The Origins of Coca: Museum Genomics Reveals Multiple Independent Domestications from Progenitor Erythroxylum gracilipes.

Coca is the natural source of cocaine as well as a sacred and medicinal plant farmed by South American Amerindians and mestizos. The coca crop comprises four closely related varieties classified into two species (Amazonian and Huánuco varieties within Erythroxylum coca Lam., and Colombian and Trujillo varieties within Erythroxylum novogranatense (D. Morris) Hieron.) but our understanding of the domestication and evolutionary history of these taxa is nominal. In this study, we use genomic data from natural history collections to estimate the geographic origins and genetic diversity of this economically and culturally important crop in the context of its wild relatives. Our phylogeographic analyses clearly demonstrate the four varieties of coca comprise two or three exclusive groups nested within the diverse lineages of the widespread, wild species Erythroxylum gracilipes; establishing a new and robust hypothesis of domestication wherein coca originated two or three times from this wild progenitor. The Colombian and Trujillo coca varieties are descended from a single, ancient domestication event in northwestern South America. Huánuco coca was domesticated more recently, possibly in southeastern Peru. Amazonian coca either shares a common domesticated ancestor with Huánuco coca, or it was the product of a third and most recent independent domestication event in the western Amazon basin. This chronology of coca domestication reveals different Holocene peoples in South America were able to independently transform the same natural resource to serve their needs; in this case, a workaday stimulant. [Erythroxylum; Erythroxylaceae; Holocene; Museomics; Neotropics; phylogeography; plant domestication; target-sequence capture.].

Erythroxylum in Focus: An Interdisciplinary Review of an Overlooked Genus.

The genus Erythroxylum contains species used by indigenous people of South America long before the domestication of plants. Two species, E. coca and E. novogranatense, have been utilized for thousands of years specifically for their tropane alkaloid content. While abuse of the narcotic cocaine has impacted society on many levels, these species and their wild relatives contain untapped resources for the benefit of mankind in the form of foods, pharmaceuticals, phytotherapeutic products, and other high-value plant-derived metabolites. In this review, we describe the current state of knowledge of members within the genus and the recent advances in the realm of molecular biology and biochemistry.

Direct observation of nanometer-scale pores of melittin in supported lipid monolayers.

Melittin is the most studied membrane-active peptide and archetype within a large and diverse group of pore formers. However, the molecular characteristics of melittin pores remain largely unknown. Herein, we show by atomic force microscopy (AFM) that lipid monolayers in the presence of melittin are decorated with numerous regularly shaped circular pores that can be distinguished from nonspecific monolayer defects. The specificity of these pores is reinforced through a statistical evaluation of depressions found in Langmuir-Blodgett monolayers in the presence and absence of melittin, which eventually allows characterization of the melittin-induced pores at a quantitative low-resolution level. We observed that the large majority of pores exhibit near-circular symmetry and a Gaussian distribution in size, with a mean diameter of ∼8.7 nm. A distinctive feature is a ring of material found around the pores, made by, on average, three positive peaks, with a height over the level of the lipidic background of ∼0.23 nm. This protruding rim is most likely due to the presence of melittin near the pore border. Although the current resolution of the AFM images in the {x, y} plane does not allow distinction of the specific organization of the peptide molecules, these results provide an unprecedented view of melittin pores formed in lipidic interfaces and open new perspectives for future structural investigations of these and other pore-forming peptides and proteins using supported monolayers.

Canonical azimuthal rotations and flanking residues constrain the orientation of transmembrane helices.

In biological membranes the alignment of embedded proteins provides crucial structural information. The transmembrane (TM) parts have well-defined secondary structures, in most cases α-helices and their orientation is given by a tilt angle and an azimuthal rotation angle around the main axis. The tilt angle is readily visualized and has been found to be functionally relevant. However, there exist no general concepts on the corresponding azimuthal rotation. Here, we show that TM helices prefer discrete rotation angles. They arise from a combination of intrinsic properties of the helix geometry plus the influence of the position and type of flanking residues at both ends of the hydrophobic core. The helical geometry gives rise to canonical azimuthal angles for which the side chains of residues from the two ends of the TM helix tend to have maximum or minimum immersion within the membrane. This affects the preferential position of residues that fall near hydrophobic/polar interfaces of the membrane, depending on their hydrophobicity and capacity to form specific anchoring interactions. On this basis, we can explain the orientation and dynamics of TM helices and make accurate predictions, which correspond well to the experimental values of several model peptides (including dimers), and TM segments of polytopic membrane proteins.

Inhibition of cholinergic contractions of rat ileum by tkopane-type alkaloids present in Schizanthus hookeri.

The relative lack of specificity of atropine as a competitive antagonist of muscarinic receptors is a frequent cause of undesirable parasympathetic side effects. Consequently, new tropane alkaloids with potentially greater selectivity are usually seen with real interest. The cholinergic antagonistic effects of a purified mixture of tropane alkaloids extracted from Schizanthus hookeri were evaluated in rat ileum. For this purpose, ileal segments were obtained from randomly selected male Sprague-Dawley rats, and the effect of 1 x 10(-4), 1 x 10(-3), and 1 x 10(-2) mg/mL of the purified mixture of alkaloids on the contractile response of the ileum induced with increasing doses of carbachol (5 x 10(-8) - 10(-4) M) was determined. The results were compared with those obtained in the presence of 3.46 x 10(-7), 3.46 x 10(-6), and 3.46 x 10(-5) mg/mL atropine as an agonist. Tropane alkaloids extracted from Schizanthus hookeri competitively antagonized acetylcholine muscarinic receptors.

Medicinal plants of Chile: evaluation of their anti-Trypanosoma cruzi activity.

The extracts of several plants of Central Chile exhibited anti-Trypanosoma cruzi trypomastigotes activity. Most active extracts were those obtained from Podanthus ovatifolius, Berberis microphylla, Kageneckia oblonga, and Drimys winteri. The active extract of Drimys winteri (IC50 51.2 microg/mL) was purified and three drimane sesquiterpenes were obtained: polygodial, drimenol, and isodrimenin. Isodrimenin and drimenol were found to be active against the trypomastigote form of T. cruzi with IC50 values of 27.9 and 25.1 microM, respectively.

Switchable bactericidal effects from novel silica-coated silver nanoparticles mediated by light irradiation.

Here we report on the triggering of antibacterial activity by a new type of silver nanoparticle coated with porous silica, Ag@silica, irradiated at their surface plasmon resonant frequency. The nanoparticles are able to bind readily to the surface of bacterial cells, although this does not affect bacterial growth since the silica shell largely attenuates the intrinsic toxicity of silver. However, upon simultaneous exposure to light corresponding to the absorption band of the nanoparticles, bacterial death is enhanced selectively on the irradiated zone. Because of the low power density used for the treatments, we discard thermal effects as the cause of cell killing. Instead, we propose that the increase in toxicity is due to the enhanced electromagnetic field in the proximity of the nanoparticles, which indirectly, most likely through induced photochemical reactions, is able to cause cell death.

A lipocentric view of peptide-induced pores.

Although lipid membranes serve as effective sealing barriers for the passage of most polar solutes, nonmediated leakage is not completely improbable. A high activation energy normally keeps unassisted bilayer permeation at a very low frequency, but lipids are able to self-organize as pores even in peptide-free and protein-free membranes. The probability of leakage phenomena increases under conditions such as phase coexistence, external stress or perturbation associated to binding of nonlipidic molecules. Here, we argue that pore formation can be viewed as an intrinsic property of lipid bilayers, with strong similarities in the structure and mechanism between pores formed with participation of peptides, lipidic pores induced by different types of stress, and spontaneous transient bilayer defects driven by thermal fluctuations. Within such a lipocentric framework, amphipathic peptides are best described as pore-inducing rather than pore-forming elements. Active peptides bound to membranes can be understood as a source of internal surface tension which facilitates pore formation by diminishing the high activation energy barrier. This first or immediate action of the peptide has some resemblance to catalysis. However, the presence of membrane-active peptides has the additional effect of displacing the equilibrium towards the pore-open state, which is then maintained over long times, and reducing the size of initial individual pores. Thus, pore-inducing peptides, regardless of their sequence and oligomeric organization, can be assigned a double role of increasing the probability of pore formation in membranes to high levels as well as stabilizing these pores after they appear.

Schizanthines N, O, and P, tropane alkaloids from the aerial parts of Schizanthus tricolor.

Three tropane alkaloids, named schizanthines N, O, and P (1-3), have been isolated from the crude alkaloid extract of the endemic Chilean plant Schizanthus tricolor. On the basis of extensive NMR studies and MS fragmentation analysis, their structures were determined to be 3α-(E)-4-hydroxysenecioyloxy-6ß-angeloyloxytropane (1), 3α-(E)-4-hydroxysenecioyloxy-6ß-senecioyloxytropane (2), and 3α-mesaconyloxy-6ß-senecioyloxytropane (3). Compounds 1 and 2 are the first isomeric alkaloids in the tropane series possessing a hydroxysenecioyl substituent as an esterifying moiety.

