Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

Detection heterogeneity in underwater visual‐census data

This study shows how capture–mark–recapture (CMR) models can provide robust estimates of detection heterogeneity (sources of bias) in underwater visual‐census data. Detection biases among observers and fish family groups were consistent between fished and unfished reef sites in Kenya, even when the overall level of detection declined between locations. Species characteristics were the greatest source of detection heterogeneity and large, highly mobile species were found to have lower probabilities of detection than smaller, site‐attached species. Fish family and functional‐group detectability were also found to be lower at fished locations, probably due to differences in local abundance. Because robust CMR models deal explicitly with sampling where not all species are detected, their use is encouraged for studies addressing reef‐fish community dynamics.     

Rapid Pharmacokinetic and Biodistribution Studies Using Cholorotoxin-Conjugated Iron Oxide Nanoparticles: A Novel Non-Radioactive Method

Background

Recent advances in nanotechnology have led to the development of biocompatible nanoparticles for in vivo molecular imaging and targeted therapy. Many nanoparticles have undesirable tissue distribution or unacceptably low serum half-lives. Pharmacokinetic (PK) and biodistribution studies can help inform decisions determining particle size, coatings, or other features early in nanoparticle development. Unfortunately, these studies are rarely done in a timely fashion because many nanotechnology labs lack the resources and expertise to synthesize radioactive nanoparticles and evaluate them in mice.

Methodology/Principal Findings

To address this problem, we developed an economical, radioactivity-free method for assessing serum half-life and tissue distribution of nanoparticles in mice. Iron oxide nanoparticles coated with chitosan and polyethylene glycol that utilize chlorotoxin as a targeting molecule have a serum half-life of 7–8 hours and the particles remain stable for extended periods of time in physiologic fluids and in vivo. Nanoparticles preferentially distribute to spleen and liver, presumably due to reticuloendothelial uptake. Other organs have very low levels of nanoparticles, which is ideal for imaging most cancers in the future. No acute toxicity was attributed to the nanoparticles.

Conclusions/Significance

We report here a simple near-infrared fluorescence based methodology to assess PK properties of nanoparticles in order to integrate pharmacokinetic data into early nanoparticle design and synthesis. The nanoparticles tested demonstrate properties that are excellent for future clinical imaging strategies and potentially suitable for targeted therapy.     

The position of localized soil compaction determines root and subsequent shoot growth responses

Plants growing in soils typically experience a mixture of loose and compact soil. The hypothesis that the proportion of a root system exposed to compact soil and/or the timing at which this exposure occurs determines shoot growth responses was tested. Broccoli (Brassica oleracea var. italica cv. Greenbelt) seedlings were grown in pot experiments with compact, loose and localized soil compaction created by either horizontal (compact subsoils 75 or 150 mm below loose topsoil) or vertical (adjacent compact and loose columns of soil) configurations of loose (1.2 Mg m(-3)) and compact (1.8 Mg m(-3)) soil. Entirely compact soil reduced leaf area by up to 54%, relative to loose soil. When compaction was localized, only the vertical columns of compact and loose soil reduced leaf area (by 30%). Neither the proportion of roots in compact soil nor the timing of exposure could explain the differing shoot growth responses to localized soil compaction. Instead, the strong relationship between total root length and leaf area (r(2)=0.92) indicated that localized soil compaction reduced shoot growth only when it suppressed total root length. This occurred when isolated root axes of the same plant were exposed to vertical columns of compact and loose soil. When a single root axis grew through loose soil into either a shallow or deep compact subsoil, compensatory root growth in the loose soil maintained total root length and thus shoot growth was unaffected. These contrasting root systems responses to localized soil compaction may explain the variable shoot growth responses observed under heterogeneous conditions.     

The effects of dietary (n-3) fatty acid supplementation on lipid dynamics and composition in rat lymphocytes and liver microsomes

Rats were fed diets devoid of (n-3) fatty acids (olive oil supplementation) or high in (n-3) fatty acids (fish oil supplementation) for a period of 10 days. In spleen lymphocytes and liver microsomes derived from animals fed fish oil diets, relatively high levels of (n-3) eicosapentaenoic (20:5), docosapentaenoic (22:5) and docosahexaenoic acids (22:6) were obtained compared to minimal levels when fed the olive oil diet. When the average lipid motional properties were examined by measuring the fluorescence anisotropy of diphenylhexatriene, no significant different was found between intact liver microsomes from animals fed the two diets. However, when lipid motion was examined in vesicles of phosphatidylcholine, isolated from the microsomes from fish oil fed animals (21.4% (n-3) fatty acids), the fluorescence anisotropy was significantly less than the corresponding phosphatidylcholine from olive oil fed animals (5.6% (n-3) fatty acids), indicating a more disordered or fluid bilayer in the presence of higher levels of (n-3) fatty acids. Phosphatidylethanolamine (n-3) fatty acids were also elevated after fish oil supplementation (41.3% of total fatty acids), compared to the level after olive oil supplementation (21.4%). The major effect of the fish oil supplementation was a replacement of (n-6) arachidonic acid by the (n-3) fatty acids and when this was 'modeled', using liposomes of synthetic lipids, 1-palmitoyl-2-arachidonyl(n-6) or docosahexaenoyl(n-3)-phosphatidylcholine, significant differences in lipid motional properties were found, with the docosahexaenoate conferring a more disordered or fluid lipid environment. Thus it appears that although lipid order/fluidity can be significantly decreased by increases in the highly unsaturated (n-3) fatty acid levels, alterations in membrane domain organization and/or phospholipid molecular species composition effectively compensated for the changes, at least as far as average lipid motional properties in the intact membranes was concerned.     

Genome-wide allelotyping indicates increased loss of heterozygosity on 9p and 14q in early age of onset colorectal cancer.

Colorectal cancer remains a significant public health challenge, despite our increased understanding of the genetic mechanisms involved in the initiation and progression of this disorder. It has become clear that multiple mechanisms lead to the tumorigenic phenotype, with familial predisposition syndromes accounting for less than 15% of all colorectal cancers. A genome-wide scan for loss of heterozygosity (LOH) was carried out with 150 highly polymorphic markers in an effort to identify additional loci involved in colorectal tumorigenesis in DNA samples from 42 colorectal cancer patients. The results confirm earlier observations that tumor DNAs from patients with hereditary nonpolyposis colon cancer (HNPCC) either maintain heterozygosity or exhibit altered or additional alleles. DNAs from patients with early onset colorectal carcinomas (diagnosed prior to age 50) revealed a higher overall degree of LOH than DNAs from patients with sporadic colorectal cancers diagnosed later in life (after age 50). While regions on 1p, 10q and 14q are suggestive, statistical analysis of LOH at these regions failed to reach significance. However, LOH at 9p did reveal a statistically significant increase in the early onset patient group, compared to the greater than age 50 group. LOH on 9p may involve inactivation of p16/CDKN2 through aberrant DNA methylation on the remaining chromosome, resulting in a situation analogous to a homozygous deletion of p16 and providing a selective growth advantage to these cells. This marker may prove to be a useful prognostic indicator for patient stratification in the design of therapy for early onset colorectal cancer patients.     

Cost-Effectiveness of a Specialist Geriatric Medical Intervention for Frail Older People Discharged from Acute Medical Units: Economic Evaluation in a Two-Centre Randomised Controlled Trial (AMIGOS)

BackgroundPoor outcomes and high resource-use are observed for frail older people discharged from acute medical units. A specialist geriatric medical intervention, to facilitate Comprehensive Geriatric Assessment, was developed to reduce the incidence of adverse outcomes and associated high resource-use in this group in the post-discharge period.ObjectiveTo examine the costs and cost-effectiveness of a specialist geriatric medical intervention for frail older people in the 90 days following discharge from an acute medical unit, compared with standard care.MethodsEconomic evaluation was conducted alongside a two-centre randomised controlled trial (AMIGOS). 433 patients (aged 70 or over) at risk of future health problems, discharged from acute medical units within 72 hours of attending hospital, were recruited in two general hospitals in Nottingham and Leicester, UK. Participants were randomised to the intervention, comprising geriatrician assessment in acute units and further specialist management, or to control where patients received no additional intervention over and above standard care. Primary outcome was incremental cost per quality adjusted life year (QALY) gained.ResultsWe undertook cost-effectiveness analysis for 417 patients (intervention: 205). The difference in mean adjusted QALYs gained between groups at 3 months was -0.001 (95% confidence interval [CI]: -0.009, 0.007). Total adjusted secondary and social care costs, including direct costs of the intervention, at 3 months were £4412 (€5624, $6878) and £4110 (€5239, $6408) for the intervention and standard care groups, the incremental cost was £302 (95% CI: 193, 410) [€385, $471]. The intervention was dominated by standard care with probability of 62%, and with 0% probability of cost-effectiveness (at £20,000/QALY threshold).ConclusionsThe specialist geriatric medical intervention for frail older people discharged from acute medical unit was not cost-effective. Further research on designing effective and cost-effective specialist service for frail older people discharged from acute medical units is needed.

