Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus.

We have recently reported that Notch 1, a member of the Notch multigene family, is essential for the development of murine T cells. Using a mouse model in which Notch 1 is inactivated in bone marrow (BM) precursors we have shown that B cells instead of T cells are found in the thymus of BM chimeras. However, it is not clear whether these B cells develop by default from a common lymphoid precursor due to the absence of Notch 1 signaling, or whether they arise as a result of perturbed migration of BM-derived B cells and/or altered homeostasis of normal resident thymic B cells. In this report we show that Notch 1-deficient thymic B cells resemble BM B cells in phenotype and turnover kinetics and are located predominantly in the medulla and corticomedullary junction. Peripheral blood lymphocyte analysis shows no evidence of recirculating Notch1(-/)- BM B cells. Furthermore, lack of T cell development is not due to a failure of Notch1(-/)- precursors to home to the thymus, as even after intrathymic reconstitution with BM cells, B cells instead of T cells develop from Notch 1-deficient precursors. Taken together, these results provide evidence for de novo ectopic B cell development in the thymus, and support the hypothesis that in the absence of Notch 1 common lymphoid precursors adopt the default cell fate and develop into B cells instead.

Molecular characterization of the processing of arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme-1/site-1 protease.

A crucial step in the life cycle of arenaviruses is the biosynthesis of the mature fusion-active viral envelope glycoprotein (GP) that is essential for virus-host cell attachment and entry. The maturation of the arenavirus GP precursor (GPC) critically depends on proteolytic processing by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we undertook a molecular characterization of the SKI-1/S1P processing of the GPCs of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the pathogenic Lassa virus (LASV). Previous studies showed that the GPC of LASV undergoes processing in the endoplasmic reticulum (ER)/cis-Golgi compartment, whereas the LCMV GPC is cleaved in a late Golgi compartment. Herein we confirm these findings and provide evidence that the SKI-1/S1P recognition site RRLL, present in the SKI-1/S1P prodomain and LASV GPC, but not in the LCMV GPC, is crucial for the processing of the LASV GPC in the ER/cis-Golgi compartment. Our structure-function analysis revealed that the cleavage of arenavirus GPCs, but not cellular substrates, critically depends on the autoprocessing of SKI-1/S1P, suggesting differences in the processing of cellular and viral substrates. Deletion mutagenesis showed that the transmembrane and intracellular domains of SKI-1/S1P are dispensable for arenavirus GPC processing. The expression of a soluble form of the protease in SKI-I/S1P-deficient cells resulted in the efficient processing of arenavirus GPCs and rescued productive virus infection. However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs displayed at the surface of SKI-I/S1P-deficient cells, indicating that GPC processing occurs in an intracellular compartment. In sum, our study reveals important differences in the SKI-1/S1P processing of viral and cellular substrates.

Psoriasis triggered by toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors.

BACKGROUND: It has been proposed that the innate immune system plays a central role in driving the autoimmune T-cell cascade leading to psoriasis; however, there is no direct evidence for this. OBSERVATIONS: We observed aggravation and spreading of a psoriatic plaque when treated topically with the toll-like receptor (TLR) 7 agonist imiquimod. The exacerbation of psoriasis was accompanied by a massive induction of lesional type I interferon activity, detected by MxA expression after imiquimod therapy. Since imiquimod induces large amounts of type I interferon production from TLR7-expressing plasmacytoid dendritic cell precursors (PDCs), the natural interferon-producing cells of the peripheral blood, we asked whether PDCs are present in psoriatic skin. We identified high numbers of PDCs in psoriatic skin lesions (up to 16% of the total dermal infiltrate) based on their coexpression of BDCA2 and CD123. By contrast, PDCs were present at very low levels in atopic dermatitis and not detected in normal human skin. CONCLUSIONS: This study shows that psoriasis can be driven by the innate immune system through TLR ligation. Furthermore, our finding that large numbers of PDCs infiltrate psoriatic skin suggests a role of lesional PDCs as type I interferon-producing targets for the TLR7 agonist imiquimod.

New pool of cortical interneuron precursors in the early postnatal dorsal white matter.

