Genotoxicity of inhaled nanosized TiO(2) in mice

In vitro studies have suggested that nanosized titanium dioxide (TiO(2)) is genotoxic. The significance of these findings with respect to in vivo effects is unclear, as few in vivo studies on TiO(2) genotoxicity exist. Recently, nanosized TiO(2) administered in drinking water was reported to increase, e.g., micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs) and DNA damage in leukocytes. Induction of micronuclei in mouse PCEs was earlier also described for pigment-grade TiO(2) administered intraperitoneally. The apparent systemic genotoxic effects have been suggested to reflect secondary genotoxicity of TiO(2) due to inflammation. However, a recent study suggested that induction of DNA damage in mouse bronchoalveolar lavage (BAL) cells after intratracheal instillation of nanosized or fine TiO(2) is independent of inflammation. We examined here, if inhalation of freshly generated nanosized TiO(2) (74% anatase, 26% brookite; 5 days, 4 h/day) at 0.8, 7.2, and (the highest concentration allowing stable aerosol production) 28.5 mg/m(3) could induce genotoxic effects in C57BL/6J mice locally in the lungs or systematically in peripheral PCEs. DNA damage was assessed by the comet assay in lung epithelial alveolar type II and Clara cells sampled immediately following the exposure. MN were analyzed by acridine orange staining in blood PCEs collected 48 h after the last exposure. A dose-dependent deposition of Ti in lung tissue was seen. Although the highest exposure level produced a clear increase in neutrophils in BAL fluid, indicating an inflammatory effect, no significant effect on the level of DNA damage in lung epithelial cells or micronuclei in PCEs was observed, suggesting no genotoxic effects by the 5-day inhalation exposure to nanosized TiO(2) anatase. Our inhalation exposure resulted in much lower systemic TiO(2) doses than the previous oral and intraperitoneal treatments, and lung epithelial cells probably received considerably less TiO(2) than BAL cells in the earlier intratracheal study.     

Spray-Dried Cellulose Nanofibers as Novel Tablet Excipient

The purpose of this study was to evaluate the potential of cellulose nanofibers (also referred as microfibrillated cellulose, nanocellulose, nanofibrillated, or nanofibrillar cellulose) as novel tabletting material. For this purpose, physical and mechanical properties of spray-dried cellulose nanofibers (CNF) were examined, and results were compared to those of two commercial grades of microcrystalline cellulose (MCC), Avicel PH101 and Avicel PH102, which are the most commonly and widely used direct compression excipients. Chemically, MCC and CNF are almost identical, but their physical characteristics, like mechanical properties and surface-to-volume ratio, differ remarkably. The novel material was characterized with respect to bulk and tapped as well as true density, moisture content, and flow properties. Tablets made of CNF powder and its mixtures with MCC with or without paracetamol as model compound were produced by direct compression and after wet granulation. The tensile strength of the tablets made in a series of applied pressures was determined, and yield pressure values were calculated from the measurements. With CNF, both wet granulation and direct compression were successful. During tablet compression, CNF particles were less prone to permanent deformation and had less pronounced ductile characteristics. Disintegration and dissolution studies showed slightly faster drug release from direct compression tablets with CNF, while wet granulated systems did not have any significant difference.     

Regulation of Phagocyte Migration by Signal Regulatory Protein-Alpha Signaling

Signaling through the inhibitory receptor signal regulatory protein-alpha (SIRPα) controls effector functions in phagocytes. However, there are also indications that interactions between SIRPα and its ligand CD47 are involved in phagocyte transendothelial migration. We have investigated the involvement of SIRPα signaling in phagocyte migration in vitro and in vivo using mice that lack the SIRPα cytoplasmic tail. During thioglycolate-induced peritonitis in SIRPα mutant mice, both neutrophil and macrophage influx were found to occur, but to be significantly delayed. SIRPα signaling appeared to be essential for an optimal transendothelial migration and chemotaxis, and for the amoeboid type of phagocyte migration in 3-dimensional environments. These findings demonstrate, for the first time, that SIRPα signaling can directly control phagocyte migration, and this may contribute to the impaired inflammatory phenotype that has been observed in the absence of SIRPα signaling.     

