose and to a lesser extent by d-galactose, indicating the involvement of the carbohydrate recognition domain of SP-D in these functions. The modulation of allergen phagocytosis by SP-D might play an important role in allergen clearance from the lung and thereby modulate the allergic inflammation of asthma.     

Interspecific hybridization contributes to high genetic diversity and apparent effective population size in an endemic population of mottled ducks (Anas fulvigula maculosa)

Under drift-mutation equilibrium, genetic diversity is expected to be correlated with effective population size (N _e ). Changes in population size and gene flow are two important processes that can cause populations to deviate from this expected relationship. In this study, we used DNA sequences from six independent loci to examine the influence of these processes on standing genetic diversity in endemic mottled ducks (Anas fulvigula) and geographically widespread mallards (A. platyrhynchos), two species known to hybridize. Mottled ducks have an estimated census size that is about two orders-of-magnitude smaller than that of mallards, yet these two species have similar levels of genetic diversity, especially at nuclear DNA. Coalescent analyses suggest that a population expansion in the mallard at least partly explains this discrepancy, but the mottled duck harbors higher genetic diversity and apparent N _e than expected for its census size even after accounting for a population decline. Incorporating gene flow into the model, however, reduced the estimated N _e of mottled ducks to 33 % of the equilibrium N _e and yielded an estimated N _e consistent with census size. We also examined the utility of these loci to distinguish among mallards, mottled ducks, and their hybrids. Most putatively pure individuals were correctly assigned to species, but the power for detecting hybrids was low. Although hybridization with mallards potentially poses a conservation threat to mottled ducks by creating a risk of extinction by hybridization, introgression of mallard alleles has helped maintain high genetic diversity in mottled ducks and might be important for the adaptability and survival of this species.     

Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke

Multi-protein complexes, termed “inflammasomes,” are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.     

Characterization of the molecular properties of KtrC,a second RCK domain that regulates a Ktr channel in Bacillus subtilis

RCK (regulating conductance of K⁺) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K⁺ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.     

A high-sensitivity method for detection and measurement of HMGB1 protein concentration by high-affinity binding to DNA hemicatenanes

Background

Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration.

Methodology/Principal Findings

In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum.

Conclusion

The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies.