What to Cut and How to Cut? Historical Lessons from Past Reductions in the Intelligence Community

Under what conditions can the Intelligence Community cut its resources while still maintaining their effectiveness? What do past eras of reduction suggest about what to cut and how best to cut? The United States Government faces pressure to reduce its overall expenditures. This will likely spur calls to reduce the country’s foreign policy and defense budget. Under the current fiscal environment, these cuts will surely include the Intelligence Community, which grew rapidly over the last ten year’s. Prudent policymakers, therefore, should start thinking now about what they can cut and how they should implement these changes while still carrying out their missions effectively. This project will help the Intelligence Community think about these problems by examining the lessons learned from previous eras of reduction. The project will produce an article-length report as well as a briefing. Team members will brief the sponsor in their Washington, DC office.Rand Corporation's Intelligence Policy Cente

The exported chaperone Hsp70-x supports virulence functions for <i>Plasmodium falciparum</i> blood stage parasites

<div><p>Malaria is caused by five different <i>Plasmodium</i> spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, <i>P</i>. <i>falciparum</i> and <i>P</i>. <i>vivax</i>, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of <i>P</i>. <i>falciparum</i> in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as <i>Pf</i>EMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in <i>P</i>. <i>falciparum</i> but not <i>P</i>. <i>vivax</i>. Although the growth of Δ<i>hsp70-x</i> parasites was unaffected, the export of <i>Pf</i>EMP1 cytoadherence proteins was delayed and <i>Δhsp70-x</i> IE had reduced adhesion. The Δ<i>hsp70-x</i> IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that <i>Pf</i>Hsp70-x is not essential for survival <i>in vitro</i>, but may be required for the efficient export and functioning of some <i>P</i>. <i>falciparum</i> exported proteins.</p></div

Transcriptional and translational changes in ΔHsp70-x indicate an up-regulation of some exported proteins.

<p>(a) Mass spectrometry based SILAC sequencing of proteins indicates several exported proteins are over- and under-expressed in ΔHsp70-x relative to CS2. ΔHsp70-x and CS2 were differentially labeled with heavy (H) and light (L) isotopic forms of isoleucine respectively. Proteins with statistically insignificant Z-score ratios (X-axis) versus—log10 (P-value) (Y-axis) and therefore in ~ 1:1 ratio represent the base of the volcano plot. Proteins at higher levels in ΔHsp70-x only and which satisfy both statistical parameters are labeled in red in the upper right. Those up-regulated proteins that only satisfy one parameter are labeled in yellow. Proteins down-regulated in ΔHsp70-x are similarly coloured and shown in the upper left. (b) Correlation plots of RNA-seq transcripts between wild type and ΔHsp70-x ring, trophozoite and schizont stages show close correlation in most transcripts (close to line x = y). Most differential genes are downregulated in ΔHsp70-x trophozoites and schizonts (points furthest from line). (c) MA plot representing RNA-seq data. The change in expression (log 2 fold change) is plotted against the average of the normalized count values (base mean), of ΔHsp70-x and CS2 transcripts at the trophozoite stage. Red dots indicate differentially expressed genes in ΔHsp70-x. (d) Summary of increased proteins and transcripts by SILAC and RNA-seq. Numbers indicate heavy/light isoleucine ratio (H/L) in SILAC ordered by the ratio, or the log2 fold change in transcripts (LogFC) in RNA-seq data, ordered by significance which takes into account number of transcripts as well. Exported proteins are denoted as PEXEL or no PEXEL (PNEP) or otherwise left blank. Known homology to protein families or potential function identified in final column, including annotated pseudogenes. Pseudogenes may produce a limited numbers of transcripts so small changes can look significant.</p

ΔHsp70-x parasites generally resist stress as well as CS2.

