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A bicyclic peptide scaffold was chemically adapted to generate diarylethene‐based photoswitchable inhibitors of serine protease Bos taurus trypsin 1 (T1). Starting from a prototype molecule—sunflower trypsin inhibitor‐1 (SFTI‐1)—we obtained light‐controllable inhibitors of T1 with Ki in the low nanomolar range, whose activity could be modulated over 20‐fold by irradiation. The inhibitory potency as well as resistance to proteolytic degradation were systematically studied on a series of 17 SFTI‐1 analogues. The hydrogen bond network that stabilizes the structure of inhibitors and possibly the enzyme–inhibitor binding dynamics were affected by isomerization of the photoswitch. The feasibility of manipulating enzyme activity in time and space was demonstrated by controlled digestion of gelatin‐based hydrogel and an antimicrobial peptide BP100‐RW. Finally, our design principles of diarylethene photoswitches are shown to apply also for the development of other serine protease inhibitors. [ABSTRACT FROM AUTHOR]

Ein bizyklisches Peptid wurde mit einem Diarylethen modifiziert, um lichtschaltbare Inhibitoren der Serinprotease Bos taurus Trypsin 1 (T1) zu erzeugen. Ausgehend von dem Sonnenblumen‐Trypsin‐Inhibitor‐1 (SFTI‐1) als Prototyp, erhielten wir lichtschaltbare Inhibitoren von T1 mit Ki im niedrigen nanomolaren Bereich, deren Aktivität durch Bestrahlung mit Licht um das 20‐Fache moduliert werden konnte. Die inhibitorische Potenz sowie die Resistenz gegen proteolytischen Abbau wurden systematisch an einer Reihe von 17 SFTI‐1‐Analoga untersucht. Die Isomerisierung des Photoschalters beeinflusst das Netzwerk aus Wasserstoffbrücken, das zur Stabilisierung der Struktur der Inhibitoren dient, und möglicherweise auch die Bindungsdynamik zwischen Enzym und Inhibitor. Die Machbarkeit einer zeitlichen und räumlichen Manipulation der Enzymaktivität wurde durch kontrollierten Verdau eines Hydrogels auf Gelatinebasis und eines antimikrobiellen Peptids BP100‐RW bewiesen. Die angewendeten Designprinzipien für Diarylethen‐Photoschalter stellen eine Grundlage für die Entwicklung anderer Serinprotease‐Inhibitoren dar. [ABSTRACT FROM AUTHOR]

The focus of this paper is to describe and document the recent effort to validate the computer code Numerical Flow . Analysis (NFA) for the prediction of hydrodynamic forces and moments associated with deepv-V planing craft. This detailed validation effort was composed of three parts. The first part focused on assessing NFA's ability to predict pressures on the surface of a 10 degree deadrise wedge during impact with an undisturbed free surface. Detailed comparisons to pressure gauges are presented here for two different drop heights, 6 and 10 inches. Results show NFA accurately predicted pressures during the slamming event. The second part examines NFA's ability to match sinkage, trim and resistance from experiments performed on constant deadrise planing hulls, detailed in Fridsma (1969). Simulations were preformed on two 20 degree deadrise hullforms of varying length to beam ratios (4 and 5) over a range of speed-length ratios (2, 3, 4, 5, and 6). Results show good agreement with experimentally measured values, as well as values calculated using Savitsky's parametric equations, detailed in Savistsky (1964). The final part of the validation study focused on assessing how well NFA was able to accurately model the complex multiphase flow associated with high Froude number flows, specifically the formation of the spray sheet. NFA simulations of a planing hull fixed at Various angles of roll (0, 10, 20, and 30 degrees) were compared to experiments from Judge (2012). Underwater photographs illustrate NFA's ability to model the formation of the spray sheet and the free surface turbulence associated with planing boat hydrodynamics. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was the evaluation of prognostic factors for long-term survival and progression-free survival (PFS) after treatment of colorectal cancer (CRC) liver metastases with magnetic resonance-guided laser-induced interstital thermotherapy (LITT)., Patients and Methods: We included 594 patients (mean age, 61.2 years) with CRC liver metastases who were treated with LITT. The statistical analysis of the long-term survival and PFS were based on the Kaplan-Meier method. The Cox regression model tested different parameters that could be of prognostic value. The tested prognostic factors were the following: sex, age, the location of primary tumor, the number of metastases, the maximal diameter and total volume of metastases and necroses, the quotient of total volumes of metastases and necroses, the time of appearance of liver metastases and location in the liver, the TNM classification of CRC, extrahepatic metastases, and neoadjuvant treatments., Results: The median survival was 25 months starting from the date of the first LITT. The 1-, 2-, 3-, 4-, and 5-year survival rates were 78%, 50.1%, 28%, 16.4%, and 7.8%, respectively. The median PFS was 13 months. The 1-, 2-, 3-, 4-, and 5-year PFS rates were 51.3%, 35.4%, 30.7%, 25.4%, and 22.3%, respectively. The number of metastases and their maximal diameter were the most important prognostic factors for both long-term survival and PFS. Long-term survival was also highly influenced by the initial involvement of the lymph nodes., Conclusions: For patients treated with LITT for CRC liver metastases, the number and size of metastases, together with the initial lymph node status, are significant prognostic factors for long-term survival.

