Tiamulin-Resistant Mutants of the Thermophilic Bacterium \u3cem\u3eThermus thermophilus\u3c/em\u3e

Tiamulin is a semisynthetic pleuromutilin antibiotic that binds to the 50S ribosomal subunit A site and whose (((2-diethylamino)ethyl)thio)-acetic acid tail extends into the P site to interfere with peptide bond formation. We have isolated spontaneous tiamulin-resistant mutants of the thermophilic bacterium Thermus thermophilus, containing either single amino acid substitutions in ribosomal protein uL3 or single base substitutions in the peptidyltransferase active site of 23S rRNA. These mutations are consistent with those found in other organisms and are in close proximity to the crystallographically determined tiamulin binding site. We also conducted a cross-resistance analysis of nine other single-base substitutions in or near the peptidyltransferase active site, previously selected for resistance to structurally unrelated antibiotics. While some of the base substitutions in 23S rRNA are positioned to directly affect tiamulin-ribosome contacts, others are some distance from the tiamulin binding site, indicating an indirect mechanism of resistance. Similarly, amino acid substitutions in uL3 are predicted to act indirectly by destabilizing rRNA conformation in the active site. We interpret these observations in light of the available ribosome X-ray crystal structures. These results provide a more comprehensive profile of tiamulin resistance caused by mutations in the bacterial ribosome

Development of Lightweight Refractory Composites Based on Aluminous Cement

Vývoj nových kompozitních materiálů je celosvětově mimořádně progresivní odvětví inženýrské činnosti. Principem kompozitů je kombinace rozdílných materiálů, kterou vzniká zcela nový materiál se specifickými vlastnostmi. Tato disertační práce přispívá do kategorie experimentálního výzkumu a vývoje speciálních kompozitů. Snahou této studie je příprava lehčených žárovzdorných kompozitů s dobrými mechanickými vlastnostmi, které by vedly k technicky ekonomickým řešením. Oheň patří k jednomu z nejnebezpečnějších aspektů betonových konstrukcí. Pokud je beton vystaven vysokým teplotám, jeho mechanické chování se dramaticky mění s nárůstem teploty. Důležitá není pouze maximální teplota, ale také doba, po kterou je konstrukce vystavena extrémní teplotě, a rychlost, s jakou teplota stoupá. To vše může vést k explozivnímu odprýskávání betonu s vážnými důsledky pro konstrukci a osoby v její blízkosti. Účelem navrhovaných kompozitů je působit jako tepelná bariéra ve volitelném tvaru a snížit tak přenos tepla z vysokoteplotního zdroje. Křemičité kamenivo a cementové kompozity na bázi portlandského cementu nejsou schopny odolat účinkům vysokých teplot, proto směsi v tomto experimentu jsou navrženy na bázi hlinitanového cementu a obsahují pouze složky, které odolávají vysokým teplotám. Pro účel výzkumu byly připraveny malé hranolové vzorky vystavené teplotám 105, 400, 600 a 1000 °C. Výstupem této práce je analýza vlivu žáruvzdorných komponentů a jejich reakce na postupné teplotní zatížení. Fyzikálními a mechanickými zkouškami byly zkoumány a vyhodnoceny různé kombinace lehčeného kameniva, provzdušňovací přísady a vláken v navržených směsích. Experimentální výsledky prokázaly pozitivní účinek použitých materiálů v žáruvzdorné kompozici. Mechanické vlastnosti navržených kompozitů dosáhly hodnot vysoké kvality, též i další užitné vlastnosti. Ani při zvýšené teplotě 1 000 °C nedošlo k nepříznivým účinkům, jako je zmíněné explozivní odprýskávání.The development of new composite materials is a worldwide extremely progressive branch of engineering activity. The principle of composite materials is a combination of different materials providing an entirely new material with specific properties. The effort of the present thesis is the preparation of lightweight refractory composites with good mechanical properties, which would reach technically economical solutions. Fire belongs to one of the most dangerous aspects of concrete structures. If concrete is subject to high temperatures, its mechanical behavior changes dramatically with the increase in temperature. It is not only the maximum temperature that is important, but also the time at which the structure is exposed to the extreme temperature and the rate at which the temperature rises. All that can lead to explosive spalling with serious consequences to the structure and people. The purpose of the proposed composite is to act as a thermal barrier in an optional form and reduce the heat transfer from a high-temperature source. Silica composites based on Portland cement and silica aggregates are not able to resist the effects of high temperatures, therefore, the mixtures in this experiment include only components that can resist high temperatures. For the experimental program, small prismatic specimens based on aluminous cement were prepared and exposed to temperatures of 105, 400, 600, and 1000 °C. One output of this study is the analysis of the influence of refractory compositions and their response to gradual temperature loading. Various compositions of lightweight aggregates, air-entraining additives, and fibers were investigated and evaluated by physical and mechanical testing. Experimental results have shown a positive effect of the used materials in a refractory composition. The mechanical properties of the designed composites have achieved values of high quality, as well as other utility properties. No adverse effects, such as explosive spalling, occurred even when the temperature had been increased to 1000 °C

Expectation-Complete Graph Representations with Homomorphisms

We investigate novel random graph embeddings that can be computed in expected polynomial time and that are able to distinguish all non-isomorphic graphs in expectation. Previous graph embeddings have limited expressiveness and either cannot distinguish all graphs or cannot be computed efficiently for every graph. To be able to approximate arbitrary functions on graphs, we are interested in efficient alternatives that become arbitrarily expressive with increasing resources. Our approach is based on Lov\'asz' characterisation of graph isomorphism through an infinite dimensional vector of homomorphism counts. Our empirical evaluation shows competitive results on several benchmark graph learning tasks.Comment: accepted for publication at ICML 202

Mechanistic insight into acrylate metabolism and detoxification in marine dimethylsulfoniopropionate-catabolizing bacteria

Dimethylsulfoniopropionate (DMSP) cleavage, yielding dimethyl sulfide (DMS) and acrylate, provides vital carbon sources to marine bacteria, is a key component of the global sulfur cycle and effects atmospheric chemistry and potentially climate. Acrylate and its metabolite acryloyl-CoA are toxic if allowed to accumulate within cells. Thus, organisms cleaving DMSP require effective systems for both the utilization and detoxification of acrylate. Here, we examine the mechanism of acrylate utilization and detoxification in Roseobacters. We propose propionate-CoA ligase (PrpE) and acryloyl-CoA reductase (AcuI) as the key enzymes involved and through structural and mutagenesis analyses, provide explanations of their catalytic mechanisms. In most cases, DMSP lyases and DMSP demethylases (DmdAs) have low substrate affinities, but AcuIs have very high substrate affinities, suggesting that an effective detoxification system for acylate catabolism exists in DMSP-catabolizing Roseobacters. This study provides insight on acrylate metabolism and detoxification and a possible explanation for the high Km values that have been noted for some DMSP lyases. Since acrylate/acryloyl-CoA is probably produced by other metabolism, and AcuI and PrpE are conserved in many organisms across all domains of life, the detoxification system is likely relevant to many metabolic processes and environments beyond DMSP catabolism

The ER-alpha mutation Y537S confers Tamoxifen-resistance via enhanced mitochondrial metabolism, glycolysis and Rho-GDI/PTEN signaling : implicating TIGAR in somatic resistance to endocrine therapy

Naturally-occurring somatic mutations in the estrogen receptor gene (ESR1) have been previously implicated in the clinical development of resistance to hormonal therapies, such as Tamoxifen. For example, the somatic mutation Y537S has been specifically associated with acquired endocrine resistance. Briefly, we recombinantly-transduced MCF7 cells with a lentiviral vector encoding ESR1 (Y537S). As a first step, we confirmed that MCF7-Y537S cells are indeed functionally resistant to Tamoxifen, as compared with vector alone controls. Importantly, further phenotypic characterization of Y537S cells revealed that they show increased resistance to Tamoxifen-induced apoptosis, allowing them to form mammospheres with higher efficiency, in the presence of Tamoxifen. Similarly, Y537S cells had elevated basal levels of ALDH activity, a marker of "stemness", which was also Tamoxifen-resistant. Metabolic flux analysis of Y537S cells revealed a hyper-metabolic phenotype, with significantly increased mitochondrial respiration and high ATP production, as well as enhanced aerobic glycolysis. Finally, to understand which molecular signaling pathways that may be hyper-activated in Y537S cells, we performed unbiased label-free proteomics analysis. Our results indicate that TIGAR over-expression and the Rho-GDI/PTEN signaling pathway appear to be selectively activated by the Y537S mutation. Remarkably, this profile is nearly identical in MCF7-TAMR cells; these cells were independently-generated , suggesting a highly conserved mechanism underlying Tamoxifen-resistance. Importantly, we show that the Y537S mutation is specifically associated with the over-expression of a number of protein markers of poor clinical outcome (COL6A3, ERBB2, STAT3, AFP, TFF1, CDK4 and CD44). In summary, we have uncovered a novel metabolic mechanism leading to endocrine resistance, which may have important clinical implications for improving patient outcomes

The Expressive Power of Path-Based Graph Neural Networks

We systematically investigate the expressive power of path-based graph neural networks. While it has been shown that path-based graph neural networks can achieve strong empirical results, an investigation into their expressive power is lacking. Therefore, we propose PATH-WL, a general class of color refinement algorithms based on paths and shortest path distance information. We show that PATH-WL is incomparable to a wide range of expressive graph neural networks, can count cycles, and achieves strong empirical results on the notoriously difficult family of strongly regular graphs. Our theoretical results indicate that PATH-WL forms a new hierarchy of highly expressive graph neural networks