Grahamines A-E, cyclobutane-centered tropane alkaloids from the aerial parts of Schizanthus grahamii.

Schizanthus grahamii is an endemic Chilean plant that is known to contain tropane alkaloids. Five new alkaloids, grahamines A-E (1-5), were isolated and characterized by extensive spectroscopic analysis. Their structures were determined to be 2-{[(3α-hydroxytropo-6ß-yl)oxy]carbonyl}-2-methyl-3-{[((6ß-angeloyloxy)-3α-yl)oxy]carbonyl}-4-phenylcyclobutanecarboxylic acid (1), 2-{[(3α-hydroxytropo-6ß-yl)oxy]carbonyl}-2-methyl-3-{[((6ß-tigloyloxy)-3α-yl)oxy]carbonyl}-4-phenylcyclobutanecarboxylic acid (2), 1-methyl-2-{[(3α-hydroxytropo-6ß-yl)oxy]carbonyl}-4-{[((6ß-angeloyloxy)-3α-yl)oxy]carbonyl}-3-phenylcyclobutanecarboxylic acid (3), 1,2-bis{[(3α-hydroxytropo-6ß-yl)oxy]carbonyl}-2-methyl-3-{[((6ß-angeloyloxy)-3α-yl)oxy]carbonyl}-4-phenylcyclobutanecarboxylate (4), and 1-{[(3α-mesaconyloxytropo-6ß-yl)oxy]carbonyl}-2-{[(3α-hydroxytropo-6ß-yl)oxy]carbonyl}-2-methyl-3-{[((6ß-angeloyloxy)-3α-yl)oxy]carbonyl}-4-phenylcyclobutanecarboxylate (5).

Chirality and numbering of substituted tropane alkaloids.

The strict application of IUPAC rules for the numbering of tropane alkaloids is not always applied by authors and there is hence a lot of confusion in the literature. In most cases, the notation of 3, 6/7-disubstituted derivatives has been chosen arbitrarily, based on NMR and MS data, without taking into account the absolute configuration of these two carbons. This paper discusses the problem and the relevance of CD and NMR to determine molecular configurations. We report on the use of (1)H-NMR anisochrony (Δδ) induced by the Mosher's chiral auxiliary reagents (R)-(-)- and (S)-(+)-α-methoxy-α-trifluoromethyl-phenylacetyl chlorides (MTPA-Cl), to determine the absolute configuration of (3R,6R)-3α-hydroxy-6ß-senecioyloxytropane, a disubstituted tropane alkaloid isolated from the aerial parts of Schizanthus grahamii (Solanaceae). These analytical tools should help future works in correctly assigning the configuration of additional 3, 6/7 disubstituted tropane derivatives.

Hygroline derivatives from Schizanthus tricolor and their anti-trypanosomatid and antiplasmodial activities.

Chemical investigation of the alkaloid extract of the aerial parts of Schizanthus tricolor led to the targeted isolation of 26 hygroline derivatives of which 20 were fully characterized. They have not yet been described in the literature and their structures were established by 1D and 2D NMR, UV and IR spectroscopy, and HRESIMS. The configuration was determined by Gauge-Independent Atomic Orbital NMR chemical shift calculations supported by the advanced statistical method DP4 plus, vibrational circular dichroism, and measurement of optical rotation. Their anti-trypanosomatid, antiplasmodial and cytotoxic activities were measured. Several compounds exhibited low micromolar activity against Plasmodium falciparum. None of the identified molecules was cytotoxic.

Pores formed by Baxα5 relax to a smaller size and keep at equilibrium.

Pores made by amphipathic cationic peptides (e.g., antimicrobials and fragments of pore-forming proteins) are typically studied by examining the kinetics of vesicle leakage after peptide addition or obtaining structural measurements in reconstituted peptide-lipid systems. In the first case, the pores have been considered transient phenomena that allow the relaxation of the peptide-membrane system. In the second, they correspond to equilibrium structures at minimum free energy. Here we reconcile both approaches by investigating the pore activity of the α5 fragment from the proapoptotic protein Bax (Baxα5) before and after equilibrium of peptide/vesicle complexes. Quenching assays on suspensions of large unilamellar vesicles suggest that in the presence of Baxα5, the vesicles maintain a leaky state for hours under equilibrium conditions. We proved and analyzed stable pores on single giant unilamellar vesicles (GUVs) in detail by monitoring the entrance of dyes added at different times after incubation with the peptide. When the GUVs came in contact with Baxα5, leakage started stochastically, was delayed for various periods of time, and in the majority of cases proceeded rapidly to completion. After hours in the presence of the peptide, the same individual GUVs that refilled completely at first instance maintained a porated state, which could be observed in subsequent leak-in events for serially added dyes. However, these long-term pores were smaller in size than the initial equilibration pores. Stable pores were also detected in GUVs made in the presence of Baxα5. The latter pores can be considered equilibrium states and may correspond to structures measured previously in bilayer stacks. Although pore formation may occur as a kinetic process, equilibrium pores may also be functionally relevant structures, especially in highly regulated systems such as the apoptotic mitochondrial pores induced by Bax.

