Agricultural intensification was associated with crop diversification in India (1947-2014).

Declines in agricultural biodiversity associated with modern farming practices may negatively affect the sustainability of agro-ecosystems, but formal knowledge of historical variation in spatio-temporal variation of agro-biodiversity is limited. We used time series of national (1947-2014) and district-level (1956-2008) crop distribution data for India to show that despite strong agricultural intensification after 1960, the average crop species diversity at the district level was stable, but increased at the country-level. While there was a decline in diversity in the major rice and wheat producing regions of northwestern India, associated with intensification of the production of these crops, diversity in western and southern India increased due to expansion of oilseeds and horticultural crops that replaced millet and sorghum. These opposite, but related, trends in crop-level diversity at the sub-national level partially canceled each other out at national level, but there nevertheless was a noticeable increase in overall crop diversity in India during this time period. Our results illustrate how patterns of change in crop diversity need to be considered at different levels of aggregation, and how a decrease in diversity associated with intensification and specialization in one area, may be associated with increased diversity elsewhere, and that support for intensive agriculture with relatively low crop diversity in some regions may be associated with an increase in crop diversity in other regions and at a higher level of aggregation.

Effect of Passive Drying on Ascorbic Acid, α-Tocopherol, and ß-Carotene in Tomato and Mango.

The effect of two passive drying methods on the degradation of ascorbic acid (vitamin C),  α-tocopherol (vitamin E), and ß-carotene (provitamin A) in ripe mango and tomato was determined. Samples of both commodities were dried outdoors in a solar drying cabinet or indoors in sealed plastic boxes containing ceramic zeolite drying beads in a bead-to-fruit ratio of 1:10. Nutrient content of the dried samples was compared to fresh samples (undried control) obtained from the same batch of fruit. Mango samples tended to reach an equilibrium weight of approximately 18% of their initial weight after 28 to 30 hr of drying in both drying methods, while tomatoes equilibrated at 5% to 6% of their initial weight after 8 to 10 hr of drying in the solar drying cabinet and after approximately 30 hr using drying beads. The content of all three nutrients in solar dried tomato, and of ß-carotene in mango were significantly (P < 0.05) lower than the control. α-Tocopherol and ß-carotene content in tomato and ß-carotene content in mango were significantly lower than the control in samples dried using drying beads. Drying method had a significant (P < 0.05) effect on nutrient content; ascorbic acid and ß-carotene content were lower in solar dried tomato than in tomato dried with drying beads. For mango, ß-carotene content was lower after solar drying as compared to drying with drying beads. Drying fruit at lower temperatures using drying beads resulted in higher overall nutrient content of the dried fruit. PRACTICAL APPLICATION: Agriculturalists in developing countries still depend largely on solar drying methods as a means of extending the shelf life and increasing the value of fruit and vegetable products. Zeolite drying beads are reusable and are capable of drying fruit relatively quickly in a controlled environment, and may reduce the degradation of certain nutrients due to heat during solar drying.

The dimerization domain in DapE enzymes is required for catalysis.

The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of the enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate.

Analysis of periplasmic sensor domains from Anaeromyxobacter dehalogenans 2CP-C: structure of one sensor domain from a histidine kinase and another from a chemotaxis protein.

Anaeromyxobacter dehalogenans is a δ-proteobacterium found in diverse soils and sediments. It is of interest in bioremediation efforts due to its dechlorination and metal-reducing capabilities. To gain an understanding on A. dehalogenans' abilities to adapt to diverse environments we analyzed its signal transduction proteins. The A. dehalogenans genome codes for a large number of sensor histidine kinases (HK) and methyl-accepting chemotaxis proteins (MCP); among these 23 HK and 11 MCP proteins have a sensor domain in the periplasm. These proteins most likely contribute to adaptation to the organism's surroundings. We predicted their three-dimensional folds and determined the structures of two of the periplasmic sensor domains by X-ray diffraction. Most of the domains are predicted to have either PAS-like or helical bundle structures, with two predicted to have solute-binding protein fold, and another predicted to have a 6-phosphogluconolactonase like fold. Atomic structures of two sensor domains confirmed the respective fold predictions. The Adeh_2942 sensor (HK) was found to have a helical bundle structure, and the Adeh_3718 sensor (MCP) has a PAS-like structure. Interestingly, the Adeh_3718 sensor has an acetate moiety bound in a binding site typical for PAS-like domains. Future work is needed to determine whether Adeh_3718 is involved in acetate sensing by A. dehalogenans.

Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p-coumaric acid and related aromatic acids.

Lignin comprises 15-25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP-binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p-coumarate, 3-phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X-ray crystal structures of protein-ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin-derived aromatic compounds. The screens and structural data provide new functional assignments for these solute-binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence-based functional annotation methods for this family of proteins.

Characterization of transport proteins for aromatic compounds derived from lignin: benzoate derivative binding proteins.

In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein-ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure-function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino-acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequence-based methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria.

Bacillus anthracis inosine 5'-monophosphate dehydrogenase in action: the first bacterial series of structures of phosphate ion-, substrate-, and product-bound complexes.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of Bacillus anthracis IMPDH, in a phosphate ion-bound (termed "apo") form and in complex with its substrate, inosine 5'-monophosphate (IMP), and product, xanthosine 5'-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known Cryptosporidium parvum IMPDH inhibitors was examined for the B. anthracis enzyme and compared with those of three bacterial IMPDHs from Campylobacter jejuni, Clostridium perfringens, and Vibrio cholerae. The structures contribute to the characterization of the active site and design of inhibitors that specifically target B. anthracis and other microbial IMPDH enzymes.

Toxicological evaluation of thiol-reactive compounds identified using a la assay to detect reactive molecules by nuclear magnetic resonance.

We have recently reported on the development of a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) to detect reactive false positive hits from high-throughput screening, in which we observed a surprisingly large number of compounds that can oxidize or form covalent adducts with protein thiols groups. In the vast majority of these cases, the covalent interactions are largely nonspecific (e.g., affect many protein targets) and therefore unsuitable for drug development. However, certain thiol-reactive species do appear to inhibit the target of interest in a specific manner. The question then arises as to the potential toxicology risks of developing a drug that can react with protein thiol groups. Here, we report on the evaluation of a large set of ALARM-reactive and -nonreactive compounds against a panel of additional proteins (aldehyde dehydrogenase, superoxide dismutase, and three cytochrome P450 enzymes). It was observed that ALARM-reactive compounds have significantly increased risks of interacting with one or more of these enzymes in vitro. Thus, ALARM NMR seems to be a sensitive tool to rapidly identify compounds with an enhanced risk of producing side effects in humans, including alcohol intolerance, the formation of reactive oxygen species, and drug-drug interactions. In conjunction with other toxicology assays, ALARM NMR should be a valuable tool for prioritizing compounds for lead optimization and animal testing.

Discovery and design of novel HSP90 inhibitors using multiple fragment-based design strategies.

The molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme. Second site leads were also identified which bound to HSP90 in two distinct conformations, an 'open' and 'closed' form. Intriguingly, linked fragment approaches targeting both of these conformations were successful in producing novel, micromolar inhibitors. Overall, this study shows that, with only a few fragment hits, multiple lead series can be generated for HSP90 due to the inherent flexibility of the active site. Thus, ample opportunities exist to use these lead series in the development of clinically useful HSP90 inhibitors for the treatment of cancers.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.

SOS-NMR: a saturation transfer NMR-based method for determining the structures of protein-ligand complexes.

