Symbiont dynamics and strain diversity in the defensive mutualism between Lagria beetles and Burkholderia.

Defensive mutualisms are often facultative in nature, and their evolutionary dynamics can be shaped by changes in local antagonist communities or arms races with coevolving antagonists. Under these conditions, selection may favour hosts that flexibly acquire symbionts producing compounds with bioactivity against current antagonists. Here, we study the prevalence, dynamics and strain diversity of Burkholderia gladioli bacteria in Lagria beetles, a recently described protective symbiosis involving vertical transmission and antifungal defense for the host eggs. In Lagria hirta, we investigate the fate of the bacteria during the host life cycle. Despite a transmission route relying solely on the females, the bacteria are present in both sexes during the larval stage, suggesting a potentially multifaceted defensive role. In L. hirta and L. villosa adults, culture-dependent and -independent techniques revealed that individual beetles harbour diverse Burkholderia strains from at least two different phylogenetic clades, yet all closely related to free-living B. gladioli. Interestingly, rearing the beetles in the laboratory strongly impacted symbiont strain profiles in both beetle species. Our findings highlight the dynamic nature of the B. gladioli-Lagria symbiosis and present this as a valuable system for studying multiple strain coinfections, as well as the evolutionary and ecological factors regulating defensive symbiosis.

Acyl dihydropyrazolo[1,5-a]pyrimidinones as metabotropic glutamate receptor 5 positive allosteric modulators.

We report the optimization of a series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from an acyl dihydropyrazolo[1,5-a]pyrimidinone class. Investigation of exocyclic amide transpositions with this unique 5,6-bicyclic core were conducted in attempt to modulate physicochemical properties and identify a suitable backup candidate with a reduced half-life. A potent and selective PAM, 1-(2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyrimidin-4(5H)-yl)ethanone (9a, VU0462807), was identified with superior solubility and efficacy in the acute amphetamine-induced hyperlocomotion (AHL) rat model with a minimum effective dose of 3mg/kg. Attempts to mitigate oxidative metabolism of the western phenoxy of 9a through extensive modification and profiling are described.

PreQ0 base, an unusual metabolite with anti-cancer activity from Streptomyces qinglanensis 172205.

PreQ0 base (7-cyano-7-deazaguanine, compound 1) is the biosynthetic precursor of queuosine-tRNA and important synthetic intermediate for bioactive compounds. It was obtained for the first time as a new natural product from a mangrove actinomycete Streptomyces qinglanensis 172205, during the course of searching for anti-cancer compounds from marine microbes. PreQ0 base showed anti-HeLa (IC50 = 62.0 µg/ml) and anti-HepG2 (IC50 = 80.6 µg/ml) activities, higher cytotoxicity than the positive control, fluorouracil. Furthermore, it exhibited weak α -glucosidase inhibitory activity, but no obvious antimicrobial and Aß1-42 fibrillation inhibitory activities. In silico analysis of the genome of the strain 172205 revealed a putative biosynthetic gene cluster directing the biosynthesis of PreQ0 base. The gene cluster only contained three Open Reading Frames (ORFs), queC, queD and queE. The absence of the key gene queF encoding 7-cyano-7-deazaguanine reductase catalyzing PreQ0 base to PreQ1 base suggested that the strain only has the capacity of accumulation of PreQ0 base as a metabolite, consistent with our observation in chemical identification.

Efficient synthesis of dichlorodenafil, an unapproved sildenafil analogue appearing in non-prescription supplements.

We have developed an efficient synthesis of dichlorodenafil (4), an unapproved sildenafil analogue isolated from dietary supplements. Our sequence employs POCl(3)-mediated chlorination of readily available chloroacetyl compound 7 followed by selective hydrolysis of the chloro-heterocycle function. Our synthesis confirms the structure of the illegal additive, and will provide regulatory agencies with ready access to authentic standard samples of dichlorodenafil (4) to aid in their mission to protect the public from unapproved and potentially harmful erectile dysfunction (ED) drug analogues that are added to herbal and dietary supplements without providing users with appropriate toxicological or pharmacological information.

Anti-influenza virus compound from Streptomyces sp. RI18.

Screening for anti-influenza virus activity in compounds isolated from Streptomyces sp. RI18, which was isolated using the membrane filter method, uncovered a novel compound, JBIR-68 (1), which contains a unique skeleton. Its structure was established by extensive NMR and MS analyses. In addition, 1 was synthesized to confirm the configuration of its sugar moiety. Compound 1 inhibited influenza virus growth in plaque assays.

