Functional analysis of single enzymes combining programmable molecular circuits with droplet-based microfluidics.

The analysis of proteins at the single-molecule level reveals heterogeneous behaviours that are masked in ensemble-averaged techniques. The digital quantification of enzymes traditionally involves the observation and counting of single molecules partitioned into microcompartments via the conversion of a profluorescent substrate. This strategy, based on linear signal amplification, is limited to a few enzymes with sufficiently high turnover rate.

Enzyme Self-Selection Using Molecular Programs.

Directed evolution provides a powerful route for enzyme engineering. State-of-the-art techniques functionally screen up to millions of enzyme variants using high throughput microfluidic sorters, whose operation remains technically challenging. Alternatively, self-selection methods, analogous to complementation strategies, open the way to even higher throughputs, but have been demonstrated only for a few specific activities.

Accurate gene consensus at low nanopore coverage.

Background: Nanopore technologies allow high-throughput sequencing of long strands of DNA at the cost of a relatively large error rate. This limits its use in the reading of amplicon libraries in which there are only a few mutations per variant and therefore they are easily confused with the sequencing noise. Consensus calling strategies reduce the error but sacrifice part of the throughput on reading typically 30 to 100 times each member of the library.

Size and structure of the sequence space of repeat proteins.

The coding space of protein sequences is shaped by evolutionary constraints set by requirements of function and stability. We show that the coding space of a given protein family-the total number of sequences in that family-can be estimated using models of maximum entropy trained on multiple sequence alignments of naturally occuring amino acid sequences. We analyzed and calculated the size of three abundant repeat proteins families, whose members are large proteins made of many repetitions of conserved portions of ∼30 amino acids.

Hybridization in the wild between Tursiops truncatus (Montagu 1821) and Delphinus delphis (Linnaeus 1758).

A case of intergeneric hybridization in the wild between a female bottlenose dolphin (Tursiops truncatus) and a short-beaked common dolphin (Delphinus delphis), considered members of 'vulnerable' and 'endangered' subpopulations in the Mediterranean, respectively, by the International Union of Conservation of Nature is described in this paper. The birth of the hybrid was registered in the Bay of Algeciras (southern Spain) in August 2016, and the animal has been tracked on frequent trips aboard dolphin-watching platforms. This unique occurrence is the result of an apparent ongoing interaction (10 years) between a female bottlenose dolphin and common dolphins.

Inferring repeat-protein energetics from evolutionary information.

Natural protein sequences contain a record of their history. A common constraint in a given protein family is the ability to fold to specific structures, and it has been shown possible to infer the main native ensemble by analyzing covariations in extant sequences. Still, many natural proteins that fold into the same structural topology show different stabilization energies, and these are often related to their physiological behavior.

Protein Repeats from First Principles.

Some natural proteins display recurrent structural patterns. Despite being highly similar at the tertiary structure level, repeating patterns within a single repeat protein can be extremely variable at the sequence level. We use a mathematical definition of a repetition and investigate the occurrences of these in sequences of different protein families.

Structural and Energetic Characterization of the Ankyrin Repeat Protein Family.

Ankyrin repeat containing proteins are one of the most abundant solenoid folds. Usually implicated in specific protein-protein interactions, these proteins are readily amenable for design, with promising biotechnological and biomedical applications. Studying repeat protein families presents technical challenges due to the high sequence divergence among the repeating units.

Repeat proteins challenge the concept of structural domains.

Structural domains are believed to be modules within proteins that can fold and function independently. Some proteins show tandem repetitions of apparent modular structure that do not fold independently, but rather co-operate in stabilizing structural forms that comprise several repeat-units. For many natural repeat-proteins, it has been shown that weak energetic links between repeats lead to the breakdown of co-operativity and the appearance of folding sub-domains within an apparently regular repeat array.

Capturing coevolutionary signals inrepeat proteins.

Background: The analysis of correlations of amino acid occurrences in globular domains has led to the development of statistical tools that can identify native contacts - portions of the chains that come to close distance in folded structural ensembles. Here we introduce a direct coupling analysis for repeat proteins - natural systems for which the identification of folding domains remains challenging.

Results: We show that the inherent translational symmetry of repeat protein sequences introduces a strong bias in the pair correlations at precisely the length scale of the repeat-unit.

Detecting repetitions and periodicities in proteins by tiling the structural space.