Dithiothreitol (DTT) Acts as a Specific,UV-inducible Cross-linker in Elucidation of Protein–RNA Interactions*

Protein–RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein–RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein–RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks, which is observable as a mass increment of 151.9966 Da (C₄H₈S₂O₂) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive sites as the most probable structure of the cross-linking products.Cross-linking of biomolecules combined with mass spectrometry (MS) has emerged as a powerful tool to characterize not only the tertiary and quaternary arrangements of individual biomolecules, but especially their interaction sites in biologically active complexes. By MS-based identification of the cross-linked parts or even the exact cross-linking sites of the respective biomolecules, proximity information can be derived. This has proven highly useful for computational approaches to problems such as docking or the arrangement of subunits (1–3).In principle, cross-linking can be achieved in two ways: (1) By using a chemical cross-linker that connects reactive groups of the respective biomolecules within a certain distance range, the range depending on the reagent used. (2) By generating a so-called zero-length cross-link that connects reactive groups of biomolecules that are already directly adjacent to one another. The latter is usually achieved by (UV) light-induced cross-linking, with or without the addition of compounds that induce the generation of radicals on reactive groups of the cross-linkable components or in close vicinity to them.Cross-linking in combination with MS analysis is nowadays frequently used in protein-protein interaction studies (4–7) but can also be applied to protein-nucleic acid complexes. Indeed much attention is currently paid to their MS-based analysis owing to the crucial cellular function of many such complexes. A large variety of studies over decades have examined chemical cross-linking between proteins and DNA, using reagents such as the genotoxic diepoxybutane, endogenous aldehydes, transition metals, nitrogen mustard, and platinum compounds, alkynitrosoureas, and formaldehyde (8). In addition, UV irradiation has been used to establish cross-links between proteins and protein-bound single-stranded DNA (ssDNA), which can then be identified by mass spectrometry (9, 10). UV cross-linking makes use of the natural sensitivity of nucleobases to UV light (11, 12). The site of cross-linking can then be determined by mass spectrometric analysis (including gas phase fragmentation of the corresponding peptide-nucleic acid conjugates) and database searching. To enhance the low yield of protein-DNA cross-linking, derivatives with higher UV reactivity, such as halonucleotides, can be employed (13–15).UV-induced cross-linking is more frequently used to monitor protein–RNA interactions. RNA is mainly present in its single-stranded form, and adopts a variety of tertiary structures in which the bases of the nucleotides are in close contact with amino acid residues of the proteins that are bound to the RNA. Several studies have used this technique to identify globally the proteins that directly interact with e.g. poly(A) mRNA in yeast and human systems, and have yielded insight into the large variety of RNA-binding proteins that exist within the cell (16–21). More detailed approaches apply UV-induced protein–RNA cross-linking in a similar manner, but extend the MS analysis toward the identification of the cross-linked amino acids together with the corresponding nucleotide moieties, allowing exact definition of the RNA-binding regions in the cross-linked proteins (22–24). To improve the yield of cross-linking, more reactive nucleoside derivatives (4-thiouridine and 6-thioguanosine) have been incorporated into RNA in growing cells. The cross-linking reaction of these derivatives with amino acids is not entirely additive, however, but is accompanied by the loss of H₂S ((22), U.Z. and H.U, unpublished results). Other cross-linking reactions between proteins and RNA have made use of nucleotide derivatives that contain a cross-linkable function at the 2′ hydroxyl group or the phosphate moiety (25–27), but have not been characterized by MS yet.Additional cross-linking agents for the analysis of protein–RNA interactions have been exploited, but have not yet found their way into modern MS-based proteome analyses. For instance, methylene blue has been described as a light-inducible cross-linker, in particular for mapping interactions of proteins with dsRNA (28). Similarly, protein–RNA interaction studies in ribosomal subunits have made use of diepoxybutane, or nitrogen mustard (29–31). The same holds true for the use of 2-iminothiolane (“Traut''s reagent”), which is a protein–RNA cross-linking reagent that combines chemical and UV-inducible features and has been extensively applied to the analysis of protein–RNA contacts in ribosomal subunits (33, 32). Here, we introduce dithiothreitol (DTT)¹ as a potent UV-inducible cross-linking reagent for the analysis of protein–RNA linkages following UV irradiation. By exhaustive mass-spectrometric analyses we found that upon UV light exposure DTT forms a covalent linkage between cysteine residues within proteins and uracil bases of RNA in close proximity. We applied this to protein–RNA complexes isolated from yeast cells and compared the protein–RNA cross-linking patterns that were obtained in the presence and absence of DTT. We found that the cross-linking reaction is surprisingly efficient and specific.     