An NMR-based alternative to traditional X-ray crystallography and NMR methods for structure-based drug design is described that enables the structure determination of ligands complexed to virtually any biomolecular target regardless of size, composition, or oligomeric state. The method utilizes saturation transfer difference (STD) NMR spectroscopy performed on a ligand complexed to a series of target samples that have been deuterated everywhere except for specific amino acid types. In this way, the amino acid composition of the ligand-binding site can be defined, and, given the three-dimensional structure of the protein target, the three-dimensional structure of the protein-ligand complex can be determined. Unlike earlier NMR methods for solving the structures of protein-ligand complexes, no protein resonance assignments are necessary. Thus, the approach has broad potential applications--especially in cases where X-ray crystallography and traditional NMR methods have failed to produce structural data. The method is called SOS-NMR for structural information using Overhauser effects and selective labeling and is validated on two protein-ligand complexes: FKBP complexed to 2-(3'-pyridyl)-benzimidazole and MurA complexed to uridine diphosphate N-acetylglucosamine.

Doping control for beta-adrenergic compounds through hair analysis.

An original procedure was developed to simultaneously test beta2-agonists (salbutamol and clenbuterol) and beta-blockers (atenolol, acebutolol, pindolol, betaxolol, propranolol, timolol, sotalol, metoprolol, tertatolol, bisoprolol, labetalol and oxprenolol) in both human and animal hair. After decontamination with methylene chloride (2 times, 2 min), a 200 mg hair strand is pulverized in a ball mill. Then, a 100 mg portion is incubated overnight in 2 mL 0.1 N HCl, at 56 degrees C, in the presence of carteolol, which was used as an internal standard. After neutralization of the acid phase with 0.1 N NaOH, a 2 mL bicarbonate buffer (pH 8.6) is added to the preparation, which is then purified by solid-phase extraction with Isolute C18 columns. Drugs are derivatized using a mixture of trimethylboroxine-ethyl acetate for 15 min at 80 degrees C to form methaneboronate derivatives. Drugs are detected using GC/MS on an HP 6890-5973 system. A 4 microL portion of the derivatized extract is injected using a pulsed mode in a 30 m HP5 MS capillary column. Linearity was observed for all compounds in the range 25 pg/mg to 10 ng/mg. Limits of detection were in the range 2 to 10 pg/mg. At 1 ng/mg, recoveries were in the range from 37 to 100%, with a within-run precision of 5.9 to 14.1% (n = 8). The application of the method can be documented by the following examples: (1) Hair from asthmatic patients (n - 11), including two cases of asthma deaths, tested positive for salbutamol in the range of 27 to 210 pg/mg. (2) A 24-year-old swimmer who tested positive in urine for salbutamnol denied the results. Hair analysis confirmed salbutamol exposure, with a concentration of 71 pg/mg. (3) A shooting specialist was assumed to chronically use metoprolol (100 mg/daily during some periods). Hair concentration of metoprolol was 8.41 ng/mg. (4) An archery specialist was assumed to chronically use sotalol (80 mg/daily, during some periods). Hair concentration of sotalol was 261 pg/mg. (5) Hair from two calves revealed chronic exposure to clenbuterol, which was used to increase the mass of the animals at a concentration of 30 and 48 pg/mg.

Are cannabinoids detected in hair after washing with Cannabio shampoo?

