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Next-generation bioprocesses of a future bio-based economy will rely on a flexible mix of readily available feedstocks. Renewable energy can be used to generate sustainable CO 2 -derived substrates. Metabolic engineering already enables the functional implementation of different pathways for the assimilation of C1 substrates in various microorganisms. In addition to feedstocks, the benchmark for all future bioprocesses will be sustainability, including the avoidance of CO 2 emissions. Here we review recent advances in the utilization of C1-compounds from different perspectives, considering both strain and bioprocess engineering technologies. In particular, we evaluate methanol as a co-feed for enabling the CO 2 emission-free production of acetyl-CoA-derived compounds. The possible metabolic strategies are analyzed using stoichiometric modeling combined with thermodynamic analysis and prospects for industrial-scale implementation are discussed., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

L-glutamate (Glu) is the major excitatory transmitter in mammalian brain. Inadequate concentration of Glu in the brain correlates to mood disorder. In industry, Glu is used as a flavour enhancer in food and in foodstuff processing. A high concentration of Glu has several effects on human health such as hypersensitive effects, headache and stomach pain. The presence of Glu in food can be detected by different analytical methods based on chromatography, or capillary electrophoresis or amperometric techniques. We have isolated and characterized a glutamate-binding protein (GluB) from the Gram-positive bacteria Corynebacterium glutamicum. Together with GluC protein, GluD protein and the cytoplasmic protein GluA, GluB permits the transport of Glu in/out of cell. In this study, we have investigated the binding features of GluB as well as the effect of temperature on its structure both in the absence and in the presence of Glu. The results have showed that GluB has a high affinity and selectivity versus Glu (nanomolar range) and the presence of the ligand induces a higher thermal stability of the protein structure., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Secondary plant metabolites are extensively used in today's food industries, for example, as coloring-agents, flavouring-agents or texturizing agents. In particular, metabolites with antioxidative properties find applications as preservatives or anti-browning agents. Today, extraction from plant material represents the major source of these metabolites, but progress in the field of metabolic engineering also enabled the microbial production of these valuable compounds as a more economic and ecological alternative. This review article presents the current state of metabolic engineering of microorganisms for production of plant metabolites with applications in the food industries. We focus on compounds, which are already used in food applications, discuss current limitations of microbial plant metabolite production, and outline strategies on how these challenges can be addressed in the future., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biosensors offer exciting possibilities for improving cells or enzymes as biocatalysts for the synthesis of small molecules. We here review recent progress in the development and the screening applications of transcription-factor-based biosensors. An example is a cofactor-dependent biosensor which provides a generalizable screen for NADPH-dependent enzymes. Another example is the use of a biosensor in combination with recombineering for strain development, thereby expanding the genome engineering techniques to deliver directly bacteria producing small molecules of interest. Biosensor-based techniques in combination with fluorescence-activated cell sorting demonstrate that the gap regarding throughput capabilities of existing methods for the generation of genetic diversity and methods for the subsequent screening can be closed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The engineering of microbial strains for the production of small molecules of biotechnological interest is a time-consuming, laborious and expensive process. This can be mostly attributed to the fact that good producers cannot be readily obtained by high-throughput screening approaches since increased product formation usually does not confer a clear phenotype to producing strain variants. Recently, advances were made in the design and construction of genetically encoded RNA aptamer-based or transcription factor-based biosensors for detecting small molecules at the single-cell level. The first promising examples for the application of these molecular biosensors in combination with fluorescent-activated cell sorting as a high-throughput screening device demonstrated the value and potential of these new tools for microbial strain development., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Many ligase-independent cloning methods have been developed to overcome problems of standard restriction cloning such as low transformation efficiency and high background of vector with no insert. Most of these methods are still enzyme based, require time-consuming incubation and multiple purification steps, and/or might have a low robustness in handling. Thus, with the aim to establish a robust enzyme/ligase-free method, we developed the phosphorothioate-based ligase-independent gene cloning (PLICing) method, which is based on a chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution. After optimization of polymerase chain reaction (PCR) and DNA cleavage conditions, PLICing performs competitively with all commercialized methods in terms of handling and transformation efficiency. In addition, PLICing is absolutely sequence independent and surpasses other concepts regarding cloning efficiency given that none of the 240 analyzed clones showed any religation event for three different model genes. A developed fast PLICing protocol does not require any purification step and can be completed within 10 min. Due to its robustness, reliability, and simplicity, PLICing should prove to be a true alternative to other well-established cloning techniques., (Copyright 2010 Elsevier Inc. All rights reserved.)

Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., omega6 in AA or omega3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (omega6), EPA (omega3), or DHA (omega3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor.

