Macrophage-mediated tissue response evoked by subchronic inhalation of lead oxide nanoparticles is associated with the alteration of phospholipases C and cholesterol transporters.

BACKGROUND: Inhalation of lead oxide nanoparticles (PbO NPs), which are emitted to the environment by high-temperature technological processes, heavily impairs target organs. These nanoparticles pass through the lung barrier and are distributed via the blood into secondary target organs, where they cause numerous pathological alterations. Here, we studied in detail, macrophages as specialized cells involved in the innate and adaptive immune response in selected target organs to unravel their potential involvement in reaction to subchronic PbO NP inhalation. In this context, we also tackled possible alterations in lipid uptake in the lungs and liver, which is usually associated with foam macrophage formation. RESULTS: The histopathological analysis of PbO NP exposed lung revealed serious chronic inflammation of lung tissues. The number of total and foam macrophages was significantly increased in lung, and they contained numerous cholesterol crystals. PbO NP inhalation induced changes in expression of phospholipases C (PLC) as enzymes linked to macrophage-mediated inflammation in lungs. In the liver, the subchronic inhalation of PbO NPs caused predominantly hyperemia, microsteatosis or remodeling of the liver parenchyma, and the number of liver macrophages also significantly was increased. The gene and protein expression of a cholesterol transporter CD36, which is associated with lipid metabolism, was altered in the liver. The amount of selected cholesteryl esters (CE 16:0, CE 18:1, CE 20:4, CE 22:6) in liver tissue was decreased after subchronic PbO NP inhalation, while total and free cholesterol in liver tissue was slightly increased. Gene and protein expression of phospholipase PLCß1 and receptor CD36 in human hepatocytes were affected also in in vitro experiments after acute PbO NP exposure. No microscopic or serious functional kidney alterations were detected after subchronic PbO NP exposure and CD68 positive cells were present in the physiological mode in its interstitial tissues. CONCLUSION: Our study revealed the association of increased cholesterol and lipid storage in targeted tissues with the alteration of scavenger receptors and phospholipases C after subchronic inhalation of PbO NPs and yet uncovered processes, which can contribute to steatosis in liver after metal nanoparticles exposure.

The most abundant maternal lncRNA Sirena1 acts post-transcriptionally and impacts mitochondrial distribution.

Tens of thousands of rapidly evolving long non-coding RNA (lncRNA) genes have been identified, but functions were assigned to relatively few of them. The lncRNA contribution to the mouse oocyte physiology remains unknown. We report the evolutionary history and functional analysis of Sirena1, the most expressed lncRNA and the 10th most abundant poly(A) transcript in mouse oocytes. Sirena1 appeared in the common ancestor of mouse and rat and became engaged in two different post-transcriptional regulations. First, antisense oriented Elob pseudogene insertion into Sirena1 exon 1 is a source of small RNAs targeting Elob mRNA via RNA interference. Second, Sirena1 evolved functional cytoplasmic polyadenylation elements, an unexpected feature borrowed from translation control of specific maternal mRNAs. Sirena1 knock-out does not affect fertility, but causes minor dysregulation of the maternal transcriptome. This includes increased levels of Elob and mitochondrial mRNAs. Mitochondria in Sirena1-/- oocytes disperse from the perinuclear compartment, but do not change in number or ultrastructure. Taken together, Sirena1 contributes to RNA interference and mitochondrial aggregation in mouse oocytes. Sirena1 exemplifies how lncRNAs stochastically engage or even repurpose molecular mechanisms during evolution. Simultaneously, Sirena1 expression levels and unique functional features contrast with the lack of functional importance assessed under laboratory conditions.

Bacillus subtilis alters the proportion of major membrane phospholipids in response to surfactin exposure.

