Total synthesis of a fully lipidated form of phosphatidyl-myo-inositol dimannoside (PIM-2) of Mycobacterium tuberculosis

Using an orthogonal protecting group approach to resolve racemic myo-inositol directly as its D-mannoside, we report a [2+n]-glycan/phosphatidylation strategy to assemble a bis-palmityl, tuberculostearyl form of phosphatidyl-myo-inositol dimannoside (PIM-2).</p

Total synthesis of a fully lipidated form of phosphatidyl-myo-inositol dimannoside (PIM-2) of Mycobacterium tuberculosis

Using an orthogonal protecting group approach to resolve racemic myo-inositol directly as its D-mannoside, we report a [2+n]-glycan/phosphatidylation strategy to assemble a bis-palmityl, tuberculostearyl form of phosphatidyl-myo-inositol dimannoside (PIM-2).</p

Preparation of azide biosynthetic surrogates of myo-Inositol

As a prelude to biomolecular incorporation studies, practical routes to a series of four regioisomeric azido-deoxy derivatives of inositol that mimic the natural myo-stereochemistry are described. Starting from commercially available myo-inositol, the regioselective and stereoselective introduction of azide functionality was achieved at the C-2, C-3, C-4 and C-5 positions via azide displacement of the corresponding O-sulfonates of suitably protected scyllo-, chiro-, epi- and neo-inositols, respectively. Notably, a final one-pot acetolysis method conveniently allowed for rapid access to pentaacetate azido-deoxy inositols. Investigations on the metabolic incorporation of these myo-inositol azide surrogates in both acetate and free alcohol forms are in progress.</p

Preparation of azide biosynthetic surrogates of myo-Inositol

As a prelude to biomolecular incorporation studies, practical routes to a series of four regioisomeric azido-deoxy derivatives of inositol that mimic the natural myo-stereochemistry are described. Starting from commercially available myo-inositol, the regioselective and stereoselective introduction of azide functionality was achieved at the C-2, C-3, C-4 and C-5 positions via azide displacement of the corresponding O-sulfonates of suitably protected scyllo-, chiro-, epi- and neo-inositols, respectively. Notably, a final one-pot acetolysis method conveniently allowed for rapid access to pentaacetate azido-deoxy inositols. Investigations on the metabolic incorporation of these myo-inositol azide surrogates in both acetate and free alcohol forms are in progress.</p

Isolation of ATS from <i>abc3</i>Δ appressoria in <i>M. oryzae</i>.

<p>(<b>A</b>) Wild-type <i>S. pombe</i> cells were treated with extracellular fluid (E/F) or appressorial extract (A/E) from the wild-type or <i>abc3</i>Δ <i>M. oryzae</i> strain for 6 h and stained with calcofluor white (CFW). Arrowheads indicate aberrant deposition of septal/cell wall material at the cell tip(s). Bars = 10 µm. (<b>B</b>) Schematic representation of the <i>S. pombe</i> cell-based assay used to guide the purification of ATS and to confirm ATS as an efflux substrate of the Abc3 transporter. Mo<i>ABC3</i> refers to <i>M. oryzae ABC3</i>.</p

Rapid and sensitive profiling of tear wax ester species using high performance liquid chromatography coupled with tandem mass spectrometry

A rapid and sensitive method was developed for quantitative profiling of wax esters (WEs) in human tear lipide. Individual WE species was separated by liquid chromatography and detected by electrospray ionisation mass spectrometry using specific multiple reaction monitoring (MRM) scanning. Palmitoyl palmitate and in-house synthesized wax esters 13C18:1(oleic acid-1,2,3,7,8,9,10-13C7)C26:0 were used as internal standards for quantitation of WEs containing saturated and unsaturated fatty acids (FA), respectively. The limit of detection was approximately 70 nmol/L. The linearity range of the liquid chromatography (LC)-MRM detection for WEs was about three orders of magnitude. Quantitative analyses of 141 individual WE in the human tear lipidome demonstrated that species comprising FA18:1 and FA16:1 each accounted for 47.7% and 24.0% (molar%) of total WE, while fatty alcohols in WEs of human tears ranged from 17 carbons to 32 carbons with predominant species represented by C24, C25 and C26.</p

ATS associates with Tef2 in <i>S. pombe</i> and <i>M. oryzae</i>.

<p>(<b>A</b>) Loss of SpTef2-function simulates ATS effect in <i>S. pombe</i>. Cell wall staining of the wild-type or <i>tef2</i>Δ <i>S. pombe</i> cells using CFW. Red arrowheads depict defective septal/cell wall deposition. Scale bar equals 10 micron. (<b>B</b>) Effect of digoxin on subcellular localization of SpTef2-RFP or Swo1-GFP in <i>S. pombe</i> cells. The strains expressing the indicated fusion proteins were stained with CFW and analysed by epifluorescence microscopy. Arrowheads show distinct aggregates of SpTef2-RFP. Bar = 10 µm. (<b>C</b>) Effect of ATS on localization of RFP-Tef2 in <i>M. oryzae</i> vegetative hyphae (upper panels; Scale Bar = 5 µm) and conidia (middle and lower panels; Bar represents 10 µm) co-stained with DAPI to aid visualization of nuclei. Arrowheads denote aberrant perinuclear aggregates and/or patches of RFP-Tef2. BF, Bright Field.</p

ATS plays a role in ion homeostasis during pathogenesis in <i>M. oryzae</i>.

<p>(<b>A</b>) ATS increases sensitivity of wild-type <i>M. oryzae</i> towards specific cations. Excess or permissive concentrations of Ca⁺², Na⁺, or Mg⁺² ion were added to the germinating wild-type conidia in the presence or absence of ATS. Arrows show delayed appressorial development (longer germ tubes) in the presence of ATS, which was otherwise seen only in the presence of excess concentration of the ions under control condition. Bar = 10 µm. (<b>B</b>) Sensitivity of the <i>abc3</i>Δ towards permissive concentratios of indicated cations. Arrows indicate delayed response in terms of longer germ tubes. Bars = 10 µm. (<b>C</b>) Effect of excess Ca⁺² on appressorial function/host penetration efficiency in <i>M. oryzae</i>. Penetration efficiency was evaluated at 28 hpi by staining callose deposits with Aniline Blue. Arrowheads depict appressoria successful in host penetration. Bar = 10 µm. (<b>D</b>) Penetration efficiency of the appressoria was calculated as % appressorial function at 28 hpi. Data represent mean ± SEM from 3 individual experiments (n = 100 each per replicate). (<b>E</b>) Rice leaf sheaths were inoculated with wild-type <i>M. oryzae</i> in the presence of residual solvent or ATS for 24 h, and stained with aniline blue (right panels) for induced callose deposits (arrow) underneath the sites of host penetration (appressorial function). Asterisk shows occasional callose deposition. Bars = 10 µm. (<b>F</b>) Quantification of appressorial function at 30 hpi. The data represents mean ± SEM from 3 individual assays.</p

SpTef2 function and the F-actin cytoskeleton in <i>S. pombe</i>.

<p>(<b>A</b>) Sensitivity of <i>tef2</i>Δ <i>S. pombe</i> cells towards Ca⁺² in the growth medium. Serial dilutions of the wild-type or <i>tef2</i>Δ cells were inoculated under indicated growth conditions. (<b>B</b>) Morphology and dynamics of GFP-labelled F-actin cytoskeleton in wild-type <i>S. pombe</i> treated with ATS, digoxin or Ca⁺². The <i>tef2</i>Δ strain was analyzed in parallel. Arrowheads show excess accumulation of F-actin patches and/or short, spooling cables at the cell end(s). The maximum projection images shown here represent the compressed z-stack sections. Bar equals 10 µm.</p

Exogenous ATS or digoxin alters the F-actin cytoskeleton in <i>M. oryzae</i>.

<p>Morphology (<b>A</b>) and dynamics (<b>B</b>) of the F-actin patches in wild type <i>M. oryzae</i> expressing Abp1-RFP and treated with ATS, digoxin, or 0.1 M CaCl₂. Arrowheads depict developing appressoria. Bars = 10 µm.</p