Despite advances in screening technologies, only a very small fraction of theoretical protein sequence can be sampled in directed evolution experiments. At the current state of random mutagenesis technologies mutation frequencies have often been adjusted to values that cause a limited number of amino acid changes (often one to four amino acid changes per protein). For harvesting the power of directed evolution algorithms it is therefore important that generated mutant libraries are rich in diversity and enriched in active population. Insufficient knowledge about protein traits, mutational robustness of protein folds and technological limitations in diversity generating methods are main challenges for managing the complexity of protein sequence space. This review covers computational and experimental advances for high quality mutant library generation that have been achieved in the past two years.

It was of interest to determine the impact of FDG-PET/CT on general therapy management and radiotherapy (RT) planning in patients with stage IV head and neck tumours. The study was conducted prospectively between March 2006 and March 2007 in 35 patients with histologically confirmed, locally advanced squamous cell carcinomas of the head and neck. Prior to primary radiochemotherapy, whole-body and head/neck FDG-PET/CT was performed. The FDG-PET information was integrated into RT planning. By comparison with anatomical imaging, the FDG-PET/CT yielded the following additional information: distant metastases in 17.1% (6/35), second primary tumours in 11.4% (4/35), and changes in nodal status based on metabolic activity, i.e. upstaging in 34.3% (12/35) or downstaging in 22.9% (8/35). As a result, treatment strategy was changed from curative to palliative in six patients, and additional curative therapy was implemented following exclusion of distant metastases in two patients with a simultaneous local second primary tumour. The discordant nodal status found with head/neck FDG-PET/CT compared with anatomical imaging led to modification of radiotherapy volume and dose in 20 patients (57.1%). From the radiotherapist's perspective FDG-PET/CT is therefore useful and justifiable in the management of stage IV head and neck tumours.

We report the clinical, structural, functional and genetic characterization of a 37-year-old Caucasian female, presenting as a sporadic case of complicated spastic paraplegia with thin corpus callosum (CC), cognitive impairment, amyotrophy of the hand muscles and a sensorimotor neuropathy and review the literature for spastic paraplegia with thin CC. Magnetic resonance imaging (MRI) examination revealed a thin CC with fronto-parietal cortical atrophy. 18Fluordesoxyglucose positron emission tomography (FDG-PET) showed reduced cortical and thalamic metabolism. By transcranial magnetic stimulation, we delineated a severe impairment of transcallosal inhibition. Sequence analysis did not reveal disease causing mutations in the genes SLC12A6 (Andermann), Spastin (SPG 4), BSCL2 (SPG 17) and Spartin (SPG 20). We reviewed the literature for HSP with thin CC and found 113 HSP patients with thin CC previously described (35 with linkage to chromosome 15q13-15). Thin CC and peripheral neuropathy often appear together in spastic paraplegia and might be indicative for combined degeneration mechanism of central and peripheral axons.

To assess the role of NOD2/CARD15 variants on the long-term outcome of allogeneic stem cell transplantation in a genetically homogeneous group, we extended our previous study (cohort I, n = 78) and typed DNA for NOD2/CARD15 single nucleotide polymorphisms (SNPs) from an additional 225 recipients and their HLA-identical sibling donors (cohort II) treated at four other European centers. Results of genotyping were compared with clinical outcome. The strong association of NOD2/CARD15 variants with transplantation-related mortality (TRM) was confirmed in univariate and multivariate analysis; TRM increased from 20% in cohort I/22% in cohort II in recipient/donor pairs without any NOD2/CARD15 variants to 47% in cohort I/32% in cohort II in the presence of one variant in either donor or recipient and further to 57% in cohort I/74% in cohort II in the presence of 2 or more variants (P < .002 in both cohorts). NOD2/CARD15 SNPs were not associated with relapse rate but had a strong impact on overall survival. In an analysis of center effects, the type of gastrointestinal decontamination was the only factor interfering with the prognostic significance of NOD2/CARD15 SNPs. Our data further support an interaction between gastrointestinal defense mechanisms, activation of the innate immune system, and specific transplant-related complications.

Background: Clinical as well as neurophysiological and neuroimaging data suggest that chronic tinnitus resembles neuropsychiatric syndromes characterized by focal brain activation. Low-frequency repetitive transcranial magnetic stimulation (rTMS) has been proposed as an efficient method in treating brain hyperexcitability disorders by reducing cortical excitability., Methods: In three patients suffering from chronic tinnitus, the effect of magnetic resonance imaging and positron emission tomography guided neuronavigated 1 Hz rTMS on auditory cortex activity was evaluated, using a sham controlled double-blind crossover design., Results: Two of three patients revealed clearly increased metabolic activity in circumscript areas of the primary auditory cortex (PAC), allowing a selective stimulation of these cortical areas with low-frequency rTMS. Considerable improvement in tinnitus was achieved in these patients., Conclusions: Neuronavigated rTMS of increased PAC activity may help to better understand the neuronal basis of chronic tinnitus and might offer a new option for treating auditory phantom perceptions like chronic tinnitus.