Mechanistic insight into acrylate metabolism and detoxification in marine dimethylsulfoniopropionate-catabolizing bacteria

Dimethylsulfoniopropionate (DMSP) cleavage, yielding dimethyl sulfide (DMS) and acrylate, provides vital carbon sources to marine bacteria, is a key component of the global sulfur cycle and effects atmospheric chemistry and potentially climate. Acrylate and its metabolite acryloyl-CoA are toxic if allowed to accumulate within cells. Thus, organisms cleaving DMSP require effective systems for both the utilization and detoxification of acrylate. Here, we examine the mechanism of acrylate utilization and detoxification in Roseobacters. We propose propionate-CoA ligase (PrpE) and acryloyl-CoA reductase (AcuI) as the key enzymes involved and through structural and mutagenesis analyses, provide explanations of their catalytic mechanisms. In most cases, DMSP lyases and DMSP demethylases (DmdAs) have low substrate affinities, but AcuIs have very high substrate affinities, suggesting that an effective detoxification system for acylate catabolism exists in DMSP-catabolizing Roseobacters. This study provides insight on acrylate metabolism and detoxification and a possible explanation for the high Km values that have been noted for some DMSP lyases. Since acrylate/acryloyl-CoA is probably produced by other metabolism, and AcuI and PrpE are conserved in many organisms across all domains of life, the detoxification system is likely relevant to many metabolic processes and environments beyond DMSP catabolism

Proteomic analysis of nitrate-dependent acetone degradation by Alicycliphilus denitrificans strain BC

Alicycliphilus denitrificans strain BC grows anaerobically on acetone with nitrate as electron acceptor. Comparative proteomics of cultures of A. denitrificans strain BC grown on either acetone or acetate with nitrate was performed to study the enzymes involved in the acetone degradation pathway. In the proposed acetone degradation pathway, an acetone carboxylase converts acetone to acetoacetate, an AMP-dependent synthetase/ligase converts acetoacetate to acetoacetyl-CoA, and an acetyl-CoA acetyltransferase cleaves acetoacetyl-CoA to two acetyl-CoA. We also found a putative aldehyde dehydrogenase associated with acetone degradation. This enzyme functioned as a -hydroxybutyrate dehydrogenase catalyzing the conversion of surplus acetoacetate to -hydroxybutyrate that may be converted to the energy and carbon storage compound, poly--hydroxybutyrate. Accordingly, we confirmed the formation of poly-?-hydroxybutyrate in acetone-grown cells of strain BC. Our findings provide insight in nitrate-dependent acetone degradation that is activated by carboxylation of acetone. This will aid studies of similar pathways found in other microorganisms degrading acetone with nitrate or sulfate as electron acceptor.This work was supported by the Technology Foundation, the Applied Science Division (STW) of the Netherlands Organization for Scientific Research (NWO) [project 08053]. Additional funding was provided by BE-BASIC [grant F08.004.01 to SA], an ERC grant [project 323009 to AJMS] and the Gravitation grant [project 024.002.002 to AJMS] of the Netherlands Ministry of Education, Culture and Science and NWO

Expectation-Complete Graph Representations with Homomorphisms

We investigate novel random graph embeddings that can be computed in expected polynomial time and that are able to distinguish all non-isomorphic graphs in expectation. Previous graph embeddings have limited expressiveness and either cannot distinguish all graphs or cannot be computed efficiently for every graph. To be able to approximate arbitrary functions on graphs, we are interested in efficient alternatives that become arbitrarily expressive with increasing resources. Our approach is based on Lov\'asz' characterisation of graph isomorphism through an infinite dimensional vector of homomorphism counts. Our empirical evaluation shows competitive results on several benchmark graph learning tasks

Physical and Mechanical Properties of Composites Made with Aluminous Cement and Basalt Fibers Developed for High Temperature Application

Present paper deals with the experimental study of the composition of refractory fiber-reinforced aluminous cement based composites and its response to gradual thermal loading. Basalt fibers were applied in doses of 0.25, 0.5, 1.0, 2.0, and 4.0% in volume. Simultaneously, binder system based on the aluminous cement was modified by fine ground ceramic powder originated from the accurate ceramic blocks production. Ceramic powder was dosed as partial replacement of used cement of 5, 10, 15, 20, and 25%. Influence of composition changes was evaluated by the results of physical and mechanical testing; compressive strength, flexural strength, bulk density, and fracture energy were determined on the different levels of temperature loading. Increased dose of basalt fibers allows reaching expected higher values of fracture energy, but with respect to results of compressive and flexural strength determination as an optimal rate of basalt fibers dose was considered 0.25% in volume. Fine ground ceramic powder application led to extensive increase of residual mechanical parameters just up to replacement of 10%. Higher replacement of aluminous cement reduced final values of bulk density but kept mechanical properties on the level of mixtures without aluminous cement replacement