Today, cannabis plants are used in shampoo preparations, in foodstuffs (e.g., oils, noodles, crackers, etc.), and in beverages (e.g., tea). These products often contain < 1% delta9-tetrahydrocannabinol (THC) in order to eliminate psychoactive effects, but some of them can include 1 to 3% of THC. Gas chromatography-mass spectrometry (GC-MS) analysis of Cannabio shampoo revealed the presence of THC (412 ng/mL) and two constituents of cannabis plants, cannabidiol (CBD, 4079 ng/mL) and cannabinol (CBN, 380 ng/mL). In order to verify if normal hygiene practices with Cannabio shampoo can result in positive tests for cannabinoids in hair, three subjects washed their hair with this shampoo once daily for two weeks. After this period, hair specimens were collected. In the three hair specimens, THC, CBD, and CBN were never detected within their limits of detection, 0.05, 0.02, and 0.01 ng/mg, respectively. We concluded that the use of Cannabio shampoo during normal hygiene practices cannot be considered as a source of potential contamination of hair. In a second experiment, drug-free hair specimens (200 mg) were incubated in 10 mL water/Cannabio shampoo (20:1, v/v) for 30 min, 2 h, and 5 h. After incubation, hair strands were washed with water and separated into two portions. One portion was extracted directly; the second was decontaminated with methylene chloride and then extracted. After an incubation period of 30 min, the analysis of hair by GC-MS did not reveal the presence of THC, CBD, and CBN in hair, regardless of whether the hair was decontaminated. After an incubation period of 2 h, specimens tested positive for CBD (0.11 ng/mg without decontamination and 0.10 ng/mg with decontamination) and CBN (0.02 ng/mg without decontamination and 0.02 ng/mg after decontamination). After an incubation period of 5 h, specimens tested positive for CBD (0.25 ng/mg without decontamination and 0.14 ng/mg after decontamination) and CBN (0.02 ng/mg without decontamination and 0.02 ng/mg after decontamination). In all cases, THC was never detected. Extensive but unrealistic use of Cannabio shampoo can cause drug-free hair to test positive for CBD and CBN but not for the primary psychoactive drug THC.

Evaluation of acetylcodeine as a specific marker of illicit heroin in human hair.

In addition to acetylmorphine (6-AM), acetylcodeine (AC) has been suggested as a marker for the use of illicit heroin. Because no procedure was available for AC testing in hair, a new method was developed for the simultaneous identification and quantitation of morphine (MOR), codeine (COD), 6-AM, and AC. After decontamination, each hair specimen was cut into 1-mm pieces. A 50-mg aliquot was incubated overnight at 50 degrees C in 1 mL Soerensen buffer (pH 7.6) in presence of 200 ng of MOR-d3, COD-d3, 6-AM-d3, and AC-d3. After pH adjustment to 8.4, the analytes were extracted in 5 mL of chloroform/isopropanol/n-heptane (25:10:65, v/v/v). The organic phase was removed and evaporated to dryness, and the residue was derivatized by silylation (BSTFA + 1% TMCS). Drugs were analyzed by gas chromatography-mass spectrometry in electron impact mode. Limits of quantitation were set to 0.1 ng/mg. Fifty hair specimens obtained from subjects who died from fatal opiate overdose were analyzed. AC was detected in 22 samples in concentrations ranging from 0.17 to 5.60 ng/mg with a mean value of 1.04 ng/mg. 6-AM was also present in these samples at concentrations ranging from 1.35 to 41.10 ng/mg with a mean value of 7.79 ng/mg. Of the 28 specimens negative for AC, 21 were positive for 6-AM at concentrations ranging from 0.18 to 7.13 ng/mg. When detected, the AC concentrations were an average of 15.5% (2.8 to 32.6%) of the 6-AM concentrations. There was a positive relationship between AC concentrations and 6-AM concentrations (r = 0.915, p = 0.001). Neither AC nor COD was identified in hair specimens collected from 20 subjects taking part in a heroin-maintenance program in Switzerland and receiving pure pharmaceutical heroin hydrochloride daily. Although it is indicative of illicit heroin use, AC would not make a suitable biomarker in place of 6-AM because of its low concentration in hair compared with that of 6-AM and its absence in about 50% of the specimens that tested positive for 6-AM.

Acute fatal poisoning with dichlorophen.

A case is presented involving an acute fatality resulting from self-administered dichlorophen, a chlorophenol fungicide. The compound was quantified using gas chromatography/mass spectrometry after extraction with methyl-tert-butyl ether, derivatization by methylation and separation on a HP5-MS capillary column. The blood concentration was 9.77 mg/l and other drugs, including ethanol, were not detected.

Colchicine poisoning: report of a fatal case and presentation of an HPLC procedure for body fluid and tissue analyses.

