Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs

Whole‐organ and whole‐body optical tissue clearing methods allowing imaging in 3 dimensions are an area of profound research interest. Originally developed to study nervous tissue, they have been successfully applied to all murine organs, yet clearing and imaging of rat peripheral organs is less advanced. Here, a modification of CUBIC clearing protocol is presented. It provides a rapid and simple approach to clear the entire adult rat organism and thus all organs within as little as 4 days. Upgraded perfusion‐based rat CUBIC protocol preserves both anatomical structure of organs and signal from proteinaceous fluorophores, and furthermore is compatible with antibody staining. Finally, it enables also volumetric cells analyses and is tailored for staining of calcium deposits within unsectioned soft tissues.      

Human sperm proteins identified by 2-dimensional electrophoresis and mass spectrometry and their relevance to a transcriptomic analysis

The aim of this study was to identify and analyse human sperm proteins from normozoospermic men using 2-dimensional electrophoresis (2-DE) and mass spectrometry (MS). We identified 73 different sperm proteins, including two less characterized human sperm proteins, Annexin A7 (ANXA7) and c14orf105. Bioinformatic analysis of detected sperm proteins revealed new carbohydrate and lipid metabolic pathways, which supply energy to motile sperm. A comparison of our data with available mRNA microarray data from the human testis allows for validation of identified sperm proteins and aids in the recognition of their physiological pathways.     

The effect of genotype,media composition,pH and sugar concentrations on oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.) doubled haploid production through oat × maize crosses

Doubled haploid (DH) technology in oat has not reached the same stage as in other cereals leading to its application in plant breeding. The objective of this investigation was to increase the effectiveness of Avena sativa L. haploid embryo germination obtained by the distant crosses with maize. Developed embryos (obtained from 22 genotypes) were transferred on five germination media: MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) with 3% sucrose, pH 5.8 (control medium), and 190-2 supplemented with 6 and 9% maltose. The pH of 190-2 was adjusted to 5.5 and 6.0. Of all tested genotypes, 591 haploid embryos were obtained, almost half of them (279) germinated. The rate of haploid embryo germination induced on 190-2 was 6.92%, while in MS it was 3.25%. The sugar and its concentration significantly affected the germination of haploid embryos. The highest percentage of haploid embryo germination (9.11%) and DH lines production (1.64%) was achieved on 190-2 with 9% maltose and pH 6.0. All DH lines are incorporated to breeding programs for the development of new cultivars.     

The Host Response to Listeria monocytogenes Mutants Defective in Genes Encoding Phospholipases C (plcA,plcB) and Actin Assembly (actA)

Several genes involved in the determination of Listeria monocytogenes pathogenesis have been identified. Among them, plcA gene encodes phosphatidylinositol-specific phospholipase C (PI-PLC), plcB gene encodes a broad-range phospholipase C (PC-PLC), and actA encodes a protein contributing to actin assembly in infected cells. The interaction of L. monocytogenes wild type (LO 28) strain and two derivative mutants, plcA^? (BUG 206) and actA^?/plcB^? (LUT 12), with macrophages and T lymphocytes was investigated in a mouse model of listeriosis. Both mutants showed evidence of attenuation. The plcA^? mutant, but not the plcB^? mutant, expressed an increase in susceptibility to the anti-listerial activity of macrophages. Both mutants showed a decreased ability to induce IL-12 production by bone marrow macrophages when co-stimulated with E. coli LPS or IFN-γ. In vivo, L. monocytogenes plcA^? mutant was found to be a more effective stimulator of T cells than the wild LO 28 strain.     

