Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore.

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa-sized microtubule-embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi-orientation.

Kindlin-2 recruits paxillin and Arp2/3 to promote membrane protrusions during initial cell spreading.

Cell spreading requires the coupling of actin-driven membrane protrusion and integrin-mediated adhesion to the extracellular matrix. The integrin-activating adaptor protein kindlin-2 plays a central role for cell adhesion and membrane protrusion by directly binding and recruiting paxillin to nascent adhesions. Here, we report that kindlin-2 has a dual role during initial cell spreading: it binds paxillin via the pleckstrin homology and F0 domains to activate Rac1, and it directly associates with the Arp2/3 complex to induce Rac1-mediated membrane protrusions.

Structural basis for the disaggregase activity and regulation of Hsp104.

The Hsp104 disaggregase is a two-ring ATPase machine that rescues various forms of non-native proteins including the highly resistant amyloid fibers. The structural-mechanistic underpinnings of how the recovery of toxic protein aggregates is promoted and how this potent unfolding activity is prevented from doing collateral damage to cellular proteins are not well understood. Here, we present structural and biochemical data revealing the organization of Hsp104 from at 3.

Structure of the MIS12 Complex and Molecular Basis of Its Interaction with CENP-C at Human Kinetochores.

Kinetochores, multisubunit protein assemblies, connect chromosomes to spindle microtubules to promote chromosome segregation. The 10-subunit KMN assembly (comprising KNL1, MIS12, and NDC80 complexes, designated KNL1C, MIS12C, and NDC80C) binds microtubules and regulates mitotic checkpoint function through NDC80C and KNL1C, respectively. MIS12C, on the other hand, connects the KMN to the chromosome-proximal domain of the kinetochore through a direct interaction with CENP-C.

Determination of local chromatin composition by CasID.

Chromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci.

Topology and structure of an engineered human cohesin complex bound to Pds5B.

The cohesin subunits Smc1, Smc3 and Scc1 form large tripartite rings which mediate sister chromatid cohesion and chromatin structure. These are thought to entrap DNA with the help of the associated proteins SA1/2 and Pds5A/B. Structural information is available for parts of cohesin, but analyses of entire cohesin complexes are limited by their flexibility.

Architecture and RNA binding of the human negative elongation factor.

Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure.

Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster.

Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A.

New Perspectives for Fixed Dose Combinations of Poorly Water-Soluble Compounds: a Case Study with Ezetimibe and Lovastatin.

Purpose: Aiming to improve the dissolution rate of ezetimibe (EZE) and lovastatin (LOV) in a fixed dose combination (FDC), co-amorphous systems and ternary solid dispersions were prepared by quench cooling and spray drying, respectively.

Methods: Formulations were characterized through X-ray diffraction, modulated differential scanning calorimetry, infrared spectroscopy, scanning electron microscopy and laser diffraction, and evaluated by 'in vitro' dissolution. Stability studies were conducted at different conditions during 30 days with the ternary solid dispersion composed of 75% of Soluplus® (ELS 1:1 75%).

A modular open platform for systematic functional studies under physiological conditions.

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination.

Agglomeration of mesoporous silica by melt and steam granulation. Part I: a comparison between disordered and ordered mesoporous silica.

The objective of this study was to compare agglomerations by melt and steam granulation of ordered, COK-12, and disordered, Syloid(®) 244 FP (244), mesoporous silica material. Poloxamer 188 (P188) and polyvinylpyrrolidone K25 (PVP) were chosen as binders for melt and steam granulation, respectively. The poorly water-soluble compound, itraconazole (ITZ), was selected for the development of an immediate-release oral dosage form.

Agglomeration of mesoporous silica by melt and steam granulation. part II: screening of steam granulation process variables using a factorial design.

The objectives of this study were to identify the key process parameters during steam granulation of disordered mesoporous silica material Syloid® 244 FP (244) and to compare two different binders: polyvinylpyrrolidone (PVP) K25 and hydroxypropylmethyl cellulose (HPMC). Itraconazole (ITZ) was selected as the model compound for the development of an oral dosage form for enhanced release. Six factors: binder content, steam amount, mixing time, impeller speed, spray pause time, and filler content were investigated using a two-level quarter-fraction factorial design of experiment (DOE) for each binder type.

Eudragit E PO as a complementary material for designing oral drug delivery systems with controlled release properties: comparative evaluation of new interpolyelectrolyte complexes with countercharged eudragit L100 copolymers.

The design of new interpolyelectrolyte complexes (IPEC) between countercharged polymers (Eudragit EPO (EPO) and Eudragit L100 (L100)) was investigated. The formation and chemical composition of three new IPECs between EPO and L100 was established by elemental analysis. The structure and solid state properties of the synthesized IPEC were investigated using Fourier transform infrared (FTIR) spectroscopy and modulated temperatre differential scanning calorimetry (MTDSC).

Drug release modification by interpolymer interaction between countercharged types of Eudragit® RL 30D and FS 30D in double-layer films.

Interpolymer interactions between the countercharged methacrylate copolymers Eudragit(®) RL 30D (polycation) and Eudragit(®) FS 30D (polyanion), were investigated in conditions mimicking the gastrointestinal environment. The formation of inter-macromolecular ionic bonds between Eudragit(®) RL 30D and Eudragit(®) FS 30D was investigated using FT-IR spectroscopy and modulated DSC. The FT-IR spectra of the tested polymeric matrices are characterized by visible changes in the observed IR region indicating the interaction between chains of two oppositely charged copolymers.