Dibromotyrosine and histamine derivatives from the tropical marine sponge Aplysina sp.

Two new compounds, 3-amino-7,8-dihydroimidazo-[1,5-c]-pyrimidin-5(6H)-one (1) and ethyl 3-(2-amino-1H-imidazol-4-yl)propylcarbamate (2), along with the previously known 7,8-dihydroimidazo-[1,5-c]-pyrimidin-5(6H)-one (3), aeroplysinin-1 (4), dibromoverongiaquinol (5), bisoxazolidinone derivative (6), aerophobins-1 (7) and -2 (8), purealidins J (9) and L, have been isolated from Aplysina sp. from the South China Sea. The structures were elucidated on the basis of 1H, 13C NMR, MS and IR analyses. The histamine-derived alkaloids 1-3 may be unknown bioconversion products of purealidin J (9), aerophobin-2 (8) and aerophobin-1 (7), respectively, when 7-9 are cleaved at C-8-C-9 in reactions of activated chemical defense in Aplysina sponge.

Ardeemins and cytochalasins from Aspergillus terreus residing in Artemisia annua.

Three new alkaloids, 15b-dehydro-5- N-acetylardeemin ( 3), 10-phenyl-[12]-cytochalasins Z16 ( 6) and Z17 ( 7), were characterized from the liquid culture of the endophytic fungus ASPERGILLUS TERREUS IFB-E030 along with six known derivatives, 5- N-acetylardeemin ( 1), 15b- ß-hydroxyl-5- N-acetylardeemin ( 2), cytochalasin E ( 4), rosellichalasin ( 5), cytochalasins Z11 ( 8), and Z13 ( 9). The structures of the new metabolites were established mainly by a combination of their 1D- and 2D-NMR spectra, single crystal X-ray diffraction, and the modified Mosher reaction. Biological assays indicated that cytochalasin Z17 ( 7) had moderate cytotoxicity against human nasopharyngeal epidermoid tumor KB cell line with an IC (50) value of 26.2 µM.

Rapid, simultaneous determination of lopinavir and ritonavir in human plasma by stacking protein precipitations and salting-out assisted liquid/liquid extraction, and ultrafast LC-MS/MS.

Lopinavir and ritonavir are co-formulated in Kaletra approved for the treatment of human immunodeficiency virus infection. A validated analytical method is mandatory for clinical development and therapeutic drug monitoring. Here we are reporting a method for rapid, simultaneous determination of lopinavir and ritonavir in human plasma with stacked protein precipitations and salting-out assisted extraction (SALLE), and ultrafast LC-MS/MS detection. With stacked protein precipitations and SALLE, the sample preparation for a 96-well plate can be completed within 20 min by an automated pipette. Due to the unique cleanliness of SALLE extracts post double protein precipitations, the extracts were injected into an ultrafast liquid chromatography and tandem mass spectrometry system (LC-MS/MS) after simple dilution. An Agilent Zobax Extend-C18 Rapid resolution HT column (1.8 microm, 2.1 mm x 30 mm) was used for the separation. A mixture of acetonitrile:water (55:45, v/v) with 0.1% formic acid was used as the mobile phase. LC ran for approximately 48 s at a flow rate of 0.5 mL min(-1), tandem mass spectrometric data collection started at 15 s and lasts for 30 s. The method was validated with reference to Industry Guidance for Bioanalytical Method Validation and then used for clinical samples. The method is ultrafast, and robust. Results of incurred samples demonstrated excellent method of reproducibility. This ultrafast analysis speed did not compromise with the data quality. To our knowledge, this is the fastest analytical method for simultaneous determination of lopinavir and ritonavir.

Determination of lopinavir cerebral spinal fluid and plasma ultrafiltrate concentrations by liquid chromatography coupled to tandem mass spectrometry.

