Molecular Mechanisms of Antibiotic Resistance in Helicobacter pylori

An estimated 4 to 5 million individuals in the Netherlands are actively infected with Helicobacter pylori. Eradication of this bacterium becomes more difficult as the prevalence of antibiotic resistance is increasing worldwide. Most H. pylori infections are now diagnosed by non-invasive testing (i.e. urea breath test, serology, stool test), and thus data on antibiotic susceptibility are lacking. Furthermore, once the antibiotic susceptibility is assessed using conventional culture-based methods by means of an E-test, agar dilution or disc-diffusion, then data are difficult to compare between different centers due to lack of standardization. Molecular-based methods are reproducible and easily standardized, and thus they can offer an attractive alternative. To develop molecular-based methods knowledge of molecular mechanisms underlying antibiotic resistance is mandatory. The research presented in this thesis aims to obtain information on molecular mechanisms of antibiotic resistance in H. pylori

Toepassing van weefselkweek en embryogenese bij vermeerdering en veredeling van tulp

In dit rapport worden de resultaten beschreven van twee projecten waarin weekfselkweekmethoden voor vermeerderding en veredeling van tulp zijn onderzocht. Vermeerdering van tulp op het veld gaat erg langzaam waardoor het lang duurt voordat nieuwe cultivars op de markt kunnen worden gebracht. Vermeerdering m.b.v. weefselkweek kan dit aanzienlijk versnellen. Het protocol dat bij aanvang van de twee projecten beschikbaar was, is aanzienlijk verbeterd. In het eerste project werd nagegaan of somatische embryogenese (vorming van embryo's uit niet-geslachtscellen) toepasbaar is voor een sneller vermeerderingssysteem van tulp. Het tweede project draaide om het verbeteren van de vermeedering van tulp in vitro, uitgaande van scheutvorming op bloemstengelplakje

Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori

Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance

Gain-of-function Nav1.8 mutations in painful neuropathy

Painful peripheral neuropathy often occurs without apparent underlying cause. Gain-of-function variants of sodium channel Nav1.7 have recently been found in ~30% of cases of idiopathic painful small-fiber neuropathy. Here, we describe mutations in Nav1.8, another sodium channel that is specifically expressed in dorsal root ganglion (DRG) neurons and peripheral nerve axons, in patients with painful neuropathy. Seven Nav1.8 mutations were identified in 9 subjects within a series of 104 patients with painful predominantly small-fiber neuropathy. Three mutations met criteria for potential pathogenicity based on predictive algorithms and were assessed by voltage and current clamp. Functional profiling showed that two of these three Na v1.8 mutations enhance the channel's response to depolarization and produce hyperexcitability in DRG neurons. These observations suggest that mutations of Nav1.8 contribute to painful peripheral neuropathy

Network topology of NaV1.7 mutations in sodium channel-related painful disorders

Background: Gain-of-function mutations in SCN9A gene that encodes the voltage-gated sodium channel NaV1.7 have been associated with a wide spectrum of painful syndromes in humans including inherited erythromelalgia, paroxysmal extreme pain disorder and small fibre neuropathy. These mutations change the biophysical properties of NaV1.7 channels leading to hyperexcitability of dorsal root ganglion nociceptors and pain symptoms. There is a need for better understanding of how gain-of-function mutations alter the atomic structure of Nav1.7. Results: We used homology modeling to build an atomic model of NaV1.7 and a network-based theoretical approach, which can predict interatomic interactions and connectivity arrangements, to investigate how pain-related NaV1.7 mutations may alter specific interatomic bonds and cause connectivity rearrangement, compared to benign variants and polymorphisms. For each amino acid substitution, we calculated the topological parameters betweenness centrality (Bct), degree (D), clustering coefficient (CCct), closeness (Cct), and eccentricity (Ect), and calculated their variation (value= mutantvalue-WTvalue). Pathogenic NaV1.7 mutations showed significantly higher variation of |Bct| compared to benign variants and polymorphisms. Using the cut-off value \uc2\ub10.26 calculated by receiver operating curve analysis, we found that Bctcorrectly differentiated pathogenic NaV1.7 mutations from variants not causing biophysical abnormalities (nABN) and homologous SNPs (hSNPs) with 76% sensitivity and 83% specificity. Conclusions: Our in-silico analyses predict that pain-related pathogenic NaV1.7 mutations may affect the network topological properties of the protein and suggest |Bct| value as a potential in-silico marker

