Antimicrobial effects of tea-tree oil and its major components on Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes

Major components of two tea-tree oil samples were identified using thin layer and gas-liquid chromatography (TLC and GLC). Using a TLC-bioautographic technique, the tea-tree oils, terpinen-4-ol, oc-terpineol and α-pinene were found to be active against Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes whereas cineole was inactive against these organisms. The MIC values of the three active compounds increased in the order α-terpineol < terpinen-4-ol < α-pinene for all three micro-organisms. MIC values of the tea-tree oils and terpinen-4-ol were lower for P. acnes than for the two staphylococci. This study supports the use of tea-tree oil in the treatment of acne, and demonstrates that terpinen-4-ol is not the sole active constituent of the oil.     

Identification and validation of genomic regions influencing kernel zinc and iron in maize

Key message

Genome-wide association study (GWAS) on 923 maize lines and validation in bi-parental populations identified significant genomic regions for kernel-Zinc and-Iron in maize.

Abstract

Bio-fortification of maize with elevated Zinc (Zn) and Iron (Fe) holds considerable promise for alleviating under-nutrition among the world’s poor. Bio-fortification through molecular breeding could be an economical strategy for developing nutritious maize, and hence in this study, we adopted GWAS to identify markers associated with high kernel-Zn and Fe in maize and subsequently validated marker-trait associations in independent bi-parental populations. For GWAS, we evaluated a diverse maize association mapping panel of 923 inbred lines across three environments and detected trait associations using high-density Single nucleotide polymorphism (SNPs) obtained through genotyping-by-sequencing. Phenotyping trials of the GWAS panel showed high heritability and moderate correlation between kernel-Zn and Fe concentrations. GWAS revealed a total of 46 SNPs (Zn-20 and Fe-26) significantly associated (P?≤?5.03?×?10^?05) with kernel-Zn and Fe concentrations with some of these associated SNPs located within previously reported QTL intervals for these traits. Three double-haploid (DH) populations were developed using lines identified from the panel that were contrasting for these micronutrients. The DH populations were phenotyped at two environments and were used for validating significant SNPs (P?≤?1?×?10^?03) based on single marker QTL analysis. Based on this analysis, 11 (Zn) and 11 (Fe) SNPs were found to have significant effect on the trait variance (P?≤?0.01, R²?≥?0.05) in at least one bi-parental population. These findings are being pursued in the kernel-Zn and Fe breeding program, and could hold great value in functional analysis and possible cloning of high-value genes for these traits in maize.

     

Sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal

A multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM) principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin was used in this study, M _r6000 Da) was correlated with the shift of resonant frequency of QCM system before and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity, and is sensitive to approx. 1 ng insulin ml^–1 with a linear response for insulin from 10 g ml^–1 to 10 ng ml^–1 in standard solutions. The technique may also be applied for the detection of other small biomolecules.     

A thermodynamically consistent constitutive equation for the elastic force-length relation of soft biological materials

Starting from the laws of thermodynamics of reversible processes, a temperature-dependent constitutive equation is derived for the elastic force-length relation of soft biological tissues. These tissues are composed of a network of fibres (mainly collagen). The equation is based on a model which uses a simplified two-dimensional representation of the alpha-helix of collagen.     

Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.     

Biogenic Synthesis, Purification, and Chemical Characterization of Anti-inflammatory Resolvins Derived from Docosapentaenoic Acid (DPAn-6)

Enzymatically oxygenated derivatives of the ω-3 fatty acids cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and cis-5,8,11,14,17-eicosapentaenoic acid, known as resolvins, have potent inflammation resolution activity (Serhan, C. N., Clish, C. B., Brannon, J., Colgan, S. P., Chiang, N., and Gronert, K. (2000) J. Exp. Med. 192, 1197–1204; Hong, S., Gronert, K., Devchand, P. R., Moussignac, R., and Serhan, C. N. (2003) J. Biol. Chem. 278, 14677–14687). Our objective was to determine whether similar derivatives are enzymatically synthesized from other C-22 fatty acids and whether these molecules possess inflammation resolution properties. The reaction of DHA, DPAn-3, and DPAn-6 with 5-, 12-, and 15-lipoxygenases produced oxylipins, which were identified and characterized by liquid chromatography coupled with tandem mass-spectrometry. DPAn-6 and DPAn-3 proved to be good substrates for 15-lipoxygenase. 15-Lipoxygenase proved to be the most efficient enzyme of the three tested for conversion of long chain polyunsaturated fatty acids to corresponding oxylipins. Since DPAn-6 is a major component of Martek DHA-S™ oil, we focused our attention on reaction products obtained from the DPAn-6 and 15-lipoxygenase reaction. (17S)-hydroxy-DPAn-6 and (10,17S)-dihydroxy-DPAn-6 were the main products of this reaction. These compounds were purified by preparatory high performance liquid chromatography techniques and further characterized by NMR, UV spectrophotometry, and tandem mass spectrometry. We tested both compounds in two animal models of acute inflammation and demonstrated that both compounds are potent anti-inflammatory agents that are active on local intravenous as well as oral administration. These oxygenated DPAn-6 compounds can thus be categorized as a new class of DPAn-6-derived resolvins.Enzymatically formed oxygenation products of C-20 and C-22 long chain polyunsaturated fatty acids (LC-PUFAs),⁴ have important biological roles in inflammation, allergies, and blood clotting and are thus believed to have therapeutic potential in several chronic immune diseases (1–10) Several biologically important products of cis-5,8,11,14-eicosatetraenoic acid/arachidonic acid (ARA), cis-5,8,11,14,17-eicosapentaenoic acid (EPA), and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) have been described (4, 11, 12). Proinflammatory oxylipins, such as leukotrienes and some prostaglandins, are derived from ARA, an ω-6 fatty acid. Interestingly, the same fatty acid also serves as a precursor to anti-inflammatory or proresolution molecules like lipoxins (13, 14). Stable analogues of lipoxins are being developed as drugs for asthma and other inflammatory airway diseases (15, 16). Oxylipins derived from ω-3 fatty acids, such as DHA and EPA, known as resolvins, are primarily anti-inflammatory in nature (17). EPA acts as a precursor to the E-series resolvins that have shown potential in the treatment of colitis, arthritis, and periodontitis (18–20). The resolvins of the D-series derived from DHA are useful as neuroprotective agents. 10,17-Dihydroxy-4,7,11,13,15,19-docosahexaenoic acid (10,17-HDHA) or neuroprotectin D1 is a resolvin that is formed endogenously in the human brain and eye and is believed to exert its protective effect against cell injury-induced oxidative stress (21–23).The main enzymes responsible for the production of these oxygenated LC-PUFA products are primarily lipoxygenases and, in addition, cyclo-oxygenases and cytochromes P450. These enzymes produce oxylipins via transcellular activity, often involving multiple cell types (24). This activity mainly results in mono-, di-, and tri-hydroxylation products of fatty acids that have varying potencies, depending on the exact structure of the compound. Lipoxygenases are non-heme, iron-containing dioxygenases that catalyze the regioselective and enantioselective oxidation of polyunsaturated fatty acids containing one or more cis,cis-1,4-pentadienoic moieties to give the corresponding hydroperoxy derivatives (25, 26). We thus considered that, in addition to DHA and EPA, other C-22 PUFAs containing such methylene interrupted double bonds may also be substrates for lipoxygenases and that resulting products may have anti-inflammatory activity similar to DHA-derived resolvins. DPAn-6 (cis-4,7,10,13,16-docosapentaenoic acid) is present in algal oils, and recent studies have demonstrated that this fatty acid has anti-inflammatory activities in vitro and, in conjunction with DHA, also has anti-inflammatory activity in vivo.⁵ Also, it has been suggested that a combination of DHA and DPAn-6 could be a beneficial natural therapy in neuroinflammatory conditions like Alzheimer disease. Specifically, in a 3×Tg-AD mouse model of Alzheimer disease, DPAn-6 was shown to reduce levels of early stage phospho-Tau epitopes, which in turn correlated with a reduction in phosphorylated c-Jun N-terminal kinase, a putative Tau kinase (27). Although the precise mechanism of action of DPAn-6 in these inflammatory milieus is not known, it suggests a possible role for oxylipin products of DPAn-6 in resolution of inflammation. Also, another LC-PUFA, DPAn-3 (cis-7,10,13,16,19-docosapentaenoic acid) usually present along with DHA and EPA in marine oils is known to be a potent inhibitor of platelet aggregation (28–30). In addition, this LC-PUFA has a potent inhibitory effect on angiogenesis through the suppression of VEGFR-2 (vascular endothelial-cell growth factor receptor 2) expression. Angiogenesis is known to contribute to tumor growth, inflammation, and microangiopathy, again pointing to the possibility that anti-inflammatory activity of DPAn-3 might be mediated through resolvin-like products as in the case of DHA and EPA (31).The purpose of this research was to determine whether oxylipins are formed from the C-22 LC-PUFAs, DPAn-6 and DPAn-3, by lipoxygenase activity; to compare them to products formed from DHA; to chemically characterize products; to purify key oxylipin products from the DPAn-6/15-lipoxygenase reaction; and to test whether these compounds have resolvin-like anti-inflammatory activity. This research also sets the stage for preparation and isolation of a wide range of other C-22 oxylipins that could be evaluated as potential anti-inflammatory compounds.     

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.     

Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue N205: Towards tetraoxane scaffolds with potential for single dose cure of malaria

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84?nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.     

Hydrophobic surfaces in oligosaccharides: linear dextrins are amphiphilic chains.

Polysaccharide chains are usually considered to be highly hydrophilic, since they have no obvious nonpolar moieties in them. Yet, it is possible to realise conformations in these chains wherein all the hydroxy groups are disposed in one side or face of the chain and the hydrogens disposed in the other. We experimentally demonstrate that such an amphiphilic surface is present in linear oligomeric dextrins, i.e., alpha-1,4-linked D-glucosides, but not in alpha-1,6-D-glucosides (dextrans) or in beta-1,4-D-glucosides (cellulose). This amphiphilicity is generated as a consequence of the stereochemical constraints, which vary with the structure of the sugar and with the type of linkage. Oligosaccharide chains that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in heteromolecular recognition process than as homomolecular self-aggregation.