A method for the determination of lopinavir (LPV) concentrations in cerebral spinal fluid (CSF) and plasma ultrafiltrate (UF) was developed and validated to analyze clinical specimens from patients receiving antiretroviral treatment with lopinavir/ritonavir. The CSF (400 microL sample volume) final calibration range for LPV was 0.313-25.0 ng/mL. The final calibration range for UF (50 microL sample volume) was 1.25-100 ng/mL. The samples were prepared using liquid-liquid extraction, concentrated, and analyzed using a reversed phase isocratic separation. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer. Isolation of LPV through chromatographic separation and proper selection of calibration matrix were important factors in achieving accurate results. Plasma UF was found to be an equivalent calibration matrix to CSF whereas plasma matrix produced a positive bias in samples with unknown concentrations. Artificial CSF media prepared chemically were biased and less superior than UF. Sources of plasma for the UF did not affect accuracy. Several CSF sources were tested for specificity of the method and LPV concentrations were accurately produced with atmospheric pressure chemical ionization source producing more accurate results than the electrospray source. The method successfully measured LPV concentrations in CSF that were previously undetectable by HPLC as well as UF from protein binding studies.

Evaluation of 384-well formatted sample preparation technologies for regulated bioanalysis.

The capabilities and limitations of 384-well formatted sample preparation technologies applied to regulated bioanalysis were evaluated by developing two assays for the simultaneous quantitation of lopinavir and ritonavir, the active ingredients of Kaletra. One method used liquid-liquid extraction (LLE), and the other used solid-phase extraction (SPE). The steps and apparatuses employed by the two methods covered most of those used for bioanalysis. Briefly, the previously validated 96-well formatted assays were adapted to the 384-format with minor modifications. Because the wells of a 384-well plate are clustered together, cross-contamination between adjacent wells was evaluated critically, along with sensitivity, assay throughput, and ruggedness. Samples (35 microL) containing plasma samples (15 microL), internal standard (10 microL), and sodium carbonate (0.5 M, 10 microL to basify the sample) were placed in a 384-well microtiter plate that may contain saquinavir or amprenavir as contamination markers. For LLE preparation, the samples were placed in a deep 384-well plate (300-microL well volume) and extracted with 150 microL of ethyl acetate. Approximately 50 microL of the extracts were removed from each well after phase separation for analysis. For SPE preparation, the fortified samples were transferred to a 384-formatted SPE plate (C18, 5 mg packing). The extracts were eluted from the plate with basified 2-propanol. The LLE or SPE extracts were dried and reconstituted for column-switching high-performance liquid chromatography with tandem mass spectrometric detection (HPLC/MS/MS). The lower limit of quantitation and the assay range were the same as the 96-well formatted assay. If combined with appropriate automation, sample preparation in the 384-well format would be up to five times more efficient than the 96-well format.

Validation and application of a high-performance liquid chromatography-tandem mass spectrometric method for simultaneous quantification of lopinavir and ritonavir in human plasma using semi-automated 96-well liquid-liquid extraction.

Kaletra is an important antiretroviral drug, which has been developed by Abbott Laboratories. It is composed of lopinavir (low-pin-a-veer) and ritonavir (ri-toe-na-veer). Both have been proved to be human immunodeficiency virus (HIV) protease inhibitors and have substantially reduced the morbidity and mortality associated with HIV-1 infection. We have developed and validated an assay, using liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC/MS/MS), for the routine quantification of lopinavir and ritonavir in human plasma, in which lopinavir and ritonavir can be simultaneously analyzed with high throughput. The sample preparation consisted of liquid-liquid extraction with a mixture of hexane: ethyl acetate (1:1, v/v), using 100 microL of plasma. Chromatographic separation was performed on a Waters Symmetry C(18) column (150 mm x 3.9 mm, particle size 5 microm) with reverse-phase isocratic using mobile phase of 70:30 (v/v) acetonitrile: 2 mM ammonium acetate aqueous solution containing 0.01% formic acid (v/v) at a flow rate of 1.0 mL/min. A Waters symmetry C(18) guard column (20 mm x 3.9 mm, particle size 5 microm) was connected prior to the analytical column, and a guard column back wash was performed to reduce the analytical column contamination using a mixture of tetrahydrofuran (THF), methanol and water (45:45:10, v/v/v). The analytical run was 4 min. The use of a 96-well plate autosampler allowed a batch size up to 73 study samples. A triple-quadrupole mass spectrometer was operated in a positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over the concentration ranges of 19-5,300 ng/mL for lopinavir and 11-3,100 ng/mL for ritonavir. A-86093 was used as an internal standard (I.S.). The relative standard deviation (RSD) were <6% for both lopinavir and ritonavir. Mean accuracies were between the designed limits (+/-15%). The robust and rapid LC/MS/MS assay has been successfully applied for routine assay to support bioavailability, bioequivalence, and pharmacokinetics studies.

Monolithic silica columns with mixed mode of hydrophilic interaction and weak anion-exchange stationary phase for pressurized capillary electrochromatography.

A silica-based monolithic column as polar stationary phase is proposed for pressurized CEC (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by 3-aminopropyltrimethoxysilane to produce a column for hydrophilic interaction applications. The amino groups on the surface of the polar stationary phase generated anodic EOF under acidic conditions and served at the same time as a weak anion-exchanger. The anion solutes such as nucleotides were separated by the mixed mode mechanism, which comprised hydrophilic interaction, weak anion-exchange, and electrophoresis. The influences of buffer concentration and organic modifier content on the separation of nucleotides by pCEC have been investigated. In addition, the monolithic silica columns were also able to separate various polar compounds such as phenols, nucleic acid bases, and nucleosides in the hydrophilic interaction CEC mode.

High-performance liquid chromatographic separation of dihydropyrimidine racemates on polysaccharide-derived chiral stationary phases.

The chromatographic behavior of a set of racemic dihydropyrimidines (DHPMs) has been evaluated on two polysaccharide-derived chiral stationary phases under normal phase conditions. One of these is coated, the other chemically immobilized. The outstanding solvent compatibility of the immobilized chiral stationary phase (CSP) permits the use of solvents such as ethyl acetate (EtOAc) that are unsuitable for coated supports, for which traditional 2-propanol:hexanes mixtures have been employed. Drastic changes in the chromatographic retention and resolution of DHPMs and, in general, in the separation performances have been observed for the two systems. From a practical point of view, EtOAc has been proven to be a better choice for the separation of this important class of compounds. By comparing molecules different in specific positions of their scaffolds, hypotheses concerning the role of individual chemical groups on retention and selectivity have been done. These effects have been quantified, in some cases, in terms of standard Gibbs energy variations. Even if no chromatographic measurements have been made under nonlinear conditions, clear indications of the potential use of immobilized chiral adsorptive media operated with EtOAc:hexanes mixtures for preparative separations of DHPMs have been evidenced.

Comparison study of Chiralpak AD-H with AD columns in chromatographic enantioseparation of dihydropyrimidinone acid and its methyl ester.

This paper reports a comparison study of the difference between Chiralpak AD-H and AD columns in enantioseparation of dihydropyrimidinone (DHP) acid and its methyl ester under normal phase LC conditions. Unlike those of the AD phase, the van't Hoff plots of retention factors for DHP acid on the AD-H phase were linear. The cyclic van't Hoff plots of selectivity factors for DHP acid on the AD-H phase were non-linear and slightly non-superimposable. No conformational transition was observed on the AD-H phase in the whole temperature range. A single-step temperature program on the AD-H phase showed that the selectivity factors of DHP acid only increased approximately 1.7% in 24 h (versus approximately 50% on the AD phase). For DHP ester, the single-step temperature program showed that the selectivity factors on the AD-H phase remained the same in 24 h while those on the AD phase increased around 3.1%. The enantioselectivity of DHP acid on the AD-H phase was lower than that on the AD phase while the enantioselectivity of DHP ester on the AD-H phase was higher than that on the AD phase. The resolution of DHP acid on the AD-H phase was about the same as that on the AD phase while the resolution of DHP ester on the AD-H phase was much higher than that on the AD phase. The results of DHP acid are opposite of what the vendor suggested while the results of DHP ester are the same as the vendor's application notes. This indicates that the differences between Chiralpak AD-H and AD columns are not only in their particle size, but also in the solvated conformations.

Jenamidines A to C: unusual alkaloids from Streptomyces sp. with specific antiproliferative properties obtained by chemical screening.

Three new naturally occurring bicyclic alkaloids, jenamidines A (1), B (2) and C (3), were discovered and isolated from the culture broth of Streptomyces sp. (strain HKI0297) via the chemical screening approach. Fermentation, isolation, structure and biological activities of these three new secondary metabolites are reported. The jenamidines have an unusual octahydro-pyrido[1,2-a]pyrimidine skeleton. Jenamidine A (1) shows antiproliferative effects against the chronic myeloid leukaemic cell line K-562. In addition, the new tricyclic sesquiterpenoid, africantriol (4) was isolated from the same strain.

Enantioseparation of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidones on chiral stationary phases based on 3,5-dimethylanilides of N-(4-alkylamino-3,5-dinitro)benzoyl L-alpha-amino acids.

Three novel chiral packing materials for high-performance liquid chromatography were prepared by covalently binding of (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]propan-amide (7), (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]-4-methylpentanamide (8), and (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonyl-amino]-2-phenylacetamide (9) to aminopropyl silica. The resulting chiral stationary phases (CSPs 1-3) proved effective for the resolution of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidone derivatives (TR 1-14). The mechanism of their enantioselection, supported by the elution order of (S)-TR 13 and (R)-TR 13 and molecular modeling of the complex of the slower running (S)-TR 13 with CSP 1 is discussed.

Vicine and convicine in common vetch (Vicia sativa) seeds enhance beta-cyanoalanine toxicity in male broiler chicks.

Two studies were conducted to investigate the effects of feeding raw and water-soaked vetch seeds to male broiler chicks on performance, organ weights, and blood parameters. Intact and ground vetch seeds were soaked in water (1:5) at room temperature for 24 hours (study 1), and (1:10) at 40 degrees C for 24, 48, and 72 hours, with water change every 12 hours (study 2). In study 1, untreated vetch contained, on dry matter basis, 0.530%, 0.731%, and 0.081% total beta-cyanoalanine (BCA), vicine, and convicine, respectively. Toxins were not appreciably reduced in soaked intact and ground vetch. Diets containing untreated, soaked intact, and soaked ground vetch, each at 0%, 20%, 40%, and 60%, were fed to 7-day-old male broilers until onset of neurotoxicity symptoms. Survival time was not only decreased by BCA level but also by those of vicine and convicine (p <.05). In study 2, 60% of untreated or treated vetch seeds were incorporated in chick diets. Although untreated vetch used in this study contained 32% less BCA but 8% and 81% more vicine and convicine, respectively, yet, the chicks on 60% untreated vetch showed toxicity symptoms earlier than those of study 1. Soaking ground vetch for 48 hours or more reduced BCA and totally removed vicine and convicine. Consequently, birds on 60% ground vetch soaked for 48 and 72 hours survived through the starter period and had mean corpuscular hemoglobin concentration and organ weights comparable to those of control at 4 days post trial. The results indicated that "high levels" of vicine or convicine or both might have shortened the birds' survival time by enhancing the neurotoxicity induced by "lower levels" of BCA.

Further insight in the photochemistry of DNA: structure of a 2-imidazolone (5-4) pyrimidone adduct derived from the mutagenic pyrimidine (6-4) pyrimidone photolesion by UV irradiation.

Pyrimidine (6-4) pyrimidone photoproducts represent one of the major mutagenic and carcinogenic class of DNA damage produced by UV exposure. At present, besides their conversion to their Dewar valence isomer, (6-4) photoproducts are generally believed to be photostable, and the observed biological properties of Paterno-Büchi-derived photoproducts are, thus far, exclusively attributed to these two types of compounds. Using a model system (2) relevant to DNA photochemistry, we have observed that the 5'-base moiety of the (6-4) thymine dimer 3, under far-UV radiation, is able to undergo a ring contraction leading to a 2-oxoimidazoline, 1. This unprecedented secondary photochemical reaction constitutes the first report of a major photomodification affecting (6-4) photoproducts and strongly questions the biological stability of the (6-4) adducts under UV light with 2-imidazolone (5-4) pyrimidone adducts being possibly another source of endogenous DNA damage.

Chiral separation of pharmacologically active dihydropyrimidinones with carboxymethyl-beta-cyclodextrin.

We report on the chiral separation of pharmacologically active dihydropyrimidinones by capillary electrophoresis (CE) using carboxymethyl-beta-cyclodextrin as chiral selector. The influence of selector concentration, pH, and the addition of varying amounts of methanol is investigated. Out of 21 compounds investigated, 19 were resolved, 13 with baseline separation.

Cytotoxic cyclopenta[b]benzofuran derivatives from the stem bark of Aglaia formosana.

A new cytotoxic cyclopenta[b]benzofuran derivative, aglaiformosanin (1) and three known cyclopenta[b]benzofuran derivatives (2 - 4) were isolated from the stem bark of Aglaia formosana. Compounds 1 - 4 exhibited potent cytotoxicity against the P-388, KB, HT-29, HL-60, and A549 cell lines. The structure of compound 1 was determined by spectroscopic analysis.