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A new approach to automating droplet freezing assays is demonstrated by comparing the ice-nucleating efficiency of a K-feldspar glass and a crystal with the same bulk composition. The method uses a pyroelectric polymer PVDF (polyvinylidene fluoride) as a thermal sensor. PVDF is highly sensitive, cheap, and readily available in a variety of sizes. As a droplet freezes latent heat is released, which is detected by the sensor. Each event is correlated with the temperature at which it occurred. The sensor has been used to detect microlitre volume droplets of water freezing, from which frozen proportion curves and nucleation rates can be quickly and automatically calculated. Our method shows glassy K-feldspar to be a poor nucleator compared to the crystalline form.

Fluorescence imaging is a versatile tool for biological and preclinical studies with steady improvements in performance thanks to instrumentation and probe developments. The sensitive detection and imaging of deep targets in vivo is especially challenging due to the diffusion and absorption of light by the tissues and to the emission of autofluorescence from intrinsic chromophores. Fluorescent inorganic nanoparticles present interesting optical properties that may significantly differ from organic dyes. In this short review, we present recent developments in the design of these nanoprobes and their use for new in vivo fluorescence modalities which provide enhanced imaging capabilities., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Energy loss due to carrier recombination is among the major factors limiting the performance of TiO2/PbS colloidal quantum dot (QD) heterojunction solar cells. In this work, enhanced photocurrent is achieved by incorporating another type of hole-transporting QDs, Zn-doped CuInS2 (Zn-CIS) QDs into the PbS QD matrix. Binary QD solar cells exhibit a reduced charge recombination associated with the spatial charge separation between these two types of QDs. A ~30% increase in short-circuit current density and a ~20% increase in power conversion efficiency are observed in binary QD solar cells compared to cells built from PbS QDs only. In agreement with the charge transfer process identified through ultrafast pump/probe spectroscopy between these two QD components, transient photovoltage characteristics of single-component and binary QDs solar cells reveal longer carrier recombination time constants associated with the incorporation of Zn-CIS QDs. This work presents a straightforward, solution-processed method based on the incorporation of another QDs in the PbS QD matrix to control the carrier dynamics in colloidal QD materials and enhance solar cell performance.

The development of sensitive multimodal contrast agents is a key issue to provide better global, multi-scale images for diagnostic or therapeutic purposes. Here we present the synthesis of Zn-Cu-In-(S, Se)/Zn(1-x)Mn(x)S core-shell quantum dots (QDs) that can be used as markers for both near-infrared fluorescence imaging and magnetic resonance imaging (MRI). We first present the synthesis of Zn-Cu-In-(S, Se) cores coated with a thick ZnS shell doped with various proportions of Mn. Their emission wavelengths can be tuned over the NIR optical window suitable for deep tissue imaging. The incorporation of manganese ions (up to a few thousand ions per QD) confers them a paramagnetic character, as demonstrated by structural analysis and electron paramagnetic resonance spectroscopy. These QDs maintain their optical properties after transfer to water using ligand exchange. They exhibit T1-relaxivities up to 1400 mM(-1) [QD] s(-1) at 7 T and 300 K. We finally show that these QDs are suitable multimodal in vivo probes and demonstrate MRI and NIR fluorescence detection of regional lymph nodes in mice.

Fluorescence imaging is a promising technique for the detection of individual cell migration. Its sensitivity is, however, limited by a high tissue autofluorescence and a poor visible light penetration depth. In order to solve this problem, the fluorescence signal peak wavelength should lie in an absorption and diffusion free region and should be distinguishable, either spectrally or temporally, from the autofluorescence background. We present, here, the synthesis and characterization of low toxicity Zn-Cu-In-Se/ZnS core/shell quantum dots. Their fluorescence emission wavelength peaks around 800 nm, where the absorption and scattering of tissues are minimal. They are coated with a new ligand, which yields small, stable, and bright individual probes in the live cell cytoplasm, even 48 h after the labeling. Furthermore, these near-infrared-emitting quantum dots have a long fluorescence lifetime component (around 150 ns) compared to autofluorescence (<5 ns). Taking the advantage of this property and coupling these probes to a time-gated detection, we demonstrate efficiently the discrimination between the signal and short lifetime fluorescence such as the autofluorescence. This technique is supported by a method we developed, to massively stain cells that preserves the quantum dot stability and brightness for 48 h.