A case involving a suicidal overdose resulting from the ingestion of colchicine tablets is presented. The drug was quantitated using liquid chromatography. The femoral blood level was 62 ng/mL, and the maximum concentration found in bile was 2921 ng/mL. Therefore, bile appears to be the sample of choice for toxicological analysis when a poisoning case involving colchicine is suspected.

Detection of codeine and phenobarbital in sweat collected with a sweat patch.

Six male and two female subjects participated in a clinical study to determine the time course, the cumulative excretion, the intrasubject variability, the influence of site application, and the concentrations of codeine or phenobarbital in sweat following administration of a single dose of the drug. The doses of codeine and phenobarbital were 90 and 100 mg. respectively. Sweat was collected by means of a Sudormed sweat patch. Patches were removed at specified times over 1 week, and the drug content was determined by gas chromatography-mass spectrometry using deuterated internal standards. Codeine was detectable at 1 h following the administration, and a plateau concentration was observed on the third day. The peak codeine concentration was observed during the 12-24-h period. Morphine was never detected in sweat. In contrast, phenobarbital was first observed 3 h after administration, and cumulative excretion was continual throughout the week. Intersubject variability was enormous, as the concentrations for the same dose were in a magnitude of 1-5. Concentrations were in the range of 2-127 and 0.5-33 ng per patch for codeine and phenobarbital, respectively. The influence of the site of patch application was evaluated by analysis of six patches, all removed at the same time (24 h) in two subjects receiving 90 mg codeine. Codeine concentrations differed by a magnitude of 1-3 according to the area of application: the upper arm, the back, and the ribs. These data suggest that the sweat patch technology can be useful for documenting drug use over a 1-week period of surveillance.

Trichloroethanol is not a metabolite of alpha chloralose.

Head space capillary gas chromatography was used to detect alpha chloralose and its potent metabolite, trichloroethanol in clinical and forensic cases. Although alpha chloralose was identified in blood and urine in all cases, trichloroethanol was never detected. In a fatal case the alpha chloralose concentration in blood was 151.3 mg/l. It was concluded that trichloroethanol is not a metabolite of alpha chloralose.

Comparison between GC-MS and the EMIT II, Abbott ADx, and Roche OnLine immunoassays for the determination of THCCOOH.

Gas chromatography-mass spectrometry (GC-MS) and immunological methods, including the Syva enzyme multiplied immunoassay technique, the Abbott fluorescence polarization immunoassay, and the Roche OnLine immunoassay, were compared for the determination of 11-nor-delta 9-tetrahydrocannabinol-9- carboxylic acid (THCCOOH). The results of all three immunoassays were not in accordance with the GC-MS results in three cases of a 72-specimen panel. Only one false negative was observed using the OnLine immunoassay. The immunological methods compared favorably and are acceptable for detecting the presence of cannabis metabolites in urine. These results support the concept that all immunoassays for cannabinoids should be considered as screening procedures. No concentration correlation between GC-MS and the immunoassays could be established because of the different cross-reactivities of the metabolites.

The detection of opiate drugs in nontraditional specimens (clothing): a report of ten cases.

We present a series of 10 fatalities involving opiate overdosage, in which morphine, codeine, and 6-monoacetylmorphine were identified and quantified, not only in postmortem biological samples, but also in pieces of underwear taken from the bodies. Small tissue samples (about 1 g) were cut off from several parts of the underwear, stored at ambient temperature until analysis, then extracted by agitation in a mixture of chloroform/2-propanol/n-heptane (60:14:26, v/v/v) and assayed using GC/MS in the single ion monitoring mode. Morphine, codeine and 6-monoacetylmorphine concentrations were in the range 0.02 to 9.27 micrograms/g. These results indicate that the impregnation of underwear by sweat and sebaceous secretions and/or urine provides detectable levels of the drugs excreted by these ways. Even in the absence of biological samples, assaying pieces of clothing may bring some evidence about the drug abuser status of their owner.