Isomeric tropane alkaloids from the aerial parts of Schizanthus tricolor.

Investigation of the aerial parts of Schizanthus tricolor yielded seven isomeric tropane alkaloids: 3alpha-(1-methylitaconyl)-6beta-senecioyloxytropane (1), 3alpha-(1-methylitaconyl)-6beta-angeloyloxytropane (2), 3alpha-(1-methylmesaconyl)-6beta-senecioyloxytropane (3), 3alpha-(1-methylmesaconyl)-6beta-angeloyloxytropane (4), 3alpha-(1-methylmesaconyl)-6beta-tigloyloxytropane (5), 3alpha-(1-methylcitraconyl)-6beta-senecioyloxytropane (6), and 3alpha-(1-methylcitraconyl)-6beta-angeloyloxytropane (7). Their structures were established by NMR including (1)H, (13)C NMR, HSQC, HMBC, COSY, and NOESY experiments, UV, IR, and mass spectrometry. Compounds 1, 6, and 7 are new to the literature. Alkaloids 1, 3, 4, and 5 and a mixture of 3, 4, and 5 were evaluated for in vitro antiplasmodial and cytotoxic activity. Compounds 1, 4, and 5 showed marginal inhibition of Plasmodium falciparum strain K1 with IC(50) values of 22.8, 24.8, and 36.0 microM and displayed no cytotoxicity on MRC-65 cells (CC(50) > 64 microM). Alkaloid 3 was inactive (IC(50) 63.5 microM). The alkaloid mixture exhibited slightly higher activity (IC(50) 17.0 microM) than the pure compounds, indicating some synergy between the different isomers.

Absolute configuration determination and conformational analysis of (-)-(3S,6S)-3alpha,6beta-diacetoxytropane using vibrational circular dichroism and DFT techniques.

The absolute configuration of semisynthetic (-)-3alpha,6beta-acetoxytropane 1, prepared from (-)-6beta-hydroxyhyoscyamine 2, has been determined using vibrational circular dichroism (VCD) spectroscopy. The vibrational spectra (IR and VCD) were calculated using DFT at the B3LYP/DGDZVP level of theory for the eight more stable conformers which account for 99.97% of the total relative abundance in the first 10 kcal/mol range. The calculated VCD spectra of all considered conformations showed two distinctive spectral ranges, one between 1300 and 1200 cm(-1), and the other one in the 1150-950 cm(-1) region. When compared with the experimental VCD spectrum, the first spectral region confirmed the calculated conformational preferences, whereas the second region showed little change with conformation, thus allowing the determination of the absolute configuration of 1 as (3S,6S)-3alpha,6beta-diacetoxytropane. Also, the bands in the second region showed similarities between 1 and 2 in both the experimental and calculated VCD spectra, suggesting that these bands are mainly related to the absolute configuration of the rigid tropane ring system, since they show conformational independency, no variations with the nature of the substituent, and are composed by closely related vibrational modes.

Influence of surface porosity and pH on bacterial adherence to hydroxyapatite and biphasic calcium phosphate bioceramics.

Hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramic materials are widely employed as bone substitutes due to their porous and osteoconductive structure. Their porosity and the lowering of surrounding pH as a result of surgical trauma may, however, predispose these materials to bacterial infections. For this reason, the influence of porosity and pH on the adherence of common Gram-positive bacteria to the surfaces of these materials requires investigation. Mercury intrusion porosimetry measurements revealed that the pore size distribution of both bioceramics had, on a logarithmic scale, a sinusoidal frequency distribution ranging from 50 to 300 nm, with a mean pore diameter of 200 nm. Moreover, total porosity was 20 % for HA and 50 % for BCP. Adherence of Staphylococcus aureus and Staphylococcus epidermidis was studied at a physiological pH of 7.4 and at a pH simulating bone infection of 6.8. Moreover, the effect of pH on the zeta potential of HA, BCP and of both staphylococci was evaluated. Results showed that when pH decreased from 7.4 to 6.8, the adherence of both staphylococci to HA and BCP surfaces decreased significantly, although at the same time the negative zeta-potential values of the ceramic surfaces and both bacteria diminished. At both pH values, the number of S. aureus adhered to the HA surface appeared to be lower than that for BCP. A decrease in pH to 6.8 reduced the adherence of both bacterial species (mean 57 %). This study provides evidence that HA and BCP ceramics do not have pores sufficiently large to allow the internalization of staphylococci. Their anti-adherent properties seemed to improve when pH value decreased, suggesting that HA and BCP bioceramics are not compromised upon orthopaedic use.

