Route Of Assimilation Research Articles

Functional Degeneracy in Paracoccus denitrificans Pd1222 Is Coordinated via RamB, Which Links Expression of the Glyoxylate Cycle to Activity of the Ethylmalonyl-CoA Pathway.

Metabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. IMPORTANCE Carbon metabolism provides organisms with energy and building blocks for cellular functions and growth. The tight regulation between degradation and assimilation of carbon substrates is central for optimal growth. Understanding the underlying mechanisms of metabolic control in bacteria is of importance for applications in health (e.g., targeting of metabolic pathways with new antibiotics, development of resistances) and biotechnology (e.g., metabolic engineering, introduction of new-to-nature pathways). In this study, we use the alphaproteobacterium P. denitrificans as model organism to study functional degeneracy, a well-known phenomenon of bacteria to use the same carbon source through two different (competing) metabolic routes. We demonstrate that two seemingly degenerate central carbon metabolic pathways are metabolically and genetically linked with each other, which allows the organism to control the switch between them in a coordinated manner during growth. Our study elucidates the molecular basis of metabolic plasticity in central carbon metabolism, which improves our understanding of how bacterial metabolism is able to partition fluxes between anabolism and catabolism.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways for a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Building on this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-informed engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route—the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic P. putida auxotroph to rewire its metabolic network to restore C2 prototrophy via the PKT shunt. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff pathway and the synthetic PKT bypass for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

On the flexibility of the cellular amination network in E coli.

Ammonium (NH4+) is essential to generate the nitrogenous building blocks of life. It gets assimilated via the canonical biosynthetic routes to glutamate and is further distributed throughout metabolism via a network of transaminases. To study the flexibility of this network, we constructed an Escherichia coli glutamate auxotrophic strain. This strain allowed us to systematically study which amino acids serve as amine sources. We found that several amino acids complemented the auxotrophy either by producing glutamate via transamination reactions or by their conversion to glutamate. In this network, we identified aspartate transaminase AspC as a major connector between many amino acids and glutamate. Additionally, we extended the transaminase network by the amino acids β-alanine, alanine, glycine, and serine as new amine sources and identified d-amino acid dehydrogenase (DadA) as an intracellular amino acid sink removing substrates from transaminase reactions. Finally, ammonium assimilation routes producing aspartate or leucine were introduced. Our study reveals the high flexibility of the cellular amination network, both in terms of transaminase promiscuity and adaptability to new connections and ammonium entry points.

An Arsenic-76 radiotracer to study the routes of assimilation, hemolymph distribution, and tissue inventories in the bioindicator organism Pomacea canaliculata

The aim of this work was to study the absorption, distribution through the hemolymph, and bioaccumulation of arsenic by the freshwater Pomacea canaliculata using a short-lived tracer (76As, t1/2: 1.07 d) with high specific activity. Arsenic travels mainly dissolved in the plasma of the snail's hemolymph. This element is transferred from the hemolymph to the tissues (87%) 4 h after the inoculation of 50 μL of a 0.04 g/L of 76As radiotracer solution, being the digestive gland, kidney, and head-foot the main places of arsenical inventories. Snails exhibited a rapid arsenic accumulation response in a wide range of concentrations (from 1 to 1000 μg/L) of the metalloid dissolved in water and in a concentration-dependent manner. Also, snails incorporated As from the digestive system when they received a single safe dose of ~2 μg of 76As inoculated in a fish food pellet. The (semi) physiologically based toxicokinetic model developed in this study is based on anatomical and physiological parameters (blood flow, irrigation, tissue volume and other). Together, these findings make P. canaliculata an excellent sentinel organism to evaluate freshwater bodies naturally contaminated with As.

Engineering the Reductive Glycine Pathway: A Promising Synthetic Metabolism Approach for C1-Assimilation.