Surfactin, an anionic lipopeptide produced by Bacillus subtilis, is an antimicrobial that targets the cytoplasmic membrane. Nowadays it appears increasingly apparent that the mechanism of resistance against these types of antibiotics consists of target site modification. This prompted us to investigate whether the surfactin non-producing strain B. subtilis 168 changes its membrane composition in response to a sublethal surfactin concentration. Here we show that the exposure of B. subtilis to surfactin at concentrations of 350 and 650 µg/ml (designated as SF350 and SF650, respectively) leads to a concentration-dependent growth arrest followed by regrowth with an altered growth rate. Analysis of the membrane lipid composition revealed modifications both in the polar head group and the fatty acid region. The presence of either surfactin concentration resulted in a reduction in the content of the major membrane phospholipid phosphatidylglycerol (PG) and increase in phosphatidylethanolamine (PE), which was accompanied by elevated levels of phosphatidic acid (PA) in SF350 cultures. The fatty acid analysis of SF350 cells showed a marked increase in non-branched high-melting fatty acids, which lowered the fluidity of the membrane interior measured as the steady-state fluorescence anisotropy of DPH. The liposome leakage of carboxyfluorescein-loaded vesicles resembling the phospholipid composition of surfactin-adapted cells showed that the susceptibility to surfactin-induced leakage is strongly reduced when the PG/PE ratio decreases and/or PA is included in the target bilayer. We concluded that the modifications of the phospholipid content of B. subtilis cells might provide a self-tolerance of the membrane active surfactin.

Cellular uptake and fate of cationic polymer-coated nanodiamonds delivering siRNA: a mechanistic study.

Gene silencing using small interfering RNAs (siRNAs) is a selective and promising approach for treatment of numerous diseases. However, broad applications of siRNAs are compromised by their low stability in a biological environment and limited ability to penetrate cells. Nanodiamonds (NDs) coated with cationic polymers can enable cellular delivery of siRNAs. Recently, we developed a new type of ND coating based on a random copolymer consisting of (2-dimethylaminoethyl) methacrylate (DMAEMA) and N-(2-hydroxypropyl) methacrylamide (HPMA) monomers. These hybrid ND-polymer particles (Cop+-FND) provide near-infrared fluorescence, form stable complexes with siRNA in serum, show low toxicity, and effectively deliver siRNA into cells in vitro and in vivo. Here, we present data on the mechanism of cellular uptake and cell trafficking of Cop+-FND : siRNA complexes and their ability to selectively suppress mRNA levels, as well as their cytotoxicity, viability and colloidal stability. We identified clathrin-mediated endocytosis as the predominant entry mechanism for Cop+-FND : siRNA into U-2 OS human bone osteosarcoma cells, with a substantial fraction of Cop+-FND : siRNA following the lysosome pathway. Cop+-FND : siRNA potently inhibited the target GAPDH gene with negligible toxicity and sufficient colloidal stability. Based on our results, we suggest that Cop+-FND : siRNA can serve as a suitable in vivo delivery system for siRNA.

Ion beam processing of DNA origami nanostructures.

DNA origami nanostructures are emerging as a bottom-up nanopatterning approach. Direct combination of this approach with top-down nanotechnology, such as ion beams, has not been considered because of the soft nature of the DNA material. Here we demonstrate that the shape of 2D DNA origami nanostructures deposited on Si substrates is well preserved upon irradiation by ion beams, modeling ion implantation, lithography, and sputtering conditions. Structural changes in 2D DNA origami nanostructures deposited on Si are analyzed using AFM imaging. The observed effects on DNA origami include structure height decrease or increase upon fast heavy ion irradiation in vacuum and in air, respectively. Slow- and medium-energy heavy ion irradiation results in the cutting of the nanostructures or crater formation with ion-induced damage in the 10 nm range around the primary ion track. In all these cases, the designed shape of the 2D origami nanostructure remains unperturbed. Present stability and nature of damages on DNA origami nanostructures enable fusion of DNA origami advantages such as shape and positioning control into novel ion beam nanofabrication approaches.

Evidence of Au(II) and Au(0) States in Bovine Serum Albumin-Au Nanoclusters Revealed by CW-EPR/LEPR and Peculiarities in HR-TEM/STEM Imaging.

Bovine serum albumin-embedded Au nanoclusters (BSA-AuNCs) are thoroughly probed by continuous wave electron paramagnetic resonance (CW-EPR), light-induced EPR (LEPR), and sequences of microscopic investigations performed via high-resolution transmission electron microscopy (HR-TEM), scanning transmission electron microscopy (STEM), and energy dispersive X-ray analysis (EDS). To the best of our knowledge, this is the first report analyzing the BSA-AuNCs by CW-EPR/LEPR technique. Besides the presence of Au(0) and Au(I) oxidation states in BSA-AuNCs, the authors observe a significant amount of Au(II), which may result from a disproportionation event occurring within NCs: 2Au(I) → Au(II) + Au(0). Based on the LEPR experiments, and by comparing the behavior of BSA versus BSA-AuNCs under UV light irradiation (at 325 nm) during light off-on-off cycles, any energy and/or charge transfer event occurring between BSA and AuNCs during photoexcitation can be excluded. According to CW-EPR results, the Au nano assemblies within BSA-AuNCs are estimated to contain 6-8 Au units per fluorescent cluster. Direct observation of BSA-AuNCs by STEM and HR-TEM techniques confirms the presence of such diameters of gold nanoclusters in BSA-AuNCs. Moreover, in situ formation and migration of Au nanostructures are observed and evidenced after application of either a focused electron beam from HR-TEM, or an X-ray from EDS experiments.