Trial Registration

ISRCTN registry ISRCTN21800480 http://www.isrctn.com/ISRCTN21800480     

Paleopediatrics: Or when did human infants really become human?

Modern human children take about twice as long as their closest biological relative, the chimpanzee, to mature. One standard explanation for the evolution of “delayed maturation” at an early stage of human evolution is that it provided the time necessary for immature individuals to learn complex skills, most notably those relating to tool-making abilities. However, after comparing dental maturational profiles of early hominids from South Africa (who apparently did make and use stone tools) (Susman [1994] Science 265:1570–1573) to those of extant humans and chimpanzees, we find no evidence to document an association between “delayed maturation” and tool-making abilities in the early stages of human evolution. This also suggests that the assumed association between prolonged childhood dependency and other behaviors often associated with the advent of tool-making such as cooperative hunting, food sharing, home bases, sexual division of labor, etc., is also suspect. Instead, we must look for other, or additional, selective pressures for the evolution of “delayed maturation,” which may postdate the australopithecine radiation. © 1995 Wiley-Liss, Inc.     

HTLV-III env gene products synthesized in E. coli are recognized by antibodies present in the sera of AIDS patients

The envelope gene of HTLV-III, the retrovirus directly linked to AIDS, encodes a protein of 856 amino acids. Our sequence analysis of the cloned HTLV-III (HXB-3) env gene and its comparison with other isolates reveal significant divergence, especially in the external portion of this protein. A large segment of the env gene (1800 bp) was inserted into the expression vector pEV-vrf3, and a corresponding 68 kd protein, which encompasses both the extracellular and the membrane-associated regions of the native protein, was produced in E. coli. Several smaller polypeptides, which appear to be internal initiation products, were also produced. All 50 AIDS patient sera obtained from different locations in the United States specifically recognized the bacterially synthesized envelope proteins, as judged by Western blots. This suggests that these proteins will be useful for the diagnosis of HTLV-III infection and possibly as a vaccine against AIDS.     

Programmatic Cost Evaluation of Nontargeted Opt-Out Rapid HIV Screening in the Emergency Department

Background

The Centers for Disease Control and Prevention recommends nontargeted opt-out HIV screening in healthcare settings. Cost effectiveness is critical when considering potential screening methods. Our goal was to compare programmatic costs of nontargeted opt-out rapid HIV screening with physician-directed diagnostic rapid HIV testing in an urban emergency department (ED) as part of the Denver ED HIV Opt-Out Trial.

Methods

This was a prospective cohort study nested in a larger quasi-experiment. Over 16 months, nontargeted rapid HIV screening (intervention) and diagnostic rapid HIV testing (control) were alternated in 4-month time blocks. During the intervention phase, patients were offered HIV testing using an opt-out approach during registration; during the control phase, physicians used a diagnostic approach to offer HIV testing to patients. Each method was fully integrated into ED operations. Direct program costs were determined using the perspective of the ED. Time-motion methodology was used to estimate personnel activity costs. Costs per patient newly-diagnosed with HIV infection by intervention phase, and incremental cost effectiveness ratios were calculated.

Results

During the intervention phase, 28,043 eligible patients were included, 6,933 (25%) completed testing, and 15 (0.2%, 95% CI: 0.1%–0.4%) were newly-diagnosed with HIV infection. During the control phase, 29,925 eligible patients were included, 243 (0.8%) completed testing, and 4 (1.7%, 95% CI: 0.4%–4.2%) were newly-diagnosed with HIV infection. Total annualized costs for nontargeted screening were $148,997, whereas total annualized costs for diagnostic HIV testing were $31,355. The average costs per HIV diagnosis were $9,932 and $7,839, respectively. Nontargeted HIV screening identified 11 more HIV infections at an incremental cost of $10,693 per additional infection.

Conclusions

Compared to diagnostic testing, nontargeted HIV screening was more costly but identified more HIV infections. More effective and less costly testing strategies may be required to improve the identification of patients with undiagnosed HIV infection in the ED.