In recent years the reductive glycine pathway (rGlyP) has emerged as a promising pathway for the assimilation of formate and other sustainable C1-feedstocks for future biotechnology. It was originally proposed as an attractive "synthetic pathway" to support formatotrophic growth due to its high ATP efficiency, linear structure, and limited overlap with native pathways in most microbial hosts. Here, we present the current state of research on this pathway including breakthroughs on its engineering. Different variants of the rGlyP are discussed, including its core module for formate to glycine conversion, as well as varying modules for substrate conversion to formate, and glycine assimilation routes. Very recently, the rGlyP has been successfully implemented for synthetic formatotrophic growth, as well as for growth on methanol, in some bacterial hosts. We discuss the engineering strategies employed in these studies, including growth-coupled selection of functional pathway modules. We also compare the rGlyP to other natural and synthetic C1-assimilation pathways. Finally, we provide an outlook on open challenges and opportunities for the rGlyP, including its engineering into more biotechnological hosts, as well as the still-to-be realized production of value-added chemicals via this pathway. We expect that further research on the rGlyP will support the efficient use of sustainable C1-substrates in bioproduction.

Engineering a Highly Efficient Carboligase for Synthetic One-Carbon Metabolism.

One of the biggest challenges to realize a circular carbon economy is the synthesis of complex carbon compounds from one-carbon (C1) building blocks. Since the natural solution space of C1–C1 condensations is limited to highly complex enzymes, the development of more simple and robust biocatalysts may facilitate the engineering of C1 assimilation routes. Thiamine diphosphate-dependent enzymes harbor great potential for this task, due to their ability to create C–C bonds. Here, we employed structure-guided iterative saturation mutagenesis to convert oxalyl-CoA decarboxylase (OXC) from Methylobacterium extorquens into a glycolyl-CoA synthase (GCS) that allows for the direct condensation of the two C1 units formyl-CoA and formaldehyde. A quadruple variant MeOXC4 showed a 100 000-fold switch between OXC and GCS activities, a 200-fold increase in the GCS activity compared to the wild type, and formaldehyde affinity that is comparable to natural formaldehyde-converting enzymes. Notably, MeOCX4 outcompetes all other natural and engineered enzymes for C1–C1 condensations by more than 40-fold in catalytic efficiency and is highly soluble in Escherichia coli. In addition to the increased GCS activity, MeOXC4 showed up to 300-fold higher activity than the wild type toward a broad range of carbonyl acceptor substrates. When applied in vivo, MeOXC4 enables the production of glycolate from formaldehyde, overcoming the current bottleneck of C1–C1 condensation in whole-cell bioconversions and paving the way toward synthetic C1 assimilation routes in vivo.

Malyl-CoA lyase provides glycine/glyoxylate synthesis in type I methanotrophs.

The biochemical routes for assimilation of one-carbon compounds in bacteria require many clarifications. In this study, the role of malyl-CoA lyase in the metabolism of the aerobic type I methanotroph Methylotuvimicrobium alcaliphilum 20Z has been investigated by gene inactivation and biochemical studies. The functionality of the enzyme has been confirmed by heterologous expression in Escherichia coli. The mutant strain lacking Mcl activity demonstrated the phenotype of glycine auxotrophy. The genes encoding malyl-CoA lyase are present in the genomes of all methanotrophs, except for representatives of the phylum Verrucomicrobium. We suppose that malyl-CoA lyase is the enzyme that provides glyoxylate and glycine synthesis in the type I methanotrophs supporting carbon assimilation via the serine cycle in addition to the major ribulose monophosphate cycle.

An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli

Engineering biotechnological microorganisms to use methanol as a feedstock for bioproduction is a major goal for the synthetic metabolism community. Here, we aim to redesign the natural serine cycle for implementation in E. coli. We propose the homoserine cycle, relying on two promiscuous formaldehyde aldolase reactions, as a superior pathway design. The homoserine cycle is expected to outperform the serine cycle and its variants with respect to biomass yield, thermodynamic favorability, and integration with host endogenous metabolism. Even as compared to the RuMP cycle, the most efficient naturally occurring methanol assimilation route, the homoserine cycle is expected to support higher yields of a wide array of products. We test the in vivo feasibility of the homoserine cycle by constructing several E. coli gene deletion strains whose growth is coupled to the activity of different pathway segments. Using this approach, we demonstrate that all required promiscuous enzymes are active enough to enable growth of the auxotrophic strains. Our findings thus identify a novel metabolic solution that opens the way to an optimized methylotrophic platform.

An Aldolase-Catalyzed New Metabolic Pathway for the Assimilation of Formaldehyde and Methanol To Synthesize 2-Keto-4-hydroxybutyrate and 1,3-Propanediol in Escherichia coli.

Formaldehyde (HCHO) is an important intermediate in the metabolism of one-carbon (C1) compounds such as methanol, formate, and methane. The ribulose monophosphate (RuMP) pathway is the most-studied HCHO assimilation route and the 3-hexulose-6-phosphate synthase (Hps) plays an important role for HCHO fixation. In this study, we proposed and selected a pyruvate-dependent aldolase to channel HCHO into 2-keto-4-hydroxybutyrate as an important intermediate for biosynthesis. By combining this reaction with three further enzymes we demonstrated a pyruvate-based C1 metabolic pathway for biosynthesis of the appealing compound 1,3-propanediol (1,3-PDO). This novel pathway is first confirmed in vitro using HCHO and pyruvate as substrates. It is then demonstrated in vivo in E. coli for 1,3-PDO production from HCHO and methanol with glucose as a cosubstrate. This de novo pathway has several decisive advantages over the known metabolic pathways for 1,3-PDO: (1) C1 carbon is directly channeled into a precursor of 1,3-PDO; (2) the use of pyruvate as an acceptor of HCHO is glycerol-independent, circumventing thus the need of coenzyme B12 as cofactor for glycerol dehydration; (3) the pathway is much shorter and more simple than the recently proposed l-homoserine-dependent pathway, thus avoiding complicated regulations involving precursors for essential amino acids. In addition to proof-of-concept we further improved the host strain by deleting a gene (frmA) responsible for the conversion of HCHO to formate, thereby increasing the production of 1,3-PDO from 298.3 ± 11.4 mg/L to 508.3 ± 9.1 mg/L and from 3.8 mg/L to 32.7 ± 0.8 mg/L with HCHO and methanol as cosubstrate of glucose fermentation, respectively. This work is the first study demonstrating a genetically engineered E. coli that can directly use HCHO or methanol for the synthesis of 2-keto-4-hydroxybutyrate and its further conversion to 1,3-PDO.

Macrobenthic invertebrates as tracers of rare earth elements in freshwater watercourses

Rare earth elements (REEs) are emergent contaminants in aquatic ecosystems in parallel with their growing use in science, technology, and industry. In this study we measured the concentration of 16 REEs in freshwater marcobenthic invertebrates from 6 watercourses in northeast Italy to determine their potential use as ecological tracers of REEs in aquatic ecosystems. The total REE concentration at the sampling sites followed this order: site 6 (7.05 mg Kg−1) > site 3 (5.76 mg Kg−1) > site 4 (3.58 mg Kg−1) > site 1 (3.0 mg Kg−1) > site 5 (2.36 mg Kg−1) > site 2 (1.95 mg Kg−1). There were no significant differences in REE concentrations across the six samplings sites (Kruskal Wallis test, p = 0.1773), but two (site 3 and 6) had higher amount of REEs and were classified with the ecological status “Moderate” sensu Water Framework Directive since affected by anthropogenic activities. Light REE were always greater than heavy REE concentrations at all six sites. A positive correlation was observed between certain REEs (La, Ce, Gd) and the density of genera Caenis and Baetis (Ehemeroptera, collector-gatherers) (ρS range 0.795–0.812), suggesting that non-predatory macrobenthic invertebrates accumulate more REEs than predatory organisms and that the intake of sediment is the most effective route of assimilation.

Assimilating cloudy and rainy microwave observations from SAPHIR on board Megha Tropiques within the ARPEGE global model

The Megha‐Tropiques satellite was launched in 2011 with a microwave sounder called SAPHIR onboard. This instrument probes the atmosphere with six channels around the 183.31 GHz water vapour absorption band. Its observations are sensitive to water vapour as well as to hydrometeors. This instrument was proven to be useful for data assimilation by different numerical weather prediction centres, in particular for clear‐sky assimilation. At Météo‐France, SAPHIR observations have been routinely assimilated in clear sky since 2015 in the ARPEGE global model. The present article introduces a framework to complement this clear‐sky assimilation route by a new cloudy and rainy assimilation route for satellite microwave brightness temperatures. This framework is based on several steps including a Bayesian inversion of the SAPHIR brightness temperatures into relative humidity retrievals, which are then assimilated within the ARPEGE global model. This study presents the methodology of assimilation, including the development of two error models, one for the Bayesian inversion, and one for the observation errors of relative humidity retrievals within the ARPEGE 4D‐Var data assimilation system. The forecast scores obtained with this methodology over a three‐month period indicate a positive impact of SAPHIR cloudy and rainy observations within the ARPEGE system, in particular on tropical temperature and wind forecasts for which the improvements range from 0.5 to 1.7% on standard deviations with respect to the ECMWF analysis and up to a +60 h lead time.

Factors determining accumulation of bisphenol A and alkylphenols at a low trophic level as exemplified by mussels Mytilus trossulus

The aim of the study was to investigate abiotic and biotic factors influencing the accumulation of endocrine disrupting compounds (EDCs) such as bisphenol A (BPA), 4-tert-octylphenol (OP) and 4-nonylphenol (NP) in mussels Mytilus trossulus from the Gulf of Gdansk (Southern Baltic). The key abiotic factor influencing BPA, OP and NP accumulation in mussels is their hydrophilicity/lipophilicity, which affects their main assimilation routes - by digestive tract for the more lipophilic OP and NP, and additionally by the gills for the less lipophilic BPA. As a result, high condition index (i.e. higher soft tissue weight) is more often correlated with high concentrations of OP and NP in mussels than with BPA. Furthermore, alkylphenols have 6–8 times greater accumulative potential than BPA. Concentration of the studied compounds was lower in females than in males following spawning, and the effect lasted longer for BPA than for alkylphenols. The influence of season and hydrological conditions on BPA, OP, NP in the mussel was more pronounced than the proximity of external sources of these compounds. An increase in water temperature in summer probably stimulated the solubility of BPA, the least lipophilic of the studied compounds, and led to increased assimilation of this compound from water (through gills). On the other hand, high OP and NP concentrations in mussels occurred in spring, which was caused by increased surface run-off and sediments resuspension.

D‐Xylose assimilation via the Weimberg pathway by solvent‐tolerant Pseudomonas taiwanensis VLB120

The natural ability of Pseudomonas taiwanensis VLB120 to use xylose as sole carbon and energy source offers a high potential for sustainable industrial biotechnology. In general, three xylose assimilation routes are reported for bacteria. To elaborate the metabolic capacity of P. taiwanensis VLB120 and to identify potential targets for metabolic engineering, an in silico/in vivo experiment was designed, allowing for discrimination between these pathways. Kinetics of glucose and xylose degradation in P. taiwanensis VLB120 was determined and the underlying stoichiometry was investigated by genome-based metabolic modelling and tracer studies using stable isotope labelling. Additionally, reverse transcription quantitative polymerase chain reaction experiments have been performed to link physiology to the genomic inventory. Based on in silico experiments, a labelling strategy was developed, ensuring a measurable and unique (13) C-labelling distribution in proteinogenic amino acids for every possible distribution between the different xylose metabolization routes. A comparison with in vivo results allows the conclusion that xylose is metabolized by P. taiwanensis VLB120 via the Weimberg pathway. Transcriptomic and physiological studies point to the biotransformation of xylose to xylonate by glucose dehydrogenase. The kinetics of this enzyme is also responsible for the preference of glucose as carbon source by cells growing in the presence of glucose and xylose.

Inorganic Nitrogen Nutrition Enhances Osmotic Stress Tolerance in Phaseolus vulgaris: Lessons from a Drought-sensitive Cultivar

The nitrogen (N) requirement of legumes can be met by inorganic N assimilation and symbiotic N2 fixation. Both N assimilation routes have diverse influences on drought tolerance because of the differences in the use of energy and the reduction in power characteristics of those pathways. We investigated N nutrition under osmotic stress and likely criteria conferring tolerance to osmotic stress in common bean. Osmotic stress tolerance in a drought-sensitive common bean cultivar (Coco blanc) was compared with beans relying on N nutrition, N2 fixation, or fertilization with urea. Osmotic stress was applied by means of 25 (mild stress), 50 (moderate stress), or 75 (severe stress) mm mannitol for 15 days. At the end of the stress period, relative water content, total N contents, total soluble sugars, total chlorophyll, total protein, and potassium were assayed. Urea-fed plants grew better and had better tolerance to osmotic stress. This was attributed to maintaining higher N use efficiency, better leaf hydration, and adequate osmoregulation.

Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae

BackgroundTo effectively convert lignocellulosic feedstocks to bio-ethanol anaerobic growth on xylose constitutes an essential trait that Saccharomyces cerevisiae strains normally do not adopt through the selective integration of a xylose assimilation route as the rate of ATP-formation is below energy requirements for cell maintenance (mATP). To enable cell growth extensive evolutionary and/or elaborate rational engineering is required. However the number of available strains meeting demands for process integration are limited. In this work evolutionary engineering in just two stages coupled to strain selection under strict anaerobic conditions was carried out with BP10001 as progenitor. BP10001 is an efficient (Yethanol = 0.35 g/g) but slow (qethanol = 0.05 ± 0.01 g/gBM/h) xylose-metabolizing recombinant strain of Saccharomyces cerevisiae that expresses an optimized yeast-type xylose assimilation pathway.ResultsBP10001 was adapted in 5 generations to anaerobic growth on xylose by prolonged incubation for 91 days in sealed flasks. Resultant strain IBB10A02 displayed a specific growth rate μ of 0.025 ± 0.002 h-1 but produced large amounts of glycerol and xylitol. In addition growth was strongly impaired at pH below 6.0 and in the presence of weak acids. Using sequential batch selection and IBB10A02 as basis, IBB10B05 was evolved (56 generations). IBB10B05 was capable of fast (μ = 0.056 ± 0.003 h-1; qethanol = 0.28 ± 0.04 g/gBM/h), efficient (Yethanol = 0.35 ± 0.02 g/g), robust and balanced fermentation of xylose. Importantly, IBB10A02 and IBB10B05 displayed a stable phenotype. Unlike BP10001 both strains displayed an unprecedented biphasic formation of glycerol and xylitol along the fermentation time. Transition from a glycerol- to a xylitol-dominated growth phase, probably controlled by CO2/HCO3-, was accompanied by a 2.3-fold increase of mATP while YATP (= 87 ± 7 mmolATP/gBM) remained unaffected. As long as glycerol constituted the main by-product energetics of anaerobic growth on xylose and glucose were almost identical.ConclusionsIn just 61 generation IBB10B05, displaying ~530% improved strain fitness, was evolved from BP10001. Its excellent xylose fermentation properties under industrial relevant conditions were proven and rendered it competitive. Based on detailed analysis of growth energetics we showed that mATP was predominantly determined by the type of polyol formed rather than, as previously assumed, substrate-specific.

Systems-Level Analysis of Nitrogen Starvation-Induced Modifications of Carbon Metabolism in a Chlamydomonas reinhardtii Starchless Mutant

To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall-deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation-induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10(2)) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicated increased succinate, malate, and Glc-6-P and decreased Fru-1,6-bisphosphate, illustrating the effect of these changes. Comparisons of independent data sets in multiple strains allowed the delineation of a sequence of events in the global N starvation response in C. reinhardtii, starting within minutes with the upregulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual upregulation of genes encoding enzymes of TAG synthesis. Finally, genome resequencing analysis indicated that (1) the deletion in sta6 extends into the neighboring gene encoding respiratory burst oxidase, and (2) a commonly used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype.

Comparison of One- and Two-dimensional Liquid Chromatography Approaches in the Label-free Quantitative Analysis of Methylocella silvestris

The proteome of the bacterium Methylocella silvestris has been characterized using reversed phase ultra high pressure liquid chromatography (UPLC) and two-dimensional reversed phase (high pH)-reversed phase (low pH) UPLC prior to mass spectrometric analysis. Variations in protein expression levels were identified with the aid of label-free quantification in a study of soluble protein extracts from the organism grown using methane, succinate, or propane as a substrate. The number of first dimensional fractionation steps has been varied for 2D analyses, and the impact on data throughput and quality has been demonstrated. Comparisons have been made regarding required experimental considerations including total loading of biological samples required, instrument time, and resulting data file sizes. The data obtained have been evaluated with respect to number of protein identifications, confidence of assignments, sequence coverage, relative levels of proteins, and dynamic range. Good qualitative and quantitative agreement was observed between the different approaches, and the potential benefits and limitations of the reversed phase-reversed phase UPLC technique in label-free analysis are discussed. A preliminary screen of the protein regulation data has also been performed, providing evidence for a possible propane assimilation route.