The migration of cortical γ-aminobutyric acidergic interneurons has been extensively studied in rodent embryos, whereas few studies have documented their postnatal migration. Combining in vivo analysis together with time-lapse imaging on cortical slices, we explored the origin and migration of cortical interneurons during the first weeks of postnatal life. Strikingly, we observed that a large pool of GAD65-GFP-positive cells accumulate in the dorsal white matter region during the first postnatal week. Part of these cells divides and expresses the transcription factor paired box 6 indicating the presence of local transient amplifying precursors. The vast majority of these cells are immature interneurons expressing the neuronal marker doublecortin and partly the calcium-binding protein calretinin. Time-lapse imaging reveals that GAD65-GFP-positive neurons migrate from the white matter pool into the overlying anterior cingulate cortex (aCC). Some interneurons in the postnatal aCC express the same immature neuronal markers suggesting ongoing migration of calretinin-positive interneurons. Finally, bromodeoxyuridine incorporation experiments confirm that a small fraction of interneurons located in the aCC are generated during the early postnatal period. These results altogether reveal that at postnatal ages, the dorsal white matter contains a pool of interneuron precursors that divide and migrate into the aCC.

Thymus-independent generation of NK1+ T cells in vitro from fetal liver precursors.

NK1.1+TCR alpha beta+ (NK1+) T cells are an unusual subset of mouse TCR alpha beta+ cells found primarily in adult thymus and liver. In contrast to conventional TCR alpha beta+ cells, NK1+ T cells have a TCR repertoire that is highly skewed to V alpha14 and to Vbeta8, -7, and -2. The developmental origin and ligand specificity of NK1+ T cells are controversial. We show here that NK1+ T cells with a typically biased V alpha and V beta repertoire develop in cytokine-supplemented suspension cultures of fetal liver established from either normal or athymic mice. Furthermore, NK1+ T cell development in fetal liver cultures is abrogated in beta2m-deficient mice (which lack MHC class I and other related molecules) and can be partially inhibited by the presence of anti-CD1 mAbs. Moreover, mixing experiments indicate that recombination-deficient SCID fetal liver cells can reconstitute NK1+ T cell development in beta2m-deficient fetal liver cultures. Collectively, our data demonstrate that NK1+ T cells can develop extrathymically from fetal liver precursors and that a beta2m-associated ligand (putatively CD1) present on nonlymphoid cells is essential for their positive selection and/or expansion.

Wnt-3a and Wnt-3 differently stimulate proliferation and neurogenesis of spinal neural precursors and promote neurite outgrowth by canonical signaling

Wnt factors regulate neural stem cell development and neuronal connectivity. Here we investigated whether Wnt-3a and Wnt-3, expressed in the developing spinal cord, regulate proliferation and the neuronal differentiation of spinal cord neural precursors (SCNP). Wnt-3a promoted a sustained increase of SCNP proliferation, whereas Wnt-3 enhanced SCNP proliferation transiently and increased neurogenesis through β-catenin signaling. Consistent with this, Wnt-3a and Wnt-3 differently regulate the expression of Cyclin-dependent kinase inhibitors. Furthermore, Wnt-3a and Wnt-3 stimulated neurite outgrowth in SCNP-derived neurons through ß-catenin and TCF4-dependent transcription. GSK-3ß inhibitors mimicked Wnt signaling and promoted neurite outgrowth in established cultures. We conclude that Wnt-3a and Wnt-3 signal through the canonical Wnt/β-catenin pathway to regulate different aspects of SCNP development. These findings may be of therapeutic interest for the treatment of neurodegenerative diseases and nerve injury.

Bulk silica-based luminescent materials by sol-gel processing of non-conventional precursors

The sol-gel synthesis of bulk silica-based luminescent materials using innocuous hexaethoxydisilane and hexamethoxydisilane monomers, followed by one hour thermal annealing in an inert atmosphere at 950oC-1150oC, is reported. As-synthesized hexamethoxydisilane-derived samples exhibit an intense blue photoluminescence band, whereas thermally treated ones emit stronger photoluminescence radiation peaking below 600 nm. For hexaethoxydisilane-based material, annealed at or above 1000oC, a less intense photoluminescence band, peaking between 780 nm and 850 nm that is attributed to nanocrystalline silicon is observed. Mixtures of both precursors lead to composed spectra, thus envisaging the possibility of obtaining pre-designed spectral behaviors by varying the mixture composition.

Cell wall precursors are required to organize the chlamydial division septum.

Members of the Chlamydiales order are major bacterial pathogens that divide at mid-cell, without a sequence homologue of the FtsZ cytokinetic tubulin and without a classical peptidoglycan cell wall. Moreover, the spatiotemporal mechanisms directing constriction in Chlamydia are not known. Here we show that the MreB actin homologue and its conserved regulator RodZ localize to the division furrow in Waddlia chondrophila, a member of the Chlamydiales order implicated in human miscarriage. RodZ is recruited to the septal site earlier than MreB and in a manner that depends on biosynthesis of the peptidoglycan precursor lipid II by the MurA enzyme. By contrast, crosslinking of lipid II peptides by the Pbp3 transpeptidase disperses RodZ from the septum. Altogether, these findings provide a cytological framework for understanding chlamydial cytokinesis driven by septal cell wall synthesis.

Tangential migration of neuronal precursors of glutamatergic neurons in the adult mammalian brain.

In a classic model of mammalian brain formation, precursors of principal glutamatergic neurons migrate radially along radial glia fibers whereas GABAergic interneuron precursors migrate tangentially. These migration modes have significant implications for brain function. Here we used clonal lineage tracing of active radial glia-like neural stem cells in the adult mouse dentate gyrus and made the surprising discovery that proliferating neuronal precursors of glutamatergic granule neurons exhibit significant tangential migration along blood vessels, followed by limited radial migration. Genetic birthdating and morphological and molecular analyses pinpointed the neuroblast stage as the main developmental window when tangential migration occurs. We also developed a partial "whole-mount" dentate gyrus preparation and observed a dense plexus of capillaries, with which only neuroblasts, among the entire population of progenitors, are directly associated. Together, these results provide insight into neuronal migration in the adult mammalian nervous system.

Optical characterization of sol-gel glasses derived from Eu3+ complex-forming precursors

Fabrication of new optical devices based upon the incorporation of rare earth ions via sol-gel methods depends on elimination of dopant ion clusters and residual hydroxyl groups from the final material. The optical absorption and/or luminescence properties of luminescent rare earth ions are influenced by the local bonding environment and the distribution of the rare-earth dopants in the matrix. Typically, dopants are incorporated into gel via dissolution of soluble species into the initial precursor sol. In this work, Eu3+ is used as optical probe, to assess changes in the local environment. Results of emission, excitation, fluorescence line narrowing and lifetimes studies of Eu3+-doped gels derived from Si(OCH3)4 and fluorinated/chelate Eu3+ precursors are presented. The precursors used in the sol-gel synthesis were: tris (6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionate) Eu(III), Eu (III) trifluoromethanesulfonate, Eu(III) acetylacetonate hydrate, Eu (III) trifluoroacetate trihidrate, tris (2,2,6,6-tetramethyl-3,5- heptanedionate) Eu(III) and Eu(NO3)3.6H2O. The results were interpreted in terms of the evolution of the Eu3+ fluorescence in systems varying from solution to the gels densified to 800ºC. The lifetimes studies indicate that the fluorinated precursors are effective at reducing the water content in densified gels.

ENZYMATIC RESOLUTION OF ANTIDEPRESSANT DRUG PRECURSORS IN AN UNDERGRADUATE LABORATORY

The use of biocatalysts in synthetic chemistry is a conventional methodology for preparing enantiomerically enriched compounds. Despite this fact, the number of experiments in chemical teaching laboratories that demonstrate the potential of enzymes in synthetic organic chemistry is limited. We describe a laboratory experiment in which students synthesized a chiral secondary alcohol that can be used in the preparation of antidepressant drugs. This experiment was conducted by individual students as part of a Drug Synthesis course held at the Pharmacy Faculty, Lisbon University. This laboratory experiment requires six laboratory periods, each lasting four hours. During the first four laboratory periods, students synthesized and characterized a racemic ester using nuclear magnetic resonance spectroscopy and gas chromatography. During the last two laboratory periods, they performed enzymatic hydrolysis resolution of the racemic ester using Candida antarctica lipase B to yield enantiomerically enriched secondary alcohol. Students successfully prepared the racemic ester with a 70%-81% overall yield in three steps. The enzymatic hydrolysis afforded (R)- secondary alcohol with good enantioselectivity (90%-95%) and reasonable yields (10%-19%). In these experiments, students were exposed to theoretical and practical concepts of aromatic acylation, ketone reduction, esterification, and enzymatic hydrolysis.

Effect of additives, precursors and process conditions on particle morphology

The objective of the thesis was to develop methods to manufacture and control calcium carbonate crystal nucleation and growth in precipitation process. The work consists of experimental part and literature part that addresses theory of nucleation, crystallization and precipitation. In the experimental part calcium carbonate was precipitated using carbonization reaction. Precipitation was carried out in presence of known morphology controlling agents (anionic polymers and sodium silicate) and by using different operation conditions. Formed material was characterized using SEM images, and its thermal stability was assessed. This work demonstrates that carbon dioxide feeding rate and concentrations of calcium hydroxide and additives can be used to control size, shape and amount of precipitating calcium carbonate.

Detection of Illicit Drugs and Drug Precursors with Cantilever-Enhanced Photoacoustic Spectroscopy

In this study, cantilever-enhanced photoacoustic spectroscopy (CEPAS) was applied in different drug detection schemes. The study was divided into two different applications: trace detection of vaporized drugs and drug precursors in the gas-phase, and detection of cocaine abuse in hair. The main focus, however, was the study of hair samples. In the gas-phase, methyl benzoate, a hydrolysis product of cocaine hydrochloride, and benzyl methyl ketone (BMK), a precursor of amphetamine and methamphetamine were investigated. In the solid-phase, hair samples from cocaine overdose patients were measured and compared to a drug-free reference group. As hair consists mostly of long fibrous proteins generally called keratin, proteins from fingernails and saliva were also studied for comparison. Different measurement setups were applied in this study. Gas measurements were carried out using quantum cascade lasers (QLC) as a source in the photoacoustic detection. Also, an external cavity (EC) design was used for a broader tuning range. Detection limits of 3.4 particles per billion (ppb) for methyl benzoate and 26 ppb for BMK in 0.9 s were achieved with the EC-QCL PAS setup. The achieved detection limits are sufficient for realistic drug detection applications. The measurements from drug overdose patients were carried out using Fourier transform infrared (FTIR) PAS. The drug-containing hair samples and drug-free samples were both measured with the FTIR-PAS setup, and the measured spectra were analyzed statistically with principal component analysis (PCA). The two groups were separated by their spectra with PCA and proper spectral pre-processing. To improve the method, ECQCL measurements of the hair samples, and studies using photoacoustic microsampling techniques, were performed. High quality, high-resolution spectra with a broad tuning range were recorded from a single hair fiber. This broad tuning range of an EC-QCL has not previously been used in the photoacoustic spectroscopy of solids. However, no drug detection studies were performed with the EC-QCL solid-phase setup.

Immunophenotype of normal and leukemic bone marrow B-precursors in a Brazilian population. A comparative analysis by quantitative fluorescence cytometry

The distinction between normal and leukemic bone marrow (BM) B-precursors is essential for the diagnosis and treatment monitoring of acute lymphoblastic leukemia (ALL). In order to evaluate the potential use of quantitative fluorescence cytometry (QFC) for this distinction, we studied 21 normal individuals and 40 patients with CD10+ ALL. We characterized the age-related changes of the CD10, CD19, TdT, CD34 and CD79a densities in normal and leukemic BM. Compared to normal adults, the B-precursors from normal children expressed significantly lower values of CD34-specific antibody binding capacity (SABC) (median value of 86.6 vs 160.2 arbitrary units (a.u.) in children and adults, respectively). No significant age-related difference was observed in the expression of the other markers in the normal BM, or in any of the markers in the leukemic BM. Based on the literature, we set the cut-off value for the normal CD10 expression at 45 x 10³ a.u. for both age groups. For the remaining markers we established the cut-off values based on the minimum-maximum values in the normal BM in each age group. The expression of CD10 was higher than the cut-off in 30 ALL cases and in 18 of them there was a concomitant aberrant expression of other markers. In 9 of the 10 CD10+ ALL with normal CD10 SABC values, the expression of at least one other marker was aberrant. In conclusion, the distinction between normal and leukemic cells by QFC was possible in 38/40 CD10+ ALL cases.

Studies on some precursors involved in meat flavour formation

The effect of some precursors on the formation of meat flavour during heating has been investigated. A comparison of the influence of three different precursors, inosine-5'-monophosphate (5'-IMP), cysteine and thiamine, added to the meat systems, showed that formation of certain heterocyclic compounds, like sulfur-containing furans, dithiolanones and thiophenes, was significantly affected by changes in the concentration of precursors. However, aliphatic compounds, such as hydrocarbons, alcohols and ketones were not changed by these additions. Inosine-5'-monophosphate was established to be more effective than cysteine or thiamine in the formation of some "meaty" volatiles, i.e. the furanthiols, when its concentration was increased 10 times in raw meat.