Cardiovascular morbidity and mortality is high in patients following renal transplantation. The present analysis assessed major cardiovascular risk factors and estimated the risk of coronary artery disease in the largest present-day comparative trial of tacrolimus vs. microemulsified cyclosporine A. In this 6-month study, 557 patients were randomly allocated to therapy with tacrolimus (n = 286) or cyclosporine A (n = 271) concomitantly with azathioprine and corticosteroids. The primary endpoint was the incidence of and time to acute rejection. Blood pressure, serum cholesterol, HDL cholesterol, triglycerides, and blood glucose were measured at baseline, and at months 1, 3, and 6. Ten-year risk of coronary heart disease was estimated according to the Framingham risk algorithm. Tacrolimus resulted in significantly lower summary measures (time-weighted average) of serum cholesterol (p = 0.0004) and mean arterial blood pressure (p = 0.0156), but in a higher summary measure of blood glucose (p = 0.0028) than cyclosporine. The summary measure of serum triglycerides was not different between treatment groups (p = 0.368). The mean 10-year coronary artery disease risk estimate was significantly lowered in men (p = 0.0032) treated with tacrolimus, but was unchanged in women. Tacrolimus and cyclosporine A microemulsion exert a compound-specific impact on cardiovascular risk factors and appear to affect the predicted rate of cardiovascular morbidity in different manners.

A detailed mathematical analysis of the diffusion process of neurotransmitter inside the synaptic cleft is presented and the spatio-temporal concentration profile is calculated. Using information about the experimentally observed time course of glutamate in the cleft the effective diffusion coefficient Dnet is estimated as Dnet approximately 20-50 nm(2) microseconds(-1), implying a strong reduction compared with free diffusion in aqueous solution. The tortuosity of the cleft and interactions with transporter molecules are assumed to affect the transmitter motion. We estimate the transporter density to be 5170 to 8900 micrometer(-2) in the synaptic cleft and its vicinity, using the experimentally observed time constant of glutamate. Furthermore a theoretical model of synaptic transmission is presented, taking the spatial distribution of post-synaptic (AMPA-) receptors into account. The transmitter diffusion and receptor dynamics are modeled by Monte Carlo simulations preserving the typically observed noisy character of post-synaptic responses. Distributions of amplitudes, rise and decay times are calculated and shown to agree well with experiments. Average open probabilities are computed from a novel kinetic model and are shown to agree with averages over many Monte Carlo runs. Our results suggest that post-synaptic currents are only weakly potentiated by clustering of post-synaptic receptors, but increase linearly with the total number of receptors. Distributions of amplitudes and rise times are used to discriminate between different morphologies, e.g. simple and perforated synapses. A skew in the miniature amplitude distribution can be caused by multiple release of pre-synaptic vesicles at perforated synapses., (Copyright 1999 Academic Press.)

We present a minimal kinetic model for excitatory synaptic transmission to cerebellar Purkinje cells. The main components are a kinetic model for a single glutamate receptor, which is calibrated with the help of patch clamp data, and a mean field approximation for the dynamics of a population of channels, which generate an EPSC. The resulting minimal model of the parallel fiber-Purkinje cell synapse is used to estimate the dynamics of glutamate in the synaptic cleft and to clarify the role of receptor desensitization in synaptic transmission. We also apply the model to different aspects of synaptic modulation, like long-term depression and potentiation by pharmacological application of ampakines. In the framework of the minimal model these effects can be understood as the result of modified receptor kinetics., (Copyright 1997 Academic Press Limited.)

In order to define which of selected ECG variables could indicate irreversibly impaired myocardial function in chronic aortic regurgitation 54 patients were stratified according to normal (> or = 50%; Group A, n = 41) or subnormal radionuclide left ventricular ejection fraction (LVEF < 50%; Group B, n = 13) late after aortic valve replacement. Preoperatively, Group B patients had a significantly greater QRS duration, greater R-peak time (RPT) prolongation in I, V5 or V6, greater RPT relative to the S-peak time of the maximum S in V1, V2 or V3 (R-peak delay) and a greater negative T-wave in I or V6, as compared with Group A. These ECG variables together with preoperative angiocardiographic LVEF and end-systolic volume index were subjected to stepwise linear discriminant analysis. The maximum RPT, angio-LVEF and the maximum RPT relative to the S-peak time of the maximum S in V1, V2 or V3 emerged as the most promising variables. Of of Group A patients 82.9% and 84.6% of Group B patients were correctly classified by the three variables. If applied separately, APT prolongation or the presence of the R-peak delay in the left-sided leads, although less sensitive, have reasonably high specificity as risk indicators of irreversibly impaired chamber function, their positive predictive value being 60 and 62.5%, respectively. In conjunction with preoperative LVEF the diagnostic contribution of the two ECG variables amounts to the greatest overall separation of postoperatively preserved from irreversibly impaired systolic function.