Investigation of the Impact of Saccharides on the Relative Activity of Trypsin and Catalase after Droplet and Spray Drying.

While using saccharides as stabilizers for therapeutic protein drying is common, the mechanisms underlying the stabilization during drying remain largely unexplored. Herein, we investigated the effect of different saccharides, trehalose dihydrate (TD), dextran (DEX), and hydroxypropyl ß-cyclodextrins (low substitution-HP and high substitution-HPB), on the relative activities of the enzymes trypsin and catalase during miniaturized drying (MD) or spray drying (SD). For trypsin, the presence of saccharides, especially HP, was beneficial, as it significantly improved the enzyme activity following MD. The HPB preserved trypsin's activity during MD and SD. Adding saccharides during MD did not show a notable improvement in catalase activities. Increasing TD was beneficial during the SD of catalase, as indicated by significantly increased activity. Molecular docking and molecular dynamics simulations oftrypsin with HP or HPB revealed the influence of their substitution on the binding affinity for the enzyme. A higher affinity of HP to bind trypsin and itself was observed during simulations. Experimentally, activity reduction was mainly observed during MD, attributable to the higher droplet temperature during MD than during SD. The activities from the experiments and aggregation propensity from molecular modeling helped elucidate the impact of the size of protein and saccharides on preserving the activity during drying.

Bidirectional interaction between oral contraception and lamotrigine in women with epilepsy - Role of progestins.

PURPOSE: To investigate the effects of various progestins in combined oral contraceptives (COCs) on lamotrigine (LTG) serum concentrations and, vice versa, the potential impact of LTG on progestin serum levels during the menstrual cycle. METHODS: Twenty women with epilepsy (WWE) undergoing LTG monotherapy and COC (LTG group; mean ± SD [median; range] age 24.2 ± 4.6 [23.0; 18-37] years) as well as fourteen controls on COC (24.9 ± 5.6 [22.5; 20-39] years) were assessed for eligibility and all agreed to participate in the study and remained for data analyses. RESULTS: LTG levels differed significantly between phases of inactive pill and active pill use (p= 0.004), particularly with drospirenon (p= 0.018) and levonorgestrel (p= 0.068) as progestogen component but not with gestoden (p= 0.593). Furthermore, the LTG group showed significantly lower progestin levels during inactive pill when compared to active pill use with respect to levonorgestrel (p= 0.042) and drospirenon (p= 0.018) but not to gestoden (p= 0.109). Progestin concentrations did not differ between patients and controls (p> 0.05). CONCLUSIONS: The findings suggest that drospirenon and levonorgestrel but not gestoden seem to reduce LTG serum concentrations when being co-administered in WWE which might be of importance concerning seizure risk. Vice versa, no effect of LTG on several progestins could be demonstrated, arguing against a potential loss of contraception safety with LTG.

Targeting actin inhibits repair of doxorubicin-induced DNA damage: a novel therapeutic approach for combination therapy.

Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin.

Persistent inhibition of pore-based cell migration by sub-toxic doses of miuraenamide, an actin filament stabilizer.

Opposed to tubulin-binding agents, actin-binding small molecules have not yet become part of clinical tumor treatment, most likely due to the fear of general cytotoxicity. Addressing this problem, we investigated the long-term efficacy of sub-toxic doses of miuraenamide, an actin filament stabilizing natural compound, on tumor cell (SKOV3) migration. No cytotoxic effects or persistent morphological changes occurred at a concentration of miuraenamide of 20 nM. After 72 h treatment with this concentration, nuclear stiffness was increased, causing reduced migration through pores in a Boyden chamber, while cell migration and chemotaxis per se were unaltered. A concomitant time-resolved proteomic approach showed down regulation of a protein cluster after 56 h treatment. This cluster correlated best with the Wnt signaling pathway. A further analysis of the actin associated MRTF/SRF signaling showed a surprising reduction of SRF-regulated proteins. In contrast to acute effects of actin-binding compounds on actin at high concentrations, long-term low-dose treatment elicits much more subtle but still functionally relevant changes beyond simple destruction of the cytoskeleton. These range from biophysical parameters to regulation of protein expression, and may help to better understand the complex biology of actin, as well as to initiate alternative regimes for the testing of actin-targeting drugs.