The notion of energy landscapes provides conceptual tools for understanding the complexities of protein folding and function. Energy landscape theory indicates that it is much easier to find sequences that satisfy the "Principle of Minimal Frustration" when the folded structure is symmetric (Wolynes, P. G.

Effect of PLGA hydrophilia on the drug release and the hypoglucemic activity of different insulin-loaded PLGA microspheres.

The effects of viscosity and hydrophilic characteristics of different PLGA polymers on the microencapsulation of insulin have been studied in vitro and in vivo after subcutaneous administration to hyperglycemic rats. Hydrophilic PLGA polymers produced a higher burst effect than the hydrophobic ones. Moreover, an incomplete insulin release was observed with the hydrophilic PLGA polymers in comparison with the hydrophobic ones.

Custom-made modification of a commercial confocal microscope to photolyze caged compounds using the conventional illumination module and its application to the observation of Inositol 1,4,5-trisphosphate-mediated calcium signals.

The flash photolysis of "caged" compounds is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. These caged compounds are inactive and become active when illuminated with ultraviolet light. This paper describes an inexpensive adaptation of an Olympus confocal microscope that uses as source of ultraviolet light the mercury lamp that comes with the microscope for conventional fluorescence microscopy.

Efficacy of a new formulation of amphotericin B in murine disseminated infections by Candida glabrata or Candida tropicalis.

Amphotericin B microspheres (M-AMB) are a new, inexpensive formulation of amphotericin B. In this study, we tested the efficacy of this new formulation for treating murine disseminated infections by Candida glabrata or Candida tropicalis. M-AMB showed a similar efficacy to that of amphotericin B deoxycholate and liposomal amphotericin B in the treatment of both disseminated murine infections.

Effect of aggregation state on the toxicity of different amphotericin B preparations.

The aim of this work was to study the effect of aggregation of amphotericin B (AMB) in their toxicity. The aggregation of AMB depends on different formulation factors such as pH and excipients, therefore three formulations with different AMB aggregation states were prepared: a monomeric form (M-AMB), a dimeric form (D-AMB) and a poly-aggregated form (P-AMB). The predominant aggregation state of each AMB formulation was characterized by spectrophotometry and their size by dynamic laser light scattering.

Efficacy of alternative dosing regimens of poly-aggregated amphotericin B.

A new poly-aggregated form of amphotericin B was formulated as a non-microencapsulated form (P-AMB) or incorporated in albumin microspheres (MP-AMB) and compared with the conventional amphotericin B formulation (D-AMB). Mice were infected with Candida albicans and treated with two different intermittent dose regimens of the different amphotericin B formulations. Efficacy and toxicity were studied by the determination of survival rate, kidney colony-forming units counts, biochemical parameters and amphotericin B concentrations in plasma and organs.

In vivo distribution and therapeutic efficacy of a novel amphotericin B poly-aggregated formulation.

Objectives: The purpose of this investigation is the study of toxicity, in vivo distribution and therapeutic activity against candidiasis of poly-aggregated amphotericin B, in two different formulations: not microencapsulated (P-AMB) or incorporated in albumin microspheres (MP-AMB).

Methods: The therapeutic efficacy and toxicity of amphotericin B formulations was studied in an immunocompetent murine model of systemic candidiasis. A pharmacokinetic study was also performed to measure the plasma, kidney, liver and spleen amphotericin B concentrations after administration of the three formulations to mice.

Efficacy of a new formulation of amphotericin B in a murine model of disseminated infection by Candida glabrata.

Objectives: Amphotericin B poly-aggregates are a new formulation of amphotericin B, which can be obtained cheaply. In this study, we tested the efficacy of this new formulation for treating a disseminated infection by Candida glabrata in a murine model.

Methods: Mice were rendered neutropenic by intraperitoneal cyclophosphamide and intravenous 5-fluorouracil administration and infected intravenously with 2 x 10(8) cfu of C.

HPLC assay for determination of amphotericin B in biological samples.

A fast and selective HPLC method for assaying amphotericin B in biological samples was developed and validated. The chromatographic separation was achieved in less than 12 min on a reverse-phase C(18) column using an acetonitrile-acetic acid-water (52:4.3:43.

Amphotericin B formulations and drug targeting.

Amphotericin B is a low-soluble polyene antibiotic which is able to self-aggregate. The aggregation state can modify its activity and pharmacokinetical characteristics. In spite of its high toxicity it is still widely employed for the treatment of systemic fungal infections and parasitic disease and different formulations are marketed.