Enhancing Employment Opportunities for Mature Workers Through Training: Case Studies of Employment Services in Massachusetts

Extension of working years among those approaching “normal” retirement ages is receiving increased attention. Much of the impetus is financial. The weakening of private pension systems is leaving increasing numbers of those approaching retirement with inadequate savings. Rising health care costs and the erosion of retiree health benefits also encourages older workers to remain in the workforce. The current recession has greatly escalated the financial concerns of those who are late in their working lives. With the deterioration of financial markets, the values of 401K portfolios have declined enormously. Further, the decline in home prices has left many with substantial reductions in home equity. In addition, some mature workers are among those with home mortgages that exceed the market values of their homes

Analysis of Retirement Risk within Massachusetts State Agencies and Use of Post-Retirement Contracting as a Workforce Planning Tool

The public sector is at risk for skills shortages resulting from the retirement of older workers, mainly Boomers, who will be exiting the labor force over the next 5 to 10 years. In Massachusetts, state agencies are recognizing the potential risk to operations resulting from these impending retirements, and agency leaders are beginning to question what can be done to retain older workers, particularly those in leadership, technical or specialized job titles. Incentivizing older workers to continue working beyond the normal retirement age is difficult within the Massachusetts Public Employee Pension System, which is designed to encourage workers to retire after reaching the maximum pension benefit. Agencies have one policy tool at their disposal to mitigate knowledge or skill loss from retirements; a mechanism within the pension laws that allows retirees to return to work as a contract employee. This policy has important implications for state agencies interested in retaining skilled employees, as well as retirees who need to maximize their post-retirement income or wish to continue to work for other reasons. This dissertation will utilize multiple methods and analyses to 1) determine the retirement risk faced by agencies in the Health and Human Services Secretariat, 2) measure the historical utilization of post-retirement contracting, 3) predict future use of such contracting, and 4) analyze the effectiveness and efficiency of the post retirement contracting policy versus other policy options for retaining experienced staff

Phospholipase A2 activity during the replication cycle of the flavivirus West Nile virus

<div><p>Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNV_KUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNV_KUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNV_KUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNV_KUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNV_KUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.</p></div

Evaluation of the effect of fatty acid synthase and PLA2 activity inhibitors on WNV_KUN replication.

<p>Vero cells were pre-treated with the chemical inhibitors and subsequently infected for an additional 24 hrs. <b>(A and B)</b> Production of viral protein was determined by western blotting with mouse monoclonal antibodies against the WNV_KUN envelope (Env) and NS5 proteins and normalized to cellular proteins GRP78 and actin. Quantitation is depicted in panel B. <b>(C)</b> Production of genomic viral RNA was determined by qRT-PCR after treatment and infection with inhibitors and virus at 24 h.p.i. <b>(D)</b> Production of infectious secreted virus was determined by plaque assay after treatment and infection with inhibitors and virus at 24 h.p.i. <b>(E)</b> Immunofluorescence analysis of WNV_KUN-infected Vero cells at 24 h.p.i. following drug treatment. Cells were stained with mouse monoclonal anti-NS1 antibodies and viewed on a Zeiss confocal microscope. In all cases statistical analysis was performed on duplicate analysis of triplicate experiments via Students <i>t-</i>test on GraphPad Prism 6. C75 (final concentration of 30μM), ACA = n-(p-amylcinnamoyl) anthranilic acid (final concentration of 20μM) and PACOCF3 = palmityl trifluromethyl ketone (final concentration of 10μM). ACA and PACOCF3 were used at a final concentration of 15μM each when treated together.</p

PLA2 enzyme inhibition induces an altered WNV_KUN replication complex morphology.

<p>Vero cells were pretreated with the vehicle solvent DMSO <b>(A and B)</b>, or inhibitors C75 (30μM) (<b>C and D</b>), ACA (20μM) <b>(E and F)</b>, PACOCF3 (15μM) <b>(G and H)</b> or ACA+PACOCF3 (15μM each) <b>(I and J)</b> and subsequently infected with WNV_KUN for an additional 24 hrs before analysis by transmission electron microscopy on a Technai F30. Arrows indicate elongated vesicles and all magnification bars represent 200nm. <b>(K)</b> Vesicle length was measured on images gathered in Adobe Photoshop CS6 and quantitation performed in GraphPad Prism 6, statistical significance was determined by Student’s <i>t-</i>test on the number of vesicles visualized in duplicate experiments.</p

Host enzyme PLA2 are activated in WNV_KUN-infected cells, and exogenous addition of lyso-PChol lipid can rescue chemical PLA2 inhibition.