High colloidal stability in aqueous conditions is a prerequisite for fluorescent nanocrystals, otherwise known as "quantum dots", intended to be used in any long-term bioimaging experiment. This essential property implies a strong affinity between the nanoparticles themselves and the ligands they are coated with. To further improve the properties of the bidentate monozwitterionic ligand previously developed in our team, we synthesized a multidentate polyzwitterionic ligand, issued from the copolymerization of a bidentate monomer and a monozwitterionic one. The nanocrystals passivated by this polymeric ligand showed an exceptional colloidal stability, regardless of the medium conditions (pH, salinity, dilution, and biological environment), and we demonstrated the affinity of the polymer exceeded by 3 orders of magnitude that of the bidentate ligand (desorption rates assessed by a competition experiment). The synthesis of the multidentate polyzwitterionic ligand proved also to be easily tunable and allowed facile functionalization of the corresponding quantum dots, which led to successful specific biomolecules targeting.

In accordance with the Regulation N 2309/93/EC, the Commission shall produce a synopsis of European registration operating procedures no later than 2001. On July 18, 2001, after a wide open consultation, the College of Commissioners adopted a Proposed Revision elaborated by the "DG Enterprises". This Proposed Revision was guided by the desire to facilitate access of citizens of the European Union to innovating and safe medications, to limit fragmentation of the interior market, prejudicial to the community pharmaceutical industry, and to prepare widening of the community. This synopsis focused essentially on registration procedures and pharmacovigilance, on patient information, on reform of the European agency for administrative protection of recorded data. The Proposed Revision is following the codecision procedure and should come into force, in a more or less amended form, at the end of 2004 at the latest.

Antiviral efficacy and serum lipids were investigated following a switch from long-term successful protease inhibitor based antiretroviral treatment (PI-ART) to abacavir-based ART in 29 patients who have been followed for 28 months thereafter. Virologic failure occurred within 3 months in 21% (6/29) of the patients, and abacavir hypersensitivity in 1 individual. The remaining 22 patients continue to have HIV RNA < 50 copies/ml after 28 months with a CD4 increase from 605 +/- 265 x 10(6)/l to 798 +/- 366 x 10(6)/l (p < 0.001). All virologic failing patients had been on long-term unsuccessful nucleoside reverse transcriptase inhibitor (NRTI) therapy before PI-ART as compared to 32% (7/22) of the virologic non-failing patients (p < 0.01). The viral strains from the virologic failing patients harboured 3-6 reverse transcriptase (RT) mutations, including the V118I mutation in 5/6 cases prior to PI-ART or at viral rebound. Only the V118I RT mutation was statistically more common among virologic failing than non-failing NRTI pretreated patients (p < 0.05). Stepwise multiple regression analysis for viral failure resulted in a model with only the V118I RT mutation entering the model (p < 0.01). The LDL cholesterol and triglyceride values decreased and the HDL cholesterol increased after the switch to abacavir-based ART (p < 0.01, p < 0.05, p < 0.05, respectively). In conclusion, viral failure was associated with prior mono- or dual-NRTI treatment and the occurrence of the V1181 RT mutation, persisting despite long term viral control. The selection process for patients suitable for treatment simplification to abacavir-based ART should contain a detailed antiretroviral treatment history.

Objective: To study the appearance of drug-induced mutations at low viraemia in treated HIV-1-infected patients., Design and Methods: Fourteen patients, who received their first (n = 5), second (n = 7) or third (n = 2) line antiretroviral combination therapy, developed a persistent low-grade viraemia after an initial decrease of the viral load (VL) to less than 500 copies/ml. The amount of HIV-1 RNA (n = 71) and reverse transcriptase (RT)/protease sequences (n = 56) were determined in longitudinally obtained plasma samples during a mean period of 16.6 months., Results: In the vast majority (93%) of patients, new primary resistance mutations were found in the RT and/or protease genes at virological failure at a median VL of 500 and 200 copies/ml, respectively. Drug-experienced patients developed mutations at a lower VL than naive patients. In one previously protease inhibitor-naive patient, primary RT and protease mutations were detected, although the VL was less than 50 copies/ml. A serial accumulation of drug resistance mutations was seen despite the VL increase being mostly modest, reaching a median of 1450 copies/ml at the end of the study, and the CD4 T cell counts continued to increase. One patient still had a VL of 300 copies/ml after 28 months, despite the presence of the multidrug-resistance Q151M mutation., Conclusion: Low viraemia after virological treatment failure can select for virus with several new drug resistance mutations, despite a concomitant increase in CD4 T cell counts. This serial accumulation of mutations is likely to exhaust future drug options

Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.

Objectives: Assessment of genotypic change in HIV protease during treatment with saquinavir (SQV) in combination with zidovudine (ZDV) and/or zalcitabine (ddC), to determine the influence of such changes on viral phenotype and response to treatment., Design: Virologic substudies of Phase III clinical trials NV14256 and SV14604., Methods: Population sequencing of HIV protease genes amplified from pre- and post-treatment plasma. Phenotyping of peripheral blood mononuclear cell (PBMC)-derived virus isolates, and genotyping of proviral DNA clones amplified from PBMC used in the expansion of virus isolates., Results: In both trials the incidence of Met90 remained at < or = 20% in subjects receiving SQV in combination with ddC (with or without ZDV) for 1 year. A Val48 substitution was observed in two out of 81 subjects after 24 weeks and in two out of 75 subjects after 48 weeks. In 12 out of 13 NV14256 subjects with viral load rebound during SQV monotherapy these substitutions were associated with the rebound. In subjects treated with SQV plus ddC, rebound was associated with SQV resistance in six out of 22 cases and ddC resistance in five out of 22 cases. The incidences of non-BRU residues at positions 10, 63 and 71 were increased significantly (P < 0.05, Fisher's exact test) after SQV treatment with or without ZDV. However, comparison of genotypic and phenotypic data showed that these changes were not associated with reduced sensitivity to SQV., Conclusions: Virological failure during combination therapy can be due to resistance to either treatment drug, emphasising the need to change both the reverse transcriptase inhibitor and the protease inhibitor. Only Val48 and Met90 correlated directly with the development of reduced drug sensitivity during treatment with SQV in vivo.

We describe a colorimetric screening method for the mutation in codon 506 of the coagulation factor V gene. The nucleotide at the site of the FV:Q506 mutation is identified in an immobilized amplified DNA template by extension of a primer with a hapten-labelled dNTP using a DNA polymerase. The incorporated hapten is detected by an antibody-alkaline phosphatase conjugate that catalyses the formation of a coloured end product. The assay is carried out in a microtiter plate format, and the procedure is identical to that of enzyme immunoassays. It unequivocally identifies the FV:Q506 mutation in heterozygous and homozygous form. The colorimetric minisequencing method gave the same result as a 3H-based minisequencing assay and restriction site analysis with Mn11 used as reference methods. Because of its simple format and numeric result, the novel colorimetric minisequencing method should be an attractive alternative for screening for the FV:Q506 mutation in clinical laboratories.

56 laryngeal paralyses were seen in newborn infants between 1970 and 1976 (25 bilateral, 31 unilateral). The aetiology was obstetric trauma in 11 cases, nuclear agenesis in 7 cases, a severe neurological disorder (spina bifida, hydrocephaly, microcephaly, lesions of the central nervous system) in 13 cases, and congenital heart disease in 3 cases. In 10 cases, the paralysis was present in isolation. The initial state was not recorded in 11 cases. The course varied according to the aetiology: 5 deaths, 4 due to the severity of neurological problems. Regression, which invariably occured before the end of the 6th month, was seen in all the cases with an obstetric aetiology and in 50% of those in which the paralysis was present in isolation. There was persistence in the majority of neurological, nuclear or central causes. However, subsequent tolerance of persistent forms was good in all those patients followed-up on a regular basis, apart from 5 who underwent surgery. Treatment consisted of intubation for forms poorly tolerated initially, for the first few weeks. Tracheotomy did not prove necessary in any case. 5 patients underwent surgery: arytenoidectomy or arytenoidopexy via an extralaryngeal approach.

The endocrine pancreas of birds contains 3 islet types and releases glucagon, insulin, somatostatin and avian pancreatic polypeptide (APP). Interactions between these hormones, and pancreatic hormone-plasma metabolite feedback mechanisms, are the main regulators of these secretions. In birds, total pancreatectomy induces a fatal hypoglycaemia associated with the disappearance of circulating glucagon, and an impaired glucose tolerance ascribed to the lack of insulin. Normoglycaemia can be restored in depancreatized birds by infusion of glucagon and insulin, provided that the G/I ratio is close to normal. Subtotal pancreatectomy provokes a diabetes characterized by a basal hyperglycaemia and an impaired glucose tolerance. However, while a transient diabetes is observed in most species, in the goose a permanent diabetes is obtained. These results point out the importance of the pancreas and especially of glucagon, in birds. This importance can also be detected in the protein metabolism, since total or subtotal pancreatectomy markedly increases the concentration of plasma aminoacids. As for lipid metabolism, in vivo and/or in vitro experiments have shown the sensitivity of the adipose tissue and liver cells to glucagon. Finally, contrary to the general opinion, insulin seems to be antilipolytic, as in mammals.

1. Total pancreatectomy in the goose, in addition to the effects previously described (fatal hypoglycaemia and impaired tolerance), increases markedly the plasma amino-acid level. This increase corresponds to a significant rise in individually tested plasma amino-acids. In addition, pancreatectomy suppresses the hyperglycaemia which normally follows arginine treatment. 2. Oral administration of arginine induces hyperglycaemia in normal, but not in depancreatized geese. Therefore, the arginine effect upon the blood glucose level could be mediated by glucagon. Plasma glucose and glucagon changes, in normal animals with oral administration or i.v. infusion (Khemiss and Sitbon, 1982), agree with this conclusion. 3. Conversely, alanine elicits hyperglycaemia in normal animals, and such hyperglycaemia persists in pancreatectomized geese. Therefore, in the goose, in contrast to the duck, alanine appears to be a strong gluconeogenic amino-acid. These experiments with alanine indicate that closely related species, such as ducks and geese, may present important physiological differences.

We have studied the pancreatic hormone-glucose feed-back mechanisms by infusing glucagon (G), insulin (I) and glucose into normal fasting geese. The controls received saline. Whilst a NaCl 9% infusion is devoid of effect, the pancreatic hormones, used at physiological doses, modify the plasma glucose level, glucagon being hyperglycaemic and insulin hypoglycaemic. In addition, a physiological increase in plasma glucose provokes a drop in plasma glucagon and a rise in plasma insulin, thus a marked decrease in the G/I ratio. The results show that the pancreatic hormone-glucose feed-back mechanisms are effective under physiological conditions.

In the goose, alanine and arginine, intravenously or orally administered, act in the same way on pancreatic hormones; they both stimulate insulin and glucagon secretions. Conversely, whereas alanine treatment has no effect on plasma gut GLI, oral arginine stimulates gut GLI secretion. Since stimulation of gut GLI secretion does not occur with i.v. arginine, it may be assumed that this secretion depends on the intestinal transit of arginine and, as already described (Sitbon and Mialhe 1979), of glucose. The results, compared with studies on a similar species (duck) and on mammals, point out that i.v. infusion of alanine stimulates IRI and GLI secretions in the goose and not in the duck. In the same way, arginine i.v. infusion, contrarily to the observation made in the duck, is without effect on gut GLI secretion in the goose. Furthermore, insulin seems to be able to inhibit the alpha cell response to arginine infusion, as in mammals, whereas this is not the case in ducks.

Measurements of plasma GLI and IRI in normal fasting geese, before and during constant I.V. infusion of saline, gave GLI/I ratios of 1.32 +/- .07 and 1.34 +/- .03 (w/w). As total pancreatectomy markedly reduces the pancreatic hormone level, leading to a mortal hypoglycaemia, we attempted to maintain plasma glucose within the normal range by constant I.V. infusion of glucagon and insulin into operated animals. The results as follows: 1. Blood glucose levels can be maintained within the normal range during experiments lasting 6 or more hours with a constant G/I ratio. 2. The G/I ratio obtained in operated animals (.96 +/- .12) is near to, but significantly lower (p less than .005) than, the GLI/I ratio measured in normal animals. This difference may be explained by the presence of a small amount of circulating gut GLI in the 2nd group.