Outer membrane and phospholipid composition of the target membrane affect the antimicrobial potential of first- and second-generation lipophosphonoxins.

Lipophosphonoxins (LPPOs) are small modular synthetic antibacterial compounds that target the cytoplasmic membrane. First-generation LPPOs (LPPO I) exhibit an antimicrobial activity against Gram-positive bacteria; however they do not exhibit any activity against Gram-negatives. Second-generation LPPOs (LPPO II) also exhibit broadened activity against Gram-negatives. We investigated the reasons behind this different susceptibility of bacteria to the two generations of LPPOs using model membranes and the living model bacteria Bacillus subtilis and Escherichia coli. We show that both generations of LPPOs form oligomeric conductive pores and permeabilize the bacterial membrane of sensitive cells. LPPO activity is not affected by the value of the target membrane potential, and thus they are also active against persister cells. The insensitivity of Gram-negative bacteria to LPPO I is probably caused by the barrier function of the outer membrane with LPS. LPPO I is almost incapable of overcoming the outer membrane in living cells, and the presence of LPS in liposomes substantially reduces their activity. Further, the antimicrobial activity of LPPO is also influenced by the phospholipid composition of the target membrane. A higher proportion of phospholipids with neutral charge such as phosphatidylethanolamine or phosphatidylcholine reduces the LPPO permeabilizing potential.

Bacillus subtilis cardiolipin protects its own membrane against surfactin-induced permeabilization.

Surfactin, a cyclic lipoheptapeptide produced by Bacillus subtilis, is a surface-active antimicrobial that targets the barrier function of lipid membranes. It inserts itself into the membrane, where it forms conductive pores. Depending on its concentration, it eventually disintegrates the membrane in a detergent-like manner. The molecular details of this activity are not yet sufficiently understood, nor are the mechanisms that the surfactin producer employs to resist its own toxic product. We have previously shown that B. subtilis modifies its membrane lipid composition upon the onset of surfactin production, mainly increasing the cardiolipin content. Here we show that the increased cardiolipin content leads to a decreased surfactin-induced leakage of liposomes reconstituted from lipids isolated from the surfactin producer. This stabilizing effect of cardiolipin is concentration-dependent. Using a propidium iodide-based cell permeabilization assay, we further confirmed that the cytoplasmic membrane of the mutant B. subtilis strain lacking cardiolipin was substantially more susceptible to the action of surfactin, even though the amount of bound surfactin was the same as in the wild-type strain. We propose that membrane remodelling; due to the increase in cardiolipin content, contributes to the surfactin tolerance of B. subtilis.

Multi-Component Antioxidative System and Robust Carbohydrate Status, the Essence of Plant Arsenic Tolerance.

Arsenic (As) contaminates the food chain and decreases agricultural production through impairing plants, particularly due to oxidative stress. To better understand the As tolerance mechanisms, two contrasting tobacco genotypes: As-sensitive Nicotiana sylvestris and As-tolerant N.tabacum, cv. 'Wisconsin' were analyzed. The most meaningful differences were found in the carbohydrate status, neglected so far in the As context. In the tolerant genotype, contrary to the sensitive one, net photosynthesis rates and saccharide levels were unaffected by As exposure. Importantly, the total antioxidant capacity was far stronger in the As-tolerant genotype, based on higher antioxidants levels (e.g., phenolics, ascorbate, glutathione) and activities and/or appropriate localizations of antioxidative enzymes, manifested as reverse root/shoot activities in the selected genotypes. Accordingly, malondialdehyde levels, a lipid peroxidation marker, increased only in sensitive tobacco, indicating efficient membrane protection in As-tolerant species. We bring new evidence of the orchestrated action of a broad spectrum of both antioxidant enzymes and molecules essential for As stress coping. For the first time, we propose robust carbohydrate metabolism based on undisturbed photosynthesis to be crucial not only for subsidizing C and energy for defense but also for participating in direct reactive oxygen species (ROS) quenching. The collected data and suggestions can serve as a basis for the selection of plant As phytoremediators or for targeted breeding of tolerant crops.

The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts.

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.

Toxicity of TiO2, ZnO, and SiO2 Nanoparticles in Human Lung Cells: Safe-by-Design Development of Construction Materials.

Rapid progress in the development of highly efficient nanoparticle-based construction technologies has not always been accompanied by a corresponding understanding of their effects on human health and ecosystems. In this study, we compare the toxicological effects of pristine TiO2, ZnO, SiO2, and coated SiO2 nanoparticles, and evaluate their suitability as additives to consolidants of weathered construction materials. First, water soluble tetrazolium 1 (WST-1) and lactate dehydrogenase (LDH) assays were used to determine the viability of human alveolar A549 cells at various nanoparticle concentrations (0-250 µg mL-1). While the pristine TiO2 and coated SiO2 nanoparticles did not exhibit any cytotoxic effects up to the highest tested concentration, the pristine SiO2 and ZnO nanoparticles significantly reduced cell viability. Second, as all developed nanoparticle-modified consolidants increased the mechanical strength of weathered sandstone, the decisive criterion for the selection of the most suitable nanoparticle additive was as low toxicity as possible. We believe that this approach would be of high importance in the industry, to identify materials representing top functional properties and low toxicity, at an early stage of the product development.

Molecular Responses in THP-1 Macrophage-Like Cells Exposed to Diverse Nanoparticles.

In the body, engineered nanoparticles (NPs) may be recognized and processed by immune cells, among which macrophages play a crucial role. We evaluated the effects of selected NPs [NM-100 (TiO2), NM-110 (ZnO), NM-200 (SiO2), and NM-300 K (Ag)] on THP-1 macrophage-like cells. The cells were exposed to subcytotoxic concentrations of NPs (1-25 µg/mL) and the expression of immunologically relevant genes (VCAM1, TNFA, CXCL8, ICAM1, CD86, CD192, and IL1B) was analyzed by RT-qPCR. The expression of selected cytokines, growth factors and surface molecules was assessed by flow cytometry or ELISA. Generation of reactive oxygen species and induction of DNA breaks were also analyzed. Exposure to diverse NPs caused substantially different molecular responses. No significant effects were detected for NM-100 treatment. NM-200 induced production of IL-8, a potent attractor and activator of neutrophils, growth factors (VEGF and IGF-1) and superoxide. NM-110 triggered a proinflammatory response, characterized by the activation of transcription factor NF-κB, an enhanced production of proinflammatory cytokines (TNF-α) and chemokines (IL-8). Furthermore, the expression of cell adhesion molecules VCAM-1 and ICAM-1 and hepatocyte growth factor (HGF), as well as superoxide production and DNA breaks, were affected. NM-300 K enhanced IL-8 production and induced DNA breaks, however, it decreased the expression of chemokine receptor (CCR2) and CD86 molecule, indicating potential immunosuppressive activity. The toxicity of ZnO and Ag NPs was probably caused by their intracellular dissolution, as indicated by transmission electron microscopy imaging. The observed effects in macrophages might further influence both innate and adaptive immune responses by promoting neutrophil recruitment via IL-8 release and enhancing the adhesion and stimulation of T cells by VCAM-1 and ICAM-1 expression.

Francisella tularensis D-Ala D-Ala Carboxypeptidase DacD Is Involved in Intracellular Replication and It Is Necessary for Bacterial Cell Wall Integrity.

D-alanyl-D-alanine carboxypeptidase, product of dacD gene in Francisella, belongs to penicillin binding proteins (PBPs) and is involved in remodeling of newly synthetized peptidoglycan. In E. coli, PBPs are synthetized in various growth phases and they are able to substitute each other to a certain extent. The DacD protein was found to be accumulated in fraction enriched in membrane proteins from severely attenuated dsbA deletion mutant strain. It has been presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are the primary virulence factors. Here we demonstrate that Francisella DacD is required for intracellular replication and virulence in mice. The dacD insertion mutant strain showed higher sensitivity to acidic pH, high temperature and high osmolarity when compared to the wild-type. Eventually, transmission electron microscopy revealed differences in mutant bacteria in both the size and defects in outer membrane underlying its SDS and serum sensitivity. Taken together these results suggest DacD plays an important role in Francisella pathogenicity.