A cohort of 59 persons with industrial handling of low levels of acrylonitrile is being studied as part of a medical surveillance programme. Previously, an extended haemoglobin adduct monitoring (N-(cyanoethyl)valine and N-(hydroxyethyl)-valine) was performed regarding the glutathione transferases hGSTM1 and hGSTT1 polymorphisms but no influence of hGSTM1 or hGSTT1 polymorphisms on specific adduct levels was found. A compilation of case reports of human accidental poisonings had pointed to significant individual differences in human acrylonitrile metabolism and toxicity. Therefore, a re-evaluation of the industrial cohort included known polymorphisms of the glutathione transferases hGSTM3 and hGSTP1 as well as of the cytochrome P450 CYP2E1. A detailed statistical analysis revealed that exposed carriers of the allelic variants of hGSTP1, hGSTP1*B/hGSTP1*C, characterized by a single nucleotide polymorphism at nucleotide 313 which results in a change from Ile to Val at codon 104, had higher levels of the acrylonitrile-specific haemoglobin adduct N-(cyanoethyl)valine compared to the carriers of the codon 113 alleles hGSTP1*A and hGSTP1*D. The single nucleotide polymorphism at codon 113 of hGSTP1 (hGSTP1*A/hGSTP1*B versus hGSTP1*C/hGSTP1*D) did not show an effect, and also no influence was seen on specific haemoglobin adduct levels of the polymorphisms of hGSTM3 or CYP2E1. The data, therefore, point to a possible influence of a human enzyme polymorphism of the GSTP1 gene at codon 104 on the detoxication of acrylonitrile which calls for experimental toxicological investigation. The study also confirmed the impact of GSTT1 polymorphism on background N-(hydroxyethyl)-valine adduct levels in haemoglobin which are caused by endogenous ethylene oxide.

A primary route of metabolism of dihalomethanes occurs via glutathione (GSH) transferase-catalyzed conjugation. Mammalian theta class GSH transferases and a group of bacterial dichloromethane dehalogenases are able to catalyze the hydrolytic dehalogenation of dihalomethanes via GSH conjugation and subsequent formation of HCHO. Dihalomethanes have been shown to induce revertants in Salmonella typhimurium TA 1535 expressing theta class GSH transferases. Two mammalian theta class GSH transferases (rat GST 5-5 and human GST T1) and the bacterial dehalogenase DM11 were compared in the in vitro conjugation of CH(3)Cl and using in vitro assays (HCHO formation) and the S. typhimurium mutagenesis assay with the dihalomethanes CH(2)Cl(2), CH(2)Br(2), CH(2)BrCl, CH(2)ICl, CH(2)I(2), and CH(2)ClF. GSTs 5-5 and T1 had similar characteristics and exhibited first-order rather than Michaelis-Menten kinetics for HCHO formation over the range of dihalomethane concentrations tested. In contrast, the DM11 enzyme displayed typical hyperbolic Michaelis-Menten kinetics for all of the compounds tested. A similar pattern was observed for the conjugation of CH(3)Cl. The reversion tests with S. typhimurium expressing DM11 or GST 5-5 showed a concentration-dependent increase in revertants for most of the dihalomethanes, and DM11 produced revertants at dihalomethane concentrations lower than GST 5-5. Collectively, the results indicate that rates of conversion of dihalomethanes to HCHO are not correlated with mutagenicity and that GSH conjugates are genotoxic. The results are compared with the conjugation and genotoxicity of haloethanes in the preceding paper in this issue [Wheeler, J. B., Stourman, N. V., Armstrong, R. N., and Guengerich, F. P. (2001) Chem. Res. Toxicol. 14, 1107-1117]. The halide order appears most important in the dihalomethane conjugation reactions catalyzed by GST 5-5 and less so in GST T1 and DM11, probably due to changes in the rate-limiting steps.

The polymorphism of human glutathione transferase hGSTT1-1 is expressed in three phenotypes. Experimentally, individuals can be classified as non-conjugators, low conjugators and 'high' conjugators depending on the enzyme activity in blood towards methylene chloride using a gas chromatographic assay. Non-conjugators do not have a functional hGSTT1 gene; however, little is known about the molecular basis of the three conjugator phenotypes. The higher hGSTT1-1 activity in high conjugators may be the result of enzyme induction or be genetically determined. Twenty-nine members of a large family, including three generations were phenotyped and genotyped with respect to hGSTT1-1. The hGSTT1-1 enzyme activity of high conjugators was twice as high as that of low conjugators. The distribution of hGSTT1-1 phenotypes strongly indicates a Mendelian intermediary inheritance, in which a gene-dosage effect results in a doubled enzyme expression in the presence of two functional alleles. The Mendelian intermediary inheritance is further supported by the findings of a semiquantitative polymerase chain reaction method designed to distinguish the three genotypes of hGSTT1 for rapid screening of large study groups.

Human hemolysate was incubated in vitro with different concentrations of dichloromethane (methylene chloride). The resulting enzymatically mediated production of formaldehyde was determined by two independent analytical methods (Nash-reaction/colorimetry or HPLC). The formation of formaldehyde from dichloromethane is influenced by the polymorphism of glutathione-S-transferase (GST) Theta, in the same way as the metabolism of methyl bromide, methyl chloride, methyl iodide and ethylene oxide. Three quarters of the population ("conjugators") possess, whereas one quarter ("non-conjugators") lack this enzyme activity in human erythrocytes. The metabolism of dichloromethane in hemolysate in vitro can be described by Michaelis-Menten kinetics; for an individual with high GST T1-1 enzyme activity, the maximum velocity of formaldehyde production was calculated to be approximately 180 pmol/min per mg Hb, the kM being approximately 60 mM dichloromethane. Carcinogenicity of dichloromethane in long-term inhalation exposure of rodents has been attributed to metabolism of the compound via the GST-dependent pathway. Extrapolation of the results to humans for risk assessment should consider the newly discovered polymorphic enzyme activity of GST Theta. Furthermore, the possible existence of a "high-risk" population among humans should be considered in epidemiological research.