Parallel evolution of cannabinoid biosynthesis.

Modulation of the endocannabinoid system is projected to have therapeutic potential in almost all human diseases. Accordingly, the high demand for novel cannabinoids stimulates the discovery of untapped sources and efficient manufacturing technologies. Here we explored Helichrysum umbraculigerum, an Asteraceae species unrelated to Cannabis sativa that produces Cannabis-type cannabinoids (for example, 4.3% cannabigerolic acid). In contrast to Cannabis, cannabinoids in H. umbraculigerum accumulate in leaves' glandular trichomes rather than in flowers. The integration of de novo whole-genome sequencing data with unambiguous chemical structure annotation, enzymatic assays and pathway reconstitution in Nicotiana benthamiana and in Saccharomyces cerevisiae has uncovered the molecular and chemical features of this plant. Apart from core biosynthetic enzymes, we reveal tailoring ones producing previously unknown cannabinoid metabolites. Orthology analyses demonstrate that cannabinoid synthesis evolved in parallel in H. umbraculigerum and Cannabis. Our discovery provides a currently unexploited source of cannabinoids and tools for engineering in heterologous hosts.

Insight into the Autosomal-Dominant Inheritance Pattern of SOD1-Associated ALS from Native Mass Spectrometry.

About 20% of all familial amyotrophic lateral sclerosis (ALS) cases are associated with mutations in superoxide dismutase (SOD1), a homodimeric protein. The disease has an autosomal-dominant inheritance pattern. It is, therefore, important to determine whether wild-type and mutant SOD1 subunits self-associate randomly or preferentially. A measure for the extent of bias in subunit association is the coupling constant determined in a double-mutant cycle type analysis. Here, cell lysates containing co-expressed wild-type and mutant SOD1 subunits were analyzed by native mass spectrometry to determine these coupling constants. Strikingly, we find a linear positive correlation between the coupling constant and the reported average duration of the disease. Our results indicate that inter-subunit communication and a preference for heterodimerization greatly increase the disease severity.

Estimating Interprotein Pairwise Interaction Energies in Cell Lysates from a Single Native Mass Spectrum.

A powerful method to determine the energetic coupling between amino acids is double mutant cycle analysis. In this method, two residues are mutated separately and in combination and the energetic effects of the mutations are determined. A deviation of the effect of the double mutation from the sum of effects of the single mutations indicates that the two residues are interacting directly or indirectly. Here, we show that double mutant cycle analysis by native mass spectrometry can be carried out for interactions in crude Escherichia coli cell extracts, thereby obviating the need for protein purification and generating binding isotherms. Our results indicate that intermolecular hydrogen bond strengths are not affected by the more crowded conditions in cell lysates.

Measuring inter-protein pairwise interaction energies from a single native mass spectrum by double-mutant cycle analysis.

The strength and specificity of protein complex formation is crucial for most life processes and is determined by interactions between residues in the binding partners. Double-mutant cycle analysis provides a strategy for studying the energetic coupling between amino acids at the interfaces of such complexes. Here we show that these pairwise interaction energies can be determined from a single high-resolution native mass spectrum by measuring the intensities of the complexes formed by the two wild-type proteins, the complex of each wild-type protein with a mutant protein, and the complex of the two mutant proteins. This native mass spectrometry approach, which obviates the need for error-prone measurements of binding constants, can provide information regarding multiple interactions in a single spectrum much like nuclear Overhauser effects (NOEs) in nuclear magnetic resonance. Importantly, our results show that specific inter-protein contacts in solution are maintained in the gas phase.Double mutant cycle (DMC) analyses can provide the interaction energies between amino acids at the interface of protein complexes. Here, the authors determine pairwise interaction energies using high-resolution native mass spectroscopy, offering a straightforward route for the DMC methodology.

Genotoxic assessment of carbon nanotubes.

Carbon nanotubes are unique one-dimensional macromolecules with promising application in biology and medicine. Since their toxicity is still under debate, here we describe an investigation of genotoxic properties of purified single-walled carbon nanotubes (SWCNT), multiwall carbon nanotubes (MWCNT), and amide-functionalized purified SWCNT. We used two different cell systems: cultured human lymphocytes where we employed cytokinesis-block micronucleus test and human fibroblasts where we investigate the induction of DNA double-strand breaks (DSBs) employing H2AX phosphorylation assay.

Using carbon nanotubes to induce micronuclei and double strand breaks of the DNA in human cells.

Carbon nanotubes are unique one-dimensional macromolecules with promising applications in biology and medicine. Since their toxicity is still under debate, here we present a study investigating the genotoxic properties of purified single wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs), and amide functionalized purified SWCNTs on cultured human lymphocytes employing cytokinesis block micronucleus assay and enumeration of gamma H2AX foci as a measure of double strand breaks (DSBs) of the DNA in normal human fibroblasts. SWCNTs induce micronuclei (MN) formation in lymphocytes and decrease the proliferation potential (CBPI) of cells. In a fibroblast cell line the same dose of SWCNTs induces gamma H2AX foci 2.7-fold higher than in a control. Amide functionalized purified SWCNTs behave differently: they do not disturb the cell proliferation potential of harvested lymphocytes, but induce micronuclei to a higher extent than SWCNTs. When applied on fibroblasts, amide functionalized SWCNTs also induce gamma H2AX foci, 3.18-fold higher than the control. The cellular effects of MWCNTs display the broad spectrum of clastogenic properties seen as the highest incidence of induced lymphocyte micronuclei and anaphase bridges among nuclei in binucleated cells. Surprisingly, the incidence of induced gamma H2AX foci was not as high as was expected by the micronucleus test, which indicates that MWCNTs act as clastogen and aneugen agents simultaneously. Biological endpoints investigated in this study indicate a close relationship between the electrochemical properties of carbon nanotubes and observed genotoxicity.

Ionization energies of LinX (n = 2, 3; X = Cl, Br, I) molecules.

Molecules of Li(n)X (n = 2, 3; X = Cl, Br, I) were examined with a magnetic sector mass spectrometer by surface ionization using a triple rhenium filament impregnated with fullerene (C60). The ionization energies obtained for Li(2)Cl, Li(2)Br and Li(2)I molecules are 3.8 +/- 0.1, 3.9 +/- 0.1 and 4.0 +/- 0.1 eV, respectively. The first ionization energy of Li(2)Cl is documented, while there are no literature data for the ionization energies of Li(2)Br and Li(2)I. The molecules of Li(3)Cl, Li(3)Br and Li(3)I were detected experimentally for the first time with ionization energies of 4.0 +/- 0.1, 4.1 +/- 0.1 and 4.1 +/- 0.1 eV, respectively. The ionization energies of Li(n)X (n = 2, 3; X = Cl, Br, I) are in correlation with the theoretical prediction of their hyperlithiated configurations.