Lipid Composition of the Viral Envelope of Three Strains of Influenza VirusNot All Viruses Are Created Equal

Although differences in the rate of virus fusion and budding from the host cell membrane have been correlated with pathogenicity, no systematic study of the contribution of differences in viral envelope composition has previously been attempted. Using rigorous virus purification, marked differences between virions and host were observed. Over 125 phospholipid species have been quantitated for three strains of influenza (HKx31-H3N2, PR8-H1N1, and VN1203-H5N1) grown in eggs. The glycerophospholipid composition of purified virions differs from that of the host or that of typical mammalian cells. Phosphatidylcholine is the major component in most mammalian cell membranes, whereas in purified virions phosphatidylethanolamine dominates. Due to its effects on membrane curvature, it is likely that the variations in its content are important to viral processing during infection. This integrated method of virion isolation with systematic analysis of glycerophospholipids provides a tool for the assessment of species-specific biomarkers of viral pathogenicity

Lipid Composition of the Viral Envelope of Three Strains of Influenza VirusNot All Viruses Are Created Equal

Although differences in the rate of virus fusion and budding from the host cell membrane have been correlated with pathogenicity, no systematic study of the contribution of differences in viral envelope composition has previously been attempted. Using rigorous virus purification, marked differences between virions and host were observed. Over 125 phospholipid species have been quantitated for three strains of influenza (HKx31-H3N2, PR8-H1N1, and VN1203-H5N1) grown in eggs. The glycerophospholipid composition of purified virions differs from that of the host or that of typical mammalian cells. Phosphatidylcholine is the major component in most mammalian cell membranes, whereas in purified virions phosphatidylethanolamine dominates. Due to its effects on membrane curvature, it is likely that the variations in its content are important to viral processing during infection. This integrated method of virion isolation with systematic analysis of glycerophospholipids provides a tool for the assessment of species-specific biomarkers of viral pathogenicity

<i>pex3²</i> is a null mutant.

<p>(<b>A</b>) A schematic of the pex3 genomic region showing the EP element insertion site and the deleted region in <i>pex3²</i>. (<b>B</b>) Immunoblot of total protein extracts from wandering third instar larvae show that Pex3 protein is undetectable in the <i>pex3²</i> mutants, but can be detected by the addition of a genomic fragment containing <i>pex3</i> or expression of a fly or human cDNA transgene. The Myc-tagged transgenic protein is a higher molecular weight than endogenous Pex3. 37.5 µg of total protein was loaded in lanes 2–8. (<b>C</b>) Immunoblot with increasing amounts of <i>pex3²</i> protein shows that Pex3 protein is undetectable. Total protein amounts loaded in lanes 2–6, were 37.5 µg, 37.5 µg, 75 µg, 112.5 µg, and 37.5 µg. The intensities of the Tubulin bands in lanes 3–6, relative to lane 2, are 30%, 94%, 124%, and 108%.(<b>D</b>) Adult fly viability measurements show that the loss of pex3 (<i>pex3²</i>) results in nonviable Drosophila 0±0% (n = 381) compared to controls (<i>pex3^rev</i>; 99.5±0.4%; n = 472), genomic rescue (72±12.9%; n = 185), and fly cDNA rescue (48.2±10.9%, n = 46). (<b>E</b>) Imaging of wandering third instar larvae shows that <i>pex3²</i> larvae are smaller than <i>pex3^rev</i> control larvae.</p

Peroxisome loss causes aberrant lipid metabolism.

<p>(<b>A</b>) Mass spectrometry (MS) analysis of larval lipids shows that the levels of the major lipid classes, Polar lipids, DAG, and TAG are unchanged in <i>pex3²</i> mutants. (<b>B</b>) However, <i>pex3²</i> mutants have elevated longer acyl chain length TAG species. (<b>C</b>) CerPE levels are decreased in <i>pex3²</i>. (<b>D</b>) Survival analysis of larvae under starvation conditions reveals that larvae lacking Pex3 (<i>pex3²</i>) are hypersensitive to starvation. Starvation sensitivity can be partially rescued by expression of genomic <i>pex3</i> or <i>pex3</i> cDNA.</p

Peroxisome biogenesis is impaired in <i>pex3²</i> mutants.

<p>Peroxisomes were visualized by confocal microscopy in hepatocyte-like, oenocyte cells of third instar larvae. <i>UAS-eYFP-PTS1</i> was driven by the <i>arm</i> GAL4 driver in various backgrounds. (<b>A</b>) Peroxisomes are abundant in oenocytes of <i>pex3^rev</i> control larvae. (<b>B</b>) eYFP-PTS1 is diffuse in the cytoplasm of <i>pex3²</i> larvae indicating that peroxisomes are absent. Some cells contain bright spots, but it is unclear if these are peroxisomes. Peroxisomes are restored when fly (<b>C</b>) or human (<b>D</b>) <i>pex3</i> cDNA are expressed in the <i>pex3²</i> background. Scale bar = 10 µm.</p

Peroxisome distribution in <i>D. melanogaster</i>.

<p>UAS-eYFP-PTS1 was expressed in various tissues and imaged in fixed samples by confocal microscopy. Peroxisomes are present in all tissues examined including, (<b>A</b>) Fat body (<i>lsp</i>><i>eYFP-PTS1</i>), (<b>B</b>) Gut (<i>act5c</i>><i>eYFP-PTS1</i>), (<b>C</b>) Oenocytes (<i>BO</i>><i>eYFP-PTS1</i>), (<b>D</b>) Muscles (<i>24B</i>><i>eYFP-PTS1</i>), and (<b>E</b>) Epidermal cells (<i>act5c</i>><i>eYFP-PTS1</i>). (<b>F</b>) Embryos expressing PMP34-Cerulean were imaged by epifluorescence microscopy. Scale bar = 20 µm.</p

Peroxisome loss causes muscle defects.

<p><i>pex3</i> knockdown in wandering third instar larval body wall muscles (<b>B</b>, <i>Mef2</i>><i>eYFP-PTS1</i>, <i>pex3.IR</i>) leads to a reduction in muscle peroxisome numbers compared to controls (<b>A</b>, <i>Mef2</i>><i>eYFP-PTS1</i>). (<b>C</b>) <i>pex3</i> knockdown in muscles (<i>Mef2</i>><i>pex3.IR</i>,<i>dcr</i>) causes some flies to die as fully formed, pharate adults trapped in the pupal case. (<b>D</b>) Viability of <i>Mef2</i>><i>pex3.IR</i>,<i>dcr</i> flies is reduced to 34.1±1%. (<b>E</b>) Climb tests of adult flies with reduced pex3 in muscles shows that <i>Mef2</i>><i>pex3.IR</i>,<i>dcr</i> flies display impaired locomotor ability, as measured by the time required to climb 5 cm (11.2±0.8 sec) compared to controls (3.3±0.2 sec). Some <i>Mef2</i>><i>pex3.IR</i>,<i>dcr</i> flies (<b>G</b>) have “crumpled” wings compared to <i>Mef2</i>><i>dcr</i> control flies (<b>F</b>), possibly due to impaired muscle function required for wing expansion. (<b>H</b>) Of the flies that successfully eclose, 88.4±2.7% have crumpled wings. Scale bar = 20 µm.</p

RNAi knockdown of pex3 reduces peroxisome numbers and impairs viability.

<p>(<b>A</b>) A schematic of the pex3 genomic region shows the area targeted by the inverted repeat. (<b>B</b>) Immunoblot of total protein extracts from wandering third instar larvae shows that Pex3 protein levels are reduced when <i>UAS-pex3.IR</i> is driven by global GAL4 drivers. The weak, global driver armadillo (<i>arm</i>) driving <i>UAS-pex3.IR</i> does not detectably reduce Pex3 levels. The medium strength global driver, daughterless (<i>Da^G32</i>), driving <i>UAS-pex3.IR</i> reduces Pex3 levels at 18°C and room temperature. The strong global driver, actin 5c (<i>act5c</i>), driving <i>UAS-pex3.IR</i> reduces the level of Pex3 even further. (<b>C</b>) Adult viability of flies expressing pex3.IR driven by global GAL4 drivers was measured. Weak knockdown (<i>arm</i>><i>pex3.IR</i>) did not appreciably decrease viability (95.3±1.0%; n = 190). At 18°C, viability of <i>Da^G32</i>><i>pex3.IR</i> flies was reduced to 25.2±1.5% (n = 330). At room temperature, viability of <i>Da^G32</i>><i>pex3.IR</i> flies fell to 1±0.5% (n = 444). At both temperatures, some flies died as pupae or pharate adults and remained trapped in the pupal case. Viability of act5c><i>pex3.IR</i> flies was also reduced (11.9±6.5%; n = 106). (<b>D</b>) Microscopy of wandering third instar larval epidermal cells shows that peroxisome numbers are severely reduced when UAS-pex3.IR is driven by the act5c GAL4 driver compared to controls lacking the RNAi transgene (<b>E</b>).</p

Peroxisome loss causes pupal lethality and eclosion defects.

<p>Peroxisome loss causes pupal lethality and eclosion defects.</p

Medication Exposure in Highly Adherent Psychiatry Patients

Medication exposure is dependent upon many factors, the single most important being if the patient took the prescribed medication as indicated. To assess medication exposure for psychotropic and other medication classes, we enrolled 115 highly adherent psychiatry patients prescribed five or more medications. In these patients, we measured 21 psychotropic and 38 nonpsychotropic medications comprising a 59 medication multiplex assay panel. Strict enrollment criteria and reconciliation of the electronic health record medication list prior to study initiation produced a patient cohort that was adherent with 91% of their prescribed medications as determined by comparing medications detected empirically in blood to the electronic health record medication list. In addition, 13% of detected medications were not in the electronic health record medication list. We found that only 53% of detected medications were within the literature-derived reference range with 41% below and 6% above the reference range specific to each medication. When psychotropic medications were analyzed near trough-level, only sertraline was found to be within the literature-derived reference range for all patients tested. Concentrations of the remaining medications indicated extensive exposure below the reference range. This is the first study to empirically and comprehensively assess medication exposure obtained in comorbid polypharmacy patients, minimizing the important behavioral factor of adherence in the study of medication exposure. These data indicate that low medication exposure is extensive and must be considered when therapeutic issues arise, including the lack of response to medication therapy