Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy.

Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage.

Electrochemical immunosensor for simultaneous determination of interleukin-1 beta and tumor necrosis factor alpha in serum and saliva using dual screen printed electrodes modified with functionalized double-walled carbon nanotubes.

Dual screen-printed carbon electrodes modified with 4-carboxyphenyl-functionalized double-walled carbon nanotubes (HOOC-Phe-DWCNTs/SPCEs) have been used as scaffolds for the preparation of electrochemical immunosensors for the simultaneous determination of the cytokines Interleukin-1ß (IL-1ß) and factor necrosis tumor α (TNF-α). IL-1ß. Capture antibodies were immobilized onto HOOC-Phe-DWCNTs/SPCEs in an oriented form making using the commercial polymeric coating Mix&Go™. Sandwich type immunoassays with amperometric signal amplification through the use of poly-HRP-streptavidin conjugates and H2O2 as HRP substrate and hydroquinone as redox mediator were implemented. Upon optimization of the experimental variables affecting the immunosensor performance, the dual immunosensor allows ranges of linearity extending between 0.5 and 100 pg/mL and from 1 to 200 pg/mL for IL-1ß and TNF-α, respectively, these ranges being adequate for the determination of the cytokines in clinical samples. The achieved limits of detection were 0.38 pg/mL (IL-1ß) and 0.85 pg/mL (TNF-α). In addition, the dual immunosensor exhibits excellent reproducibility of the measurements, storage stability of the anti-IL-Phe-DWCNTs/SPCE and anti-TNF-Phe-DWCNTs/SPCE conjugates, and selectivity as well as negligible cross-talking. The dual immunosensor was applied to the simultaneous determination of IL-1ß and TNF-α in human serum spiked at clinically relevant concentration levels and in real saliva samples.

Covalent functionalization of N-doped graphene by N-alkylation.

Nitrogen doped graphene was modified by N-alkylation using a combination of phase transfer catalysis and microwave irradiation. The resulting derivatives of N-doped graphene were analysed showing that the bandgap of the material varied depending on the alkylation agent used.

A photoresponsive graphene oxide-C60 conjugate.

An all-carbon donor-acceptor hybrid combining graphene oxide (GO) and C60 has been prepared. Laser flash photolysis measurements revealed the occurrence of photoinduced electron transfer from the GO electron donor to the C60 electron acceptor in the conjugate.

Increased contribution of L-arginine-nitric oxide pathway in aorta of mice lacking the gene for vimentin.

Experiments were designed to investigate endothelial function in the aorta of mice lacking the gene for the cytoskeleton protein vimentin (vim -/- ). Rings with and without endothelium from wild-type (vim +/+ ), heterozygous (vim +/- ), and homozygous (vim -/- ) mice were suspended in organ chambers to record of changes in isometric tension. During phenylephrine contraction, acetylcholine evoked comparable endothelium-dependent relaxations in the three groups. In the presence of Nomega-nitro-L-arginine, acetylcholine caused endothelium-dependent contractions, which were greater in vim -/- than in vim +/+ and vim +/- aortas. Indomethacin did not affect relaxation to acetylcholine in vim +/+ or in vim +/-, but it significantly increased the maximal response in vim -/- (67 +/- 7 vs. 102 +/- 4%). Response to acetylcholine in vim -/- aortas was not affected by cyclooxygenase type 2 inhibitor NS-398, the thromboxane receptor antagonist SQ-29,548, or superoxide dismutase. Relaxations to sodium nitroprusside were not different between vim +/+ and vim -/- mice and were not affected by cyclooxygenase inhibition. Cyclic guanosine monophosphate levels, which were increased to a comparable level by acetylcholine in vim +/+ and vim -/-, were augmented by indomethacin in vim -/- aortas but not in vim +/+ aortas. Expression of endothelial nitric oxide synthase was not different between vim +/+ and vim -/- preparations. These results suggest that despite comparable endothelium-dependent responses to acetylcholine, endothelial cells from vim -/- mice release a cyclooxygenase product that compensates the augmented contribution of nitric oxide.