Micropropagation of flower bulbs : Lily and narcissus

For most bulbous crops, artificial (vegetative) propagation methods have been developed, such as scaling (lily), scooping (hyacinth), and chipping (narcissus). Because the speed of these methods is often low, introduction of newly bred cultivars (either produced by conventional breeding or by genetic modification) or of pathogen-free bulbs (produced by meristem culture) requires a long period of time. In tulip, for which no artificial propagation method exists, this can even take 20–25 yr. Micropropagation considerably shortens this period. Furthermore, because of the large number of propagation cycles in the field, conventionally produced bulbs may become easily infected. Micropropagation produces starting material that is completely or predominantly pathogen-free

Phase change in lily bulblets regenerated in vitro

During the development of the lily (Lilium), three phases can be distinguished: the juvenile, the vegetative adult and the flowering phase. Juvenile bulblets sprout with one or a few leaves whereas vegetative adult bulblets sprout with a stem with elongated internodes. The transition to the vegetative adult phase was studied in lily (Lilium x cv. Star Gazer) bulblets regenerating on bulb scale segments in vitro. The phase change was marked by the development of a tunica-corpus structure in the apical meristem which leads to the formation of an actively growing stem primordium. This structure is absent in juvenile bulblets. Juvenile bulblets first developed competence for phase change during a culture period of at least 6 weeks at 25degreesC. Subsequent induction of the phase change occurred during a period of 2 weeks at lower temperature (15degreesC). A major factor influencing phase transition was bulblet weight. Small bulblets never formed a stem whereas large bulblets always formed a stem under inducing conditions. Large bulblets more often formed a stem than small ones but the relation between bulb growth and phase transition was not absolute. A high sucrose concentration, a large explant and a prolonged period for competence development stimulated bulb growth but also phase transition independently of growth. Lowering the concentration of MS-minerals reduced bulb growth but did not affect phase transition. Under these conditions, phase change was correlated with a low phosphorus content

Contribution of explant carbohydrate reserves and sucrose in the medium to bulb growth of lily regenerated on scale segments in vitro

Bulb size is an important factor determining phase change in Lilium: phase change only occurs in bulblets over a certain threshold weight. After phase change has occurred, bulblets sprout with a stem with many leaves. Juvenile bulblets sprout with only a few leaves. The factors contributing to bulb size were studied during in vitro regeneration of bulblets on scale segments. The larger the explants, the larger the regenerated bulblets. Explant size influenced bulb growth during the complete culture period. Bulb growth was stimulated by a high sucrose concentration. The contribution of the medium and the explant reserves to bulb growth were studied in large and small explants using labelled sucrose. Sucrose was mainly taken up through the cut surfaces. In freshly cut explants, the rate of uptake was correlated with the size of the contact area, but at later stages, when regenerating organs were present, the difference in uptake rate of small and large explants almost disappeared. Small explants had a larger sink activity than large ones. Explants with regenerating organs took up more sucrose than freshly cut explants. Sucrose uptake and bulb growth were rather constant in the later phases and doubled when the sucrose concentration was doubled. Partitioning of label from the sucrose over the various organs, was also rather constant in time: approximately 25% was accumulated in the bulblets, 35-45% in the explant and 30-35% was converted to CO2. About half of the label in the explant was recovered at the proximal side where regeneration takes place. Sucrose, once incorporated in the storage pools in the explant, either remained in the explant or was converted to CO2. Redistribution to the growing bulblets hardly occurred. The percentage of bulb growth that could be attributed to uptake of medium components was constant over the regeneration period: 45-50% for large and 65-75% for small explants

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature ( 5 degreesC). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 degreesC or 15 degreesC, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales