Trophic exchange in Pardosa hortensis (Lycosidae, Araneae)

Young Pardosa hortensis not only need their mother's help to leave their cocoon once developed (about 20 days), as they are unable to open it by themselves, but also during the period they are carried around in the cocoon. Using radioactive markers, indirect evidence revealed that the mother periodically opens the cocoon to feed her young and reseals it with fresh silk. Water is probably the main component in trophic exchange.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

Sphingosine 1-phosphate inhibits cell migration in C2C12 myoblasts

This study shows that sphingosine 1-phosphate (S1P) exerts an anti-migratory action in C2C12 myoblasts by reducing directional cell motility and fully abrogating the chemotactic response to insulin-like growth factor-1. The anti-migratory response to S1P required ligation to S1P(2), being attenuated in myoblasts where the receptor was down-regulated by specific antisense oligodeoxyribonucleotides or small interfering RNA (siRNA) and conversely potentiated in S1P(2)-overexpressing myoblasts. The investigation of RhoA and Rac GTPases, critically implicated in cell motility regulation, demonstrated that RhoA was rapidly activated by S1P, while Rac1 was unaffected within the first 5 min but stimulated thereafter. RhoA, but not Rac activation, was identified as a S1P(2)-dependent pathway in experiments in which receptor expression was attenuated by siRNA treatment or up-regulated by S1P(2)-encoding plasmid transfection. Finally, by expression of the dominant negative mutant of RhoA, the GTPase was found implicated in the anti-migratory action of S1P, whereas modulation of Rac1 functionality unaffected the anti-chemotactic effect of S1P, ruling out a role for this protein in the biological response. Since S1P was previously shown to inhibit myoblast proliferation and stimulate myogenesis, the here identified novel biological activity is in favour of a complex physiological role of the sphingolipid in the process of muscle repair.     

Association of sugar-based carboranes with cationic liposomes: an electron spin resonance and light scattering study

The possibility of cationic (di-oleoyltrimethylammonium propane, DOTAP)/(l-α-dioleoylphosphatidyl-ethanolamine, DOPE) liposomes to act as carriers of boronated compounds such as 1,2-dicarba-closo-dodecaboran(12)-1-ylmethyl](β-d-galactopyranosyl)-(1→4)-β-d-glucopyranoside and 1,2-di-(β-d-gluco-pyranosyl-ox)methyl-1,2-dicarba-closo-dodeca-borane(12) has been investigated by Electron Spin Resonance (ESR) of n-doxyl stearic acids (n-DSA) and Quasi-Elastic Light Scattering (QELS). Both these carboranes have potential use in Boron Neutron Capture Therapy (BNCT), which is a targeted therapy for the treatment of radiation resistant tumors. They were shown to give aggregation both in plain water and in saline solution. Carborane aggregates were, however, disrupted when DOTAP/DOPE liposome solutions were used as dispersing agents. The computer analysis of the ESR spectra from carborane-loaded liposomes allowed to establish an increase of the order degree in the liposome bilayer with increasing carborane concentration, together with a decreased mobility. The same discontinuities of both correlation time and order parameter with respect to temperature variations were observed in carborane-containing and carborane-free liposomes. This suggested that a homogeneous dispersion of nitroxides and carboranes occurred in the liposome bilayer. The ESR line shape analysis proved that no dramatic changes were induced in the liposome environment by carborane insertion. QELS data showed that the overall liposome structure was preserved, with a slight decrease in the mean hydrodynamic radius and increase in polydispersity caused by the guest molecules.     

Nitroxides and malignant human tissues: electron spin resonance in colorectal neoplastic and healthy tissues

Healthy and neoplastic colorectal human tissues of as many as 12 patients have been studied, immediately after surgery, by electron spin resonance (ESR) of stable nitroxides at physiological temperature. Cells were maintained in a living state using the McCoy's 5A culture medium. The very low concentration changes of hydrophilic and lipophilic nitroxides allowed us to establish that the response to the oxidative stress induced by the occurrence of nitroxides in healthy and tumor cells was very weak, thus suggesting these compounds are good candidates for contrast enhancement agents in magnetic resonance imaging of colorectal tumor. The analysis of the computed ESR line shape of lipophilic nitroxides in both healthy and malignant cells of the same patient agreed for an unmodified physical status of the membranes where they were mainly localized. The results reported here proved that the comparison between ESR results must be made in tissues from the same patient and that the physical status of the membranes depended more on the patient history than on changes in the colorectal cell membrane fluidity induced by the neoplastic process.     

A role for calcium in sphingosine 1-phosphate-induced phospholipase D activity in C2C12 myoblasts

Receptor-regulated phospholipase D (PLD) is a key signaling pathway implicated in the control of fundamental biological processes. Here evidence is presented that in addition to protein kinase C (PKC) and Rho GTPases, Ca(2+) response evoked by sphingosine 1-phosphate (S1P) also participates to the enzyme regulation. Ca(2+) was found critical for PKC(alpha)-mediated PLD activation. Moreover, S1P-induced PLD activity resulted diminished by calmodulin inhibitors such as W-7 and CGS9343B implicating its involvement in the process. A plausible candidate for Ca(2+)-dependent PLD regulation by S1P was represented by calcineurin, in view of the observed reduction of the stimulatory effect by cyclosporin A. In contrast, monomeric GTP-binding protein Ral was translocated to membranes by S1P in a Ca(2+)-independent manner, ruling out its possible role in agonist-mediated regulation of PLD.     