Flexible Structure of Peptide-Bound Filamin A Mechanosensor Domain Pair 20–21

Filamins (FLNs) are large, multidomain actin cross-linking proteins with diverse functions. Besides regulating the actin cytoskeleton, they serve as important links between the extracellular matrix and the cytoskeleton by binding cell surface receptors, functioning as scaffolds for signaling proteins, and binding several other cytoskeletal proteins that regulate cell adhesion dynamics. Structurally, FLNs are formed of an amino terminal actin-binding domain followed by 24 immunoglobulin-like domains (IgFLNs). Recent studies have demonstrated that myosin-mediated contractile forces can reveal hidden protein binding sites in the domain pairs IgFLNa18–19 and 20–21, enabling FLNs to transduce mechanical signals in cells. The atomic structures of these mechanosensor domain pairs in the resting state are known, as well as the structures of individual IgFLN21 with ligand peptides. However, little experimental data is available on how interacting protein binding deforms the domain pair structures. Here, using small-angle x-ray scattering-based modelling, x-ray crystallography, and NMR, we show that the adaptor protein migfilin-derived peptide-bound structure of IgFLNa20–21 is flexible and adopts distinctive conformations depending on the presence or absence of the interacting peptide. The conformational changes reported here may be common for all peptides and may play a role in the mechanosensor function of the site.     

Sex Hormones and Sexual Function in Obese Men Losing Weight

Objective: To study the impact of a weight‐loss program on sex hormones and sexual function among 38 middle‐aged obese men (BMI ≥35 kg/m²). Research Methods and Procedures: A randomized controlled clinical trial was conducted. The treatment group (n = 19) participated in a 4‐month weight‐loss program including 10 weeks on a very‐low‐energy diet (VLED) and 17 behavior modification visits. There was no intervention in the control group (n = 19). Both groups were followed for 8 months, i.e., 22 weeks after the active weight loss in the treatment group. The outcome measures (weight, sex hormones, sexual function, leptin, and metabolic variables) were obtained at baseline and at three time‐points during follow‐up. Results: The mean weight loss in the treatment group was 21 kg at the end of the 10‐week VLED. At the end of follow‐up, the maintained weight loss was 17 kg of baseline weight. The control group was weight stable throughout the study. In the treatment group, increases in sex hormone‐binding globulin, testosterone, and high‐density lipoprotein‐cholesterol, as well as decreases in insulin and leptin, were maintained until the end of follow‐up, although with VLED, the level of several hormones and metabolic variables improved transiently during the rapid weight loss. There were no significant changes in the questionnaire scores on sexual function in either group. Discussion: We conclude that obese men lose weight and increase their serum testosterone level on a weight‐loss program with VLED and behavior modification. However, they do not change their sexual function scores.     

Functional group richness: implications of biodiversity for light use and lipid yield in microalgae

Currently, very few studies address the relationship between diversity and biomass/lipid production in primary producer communities for biofuel production. Basic studies on the growth of microalgal communities, however, provide evidence of a positive relationship between diversity and biomass production. Recent studies have also shown that positive diversity–productivity relationships are related to an increase in the efficiency of light use by diverse microalgal communities. Here, we hypothesize that there is a relationship between diversity, light use, and microalgal lipid production in phytoplankton communities. Microalgae from all major freshwater algal groups were cultivated in treatments that differed in species richness and functional group richness. Polycultures with high functional group richness showed more efficient light use and higher algal lipid content with increasing species richness. There was a clear correlation between light use and lipid production in functionally diverse communities. Hence, a powerful and cost‐effective way to improve biofuel production might be accomplished by incorporating diversity related, resource‐use‐dynamics into algal biomass production.