Essential role of glucose transporter GLUT3 for post-implantation embryonic development

Deletion of glucose transporter gene Slc2a3 (GLUT3) has previously been reported to result in embryonic lethality. Here, we define the exact time point of growth arrest and subsequent death of the embryo. Slc2a3−/− morulae and blastocysts developed normally, implanted in vivo, and formed egg-cylinder-stage embryos that appeared normal until day 6·0. At day 6·5, apoptosis was detected in the ectodermal cells of Slc2a3−/− embryos resulting in severe disorganization and growth retardation at day 7·5 and complete loss of embryos at day 12·5. GLUT3 was detected in placental cone, in the visceral ectoderm and in the mesoderm of 7·5-day-old wild-type embryos. Our data indicate that GLUT3 is essential for the development of early post-implanted embryos

Current applications and future potential for bioinorganic chemistry in the development of anticancer drugs

This review illustrates notable recent progress in the field of medicinal bioinorganic chemistry as many new approaches to the design of innovative metal-based anticancer drugs are emerging. Current research addressing the problems associated with platinum drugs has focused on other metal-based therapeutics that have different modes of action and on prodrug and targeting strategies in an effort to diminish the side-effects of cisplatin chemotherapy

An Unusual Ligand Coordination Gives Rise to a New Family of Rhodium Metalloinsertors with Improved Selectivity and Potency

Rhodium metalloinsertors are octahedral complexes that bind DNA mismatches with high affinity and specificity and exhibit unique cell-selective cytotoxicity, targeting mismatch repair (MMR)-deficient cells over MMR-proficient cells. Here we describe a new generation of metalloinsertors with enhanced biological potency and selectivity, in which the complexes show Rh–O coordination. In particular, it has been found that both Δ- and Λ-[Rh(chrysi)(phen)(DPE)]2+ (where chrysi =5,6 chrysenequinone diimmine, phen =1,10-phenanthroline, and DPE = 1,1-di(pyridine-2-yl)ethan-1-ol) bind to DNA containing a single CC mismatch with similar affinities and without racemization. This is in direct contrast with previous metalloinsertors and suggests a possible different binding disposition for these complexes in the mismatch site. We ascribe this difference to the higher pK_a of the coordinated immine of the chrysi ligand in these complexes, so that the complexes must insert into the DNA helix with the inserting ligand in a buckled orientation; spectroscopic studies in the presence and absence of DNA along with the crystal structure of the complex without DNA support this assignment. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than cisplatin and N-methyl-N′-nitro-nitrosoguanidine (MNNG, a common DNA-alkylating chemotherapeutic agent). Moreover, the activities of the new metalloinsertors are coupled with high levels of selective cytotoxicity for MMR-deficient versus proficient colorectal cancer cells

