Collection and identification of an unknown component from Eugenia uniflora essential oil exploiting a multidimensional preparative three-GC system employing apolar, mid-polar and ionic liquid stationary phases.

The present research deals with the collection and structural elucidation of an unknown component, accounting for about 35% of the essential oil obtained upon distillation of the leaves of Eugenia uniflora L., harvested during summer (January, 2017) in Paraná State (Southern Brazil). A multidimensional gas chromatographic preparative system, based on the coupling of three GC systems equipped with apolar, PEG and ionic liquid-based stationary phases, was successfully applied for the isolation of the chromatographic band relative to the unknown molecule. The use of wide-bore columns allowed for an increased sample capacity compared to conventional micro-bore columns, thus the injection of a neat sample was feasible, greatly reducing the total collection time. A higher chromatographic efficiency was afforded by the use of a multidimensional approach in the heart-cut mode, exploiting the different selectivity of three stationary phases, which ensured the attainment of a highly pure fraction. In only five runs, more than 3 milligrams were collected, with an average purity greater then 95%. Finally, the unknown component was subjected to nuclear magnetic resonance spectroscopy, mass spectrometry and condensed phase Fourier-transform infrared spectroscopy, leading to the identification of 6-ethenyl-6-methyl-3,5-di(prop-1-en-2-yl)cyclohex-2-en-1-one. The presented approach has been demonstrated to be effective for the isolation and structural elucidation of unknown molecules in complex samples, which will allow for further in-depth studies, like biological evaluation or pharmacological tests.

Multidimensional Gas Chromatography Coupled to Combustion-Isotope Ratio Mass Spectrometry/Quadrupole MS with a Low-Bleed Ionic Liquid Secondary Column for the Authentication of Truffles and Products Containing Truffle.

Truffles are among the most expensive foods available in the market, usually used as flavoring additives for their distinctive aroma. The most valuable species is Tuber magnatum Pico, better known as "Alba white truffle", in which bis(methylthio)methane is the key aroma compound. Given the high economical value of genuine white truffles, analytical approaches are required to be able to discriminate between natural or synthetic truffle aroma. Gas chromatography coupled to combustion-isotope ratio mass spectrometry (GC-C-IRMS), exploiting the 13C/12C ratio abundance of the key flavorings compounds in foods, has been a recognized technique for authenticity and traceability purposes; however, a number of issues have greatly limited its widespread use so far. In the present research, a high-efficiency HS-SPME MDGC-C-IRMS with simultaneous quadrupole MS detection has been applied for the evaluation of bis(methylthio)methane, resolving the coelution occurring with other components. With the aim to minimize the effect of column bleeding on δ13C measurement, a medium polarity ionic liquid-based stationary phase was preferred to a polyethylene glycol one, as the secondary column. In total, 24 genuine white truffles harvested in Italy were analyzed, attaining a δ13C values between -42.6‰ and -33.9‰, with a maximum standard deviation lower than 0.7‰. Two commercial intact truffles and 14 commercial samples of pasta, sauce, olive oil, cream, honey, and fresh cheese flavored with truffle aroma were analyzed, and the results from δ13C measurement were evaluated in comparison with those of genuine "white truffle" range and commercial synthetic bis(methylthio)methane standard.

Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter.

Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.

Protease signaling regulates apical cell extrusion, cell contacts, and proliferation in epithelia.

Mechanisms that sense and regulate epithelial morphogenesis, integrity, and homeostasis are incompletely understood. Protease-activated receptor 2 (Par2), the Par2-activating membrane-tethered protease matriptase, and its inhibitor, hepatocyte activator inhibitor 1 (Hai1), are coexpressed in most epithelia and may make up a local signaling system that regulates epithelial behavior. We explored the role of Par2b in matriptase-dependent skin abnormalities in Hai1a-deficient zebrafish embryos. We show an unexpected role for Par2b in regulation of epithelial apical cell extrusion, roles in regulating proliferation that were opposite in distinct but adjacent epithelial monolayers, and roles in regulating cell-cell junctions, mobility, survival, and expression of genes involved in tissue remodeling and inflammation. The epidermal growth factor receptor Erbb2 and matrix metalloproteinases, the latter induced by Par2b, may contribute to some matriptase- and Par2b-dependent phenotypes and be permissive for others. Our results suggest that local protease-activated receptor signaling can coordinate cell behaviors known to contribute to epithelial morphogenesis and homeostasis.

Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo.

Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful for imaging apoptosis in cultured cells, their in vivo application has been handicapped by poor signal to noise. Here, we report the design and characterization of a GFP-based fluorogenic protease reporter, dubbed ZipGFP. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.

Sept7b is essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis.

The conserved septin family of filamentous small GTPases plays important roles in mitosis, cell migration and cell morphogenesis by forming scaffolds and diffusion barriers. Recent studies in cultured cells in vitro indicate that a septin complex of septin 2, 7 and 9 is required for ciliogenesis and cilia function, but septin function in ciliogenesis in vertebrate organs in vivo is not understood. We show that sept7b is expressed in ciliated cells in different tissues during early zebrafish development. Knockdown of sept7b by using morpholino antisense oligonucleotides caused misorientation of basal bodies and cilia, reduction of apical actin and the shortening of motile cilia in Kupffer's vesicle and pronephric tubules. This resulted in pericardial and yolk sac edema, body axis curvature and hydrocephaly. Notably, in sept7b morphants we detected strong left-right asymmetry defects in the heart and lateral plate mesoderm (situs inversus), reduced fluid flow in the kidney, the formation of kidney cysts and loss of glomerular filtration barrier function. Thus, sept7b is essential during zebrafish development for pronephric function and ciliogenesis, and loss of expression of sept7b results in defects that resemble human ciliopathies.

Adherens junction function and regulation during zebrafish gastrulation.

The adherens junction (AJ) comprises multi-protein complexes required for cell-cell adhesion in embryonic development and adult tissue homeostasis. Mutations in key proteins and mis-regulation of AJ adhesive properties can lead to pathologies such as cancer. In recent years, the zebrafish has become an excellent model organism to integrate cell biology in the context of a multicellular organization. The combination of classical genetic approaches with new tools for live imaging and biophysical approaches has revealed new aspects of AJ biology, particularly during zebrafish gastrulation. These studies have resulted in progress in understanding the relationship between cell-cell adhesion, cell migration and plasma membrane blebbing.

Vaccinia virus-induced microtubule-dependent cellular rearrangements.

Although infection with vaccinia virus (VV) is known to affect the cytoskeleton, it is not known how this affects the cellular architecture or whether the attenuated modified VV ankara (MVA) behaves similar to wild-type VV (wtVV). In the present study, we therefore compared effects of wtVV and MVA infection on the cellular architecture. WtVV-infection induces cell rounding early in infection, which coincides with the retraction of microtubules (MTs) and intermediate filaments from the cellular periphery, whereas mitochondria and late endosomes cluster around the nucleus. Nocodazole treatment demonstrates that cell rounding and organelle clustering require intact MTs. At the onset of virus assembly late in infection, cells reflatten, a process that coincides with the regrowth of MTs into the cellular periphery. We find that the actin network undergoes several rearrangements that occur sequentially in time and that closely follow the cell-shape changes. Unexpectedly, these actin changes are blocked or reversed upon nocodazole treatment, indicating that intact MTs are also responsible for the wtVV-induced actin rearrangements. Finally, MVA infection does not induce any of these cellular changes. Because this virus lacks a substantial number of VV genes, MVA opens up a system to search for the molecules involved in wtVV-induced cellular changes; in particular, those that may regulate actin/MT interactions.

The deubiquitinating enzyme Doa4p protects cells from DNA topoisomerase I poisons.

DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36 degrees C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4Delta cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage.