Multimodality treatment of locally advanced soft-tissue sarcomas of the extremities

Background

Adjuvant radiotherapy (RTE) still has a fundamental role as a post-operative treatment of locally advanced soft tissues sarcomas of the extremities. Moreover the employment of combined modalities in locally advanced soft tissues sarcomas of the extremities allow to maximize the chance of local cure even in difficult presentation cases, and possibly improve survival, especially in high-risk disease patients. In patients with sarcomas of the extremities in which definitive surgery has not been radical (with positive or “close” margins) radiotherapy can improve the results in terms of Disease Free Survival (DFS) and, together with chemotherapy, of Overall Survival (OS). We recommend radiotherapy in case of deep tumor location, inadequate surgical margins and grade 3 tumour; for positive or “marginal (or close)” excision (that means inadequate surgery) or in selected patients with a bad prognosis, we believe that a multidisciplinary approach can be preferable.

Introduction

Adjuvant radiotherapy (RTE) still has a fundamental role as a post-operative treatment. In patients with sarcomas of the extremities in whom definitive surgery has been or not radical (positive or “close” margins), radiotherapy with chemotherapy can improve the results in terms of Disease Free Survival (DFS) and Overall Survival (OS), while RTE alone seems to improve local control.

Materials and methods

From 1/2000 to 12/2005 we treated 34 patients affected by locally advanced sarcomas of the upper or lower extremities with radiotherapy (doses ranging from 54 to 66 Gy) and chemotherapy in 18/34 with an adjuvant scheme that consisted in Epirubicine (120 mg/m²) plus Ifosfamide (7000–9000 mg/m²).

Results

Disease Free Survival (DFS) and the Overall Survival (OS) rates were 76% and 82%, respectively. Eighteen patients developed one or more long-term side effects. Most of these complications were mild: all patients experienced only erithema, edema, local sclerosis or moderate pain.

Conclusion

Radiotherapy has an important role as a post-operative treatment also when surgery was non-radical. It improves local control more in patients with high-grade sarcomas of the extremity with positive or close margins. It is still difficult to assess the role of adjuvant chemotherapy.     

Networks and dominance hierarchies: does interspecific aggression explain flower partitioning among stingless bees?

1. The distribution of consumers among resources (trophic interaction network) may be shaped by asymmetric competition. Dominance hierarchy models predict that asymmetric interference competition leads to a domination of high quality resources by hierarchically superior species. 2. In order to determine the competitive dominance hierarchy and its effect on flower partitioning in a local stingless bee community in Borneo, interspecific aggressions were tested among eight species in arena experiments. 3. All species tested were strongly mutually aggressive in the arena, and the observed interactions were often lethal for one or both opponents. Aggression significantly increased with body size differences between fighting pairs and was asymmetric: larger aggressors were superior over smaller species. Additional aggression tests involved dummies with surface extracts, and results suggest that species‐ and colony‐specific surface profiles are important in triggering the aggressive behaviour. 4. Sixteen stingless bee species were observed foraging on 41 species of flowering plants. The resulting bee–flower interaction network showed a high degree of generalisation (network‐level specialisation H₂’ = 0.11), corresponding to a random, opportunistic distribution of bee species among available flower species. 5. Aggressions on flowers were rare and only occurred at a low level. The dominance hierarchy obtained in the arena experiments did not correlate significantly with plant quality, estimated as the number of flowers per plant or as total bee visitation rate. 6. Our findings suggest that asymmetries in interference competition do not necessarily translate into actual resource partitioning in the context of complex interacting communities.     

Missed Opportunities: Barriers to HIV Testing during Pregnancy from a Population Based Cohort Study in Rural Uganda

The aim was to assess population-level HIV-testing uptake among pregnant women, key for access to prevention-of mother to child transmission (PMTCT) services, and to identify risk factors for not being HIV tested,The study was conducted May 2008–May 2010 in the Iganga/Mayuge Health and Demographic Surveillance Site (HDSS), Eastern Uganda, during regular surveillance of 68,000 individuals. All women identified to be pregnant May–July 2008 (n = 881) were interviewed about pregnancy-related issues and linked to the HDSS database for socio-demographic data. Women were followed-up via antenatal care (ANC) register reviews at the health facilities to collect data related to ANC services received, including HIV testing. Adjusted relative risk (aRR), and 95% confidence intervals (CI) for not being HIV tested were calculated using multivariable binomial regression among the 544 women who remained after record review.Despite high ANC attendance (96%), the coverage of HIV testing was 64%. Only 6% of pregnant women who sought ANC at a facility without HIV testing services were referred for testing and only 20% received counseling regarding HIV. At ANC facilities with HIV testing services, 85% were tested. Only 4% of the women tested had been couple tested for HIV. Living more than three kilometers away from a health facility with HIV testing services was associated with not being tested both among the poorest (aRR,CI; 1.44,1.02–2.04) and the least poor women (aRR,CI;1.72,1.12–2.63).The lack of onsite HIV testing services and distant ANC facilities lead to missed opportunities for PMTCT, especially for the poorest women. Referral systems for HIV testing need to be improved and testing should be expanded to lower level health facilities. This is in order to ensure that the policy of HIV testing during pregnancy is implemented more effectively and that testing is accessible for all.     

Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure

The pathological form of prion protein (PrP^Sc), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP^Sc extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90–231 (hPrP_90–231) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP^Sc. In the present study we demonstrate that hPrP_90–231, pre-incubated with 10 mM Ca⁺⁺ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP_90–231 bearing pathogenic mutations (D202N and E200K). We also report that Ca⁺⁺ binding to hPrP_90–231 induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP_90–231 cytotoxicity. Finally, by in silico structural analysis, we propose that Ca⁺⁺ binding to hPrP_90–231 modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.     

Rapid DNA fingerprinting of pathogens by flow cytometry

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.     

Flow cytometry-based minisequencing: a new platform for high-throughput single-nucleotide polymorphism scoring

Single-nucleotide polymorphisms (SNPs) are the most abundant type of human genetic variation. These variable sites are present at high density in the genome, making them powerful tools for mapping and diagnosing disease-related alleles. We have developed a sensitive and rapid flow cytometry-based assay for the multiplexed analysis of SNPs based on polymerase-mediated primer extension, or minisequencing, using microspheres as solid supports. The new method involves subnanomolar concentrations of sample in small volumes ( approximately 10 microl) which can be analyzed at rates of one sample per minute or faster, without a wash step. Further, genomic analysis using multiplexing microsphere arrays (GAMMArrays), enables the simultaneous analysis of dozens, and potentially hundreds of SNPs per sample. We have tested the new method by genotyping the Glu69 variant from the HLA DPB1 locus, a SNP associated with chronic beryllium disease, as well as HLA DPA1 alleles using the multiplexed method. The results demonstrate the sensitivity and accuracy of flow cytometry-based minisequencing, a powerful new tool for genome- and global-scale SNP analysis.     

Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA ( approximately 450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.