High-precision heteronuclear 2D NMR experiments using 10-ppm spectral window to resolve carbon overlap.

The acquisition of a complementary heteronuclear 2D NMR experiment with 10-ppm carbon window allows chemists to improve by a factor 20-25 the spectral resolution and determine carbon chemical shifts with five figures from 2D spectra.

Stable-bond polymeric reversed-phase/weak anion-exchange mixed-mode stationary phases obtained by simultaneous functionalization and crosslinking of a poly(3-mercaptopropyl)methylsiloxane-film on vinyl silica via thiol-ene double click reaction.

A polymeric reversed-phase/weak anion exchange (Poly-RP/WAX) mixed-mode stationary phase has been prepared by coating of a poly(3-mercaptopropyl)methylsiloxane film on vinyl-modified silica (100 Å, 5 µm) and simultaneous in situ functionalization with N-(10-undecenoyl)-3-aminoquinuclidine as well as crosslinking to the vinyl silica surface by solventless thiol-ene double click reaction. Such bonding chemistry showed greatly enhanced stability compared to brush-type analogs with bifunctional siloxane bonding to silica. Solid-state 29Si-CP/MAS NMR confirmed the immobilization of the siloxane layer. pH-Dependent ζ-potential determinations revealed a high anion-exchange capacity over the entire pH range with a maximum around pH 5. Oxidation of residual thiols yielded a zwitterionic Poly-RP/WAX/SCX mixed-mode phase with sulfonic acid endcapping and shifted the still net positive surface charge to lower ζ-potentials. It allowed a faster elution of strongly retained anionic species in particular of multiply negatively charged analytes such as oligonucleotides. Chromatographic tests under RPLC and HILIC elution mode with various test substances documented the multimodal utility and complementarity in retention profiles compared to RP, HILIC and commercial mixed-mode phases.

N-Propyl-N'-2-pyridylurea-modified silica as mixed-mode stationary phase with moderate weak anion exchange capacity and pH-dependent surface charge reversal.

Herein, we present a novel silica-based stationary phase modified with N-propyl-N'-2-pyridylurea selector. Due to the weakly basic properties of the pyridine selector and the presence of residual silanols after selector immobilization, a zwitterionic surface with a pI observed at approximately pH 5.5 was measured by electrophoretic light scattering in pH-dependent ζ-potential determinations. The capability of the new N-propyl-N'-2-pyridylurea-modified silica to serve as mixed-mode stationary phase was investigated. For this purpose, it was characterized under RP and HILIC conditions using test mixtures. Subsequent classification of this stationary phase in comparison to in-house and commercial benchmarks was carried by principal component analysis of resultant retention factors from chromatographic tests. The results show a relatively unique mixed-mode character amongst the tested stationary phases. The chromatographic retention characteristics of acidic compounds matched well the ζ-potential determinations. The application of anion-exchange at low pH values (e.g. pH 5) and ion exclusion chromatography at pH 7 for the separation of uridine 5'-mono-, di- and triphosphate demonstrated a pH-dependent umpolung of the stationary phase surface. The combination of these separation principles in a pH gradient from 5 to 7 gave rise to weak anion-exchange selectivity with a charge-inducted elution due to repulsive interactions at higher pH and resulted in a significant faster separation with improved peak shape under mild elution conditions.

Two New Hygroline and Tropane Alkaloids Isolated from Schizanthus hookeri and S. tricolor (Solanaceae).

Two new hygroline and tropane alkaloids, 4-hydroxybenzenepropanoylhygroline (1) and 3α,4ß-dihydroxy-6ß-angeloyoxytropane (2) have been isolated from the aerial parts of Schizanthus hookeri and S. tricolor, respectively, two plants indigenous from Chile. Their structures were elucidated by spectroscopic methods and high resolution mass spectrometry. Their antiparasitic activity and cytotoxicity were measured.