Application of nested PCR and mass spectrometry for DNA-based virus detection: HBV-DNA detected in the majority of isolated anti-HBc positive sera

DNA preparations from three different groups of serum samples were examined for HBV-DNA via a nested polymerase chain reaction assay (lower detection limit: 10 viral genomes in 100 microliters serum): Group I consisted of 11 uninfected control sera, group II consisted of sera obtained from 11 HBV infected patients and group III consisted of 21 isolated anti-HBc positive samples. The 21 samples from group III were HBV-DNA negative according to a conventional non-nested PCR assay and hybridization with a 32P-labelled probe. Using nested PCR and mass spectrometry, HBV-DNA was detected in none of group I and in all of group II samples. In 11 out of 21 (52%) of the isolated anti-HBc positive sera from group III, HBV-DNA was detected. No correlation was observed between HBV-DNA positivity and anti-HBc titers. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided a fast, sensitive and non-radioactive assay for the detection of PCR products without the need for gel electrophoresis or hybridization with labelled probes.     

Investigation of the presynaptic effects of quinine and quinidine on the release and uptake of monoamines in rat brain tissue

Quinine and quinidine are reported to potentiate the behavioural effects of serotonergic agents and monoamine uptake inhibitors. We have therefore investigated the presynaptic actions of quinine and quinidine on monoamine uptake and release in rat brain tissue in vitro. Quinidine evoked the release of [3H]5-HT, [3H]noradrenaline and [3H]dopamine from pre-loaded rat brain slices in a concentration dependent manner with EC50 values of 175, 486 and 150 microM, respectively. Quinine induced [3H]monoamine release with similar potencies. Both quinine and quinidine also inhibited the active uptake of [3H]5-HT, [3H]noradrenaline and [3H]dopamine into rat brain synaptosomes with IC50 values in the range 0.13-12.4 microM. The potency of each drug to inhibit [3H]5-HT uptake was significantly higher than that for [3H]noradrenaline or [3H]dopamine. The relative potency of quinidine compared to quinine was more marked in the case of [3H]5-HT (58-fold) than for [3H]noradrenaline (3-fold) or [3H]dopamine (4-fold). The inhibition of [3H]5-HT uptake by quinine and quinidine was competitive in nature and corresponded with the potencies of these drugs to inhibit [3H]paroxetine binding. No correlation was observed between the potencies of quinine and quinidine to induce the release of [3H]monoamines and to inhibit their uptake, suggesting that these effects are mediated by two distinct mechanisms. We conclude that the presynaptic actions of quinine and quinidine on monoamine uptake and release may be implicated in their potentiation of the effects of serotonergic agents and uptake blockers.     

Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of Candida spp. (50 C. albicans, 50 C. glabrata, 10 C. kefyr, 20 C. krusei, 10 C. lusitaniae, 20 C. parapsilosis, and 20 C. tropicalis isolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of C. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.     

A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity

A novel chitinase gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I-IV and are designated as class V chitinases. Comparison of the chitinase class V peptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratia marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinase class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Superose chromatography and gel filtration. In vitro assays demonstrated that class V chitinases have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitinase acts synergistically with tobacco class I beta-1,3-glucanase against Fusarium solani germlings.     

Using column generation to solve parallel machine scheduling problems with minmax objective functions

In this paper we consider the parallel machine scheduling problem of minimizing an objective function of the minmax type, like maximum lateness, subject to release dates, deadlines, and/or generalized precedence constraints. We use a destructive strategy to compute a lower bound. Here we test the feasibility of a decision problem by applying column generation to compute a bound on the number of machines that we need to feasibly accommodate all jobs. After having derived the lower bound, we try to find a matching upper bound by identifying a feasible schedule with objective function value equal to this lower bound. Our computational results show that our lower bound is so strong that this is almost always possible. We are able to solve problems with up to 160 jobs and 10 machines in 10 minutes on average.     

Finding a robust assignment of flights to gates at Amsterdam Airport Schiphol

In this paper we investigate the gate assignment problem as it appears at Amsterdam Airport Schiphol (AAS). Currently, the gate planners spend many hours on adjusting the automatically generated planning during the day of operation to make it proof against small deviations from the schedule. To alleviate this problem, we aim at finding a robust solution, given the planned arrivals and departures for the next day. We present a completely new integer linear programming formulation that is based on so-called gate plans. Each gate plan consists of a subset of the flights that can be assigned to a single gate of the corresponding type; gates with identical characteristics are aggregated in gate types. The gate assignment problem then boils down to selecting the best subset of gate plans such that each flight belongs to one selected gate plan, and such that the number of selected gate plans for a certain type of gate is equal to the number of gates of this type. In the first phase, we solve the LP-relaxation through column generation, and we describe specific features to find a very good solution to the ILP quickly. This solution is then handed to the planners at AAS in order to assign gate plans to physical gates. This consists of a number of relatively small problems that can be solved by hand and in which additional operational constraints can be incorporated. We also present the possibility of directly assigning flights to physical gates using the column generation formulation, where we then take into account other criteria as well. Computational results with real-life data provided by AAS are promising and indicate that the algorithm is able to solve real-life instances within rather small running times.     