Quantifying electron fluxes in methanogenic microbial communities

Anaerobic digestion is a widely applied process in which close interactions between different microbial groups result in the formation of renewable energy in the form of biogas. Nevertheless, the regulatory mechanisms of the electron transfer between acetogenic bacteria and methanogenic archaea in the final steps of the anaerobic digestion process are not fully understood. The electron flux of each syntrophic partner is defined as the product of the biomass-specific electron transfer rate and the individual biomass concentration. Therefore, to investigate how these biomass-specific electron fluxes are regulated, individual biomass concentrations need to be determined. So far, the lack of experimental techniques has posed a major obstacle to measuring individual biomass concentrations and thus our quantitative understanding of interspecies electron transfer in methanogenic environments remained limited. Novel molecular tools hold the promise for an accurate determination of individual biomass concentrations. This thesis aimed to elucidate the kinetic and thermodynamic control mechanisms of interspecies hydrogen transfer in defined and non-defined methanogenic communities. For this to achieve, interspecies electron fluxes were quantified by a combination of direct measurement and modelling. The chapters of this thesis present advances in the measurement of individual biomass concentrations and address important questions on energy sharing in syntrophic communities and the principles of microbial survival at the thermodynamic limits of life. Chapter 2 describes a novel qPCR method to quantify individual biomass concentrations in a syntrophic methanogenic coculture of Desulfovibrio sp. G11 and Methanospirillum hungatei JF1. Existing qPCR methods usually rely on several conversion factors such as gene copy numbers of the cell and cell concentrations, introducing potential error sources and inaccuracies. The presented qPCR approach, in contrast, benefits from the direct correlation of the qPCR signal to the individual biomass concentrations and thus from higher accuracy. The accurate measurement of individual biomass concentrations in syntrophic methanogenic communities finally allows to validate biomass-specific conversion rates and to improve the description of anaerobic systems. In Chapter 3, the kinetic and thermodynamic effect of an imposed change in the hydrogen partial pressure on lactate conversion was studied using the syntrophic coculture of Desulfovibrio sp. G11 and Methanobrevibacter arboriphilus DH1. The biomass-specific lactate consumption rate increased three-fold as the hydrogen partial pressures decreased from 1200 ppm to 250 ppm. Since all partial reactions were exergonic, the observed inhibitory effect of hydrogen on lactate conversion was not due to thermodynamics but was rather an effect of reaction kinetics. An adequate consideration of mass-transfer phenomena allowed for the determination of several kinetic parameters including the 50 % hydrogen inhibition constant of lactate conversion (KiH2,Lacox), the maximum biomass-specific lactate consumption rate of Desulfovibrio sp. G11 (qLac,max) and the affinity constant for hydrogen uptake of Methanobrevibacter arboriphilus DH1 (KS,H2). Both, the KS,H2 and KiH2,Lacox, were considerably higher than the hydrogen partial pressure prevailing during syntrophic lactate degradation. These results demonstrate that the acetogen was operating at qLac,max whereas the hydrogenotrophic methanogen was working far below its maximum capacity due to hydrogen limitation. The tight coupling of syntrophic partner organisms suggests that these microbial consortia are susceptible to small environmental changes. However, the overcapacity of hydrogenotrophic methanogens reflects the actual robustness of methanogenic ecosystems and shows first indication that coculture growth is rather restricted by the acetogen and not the methanogen. In Chapter 4, the novel qPCR approach described in Chapter 2 was applied for the direct measurement of individual biomass concentrations in the syntrophic coculture of Desulfovibrio sp. G11 and Methanospirillum hungatei JF1 grown on lactate and formate. This methodology enabled not only to accurately validate model-derived biomass-specific rates and growth yields, but also to prove that the acetogen is the growth-limiting partner during syntrophic lactate conversion. The observed overcapacity of hydrogenotrophic methanogens reflects the robustness of syntrophic methanogenic communities which is in contrast to the expectation that these consortia are susceptible even to slight imbalances. In addition, the measurement of biomass-specific rates revealed different growth strategies of the syntrophic partners during syntrophic lactate conversion. The biomass-specific electron transfer rate of the hydrogenotrophic methanogen was three-fold higher compared to its acetogenic partner. This is due to the low methanogenic biomass yield per electron mole of substrate which is determined by thermodynamics. In Chapter 5, the kinetic and thermodynamic control mechanisms of electron transfer were investigated using chemostat grown non-defined methanogenic enrichments on butyrate and ethanol. It was shown that elevated hydrogen partial pressures have an inhibitory effect on butyrate and ethanol conversion. Compared to the Anaerobic Digestion Model No. 1, a ten times lower hydrogen inhibition constant on butyrate conversion was found in this study, indicating a much stronger inhibition by hydrogen. The distinct microbial groups of the enrichment followed different growth strategies during syntrophic butyrate and ethanol conversion, which is in line with previous studies in a syntrophic coculture on lactate (Chapter 4). The hydrogenotrophic methanogens exhibited a 2-fold higher biomass-specific electron transfer rate compared to the butyrate-utilizing acetogen. The overcapacity of hydrogenotrophic methanogens previously observed in defined cocultures on lactate (Chapter 3 and 4), was also noted in the non-defined enrichments on butyrate and ethanol (Chapter 5). These results significantly contribute to a better understanding of the regulatory mechanisms in anaerobic digestion processes, implying that syntrophic methanogenic ecosystems are not as easily affected by environmental perturbations as previously believed. In addition to these kinetic limitations, Chapter 5 demonstrated the need to consider thermodynamic restrictions during syntrophic butyrate conversion, since the biomass-specific butyrate consumption rate (qBut) decreased significantly and remained close to zero when anaerobic butyrate conversion became endergonic. More insight was gained on how the syntrophic partner organisms share the little energy available in anaerobic methanogenic ecosystems. During syntrophic butyrate conversion, an unequal energy distribution between the butyrate-utilizing species (17 %), the hydrogenotrophic methanogens (9 – 10 %) and the acetoclastic methanogens (73 – 74 %) was found. These findings are consistent with previous coculture studies on lactate where the smallest fraction of the total energy (17 – 21 %) was attributed to the hydrogenotrophic methanogen (Chapter 3 and 4). As a result, hydrogenotrophic methanogens showed a low biomass yield that requires a large qe to equalize specific growth rates in the coculture. The observed growth strategies are a direct consequence of energy disproportion and illustrate the impact of thermodynamics on growth kinetics. As another interesting observation, butanol was formed at bicarbonate limiting conditions of hydrogenotrophic methanogenesis and increasing hydrogen partial pressures (> 390 ppm). These observations indicate that the hydrogen partial pressure may not only play a key role in the kinetic and thermodynamic regulation of syntrophic methanogenic conversions, but is also of great importance for shifting the electron fluxes towards reduced product formation.BiotechnologyApplied Science

