Identification and validation of the mode of action of the chalcone anti-mycobacterial compounds.

OBJECTIVES: The search for new TB drugs has become one of the great challenges for modern medicinal chemistry. An improvement in the outcomes of TB chemotherapy can be achieved by the development of new, shorter, cheap, safe and effective anti-TB regimens. METHODS: Chalcones (1a-1o) were synthesized and evaluated for their antimycobacterial activity against Mycobacterium bovis BCG using growth inhibition assays. Compound 1a was selected as a 'hit' compound. The mode of action of compound 1a, was identified by mycolic acid methyl esters (MAMEs) and fatty acid methyl esters (FAMEs) analysis using thin layer chromatography. Dose dependent experiments were conducted by over-expressing components of FAS-II in M. bovis BCG to confirm the target. Ligand binding using intrinsic tryptophan assay and molecular docking were used to further validate the target. RESULTS: MAMEs and FAMEs analysis showed dose-dependent reduction of MAMEs with the overall abundance of FAMEs suggesting that compound 1a targets mycolic acid biosynthesis. Direct binding of 1a to InhA was observed using an intrinsic tryptophan fluorescence binding assay, and a 2-fold IC50 shift was observed with an InhA overexpressing strain confirming InhA as the cellular target. CONCLUSION: The chalcone 1a exhibits potent antimycobacterial activity, displays a good safety profile and is a direct inhibitor of InhA, a key component in mycolic acid synthesis, validating this series for further anti-TB drug development.

The drugs don't work: evaluation of educational theatre to gauge and influence public opinion on antimicrobial resistance.

Increased public awareness of antimicrobial resistance (AMR) is a key component of effective antimicrobial stewardship strategies. Educational theatre combined with an expert panel was used to engage the public about AMR through delivery of a play entitled 'The drugs don't work'. Audience knowledge and understanding of AMR were measured by pre- and post-play questionnaires. Performance of the play and discussion with the expert panel significantly improved audience knowledge and understanding of AMR, including antibiotic misuse and prescribing. Educational theatre provides a positive learning experience and is an innovative method of public engagement to disseminate important public health messages.

Quantifying airborne dispersal routes of pathogens over continents to safeguard global wheat supply.

Infectious crop diseases spreading over large agricultural areas pose a threat to food security. Aggressive strains of the obligate pathogenic fungus Puccinia graminis f.sp. tritici (Pgt), causing the crop disease wheat stem rust, have been detected in East Africa and the Middle East, where they lead to substantial economic losses and threaten livelihoods of farmers. The majority of commercially grown wheat cultivars worldwide are susceptible to these emerging strains, which pose a risk to global wheat production, because the fungal spores transmitting the disease can be wind-dispersed over regions and even continents 1-11 . Targeted surveillance and control requires knowledge about airborne dispersal of pathogens, but the complex nature of long-distance dispersal poses significant challenges for quantitative research 12-14 . We combine international field surveys, global meteorological data, a Lagrangian dispersion model and high-performance computational resources to simulate a set of disease outbreak scenarios, tracing billions of stochastic trajectories of fungal spores over dynamically changing host and environmental landscapes for more than a decade. This provides the first quantitative assessment of spore transmission frequencies and amounts amongst all wheat producing countries in Southern/East Africa, the Middle East and Central/South Asia. We identify zones of high air-borne connectivity that geographically correspond with previously postulated wheat rust epidemiological zones (characterized by endemic disease and free movement of inoculum) 10,15 , and regions with genetic similarities in related pathogen populations 16,17 . We quantify the circumstances (routes, timing, outbreak sizes) under which virulent pathogen strains such as 'Ug99' 5,6 pose a threat from long-distance dispersal out of East Africa to the large wheat producing areas in Pakistan and India. Long-term mean spore dispersal trends (predominant direction, frequencies, amounts) are summarized for all countries in the domain (Supplementary Data). Our mechanistic modelling framework can be applied to other geographic areas, adapted for other pathogens and used to provide risk assessments in real-time 3 .