The “Brazilian” Bikini-Shaped Lip-Reduction Technique: New Developments in Cheiloplasty

The lips, one of the most illustrious facial features, have a key role in forming facial expressions. In the past, hypertrophic lips were an aesthetic issue among certain ethnic groups. Although full lips are a desirable feature sought by many people, the current world of fashion tends emphasize equilibrium and significant matching of features, encouraging people to seek refinement through cosmetic surgery. The purpose is to reach the standard level of perceived attractiveness in current society. This article aims to present a novel lip reduction technique that restores an attractive labial contour by shifting the shape toward a more "Brazilian way" and resulting in more aesthetically appealing lips. The technique described in this report was performed on more than 40 patients between 2008 and 2010. The major difference between this technique and others is that it transforms the shape of the bikini lines to a more "Brazilian" way. The upper resection is more conservative, whereas the lower resection is less triangular and more curved, displaying more of the bilateral "bands" on the lips. The reported patients did not present any infections or any other complications. The nature of human beings urges them to seek routes of assimilation into their society. This also applies to the rules of perceived attractiveness. The technique presented in this article has recaptured specific attention to the resultant lip contour, altering the shape of the marks on the lower lip. The new technique yields a harmonious relationship between the upper and lower lips. The aesthetic results and patient satisfaction attained through this novel technique of lip reduction have been very satisfactory. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors at www.springer.com/00266.

Oxalyl-Coenzyme A Reduction to Glyoxylate Is the Preferred Route of Oxalate Assimilation in Methylobacterium extorquens AM1

Oxalate catabolism is conducted by phylogenetically diverse organisms, including Methylobacterium extorquens AM1. Here, we investigate the central metabolism of this alphaproteobacterium during growth on oxalate by using proteomics, mutant characterization, and (13)C-labeling experiments. Our results confirm that energy conservation proceeds as previously described for M. extorquens AM1 and other characterized oxalotrophic bacteria via oxalyl-coenzyme A (oxalyl-CoA) decarboxylase and formyl-CoA transferase and subsequent oxidation to carbon dioxide via formate dehydrogenase. However, in contrast to other oxalate-degrading organisms, the assimilation of this carbon compound in M. extorquens AM1 occurs via the operation of a variant of the serine cycle as follows: oxalyl-CoA reduction to glyoxylate and conversion to glycine and its condensation with methylene-tetrahydrofolate derived from formate, resulting in the formation of C3 units. The recently discovered ethylmalonyl-CoA pathway operates during growth on oxalate but is nevertheless dispensable, indicating that oxalyl-CoA reductase is sufficient to provide the glyoxylate required for biosynthesis. Analysis of an oxalyl-CoA synthetase- and oxalyl-CoA-reductase-deficient double mutant revealed an alternative, although less efficient, strategy for oxalate assimilation via one-carbon intermediates. The alternative process consists of formate assimilation via the tetrahydrofolate pathway to fuel the serine cycle, and the ethylmalonyl-CoA pathway is used for glyoxylate regeneration. Our results support the notion that M. extorquens AM1 has a plastic central metabolism featuring multiple assimilation routes for C1 and C2 substrates, which may contribute to the rapid adaptation of this organism to new substrates and the eventual coconsumption of substrates under environmental conditions.

Generation of Se‐fortified broccoli as functional food: impact of Se fertilization on S metabolism

Selenium (Se)-fortified broccoli (Brassica oleracea var. italica) has been proposed as a functional food for cancer prevention, based on its high glucosinolate (GSL) content and capacity for Se accumulation. However, as selenate and sulphate share the initial assimilation route, Se fertilization could interfere with sulphur metabolism and plant growth. Consequently, GSL accumulation could be compromised. To evaluate these potentially adverse effects of Se fertilization, we performed a comprehensive study on sand-grown young broccoli plants (weekly selenate applications of 0.8 µmol plant(-1) via the root) and field-grown adult broccoli plants during head formation (single foliar selenate application: 25.3 or 253 µmol plant(-1) ). The results show that under these conditions, Se application does not affect plant growth, contents of cysteine, glutathione, total GSL, glucoraphanin (major aliphatic GSL) or the expression of BoMYB28 (encoding a functionally confirmed master regulator for aliphatic GSL biosynthesis). Conversely, due to the changed expression of sulphate transporters (BoSULTR1;1, 1;2, 2;1, and 2;2), sulphate and total S contents increased in the shoot of young plants while decreasing in the root. We conclude that broccoli can be fertilized with Se without reduction in GSL content, even with Se accumulation exceeding the level recommended for human consumption.