Data Quality Improvement in Clinical Databases Using Statistical Quality Control: Review and Case Study.

Ensuring the quality of data being collected in clinical and medical contexts is a concern for data managers and users. Quality assurance frameworks, systematic audits, and correction procedures have been proposed to enhance the accuracy and completeness of databases. Following an overview of the undertaken approaches, particularly statistical methods, the authors promote acceptance sampling plans (ASPs) and statistical process control (SPC) tools, including control charts and root cause analysis, as the technical core of the data quality improvement mechanism. They review ASP and SPC techniques and discuss their implementation in data quality evaluation and improvement. Two case studies are presented in which the authors apply some of the techniques to databases maintained by a local hospital. Finally, guidelines are proposed for which techniques are appropriate with regard to dataflow and database specifications.

Comparison of a commercial urinary neopterin radioimmunoassay with high performance liquid chromatography.

BACKGROUND: The determination of urinary neopterin using high performance liquid chromatography (HPLC) has routinely been performed in Innsbruck since the middle of the 1980s. The aim was to compare the HPLC method with a commercially available radioimmunoassay. METHODS: Neopterin to creatinine ratios were calculated in the urine of 33 healthy volunteers (33±7.3 years) collected at eight given time points. Neopterin and creatinine were simultaneously determined by HPLC and by radioimmunoassay (RIA; BRAHMS; neopterin) and an enzymatic method (Roche; creatinine), respectively. Assays were compared with Bland-Altman plots and Passing-Bablok regression analysis. RESULTS: Neopterin to creatinine ratios ranged from 62 to 490 µmol/mol (HPLC) and from 17 to 425 µmol/mol (RIA-enzymatic combined). The ratios were on average 17.3% lower using the combined RIA-enzymatic method compared to the HPLC method. Neopterin concentrations measured by RIA were only slightly lower (on average 4.9%) than by the HPLC and the regression analysis revealed a good accordance with linearity. In contrast, creatinine concentrations by the enzymatic method were on average 12.8% higher than by HPLC. CONCLUSIONS: There is a marked bias between HPLC and the combined RIA-enzymatic method for measurement of urinary neopterin to creatinine ratios which has to be considered when methods are switched during the follow-up of patients.

Complexation of europium and uranium by humic acids analyzed by capillary electrophoresis-inductively coupled plasma mass spectrometry.

Investigations of the mobility of radioactive and nonradioactive substances in the environment are important tasks for the development of a future disposal in deep geological formations. Dissolved organic matter (DOM) can play an important role in the mobilization of metal ions due to complexation. In this study, we investigate the complexation behavior of humic acid (HA) as a model substance for DOM and its influence on the migration of europium as homologue for the actinide americium and uranium as the principal component of nuclear fuel. As speciation technique, capillary electrophoresis (CE) was hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). For the study, 0.5 mg·L⁻¹ of the metals and 25 mg·L⁻¹ of (purified Aldrich) HA and an aqueous solution sodium-perchlorate with an ionic strength of 10 mM at pH 5 were used. CE-ICP-MS clearly shows the different speciation of the triple positively charged europium and the double positively charged uranyl cation with HA.

Quantitative levonorgestrel plasma level measurements in patients with regular and prolonged use of the levonorgestrel-releasing intrauterine system.

BACKGROUND: The levonorgestrel-releasing intrauterine system (LNG-IUS) is well accepted as an easy-to-use contraceptive with an excellent side-effect profile. It contains a reservoir of 52 mg of levonorgestrel (LNG) with continuous release of the steroid. Its contraceptive use is approved for 5 years. The aim of this study was to determine the plasma concentration of LNG and its variation with time in patients with in-dwelling LNG-IUS Mirena®. STUDY DESIGN: In this study, we determined LNG plasma concentrations in 110 women with LNG-IUS at different time points of use. Time from insertion of the system in the study population ranged from 20 days to 11.1 years. Quantitative LNG levels were determined using a validated liquid chromatography-tandem mass spectrometry assay. RESULTS: The mean±SD LNG plasma level in all women was 147±59 pg/mL. A highly significant negative correlation between LNG plasma level and LNG-IUS time of use could be demonstrated. In the first year of use, LNG plasma level was as high as 191±71 pg/mL, decreasing to 157±68 pg/mL in the second year and 134±41 pg/mL in the third year. Even after exceeding the recommended period of LNG-IUS use, systemic LNG concentrations were detectable: 133±38 pg/mL in the sixth year, 133±48 pg/mL in the seventh year and 117±45 pg/mL in the eighth year. Furthermore, a significant negative correlation between LNG plasma level and body mass index could be shown. CONCLUSION: Systemic LNG concentrations can be found in all patients with LNG-IUS IUS. However, concentrations are much lower than in other forms of LNG application. Moreover, this study demonstrates that a systemic effect of LNG-IUS can also be found after the recommended contraceptive lifespan of 5 years.