Antibiotic stewardship in low- and middle-income countries: the same but different?

BACKGROUND: Antibiotic resistance (ABR) is a quickly worsening problem worldwide, also in low- and middle-income countries (LMICs). Appropriate antibiotic use in humans and animals, i.e. antibiotic stewardship (ABS), is one of the cornerstones of the World Health Organization's global action plan for ABR. Many LMICs are in the process of developing stewardship programs. AIMS: We highlight challenges for ABS initiatives in LMICs, give an outline of (inter)national recommendations and demonstrate examples of effective, contextualized stewardship interventions. SOURCES: We searched PubMed for articles on ABS interventions in humans in LMICs. Relevant websites and experts were consulted for additional sources. CONTENT: Evidence on effective and feasible stewardship interventions in LMICs is limited, and challenges for implementation of interventions are numerous. Nevertheless, several initiatives at the international and local levels in Latin America, Africa and Asia have shown that ABS effective interventions are feasible in LMICs, although contextualization is essential. IMPLICATIONS: Specific guidance for setting up antimicrobial stewardship programs in LMICs should be developed. Strategic points might need to be progressively addressed in LMICs, such as (a) ensuring availability of diagnostic testing, (b) providing dedicated education in ABR both for healthcare workers and the general public, (c) creating or strengthening (inter)national agencies towards better regulations and audit on production, distribution and dispensing of drugs, (d) strengthening healthcare facilities, (e) exploring a broader synergism between policy makers, academia, professional bodies and civil society and (f) designing and studying easy and scalable ABS interventions for both hospital and community settings.

Exploring the consequences of mitochondrial differences arising through hybridization of sunfish.

Previous studies have shown evidence of genomic incompatibility and mitochondrial enzyme dysfunction in hybrids of bluegill (Lepomis macrochirus Rafinesque) and pumpkinseed (Lepomis gibbosus Linnaeus) sunfish (Davies et al., 2012 Physiol. Biochem. Zool. 85, 321-331). We assessed if these differences in mitochondria had an impact on metabolic processes that depend on mitochondrial function, specifically hypoxia tolerance and recovery from burst exercise. Bluegill, pumpkinseed, and their hybrids showed no difference in the critical oxygen tension (Pcrit) and no differences in tissue metabolites measured after exposure to 10% O2 for 30min. In contrast, loss of equilibrium (LOE) measurements showed that hybrids had reduced hypoxia tolerance and lacked the size-dependence in hypoxia tolerance seen in the parental species. However, we found no evidence of systematic differences in metabolite levels in fish after LOE. Furthermore, there were abundant glycogen reserves at the point of loss of equilibrium. The three genotypes did not differ in metabolite status at rest, showed an equal disruption at exhaustion, and similar metabolic profiles throughout recovery. Thus, we found no evidence of a mitochondria dysfunction in hybrids, and mitochondrial differences and oxidative metabolism did not explain the variation in hypoxia tolerance seen in the hybrid and two parental species.

Differential scattering cross-sections for the different product vibrational States in the ion-molecule reaction Ar(+)+N2.

The charge transfer reaction Ar(+)+N(2)→Ar+N(2)(+) has been investigated in a crossed-beam experiment in combination with three-dimensional velocity map imaging. Angular-differential state-to-state cross sections were determined as a function of the collision energy. We found that scattering into the first excited vibrational level dominates as expected, but only for scattering in the forward direction. Higher vibrational excitations up to v'=6 have been observed for larger scattering angles. For decreasing collision energy, scattering into higher scattering angles becomes increasingly important for all kinematically allowed quantum states. Our detailed measurements indicate that a quantitative agreement between experiment and theory for this basic ion-molecule reaction now comes within reach.

Pulse synthesis in the single-cycle regime from independent mode-locked lasers using attosecond-precision feedback.