<p>(a) Growth of ΔHsp70-x and CS2 parasites for 1.5 cell cycles after heat shock treatment of ring stage parasites indicated that growth of the mutant ΔHsp70-x and CS2 were similarly reduced. The ring-stage parasites were heat shocked at indicated temperatures for six hours and the percentage growth relative to parasite growth at 37°C is shown. The parasite lactate dehydrogenase (LDH) assay was used to measured growth and was performed twice in triplicate. (b) Percentage of blood stage parasite growth in 5% CO₂ in normal air (20% O₂) as a proxy for oxidative stress, relative to non-stressed cultures in 5% CO₂, 1% O₂. Both CS2 and ΔHsp70-x have reduced growth in oxidative stress conditions at 48 h with ΔHsp70-x not recovering as well as some of the CS2 replicates at 96 h. Growth was measured by LDH assay and performed in triplicate, showing the mean with 95% CI * p = 0.02. (c) Growth in media with reduced levels of isoleucine as measured by LDH assay at 0, 48 and 72 h after start of growth period, <i>n</i> = 2. For most isoleucine concentrations there was no difference between the ΔHsp70-x and CS2 parasites. (d) With hypoxanthine reduced to 1% (0.4 μM) and 0.5% (0.2 μM) of standard RPMI culture conditions, the growth of ΔHsp70-x was slightly reduced compared to CS2 as indicated by the dashed line at 100% (<i>n</i> = 3). Growth was measured by LDH activity after two cycles and was performed with two clones of ΔHsp70-x whose data were combined because they were not significantly different to each other. T-test comparing ΔHsp70-x growth to CS2 p = *** > 0.0001, ** 0.0008. (e) Growth of parasite lines in different aged erythrocytes as measured by LDH activity after 2.5 cell cycles indicates both parasite lines grew less well in older erythrocytes. The erythrocytes were stored at 4°C for the indicated number of days prior to start of the assay. Parasite LDH was measured five days after the start of the assay and is reported as % growth compared to parasites grown in 3 day old blood, <i>n</i> = 6. (f) Data from (e) showing the growth of ΔHsp70-x clones compared to control CS2 parasites normalised to 100% for each erythrocyte age group indicates no difference.</p

Deletion of <i>hsp70-x</i> does not affect parasite growth in the blood stage cell cycle.

<p>(a) Measurement of the fold increase cell proliferation per blood stage cycle of ΔHsp70-x and CS2 parasites as assessed by parasite lactate dehydrogenase (LDH) activity indicates the growth of ΔHsp70-x parasites was not significantly (n.s.) slower than CS2. Mann-Whitney p = 0.7, <i>n</i> = 36. (b) Light microscopy counts of the mean number of merozoites per schizont in ΔHsp70-x and CS2 parasite lines indicated there was no significant difference. (c) Light microscopy images of four hour synchronized Giemsa stained parasites indicating both lines were morphologically similar. (d) Measurement of the DNA content of ethidium bromide (EtBR) stained ΔHsp70-x and CS2 IE by flow cytometry indicates similar DNA content in the majority of cells at each time point. Parasites were synchronized to a four-hour window and the red trace indicates ΔHsp70-x DNA content compared to CS2 in purple.</p

The expression of cytoskeletal binding proteins KAHRP is increased in ΔHsp70-x.

<p>(a) Immuno fluorescence microscopy of 16-24hpi parasites indicates expression of the exported, knob localized KAHRP protein but not RESA appears higher in ΔHsp70-x. n = number of cells counted. (b) Mean fluorescence intensity (MFI) measurements of the erythrocyte compartment confirmed the KAHRP signal is higher in ΔHsp70-x compared to CS2 (Mann Whitney test). (c) Western blot analysis of magnet binding (trophozoites) and unbound (ring stage) parasites indicates KAHRP but not RESA or SBP1 appears to be more highly expressed in two ΔHsp70-x clonal lines. Whole purified trophozoites and saponin treated ring stage parasites were analyzed. (d) Densitometry of KAHRP levels from three blots normalized to Hsp70-1 and EXP2 loading controls, indicated that although KAHRP expression was always higher in ΔHsp70-x than CS2 this was not significant (p = 0.1, pairwise comparison, Mann Whitney test).</p