Kinetic and thermodynamic control of butyrate conversion in non-defined methanogenic communities

Many anaerobic conversions proceed close to thermodynamic equilibrium and the microbial groups involved need to share their low energy budget to survive at the thermodynamic boundary of life. This study aimed to investigate the kinetic and thermodynamic control mechanisms of the electron transfer during syntrophic butyrate conversion in non-defined methanogenic communities. Despite the rather low energy content of butyrate, results demonstrate unequal energy sharing between the butyrate-utilizing species (17 %), the hydrogenotrophic methanogens (9–10 %), and the acetoclastic methanogens (73–74 %). As a key finding, the energy disproportion resulted in different growth strategies of the syntrophic partners. Compared to the butyrate-utilizing partner, the hydrogenotrophic methanogens compensated their lower biomass yield per mole of electrons transferred with a 2-fold higher biomass-specific electron transfer rate. Apart from these thermodynamic control mechanisms, experiments revealed a ten times lower hydrogen inhibition constant on butyrate conversion than proposed by the Anaerobic Digestion Model No. 1, suggesting a much stronger inhibitory effect of hydrogen on anaerobic butyrate conversion. At hydrogen partial pressures exceeding 40 Pa and at bicarbonate limited conditions, a shift from methanogenesis to reduced product formation was observed which indicates an important role of the hydrogen partial pressure in redirecting electron fluxes towards reduced products such as butanol. The findings of this study demonstrate that a careful consideration of thermodynamics and kinetics is required to advance our current understanding of flux regulation in energy-limited syntrophic ecosystems.BT/BiotechnologyApplied Science

Absolute Quantification of Individual Biomass Concentrations in a Methanogenic Coculture

Identification of individual biomass concentrations is a crucial step towards an improved understanding of anaerobic digestion processes and mixed microbial conversions in general. The knowledge of individual biomass concentrations allows for the calculation of biomass specific conversion rates which form the basis of anaerobic digestion models. Only few attempts addressed the absolute quantification of individual biomass concentrations in methanogenic microbial ecosystems which has so far impaired the calculation of biomass specific conversion rates and thus model validation. This study proposes a quantitative PCR (qPCR) approach for the direct determination of individual biomass concentrations in methanogenic microbial associations by correlating the native qPCR signal (cycle threshold, Ct) to individual biomass concentrations (mg dry matter/L). Unlike existing methods, the proposed approach circumvents error-prone conversion factors that are typically used to convert gene copy numbers or cell concentrations into actual biomass concentrations. The newly developed method was assessed and deemed suitable for the determination of individual biomass concentrations in a defined coculture of Desulfovibrio sp. G11 and Methanospirillum hungatei JF1. The obtained calibration curves showed high accuracy, indicating that the new approach is well suited for any engineering applications where the knowledge of individual biomass concentrations is required.BT/BiotechnologyApplied Science