Simultaneous online SPE-LC-MS/MS quantification of six widely used synthetic progestins in human plasma.

Co-administration of synthetic progestin containing hormonal contraceptives (HCs) and antiepileptic drugs (AEDs) is a common clinical situation which needs specific considerations due to drug interactions. Several studies have demonstrated that lamotrigine plasma levels are significantly decreased during co-medication with HCs, and that this interaction is associated with increased seizure frequency in most of the cases. Additionally, an increase in contraceptive failure and unintended pregnancy could be observed during co-medication. Hence, monitoring of progestin plasma levels in patients with AED co-medication is of interest. A rapid and reliable online solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (online SPE-LC-MS/MS) method using gradient elution in the LC domain was established and validated for the simultaneous quantitative determination of gestodene, dienogest, drospirenone, etonogestrel, cyproterone acetate, and levonorgestrel in human plasma. The online SPE-LC-MS/MS method covered a quantification concentration range of 5-100 ng/ml for dienogest, 1-100 ng/ml for etonogestrel and 2-100 ng/ml for all other analytes. Stable isotope-labeled internal standards were used for analyte quantification based on selected reaction monitoring experiments. Inter- and intra-assay precision and accuracy were determined from quality control (QC) samples at the lower limits of quantification and at low, medium, and high concentration levels within the calibration range. Inter-assay reproducibility at the QC levels was better than 10% (relative standard deviation, RSD), accuracy at these levels ranged from -3.7% to 11.3%. Total extraction efficiency, tested at three concentrations, ranged from 92.5% to 106.4%. Matrix interferences were excluded by post-column infusion experiments. To prove the applicability of the assay in clinical cohorts, a sample set (n = 298) stemming from study patients under AED/oral HC co-medication was screened for progestin plasma levels. This method has to be considered a research-use-only assay and must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the IVD directive (98/79/EG) of the European Community.

An ultra-sensitive online SPE-LC-MS/MS method for the quantification of levonorgestrel released from intrauterine devices.

A selective and sensitive liquid chromatography-tandem mass spectrometry method for the determination of very low levonorgestrel (D-(-)-norgestrel) serum levels such as those found in patients using levonorgestrel-releasing intrauterine devices (IUDs) was developed. To achieve the sub-nanomolar sensitivity needed to measure such serum levels, a diethyl ether extraction sample preparation protocol was applied prior to the online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) assay. Analyte quantification from the selected reaction monitoring experiments relied on the use of sixfold deuterated norgestrel as internal standard. The final method was linear up to 1.50 ng/ml with a lower limit of quantification (LLOQ) of 0.05 ng/ml. It was found to be precise and accurate with imprecision <8% and bias <6% assessed at three control levels. Total analyte recovery measured in patient pools at three concentration levels was found to exceed 92%. Matrix interferences were excluded by post-column analyte infusion experiments. As a proof of concept, a set of IUD patient serum samples was screened for their levonorgestrel content. A total of 97.5% (n = 94) of the samples did show serum levels exceeding the LLOQ, proving the applicability of the assay in relevant clinical cohorts. This method must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the in vitro diagnostic directive (98/79/EG) of the European community.

Cigarette smoke--an aging accelerator?

Cigarette smoking reduces life span by an average of 7 years, and tobacco consumption accounts for a shortening of disease free life by 14 years. The exact mechanisms by which smoking causes disease and death are generally not well understood, but evidence continues to mount that cigarette smoking exhausts cellular defense and repair functions, leading to an accumulation of damage e.g. mutations and malfunctioning proteins. In this review, we make an attempt to ascribe many of the deleterious effects of smoking on human health to a general principle, namely the acceleration of aging processes by cigarette smoke chemicals.