Boronphenylalanine insertion in cationic liposomes for Boron Neutron Capture Therapy

Cationic liposomes are widely used as carriers of biomolecules specifically targeted to the cell nucleus. p-Boronphenylalanine (BPA) is a powerful anti-tumor agent for Boron Neutron Capture Therapy (BNCT). In this paper, (1)H and (13)C NMR was used to study the insertion of BPA in mixed liposomes, made up by the positively charged 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The boronated drug was distributed between the water phase and the liposomes. The location site of BPA into the lipid bilayer was investigated and the boron-substituted aromatic ring was found inserted in the hydrophobic region, whereas the amino acidic group was oriented towards the aqueous environment. Further information was given by proton spin-lattice relaxation rates.     

Association of sugar-based carboranes with cationic liposomes: an electron spin resonance and light scattering study

The possibility of cationic (di-oleoyltrimethylammonium propane, DOTAP)/(L-alpha-dioleoylphosphatidyl-ethanolamine, DOPE) liposomes to act as carriers of boronated compounds such as 1,2-dicarba-closo-dodecaboran(12)-1-ylmethyl](beta-D-galactopyranosyl)-(1-->4)-beta-D-glucopyranoside and 1,2-di-(beta-D-gluco-pyranosyl-ox)methyl-1,2-dicarba-closo-dodeca-borane(12) has been investigated by Electron Spin Resonance (ESR) of n-doxyl stearic acids (n-DSA) and Quasi-Elastic Light Scattering (QELS). Both these carboranes have potential use in Boron Neutron Capture Therapy (BNCT), which is a targeted therapy for the treatment of radiation resistant tumors. They were shown to give aggregation both in plain water and in saline solution. Carborane aggregates were, however, disrupted when DOTAP/DOPE liposome solutions were used as dispersing agents. The computer analysis of the ESR spectra from carborane-loaded liposomes allowed to establish an increase of the order degree in the liposome bilayer with increasing carborane concentration, together with a decreased mobility. The same discontinuities of both correlation time and order parameter with respect to temperature variations were observed in carborane-containing and carborane-free liposomes. This suggested that a homogeneous dispersion of nitroxides and carboranes occurred in the liposome bilayer. The ESR line shape analysis proved that no dramatic changes were induced in the liposome environment by carborane insertion. QELS data showed that the overall liposome structure was preserved, with a slight decrease in the mean hydrodynamic radius and increase in polydispersity caused by the guest molecules.     

Realtime PCR Is More Sensitive than Multiplex PCR for Diagnosis and Serotyping in Children with Culture Negative Pneumococcal Invasive Disease

Background

Pneumococcal serotyping is usually performed by Quellung reaction, considered the gold standard test. However the method cannot be used on culture-negative samples. Molecular methods can be a useful alternative. The aim of the study was to evaluate the use of Multiplex-sequential-PCR (MS-PCR) or Realtime-PCR on blood samples for diagnosis and serotyping of invasive pneumococcal disease (IPD) in a pediatric clinical setting.

Methodology/Principal Findings

Sensitivity and specificity of MS-PCR and Realtime-PCR have been evaluated both on 46 well characterized pneumococcal isolates and on 67 clinical samples from children with culture-negative IPD. No difference in sensitivity and specificity between MS-PCR and Realtime PCR was found when the methods were used on isolates: both methods could type 100% isolates and the results were always consistent with culture-based methods. On the contrary, when used on clinical samples 43/67 (64.2%) were typeable by MS-PCR and 61/67 (91.0%) by Realtime-PCR (p = 0.0004,K Cohen 0.3, McNemar''s p<0.001). Non-typeability by MS-PCR was associated in 18/20 cases (90.0%) with low bacterial load. The difference between the two methods was present both when they were used on normally sterile fluids (respectively 31/33 (93.9%) typeable samples for Realtime-PCR and 24/33 (72.7%) for MS-PCR, p = 0.047, 95%CL 0.03–0.98; K Cohen 0.3; McNemar''s p = 0.0016) and when they were used on nasopharyngeal swabs (respectively 30/34 (88.2%) typeable samples for Realtime-PCR and 19/34 (55.9%) for MS-PCR, p = 0.007, 95%CL 0.04–0.66); the presence of multiple pneumococcal serotypes in nasopharyngeal swabs was found more frequently by Realtime PCR (19/30; 63.3%) than by Multiplex-sequential PCR (3/19; 15.8%; p = 0.003;95%CL 1.87–39.97).

Conclusions/Significance

In conclusion, both MS-PCR and Realtime PCR can be used for pneumococcal serotyping of most serotypes/serogroups directly on clinical samples from culture-negative patients but Realtime-PCR appears more sensitive.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.