Quantitative aspects of two methods to dissolve collagen-free muscle proteins from acetone dry powders of Guelders ring sausage

Summary The purpose of this study was to find experimental conditions for the complete solubility of collagen-free muscle proteins (CFMP) using acetone powder of Guelders ring sausage. Preliminary experiments were carried out to choose the best procedure for preparing the acetone dry powder. Two different methods of acetone extraction of minced sausage were compared. The acetone dry mass (ADM) method using continuous extraction in a Soxhlet [2] apparatus gave better results than the acetone powder (ACP) method, which used a blender [1]. The ADM method was used for further investigations. ADM was extracted with two types of sodium dodecyl sulphate (SDS), containing solvents A and B. Solvent A contains a Tris-boric acid buffer (pH 8.2) with 1.5% (m/v) SDS and 0.05% (m/v) dithioerythreitol [3]. Solvent B is a borate-chloric acid buffer (pH 9.0) with 2.0% (m/v) SDS and 1.0% (m/v) mercapto-ethanol [2]. Both solvents showed a linear relationship between the quantities of CFMP in ADM and the dissolved CFMP. The linear relationships were found between quantities of 10.0 and 30.0 mg (solution A) and of 5.0 and 30.0 mg ADM (solution B) per ml solvent. The solubility of CFMP was better in solvent B than in solution A. Completely dissolved CFMP from ADM was only obtained in the case of 5.0 mg ADM in 1.0 ml solution B. These conditions will be used in liquid chromatography experiments, the results of which will be reported later.

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Quantitative Aspekte zweier Verfahren für das Auflösen kollagenfreier Muskelproteine aus acetontrockenen Pulvern der Gelderschen Rauchwurst

Zusammenfassung Der Zweck dieser Untersuchungen ist, die experimentellen Bedingungen für die vollstän-dige Löslichkeit des kollagenfreien Muskelproteins (CFMP) aus dem Acetonpulver der Gelderschen Rauchwurst zu finden. Durch Vorversuche wurde die beste Arbeitsweise für das Zubereiten des Acetonpulvers gewählt. Zwei verschiedene Extraktionsverfahren mit zerkleinertem Wurstmaterial wurden miteinander verglichen. Die Methode mit der Acetontrockenmasse (ADM) mittels kontinuierlicher Extraktion [2] führte zu besseren Ergebnissen als die Methode mit Acetonpulver (ACP), wozu ein Mischgerät [1] verwendet wurde. Die ADM-Methode wurde für weitere Untersuchungen angewendet. ADM wurde mit zwei verschiedenen Extraktionslösungen von Natrium-Dodecylsulfat (SDS) (A und B) extrahiert. Lösung A enthalt einen Tris-Borsäure Puffer (pH 8,2) mit SDS (1,5%) und Dithioerithritol (0,05%) [3]. Die Lösung B enthält einen Borat-Salzsäure Puffer (pH 9,0) mit SDS (2,0%) und Mercapto-Ethanol (1,0%) [2]. Beide Extraktionslösungen zeigen ein lineares Verhalten zwischen den Mengen vom CFMP in ADM und in aufgelöstem CFMP. Diese Linearität wurde von 10,030,0 mg ADM (Lösungsmittel A) und von 5,030,0 mg ADM (Lösungsmittel B) gefunden. Die Lös-lichkeit in Lösung B ist gegeniiber Lösung A besser. Ein vollständig gelöstes CFMP aus ADM wurde nur bei der Extraktion von 5,0 mg ADM in 1,0 ml der Losung B erhalten. Diese Bedingung soll in unseren künftigen flüssigchromatographischen Experimenten verwendet werden.

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Supported by a grant from the Hoofdinspectie Levensmiddelen of the Ministry of Welzijn, Volksgezondheid en Cultuur     

The structure of a cast dental Ni-Cr-Be alloy

The structure of a typical dental Ni-Cr-Be alloy with 1.8 wt% Be has been investigated by SEM and TEM as well as by quantitative X-ray microanalyses in both instruments. Due to its low atomic weight the atomic fraction of Be is as high as 0.10. During solidification beryllium segregates substantially, and a large volume fraction of the casting is made up of a eutectic with coarse ( 1 µm diameter) alternating rods of fcc Ni-Cr and NiBe with a CsCl-type structure (ordered bcc). Smaller ( 0.1 µm diameter) rods of NiBe are precipitated in matrix in the solid state. Microanalyses of the NiBe rods show that they have a low chromium content ( 1.5 wt%). The cube boundary planes of the ordered b cc and fcc structures have a slight difference in orientation of about 7° which is most probably due to a small coherency misfit of the two types of lattices. The 100 directions in cube boundary plane of the fcc structure are nearly parallel to the 110 directions of the ordered bcc cube boundary plane. Sometimes another and more complex relationship between the two lattices occurs. The alloy contains 3.9 wt% Al which gives rise to numerous small ( 10 nm), spherical, ordered particles of Ni₃Al both in matrix as well as in the fcc eutectic rods.     

Exogenous Integrin αIIbβ3 Inhibitors Revisited: Past,Present and Future Applications

The integrin αIIbβ3 is the most abundant integrin on platelets. Upon platelet activation, the integrin changes its conformation (inside-out signalling) and outside-in signalling takes place leading to platelet spreading, platelet aggregation and thrombus formation. Bloodsucking parasites such as mosquitoes, leeches and ticks express anticoagulant and antiplatelet proteins, which represent major sources of lead compounds for the development of useful therapeutic agents for the treatment of haemostatic disorders or cardiovascular diseases. In addition to hematophagous parasites, snakes also possess anticoagulant and antiplatelet proteins in their salivary glands. Two snake venom proteins have been developed into two antiplatelet drugs that are currently used in the clinic. The group of proteins discussed in this review are disintegrins, low molecular weight integrin-binding cysteine-rich proteins, found in snakes, ticks, leeches, worms and horseflies. Finally, we highlight various oral antagonists, which have been tested in clinical trials but were discontinued due to an increase in mortality. No new αIIbβ3 inhibitors are developed since the approval of current platelet antagonists, and structure-function analysis of exogenous disintegrins could help find platelet antagonists with fewer adverse side effects.     

Molecular Genetics of Conjunctival Melanoma and Prognostic Value of TERT Promoter Mutation Analysis

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.