We report the synthesis of a nearly single-cycle (3.7 fs), ultrafast optical pulse train at 78 MHz from the coherent combination of a passively mode-locked Ti:sapphire laser (6 fs pulses) and a fiber supercontinuum (1-1.4 µm, with 8 fs pulses). The coherent combination is achieved via orthogonal, attosecond-precision synchronization of both pulse envelope timing and carrier envelope phase using balanced optical cross-correlation and balanced homodyne detection, respectively. The resulting pulse envelope, which is only 1.1 optical cycles in duration, is retrieved with two-dimensional spectral shearing interferometry (2DSI). To our knowledge, this work represents the first stable synthesis of few-cycle pulses from independent laser sources.

Efficacy of antiglaucoma fixed combination therapy versus unfixed components in reducing intraocular pressure: a systematic review.

AIM: To evaluate the efficacy of the fixed combination ocular hypotensive therapies compared with their non-fixed components used concomitantly for lowering intraocular pressure (IOP) in glaucoma and ocular hypertension. METHODS: A systematic review of the literature, up to May 2007, without limits on year or language of publication was performed. Seven randomised controlled trials (n = 2,083 eyes) were identified. Assessment of methodological quality was made using standardised criteria. Results were pooled quantitatively using meta-analysis methods, and statistical analysis was performed using STATA software. The difference in mean intraocular pressure (mm Hg) from baseline between the fixed combination and non-fixed component therapies was compared. Non-inferiority in terms of efficacy was set at an upper confidence limit of < or =1.5 mm Hg for all time points (hour (Hr)0, Hr2 and Hr8) and evaluated at 12 weeks. Safety was evaluated from data on adverse events as reported in the included studies. RESULTS: Of the 679 abstracts identified, seven randomised controlled trials met the selection criteria. The quality scores of included studies were high (mean of 29.4, maximum score 30). The mean differences (95% CI) and p values at 12 weeks were as follows: 0.200 mm Hg, (CI -0.106 to 0.507), p = 0.20 for Hr0, 0.393 mm Hg (CI 0.038 to 0.747), p = 0.03 for Hr2 and 0.501 mm Hg (CI 0.156 to 0.846), p = 0.004 for Hr8. Although both Hr2 and Hr8 showed statistical significance favouring the non-fixed combinations, the non-inferiority measure < or =1.5 mm Hg upper confidence limit was not exceeded. CONCLUSIONS: Fixed combination therapies are equally safe and effective at lowering IOP as their non-fixed components administered concomitantly.

Laser cooling of trapped ytterbium ions with an ultraviolet diode laser.

We demonstrate an ultraviolet diode laser system for cooling of trapped ytterbium ions. The laser power and linewidth are comparable to those of previous systems based on resonant frequency doubling, but the system is simpler, more robust, and less expensive. We use the laser system to cool small numbers of ytterbium ions confined in a linear Paul trap. From the observed spectra, we deduce final temperatures of < 270 mK.

Selective labeling of central and peripheral sensory neurons in the developing zebrafish using P2X(3) receptor subunit transgenes.

The two paralogous P2X receptor subunit genes p2rx3.1 and p2rx3.2 are selectively expressed in overlapping, but unique, patterns of sensory neurons in the developing zebrafish. We constructed a series of transgenes derived from both genes using the recombineering technique. Transgenes utilizing either enhanced green fluorescent protein or monomeric red fluorescent protein-1 were shown to be expressed with the same spatial and temporal patterns as the native genes. The p2rx3.1-derived transgenes labeled the vast majority of the Rohon-Beard neurons in the spinal cord and neurons of the trigeminal ganglia. The p2rx3.2-derived transgene labeled fewer Rohon-Beard and trigeminal neurons than what was observed for the p2rx3.1-derived transgenes, but was also detected in neurons of the epibranchial ganglia. Three distinct populations of sensory neurons were detected: those expressing only one or the other paralog, and those expressing both paralogs. The fluorescent proteins encoded by the transgenes allowed for visualization of the neuronal somas as well as their peripheral and central projections. These reagents should prove extremely useful in providing the basis for future studies aimed at elucidating the developmental and physiological attributes of sensory neurons.

