Enhanced EGFP Fluorescence Emission in Presence of PEG Aqueous Solutions and PIB1000-PEG6000-PIB1000 Copolymer Vesicles.

An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a "pure" PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1 : 10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to "wrap" the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.

Marker-independent identification of glioma-initiating cells.

Tumor-initiating cells with stem cell properties are believed to sustain the growth of gliomas, but proposed markers such as CD133 cannot be used to identify these cells with sufficient specificity. We report an alternative isolation method purely based on phenotypic qualities of glioma-initiating cells (GICs), avoiding the use of molecular markers. We exploited intrinsic autofluorescence properties and a distinctive morphology to isolate a subpopulation of cells (FL1(+)) from human glioma or glioma cultures. FL1(+) cells are capable of self-renewal in vitro, tumorigenesis in vivo and preferentially express stem cell genes. The FL1(+) phenotype did not correlate with the expression of proposed GIC markers. Our data propose an alternative approach to investigate tumor-initiating potential in gliomas and to advance the development of new therapies and diagnostics.

Human muscular fetal cells: a potential cell source for muscular therapies.

Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30 years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1 H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5 years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4 days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineering.

Sham nepotism as a result of intrinsic differences in brood viability in ants.

In animal societies, cooperation for the common wealth and latent conflicts due to the selfish interests of individuals are in delicate balance. In many ant species, colonies contain multiple breeders and workers interact with nestmates of varying degrees of relatedness. Therefore, workers could increase their inclusive fitness by preferentially caring for their closest relatives, yet evidence for nepotism in insect societies remains scarce and controversial. We experimentally demonstrate that workers of the ant Formica exsecta do not discriminate between highly related and unrelated brood, but that brood viability differs between queens. We further show that differences in brood viability are sufficient to explain a relatedness pattern that has previously been interpreted as evidence for nepotism. Hence, our findings support the view that nepotism remains elusive in social insects and emphasize the need for further controlled experiments.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

Pathological complete response after neoadjuvant chemotherapy is an independent predictive factor irrespective of simplified breast cancer intrinsic subtypes: a landmark and two-step approach analyses from the EORTC 10994/BIG 1-00 phase III trial.

BACKGROUND: Pathological complete response (pCR) following chemotherapy is strongly associated with both breast cancer subtype and long-term survival. Within a phase III neoadjuvant chemotherapy trial, we sought to determine whether the prognostic implications of pCR, TP53 status and treatment arm (taxane versus non-taxane) differed between intrinsic subtypes. PATIENTS AND METHODS: Patients were randomized to receive either six cycles of anthracycline-based chemotherapy or three cycles of docetaxel then three cycles of eprirubicin/docetaxel (T-ET). pCR was defined as no evidence of residual invasive cancer (or very few scattered tumour cells) in primary tumour and lymph nodes. We used a simplified intrinsic subtypes classification, as suggested by the 2011 St Gallen consensus. Interactions between pCR, TP53 status, treatment arm and intrinsic subtype on event-free survival (EFS), distant metastasis-free survival (DMFS) and overall survival (OS) were studied using a landmark and a two-step approach multivariate analyses. RESULTS: Sufficient data for pCR analyses were available in 1212 (65%) of 1856 patients randomized. pCR occurred in 222 of 1212 (18%) patients: 37 of 496 (7.5%) luminal A, 22 of 147 (15%) luminal B/HER2 negative, 51 of 230 (22%) luminal B/HER2 positive, 43 of 118 (36%) HER2 positive/non-luminal, 69 of 221(31%) triple negative (TN). The prognostic effect of pCR on EFS did not differ between subtypes and was an independent predictor for better EFS [hazard ratio (HR) = 0.40, P < 0.001 in favour of pCR], DMFS (HR = 0.32, P < 0.001) and OS (HR = 0.32, P < 0.001). Chemotherapy arm was an independent predictor only for EFS (HR = 0.73, P = 0.004 in favour of T-ET). The interaction between TP53, intrinsic subtypes and survival outcomes only approached statistical significance for EFS (P = 0.1). CONCLUSIONS: pCR is an independent predictor of favourable clinical outcomes in all molecular subtypes in a two-step multivariate analysis. CLINICALTRIALSGOV: EORTC 10994/BIG 1-00 Trial registration number NCT00017095.

Clinical assessment of fluorescence cytoscopy during transurethral bladder resection in superficial bladder cancer.

The prognosis of superficial bladder cancer in terms of recurrence and disease progression is related to bladder tumor multiplicity and the presence of concomitant "plane" tumors such as high-grade dysplasia and carcinoma in situ. This study in 33 patients aimed to demonstrate the role of fluorescence cystoscopy in transurethral resection of superficial bladder cancer. The method is based on the detection of protoporphyrin-IX-induced fluorescence in urothelial cancer cells by topical administration of 5-aminolevulinic acid. The sensitivity and the specificity of this procedure on apparently normal mucosa in superficial bladder cancer are estimated to be 82.9% and 81.3%, respectively. Thus, fluorescence cytoscopy is a simple and reliable method for mapping the bladder mucosa, especially in the case of multifocal bladder disease, and it facilitates the screening of occult dysplasia.

Relaxivity of Gd-based contrast agents on X nuclei with long intrinsic relaxation times in aqueous solutions.

The relaxivity of commercially available gadolinium (Gd)-based contrast agents was studied for X-nuclei resonances with long intrinsic relaxation times ranging from 6 s to several hundred seconds. Omniscan in pure 13C formic acid had a relaxivity of 2.9 mM(-1) s(-1), whereas its relaxivity on glutamate C1 and C5 in aqueous solution was approximately 0.5 mM(-1) s(-1). Both relaxivities allow the preparation of solutions with a predetermined short T1 and suggest that in vitro substantial sensitivity gains in their measurement can be achieved. 6Li has a long intrinsic relaxation time, on the order of several minutes, which was strongly affected by the contrast agents. Relaxivity ranged from approximately 0.1 mM(-1) s(-1) for Omniscan to 0.3 for Magnevist, whereas the relaxivity of Gd-DOTP was at 11 mM(-1) s(-1), which is two orders of magnitude higher. Overall, these experiments suggest that the presence of 0.1- to 10-microM contrast agents should be detectable, provided sufficient sensitivity is available, such as that afforded by hyperpolarization, recently introduced to in vivo imaging.

Intrinsic coordinate system for the tibial plateau

Determination of the sub-chondral bone density, or more precisely the internal density spot, can be used to evaluate the capability of a knee to sustain normal kinematics. To use this technique as a mean of knee kinematics control, the position of the internal density spot must be determined in a reproducible way. This paper presents a definition of an intrinsic polar coordinate system, allowing to measure the position of the internal density spot of the tibial plateau. Tests of reproducibility gave good results and justify the use of this coordinate system for comparison of the internal density spot position between left and right paired knees.

HRM Practices, Intrinsic Motivators, and Organizational Performance in the Public Sector

This article aims to determine the impact of human resource management (HRM) practices on public service motivation (PSM) and organizational performance. Based on a survey of Swiss cantonal public employees (N = 3,131), this study shows that several HRM practices may be considered as organizational antecedents of PSM and strong predictors of perceived organizational performance. Fairness, job enrichment, individual appraisal, and professional development are HRM practices that are positively and significantly associated with PSM and perceived organizational performance. Moreover, these results suggest that HRM practices are stronger predictors than either PSM or organizational commitment when explaining the individual perception of organizational performance.

Thrombin-sensitive dual fluorescence imaging and therapeutic agent for detection and treatment of synovial inflammation in murine rheumatoid arthritis.

We have developed a thrombin-sensitive polymeric photosensitizer prodrug (T-PS) to selectively image and eradicate inflammatory lesions in rheumatoid arthritis (RA). Thrombin is a serine protease up-regulated in synovial tissues of rheumatoid arthritis (RA) patients. T-PS consists of a polymeric backbone, to which multiple photosensitizer (PS) units are tethered via short thrombin-cleavable peptide linkers. Fluorescence emission and phototoxicity of the prodrug are efficiently quenched due to the interaction of neighboring photosensitizer units. The prodrug is passively delivered to the inflammation site via the enhanced permeability and retention (EPR) effect. Subsequent site-selective proteolytic cleavage of the peptide linkers restores its photoactivity by increasing the mutual distance between PS. Whole animal imaging in murine collagen-induced arthritis, an experimental model of RA revealed a dose-dependent fluorescence increase in arthritic paws after systemic prodrug injection. In addition, administration of T-PS resulted in much higher fluorescence selectivity for arthritic joints as compared to the free PS. Irradiation of the arthritic joints induced light dose dependent phototoxic effects such as apoptosis, vascular damage and local hemorrhage. Long-term observations showed complete regression of the latter. Irradiated non-arthritic tissues or non-irradiated arthritic tissues showed no histological effects after photodynamic therapy with T-PS. This illustrates that T-PS can localize inflammatory lesions with excellent selectivity and induce apoptosis and vascular shut down after irradiation.

Hypofractionated radiotherapy in the treatment of diffuse intrinsic pontine glioma in children: a single institution's experience

Objective: To demonstrate our institutional experience in the treatment ofdiffuse intrinsic pontine glioma (DIPG) with an hypofractionated external beam radiotherapy schedule.Materials and Methods: Between April 1996 and January 2004, 22 patients, ages 2.9-12.5 years, with newly diagnosed DIPG were treated by hypofractionated radiation therapy delivering a total dose of 45 Gy in daily fraction of 3 Gy, given over 3 weeks. No other treatment was applied concomittently.Results: Fourteen of the 22 patients received the prescribed dose of 45 Gy in 15 fractions of 3 Gy, two patients received a total dose of 60 and 45 Gy with a combination of two different beams (photons and neutrons), in 5 cases the daily fraction was modified to 2 Gy because of bad tolerance and one patient died due to serious intracranial hypertension after 2 fractions of 3 Gy and one of 2 Gy. Fourteen patients of 22 patients/of the total showed a clinical improvement, usually starting in the second week of treatment. No grade 3 or 4 acute toxicity from radiotherapy was observed. No treatment interruption was needed. In six patients, steroids could be discontinued within one month after the end of radiotherapy. The median time to progression and the median overall survival were 5.7 months and 7.6 months, respectively.Conclusion: External radiotherapy with a radical hypofractionated regimen is feasible and well tolerated in children with newly diagnosed DIPG. This regimen does not seem however to change the overall survival in this setting. It could represent an alternative option of short duration to more protracted regimens.

Correlation of photodynamic activity and fluorescence signaling for free and pegylated mTHPC in mesothelioma xenografts

Rapport de synthèse : Introduction : Les premières applications cliniques de la thérapie photodynamique (PDT) remontent à plus d'une vingtaine d'années. Basée sur l'activation d'un médicament photosensibilisateur par une source lumineuse à une longueur d'onde spécifique, la PDT permet la destruction sélective de tissus contenant le produit actif. Ce procédé a été expérimenté dans le traitement de cancers en raison de la propriété du médicament à se concentrer dans les tumeurs tout en épargnant les structures normales contigües. Cependant, les photosensibilisateurs utilisés jusqu'à ce jour n'ont pas démontré une accumulation exclusive dans les tissus néoplasiques mais également dans les structures saines avoisinantes induisant une destruction tissulaire non sélective. Notamment, d'importantes complications ont été rapportées suite à l'utilisation de la PDT dans la cavité thoracique après la résection de mésothéliomes pleuraux, et ce malgré l'arrivée de photosensibilisateurs de secondes générations. De ce fait, plusieurs études expérimentales ont été menées afin d'améliorer la sélectivité tumorale du médicament en modulant différentes conditions de traitement et en modifiant la structure du photosensibilisateur par pégylation. Le but de cette étude expérimentale est de corréler l'activité photodynamique, la phototoxicité et la distribution du m-tetrahydroxyphenylchlorin (mTHPC) et de sa forme pégylée, le PEG-mTHPC. De ce fait, un modèle de souris nues porteur de xenogreffes de mésothéliome humain a été utilisé pour étudier les deux photosensibilisateurs. De récents travaux avec ce modèle ont montré que la mesure de la concentration tissulaire du mTHPC et de sa forme pégylée par HPLC restait limitée afin de prédire l'activité photodynamique. De ce fait, nous pensons que les mesures de fluorescence peuvent être plus appropriée. Le signalement fluorescent est mesuré dans le tissu tumoral et dans une région contrôle de la peau afin d'étudier la distribution et l'intensité des deux sensibilisateurs. Méthode : Des souris nues (cd1nu/nu mice) de 8 semaines ont été transplantées avec des fragments de mésothéliome malin humain (H-meso-1). Ces derniers ont été obtenus à partir d'une suspension cellulaire. Au moins trois passages ont été faits dans les animaux, avant que le traitement soit initié. Deux groupes de 6 souris chacun ont été utilisés pour l'injection intraveineuse par la queue du mTHPC à 0.15 mg/kg et du PEG-mTHPC à dose équimolaire. Après trois jour, la tumeur ainsi qu'une région contrôle de la cuisse ont été illuminées sur une surface d'un diamètre de 1.2 cm et pendant 133 secondes avec un laser à une longueur d'onde à 652 nm (fluence 20 J/cm2, fluence rate 150 mW/cm2). Les animaux ont été ensuite sacrifiés 72 heures après l'illumination. L'étendue de la nécrose tumorale et de la région contrôle ont été déterminées en aveugle par histomorphometrie par un pathologue (HJA). La fluorescence microscopique a été évaluée dans 12 souris à une concentration de 0.15 et 0.5 mg/kg pour le mTHPC, et à doses équimolaires pour le PEG-mTHPC. Trois animaux ont été injectés avec le mTHPC à 0.15 mg/kg, 3 autres à dose équimolaire avec la forme pégylée et 6 souris avec le mTHPC à 0.5 mg/kg et à dose équimolaire. Les animaux ont été sacrifiés 72 heures après injection. L'intensité fluorescente des sensibilisateurs a été mesurée dans la tumeur et la région contrôle. Suite à cela, les coupes ont été fixées par H&E et superposées aux images fluorescentes, afin de localiser la distribution des deux photosensibilisateurs dans les différents compartiments tissulaires. Six souris transplantées n'ayant ni été injectées avec les sensibilisateurs ou illuminées ont servi de groupe contrôle. Résultats : Trois jours après l'illumination, la PDT provoque une nécrose tumorale de 10 ±5.4 mm2 pour le mTHPC à 0.15mg/kg et 5.2 ± 4.6 mm2 pour sa forme pégylée à dose équimolaire. Cependant, la nécrose tumorale induite par les deux formulations du sensibilisateur est significativement plus élevée que dans le groupe contrôle (0.33 ± 0.58 mm2) (P=0.02). Toutefois, le mTHPC pégylé provoque une photosensibilité cutanée moins importante que la forme non-pegylée. Dans les deux groupes, aucune nécrose n'a été observée dans la cuisse des animaux. Trois jours après l'injection du mTHPC et de la forme pégylée à 0.15 mg/kg, aucune activité fluorescente n'a été détectée. Cependant, à 0.5 mg/kg, la fluorescence microscopique révèle une distribution hétérogène des deux photo-sensibilisateurs dans le tissu tumoral avec une accumulation prédominante dans les régions peri-vasculaires. Les deux médicaments montrent une distribution intracellulaire homogène dans le cytoplasme et une absence de signalement dans le nucleus. La mesure de l'intensité fluorescente du mTHPC à 0.5mg/kg ne montre pas de différence significative entre le tissu tumoral et la région contrôle. Par contre, le PEG-mTHPC montre une intensité fluorescente supérieure dans le tissu tumoral que dans la peau (ratio tumeur- peau 0.94 pour le mTHPC et 1.73 pour le PEG-mTHPC). Conclusion : L'utilisation du mTHPC à 0.15mg/kg induit une nécrose tumorale similaire à celle du PEG-mTHPC à dose équimolaire. Cependant, ce dernier démontre une photo-toxicité plus atténuée de la peau. La fluorescence microscopique permet de localiser les deux sensibilisateurs dans les différents compartiments tissulaires à partir d'une dose de 0.5 mg/kg. Le PEG-mTHPC induit un signalement fluorescent supérieur dans le tissu tumoral par rapport à la peau. La mesure du signalement fluorescent a le potentiel de prédire l'activité photodynamique du mTHPC et de sa forme pégylée dans les xénogreffes de mésothéliome humain dans un modèle de souris nue.