Antrodia gossypium,Phlebiopsis gigantea and Heterobasidion parviporum: in vitro growth and Norway spruce wood block decay

Polymerase chain reaction-amplified and sequenced isolates of Antrodia gossypium, Phlebiopsis gigantea and Heterobasidion parviporum from decaying Norway spruce wood blocks after three and six months, which exhibited linear growth, were investigated. P. gigantea strains showed the fastest growth, whereas A. gossypium growth was five times slower. The differences between the mean daily increment of A. gossypium and the other examined isolates (except Hp2) were statistically significant. There were also significant differences in wood decay between densities over time. These results were confirmed by the decay acceleration index (DAI) and decay activity index, which were positively correlated with wood density regardless of the fungus species. The registered P. gigantea strains (Rotstop and PG Suspension) exhibited a strong decomposition ability (28% after six months); the weight loss caused by A. gossypium after six months of decay (15.2%) was similar to the results of P. gigantea (GB) after just three months (13.2%). All tested H. parviporum isolates showed rather rapid growth and equally strong wood decay (20–25%) compared to those of P. gigantea. DAI showed that A. gossypium may significantly contribute to wood decomposition over time, particularly in less dense wood samples. The use of both saprotrophs as biological agents against root pathogens is discussed.     

Prenyl Ammonium Salts – New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model

PurposePrenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents.MethodsAP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells.ResultsAll the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation—considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.ConclusionObtained results indicate that APs have a potential as non-viral vectors for cell transfection.     

Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins

mRNA 5'-cap recognition by the eukaryotic translation initiation factor eIF4E has been exhaustively characterized with the aid of a novel fluorometric, time-synchronized titration method, and X-ray crystallography. The association constant values of recombinant eIF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m(7)GTP (approximately 1.1 x 10(8) M(-1)). The affinity of the cap analogues for eIF4E correlates with their ability to inhibit in vitro translation. The association constants yield contributions of non-covalent interactions involving single structural elements of the cap to the free energy of binding, giving a reliable starting point to rational drug design. The free energy of 7-methylguanine stacking and hydrogen bonding (-4.9 kcal/mol) is separate from the energies of phosphate chain interactions (-3.0, -1.9, -0.9 kcal/mol for alpha, beta, gamma phosphates, respectively), supporting two-step mechanism of the binding. The negatively charged phosphate groups of the cap act as a molecular anchor, enabling further formation of the intermolecular contacts within the cap-binding slot. Stabilization of the stacked Trp102/m(7)G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostatically steered eIF4E-cap association is accompanied by additional hydration of the complex by approximately 65 water molecules, and by ionic equilibria shift. Temperature dependence reveals the enthalpy-driven and entropy-opposed character of the m(7)GTP-eIF4E binding, which results from dominant charge-related interactions (DeltaH degrees =-17.8 kcal/mol, DeltaS degrees= -23.6 cal/mol K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to eIF4E, all the association constants were approximately 10(7) M(-1), in decreasing order: eIF4GI>4E-BP1>eIF4GII approximately 4E-BP1(P-Ser65) approximately 4E-BP1(P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to eIF4E. Enhancement of the eIF4E affinity for cap occurs after binding to eIF4G peptides.     

Coexpression of CD1a, langerin and Birbeck's granules in Langerhans cell histiocytoses (LCH) in children: ultrastructural and immunocytochemical studies

Langerhans cell histiocytoses (LCH) represent rare diseases of unclear etiology and pathogenesis. Most of the cases include children, 1 to 15 years of age, and various organs are involved (bones, skin, liver, lymph nodes, bone marrow and other). The diagnosis of LCH used to be established by biopsy of the inflamed tissue and demonstration of expression of markers specific for Langerhans cells: CD1a and langerin. The diagnosis can be ultimately confirmed by demonstration of Birbeck's granules in the electron microscopy. The present study was aimed at immunocytochemical demonstration, in the examined LCH material (skin, bones, lymph nodes), of the specific antigen expression and at comparing it with the presence of Birbeck's granules. In the examined 11 cases co-expression of CD1a with langerin and with the presence of Birbeck's granules was noted. Also in all examined biopsies the expression of S-100 protein on inflammatory cells was found. The results corroborate the usefulness of immunocytochemical studies on CD 1 a and langerin expression in diagnosis of LCH.