The investigation of the behavior of a long period grating sensor with a copper sensitive coating fabricated by layer-by-layer electrostatic adsorption.

Sensors based on changes of refractive index in response to sorption of an analyte on the coating or film of a long period grating fiber (LPG) fiber have recently been reported. In most prior work the coating or film swelled during interaction with the analyte. The swelling mechanism produced a kinetic response that slowed both the sensor's time for steady-state measurement and the reversibility of the sensor. Here, the analytical utility of fabricating these nanometer thin films using the layer-by-layer (LBL) electrostatic assembly method is evaluated using Cu(II) as the test analyte and Cibacron Blue as the reagent immobilized in the LBL assembly; a generation-4 poly(amidoamine) dendrimer served as the spacer in the assembly. Detection of 1.3mgCu(II)L(-1) was observed when six bilayers comprised the coating. The stable response was achieved with 0.6mgL(-1) in less than 1min. When 0.1M HCl was used as the rinsing solution, this LPG sensor was reversible and the signal to similar concentrations of Cu(II) reproducible.

Pollen limitation or mate search need not induce an Allee effect.

When a process modelling the availability of gametes is included explicitly in population models a critical depensation or Allee effect usually results. Non-spatial models cannot describe clumping and so small populations must be assumed very diffuse. Consequently individuals in small populations experience low contact rates and so reproduction is limited. In Nature invasions into new territory are unlikely to be as diffuse as those described by non-spatial models. We develop pair approximations to a probabilistic cellular automata model with independent pollination and seed setting processes (equivalently mate search and reproduction processes). Each process can be either global (population-wide) or local (within a small neighbourhood) or a mixture of the two. When either process is global the resulting model recaptures the Allee effect found in non-spatial models. However, if both processes are at least partially local we obtain a model in which Allee effects can be avoided altogether if individuals are suitably strong pollinators and colonisers. The Allee effect disappears because small populations are dramatically more clumped when colonisation is local and less wasteful of pollen when pollination is local.

Space mediates coexistence of females and hermaphrodites.

In gynodioecious populations of flowering plants females and hermaphrodites coexist. Gynodioecy is widespread and occurs in both asexual and sexual species but does not admit a satisfactory explanation from classical sex ratio theory. In sexual populations male fertility restoring genes have evolved to counter non-nuclear male sterility mutations. In pseudogamous asexual populations pollen retention and increased self-fertilization can make male sterility costly. Both of these mechanisms can promote coexistence. However, it remains unclear how either of these mechanisms could evolve if coexistence was not initially possible. In the absence of these adaptations non-spatial models predict that females either fail to invade hermaphrodite populations or else displace them until pollen shortage drives the population to extinction. We develop a pair approximation to a probabilistic cellular automata model in which females and hermaphrodites interact on a regular lattice. The model features independent pollination and colonization processes which take place on different timescales. The timescale separation is exploited to obtain, with perturbation methods, a more manageable aggregated pair approximation. We present both the mean field model which recreates the classical non-spatial predictions and the pair approximation, which strikingly predicts different invasion criteria and coexistence under a wide range of parameters. The pair approximation is shown to correspond well qualitatively with simulation behaviour.

Endosperm triploidy has a selective advantage during ongoing parental conflict by imprinting.

The endosperm of the flowering plant mediates the supply of maternal resources for embryogenesis. An endosperm formed in sexual reproduction between diploid parents is typically triploid, with a 2 : 1 ratio of maternal genetic material (denoted as 2m : 1p). Variation from this ratio affects endosperm size, indicating parent-specific expression of genes involved in endosperm growth and development. The presence of paternally or maternally imprinted genes can be explained by parental conflict over the transfer of nutrients from maternal to offspring tissue. Genomic imprinting can, for example, provide the male parent of an embryo in a mixed-paternity seed pod, with an opportunity for expressing its preference for a disproportionate allocation of resources to its embryo. It has been argued that a diploid 1m : 1p endosperm was ancestral and the 2m : 1p endosperm evolved after parental conflict, to improve maternal control over seed provisioning. We present a population genetic model, which instead places the origin of triploidy early in the parental conflict over resource allocation. We find that there is an advantage to having a triploid endosperm as the parental conflict continues. This advantage can help to explain why the 2m : 1p endosperm prevails among flowering plants.