PLGA nanoparticles and nanosuspensions with amphotericin B: Potent in vitro and in vivo alternatives to Fungizone and AmBisome.

This paper describes the development of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) and nanosuspensions with the polyene antibiotic amphotericin B (AmB). The nanoformulations were prepared using nanoprecipitation and were characterised with respect to size, zeta potential, morphology, drug crystallinity and content. Standard in vitro sensitivity tests were performed on MRC-5 cells, red blood cells, Leishmania infantum promastigotes and intracellular amastigotes and the fungal species Candida albicans, Aspergillus fumigatus and Trichophyton rubrum.

Effect of compression on non-isothermal crystallization behaviour of amorphous indomethacin.

Purpose: To evaluate the effect of tablet compression on the physical stability of amorphous indomethacin.

Methods: The amorphous indomethacin generated by melt cooling, rapid (5°C/min) or slow (0.2°C/min) cooling, was evaluated by PXRD, mDSC and FTIR analysis.

Solution calorimetry as an alternative approach for dissolution testing of nanosuspensions.

The formulation of poorly soluble drugs as nanocrystals/nanosuspensions has rapidly evolved during the past decade into a mature drug-delivery strategy. The major characteristic of these systems is the high drug dissolution rate, enabling bioavailability enhancement after oral administration. It is therefore of great importance to have access to analytical methodology that is able to accurately monitor the extreme fast dissolution process of such formulations.

Influence of preparation methods on solid state supersaturation of amorphous solid dispersions: a case study with itraconazole and eudragit e100.

Purpose: The present study aims to determine the drug / polymer miscibility level as a function of the preparation method for an amorphous solid dispersion model system containing itraconazole and eudragit E100. This value was compared to the theoretical crystalline drug solubility in the amorphous polymer and the miscibility of the amorphous drug in the amorphous polymer.

Methods: The amorphous solid dispersions were prepared via spray drying and film casting in order to evaluate the influence of the solvent drying rate.

Spray drying from complex solvent systems broadens the applicability of Kollicoat IR as a carrier in the formulation of solid dispersions.

The presented study aims to explore the feasibility of preparing solid dispersions of the poorly soluble drug, itraconazole, with Kollicoat IR via spray drying, in order to broaden the application window of the polymer. In order to circumvent the need for a common solvent, Kollicoat IR was dissolved in a 50/50 (v/v) water/ethanol mixture and itraconazole was dissolved in a 50/50 (v/v) dichloromethane/ethanol mixture. In a first approach these two solutions were simultaneously spray dried via a spray nozzle with two inlets.

Influence of polyethylene glycol chain length on compatibility and release characteristics of ternary solid dispersions of itraconazole in polyethylene glycol/hydroxypropylmethylcellulose 2910 E5 blends.

The present study aims to elucidate the influence of the polyethylene glycol chain length on the miscibility of PEG/HPMC 2910 E5 polymer blends, the influence of polymer compatibility on the degree of molecular dispersion of itraconazole, and in vitro dissolution. PEG 2000, 6000, 10,000 and 20,000 were included in the study. Solid dispersions were prepared by spray drying and characterized with MDSC, XRPD and in vitro dissolution testing.

The effect of pressurized carbon dioxide as a temporary plasticizer and foaming agent on the hot stage extrusion process and extrudate properties of solid dispersions of itraconazole with PVP-VA 64.

The aim of the current research project was to explore the possibilities of combining pressurized carbon dioxide with hot stage extrusion during manufacturing of solid dispersions of itraconazole and polyvinylpyrrolidone-co-vinyl acetate 64 (PVP-VA 64) and to evaluate the ability of the pressurized gas to act as a temporary plasticizer as well as to produce a foamed extrudate. Pressurized carbon dioxide was injected into a Leistritz Micro 18 intermeshing co-rotating twin-screw melt extruder using an ISCO 260D syringe pump. The physicochemical characteristics of the extrudates with and without injection of carbon dioxide were evaluated with reference to the morphology of the solid dispersion and dissolution behaviour and particle properties.

[The underpaid nursing profession: myth or reality].

The originality of this research lies in its object, nurses salaries, a subject that is often discussed amongst professionals, yet rarely broached in writing. The concepts of salaries and salaried workers are examined, then, through literature, a list of salary data from 1845 to 1945 is made. The study of nursing personnel salaries from 1945 to 2001 begins with a reconstruction of salary charts, continuing with an internal analysis of the private and semi-public sector, then alternatively with the teachers.

Comparative evaluation of co-processed lactose and microcrystalline cellulose with their physical mixtures in the formulation of folic acid tablets.

In this study, a new co-processed filler-binder ingredient for direct compression, MicroceLac 100, was compared with three different lactoses mixed with microcrystalline cellulose. The aim was to improve a folic acid tablet formulation. Therefore, the influence of drug addition to these mixtures was studied with regard to flow and binding properties.

Comparison of external radiotherapy, laser microsurgery and partial laryngectomy for the treatment of T1N0M0 glottic carcinomas: a retrospective evaluation.

Purpose: The aim of this study was to retrospectively compare the efficacy and functional results of three treatment options for T1N0M0 glottic carcinomas applied in a single institution.

Materials And Methods: One hundred six charts of patients with biopsy-proven T1N0M0 glottic carcinomas treated between 1979 and 1995 were reviewed. There were 81 T1a and 25 T1b tumors.