Healthy mice with an altered c-myc gene: role of the 3' untranslated region revisited.

c-Myc is a protooncogene involved in the control of cellular proliferation, differentiation and apoptosis. Like many other early response genes, regulation of c-myc expression is mainly controlled at the level of mRNA stability. Multiple cis-acting destabilizing elements have been described that are located both in the protein-coding region and in the 3' untranslated region (3' UTR). However, it is not known when they function during development and whether they act as partly redundant or independent elements to regulate c-myc mRNA level of expression. To begin to address these questions, we created a series of c-myc alleles modified in the 3' UTR, using homologous recombination and the Cre/loxP system, and analysed the consequences of these modifications in ES cells and transgenic animals. We found that deletion of the complete 3' UTR, including runs of Us and AU-rich elements proposed, on the basis of cell-culture assays, to be involved in the control of c-myc mRNA stability, did not alter the steady-state level of c-myc mRNA in any of the various situations analysed in vivo. Moreover, mice homozygous for the 3' UTR-deleted gene were perfectly healthy and fertile. Our results therefore strongly suggest that the 3' UTR of c-myc mRNA does not play a major role in the developmental control of c-myc expression.

C(60)-based triads with improved electron-acceptor properties: pyrazolylpyrazolino[60]fullerenes.

A series of triad pyrazolylpyrazolino[60]fullerenes has been prepared in one pot from suitably functionalized hydrazones by 1,3-dipolar cycloaddition reactions under microwave irradiation. The electrochemical properties of the compounds obtained were investigated by cyclic voltammmetry, and they show better electron acceptor character than the parent C(60) in all cases. Fluorescence experiments and time-resolved transition spectroscopy indicate the existence of photoinduced charge-transfer processes with the C(60) triplet acting as the acceptor.

Teratocarcinomas induced by embryonic stem (ES) cells lacking vimentin: an approach to study the role of vimentin in tumorigenesis.

Vimentin is a class III intermediate filament protein widely expressed in the developing embryo and in cells of mesenchymal origin in the adult. Vimentin knock-out mice develop and reproduce without any obvious defect. This is an unexpected finding in view of the high degree of conservation of the vimentin gene among vertebrates. However, it does not exclude the possibility of a role for vimentin in pathological conditions, like tumorigenesis. To address this question directly, we have used a teratocarcinoma model involving the injection of ES cells into syngeneic mice. ES cells lacking vimentin were generated from 129/Sv Vim-/- mice with high efficiency. The absence of vimentin did not affect ES cell morphology, viability or growth rate in vitro. Tumours were induced by subcutaneous injection of either Vim-/- or Vim+/+ ES cells into Vim+/+ and Vim-/- mice, in order to analyse the effect of the absence of vimentin in either the tumorigenic cells or the host mice. No significant differences were found in either tumour incidence, size or vascularization of teratocarcinomas obtained with all possible combinations. Vim-/- ES-derived tumours showed the same cellular composition typical of teratocarcinomas induced by wild-type ES cells together with a very similar apoptotic pattern. Taken together, these results demonstrate that in this model vimentin is not essential for efficient tumour growth and differentiation in vivo.

Modification of regioselectivity in cycloadditions to C70 under microwave irradiation

The regioselectivity of the cycloaddition of N-methylazomethine ylide to C70 can be modified by using microwave irradiation as the source of energy. Under microwave irradiation and by choosing the appropriate solvent and irradiation power, the 5-6 isomer is the major product, a situation that is in contrast to conventional heating where the 1-2 isomer predominates. Moreover, isomer 7-21, which represents 13% of monoadducts under classical heating, is not formed under microwave irradiation and with ODCB as solvent. Theoretical calculations predict an asynchronous mechanism and suggest that the modification of the regiochemical outcome is related to the relative energies and hardnesses of the transition structures involved.

Comparative anatomy of the cerebellar cortex in mice lacking vimentin, GFAP, and both vimentin and GFAP.

In the cerebellum of adult mammals, glial fibrillary acidic protein (GFAP) and vimentin (VIM) are coexpressed in Golgi epithelial cells (GEC), also known as Bergmann glia. In this study we used three transgenic knockout mice (GFAP, VIM and double GFAP and VIM) to analyze the involvement of these proteins in the building of glial filaments and in neuron-glia interactions. The cerebella of VIM, GFAP, and GFAP/VIM mutant mice were processed by the rapid Golgi method and also for electron microscopy. In VIM mutant mice, Bergmann fibers are hypertrophic with thickened appendages. In the electron microscope they appear as large glial profiles devoid of glial filaments, with embedded dendritic thorns and parallel fiber boutons. In addition, signs of degeneration are observed in Purkinje cells. In GFAP mutant mice, GEC exhibit fine, delicate processes, as those seen in wild-type animals, however, a large accumulation of lamellae and granular appendages was observed along their surfaces, which came into contact with each other. The electron microscope exhibited fine and scarce astroglial profiles containing some glial filaments, a stunted glia limitans, and the presence of large extracellular spaces. In double mutant mice, the two phenotypes are expressed but appear attenuated, with a total absence of glial filaments and the general appearance of immaturity for GEC. In conclusion, it appears that the absence of each of the proteins yields a specific phenotype and that the defects are not necessarily additive.

Synthesis and properties of isoxazolo[60]fullerene-donor dyads.

A series of isoxazolo[60]fullerenes has been prepared in one pot from aldoximes under microwave irradiation. Several donors and acceptors were used as substituents. The absorption and emission spectra of these compounds in polar solvents suggest a weak charge-transfer interaction between the oxygen atom of the isoxazoline moiety and the C(60) cage, as well as a stronger interaction between the donor and the fullerene cage when the attached groups are p-N,N-dimethylaniline or ferrocene. The electrochemical properties of the compounds were investigated and they show the same or better acceptor character than C(60) in all cases. Theoretical calculations support the results obtained. Solvent effects in the (1)H NMR spectra have been determined and provide useful information concerning the polarization of dyads.

Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts.

Loss of a vimentin network due to gene disruption created viable mice that did not differ overtly from wild-type littermates. Here, primary fibroblasts derived from vimentin-deficient (-/-) and wild-type (+/+) mouse embryos were cultured, and biological functions were studied in in vitro systems resembling stress situations. Stiffness of -/- fibroblasts was reduced by 40% in comparison to wild-type cells. Vimentin-deficient cells also displayed reduced mechanical stability, motility and directional migration towards different chemo-attractive stimuli. Reorganization of collagen fibrils and contraction of collagen lattices were severely impaired. The spatial organization of focal contact proteins, as well as actin microfilament organization was disturbed. Thus, absence of a vimentin filament network does not impair basic cellular functions needed for growth in culture, but cells are mechanically less stable, and we propose that therefore they are impaired in all functions depending upon mechanical stability.

Retention of the fetoacinar pancreatic (FAP) protein to the endoplasmic reticulum of tumor cells.

The fetoacinar pancreatic (FAP) protein is a specific component of the human exocrine pancreas associated with the differentiation and proliferation of acinar cells. FAP expression is enhanced in cases of pancreatic exocrine cancer and it is found in relatively high concentrations in pathological pancreatic juices. However, tumor cell lines do not secrete FAP into the culture medium. In this paper we analyze the intracellular localization of FAP in cell lines and compare some biological properties of the tumoral FAP with the normal adult and fetal forms. Immunocytological experiments performed using Mab J28 which characterizes FAP, gave a staining pattern suggestive of FAP localization in the ER. Subcellular fractionation corroborated this localization and established that FAP is tightly associated with the microsomal membranes. The absence of reactivity of the tumoral FAP with wheat germ agglutinin lectin and its strong reactivity with concanavalin A is consistent with the idea that FAP in tumor cells does not reach the Golgi apparatus and it is consequently retained in the endoplasmic reticulum (ER). FAP contained in hepatic metastasis derived from pancreatic adenocarcinoma appeared to be similar, if not identical, to that expressed by cell lines. This supports the hypothesis that FAP retention in the ER of malignant cells is a physiological phenomenon and not the result of a modification of cell lines due to the culture conditions. FAP expressed by cancer cell lines and metastases appeared by sodium dodecyl sulfate polyacrylamide gel electrophoresis a homogeneous protein with a M(r) of 120,000. Instead, the secreted mature protein consists of a main component of M(r) 110,000 and shows pronounced polymorphism (dispersion from M(r) 110,000 to 80,000). Increased size of the ER-retained protein is likely due to elongation of the peptide chain. Defective processing in the ER as a result of amino acid mutation could therefore explain the behavior of this protein in tumors.