Molecular characterization of the zebrafish P2X receptor subunit gene family.

P2X receptors are non-selective cation channels gated by extracellular ATP and are encoded by a family of seven subunit genes in mammals. These receptors exhibit high permeabilities to calcium and in the mammalian nervous system they have been linked to modulation of neurotransmitter release. Previously, three complementary DNAs (cDNAs) encoding members of the zebrafish gene family have been described. We report here the cloning and characterization of an additional six genes of this family. Sequence analysis of all nine genes suggests that six are orthologs of mammalian genes, two are paralogs of previously described zebrafish subunits, and one remains unclassified. All nine subunits were physically mapped onto the zebrafish genome using radiation hybrid analysis. Of the nine gene products, seven give functional homo-oligomeric receptors when recombinantly expressed in human embryonic kidney cell line 293 cells. In addition, these subunits can form hetero-oligomeric receptors with phenotypes distinct from the parent subunits. Analysis of gene expression patterns was carried out using in situ hybridization, and seven of the nine genes were found to be expressed in embryos at 24 and 48 h post-fertilization. Of the seven that were expressed, six were present in the nervous system and four of these demonstrated considerable overlap in cells present in the sensory nervous system. These results suggest that P2X receptors might play a role in the early development and/or function of the sensory nervous system in vertebrates.

Itih-4, a serine protease inhibitor regulated in interleukin-6-dependent liver formation: role in liver development and regeneration.

Inter-alpha-trypsin inhibitor-4 (Itih-4) is a liver-restricted member of the serine protease inhibitor family with diverse functions as an anti-apoptotic and matrix stabilizing molecule that are important throughout development. We investigate the functional role of Itih-4 in liver formation, regeneration (LR) and examine its role in calcium and hyaluronic acid binding. Itih-4 expression is prominent in early liver development at E9 and later at E16, being restricted to hepatoblasts, immature hepatocytes, and differentiated hepatocytes. We note a marked and differential increase in Itih-4 labeling in proliferating hepatocytes, compared with bile duct cells in liver explant cultures treated with interleukin-6 (IL-6). After partial hepatectomy, maximal Itih-4 expression occurs in a bimodal manner at 30 min and at 12 hr, with a predominant centrizonal distribution. There is no detectable binding of glutathione transferase-fusion Itih-4 protein to calcium and hyaluronic acid, indicating a possible requirement for posttranslational modifications for these functions. These results suggest that in LR, Itih-4 expression corresponds to that of immediate early genes and may contribute to the entry of normally quiescent hepatocytes into the early stages of the cell cycle. The markedly high expression of Itih-4 in early liver development and in explants treated with IL-6 suggests a prominent role for Itih-4 at key points in liver formation.

Solution structure and backbone dynamics of the defunct domain of calcium vector protein.

CaVP (calcium vector protein) is a Ca(2+) sensor of the EF-hand protein family which is highly abundant in the muscle of Amphioxus. Its three-dimensional structure is not known, but according to the sequence analysis, the protein is composed of two domains, each containing a pair of EF-hand motifs. We determined recently the solution structure of the C-terminal domain (Trp81-Ser161) and characterized the large conformational and dynamic changes induced by Ca(2+) binding. In contrast, the N-terminal domain (Ala1-Asp86) has lost the capacity to bind the metal ion due to critical mutations and insertions in the two calcium loops. In this paper, we report the solution structure of the N-terminal domain and its backbone dynamics based on NMR spectroscopy, nuclear relaxation, and molecular modeling. The well-resolved three-dimensional structure is typical of a pair of EF-hand motifs, joined together by a short antiparallel beta-sheet. The tertiary arrangement of the two EF-hands results in a closed-type conformation, with near-antiparallel alpha-helices, similar to other EF-hand pairs in the absence of calcium ions. To characterize the internal dynamics of the protein, we measured the (15)N nuclear relaxation rates and the heteronuclear NOE effect in (15)N-labeled N-CaVP at a magnetic field of 11.74 T and 298 K. The domain is mainly monomeric in solution and undergoes an isotropic Brownian rotational diffusion with a correlation time of 7.1 ns, in good agreement with the fluorescence anisotropy decay measurements. Data analysis using a model-free procedure showed that the amide backbone groups in the alpha-helices and beta-strands undergo highly restricted movements on a picosecond to nanosecond time scale. The amide groups in Ca(2+) binding loops and in the linker fragment also display rapid fluctuations with slightly increased amplitudes.

The first transmembrane domain of the P2X receptor subunit participates in the agonist-induced gating of the channel.

Based on pharmacological properties, the P2X receptor family can be subdivided into those homo-oligomers that are sensitive to the ATP analog alphabeta-methylene ATP(alphabetameATP) (P2X(1) and P2X(3)) and those that are not (P2X(2), P2X(4), P2X(5), P2X(6), and P2X(7)). We exploited this dichotomy through the construction of chimeric receptors and site-directed mutagenesis in order to identify domains responsible for these differences in the abilities of extracellular agonists to gate P2X receptors. Replacement of the extracellular domain of the alphabetameATP-sensitive rat P2X(1) subunit with that of the alphabetameATP-insensitive rat P2X(2) subunit resulted in a receptor that was still alphabetameATP-sensitive, suggesting a non-extracellular domain was responsible for the differential gating of P2X receptors by various agonists. Replacement of the first transmembrane domain of the rat P2X(2) subunit with one from an alphabetameATP-sensitive subunit (either rat P2X(1) or P2X(3) subunit) converted the resulting chimera to alphabetameATP sensitivity. This conversion did not occur when the first transmembrane domain came from a non-alphabetameATP-sensitive subunit. Site-directed mutagenesis indicated that the C-terminal portion of the first transmembrane domain was important in determining the agonist selectivity of channel gating for these chimeras. These results suggest that the first transmembrane domain plays an important role in the agonist operation of the P2X receptor.

Gene structure, chromosomal localization, cDNA cloning and expression of the mouse ATP-gated ionotropic receptor P2X5 subunit.

The P2X receptors are oligomeric ligand-gated ion channels operated by extracellular ATP. Here we report the genomic and cDNA sequence of the mouse P2X(5) subunit, as well as its genomic organization, chromosomal localization and expression in select tissues. The mouse gene spans approximately 13 kb of DNA and contains thirteen exons. The cDNA encodes a 455 amino acid protein with 95% identity to the rat subunit. The P2X(5) subunit gene encodes a 2.6 kb mRNA that was found to in a number of tissues, with highest levels detected in heart and kidney. Results from rapid amplification of cDNA ends (RACE) PCR suggest that there are multiple transcriptional start sites located approximately 30-70 bp upstream from the start codon. The gene was localized to band B5 on Chromosome 11 using fluorescence in-situ hybridization (FISH), a region that has a high degree of synteny with human Chromosome 17. These results provide the initial information useful for further investigation into the functional role(s) of the P2X(5) subunit in physiological processes.

Backbone dynamics of the regulatory domain of calcium vector protein, studied by (15)N relaxation at four fields, reveals unique mobility characteristics of the intermotif linker.

UNLABELLED: CaVP is a calcium-binding protein from amphioxus. It has a modular composition with two domains, but only the two EF-hand motifs localized in the C-terminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca(2+)-saturated form and characterized the stepwise ion binding. This paper reports the (15)N nuclear relaxation rates of the Ca(2+)-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide H(N)-N vectors. Fitting the spectral density values at eight frequencies by a model-free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca(2+) binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP characterized here is discussed in relation with other well-characterized Ca(2+)-binding proteins. SUPPLEMENTAL MATERIAL: See www.proteinscience.org