<p><b>(A)</b> Summary of the homeostatic change in phospholipids as quantified by LC/MS revealing a correlation of present PChol and lyso-PChol lipid species. <b>(B)</b> Cellular PLA2 activity level in mock- and WNV_KUN-infected cells from various cell lineages showing increased PLA2 activity during viral infection for all cell types (n = 3 independent experiments). <b>(C)</b> Visualisation of exogenous administered fluorophore-tagged lyso-PChol lipid moieties (lyso-PChol488; 5μM), co-stained with antibodies recognising viral NS3 protein. Bar = 2μm (images i-ii, vi-vii) and bar = 1μm for inset images (iii-v, xiii-x). Arrows indicate colocalisation. <b>(D)</b> Visualisation of lyso-PChol488 (5μM) co-stained with antibodies recognising replication intermediates (dsRNA). Bar = 5μm (images xii-xii, xvi-xvii) and bar = 2μm for inset images (xiii-xv, xviii-xx). Arrows indicate colocalisation. For rescue experiments, vero cells were infected with WNV_KUN for an hour and ACA (20μM) drug-treated until 8 h.p.i. Exogenous lyso-PChol (1.5μM) was subsequently added and cells subject to analysis at 24 h.p.i. <b>(E)</b> Restored production of secreted infectious virus particles following the addition of lyso-PChol as determined by plaque assay (n = 3 independent experiments). <b>(F and G)</b> Western blot analysis and quantification of viral protein levels during PLA2 enzyme inhibition and lyso-PChol addition. Envelope (Env) and NS5 proteins were normalized to the cellular protein Calnexin (n = 3 independent experiments).</p

Expression and activation of specific PLA2 isoforms during viral replication.

<p><b>(A)</b> Schematic representation of the 20 known PLA2 genes/isoforms, and their subcellular localisation, that are involved in the hydrolysis of PChol to lyso-PChol. (based on databases: KEGG, GeneCards, Reactome, Human Protein Atlas). <b>(B and C)</b> Western blot analysis and quantification of the phosphorylation status of PLA2G4A (p-PLA2G4A) during N-Ethylmaleimide (NME; 100μM for 30mins) control-treated and WNV_KUN-infected cells (n = 3 independent experiments). <b>(D and E)</b> Confocal microscopy and quantification of enzyme translocation and phosphorylation of PLA2G4A and in NME control-treated and WNV_KUN-infected cells. Images i-iii are based on antibodies recognizing total PLA2G4A protein, whereas images iv-ix are based on antibodies recognizing phosphorylated (activated) PLA2G4A (n = 4 independent experiments). Bar = 20um. Arrowheads indicate enzyme translocation towards the plasma membrane. <b>(F)</b> Confocal microscopy showing the partial sequestering of PLA2G4A protein populations to sites of viral protein expression (NS3 protein). Bar = 20μm for images x-xi and bar = 5μm for images xii-xiv. <b>(G to K)</b> The effect of targeted siRNA-mediated gene silencing of PLA2G4A or PLA2G4C-encoded enzymes on WNV replication. <b>(G)</b> Representative Western Blots and <b>(H)</b> quantification of viral protein levels. <b>(I)</b> Virus production and <b>(J)</b> viral RNA genomes. <b>(K)</b> PLA2G4A and PLA2G4C protein expression levels (n = 3 independent experiments).</p

LC/MS analysis of WNV_KUN-infected cells reveals a homeostatic change in phospholipid levels.

<p>Vero cells were mock- or WNV_KUN-infected and harvested at 24 h.p.i. Levels of phospholipids were quantitated by LC/MS and the fold change plotted on GraphPad Prism 6. The analysis was performed on triplicate samples from triplicate experiments. <b>(A)</b> Lyso-phosphatidylcholine (Lyso-PChol), <b>(B)</b> Phosphatidylcholine (PChol), <b>(C)</b> Phosphatidylethanolamine (PE), <b>(D)</b> Phosphatidylserine (PS), <b>(E)</b> Phosphatidylinositol (PI). <b>(F)</b> Summary of significant changes in individual phospholipid species, and <b>(G)</b> Summary of fold change per phospholipid class.</p