Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction–high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S- -cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l⁻¹ and 25 to 5000 μg l⁻¹ for plasma and urine samples, respectively, a limit of detection of 0.17 μg l⁻¹, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.     

Region‐specific effects of oestradiol on adipose‐derived stem cell differentiation in post‐menopausal women

The goal of this study was to determine the effect of acute transdermal 17β‐oestradiol (E₂) on the adipogenic potential of subcutaneous adipose‐derived stem cells (ASC) in post‐menopausal women. Post‐menopausal women (n = 11; mean age 57 ± 4.5 years) were treated for 2 weeks, in a randomized, cross‐over design, with transdermal E₂ (0.15 mg) or placebo patches. Biopsies of abdominal (AB) and femoral (FEM) subcutaneous adipose tissue (SAT) were obtained after each treatment and mature adipocytes were analysed for cell size and ASC for their capacity for proliferation (growth rate), differentiation (triglyceride accumulation) and susceptibility to tumour necrosis factor alpha‐induced apoptosis. Gene expression of oestrogen receptors α and β (ESR1 and ESR2), perilipin 1 and hormone‐sensitive lipase (HSL), was also assessed. In FEM SAT, but not AB SAT, 2 weeks of E₂ significantly (P = 0.03) increased ASC differentiation and whole SAT HSL mRNA expression (P = 0.03) compared to placebo. These changes were not associated with mRNA expression of oestrogen receptors α and β, but HSL expression was significantly increased in FEM SAT with transdermal E₂ treatment. Adipose‐derived stem cells proliferation and apoptosis did not change in either SAT depot after E₂ compared with placebo. Short‐term E₂ appeared to increase the adipogenic potential of FEM, but not AB, SAT in post‐menopausal women with possible implications for metabolic disease. Future studies are needed to determine longer term impact of E₂ on regional SAT accumulation in the context of positive energy imbalance.     

The Severity of Pandemic H1N1 Influenza in the United States,from April to July 2009: A Bayesian Analysis

Background

Accurate measures of the severity of pandemic (H1N1) 2009 influenza (pH1N1) are needed to assess the likely impact of an anticipated resurgence in the autumn in the Northern Hemisphere. Severity has been difficult to measure because jurisdictions with large numbers of deaths and other severe outcomes have had too many cases to assess the total number with confidence. Also, detection of severe cases may be more likely, resulting in overestimation of the severity of an average case. We sought to estimate the probabilities that symptomatic infection would lead to hospitalization, ICU admission, and death by combining data from multiple sources.

Methods and Findings

We used complementary data from two US cities: Milwaukee attempted to identify cases of medically attended infection whether or not they required hospitalization, while New York City focused on the identification of hospitalizations, intensive care admission or mechanical ventilation (hereafter, ICU), and deaths. New York data were used to estimate numerators for ICU and death, and two sources of data—medically attended cases in Milwaukee or self-reported influenza-like illness (ILI) in New York—were used to estimate ratios of symptomatic cases to hospitalizations. Combining these data with estimates of the fraction detected for each level of severity, we estimated the proportion of symptomatic patients who died (symptomatic case-fatality ratio, sCFR), required ICU (sCIR), and required hospitalization (sCHR), overall and by age category. Evidence, prior information, and associated uncertainty were analyzed in a Bayesian evidence synthesis framework. Using medically attended cases and estimates of the proportion of symptomatic cases medically attended, we estimated an sCFR of 0.048% (95% credible interval [CI] 0.026%–0.096%), sCIR of 0.239% (0.134%–0.458%), and sCHR of 1.44% (0.83%–2.64%). Using self-reported ILI, we obtained estimates approximately 7–9× lower. sCFR and sCIR appear to be highest in persons aged 18 y and older, and lowest in children aged 5–17 y. sCHR appears to be lowest in persons aged 5–17; our data were too sparse to allow us to determine the group in which it was the highest.

Conclusions

These estimates suggest that an autumn–winter pandemic wave of pH1N1 with comparable severity per case could lead to a number of deaths in the range from considerably below that associated with seasonal influenza to slightly higher, but with the greatest impact in children aged 0–4 and adults 18–64. These estimates of impact depend on assumptions about total incidence of infection and would be larger if incidence of symptomatic infection were higher or shifted toward adults, if viral virulence increased, or if suboptimal treatment resulted from stress on the health care system; numbers would decrease if the total proportion of the population symptomatically infected were lower than assumed. Please see later in the article for the Editors'' Summary     

Role of the stable signal peptide of Junín arenavirus envelope glycoprotein in pH-dependent membrane fusion

The envelope glycoprotein of the arenaviruses (GP-C) is unusual in that the mature complex retains the cleaved, 58-amino-acid signal peptide. Association of this stable signal peptide (SSP) has been shown to be essential for intracellular trafficking and proteolytic maturation of the GP-C complex. We identify here a specific and previously unrecognized role of SSP in pH-dependent membrane fusion. Amino acid substitutions that alter the positive charge at lysine K33 in SSP affect the ability of GP-C to mediate cell-cell fusion and the threshold pH at which membrane fusion is triggered. Based on the presumed location of K33 at or near the luminal domain of SSP, we postulate that SSP interacts with the membrane-proximal or transmembrane regions of the G2 fusion protein. This unique organization of the GP-C complex may suggest novel strategies for intervention in arenavirus infection.