Preparation and characterization of spray-dried submicron particles for pharmaceutical application

The versatile use of submicron-sized particles (0.1–1?μm) requires new manufacturing methods. One possibility for the preparation of submicron-sized particles is spray drying. However, the generation of small droplets at a high production rate and the precipitation of submicron particles are quite challenging. In order to produce a sufficient amount of fine and uniform droplets, a two-fluid nozzle with internal mixing was combined with a cyclone droplet separator. The precipitation of particles was realized with an electrostatic precipitator. Considering the difficulty of electrostatic precipitation concerning explosion risks and to make it capable using organic solvents, the spray dryer was integrated in a pressure resistant vessel. Based on previous experiments, the now presented design is compact and the electrostatic precipitator is shortened. In addition, enhanced drying conditions ensured a controlled and reproducible preparation of submicron-sized particles. Thus, high separation efficiencies were shown. Spray-drying experiments were conducted with the model substance mannitol. With the cyclone droplet separator, a fine and uniform spray with a droplet size smaller 2?μm was produced. This robust atomizing technique is capable for high concentrations. For a 10?wt% mannitol solution, particles in the submicron range d_50,3?=?0.7?μm were produced.     

Algorithms for compact letter displays: Comparison and evaluation

Multiple pairwise comparisons are one of the most frequent tasks in applied statistics. In this context, letter displays may be used for a compact presentation of results of multiple comparisons. A heuristic previously proposed for this task is compared with two new algorithmic approaches. The latter rely on the equivalence of computing compact letter displays to computing clique covers in graphs, a problem that is well-studied in theoretical computer science. A thorough discussion of the three approaches aims to give a comparison of the algorithms’ advantages and disadvantages. The three algorithms are compared in a series of experiments on simulated and real data, e.g., using data from wheat and triticale yield trials.     

A strategy of plasma protein quantitation by selective reaction monitoring of an intact protein

Immunoassays are used extensively in the quantitative analysis of proteins in plasma, urine, and other biological matrixes to support preclinical and clinical studies. Although immunoassays are both sensitive and rapid, difficulties during development of these assays are compounded by the need to have a specific antibody or antigen to the protein of interest. Furthermore, calibration curves of immunoassays are inherently nonlinear, and the technique often detects many structurally related components in addition to the analyte of interest. We have developed a novel strategy of analyzing protein concentrations in plasma by utilizing 96-well solid-phase extraction and LC-MS/MS detection of the intact protein. This strategy has been successfully applied in method development and assay validation of quantitatively analyzing protein rK5 concentrations in monkey plasma samples. Additional techniques such as precolumn regeneration and column heating were also incorporated into the assay. Total run time for each sample was approximately 15 min. An LLOQ of 99.2 ng/mL from a sample volume of 50 microL, corresponding to only 380 fmol (3.97 ng) of the rK5 analyte being injected onto the analytical column (assuming 100% extraction recovery), was obtained. The validated linear dynamic range was between 99.2 and 52 920.0 ng/mL, with a correlation coefficient (r(2)) ranging from 0.9972 and 0.9994. The intraassay CV for this assay was between 0.6 and 3.8%, and the interassay CV was between 1.7 and 3.2%. Interassay mean accuracies were between 101.5 and 104.7%. The assay has proven rugged and specific and has been employed to generate data in support of preclinical studies. This strategy for rK5 assay could be used for the development of bioanalytical assays to provide preclinical and clinical support for other protein drug candidates and, furthermore, for the validation of biomarkers discovered from proteomic research.     

Single-molecule assays of calmodulin target binding detected with a calmodulin energy-transfer construct

We have detected single-molecule binding interactions of a target peptide with the calcium-signaling protein calmodulin (CaM) immobilized in an agarose gel, and we have demonstrated the application of a single-molecule binding assay to measure the binding strength of CaM with the CaM-binding domain of calmodulin-dependent protein kinase II (CaMKII). The results demonstrate the potential for ultrasensitive assays of CaM-target interactions and the measurement of a picomolar dissociation constant. To detect single-molecule protein interactions, single-molecule assays require that the analyte molecule be confined to the focal spot of the objective for the time scale of the measurement. We demonstrate the deleterious effect of surface immobilization on CaM. As an alternative to surface immobilization, we have constructed a CaM/maltose binding protein fusion protein, which renders CaM translationally immobile in a low weight percent agarose gel. The target binding functionality of CaM assayed in agarose gels is in good agreement with solution assays. The utility of the construct for detecting interactions with CaM targets was demonstrated in a single-molecule assay of binding interactions of MBP-CaM with the CaMKII CaM-binding domain peptide. A value of 103 +/- 35 pM for the dissociation constant of this interaction was determined by simple counting of fluorescent molecules.     

Variable time-delay estimation for anesthesia control during intensive care

The presence of artifacts plays a crucial role in automatic sedation systems and may introduce variable time delays (TDs) in the closed-loop-control structures. This paper presents a successful procedure to estimate the varying TD of the bispectral index (BIS) monitor used in closed-loop control during intensive care. The TD estimation (TDE) is based on the cross-correlation analysis technique and the method is validated with real measured signals of propofol and BIS. Extended prediction self-adaptive control is used in combination with a Smith predictor to reduce the computational burden imposed by the variable TD. The conclusion is that an online TDE of the BIS monitor improves the performance of the closed-loop system for reference tracking, disturbance rejection, and overall stability.     

Pre-natal social stress and post-natal pain affect the developing pig reproductive axis

This study assessed the effect of pre-natal social stress and post-natal pain on the reproductive development of young (approximately day 40) pigs. Male pigs carried by sows that were stressed by mixing with unfamiliar older sows for two 1-week periods during mid-pregnancy had lower plasma testosterone (0.54 vs 0.86 ng/ml, S.E.D.=0.11; P=0.014) and oestradiol (E(2); 22.9 vs 38.7 pg/ml, S.E.D.=7.80; P=0.021) concentrations compared with males carried by unstressed control sows. Although there was no effect of pre-natal stress on female E(2) concentrations, female pigs carried by stressed sows had fewer primordial ovarian follicles (log -4.32/μm(2) vs -4.00/μm(2), s.e.d.=0.136; P=0.027). Tail amputation on day 3 after birth reduced E(2) concentrations in female (4.78 vs 6.84 pg/ml, s.e.d.=0.86; P=0.03) and in male (25.6 vs 34.9 pg/ml, S.E.D.=3.56; P=0.021) pigs and reduced both testis weight (0.09% of body weight vs 0.10% of body weight, S.E.D.=0.003; P=0.01) and the percentage of proliferating Leydig cells (1.97 vs 2.12, S.E.D.=0.114; P=0.036) compared with sham-amputated littermate controls. There was a significant (P=0.036) interaction between the effects of pre-natal stress and post-natal pain on testicular expression of the steroidogenic enzyme 17α-hydroxylase, such that amputation increased expression in pigs born to control sows, but reduced expression in animals born to stressed sows. This study shows that stressful procedures associated with routine animal husbandry can disrupt the developing reproductive axis.